**Adult Human T Cell Leukemia**

Jean-Philippe Herbeuval

*CNRS UMR 8147, Université Paris Descartes France* 

#### **1. Introduction**

Human T cell leukemia virus 1 (HTLV-1), the first characterized human retrovirus, has been identified as the causative agent for adult T-cell leukemia/lymphoma (ATLL). This aggressive lymphoid proliferation is associated with a bad prognosis due to the resistance of HTLV-1-infected cells to most classical chemotherapeutic agents.

HTLV-1 is transmitted intravenously, by sexual contact, or through breast-feeding from mother to child, and epidemiological evidence predicts that ATLL development occurs following childhood infection. ATLL exhibits diverse clinical features: the acute, the subacute or smoldering, the chronic forms and the ATL lymphoma. In the two most aggressive forms (acute leukemia and lymphoma), the tumor syndrome comprises massive lymphadenopathy, hepatosplenomegaly, lytic bone lesions and multiple visceral lesions with skin and lung infiltration.

HTLV-1 virions infect CD4+ T cells, which represent the main target for HTLV-1 infection in peripheral blood. HTLV-1 associated diseases occur after long periods of virus latency. For years it has been thought that unlike other retroviruses, free virions were poorly infectious. However, a recent study reported that freshly isolated myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) are efficiently and productively infected by cell-free HTLV-1. Furthermore, infected mDC and pDC were able to transfer virions to autologous CD4+ T cells, clearly demonstrating that cell free HTLV-1 can be infectious and target dendritic cells. Innate immune response against HTLV-1 is poorly documented.

We describe here immune response against HTLV-1 and physiological consequences.

#### **2. The Human T cell leukemia virus 1 (HTLV-1)**

In 1980 the group of Robert C. Gallo characterized the first human retrovirus, the Human T cell leukemia virus 1 (HTLV-1) (Poiesz, Ruscetti et al., 1980). This virus was recovered from the peripheral blood cells of a patient suffering from adult T-cell-leukemia/lymphoma (ATLL). This form of leukemia is a severe T-cell lymphoma proliferation with bad prognostic due to the resistance of HTLV-1-infected cells to most classical chemotherapeutic agents.

We first describe here the epidemiology, the genomic of HTLV-1 virus and its receptor complex.

#### **2.1 HTLV-1 genomic characteristics**

HTLV-1 is classified as a complex retrovirus, in the genus delta-retrovirus of the subfamily Orthoretrovirinae. HTLV-1 retrovirus genetic material is composed by a

Adult Human T Cell Leukemia 3

serologic is confirmed by immunoblot of specific antibodies and polymerase chain reaction

The number of HTLV-1 infected people is elevated and the most recent studies estimated at 15-20 millions people infected worldwide (Verdonck, Gonzalez et al., 2007). Epidemiologic studies revealed that density of infected individuals were in Malaysia, Caribbean, Africa (Gabon, Cameroun), South America (Brazil, Guyana, Colombia) and South Japan (Figure 2). However, these numbers are only estimations and probably do not reflect the reality. Indeed, most of infected people are not diagnosed due to the complex and expensive methods of diagnosis. Thus, number of people really infected might be higher especially in

Among the HTLV-1-infected population, around 3 to 6% develop the ATLL syndrome. HTLV-1 infection is highly concentrated in some regions especially in South Japan where the prevalence can reach as high as 37% in a selected population (Mueller, Okayama et al., 1996; Yamaguchi, 1994). The reasons for HTLV-1 clustering, the high ubiquity in southwestern Japan but low prevalence in neighboring regions of Korea, China and Eastern Russia are still unknown. For nonendemic geographic regions, HTLV-1 is mainly found in immigrants. The contamination is largely due to sexual contacts with sex workers. However, the prevalence in Europe and North America remains extremly low and does not exceed

(PCR) of genomic DNA from cells of infected patients.

0,01% (Proietti, Carneiro-Proietti et al., 2005).

Fig. 2. Origin, spread, and prevalence of HTLV-1.

developed countries.

diploid single strain RNA (Figure 1). The length of the HTLV-1 genome is 9.032 basepair (bp). The group antigens are similar to other retroviruses-(gag), polymerase (pol), and envelope (env) genes are flanked by long terminal repeats (LTR). The LTR consists of U3, R and U5 regions. The U3 region of HTLV-1 controls the virus transcription. It contains essential elements such as the TATA box, which is necessary for viral transcription, a sequence that causes termination and polyadenylation of the RNA messenger and Tax responsive elements (TRE) involved in Tax protein transcription which regulates the transcription of the HTLV-1 provirus. The R region overlaps the 3´ of the U3 region and contains the majority of the Rex response element. The "gag" gene encodes the virus core protein, which is initially synthesized with approximatively molecular weight of 53 kD. During viral maturation this precursor is cleaved to form the matured matrix P19 (MA), the capsid P24 (CA) and the nucleocapsid P15 (NC).

HTLV-1 virions are enveloped into a lipidic membrane and a nucleocapsid that protect the genetic material, the ribonucleic acid (RNA). The lipidic membrane is derived from cellular plasma membrane. The envelope proteins are constituted by the glycoprotein (gp) 21 (Transmembrane subunit, TM) and gp46 (Surface subunit, SU), which are coded by env and are integrated to the lipidic membrane. Matrix protein p19 and p24 are coded by gag and constitute the intern core of viral envelope. The nucleocapsid p15 is also coded by gag and is enveloping the genetic material composed of a diploid single stranded RNA (Figure 1).

Fig. 1. Genomic and proteic structure of HTLV-1 virion.

#### **2.2 HTLV-1 epidemiology**

Over the course of more than 30 years, the epidemiology of HTLV-1 has matured. Epidemiologic studies are based on serologic diagnosis by detection of specific antibodies using enzyme-linked immunosorbent assay (ELISA) or by agglutination assay. Thus, the

diploid single strain RNA (Figure 1). The length of the HTLV-1 genome is 9.032 basepair (bp). The group antigens are similar to other retroviruses-(gag), polymerase (pol), and envelope (env) genes are flanked by long terminal repeats (LTR). The LTR consists of U3, R and U5 regions. The U3 region of HTLV-1 controls the virus transcription. It contains essential elements such as the TATA box, which is necessary for viral transcription, a sequence that causes termination and polyadenylation of the RNA messenger and Tax responsive elements (TRE) involved in Tax protein transcription which regulates the transcription of the HTLV-1 provirus. The R region overlaps the 3´ of the U3 region and contains the majority of the Rex response element. The "gag" gene encodes the virus core protein, which is initially synthesized with approximatively molecular weight of 53 kD. During viral maturation this precursor is cleaved to form the matured matrix P19 (MA),

HTLV-1 virions are enveloped into a lipidic membrane and a nucleocapsid that protect the genetic material, the ribonucleic acid (RNA). The lipidic membrane is derived from cellular plasma membrane. The envelope proteins are constituted by the glycoprotein (gp) 21 (Transmembrane subunit, TM) and gp46 (Surface subunit, SU), which are coded by env and are integrated to the lipidic membrane. Matrix protein p19 and p24 are coded by gag and constitute the intern core of viral envelope. The nucleocapsid p15 is also coded by gag and is enveloping the genetic material composed of a diploid single stranded RNA (Figure 1).

Over the course of more than 30 years, the epidemiology of HTLV-1 has matured. Epidemiologic studies are based on serologic diagnosis by detection of specific antibodies using enzyme-linked immunosorbent assay (ELISA) or by agglutination assay. Thus, the

the capsid P24 (CA) and the nucleocapsid P15 (NC).

Fig. 1. Genomic and proteic structure of HTLV-1 virion.

**2.2 HTLV-1 epidemiology** 

serologic is confirmed by immunoblot of specific antibodies and polymerase chain reaction (PCR) of genomic DNA from cells of infected patients.

The number of HTLV-1 infected people is elevated and the most recent studies estimated at 15-20 millions people infected worldwide (Verdonck, Gonzalez et al., 2007). Epidemiologic studies revealed that density of infected individuals were in Malaysia, Caribbean, Africa (Gabon, Cameroun), South America (Brazil, Guyana, Colombia) and South Japan (Figure 2). However, these numbers are only estimations and probably do not reflect the reality. Indeed, most of infected people are not diagnosed due to the complex and expensive methods of diagnosis. Thus, number of people really infected might be higher especially in developed countries.

Among the HTLV-1-infected population, around 3 to 6% develop the ATLL syndrome. HTLV-1 infection is highly concentrated in some regions especially in South Japan where the prevalence can reach as high as 37% in a selected population (Mueller, Okayama et al., 1996; Yamaguchi, 1994). The reasons for HTLV-1 clustering, the high ubiquity in southwestern Japan but low prevalence in neighboring regions of Korea, China and Eastern Russia are still unknown. For nonendemic geographic regions, HTLV-1 is mainly found in immigrants. The contamination is largely due to sexual contacts with sex workers. However, the prevalence in Europe and North America remains extremly low and does not exceed 0,01% (Proietti, Carneiro-Proietti et al., 2005).

Fig. 2. Origin, spread, and prevalence of HTLV-1.

Adult Human T Cell Leukemia 5

polysaccharide side chains confer to HSPG members electrostatic properties that allow binding to a very large range of proteins, including cytokines, receptors, hormones, chemokines and extracellular matrix proteins. HSPG enhances infection by facilitating the attachment of the particles on target cells and/or allowing their clustering at the cell surface before specific interactions between viral proteins and their receptors that lead to fusion. HSPG had been showed to bind the HIV-1 protein gp120, therefore facilitating HIV-1 infection. Studies demonstrated that inhibition of HSPG dramatically reduced syncitium formation and infection in CD4+ T cells (Lambert, Bouttier et al., 2009). Furthermore, inhibition of HSPG also reduced infection of dendritic cells. Thus, a model involving three

More recently, one study proposed another model for HTLV-1 entry into target cells (Pais-Correia, Sachse et al.). This model proposes that HTLV-1-infected T lymphocytes transiently store viral particles as carbohydrate-rich extracellular assemblies. These carbohydrate assemblies are attached to cell surface and held together by virally-induced extracellular matrix components. This extracellular matrix is made of protein such as collagen, agrin, galectin-3 and tetherin. It should be noted that HSPG is probably a protein of the HTLV-1 extracellular assemblies. This kind of structure was first discovered for bacteria and called "biofilm". Authors showed that extracellular HTLV-1 biofilms adhere to other cells facilitating viral binding and infection. This form of viral infection is extremely efficient due

Thus, HTLV-1 may use several strategies to infect target cells. However, further studies are

partners had been proposed (Figure 3).

Fig. 3. Model for HTLV-1 receptor complex.

to high concentration of extracellular viruses on cell surface.

needed to clarify the entry of HTLV-1 in patients.

From Ghez, Lepelletier et al., 2006.

HTLV-1 is transmitted intravenously, by sexual contact, or through breast-feeding from mother to child, and epidemiological evidence predicts that ATLL development occurs following childhood infection. Mother to child transmission occurs very frequently (around 20%) and is related to mother viral load and prolonged breast-feeding. Indeed, it is now well accepted that HTLV-1 could be transmitted through mother's milk and is one of the major factor in vertical transmission. Thus, screening of HTLV-1 among blood donors had been extended and breast-feeding among HTLV-1-infected women had been refrained in Japan decreasing vertical transmission.

Finally, it is also possible that HTLV-1 could be transmitted by saliva, which contains HTLV-1 antibodies and proviral DNA. However, there is no clear study demonstrating this way of contamination (Fujino and Nagata, 2000).

Origin and spread hypothesis based on phylogenetic and anthropological data. HTLV-1 originated in African primates and migrated to Asia where it evolved into STLV-1. This early STLV-1 lineage spread to India, Japan, Indonesia, and back to Africa (arrows 1). It crossed the simian–human barrier in Indonesian human beings who migrated to Malesia, resulting in the HTLV-1c subtype (arrows 2). In Africa, STLV-1 evolved through several interspecies transmissions into HTLV-1a, HTLV-1b, and HTLV-1d, HTLV-1e, and HTLV-1f (arrows 3). Because of the slave trade and increased mobility, HTLV-1a was introduced in the New World, Japan, the Middle East, and North Africa (arrows 4). Colours indicate current prevalence estimates based on population surveys and on studies in pregnant women and blood donors. In some countries, HTLV-1 infection is limited to certain population groups or areas. (Verdonck, Gonzalez et al., 2007).

#### **2.3 HTLV-1 receptor complex**

For years the HTLV-1 receptor remained unknown and a real mystery. Serious evidences indicated that HTLV-1 entry requires the viral envelope glycoprotein (Env), the surface subunit gp46 and the transmembrane subunit gp21, generated from the clivage of a precursor gp61. Mutation in any of this proteins or use of blocking antibodies dramatically reduced HTLV-1 infection. Thus, one study demonstrated that glucose transporter GLUT1 was the receptor for HTLV-1 (Manel, Kim et al., 2003). GLUT1 matched all requirement for HTLV-1 entry. GLUT1 is overexpressed by activated T cells, which are targets of HTLV-1. Using small interfering RNA siRNA strategy they demonstrated that downregulation of GLUT1 in cell lines reduced HTLV-1 infection. Furthermore, GLUT1 transfection of GLUT1 negative cells restored HTLV-1 infection, demonstrating that GLUT1 is an essential component of HTLV-1 receptor. More recently, it has been suggested that two other molecules are involved in HTLV-1 infection of target cells: neuropilin 1 (NRP-1) and Heparan Sulfate Proteoglycans (HSPG) (Ghez, Lepelletier et al.).

The Neuropilin-1 was initially identified as a embryonic neurons guidance factor. NRP-1 is a glycoprotein receptor for Semaphorin 3a and VEGF (Vascular endothelial growth factor). It also has been showed that NRP-1 was a key molecule in angiogenesis and is also implicated in the regulation of immune response (Tordjman, Lepelletier et al., 2002). It has been showed that NRP-1 directly binds HTLV-1 virus. The interaction appeared functionnaly relevant since NRP-1 overexpression enhanced syncytium formation *in vitro*. Furthermore, confocal analysis revealed a strong polarisation of NRP-1 and viral glycoprotein Env at the interface of an infected cell and a target T cell (Ghez, Lepelletier et al., 2006).

HSPG family members are composed of a core protein associated with one or several sulphated polysaccharide side chains (i.e. sulfate glycosaminoglycans). Sulphated

HTLV-1 is transmitted intravenously, by sexual contact, or through breast-feeding from mother to child, and epidemiological evidence predicts that ATLL development occurs following childhood infection. Mother to child transmission occurs very frequently (around 20%) and is related to mother viral load and prolonged breast-feeding. Indeed, it is now well accepted that HTLV-1 could be transmitted through mother's milk and is one of the major factor in vertical transmission. Thus, screening of HTLV-1 among blood donors had been extended and breast-feeding among HTLV-1-infected women had been refrained in Japan

Finally, it is also possible that HTLV-1 could be transmitted by saliva, which contains HTLV-1 antibodies and proviral DNA. However, there is no clear study demonstrating this

Origin and spread hypothesis based on phylogenetic and anthropological data. HTLV-1 originated in African primates and migrated to Asia where it evolved into STLV-1. This early STLV-1 lineage spread to India, Japan, Indonesia, and back to Africa (arrows 1). It crossed the simian–human barrier in Indonesian human beings who migrated to Malesia, resulting in the HTLV-1c subtype (arrows 2). In Africa, STLV-1 evolved through several interspecies transmissions into HTLV-1a, HTLV-1b, and HTLV-1d, HTLV-1e, and HTLV-1f (arrows 3). Because of the slave trade and increased mobility, HTLV-1a was introduced in the New World, Japan, the Middle East, and North Africa (arrows 4). Colours indicate current prevalence estimates based on population surveys and on studies in pregnant women and blood donors. In some countries, HTLV-1 infection is limited to certain

For years the HTLV-1 receptor remained unknown and a real mystery. Serious evidences indicated that HTLV-1 entry requires the viral envelope glycoprotein (Env), the surface subunit gp46 and the transmembrane subunit gp21, generated from the clivage of a precursor gp61. Mutation in any of this proteins or use of blocking antibodies dramatically reduced HTLV-1 infection. Thus, one study demonstrated that glucose transporter GLUT1 was the receptor for HTLV-1 (Manel, Kim et al., 2003). GLUT1 matched all requirement for HTLV-1 entry. GLUT1 is overexpressed by activated T cells, which are targets of HTLV-1. Using small interfering RNA siRNA strategy they demonstrated that downregulation of GLUT1 in cell lines reduced HTLV-1 infection. Furthermore, GLUT1 transfection of GLUT1 negative cells restored HTLV-1 infection, demonstrating that GLUT1 is an essential component of HTLV-1 receptor. More recently, it has been suggested that two other molecules are involved in HTLV-1 infection of target cells: neuropilin 1 (NRP-1) and

The Neuropilin-1 was initially identified as a embryonic neurons guidance factor. NRP-1 is a glycoprotein receptor for Semaphorin 3a and VEGF (Vascular endothelial growth factor). It also has been showed that NRP-1 was a key molecule in angiogenesis and is also implicated in the regulation of immune response (Tordjman, Lepelletier et al., 2002). It has been showed that NRP-1 directly binds HTLV-1 virus. The interaction appeared functionnaly relevant since NRP-1 overexpression enhanced syncytium formation *in vitro*. Furthermore, confocal analysis revealed a strong polarisation of NRP-1 and viral glycoprotein Env at the interface

HSPG family members are composed of a core protein associated with one or several sulphated polysaccharide side chains (i.e. sulfate glycosaminoglycans). Sulphated

decreasing vertical transmission.

**2.3 HTLV-1 receptor complex** 

way of contamination (Fujino and Nagata, 2000).

population groups or areas. (Verdonck, Gonzalez et al., 2007).

Heparan Sulfate Proteoglycans (HSPG) (Ghez, Lepelletier et al.).

of an infected cell and a target T cell (Ghez, Lepelletier et al., 2006).

polysaccharide side chains confer to HSPG members electrostatic properties that allow binding to a very large range of proteins, including cytokines, receptors, hormones, chemokines and extracellular matrix proteins. HSPG enhances infection by facilitating the attachment of the particles on target cells and/or allowing their clustering at the cell surface before specific interactions between viral proteins and their receptors that lead to fusion. HSPG had been showed to bind the HIV-1 protein gp120, therefore facilitating HIV-1 infection. Studies demonstrated that inhibition of HSPG dramatically reduced syncitium formation and infection in CD4+ T cells (Lambert, Bouttier et al., 2009). Furthermore, inhibition of HSPG also reduced infection of dendritic cells. Thus, a model involving three partners had been proposed (Figure 3).

Fig. 3. Model for HTLV-1 receptor complex. From Ghez, Lepelletier et al., 2006.

More recently, one study proposed another model for HTLV-1 entry into target cells (Pais-Correia, Sachse et al.). This model proposes that HTLV-1-infected T lymphocytes transiently store viral particles as carbohydrate-rich extracellular assemblies. These carbohydrate assemblies are attached to cell surface and held together by virally-induced extracellular matrix components. This extracellular matrix is made of protein such as collagen, agrin, galectin-3 and tetherin. It should be noted that HSPG is probably a protein of the HTLV-1 extracellular assemblies. This kind of structure was first discovered for bacteria and called "biofilm". Authors showed that extracellular HTLV-1 biofilms adhere to other cells facilitating viral binding and infection. This form of viral infection is extremely efficient due to high concentration of extracellular viruses on cell surface.

Thus, HTLV-1 may use several strategies to infect target cells. However, further studies are needed to clarify the entry of HTLV-1 in patients.

Adult Human T Cell Leukemia 7

basophilic cytoplasm, a multilobed nucleus with a flower shape (Figure 4). ATL cells express most of T cell markers (CD2, CD3, CD4, CD45RO) and more rarely CD8. However, ATL cells also express the alpha chain of IL-2 receptor, CD25, and T cell activation markers such as major histocompatibility complex HLA-DR and HLA-DQ. ATL is well known to infiltrate various organs and tissues, such as the skin, lungs, liver, gastrointestinal tract, central nervous system and bone. This infiltrative tendency of leukemic cells is possibly attributable to the expression of various surface molecules, such as chemokine receptors and adhesion molecules. Skin-homing memory T-cells uniformly express CCR4, and its ligands are thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). CCR4 is expressed on most ATL cells. In addition, TARC and MDC are expressed in skin lesions in ATL patients. Thus, CCR4 expression should be implicated in the skin infiltration (Yoshie, 2005). On the other hand, CCR7 expression is associated with lymph node involvement (Kohno, Moriuchi et al., 2000). OX40 is a member of the tumor necrosis factor family and was reported to be expressed on ATL cells (Imura, Hori et al., 1997;

Fig. 4. Typical "flower cell" in the peripheral blood of an acute ATL patient observed on microscope**.** In the peripheral blood of an acute ATL patient, leukemic cells with

Adult T cell leukemia is an agressive malignant disease that results from HTLV-1 infection. The prognostic is directly correlated to the ATL subtype. Since ATL is a neoplasm of mature T cells, it has been treated with chemotherapies for non-Hodgkin's lymphoma. Conventional chemoterapy (LSG15) had only transcient effect and the prognostic remains poor. The

Therefore, new therapeutic strategies were tested to adapt treatment to ATL subtype reviewed by Kimiharu Uozumi (Uozumi). New strategies can be divided in 3 main groups:

Among new chemical anti-tumor agents the combined use of anti-retroviral drug AZT and recombinant interferon alpha (IFN-) showed promising results (Hermine, Bouscary et al., 1995). The MST was increased in most patients and this therapy constitutes one of the most

Kunitomi, Hori et al., 2002).

multilobulated nuclei (Matsuoka, 2005).

median survival time (MST) never exceeded 10 month.

chemical anti-tumor agents, monoclonal antibodies and vaccination.

**3.4 Treatment of ATL** 

efficient at the present time.

#### **3. The adult T-cell leukemia/lymphoma (ATLL)**

Adult T cell leukemia/lymphoma (ATLL) had been shown to be a consequence of HTLV-1 infection (Hinuma, Gotoh et al., 1982). HTLV-1 infection is also responsible for myelopathy/tropical spastic paraparesis (HAM/TSP) (de The, Gazzolo et al., 1985; Gessain, Barin et al., 1985), uveitis and infective dermatitis in children (Manns et al., 1999). We focus in this section on the complex T-cell leukemia/lymphoma induced by HTLV-1 infection.

#### **3.1 HTLV-1 induces T cell leukemia**

In the late seventies, a group of leukemia patients with characteristic clinical features and particular geographical distribution were identified. Uchiyama *et al* proposed adult T cell leukemia (ATL) as a new disease. In 1980 the group of Robert C. Gallo characterized the first human retrovirus responsible for ATL, the Human T cell leukemia virus 1 (HTLV-1) (Poiesz, Ruscetti et al., 1980).

Since then, HTLV-1 has been identified as the causative agent for two major syndromes: the adult T-cell leukemia (ATL) (Poiesz, Ruscetti et al., 1981; Robert-Guroff, Nakao et al., 1982) and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Jacobson, 1996). More recently, HTLV-1 also has been shown as the causative agent for uveitis and infective dermatitis in children (Manns, Miley et al., 1999). Among the HTLV-1-infected population, around 3 to 6% develop the ATL syndrome. At the present time, it is not known why some infected patients develop ATL and others do not.

#### **3.2 ATL syndrome**

The ATL exhibits diverse clinical features and outcome is directly correlated to ATL subtype. The malignancy ranges from a very indolent and slowly progressive lymphoma to a very aggressive and nearly uniformaly lethal proliferative lymphoma. The incidence of ATL is estimated to be 61/100,000 HTLV-1 carriers, and the crude lifetime risk for developing ATL is 7.3% for males and 3.8% for females. Four types of ATL had been described based on the number of abnormal T cells in peripheral blood, tumor lesions in organs, serum lactic acid dehydrogenase (LDH) level and clinical course: the sub-acute or smoldering (5%), the acute (55%), the chronic forms (20%) and the ATL lymphoma (20%).

Patients with smoldering ATL exhibit skin lesions, minimal lymph node enlargement and few leukemic cells in blood. Chronic ATL is characterized by mild symptoms and longer clinical course. In the two most aggressive forms (acute leukemia and lymphoma), the tumor syndrome comprises massive lymphadenopathy, hepatosplenomegaly, lytic bone lesions and multiple visceral lesions with skin and lung infiltration. Acute ATL is also characterized by general malaise, fever, cough, high lactate deshydrogenase (LDH) serum levels, and appearance of multilobulated nuclei leukemic cells. This aggressive lymphoid proliferation is associated with a bad prognosis due to the resistance of HTLV-1-infected cells to most classical chemotherapeutic agents. The cancer is thought to be due to the prooncogenic effect of viral DNA incorporated into host lymphocytes DNA, and chronic stimulation of the lymphocytes at the cytokine level may play a role in development of malignancy. The time between infection and onset of cancer also varies geographically. It is believed to be about sixty years in Japan, and less than forty years in the Caribbean.

#### **3.3 The ATL cell**

The ATL cell is easily characterized by histological and/or cytological infiltration by flower cells (Matsuoka, 2005) that are malignant activated lymphocytes with convoluted nuclei and

Adult T cell leukemia/lymphoma (ATLL) had been shown to be a consequence of HTLV-1 infection (Hinuma, Gotoh et al., 1982). HTLV-1 infection is also responsible for myelopathy/tropical spastic paraparesis (HAM/TSP) (de The, Gazzolo et al., 1985; Gessain, Barin et al., 1985), uveitis and infective dermatitis in children (Manns et al., 1999). We focus in this section on the complex T-cell leukemia/lymphoma induced by HTLV-1 infection.

In the late seventies, a group of leukemia patients with characteristic clinical features and particular geographical distribution were identified. Uchiyama *et al* proposed adult T cell leukemia (ATL) as a new disease. In 1980 the group of Robert C. Gallo characterized the first human retrovirus responsible for ATL, the Human T cell leukemia virus 1 (HTLV-1) (Poiesz,

Since then, HTLV-1 has been identified as the causative agent for two major syndromes: the adult T-cell leukemia (ATL) (Poiesz, Ruscetti et al., 1981; Robert-Guroff, Nakao et al., 1982) and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Jacobson, 1996). More recently, HTLV-1 also has been shown as the causative agent for uveitis and infective dermatitis in children (Manns, Miley et al., 1999). Among the HTLV-1-infected population, around 3 to 6% develop the ATL syndrome. At the present time, it is not known

The ATL exhibits diverse clinical features and outcome is directly correlated to ATL subtype. The malignancy ranges from a very indolent and slowly progressive lymphoma to a very aggressive and nearly uniformaly lethal proliferative lymphoma. The incidence of ATL is estimated to be 61/100,000 HTLV-1 carriers, and the crude lifetime risk for developing ATL is 7.3% for males and 3.8% for females. Four types of ATL had been described based on the number of abnormal T cells in peripheral blood, tumor lesions in organs, serum lactic acid dehydrogenase (LDH) level and clinical course: the sub-acute or smoldering (5%), the acute (55%), the chronic forms (20%) and the ATL lymphoma (20%). Patients with smoldering ATL exhibit skin lesions, minimal lymph node enlargement and few leukemic cells in blood. Chronic ATL is characterized by mild symptoms and longer clinical course. In the two most aggressive forms (acute leukemia and lymphoma), the tumor syndrome comprises massive lymphadenopathy, hepatosplenomegaly, lytic bone lesions and multiple visceral lesions with skin and lung infiltration. Acute ATL is also characterized by general malaise, fever, cough, high lactate deshydrogenase (LDH) serum levels, and appearance of multilobulated nuclei leukemic cells. This aggressive lymphoid proliferation is associated with a bad prognosis due to the resistance of HTLV-1-infected cells to most classical chemotherapeutic agents. The cancer is thought to be due to the prooncogenic effect of viral DNA incorporated into host lymphocytes DNA, and chronic stimulation of the lymphocytes at the cytokine level may play a role in development of malignancy. The time between infection and onset of cancer also varies geographically. It is

believed to be about sixty years in Japan, and less than forty years in the Caribbean.

The ATL cell is easily characterized by histological and/or cytological infiltration by flower cells (Matsuoka, 2005) that are malignant activated lymphocytes with convoluted nuclei and

**3. The adult T-cell leukemia/lymphoma (ATLL)** 

why some infected patients develop ATL and others do not.

**3.1 HTLV-1 induces T cell leukemia** 

Ruscetti et al., 1980).

**3.2 ATL syndrome** 

**3.3 The ATL cell** 

basophilic cytoplasm, a multilobed nucleus with a flower shape (Figure 4). ATL cells express most of T cell markers (CD2, CD3, CD4, CD45RO) and more rarely CD8. However, ATL cells also express the alpha chain of IL-2 receptor, CD25, and T cell activation markers such as major histocompatibility complex HLA-DR and HLA-DQ. ATL is well known to infiltrate various organs and tissues, such as the skin, lungs, liver, gastrointestinal tract, central nervous system and bone. This infiltrative tendency of leukemic cells is possibly attributable to the expression of various surface molecules, such as chemokine receptors and adhesion molecules. Skin-homing memory T-cells uniformly express CCR4, and its ligands are thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). CCR4 is expressed on most ATL cells. In addition, TARC and MDC are expressed in skin lesions in ATL patients. Thus, CCR4 expression should be implicated in the skin infiltration (Yoshie, 2005). On the other hand, CCR7 expression is associated with lymph node involvement (Kohno, Moriuchi et al., 2000). OX40 is a member of the tumor necrosis factor family and was reported to be expressed on ATL cells (Imura, Hori et al., 1997; Kunitomi, Hori et al., 2002).

Fig. 4. Typical "flower cell" in the peripheral blood of an acute ATL patient observed on microscope**.** In the peripheral blood of an acute ATL patient, leukemic cells with multilobulated nuclei (Matsuoka, 2005).

#### **3.4 Treatment of ATL**

Adult T cell leukemia is an agressive malignant disease that results from HTLV-1 infection. The prognostic is directly correlated to the ATL subtype. Since ATL is a neoplasm of mature T cells, it has been treated with chemotherapies for non-Hodgkin's lymphoma. Conventional chemoterapy (LSG15) had only transcient effect and the prognostic remains poor. The median survival time (MST) never exceeded 10 month.

Therefore, new therapeutic strategies were tested to adapt treatment to ATL subtype reviewed by Kimiharu Uozumi (Uozumi). New strategies can be divided in 3 main groups: chemical anti-tumor agents, monoclonal antibodies and vaccination.

Among new chemical anti-tumor agents the combined use of anti-retroviral drug AZT and recombinant interferon alpha (IFN-) showed promising results (Hermine, Bouscary et al., 1995). The MST was increased in most patients and this therapy constitutes one of the most efficient at the present time.

Adult Human T Cell Leukemia 9

infection determines the preferential infection of dendritic cell subsets but these

Furthermore, the proportion of blood and secondary lymphoid organs HTLV-1+ DC is proportional to the total proviral DNA load in the blood, providing a correlation of proviral DNA load and the frequency of effector/memory Tax- CD8+ T cells (Nagai,

HTLV Tax oncogene may be released and act as a cytokine on neighboring cells in the CNS inducing NF-kB nuclear localization and immunoglobulin light chain, IL-2Ra, IL-1b, IL-6, TNF-, and TNF- expression (Lindholm et al., 1990; Lindholm et al., 1992; Marriott et al., 1992; Dhib-Jalbut et al., 1994). The cytokine like effects of Tax may induce a signaling

Interestingly, one of the members of the interferon regulatory factor (IRF) family – IRF-4 – was shown to be highly expressed in cells derived from patients with ATL and in HTLV-1 infected cell lines (Imaizumi, Kohno et al., 2001; Mamane, Grandvaux et al., 2002; Mamane, Loignon et al., 2005; Sharma, Grandvaux et al., 2002; Sharma, Mamane et al., 2000; Yamagata et al., 1996). A detailed analysis of IRF-4 has implicated the viral Tax protein in mediating activation of the Sp1, NF-kB and NF-AT pathways leading to a feed back loop mediated by Tax (Grumont and Gerondakis, 2000; Sharma, Grandvaux et al., 2002; Sharma, Mamane et

Induction of tumor cell death by apoptotic molecules is one of strategy that could be used to selectively reduce cancer cell proliferation without damaging normal tissue. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a TNF superfamily member, has been shown to induce apoptosis of the vast majority of tumor cell lines (Wiley, Schooley et al., 1995). TRAIL-induced apoptosis is finely regulated by the expression of two groups of receptors. Three receptors do not induce apoptosis (Decoy Receptors, DcR) and two activate apoptosis of target cells (Death Receptor 4 and 5, DR4, DR5) (Sheikh, Burns et al., 1998; Sheridan, Marsters et al., 1997; Wu, Burns et al., 1997). The two biologically active forms of TRAIL, membrane-bound (mTRAIL) and soluble TRAIL (sTRAIL), are regulated by type I interferon (interferon-alpha and beta: IFN- and IFN-)(Ehrlich, Infante-Duarte et al., 2003; Sato, Hida et al., 2001; Tecchio, Huber et al., 2004). TRAIL active form consist of a trimer stabilized by a zinc molecule. TRAIL is secreted by leukocytes, including T lymphocytes (Kayagaki, Yamaguchi et al., 1999), natural killer cells (Smyth, Cretney et al., 2001), dendritic cells (Vidalain, Azocar et al., 2000; Vidalain, Azocar et al., 2001), monocytes and macrophages (Herbeuval, Lambert et al., 2003). TRAIL can activate both intrinsic or extrinsic apoptosis pathway. DR4 and DR5 induced apoptosis through the formation of a death inducing signaling complex (DISC) containing the death receptor, adaptor proteins such as Fas-associated death domain (FADD), and initiator caspases such as pro-caspase-8 or pro-caspase-10 (Bellail, Tse et al., 2009; Jin, Kurakin et al., 2004; Walczak and Haas, 2008). Consequently, pro-caspase-8 or pro-caspase-10 are activated by autoproteolytic processing, which then cleave and activate downstream effector caspases (Gomez-Benito, Martinez-Lorenzo et al., 2007), such as caspase-3 (extrinsic pathway). Additionally, the Bcl-2 interacting protein Bid is also cleaved by caspase-8. Truncated-Bid causes the loss of mitochondrial membrane potential and caspase-9 cleavage, resulting in apoptosis. Very little is known concerning death and decoy receptors regulation. Among these receptors, death

cascade by binding to a specific cell surface receptor (DC1 and/or DC2 NP-1).

consequences of event remain unknown.

**4.2 ATL and TRAIL-induced apoptosis** 

Kubota et al., 2001).

al., 2000).

Similarly the combined use of arsenic trioxid and interferon alpha exhibits an anti-leukemia effect in very poor prognosis ATL patients despite a significant toxicity (Hermine, Dombret et al., 2004).

Treatment using monoclonal antibodies and recombinant cytokines are also very promising. We will describe later in section 4 the use of TNF-related Apoptosis Inducing Ligand (TRAIL), a Tumor Necrosis Factor (TNF) superfamily member (Wiley, Schooley et al., 1995), as a new therapeutical strategy to induce ATL cells apoptosis.

#### **4. HTLV-1 and immune response**

HTLV-1 is a retrovirus and therefore is recognised by the immune system as foreign agent. Immune system is activated after infection and produce specific anti-HTL-1 antibodies. Most of immune cells respond to HTLV-1 virions. However, because symptoms occur after a long period of latency it is extremely hard to study acute infection. Thus, most of immunologic studies are performed using samples from patients infected since several years. We review in this section the interactions between immune cells and HTLV-1 and provide some new features concerning innate immune response.

#### **4.1 Immune cell activation by HTLV-1**

HTLV-1 like HIV-1 is a retrovirus and induces a chronic disease. Although a large number of studies have indicated that initial virus infection involves majority viral invasion of CD4+ T cells, which represent an important target for HTLV-1 infection in the peripheral blood, additional evidence has demonstrated that HTLV-1 can infect several additional cellular compartments *in vivo*, including CD8+ T lymphocytes, monocytes, dendritic cells, B lymphocytes residing in the peripheral blood and lymphoid organs or resident central nervous system (CNS) astrocytes (Koyanagi, Itoyama et al., 1993; Macatonia, Cruickshank et al., 1992; Nagai, Kubota et al., 2001; Richardson, Edwards et al., 1990).

Transient phase of reverse transcription of viral RNA is followed by a persistent phase of clonal expansion within the CD4+ and CD8+ T cell populations (Mansky, 2000; Mortreux, Leclercq et al., 2001). Very little viral gene expression and low amounts of infectious virus production of HTLV-1 infected monocyte/macrophage lineage and dendritic cells are likely attributable to their postmitotic status and relatively short lifetime (Banchereau and Steinman, 1998; Valledor, Borras et al., 1998).

This differential viral gene expression between T and dendritic cells depending of viral clonal expansion and expression drives the HTLV-1 immune response. Although dendritic cells have a low level of viral gene expression, recent evidence has suggested that HTLV-1 infected dendritic cells exhibit an enhance capacity to stimulate antigen-specific T cell activation (Makino, Shimokubo et al., 1999). Furthermore, the Th1-type cytokines IL-1b, interferon- (IFN-), and TNF- were overexpressed in asymptomatic carriers and patients with HAM/TSP, while the Th2/Th3-type cytokine transformin growth factor (TGF-) was overexpressed in patients with ATL (Tendler, Greenberg et al., 1991). Several events may lead to stimulation of a Th1 or a Th2/Th3 T cell response besides the subset of dendritic cells (DC1 and DC2) that are first encountered antigen, or depending of the pathogen, recognition receptors and site of exposure (Pulendran, Palucka et al., 2001). Furthermore, the type of T cell response is dependent of both DC ontogeny, but also of the dendritic cell activating stimulus (Grabbe, Kampgen et al., 2000). Consequently, the initial route of

Similarly the combined use of arsenic trioxid and interferon alpha exhibits an anti-leukemia effect in very poor prognosis ATL patients despite a significant toxicity (Hermine, Dombret

Treatment using monoclonal antibodies and recombinant cytokines are also very promising. We will describe later in section 4 the use of TNF-related Apoptosis Inducing Ligand (TRAIL), a Tumor Necrosis Factor (TNF) superfamily member (Wiley, Schooley et al., 1995),

HTLV-1 is a retrovirus and therefore is recognised by the immune system as foreign agent. Immune system is activated after infection and produce specific anti-HTL-1 antibodies. Most of immune cells respond to HTLV-1 virions. However, because symptoms occur after a long period of latency it is extremely hard to study acute infection. Thus, most of immunologic studies are performed using samples from patients infected since several years. We review in this section the interactions between immune cells and HTLV-1 and

HTLV-1 like HIV-1 is a retrovirus and induces a chronic disease. Although a large number of studies have indicated that initial virus infection involves majority viral invasion of CD4+ T cells, which represent an important target for HTLV-1 infection in the peripheral blood, additional evidence has demonstrated that HTLV-1 can infect several additional cellular compartments *in vivo*, including CD8+ T lymphocytes, monocytes, dendritic cells, B lymphocytes residing in the peripheral blood and lymphoid organs or resident central nervous system (CNS) astrocytes (Koyanagi, Itoyama et al., 1993; Macatonia, Cruickshank et

Transient phase of reverse transcription of viral RNA is followed by a persistent phase of clonal expansion within the CD4+ and CD8+ T cell populations (Mansky, 2000; Mortreux, Leclercq et al., 2001). Very little viral gene expression and low amounts of infectious virus production of HTLV-1 infected monocyte/macrophage lineage and dendritic cells are likely attributable to their postmitotic status and relatively short lifetime (Banchereau and

This differential viral gene expression between T and dendritic cells depending of viral clonal expansion and expression drives the HTLV-1 immune response. Although dendritic cells have a low level of viral gene expression, recent evidence has suggested that HTLV-1 infected dendritic cells exhibit an enhance capacity to stimulate antigen-specific T cell activation (Makino, Shimokubo et al., 1999). Furthermore, the Th1-type cytokines IL-1b, interferon- (IFN-), and TNF- were overexpressed in asymptomatic carriers and patients with HAM/TSP, while the Th2/Th3-type cytokine transformin growth factor (TGF-) was overexpressed in patients with ATL (Tendler, Greenberg et al., 1991). Several events may lead to stimulation of a Th1 or a Th2/Th3 T cell response besides the subset of dendritic cells (DC1 and DC2) that are first encountered antigen, or depending of the pathogen, recognition receptors and site of exposure (Pulendran, Palucka et al., 2001). Furthermore, the type of T cell response is dependent of both DC ontogeny, but also of the dendritic cell activating stimulus (Grabbe, Kampgen et al., 2000). Consequently, the initial route of

as a new therapeutical strategy to induce ATL cells apoptosis.

provide some new features concerning innate immune response.

al., 1992; Nagai, Kubota et al., 2001; Richardson, Edwards et al., 1990).

**4. HTLV-1 and immune response** 

**4.1 Immune cell activation by HTLV-1** 

Steinman, 1998; Valledor, Borras et al., 1998).

et al., 2004).

infection determines the preferential infection of dendritic cell subsets but these consequences of event remain unknown.

Furthermore, the proportion of blood and secondary lymphoid organs HTLV-1+ DC is proportional to the total proviral DNA load in the blood, providing a correlation of proviral DNA load and the frequency of effector/memory Tax- CD8+ T cells (Nagai, Kubota et al., 2001).

HTLV Tax oncogene may be released and act as a cytokine on neighboring cells in the CNS inducing NF-kB nuclear localization and immunoglobulin light chain, IL-2Ra, IL-1b, IL-6, TNF-, and TNF- expression (Lindholm et al., 1990; Lindholm et al., 1992; Marriott et al., 1992; Dhib-Jalbut et al., 1994). The cytokine like effects of Tax may induce a signaling cascade by binding to a specific cell surface receptor (DC1 and/or DC2 NP-1).

Interestingly, one of the members of the interferon regulatory factor (IRF) family – IRF-4 – was shown to be highly expressed in cells derived from patients with ATL and in HTLV-1 infected cell lines (Imaizumi, Kohno et al., 2001; Mamane, Grandvaux et al., 2002; Mamane, Loignon et al., 2005; Sharma, Grandvaux et al., 2002; Sharma, Mamane et al., 2000; Yamagata et al., 1996). A detailed analysis of IRF-4 has implicated the viral Tax protein in mediating activation of the Sp1, NF-kB and NF-AT pathways leading to a feed back loop mediated by Tax (Grumont and Gerondakis, 2000; Sharma, Grandvaux et al., 2002; Sharma, Mamane et al., 2000).

#### **4.2 ATL and TRAIL-induced apoptosis**

Induction of tumor cell death by apoptotic molecules is one of strategy that could be used to selectively reduce cancer cell proliferation without damaging normal tissue. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a TNF superfamily member, has been shown to induce apoptosis of the vast majority of tumor cell lines (Wiley, Schooley et al., 1995). TRAIL-induced apoptosis is finely regulated by the expression of two groups of receptors. Three receptors do not induce apoptosis (Decoy Receptors, DcR) and two activate apoptosis of target cells (Death Receptor 4 and 5, DR4, DR5) (Sheikh, Burns et al., 1998; Sheridan, Marsters et al., 1997; Wu, Burns et al., 1997). The two biologically active forms of TRAIL, membrane-bound (mTRAIL) and soluble TRAIL (sTRAIL), are regulated by type I interferon (interferon-alpha and beta: IFN- and IFN-)(Ehrlich, Infante-Duarte et al., 2003; Sato, Hida et al., 2001; Tecchio, Huber et al., 2004). TRAIL active form consist of a trimer stabilized by a zinc molecule. TRAIL is secreted by leukocytes, including T lymphocytes (Kayagaki, Yamaguchi et al., 1999), natural killer cells (Smyth, Cretney et al., 2001), dendritic cells (Vidalain, Azocar et al., 2000; Vidalain, Azocar et al., 2001), monocytes and macrophages (Herbeuval, Lambert et al., 2003). TRAIL can activate both intrinsic or extrinsic apoptosis pathway. DR4 and DR5 induced apoptosis through the formation of a death inducing signaling complex (DISC) containing the death receptor, adaptor proteins such as Fas-associated death domain (FADD), and initiator caspases such as pro-caspase-8 or pro-caspase-10 (Bellail, Tse et al., 2009; Jin, Kurakin et al., 2004; Walczak and Haas, 2008). Consequently, pro-caspase-8 or pro-caspase-10 are activated by autoproteolytic processing, which then cleave and activate downstream effector caspases (Gomez-Benito, Martinez-Lorenzo et al., 2007), such as caspase-3 (extrinsic pathway). Additionally, the Bcl-2 interacting protein Bid is also cleaved by caspase-8. Truncated-Bid causes the loss of mitochondrial membrane potential and caspase-9 cleavage, resulting in apoptosis. Very little is known concerning death and decoy receptors regulation. Among these receptors, death

Adult Human T Cell Leukemia 11

Fig. 5. TRAIL apoptotic pathway. Membrane (mTRAIL) or soluble TRAIL (sTRAIL) bind to 3 decoy receptors (DcR1, DcR2 and OPG) and 2 death receptors (DR4 and DR5) which

HTLV-1 targets CD4+ T cells which represent an important target for HTLV-1 infection in the peripheral blood. However, there are some additional evidence that showed that HTLV-1 can also infect including CD8+ T lymphocytes, monocytes, B lymphocytes, astrocytes (Richardson, Edwards et al., 1990) and dendritic cells (DC) *in vivo.* Myeloid dendritic cells do not exhibit high viral gene expression, but recent work suggested that HTLV-1-infected dendritic cells show better capacity to stimulate antigen-specific T cell activation (Makino, Shimokubo et al., 1999). Moreover, the proportion of lymphoid organs containing HTLV-1 positive dendritic cells is proportional to the total proviral DNA load in the blood, providing a correlation of proviral DNA load and the frequency of effector/memory Tax-

More recently, findings demonstrated a central role to myeloid dendritic (mDC) and plasmacytoid dendritic cells (pDC) in HTLV-1 infection. For years it has been thought that unlike other retroviruses such as HIV-1, free virions were poorly infectious (Donegan, Lee et al., 1994). However, a recent study reported that freshly isolated mDC and pDC are efficiently and productively infected by cell-free HTLV-1 (Jones, Petrow-Sadowski et al.,

activate the caspase pathway leading to apoptosis.

**4.3 Myeloid dendritic cells and HTLV-1 infection** 

CD8+ T cells (Nagai, Kubota et al., 2001).

receptor is the most studied, and authors showed that DR5 transcription is regulated (at least partially) by the protooncogene p53 (Sheikh, Burns et al., 1998; Wu, Burns et al., 1997). It should be noticed that TRAIL death receptors 4 and 5 not only induce apoptosis but may also play a crucial role in inflammatory responses (Collison, Foster et al., 2009). Figure 5 illustrates TRAIL pathway and regulation.

TRAIL is a very promising candidate for cancer treatment due to its sophisticated way of inducing apoptosis. While the vast majority of normal cells express decoy receptors and are therefore protected from TRAIL-mediated apoptosis, tumor cells generally express death receptors. Indeed, TRAIL induces apoptosis in human tumor cell lines (Griffith, Chin et al., 1998) but not in normal cells (Gura, 1997). TRAIL also induces apoptosis of infected cells. For example, plasma TRAIL has been reported to be an early pathogenic marker in acute HIV-1 infection and is correlated to viral load in chronic disease (Gasper-Smith, Crossman et al., 2008; Herbeuval, Nilsson et al., 2009). HIV-1 upregulates DR5 expression on the membrane of CD4+ T cells *in vitro* (Herbeuval, Boasso et al., 2005; Herbeuval, Grivel et al., 2005) making them prone to TRAIL-mediated apoptosis (Lichtner, Maranon et al., 2004). Furthermore, the percentage of CD4+ T cells co-expressing TRAIL and DR5 are elevated in the blood of viremic progressors (Herbeuval, Grivel et al., 2005). Thus, TRAIL does not exhibit cytotoxic effects on normal cells and tissues and is potentially efficient to eradicate a large panel of cancer cells. Several clinical trial are currently evaluating TRAIL anti tumor effect, alone or in combination with other chemotherapeutic drugs.

Thus, it remained pertinent to determine whether ATL cells were sensitive to TRAILmediated apoptosis. One study characterized the sensitivity of ATL cells to TRAIL cytotoxicity. Authors tested several cell lines and also primary cells from both chronic and acute ATL. Unfortunately, the vast majority of primary ATL cells or cell lines appears to be resistant to TRAIL induced cell death (Matsuda, Almasan et al., 2005). This resistance was due to multiple parameters, including the lack of DR4 and DR5 expression, abrogation of death signal upstream caspase-8, attenuation of both extrinsic and intrinsic apoptotic pathways. More recently, it has been shown that the resistance upstream caspase 8 was due to an over expression of the cellular caspase-8 (FLICE)-inhibitory protein (c-FLIP) that blocks caspase recruitment and apoptosis. However, other study show that ATL cells might be sensitive to TRAIL-induced apoptosis (Hasegawa, Yamada et al., 2005), therefore TRAIL effect in ATL should be clarified.

Surprisingly, most of ATL cells expressed TRAIL on their surface. This finding suggested that constitutive expression of TRAIL would participate in the development of TRAILresistant clones observed in patients. The natural resistance of ATL cells would have excluded the use of TRAIL as therapeutic agent. However, a recent study demonstrated that the herbal compound Rocaglamide restores TRAIL sensibility in ATL cells. Indeed, Rocaglamide induces suppression of c-FLIP expression in ATL cells that sensitizes these cells to TRAIL-mediated apoptosis. Authors suggest the use of Rocaglamide as an adjuvant to TRAIL as new therapeutic strategies against HTLV-1-mediated ATL (Bleumink, Kohler et al.). It has also been observed that the use of a combination of a p53 activator, Nutlin-3a, and TRAIL synergized to induce ATL cell apoptosis (Hasegawa, Yamada et al., 2009). This could be explained by the fact that p53 regulates TRAIL death receptor 5 on cell surface. Thus, Nutlin-3a treated ATL cells would express DR5 and then become sensitive to TRAILmediated apoptosis.

Therefore, the understanding of ATL sensitivity to TRAIL-mediated apoptosis appears to be crucial to develop new therapeutic options.

receptor is the most studied, and authors showed that DR5 transcription is regulated (at least partially) by the protooncogene p53 (Sheikh, Burns et al., 1998; Wu, Burns et al., 1997). It should be noticed that TRAIL death receptors 4 and 5 not only induce apoptosis but may also play a crucial role in inflammatory responses (Collison, Foster et al., 2009). Figure 5

TRAIL is a very promising candidate for cancer treatment due to its sophisticated way of inducing apoptosis. While the vast majority of normal cells express decoy receptors and are therefore protected from TRAIL-mediated apoptosis, tumor cells generally express death receptors. Indeed, TRAIL induces apoptosis in human tumor cell lines (Griffith, Chin et al., 1998) but not in normal cells (Gura, 1997). TRAIL also induces apoptosis of infected cells. For example, plasma TRAIL has been reported to be an early pathogenic marker in acute HIV-1 infection and is correlated to viral load in chronic disease (Gasper-Smith, Crossman et al., 2008; Herbeuval, Nilsson et al., 2009). HIV-1 upregulates DR5 expression on the membrane of CD4+ T cells *in vitro* (Herbeuval, Boasso et al., 2005; Herbeuval, Grivel et al., 2005) making them prone to TRAIL-mediated apoptosis (Lichtner, Maranon et al., 2004). Furthermore, the percentage of CD4+ T cells co-expressing TRAIL and DR5 are elevated in the blood of viremic progressors (Herbeuval, Grivel et al., 2005). Thus, TRAIL does not exhibit cytotoxic effects on normal cells and tissues and is potentially efficient to eradicate a large panel of cancer cells. Several clinical trial are currently evaluating TRAIL anti tumor

Thus, it remained pertinent to determine whether ATL cells were sensitive to TRAILmediated apoptosis. One study characterized the sensitivity of ATL cells to TRAIL cytotoxicity. Authors tested several cell lines and also primary cells from both chronic and acute ATL. Unfortunately, the vast majority of primary ATL cells or cell lines appears to be resistant to TRAIL induced cell death (Matsuda, Almasan et al., 2005). This resistance was due to multiple parameters, including the lack of DR4 and DR5 expression, abrogation of death signal upstream caspase-8, attenuation of both extrinsic and intrinsic apoptotic pathways. More recently, it has been shown that the resistance upstream caspase 8 was due to an over expression of the cellular caspase-8 (FLICE)-inhibitory protein (c-FLIP) that blocks caspase recruitment and apoptosis. However, other study show that ATL cells might be sensitive to TRAIL-induced apoptosis (Hasegawa, Yamada et al., 2005), therefore TRAIL

Surprisingly, most of ATL cells expressed TRAIL on their surface. This finding suggested that constitutive expression of TRAIL would participate in the development of TRAILresistant clones observed in patients. The natural resistance of ATL cells would have excluded the use of TRAIL as therapeutic agent. However, a recent study demonstrated that the herbal compound Rocaglamide restores TRAIL sensibility in ATL cells. Indeed, Rocaglamide induces suppression of c-FLIP expression in ATL cells that sensitizes these cells to TRAIL-mediated apoptosis. Authors suggest the use of Rocaglamide as an adjuvant to TRAIL as new therapeutic strategies against HTLV-1-mediated ATL (Bleumink, Kohler et al.). It has also been observed that the use of a combination of a p53 activator, Nutlin-3a, and TRAIL synergized to induce ATL cell apoptosis (Hasegawa, Yamada et al., 2009). This could be explained by the fact that p53 regulates TRAIL death receptor 5 on cell surface. Thus, Nutlin-3a treated ATL cells would express DR5 and then become sensitive to TRAIL-

Therefore, the understanding of ATL sensitivity to TRAIL-mediated apoptosis appears to be

illustrates TRAIL pathway and regulation.

effect in ATL should be clarified.

mediated apoptosis.

crucial to develop new therapeutic options.

effect, alone or in combination with other chemotherapeutic drugs.

Fig. 5. TRAIL apoptotic pathway. Membrane (mTRAIL) or soluble TRAIL (sTRAIL) bind to 3 decoy receptors (DcR1, DcR2 and OPG) and 2 death receptors (DR4 and DR5) which activate the caspase pathway leading to apoptosis.

#### **4.3 Myeloid dendritic cells and HTLV-1 infection**

HTLV-1 targets CD4+ T cells which represent an important target for HTLV-1 infection in the peripheral blood. However, there are some additional evidence that showed that HTLV-1 can also infect including CD8+ T lymphocytes, monocytes, B lymphocytes, astrocytes (Richardson, Edwards et al., 1990) and dendritic cells (DC) *in vivo.* Myeloid dendritic cells do not exhibit high viral gene expression, but recent work suggested that HTLV-1-infected dendritic cells show better capacity to stimulate antigen-specific T cell activation (Makino, Shimokubo et al., 1999). Moreover, the proportion of lymphoid organs containing HTLV-1 positive dendritic cells is proportional to the total proviral DNA load in the blood, providing a correlation of proviral DNA load and the frequency of effector/memory Tax-CD8+ T cells (Nagai, Kubota et al., 2001).

More recently, findings demonstrated a central role to myeloid dendritic (mDC) and plasmacytoid dendritic cells (pDC) in HTLV-1 infection. For years it has been thought that unlike other retroviruses such as HIV-1, free virions were poorly infectious (Donegan, Lee et al., 1994). However, a recent study reported that freshly isolated mDC and pDC are efficiently and productively infected by cell-free HTLV-1 (Jones, Petrow-Sadowski et al.,

Adult Human T Cell Leukemia 13

phosphorylation and nuclear translocation of the transcription factor IRF-7 (Honda, Yanai et al., 2005). This last step was shown to depend on the kinases IRAK-1 (Uematsu, Sato et al., 2005) and IkB kinase-a (IKK-a) (Hoshino, Sugiyama et al., 2006) in mouse pDC. It has been recently shown that the PI3-kinase pathway was critical to control the nuclear translocation of IRF-7 and the subsequent production of type I IFN (Guiducci, Ghirelli et al., 2008). At the present time, it is not known whether additional molecular pathways are involved and

PDC express a panel of surface receptors but their function remain largely unknown. The best characterized is the lectin BDCA-2 (Blood dendritic cell antigen-2) (Dzionek, Inagaki et al., 2002; Dzionek, Sohma et al., 2001). BDCA-2 mediates antigen uptake and inhibits pDC production of type 1 IFN induced by *influenza* virus (Dzionek, Sohma et al., 2001). This inhibition is mediated by the induction of a B cell receptor-like signaling cascade (Cao, Zhang et al., 2007). Neuropilin 1 (NRP1), also called BDCA-4, is another surface receptor constitutively expressed at high levels on human pDC. NRP1 is involved in the interaction between myeloid DC and T cells within the immune synapse (Tordjman, Lepelletier et al., 2002). However, its role in pDC function remains unknown. Recently, we have shown that NRP1 was a coreceptor for the HTLV-1 virus and might be involved in viral entry (Ghez, Lepelletier et al., 2006). Thus HTLV-1 could provide a link between the molecular pathways

downstream of NRP1 and the physiopathology of pDC in HTLV-related diseases.

erythematosus and psoriasis (Nestle, Conrad et al., 2005).

**5.2 pDC and HTLV-1 infection** 

There is increasing evidence that pDC are involved in several disease settings. They were observed *in situ* in a variety of pathological conditions, such as HPV-related cervical cancer, skin melanoma (Salio, Cella et al., 2003), psoriasis (Nestle, Conrad et al., 2005) or allergic contact dermatitis (Bangert, Friedl et al., 2003) and in the nasal mucosa as early as 6 hours after allergen challenge, suggesting an active recruitment of blood pDC at the site of inflammation. Moreover, a dysregulated TLR-induced IFN response has been linked to autoimmune diseases (Colonna, 2006; Marshak-Rothstein, 2006), particularly lupus

Three molecules have been characterized for HTLV-1 entry into cells, heparin sulfate proteoglycans (HSPG) (Jones, Petrow-Sadowski et al., 2005) and NRP-1 (also called BDCA-4) for the initial virus binding to target cells (Ghez, Lepelletier et al., 2006), and glucose transporter 1 (GLUT-1) for the post-attachment and the viral fusion (Manel, Kim et al., 2003; Takenouchi, Jones et al., 2007). Interestingly, NRP-1 is expressed by mDC and T cells but cells expressing the highest level of BDCA-4 in blood are pDC (Grouard, Rissoan et al., 1997; Siegal, Kadowaki et al., 1999), strongly suggesting that HTLV-1 could interact with pDC. Nevertheless, HTLV-1-induced immune response by professional "sentinel" pDC has not been reported. Viral activation of pDC can be regulated by either of two Toll-like Receptors (TLR), TLR7 or TLR9, which are considered to be the receptors that human pDC use for recognition of RNA/retroviruses and DNA, respectively. HIV-activated pDC were recently reported to express the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Chaperot, Blum et al., 2006; Hardy, Graham et al., 2007; Stary, Klein et al., 2009). TRAIL has been shown to induce apoptosis of cancer (Herbeuval, Lambert et al., 2003; Walczak, Miller et al., 1999) and infected cells expressing death receptor-4 or -5 (DR4, DR5). We recently demonstrated that HTLV-1 stimulated pDC expressed TRAIL and acquired cytotoxic activity, transforming them into a new subset of killer innate immune cells, which

modulated HTLV-1 diseases.

2008). Furthermore, infected mDC and pDC were able to transfer virions to autologous CD4+ T cells, clearly demonstrating that cell free HTLV-1 can be infectious and target dendritic cells (Jones, Petrow-Sadowski et al., 2008).

#### **5. HTLV-1 and plasmacytoid dendritic cell response**

Plasmacytoid dendritic cells were discovered in 1997 as professional IFN-alpha producers and innate immune cells (Grouard, Rissoan et al., 1997). These cells are rare but play a central role in host defense against viruses and bacteria by producing cytokines and antiviral factors. The role of pDC in HTLV-1 infection remained unknown until recent years probably because of the extreme difficulty of studying HTLV-1 acute infection. We describe here recent data providing some new features in the understanding of HTLV-1 innate immune response.

#### **5.1 The Plasmacytoid dendritic cell (pDC)**

PDC are cells of hemopoietic origin that are found at steady state in the blood, thymus and peripheral lymphoid tissues. Early studies described pDC as being oval-shaped with typical plasmacytoid morphology. The ability of plasmacytoid-derived DC (also named DC2) to induce a Th2 differentiation of naïve CD4 T cells formed the basis for the concept of type 1 and type 2 DC (Review Nat immunol, 2001). The role of these DC in mouse and human was studied in different models and is not completely elucidated (Liu, 2005). A little later, it was shown that pDC were specialized in the production of type I IFN (Siegal, Kadowaki et al., 1999). They are the principal source of type I IFN in human blood and very rapidly produce all type I IFN isoforms in response to microbial stimuli, such as virus (Cella, Jarrossay et al., 1999; Siegal, Kadowaki et al., 1999), CpG-containing oligonucleotides (Kadowaki, Antonenko et al., 2000), or the synthetic molecules imidazoquinolines (Gibson, Lindh et al., 2002). PDC-derived type I IFN has direct anti-viral activity against a variety of virus, including HIV, and has important adjuvant functions on other immune cell-types, such as NK cells, T cells, macrophages and DC. Thus, pDC activation triggers a dual type of response: type I IFN production and DC differentiation (Colonna, Trinchieri et al., 2004; Yang, Lian et al., 2005).

PDC and plasmacytoid-derived DC express the Toll-Like receptor TLR7 and TLR9 (Jarrossay, Napolitani et al., 2001; Kadowaki and Liu, 2002) and respond to their respective ligands, imidazoquinolines (Hemmi, Kaisho et al., 2002) and single strand RNA (Diebold, Kaisho et al., 2004; Heil, Hemmi et al., 2004; Lund, Alexopoulou et al., 2004) for TLR7, CpGcontaining oligonucleotides (Hemmi, Takeuchi et al., 2000) and DNA viruses (Lund, Sato et al., 2003) for TLR9. They do not express TLR2, TLR3 and TLR4, and do not respond to such ligands as peptidoglycan, LPS (lipopolysaccharides) or double-stranded RNA (Jarrossay, Napolitani et al., 2001; Kadowaki and Liu, 2002). Activation of pDC through TLR7 and TLR9 can trigger both types of response, including large quantities of type I IFN production and/or DC differentiation (Liu, 2005). Synthetic CpG-containing oligonucleotides of the types A and B (CpG-A, CpG-B) selectively induce type I IFN production and DC differentiation, respectively (Duramad, Fearon et al., 2005) while some viral stimuli, such as *influenza* virus (Flu), herpes simplex virus (HSV) or CpG-C can induce simultaneously both responses (Liu, 2005). Two factors seem to be key for the induction of large quantities of type I IFN in pDC: 1) the ability of the TLR ligands to bind its receptor in the early endosomal compartments (Guiducci, Ott et al., 2006; Honda, Ohba et al., 2005); 2) the

2008). Furthermore, infected mDC and pDC were able to transfer virions to autologous CD4+ T cells, clearly demonstrating that cell free HTLV-1 can be infectious and target

Plasmacytoid dendritic cells were discovered in 1997 as professional IFN-alpha producers and innate immune cells (Grouard, Rissoan et al., 1997). These cells are rare but play a central role in host defense against viruses and bacteria by producing cytokines and antiviral factors. The role of pDC in HTLV-1 infection remained unknown until recent years probably because of the extreme difficulty of studying HTLV-1 acute infection. We describe here recent data providing some new features in the understanding of HTLV-1 innate

PDC are cells of hemopoietic origin that are found at steady state in the blood, thymus and peripheral lymphoid tissues. Early studies described pDC as being oval-shaped with typical plasmacytoid morphology. The ability of plasmacytoid-derived DC (also named DC2) to induce a Th2 differentiation of naïve CD4 T cells formed the basis for the concept of type 1 and type 2 DC (Review Nat immunol, 2001). The role of these DC in mouse and human was studied in different models and is not completely elucidated (Liu, 2005). A little later, it was shown that pDC were specialized in the production of type I IFN (Siegal, Kadowaki et al., 1999). They are the principal source of type I IFN in human blood and very rapidly produce all type I IFN isoforms in response to microbial stimuli, such as virus (Cella, Jarrossay et al., 1999; Siegal, Kadowaki et al., 1999), CpG-containing oligonucleotides (Kadowaki, Antonenko et al., 2000), or the synthetic molecules imidazoquinolines (Gibson, Lindh et al., 2002). PDC-derived type I IFN has direct anti-viral activity against a variety of virus, including HIV, and has important adjuvant functions on other immune cell-types, such as NK cells, T cells, macrophages and DC. Thus, pDC activation triggers a dual type of response: type I IFN production and DC differentiation (Colonna, Trinchieri et al., 2004;

PDC and plasmacytoid-derived DC express the Toll-Like receptor TLR7 and TLR9 (Jarrossay, Napolitani et al., 2001; Kadowaki and Liu, 2002) and respond to their respective ligands, imidazoquinolines (Hemmi, Kaisho et al., 2002) and single strand RNA (Diebold, Kaisho et al., 2004; Heil, Hemmi et al., 2004; Lund, Alexopoulou et al., 2004) for TLR7, CpGcontaining oligonucleotides (Hemmi, Takeuchi et al., 2000) and DNA viruses (Lund, Sato et al., 2003) for TLR9. They do not express TLR2, TLR3 and TLR4, and do not respond to such ligands as peptidoglycan, LPS (lipopolysaccharides) or double-stranded RNA (Jarrossay, Napolitani et al., 2001; Kadowaki and Liu, 2002). Activation of pDC through TLR7 and TLR9 can trigger both types of response, including large quantities of type I IFN production and/or DC differentiation (Liu, 2005). Synthetic CpG-containing oligonucleotides of the types A and B (CpG-A, CpG-B) selectively induce type I IFN production and DC differentiation, respectively (Duramad, Fearon et al., 2005) while some viral stimuli, such as *influenza* virus (Flu), herpes simplex virus (HSV) or CpG-C can induce simultaneously both responses (Liu, 2005). Two factors seem to be key for the induction of large quantities of type I IFN in pDC: 1) the ability of the TLR ligands to bind its receptor in the early endosomal compartments (Guiducci, Ott et al., 2006; Honda, Ohba et al., 2005); 2) the

dendritic cells (Jones, Petrow-Sadowski et al., 2008).

**5.1 The Plasmacytoid dendritic cell (pDC)** 

immune response.

Yang, Lian et al., 2005).

**5. HTLV-1 and plasmacytoid dendritic cell response** 

phosphorylation and nuclear translocation of the transcription factor IRF-7 (Honda, Yanai et al., 2005). This last step was shown to depend on the kinases IRAK-1 (Uematsu, Sato et al., 2005) and IkB kinase-a (IKK-a) (Hoshino, Sugiyama et al., 2006) in mouse pDC. It has been recently shown that the PI3-kinase pathway was critical to control the nuclear translocation of IRF-7 and the subsequent production of type I IFN (Guiducci, Ghirelli et al., 2008). At the present time, it is not known whether additional molecular pathways are involved and modulated HTLV-1 diseases.

PDC express a panel of surface receptors but their function remain largely unknown. The best characterized is the lectin BDCA-2 (Blood dendritic cell antigen-2) (Dzionek, Inagaki et al., 2002; Dzionek, Sohma et al., 2001). BDCA-2 mediates antigen uptake and inhibits pDC production of type 1 IFN induced by *influenza* virus (Dzionek, Sohma et al., 2001). This inhibition is mediated by the induction of a B cell receptor-like signaling cascade (Cao, Zhang et al., 2007). Neuropilin 1 (NRP1), also called BDCA-4, is another surface receptor constitutively expressed at high levels on human pDC. NRP1 is involved in the interaction between myeloid DC and T cells within the immune synapse (Tordjman, Lepelletier et al., 2002). However, its role in pDC function remains unknown. Recently, we have shown that NRP1 was a coreceptor for the HTLV-1 virus and might be involved in viral entry (Ghez, Lepelletier et al., 2006). Thus HTLV-1 could provide a link between the molecular pathways downstream of NRP1 and the physiopathology of pDC in HTLV-related diseases.

There is increasing evidence that pDC are involved in several disease settings. They were observed *in situ* in a variety of pathological conditions, such as HPV-related cervical cancer, skin melanoma (Salio, Cella et al., 2003), psoriasis (Nestle, Conrad et al., 2005) or allergic contact dermatitis (Bangert, Friedl et al., 2003) and in the nasal mucosa as early as 6 hours after allergen challenge, suggesting an active recruitment of blood pDC at the site of inflammation. Moreover, a dysregulated TLR-induced IFN response has been linked to autoimmune diseases (Colonna, 2006; Marshak-Rothstein, 2006), particularly lupus erythematosus and psoriasis (Nestle, Conrad et al., 2005).

#### **5.2 pDC and HTLV-1 infection**

Three molecules have been characterized for HTLV-1 entry into cells, heparin sulfate proteoglycans (HSPG) (Jones, Petrow-Sadowski et al., 2005) and NRP-1 (also called BDCA-4) for the initial virus binding to target cells (Ghez, Lepelletier et al., 2006), and glucose transporter 1 (GLUT-1) for the post-attachment and the viral fusion (Manel, Kim et al., 2003; Takenouchi, Jones et al., 2007). Interestingly, NRP-1 is expressed by mDC and T cells but cells expressing the highest level of BDCA-4 in blood are pDC (Grouard, Rissoan et al., 1997; Siegal, Kadowaki et al., 1999), strongly suggesting that HTLV-1 could interact with pDC. Nevertheless, HTLV-1-induced immune response by professional "sentinel" pDC has not been reported. Viral activation of pDC can be regulated by either of two Toll-like Receptors (TLR), TLR7 or TLR9, which are considered to be the receptors that human pDC use for recognition of RNA/retroviruses and DNA, respectively. HIV-activated pDC were recently reported to express the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Chaperot, Blum et al., 2006; Hardy, Graham et al., 2007; Stary, Klein et al., 2009). TRAIL has been shown to induce apoptosis of cancer (Herbeuval, Lambert et al., 2003; Walczak, Miller et al., 1999) and infected cells expressing death receptor-4 or -5 (DR4, DR5). We recently demonstrated that HTLV-1 stimulated pDC expressed TRAIL and acquired cytotoxic activity, transforming them into a new subset of killer innate immune cells, which

Adult Human T Cell Leukemia 15

subset of killer cells was called Interferon-producing Killer pDC (ie IKpDC). Using cell free purified HTLV-1 particles, we stimulated isolated pDC from healthy donors and cells were analyzed using three dimensional (3D) microscopy. Microscopy revealed some surprising results. We found high levels of TRAIL in non activated pDC. This result was not expected as it has never been observed before. However, it was not clear whether TRAIL was located on membrane or in cytoplasm. Thus, using the ImageJ tool "3D interactive surface plot", we demonstrated that TRAIL was located in the cytoplasm of resting pDC (Colisson, Barblu et al.). In contrast, HTLV-1 stimulated pDC showed a relocalization of TRAIL from cytoplasm to plasma membrane (Figure 6). HTLV-1 exposure induced a relocalization of intracellular stock of TRAIL to the membrane, conferring a killer activity to pDC. Surprinsingly, we did not detect HTLV-1 viruses in pDC. However, chloroquine treated pDC revealed some HTLV-1 particles in cytoplasm. In fact, this latest result provides some indication concerning

Fig. 6. 3D microscopy of HTLV-1 activated pDC. 3D interactive surface plot was used to precisely delimitated TRAIL in pDC. Upper picture show intracellular TRAIL (green) inside pDC plasma membrane. Bottom picture shows TRAIL relocalization to the membrane that

Nucleus

Because pDC express high levels of NRP-1, which is a member of the HTLV-1 complex receptor, they may be productively infected by HTLV-1. However, pDC could aslo activate the endocytosis pathway under viral exposure. Endocytosis pathway is charcaterized by formation of endosomes in which pH get low activating multiple protease. Viral particles are degradated by low pH proteases and genetic material is released into the vesicles. Thus,

appears green in HTLV-1 exposed pDC. HTLV-1 viruses (red) binds to plasma cell

membrane. Adapated from Colisson, Barblu et al 2010.

**5.4 HTLV-1 activated endocytosis pathway in pDC** 

the pathway by which HTLV-1 particles activate pDC.

Membrane

Cytoplasm

may play a central role in viral immunopathogenesis and tumor development (Hardy, Graham et al., 2007).

The role of pDC in HTLV-1 infection was unknown until Jones *et al* reported that freshly purified mDC and pDC could be productively infected by HTLV-1 free viruses (Jones, Petrow-Sadowski et al., 2008). Authors clearly demonstrated that infected pDC and mDC could also infect CD4+ T cells *in vitro*. These findings were major discovery for two reasons: first, they showed that, unlike researcher thought for years, free particles of HTLV-1 could directly infect T cells, and secondly because it was the first demonstration of pDC-HTLV-1 interaction.

However, the infection of target cells by HTLV-1 is not totally understood and need to be clarified. HTLV-1 infection is a sequential process that potentially involves the recruitment of at least three molecules.

#### **5.3 pDC activation by cell free HTLV-1 virions**

Due to abvious evidences that pDC and HTLV-1 could interact, we decided to study pDC response against HTLV-1. The first parameter we tested was the IFN- production, which is characteristic of the innate immune response. Our first results were disappointing. Using supernatants from chronically HTLV-1-infected cell lines we stimulated pDC *in vitro*. The IFN produced by pDC exposed to supernatants from MT-2 cell line remained very low compared to *Influenza* A (Flu) stimulation. The difference between the two stimulation was the purity of the viruses: Flu was a purified virus while HTLV-1 stimulation was made from supernatants. Thus, we decided to purify HTLV-1 by ultracentrifugation. Pelets were collected and the quantity of viruses was determined using p19 ELISA. Thus, we could calculate the concentration of purified HTLV-1 and use a large range of viral concentration to stimulate pDC. Therefore, we showed that purified cell free HTLV-1 particles could induce massive IFN by pDC, similarly to Influenza A virus (Flu) or HIV stimulation. For the first time, we clearly demonstrated that free HTLV-1 particles, as other retroviruses, could generate an IFN response by pDC (Colisson, Barblu et al, 2010). We also tested IL-10 and TNF- production by HTLV-1-exposed pDC and found high levels of IL-10 and TNF- production.

We and others reported that Flu or HIV-1 activation of pDC resulted in cytokine production but also activation markers (CD40, HLADR), maturation markers (CD80, CD86) and migration marker CCR7 (Beignon, McKenna et al., 2005; Chaperot, Blum et al., 2006; Fonteneau, Larsson et al., 2004). We found that HTLV-1, like other retroviruses, induced activation and maturation marker expression by pDC. However, it remained unclear whether the lymphoid migration marker CCR7 was expressed by pDC after HTLV-1 exposure. This might have essential consequences in immunopathogenesis. Indeed, HIV-1 induces migration of pDC from the blood to lymphoid organs by upregulating CCR7 expression on cell surface. Thus, activated pDC migrate to tonsils and other lymphoid organs and participate to CD4+ T cell depletion in tissues (Stary, Klein et al., 2009). We observed that CCR7 was not or weakly expressed by pDC after HTLV-1 exposure. Consequently, we could imagine that pDC do not migrate to lymphoid tissues in HTLV-1 infected patients, in contrast to HIV-1 patients. Further studies are needed to better characterized *in vitro* and *in vivo* CCR7 expression and pDC migration in patients.

We next wanted to better characterize pDC activation by HTLV-1. We previously reported that HIV-1 induced pDC transformation into TRAIL-expressing pDC, which were able to induce apoptosis of CD4+ T cells expressing DR5 (Hardy, Graham et al., 2007). This new

may play a central role in viral immunopathogenesis and tumor development (Hardy,

The role of pDC in HTLV-1 infection was unknown until Jones *et al* reported that freshly purified mDC and pDC could be productively infected by HTLV-1 free viruses (Jones, Petrow-Sadowski et al., 2008). Authors clearly demonstrated that infected pDC and mDC could also infect CD4+ T cells *in vitro*. These findings were major discovery for two reasons: first, they showed that, unlike researcher thought for years, free particles of HTLV-1 could directly infect T cells, and secondly because it was the first demonstration of pDC-HTLV-1

However, the infection of target cells by HTLV-1 is not totally understood and need to be clarified. HTLV-1 infection is a sequential process that potentially involves the recruitment

Due to abvious evidences that pDC and HTLV-1 could interact, we decided to study pDC response against HTLV-1. The first parameter we tested was the IFN- production, which is characteristic of the innate immune response. Our first results were disappointing. Using supernatants from chronically HTLV-1-infected cell lines we stimulated pDC *in vitro*. The IFN produced by pDC exposed to supernatants from MT-2 cell line remained very low compared to *Influenza* A (Flu) stimulation. The difference between the two stimulation was the purity of the viruses: Flu was a purified virus while HTLV-1 stimulation was made from supernatants. Thus, we decided to purify HTLV-1 by ultracentrifugation. Pelets were collected and the quantity of viruses was determined using p19 ELISA. Thus, we could calculate the concentration of purified HTLV-1 and use a large range of viral concentration to stimulate pDC. Therefore, we showed that purified cell free HTLV-1 particles could induce massive IFN by pDC, similarly to Influenza A virus (Flu) or HIV stimulation. For the first time, we clearly demonstrated that free HTLV-1 particles, as other retroviruses, could generate an IFN response by pDC (Colisson, Barblu et al, 2010). We also tested IL-10 and TNF- production by

We and others reported that Flu or HIV-1 activation of pDC resulted in cytokine production but also activation markers (CD40, HLADR), maturation markers (CD80, CD86) and migration marker CCR7 (Beignon, McKenna et al., 2005; Chaperot, Blum et al., 2006; Fonteneau, Larsson et al., 2004). We found that HTLV-1, like other retroviruses, induced activation and maturation marker expression by pDC. However, it remained unclear whether the lymphoid migration marker CCR7 was expressed by pDC after HTLV-1 exposure. This might have essential consequences in immunopathogenesis. Indeed, HIV-1 induces migration of pDC from the blood to lymphoid organs by upregulating CCR7 expression on cell surface. Thus, activated pDC migrate to tonsils and other lymphoid organs and participate to CD4+ T cell depletion in tissues (Stary, Klein et al., 2009). We observed that CCR7 was not or weakly expressed by pDC after HTLV-1 exposure. Consequently, we could imagine that pDC do not migrate to lymphoid tissues in HTLV-1 infected patients, in contrast to HIV-1 patients. Further studies are needed to better

HTLV-1-exposed pDC and found high levels of IL-10 and TNF- production.

characterized *in vitro* and *in vivo* CCR7 expression and pDC migration in patients.

We next wanted to better characterize pDC activation by HTLV-1. We previously reported that HIV-1 induced pDC transformation into TRAIL-expressing pDC, which were able to induce apoptosis of CD4+ T cells expressing DR5 (Hardy, Graham et al., 2007). This new

Graham et al., 2007).

of at least three molecules.

**5.3 pDC activation by cell free HTLV-1 virions** 

interaction.

subset of killer cells was called Interferon-producing Killer pDC (ie IKpDC). Using cell free purified HTLV-1 particles, we stimulated isolated pDC from healthy donors and cells were analyzed using three dimensional (3D) microscopy. Microscopy revealed some surprising results. We found high levels of TRAIL in non activated pDC. This result was not expected as it has never been observed before. However, it was not clear whether TRAIL was located on membrane or in cytoplasm. Thus, using the ImageJ tool "3D interactive surface plot", we demonstrated that TRAIL was located in the cytoplasm of resting pDC (Colisson, Barblu et al.). In contrast, HTLV-1 stimulated pDC showed a relocalization of TRAIL from cytoplasm to plasma membrane (Figure 6). HTLV-1 exposure induced a relocalization of intracellular stock of TRAIL to the membrane, conferring a killer activity to pDC. Surprinsingly, we did not detect HTLV-1 viruses in pDC. However, chloroquine treated pDC revealed some HTLV-1 particles in cytoplasm. In fact, this latest result provides some indication concerning the pathway by which HTLV-1 particles activate pDC.

Fig. 6. 3D microscopy of HTLV-1 activated pDC. 3D interactive surface plot was used to precisely delimitated TRAIL in pDC. Upper picture show intracellular TRAIL (green) inside pDC plasma membrane. Bottom picture shows TRAIL relocalization to the membrane that appears green in HTLV-1 exposed pDC. HTLV-1 viruses (red) binds to plasma cell membrane. Adapated from Colisson, Barblu et al 2010.

#### **5.4 HTLV-1 activated endocytosis pathway in pDC**

Because pDC express high levels of NRP-1, which is a member of the HTLV-1 complex receptor, they may be productively infected by HTLV-1. However, pDC could aslo activate the endocytosis pathway under viral exposure. Endocytosis pathway is charcaterized by formation of endosomes in which pH get low activating multiple protease. Viral particles are degradated by low pH proteases and genetic material is released into the vesicles. Thus,

Adult Human T Cell Leukemia 17

Adult T cell leukemia induced by HTLV-1 infection exhibits diverse clinical features. The outcome is directly correlated to ATL subtype, that could range from a very indolent and slowly progressive lymphoma to a very aggressive and nearly uniformaly lethal proliferative lymphoma. Thus, knowledge about HTLV-1 infection and propagation remains

An important challenge would be to link the pDC phenotype to the different HTLV-1 associated pathologies (ATLL). It would be interesting to determine whether IKpDC persist during chronic infection in order to generate new HTLV-1 progression markers. The characterization of IKpDC *in vivo* opens new area of dendritic cells research in HTLV-1 and other retrovirus-induced immunopathogenesis and in tumor cell biology. Considered together, our data highlight a dual role for pDC in HTLV-1 disease. pDC that become infected may participate in viral spread in the host (Jones, Petrow-Sadowski et al., 2008) and concomitantly express TRAIL, which may select the transformed CD4+ T cell clone, leading to ATLL years later. In this context, it will be of great interest to test TRAIL sensitivity of the persistent clones after HTLV-1 infection that may subsequently be transformed to lymphoma/leukemia. Thus, pDC investigation in HTLV-1 disease will be crucial for understanding complex HTLV-1-associated pathologies. However, detection of primary infection in humans is currently not feasible due to the high latency of HTLV-1 virus before disease symptoms appearance. An alternative way to characterize and understand the early steps of HTLV-1 infection is the development of the pathogenic simian model (STLV-1). However, in addition to selection of TRAIL-resistant clones, one could hypothesize that similar to HIV-1 infection, pDC may participate in and contribute to the immune

HTLV-1 free particles generate an immune response by professional virus "sentinel" pDC. We then identify and describe the mechanism by which purified HTLV-1 virions stimulate pDC and transform them into functional killer cells. We show that pDC response and activation to HTLV-1 is strictly virus-dose dependent. Finally, purified HTLV-1 particles induced TLR7-mediated relocalization of intracellular TRAIL to the pDC membrane. In conclusion, the physiological function of pDC during the different stages of HTLV-1 infection will represent a new field of investigation and may lead to new therapeutic

I would like to thank Pr Olivier Hermine, UMR 8147 Hopital Necker, Paris, for his precious

Banchereau, J. and R. M. Steinman (1998). "Dendritic cells and the control of immunity."

Bangert, C., J. Friedl, et al. (2003). "Immunopathologic features of allergic contact dermatitis

in humans: participation of plasmacytoid dendritic cells in the pathogenesis of the

advice and critics and Lucie Barblu for helping me on this manuscript.

disease?" *J Invest Dermatol* 121 (6): 1409-18

essential to better understand pathogenesis consequences.

**6. Conclusion** 

suppression that occurs in ATLL.

strategies.

**7. Acknowledgment**

*Nature* 392 (6673): 245-52

**8. References** 

viral RNA or DNA activate their respective receptors TLR7 or TLR9. Chloroquine inhibits endocytosis by reducing pH acidification in endosomes.

We demonstrated using chloroquine and TLR7 inhibitor that HTLV-1 activated the endocytosis pathway in pDC as demonstrated for other retrovirus like HIV-1 (Beignon, McKenna et al., 2005; Hardy, Graham et al., 2007). Viral particles, after initial binding to HTLV-1 receptor complex, entered into the endosome, which became pH low. This acidification activates endosomal protease that destroyed virus envelop and capsid, leading to single strand RNA (ssRNA) release into the vesicles. This viral ssRNA activates TLR7, which in turn recrutes the adaptor protein MyD88, a central molecule in most of TLRdependent. The recruitment of MyD88 starts a cascade of activation leading to IFN production (due to the recruitment of Inteferon Regulatory factor 7, IRF7). We also showed that TRAIL expression, activation markers (CD40, HLADR) and maturation markers (CD80, CD86) were regulated by TLR7 activation in pDC. These results place TLR7 as the central molecule of HTLV-1-induced pDC response (Figure 7). Thus, endocytosis seems to be the major pathway involved in pDC activation by HTLV-1. However, it should be noticed that our findings do not exclude the possibility that pDC could get productively infected. Our study focused on short time experiments that did not allow us to detect newly synthesized viruses. Jones *et al* showed that coculture of mDC and pDC with HTLV-1 could induce CD4+ T cell infection, while free viruses alone could not infect T cells (Jones, Petrow-Sadowski et al., 2008). Further experiments are needed to determine what is the proportion of infected pDC versus activated IKpDC.

Fig. 7. Transformation of pDC into IKpDC by HTLV-1.

#### **6. Conclusion**

16 T-Cell Leukemia

viral RNA or DNA activate their respective receptors TLR7 or TLR9. Chloroquine inhibits

We demonstrated using chloroquine and TLR7 inhibitor that HTLV-1 activated the endocytosis pathway in pDC as demonstrated for other retrovirus like HIV-1 (Beignon, McKenna et al., 2005; Hardy, Graham et al., 2007). Viral particles, after initial binding to HTLV-1 receptor complex, entered into the endosome, which became pH low. This acidification activates endosomal protease that destroyed virus envelop and capsid, leading to single strand RNA (ssRNA) release into the vesicles. This viral ssRNA activates TLR7, which in turn recrutes the adaptor protein MyD88, a central molecule in most of TLRdependent. The recruitment of MyD88 starts a cascade of activation leading to IFN production (due to the recruitment of Inteferon Regulatory factor 7, IRF7). We also showed that TRAIL expression, activation markers (CD40, HLADR) and maturation markers (CD80, CD86) were regulated by TLR7 activation in pDC. These results place TLR7 as the central molecule of HTLV-1-induced pDC response (Figure 7). Thus, endocytosis seems to be the major pathway involved in pDC activation by HTLV-1. However, it should be noticed that our findings do not exclude the possibility that pDC could get productively infected. Our study focused on short time experiments that did not allow us to detect newly synthesized viruses. Jones *et al* showed that coculture of mDC and pDC with HTLV-1 could induce CD4+ T cell infection, while free viruses alone could not infect T cells (Jones, Petrow-Sadowski et al., 2008). Further experiments are needed to determine what is the proportion

endocytosis by reducing pH acidification in endosomes.

of infected pDC versus activated IKpDC.

Fig. 7. Transformation of pDC into IKpDC by HTLV-1.

Adult T cell leukemia induced by HTLV-1 infection exhibits diverse clinical features. The outcome is directly correlated to ATL subtype, that could range from a very indolent and slowly progressive lymphoma to a very aggressive and nearly uniformaly lethal proliferative lymphoma. Thus, knowledge about HTLV-1 infection and propagation remains essential to better understand pathogenesis consequences.

An important challenge would be to link the pDC phenotype to the different HTLV-1 associated pathologies (ATLL). It would be interesting to determine whether IKpDC persist during chronic infection in order to generate new HTLV-1 progression markers. The characterization of IKpDC *in vivo* opens new area of dendritic cells research in HTLV-1 and other retrovirus-induced immunopathogenesis and in tumor cell biology. Considered together, our data highlight a dual role for pDC in HTLV-1 disease. pDC that become infected may participate in viral spread in the host (Jones, Petrow-Sadowski et al., 2008) and concomitantly express TRAIL, which may select the transformed CD4+ T cell clone, leading to ATLL years later. In this context, it will be of great interest to test TRAIL sensitivity of the persistent clones after HTLV-1 infection that may subsequently be transformed to lymphoma/leukemia. Thus, pDC investigation in HTLV-1 disease will be crucial for understanding complex HTLV-1-associated pathologies. However, detection of primary infection in humans is currently not feasible due to the high latency of HTLV-1 virus before disease symptoms appearance. An alternative way to characterize and understand the early steps of HTLV-1 infection is the development of the pathogenic simian model (STLV-1). However, in addition to selection of TRAIL-resistant clones, one could hypothesize that similar to HIV-1 infection, pDC may participate in and contribute to the immune suppression that occurs in ATLL.

HTLV-1 free particles generate an immune response by professional virus "sentinel" pDC. We then identify and describe the mechanism by which purified HTLV-1 virions stimulate pDC and transform them into functional killer cells. We show that pDC response and activation to HTLV-1 is strictly virus-dose dependent. Finally, purified HTLV-1 particles induced TLR7-mediated relocalization of intracellular TRAIL to the pDC membrane. In conclusion, the physiological function of pDC during the different stages of HTLV-1 infection will represent a new field of investigation and may lead to new therapeutic strategies.

#### **7. Acknowledgment**

I would like to thank Pr Olivier Hermine, UMR 8147 Hopital Necker, Paris, for his precious advice and critics and Lucie Barblu for helping me on this manuscript.

#### **8. References**

Banchereau, J. and R. M. Steinman (1998). "Dendritic cells and the control of immunity." *Nature* 392 (6673): 245-52

Bangert, C., J. Friedl, et al. (2003). "Immunopathologic features of allergic contact dermatitis in humans: participation of plasmacytoid dendritic cells in the pathogenesis of the disease?" *J Invest Dermatol* 121 (6): 1409-18

Adult Human T Cell Leukemia 19

Fujino, T. and Y. Nagata (2000). "HTLV-I transmission from mother to child." *J Reprod* 

Gasper-Smith, N., D. M. Crossman, et al. (2008). "Induction of plasma (TRAIL), TNFR-2, Fas

Gessain, A., F. Barin, et al. (1985). "Antibodies to human T-lymphotropic virus type-I in

Ghez, D., Y. Lepelletier, et al. "Current concepts regarding the HTLV-1 receptor complex."

Ghez, D., Y. Lepelletier, et al. (2006). "Neuropilin-1 is involved in human T-cell

Gibson, S. J., J. M. Lindh, et al. (2002). "Plasmacytoid dendritic cells produce cytokines and

Gomez-Benito, M., M. J. Martinez-Lorenzo, et al. (2007). "Membrane expression of DR4, DR5

Grabbe, S., E. Kampgen, et al. (2000). "Dendritic cells: multi-lineal and multi-functional."

Griffith, T. S., W. A. Chin, et al. (1998). "Intracellular regulation of TRAIL-induced apoptosis

Grouard, G., M. C. Rissoan, et al. (1997). "The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand." *J Exp Med* 185 (6): 1101-11 Grumont, R. J. and S. Gerondakis (2000). "Rel induces interferon regulatory factor 4 (IRF-4)

Guiducci, C., C. Ghirelli, et al. (2008). "PI3K is critical for the nuclear translocation of IRF-7

Guiducci, C., G. Ott, et al. (2006). "Properties regulating the nature of the plasmacytoid

Gura, T. (1997). "How TRAIL kills cancer cells, but not normal cells." *Science* 277 (5327): 768 Hardy, A. W., D. R. Graham, et al. (2007). "HIV turns plasmacytoid dendritic cells (pDC)

receptor 7-induced IFN-alpha." *Proc Natl Acad Sci U S A* 104 (44): 17453-8 Hasegawa, H., Y. Yamada, et al. (2005). "Sensitivity of adult T-cell leukaemia lymphoma

Hasegawa, H., Y. Yamada, et al. (2009). "Activation of p53 by Nutlin-3a, an antagonist of

mature in response to the TLR7 agonists, imiquimod and resiquimod." *Cell Immunol*

and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells

expression in lymphocytes: modulation of interferon-regulated gene expression by

and type I IFN production by human plasmacytoid predendritic cells in response to

dendritic cell response to Toll-like receptor 9 activation." *J Exp Med* 203 (8):

into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like

cells to tumour necrosis factor-related apoptosis-inducing ligand." *Br J Haematol* 128

MDM2, induces apoptosis and cellular senescence in adult T-cell leukemia cells."

patients with tropical spastic paraparesis." *Lancet* 2 (8452): 407-10

lymphotropic virus type 1 entry." *J Virol 80 (14): 6844-54* 

to Apo2L/TRAIL." *Exp Cell Res* 313 (11): 2378-88

in human melanoma cells." *J Immunol* 161 (6): 2833-40

rel/nuclear factor kappaB." *J Exp Med* 191 (8): 1281-92

ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1) transmission: implications for HIV-1 vaccine design." *J Virol* 82 (15):

*Immunol* 47 (2): 197-206

7700-10

*Retrovirology* 7: 99

218 (1-2): 74-86

*Immunol Today* 21 (9): 431-3

TLR activation." *J Exp Med*

*Leukemia* 23 (11): 2090-101

1999-2008

(2): 253-65


Beignon, A. S., K. McKenna, et al. (2005). "Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor- viral RNA interactions." *J Clin Invest* Bellail, A. C., M. C. Tse, et al. (2009). "DR5-mediated DISC controls caspase-8 cleavage and

Bleumink, M., R. Kohler, et al. "Rocaglamide breaks TRAIL resistance in HTLV-1-associated

Cao, W., L. Zhang, et al. (2007). "BDCA2/Fc epsilon RI gamma complex signals through a

Cella, M., D. Jarrossay, et al. (1999). "Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon." *Nat Med* 5 (8): 919-23 Chaperot, L., A. Blum, et al. (2006). "Virus or TLR agonists induce TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells." *J Immunol* 176 (1): 248-55 Colisson, R., L. Barblu, et al. "Free HTLV-1 induces TLR7-dependent innate immune

Collison, A., P. S. Foster, et al. (2009). "Emerging role of tumour necrosis factor-related

Colonna, M. (2006). "Toll-like receptors and IFN-alpha: partners in autoimmunity." *J Clin* 

Colonna, M., G. Trinchieri, et al. (2004). "Plasmacytoid dendritic cells in immunity." *Nat* 

de The, G., L. Gazzolo, et al. (1985). "Viruses as risk factors or causes of human leukaemias

Diebold, S. S., T. Kaisho, et al. (2004). "Innate antiviral responses by means of TLR7 mediated recognition of single-stranded RNA." *Science* 303 (5663): 1529-31 Donegan, E., H. Lee, et al. (1994). "Transfusion transmission of retroviruses: human T-

Duramad, O., K. L. Fearon, et al. (2005). "Inhibitors of TLR-9 act on multiple cell subsets in

Dzionek, A., Y. Inagaki, et al. (2002). "Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions." *Hum Immunol* 63 (12): 1133-48 Dzionek, A., Y. Sohma, et al. (2001). "BDCA-2, a novel plasmacytoid dendritic cell-specific

Ehrlich, S., C. Infante-Duarte, et al. (2003). "Regulation of soluble and surface-bound TRAIL

Fonteneau, J. F., M. Larsson, et al. (2004). "Human immunodeficiency virus type 1 activates

*Journal of immunology* (Baltimore, Md.: 1950) 174 (9): 5193-5200

interferon alpha/beta induction." *J Exp Med* 194 (12): 1823-34

of myeloid dendritic cells." *J Virol* 78 (10): 5223-32

in human T cells, B cells, and monocytes." *Cytokine* 24 (6): 244-53

lymphotropic virus types I and II compared with human immunodeficiency virus

mouse and man in vitro and prevent death in vivo from systemic inflammation."

type II C-type lectin, mediates antigen capture and is a potent inhibitor of

plasmacytoid dendritic cells and concomitantly induces the bystander maturation

adult T-cell leukemia/lymphoma by translational suppression of c-FLIP

novel BCR-like pathway in human plasmacytoid dendritic cells." *PLoS Biol* 5 (10):

response and TRAIL relocalization in killer plasmacytoid dendritic cells." *Blood* 115

apoptosis-inducing ligand (TRAIL) as a key regulator of inflammatory responses."

initiation of apoptosis in human glioblastomas." *J Cell Mol Med*

expression." *Cell Death Differ* 18 (2): 362-70

*Clin Exp Pharmacol Physiol* 36 (11): 1049-53

and lymphomas?" *Leuk Res* 9 (6): 691-6

type 1." *Transfusion* 34 (6): 478-83

e248

(11): 2177-85

*Invest* 116 (9): 2319-22

*Immunol* 5 (12): 1219-26


Adult Human T Cell Leukemia 21

Jin, T. G., A. Kurakin, et al. (2004). "Fas-associated protein with death domain (FADD)-

Jones, K. S., C. Petrow-Sadowski, et al. (2005). "Heparan sulfate proteoglycans mediate

Jones, K. S., C. Petrow-Sadowski, et al. (2008). "Cell-free HTLV-1 infects dendritic cells

Kadowaki, N., S. Antonenko, et al. (2000). "Natural interferon alpha/beta-producing cells

Kadowaki, N. and Y. J. Liu (2002). "Natural type I interferon-producing cells as a link between innate and adaptive immunity." *Hum Immunol* 63 (12): 1126-32 Kayagaki, N., N. Yamaguchi, et al. (1999). "Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity." *J Immunol* 162 (5): 2639-47 Kohno, T., R. Moriuchi, et al. (2000). "Identification of genes associated with the progression

Koyanagi, Y., Y. Itoyama, et al. (1993). "In vivo infection of human T-cell leukemia virus type

Kunitomi, A., T. Hori, et al. (2002). "OX40 signaling renders adult T-cell leukemia cells

Lambert, S., M. Bouttier, et al. (2009). "HTLV-1 uses HSPG and neuropilin 1 for entry by

Lichtner, M., C. Maranon, et al. (2004). "HIV type 1-infected dendritic cells induce apoptotic

Liu, Y. J. (2005). "IPC: professional type 1 interferon-producing cells and plasmacytoid

Lund, J., A. Sato, et al. (2003). "Toll-like receptor 9-mediated recognition of Herpes simplex

Lund, J. M., L. Alexopoulou, et al. (2004). "Recognition of single-stranded RNA viruses by

Macatonia, S. E., J. K. Cruickshank, et al. (1992). "Dendritic cells from patients with tropical

Makino, M., S. Shimokubo, et al. (1999). "The role of human T-lymphotropic virus type 1

Mamane, Y., N. Grandvaux, et al. (2002). "Repression of IRF-4 target genes in human T cell

Mamane, Y., M. Loignon, et al. (2005). "Repression of DNA repair mechanisms in IRF-4-

spastic paraparesis are infected with HTLV-1 and stimulate autologous lymphocyte

(HTLV-1)-infected dendritic cells in the development of HTLV-1-associated

expressing and HTLV-I-infected T lymphocytes." *J Interferon Cytokine Res* 25 (1):

virus-2 by plasmacytoid dendritic cells." *J Exp Med* 198 (3): 513-20

Toll-like receptor 7." *Proc Natl Acad Sci U S A* 101 (15): 5598-603

myelopathy/tropical spastic paraparesis." *J Virol* 73 (6): 4575-81

death in infected and uninfected primary CD4 T lymphocytes." *AIDS Res Hum* 

link innate and adaptive immunity." *J Exp Med* 192 (2): 219-26

of adult T cell leukemia (ATL)." *Jpn J Cancer Res* 91 (11): 1103-10

resistant to Fas-induced apoptosis." *Int J Hematol* 76 (3): 260-6

dendritic cell precursors." *Annu Rev Immunol* 23: 275-306

proliferation." *AIDS Res Hum Retroviruses* 8 (9): 1699-706

leukemia virus-1 infection." *Oncogene* 21 (44): 6751-65

55594-601

429-36

cells." *J Virol* 79 (20): 12692-702

I in non-T cells." *Virology* 196 (1): 25-33

molecular mimicry of VEGF165." *Blood*

*Retroviruses* 20 (2): 175-82

43-51

independent recruitment of c-FLIPL to death receptor 5." *J Biol Chem* 279 (53):

attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T

leading to transmission and transformation of CD4(+) T cells." *Nat Med* 14 (4):


Heil, F., H. Hemmi, et al. (2004). "Species-specific recognition of single-stranded RNA via

Hemmi, H., T. Kaisho, et al. (2002). "Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway." *Nat Immunol* 3 (2): 196-200 Hemmi, H., O. Takeuchi, et al. (2000). "A Toll-like receptor recognizes bacterial DNA."

Herbeuval, J. P., A. Boasso, et al. (2005). "TNF-related apoptosis-inducing ligand (TRAIL) in

Herbeuval, J. P., J. C. Grivel, et al. (2005). "CD4+ T-cell death induced by infectious and

Herbeuval, J. P., C. Lambert, et al. (2003). "Macrophages From Cancer Patients: Analysis of

Herbeuval, J. P., J. Nilsson, et al. (2009). "HAART reduces death ligand but not death

Hermine, O., D. Bouscary, et al. (1995). "Brief report: treatment of adult T-cell leukemialymphoma with zidovudine and interferon alfa." *N Engl J Med* 332 (26): 1749-51 Hermine, O., H. Dombret, et al. (2004). "Phase II trial of arsenic trioxide and alpha interferon

Hinuma, Y., Y. Gotoh, et al. (1982). "A retrovirus associated with human adult T-cell

Honda, K., Y. Ohba, et al. (2005). "Spatiotemporal regulation of MyD88-IRF-7 signalling for

Honda, K., H. Yanai, et al. (2005). "IRF-7 is the master regulator of type-I interferon-

Hoshino, K., T. Sugiyama, et al. (2006). "IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9." *Nature* 440 (7086): 949-953 Imaizumi, Y., T. Kohno, et al. (2001). "Possible involvement of interferon regulatory factor 4

Imura, A., T. Hori, et al. (1997). "OX40 expressed on fresh leukemic cells from adult T-cell

Jacobson, S. (1996). "Cellular immune responses to HTLV-I: immunopathogenic role in

Jarrossay, D., G. Napolitani, et al. (2001). "Specialization and complementarity in microbial

(IRF4) in a clinical subtype of adult T-cell leukemia." *Jpn J Cancer Res* 92 (12):

leukemia patients mediates cell adhesion to vascular endothelial cells: implication for the possible involvement of OX40 in leukemic cell infiltration." *Blood* 89 (8):

HTLV-I-associated neurologic disease." *J Acquir Immune Defic Syndr Hum Retrovirol*

molecule recognition by human myeloid and plasmacytoid dendritic cells." *Eur J* 

immunodeficiency virus-infected macaques." *Aids* 23 (1): 35-40

robust type-I interferon induction." *Nature* 434 (7036): 1035-40

dependent immune responses." *Nature* 434 (7034): 772-7

leukemia: in vitro activation." *Gann* 73 (2): 341-4

HIV-1-infected patients and its in vitro production by antigen-presenting cells."

noninfectious HIV-1: role of type 1 interferon-dependent, TRAIL/DR5-mediated

TRAIL, TRAIL Receptors, and Colon Tumor Cell Apoptosis." *J Natl Cancer Inst* 95

receptors in lymphoid tissue of HIV-infected patients and simian

in patients with relapsed/refractory adult T-cell leukemia/lymphoma." *Hematol J* 5

toll-like receptor 7 and 8." *Science* 303 (5663): 1526-9

*Nature* 408 (6813): 740-5

*Blood* 105 (6): 2458-64

(8): 611-621

(2): 130-4

1284-92

2951-8

13 Suppl 1: S100-6

*Immunol* 31 (11): 3388-93

apoptosis." *Blood* 106 (10): 3524-31


Adult Human T Cell Leukemia 23

Sato, K., S. Hida, et al. (2001). "Antiviral response by natural killer cells through TRAIL gene

Sharma, S., N. Grandvaux, et al. (2002). "Regulation of IFN regulatory factor 4 expression in human T cell leukemia virus-I-transformed T cells." *J Immunol* 169 (6): 3120-30 Sharma, S., Y. Mamane, et al. (2000). "Activation and regulation of interferon regulatory

Sheikh, M. S., T. F. Burns, et al. (1998). "p53-dependent and -independent regulation of the

Sheridan, J. P., S. A. Marsters, et al. (1997). "Control of TRAIL-induced apoptosis by a family

Siegal, F. P., N. Kadowaki, et al. (1999). "The nature of the principal type 1 interferon-

Smyth, M. J., E. Cretney, et al. (2001). "Tumor necrosis factor-related apoptosis-inducing

Stary, G., I. Klein, et al. (2009). "Plasmacytoid dendritic cells express TRAIL and induce CD4+ T-cell apoptosis in HIV-1 viremic patients." *Blood* 114 (18): 3854-63 Takenouchi, N., K. S. Jones, et al. (2007). "GLUT1 is not the primary binding receptor but is

Tecchio, C., V. Huber, et al. (2004). "IFNalpha-stimulated neutrophils and monocytes release

Tordjman, R., Y. Lepelletier, et al. (2002). "A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response." *Nat Immunol* 3 (5): 477-82 Uematsu, S., S. Sato, et al. (2005). "Interleukin-1 receptor-associated kinase-1 plays an

Valledor, A. F., F. E. Borras, et al. (1998). "Transcription factors that regulate

Verdonck, K., E. Gonzalez, et al. (2007). "Human T-lymphotropic virus 1: recent knowledge

Vidalain, P. O., O. Azocar, et al. (2000). "Measles virus induces functional TRAIL production

Vidalain, P. O., O. Azocar, et al. (2001). "Measle virus-infected dendritic cells develop immunosuppressive and cytotoxic activities." *Immunobiology* 204 (5): 629-38 Walczak, H. and T. L. Haas (2008). "Biochemical analysis of the native TRAIL death-

displaying apoptotic activity on leukemic cells." *Blood* 103 (10): 3837-44 Tendler, C. L., S. J. Greenberg, et al. (1991). "Cytokine induction in HTLV-I associated

factor 4 in HTLV type 1-infected T lymphocytes." *AIDS Res Hum Retroviruses* 16

death receptor KILLER/DR5 gene expression in response to genotoxic stress and

ligand (TRAIL) contributes to interferon gamma-dependent natural killer cell

associated with cell-to-cell transmission of human T-cell leukemia virus type 1." *J* 

a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand)

myelopathy and adult T-cell leukemia: alternate molecular mechanisms underlying

essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha}

induction by IFN-alpha/beta." *Eur J Immunol* 31 (11): 3138-46

tumor necrosis factor alpha." *Cancer Res* 58 (8): 1593-8

of signaling and decoy receptors." *Science* 277 (5327): 818-21.

producing cells in human blood." *Science* 284 (5421): 1835-7

protection from tumor metastasis." *J Exp Med* 193 (6): 661-70

retroviral pathogenesis." *J Cell Biochem* 46 (4): 302-11

Uozumi, K. "Treatment of adult T-cell leukemia." *J Clin Exp Hematop* 50 (1): 9-25

about an ancient infection." *Lancet Infect Dis* 7 (4): 266-81

inducing signaling complex." *Methods Mol Biol* 414: 221-39

by human dendritic cells." *J Virol* 74 (1): 556-9

monocyte/macrophage differentiation." *J Leukoc Biol* 63 (4): 405-17

induction." *J ex Med* 201 (6): 915-923

(16): 1613-22

*Virol* 81 (3): 1506-10


Manel, N., F. J. Kim, et al. (2003). "The ubiquitous glucose transporter GLUT-1 is a receptor

Manns, A., W. J. Miley, et al. (1999). "Quantitative proviral DNA and antibody levels in the

Mansky, L. M. (2000). "In vivo analysis of human T-cell leukemia virus type 1 reverse

Marshak-Rothstein, A. (2006). "Toll-like receptors in systemic autoimmune disease." *Nat Rev* 

Matsuda, T., A. Almasan, et al. (2005). "Resistance to Apo2 ligand (Apo2L)/tumor necrosis

Matsuoka, M. (2005). "Human T-cell leukemia virus type I (HTLV-I) infection and the onset

Mortreux, F., I. Leclercq, et al. (2001). "Somatic mutation in human T-cell leukemia virus

Mueller, N., A. Okayama, et al. (1996). "Findings from the Miyazaki Cohort Study." *J Acquir* 

Nagai, M., R. Kubota, et al. (2001). "Increased activated human T cell lymphotropic virus

Nestle, F. O., C. Conrad, et al. (2005). "Plasmacytoid predendritic cells initiate psoriasis

Pais-Correia, A. M., M. Sachse, et al. "Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses." *Nat Med* 16 (1): 83-9 Poiesz, B. J., F. W. Ruscetti, et al. (1980). "T-cell lines established from human T-lymphocytic

Poiesz, B. J., F. W. Ruscetti, et al. (1981). "Isolation of a new type C retrovirus (HTLV) in

Proietti, F. A., A. B. Carneiro-Proietti, et al. (2005). "Global epidemiology of HTLV-I infection

Pulendran, B., K. Palucka, et al. (2001). "Sensing pathogens and tuning immune responses."

Richardson, J. H., A. J. Edwards, et al. (1990). "In vivo cellular tropism of human T-cell

Robert-Guroff, M., Y. Nakao, et al. (1982). "Natural antibodies to human retrovirus HTLV in a cluster of Japanese patients with adult T cell leukemia." *Science* 215 (4535): 975-8 Salio, M., M. Cella, et al. (2003). "Plasmacytoid dendritic cells prime IFN-gamma-secreting

melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions."

through interferon-alpha production." *J Exp Med* 202 (1): 135-43

factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-

type 1 provirus and flanking cellular sequences during clonal expansion in vivo." *J* 

type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with

neoplasias by direct response to T-cell growth factor." *Proc Natl Acad Sci U S A* 77

primary uncultured cells of a patient with Sezary T-cell leukaemia." *Nature* 294

natural history of HTLV-I infection." *J Infect Dis* 180 (5): 1487-93

transcription accuracy." *J Virol* 74 (20): 9525-31

infected T-cell lines." *J Virol* 79 (3): 1367-78

of adult T-cell leukemia (ATL)." *Retrovirology* 2: 27

*Immune Defic Syndr Hum Retrovirol* 13 Suppl 1: S2-7

HTLV-I provirus load." *J Infect Dis* 183 (2): 197-205

and associated diseases." *Oncogene* 24 (39): 6058-68

leukemia virus type 1." *J Virol* 64 (11): 5682-7

for HTLV." Cell 115 (4): 449-59

*Immunol* 6 (11): 823-35

*Natl Cancer Inst* 93 (5): 367-77

(11): 6815-9

(5838): 268-71

*Science* 293 (5528): 253-6

*Eur J Immunol* 33 (4): 1052-62


**2** 

*Iran* 

**Adult T-Cell Leukemia** 

Mohammad R. Abbaszadegan and Mehran Gholamin *Division of Human Genetics, Immunology Research Center* 

**Human T-Cell Lymphotropic Virus (HTLV-1) and** 

Human T-cell Lymphotropic Viruses (HTLVs) and Simian T-cell Lymphotropic Viruses (STLVs) are anciently related primate T-cell leukemia viruses (PTLVs) that share molecular and virological features. Human T-cell Lymphotropic Virus (HTLV-1) is believed to be repeatedly transmitted in separate independent events from simians to humans beginning 50,000 ± 10,000 years ago; this course has resulted in the formation of several viral subtypes around the world. There are four known strains of HTLV, of which HTLV-1 and HTLV-2 are the most prevalent worldwide. Newer HTLVs, HTLV-3 and 4 have been identified recently

HTLV-1, the first human retrovirus was discovered by two independent investigating groups in 1980 and 1981. A geographical clustering of leukaemias in southwestern Japan led to the description of a unique clinical entity termed adult T-cell leukemia (ATL), where Japanese investigators identified HTLV-1 as an etiologic agent of newly described ATL, and

HTLV-1 belongs to the Deltaretrovirus genera of the Orthoretrovirinae subfamily, the first discovered human retrovirus, isolated in the early 1980s from peripheral blood samples of a patient with cutaneous T-cell lymphoma (Poiesz et al, 1980). It is the etiologic agent of two predominant distinct human diseases, ATL or adult T-cell leukemia lymphoma (ATLL) and a chronic, progressive demyelinating disorder known as HTLV-1-associated

The major findings that support the etiologic association of HTLV-1 are: 1) All patients with ATL have antibodies against HTLV-1, 2) The areas of high incidence of ATL patients correspond closely with those of high incidence of HTLV-1 carriers, 3) HTLV-1 immortalizes human T cells *in vitro*, 4) Monoclonal integration of HTLV-1 proviral DNA was demonstrated in ATL cells. Thus, HTLV-1 is the first retrovirus directly associated with human malignancy (Takatsuki 2005). HTLV-1 is a complex leukemogenic retrovirus with a single stranded positive sense RNA genome that expresses unique proteins with oncogenic potential. HTLV-1 can infect T cells, B cells, monocytes, dendritic cells and endothelial cells

HTLV-2 was identified in a CD8+ T cell line derived from a patient with a variant form of hairy T cell leukemia. Since then, HTLV-2 has not been associated with leukemia/lymphoma; nevertheless, it has been associated with a few sporadic cases of

with equal efficiency; yet, it can transform only primary T cells (Hanon et al. 2000).

the U.S. investigators detected HTLV-1 retrovirus in human cell lines (Yoshida 2010).

myelopathy/tropical spastic paraparesis (HAM/TSP)(Zanjani et al. 2010).

from bush meat hunters in central Africa(Matsuoka and Jeang 2007).

**1. Introduction**

*Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad* 


### **Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia**

Mohammad R. Abbaszadegan and Mehran Gholamin

*Division of Human Genetics, Immunology Research Center Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad Iran* 

#### **1. Introduction**

24 T-Cell Leukemia

Walczak, H., R. E. Miller, et al. (1999). "Tumoricidal activity of tumor necrosis factor-related

Wiley, S. R., K. Schooley, et al. (1995). "Identification and characterization of a new member

Wu, G. S., T. F. Burns, et al. (1997). "KILLER/DR5 is a DNA damage-inducible p53-regulated

Yamaguchi, K. (1994). "Human T-lymphotropic virus type I in Japan." *Lancet* 343 (8891):

Yang, G. X., Z. X. Lian, et al. (2005). "Plasmacytoid dendritic cells of different origins have

Yoshie, O. (2005). "Expression of CCR4 in adult T-cell leukemia." *Leuk Lymphoma* 46 (2):

distinct characteristics and function: studies of lymphoid progenitors versus

apoptosis-inducing ligand in vivo." *Nat Med* 5 (2): 157-63

death receptor gene." *Nat Genet* 17 (2): 141-3

myeloid progenitors." *J Immunol* 175 (11): 7281-7

213-6

185-90

of the TNF family that induces apoptosis." *Immunity* 3 (6): 673-82

Human T-cell Lymphotropic Viruses (HTLVs) and Simian T-cell Lymphotropic Viruses (STLVs) are anciently related primate T-cell leukemia viruses (PTLVs) that share molecular and virological features. Human T-cell Lymphotropic Virus (HTLV-1) is believed to be repeatedly transmitted in separate independent events from simians to humans beginning 50,000 ± 10,000 years ago; this course has resulted in the formation of several viral subtypes around the world. There are four known strains of HTLV, of which HTLV-1 and HTLV-2 are the most prevalent worldwide. Newer HTLVs, HTLV-3 and 4 have been identified recently from bush meat hunters in central Africa(Matsuoka and Jeang 2007).

HTLV-1, the first human retrovirus was discovered by two independent investigating groups in 1980 and 1981. A geographical clustering of leukaemias in southwestern Japan led to the description of a unique clinical entity termed adult T-cell leukemia (ATL), where Japanese investigators identified HTLV-1 as an etiologic agent of newly described ATL, and the U.S. investigators detected HTLV-1 retrovirus in human cell lines (Yoshida 2010).

HTLV-1 belongs to the Deltaretrovirus genera of the Orthoretrovirinae subfamily, the first discovered human retrovirus, isolated in the early 1980s from peripheral blood samples of a patient with cutaneous T-cell lymphoma (Poiesz et al, 1980). It is the etiologic agent of two predominant distinct human diseases, ATL or adult T-cell leukemia lymphoma (ATLL) and a chronic, progressive demyelinating disorder known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)(Zanjani et al. 2010).

The major findings that support the etiologic association of HTLV-1 are: 1) All patients with ATL have antibodies against HTLV-1, 2) The areas of high incidence of ATL patients correspond closely with those of high incidence of HTLV-1 carriers, 3) HTLV-1 immortalizes human T cells *in vitro*, 4) Monoclonal integration of HTLV-1 proviral DNA was demonstrated in ATL cells. Thus, HTLV-1 is the first retrovirus directly associated with human malignancy (Takatsuki 2005). HTLV-1 is a complex leukemogenic retrovirus with a single stranded positive sense RNA genome that expresses unique proteins with oncogenic potential. HTLV-1 can infect T cells, B cells, monocytes, dendritic cells and endothelial cells with equal efficiency; yet, it can transform only primary T cells (Hanon et al. 2000).

HTLV-2 was identified in a CD8+ T cell line derived from a patient with a variant form of hairy T cell leukemia. Since then, HTLV-2 has not been associated with leukemia/lymphoma; nevertheless, it has been associated with a few sporadic cases of

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 27

of seropositive people in the world. In non-endemic areas, certain groups should be considered as at risk, such as immigrants from endemic areas, the sexual partners and descendents of people known to be infected, sex professionals and drug users(Romanelli,

HTLV-1 carriers are mostly asymptomatic in their life spans. The lifetime risks of developing ATL and HAM/TSP are about 2.5 to 5% and 0.3 to 2%, respectively (Silva et al. 2007). Among HTLV-1 infected individuals in Japan, a small proportion of carriers (6% for males and 2% for females) develop ATL. The majority of HTLV-1 carriers do not develop HTLV-1-associated diseases. The latency period from the initial infection until onset of ATL is about 60 years in Japan and 40 years in Jamaica. These determinations indicate a multistep

HTLV-1 virions are complex type C particles, spherical, enveloped and 100–110 nm in diameter. The inner membrane of the virion envelope is lined by the viral matrix protein (MA). This structure encloses the viral capsid (CA), which carries two identical strands of the genomic RNA as well as functional protease (Pro), integrase (IN), and reverse transcriptase (RT) enzymes. A newly synthesized viral particle attaches to the target cell receptor through the viral envelope (Env) and enters via fusion, which is followed by the uncoating of the capsid and the release of its contents into the cell cytoplasm. The viral genome consists of a linear, positive sense, ssRNA held together by hydrogen bonds. Each monomer has about 9032 nucleotides. The 3' terminal viral genome is polyadenylated and its 5'- terminal is capped. Each unit is associated with a specific molecule of tRNA that is base paired to a region, primer binding site, near the 5' end of the RNA. Proviral forms are flanked at both termini by long terminal repeats (LTRs) of 754 nucleotides. The genomic structure encodes structural and enzymatic proteins: gag, pol, env, reverse transcriptase, protease, and integrase. In addition, HTLV-1 has a region at the 3' end of the virus, called pX, which encodes four partially overlapping reading frames (ORFs). These ORFs code for regulatory proteins which impact the expression and replication of the virus (Figure 1)

The viral RNA is reverse transcribed into double stranded DNA by the RT. This double stranded DNA is then transported to the nucleus and becomes integrated into the host chromosome forming the provirus. The provirus contains the promoter and enhancer elements for transcription initiation in the long terminal repeats (LTR); the polyadenylation

The initial round of HTLV-1 transcription is dependent on cellular factors. The complex retroviral genome codes for the structural proteins Gag (capsid, nucleocapsid, and matrix), Pro, polymerase (Pol) and Env from unspliced/singly spliced mRNAs. Alternatively spliced mRNA transcripts encode regulatory and accessory proteins. The two regulatory genes rex and tax are encoded by open reading frames (ORF) III and IV, respectively, and share a common doubly spliced transcript. Tax is the transactivator gene, which increases the rate of viral LTR-mediated transcription and modulates the transcription of numerous cellular genes involved in cell proliferation and differentiation, cell cycle control and DNA repair. Tax has displayed oncogenic potential in several experimental systems and is essential for HTLV-1 and HTLV-2-mediated transformation of primary human T cells. Rex acts posttranscriptionally by preferentially binding, stabilizing exporting intron-containing viral

signal for plus strand transcription are located in the 3'LTR (Kannian 2010).

Caramelli and Proietti 2010).

leukemogenic mechanism in the generation of ATL.

**3. Genomic structure of HTLV-1**

(Boxus and Willems 2009).

neurological disorders and chronic encephalomyelopathy (Hjelle et al. 1992). The clinical symptoms presented are similar to those of HAM/TSP. The prevalence of HTLV-2 associated myelopathy was reported to be 1% compared to 3.7% for HTLV-1 associated HAM/TSP in the United State. Although other neurological disorders have been reported, their clear association with HTLV-2 is hampered by confounding factors such as intravenous drug use or concomitant HIV infection. To date, HTLV-3 and HTLV-4 have not been associated with any known clinical conditions (Kannian 2010).

In 1985, Gessain et al., demonstrated that 68% of patients with tropical spastic paraparesis (TSP) in Martinique had positive serology for HTLV-1. In 1986, a similar neurological condition was described in Japan and named HTLV-1 associated myelopathy (HAM). Later, Román and Osame (1988) concluded that they were dealing with the same disease, and the term HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP) came to be used. Since then, countless other diseases have been correlated with this infection: uveitis, Sjögren's syndrome, infectious dermatitis, polymyositis, arthropathies, thyroiditis, polyneuropathies, lymphocytic alveolitis, cutaneous T-cell lymphoma, strongyloidiasis, scabies, Hansen's disease and tuberculosis. The importance of the possible clinical manifestations of the HTLV virus has now become clear in several different medical specialties such as oncology, neurology, internal medicine, dermatology, and ophthalmology (Romanelli, Caramelli and Proietti 2010).

Only HTLV-1-infected individuals develop ATL, and all ATL cells contain integrated HTLV-1 provirus, supporting the causal etiology of the virus for leukaemogenesis. Nevertheless, only a small minority of HTLV-1-infected individuals progress to ATL. Indeed, the cumulative risks of developing ATL among virus carriers are estimated to be approximately 6.6% for males and 2.1% for females (Matsuoka and Jeang 2007).

A long period of latency from HTLV-1 infection to ATL development suggests a multistep process of T-lymphocyte transformation. In ATL patients, the malignant cells typically consist of oligoclonal or monoclonal outgrowths of CD4+ and CD25+ T lymphocytes carrying a complete or defective provirus of HTLV-1. Four clinical subtypes of ATL include acute, lymphoma, chronic and smoldering (Noula Shembade 2010).

#### **2. Worldwide distribution**

Approximately 15-25 million people worldwide are infected with HTLV-1. The virus is endemic in southwestern Japan, Africa, the Caribbean Islands and South America and is frequently found in Melanesia, Papua New Guinea, Solomon Islands and Australian aborigines. HTLV-1 is also prevalent in certain populations in the Middle East (Iran) and India. HTLV-2 is more prevalent among intravenous drug users (IDUs), and is endemic among IDUs in the USA, Europe, South America and Southeast Asia. HTLV-3 and HTLV-4 have been identified only in African primate hunters (Kannian 2010). HTLV-1 infection is endemic in northeastern Iran (Khorasan province) and the prevalence of HTLV-1 infection is estimated to be 2-3% in the whole population and 0.78% in blood donors (Abbaszadegan et al. 2003; Safai et al. 1996).

High prevalence rates in the general population are observed in the South of Japan (10%), in Jamaica and Trinidad and Tobago (6%). In South America (Argentina, Brazil, Colombia and Peru) a 2% prevalence of seropositivity was observed among blood donors. It is known that the prevalence of HITLV-1 in population of blood donors represents an underestimation of prevalence in the general population. In absolute terms, Brazil may have the largest number of seropositive people in the world. In non-endemic areas, certain groups should be considered as at risk, such as immigrants from endemic areas, the sexual partners and descendents of people known to be infected, sex professionals and drug users(Romanelli, Caramelli and Proietti 2010).

HTLV-1 carriers are mostly asymptomatic in their life spans. The lifetime risks of developing ATL and HAM/TSP are about 2.5 to 5% and 0.3 to 2%, respectively (Silva et al. 2007). Among HTLV-1 infected individuals in Japan, a small proportion of carriers (6% for males and 2% for females) develop ATL. The majority of HTLV-1 carriers do not develop HTLV-1-associated diseases. The latency period from the initial infection until onset of ATL is about 60 years in Japan and 40 years in Jamaica. These determinations indicate a multistep leukemogenic mechanism in the generation of ATL.

#### **3. Genomic structure of HTLV-1**

26 T-Cell Leukemia

neurological disorders and chronic encephalomyelopathy (Hjelle et al. 1992). The clinical symptoms presented are similar to those of HAM/TSP. The prevalence of HTLV-2 associated myelopathy was reported to be 1% compared to 3.7% for HTLV-1 associated HAM/TSP in the United State. Although other neurological disorders have been reported, their clear association with HTLV-2 is hampered by confounding factors such as intravenous drug use or concomitant HIV infection. To date, HTLV-3 and HTLV-4 have not

In 1985, Gessain et al., demonstrated that 68% of patients with tropical spastic paraparesis (TSP) in Martinique had positive serology for HTLV-1. In 1986, a similar neurological condition was described in Japan and named HTLV-1 associated myelopathy (HAM). Later, Román and Osame (1988) concluded that they were dealing with the same disease, and the term HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP) came to be used. Since then, countless other diseases have been correlated with this infection: uveitis, Sjögren's syndrome, infectious dermatitis, polymyositis, arthropathies, thyroiditis, polyneuropathies, lymphocytic alveolitis, cutaneous T-cell lymphoma, strongyloidiasis, scabies, Hansen's disease and tuberculosis. The importance of the possible clinical manifestations of the HTLV virus has now become clear in several different medical specialties such as oncology, neurology, internal medicine, dermatology, and

Only HTLV-1-infected individuals develop ATL, and all ATL cells contain integrated HTLV-1 provirus, supporting the causal etiology of the virus for leukaemogenesis. Nevertheless, only a small minority of HTLV-1-infected individuals progress to ATL. Indeed, the cumulative risks of developing ATL among virus carriers are estimated to be

A long period of latency from HTLV-1 infection to ATL development suggests a multistep process of T-lymphocyte transformation. In ATL patients, the malignant cells typically consist of oligoclonal or monoclonal outgrowths of CD4+ and CD25+ T lymphocytes carrying a complete or defective provirus of HTLV-1. Four clinical subtypes of ATL include

Approximately 15-25 million people worldwide are infected with HTLV-1. The virus is endemic in southwestern Japan, Africa, the Caribbean Islands and South America and is frequently found in Melanesia, Papua New Guinea, Solomon Islands and Australian aborigines. HTLV-1 is also prevalent in certain populations in the Middle East (Iran) and India. HTLV-2 is more prevalent among intravenous drug users (IDUs), and is endemic among IDUs in the USA, Europe, South America and Southeast Asia. HTLV-3 and HTLV-4 have been identified only in African primate hunters (Kannian 2010). HTLV-1 infection is endemic in northeastern Iran (Khorasan province) and the prevalence of HTLV-1 infection is estimated to be 2-3% in the whole population and 0.78% in blood donors (Abbaszadegan et

High prevalence rates in the general population are observed in the South of Japan (10%), in Jamaica and Trinidad and Tobago (6%). In South America (Argentina, Brazil, Colombia and Peru) a 2% prevalence of seropositivity was observed among blood donors. It is known that the prevalence of HITLV-1 in population of blood donors represents an underestimation of prevalence in the general population. In absolute terms, Brazil may have the largest number

approximately 6.6% for males and 2.1% for females (Matsuoka and Jeang 2007).

acute, lymphoma, chronic and smoldering (Noula Shembade 2010).

**2. Worldwide distribution** 

al. 2003; Safai et al. 1996).

been associated with any known clinical conditions (Kannian 2010).

ophthalmology (Romanelli, Caramelli and Proietti 2010).

HTLV-1 virions are complex type C particles, spherical, enveloped and 100–110 nm in diameter. The inner membrane of the virion envelope is lined by the viral matrix protein (MA). This structure encloses the viral capsid (CA), which carries two identical strands of the genomic RNA as well as functional protease (Pro), integrase (IN), and reverse transcriptase (RT) enzymes. A newly synthesized viral particle attaches to the target cell receptor through the viral envelope (Env) and enters via fusion, which is followed by the uncoating of the capsid and the release of its contents into the cell cytoplasm. The viral genome consists of a linear, positive sense, ssRNA held together by hydrogen bonds. Each monomer has about 9032 nucleotides. The 3' terminal viral genome is polyadenylated and its 5'- terminal is capped. Each unit is associated with a specific molecule of tRNA that is base paired to a region, primer binding site, near the 5' end of the RNA. Proviral forms are flanked at both termini by long terminal repeats (LTRs) of 754 nucleotides. The genomic structure encodes structural and enzymatic proteins: gag, pol, env, reverse transcriptase, protease, and integrase. In addition, HTLV-1 has a region at the 3' end of the virus, called pX, which encodes four partially overlapping reading frames (ORFs). These ORFs code for regulatory proteins which impact the expression and replication of the virus (Figure 1) (Boxus and Willems 2009).

The viral RNA is reverse transcribed into double stranded DNA by the RT. This double stranded DNA is then transported to the nucleus and becomes integrated into the host chromosome forming the provirus. The provirus contains the promoter and enhancer elements for transcription initiation in the long terminal repeats (LTR); the polyadenylation signal for plus strand transcription are located in the 3'LTR (Kannian 2010).

The initial round of HTLV-1 transcription is dependent on cellular factors. The complex retroviral genome codes for the structural proteins Gag (capsid, nucleocapsid, and matrix), Pro, polymerase (Pol) and Env from unspliced/singly spliced mRNAs. Alternatively spliced mRNA transcripts encode regulatory and accessory proteins. The two regulatory genes rex and tax are encoded by open reading frames (ORF) III and IV, respectively, and share a common doubly spliced transcript. Tax is the transactivator gene, which increases the rate of viral LTR-mediated transcription and modulates the transcription of numerous cellular genes involved in cell proliferation and differentiation, cell cycle control and DNA repair. Tax has displayed oncogenic potential in several experimental systems and is essential for HTLV-1 and HTLV-2-mediated transformation of primary human T cells. Rex acts posttranscriptionally by preferentially binding, stabilizing exporting intron-containing viral

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 29

unspliced and spliced mRNAs) of the HTLV-1 basic leucine zipper factor (HBZ). HBZ interacts with cellular factors JunB, c-Jun, JunD, cAMP response element binding (CREB) and CREB binding protein (CBP)/p300to modulate both viral and cellular gene transcription. HBZ also plays a crucial role in T cell proliferation. Although research data indicate that Tax, among all the viral proteins, is the viral oncoprotein, but emerging data

HTLV-1 can infect various cell types, including T-lymphocytes, B-lymphocytes, monocytes, dendritic cells (DC) and fibroblasts. Glucose transporter 1 (GLUT-1) is ubiquitously expressed cell surface receptor targeted by HTLV-1 (Manel et al. 2003). Other cell surface receptors such as neuropilin 1 (NRP1) and surface heparan sulfate proteoglycans (HSPGs) have been reported to be target of HTLV-1 and are required for efficient entry (Boxus and Willems 2009). There is also evidence for cell-type specific receptors since a recent study has reported that HTLV-1 enters DCs by binding to the receptor DC-SIGN (Noula Shembade 2010.). However, the HTLV-1 provirus is mainly detected in CD4-positive lymphocytes, with about 10% in CD8-positive T-lymphocytes. This situation possibly arises because of Tax transformation of CD4-positive T-lymphocytes in vivo causing enhanced proliferation and suppressed apoptosis. In HTLV-1-infected individuals, no virions are detected in the serum. In addition, the infectivity of free virions is very poor compared with that of infected cells suggesting that HTLV-1 spreads through cell-to-cell transmission, rather than by free virions. *In vitro* analyses of HTLV-1 infected cells revealed that HTLV-1-infected cells form "virological synapses" with uninfected cells. The viral proteins Gag and Env, viral RNA and microtubules are accumulated as an infected cell is in contact with a target cell, and the viral complex subsequently transfers into the target cell. HTLV-1 also spreads in a cell- to-cell

In either route, HTLV-1-infected cells are essential for transmission. This was supported by the findings that fresh frozen plasma from carriers did not cause transmission and freeze-

Transplacental transmission is also suspected. Cellular blood products are the main source of transfusion- associated HTLV transmission, whereas fresh frozen plasma, cryoprecipitate, or coagulation factor concentrates appear not to cause infection (Abbaszadegan et al. 2003). The efficiency of virus transmission is from males to females during sexually active years causing a higher seroprevalence of about more than twice in females. The HTLV-1 infection tends to be more within family members and three to four times greater than its rate in general population. It is proposed that repeated close contact and shared environment could be significant in HTLV-1 transmission (Rafatpanah et al. 2006). Viral antigens expressed by infected cell are quickly targeted by cytotoxic T cells; hence the viral load is maintained predominantly by cells harboring silent provirus spread by mitotic transmission. HTLV-1 transmission by free virions is very inefficient, at least in T cells, however, recent studies indicate that cell-free HTLV-1 virions are highly infectious

HTLV-1 is mainly transmitted via three routes: 1) mother- to-infant transmission, mainly through breast feeding; 2) sexual transmission, mainly from male-to- female; and 3)

suggests a supporting role for HBZ in the oncogenic process (Kannian 2010).

manner via such virological synapses *in vivo* (Igakura et al. 2003).

for DCs (Noula Shembade 2010).

thawing of breast milk reduced vertical transmission (Matsuoka 2005).

**4.1 Transmission of HTLV-1 occurs through three main routes** 

parenteral transmission (blood transfusion or intravenous drug use).

**4. Transmission** 

Fig. 1. HTLV-1 genome structure and gene product

mRNAs from the nucleus to the cytoplasm. The accessory genes, p12/p8 encoded by ORF I and p30/p13 encoded by ORF II is not necessary in standard immortalization assays in culture. However, these genes are essential for initiation of viral infection and the establishment of persistence in animal models. P8 is a proteolytic cleavage product of the p12 parent molecule, whereas the p13 polypeptide, comprised of the carboxy terminus of p30, is expressed from a distinct mRNA. These accessory proteins may also play a role in gene regulation and contribute to the productive infection of quiescent T lymphocytes *in vitro*. The minus strand of the proviral genome encodes several isoforms (generated from unspliced and spliced mRNAs) of the HTLV-1 basic leucine zipper factor (HBZ). HBZ interacts with cellular factors JunB, c-Jun, JunD, cAMP response element binding (CREB) and CREB binding protein (CBP)/p300to modulate both viral and cellular gene transcription. HBZ also plays a crucial role in T cell proliferation. Although research data indicate that Tax, among all the viral proteins, is the viral oncoprotein, but emerging data suggests a supporting role for HBZ in the oncogenic process (Kannian 2010).

#### **4. Transmission**

28 T-Cell Leukemia

Fig. 1. HTLV-1 genome structure and gene product

mRNAs from the nucleus to the cytoplasm. The accessory genes, p12/p8 encoded by ORF I and p30/p13 encoded by ORF II is not necessary in standard immortalization assays in culture. However, these genes are essential for initiation of viral infection and the establishment of persistence in animal models. P8 is a proteolytic cleavage product of the p12 parent molecule, whereas the p13 polypeptide, comprised of the carboxy terminus of p30, is expressed from a distinct mRNA. These accessory proteins may also play a role in gene regulation and contribute to the productive infection of quiescent T lymphocytes *in vitro*. The minus strand of the proviral genome encodes several isoforms (generated from HTLV-1 can infect various cell types, including T-lymphocytes, B-lymphocytes, monocytes, dendritic cells (DC) and fibroblasts. Glucose transporter 1 (GLUT-1) is ubiquitously expressed cell surface receptor targeted by HTLV-1 (Manel et al. 2003). Other cell surface receptors such as neuropilin 1 (NRP1) and surface heparan sulfate proteoglycans (HSPGs) have been reported to be target of HTLV-1 and are required for efficient entry (Boxus and Willems 2009). There is also evidence for cell-type specific receptors since a recent study has reported that HTLV-1 enters DCs by binding to the receptor DC-SIGN (Noula Shembade 2010.). However, the HTLV-1 provirus is mainly detected in CD4-positive lymphocytes, with about 10% in CD8-positive T-lymphocytes. This situation possibly arises because of Tax transformation of CD4-positive T-lymphocytes in vivo causing enhanced proliferation and suppressed apoptosis. In HTLV-1-infected individuals, no virions are detected in the serum. In addition, the infectivity of free virions is very poor compared with that of infected cells suggesting that HTLV-1 spreads through cell-to-cell transmission, rather than by free virions. *In vitro* analyses of HTLV-1 infected cells revealed that HTLV-1-infected cells form "virological synapses" with uninfected cells. The viral proteins Gag and Env, viral RNA and microtubules are accumulated as an infected cell is in contact with a target cell, and the viral complex subsequently transfers into the target cell. HTLV-1 also spreads in a cell- to-cell manner via such virological synapses *in vivo* (Igakura et al. 2003).

In either route, HTLV-1-infected cells are essential for transmission. This was supported by the findings that fresh frozen plasma from carriers did not cause transmission and freezethawing of breast milk reduced vertical transmission (Matsuoka 2005).

Transplacental transmission is also suspected. Cellular blood products are the main source of transfusion- associated HTLV transmission, whereas fresh frozen plasma, cryoprecipitate, or coagulation factor concentrates appear not to cause infection (Abbaszadegan et al. 2003).

The efficiency of virus transmission is from males to females during sexually active years causing a higher seroprevalence of about more than twice in females. The HTLV-1 infection tends to be more within family members and three to four times greater than its rate in general population. It is proposed that repeated close contact and shared environment could be significant in HTLV-1 transmission (Rafatpanah et al. 2006). Viral antigens expressed by infected cell are quickly targeted by cytotoxic T cells; hence the viral load is maintained predominantly by cells harboring silent provirus spread by mitotic transmission. HTLV-1 transmission by free virions is very inefficient, at least in T cells, however, recent studies indicate that cell-free HTLV-1 virions are highly infectious for DCs (Noula Shembade 2010).

#### **4.1 Transmission of HTLV-1 occurs through three main routes**

HTLV-1 is mainly transmitted via three routes: 1) mother- to-infant transmission, mainly through breast feeding; 2) sexual transmission, mainly from male-to- female; and 3) parenteral transmission (blood transfusion or intravenous drug use).

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 31

of contaminated blood is about 44%. Thus, it is essential to have an efficient blood screening system for HTLV-1 in endemic areas to limit the HTLV-1 transmission. Whole blood components, platelets and packed red blood cells, but not fresh frozen plasma, are the sources of virus transmission. White blood cells are reservoirs of HTLV-1. The probability of transmission of HTLV-1 decreases, if infected units of blood are stored for more than one week HTLV-1. Approximately 12% of HTLV infections occur by blood transfusion. Unlike HIV-1, whole cell transfusion is required for transmission of the virus, with a seroconversion rate of approximately 50%. The development of HAM/TSP has been noted as early as six months after transfusion of an individual with infected blood. In 1988, concerns about transmission of HTLV through blood components led to mandatory blood donor screening for HTLV resulting in a significant decrease in transmission via this mode. Cell-free infection with HTLV-1 is very inefficient; and efficient transmission depends on cell-to-cell transfer through direct cell contact, polarization of the microtubule-organizing center (MTOC), which is triggered by Tax, and the formation of a virological synapse, which allows the entry of viral particles, viral proteins and genomic RNA into fresh target cells. Similar to HIV-1 infection, dendritic cells (DCs) have been demonstrated to play a biphasic role in cell-to-cell transmission of HTLV-1. DCs can capture and transfer the virions to fresh T cells in a trans fashion or transmit de novo synthesized virions upon infection to fresh T cells in a cis fashion (Kannian 2010). Transmission via organ transplantation has been described and is associated with rapidly progressing HAM/TSP, possibly because of the immunosuppression that transplant

**Contaminated needles from Drug addicts:** HTLV-1 can also be transmitted by sharing of needles among drug addicts (Rafatpanah et al. 2006). In the United States, HTLV infection of intravenous drug users (IVDUs) was first reported from Queens, New York, and was mainly attributed to HTLV-II (Robert-Guroff et al. 1986). Subsequently, others have reported prevalence rates of HTLV antibody among IVDUs ranging from 0.4% to 24.0%

HTLV-1 is usually detected by carrying out laboratory tests because of clinical suspicion, screening at the blood bank or due to concerns by family members of HTLV-1 positive patients. The antibodies can be detected by enzyme-linked immunosorbent assay (ELISA). ELISA kits have high sensitivity and low specicity; thus, it may not be a reliable screening tool. Therefore, positive ELISA results should be conrmed by western blot analysis and or

For the diagnosis of HTLV-1/2 infection, the rst immunoassays used HTLV-1 whole-viral lysate as the only antigen. Then, assays were based on recombinant and/or synthetic peptide antigens only or in combination with viral lysates. Furthermore, HTLV-2 specic antigens were included, which improved the sensitivity for detection of HTLV- 2 antibodies. At present, the initial diagnosis of HTLV-1/2 infection is based mainly on screening for antibodies by ELISA. Even the lack of Food and Drug Administration (FDA) licensure for HTLV-1/2 Western blot (WB) assay, it is generally applied to all repeatedly reactive samples for further conrmation of HTLV-1/2 infection (CDC, 1988). The WB assay reduces the number of false positive transmembrane results thereby increasing the specicity for serological conrmation of HTLV-1/2. This assay contains viral lysates and recombinant

patients undergo (Romanelli, Caramelli and Proietti 2010).

polymerase chain reaction (PCR) (Andrade et al. 2010).

(Lee et al. 1990).

**5. Diagnosis**

**Mother to child:** One of the main modes of HTLV-1 transmition from mother to child is by breast feeding. Studies in Japan showed that the prevalence of HTLV-1 infection in children of carrier mothers was significantly higher (21%) than in children in the general population (1%). More than 85% of infected mothers had infected their children. The length of breastfeeding affects the risk of HTLV-1 transmission. The duration of breast-feeding affects the risk of HTLV-1 transmission. HTLV-1 antigen in cord blood lymphocytes of babies born to healthy carriers raised a possibility to consider intrauterine transmission as an alternative pathway. However, the HTLV-1 provirus in the cord blood circulation is derived from migrated maternal cells that are not a part of blood circulation of the baby. Thus, intrauterine transmission could not be a major pathway of transmission.

Only ~3%–4% of children become infected if they are not breast-fed or are breast- fed for <6 months, and the transmission risk increases with the duration of breast-feeding. The cumulative risk of infection in children who are regularly breast-fed is ~20%. Furthermore, the transmission risk increases with the amount of provirus in breast milk. Breast milk proviral levels also correlate with proviral levels in maternal peripheral blood mononuclear cells (PBMCs) and with antibody titers. It is therefore not surprising that the transmission risk correlates with proviral and antibody levels in maternal peripheral blood. However, this is contradictory to the fact that the majority of children who are breastfed for long periods do not become infected. HIV can also be transmitted by breastfeeding, however, it is more commonly transmitted in utero or perinatally, As with HTLV-1, transmission occurs in proportion to the duration of breast-feeding, and the risk increases when mothers have high HIV provirus levels in breast milk, which are also directly correlated with peripheral blood provirus levels. Mother- to-child in utero and perinatal HIV transmission was more likely when children were more concordant with their mothers in HLA class I type. Children with HLA class I type A\*02 (later reported to be the A2 supertype) have a decreased risk of early infection. HLA class I type variability is not associated with HIV transmission via breast-feeding, whereas, with HTLV-1, vertical transmission almost always occurs via breast-feeding. However, unlike HIV, HTLV-1 is transmitted primarily by cell- to-cell contact rather than by free virus. We reasoned that factors affecting cellular immunity might be more important for the vertical transmission of HTLV-1 than for HIV (Biggar et al. 2006).

Transmission rates are 16% for children born to infected mothers, 27% for children nursed by infected mothers for more than three months and 5% for children nursed by infected mothers for less than three months (Ureta-Vidal et al. 1999). It is Interesting that HTLV is transmitted to about 13% of bottle-fed children from their infected mother suggesting a different route than breast-feeding. The infants seroconvert within 1-3 years of age (Ureta-Vidal et al. 1999). The infants seroconvert within 1-3 years of age (Ureta-Vidal et al. 1999).

**Sexual:** HTLV-1 can also be transmitted through sexual contact. Heterosexual transmission is able to introduce HTLV-1 infection into previously uninfected groups. Transmission from man to woman is more frequent (60%) than woman to man (0.4%). Like HIV, HTLV-1 can be transmitted through homosexual activity. Predisposing factors associated with sexual transmission include the presence of genital ulcers, high viral loads and high antibody titers in the donor (Kaplan et al. 1996). Sexual transmission is a more common mode in non-drug user sexual partners of IDUs than parenteral transmission. Among IDUs, blood and blood products are the most significant source of infection (Roucoux and Murphy 2004).

**Blood transfusion:** is the third mode of HTLV-1 transmission. The proviral DNA in donor's blood lymphocytes acts as an infectious agent. The probability of seroconversion in a recipient of contaminated blood is about 44%. Thus, it is essential to have an efficient blood screening system for HTLV-1 in endemic areas to limit the HTLV-1 transmission. Whole blood components, platelets and packed red blood cells, but not fresh frozen plasma, are the sources of virus transmission. White blood cells are reservoirs of HTLV-1. The probability of transmission of HTLV-1 decreases, if infected units of blood are stored for more than one week HTLV-1. Approximately 12% of HTLV infections occur by blood transfusion. Unlike HIV-1, whole cell transfusion is required for transmission of the virus, with a seroconversion rate of approximately 50%. The development of HAM/TSP has been noted as early as six months after transfusion of an individual with infected blood. In 1988, concerns about transmission of HTLV through blood components led to mandatory blood donor screening for HTLV resulting in a significant decrease in transmission via this mode. Cell-free infection with HTLV-1 is very inefficient; and efficient transmission depends on cell-to-cell transfer through direct cell contact, polarization of the microtubule-organizing center (MTOC), which is triggered by Tax, and the formation of a virological synapse, which allows the entry of viral particles, viral proteins and genomic RNA into fresh target cells. Similar to HIV-1 infection, dendritic cells (DCs) have been demonstrated to play a biphasic role in cell-to-cell transmission of HTLV-1. DCs can capture and transfer the virions to fresh T cells in a trans fashion or transmit de novo synthesized virions upon infection to fresh T cells in a cis fashion (Kannian 2010). Transmission via organ transplantation has been described and is associated with rapidly progressing HAM/TSP, possibly because of the immunosuppression that transplant patients undergo (Romanelli, Caramelli and Proietti 2010).

**Contaminated needles from Drug addicts:** HTLV-1 can also be transmitted by sharing of needles among drug addicts (Rafatpanah et al. 2006). In the United States, HTLV infection of intravenous drug users (IVDUs) was first reported from Queens, New York, and was mainly attributed to HTLV-II (Robert-Guroff et al. 1986). Subsequently, others have reported prevalence rates of HTLV antibody among IVDUs ranging from 0.4% to 24.0% (Lee et al. 1990).

#### **5. Diagnosis**

30 T-Cell Leukemia

**Mother to child:** One of the main modes of HTLV-1 transmition from mother to child is by breast feeding. Studies in Japan showed that the prevalence of HTLV-1 infection in children of carrier mothers was significantly higher (21%) than in children in the general population (1%). More than 85% of infected mothers had infected their children. The length of breastfeeding affects the risk of HTLV-1 transmission. The duration of breast-feeding affects the risk of HTLV-1 transmission. HTLV-1 antigen in cord blood lymphocytes of babies born to healthy carriers raised a possibility to consider intrauterine transmission as an alternative pathway. However, the HTLV-1 provirus in the cord blood circulation is derived from migrated maternal cells that are not a part of blood circulation of the baby. Thus,

Only ~3%–4% of children become infected if they are not breast-fed or are breast- fed for <6 months, and the transmission risk increases with the duration of breast-feeding. The cumulative risk of infection in children who are regularly breast-fed is ~20%. Furthermore, the transmission risk increases with the amount of provirus in breast milk. Breast milk proviral levels also correlate with proviral levels in maternal peripheral blood mononuclear cells (PBMCs) and with antibody titers. It is therefore not surprising that the transmission risk correlates with proviral and antibody levels in maternal peripheral blood. However, this is contradictory to the fact that the majority of children who are breastfed for long periods do not become infected. HIV can also be transmitted by breastfeeding, however, it is more commonly transmitted in utero or perinatally, As with HTLV-1, transmission occurs in proportion to the duration of breast-feeding, and the risk increases when mothers have high HIV provirus levels in breast milk, which are also directly correlated with peripheral blood provirus levels. Mother- to-child in utero and perinatal HIV transmission was more likely when children were more concordant with their mothers in HLA class I type. Children with HLA class I type A\*02 (later reported to be the A2 supertype) have a decreased risk of early infection. HLA class I type variability is not associated with HIV transmission via breast-feeding, whereas, with HTLV-1, vertical transmission almost always occurs via breast-feeding. However, unlike HIV, HTLV-1 is transmitted primarily by cell- to-cell contact rather than by free virus. We reasoned that factors affecting cellular immunity might be more important for the vertical

Transmission rates are 16% for children born to infected mothers, 27% for children nursed by infected mothers for more than three months and 5% for children nursed by infected mothers for less than three months (Ureta-Vidal et al. 1999). It is Interesting that HTLV is transmitted to about 13% of bottle-fed children from their infected mother suggesting a different route than breast-feeding. The infants seroconvert within 1-3 years of age (Ureta-Vidal et al. 1999). The infants seroconvert within 1-3 years of age (Ureta-Vidal et al. 1999). **Sexual:** HTLV-1 can also be transmitted through sexual contact. Heterosexual transmission is able to introduce HTLV-1 infection into previously uninfected groups. Transmission from man to woman is more frequent (60%) than woman to man (0.4%). Like HIV, HTLV-1 can be transmitted through homosexual activity. Predisposing factors associated with sexual transmission include the presence of genital ulcers, high viral loads and high antibody titers in the donor (Kaplan et al. 1996). Sexual transmission is a more common mode in non-drug user sexual partners of IDUs than parenteral transmission. Among IDUs, blood and blood

products are the most significant source of infection (Roucoux and Murphy 2004).

**Blood transfusion:** is the third mode of HTLV-1 transmission. The proviral DNA in donor's blood lymphocytes acts as an infectious agent. The probability of seroconversion in a recipient

intrauterine transmission could not be a major pathway of transmission.

transmission of HTLV-1 than for HIV (Biggar et al. 2006).

HTLV-1 is usually detected by carrying out laboratory tests because of clinical suspicion, screening at the blood bank or due to concerns by family members of HTLV-1 positive patients. The antibodies can be detected by enzyme-linked immunosorbent assay (ELISA). ELISA kits have high sensitivity and low specicity; thus, it may not be a reliable screening tool. Therefore, positive ELISA results should be conrmed by western blot analysis and or polymerase chain reaction (PCR) (Andrade et al. 2010).

For the diagnosis of HTLV-1/2 infection, the rst immunoassays used HTLV-1 whole-viral lysate as the only antigen. Then, assays were based on recombinant and/or synthetic peptide antigens only or in combination with viral lysates. Furthermore, HTLV-2 specic antigens were included, which improved the sensitivity for detection of HTLV- 2 antibodies. At present, the initial diagnosis of HTLV-1/2 infection is based mainly on screening for antibodies by ELISA. Even the lack of Food and Drug Administration (FDA) licensure for HTLV-1/2 Western blot (WB) assay, it is generally applied to all repeatedly reactive samples for further conrmation of HTLV-1/2 infection (CDC, 1988). The WB assay reduces the number of false positive transmembrane results thereby increasing the specicity for serological conrmation of HTLV-1/2. This assay contains viral lysates and recombinant

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 33

eliminate foreign pathogens, and like other successful pathogens, HTLV-1 must have

Like HIV, HTLV-1 mainly infects CD4 T cells, which are the central regulators of the acquired immune response. To establish persistent infection, HTLV-1 perturbs the regulation of CD4 T cells, sometimes leading to diseases such as ATL or chronic inflammatory diseases such as HAM/TSP, uveitis, arthritis, and alveolitis. Since the discovery of HTLV-1, extensive studies have been performed using various experimental approaches to elucidate the exact pathogenesis of this virus. However, the nature of HTLV-1 pathogenesis still remains elusive. This problem is a serious obstacle to establishing effective therapies for HTLV-1 associated diseases. Precise insight into HTLV-1 mediated pathogenesis requires careful consideration of the host cells and the effect HTLV-1 has on them. A better understanding of the interactions between HTLV-1 and the host immune system should provide additional clues to effective therapies for HTLV-1- associated

The host immune system, especially the cellular response, against HTLV-1 exerts critical control over virus replication and the proliferation of infected cells. CTLs against the virus have been extensively studied, and Tax protein was found to be the dominant antigen recognized by CTLs in vivo. HTLV-1-specific CD8-positive CTLs are abundant and chronically activated. The paradox is that the frequency of Tax-specific CTLs is much higher in HAM/TSP patients than in carriers. Since the provirus load is higher in HAM/TSP patients, this finding suggests that the CTLs in HAM/TSP cannot control the number of infected cells. One explanation for this is that the CTLs in HAM/TSP patients show less efficient cytolytic activity toward infected cells, whereas CTLs in carriers can suppress the proliferation of infected cells. Hence, the gene expression profiles of circulating CD4+ and CD8+ lymphocytes were compared between carriers with high and low provirus loads. The results revealed that CD8+ lymphocytes from individuals with a low HTLV-1 provirus load show higher expressions of genes associated with cytolytic activities or antigen recognition than those from carriers with a high provirus load. Thus, CD8+ T-lymphocytes in individuals with a low provirus load successfully control the number of HTLV-1-infected cells due to their higher CTL activities. Thus, the major determinant of the provirus load is thought to be the CTL response to HTLV-1. As mentioned above, the provirus load is considered to be controlled by host factors. Considering that the cellular immune responses are critically implicated in the control of HTLV-1 infection, human leukocyte antigen (HLA) should be a candidate for such a host genetic factor. From analyses of HAM/TSP patients and asymptomatic carriers, HLA-A02, and Cw08 are independently associated with a lower provirus load and a lower risk of HAM/TSP. In addition, polymorphisms of other genes including TNF-α, SDF-1, HLA- B54, HLA-DRB-10101 and IL-15 are also associated with the provirus load, however with a lower significance than with HLA-A02, and Cw08. Regarding the onset of ATL, only a polymorphism of TNF- α gene was reported to show an association. However, familial clustering of ATL cases is a well-known phenomenon, strongly suggesting that genetic factors are implicated in the onset of ATL. Spontaneous remission is more frequently observed in patients with ATL than those with other hematological malignancies. Usually, this phenomenon is associated with infectious diseases, suggesting that immune activation of the host enhances the immune response against ATL cells. If the immune response against HTLV-1 is implicated in spontaneous remission, this suggests the

strategies for escaping the host immune response.

diseases (Satou and Matsuoka 2010).

proteins. MTA-1 is a unique HTLV-1 envelope recombinant protein (rgp46-I), K-55 is a unique HTLV-2 envelope recombinant protein (rgp46-II), and GD21 is a common yet specic HTLV-1 and HTLV-2 epitope recombinant envelope protein. An HTLV-1 positive sample was considered when there were bands for the gag proteins p19 and p24, and the env proteins GD21 and rgp46-I; HTLV-2 positive if p24, GD21, and rgp46-II bands were present; an indeterminate sample when there were specic bands for the virus that did not meet the HTLV-1/2 positivity criteria, and a negative result for those samples that did not exhibit any specic band. In some cases, however, it is necessary to perform a complementary assay such as a nested-polymerase chain reaction (nested-PCR) in order to conrm true HTLV-1/2 infection and to obtain a conclusive diagnosis. When WB is used for conrmation, a signicant proportion of the samples reports indeterminate results, ranging from0.02% in non-endemic areas to 50% in endemic ones , although it has been observed that indeterminate samples could result in true HTLV-1/2 infection, even in non-endemic areas. Several studies have shown that most low-risk HTLV-seroindeterminate and asymptomatic individuals are negative for HTLV-1/2 infection after testing with a highly sensitive nested-PCR. It is known that the use of highly efcient screening assays may reduce signicantly false reactive results, diminishing the amount of samples further submitted to WB and/or nested-PCR analysis for conrmation. One of the strategies proposed to reduce the number of samples requiring conrmatory testing is the use of a dual ELISA algorithm (Yoshida 2010).

A pitfall in ELISA-based immunoassay may exist in HTLV-1 detection due to truncated MTA-1 envelope glycoprotein. This report describes experiments designed to determine whether some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. Recombinant envelope glycoprotein is used in production of diagnostic enzyme-linked immunosorbent assay (ELISA) kit. There are some reports that a significant percentage of Iranian HTLV-1 infected patients showed no seroreactivity with MTA-1 peptide, while HTLV-1 had been confirmed by PCR detection methods or ELISA kits containing a cocktail of HTLV-1 specific peptides. Some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. This is because of an insertion of a cytosine in position 271 causing a stop codon in the MTA-1 protein translation. SDS-PAGE analysis also failed to reveal the presence of the desired protein. Subjects with a mutant HTLV-1 env gene were shown to be seronegative using ELISA, but positive with PCR (Abbaszadegan et al. 2008).

Three diagnostic criteria for ATL have been defined. The first is the presence of morphologically proven lymphoid malignancy with T-cell surface antigens (typically CD4+, CD25+). These abnormal T lymphocytes have hyperlobulated nuclei in acute ATL and are known as "flower cells." On the other hand, in the indolent types of ATL, smoldering and chronic types, the abnormality of the nuclear shape is generally milder than that in the acute form of the disease. The second criteria is the presence of antibodies to HTLV-1 in the sera, and the third is the demonstration of monoclonal integration of HTLV-1 provirus in tumor cells by Southern blotting (Yasunaga and Matsuoka 2007).

#### **6. HTLV-1 and the host immune system**

HTLV-1 is a complex retrovirus that may have been transmitted to humans from monkeys more than ten thousands years ago. The human host has several immune mechanisms that

proteins. MTA-1 is a unique HTLV-1 envelope recombinant protein (rgp46-I), K-55 is a unique HTLV-2 envelope recombinant protein (rgp46-II), and GD21 is a common yet specic HTLV-1 and HTLV-2 epitope recombinant envelope protein. An HTLV-1 positive sample was considered when there were bands for the gag proteins p19 and p24, and the env proteins GD21 and rgp46-I; HTLV-2 positive if p24, GD21, and rgp46-II bands were present; an indeterminate sample when there were specic bands for the virus that did not meet the HTLV-1/2 positivity criteria, and a negative result for those samples that did not exhibit any specic band. In some cases, however, it is necessary to perform a complementary assay such as a nested-polymerase chain reaction (nested-PCR) in order to conrm true HTLV-1/2 infection and to obtain a conclusive diagnosis. When WB is used for conrmation, a signicant proportion of the samples reports indeterminate results, ranging from0.02% in non-endemic areas to 50% in endemic ones , although it has been observed that indeterminate samples could result in true HTLV-1/2 infection, even in non-endemic areas. Several studies have shown that most low-risk HTLV-seroindeterminate and asymptomatic individuals are negative for HTLV-1/2 infection after testing with a highly sensitive nested-PCR. It is known that the use of highly efcient screening assays may reduce signicantly false reactive results, diminishing the amount of samples further submitted to WB and/or nested-PCR analysis for conrmation. One of the strategies proposed to reduce the number of samples requiring conrmatory testing is the use of a

A pitfall in ELISA-based immunoassay may exist in HTLV-1 detection due to truncated MTA-1 envelope glycoprotein. This report describes experiments designed to determine whether some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. Recombinant envelope glycoprotein is used in production of diagnostic enzyme-linked immunosorbent assay (ELISA) kit. There are some reports that a significant percentage of Iranian HTLV-1 infected patients showed no seroreactivity with MTA-1 peptide, while HTLV-1 had been confirmed by PCR detection methods or ELISA kits containing a cocktail of HTLV-1 specific peptides. Some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. This is because of an insertion of a cytosine in position 271 causing a stop codon in the MTA-1 protein translation. SDS-PAGE analysis also failed to reveal the presence of the desired protein. Subjects with a mutant HTLV-1 env gene were shown to be seronegative using ELISA, but positive with PCR

Three diagnostic criteria for ATL have been defined. The first is the presence of morphologically proven lymphoid malignancy with T-cell surface antigens (typically CD4+, CD25+). These abnormal T lymphocytes have hyperlobulated nuclei in acute ATL and are known as "flower cells." On the other hand, in the indolent types of ATL, smoldering and chronic types, the abnormality of the nuclear shape is generally milder than that in the acute form of the disease. The second criteria is the presence of antibodies to HTLV-1 in the sera, and the third is the demonstration of monoclonal integration of HTLV-1 provirus in tumor

HTLV-1 is a complex retrovirus that may have been transmitted to humans from monkeys more than ten thousands years ago. The human host has several immune mechanisms that

dual ELISA algorithm (Yoshida 2010).

(Abbaszadegan et al. 2008).

cells by Southern blotting (Yasunaga and Matsuoka 2007).

**6. HTLV-1 and the host immune system** 

eliminate foreign pathogens, and like other successful pathogens, HTLV-1 must have strategies for escaping the host immune response.

Like HIV, HTLV-1 mainly infects CD4 T cells, which are the central regulators of the acquired immune response. To establish persistent infection, HTLV-1 perturbs the regulation of CD4 T cells, sometimes leading to diseases such as ATL or chronic inflammatory diseases such as HAM/TSP, uveitis, arthritis, and alveolitis. Since the discovery of HTLV-1, extensive studies have been performed using various experimental approaches to elucidate the exact pathogenesis of this virus. However, the nature of HTLV-1 pathogenesis still remains elusive. This problem is a serious obstacle to establishing effective therapies for HTLV-1 associated diseases. Precise insight into HTLV-1 mediated pathogenesis requires careful consideration of the host cells and the effect HTLV-1 has on them. A better understanding of the interactions between HTLV-1 and the host immune system should provide additional clues to effective therapies for HTLV-1- associated diseases (Satou and Matsuoka 2010).

The host immune system, especially the cellular response, against HTLV-1 exerts critical control over virus replication and the proliferation of infected cells. CTLs against the virus have been extensively studied, and Tax protein was found to be the dominant antigen recognized by CTLs in vivo. HTLV-1-specific CD8-positive CTLs are abundant and chronically activated. The paradox is that the frequency of Tax-specific CTLs is much higher in HAM/TSP patients than in carriers. Since the provirus load is higher in HAM/TSP patients, this finding suggests that the CTLs in HAM/TSP cannot control the number of infected cells. One explanation for this is that the CTLs in HAM/TSP patients show less efficient cytolytic activity toward infected cells, whereas CTLs in carriers can suppress the proliferation of infected cells. Hence, the gene expression profiles of circulating CD4+ and CD8+ lymphocytes were compared between carriers with high and low provirus loads. The results revealed that CD8+ lymphocytes from individuals with a low HTLV-1 provirus load show higher expressions of genes associated with cytolytic activities or antigen recognition than those from carriers with a high provirus load. Thus, CD8+ T-lymphocytes in individuals with a low provirus load successfully control the number of HTLV-1-infected cells due to their higher CTL activities. Thus, the major determinant of the provirus load is thought to be the CTL response to HTLV-1. As mentioned above, the provirus load is considered to be controlled by host factors. Considering that the cellular immune responses are critically implicated in the control of HTLV-1 infection, human leukocyte antigen (HLA) should be a candidate for such a host genetic factor. From analyses of HAM/TSP patients and asymptomatic carriers, HLA-A02, and Cw08 are independently associated with a lower provirus load and a lower risk of HAM/TSP. In addition, polymorphisms of other genes including TNF-α, SDF-1, HLA- B54, HLA-DRB-10101 and IL-15 are also associated with the provirus load, however with a lower significance than with HLA-A02, and Cw08. Regarding the onset of ATL, only a polymorphism of TNF- α gene was reported to show an association. However, familial clustering of ATL cases is a well-known phenomenon, strongly suggesting that genetic factors are implicated in the onset of ATL. Spontaneous remission is more frequently observed in patients with ATL than those with other hematological malignancies. Usually, this phenomenon is associated with infectious diseases, suggesting that immune activation of the host enhances the immune response against ATL cells. If the immune response against HTLV-1 is implicated in spontaneous remission, this suggests the

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 35

59%). Such ATL cells are thought to suppress the immune response via expression of immunoregulatory molecules on their surfaces, and production of immunosuppressive

In 1977, Takatsuki et al. reported ATL as a distinct clinical entity. This disease is characterized by its aggressive clinical course, infiltrations into skin, liver, gastrointestinal tract and lung, hypercalcemia and the presence of leukemic cells with multilobulated nuclei, flower cell (Figure 2). The linkage between ATL and HTLV-1 was proven by Hinuma et al., who demonstrated the presence of an antibody against HTLV-1 in patient sera. Thereafter, Seiki et al. determined the whole sequence of HTLV-1 and revealed the presence of a unique region, designated pX. The pX region encodes several accessory genes, which control viral

Fig. 2. Typical "**flower cell**", Morphological findings of typical ATL cells, leukemic cells with

Several molecular biologic studies have reported that various cellular dysfunctions induced by viral genes (eg, tax and HBZ), genetic and epigenetic alterations, and the host immune system may be involved in the leukemogenesis of ATL. Clinical and epidemiologic studies have also reported a variety of possible risk factors for ATL, including vertical transmission of HTLV-1 infection, male gender, a long latent period, increased leukocyte counts or abnormal lymphocyte counts, and higher levels of anti– HTLV-1 antibody titers and soluble interleukin-2 receptor. However, there are no clear determinants that separate those who develop ATL from those who remain healthy carriers. Recently, HTLV-1 proviral load levels have been evaluated as important predictors of development of ATL and HAM/TSP. Some cross-sectional studies showed that HTLV-1 proviral load levels were higher in ATL and HAM/TSP compared with asymptomatic HTLV-1 carriers. In conclusion, the cohort study of 1218 asymptomatic HTLV-1 carriers provided detailed distributions for HTLV-1 proviral loads regarding the host-specic characteristics and the associations with the development of ATL. A higher proviral load levels (especially > 4 copies/100 PBMCs), advanced age, family history of ATL, and having the rst opportunity to learn of HTLV-1 infection during treatment of other diseases are independent risk factors for progression from carrier status to ATL.

cytokines (Matsuoka 2005).

multilobulated nuclei was Shawn.

**7. Mechanism of oncogenesis by HTLV 1**

replication and the proliferation of infected cells (Matsuoka 2005).

possibility of immunotherapy for ATL patients by the induction of an immune response to HTLV-1, for example via antigen-stimulated dendritic cells. Immunodeficiency in ATL patients is pronounced, and results in frequent opportunistic infections by various pathogens, including Pneumocystis carinii, cytomegalovirus, fungus, Strongyloides and bacteria, due to the inevitable impairment of the T-cell functions. To a lesser extent, impaired cell-mediated immunity has also been demonstrated in HTLV-1 carriers. Such immunodeficiency in the carrier state may be associated with the leukemogenesis of ATL by allowing the proliferation of HTLV-1-infected cells. A prospective study of HTLV-1 infected individuals found that carriers who later develop ATL have a higher anti-HTLV-1 antibody and a low anti-Tax antibody level for up to 10 years preceding their diagnosis. This finding indicates that HTLV-1 carriers with a higher anti-HTLV-1 titer, which is roughly correlated with the HTLV-1 provirus load and a lower anti-Tax reactivity, may be at the greatest risk of developing ATL. The anti-HTLV-1 antibody and soluble IL-2 receptor (sIL-2R) levels are correlated with the HTLV-1 provirus load, and a high antibody titer and high sIL-2R level are risk factors for developing ATL among carriers. Taken together, these findings suggest that a higher proliferation of HTLV-1-infected cells and a low immune response against Tax may be associated with the onset of ATL. Given these findings, potentiation of CTLs against Tax via a vaccine strategy may be useful for preventing the onset of ATL. EBV-associated lymphomas frequently develop in individuals with an immunodeficient state associated with transplantation or AIDS. This has also been reported in an ATL patient. Does such an immunodeficient state influence the onset of ATL? Among 24 patients with post- transplantation lymphoproliferative disorders (PT-LPDs) after renal transplantation in Japan, 5 cases of ATL have been reported. Considering that most PT-LPDs are of B- cell origin in Western countries, this frequency of ATL in Japan is quite high. Although the high HTLV-1 seroprevalence is due to blood transfusion during hemodialysis, the immunodeficient state during renal transplantation apparently promotes the onset of ATL. In addition, when experimental allogeneic transplantation was performed to 12 rhesus monkeys and immunosuppressive agents (cyclosporine, prednisolone or lymphocytespecific monoclonal antibodies) were administered to prevent rejection, 4 of the 7 monkeys that died during the experiment showed PT-LPDs. Importantly, the STLV pro- virus was detected in all PT-LPD samples. These observations emphasize that transplantation into HTLV-1- infected individuals or from HTLV-1 positive donors require special attention. Although the mechanism of immunodeficiency remains unknown, some previous reports have provided important clues. One mechanism for immunodeficiency is that HTLV-1 infects CD8-positive T-lymphocytes, which may impair their functions. Indeed, the immune response against Tax via HTLV-1-infected CD8-positive T-cells renders these cells susceptible to fratricide mediated by autologous HTLV-1-specific CD8-positive Tlymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-1. Another mechanism for immunodeficiency is based on the observation that the number of naive T-cells decreases in individuals infected with HTLV-1 via decreased thymopoiesis [48]. In addition, CD4+ and CD25+ T-lymphocytes are classified as immunoregulatory T-cells that control the host immune system. Regulatory T-cells suppress the immune reaction via the expression of immunoregulatory molecules on their surfaces. The FOXP3 gene has been identified as a master gene that controls gene expressions specific to regulatory T-cells. FOXP3 gene transcription can be detected in some ATL cases (10/17; 59%). Such ATL cells are thought to suppress the immune response via expression of immunoregulatory molecules on their surfaces, and production of immunosuppressive cytokines (Matsuoka 2005).

### **7. Mechanism of oncogenesis by HTLV 1**

34 T-Cell Leukemia

possibility of immunotherapy for ATL patients by the induction of an immune response to HTLV-1, for example via antigen-stimulated dendritic cells. Immunodeficiency in ATL patients is pronounced, and results in frequent opportunistic infections by various pathogens, including Pneumocystis carinii, cytomegalovirus, fungus, Strongyloides and bacteria, due to the inevitable impairment of the T-cell functions. To a lesser extent, impaired cell-mediated immunity has also been demonstrated in HTLV-1 carriers. Such immunodeficiency in the carrier state may be associated with the leukemogenesis of ATL by allowing the proliferation of HTLV-1-infected cells. A prospective study of HTLV-1 infected individuals found that carriers who later develop ATL have a higher anti-HTLV-1 antibody and a low anti-Tax antibody level for up to 10 years preceding their diagnosis. This finding indicates that HTLV-1 carriers with a higher anti-HTLV-1 titer, which is roughly correlated with the HTLV-1 provirus load and a lower anti-Tax reactivity, may be at the greatest risk of developing ATL. The anti-HTLV-1 antibody and soluble IL-2 receptor (sIL-2R) levels are correlated with the HTLV-1 provirus load, and a high antibody titer and high sIL-2R level are risk factors for developing ATL among carriers. Taken together, these findings suggest that a higher proliferation of HTLV-1-infected cells and a low immune response against Tax may be associated with the onset of ATL. Given these findings, potentiation of CTLs against Tax via a vaccine strategy may be useful for preventing the onset of ATL. EBV-associated lymphomas frequently develop in individuals with an immunodeficient state associated with transplantation or AIDS. This has also been reported in an ATL patient. Does such an immunodeficient state influence the onset of ATL? Among 24 patients with post- transplantation lymphoproliferative disorders (PT-LPDs) after renal transplantation in Japan, 5 cases of ATL have been reported. Considering that most PT-LPDs are of B- cell origin in Western countries, this frequency of ATL in Japan is quite high. Although the high HTLV-1 seroprevalence is due to blood transfusion during hemodialysis, the immunodeficient state during renal transplantation apparently promotes the onset of ATL. In addition, when experimental allogeneic transplantation was performed to 12 rhesus monkeys and immunosuppressive agents (cyclosporine, prednisolone or lymphocytespecific monoclonal antibodies) were administered to prevent rejection, 4 of the 7 monkeys that died during the experiment showed PT-LPDs. Importantly, the STLV pro- virus was detected in all PT-LPD samples. These observations emphasize that transplantation into HTLV-1- infected individuals or from HTLV-1 positive donors require special attention. Although the mechanism of immunodeficiency remains unknown, some previous reports have provided important clues. One mechanism for immunodeficiency is that HTLV-1 infects CD8-positive T-lymphocytes, which may impair their functions. Indeed, the immune response against Tax via HTLV-1-infected CD8-positive T-cells renders these cells susceptible to fratricide mediated by autologous HTLV-1-specific CD8-positive Tlymphocytes. Fratricide among virus-specific CTLs could impair the immune control of HTLV-1. Another mechanism for immunodeficiency is based on the observation that the number of naive T-cells decreases in individuals infected with HTLV-1 via decreased thymopoiesis [48]. In addition, CD4+ and CD25+ T-lymphocytes are classified as immunoregulatory T-cells that control the host immune system. Regulatory T-cells suppress the immune reaction via the expression of immunoregulatory molecules on their surfaces. The FOXP3 gene has been identified as a master gene that controls gene expressions specific to regulatory T-cells. FOXP3 gene transcription can be detected in some ATL cases (10/17;

In 1977, Takatsuki et al. reported ATL as a distinct clinical entity. This disease is characterized by its aggressive clinical course, infiltrations into skin, liver, gastrointestinal tract and lung, hypercalcemia and the presence of leukemic cells with multilobulated nuclei, flower cell (Figure 2). The linkage between ATL and HTLV-1 was proven by Hinuma et al., who demonstrated the presence of an antibody against HTLV-1 in patient sera. Thereafter, Seiki et al. determined the whole sequence of HTLV-1 and revealed the presence of a unique region, designated pX. The pX region encodes several accessory genes, which control viral replication and the proliferation of infected cells (Matsuoka 2005).

Fig. 2. Typical "**flower cell**", Morphological findings of typical ATL cells, leukemic cells with multilobulated nuclei was Shawn.

Several molecular biologic studies have reported that various cellular dysfunctions induced by viral genes (eg, tax and HBZ), genetic and epigenetic alterations, and the host immune system may be involved in the leukemogenesis of ATL. Clinical and epidemiologic studies have also reported a variety of possible risk factors for ATL, including vertical transmission of HTLV-1 infection, male gender, a long latent period, increased leukocyte counts or abnormal lymphocyte counts, and higher levels of anti– HTLV-1 antibody titers and soluble interleukin-2 receptor. However, there are no clear determinants that separate those who develop ATL from those who remain healthy carriers. Recently, HTLV-1 proviral load levels have been evaluated as important predictors of development of ATL and HAM/TSP. Some cross-sectional studies showed that HTLV-1 proviral load levels were higher in ATL and HAM/TSP compared with asymptomatic HTLV-1 carriers. In conclusion, the cohort study of 1218 asymptomatic HTLV-1 carriers provided detailed distributions for HTLV-1 proviral loads regarding the host-specic characteristics and the associations with the development of ATL. A higher proviral load levels (especially > 4 copies/100 PBMCs), advanced age, family history of ATL, and having the rst opportunity to learn of HTLV-1 infection during treatment of other diseases are independent risk factors for progression from carrier status to ATL.

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 37

(Kiyokawa et al. 1987). In hypercalcemic patients, the number of osteoclasts increases in the bone. RANK ligand, which is expressed on osteoblasts, and M-CSF act synergistically on hematopoietic precursor cells, and induce the differentiation into osteoclasts (Arai et al. 1999). ATL cells from hypercalcemic ATL patients express RANK ligand, and induced the differentiation of hematopoietic stem cells into osteoclasts when ATL cells were co-cultured with hematopoietic stem cells (Nosaka et al. 2002). In addition, the serum level of parathyroid hormone-related peptide (PTH-rP) is also elevated in most of hypercalcemic ATL patients. PTH- rP indirectly increases the number of osteoclasts, as well as activating them (Watanabe et al. 1990), which is also implicated in mechanisms of hypercalcemia.

Tax, a transactivator protein, triggers a plethora of events like cell signaling, cell cycle regulation and interference with checkpoint control and inhibition of DNA repair. Tax is expressed from a doubly spliced mRNA transcript. Although Tax shares the same mRNA transcript with Rex, translation of Tax is favored over Rex due to a stronger Kozak sequence. Tax made in the cytoplasm is translocated into the nucleus, where it binds to its response

The tax gene plays central roles in viral gene transcription, viral replication and the proliferation of HTLV-1-infected cells. Tax enhances viral gene transcription from the 5'-LTR via interaction with cyclic AMP responsive element binding protein (CREB). Tax also interacts with cellular factors and activates transcriptional pathways, such as NF-κB, AP-1 and SRF (Yoshida 2001). For example, activation of NF-κB induces the transcription of various cytokines and their receptor genes, as well as anti-apoptotic genes such as bcl- xL and survivin (Tsukahara et al. 1999). The activation of NF-κB has been demonstrated to be critical for tumorigenesis both in vitro and in vivo (Mori et al. 1999). On the other hand, Tax variant without activation of NF-κB has also been reported to immortalize primary T-lymphocytes in vitro, suggesting that mechanisms of immortalization are complex. In addition to NF-κB, activation of other transcriptional pathways such as CREB by Tax should be implicated in the immortalization and leukemogenesis. Tax also interferes with the functions of p53, p16 and MAD1 (Ariumi et al. 2000). These interactions enable HTLV-1-infected cells to escape from apoptosis, and also induce genetic instability. Although inactivation of p53 function by Tax is reported to be mediated by p300/CBP or NF- κB activation (Pise-Masison et al. 2000), Tax can still repress p53's activity in spite of loss of p300/CBP binding or in cells lacking NF- κB activation (Miyazato et al. 2005), indicating the mechanism of p53 inactivation by Tax needs further investigation. Although Tax promotes the proliferation of infected cells, it is also the major target of cytotoxic T-lymphocytes (CTLs) *in vivo*. Moreover, excess expression of Tax protein is considered to be harmful to infected cells. Therefore, HTLV-1 has redundant mechanisms to suppress Tax expression. Rex binds to Rex-responsive element (RxRE) in the U3 and R regions of the 3'-LTR, and enhances the transport of the unspliced gag/pol and the singly spliced env transcripts. By this mechanism, double-spliced tax/rex mRNA decreases, resulting in suppressed expression of Tax (Inoue, Yoshida and Seiki 1987). Additionally, p30 binds to tax/rex transcripts, and retains them in the nucleus. The HBZ gene is encoded by the complementary strand of HTLV-1, and contains a leucine zipper domain. HBZ directly interacts with c-Jun or JunB (Basbous et al. 2003), or enhances their degradation, resulting in the suppression of Tax-mediated viral transcription from the LTR. Transforming growth factor-β (TGF-β) is an inhibitory cytokine that plays important roles in development, the

element and activates viral LTR-mediated transcription. (Boxus and Willems 2009).

**7.3 Role of Tax in HTLV-1 induced oncogenesis**

Further large-scale epidemiologic studies are needed to clearly identify the determinants of ATL for early detection and rapid cure for HTLV-1–associated diseases(Silva et al. 2007). Genetic and immunological factors in the host are the principal determinants of the emergence of associated diseases(Romanelli, Caramelli and Proietti 2010).

#### **7.1 Etiology of ATL**

The most important aspect of the new retroviral isolation was not just novelty but an etiology for a human leukemia. However, etiological proof for a human disease is generally not easy unless an animal model is available. The most critical question thereafter was whether 'close association of HTLV-1 with ATL' reects its causative role or whether the virus was just a passenger. The nature of provirus integration of the retroviruses provided a critical tool for the discrimination. The retroviral genomes are generally reverse-transcribed into provirus DNA, and the proviral genomes are integrated into host cell DNA at random sites. Since a tumor originates from unlimited expansion of a single malignant cell, the site for the proviral integration into tumor cells would be uniform in individuals if the retroviral infection plays a causative role; but if the virus fortuitously infects leukemic cells, then the integration sites would be random. Southern blot analysis of patients' leukemic cell DNA clearly indicated clonal integration in each patient revealing two distinct bands with cellular flanking sequences. This nding clearly supported the virus playing a causative role in ATL. Virtually all ATL cases were clonally infected leukemic cells; therefore, the conclusion for a 'causative role' became generally accepted. As controls, the sites for the integration in viral carriers are random except only in a few cases which show clonal integration with higher viral burden (Yoshida 2005).

#### **7.2 Pathogenesis of HTLV-1 infection**

ATL cells are derived from activated helper T-lymphocytes, which play central roles in the immune system by elaborating cytokines and expressing immunoregulatory molecules. ATL cells are known to retain such features and this cytokine production or surface molecule expression may modify the pathogenesis. ATL is well known to infiltrate various organs and tissues, such as the skin, lungs, liver, gastrointestinal tract, central nervous system and bone (Takatsuki 1995). This infiltrative tendency of leukemic cells is possibly attributable to the expressions of various surface molecules, such as chemokine receptors and adhesion molecules. Skin-homing memory T-cells uniformly expresses CCR4, and its ligands are thymus and activation-regulated chemokine (TARC) and macrophage- derived chemokine (MDC). CCR4 is expressed on most ATL cells. In addition, TARC and MDC are expressed in skin lesions in ATL patients. Thus, CCR4 expression should be implicated in the skin infiltration (Yoshie et al. 2002). On the other hand, CCR7 expression is associated with lymph node involvement (Hasegawa et al. 2000). OX40 is a member of the tumor necrosis factor family, and was reported to be expressed on ATL cells (Higashimura et al. 1996). It was also identified as a gene associated with the adhesion of ATL cells to endothelial cells by a functional cloning system using a monoclonal antibody that inhibited the attachment of ATL cells (Imura et al. 1996). Thus, OX40 is also implicated in the cell adhesion and infiltration of ATL cells. Thus, ATL cells express various molecules that can modify their phenotypic properties, thereby modifying the clinical disease manifestation, and facilitating the survival of ATL cells (Matsuoka 2003). Hypercalcemia is frequently complicated in patients with acute ATL (more than 70% during the whole clinical course)

Further large-scale epidemiologic studies are needed to clearly identify the determinants of ATL for early detection and rapid cure for HTLV-1–associated diseases(Silva et al. 2007). Genetic and immunological factors in the host are the principal determinants of the

The most important aspect of the new retroviral isolation was not just novelty but an etiology for a human leukemia. However, etiological proof for a human disease is generally not easy unless an animal model is available. The most critical question thereafter was whether 'close association of HTLV-1 with ATL' reects its causative role or whether the virus was just a passenger. The nature of provirus integration of the retroviruses provided a critical tool for the discrimination. The retroviral genomes are generally reverse-transcribed into provirus DNA, and the proviral genomes are integrated into host cell DNA at random sites. Since a tumor originates from unlimited expansion of a single malignant cell, the site for the proviral integration into tumor cells would be uniform in individuals if the retroviral infection plays a causative role; but if the virus fortuitously infects leukemic cells, then the integration sites would be random. Southern blot analysis of patients' leukemic cell DNA clearly indicated clonal integration in each patient revealing two distinct bands with cellular flanking sequences. This nding clearly supported the virus playing a causative role in ATL. Virtually all ATL cases were clonally infected leukemic cells; therefore, the conclusion for a 'causative role' became generally accepted. As controls, the sites for the integration in viral carriers are random except only in a few cases which show clonal integration with higher

ATL cells are derived from activated helper T-lymphocytes, which play central roles in the immune system by elaborating cytokines and expressing immunoregulatory molecules. ATL cells are known to retain such features and this cytokine production or surface molecule expression may modify the pathogenesis. ATL is well known to infiltrate various organs and tissues, such as the skin, lungs, liver, gastrointestinal tract, central nervous system and bone (Takatsuki 1995). This infiltrative tendency of leukemic cells is possibly attributable to the expressions of various surface molecules, such as chemokine receptors and adhesion molecules. Skin-homing memory T-cells uniformly expresses CCR4, and its ligands are thymus and activation-regulated chemokine (TARC) and macrophage- derived chemokine (MDC). CCR4 is expressed on most ATL cells. In addition, TARC and MDC are expressed in skin lesions in ATL patients. Thus, CCR4 expression should be implicated in the skin infiltration (Yoshie et al. 2002). On the other hand, CCR7 expression is associated with lymph node involvement (Hasegawa et al. 2000). OX40 is a member of the tumor necrosis factor family, and was reported to be expressed on ATL cells (Higashimura et al. 1996). It was also identified as a gene associated with the adhesion of ATL cells to endothelial cells by a functional cloning system using a monoclonal antibody that inhibited the attachment of ATL cells (Imura et al. 1996). Thus, OX40 is also implicated in the cell adhesion and infiltration of ATL cells. Thus, ATL cells express various molecules that can modify their phenotypic properties, thereby modifying the clinical disease manifestation, and facilitating the survival of ATL cells (Matsuoka 2003). Hypercalcemia is frequently complicated in patients with acute ATL (more than 70% during the whole clinical course)

emergence of associated diseases(Romanelli, Caramelli and Proietti 2010).

**7.1 Etiology of ATL**

viral burden (Yoshida 2005).

**7.2 Pathogenesis of HTLV-1 infection**

(Kiyokawa et al. 1987). In hypercalcemic patients, the number of osteoclasts increases in the bone. RANK ligand, which is expressed on osteoblasts, and M-CSF act synergistically on hematopoietic precursor cells, and induce the differentiation into osteoclasts (Arai et al. 1999). ATL cells from hypercalcemic ATL patients express RANK ligand, and induced the differentiation of hematopoietic stem cells into osteoclasts when ATL cells were co-cultured with hematopoietic stem cells (Nosaka et al. 2002). In addition, the serum level of parathyroid hormone-related peptide (PTH-rP) is also elevated in most of hypercalcemic ATL patients. PTH- rP indirectly increases the number of osteoclasts, as well as activating them (Watanabe et al. 1990), which is also implicated in mechanisms of hypercalcemia.

#### **7.3 Role of Tax in HTLV-1 induced oncogenesis**

Tax, a transactivator protein, triggers a plethora of events like cell signaling, cell cycle regulation and interference with checkpoint control and inhibition of DNA repair. Tax is expressed from a doubly spliced mRNA transcript. Although Tax shares the same mRNA transcript with Rex, translation of Tax is favored over Rex due to a stronger Kozak sequence. Tax made in the cytoplasm is translocated into the nucleus, where it binds to its response element and activates viral LTR-mediated transcription. (Boxus and Willems 2009).

The tax gene plays central roles in viral gene transcription, viral replication and the proliferation of HTLV-1-infected cells. Tax enhances viral gene transcription from the 5'-LTR via interaction with cyclic AMP responsive element binding protein (CREB). Tax also interacts with cellular factors and activates transcriptional pathways, such as NF-κB, AP-1 and SRF (Yoshida 2001). For example, activation of NF-κB induces the transcription of various cytokines and their receptor genes, as well as anti-apoptotic genes such as bcl- xL and survivin (Tsukahara et al. 1999). The activation of NF-κB has been demonstrated to be critical for tumorigenesis both in vitro and in vivo (Mori et al. 1999). On the other hand, Tax variant without activation of NF-κB has also been reported to immortalize primary T-lymphocytes in vitro, suggesting that mechanisms of immortalization are complex. In addition to NF-κB, activation of other transcriptional pathways such as CREB by Tax should be implicated in the immortalization and leukemogenesis. Tax also interferes with the functions of p53, p16 and MAD1 (Ariumi et al. 2000). These interactions enable HTLV-1-infected cells to escape from apoptosis, and also induce genetic instability. Although inactivation of p53 function by Tax is reported to be mediated by p300/CBP or NF- κB activation (Pise-Masison et al. 2000), Tax can still repress p53's activity in spite of loss of p300/CBP binding or in cells lacking NF- κB activation (Miyazato et al. 2005), indicating the mechanism of p53 inactivation by Tax needs further investigation. Although Tax promotes the proliferation of infected cells, it is also the major target of cytotoxic T-lymphocytes (CTLs) *in vivo*. Moreover, excess expression of Tax protein is considered to be harmful to infected cells. Therefore, HTLV-1 has redundant mechanisms to suppress Tax expression. Rex binds to Rex-responsive element (RxRE) in the U3 and R regions of the 3'-LTR, and enhances the transport of the unspliced gag/pol and the singly spliced env transcripts. By this mechanism, double-spliced tax/rex mRNA decreases, resulting in suppressed expression of Tax (Inoue, Yoshida and Seiki 1987). Additionally, p30 binds to tax/rex transcripts, and retains them in the nucleus. The HBZ gene is encoded by the complementary strand of HTLV-1, and contains a leucine zipper domain. HBZ directly interacts with c-Jun or JunB (Basbous et al. 2003), or enhances their degradation, resulting in the suppression of Tax-mediated viral transcription from the LTR. Transforming growth factor-β (TGF-β) is an inhibitory cytokine that plays important roles in development, the

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 39

Fig. 3. Pleiotropic actions of Tax. Pleiotropic actions of Tax proteins are summarized

advantage for their survival since they can escape from CTLs (Figure 4) (Matsuoka 2005).

Some ATL cells can proliferate without functional Tax protein, suggesting that somatic (genetic and epigenetic) alterations cause transcriptional or functional changes to the host

As mentioned above, Tax expression confers advantages and disadvantages on HTLV-1-infected cells. Although the proliferation of infected cells is promoted by Tax expression, CTLs attack the Tax-expressing cells since Tax is their major target. In HTLV-1-infected cells, Rex, p30 and HBZ suppress Tax expression. On the other hand, loss of Tax expression is frequently observed in leukemic cells. Three mechanisms have been identified for inactivation of Tax expression: 1) genetic changes of the tax gene (non- sense mutations, deletions or insertions); 2) DNA methylation of the 5'-LTR; and 3) deletion of the 5'-LTR. Among fresh leukemic cells isolated from ATL patients, about 60% of cases do not express the tax gene transcript. Interestingly, ATL cells with genetic changes of the tax gene expressed its transcripts, suggesting that ATL cells do not silence the transcription when the tax gene is abortive. Loss of Tax expression gives ATL cells

**7.4 Inactivation of Tax expression in ATL cells** 

immune system and oncogenesis. Since TGF-β generally suppresses the growth of tumor cells, most tumor cells acquire escape mechanisms that inhibit TGF-β signaling, including mutations in its receptor and in the Smad molecules that transduce the signal from the receptor. Tax has also been reported to inhibit TGF-β signaling by binding to Smad2, 3 and 4 or CBP/p300 (Mori et al. 2001). Inhibition of TGF-β signaling enables HTLV-1-infected cells to escape TGF-βmediated growth inhibition. ATL cells have been reported to show remarkable chromosomal abnormalities (Sanada et al. 1986), implicated in the disease progression. Tax has been reported to interact with the checkpoint protein MAD1, which forms a complex with MAD2 and controls the mitotic checkpoint. This functional hindrance of MAD1 by Tax protein causes chromosomal instability, suggesting the involvement of this mechanism in oncogenesis. Recently, Tax has been reported to interact with Cdc20 and activate Cdc20- associated anaphase-promoting complex, an E3 ubiquitin ligase that controls the metaphase-to-anaphase transition, thereby resulting in mitotic abnormalities (Liu et al. 2005). In contrast to HTLV-1, HTLV-2 promotes the proliferation of CD8-positive T-lymphocytes in vivo. Although it was first discovered in a patient with variant hairy cell leukemia, HTLV-2 is less likely to have oncogenic properties since there is no obvious association between HTLV-2 infections and cancers. Regardless of the homology of their tax sequences, the oncogenic potential of Tax1 (HTLV-1 Tax) is more prominent than that of Tax2 (HTLV- 2 Tax). The most striking difference is that Tax2 lacks the binding motif at C-terminal end to PDZ domain proteins, while Tax 1 retains it. When the PDZ domain of Tax1 is added to Tax2, the latter acquires oncogenic properties in the rat fibroblast cell line Rat-1, indicating that this domain is responsible for the transforming activity of HTLV-1. To understand the pleiotropic actions of Tax protein more clearly, transcriptome analyses are essential. The transcriptional changes induced by Tax expression have been studied using DNA microarrays, which revealed that Tax upregulated the expression of the mixed-lineage kinase MLK3. MLK3 is involved in NF-κB activation by Tax as well as NIK and MEKK1. In addition to transcriptional changes, Tax is also well known to interact with cellular proteins and impair or alter their functions. For example, proteomic analyses of Tax-associated complexes showed that Tax could interact with cellular proteins, including the active forms of small GTPases, such as Cdc42, RhoA and Rac1, which should be implicated in the migration, invasion and adhesion of T-cells, as well as in the activation of the Jun-kinase (JNK) pathway (figure 3) (Matsuoka 2005).

Tax1 upregulates the expression of genes encoding cytokines, chemokines, cell surface ligands, and their receptors, in an NF-κB, AP-1, CREB/ATF and/or NFAT dependent manner. They include IL-2 receptor (IL-2R) α-chain, IL-9, IL-13, IL-15/IL-15R, IL-21/IL-21R, IL-8, CCL2, CCL5, CCL22, CCR9, CXCR7, CD40, OX40/OX40L, and 4-1BB/4-1BBL. Among these, the IL-2R α-chain is crucially important for T-cell immortalization by Tax, since the immortalized cells are dependent on IL-2 for their growth.

ATL cells are well known to inltrate into various organs or tissues, frequently invading skin or lymphoid tissues. Analysis of chemokine receptor expression revealed that CCR4 was frequently expressed on HTLV-1-transformed cell lines and fresh ATL cells. CCR4 positive T lymphocytes contain skin-seeking memory T cells, accounting for frequent inltration of ATL cells into skin. On the other hand, expression of CCR7 was reported to be associated with the involvement of lymphoid tissues, and lymph node enlargement. A subtraction strategy between ATL cell lines and activated T cells identied I-309 as a secreted chemokine from ATL cells, and I-309 expression was remarkably enhanced in ATL cell lines. I-309 showed anti apoptotic effect via its receptor CCR8, invoking that an autocrine mechanism via I-309/CCR8 allowed ATL cells to survive in vivo (Matsuoka 2003).

immune system and oncogenesis. Since TGF-β generally suppresses the growth of tumor cells, most tumor cells acquire escape mechanisms that inhibit TGF-β signaling, including mutations in its receptor and in the Smad molecules that transduce the signal from the receptor. Tax has also been reported to inhibit TGF-β signaling by binding to Smad2, 3 and 4 or CBP/p300 (Mori et al. 2001). Inhibition of TGF-β signaling enables HTLV-1-infected cells to escape TGF-βmediated growth inhibition. ATL cells have been reported to show remarkable chromosomal abnormalities (Sanada et al. 1986), implicated in the disease progression. Tax has been reported to interact with the checkpoint protein MAD1, which forms a complex with MAD2 and controls the mitotic checkpoint. This functional hindrance of MAD1 by Tax protein causes chromosomal instability, suggesting the involvement of this mechanism in oncogenesis. Recently, Tax has been reported to interact with Cdc20 and activate Cdc20- associated anaphase-promoting complex, an E3 ubiquitin ligase that controls the metaphase-to-anaphase transition, thereby resulting in mitotic abnormalities (Liu et al. 2005). In contrast to HTLV-1, HTLV-2 promotes the proliferation of CD8-positive T-lymphocytes in vivo. Although it was first discovered in a patient with variant hairy cell leukemia, HTLV-2 is less likely to have oncogenic properties since there is no obvious association between HTLV-2 infections and cancers. Regardless of the homology of their tax sequences, the oncogenic potential of Tax1 (HTLV-1 Tax) is more prominent than that of Tax2 (HTLV- 2 Tax). The most striking difference is that Tax2 lacks the binding motif at C-terminal end to PDZ domain proteins, while Tax 1 retains it. When the PDZ domain of Tax1 is added to Tax2, the latter acquires oncogenic properties in the rat fibroblast cell line Rat-1, indicating that this domain is responsible for the transforming activity of HTLV-1. To understand the pleiotropic actions of Tax protein more clearly, transcriptome analyses are essential. The transcriptional changes induced by Tax expression have been studied using DNA microarrays, which revealed that Tax upregulated the expression of the mixed-lineage kinase MLK3. MLK3 is involved in NF-κB activation by Tax as well as NIK and MEKK1. In addition to transcriptional changes, Tax is also well known to interact with cellular proteins and impair or alter their functions. For example, proteomic analyses of Tax-associated complexes showed that Tax could interact with cellular proteins, including the active forms of small GTPases, such as Cdc42, RhoA and Rac1, which should be implicated in the migration, invasion and adhesion of T-cells, as well as in the activation of the

Tax1 upregulates the expression of genes encoding cytokines, chemokines, cell surface ligands, and their receptors, in an NF-κB, AP-1, CREB/ATF and/or NFAT dependent manner. They include IL-2 receptor (IL-2R) α-chain, IL-9, IL-13, IL-15/IL-15R, IL-21/IL-21R, IL-8, CCL2, CCL5, CCL22, CCR9, CXCR7, CD40, OX40/OX40L, and 4-1BB/4-1BBL. Among these, the IL-2R α-chain is crucially important for T-cell immortalization by Tax, since the

ATL cells are well known to inltrate into various organs or tissues, frequently invading skin or lymphoid tissues. Analysis of chemokine receptor expression revealed that CCR4 was frequently expressed on HTLV-1-transformed cell lines and fresh ATL cells. CCR4 positive T lymphocytes contain skin-seeking memory T cells, accounting for frequent inltration of ATL cells into skin. On the other hand, expression of CCR7 was reported to be associated with the involvement of lymphoid tissues, and lymph node enlargement. A subtraction strategy between ATL cell lines and activated T cells identied I-309 as a secreted chemokine from ATL cells, and I-309 expression was remarkably enhanced in ATL cell lines. I-309 showed anti apoptotic effect via its receptor CCR8, invoking that an autocrine mechanism via I-309/CCR8 allowed ATL cells to survive in vivo (Matsuoka 2003).

Jun-kinase (JNK) pathway (figure 3) (Matsuoka 2005).

immortalized cells are dependent on IL-2 for their growth.

Fig. 3. Pleiotropic actions of Tax. Pleiotropic actions of Tax proteins are summarized

#### **7.4 Inactivation of Tax expression in ATL cells**

As mentioned above, Tax expression confers advantages and disadvantages on HTLV-1-infected cells. Although the proliferation of infected cells is promoted by Tax expression, CTLs attack the Tax-expressing cells since Tax is their major target. In HTLV-1-infected cells, Rex, p30 and HBZ suppress Tax expression. On the other hand, loss of Tax expression is frequently observed in leukemic cells. Three mechanisms have been identified for inactivation of Tax expression: 1) genetic changes of the tax gene (non- sense mutations, deletions or insertions); 2) DNA methylation of the 5'-LTR; and 3) deletion of the 5'-LTR. Among fresh leukemic cells isolated from ATL patients, about 60% of cases do not express the tax gene transcript. Interestingly, ATL cells with genetic changes of the tax gene expressed its transcripts, suggesting that ATL cells do not silence the transcription when the tax gene is abortive. Loss of Tax expression gives ATL cells advantage for their survival since they can escape from CTLs (Figure 4) (Matsuoka 2005).

Some ATL cells can proliferate without functional Tax protein, suggesting that somatic (genetic and epigenetic) alterations cause transcriptional or functional changes to the host

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 41

methylation has been identified in the genome of ATL cells compared with that of PBMCs from carriers: hypomethylation is associated with aberrant expression of the MEL1S gene (Yoshida et al. 2004), while hypermethylation silences transcription of the p16 (Nosaka et al. 2000), EGR3 and KLF4 genes as well as many others. It is reasonable to consider that other currently unidentified genes are involved in such alterations of the genome in ATL cells, and play roles in leukemogenesis. Transcriptome analyses using DNA microarrays have revealed transcriptional changes that are specific to ATL cells. Among 192 up-regulated genes, the expressions of the tumor suppressor in lung cancer 1 (TSLC1), caveolin 1 and prostaglandin D2 synthase genes were increased more than 30-fold in fresh ATL cells compared with normal CD4+ and CD4+, CD45RO+ T-cells (Sasaki et al. 2005). TSLC1 is a cell adhesion molecule that acts as a tumor suppressor in lung cancer. Although TSLC1 is not expressed on normal T- lymphocytes, all acute ATL cells show ectopic TSLC1 expression. Enforced expression of TSLC1 enhances both the self-aggregation and adhesion abilities to vascular endothelial cells in ATL cells. Thus, TSLC1 expression is implicated in the adhesion or infiltration of ATL cells. A retrovirus cDNA library screening from ATL cells, a gene with oncogenic potency was identified in NIH3T3 cells, and designated the Tgat gene. Ectopic expression of the Tgat gene is observed in aggressive forms of ATL, and in vitro experiments showed that its expression is associated with an invasive phenotype

The pathogenesis of ATL involves four stages: infection, polyclonal proliferation, clinical latency and tumorigenesis. HTLV-1 induced ATL after a long latent period. Previous studies suggested the significance of the tax gene. However, Tax is not expressed in approximately 60% of ATL cases by three mechanisms: 1) deletion of 5' long terminal repeat (LTR), 2) DNA methylation of 5 LTR, and 3) genetic changes of the tax gene. Recent studies, demonstrated that the HTLV-1 basic leucine zipper factor (HBZ) encoded by the virus in an antisense orientation may play a critical role in the malignant proliferation of ATL cells. The expression of HBZ gene is detected in all ATL cases, and this is due to the usage of the promoter in the 3' LTR of HTLV-1 gene which is not inactivated in the ATL cells. HBZ interacts with various host factors, including c-Jun, JunB, JunD, and p65. Thus, HBZ modulates cellular signal pathways in addition to promoted proliferation. These findings indicate that HBZ is an essential viral gene for oncogenesis by HTLV-1(Boxus

Short hairpin RNA mediated knockdowns of HBZ expression in both ATL and HTLV-1 transformed cell lines reduce their proliferation. Moreover, transgenic mice expressing HBZ under the control of the CD4 promoter/enhancer display increased numbers of CD4 positive T-cells in the spleen, and augmented proliferation of thymocytes after anti-CD3 stimulation. Thus, these findings indicate that HBZ has a growth promoting activity, and could be involved in the malignant proliferation of ATL cells *in vivo*, although the precise molecular mechanism for these findings is still unclear. HTLV-2 also encodes a HBZ like protein, designated as the antisense protein of HTLV-2 (APH-2). Interestingly, unlike HBZ, APH-2 does not have a leucine zipper motif which is essential for various HBZ functions. Thus, it is important to study whether the HTLV-2 APH-2 protein has a growth promoting activity in T-cells like HBZ in order to understand better how these two viruses show

(Matsuoka 2005).

and Willems 2009).

**7.5 Role of HBZ in HTLV-1-induced oncogenesis**

distinct pathogenicities (Higuchi and Fujii 2009).

genes. The mutation rate of the p53 gene in ATL cells has been reported to be 36% (4/11) and 30% (3/10) (Nishimura et al. 1995). The p16 gene is an inhibitor of cyclin-dependent kinase 4/6, and blocks the cell cycle. Deletion and aberrant methylation of the p16 gene has also been reported in ATL cells. In addition, genetic changes in the p27KIP1, RB1/p105 and RB2/p130 genes have been reported in ATL, although they are relatively rare: 2/42 (4.8%) for the p27KIP1 gene; 2/40 (5%) for the RB1/p105 gene; and 1/41 (2.4%) for the RB2/p130 gene) (Morosetti et al. 1995). The fact that higher frequencies of genetic changes in these tumor suppressor genes are observed among aggressive forms of ATL suggests that such genetic changes are implicated in disease progression. Fas antigen was the first identified death receptor. It transduces the death signal by binding of its ligand, Fas ligand (FasL). ATL cells highly express Fas antigen on their cell surface (Nagata 1999), and are highly susceptible to death signals mediated by agonistic antibodies to Fas antigen, such as CH-11. Genetic changes of Fas gene in ATL cells, which confer resistance to the Fas-mediated signal, have been reported (Tamiya et al. 1998). Normal activated Tlymphocytes express FasL as well as Fas antigen. Apoptosis induced by autocrine mechanisms is designated activation-induced cell death (AICD) and this controls the

Fig. 4. Natural course from the infection of HTLV-I to onset of ATL

immune response (Krueger et al. 2003). Although ATL cells express Fas antigen, they do not produce FasL, thereby enabling ATL cells to escape from AICD. Attempts to isolate hypermethylated genes from ATL cells identified the EGR3 gene as a hypermethylated gene compared to PBMCs from carriers (Yasunaga et al. 2004). EGR3 is a transcriptional factor with a zinc finger domain, which is essential for transcription of the FasL gene. The finding that EGR3 gene transcription is silenced in ATL cells could account for the loss of FasL expression, and the escape of ATL cells from AICD. Thus, alterations of the Fas (genetic) and EGR3 (epigenetic) genes are examples of ATL cell evolution *in vivo*. Disordered DNA

genes. The mutation rate of the p53 gene in ATL cells has been reported to be 36% (4/11) and 30% (3/10) (Nishimura et al. 1995). The p16 gene is an inhibitor of cyclin-dependent kinase 4/6, and blocks the cell cycle. Deletion and aberrant methylation of the p16 gene has also been reported in ATL cells. In addition, genetic changes in the p27KIP1, RB1/p105 and RB2/p130 genes have been reported in ATL, although they are relatively rare: 2/42 (4.8%) for the p27KIP1 gene; 2/40 (5%) for the RB1/p105 gene; and 1/41 (2.4%) for the RB2/p130 gene) (Morosetti et al. 1995). The fact that higher frequencies of genetic changes in these tumor suppressor genes are observed among aggressive forms of ATL suggests that such genetic changes are implicated in disease progression. Fas antigen was the first identified death receptor. It transduces the death signal by binding of its ligand, Fas ligand (FasL). ATL cells highly express Fas antigen on their cell surface (Nagata 1999), and are highly susceptible to death signals mediated by agonistic antibodies to Fas antigen, such as CH-11. Genetic changes of Fas gene in ATL cells, which confer resistance to the Fas-mediated signal, have been reported (Tamiya et al. 1998). Normal activated Tlymphocytes express FasL as well as Fas antigen. Apoptosis induced by autocrine mechanisms is designated activation-induced cell death (AICD) and this controls the

Fig. 4. Natural course from the infection of HTLV-I to onset of ATL

immune response (Krueger et al. 2003). Although ATL cells express Fas antigen, they do not produce FasL, thereby enabling ATL cells to escape from AICD. Attempts to isolate hypermethylated genes from ATL cells identified the EGR3 gene as a hypermethylated gene compared to PBMCs from carriers (Yasunaga et al. 2004). EGR3 is a transcriptional factor with a zinc finger domain, which is essential for transcription of the FasL gene. The finding that EGR3 gene transcription is silenced in ATL cells could account for the loss of FasL expression, and the escape of ATL cells from AICD. Thus, alterations of the Fas (genetic) and EGR3 (epigenetic) genes are examples of ATL cell evolution *in vivo*. Disordered DNA

methylation has been identified in the genome of ATL cells compared with that of PBMCs from carriers: hypomethylation is associated with aberrant expression of the MEL1S gene (Yoshida et al. 2004), while hypermethylation silences transcription of the p16 (Nosaka et al. 2000), EGR3 and KLF4 genes as well as many others. It is reasonable to consider that other currently unidentified genes are involved in such alterations of the genome in ATL cells, and play roles in leukemogenesis. Transcriptome analyses using DNA microarrays have revealed transcriptional changes that are specific to ATL cells. Among 192 up-regulated genes, the expressions of the tumor suppressor in lung cancer 1 (TSLC1), caveolin 1 and prostaglandin D2 synthase genes were increased more than 30-fold in fresh ATL cells compared with normal CD4+ and CD4+, CD45RO+ T-cells (Sasaki et al. 2005). TSLC1 is a cell adhesion molecule that acts as a tumor suppressor in lung cancer. Although TSLC1 is not expressed on normal T- lymphocytes, all acute ATL cells show ectopic TSLC1 expression. Enforced expression of TSLC1 enhances both the self-aggregation and adhesion abilities to vascular endothelial cells in ATL cells. Thus, TSLC1 expression is implicated in the adhesion or infiltration of ATL cells. A retrovirus cDNA library screening from ATL cells, a gene with oncogenic potency was identified in NIH3T3 cells, and designated the Tgat gene. Ectopic expression of the Tgat gene is observed in aggressive forms of ATL, and in vitro experiments showed that its expression is associated with an invasive phenotype (Matsuoka 2005).

#### **7.5 Role of HBZ in HTLV-1-induced oncogenesis**

The pathogenesis of ATL involves four stages: infection, polyclonal proliferation, clinical latency and tumorigenesis. HTLV-1 induced ATL after a long latent period. Previous studies suggested the significance of the tax gene. However, Tax is not expressed in approximately 60% of ATL cases by three mechanisms: 1) deletion of 5' long terminal repeat (LTR), 2) DNA methylation of 5 LTR, and 3) genetic changes of the tax gene. Recent studies, demonstrated that the HTLV-1 basic leucine zipper factor (HBZ) encoded by the virus in an antisense orientation may play a critical role in the malignant proliferation of ATL cells. The expression of HBZ gene is detected in all ATL cases, and this is due to the usage of the promoter in the 3' LTR of HTLV-1 gene which is not inactivated in the ATL cells. HBZ interacts with various host factors, including c-Jun, JunB, JunD, and p65. Thus, HBZ modulates cellular signal pathways in addition to promoted proliferation. These findings indicate that HBZ is an essential viral gene for oncogenesis by HTLV-1(Boxus and Willems 2009).

Short hairpin RNA mediated knockdowns of HBZ expression in both ATL and HTLV-1 transformed cell lines reduce their proliferation. Moreover, transgenic mice expressing HBZ under the control of the CD4 promoter/enhancer display increased numbers of CD4 positive T-cells in the spleen, and augmented proliferation of thymocytes after anti-CD3 stimulation. Thus, these findings indicate that HBZ has a growth promoting activity, and could be involved in the malignant proliferation of ATL cells *in vivo*, although the precise molecular mechanism for these findings is still unclear. HTLV-2 also encodes a HBZ like protein, designated as the antisense protein of HTLV-2 (APH-2). Interestingly, unlike HBZ, APH-2 does not have a leucine zipper motif which is essential for various HBZ functions. Thus, it is important to study whether the HTLV-2 APH-2 protein has a growth promoting activity in T-cells like HBZ in order to understand better how these two viruses show distinct pathogenicities (Higuchi and Fujii 2009).

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 43

with allogeneic stem cell transplantation with reduced conditioning intensity (RIST) from HLA-matched sibling donors (Okamura et al. 2005). Among 9 patients in whom ATL relapsed after transplantation, 3 achieved a second complete remission after rapid discontinuation of cyclosporine A. This finding strongly suggests the presence of a graftversus-ATL effect in these patients. In addition, Tax peptide-recognizing cells were detected by a tetramer assay (HLA-A2/Tax 11–19 or HLA-A24/Tax 301–309) in patients after allogeneic stem cell transplantation (Harashima et al. 2004). In 8 patients, the provirus became undetectable by real- time PCR. Among these, 2 patients who received grafts from HTLV-1-positive donors also became provirus-negative after RIST. Since the provirus load is relatively constant in HTLV-1-infected individuals (Etoh et al. 1999), this finding indicates an enhanced immune response against HTLV-1 after RIST, which suppresses the provirus load. This may account for the effectiveness of allogeneic stem cell transplantation to ATL. However, Tax expression is frequently lost in ATL cells as described above. Many questions arise, such as whether the tax gene status is correlated with the effect of allogeneic stem cell transplantation, and whether the effectiveness of the anti-HTLV-1 immune response is against leukemic cells or non-leukemic HTLV-1-infected cells. Nevertheless, these data suggest that potentiation of the immune response against viral proteins such as Tax may be an attractive way to treat ATL patients. Such strategies may enable preventive treatment of high-risk HTLV-1 carriers, such as those with familial ATL history, predisposing genetic

The leukemic phase of ATL tends to spare the bone marrow; accentuated anemia and thrombocytopenia are not observed. White blood cell counts are always elevated and can be as high as 100,000/mm³. Heightened leukocyte counts and elevated lactate dehydrogenase (DHL) and calcium levels are markers of worse prognosis. Atypical lymphocytes that are pleomorphic and lobulated and have significant nuclear abnormalities (flower cells) are found in peripheral blood. If left untreated it is rapidly fatal, with death caused by pulmonary complications, opportunistic infections, sepsis and uncontrolled hypercalcemia. The chronic and indolent forms of ATL are less common, but after a number of years they will evolve into the acute form. Treatment of the indolent and chronic forms can be postponed until they evolve into the acute form; despite a less aggressive clinical course, prognosis for survival over the long term is poor. Some studies, with small patient samples and short follow-up periods, have demonstrated a satisfactory response and moderate toxicity using zidovudine in combination with interferon alpha, and both of these in combination with arsenic. In the more aggressive acute and lymphomatous forms, treatment should be started as early as possible using, CHOP chemotherapy regimens (cyclophosphamide, doxorubicin, vincristine and prednisolone). More powerful regimens such as VCAP (vincristine, cyclophosphamide, doxorubicin and prednisolone) or AMP (doxorubicin, ranimustine and prednisolone), offer a better response and prognosis, but mortality is higher. Other treatment options described in the literature include allogeneic stem cell transplant, inhibition of the NF-kappa Beta protein and monoclonal antibodies

Regardless of the extensive progress in virology, immunology and molecular biology of ATL and HTLV-1, the prognosis of patients with ATL remains poor. ATL is generally treated with aggressive combination chemotherapy, but long-term success has been less than 10%. The acute form, with hypercalcemia, high LDH levels and an elevated white blood cell count shows a particularly poor prognosis. Although G-CSF supported

factors to ATL, a higher provirus load, etc (Matsuoka 2005).

(Romanelli, Caramelli and Proietti 2010).

Both the HBZ and Tax genes are found in the genome of the simian T-cell leukemia virus type 1 (STLV-1), which shares a common ancestor with HTLV-1, indicating that HBZ has not been recently acquired; that is once the virus adapted to humans (STLV-1 and HTLV-1 are considered to have diverged around 50 000 years ago. it did not tolerate genetic drift resulting in its silencing. In the HTLV-2 genome, a human retrovirus related to HTLV-1, Tax also exists but, surprisingly, HTLV-2 lacks the HBZ-ORF. Moreover, in contrast to HTLV-1 and STLV-1, which both cause lymphoid malignancy in the host, no association between HTLV-2 infection and cancer has been yet evidenced. There has been only one reported case of a patient carrying HTLV-2 who developed a variant of hairy cell leukemia (Mesnard, Barbeau and Devaux 2006).

#### **8. ATL treatment: Current state and new strategies**

In spite of intensive chemotherapies, the prognosis of ATL patients has not improved. The median survival time of acute or lymphoma-type ATL was reported to be *13 months* with the most intensive chemotherapy. Such a poor prognosis might be due to: 1) the resistance of ATL cells to anti-cancer drugs; and 2) the immunodeficient state and complicated opportunistic infections. One mechanism of resistance to anti-cancer drugs is the activated NF-κB pathway in ATL cells (Mori et al. 1999), which increases the transcription of antiapoptotic genes such as bcl-xL and survivin.

A proteasome inhibitor, Bortezomib, is currently used for the treatment of multiple myeloma. One of its mechanisms is suppression of the NF-κB pathway by inhibiting the proteasomal degradation of IκB protein. Several groups have shown that Bortezomib is effective against ATL cells both *in vitro* and *in vivo* (Mitra-Kaushik et al. 2004). The sensitivity to Bortezomib is well correlated with the extent of NF-κB activation. Depsipeptide is a histone deacetylase inhibitor, and a clinical trial on its use in cutaneous Tcell lymphoma has commenced. This drug also inhibits the activation of NF-κB and AP-1 in ATL cells, and it induces apoptosis (Mori et al. 2004).

An alternative approach to the therapy of ATL is to target cell-surface markers on the malignant cells with monoclonal antibodies. Anti- CD25 (anti-Tac) monoclonal antibody, which was first administered to patients with ATL in the late 1980s, was reported to be effective in some patients, with a complete response in 2 of 19 patients and a partial response in 4 of 19 patients (Waldmann et al. 1993). Another antibody, anti- CD52 monoclonal antibody (Campath-1H), is being evaluated in a phase II clinical trial by the National Institutes of Health (Protocol 03-C-0194). Humanized anti-CD2 antibody (MEDI-507) has also been shown to be effective *in vivo* (Zhang et al. 2003). Bortezomib effect could be enhanced by combined use of anti-CD25 antibody (Tan and Waldmann 2002). During chemotherapy for ATL, chemotherapeutic agents worsens the immunodeficient state of ATL patients. Antibody therapy against ATL cells has advantages due to its decreased adverse effects.

As described above, most ATL cells express CCR4 antigen on their surfaces, and a humanized antibody against CCR4 is being developed as an anti-ATL agent (Ishida et al. 2004). Advances in the treatment of ATL were brought about by allogeneic bone marrow or stem cell transplantation (Borg et al. 1996; Yamada et al. 2001). Absence of graft-versus-host disease (GVHD) was linked with relapse of ATL, suggesting that GVHD or graft-versus-ATL may be implicated in the clinical effects of allogeneic stem cell transplantation (Borg et al. 1996). Furthermore, 16 patients with ATL, who were over 50 years of age, were treated

Both the HBZ and Tax genes are found in the genome of the simian T-cell leukemia virus type 1 (STLV-1), which shares a common ancestor with HTLV-1, indicating that HBZ has not been recently acquired; that is once the virus adapted to humans (STLV-1 and HTLV-1 are considered to have diverged around 50 000 years ago. it did not tolerate genetic drift resulting in its silencing. In the HTLV-2 genome, a human retrovirus related to HTLV-1, Tax also exists but, surprisingly, HTLV-2 lacks the HBZ-ORF. Moreover, in contrast to HTLV-1 and STLV-1, which both cause lymphoid malignancy in the host, no association between HTLV-2 infection and cancer has been yet evidenced. There has been only one reported case of a patient carrying HTLV-2 who developed a variant of hairy cell leukemia (Mesnard,

In spite of intensive chemotherapies, the prognosis of ATL patients has not improved. The median survival time of acute or lymphoma-type ATL was reported to be *13 months* with the most intensive chemotherapy. Such a poor prognosis might be due to: 1) the resistance of ATL cells to anti-cancer drugs; and 2) the immunodeficient state and complicated opportunistic infections. One mechanism of resistance to anti-cancer drugs is the activated NF-κB pathway in ATL cells (Mori et al. 1999), which increases the transcription of anti-

A proteasome inhibitor, Bortezomib, is currently used for the treatment of multiple myeloma. One of its mechanisms is suppression of the NF-κB pathway by inhibiting the proteasomal degradation of IκB protein. Several groups have shown that Bortezomib is effective against ATL cells both *in vitro* and *in vivo* (Mitra-Kaushik et al. 2004). The sensitivity to Bortezomib is well correlated with the extent of NF-κB activation. Depsipeptide is a histone deacetylase inhibitor, and a clinical trial on its use in cutaneous Tcell lymphoma has commenced. This drug also inhibits the activation of NF-κB and AP-1 in

An alternative approach to the therapy of ATL is to target cell-surface markers on the malignant cells with monoclonal antibodies. Anti- CD25 (anti-Tac) monoclonal antibody, which was first administered to patients with ATL in the late 1980s, was reported to be effective in some patients, with a complete response in 2 of 19 patients and a partial response in 4 of 19 patients (Waldmann et al. 1993). Another antibody, anti- CD52 monoclonal antibody (Campath-1H), is being evaluated in a phase II clinical trial by the National Institutes of Health (Protocol 03-C-0194). Humanized anti-CD2 antibody (MEDI-507) has also been shown to be effective *in vivo* (Zhang et al. 2003). Bortezomib effect could be enhanced by combined use of anti-CD25 antibody (Tan and Waldmann 2002). During chemotherapy for ATL, chemotherapeutic agents worsens the immunodeficient state of ATL patients. Antibody therapy against ATL cells has advantages due to its

As described above, most ATL cells express CCR4 antigen on their surfaces, and a humanized antibody against CCR4 is being developed as an anti-ATL agent (Ishida et al. 2004). Advances in the treatment of ATL were brought about by allogeneic bone marrow or stem cell transplantation (Borg et al. 1996; Yamada et al. 2001). Absence of graft-versus-host disease (GVHD) was linked with relapse of ATL, suggesting that GVHD or graft-versus-ATL may be implicated in the clinical effects of allogeneic stem cell transplantation (Borg et al. 1996). Furthermore, 16 patients with ATL, who were over 50 years of age, were treated

Barbeau and Devaux 2006).

decreased adverse effects.

**8. ATL treatment: Current state and new strategies**

apoptotic genes such as bcl-xL and survivin.

ATL cells, and it induces apoptosis (Mori et al. 2004).

with allogeneic stem cell transplantation with reduced conditioning intensity (RIST) from HLA-matched sibling donors (Okamura et al. 2005). Among 9 patients in whom ATL relapsed after transplantation, 3 achieved a second complete remission after rapid discontinuation of cyclosporine A. This finding strongly suggests the presence of a graftversus-ATL effect in these patients. In addition, Tax peptide-recognizing cells were detected by a tetramer assay (HLA-A2/Tax 11–19 or HLA-A24/Tax 301–309) in patients after allogeneic stem cell transplantation (Harashima et al. 2004). In 8 patients, the provirus became undetectable by real- time PCR. Among these, 2 patients who received grafts from HTLV-1-positive donors also became provirus-negative after RIST. Since the provirus load is relatively constant in HTLV-1-infected individuals (Etoh et al. 1999), this finding indicates an enhanced immune response against HTLV-1 after RIST, which suppresses the provirus load. This may account for the effectiveness of allogeneic stem cell transplantation to ATL. However, Tax expression is frequently lost in ATL cells as described above. Many questions arise, such as whether the tax gene status is correlated with the effect of allogeneic stem cell transplantation, and whether the effectiveness of the anti-HTLV-1 immune response is against leukemic cells or non-leukemic HTLV-1-infected cells. Nevertheless, these data suggest that potentiation of the immune response against viral proteins such as Tax may be an attractive way to treat ATL patients. Such strategies may enable preventive treatment of high-risk HTLV-1 carriers, such as those with familial ATL history, predisposing genetic factors to ATL, a higher provirus load, etc (Matsuoka 2005).

The leukemic phase of ATL tends to spare the bone marrow; accentuated anemia and thrombocytopenia are not observed. White blood cell counts are always elevated and can be as high as 100,000/mm³. Heightened leukocyte counts and elevated lactate dehydrogenase (DHL) and calcium levels are markers of worse prognosis. Atypical lymphocytes that are pleomorphic and lobulated and have significant nuclear abnormalities (flower cells) are found in peripheral blood. If left untreated it is rapidly fatal, with death caused by pulmonary complications, opportunistic infections, sepsis and uncontrolled hypercalcemia. The chronic and indolent forms of ATL are less common, but after a number of years they will evolve into the acute form. Treatment of the indolent and chronic forms can be postponed until they evolve into the acute form; despite a less aggressive clinical course, prognosis for survival over the long term is poor. Some studies, with small patient samples and short follow-up periods, have demonstrated a satisfactory response and moderate toxicity using zidovudine in combination with interferon alpha, and both of these in combination with arsenic. In the more aggressive acute and lymphomatous forms, treatment should be started as early as possible using, CHOP chemotherapy regimens (cyclophosphamide, doxorubicin, vincristine and prednisolone). More powerful regimens such as VCAP (vincristine, cyclophosphamide, doxorubicin and prednisolone) or AMP (doxorubicin, ranimustine and prednisolone), offer a better response and prognosis, but mortality is higher. Other treatment options described in the literature include allogeneic stem cell transplant, inhibition of the NF-kappa Beta protein and monoclonal antibodies (Romanelli, Caramelli and Proietti 2010).

Regardless of the extensive progress in virology, immunology and molecular biology of ATL and HTLV-1, the prognosis of patients with ATL remains poor. ATL is generally treated with aggressive combination chemotherapy, but long-term success has been less than 10%. The acute form, with hypercalcemia, high LDH levels and an elevated white blood cell count shows a particularly poor prognosis. Although G-CSF supported

Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 45

Biggar, R. J., J. Ng, N. Kim, M. Hisada, H. C. Li, B. Cranston, B. Hanchard, and E. M.

Borg, A., J. A. Yin, P. R. Johnson, J. Tosswill, M. Saunders, and D. Morris. 1996. "Successful

Boxus, M., and L. Willems. 2009. "Mechanisms of HTLV-1 persistence and transformation."

Etoh, K., K. Yamaguchi, S. Tokudome, T. Watanabe, A. Okayama, S. Stuver, N. Mueller, K.

Hanon, E., J. C. Stinchcombe, M. Saito, B. E. Asquith, G. P. Taylor, Y. Tanaka, J. N. Weber, G.

Harashima, N., K. Kurihara, A. Utsunomiya, R. Tanosaki, S. Hanabuchi, M. Masuda, T.

Hasegawa, H., T. Nomura, M. Kohno, N. Tateishi, Y. Suzuki, N. Maeda, R. Fujisawa, O.

Higashimura, N., N. Takasawa, Y. Tanaka, M. Nakamura, and K. Sugamura. 1996.

Higuchi, M., and M. Fujii. 2009. "Distinct functions of HTLV-1 Tax1 from HTLV-2 Tax2

Hjelle, B., O. Appenzeller, R. Mills, S. Alexander, N. Torrez-Martinez, R. Jahnke, and G.

Igakura, T., J. C. Stinchcombe, P. K. Goon, G. P. Taylor, J. N. Weber, G. M. Griffiths, Y.

Imura, A., T. Hori, K. Imada, T. Ishikawa, Y. Tanaka, M. Maeda, S. Imamura, and T.

activated T cells to vascular endothelial cells." *J Exp Med* 183(5):2185-95.

hematopoietic stem cell transplantation." *Cancer Res* 64(1):391-9.

contribute key roles to viral pathogenesis." *Retrovirology* 6:117.

allogeneic bone marrow transplantation." *Br J Haematol* 94(4):713-5.

278(44):43620-7.

193(2):277-82.

13(5):657-64.

95(1):30-8.

227-31.

*Lancet* 339(8794):645-6.

299(5613):1713-6.

*Br J Cancer* 101(9):1497-501.

donors." *Int J Cancer* 81(6):859-64.

factors JunB and c-Jun and modulates their transcriptional activity." *J Biol Chem*

Maloney. 2006. "Human leukocyte antigen concordance and the transmission risk via breast-feeding of human T cell lymphotropic virus type I." *J Infect Dis*

treatment of HTLV-1-associated acute adult T-cell leukaemia lymphoma by

Takatsuki, and M. Matsuoka. 1999. "Rapid quantification of HTLV-I provirus load: detection of monoclonal proliferation of HTLV-I-infected cells among blood

M. Griffiths, and C. R. Bangham. 2000. "Fratricide among CD8(+) T lymphocytes naturally infected with human T cell lymphotropic virus type I." *Immunity*

Ohashi, F. Fukui, A. Hasegawa, T. Masuda, Y. Takaue, J. Okamura, and M. Kannagi. 2004. "Graft-versus-Tax response in adult T-cell leukemia patients after

Yoshie, and S. Fujita. 2000. "Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells." *Blood*

"Induction of OX40, a receptor of gp34, on T cells by trans-acting transcriptional activator, Tax, of human T-cell leukemia virus type I." *Jpn J Cancer Res* 87(3):

Ross. 1992. "Chronic neurodegenerative disease associated with HTLV-II infection."

Tanaka, M. Osame, and C. R. Bangham. 2003. "Spread of HTLV-I between lymphocytes by virus-induced polarization of the cytoskeleton." *Science*

Uchiyama. 1996. "The human OX40/gp34 system directly mediates adhesion of

combination chemotherapy with eight drugs improved the survival (mean survival time 13 months), the prognosis of aggressive ATL remains poor with deaths usually being the result of severe infection or hypercalcemia, often associated with drug resistance. After successful allogeneic bone marrow transplantation (alloBMT) for a patient with ATL was reported, more patients with ATL were treated with alloBMT. The low risk of relapse in cases with graftversus-host disease, suggested that graft-versus-leukemia was effective against ATL cells. CTLs attack ATL cells via Fas ligand, perforin or granzyme. These results are consistent with the nding that ATL cells are highly susceptible to the signal via Fas antigen. Thus, the signal through Fas antigen might be a good target in therapy against ATL (Matsuoka 2003).

In a phase II study, combination of zidovudine and interferon-alpha presented promising results. Chronic ATL has a relatively better out-come, but poor long-term survival is noted when patients are managed with a watchful-waiting policy or with chemotherapy. In ATL cell lines, arsenic trioxide shuts off constitutive NF-B activation and potentiates interferon-alpha apoptotic effects through proteasomal degradation of Tax. In conclusion, treatment of chronic ATL with arsenic, interferon- alpha, and zidovudine is feasible and exhibits an impressive response rate with moderate toxicity. Viral replication (AZT) and Tax degradation (As/IFN) may eradicate the disease through this treatment. These clinical results strengthen the concept of oncogene-targeted cancer therapy (Kchour et al. 2009).

Key Words: HTLV-1, ATL, Leukemia, Molecular pathogenesis, Diagnosis, Novel treatements, Oncogenesis, Mutation, Arsenic, Interferon-alpha, Zidovudine

#### **9. References**


combination chemotherapy with eight drugs improved the survival (mean survival time 13 months), the prognosis of aggressive ATL remains poor with deaths usually being the result of severe infection or hypercalcemia, often associated with drug resistance. After successful allogeneic bone marrow transplantation (alloBMT) for a patient with ATL was reported, more patients with ATL were treated with alloBMT. The low risk of relapse in cases with graftversus-host disease, suggested that graft-versus-leukemia was effective against ATL cells. CTLs attack ATL cells via Fas ligand, perforin or granzyme. These results are consistent with the nding that ATL cells are highly susceptible to the signal via Fas antigen. Thus, the signal through Fas antigen might be a good target in therapy against ATL

In a phase II study, combination of zidovudine and interferon-alpha presented promising results. Chronic ATL has a relatively better out-come, but poor long-term survival is noted when patients are managed with a watchful-waiting policy or with chemotherapy. In ATL cell lines, arsenic trioxide shuts off constitutive NF-B activation and potentiates interferon-alpha apoptotic effects through proteasomal degradation of Tax. In conclusion, treatment of chronic ATL with arsenic, interferon- alpha, and zidovudine is feasible and exhibits an impressive response rate with moderate toxicity. Viral replication (AZT) and Tax degradation (As/IFN) may eradicate the disease through this treatment. These clinical results strengthen the concept

Key Words: HTLV-1, ATL, Leukemia, Molecular pathogenesis, Diagnosis, Novel

Abbaszadegan, M. R., M. Gholamin, A. Tabatabaee, R. Farid, M. Houshmand, and M.

Abbaszadegan, M. R., N. Jafarzadeh, M. Sankian, A. Varasteh, M. Mahmoudi, M.

Andrade, R. G., M. A. Ribeiro, M. S. Namen-Lopes, S. M. Silva, F. V. Basques, J. G. Ribas, A.

Arai, F., T. Miyamoto, O. Ohneda, T. Inada, T. Sudo, K. Brasel, T. Miyata, D. M. Anderson,

Ariumi, Y., A. Kaida, J. Y. Lin, M. Hirota, O. Masui, S. Yamaoka, Y. Taya, and K.

Basbous, J., C. Arpin, G. Gaudray, M. Piechaczyk, C. Devaux, and J. M. Mesnard. 2003. "The

Abbaszadegan. 2003. "Prevalence of human T-lymphotropic virus type 1 among

Sadeghizadeh, F. Khatami, and N. Mehramiz. 2008. "Truncated MTA-1: a pitfall in ELISA-based immunoassay of HTLV-1 infection." *J Biomed Biotechnol* 2008:

B. Carneiro-Proietti, and M. L. Martins. 2010. "Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in

and T. Suda. 1999. "Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB

Shimotohno. 2000. "HTLV-1 tax oncoprotein represses the p53-mediated transactivation function through coactivator CBP sequestration." *Oncogene* 19(12):

HBZ factor of human T-cell leukemia virus type I dimerizes with transcription

treatements, Oncogenesis, Mutation, Arsenic, Interferon-alpha, Zidovudine

blood donors from Mashhad, Iran." *J Clin Microbiol* 41(6):2593-5.

screening for blood donors." *Rev Soc Bras Med Trop* 43(2):111-5.

(RANK) receptors." *J Exp Med* 190(12):1741-54.

of oncogene-targeted cancer therapy (Kchour et al. 2009).

(Matsuoka 2003).

**9. References** 

846371.

1491-9.

factors JunB and c-Jun and modulates their transcriptional activity." *J Biol Chem* 278(44):43620-7.


Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 47

Mesnard, J. M., B. Barbeau, and C. Devaux. 2006. "HBZ, a new important player in the

Mitra-Kaushik, S., J. C. Harding, J. L. Hess, and L. Ratner. 2004. "Effects of the proteasome

Miyazato, A., S. Sheleg, H. Iha, Y. Li, and K. T. Jeang. 2005. "Evidence for NF-kappaB- and

Mori, N., M. Fujii, S. Ikeda, Y. Yamada, M. Tomonaga, D. W. Ballard, and N. Yamamoto.

Mori, N., T. Matsuda, M. Tadano, T. Kinjo, Y. Yamada, K. Tsukasaki, S. Ikeda, Y. Yamasaki,

Mori, N., M. Morishita, T. Tsukazaki, C. Z. Giam, A. Kumatori, Y. Tanaka, and N.

Morosetti, R., N. Kawamata, A. F. Gombart, C. W. Miller, Y. Hatta, T. Hirama, J. W. Said, M.

Nishimura, S., N. Asou, H. Suzushima, T. Okubo, T. Fujimoto, M. Osato, H. Yamasaki, L.

Nosaka, K., M. Maeda, S. Tamiya, T. Sakai, H. Mitsuya, and M. Matsuoka. 2000. "Increasing

Nosaka, K., T. Miyamoto, T. Sakai, H. Mitsuya, T. Suda, and M. Matsuoka. 2002.

Noula Shembade, Edward W Harhaj. 2010. "Role of post-translational modifications of

Okamura, J., A. Utsunomiya, R. Tanosaki, N. Uike, S. Sonoda, M. Kannagi, M. Tomonaga,

with CREB-binding protein/p300." *Blood* 97(7):2137-44.

Nagata, S. 1999. "Fas ligand-induced apoptosis." *Annu Rev Genet* 33:29-55.

inhibitor PS-341 on tumor growth in HTLV-1 Tax transgenic mice and Tax tumor

CBP-independent repression of p53's transcriptional activity by human T-cell leukemia virus type 1 Tax in mouse embryo and primary human fibroblasts." *J* 

1999. "Constitutive activation of NF-kappaB in primary adult T-cell leukemia cells."

Y. Tanaka, T. Ohta, T. Iwamasa, M. Tomonaga, and N. Yamamoto. 2004. "Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells." *J Virol*

Yamamoto. 2001. "Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction

Tomonaga, and H. P. Koeffler. 1995. "Alterations of the p27KIP1 gene in non-Hodgkin's lymphomas and adult T-cell leukemia/lymphoma." *Blood* 86(5):

Lisha, and K. Takatsuki. 1995. "p53 gene mutation and loss of heterozygosity are associated with increased risk of disease progression in adult T cell leukemia."

methylation of the CDKN2A gene is associated with the progression of adult T-cell

"Mechanism of hypercalcemia in adult T-cell leukemia: overexpression of receptor activator of nuclear factor kappaB ligand on adult T-cell leukemia cells." *Blood*

HTLV-1 Tax in NF-κB activation." *World Journal of Biological Chemistry* 26(1):

M. Harada, N. Kimura, M. Masuda, F. Kawano, Y. Yufu, H. Hattori, H. Kikuchi, and Y. Saburi. 2005. "Allogeneic stem-cell transplantation with reduced

mystery of adult T-cell leukemia." *Blood* 108(13):3979-82.

transplants." *Blood* 104(3):802-9.

*Virol* 79(14):9346-50.

*Blood* 93(7):2360-8.

78(9):4582-90.

1924-30.

*Leukemia* 9(4):598-604.

99(2):634-40.

13-20.

leukemia." *Cancer Res* 60(4):1043-8.


Inoue, J., M. Yoshida, and M. Seiki. 1987. "Transcriptional (p40x) and post-transcriptional

Ishida, T., S. Iida, Y. Akatsuka, T. Ishii, M. Miyazaki, H. Komatsu, H. Inagaki, N. Okada, T.

Kannian, Priya. 2010. "Human T Lymphotropic Virus Type 1 (HTLV-1): Molecular Biology

Kaplan, J. E., R. F. Khabbaz, E. L. Murphy, S. Hermansen, C. Roberts, R. Lal, W. Heneine, D.

Karin, M. 2006. "Nuclear factor-kappaB in cancer development and progression." *Nature*

Kchour, G., M. Tarhini, M. M. Kooshyar, H. El Hajj, E. Wattel, M. Mahmoudi, H. Hatoum,

Kiyokawa, T., K. Yamaguchi, M. Takeya, K. Takahashi, T. Watanabe, T. Matsumoto, S. Y.

Krueger, A., S. C. Fas, S. Baumann, and P. H. Krammer. 2003. "The role of CD95 in the

Lee, H. H., S. H. Weiss, L. S. Brown, D. Mildvan, V. Shorty, L. Saravolatz, A. Chu, H. M.

Liu, B., S. Hong, Z. Tang, H. Yu, and C. Z. Giam. 2005. "HTLV-I Tax directly binds the

Manel, N., F. J. Kim, S. Kinet, N. Taylor, M. Sitbon, and J. L. Battini. 2003. "The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV." *Cell* 115(4):449-59. Matsuoka, M. 2003. "Human T-cell leukemia virus type I and adult T-cell leukemia."

Matsuoka, M. 2005. "Human T-cell leukemia virus type I (HTLV-I) infection and the onset of

Matsuoka, M., and K. T. Jeang. 2007. "Human T-cell leukaemia virus type 1 (HTLV-1) infectivity and cellular transformation 5." *Nat Rev Cancer* 7(4):270-80.

regulation of peripheral T-cell apoptosis." *Immunol Rev* 193:58-69.

leukemia virus type I genes." *Proc Natl Acad Sci U S A* 84(11):3653-7.

7529-39.

12(2):193-201.

441(7092):431-6.

113(26):6528-32.

cell leukemia." *Cancer* 59(6):1187-91.

*Proc Natl Acad Sci U S A* 102(1):63-8.

*Oncogene* 22(33):5131-40.

regions of the USA." *J Infect Dis* 162(2):347-52.

adult T-cell leukemia (ATL)." *Retrovirology* 2:27.

and Oncogenesis " *Viruses* 2.

(p27x-III) regulators are required for the expression and replication of human T-cell

Fujita, K. Shitara, S. Akinaga, T. Takahashi, A. Utsunomiya, and R. Ueda. 2004. "The CC chemokine receptor 4 as a novel specific molecular target for immunotherapy in adult T-Cell leukemia/lymphoma." *Clin Cancer Res* 10(22):

Wright, L. Matijas, R. Thomson, D. Rudolph, W. M. Switzer, S. Kleinman, M. Busch, and G. B. Schreiber. 1996. "Male-to-female transmission of human T-cell lymphotropic virus types I and II: association with viral load. The Retrovirus Epidemiology Donor Study Group." *J Acquir Immune Defic Syndr Hum Retrovirol*

H. Rahimi, M. Maleki, H. Rafatpanah, S. A. Rezaee, M. T. Yazdi, A. Shirdel, H. de The, O. Hermine, R. Farid, and A. Bazarbachi. 2009. "Phase 2 study of the efficacy and safety of the combination of arsenic trioxide, interferon alpha, and zidovudine in newly diagnosed chronic adult T-cell leukemia/lymphoma (ATL)." *Blood*

Lee, and K. Takatsuki. 1987. "Hypercalcemia and osteoclast proliferation in adult T-

Ginzburg, N. Markowitz, D. C. Des Jarlais, and et al. 1990. "Patterns of HIV-1 and HTLV-I/II in intravenous drug abusers from the middle atlantic and central

Cdc20-associated anaphase-promoting complex and activates it ahead of schedule."


Human T-Cell Lymphotropic Virus (HTLV-1) and Adult T-Cell Leukemia 49

Tamiya, S., K. Etoh, H. Suzushima, K. Takatsuki, and M. Matsuoka. 1998. "Mutation of CD95 (Fas/Apo-1) gene in adult T-cell leukemia cells." *Blood* 91(10):3935-42. Tan, C., and T. A. Waldmann. 2002. "Proteasome inhibitor PS-341, a potential therapeutic

Tsukahara, T., M. Kannagi, T. Ohashi, H. Kato, M. Arai, G. Nunez, Y. Iwanaga, N.

Waldmann, T. A., J. D. White, C. K. Goldman, L. Top, A. Grant, R. Bamford, E. Roessler, I. D.

Watanabe, T., K. Yamaguchi, K. Takatsuki, M. Osame, and M. Yoshida. 1990. "Constitutive

Yamada, Y., M. Tomonaga, H. Fukuda, S. Hanada, A. Utsunomiya, M. Tara, M. Sano, S.

Yasunaga, J., and M. Matsuoka. 2007. "Human T-cell leukemia virus type I induces adult T-

Yasunaga, J., Y. Taniguchi, K. Nosaka, M. Yoshida, Y. Satou, T. Sakai, H. Mitsuya, and M.

Yoshida, M. 2001. "Multiple viral strategies of HTLV-1 for dysregulation of cell growth

Yoshida, M. 2005. "Discovery of HTLV-1, the first human retrovirus, its unique regulatory mechanisms, and insights into pathogenesis." *Oncogene* 24(39):5931-7. Yoshida, M. 2010. "Molecular approach to human leukemia: isolation and characterization of

Yoshida, M., K. Nosaka, J. Yasunaga, I. Nishikata, K. Morishita, and M. Matsuoka. 2004.

hypomethylation in adult T-cell leukemia cells." *Blood* 103(7):2753-60. Yoshie, O., R. Fujisawa, T. Nakayama, H. Harasawa, H. Tago, D. Izawa, K. Hieshima, Y.

apoptosis-resistant T-cell transfectants with Tax." *J Virol* 73(10):7981-7. Ureta-Vidal, A., C. Angelin-Duclos, P. Tortevoye, E. Murphy, J. F. Lepere, R. P. Buigues, N.

Yamamoto, K. Ohtani, M. Nakamura, and M. Fujii. 1999. "Induction of Bcl-x(L) expression by human T-cell leukemia virus type 1 Tax through NF-kappaB in

Jolly, M. Joubert, G. Carles, J. F. Pouliquen, G. de The, J. P. Moreau, and A. Gessain. 1999. "Mother-to-child transmission of human T-cell-leukemia/lymphoma virus type I: implication of high antiviral antibody titer and high proviral load in carrier

Horak, S. Zaknoen, C. Kasten-Sportes, and et al. 1993. "The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-

expression of parathyroid hormone-related protein gene in human T cell leukemia virus type 1 (HTLV-1) carriers and adult T cell leukemia patients that can be trans-

Ikeda, K. Takatsuki, M. Kozuru, K. Araki, F. Kawano, M. Niimi, K. Tobinai, T. Hotta, and M. Shimoyama. 2001. "A new G-CSF-supported combination chemotherapy, LSG15, for adult T-cell leukaemia-lymphoma: Japan Clinical

cell leukemia: from clinical aspects to molecular mechanisms." *Cancer Control*

Matsuoka. 2004. "Identification of aberrantly methylated genes in association with

the first human retrovirus HTLV-1 and its impact on tumorigenesis in adult T-cell

"Aberrant expression of the MEL1S gene identified in association with

Tatsumi, K. Matsushima, H. Hasegawa, A. Kanamaru, S. Kamihira, and Y. Yamada.

agent for adult T-cell leukemia." *Cancer Res* 62(4):1083-6.

induced adult T-cell leukemia." *Blood* 82(6):1701-12.

activated by HTLV-1 tax gene." *J Exp Med* 172(3):759-65.

Oncology Group Study 9303." *Br J Haematol* 113(2):375-82.

adult T-cell leukemia." *Cancer Res* 64(17):6002-9.

leukemia." *Proc Jpn Acad Ser B Phys Biol Sci* 86(2):117-30.

control." *Annu Rev Immunol* 19:475-96.

mothers." *Int J Cancer* 82(6):832-6.

14(2):133-40.

conditioning intensity as a novel immunotherapy and antiviral therapy for adult Tcell leukemia/lymphoma." *Blood* 105(10):4143-5.


Takatsuki, K. 1995. "Adult T-cell leukemia." *Intern Med* 34(10):947-52.

Takatsuki, K. 2005. "Discovery of adult T-cell leukemia." *Retrovirology* 2:16.

Pise-Masison, C. A., R. Mahieux, H. Jiang, M. Ashcroft, M. Radonovich, J. Duvall, C.

Rafatpanah, H., R. Farid, G. Golanbar, and F. Jabbari Azad. 2006. "HTLV-I Infection: virus

Robert-Guroff, M., S. H. Weiss, J. A. Giron, A. M. Jennings, H. M. Ginzburg, I. B. Margolis,

Romanelli, L. C., P. Caramelli, and A. B. Proietti. 2010. "[Human T cell lymphotropic virus

Roucoux, D. F., and E. L. Murphy. 2004. "The epidemiology and disease outcomes of human

Sabouri, A. H., M. Saito, K. Usuku, S. N. Bajestan, M. Mahmoudi, M. Forughipour, Z.

Safai, B., J. L. Huang, E. Boeri, R. Farid, J. Raafat, P. Schutzer, R. Ahkami, and G. Franchini.

Sanada, I., K. Nakada, S. Furugen, E. Kumagai, K. Yamaguchi, M. Yoshida, and K.

Satou, Y., and M. Matsuoka. 2010. "HTLV-1 and the host immune system: how the virus

Silva, M. T., R. C. Harab, A. C. Leite, D. Schor, A. Araujo, and M. J. Andrada-Serpa. 2007.

marker for acute-type adult T-cell leukemia." *Blood* 105(3):1204-13.

Takatsuki, K. 1995. "Adult T-cell leukemia." *Intern Med* 34(10):947-52. Takatsuki, K. 2005. "Discovery of adult T-cell leukemia." *Retrovirology* 2:16.

cell leukemia/lymphoma." *Blood* 105(10):4143-5.

3133-7.

340-7.

on p53 phosphorylation." *Mol Cell Biol* 20(10):3377-86.

T-lymphotropic virus type II." *AIDS Rev* 6(3):144-54.

HTLV-1-infected individuals." *J Gen Virol* 86(Pt 3):773-81.

*AIDS Res Hum Retroviruses* 12(12):1185-90.

*Hematop* 50(1):1-8.

44(5):689-92.

infected individuals." *Iran J Allergy Asthma Immunol* 5(4):153-66.

conditioning intensity as a novel immunotherapy and antiviral therapy for adult T-

Guillerm, and J. N. Brady. 2000. "Inactivation of p53 by human T-cell lymphotropic virus type 1 Tax requires activation of the NF-kappaB pathway and is dependent

structure, immune response to the virus and genetic association studies in HTLV-I-

W. A. Blattner, and R. C. Gallo. 1986. "Prevalence of antibodies to HTLV-I, -II, and - III in intravenous drug abusers from an AIDS endemic region." *JAMA* 255(22):

(HTLV)1: When should infection be suspected?]." *Rev Assoc Med Bras* 56(3):

Sabouri, Z. Abbaspour, M. E. Goharjoo, E. Khayami, A. Hasani, S. Izumo, K. Arimura, R. Farid, and M. Osame. 2005. "Differences in viral and host genetic risk factors for development of human T-cell lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis between Iranian and Japanese

1996. "Prevalence of HTLV type I infection in Iran: a serological and genetic study."

Takatsuki. 1986. "Chromosomal abnormalities in a patient with smoldering adult Tcell leukemia: evidence for a multistep pathogenesis." *Leuk Res* 10(12):1377-82. Sasaki, H., I. Nishikata, T. Shiraga, E. Akamatsu, T. Fukami, T. Hidaka, Y. Kubuki, A.

Okayama, K. Hamada, H. Okabe, Y. Murakami, H. Tsubouchi, and K. Morishita. 2005. "Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular

disrupts immune regulation, leading to HTLV-1 associated diseases." *J Clin Exp* 

"Human T lymphotropic virus type 1 (HTLV-1) proviral load in asymptomatic carriers, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other neurological abnormalities associated with HTLV-1 infection." *Clin Infect Dis*


**3** 

*Japan* 

**of Human T-Cells** 

*Yushima, Bunkyo-ku, Tokyo* 

**Roles of HTLV-1 Tax in Leukemogenesis** 

Mariko Mizuguchi, Toshifumi Hara and Masataka Nakamura *Human Gene Sciences Center, Tokyo Medical and Dental University,* 

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1), a member of the delta-retrovirus family, is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia (ATL) (Hinuma et al., 1981, Poiesz et al., 1980, Yoshida et al., 1982) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985, Osame et al., 1986). ATL is characterized by an aggressive CD4+ T-cell malignancy with resistance to anticancer therapeutics. It is currently estimated that HTLV-1 infects 10-20 million people in the world, endemically southwestern Japan, Africa, South America and the Caribbean basin (Proietti et al., 2005). HTLV-1 transmission mainly occurs from mother to child through breast milk followed by infection to child cells in a cell-cell contact manner (Kinoshita et al., 1987). Approximately 2-5% of HTLV-1-infected individuals develop ATL after a long latent period. The average Japanese ATL patients are 60 years old. Accumulation of genetic and epigenetic changes in provirus and host genes during the latent period is thought to be essential for immortalization and transformation of T-cells. However the pathogenesis of

Like other retroviruses, HTLV-1 provirus genome structure genes, gag, pro, pol, and env are flanked by 5' and 3' long terminal repeat (LTR). Besides the prototype genes, the HTLV-1 genome has the 1.6 kb pX region in the 3' terminal region. The pX region codes for several non-structural molecules Tax1, Rex, p12, p13, p30, p21 and HBZ by combination of the reading frames and alternative splicing (Figure 1) (Nicot et al., 2005). Tax1 was initially identified as a trans-acting transcriptional activator of the HTLV-1 promoter in LTR, leading to virus replication (Fujisawa et al., 1985, Sodroski et al., 1984). Tax1 has the ability to modulate transcription of cellular genes through activation of at least three cellular transcriptional factors NF-B, CREB/ATF and AP-1 (Yoshida, 2001). Tax1-mediated dysregulation of gene expression is believed to be implicated in cellular immortalization and transformation through multistep processes. Cell immortaliztion and transformation generally require at least three steps: cell growth promotion, prevention of apoptosis and escape from senescence. Involvement of Tax1 in three steps has been studies intensively and extensively; Introduction of the Tax1 gene induces phenotypic transformation in fibroblast cell lines (Tanaka et al., 1990), neoplastic transformation of primary rat fibroblast in cooperation with the ras oncogene, persistent interleukin (IL) 2-dependent growth of primary T-cells in vitro (Akagi et al., 1995, Grassmann et al., 1989), and development of tumors and

**1. Introduction** 

ATL by HTLV-1 remains incompletely understood.

2002. "Frequent expression of CCR4 in adult T-cell leukemia and human T-cell leukemia virus type 1-transformed T cells." *Blood* 99(5):1505-11.


### **Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells**

Mariko Mizuguchi, Toshifumi Hara and Masataka Nakamura *Human Gene Sciences Center, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo* 

*Japan* 

#### **1. Introduction**

50 T-Cell Leukemia

Zanjani, D. S., M. Shahabi, N. Talaei, M. Afzalaghaee, F. Tehranian, and R. Bazargani. 2010.

Zhang, Z., M. Zhang, J. V. Ravetch, C. Goldman, and T. A. Waldmann. 2003. "Effective

leukemia virus type 1-transformed T cells." *Blood* 99(5):1505-11.

tax, env, and gag Sequences." *AIDS Res Hum Retroviruses*.

monoclonal antibody, MEDI-507." *Blood* 102(1):284-8.

2002. "Frequent expression of CCR4 in adult T-cell leukemia and human T-cell

"Molecular Analysis of Human T Cell Lymphotropic Virus Type 1 and 2 (HTLV-1/2) Seroindeterminate Blood Donors from Northeast Iran: Evidence of Proviral

therapy for a murine model of adult T-cell leukemia with the humanized anti-CD2

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1), a member of the delta-retrovirus family, is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia (ATL) (Hinuma et al., 1981, Poiesz et al., 1980, Yoshida et al., 1982) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985, Osame et al., 1986). ATL is characterized by an aggressive CD4+ T-cell malignancy with resistance to anticancer therapeutics. It is currently estimated that HTLV-1 infects 10-20 million people in the world, endemically southwestern Japan, Africa, South America and the Caribbean basin (Proietti et al., 2005). HTLV-1 transmission mainly occurs from mother to child through breast milk followed by infection to child cells in a cell-cell contact manner (Kinoshita et al., 1987). Approximately 2-5% of HTLV-1-infected individuals develop ATL after a long latent period. The average Japanese ATL patients are 60 years old. Accumulation of genetic and epigenetic changes in provirus and host genes during the latent period is thought to be essential for immortalization and transformation of T-cells. However the pathogenesis of ATL by HTLV-1 remains incompletely understood.

Like other retroviruses, HTLV-1 provirus genome structure genes, gag, pro, pol, and env are flanked by 5' and 3' long terminal repeat (LTR). Besides the prototype genes, the HTLV-1 genome has the 1.6 kb pX region in the 3' terminal region. The pX region codes for several non-structural molecules Tax1, Rex, p12, p13, p30, p21 and HBZ by combination of the reading frames and alternative splicing (Figure 1) (Nicot et al., 2005). Tax1 was initially identified as a trans-acting transcriptional activator of the HTLV-1 promoter in LTR, leading to virus replication (Fujisawa et al., 1985, Sodroski et al., 1984). Tax1 has the ability to modulate transcription of cellular genes through activation of at least three cellular transcriptional factors NF-B, CREB/ATF and AP-1 (Yoshida, 2001). Tax1-mediated dysregulation of gene expression is believed to be implicated in cellular immortalization and transformation through multistep processes. Cell immortaliztion and transformation generally require at least three steps: cell growth promotion, prevention of apoptosis and escape from senescence. Involvement of Tax1 in three steps has been studies intensively and extensively; Introduction of the Tax1 gene induces phenotypic transformation in fibroblast cell lines (Tanaka et al., 1990), neoplastic transformation of primary rat fibroblast in cooperation with the ras oncogene, persistent interleukin (IL) 2-dependent growth of primary T-cells in vitro (Akagi et al., 1995, Grassmann et al., 1989), and development of tumors and

Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 53

expression of cell cycle regulators have been examined. Tax1 up-regulates expression of genes for cyclin D2, cyclin E, E2F1, CDK2, CDK4 and CDK6, while Tax1 reduced expression of genes for CDK inhibitors p19INK4d and p27Kip1 in resting Kit 225 cells (Iwanaga et al., 2001, Ohtani et al., 2000). These results indicate that Tax1-dependent deregulation of cell cycle regulators is

E2F plays crucial roles in induction of the S phase by regulating expression of genes that encode a set of molecules involved in DNA replication and cell cycle progression (Figure 2) (Nevins et al., 1997). Thus it is important to understand how Tax1 affects E2F activity. Tax1 dependnt phosphorylation of pRb results in activation of E2F1 (Iwanaga et al., 2001). Active E2F1 enhances own transcription by direct interaction to the E2F promoter in Kit 225 cells, whereas the E2F gene promoter is not activated by Tax1 in rat embryonic fibroblast REF52 cells (Ohtani et al., 2000). This finding suggests that Tax1 induces a positive feedback loop of E2F in a cell lineage-dependent manner. Indeed Tax1 increases transcript levels of genes carrying the E2F binding sites in their promoters. The HsOrc1, DHFR, DNA polymerase and Cdc6 gene are examples, all of which are necessary for DNA replication in S phase (Ohtani et al., 2000). The activity of Tax1 to activate NF-B and/or NFAT is indispensable for E2F activation (Ohtani et al., 2000). Tax1 also trans-activated promoters with E2F sites of cell cycle regulatory genes such as c-myc, cyclin D2, cyclin E and cyclin A (Huang et al., 2001, Ohtani et al., 2000, Santiago et al., 1999). These results demonstrate that Tax1 induces

Surprisingly and interestingly, primary T-cells or Kit 225 cells transduced with Tax1 show the cellular G1/S entry, but proliferation of such cells is not observed (Iwanaga et al., 2001,

cell cycle progression, partly by releasing active E2F molecules.

directly associated with abnormal cell cycle progression.

Fig. 2. Regulation of G1/S transition

**2.2 Activation of E2F** 

**2.3 Interfere with mitosis** 

leukemia in mice (Grassmann et al., 1989, Nerenberg et al., 1987). Tax1 exertion may be important for the early stage of the development of ATL, because some ATL cells do not express Tax1. The disturbance of normal cellular environment by Tax1 may be an initial step of ATL development. This chapter focuses on recent advances in molecular basis of Tax1 implication in leukemogenesis.

Fig. 1. Structure of HTLV-1 proviral genome

#### **2. Effect of Tax1 on cell growth promotion**

#### **2.1 Cell cycle progression**

Dysregulated cell cycle progression is potential for cellular transformation (Trimarchi and Lees, 2002). Cell growth is primarily controlled by the cell cycle, which in divided into five phases for convenience: the first gap (G1) phase, the DNA synthetic (S) phase, the second gap (G2) phase, the mitotic (M) phase and the resting (G0) phase. Mitogenic stimulation induces cell cycle progression by going through the restriction point between G1 and S phases (Trimarchi and Lees, 2002). Once they pass the restriction point, cells are destined to undergo one round of the cell cycle without further mitogenic stimulation. Most somatic cells usually stay at G0 or G1 phase. G1 cyclins and cyclin-dependent kinase (CDK) complexes (cyclin D1-CDK4, 6 and cyclin E-CDK2) control G1 to S transition (Dyson, 1998, Nevins, 1998, Trimarchi and Lees, 2002). Mitogenic stimulation activates cyclin-CDK complexes, which phosphorylates the retinoblastoma tumor suppressor protein (pRb), releasing active E2F that functions as a transcription factor to produce gene products required for G0/G1 to S transition (Figure 2).

 Previous studies including our findings indicate that Tax1 is directly implicated in cell cycle control (Liang et al., 2002, Neuveut et al., 1998, Ohtani et al., 2000, Schmitt et al., 1998). Tax1 induces cell cycle progression from G0/G1 to S phases in normal peripheral blood lymphocytes (PBLs) and IL-2-dependent human T-cell line Kit 225 cells (Iwanaga et al., 2001, Ohtani et al., 2000). The advantage of Kit 225 cells is that their growth is arrested at the G1 phase by depletion of IL-2 without significant apoptosis, and growth promotion can be reinduced by the addition of IL-2 (Hori et al., 1987). Tax1 is known not to have the ability to bind directly to DNA elements and to perturb expression of a lot of cellular genes through interaction with cellular transcription factors NF-B, CREB and AP-1 (Yoshida, 2001). Ectopic introduction of Tax1 into resting Kit 225 cells by recombinant adenoviruses revealed that a Tax1 mutant lacking the ability to activate NF-B fails to cell cycle progression, suggesting that NF-B is important for Tax1-mediated cell cycle progression (Iwanaga et al., 2001). To address the molecular mechanism underlying Tax1-induced cell cycle progression, effects of Tax1 on

leukemia in mice (Grassmann et al., 1989, Nerenberg et al., 1987). Tax1 exertion may be important for the early stage of the development of ATL, because some ATL cells do not express Tax1. The disturbance of normal cellular environment by Tax1 may be an initial step of ATL development. This chapter focuses on recent advances in molecular basis of Tax1

Dysregulated cell cycle progression is potential for cellular transformation (Trimarchi and Lees, 2002). Cell growth is primarily controlled by the cell cycle, which in divided into five phases for convenience: the first gap (G1) phase, the DNA synthetic (S) phase, the second gap (G2) phase, the mitotic (M) phase and the resting (G0) phase. Mitogenic stimulation induces cell cycle progression by going through the restriction point between G1 and S phases (Trimarchi and Lees, 2002). Once they pass the restriction point, cells are destined to undergo one round of the cell cycle without further mitogenic stimulation. Most somatic cells usually stay at G0 or G1 phase. G1 cyclins and cyclin-dependent kinase (CDK) complexes (cyclin D1-CDK4, 6 and cyclin E-CDK2) control G1 to S transition (Dyson, 1998, Nevins, 1998, Trimarchi and Lees, 2002). Mitogenic stimulation activates cyclin-CDK complexes, which phosphorylates the retinoblastoma tumor suppressor protein (pRb), releasing active E2F that functions as a transcription factor to produce gene products

 Previous studies including our findings indicate that Tax1 is directly implicated in cell cycle control (Liang et al., 2002, Neuveut et al., 1998, Ohtani et al., 2000, Schmitt et al., 1998). Tax1 induces cell cycle progression from G0/G1 to S phases in normal peripheral blood lymphocytes (PBLs) and IL-2-dependent human T-cell line Kit 225 cells (Iwanaga et al., 2001, Ohtani et al., 2000). The advantage of Kit 225 cells is that their growth is arrested at the G1 phase by depletion of IL-2 without significant apoptosis, and growth promotion can be reinduced by the addition of IL-2 (Hori et al., 1987). Tax1 is known not to have the ability to bind directly to DNA elements and to perturb expression of a lot of cellular genes through interaction with cellular transcription factors NF-B, CREB and AP-1 (Yoshida, 2001). Ectopic introduction of Tax1 into resting Kit 225 cells by recombinant adenoviruses revealed that a Tax1 mutant lacking the ability to activate NF-B fails to cell cycle progression, suggesting that NF-B is important for Tax1-mediated cell cycle progression (Iwanaga et al., 2001). To address the molecular mechanism underlying Tax1-induced cell cycle progression, effects of Tax1 on

implication in leukemogenesis.

Fig. 1. Structure of HTLV-1 proviral genome

required for G0/G1 to S transition (Figure 2).

**2.1 Cell cycle progression** 

**2. Effect of Tax1 on cell growth promotion** 

expression of cell cycle regulators have been examined. Tax1 up-regulates expression of genes for cyclin D2, cyclin E, E2F1, CDK2, CDK4 and CDK6, while Tax1 reduced expression of genes for CDK inhibitors p19INK4d and p27Kip1 in resting Kit 225 cells (Iwanaga et al., 2001, Ohtani et al., 2000). These results indicate that Tax1-dependent deregulation of cell cycle regulators is directly associated with abnormal cell cycle progression.

Fig. 2. Regulation of G1/S transition

#### **2.2 Activation of E2F**

E2F plays crucial roles in induction of the S phase by regulating expression of genes that encode a set of molecules involved in DNA replication and cell cycle progression (Figure 2) (Nevins et al., 1997). Thus it is important to understand how Tax1 affects E2F activity. Tax1 dependnt phosphorylation of pRb results in activation of E2F1 (Iwanaga et al., 2001). Active E2F1 enhances own transcription by direct interaction to the E2F promoter in Kit 225 cells, whereas the E2F gene promoter is not activated by Tax1 in rat embryonic fibroblast REF52 cells (Ohtani et al., 2000). This finding suggests that Tax1 induces a positive feedback loop of E2F in a cell lineage-dependent manner. Indeed Tax1 increases transcript levels of genes carrying the E2F binding sites in their promoters. The HsOrc1, DHFR, DNA polymerase and Cdc6 gene are examples, all of which are necessary for DNA replication in S phase (Ohtani et al., 2000). The activity of Tax1 to activate NF-B and/or NFAT is indispensable for E2F activation (Ohtani et al., 2000). Tax1 also trans-activated promoters with E2F sites of cell cycle regulatory genes such as c-myc, cyclin D2, cyclin E and cyclin A (Huang et al., 2001, Ohtani et al., 2000, Santiago et al., 1999). These results demonstrate that Tax1 induces cell cycle progression, partly by releasing active E2F molecules.

#### **2.3 Interfere with mitosis**

Surprisingly and interestingly, primary T-cells or Kit 225 cells transduced with Tax1 show the cellular G1/S entry, but proliferation of such cells is not observed (Iwanaga et al., 2001,

Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 55

Marzec et al., 2008, Zeng et al., 2007). The JAK/STAT pathway is one of the major cytokine signaling pathways. JAK-mediated phosphorylation of receptor subunits increases phosphorylation and dimerization of STATs, resulting in activation of downstream genes essential for cell growth and immunity (Levy and Darnell, 2002). JAK and STAT proteins are unphosphorylated and inactive in normal quiescent lymphocytes. STAT3 and STAT5 in HTLV-1 infected T-cells are reported to be constitutively activated (Hall and Fujii, 2005, Migone et al., 1995). Persistent activation of STAT3 is shown to increase proliferation, survival, angiogenesis and metastasis in various human cancers (Yu et al., 2009). IL-21 preferentially activates STAT1 and STAT3, while IL-2 and IL-15 primarily activate STAT5 (Asao et al., 2001, Zeng et al., 2007). Co-operation of intrinsic cell cycle promotion with cytokine-dependent signal transduction may be essential for cell

The pathway involving phosphoinositide 3-kinase (PI3K) and its downstream kinase Akt provides cell survival and growth signals in T-cells (Cantley, 2002). PI3K primarily phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messenger, phosphatidylinositol-3,4,5-trisphosphate (PIP3), which form a complex with Akt and phosphoinositide-dependent protein kinese 1 (PDK1) on the plasma membrane, where Akt is activated by phosphorylation by PDK1 and mTOR complex 2 (mTORC2) (Sarbassov et al., 2005). Active Akt phosphorylates several cellular proteins for cell survival and cell cycle entry. Tensin homolog deleted on chromosome 10 (PTEN) and Src homology 2 domain containing inositol polyphosphate phosphatase-1 (SHIP-1) inhibit the pathway by phosphorylation of PIP3 (Cantley and Neel, 1999, Rohrschneider et al., 2000). The PI3K/Akt pathway is constitutively active in HTLV-1 transformed cells and ATL cells (Fukuda et al., 2005, Peloponese and Jeang, 2006). Tax1 induces the phosphorylation of Akt that is linked to NF-B activation and p53 inhibition (Jeong et al., 2005). Inhibition of PI3K or Akt induces cell cycle arrest and apoptosis with accumulation of p27Kip1 and caspase-9 activation in HTLV-1 transformed T-cells (Jeong et al., 2008). In addition, Tax1 down-regulates transcription for PTEN and SHIP-1 through NF-B-mediated inhibition of the transcriptional coactivator p300 (Fukuda et al., 2009). These findings indicate that the PI3K/Akt pathway activated by Tax1 is involved in cell cycle progression and survive.

Tax1 inactivates p53 (Tabakin-Fix et al., 2006). The transcription factor p53 is critical for prevention of abnormal cell proliferation. When DNA is damaged by radiation, ultraviolet and carcinostatic, cells express high amount of active p53, resulting in expression of genes essential for cell cycle arrest, DNA repair or apoptosis (Figure 2). The p53 gene is mutated in roughly 50% of various human cancers (Grassmann et al., 2005). Mutation of p53 is poorly defined in ATL cells. Tax1 neither binds p53 nor represses p53 gene expression. Two major findings have been reported regarding inactivation of p53 by Tax1. First, Tax1 and p53 competes with each other for binding to the coactivator CREB binding protein (CBP)/p300 and p53 loses the ability to activate transcription (Ariumi et al., 2000, Van Orden et al., 1999). Second, Tax1-mediated p53 inactivation is dependent on NF-B activation. Tax1 facilitates the formation of functionally inactive complexes containing p65 (RelA) and p53, and this interaction requires p53 phosphorylation at serine-15, a site is preferentially phosphorylated in Tax1-expressing cells (Pise-Masison et al., 2000). Tax1-mediated interference with tumor suppressor p53 has been thought to facilitate resistance to apoptosis. Apoptosis is an

proliferation induced by Tax1.

**4. Modification of apoptosis by Tax1** 

Ohtani et al., 2000), perhaps suggesting blockage of mitosis by Tax1. Similarly induction of Tax1 in PA18G-BHK-21 cells, which are Tax1-inducible syrian hamster kidney cell line, revealed cell cycle transition from G1 to S phase, but further progression to mitosis was not seen (Liang et al., 2002). In addition, Tax1-transduced cells show nuclear abnormalities and cytokinesis defects, which are similar to symptoms observed in ATL patients (Jin et al., 1998, Majone et al., 1993, Semmes and Jeang, 1996). However the exact roles of Tax1 in entire cell cycle progression will be elucidated by future studies.

#### **3. Deregulation of cellular signaling by Tax1**

#### **3.1 Induction of cytokines and their receptors**

Growth stimuli for T-cells are usually delivered by cytokines, in particular IL-2 acts as an effective growth factor for T-cells (Asao et al., 1994, Tanaka et al., 1994). Cytokines, which are expressed inducibly and transiently, bind to their specific receptors, transducing intracellular signalings important not only for cellular proliferation, but for differentiation and survival of lymphocytes (Rochman et al., 2009). Expression of cytokines is crucial for proliferation of lymphocytes. The -chain of IL-2 receptor (IL-2R) is also induced by immune stimulation and its gene is the first identified cellular gene that is activated by Tax1 (Ballard et al., 1988, Ruben et al., 1988). Together with IL-2R IL-2Rand the common -chain form the high affinity IL-2 receptor complex that is an actual growth signal transducer of T-cells (Takeshita et al., 1992). Furthermore, transient transfection studies showed that the IL-2 promoter is activated by Tax1 in an NF-AT and NF-B pathwaydependent manner (Good et al., 1996, Hoyos et al., 1989, McGuire et al., 1993). These led to the hypothesis that Tax1 makes T-cells proliferative through autocrine and/or paracrine action of induced IL-2 and IL-2R. However recent studies revealed that Tax1-expressing Tcells do not produce either the IL-2 mRNA or protein (Akagi and Shimotohno, 1993, Chung et al., 2003). Hence, the IL-2/IL-2R autocrine loop mediated by Tax1 in transformation of Tcells has been reconsidered.

Tax1 trans-activates transcription of genes for other cytokines related to T-cell growth such as IL-9, IL-13, IL-15 and IL-21 (Azimi et al., 1998, Chen et al., 2008, Mizuguchi et al., 2009, Silbermann et al., 2008, Waldele et al., 2004). Notably, IL-21 is produced by activated CD4+ T-cells and effectively promotes proliferation of T-cells in co-operation with IL-15 (Onoda et al., 2007, Parrish-Novak et al., 2000). IL-21 is similar to IL-2 and IL-15 in terms of biological activity and receptor constitution, which is composed of itself specific receptor(s) and the common -chain (Asao et al., 2001, Onoda et al., 2007, Parrish-Novak et al., 2000). The common -chain is a target of Tax1 at transcription (Ohbo et al., 1995). These observations suggest that incomplete progression of the cell cycle by Tax1 may be complemented by action of cytokines and their receptors induced by Tax1. Coordination between IL-21 and IL-15 induced by Tax1 may deliver more effective growth signals in T-cells. This notion does not exclude the possibility of implication of IL-2 in Tax1-mediated cell growth. IL-21 may function as a powerful inducer for T-cell growth in the presence of IL-2, which is released in immune responses to HTLV-1 infection.

#### **3.2 Intracellular signaling**

Cytokines deliver more effective growth signals in T-cells. Interaction of cytokines with their receptors activates Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and PI3 kinase growth signaling pathways (Kelly-Welch et al., 2003,

Ohtani et al., 2000), perhaps suggesting blockage of mitosis by Tax1. Similarly induction of Tax1 in PA18G-BHK-21 cells, which are Tax1-inducible syrian hamster kidney cell line, revealed cell cycle transition from G1 to S phase, but further progression to mitosis was not seen (Liang et al., 2002). In addition, Tax1-transduced cells show nuclear abnormalities and cytokinesis defects, which are similar to symptoms observed in ATL patients (Jin et al., 1998, Majone et al., 1993, Semmes and Jeang, 1996). However the exact roles of Tax1 in entire cell

Growth stimuli for T-cells are usually delivered by cytokines, in particular IL-2 acts as an effective growth factor for T-cells (Asao et al., 1994, Tanaka et al., 1994). Cytokines, which are expressed inducibly and transiently, bind to their specific receptors, transducing intracellular signalings important not only for cellular proliferation, but for differentiation and survival of lymphocytes (Rochman et al., 2009). Expression of cytokines is crucial for proliferation of lymphocytes. The -chain of IL-2 receptor (IL-2R) is also induced by immune stimulation and its gene is the first identified cellular gene that is activated by Tax1 (Ballard et al., 1988, Ruben et al., 1988). Together with IL-2R IL-2Rand the common -chain form the high affinity IL-2 receptor complex that is an actual growth signal transducer of T-cells (Takeshita et al., 1992). Furthermore, transient transfection studies showed that the IL-2 promoter is activated by Tax1 in an NF-AT and NF-B pathwaydependent manner (Good et al., 1996, Hoyos et al., 1989, McGuire et al., 1993). These led to the hypothesis that Tax1 makes T-cells proliferative through autocrine and/or paracrine action of induced IL-2 and IL-2R. However recent studies revealed that Tax1-expressing Tcells do not produce either the IL-2 mRNA or protein (Akagi and Shimotohno, 1993, Chung et al., 2003). Hence, the IL-2/IL-2R autocrine loop mediated by Tax1 in transformation of T-

Tax1 trans-activates transcription of genes for other cytokines related to T-cell growth such as IL-9, IL-13, IL-15 and IL-21 (Azimi et al., 1998, Chen et al., 2008, Mizuguchi et al., 2009, Silbermann et al., 2008, Waldele et al., 2004). Notably, IL-21 is produced by activated CD4+ T-cells and effectively promotes proliferation of T-cells in co-operation with IL-15 (Onoda et al., 2007, Parrish-Novak et al., 2000). IL-21 is similar to IL-2 and IL-15 in terms of biological activity and receptor constitution, which is composed of itself specific receptor(s) and the common -chain (Asao et al., 2001, Onoda et al., 2007, Parrish-Novak et al., 2000). The common -chain is a target of Tax1 at transcription (Ohbo et al., 1995). These observations suggest that incomplete progression of the cell cycle by Tax1 may be complemented by action of cytokines and their receptors induced by Tax1. Coordination between IL-21 and IL-15 induced by Tax1 may deliver more effective growth signals in T-cells. This notion does not exclude the possibility of implication of IL-2 in Tax1-mediated cell growth. IL-21 may function as a powerful inducer for T-cell growth in the presence of IL-2, which is released in

Cytokines deliver more effective growth signals in T-cells. Interaction of cytokines with their receptors activates Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and PI3 kinase growth signaling pathways (Kelly-Welch et al., 2003,

cycle progression will be elucidated by future studies.

**3. Deregulation of cellular signaling by Tax1 3.1 Induction of cytokines and their receptors** 

cells has been reconsidered.

immune responses to HTLV-1 infection.

**3.2 Intracellular signaling** 

Marzec et al., 2008, Zeng et al., 2007). The JAK/STAT pathway is one of the major cytokine signaling pathways. JAK-mediated phosphorylation of receptor subunits increases phosphorylation and dimerization of STATs, resulting in activation of downstream genes essential for cell growth and immunity (Levy and Darnell, 2002). JAK and STAT proteins are unphosphorylated and inactive in normal quiescent lymphocytes. STAT3 and STAT5 in HTLV-1 infected T-cells are reported to be constitutively activated (Hall and Fujii, 2005, Migone et al., 1995). Persistent activation of STAT3 is shown to increase proliferation, survival, angiogenesis and metastasis in various human cancers (Yu et al., 2009). IL-21 preferentially activates STAT1 and STAT3, while IL-2 and IL-15 primarily activate STAT5 (Asao et al., 2001, Zeng et al., 2007). Co-operation of intrinsic cell cycle promotion with cytokine-dependent signal transduction may be essential for cell proliferation induced by Tax1.

The pathway involving phosphoinositide 3-kinase (PI3K) and its downstream kinase Akt provides cell survival and growth signals in T-cells (Cantley, 2002). PI3K primarily phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messenger, phosphatidylinositol-3,4,5-trisphosphate (PIP3), which form a complex with Akt and phosphoinositide-dependent protein kinese 1 (PDK1) on the plasma membrane, where Akt is activated by phosphorylation by PDK1 and mTOR complex 2 (mTORC2) (Sarbassov et al., 2005). Active Akt phosphorylates several cellular proteins for cell survival and cell cycle entry. Tensin homolog deleted on chromosome 10 (PTEN) and Src homology 2 domain containing inositol polyphosphate phosphatase-1 (SHIP-1) inhibit the pathway by phosphorylation of PIP3 (Cantley and Neel, 1999, Rohrschneider et al., 2000). The PI3K/Akt pathway is constitutively active in HTLV-1 transformed cells and ATL cells (Fukuda et al., 2005, Peloponese and Jeang, 2006). Tax1 induces the phosphorylation of Akt that is linked to NF-B activation and p53 inhibition (Jeong et al., 2005). Inhibition of PI3K or Akt induces cell cycle arrest and apoptosis with accumulation of p27Kip1 and caspase-9 activation in HTLV-1 transformed T-cells (Jeong et al., 2008). In addition, Tax1 down-regulates transcription for PTEN and SHIP-1 through NF-B-mediated inhibition of the transcriptional coactivator p300 (Fukuda et al., 2009). These findings indicate that the PI3K/Akt pathway activated by Tax1 is involved in cell cycle progression and survive.

#### **4. Modification of apoptosis by Tax1**

Tax1 inactivates p53 (Tabakin-Fix et al., 2006). The transcription factor p53 is critical for prevention of abnormal cell proliferation. When DNA is damaged by radiation, ultraviolet and carcinostatic, cells express high amount of active p53, resulting in expression of genes essential for cell cycle arrest, DNA repair or apoptosis (Figure 2). The p53 gene is mutated in roughly 50% of various human cancers (Grassmann et al., 2005). Mutation of p53 is poorly defined in ATL cells. Tax1 neither binds p53 nor represses p53 gene expression. Two major findings have been reported regarding inactivation of p53 by Tax1. First, Tax1 and p53 competes with each other for binding to the coactivator CREB binding protein (CBP)/p300 and p53 loses the ability to activate transcription (Ariumi et al., 2000, Van Orden et al., 1999). Second, Tax1-mediated p53 inactivation is dependent on NF-B activation. Tax1 facilitates the formation of functionally inactive complexes containing p65 (RelA) and p53, and this interaction requires p53 phosphorylation at serine-15, a site is preferentially phosphorylated in Tax1-expressing cells (Pise-Masison et al., 2000). Tax1-mediated interference with tumor suppressor p53 has been thought to facilitate resistance to apoptosis. Apoptosis is an

Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 57

the hTERT promoter parallels Tax1-mediated cell cycle progression (Matsumura-Arioka et al., 2005). In leukemia cells, Tax1 may be negatively associated with or independent of regulation of hTERT expression, rather epigenetic changes in the promoter in leukemia cells may significantly contribute to constitutive activation of the hTERT promoter. It may be important that strict repression of telomerase expression in normal T-cells to avoid undesirable immune responses and malignant transformation. An element involved in repression in the promoter is

HTLV-2 is close to HTLV-1 in genetic and biological terms, showing ~70% sequence homology with each other (Feuer and Green, 2005). HTLV-2 encodes Tax2, which shows ~75% sequence homology to Tax1 (Feuer and Green, 2005). Tax1 and Tax2 have been shown to important roles in immortalization of T-cells in an IL-2 dependent manner, though HTLV-2 has not been linked with development of hematological malignant diseases (Feuer and Green, 2005). Differences in functional domains between Tax1 and Tax2 has been demonstrated. Tax1 possesses a leucine zipper like region (LZR) within amino acids 225-232 and the PDZ binding motif (PBM) at C-terminus, which are responsible for Tax1-mediated

p100 processing and p52 nuclear translocation (Higuchi et al., 2007, Shoji et al., 2009).

The NF-B pathway is tightly controlled in normal T-cells, and transiently activated upon immune stimulation. On the other hand, aberrant NF-B activation is implicated in many types of cancer, especially hematological malignancies such as leukemia, lymphoma and myeloma (Karin, 2006). In HTLV-1 infected T-cells, NF-B is constitutively activated, which is also thought to be linked to immortalization of T-cell by HTLV-1 and HTLV-2 (Mori et al., 1999, Robek and Ratner, 1999, Ross et al., 2000). Tax1 activates both the canonical (mainly consisting of the p50 and p65 subunits) and non-canonical (mainly consisting of the p52 and RelB subunits) NF-B pathways. These are consequences of interaction of Tax1 with IKK complex. In contrast, although Tax2 can activate the canonical pathway to a level comparable to Tax1, Tax2 rarely induces the p100 processing because of lacking the LZR

Recent studies demonstrate that Tax2 induces expression of IL-2, but Tax1 fails to induce IL-2 production (Figure 3) (Niinuma et al., 2005). This finding prompted us to search for

suggested (Gabet et al., 2003, Hara et al., 2008).

and PBM regions (Higuchi et al., 2007, Shoji et al., 2009).

Fig. 3. Differential cytokine expression by Tax

**6. HTLV-1 Tax and HTLV-2 Tax** 

important mechanism with intrinsic active processes of programmed cell death, by which cells keep themselves from uncontrolled cell death. As p53 is one of pivotal molecules to trigger apoptosis, Tax1-mediated inactivation of p53 may predispose HTLV-1 infected cells to survive. In addition to Tax1-mediated inactivation of p53, Tax1 induces anti-apoptotic molecules such as Bcl-XL, XIAP and survivin (Tsukahara et al., 1999, Yoshida, 2001). In tumor cells, prevention of apoptosis is essential for their continuous growth. Anti-apoptotic effects of Tax1 may lead to cellular immortalization and contribute to accumulation of genetic mutations.

Conversely, perevious studies reported that Tax1 expression is closely linked to the induction of apoptosis (Chlichlia et al., 1995, Chlichlia et al., 1997, Kao et al., 2000). Tax1 mediated apoptosis occurs in Tax1-inducible cell line JPX-9 by activation of the Fas/FasL pathway (Chen et al., 1997). Tax1 has been reported to sensitize cells to apoptosis induced by DNA damaging agents. The results from human cDNA expression array analysis with HTLV-1 infected Tax1-expressing T-cells (C81) treated with irradiation show up-regulation of various genes for cell cycle inducers and inhibitors, anti- and pro-apoptotic molecules (de la Fuente et al., 2003). Upon irradiation, S and G2/M phase-enriched population increases in cell numbers with apoptosis, while little, if any, or no induction of apoptosis is associated with G0/G1 population (de la Fuente et al., 2003). The apparent paradox of the opposite effects of Tax1 on cell death remains to be elucidated. The choice between proliferation and cell death by Tax1 may be influenced by cell cycle state or intracellular status.

#### **5. Immortalization by Tax1**

Telomeres are DNA-protein complex structures located at the end of chromosomes (Blackburn, 1991). The structures are thought to contribute to the stabilization of linear chromosomes (Blackburn, 1991, Counter et al., 1992). Each cell division leads to the shortening of telomere length by the end-replication problem (Olovnikov, 1973, Watson, 1972). The shortening of telomeres results in chromosome instability, which is closely related to cellular senescence (Allsopp et al., 1992). Thus most human somatic cells have a limited replicative life span due to shortening of the telomere length. To avoid telomere shortening, transformed cells and germline cells appear to have certain compensatory mechanisms (Counter et al., 1994, Kim et al., 1994). One mechanism synthesizing terminal telomere sequences is mediated by the reibonucleoprotein enzyme telomerase, whose activity is restricted by expression of its catalytic subunit human telomerase reverse transcriptase (hTERT) (Meyerson et al., 1997, Nakamura et al., 1997). Development and maintenance of ATL probably require telomerase activity and indeed ATL cells carry telomerase activity (Uchida et al., 1999). Therefore activation of telomerase seems to be one of key events in development of ATL.

Effects of Tax1 on expression of telomerase in human T-cells remains controversial. An early report concerning this issue suggested that Tax1 reduced telomerase activity in human T-cell line Jurkat cells and Tax1 negatively regulated hTERT promoter activity (Gabet et al., 2003). In contrast, other group showed that Tax1 stimulated the endogenous hTERT promoter through NF-B activation (Sinha-Datta et al., 2004). Recent studies may provide systemic solution to the discrepancy. Tax1 activates hTERT gene expression only in resting T-cells, while hTERT expression is not significantly changed by Tax1 in growing cells (Hara et al., 2008). Thus, the cell cycle state may differentially influence action of Tax1 on hTERT expression in human Tcells. The activity of Tax1 to promote cell cycle progression may be critically linked to regulation of expression of the hTERT gene, because kinetics of Tax1-mediated activation of the hTERT promoter parallels Tax1-mediated cell cycle progression (Matsumura-Arioka et al., 2005). In leukemia cells, Tax1 may be negatively associated with or independent of regulation of hTERT expression, rather epigenetic changes in the promoter in leukemia cells may significantly contribute to constitutive activation of the hTERT promoter. It may be important that strict repression of telomerase expression in normal T-cells to avoid undesirable immune responses and malignant transformation. An element involved in repression in the promoter is suggested (Gabet et al., 2003, Hara et al., 2008).

#### **6. HTLV-1 Tax and HTLV-2 Tax**

56 T-Cell Leukemia

important mechanism with intrinsic active processes of programmed cell death, by which cells keep themselves from uncontrolled cell death. As p53 is one of pivotal molecules to trigger apoptosis, Tax1-mediated inactivation of p53 may predispose HTLV-1 infected cells to survive. In addition to Tax1-mediated inactivation of p53, Tax1 induces anti-apoptotic molecules such as Bcl-XL, XIAP and survivin (Tsukahara et al., 1999, Yoshida, 2001). In tumor cells, prevention of apoptosis is essential for their continuous growth. Anti-apoptotic effects of Tax1 may lead to

Conversely, perevious studies reported that Tax1 expression is closely linked to the induction of apoptosis (Chlichlia et al., 1995, Chlichlia et al., 1997, Kao et al., 2000). Tax1 mediated apoptosis occurs in Tax1-inducible cell line JPX-9 by activation of the Fas/FasL pathway (Chen et al., 1997). Tax1 has been reported to sensitize cells to apoptosis induced by DNA damaging agents. The results from human cDNA expression array analysis with HTLV-1 infected Tax1-expressing T-cells (C81) treated with irradiation show up-regulation of various genes for cell cycle inducers and inhibitors, anti- and pro-apoptotic molecules (de la Fuente et al., 2003). Upon irradiation, S and G2/M phase-enriched population increases in cell numbers with apoptosis, while little, if any, or no induction of apoptosis is associated with G0/G1 population (de la Fuente et al., 2003). The apparent paradox of the opposite effects of Tax1 on cell death remains to be elucidated. The choice between proliferation and

Telomeres are DNA-protein complex structures located at the end of chromosomes (Blackburn, 1991). The structures are thought to contribute to the stabilization of linear chromosomes (Blackburn, 1991, Counter et al., 1992). Each cell division leads to the shortening of telomere length by the end-replication problem (Olovnikov, 1973, Watson, 1972). The shortening of telomeres results in chromosome instability, which is closely related to cellular senescence (Allsopp et al., 1992). Thus most human somatic cells have a limited replicative life span due to shortening of the telomere length. To avoid telomere shortening, transformed cells and germline cells appear to have certain compensatory mechanisms (Counter et al., 1994, Kim et al., 1994). One mechanism synthesizing terminal telomere sequences is mediated by the reibonucleoprotein enzyme telomerase, whose activity is restricted by expression of its catalytic subunit human telomerase reverse transcriptase (hTERT) (Meyerson et al., 1997, Nakamura et al., 1997). Development and maintenance of ATL probably require telomerase activity and indeed ATL cells carry telomerase activity (Uchida et al., 1999). Therefore activation of telomerase seems to be one

Effects of Tax1 on expression of telomerase in human T-cells remains controversial. An early report concerning this issue suggested that Tax1 reduced telomerase activity in human T-cell line Jurkat cells and Tax1 negatively regulated hTERT promoter activity (Gabet et al., 2003). In contrast, other group showed that Tax1 stimulated the endogenous hTERT promoter through NF-B activation (Sinha-Datta et al., 2004). Recent studies may provide systemic solution to the discrepancy. Tax1 activates hTERT gene expression only in resting T-cells, while hTERT expression is not significantly changed by Tax1 in growing cells (Hara et al., 2008). Thus, the cell cycle state may differentially influence action of Tax1 on hTERT expression in human Tcells. The activity of Tax1 to promote cell cycle progression may be critically linked to regulation of expression of the hTERT gene, because kinetics of Tax1-mediated activation of

cellular immortalization and contribute to accumulation of genetic mutations.

cell death by Tax1 may be influenced by cell cycle state or intracellular status.

**5. Immortalization by Tax1** 

of key events in development of ATL.

HTLV-2 is close to HTLV-1 in genetic and biological terms, showing ~70% sequence homology with each other (Feuer and Green, 2005). HTLV-2 encodes Tax2, which shows ~75% sequence homology to Tax1 (Feuer and Green, 2005). Tax1 and Tax2 have been shown to important roles in immortalization of T-cells in an IL-2 dependent manner, though HTLV-2 has not been linked with development of hematological malignant diseases (Feuer and Green, 2005). Differences in functional domains between Tax1 and Tax2 has been demonstrated. Tax1 possesses a leucine zipper like region (LZR) within amino acids 225-232 and the PDZ binding motif (PBM) at C-terminus, which are responsible for Tax1-mediated p100 processing and p52 nuclear translocation (Higuchi et al., 2007, Shoji et al., 2009).

The NF-B pathway is tightly controlled in normal T-cells, and transiently activated upon immune stimulation. On the other hand, aberrant NF-B activation is implicated in many types of cancer, especially hematological malignancies such as leukemia, lymphoma and myeloma (Karin, 2006). In HTLV-1 infected T-cells, NF-B is constitutively activated, which is also thought to be linked to immortalization of T-cell by HTLV-1 and HTLV-2 (Mori et al., 1999, Robek and Ratner, 1999, Ross et al., 2000). Tax1 activates both the canonical (mainly consisting of the p50 and p65 subunits) and non-canonical (mainly consisting of the p52 and RelB subunits) NF-B pathways. These are consequences of interaction of Tax1 with IKK complex. In contrast, although Tax2 can activate the canonical pathway to a level comparable to Tax1, Tax2 rarely induces the p100 processing because of lacking the LZR and PBM regions (Higuchi et al., 2007, Shoji et al., 2009).

Recent studies demonstrate that Tax2 induces expression of IL-2, but Tax1 fails to induce IL-2 production (Figure 3) (Niinuma et al., 2005). This finding prompted us to search for

Fig. 3. Differential cytokine expression by Tax

Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 59

Ariumi, Y., Kaida, A., Lin, J. Y., Hirota, M., Masui, O., Yamaoka, S., Taya, Y. & Shimotohno,

Asao, H., Tanaka, N., Ishii, N., Higuchi, M., Takeshita, T., Nakamura, M., Shirasawa, T. &

Azimi, N., Brown, K., Bamford, R. N., Tagaya, Y., Siebenlist, U. & Waldmann, T. A. 1998.

Cantley, L. C. & Neel, B. G. 1999. New insights into tumor suppression: PTEN suppresses

Chen, J., Petrus, M., Bryant, B. R., Phuc Nguyen, V., Stamer, M., Goldman, C. K., Bamford,

Chen, X., Zachar, V., Zdravkovic, M., Guo, M., Ebbesen, P. & Liu, X. 1997. Role of the

Chlichlia, K., Moldenhauer, G., Daniel, P. T., Busslinger, M., Gazzolo, L., Schirrmacher, V. &

Chung, H. K., Young, H. A., Goon, P. K., Heidecker, G., Princler, G. L., Shimozato, O.,

Counter, C. M., Avilion, A. A., Lefeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B.

De La Fuente, C., Wang, L., Wang, D., Deng, L., Wu, K., Li, H., Stein, L. D., Denny, T.,

human ovarian carcinoma. *Proc Natl Acad Sci USA,* 91, 2900-4.

HTLV-1 Tax expressing cells. *Mol Cell Biochem,* 245, 99-113.

Dyson, N. 1998. The regulation of E2F by pRB-family proteins. *Genes Dev,* 12, 2245-62.

lymphotropic virus type I Tax transactivator. *J Gen Virol,* 78, 3277-85. Chlichlia, K., Busslinger, M., Peter, M. E., Walczak, H., Krammer, P. H., Schirrmacher, V. &

Blackburn, E. H. 1991. Structure and function of telomeres. *Nature,* 350, 569-73. Cantley, L. C. 2002. The phosphoinositide 3-kinase pathway. *Science,* 296, 1655-7.

leukemia cells by a paracrine mechanism. *Blood,* 111,5163-72.

function through coactivator CBP sequestration. *Oncogene,* 19, 1491-9. Asao, H., Okuyama, C., Kumaki, S., Ishii, N., Tsuchiya, S., Foster, D. & Sugamura, K. 2001.

*Immunol,* 167, 1-5.

receptor α gene. *Science,* 241, 1652-5.

*Natl Acad Sci USA,* 96, 4240-5.

death. *Oncogene,* 14, 2265-72.

cell lines. *Blood,* 102, 4130-6.

activation and apoptosis. *Oncogene,* 10, 269-77.

201-6.

K. 2000. HTLV-1 tax oncoprotein represses the p53-mediated trans-activation

The common -chain is an indispensable subunit of the IL-21 receptor complex. *J* 

Sugamura, K. 1994. Interleukin 2-induced activation of JAK3: possible involvement in signal transduction for c-myc induction and cell proliferation. *FEBS Lett,* 351,

Human T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-B site. *Proc Natl Acad Sci USA,* 95, 2452-7. Ballard, D. W., Bohnlein, E., Lowenthal, J. W., Wano, Y., Franza, B. R. & Greene, W. C. 1988.

HTLV-I tax induces cellular proteins that activate the B element in the IL-2

tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. *Proc* 

R., Morris, J. C., Janik, J. E. & Waldmann, T. A. 2008. Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell

Fas/Fas ligand pathway in apoptotic cell death induced by the human T cell

Khazaie, K. 1997. ICE-proteases mediate HTLV-I Tax-induced apoptotic T-cell

Khazaie, K. 1995. Immediate effects of reversible HTLV-1 tax function: T-cell

Taylor, G. P., Bangham, C. R. & Derse, D. 2003. Activation of interleukin-13 expression in T cells from HTLV-1-infected individuals and in chronically infected

& Bacchetti, S. 1992. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. *EMBO J,* 11, 1921-9. Counter, C. M., Hirte, H. W., Bacchetti, S. & Harley, C. B. 1994. Telomerase activity in

Coffman, F., Kehn, K., Baylor, S., Maddukuri, A., Pumfery, A. & Kashanchi, F. 2003. Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in

another factor(s), which is differently induced between Tax1 and Tax2. In contrast to IL-2 induction, Tax1 induced IL-21 expression in CD4+ T-cells, but Tax2 did not (Figure 3) (Miuguchi et al., 2009). The IL-21 promoter NF-B binding site is activated by the p52/RelB complex by direct binding in a Tax1-dependent manner, probably reflecting the difference in NF-B activation between Tax1 and Tax2. The functional differences between Tax1 and Tax2 causes different profiles of cytokine production, and may be related to phathogenesis between HTLV-1 and HTLV-2.

#### **7. Conclusion**

HTLV-1 is the first human retrovirus, which causes leukemia/lymphoma. Before the discovery of HTLV-1, many oncogenic retroviruses have been found, which induce malignant tumors in animals such as avian and rodent (Maeda et al., 2008). Most animal oncogenic retroviruses carry oncogenes, but they are totally different from the HTLV-1 oncogene for Tax1. Oncogenes, called v-onc, in animal retroviruses are usually derived from host cells, while Tax1 has no identity in host cells in terms of the origin of oncogene. Action of oncogenes are also different, unlikely to animal oncogenes, whose products are directly integrated cellular signaling pathways with dysregulated activities, Tax1 acts as transcriptional modulator that indirectly affects transcription of cellular genes related to immortalization and transformation. Therefore literature studies concerning animal retrovirus oncogenes was not much helpful in analyzes of mode of action of Tax1 in leukemogenesis.

Tax1 is a molecule of HTLV-1 products that shows strong immunogenicity (Kannagi et al., 1991). Human CD8+ T-cells target Tax1-expressing cells. It is probably expected that most Tax1-expressing cells are killed by this mechanism. Thus Tax1 functions as a molecule both advantageous and disadvantageous to virus survive in vivo. Only cells escaped from immune attack further require genetic and epigenetic changes to become full transformants. These are may be reasons why ATL occurs after the long latent period at low frequency. In summary, Tax1 provides cells abilities of cell growth promotion, apoptosis prevention and senescence avoidance, but Tax1 alone may be insufficient for ATL development.

#### **8. Acknowledgements**

We are indebted to H. Asao, M. Higuchi and M. Fujii for support and discussion. This study was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

#### **9. References**


another factor(s), which is differently induced between Tax1 and Tax2. In contrast to IL-2 induction, Tax1 induced IL-21 expression in CD4+ T-cells, but Tax2 did not (Figure 3) (Miuguchi et al., 2009). The IL-21 promoter NF-B binding site is activated by the p52/RelB complex by direct binding in a Tax1-dependent manner, probably reflecting the difference in NF-B activation between Tax1 and Tax2. The functional differences between Tax1 and Tax2 causes different profiles of cytokine production, and may be related to phathogenesis

HTLV-1 is the first human retrovirus, which causes leukemia/lymphoma. Before the discovery of HTLV-1, many oncogenic retroviruses have been found, which induce malignant tumors in animals such as avian and rodent (Maeda et al., 2008). Most animal oncogenic retroviruses carry oncogenes, but they are totally different from the HTLV-1 oncogene for Tax1. Oncogenes, called v-onc, in animal retroviruses are usually derived from host cells, while Tax1 has no identity in host cells in terms of the origin of oncogene. Action of oncogenes are also different, unlikely to animal oncogenes, whose products are directly integrated cellular signaling pathways with dysregulated activities, Tax1 acts as transcriptional modulator that indirectly affects transcription of cellular genes related to immortalization and transformation. Therefore literature studies concerning animal retrovirus oncogenes was not much helpful in analyzes of mode of action of Tax1 in

Tax1 is a molecule of HTLV-1 products that shows strong immunogenicity (Kannagi et al., 1991). Human CD8+ T-cells target Tax1-expressing cells. It is probably expected that most Tax1-expressing cells are killed by this mechanism. Thus Tax1 functions as a molecule both advantageous and disadvantageous to virus survive in vivo. Only cells escaped from immune attack further require genetic and epigenetic changes to become full transformants. These are may be reasons why ATL occurs after the long latent period at low frequency. In summary, Tax1 provides cells abilities of cell growth promotion, apoptosis prevention and

We are indebted to H. Asao, M. Higuchi and M. Fujii for support and discussion. This study was supported in part by Grants-in-Aid for scientific research from the Ministry of

Akagi, T., Ono, H. & Shimotohno, K. 1995. Characterization of T cells immortalized by Tax1

Akagi, T. & Shimotohno, K. 1993. Proliferative response of Tax1-transduced primary human

Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B.,

T cells to anti-CD3 antibody stimulation by an interleukin-2-independent pathway.

Greider, C. W. & Harley, C. B. 1992. Telomere length predicts replicative capacity of

senescence avoidance, but Tax1 alone may be insufficient for ATL development.

Education, Culture, Sports, Science and Technology of Japan.

of human T-cell leukemia virus type 1. *Blood,* 86, 4243-9.

human fibroblasts. *Proc Natl Acad Sci USA,* 89, 10114-8.

between HTLV-1 and HTLV-2.

**7. Conclusion** 

leukemogenesis.

**8. Acknowledgements** 

*J Virol,* 67, 1211-7.

**9. References** 


Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 61

Huang, Y., Ohtani, K., Iwanaga, R., Matsumura, Y. & Nakamura, M. 2001. Direct trans-

Iwanaga, R., Ohtani, K., Hayashi, T. & Nakamura, M. 2001. Molecular mechanism of cell

Jeong, S. J., Dasgupta, A., Jung, K. J., Um, J. H., Burke, A., Park, H. U. & Brady, J. N. 2008.

Jeong, S. J., Pise-Masison, C. A., Radonovich, M. F., Park, H. U. & Brady, J. N. 2005.

Jin, D. Y., Spencer, F. & Jeang, K. T. 1998. Human T cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1. *Cell,* 93,81-91. Kannagi, M., Harada, S., Maruyama, I., Inoko, H., Igarashi, H., Kuwashima, G., Sato, S.,

Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello,

I from Carrier Mothers to Their Children. *Jap J Cancer Res,* 78, 674-680. Levy, D. E. & Darnell, J. E., Jr. 2002. Stats: transcriptional control and biological impact. *Nat* 

Liang, M. H., Geisbert, T., Yao, Y., Hinrichs, S. H. & Giam, C. Z. 2002. Human T-

Maeda, N., Fan, H. & Yoshikai, Y. 2008. Oncogenesis by retroviruses: old and new

Majone, F., Semmes, O. J. & Jeang, K. T. 1993. Induction of micronuclei by HTLV-I Tax: a

Marzec, M., Liu, X., Kasprzycka, M., Witkiewicz, A., Raghunath, P. N., El-Salem, M.,

Matsumura-Arioka, Y., Ohtani, K., Hara, T., Iwanaga, R. & Nakamura, M. 2005. Identification

in association with cell growth in human T cells. *Int Immunol,* 17, 207-15. Mcguire, K. L., Curtiss, V. E., Larson, E. L. & Haseltine, W. A. 1993. Influence of human T-

interleukin-13 signaling connections maps. *Science,* 300, 1527-8.

cell leukemia virus type I. *Oncogene,* 20, 1094-102.

HTLV-1-transformed cells. *Oncogene,* 24, 6719-28.

virus type I. *Oncogene,* 20, 2055-67.

*Rev Mol Cell Biol,* 3, 651-62.

mitosis. *J Virol,* 76, 4022-33.

*Blood,* 111, 2181-9.

1590-9.

paradigms. *Rev Med Virol,* 18, 387-405.

cellular assay for function. *Virology,* 193, 456-9.

transformed cells. *Virology,* 370, 264-72.

activation of the human cyclin D2 gene by the oncogene product Tax of human T-

cycle progression induced by the oncogene product Tax of human T-cell leukemia

PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-

Activated AKT regulates NF-B activation, p53 inhibition and cell survival in

Morita, M., Kidokoro, M., Sugimoto, M. & ET AL. 1991. Predominant recognition of human T cell leukemia virus type I (HTLV-I) pX gene products by human CD8+ cytotoxic T cells directed against HTLV-I-infected cells. *Int Immunol,* 3, 761-7. Kao, S. Y., Lemoine, F. J. & Mariott, S. J. 2000. HTLV-1 Tax protein sensitizes cells to apoptotic cell death induced by DNA damaging agents. *Oncogene,* 19, 2240-8. Karin, M. 2006. Nuclear factor-ĸB in cancer development and progression. *Nature,* 441, 431-6. Kelly-Welch, A. E., Hanson, E. M., Boothby, M. R. & Keegan, A. D. 2003. Interleukin-4 and

G. M., Wright, W. E., Weinrich, S. L. & Shay, J. W. 1994. Specific association of human telomerase activity with immortal cells and cancer. *Science,* 266, 2011-5. Kinoshita, K., Amagasaki, T., Hino, S., Doi, H., Yamanouchi, K., Ban, N., Momita, S., Ikeda,

S., Kamihira, S., Ichimaru, M., Katamine, S., Miyamoto, T., Tsuji, Y., Ishimaru, T., Yamabe, T., Ito, M., Kamura, S. & Tsuda, T. 1987. Milk-Borne Transmission of Htlv-

lymphotropic virus type 1 oncoprotein tax promotes S-phase entry but blocks

Robertson, E., Odum, N. & Wasik, M. A. 2008. IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes.

of two distinct elements mediating activation of telomerase (hTERT) gene expression

cell leukemia virus type I tax and rex on interleukin-2 gene expression. *J Virol,* 67,


Feuer, G. & Green, P. L. 2005. Comparative biology of human T-cell lymphotropic virus type

Fujisawa, J., Seiki, M., Kiyokawa, T. & Yoshida, M. 1985. Functional activation of the long

Fukuda, R., Hayashi, A., Utsunomiya, A., Nukada, Y., Fukui, R., Itoh, K., Tezuka, K.,

Gabet, A. S., Mortreux, F., Charneau, P., Riou, P., Duc-Dodon, M., Wu, Y., Jeang, K. T. & Wattel, E. 2003. Inactivation of hTERT transcription by Tax. *Oncogene,* 22, 3734-41. Gessain, A., Barin, F., Vernant, J. C., Gout, O., Maurs, L., Calender, A. & De The, G. 1985.

Good, L., Maggirwar, S. B. & Sun, S. C. 1996. Activation of the IL-2 gene promoter by HTLV-

Grassmann, R., Aboud, M. & Jeang, K. T. 2005. Molecular mechanisms of cellular

Grassmann, R., Dengler, C., Muller-Fleckenstein, I., Fleckenstein, B., Mcguire, K., Dokhelar,

Hall, W. W. & Fujii, M. 2005. Deregulation of cell-signaling pathways in HTLV-1 infection.

Hara, T., Matsumura-Arioka, Y., Ohtani, K. & Nakamura, M. 2008. Role of human T-cell

transcriptase (hTERT) gene in human T-cells. *Cancer Sci,* 99, 1155-63. Higuchi, M., Tsubata, C., Kondo, R., Yoshida, S., Takahashi, M., Oie, M., Tanaka, Y.,

independent growth transformation of a T-cell line. *J Virol,* 81,11900-7. HINUMA, Y., NAGATA, K., HANAOKA, M., NAKAI, M., MATSUMOTO, T., KINOSHITA,

Hori, T., Uchiyama, T., Tsudo, M., Umadome, H., Ohno, H., Fukuhara, S., Kita, K. & Uchino,

Hoyos, B., Ballard, D. W., Bohnlein, E., Siekevitz, M. & Greene, W. C. 1989. B-specific DNA

cell leukemia/lymphoma virus. *Blood,* 70, 1069-72.

transformation by HTLV-1 Tax. *Oncogene,* 24, 5976-85.

terminal repeat of human T-cell leukemia-virus type-I by a trans-acting factor. *Proc* 

Ohashi, K., Mizuno, K., Sakamoto, M., Hamanoue, M. & Tsuji, T. 2005. Alteration of phosphatidylinositol 3-kinase cascade in the multilobulated nuclear formation of adult T cell leukemia/lymphoma (ATLL). *Proc Natl Acad Sci USA,* 102, 15213-8. Fukuda, R. I., Tsuchiya, K., Suzuki, K., Itoh, K., Fujita, J., Utsunomiya, A. & Tsuji, T. 2009.

Human T-cell leukemia virus type I tax down-regulates the expression of phosphatidylinositol 3,4,5-trisphosphate inositol phosphatases via the NF-B

Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic

I tax involves induction of NF-AT complexes bound to the CD28-responsive

M. C., Sodroski, J. G. & Haseltine, W. A. 1989. Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I Xregion genes transduced by a Herpesvirus saimiri vector. *Proc Natl Acad Sci USA,*

leukemia virus type I Tax in expression of the human telomerase reverse

Mahieux, R., Matsuoka, M. & Fujii, M. 2007. Cooperation of NF-B2/p100 activation and the PDZ domain binding motif signal in human T-cell leukemia virus type 1 (HTLV-1) Tax1 but not HTLV-2 Tax2 is crucial for interleukin-2-

K. I., SHIRAKAWA, S. & MIYOSHI, I. 1981. Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. *Proc Natl* 

H. 1987. Establishment of an interleukin 2-dependent human T cell line from a patient with T cell chronic lymphocytic leukemia who is not infected with human T

binding proteins: role in the regulation of human interleukin-2 gene expression.

1 (HTLV-1) and HTLV-2. *Oncogene,* 24, 5996-6004.

*Natl Acad Sci USA,* 82, 2277-2281.

pathway. *J Biol Chem,* 284, 2680-9.

paraparesis. *Lancet,* 2, 407-10.

element. *EMBO J,* 15, 3744-50.

86, 3351-5.

*Oncogene,* 24, 5965-75.

*Acad Sci USA,* 78, 6476–80.

*Science,* 244, 457-60.


Roles of HTLV-1 Tax in Leukemogenesis of Human T-Cells 63

Parrish-Novak, J., Dillon, S. R., Nelson, A., Hammond, A., Sprecher, C., Gross, J. A.,

Peloponese, J. M., Jr. & Jeang, K. T. 2006. Role for Akt/protein kinase B and activator

Pise-Masison, C. A., Mahieux, R., Jiang, H., Ashcroft, M., Radonovich, M., Duvall, J.,

Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D. & Gallo, R. C. 1980.

Proietti, F. A., Carneiro-Proietti, A. B., Catalan-Soares, B. C. & Murphy, E. L. 2005. Global epidemiology of HTLV-I infection and associated diseases. *Oncogene,* 24, 6058-68. Robek, M. D. & Ratner, L. 1999. Immortalization of CD4+ and CD8+ T lymphocytes by

Rochman, Y., Spolski, R. & Leonard, W. J. 2009. New insights into the regulation of T cells

Rohrschneider, L. R., Fuller, J. F., Wolf, I., Liu, Y. & Lucas, D. M. 2000. Structure, function,

Ross, T. M., Narayan, M., Fang, Z. Y., Minella, A. C. & Green, P. L. 2000. Human T-cell

Santiago, F., Clark, E., Chong, S., Molina, C., Mozafari, F., Mahieux, R., Fujii, M., Azimi, N.

Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. 2005. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. *Science,* 307, 1098-101. Schmitt, I., Rosin, O., Rohwer, P., Gossen, M. & Grassmann, R. 1998. Stimulation of cyclin-

Semmes, O. J. & Jeang, K. T. 1996. Localization of human T-cell leukemia virus type 1 tax to

Shoji, T., Higuchi, M., Kondo, R., Takahashi, M., Oie, M., Tanaka, Y., Aoyagi, Y. & Fujii, M.

activation fail to transform primary human T cells. *J Virol,* 74, 2655-62. Ruben, S., Poteat, H., Tan, T. H., Kawakami, K., Roeder, R., Haseltine, W. & Rosen, C. A.

*Nature,* 408, 57-63.

77, 7415-9.

6347-57.

1 tax oncoprotein. *J Biol Chem,* 281, 8927-38.

phosphorylation. *Mol Cell Biol,* 20, 3377-86.

molecular clone. *J Virol,* 73, 4856-65.

by c family cytokines. *Nat Rev Immunol,* 9, 480-90.

and biology of SHIP proteins. *Genes Dev,* 14, 505-20.

by HTLV-I tax gene product. *Science,* 241, 89-92.

virus type 1-infected cells. *J Virol,* 73, 9917-27.

Johnston, J., Madden, K., Xu, W., West, J., Schrader, S., Burkhead, S., Heipel, M., Brandt, C., Kuijper, J. L., Kramer, J., Conklin, D., Presnell, S. R., Berry, J., Shiota, F., Bort, S., Hambly, K., Mudri, S., Clegg, C., Moore, M., Grant, F. J., Lofton-Day, C., Gilbert, T., Rayond, F., Ching, A., Yao, L., Smith, D., Webster, P., Whitmore, T., Maurer, M., Kaushansky, K., Holly, R. D. & Foster, D. 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

protein-1 in cellular proliferation induced by the human T-cell leukemia virus type

Guillerm, C. & Brady, J. N. 2000. Inactivation of p53 by human T-cell lymphotropic virus type 1 Tax requires activation of the NF-B pathway and is dependent on p53

Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. *Proc Natl Acad Sci USA,*

human T-cell leukemia virus type 1 Tax mutants expressed in a functional

leukemia virus type 2 tax mutants that selectively abrogate NFB or CREB/ATF

1988. Cellular transcription factors and regulation of IL-2 receptor gene expression

& Kashanchi, F. 1999. Transcriptional up-regulation of the cyclin D2 gene and acquisition of new cyclin-dependent kinase partners in human T-cell leukemia

dependent kinase activity and G1- to S-phase transition in human lymphocytes by the human T-cell leukemia/lymphotropic virus type 1 Tax protein. *J Virol,* 72, 633-40.

subnuclear compartments that overlap with interchromatin speckles. *J Virol,* 70,

2009. Identification of a novel motif responsible for the distinctive transforming


Meyerson, M., Counter, C. M., Eaton, E. N., Ellisen, L. W., Steiner, P., Caddle, S. D., Ziaugra,

Mizuguchi, M., Asao, H., Hara, T., Higuchi, M., Fujii, M. & Nakamura, M. 2009.

Mori, N., Fujii, M., Ikeda, S., Yamada, Y., Tomonaga, M., Ballard, D. W. & Yamamoto, N.

Nakamura, T. M., Morin, G. B., Chapman, K. B., Weinrich, S. L., Andrews, W. H., Lingner, J.,

Nerenberg, M., Hinrichs, S. H., Reynolds, R. K., Khoury, G. & Jay, G. 1987. The tat gene of

Neuveut, C., Low, K. G., Maldarelli, F., Schmitt, I., Majone, F., Grassmann, R. & Jeang, K. T.

Nevins, J. R. 1998. Toward an understanding of the functional complexity of the E2F and

Nevins, J. R., Leone, G., Degregori, J. & Jakoi, L. 1997. Role of the Rb/E2F pathway in cell

Nicot, C., Harrod, R. L., Ciminale, V. & Franchini, G. 2005. Human T-cell leukemia/lymphoma virus type 1 nonstructural genes and their functions. *Oncogene,* 24, 6026-6034. Niinuma, A., Higuchi, M., Takahashi, M., Oie, M., Tanaka, Y., Gejyo, F., Tanaka, N.,

Ohbo, K., Takasawa, N., Ishii, N., Tanaka, N., Nakamura, M. & Sugamura, K. 1995.

Ohtani, K., Iwanaga, R., Arai, M., Huang, Y., Matsumura, Y. & Nakamura, M. 2000. Cell

Onoda, T., Rahman, M., Nara, H., Araki, A., Makabe, K., Tsumoto, K., Kumagai, I., Kudo, T.,

Osame, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M. & Tara, M. 1986. HTLV-I associated myelopathy, a new clinical entity. *Lancet,* 1, 1031-2.

proliferation of CD4+ T cell subsets. *Int Immunol,* 19, 1191-9.

HTLV-I. *Science,* 269, 79-81.

mice. *Science,* 237, 1324-9.

fission yeast and human. *Science,* 277, 955-9.

cyclin D-cdk and p110Rb. *Mol Cell Biol,* 18, 3620-32.

retinoblastoma families. *Cell Growth Differ,* 9, 585-93.

growth control. *J Cell Physiol,* 173, 233-6.

type 1 virus. *J Virol,* 79, 11925-34.

phenomenon. *J Theor Biol,* 41, 181-90.

*Biol Chem,* 270, 7479-86.

11.

93, 2360-8.

L., Beijersbergen, R. L., Davidoff, M. J., Liu, Q., Bacchetti, S., Haber, D. A. & Weinberg, R. A. 1997. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. *Cell,* 90, 785-95. Migone, T. S., Lin, J. X., Cereseto, A., Mulloy, J. C., O'shea, J. J., Franchini, G. & Leonard, W.

J. 1995. Constitutively activated Jak-STAT pathway in T cells transformed with

Transcriptional activation of the interleukin-21 gene and its receptor gene by human T-cell leukemia virus type 1 Tax in human T-cells. *J Biol Chem,* 284, 25501-

1999. Constitutive activation of NF-B in primary adult T-cell leukemia cells. *Blood,*

Harley, C. B. & Cech, T. R. 1997. Telomerase catalytic subunit homologs from

human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic

1998. Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of

Sugamura, K., Xie, L., Green, P. L. & Fujii, M. 2005. Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic

Functional analysis of the human interleukin 2 receptor chain gene promoter. *J* 

type-specific E2F activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I. *J Biol Chem,* 275, 11154-63. Olovnikov, A. M. 1973. A theory of marginotomy. The incomplete copying of template

margin in enzymic synthesis of polynucleotides and biological significance of the

Ishii, N., Tanaka, N., Sugamura, K., Hayasaka, K. & Asao, H. 2007. Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven


**4** 

*Japan* 

**Host Immune System Abnormalities Among** 

Human T-cell lymphotropic virus type 1 (HTLV-1) is a human retrovirus that causes persistent infection in the host. While most infected persons remain asymptomatic carriers (ACs), 3–5% develop a T-cell malignancy termed adult T-cell leukemia (ATL) (Uchiyama et al., 1977), and another 0.25–3% develop a chronic progressive inflammatory neurologic disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al. 1986). Although HTLV-1-associated disorders have been extensively studied, the exact mechanism by which they are induced by HTLV-1 is not completely understood. The proviral load of HTLV-1 could contribute to the development of these disorders, since the circulating number of HTLV-1-infected T cells in the peripheral blood is associated with the risk of developing HAM/TSP and ATL (Iwanaga et al., 2010; Nagai et al. 1998). However, more detail on the precise immune mechanisms controlling

HTLV-1 preferentially infects CD4+ T cells, the central regulators of the acquired immune system (Richardson et al., 1990). This is known to induce a variety of abnormalities, such as proliferation, cellular activation, and proinflammatory changes (Boxus et al., 2009; Satou et al., 2010; Yamano et al. 2009). These abnormalities, in turn, may deregulate the balance of

HTLV-1 also causes abnormalities among uninfected immune cells. Patients with HTLV-1 associated disorders demonstrate abnormalities in both the amount and function of CD8+ cytotoxic T lymphocytes (CTL), an important component of host immune response against HTLV-1 (Bangham 2009; Kannagi et al., 2011; Matsuura et al., 2010). Patients with ATL and HAM/TSP may also experience reductions in the amount and efficacy of cellular components of innate immunity, which is vital in regulating the immune response against general viral infections and cancers (Azakami et al., 2009; Matsuura et al., 2010). In this chapter, we have summarized the host immune system abnormalities that are associated

**1. Introduction** 

HTLV-1-infected cells is still needed.

the host immune system.

with HTLV-1 infection.

**Patients with Human T-Lymphotropic** 

*Department of Rare Diseases Research, Institute of Medical Science,* 

**Virus Type 1 (HTLV-1)-** 

**Associated Disorders** 

Hitoshi Ando and Yoshihisa Yamano

*St. Marianna University School of Medicine,* 

Tomoo Sato, Natsumi Araya, Naoko Yagishita,

activity of human T-cell leukemia virus (HTLV) type 1 Tax1 protein from HTLV-2 Tax2. *Retrovirology,* 6, 83.


### **Host Immune System Abnormalities Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders**

Tomoo Sato, Natsumi Araya, Naoko Yagishita, Hitoshi Ando and Yoshihisa Yamano *Department of Rare Diseases Research, Institute of Medical Science, St. Marianna University School of Medicine, Japan* 

#### **1. Introduction**

64 T-Cell Leukemia

Silbermann, K., Schneider, G. & Grassmann, R. 2008. Stimulation of interleukin-13

Tabakin-Fix, Y., Azran, I., Schavinky-Khrapunsky, Y., Levy, O. & Aboud, M. 2006.

Takeshita, T., Asao, H., Ohtani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H.,

Tanaka, A., Takahashi, C., Yamaoka, S., Nosaka, T., Maki, M. & Hatanaka, M. 1990.

Tanaka, N., Asao, H., Ohbo, K., Ishii, N., Takeshita, T., Nakamura, M., Sasaki, H. &

the interleukin 2 receptor β and γ chains. *Proc Natl Acad Sci USA,* 91, 7271-5. Trimarchi, J. M. & Lees, J. A. 2002. Sibling rivalry in the E2F family. *Nat Rev Mol Cell Biol,* 3,

Tsukahara, T., Kannagi, M., Ohashi, T., Kato, H., Arai, M., Nunez, G., Iwanaga, Y.,

Uchida, N., Otsuka, T., Arima, F., Shigematsu, H., Fukuyama, T., Maeda, M., Sugio, Y., Itoh,

Van Orden, K., Yan, J. P., Ulloa, A. & Nyborg, J. K. 1999. Binding of the human T-cell

Waldele, K., Schneider, G., Ruckes, T. & Grassmann, R. 2004. Interleukin-13 overexpression

Yoshida, M. 2001. Multiple viral strategies of HTLV-1 for dysregulation of cell growth

Yoshida, M., Miyoshi, I. & Hinuma, Y. 1982. Isolation and characterization of retrovirus

Yu, H., Pardoll, D. & Jove, R. 2009. STATs in cancer inflammation and immunity: a leading

Zeng, R., Spolski, R., Casas, E., Zhu, W., Levy, D. E. & Leonard, W. J. 2007. The molecular

resistant T-cell transfectants with Tax. *J Virol,* 73,7981-7.

progression of adult T-cell leukemia. *Leuk Res,* 23, 311-6.

Watson, J. D. 1972. Origin of concatemeric T7 DNA. *Nat New Biol,* 239, 197-201.

basis of IL-21-mediated proliferation. *Blood,* 109, 4135-42.

transcriptional control. *Oncogene,* 18, 3766-72.

virus-infected lymphocytes. *J Virol,* 78, 6081-90.

control. *Annu Rev Immunol,* 19, 475-496.

role for STAT3. *Nat Rev Cancer,* 9, 798-809.

*Proc Natl Acad Sci USA,* 79, 2031-5.

mechanisms and clinical implications. *Carcinogenesis,* 27, 673-81.

Tax2. *Retrovirology,* 6, 83.

*Science,* 225, 381-385.

11-20.

receptor. *Science,* 257, 379-82.

vitro. *Proc Natl Acad Sci USA,* 87, 1071-5.

activity of human T-cell leukemia virus (HTLV) type 1 Tax1 protein from HTLV-2

expression by human T-cell leukemia virus type 1 oncoprotein Tax via a dually active promoter element responsive to NF-B and NFAT. *J Gen Virol,* 89, 2788-98. Sinha-Datta, U., Horikawa, I., Michishita, E., Datta, A., Sigler-Nicot, J. C., Brown, M.,

Kazanji, M., Barrett, J. C. & Nicot, C. 2004. Transcriptional activation of hTERT through the NF-B pathway in HTLV-I-transformed cells. *Blood,* 104, 2523-31. Sodroski, J. G., Rosen, C. A. & Haseltine, W. A. 1984. Trans-acting transcriptional activation

of the long terminal repeat of human T-lymphotropic viruses in infected-cells.

Functional inactivation of p53 by human T-cell leukemia virus type 1 Tax protein:

Nakamura, M. & Sugamura, K. 1992. Cloning of the chain of the human IL-2

Oncogenic transformation by the tax gene of human T-cell leukemia virus type I in

Sugamura, K. 1994. Physical association of JAK1 and JAK2 tyrosine kinases with

Yamamoto, N., Ohtani, K., Nakamura, M. & Fujii, M. 1999. Induction of Bcl-xL expression by human T-cell leukemia virus type 1 Tax through NF-B in apoptosis-

Y. & Niho, Y. 1999. Correlation of telomerase activity with development and

leukemia virus Tax protein to the coactivator CBP interferes with CBP-mediated

by tax transactivation: a potential autocrine stimulus in human T-cell leukemia

from cell lines of human adult T-cell leukemia and its implication in the disease.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a human retrovirus that causes persistent infection in the host. While most infected persons remain asymptomatic carriers (ACs), 3–5% develop a T-cell malignancy termed adult T-cell leukemia (ATL) (Uchiyama et al., 1977), and another 0.25–3% develop a chronic progressive inflammatory neurologic disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al. 1986). Although HTLV-1-associated disorders have been extensively studied, the exact mechanism by which they are induced by HTLV-1 is not completely understood. The proviral load of HTLV-1 could contribute to the development of these disorders, since the circulating number of HTLV-1-infected T cells in the peripheral blood is associated with the risk of developing HAM/TSP and ATL (Iwanaga et al., 2010; Nagai et al. 1998). However, more detail on the precise immune mechanisms controlling HTLV-1-infected cells is still needed.

HTLV-1 preferentially infects CD4+ T cells, the central regulators of the acquired immune system (Richardson et al., 1990). This is known to induce a variety of abnormalities, such as proliferation, cellular activation, and proinflammatory changes (Boxus et al., 2009; Satou et al., 2010; Yamano et al. 2009). These abnormalities, in turn, may deregulate the balance of the host immune system.

HTLV-1 also causes abnormalities among uninfected immune cells. Patients with HTLV-1 associated disorders demonstrate abnormalities in both the amount and function of CD8+ cytotoxic T lymphocytes (CTL), an important component of host immune response against HTLV-1 (Bangham 2009; Kannagi et al., 2011; Matsuura et al., 2010). Patients with ATL and HAM/TSP may also experience reductions in the amount and efficacy of cellular components of innate immunity, which is vital in regulating the immune response against general viral infections and cancers (Azakami et al., 2009; Matsuura et al., 2010). In this chapter, we have summarized the host immune system abnormalities that are associated with HTLV-1 infection.

Host Immune System Abnormalities

HTLV-1-infected CD4+CD25+CCR4+ T cells.

an overproduction of IFN-γ (Figure 1).

cells lose this regulatory function (Shimauchi et al., 2008).

**2.3 HTLV-1 may induce plasticity of Foxp3+ cells into exFoxp3+ cell** 

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 67

Foxp3 expression and inhibit the suppressive function of Treg cells (Yamano et al., 2005). Furthermore, because of a Tax-induced defect in TGF-β signaling, HAM/TSP patients experience reductions in Foxp3 expression and impairment of Treg function (Grant et al., 2008). Moreover, a significant reduction in CD4+CD25+Foxp3+ Treg cells was demonstrated in HTLV-1-*tax*-expressing transgenic mice, which develop an inflammatory arthropathy (Ohsugi et al., 2011). Thus, HAM/TSP patients display a decreased ratio of Foxp3+ Treg cells within

Importantly, a more detailed flow cytometric analysis of Foxp3 expression in CD4+CD25+CCR4+ T cells demonstrated that the frequency of "Foxp3– population" was extraordinary high in HAM/TSP patients (Yamano et al., 2009). Moreover, an analysis of proinflammatory cytokine expression in this Foxp3–CD4+CD25+CCR4+ T cell subset demonstrated that these cells were unique because, in healthy individuals, they produced multiple proinflammatory cytokines such as IL-2, IL-17, and few interferon (IFN)-γ, while Foxp3+CD4+CD25+CCR4+ T cells (Treg cells) did not. Furthermore, HAM/TSP patients were found to exhibit only a few Foxp3+CD4+CD25+CCR4+ T cells that did not produce such cytokines. Rather, these patients had an increased number of Foxp3–CD4+CD25+CCR4+ T cells, which were found to overproduce IFN-γ. Further, given the increase of clinical diseases and severity of HAM/TSP observed in these patients, it appears likely that the frequency of these IFN-γ-producing Foxp3–CD4+CD25+CCR4+ T cells may have a functional consequence (Yamano et al., 2009). Thus, while the CD4+CD25+CCR4+ T cell population in healthy patients mainly comprises suppressive T cell subsets such as Treg and Th2, HAM/TSP patients possess an increased proportion of IFN-γ-producing Foxp3– CD4+CD25+CCR4+ T cells, which are rarely encountered in healthy individuals and lead to

Although Foxp3 expression is decreased by CD4+CD25+ (CCR4+) T cells in HAM/TSP patients (Hayashi et al., 2008; Michaelsson et al., 2008; Oh et al., 2006; Ramirez et al., 2010; Yamano et al., 2005), it is increased by CD4+CD25+(CCR4+) ATL cells in most ATL patients (Karube et al., 2004; Roncador et al., 2005) (Figure 1). Therefore, it has been hypothesized that ATL cells may be derived from Treg cells (Kohno et al., 2005). Interestingly, some ATL cells exhibit immunosuppressive functions similar to those of Treg cells, which may contribute to the cellular immunodeficiency that has been clinically observed in ATL patients (Chen et al., 2006; Kohno et al., 2005; Matsubar et al., 2006); however, some ATL

In HTLV-1-seronegative healthy individuals, CD4+CD25+CCR4+ T cells mainly include suppressive T cell subsets such as Treg and Th2 (Yoshie et al., 2001). In ATL patients, most of this subset develops leukemogenesis by maintaining the Foxp3+ Treg phenotype (Figure 1). However, as mentioned above, T cells of this subset become Th1-like cells that overproduce IFN-γ in HAM/TSP patients (Figure 1). Since HTLV-1 may preferentially transmit to CCR4+CD4+ T cells, these findings suggest that HTLV-1 may intracellularly induce T-cell plasticity of Treg cells into IFN-γ+ T cells. Indeed, one recent report indicated that loss of Foxp3 in Treg cells and acquisition of IFN-γ may result in the conversion of suppressor T cells into highly autoaggressive lymphocytes (exFoxp3+ cells), which can favor the development of autoimmune conditions (Tsuji et al., 2009; Zhou et al., 2009). Importantly, Toulza et al. (2008) demonstrated that the rate of CTL-mediated lysis was

#### **2. Abnormality of HTLV-1-infected CD4+ T cells**

#### **2.1 CD4<sup>+</sup> CD25<sup>+</sup> CCR4+ T Cells are a major reservoir of HTLV-1-infected T cells, which increase in HAM/TSP and ATL patients**

HTLV-1 mainly infects CD4+ T helper (Th) cells, which play a central role in adaptive immune responses (Richardson et al., 1990). CD4+ Th cells recruit and activate other immune cells, including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils, and basophils (Zhu et al., 2010). Based on their function, their pattern of cytokine secretion, and their expression of specific transcription factors and chemokine receptors, CD4+ Th cells, differentiated from naïve CD4+ T cells, are classified into 4 major lineages: Th1, Th2, Th17, and T regulatory (Treg) cells. To understand the effects of HTLV-1 infection on the function of CD4 Th cells, it is necessary to know which Th population HTLV-1 infects.

It was recently shown that the chemokine receptor CCR4 is expressed on HTLV-1-infected leukemia cells in ATL patients (Yoshie et al., 2002). CCR4 is selectively expressed on suppressive T cell subsets, such as Treg and Th2 cells, in HTLV-1-seronegative healthy individuals (Yoshie et al., 2001). Using molecular and immunological techniques, we also demonstrated that CD4+CD25+CCR4+ T cells were the predominant viral reservoir in both ACs and HAM/TSP patients, and that this T cell subset was increased in HAM/TSP patients (Yamano et al., 2009). Thus, CD4+CD25+CCR4+ T cells are a major population of HTLV-1-infected T cells, which increase in number in both HAM/TSP and ATL patients.

The molecular mechanism of HTLV-1 tropism to CCR4 expressing CD4+ T cells was recently uncovered (Hieshima et al., 2008). HTLV-1 Tax, a transcriptional regulator encoded by the HTLV-1 genome, does not induce expression of CCR4, but it does induce expression of CCL22, the ligand for CCR4. Because HTLV-1-infected T cells selectively interact with CCR4+CD4+ T cells, this results in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.

#### **2.2 Differences in the fates of CD4+ CD25<sup>+</sup> CCR4<sup>+</sup> T cells in HAM/TSP and ATL patients**

Among CD4+ Th cells, the major reservoir of HTLV-1 is CD4+CD25+CCR4+ T cells, including suppressive T cell subsets such as Treg and Th2 under healthy conditions. The exact mechanism by which HTLV-1 induces the deregulation of the host immune system is not completely understood. However, the recent discovery of Treg cells has provided new opportunities and generated increased interest in this issue. In healthy individuals, Treg cells suppress the proliferation of, and cytokine production by, pathogenic T cells, and thereby plays a key role in the maintenance of immune system homeostasis (Sakaguchi et al., 1995). Treg cells can be identified *ex vivo* by the intracellular expression of the transcriptional regulator Foxp3 (Hori et al., 2003), which is critical for the development and function of Treg cells in both mice and humans.

Significant reductions in Foxp3 expression and/or Treg cell function have been observed in several human autoimmune diseases (Sakaguchi et al., 2008), suggesting that defects in Foxp3 expression and/or Treg function may precipitate the loss of immunologic tolerance. Recently, significant reductions in Foxp3 expression and Treg cell function have also been observed in CD4+CD25+ T cells and/or CD4+CD25+CCR4+ T cells from patients with HAM/TSP (Hayashi et al., 2008; Michaelsson et al., 2008; Oh et al., 2006; Ramirez et al., 2010; Yamano et al., 2005). Furthermore, decreased expression levels of the Treg-associated immune suppressive molecules CTLA-4 and GITR were also observed on CD4+CD25+ T cells in HAM/TSP patients (Ramirez et al., 2010; Yamano et al., 2005). Notably, overexpression of HTLV-1 *tax* can reduce

HTLV-1 mainly infects CD4+ T helper (Th) cells, which play a central role in adaptive immune responses (Richardson et al., 1990). CD4+ Th cells recruit and activate other immune cells, including B cells, CD8 T cells, macrophages, mast cells, neutrophils, eosinophils, and basophils (Zhu et al., 2010). Based on their function, their pattern of cytokine secretion, and their expression of specific transcription factors and chemokine receptors, CD4+ Th cells, differentiated from naïve CD4+ T cells, are classified into 4 major lineages: Th1, Th2, Th17, and T regulatory (Treg) cells. To understand the effects of HTLV-1 infection on the function of CD4 Th cells, it is necessary to know which Th population

It was recently shown that the chemokine receptor CCR4 is expressed on HTLV-1-infected leukemia cells in ATL patients (Yoshie et al., 2002). CCR4 is selectively expressed on suppressive T cell subsets, such as Treg and Th2 cells, in HTLV-1-seronegative healthy individuals (Yoshie et al., 2001). Using molecular and immunological techniques, we also demonstrated that CD4+CD25+CCR4+ T cells were the predominant viral reservoir in both ACs and HAM/TSP patients, and that this T cell subset was increased in HAM/TSP patients (Yamano et al., 2009). Thus, CD4+CD25+CCR4+ T cells are a major population of HTLV-1-infected T cells, which increase in number in both HAM/TSP and ATL patients. The molecular mechanism of HTLV-1 tropism to CCR4 expressing CD4+ T cells was recently uncovered (Hieshima et al., 2008). HTLV-1 Tax, a transcriptional regulator encoded by the HTLV-1 genome, does not induce expression of CCR4, but it does induce expression of CCL22, the ligand for CCR4. Because HTLV-1-infected T cells selectively interact with CCR4+CD4+ T

cells, this results in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.

**CD25<sup>+</sup>**

**CCR4<sup>+</sup>**

Among CD4+ Th cells, the major reservoir of HTLV-1 is CD4+CD25+CCR4+ T cells, including suppressive T cell subsets such as Treg and Th2 under healthy conditions. The exact mechanism by which HTLV-1 induces the deregulation of the host immune system is not completely understood. However, the recent discovery of Treg cells has provided new opportunities and generated increased interest in this issue. In healthy individuals, Treg cells suppress the proliferation of, and cytokine production by, pathogenic T cells, and thereby plays a key role in the maintenance of immune system homeostasis (Sakaguchi et al., 1995). Treg cells can be identified *ex vivo* by the intracellular expression of the transcriptional regulator Foxp3 (Hori et al., 2003), which is critical for the development and

Significant reductions in Foxp3 expression and/or Treg cell function have been observed in several human autoimmune diseases (Sakaguchi et al., 2008), suggesting that defects in Foxp3 expression and/or Treg function may precipitate the loss of immunologic tolerance. Recently, significant reductions in Foxp3 expression and Treg cell function have also been observed in CD4+CD25+ T cells and/or CD4+CD25+CCR4+ T cells from patients with HAM/TSP (Hayashi et al., 2008; Michaelsson et al., 2008; Oh et al., 2006; Ramirez et al., 2010; Yamano et al., 2005). Furthermore, decreased expression levels of the Treg-associated immune suppressive molecules CTLA-4 and GITR were also observed on CD4+CD25+ T cells in HAM/TSP patients (Ramirez et al., 2010; Yamano et al., 2005). Notably, overexpression of HTLV-1 *tax* can reduce

 **T cells in HAM/TSP and ATL patients** 

 **T cells**

 **T Cells are a major reservoir of HTLV-1-infected T cells, which** 

**2. Abnormality of HTLV-1-infected CD4+**

**CCR4+**

**2.2 Differences in the fates of CD4+**

function of Treg cells in both mice and humans.

**increase in HAM/TSP and ATL patients** 

**2.1 CD4<sup>+</sup>**

HTLV-1 infects.

**CD25<sup>+</sup>**

Foxp3 expression and inhibit the suppressive function of Treg cells (Yamano et al., 2005). Furthermore, because of a Tax-induced defect in TGF-β signaling, HAM/TSP patients experience reductions in Foxp3 expression and impairment of Treg function (Grant et al., 2008). Moreover, a significant reduction in CD4+CD25+Foxp3+ Treg cells was demonstrated in HTLV-1-*tax*-expressing transgenic mice, which develop an inflammatory arthropathy (Ohsugi et al., 2011). Thus, HAM/TSP patients display a decreased ratio of Foxp3+ Treg cells within HTLV-1-infected CD4+CD25+CCR4+ T cells.

Importantly, a more detailed flow cytometric analysis of Foxp3 expression in CD4+CD25+CCR4+ T cells demonstrated that the frequency of "Foxp3– population" was extraordinary high in HAM/TSP patients (Yamano et al., 2009). Moreover, an analysis of proinflammatory cytokine expression in this Foxp3–CD4+CD25+CCR4+ T cell subset demonstrated that these cells were unique because, in healthy individuals, they produced multiple proinflammatory cytokines such as IL-2, IL-17, and few interferon (IFN)-γ, while Foxp3+CD4+CD25+CCR4+ T cells (Treg cells) did not. Furthermore, HAM/TSP patients were found to exhibit only a few Foxp3+CD4+CD25+CCR4+ T cells that did not produce such cytokines. Rather, these patients had an increased number of Foxp3–CD4+CD25+CCR4+ T cells, which were found to overproduce IFN-γ. Further, given the increase of clinical diseases and severity of HAM/TSP observed in these patients, it appears likely that the frequency of these IFN-γ-producing Foxp3–CD4+CD25+CCR4+ T cells may have a functional consequence (Yamano et al., 2009). Thus, while the CD4+CD25+CCR4+ T cell population in healthy patients mainly comprises suppressive T cell subsets such as Treg and Th2, HAM/TSP patients possess an increased proportion of IFN-γ-producing Foxp3– CD4+CD25+CCR4+ T cells, which are rarely encountered in healthy individuals and lead to an overproduction of IFN-γ (Figure 1).

Although Foxp3 expression is decreased by CD4+CD25+ (CCR4+) T cells in HAM/TSP patients (Hayashi et al., 2008; Michaelsson et al., 2008; Oh et al., 2006; Ramirez et al., 2010; Yamano et al., 2005), it is increased by CD4+CD25+(CCR4+) ATL cells in most ATL patients (Karube et al., 2004; Roncador et al., 2005) (Figure 1). Therefore, it has been hypothesized that ATL cells may be derived from Treg cells (Kohno et al., 2005). Interestingly, some ATL cells exhibit immunosuppressive functions similar to those of Treg cells, which may contribute to the cellular immunodeficiency that has been clinically observed in ATL patients (Chen et al., 2006; Kohno et al., 2005; Matsubar et al., 2006); however, some ATL cells lose this regulatory function (Shimauchi et al., 2008).

#### **2.3 HTLV-1 may induce plasticity of Foxp3+ cells into exFoxp3+ cell**

In HTLV-1-seronegative healthy individuals, CD4+CD25+CCR4+ T cells mainly include suppressive T cell subsets such as Treg and Th2 (Yoshie et al., 2001). In ATL patients, most of this subset develops leukemogenesis by maintaining the Foxp3+ Treg phenotype (Figure 1). However, as mentioned above, T cells of this subset become Th1-like cells that overproduce IFN-γ in HAM/TSP patients (Figure 1). Since HTLV-1 may preferentially transmit to CCR4+CD4+ T cells, these findings suggest that HTLV-1 may intracellularly induce T-cell plasticity of Treg cells into IFN-γ+ T cells. Indeed, one recent report indicated that loss of Foxp3 in Treg cells and acquisition of IFN-γ may result in the conversion of suppressor T cells into highly autoaggressive lymphocytes (exFoxp3+ cells), which can favor the development of autoimmune conditions (Tsuji et al., 2009; Zhou et al., 2009). Importantly, Toulza et al. (2008) demonstrated that the rate of CTL-mediated lysis was

Host Immune System Abnormalities

Jacobson, 2002; Kannagi, 2007).

**3.1 HTLV-1-specific cytotoxic T lymphocytes** 

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 69

or cancer antigen in association with major histocompatibility complex (MHC) class I molecules and by suppressing viral replication and tumor development via IFN-γ secretion. Elucidating the role of HTLV-1–specific CD8+ CTLs has been considered a priority issue in studies of host defense mechanisms involved in HTLV-1 infection (Bangham, 2008;

T-cell receptors (TCR) on CTLs recognize peptide fragments derived from viral and tumor antigens that are presented on MHC class I molecules by antigen-presenting cells or virusinfected cells. After TCR binds to the peptide-MHC complex, CTLs are activated and fulfill an effector function. There are 3 main effector mechanisms by which the CD8+ CTL kills virus-infected or tumor cells. One is to release perforin and granzymes. Perforin forms pores in the plasma membrane of the target cells, allowing entry of granzymes; caspases are then activated, leading to apoptosis. Apoptosis may also be induced via a Fas-FasL interaction between CTLs and target cells. Finally, CD8+ cells can produce IFN-, which has indirect

The Tax protein is an immunodominant antigen in HTLV-1 infections. Therefore, CTL activity is predominantly restricted to products of the HTLV-1 Tax gene, although HTLV-1 Env, Pol, Rof, Tof, and HBZ (Elovaara et al., 1993; Hilburn et al., 2011; Macnamara et al., 2010; Pique et al., 2000) could also be target proteins of HTLV-1-specific CTL. In a study that utilized properties of the CTL antigen recognition system, human MHC class I HLA-A2(\*0201) tetramers loaded with HTLV-1 Tax peptide were used to detect HTLV-1 Tax specific HLA-A2-restricted CD8+ cells (Bieganowska et al., 1999, Greten et al., 1998). This technique facilitates quantification of the frequency of antigen-specific T cells, as well as direct characterization of these cells. HLA genotype determines which part of the viral protein is presented as an antigen peptide. For HLA-A\*0201 and HLA-A\*2402, for example,

An increasing number of studies in patients with HTLV-1-associated disorders have documented an association between the disorders and abnormalities in both the frequency of CTLs and their response to HTLV-1. When peripheral blood mononuclear cells (PBMCs) from HTLV-1 carriers are stimulated with autologous HTLV-1–infected cells *in vitro,* proliferation of HTLV-1-specific CD8+ CTLs is often observed in the presence of IL-2. An increased level of HTLV-1–specific CTL responses occurs in all HAM/TSP patients and in some asymptomatic HTLV-1 carriers; however, HTLV-1–specific CTL responses are rarely induced in PBMC cultures from ATL patients (Jacobson et al., 1990; Kannagi et al., 1984, Parker et al., 1992). HTLV-1–specific CTLs are also present in ATL patients but do not expand sufficiently (Arnulf et al., 2004). Impairment of the HTLV-1 specific CTL response was observed in some individuals during the earlier stages of HTLV-1 infection (AC and smoldering ATL), as well as in advanced ATL patients (Shimizu et al., 2009). This observation suggests that the T-cell insufficiency in ATL patients is present prior to disease onset. In addition, a recent report indicated that, in comparison to ACs, ATL patients have a smaller and less diverse population of HTLV-1 specific CD8+ T cells, as well as lower anti-HTLV-1 CD8+ T cell expression of perforin and granzyme B (Kozako et al., 2006). Thus, the decreased number and functional impairment of CTLs might contribute to the onset and

cytolytic effects by promoting NK cell activity and macrophage activation.

the major epitopes are the Tax 11-19 and Tax 301-309 amino acids, respectively.

**3.2 Abnormal CTL response in patients with ATL** 

progression of ATL.

negatively correlated with the number of HTLV-1-Tax- CD4+Foxp3+ cells, but not with the number of Tax+ CD4+Foxp3+ cells, suggesting that HTLV-1-infected Treg cells lose their regulatory function, while HTLV-1-uninfected Treg cells contribute substantially to immune control of HTLV-1 infection. Additionally, functional impairment of CD4+Foxp3+ Treg cells was observed in mice that were transgenic mice for the *HTLV-1 bZIP factor* (*HBZ*) gene, which encodes the minus strand of HTLV-1 (Satou et al., 2011). These findings support the hypothesis that HTLV-1 may be one of the exogenous retrovirus genes responsible for immune dysregulation through interference of CD4+CD25+ Treg cell function. This hypothesis is currently under investigation to elucidate the precise molecular mechanisms by which HTLV-1 influences the fate and function of CD4+CD25+CCR4+ T cells, especially Foxp3+ Treg cells.

Fig. 1. Cellular components of CD4+CD25+CCR4+ T cells in healthy individuals, asymptomatic carriers, ATL, and HAM/TSP patients.

#### **3. Abnormality of cytotoxic T lymphocyte (CTL) response**

CD8+ Cytotoxic T lymphocyte (CTL) responses are an effective host defense system against all virus infections and malignancies. CTLs act by killing autologous cells that express viral or cancer antigen in association with major histocompatibility complex (MHC) class I molecules and by suppressing viral replication and tumor development via IFN-γ secretion. Elucidating the role of HTLV-1–specific CD8+ CTLs has been considered a priority issue in studies of host defense mechanisms involved in HTLV-1 infection (Bangham, 2008; Jacobson, 2002; Kannagi, 2007).

#### **3.1 HTLV-1-specific cytotoxic T lymphocytes**

68 T-Cell Leukemia

negatively correlated with the number of HTLV-1-Tax- CD4+Foxp3+ cells, but not with the number of Tax+ CD4+Foxp3+ cells, suggesting that HTLV-1-infected Treg cells lose their regulatory function, while HTLV-1-uninfected Treg cells contribute substantially to immune control of HTLV-1 infection. Additionally, functional impairment of CD4+Foxp3+ Treg cells was observed in mice that were transgenic mice for the *HTLV-1 bZIP factor* (*HBZ*) gene, which encodes the minus strand of HTLV-1 (Satou et al., 2011). These findings support the hypothesis that HTLV-1 may be one of the exogenous retrovirus genes responsible for immune dysregulation through interference of CD4+CD25+ Treg cell function. This hypothesis is currently under investigation to elucidate the precise molecular mechanisms by which HTLV-1 influences the fate and function of CD4+CD25+CCR4+ T cells, especially

Fig. 1. Cellular components of CD4+CD25+CCR4+ T cells in healthy individuals,

CD8+ Cytotoxic T lymphocyte (CTL) responses are an effective host defense system against all virus infections and malignancies. CTLs act by killing autologous cells that express viral

**3. Abnormality of cytotoxic T lymphocyte (CTL) response** 

asymptomatic carriers, ATL, and HAM/TSP patients.

Foxp3+ Treg cells.

T-cell receptors (TCR) on CTLs recognize peptide fragments derived from viral and tumor antigens that are presented on MHC class I molecules by antigen-presenting cells or virusinfected cells. After TCR binds to the peptide-MHC complex, CTLs are activated and fulfill an effector function. There are 3 main effector mechanisms by which the CD8+ CTL kills virus-infected or tumor cells. One is to release perforin and granzymes. Perforin forms pores in the plasma membrane of the target cells, allowing entry of granzymes; caspases are then activated, leading to apoptosis. Apoptosis may also be induced via a Fas-FasL interaction between CTLs and target cells. Finally, CD8+ cells can produce IFN-, which has indirect cytolytic effects by promoting NK cell activity and macrophage activation.

The Tax protein is an immunodominant antigen in HTLV-1 infections. Therefore, CTL activity is predominantly restricted to products of the HTLV-1 Tax gene, although HTLV-1 Env, Pol, Rof, Tof, and HBZ (Elovaara et al., 1993; Hilburn et al., 2011; Macnamara et al., 2010; Pique et al., 2000) could also be target proteins of HTLV-1-specific CTL. In a study that utilized properties of the CTL antigen recognition system, human MHC class I HLA-A2(\*0201) tetramers loaded with HTLV-1 Tax peptide were used to detect HTLV-1 Tax specific HLA-A2-restricted CD8+ cells (Bieganowska et al., 1999, Greten et al., 1998). This technique facilitates quantification of the frequency of antigen-specific T cells, as well as direct characterization of these cells. HLA genotype determines which part of the viral protein is presented as an antigen peptide. For HLA-A\*0201 and HLA-A\*2402, for example, the major epitopes are the Tax 11-19 and Tax 301-309 amino acids, respectively.

#### **3.2 Abnormal CTL response in patients with ATL**

An increasing number of studies in patients with HTLV-1-associated disorders have documented an association between the disorders and abnormalities in both the frequency of CTLs and their response to HTLV-1. When peripheral blood mononuclear cells (PBMCs) from HTLV-1 carriers are stimulated with autologous HTLV-1–infected cells *in vitro,* proliferation of HTLV-1-specific CD8+ CTLs is often observed in the presence of IL-2. An increased level of HTLV-1–specific CTL responses occurs in all HAM/TSP patients and in some asymptomatic HTLV-1 carriers; however, HTLV-1–specific CTL responses are rarely induced in PBMC cultures from ATL patients (Jacobson et al., 1990; Kannagi et al., 1984, Parker et al., 1992). HTLV-1–specific CTLs are also present in ATL patients but do not expand sufficiently (Arnulf et al., 2004). Impairment of the HTLV-1 specific CTL response was observed in some individuals during the earlier stages of HTLV-1 infection (AC and smoldering ATL), as well as in advanced ATL patients (Shimizu et al., 2009). This observation suggests that the T-cell insufficiency in ATL patients is present prior to disease onset. In addition, a recent report indicated that, in comparison to ACs, ATL patients have a smaller and less diverse population of HTLV-1 specific CD8+ T cells, as well as lower anti-HTLV-1 CD8+ T cell expression of perforin and granzyme B (Kozako et al., 2006). Thus, the decreased number and functional impairment of CTLs might contribute to the onset and progression of ATL.

Host Immune System Abnormalities

contributing factor in the immunopathogenesis of HAM/TSP.

**4. Abnormality of innate immunity** 

**4.1 Dendritic cells and HTLV-1** 

leading to pathogenesis.

**4.2 Natural killer cells and HTLV-1** 

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 71

Tax-specific CD8+ CTL clones secrete various inflammatory cytokines, chemokines, and matrix metalloproteinases (MMP), such as IFN-γ, TNF-α, monocyte inflammatory protein (MIP)-1α, MIP-1β, interleukin(IL)-16, and MMP-9 (Biddison et al., 1997). TNF-α induces cytotoxic damage to endothelial cells, thus decreasing the integrity of the blood-brain barrier. It can also directly injure oligodendrocytes. MIP-1α and 1β can enhance transendothelial migration of lymphocytes into the central nervous system. IL-16 is a chemoattractant for CD4+ cells, which are the major source of IL-2 required by IL-2 non-producer CD8+ cells for proliferation. Therefore, HTLV-1-specific CD8+ CTLs are an important source of proinflammatory soluble mediators that may contribute significantly to the pathogenesis of HAM/TSP. These observations continue to support the hypothesis that HTLV-1-specific CD8+ CTLs are a major

Besides CTLs, there are several cell populations in the human immune system that have cytolytic activity against virus-infected cells, including natural killer (NK) cells, natural killer T (NKT) cells, and γδ T cells, which are cellular components of innate immunity. Dendritic cells (DCs) play an important role in the activation of these cell populations and CTLs. There is little evidence suggesting a role for γδ T cells in the pathogenesis of HTLV-1-associated disorders. Thus, this section focuses solely on the roles of DCs, NK cells, and NKT cells in HTLV-1-

Immature DCs are located in peripheral tissues and can effectively capture antigens, leading to their maturation via the expression of MHC class I/II and co-stimulatory molecules such as CD80, CD86, and CD40. Mature DCs are professional antigen-presenting cells that are uniquely able to prime naïve T cells. There are 2 main subsets of DCs: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). These cells play important roles in the regulation of innate and adaptive immunity. mDCs can induce the activation of invariant NKT (iNKT) cells via surface expression of the CD1d/glycolipid complex. After antigen capture, pDCs secrete type 1 IFN, which induces the activation of NK cells and promotes the activation of iNKT cells by mDCs. An *in vitro* study indicated that cell-free HTLV-1 effectively infects DCs, leading to the transmission and transformation of CD4+ T cells (Jones et al. 2008). In addition to suggesting a mechanism for HTLV-1 transmission, this study also indicated that HTLV-1 infection of DCs plays a role in the pathogenesis of HTLV-1-associated disorders. In fact, HTLV-1 infected DCs are observed in the peripheral blood of HTLV-1-infected individuals (Hishizawa et al., 2004; Macatonia et al., 1992), and infected pDCs have an impaired ability to produce type I IFN (Azakami et al., 2009; Hishizawa et al., 2004). In addition, we recently reported that the frequency of mDCs and pDCs is significantly lower in patients with both HAM/TSP and ATL (Azakami et al., 2009). Cumulatively, these studies imply that decreases in the number and functionality of DCs interfere with innate immunity, thus

NK cells are major components of the innate immune system and account for 10–15% of PBMCs in normal individuals. They have direct and indirect cytolytic activity against tumor

associated diseases, by comparing with the role of these cells in HIV-1 infection.

Furthermore, Tax-specific CTL responses were strongly activated in some ATL patients who achieved complete remission after hematopoietic stem cell transplantation (HSCT), but were not observed in the same patients before transplantation (Harashima et al., 2004). This suggests that HTLV-1-specific CTLs, including Tax-specific CTLs, play an important role in surveillance against HTLV-1 leukemogenesis.

#### **3.3 Abnormal CTL response in patients with HAM/TSP**

One of the most striking features of the adaptive immune system in HAM/TSP patients is the larger number of HTLV-1-specific CD8+ CTLs (Elovaara et al., 1993; Greten et al., 1998; Jacobson et al., 1990; Kubota et al., 2002; Nagai et al., 2001a; Parker et al., 1992). While HTLV-1 specific CTLs are also detectable in the PBMC of ACs (Parker et al., 1992), the magnitude and frequency of these responses are clearly higher in patients with HAM/TSP, particularly in the CSF (Elovaara et al., 1993; Nagai et al. 2001a). In addition, the HTLV-1 proviral load of HAM/TSP patients may be 5- to 16-fold higher than that of ACs (Hashimoto et al., 1998; Kubota et al., 1993; Nagai et al., 1998). While some studies have found a positive correlation between the frequency of HTLV-1-specific CD8+ T cells and HTLV-1 proviral load has been detected in PBMCs from HAM/TSP patients (Kubota et al., 2000, Nagai et al., 2001b, Yamano et al., 2002), this result is not ubiquitous (Wodarz et al., 2001). Thus, the cytolytic activity of CTLs, rather than their frequency, might be impaired in HAM/TSP patients.

There are some methods to measure CTL cytolytic activity. One is the sensitive CD107a mobilization assay, which quantifies the amount of lysosomal membrane protein LAMP-1 (CD107a) present on the CTL surface (CD107a) (Betts et al. 2003). Among studies that have used this method to evaluate CTL function, results are conflicting; while one reported that HTLV-1-specific CTLs of HAM/TSP patients had significantly lower CD107a staining than those of ACs (Sabouri et al., 2008), another study reported the opposite (Abdelbary et al., 2011). Furthermore, higher expression of CD107a/IFN-was induced by tax peptide stimulation in the CD8+ T cells of HAM/TSP patients than in those of ACs (Enose-Akahata et al., 2008). Thus, it is not yet clear whether the cytolytic activity of HTLV-1-specific CTL in HAM/TSP patients is insufficient. However, these findings suggest that quantity of HTLV-1-infected cells is not determined by HTLV-1-specific CTL alone; additional factors, such as innate immunity and the proliferative ability of infected cells, must be relevant.

#### **3.4 Pathogenic Role of CTL in HAM/TSP**

In HAM/TSP patients, HTLV-1-specific CD8+ CTL levels are extraordinarily high in peripheral blood, and even higher in cerebrospinal fluid (CSF) (Elovaara et al., 1993; Greten et al., 1998; Jacobson et al., 1990; Kubota et al., 2002; Parker et al., 1994; Nagai et al., 2001; Yamano et al., 2002). Immunohistochemical analysis of affected spinal cord lesions in early-stage HAM/TSP patients revealed the presence of infiltrating CD4+ and CD8+ lymphocytes, among which CD8+ cells become increasingly dominant over the duration of the illness (Umehara et al., 1993). The expression of HLA class I antigens (Moore et al., 1989) and the existence of HTLV-1 specific CD8+ CTLs have also been found in such lesions (Levin et al., 1997). In addition, the infiltration of CD8+ CTLs in the affected spinal cord was characterized as positive for TIA-1 that is a marker of CTL (Umehara et al. 1994, Anderson et al. 1990). The number of TIA-1+ cells was clearly related to the amount of the proviral DNA *in situ*, and the number of infiltrating CD8+ cells appears to correlate with the presence of apoptotic cells.

Tax-specific CD8+ CTL clones secrete various inflammatory cytokines, chemokines, and matrix metalloproteinases (MMP), such as IFN-γ, TNF-α, monocyte inflammatory protein (MIP)-1α, MIP-1β, interleukin(IL)-16, and MMP-9 (Biddison et al., 1997). TNF-α induces cytotoxic damage to endothelial cells, thus decreasing the integrity of the blood-brain barrier. It can also directly injure oligodendrocytes. MIP-1α and 1β can enhance transendothelial migration of lymphocytes into the central nervous system. IL-16 is a chemoattractant for CD4+ cells, which are the major source of IL-2 required by IL-2 non-producer CD8+ cells for proliferation. Therefore, HTLV-1-specific CD8+ CTLs are an important source of proinflammatory soluble mediators that may contribute significantly to the pathogenesis of HAM/TSP. These observations continue to support the hypothesis that HTLV-1-specific CD8+ CTLs are a major contributing factor in the immunopathogenesis of HAM/TSP.

### **4. Abnormality of innate immunity**

70 T-Cell Leukemia

Furthermore, Tax-specific CTL responses were strongly activated in some ATL patients who achieved complete remission after hematopoietic stem cell transplantation (HSCT), but were not observed in the same patients before transplantation (Harashima et al., 2004). This suggests that HTLV-1-specific CTLs, including Tax-specific CTLs, play an important role in

One of the most striking features of the adaptive immune system in HAM/TSP patients is the larger number of HTLV-1-specific CD8+ CTLs (Elovaara et al., 1993; Greten et al., 1998; Jacobson et al., 1990; Kubota et al., 2002; Nagai et al., 2001a; Parker et al., 1992). While HTLV-1 specific CTLs are also detectable in the PBMC of ACs (Parker et al., 1992), the magnitude and frequency of these responses are clearly higher in patients with HAM/TSP, particularly in the CSF (Elovaara et al., 1993; Nagai et al. 2001a). In addition, the HTLV-1 proviral load of HAM/TSP patients may be 5- to 16-fold higher than that of ACs (Hashimoto et al., 1998; Kubota et al., 1993; Nagai et al., 1998). While some studies have found a positive correlation between the frequency of HTLV-1-specific CD8+ T cells and HTLV-1 proviral load has been detected in PBMCs from HAM/TSP patients (Kubota et al., 2000, Nagai et al., 2001b, Yamano et al., 2002), this result is not ubiquitous (Wodarz et al., 2001). Thus, the cytolytic activity of CTLs, rather than their frequency, might be impaired in

There are some methods to measure CTL cytolytic activity. One is the sensitive CD107a mobilization assay, which quantifies the amount of lysosomal membrane protein LAMP-1 (CD107a) present on the CTL surface (CD107a) (Betts et al. 2003). Among studies that have used this method to evaluate CTL function, results are conflicting; while one reported that HTLV-1-specific CTLs of HAM/TSP patients had significantly lower CD107a staining than those of ACs (Sabouri et al., 2008), another study reported the opposite (Abdelbary et al., 2011). Furthermore, higher expression of CD107a/IFN-was induced by tax peptide stimulation in the CD8+ T cells of HAM/TSP patients than in those of ACs (Enose-Akahata et al., 2008). Thus, it is not yet clear whether the cytolytic activity of HTLV-1-specific CTL in HAM/TSP patients is insufficient. However, these findings suggest that quantity of HTLV-1-infected cells is not determined by HTLV-1-specific CTL alone; additional factors, such as

In HAM/TSP patients, HTLV-1-specific CD8+ CTL levels are extraordinarily high in peripheral blood, and even higher in cerebrospinal fluid (CSF) (Elovaara et al., 1993; Greten et al., 1998; Jacobson et al., 1990; Kubota et al., 2002; Parker et al., 1994; Nagai et al., 2001; Yamano et al., 2002). Immunohistochemical analysis of affected spinal cord lesions in early-stage HAM/TSP patients revealed the presence of infiltrating CD4+ and CD8+ lymphocytes, among which CD8+ cells become increasingly dominant over the duration of the illness (Umehara et al., 1993). The expression of HLA class I antigens (Moore et al., 1989) and the existence of HTLV-1 specific CD8+ CTLs have also been found in such lesions (Levin et al., 1997). In addition, the infiltration of CD8+ CTLs in the affected spinal cord was characterized as positive for TIA-1 that is a marker of CTL (Umehara et al. 1994, Anderson et al. 1990). The number of TIA-1+ cells was clearly related to the amount of the proviral DNA *in situ*, and the number of infiltrating CD8+

innate immunity and the proliferative ability of infected cells, must be relevant.

surveillance against HTLV-1 leukemogenesis.

**3.4 Pathogenic Role of CTL in HAM/TSP** 

cells appears to correlate with the presence of apoptotic cells.

HAM/TSP patients.

**3.3 Abnormal CTL response in patients with HAM/TSP** 

Besides CTLs, there are several cell populations in the human immune system that have cytolytic activity against virus-infected cells, including natural killer (NK) cells, natural killer T (NKT) cells, and γδ T cells, which are cellular components of innate immunity. Dendritic cells (DCs) play an important role in the activation of these cell populations and CTLs. There is little evidence suggesting a role for γδ T cells in the pathogenesis of HTLV-1-associated disorders. Thus, this section focuses solely on the roles of DCs, NK cells, and NKT cells in HTLV-1 associated diseases, by comparing with the role of these cells in HIV-1 infection.

#### **4.1 Dendritic cells and HTLV-1**

Immature DCs are located in peripheral tissues and can effectively capture antigens, leading to their maturation via the expression of MHC class I/II and co-stimulatory molecules such as CD80, CD86, and CD40. Mature DCs are professional antigen-presenting cells that are uniquely able to prime naïve T cells. There are 2 main subsets of DCs: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). These cells play important roles in the regulation of innate and adaptive immunity. mDCs can induce the activation of invariant NKT (iNKT) cells via surface expression of the CD1d/glycolipid complex. After antigen capture, pDCs secrete type 1 IFN, which induces the activation of NK cells and promotes the activation of iNKT cells by mDCs. An *in vitro* study indicated that cell-free HTLV-1 effectively infects DCs, leading to the transmission and transformation of CD4+ T cells (Jones et al. 2008). In addition to suggesting a mechanism for HTLV-1 transmission, this study also indicated that HTLV-1 infection of DCs plays a role in the pathogenesis of HTLV-1-associated disorders. In fact, HTLV-1 infected DCs are observed in the peripheral blood of HTLV-1-infected individuals (Hishizawa et al., 2004; Macatonia et al., 1992), and infected pDCs have an impaired ability to produce type I IFN (Azakami et al., 2009; Hishizawa et al., 2004). In addition, we recently reported that the frequency of mDCs and pDCs is significantly lower in patients with both HAM/TSP and ATL (Azakami et al., 2009). Cumulatively, these studies imply that decreases in the number and functionality of DCs interfere with innate immunity, thus leading to pathogenesis.

#### **4.2 Natural killer cells and HTLV-1**

NK cells are major components of the innate immune system and account for 10–15% of PBMCs in normal individuals. They have direct and indirect cytolytic activity against tumor

Host Immune System Abnormalities

**5. Conclusion**

the pathogenesis of HAM/TSP and ATL.

pathogenesis of HTLV-1-associated disorders.

**6. Acknowledgments** 

**7. References** 

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 73

from ACs (Azakami et al., 2009). These results suggest that iNKT cells contribute to the immune defense against HTLV-1, and that iNKT cell depletion plays an important role in

Advances in our understanding of the immune system enhance studies of virus-host relationships. Although HTLV-1 causes 2 different diseases (ATL and HTM/TSP), CD4+CD25+CCR4+ T cells are the common viral reservoir in both disorders. According to recent studies, however, characteristics of CD4+CD25+CCR4+ T cells are completely different in the 2 diseases: Foxp3+ leukemic cells are found in ATL patients, while Foxp3- IFN- producing cells are found in HAM/TSP patients. The host immune system plays a crucial role in controlling these HTLV-1-infected cells. HTLV-1-specific CTL is activated in patients with HAM/TSP, but not in those with ATL, indicating that impairment of acquired immunity is not universal. However, both ATL and HAM/TSP patients are known to experience decreases in innate immunity via the functional impairment of DCs, NK cells, and iNKT cells, as well as lower overall population numbers of these cell types. These conditions may contribute to inadequate viral control and play an important role in the

This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor, and Welfare; the Uehara Memorial Foundation; the Nagao Takeshi Nanbyo Foundation; the Kanagawa Nanbyo Foundation; the Mishima Kaiun Memorial Foundation; the Takeda Science Foundation; the ITSUU Laboratory Research Foundation; the

Abdelbary, N.H., Abdullah, H.M., Matsuzaki, T., Hayashi, D., Tanaka, Y., Takashima, H.,

Anderson, P., Nagler-Anderson, C., O'Brien, C., Levine, H., Watkins, S., Slayter, H.S., Blue,

Arnulf, B., Thorel, M., Poirot, Y., Tamouza, R., Boulanger, E., Jaccard, A., Oksenhendler, E.,

Asquith, B., Mosley, A.J., Barfield, A., Marshall, S.E., Heaps, A., Goon, P., Hanon, E., Tanaka,

Izumo, S. & Kubota, R. 2011. Reduced Tim-3 expression on human T-lymphotropic virus type I (HTLV-I) Tax-specific cytotoxic T lymphocytes in HTLV-I infection.

M.L. & Schlossman, S.F. 1990. A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T

Hermine, O. & Pique, C. 2004. Loss of the ex vivo but not the reinducible CD8+ Tcell response to Tax in human T-cell leukemia virus type 1-infected patients with

Y., Taylor, G.P. & Bangham, C.R. 2005. A functional CD8+ cell assay reveals individual variation in CD8+ cell antiviral efficacy and explains differences in

Foundation for Total Health Promotion; and the Sankyo Foundation of Life Science.

*Journal of Infectious Diseases*, 203, 7, 948-959

lymphocytes. *Journal of Immunology*, 144, 2, 574–582

adult T-cell leukemia/lymphoma. *Leukemia*, 18, 1, 126-132

cells and virus-infected cells by producing perforins, granzymes, and IFN-γ. Human NK cells can be divided into 2 subsets on the basis of their cell-surface markers: CD56+CD16+ and CD56brightCD16– NK cells. CD56+CD16+ NK cells are the major population of NK cells and have natural cytotoxic activity. CD56brightCD16– NK cells are not cytotoxic but have the capacity to produce large amounts of IFN-γ upon activation. The activity of NK cells is regulated by a balance between positive and negative signals from different activating and inhibitory NK receptors. CD94/NKG2 receptor family is expressed on CD8+ T cells and T cells as well as NK cells, and is involved in the pathogenesis of HAM/TSP by modulating the activities of those cell populations (Saito et al. 2003, Mosley et al. 2005).

In both HIV-1- and HTLV-1-infected individuals, the number and function of NK cell subsets are impaired (Fortis et al., 2005). Multiple investigators have reported that the numbers of CD56+CD16+ NK cells in HAM/TSP and ATL patients are significantly lower than those observed in healthy controls (Azakami et al., 2009; Yu et al., 1991). Furthermore, NK cell activity was also lower in HAM/TSP patients than in healthy controls (Yu et al., 1991). When primary CD4+ T cells are infected by HTLV-1, they can escape from NK cellmediated cytotoxicity; HTLV-1 p12I downregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and -2 on the surface of infected CD4+ T cells, resulting in a reduced adherence of NK cells to HTLV-1-infected CD4+ T cells (Banerjee et al., 2007).

#### **4.3 Natural killer T cells and HTLV-1**

Natural killer T (NKT) cells, a unique T cell subpopulation, constitute a subset of lymphocytes that share the features of innate and adaptive immune cells. Unlike conventional T cells, NKT cells express a TCR that recognizes glycolipids instead of protein antigens. Moreover, these cells share properties and receptors with NK cells. They rapidly produce granzymes and perforins upon stimulation. Among the CD3+ T cells in human blood, 10–25% express NK cell surface molecules such as CD161, and these cells are classified as NKT cells. A small population of T cells within this NKT cell subset expresses a highly conserved Vα24Jα18 TCR chain that preferentially associates with Vβ11; these T cells are referred to as iNKT cells. Activation of human iNKT cells requires the presentation of glycolipids such as α-galactosylceramide (α-GalCer) on the MHC class I-like molecule CD1d. α-GalCer induces the rapid production of cytokines and potent antitumor and antipathogen responses by iNKT cells. CD4– iNKT cells preferentially induce the Th1 response and are more important than CD4+ iNKT cells in controlling viral infection and cancer (Kim et al., 2002).

HIV-1-infected subjects have fewer iNKT cells in their peripheral blood than healthy donors (Sandberg et al., 2002; van der Vliet et al., 2002). The proliferative potential and INF-γ production of residual iNKT cells are impaired in HIV-1-infected individuals (Moll et al., 2009); likewise, patients with HTLV-1-associated disorders have a decreased frequency of iNKT cells in their peripheral blood (Azakami et al., 2009). Interestingly, in contrast to patterns observed in HIV-1 infections, HTLV-1 infection leads to preferential decreases of CD4– iNKT cells (Azakami et al., 2009). The production of perforin in iNKT cells is impaired in both ACs and HAM/TSP patients (Azakami et al., 2009). In addition, there is an inverse correlation between the frequency of iNKT cells and the HTLV-1 proviral load in the peripheral blood of HTLV-1-infected individuals (Azakami et al., 2009). Notably, *in vitro* stimulation of peripheral blood cells with α-GalCer leads to an increase in the number of iNKT cells and a subsequent decrease in the number of HTLV-1-infected T cells in samples from ACs (Azakami et al., 2009). These results suggest that iNKT cells contribute to the immune defense against HTLV-1, and that iNKT cell depletion plays an important role in the pathogenesis of HAM/TSP and ATL.

### **5. Conclusion**

72 T-Cell Leukemia

cells and virus-infected cells by producing perforins, granzymes, and IFN-γ. Human NK cells can be divided into 2 subsets on the basis of their cell-surface markers: CD56+CD16+ and CD56brightCD16– NK cells. CD56+CD16+ NK cells are the major population of NK cells and have natural cytotoxic activity. CD56brightCD16– NK cells are not cytotoxic but have the capacity to produce large amounts of IFN-γ upon activation. The activity of NK cells is regulated by a balance between positive and negative signals from different activating and inhibitory NK receptors. CD94/NKG2 receptor family is expressed on CD8+ T cells and T cells as well as NK cells, and is involved in the pathogenesis of HAM/TSP by modulating

In both HIV-1- and HTLV-1-infected individuals, the number and function of NK cell subsets are impaired (Fortis et al., 2005). Multiple investigators have reported that the numbers of CD56+CD16+ NK cells in HAM/TSP and ATL patients are significantly lower than those observed in healthy controls (Azakami et al., 2009; Yu et al., 1991). Furthermore, NK cell activity was also lower in HAM/TSP patients than in healthy controls (Yu et al., 1991). When primary CD4+ T cells are infected by HTLV-1, they can escape from NK cellmediated cytotoxicity; HTLV-1 p12I downregulates the expression of intercellular adhesion molecule-1 (ICAM-1) and -2 on the surface of infected CD4+ T cells, resulting in a reduced

Natural killer T (NKT) cells, a unique T cell subpopulation, constitute a subset of lymphocytes that share the features of innate and adaptive immune cells. Unlike conventional T cells, NKT cells express a TCR that recognizes glycolipids instead of protein antigens. Moreover, these cells share properties and receptors with NK cells. They rapidly produce granzymes and perforins upon stimulation. Among the CD3+ T cells in human blood, 10–25% express NK cell surface molecules such as CD161, and these cells are classified as NKT cells. A small population of T cells within this NKT cell subset expresses a highly conserved Vα24Jα18 TCR chain that preferentially associates with Vβ11; these T cells are referred to as iNKT cells. Activation of human iNKT cells requires the presentation of glycolipids such as α-galactosylceramide (α-GalCer) on the MHC class I-like molecule CD1d. α-GalCer induces the rapid production of cytokines and potent antitumor and antipathogen responses by iNKT cells. CD4– iNKT cells preferentially induce the Th1 response and are more important than CD4+ iNKT cells in controlling viral infection and

HIV-1-infected subjects have fewer iNKT cells in their peripheral blood than healthy donors (Sandberg et al., 2002; van der Vliet et al., 2002). The proliferative potential and INF-γ production of residual iNKT cells are impaired in HIV-1-infected individuals (Moll et al., 2009); likewise, patients with HTLV-1-associated disorders have a decreased frequency of iNKT cells in their peripheral blood (Azakami et al., 2009). Interestingly, in contrast to patterns observed in HIV-1 infections, HTLV-1 infection leads to preferential decreases of CD4– iNKT cells (Azakami et al., 2009). The production of perforin in iNKT cells is impaired in both ACs and HAM/TSP patients (Azakami et al., 2009). In addition, there is an inverse correlation between the frequency of iNKT cells and the HTLV-1 proviral load in the peripheral blood of HTLV-1-infected individuals (Azakami et al., 2009). Notably, *in vitro* stimulation of peripheral blood cells with α-GalCer leads to an increase in the number of iNKT cells and a subsequent decrease in the number of HTLV-1-infected T cells in samples

the activities of those cell populations (Saito et al. 2003, Mosley et al. 2005).

adherence of NK cells to HTLV-1-infected CD4+ T cells (Banerjee et al., 2007).

**4.3 Natural killer T cells and HTLV-1** 

cancer (Kim et al., 2002).

Advances in our understanding of the immune system enhance studies of virus-host relationships. Although HTLV-1 causes 2 different diseases (ATL and HTM/TSP), CD4+CD25+CCR4+ T cells are the common viral reservoir in both disorders. According to recent studies, however, characteristics of CD4+CD25+CCR4+ T cells are completely different in the 2 diseases: Foxp3+ leukemic cells are found in ATL patients, while Foxp3- IFN- producing cells are found in HAM/TSP patients. The host immune system plays a crucial role in controlling these HTLV-1-infected cells. HTLV-1-specific CTL is activated in patients with HAM/TSP, but not in those with ATL, indicating that impairment of acquired immunity is not universal. However, both ATL and HAM/TSP patients are known to experience decreases in innate immunity via the functional impairment of DCs, NK cells, and iNKT cells, as well as lower overall population numbers of these cell types. These conditions may contribute to inadequate viral control and play an important role in the pathogenesis of HTLV-1-associated disorders.

#### **6. Acknowledgments**

This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology; the Japanese Ministry of Health, Labor, and Welfare; the Uehara Memorial Foundation; the Nagao Takeshi Nanbyo Foundation; the Kanagawa Nanbyo Foundation; the Mishima Kaiun Memorial Foundation; the Takeda Science Foundation; the ITSUU Laboratory Research Foundation; the Foundation for Total Health Promotion; and the Sankyo Foundation of Life Science.

#### **7. References**


Host Immune System Abnormalities

1735–1743

72

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 75

Gessain, A., Barin, F. & Vernant, J.C. 1985. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. *Lancet*, 2, 8452, 407–410 Goon, P.K., Igakura, T., Hanon, E., Mosley, A.J., Barfield, A., Barnard, A.L., Kaftantzi, L.,

Grant, C., Oh, U., Yao, K., Yamano, Y. & Jacobson, S. 2008. Dysregulation of TGF-beta

Greten, T.F., Slansky, J.E., Kubota, R., Soldan, S.S., Jaffee, E.M., Leist, T.P., Pardoll, D.M.,

Harashima, N., Kurihara, K., Utsunomiya, A. Tanosaki, R., Hanabuchi, S., Masuda, M.,

Hayashi, D., Kubota, R., Takenouchi, N., Tanaka, Y., Hirano, R., Takashima, H., Osame, M.,

Hieshima, K., Nagakubo, D., Nakayama, T., Shirakawa, A.K., Jin, Z., & Yoshie, O. 2008. Tax-

Hilburn, S., Rowan, A., Demontis, M.A., MacNamara, A., Asquith, B., Bangham, C.R. &

Hori, S., Nomura, T. & Sakaguchi, S. 2003. Control of regulatory T cell development by the

Iwanaga, M., Watanabe, T., Utsunomiya, A., Okayama, A., Uchimaru, K., Koh, K.R., Ogata,

transcription factor Foxp3. *Science*, 299, 5609, 1057–1061

CCR4-expressing CD4+ T cells. *Journal of Immunol*ogy, 180, 2, 931-9

hematopoietic stem cell transplantation. *Cancer Research*, 64, 391–399 Hashimoto, K., Higuchi, I., Osame, M. & Izumo, S. 1998. Quantitative in situ PCR assay of

neuroinflammatory disease. *Blood*,111, 12, 5601–5609

National Academy of Sciences U.S.A., 95, 13, 7568–7573

*Neuroimmunology*, 200, 1-2, 115–124

*Haematology*, 125, 5, 568-575

Tanaka, Y., Taylor, G.P. Weber, J.N. & Bangham, C.R. 2004. Human T cell lymphotropic virus type I (HTLV-I)-specific CD4+ T cells: immunodominance hierarchy and preferential infection with HTLV-I. *Journal of Immunology*, 172, 3,

signaling and regulatory and effector T-cell function in virus-induced

Jacobson, S. & Schneck, J.P. 1998. Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proceedings of the

Ohashi, T., Fukui, F., Hasegawa, A., Masuda, T., Takaue, Y., Okamura, J. & Kannagi, M. 2004. Graft-versus-Tax response in adult T-cell leukemia patients after

HTLV-1 infected cells in peripheral blood lymphocytes of patients with ATL, HAM/TSP and asymptomatic carriers. *Journal of the Neurological Sciences*, 159, 1, 67–

Izumo, S. & Arimura, K. 2008. Reduced Foxp3 expression with increased cytomegalovirus-specific CTL in HTLV-I-associated myelopathy. *Journal of* 

inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to

Taylor, G.P. 2011. In vivo expression of human T-lymphotropic virus type 1 basic leucine-zipper protein generates specific CD8+ and CD4+ T-lymphocyte responses that correlate with clinical outcome. *Journal of Infectious Diseases*, 203, 4, 529-36 Hishizawa M, Imada K, Kitawaki T, Ueda M, Kadowaki N, Uchiyama T. 2004. Depletion

and impaired interferon-alpha-producing capacity of blood plasmacytoid dendritic cells in human T-cell leukaemia virus type I-infected individuals. *British Journal of* 

M., Kikuchi, H., Sagara, Y., Uozumi, K., Mochizuki, M., Tsukasaki, K., Saburi, Y., Yamamura, M., Tanaka, J., Moriuchi, Y., Hino, S., Kamihira, S. & Yamaguchi, K. 2010. Human T-cell leukemia virus type I (HTLV-1) proviral load and disease

human T-lymphotropic virus type 1 proviral load. *Journal of General Virology*, 86, 5, 1515–23


Azakami, K., Sato, T., Araya, N., Utsunomiya, A., Kubota, R., Suzuki, K., Hasegawa, D.,

Banerjee, P., Feuer, G., Barker, E. 2007. Human T-cell leukemia virus type 1 (HTLV-1) p12I

Betts, M.R., Brenchley, J.M., Price, D.A., De Rosa, S.C., Douek, D.C., Roederer, M. & Koup,

Biddison, W.E., Kubota, R., Kawanishi, T., Taub, D.D., Cruikshank, W.W., Center, D.M.,

Bieganowska, K., Hollsberg, P., Buckle, G.J., Lim, D.G., Greten, T.F., Schneck, J., Altman,

Chen, S., Ishii, N., Ine, S., Ikeda, S., Fujimura, T., Ndhlovu, L.C., Soroosh, P., Tada, K.,

Elovaara, I., Koenig, S., Brewah, A.Y., Woods, R.M., Lehky, T. & Jacobson, S. 1993. High

Enose-Akahata, Y., Oh, U., Grant, C. & Jacobson, S. 2008. Retrovirally induced CTL

Fortis, C. & Poli, G. 2005. Dendritic cells and natural killer cells in the pathogenesis of HIV

virus-associated myelopathy. *Journal of Immunology*, 162, 3, 1765–1771. Boxus, M. & Willems, L. 2009. Mechanisms of HTLV-1 persistence and transformation.

metalloproteinase. *Journal of Immunology*, 159, 4, 2018–2025

Bangham, C.R. 2008. HTLV-1 infection: role of CTL efficiency. *Blood*, 112, 6, 2176-2177 Bangham, C.R. 2009. CTL quality and the control of human retroviral infections. *European* 

1515–23

65–78

2, 269–277

2400–2410

disorders. *Blood*, 114, 15, 3208-3215

*Journal of Immunology*, 39, 7, 1700-1712

*British Journal of Cancer*, 101, 9, 1497-1501

*Journal of Experimental Medicine*, 177, 6, 1567–1573

infection. *Immunologic Research*, 33: 1, 1-21

human T-lymphotropic virus type 1 proviral load. *Journal of General Virology*, 86, 5,

Izumi, T., Fujita, H., Aratani, S., Fujii, R., Yagishita, N., Kamijuku, H., Kanekura, T., Seino, K., Nishioka, K., Nakajima, T. & Yamano, Y. 2009. Severe loss of invariant NKT cells exhibiting anti-HTLV-1 activity in patients with HTLV-1-associated

down-modulates ICAM-1 and -2 and reduces adherence of natural killer cells, thereby protecting HTLV-1-infected primary CD4+ T cells from autologous natural killer cell-mediated cytotoxicity despite the reduction of major histocompatibility complex class I molecules on infected cells. *Journal of Virology*, 81, 18 9707-9717

R.A. 2003. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. *Journal of Immunological Methods*, 281, 1-2,

Connor, E.W., Utz, U. & Jacobson, S. 1997. Human T cell leukemia virus type I (HTLV-I)-specific CD8+ CTL clones from patients with HTLV-I-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix

J.D., Jacobson, S., Ledis, S.L., Hanchard, B., Chin, J., Morgan, O., Roth, P.A. & Hafler, D.A. 1999. Direct analysis of viral-specific CD8+ T cells with soluble HLA-A2/Tax11-19 tetramer complexes in patients with human T cell lymphotropic

Harigae, H., Kameoka, J., Noriyuki, K., Sasaki, T. & Sugamura, K. 2006. Regulatory T cell-like activity of Foxp3+ adult T cell leukemia cells. *International Immunology* 18,

human T cell lymphotropic virus type 1 (HTLV-1)-specific precursor cytotoxic T lymphocyte frequencies in patients with HTLV-1-associated neurological disease.

degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-Iassociated neurologic disease. *Blood*, 112, 6,


Host Immune System Abnormalities

1699-1706

*Hematology,* 84, 1, 63–69

*of Neuroimmune Pharmacology*, 5, 3, 310-25

paraparesis. *BMC Immunology,* 9, 41

*Neurovirology*, 4, 6, 586-593

812

*Journal of Infectious Diseases*, 183, 2, 197–205

Journal of Neurovirology, 8, 1, 53–57

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 77

Kubota, R., Soldan, S.S., Martin, R. & Jacobson, S. 2002. Selected cytotoxic T lymphocytes

Levin, M.C., Lehky, T.J., Flerlage, A.N., Katz, D., Kingma, D.W., Jaffe, E.S., Heiss, J.D.,

Macnamara, A., Rowan, A., Hilburn, S., Kadolsky, U., Fujiwara, H., Suemori, K., Yasukawa,

Matsuura, E., Yamano, Y. & Jacobson, S. 2010. Neuroimmunity of HTLV-I Infection. *Journal* 

Michaëlsson, J., Barbosa, H.M., Jordan, K.A., Chapman, J.M., Brunialti, M.K., Neto, W.K.,

Moll, M., Kuylenstierna, C., Gonzalez, V.D., Andersson, S.K., Bosnjak, L., Sönnerborg, A.,

Mosley, A.J., Asquith, B. & Bangham, C.R. 2005. Cell-mediated immune response to human

Nagai, M., Usuku, K., Matsumoto, W., Kodama, D., Takenouchi, N., Moritoyo, T.,

Nagai, M., Kubota, R., Greten, T.F., Schneck, J.P., Leist, T.P. & Jacobson, S. 2001a. Increased

Nagai, M., Yamano, Y., Brennan, M.B., Mora, C.A. & Jacobson, S. 2001b. Increased HTLV-I

infection. *European Journal of Immunology*, 39, 3, 902-911

T-lymphotropic virus type I. *Viral Immunology*, 18, 2, 293-305

determines outcome in HTLV-1 infection. *PLoS Pathogens*, 6, 9, e1001117 Matsubar, Y., Hori, T., Morita, R., Sakaguchi, S. & Uchiyama, T. 2006. Delineation of

with high specificity for HTLV-I in cerebrospinal fluid from a HAM/TSP patient.

Patronas, N., McFarland, H.F. & Jacobson, S. 1997. Immunologic analysis of a spinal cord-biopsy specimen from a patient with human T-cell lymphotropic virus type Iassociated neurologic disease. *New England Journal of Medicine*, 336, 12, 839–845 Macatonia, S.E., Cruickshank, J.K., Rudge, P. & Knight, S.C. 1992. Dendritic cells from

patients with tropical spastic paraparesis are infected with HTLV-1 and stimulate autologous lymphocyte proliferation. *AIDS Research and Human Retroviruses*, 8, 9,

M., Taylor, G., Bangham, C.R. & Asquith, B. 2010. HLA class I binding of HBZ

immunoregulatory properties of adult T-cell leukemia cells. *International Journal of* 

Nukui, Y., Sabino, E.C., Chieia, M.A., Oliveira, A.S.B., Nixon, D.F. & Kallas, E.G. 2008. The frequency of CD127low expressing CD4+CD25high T regulatory cells is inversely correlated with human T lymphotrophic virus type-1 (HTLV-1) proviral load in HTLV-1-infection and HTLV-1-associated myelopathy/tropical spastic

Quigley, M.F. & Sandberg, J.K. 2009.Severe functional impairment and elevated PD-1 expression in CD1d-restricted NKT cells retained during chronic HIV-1

Hashiguchi, S., Ichinose, M., Bangham, C.R., Izumo, S. & Osame, M. 1998. Analysis of HTLV-I proviral load in 202 HAM/TSP patients and 243 asymptomatic HTLV-I carriers: high proviral load strongly predisposes to HAM/TSP. *Journal of* 

activated human T cell lymphotropic virus type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load.

proviral load and preferential expansion of HTLV-I Tax-specific CD8+ T cells in cerebrospinal fluid from patients with HAM/TSP. *Annals of Neurology*, 50, 6, 807–

progression in asymptomatic HTLV-1 carriers: a nationwide prospective study in Japan. *Blood*, 116, 8, 1211-1219


Jacobson, S., Shida, H., McFarlin, D.E., Fauci, A.S. & Koenig, S. 1990. Circulating CD8+

Jacobson, S. 2002. Immunopathogenesis of human T cell lymphotropic virus type Iassociated neurologic disease. *Journal of Infectious Diseases*, 186 Suppl, S187–92 Jones, K.S., Petrow-Sadowski, C., Huang, Y.K., Bertolette, D.C. & Ruscetti, F.W. 2008. Cell-

Kannagi, M., Sugamura, K., Kinoshita, K., Uchino, H. & Hinuma, Y. 1984. Specific cytolysis

Kannagi, M. Immunologic control of human T-cell leukemia virus type I and adult T-cell

Kannagi, M., Hasegawa, A., Kinpara, S., Shimizu, Y., Takamori, A. & Utsunomiya, A. 2011.

Karube, K., Ohshima, K., Tsuchiya, T., Yamaguchi, T., Kawano, R., Suzumiya, J.,

Kim, C.H., Butcher, E.C. & Johnston, B. 2002. Distinct subsets of human Valpha24-invariant

Kohno, T., Yamada, Y., Akamatsu, N., Kamihira, S., Imaizumi, Y., Tomonaga, M. &

Kozako, T., Arima, N., Toji, S., Masamoto, I., Akimoto, M., Hamada, H., Che, X.F., Fujiwara,

Kubota, R., Fujiyoshi, T., Izumo, S., Yashiki, S., Maruyama, I., Osame, M. & Sonoda, S. 1993.

HTLV-I-associated myelopathy. *Journal of Neuroimmunology*, 42, 2, 147–154 Kubota, R., Nagai, M., Kawanishi, T., Osame, M. & Jacobson, S. 2000. Increased HTLV type 1

leukemia. 2007. International Journal of Hematology, 86, 2, 113–117

leukemia/lymphoma patient. *Journal of Immunology,* 133, 2, 1037-1041. Kannagi, M., Harada, S., Maruyama, I. Inoko, H., Igarashi, H., Kuwashima, G., Sato, S.,

Japan. *Blood*, 116, 8, 1211-1219

neurological disease. *Nature*, 348, 6298, 245–248

CD4(+) T cells. *Nature Medicine*, 14, 4, 429-436

cells. *International Immunology,* 3, 8, 761–7

acquired immunity. *Cancer Science*, 102, 4, 670-6

cells. *British Journal of Haematology,* 126, 1, 81–84

*Immunology*, 23, 11, 516-519

*of Immunology*, 177, 8, 5718–5726

*Retroviruses*, 16, 16, 1705–1709

8, 527–533

progression in asymptomatic HTLV-1 carriers: a nationwide prospective study in

cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated

free HTLV-1 infects dendritic cells leading to transmission and transformation of

of fresh tumor cells by an autologous killer T cell line derived from an adult T cell

Morita, M., Kidokoro, M., Sugimoto, M., Funahashi, S., Osame, M. & Shida, H. 1991. Predominant recognition of human T cell leukemia virus type I (HTLV-I) pX gene products by human CD8+ cytotoxic T cells directed against HTLV- I-infected

Double control systems for human T-cell leukemia virus type 1 by innate and

Utsunomiya, A., Harada, M. & Kikuchi, M. 2004. Expression of FoxP3, a key molecule in CD4CD25 regulatory T cells, in adult T-cell leukaemia/lymphoma

NKT cells: cytokine responses and chemokine receptor expression. *Trends in* 

Matsuyama, T. 2005. Possible origin of adult T-cell leukemia/lymphoma cells from human T lymphotropic virus type-1-infected regulatory T cells. *Cancer Science, 96*,

H., Matsushita, K., Tokunaga, M., Haraguchi, K., Uozumi, K., Suzuki, S., Takezaki, T. & Sonoda, S. 2006. Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients. *Journal* 

Fluctuation of HTLV-I proviral DNA in peripheral blood mononuclear cells of

tax specific CD8+ cells in HTLV type 1-associated myelopathy/tropical spastic paraparesis: correlation with HTLV type 1 proviral load. *AIDS Research and Human* 


Host Immune System Abnormalities

Among Patients with Human T-Lymphotropic Virus Type 1 (HTLV-1)- Associated Disorders 79

Satou, Y & Matsuoka, M. 2010. HTLV-1 and the host immune system: how the virus

Satou, Y., Yasunaga, J., Zhao, T., Yoshida, M., Miyazato, P., Takai, K., Shimizu, K., Ohshima,

Shimauchi, T., Kabashima, K. & Tokura, Y. 2008. Adult T-cell leukemia/lymphoma cells

Shimizu, Y., Takamori, A., Utsunomiya, A., Kurimura, M., Yamano, Y., Hishizawa, M.,

Toulza, F., Heaps, A., Tanaka, Y., Taylor, G.P. & Bangham, C.R. 2008. High frequency of

Tsuji, M., Komatsu, N., Kawamoto, S., Suzuki, K., Kanagawa, O., Honjo, T., Hori, S. &

Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K. & Uchino, H. Adult T-cell leukemia: clinical and hematologic features of 16 cases. 1977. *Blood*, 50, 3, 481-92 Umehara, F., Izumo, S., Nakagawa, M., Ronquillo, A.T., Takahashi, K., Matsumuro, K., Sato,

Umehara, F., Nakamura, A., Izumo, S., Kubota, R., Ijichi, S., Kashio, N., Hashimoto, K.,

van der Vliet, H.J., von Blomberg, B.M., Hazenberg, M.D., Nishi, N., Otto, S.A., van

Wodarz, D., Nowak, M.A. & Bangham, C.R. 1999. The dynamics of HTLV-I and the CTL

Wodarz, D., Hall, S.E., Usuku, K., Osame, M., Ogg, G.S., McMichael, A.J., Nowak, M.A. &

*Clinical and Experimental Hematopathology*. 50, 1, 1-8

smoldering stages. *Cancer Science,* 100, 3, 481–489

T cells in gut Peyer's patches. *Science* 323, 5920, 1488–1492

CTL response. *Blood* 111, 10, 5047–5053

*Experimental Neurology*, 52, 4, 424–430

*Experimental Neurology*, 53, 6, 617–624

response. *Immunology Today,* 20, 5, 220–227

168, 3, 1490-1495

vivo. *PLoS Pathogens,* 7, 2, e1001274

*Science,* 99, 1, 98–106

disrupts immune regulation, leading to HTLV-1 associated diseases. *Journal of* 

K., Green, P.L., Ohkura, N., Yamaguchi, T., Ono, M., Sakaguchi, S. & Matsuoka, M. 2011. HTLV-1 bZIP factor induces T-cell lymphoma and systemic inflammation in

from blood and skin tumors express cytotoxic T lymphocyte-associated antigen-4 and Foxp3 but lack suppressor activity toward autologous CD8+ T cells. *Cancer* 

Hasegawa, A., Kondo, F., Kurihara, K., Harashima, N., Watanabe, T., Okamura, J., Masuda, T. & Kannagi, M. 2009. Impaired Tax-specific T-cell responses with insufficient control of HTLV-1 in a subgroup of individuals at asymptomatic and

CD4+FoxP3+ cells in HTLV-1 infection: inverse correlation with HTLV-1-specific

Fagarasan, S. 2009. Preferential generation of follicular B helper T cells from Foxp3+

E. & Osame M. 1993. Immunocytochemical analysis of the cellular infiltrate in the spinal cord lesions in HTLV-Iassociated myelopathy. *Journal of Neuropathology and* 

Usuku, K., Sato, E. & Osame, M. 1994. Apoptosis of T lymphocytes in the spinal cord lesions in HTLV-I-associated myelopathy: a possible mechanism to control viral infection in the central nervous system. *Journal of Neuropathology and* 

Benthem, B.H., Prins, M., Claessen, F.A., van den Eertwegh, A.J., Giaccone, G., Miedema, F., Scheper, R.J. & Pinedo, H.M. 2002. Selective decrease in circulating V alpha 24+V beta 11+ NKT cells during HIV type 1 infection. *Journal of Immunology*,

Bangham, C.R.M.. 2001. Cytotoxic T-cell abundance and virus load in human immunodeficiency virus type 1 and human T-cell leukaemia virus type 1. Proceedings of the Royal Society of London B, 268, 1473, 1215–21Yamano, Y., Nagai, M., Brennan, M. Mora, C.A., Soldan, S.S., Tomaru, U., Takenouchi, N., Izumo, S., Osame, M. & Jacobson, S. 2002. Correlation of human T-cell


Oh, U., Grant, C., Griffith, C., Fugo, K., Takenouchi, N. & Jacobson, S. 2006. Reduced Foxp3

Ohsugi, T. & Kumasaka, 2011. T. Low CD4/CD8 T-cell ratio associated with inflammatory

Osame, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M. & Tara, M.

Parker, C.E., Daenke, S., Nightingale, S. & Bangham, C.R. 1992. Activated, HTLV-1-specific

Pique, C., Ureta-Vidal, A., Gessain, A., Chancerel, B., Gout, O., Tamouza, R., Agis, F. &

against viral peptides. 2000. *Journal of Experimental Medicine*, 191, 3, 567–72 Ramirez, J.M., Brembilla, N.C., Sorg, O., Chicheportiche, R., Matthes, T., Dayer, J.M., Saurat,

Richardson, J.H., Edwards, A.J., Cruickshank, J.K., Rudge, P. & Dalgleish, A.G. In vivo

Roncador, G., Garcia, J.F., Maestre, L., Lucas, E., Menarguez, J., Ohshima, K., Nakamura, S.,

Sabouri, A.H., Usuku, K., Hayashi, D., Izumo, S., Ohara, Y., Osame, M. & Saito, M. 2008

T cells in HTLV-1-associated neurologic disease. *Blood,* 112, 6, 2411–2420 Saito, M., Braud, V.M., Goon, P., Hanon, E., Taylor, G.P., Saito, A., Eiraku, N., Tanaka, Y.,

Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. & Toda, M. 1995. Immunologic self-

Sakaguchi, S., Yamaguchi, T., Nomura, T., & Ono, M. 2008. Regulatory T cells and immune

Sandberg, J.K., Fast, N.M., Palacios, E.H., Fennelly, G., Dobroszycki, J., Palumbo, P., Wiznia,

Sato, T., Araya, N. & Yamano, Y. 2011. Human T-lymphotropic virus type 1 (HTLV-1) and

autoimmune diseases. *Journal of Immunology,* 155, 3, 1151–1164

innate immunity. *Inflammation and Regeneration*, 31, 1, 110-115

with tropical spastic paraparesis. *Virology*. 188, 2, 628-636

cells. *European Journal of Immunology,* 40, 9, 2450–2459

leukaemia/lymphoma. *Leukemia,* 19, 12, 2247–2253

6, 4, e18518

11, 5682–5687

102, 2, 577-584

tolerance. *Cell,* 133, 5, 775–787

infection. *Journal of Virology*, 76, 15, 7528-7534

1032

protein expression is associated with inflammatory disease during human t lymphotropic virus type 1 Infection. *Journal of Infectious Diseases*, 193, 11, 1557–1566

arthropathy in human T-cell leukemia virus type I Tax transgenic mice. *PLoS One*,

1986. HTLV-I associated myelopathy, a new clinical entity. *Lancet* 1, 8488, 1031–

cytotoxic T-lymphocytes are found in healthy seropositives as well as in patients

Dokhélar, M.C. Evidence for the chronic in vivo production of human T cell leukemia virus type I Rof and Tof proteins from cytotoxic T lymphocytes directed

J.H., Roosnek, E., & Chizzolini, C. 2010. Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper

cellular tropism of human T-cell leukemia virus type 1. 1990. *Journal of Virology*, 64,

Banham, A.H., Piris, M.A. FOXP3, a selective marker for a subset of adult T-cell

Impaired function of human T-lymphotropic virus type 1 (HTLV-1)-specific CD8+

Usuku, K., Weber, J.N., Osame, M. & Bangham, C.R. 2003. Low frequency of CD94/NKG2A+ T lymphocytes in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis, but not in asymptomatic carriers. *Blood*,

tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various

A., Grant, R.M., Bhardwaj, N., Rosenberg, M.G. & Nixon, D.F. 2002. Selective loss of innate CD4(+) V alpha 24 natural killer T cells in human immunodeficiency virus


**5** 

*Japan* 

**Constitutive Activation of the JAK/STAT and** 

Adult T-cell leukemia/lymphoma (ATLL) caused by the retrovirus human T-cell leukemia virus type 1 (HTLV-1) infection is one of aggressive mature CD4+ T-cell neoplasms with a marked expansion of leukemic cells during the acute phase. ATLL is endemic in several regions of the world, especially in southwest Japan, the Caribbean basin, and parts of Central Africa. ATLL is divided into four clinical subtypes: acute, chronic, smoldering, and lymphoma type, based on the number of leukemic cells in peripheral blood, serum lactic acid dehydrogenase level, tumor lesions in various organs, and clinical course. The acute and lymphoma types still have an extremely poor prognosis, despite the advance in chemotherapy. Chemotherapy for ATLL has limited efficacy with median survivals of

The HTLV-1 genome encodes not only structural proteins, but also non-structural proteins such as Tax, Rex, p13, p12, p30, p21Rex and HTLV-1 bZIP factor (HBZ). The functional analysis of the viral proteins such as Tax has shed light on the pathogenesis of ATLL. Tax is a crucial viral protein encoded by the pX region, which can induce viral replication and a variety of cellular genes associated with cytokine production, inhibition of apoptosis and cell cycle dysregulation (Arima 1999, Azimi 1998, Geleziunas 1998, Kanno 1994, Mori 1996b). Tax-induced gene regulation, which is linked to malignant transformation of HTLV-1-infected T-cells, has been shown to be mediated by CREB/ATF, NF-B and SRF pathways. Constitutive activation of NF-B is one of common features of HTLV-1–transformed T-cells and ATLL leukemic cells, since inhibition of NF-B activity reduces cell growth and induces apoptosis of cells, suggesting a central role of NF-B in their proliferation and survival. Moreover, Tax binds to the upstream kinase, the mitogen-activated protein kinase/ERK kinase kinase-1 and enhance its kinase activity (Harhaj 1999, Huang 2002, Jin 1999). Nevertheless, ATLL develops in a period 40 to 60 years after initial infection, indicating that the development of ATLL requires a multistep oncogenic process including accumulation of genetic mutations. HTLV-1 infection alone is not sufficient to induce neoplastic transformation of infected cells. In fact, viral gene expression is at extremely low levels in primary ATLL cells (Franchini 1984). Thus,

the mechanism which develops ATLL still remains unclear.

**1. Introduction** 

approximate one year.

**Toll-Like Receptor Signaling Pathways in** 

**Adult T-Cell Leukemia/Lymphoma** 

Hiroaki Morimoto and Junichi Tsukada *Cancer Chemotherapy Center and Hematology* 

Takehiro Higashi, Takefumi Katsuragi, Atsushi Iwashige,

*University of Occupational and Environmental Health, Kitakyushu* 

lymphotropic virus type 1 (HTLV-1) mRNA with proviral DNA load, virusspecific CD8(+) T cells, and disease severity in HTLV-1-associated myelopathy (HAMTSP). *Blood* 99, 1, 88–94


### **Constitutive Activation of the JAK/STAT and Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma**

Takehiro Higashi, Takefumi Katsuragi, Atsushi Iwashige, Hiroaki Morimoto and Junichi Tsukada *Cancer Chemotherapy Center and Hematology University of Occupational and Environmental Health, Kitakyushu* 

*Japan* 

#### **1. Introduction**

80 T-Cell Leukemia

Yamano, Y., Takenouchi, N., Li, H.C., Tomaru, U., Yao, K., Grant, C.W., Maric, D.A. &

Yamano, Y., Araya, N., Sato, T., Utsunomiya, A., Azakami, K., Hasegawa, D., Izumi, T.,

Yu, F., Itoyama, Y., Fujihara, K. & Goto, I. 1991. Natural killer (NK) cells in HTLV-I-

Yoshie, O., Imai, T. & Nomiyama, H. 2001. Chemokines in immunity. *Advances in* 

Yoshie, O., Fujisawa, R., Nakayama, T., Harasawa, H., Tago, H., Izawa, D., Hieshima, K.,

Zhu, J. & Paul, W.E. 2010. Heterogeneity and plasticity of T helper cells. Cell Research, 20, 1,

T-cell leukemia virus type 1-transformed T cells. *Blood,* 99, 5, 1505–11 Zhou, X., Bailey-Bucktrout, S.L., Jeker, L.T., Penaranda, C., Martinez-Llordella, M., Ashby,

*Blood* 99, 1, 88–94

8, e6517

4-12

*Investigations*, 115, 5, 1361–1368

Neuroimmunology, 33, 2, 121-128

vivo. *Nature Immunology,* 10, 9, 1000–1007

*Immunology,* 78, 57–110

lymphotropic virus type 1 (HTLV-1) mRNA with proviral DNA load, virusspecific CD8(+) T cells, and disease severity in HTLV-1-associated myelopathy (HAMTSP).

Jacobson, S. 2005. Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease. *Journal of Clinical* 

Fujita, H., Aratani, S., Yagishita, N., Fujii, R., Nishioka, K., Jacobson, S. & Nakajima, T. 2009. Abnormally high levels of virus-infected IFN-gamma+ CCR4+ CD4+ CD25+ T cells in a retrovirus-associated neuroinflammatory disorder. *PLoS One, 4*,

associated myelopathy/tropical spastic paraparesis-decrease in NK cell subset populations and activity in HTLV-I seropositive individuals. Journal of

Tatsumi, Y., Matsushima, K., Hasegawa, H., Kanamaru, A., Kamihira, S. & Yamada, Y. 2002. Frequent expression of CCR4 in adult T-cell leukemia and human

M., Nakayama, M., Rosenthal, W. & Bluestone, J.A. 2009. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in Adult T-cell leukemia/lymphoma (ATLL) caused by the retrovirus human T-cell leukemia virus type 1 (HTLV-1) infection is one of aggressive mature CD4+ T-cell neoplasms with a marked expansion of leukemic cells during the acute phase. ATLL is endemic in several regions of the world, especially in southwest Japan, the Caribbean basin, and parts of Central Africa. ATLL is divided into four clinical subtypes: acute, chronic, smoldering, and lymphoma type, based on the number of leukemic cells in peripheral blood, serum lactic acid dehydrogenase level, tumor lesions in various organs, and clinical course. The acute and lymphoma types still have an extremely poor prognosis, despite the advance in chemotherapy. Chemotherapy for ATLL has limited efficacy with median survivals of approximate one year.

The HTLV-1 genome encodes not only structural proteins, but also non-structural proteins such as Tax, Rex, p13, p12, p30, p21Rex and HTLV-1 bZIP factor (HBZ). The functional analysis of the viral proteins such as Tax has shed light on the pathogenesis of ATLL. Tax is a crucial viral protein encoded by the pX region, which can induce viral replication and a variety of cellular genes associated with cytokine production, inhibition of apoptosis and cell cycle dysregulation (Arima 1999, Azimi 1998, Geleziunas 1998, Kanno 1994, Mori 1996b). Tax-induced gene regulation, which is linked to malignant transformation of HTLV-1-infected T-cells, has been shown to be mediated by CREB/ATF, NF-B and SRF pathways. Constitutive activation of NF-B is one of common features of HTLV-1–transformed T-cells and ATLL leukemic cells, since inhibition of NF-B activity reduces cell growth and induces apoptosis of cells, suggesting a central role of NF-B in their proliferation and survival. Moreover, Tax binds to the upstream kinase, the mitogen-activated protein kinase/ERK kinase kinase-1 and enhance its kinase activity (Harhaj 1999, Huang 2002, Jin 1999). Nevertheless, ATLL develops in a period 40 to 60 years after initial infection, indicating that the development of ATLL requires a multistep oncogenic process including accumulation of genetic mutations. HTLV-1 infection alone is not sufficient to induce neoplastic transformation of infected cells. In fact, viral gene expression is at extremely low levels in primary ATLL cells (Franchini 1984). Thus, the mechanism which develops ATLL still remains unclear.

Constitutive Activation of the JAK/STAT

prognostic significance for STAT3 (Benekli 2002).

proliferation with constitutive JAK-STAT activity.

with poor response to therapy and overall prognosis.

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 83

Recent reports have emphasized the significance of STATs in oncogenesis (Akira 1997) and leukemogenesis (Bowman 2000, Lin 2000, Levy 2000, Coffer 2000, Akira 1997). Many oncoproteins can activate STATs. In contrast to the normal cellular response, which shows rapid and transient activation of STATs, aberrant activation of JAK-STAT signaling contributes to malignant transformation. The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) induces JAK-STAT signaling, involving Jak1 and Jak3 (Danial 1995). Interestingly, constitutive expression of a dominant-active STAT3 induces neoplastic transformation (Bromberg 1999). STAT1 and STAT5 are active in BCR-ABL-positive leukemias (Carlesso 1996, Shuai 1996, Frank 1996) and STAT1, STAT3 and STAT5 are constitutively activated in acute leukemia blasts (Gouilleux-Gruart 1996, Weber-Nordt 1996, Xia 1998, Spiekermann 2001). A constitutively active form of STAT3 can transform cells (Bromberg 1999). Constitutive activation of Jaks and STATs has been observed in murine pre-B lymphocytes transformed with the A-MuLV(Danial 1995), human B cells transformed with Epstein-Barr virus (Gouilleux-Gruart 1996) and murine erythroleukemia induced by spleen focus-forming virus (Ohashi 1995). Primary acute leukemia cells also show constitutive activation of STATs (Gouilleux-Gruart 1996, Weber-Nordt 1996, Xia 1998, Spiekermann 2001). Moreover, constitutive STAT3 activation in acute myeloid leukemia blasts has been reported to be associated with short disease-free survival, showing a

HLTV-1 infects and immortalizes primary human T-cells. In early stage, the viral regulatory proteins Tax and Rex are involved in the up-regulation of IL-2 and IL-2R. In some ATL cases, IL-2 and IL-15 can induce growth of ATLL cells (Arima 1996, Maeda 1987, Yamada 1998) (Fig. 1). Phosphorylation of STAT3 and STAT5 in ATL cells is induced by IL-2, IL-15 and IL-21 (Ueda 2005). A paracrine growth loop that involves Tax-induced IL-9 production in ATL cells

However, constitutive activation of the JAK-STAT pathway is generally correlated with IL-2 independence. Transformation of T-cells by HTLV-1 is associated with constitutive activation of the JAK-STAT pathway (Migone 1995, Xu 1995) (Fig1). Migone *et al*. (Migone 1995) demonstrated activation of Jak1, Jak3, STAT3 and STAT5 correlated with the transition from an IL-2-dependent to an IL-2-independent phase in HTLV-1-transformed cells. Spontaneous phosphorylation of Jak2 and Jak3 has also been observed in T-cells transformed with HTLV-1 (Xu 1995). Leukemia cells obtained from ATL patients also showed constitutive activation of STATs (Takemoto 1997, Tsukada 2000). Takemoto *et al*.(Takemoto 1997) observed constitutive activation of STAT1, STAT3 and STAT5 in leukemic cells of ATL patients, and demonstrated the association of leukemic cell

In addition, no gain-of-function mutations of the Jak1 and Jak3 in primary ATLL cells has been detected (Kameda 2010). These data are contrast to the results obtained from acute Tlymphoblastic leukemia (T-ALL). Flex *et al*. demonstrated that *JAK1* gene mutations occur in ALL and are more frequently observed among adult individuals with involvement of the T cell lineage (Flex 2008). The mutations promote gain of kinase function, and are associated

**2.1 Unique function of the Jak-STAT signaling pathway in HTLV-1-infected T-cells**  HTLV-1 infection up-regulates expression of the suppressor of cytokine signaling 1 (SOCS1). HTLV-1-induced SOCS1 inhibits the type I IFN antiviral response against HTLV-1 by

and expression of IL-9 receptor on monocytes has been also observed (Chen 2008).

The general subject of signaling pathways in ATLL cells and HTLV-1-transformed cells has been covered by many excellent original reports and reviews. In this review, authors focus on recent advances of two signaling pathways in ATLL cells and HTLV-1–transformed Tcells; the JAK (Janus kinase)-STAT (signal transducer and activator of transcription) and TLR (Toll-like receptor) signaling pathways.

#### **2. Constitutive activation of the JAK-STAT signaling pathway in ATLL cells and HTLV-1-transformed T-cells**

STAT proteins play important roles in regulating cellular response to a variety of cytokines. STATs are latent cytosolic transcriptional factors that are activated by tyrosine phosphorylation in response to cytokines. The four mammalian members of the JAK family (Jak1, Jak2, Jak3, and Tyk2) are non-receptor tyrosine kinases functioning as signal transducers, that control activation of STATs. Jaks associate constitutively with cytokine receptors, and promote signals by phosphorylating tyrosine residues of activated receptors to allow the recruitment and activation of STAT proteins. STATs can form homo- or heterodimers in which amino acid sequence containing a phospho-tyrosine residue in one partner binds to the SH2 domain in the other vice versa, leading to nuclear translocation of STAT dimers and their participation in transcriptional regulation of various cytokine responsive genes (Darnell 1997, Ihle 2001, Leonard 1998).

Fig. 1. The JAK-STAT signaling pathway in ATLL cells and HTLV-1-transformed T-cells.

The general subject of signaling pathways in ATLL cells and HTLV-1-transformed cells has been covered by many excellent original reports and reviews. In this review, authors focus on recent advances of two signaling pathways in ATLL cells and HTLV-1–transformed Tcells; the JAK (Janus kinase)-STAT (signal transducer and activator of transcription) and

**2. Constitutive activation of the JAK-STAT signaling pathway in ATLL cells** 

Fig. 1. The JAK-STAT signaling pathway in ATLL cells and HTLV-1-transformed T-cells.

STAT proteins play important roles in regulating cellular response to a variety of cytokines. STATs are latent cytosolic transcriptional factors that are activated by tyrosine phosphorylation in response to cytokines. The four mammalian members of the JAK family (Jak1, Jak2, Jak3, and Tyk2) are non-receptor tyrosine kinases functioning as signal transducers, that control activation of STATs. Jaks associate constitutively with cytokine receptors, and promote signals by phosphorylating tyrosine residues of activated receptors to allow the recruitment and activation of STAT proteins. STATs can form homo- or heterodimers in which amino acid sequence containing a phospho-tyrosine residue in one partner binds to the SH2 domain in the other vice versa, leading to nuclear translocation of STAT dimers and their participation in transcriptional regulation of various cytokine

TLR (Toll-like receptor) signaling pathways.

responsive genes (Darnell 1997, Ihle 2001, Leonard 1998).

**and HTLV-1-transformed T-cells** 

Recent reports have emphasized the significance of STATs in oncogenesis (Akira 1997) and leukemogenesis (Bowman 2000, Lin 2000, Levy 2000, Coffer 2000, Akira 1997). Many oncoproteins can activate STATs. In contrast to the normal cellular response, which shows rapid and transient activation of STATs, aberrant activation of JAK-STAT signaling contributes to malignant transformation. The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) induces JAK-STAT signaling, involving Jak1 and Jak3 (Danial 1995). Interestingly, constitutive expression of a dominant-active STAT3 induces neoplastic transformation (Bromberg 1999). STAT1 and STAT5 are active in BCR-ABL-positive leukemias (Carlesso 1996, Shuai 1996, Frank 1996) and STAT1, STAT3 and STAT5 are constitutively activated in acute leukemia blasts (Gouilleux-Gruart 1996, Weber-Nordt 1996, Xia 1998, Spiekermann 2001). A constitutively active form of STAT3 can transform cells (Bromberg 1999). Constitutive activation of Jaks and STATs has been observed in murine pre-B lymphocytes transformed with the A-MuLV(Danial 1995), human B cells transformed with Epstein-Barr virus (Gouilleux-Gruart 1996) and murine erythroleukemia induced by spleen focus-forming virus (Ohashi 1995). Primary acute leukemia cells also show constitutive activation of STATs (Gouilleux-Gruart 1996, Weber-Nordt 1996, Xia 1998, Spiekermann 2001). Moreover, constitutive STAT3 activation in acute myeloid leukemia blasts has been reported to be associated with short disease-free survival, showing a prognostic significance for STAT3 (Benekli 2002).

HLTV-1 infects and immortalizes primary human T-cells. In early stage, the viral regulatory proteins Tax and Rex are involved in the up-regulation of IL-2 and IL-2R. In some ATL cases, IL-2 and IL-15 can induce growth of ATLL cells (Arima 1996, Maeda 1987, Yamada 1998) (Fig. 1). Phosphorylation of STAT3 and STAT5 in ATL cells is induced by IL-2, IL-15 and IL-21 (Ueda 2005). A paracrine growth loop that involves Tax-induced IL-9 production in ATL cells and expression of IL-9 receptor on monocytes has been also observed (Chen 2008).

However, constitutive activation of the JAK-STAT pathway is generally correlated with IL-2 independence. Transformation of T-cells by HTLV-1 is associated with constitutive activation of the JAK-STAT pathway (Migone 1995, Xu 1995) (Fig1). Migone *et al*. (Migone 1995) demonstrated activation of Jak1, Jak3, STAT3 and STAT5 correlated with the transition from an IL-2-dependent to an IL-2-independent phase in HTLV-1-transformed cells. Spontaneous phosphorylation of Jak2 and Jak3 has also been observed in T-cells transformed with HTLV-1 (Xu 1995). Leukemia cells obtained from ATL patients also showed constitutive activation of STATs (Takemoto 1997, Tsukada 2000). Takemoto *et al*.(Takemoto 1997) observed constitutive activation of STAT1, STAT3 and STAT5 in leukemic cells of ATL patients, and demonstrated the association of leukemic cell proliferation with constitutive JAK-STAT activity.

In addition, no gain-of-function mutations of the Jak1 and Jak3 in primary ATLL cells has been detected (Kameda 2010). These data are contrast to the results obtained from acute Tlymphoblastic leukemia (T-ALL). Flex *et al*. demonstrated that *JAK1* gene mutations occur in ALL and are more frequently observed among adult individuals with involvement of the T cell lineage (Flex 2008). The mutations promote gain of kinase function, and are associated with poor response to therapy and overall prognosis.

#### **2.1 Unique function of the Jak-STAT signaling pathway in HTLV-1-infected T-cells**

HTLV-1 infection up-regulates expression of the suppressor of cytokine signaling 1 (SOCS1). HTLV-1-induced SOCS1 inhibits the type I IFN antiviral response against HTLV-1 by

Constitutive Activation of the JAK/STAT

2006a, Tomita 2006b).

**transformed with HTLV-1** 

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 85

co-immunopreciptates with Jak3 in HTLV-1-transformed cell lines HUT102, MT2, MS9 and MS68. Phosphorylation of STAT5 and STAT3 are inhibited by cell treatment with the Src kinase inhibitor PP2 or by ectopic expression of a dominant negative Lyn kinase protein (Shuh 2011). Dequelin, a naturally occurring retinoid shows anti-proliferative effect on HTLV-1-transformed cells, MT-2 and KUT-1 in part through the down-regulation of survivin and constitutive phosphorylation of STAT3. In contrast, STAT5 phosphorylation is not affected by Deguelin (Ito 2010). roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) inhibits STAT5 activity required for the survival of MT-2 cells. A dominant negative STAT5 expression induces apoptosis and reduces the abundance of an anti-apoptotic protein XIAP in MT-2 cells. In ChIP assay, interaction of STAT5, but not STAT1 with the XIAP promoter has been observed. Interaction of STAT5 and PDGF receptor is also prevented by roscovitine (Mohapatra 2003). Curcumin (diferuloylmethane), a naturally occurring yellow pigment isolated from the rhizomes of the plant *Curcuma longa*, induces apoptosis and anti-proliferative response in HTLV-1-transformed cells; MT-2, HUT102 and SLB-1. These responses are associated with inhibition of constitutive phosphorylation of Jak3, Tyk2, STAT3 and STAT5. Additionally, AP-1, especially JunD and NF-B activity in HTLV-1-transfromed T-cells and primary ATLL cells are also inhibited by curcumin (Tomita

In a clinical study, Berkowitz et al. reported a single institute open-label phase II trial of intravenous daclizumab, a humanized monoclonal antibody that binds specifically to the alpha (CD25) subunit of the high-affinity IL-2R in ATLL patients. No responses were observed in aggressive acute or lymphoma type of ATLL. Partial responses were observed

In addition, Tasocitinib (CP-690,550), a potent and selective Jak3 inhibitor is an orally active immunosuppressant undergoing clinical trials for the treatment of autoimmune diseases and transplant rejection. It is interesting to note that Tasocitinib (CP-690,550) inhibits proliferation of peripheral blood mononuclear cells (PBMCs) from patients with chronic and smoldering form of ATLL or with HAM/TSP that manifest constitutive Jak3/STAT5 activation. This agent prolongs the survival of transgenic mice bearing human CD8 T-cell leukemia with IL-15/IL-15R autocrine growth loop required for leukemia cell survival (Ju 2011). These results suggest clinical effect of CP-690,550 on chronic and smoldering ATLL.

STAT4 is a crucial mediator of IL-12-stimulated gene regulation (Jacobson 1995, Bacon 1995b). In fact, the development of type-1 helper T (Th1) cells and production of IFN-γ in response to IL-12 are disrupted in STAT4-deficient mice (Thierfelder 1996, Kaplan 1996). STAT4 is phosphorylated on tyrosine by Jak2 and Tyk2 (Bacon 1995a, Cho 1996). Moreover, IL-12 activates the p38/MKK6 signaling pathway that in turn phosphorylates STAT4 on serine (Visconti 2000). Activation of p38 and its upstream activator MKK6 is an important step for IL-12-induced STAT4 transcriptional activity (Visconti 2000, Zhang 2000). In fact, previous studies indicated that IFN-γ production is blocked by a p38 inhibitor (Zhang 2000, Rincon 1998). Transgenic mice expressing a dominant-negative p38 showed impaired Th1 differentiation (Rincon 1998). The expression of STAT4 is observed in limited types of tissues such as testis, spleen, lung, bone marrow, thymus and muscle (Zhong 1994, Yamamoto 1994). Several T-cell lines including EL4 and DA2 contain no

in 36% of patients with chronic and smoldering ATLL (Berkowitz JL 2010).

**2.1.2 Constitutive tyrosine and serine phosphorylation of STAT4 in T-cells** 

targeting IRF3 for SOCS1-induced proteasome degradation (Oliere 2010). As an adaptor, SOCS1 brings target proteins to the elongin B/C-Cullin E3 ligase complex for ubiquitination. It may represent an immune evasion strategy and survival advantage to HTLV-1-infected cells. HTLV-1 inhibits IFN-induced phosphorylation of STAT2 and Tyk2 (Feng 2008). Zhang *et al*. further indicate that Tax interferes with IFN--induced JAK-STAT signaling by completion with STAT2 for CBP/p300 binding (Zhang 2008) (Fig2).

Fig. 2. Constitutive association of MyD88 with IRAK in HTLV-1-transformed T-cells.

The viral p12 protein from the pX open reading frame I (ORFI) activates Jak1/3 and STAT5, and decrease the IL-2-requirement for T-cell proliferation *via* binding to the cytoplasmic domain of IL-2R chain (Nicot 2001). Although the IL-2-Jak-STAT pathway is not associated with viral gene expression, viral RNA encapsidation, the maturation of the viral particle, cell-cell adherence or Gag polarization, p12 enhances viral transmission through activation of the IL-2-Jak-STAT pathway (Taylor 2009).

#### **2.1.1 Inhibition of the JAK-STAT signaling pathway in ATLL cells and HTLV-1 transformed T-cells; therapeutic approach**

Several recent studies reported inhibition of constitutive activation of the JAK-STAT signaling pathway in ATLL cells and HTLV-1-transformed T-cells. Src-related kinase Lyn

targeting IRF3 for SOCS1-induced proteasome degradation (Oliere 2010). As an adaptor, SOCS1 brings target proteins to the elongin B/C-Cullin E3 ligase complex for ubiquitination. It may represent an immune evasion strategy and survival advantage to HTLV-1-infected cells. HTLV-1 inhibits IFN-induced phosphorylation of STAT2 and Tyk2 (Feng 2008). Zhang *et al*. further indicate that Tax interferes with IFN--induced JAK-STAT

signaling by completion with STAT2 for CBP/p300 binding (Zhang 2008) (Fig2).

Fig. 2. Constitutive association of MyD88 with IRAK in HTLV-1-transformed T-cells.

**2.1.1 Inhibition of the JAK-STAT signaling pathway in ATLL cells and HTLV-1-**

of the IL-2-Jak-STAT pathway (Taylor 2009).

**transformed T-cells; therapeutic approach** 

The viral p12 protein from the pX open reading frame I (ORFI) activates Jak1/3 and STAT5, and decrease the IL-2-requirement for T-cell proliferation *via* binding to the cytoplasmic domain of IL-2R chain (Nicot 2001). Although the IL-2-Jak-STAT pathway is not associated with viral gene expression, viral RNA encapsidation, the maturation of the viral particle, cell-cell adherence or Gag polarization, p12 enhances viral transmission through activation

Several recent studies reported inhibition of constitutive activation of the JAK-STAT signaling pathway in ATLL cells and HTLV-1-transformed T-cells. Src-related kinase Lyn co-immunopreciptates with Jak3 in HTLV-1-transformed cell lines HUT102, MT2, MS9 and MS68. Phosphorylation of STAT5 and STAT3 are inhibited by cell treatment with the Src kinase inhibitor PP2 or by ectopic expression of a dominant negative Lyn kinase protein (Shuh 2011). Dequelin, a naturally occurring retinoid shows anti-proliferative effect on HTLV-1-transformed cells, MT-2 and KUT-1 in part through the down-regulation of survivin and constitutive phosphorylation of STAT3. In contrast, STAT5 phosphorylation is not affected by Deguelin (Ito 2010). roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) inhibits STAT5 activity required for the survival of MT-2 cells. A dominant negative STAT5 expression induces apoptosis and reduces the abundance of an anti-apoptotic protein XIAP in MT-2 cells. In ChIP assay, interaction of STAT5, but not STAT1 with the XIAP promoter has been observed. Interaction of STAT5 and PDGF receptor is also prevented by roscovitine (Mohapatra 2003). Curcumin (diferuloylmethane), a naturally occurring yellow pigment isolated from the rhizomes of the plant *Curcuma longa*, induces apoptosis and anti-proliferative response in HTLV-1-transformed cells; MT-2, HUT102 and SLB-1. These responses are associated with inhibition of constitutive phosphorylation of Jak3, Tyk2, STAT3 and STAT5. Additionally, AP-1, especially JunD and NF-B activity in HTLV-1-transfromed T-cells and primary ATLL cells are also inhibited by curcumin (Tomita 2006a, Tomita 2006b).

In a clinical study, Berkowitz et al. reported a single institute open-label phase II trial of intravenous daclizumab, a humanized monoclonal antibody that binds specifically to the alpha (CD25) subunit of the high-affinity IL-2R in ATLL patients. No responses were observed in aggressive acute or lymphoma type of ATLL. Partial responses were observed in 36% of patients with chronic and smoldering ATLL (Berkowitz JL 2010).

In addition, Tasocitinib (CP-690,550), a potent and selective Jak3 inhibitor is an orally active immunosuppressant undergoing clinical trials for the treatment of autoimmune diseases and transplant rejection. It is interesting to note that Tasocitinib (CP-690,550) inhibits proliferation of peripheral blood mononuclear cells (PBMCs) from patients with chronic and smoldering form of ATLL or with HAM/TSP that manifest constitutive Jak3/STAT5 activation. This agent prolongs the survival of transgenic mice bearing human CD8 T-cell leukemia with IL-15/IL-15R autocrine growth loop required for leukemia cell survival (Ju 2011). These results suggest clinical effect of CP-690,550 on chronic and smoldering ATLL.

#### **2.1.2 Constitutive tyrosine and serine phosphorylation of STAT4 in T-cells transformed with HTLV-1**

STAT4 is a crucial mediator of IL-12-stimulated gene regulation (Jacobson 1995, Bacon 1995b). In fact, the development of type-1 helper T (Th1) cells and production of IFN-γ in response to IL-12 are disrupted in STAT4-deficient mice (Thierfelder 1996, Kaplan 1996). STAT4 is phosphorylated on tyrosine by Jak2 and Tyk2 (Bacon 1995a, Cho 1996). Moreover, IL-12 activates the p38/MKK6 signaling pathway that in turn phosphorylates STAT4 on serine (Visconti 2000). Activation of p38 and its upstream activator MKK6 is an important step for IL-12-induced STAT4 transcriptional activity (Visconti 2000, Zhang 2000). In fact, previous studies indicated that IFN-γ production is blocked by a p38 inhibitor (Zhang 2000, Rincon 1998). Transgenic mice expressing a dominant-negative p38 showed impaired Th1 differentiation (Rincon 1998). The expression of STAT4 is observed in limited types of tissues such as testis, spleen, lung, bone marrow, thymus and muscle (Zhong 1994, Yamamoto 1994). Several T-cell lines including EL4 and DA2 contain no

Constitutive Activation of the JAK/STAT

(Takeuchi 2007).

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 87

same study group reported that AZD1152, a selective inhibitor for Aurora B kinase had no

The TLR comprise a subfamily within the larger superfamily of interleukin (IL) receptors, based on similarity within their cytoplasmic regions (Dunne 2003, Matsushima 2007, McGettrick 2007, Takeda 2003) The extracellular region of the IL-1 receptors (IL-1Rs) possesses three immunoglobulin-like domains, those of TLRs are characterized by the presence of 16-28 leucine-rich repeats. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which, in turn, promotes its association with the IL-1R–associated kinase (IRAK) *via* an interaction between the respective death domains of each molecule. This is followed by autophosphorylation of IRAK that results in dissociation from the receptor complex and its subsequent interaction with tumor-necrosis-factor (TNF) receptor–associated factor-6 (TRAF-6). Emanating from TRAF-6, two signaling pathways diverge, one eventually leading to NF-B activation, and another to mitogen-activated protein (MAP) kinase activation

In this regard, our study demonstrated an alternative mechanism of NF-B activation through the TLR signaling cascade including MyD88 and IRAK in HTLV-1-–transformed Tcells and ATLL cells (Fig. 2). MyD88 and IRAK1 are constitutively active in HTLV-1 transformed T-cells, but not in HTLV-1-negative T-cells (Mizobe 2007). MyD88, originally isolated as a myeloid differentiation primary response gene product, possesses its Cterminal domain, which is highly homologous to the cytoplasmic regions of the TLR family of proteins (Dunne 2003, Takeda 2003). However, unlike members of the TLR family, MyD88 contains no transmembrane domain. MyD88 acts an adaptor molecule of most TLRs and receptors for IL-1 and IL-18 to recruit IRAK to the TLR complex, thereby regulating activation of various transcription factors involved in inflammatory responses, such as NF- B and C/EBP (NF-IL6) (Akira 2003a, Akira 2001, Akira 2003b, Boch 2003, Burns 1998,

Expression of a dominant negative MyD88 (MyD88dn) lacking its death domain (DD), MyD88dn induces apoptosis and anti-proliferative response in HTLV-1-transformed T-cells. In HTLV-1-transformed T-cells, MyD88dn protein expression inhibits constitutive activation of C/EBP (NF-IL6) and NF-B, and proinflamatory cytokine gene promoters such as IL-1, IFN- and TNF-. Furthermore, Tax synergistically activates NF-B with MyD88 (Mizobe 2007). The synergy may suggest ligand-independent activation of MyD88 in HTLV-1 transformed cells (Fig. 2). However, NF-B activation has been observed even in ATLL cells lacking detectable Tax expression. The mechanism for activation of NF-B in ATLL cells is not still clear. A recent study reported contribution of elevated CD30 expression to constitutive activation of NF-B in ATLL cells (Higuchi 2005). In this regard, the noncanonical pathway for NF-B activation, induced by B-cell activation factor (Claudio 2002, Kayagaki 2002), lymphotoxin- (Dejardin 2002, Saitoh 2002), CD40 (Coope 2002), TNF-like weak inducer of apoptosis (Saitoh 2003) or CD30 (Higuchi 2005) may be also involved in

In addition, MT-2 cells express TLR-1, 6 and 10 mRNA. Several recent reports have indicated unique expression profiles of TLRs on different subsets of T-cells. Gelman *et al*. (Gelman 2004) reported that TLR-3, -5 and -9 are expressed selectively on activated human CD4+ T cells, and that treatment of activated human CD4+ T-cells, with dsRNA synthetic analogs, poly(I:C) and CpGoligodeoxynucleotides (CpG DNA), directly enhance their

effect on NF-B activity in MT-4 and HUT102 cells (Tomita 2010).

Dunne 2003, Jefferies 2001, Muzio 2000, O'Neill 2003, Takeda 2003).

constitutive NF-B activation in ATLL cells (Hironaka 2004).

STAT4 transcripts (Yamamoto 1994). However, our study showed that tyrosinephosphorylated STAT4 was detected in HTLV-1-transformed cell lines. In addition, STAT4 protein was constitutively phosphorylated on serine as well as on tyrosine in HTLV-1-transformed cell lines (Higashi 2005).

The relevance of phosphorylation of serine in STAT4 has been recently reported. Serine phosphorylation of STAT4 is dispensable for nuclear translocation or DNA binding of STAT4, but is indispensable for its maximal transcriptional activity (Visconti 2000). Serine phosphorylation of STAT4 is required for IL-12-induced IFN-γ production and IL-12 mediated Th1 development, but not for IL-12-induced cell proliferation (Morinobu 2002). Furthermore, they have shown that serine phosphorylation of STAT4 is partially dependent on precedent tyrosine phosphorylation of STAT4, whereas tyrosine phosphorylation of STAT4 can be seen even in the absence of serine phosphorylation. In contrast, it has been shown that in leukemic cells from chronic lymphocytic leukemia patients, STAT1 and STAT3 are constitutively phosphorylated on serine, but not on tyrosine residue (Frank 1997). In the other leukemias such as AML and ALL, serine phosphorylation of the STATs was occasionally seen (Frank 1997, Hayakawa 1998). Thus, STATs may have selective effects on gene expression of leukemia cells in a manner dependent upon serine phosphorylation. We observed that IFN-γ, but not IL-12 or IFN-α was produced in HTLV-1-transformed cells.

#### **2.1.3 Constitutive association of MyD88 to IRAK in HTLV-1 -transformed cells**

Aberrant cytokine gene expression is a hallmark of ATLL cells (Franchini 1995, Grossman 1997, Kanno 1994, Mori 1999, Mori 1996a, Mori 1996b, Siekevitz 1987, Yamada 1996). The cytokine gene promoters possess enhancer elements for NF-B and/or C/EBP (NF-IL6) (Azimi 1998, Faggioli 1996, Mercurio 1997, Perkins 1997, Schmitz 1995, Washizu 1998). C/EBP (NF-IL6), β isoform of CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors (Tsukada 2011) was originally identified as a nuclear factor that binds to IL-1-responsive element in the *IL6* gene (Akira 1990). Moreover, inhibition of NF-kB activity results in enhanced apoptosis and growth suppression of primary ATLL cells and HTLV-1-transformed T-cells, indicating a central role for NF-B in their survival and proliferation. Antisense oligonucleotides to RelA/p65 inhibit Tax-transformed tumor cell growth (Kitajima 1992). Sodium salicylate and cyclopentenone prostaglandins suppress proliferation of Tax-transgenic mouse spleen cells (Portis 2001). Bay-7082, an inhibitor of IB phosphorylation induces apoptosis of HTLV-1-transformed T-cell lines and primary ATLL cells *via* reduced expression of the anti-apoptotic gene BCL-XL (Mori 2002). *Ex vivo*  treatment of PBMCs with dehydroxymethylepoxyquinomicin selectively purges HTLV-1 infected cells without toxicity to normal cells in HTLV-1 carriers (Watanabe 2005). More recently, activation of the classical pathway of NF-B by the HBZ has been reported (Zhao 2009). HBZ does not affect the alternative pathway of NF-B, but induces polyubiquitination and degradation of p65. Yasunaga *et al*. demonstrated that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses Tax-induced NF-B activation (Yasunaga 2011). Several agents such as Bidens pilosa, a plant found in tropical and subtropical regions (Nakama 2011) and hippuristanol, an eukaryotic translation initiation inhibitor from the coral Isis hippuris (Tsumuraya 2011) also show inhibitory effect on ATLL cells through suppression of NF-B actitivty. On the other hand, pan-aurora kinase inhibitor has been shown to have anti-proliferative effect on HTLV-1-transformed T-cells and primary ATLL cells through the suppression of NF-B activity (Tomita 2009). However, the

STAT4 transcripts (Yamamoto 1994). However, our study showed that tyrosinephosphorylated STAT4 was detected in HTLV-1-transformed cell lines. In addition, STAT4 protein was constitutively phosphorylated on serine as well as on tyrosine in

The relevance of phosphorylation of serine in STAT4 has been recently reported. Serine phosphorylation of STAT4 is dispensable for nuclear translocation or DNA binding of STAT4, but is indispensable for its maximal transcriptional activity (Visconti 2000). Serine phosphorylation of STAT4 is required for IL-12-induced IFN-γ production and IL-12 mediated Th1 development, but not for IL-12-induced cell proliferation (Morinobu 2002). Furthermore, they have shown that serine phosphorylation of STAT4 is partially dependent on precedent tyrosine phosphorylation of STAT4, whereas tyrosine phosphorylation of STAT4 can be seen even in the absence of serine phosphorylation. In contrast, it has been shown that in leukemic cells from chronic lymphocytic leukemia patients, STAT1 and STAT3 are constitutively phosphorylated on serine, but not on tyrosine residue (Frank 1997). In the other leukemias such as AML and ALL, serine phosphorylation of the STATs was occasionally seen (Frank 1997, Hayakawa 1998). Thus, STATs may have selective effects on gene expression of leukemia cells in a manner dependent upon serine phosphorylation. We observed that IFN-γ, but not IL-12 or IFN-α

**2.1.3 Constitutive association of MyD88 to IRAK in HTLV-1 -transformed cells** 

Aberrant cytokine gene expression is a hallmark of ATLL cells (Franchini 1995, Grossman 1997, Kanno 1994, Mori 1999, Mori 1996a, Mori 1996b, Siekevitz 1987, Yamada 1996). The cytokine gene promoters possess enhancer elements for NF-B and/or C/EBP (NF-IL6) (Azimi 1998, Faggioli 1996, Mercurio 1997, Perkins 1997, Schmitz 1995, Washizu 1998). C/EBP (NF-IL6), β isoform of CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors (Tsukada 2011) was originally identified as a nuclear factor that binds to IL-1-responsive element in the *IL6* gene (Akira 1990). Moreover, inhibition of NF-kB activity results in enhanced apoptosis and growth suppression of primary ATLL cells and HTLV-1-transformed T-cells, indicating a central role for NF-B in their survival and proliferation. Antisense oligonucleotides to RelA/p65 inhibit Tax-transformed tumor cell growth (Kitajima 1992). Sodium salicylate and cyclopentenone prostaglandins suppress proliferation of Tax-transgenic mouse spleen cells (Portis 2001). Bay-7082, an inhibitor of IB phosphorylation induces apoptosis of HTLV-1-transformed T-cell lines and primary ATLL cells *via* reduced expression of the anti-apoptotic gene BCL-XL (Mori 2002). *Ex vivo*  treatment of PBMCs with dehydroxymethylepoxyquinomicin selectively purges HTLV-1 infected cells without toxicity to normal cells in HTLV-1 carriers (Watanabe 2005). More recently, activation of the classical pathway of NF-B by the HBZ has been reported (Zhao 2009). HBZ does not affect the alternative pathway of NF-B, but induces polyubiquitination and degradation of p65. Yasunaga *et al*. demonstrated that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses Tax-induced NF-B activation (Yasunaga 2011). Several agents such as Bidens pilosa, a plant found in tropical and subtropical regions (Nakama 2011) and hippuristanol, an eukaryotic translation initiation inhibitor from the coral Isis hippuris (Tsumuraya 2011) also show inhibitory effect on ATLL cells through suppression of NF-B actitivty. On the other hand, pan-aurora kinase inhibitor has been shown to have anti-proliferative effect on HTLV-1-transformed T-cells and primary ATLL cells through the suppression of NF-B activity (Tomita 2009). However, the

HTLV-1-transformed cell lines (Higashi 2005).

was produced in HTLV-1-transformed cells.

same study group reported that AZD1152, a selective inhibitor for Aurora B kinase had no effect on NF-B activity in MT-4 and HUT102 cells (Tomita 2010).

The TLR comprise a subfamily within the larger superfamily of interleukin (IL) receptors, based on similarity within their cytoplasmic regions (Dunne 2003, Matsushima 2007, McGettrick 2007, Takeda 2003) The extracellular region of the IL-1 receptors (IL-1Rs) possesses three immunoglobulin-like domains, those of TLRs are characterized by the presence of 16-28 leucine-rich repeats. Engagement of IL-1R or TLR with their cognate ligands causes an adaptor protein MyD88 to be recruited to the receptor complex, which, in turn, promotes its association with the IL-1R–associated kinase (IRAK) *via* an interaction between the respective death domains of each molecule. This is followed by autophosphorylation of IRAK that results in dissociation from the receptor complex and its subsequent interaction with tumor-necrosis-factor (TNF) receptor–associated factor-6 (TRAF-6). Emanating from TRAF-6, two signaling pathways diverge, one eventually leading to NF-B activation, and another to mitogen-activated protein (MAP) kinase activation (Takeuchi 2007).

In this regard, our study demonstrated an alternative mechanism of NF-B activation through the TLR signaling cascade including MyD88 and IRAK in HTLV-1-–transformed Tcells and ATLL cells (Fig. 2). MyD88 and IRAK1 are constitutively active in HTLV-1 transformed T-cells, but not in HTLV-1-negative T-cells (Mizobe 2007). MyD88, originally isolated as a myeloid differentiation primary response gene product, possesses its Cterminal domain, which is highly homologous to the cytoplasmic regions of the TLR family of proteins (Dunne 2003, Takeda 2003). However, unlike members of the TLR family, MyD88 contains no transmembrane domain. MyD88 acts an adaptor molecule of most TLRs and receptors for IL-1 and IL-18 to recruit IRAK to the TLR complex, thereby regulating activation of various transcription factors involved in inflammatory responses, such as NF- B and C/EBP (NF-IL6) (Akira 2003a, Akira 2001, Akira 2003b, Boch 2003, Burns 1998, Dunne 2003, Jefferies 2001, Muzio 2000, O'Neill 2003, Takeda 2003).

Expression of a dominant negative MyD88 (MyD88dn) lacking its death domain (DD), MyD88dn induces apoptosis and anti-proliferative response in HTLV-1-transformed T-cells. In HTLV-1-transformed T-cells, MyD88dn protein expression inhibits constitutive activation of C/EBP (NF-IL6) and NF-B, and proinflamatory cytokine gene promoters such as IL-1, IFN- and TNF-. Furthermore, Tax synergistically activates NF-B with MyD88 (Mizobe 2007). The synergy may suggest ligand-independent activation of MyD88 in HTLV-1 transformed cells (Fig. 2). However, NF-B activation has been observed even in ATLL cells lacking detectable Tax expression. The mechanism for activation of NF-B in ATLL cells is not still clear. A recent study reported contribution of elevated CD30 expression to constitutive activation of NF-B in ATLL cells (Higuchi 2005). In this regard, the noncanonical pathway for NF-B activation, induced by B-cell activation factor (Claudio 2002, Kayagaki 2002), lymphotoxin- (Dejardin 2002, Saitoh 2002), CD40 (Coope 2002), TNF-like weak inducer of apoptosis (Saitoh 2003) or CD30 (Higuchi 2005) may be also involved in constitutive NF-B activation in ATLL cells (Hironaka 2004).

In addition, MT-2 cells express TLR-1, 6 and 10 mRNA. Several recent reports have indicated unique expression profiles of TLRs on different subsets of T-cells. Gelman *et al*. (Gelman 2004) reported that TLR-3, -5 and -9 are expressed selectively on activated human CD4+ T cells, and that treatment of activated human CD4+ T-cells, with dsRNA synthetic analogs, poly(I:C) and CpGoligodeoxynucleotides (CpG DNA), directly enhance their

Constitutive Activation of the JAK/STAT

1168-75, 0301-472X.

survival. *Blood,* 99, 1, 252-7.

(ATL). *J Clin Oncol,* 28, 15s, suppl; abstr 8043.

*Biochem Biophys Res Commun,* 303, 2, 525-31, 0006-291X.

JE, Jr. (1999) Stat3 as an oncogene. *Cell,* 98, 3, 295-303.

*J Biol Chem,* 273, 20, 12203-9, 0021-9258..

0027-8424

399-404.

2474-88.

1551-7, 0022-1767..

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 89

Azimi, N, Brown, K, Bamford, RN, Tagaya, Y, Siebenlist, U & Waldmann, TA (1998) Human

Bacon, CM, McVicar, DW, Ortaldo, JR, Rees, RC, O'Shea, JJ & Johnston, JA (1995a)

Bacon, CM, Petricoin, EF, 3rd, Ortaldo, JR, Rees, RC, Larner, AC, Johnston, JA & O'Shea, JJ

Benekli, M, Xia, Z, Donohue, KA, Ford, LA, Pixley, LA, Baer, MR, Baumann, H & Wetzler,

Berkowitz JL, JJ, Stewart DM, Fioravanti S, Jaffe ES, Fleisher TA, Urquhart N,Wharfe JH,

Boch, JA, Yoshida, Y, Koyama, Y, Wara-Aswapati, N, Peng, H, Unlu, S & Auron, PE (2003)

Bowman, T, Garcia, R, Turkson, J & Jove, R (2000) STATs in oncogenesis. *Oncogene,* 19, 21,

Bromberg, JF, Wrzeszczynska, MH, Devgan, G, Zhao, Y, Pestell, RG, Albanese, C & Darnell,

Burns, K, Martinon, F, Esslinger, C, Pahl, H, Schneider, P, Bodmer, JL, Di Marco, F, French, L

Carlesso, N, Frank, DA & Griffin, JD (1996) Tyrosyl phosphorylation and DNA binding

hematopoietic cell lines transformed by Bcr/Abl. *J Exp Med,* 183, 3, 811-20. Caron, G, Duluc, D, Fremaux, I, Jeannin, P, David, C, Gascan, H & Delneste, Y (2005) Direct

Chen, J, Petrus, M, Bryant, BR, Phuc Nguyen, V, Stamer, M, Goldman, CK, Bamford, R,

Cho, SS, Bacon, CM, Sudarshan, C, Rees, RC, Finbloom, D, Pine, R & O'Shea, JJ (1996)

induced tyrosine and serine phosphorylation. *J Immunol,* 157, 11, 4781-9.

by a paracrine mechanism. *Blood,* 111, 10, 5163-72, 1528-0020.

human lymphocytes. *Proc Natl Acad Sci U S A,* 92, 16, 7307-11.

primary adult T-cell leukemia cells and its clinical implications. *Exp Hematol,* 27, 7,

T cell lymphotropic virus type I Tax protein trans-activates interleukin 15 gene transcription through an NF-B site. *Proc Natl Acad Sci U S A,* 95, 5, 2452-7,

Interleukin 12 (IL-12) induces tyrosine phosphorylation of JAK2 and TYK2: differential use of Janus family tyrosine kinases by IL-2 and IL-12. *J Exp Med,* 181, 1,

(1995b) Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in

M (2002) Constitutive activity of signal transducer and activator of transcription 3 protein in acute myeloid leukemia blasts is associated with short disease-free

Waldmann TA, Morris JC (2010) Phase II trial of daclizumab in human T-cell lymphotropic virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma

Characterization of a cascade of protein interactions initiated at the IL-1 receptor.

& Tschopp, J (1998) MyD88, an adapter protein involved in interleukin-1 signaling.

activity of signal transducers and activators of transcription (STAT) proteins in

stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN- production by memory CD4+ T cells. *J Immunol,* 175, 3,

Morris, JC, Janik, JE & Waldmann, TA (2008) Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell leukemia cells

Activation of STAT4 by IL-12 and IFN-: evidence for the involvement of ligand-

survival without affecting proliferation. A TLR-5 ligand flagellin and a TLR7/8 ligand R-848 promotes proliferation and to upregulate production of IFN-, IL-8 and IL-10, but not IL-4, in human CD4+ T-cells (Caron 2005). In particular, engagement of TLR-5 with flagellin enhances the suppressive capacity and FOXP3 expression in Treg cells (Crellin 2005). Direct modulation of Treg function by TLR2 ligands has been also reported (Oberg 2010). On the other hand, IRF-5 (P68) with a mutation of Ala to Pro at amino acid 68 (G202C; position relative to translation start codon) suppresses TLR-mediated IL-6 and IL-12p40 induction. The mutation has been identified in peripheral blood of ATLL patients (Yang 2009).

A more recent report has emphasized the significance of MyD88 in the pathogenesis and therapeutic approach for lymphoma. Ngo *et al*. identified a single leucine-to-proline substitution at amino acid position 265 of MyD88 protein (L265P) in 29% of activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) biopsy samples. This mutation occurs at an evolutionally invariant residue in the hydrophobic core and is rare or absent in the other DLBCL subtypes. They further demonstrated that in ABC DLBCL with L265P mutation, MyD88 L265P rescued the cell after MyD88 knockdown, but wild-type MyD88 was ineffective, showing that the L265P is a gain-of-function mutation and ABC DLBCL with L265P mutation depends upon the MyD88 signaling pathway. A selective small molecule inhibitor of IRAK1 and IRAK4 killed the ABC DLBCL cells. Moreover, in ABC DLBCL cell lines, MyD88 knockdown diminishes the secretion of IL-6 and IL-10 and phosphorylation of STAT3 (Ngo 2011).

#### **3. Conclusion**

Investigations have led to the demonstration of the several regulatory mechanisms presented in this review. Recent reports have provided detailed insight into the crucial functional roles of JAK-STAT and MyD88-TLR in ATLL. An important goal of such approaches would be the identification of unique targets for clinical intervention. The fact that the two signaling pathway are attractive targets for leukemia therapies further argues the importance of constitutive activation of these factors in ATLL cells.

#### **4. References**

Akira, S (1997) IL-6-regulated transcription factors. *Int J Biochem Cell Biol,* 29, 12, 1401-18.


survival without affecting proliferation. A TLR-5 ligand flagellin and a TLR7/8 ligand R-848 promotes proliferation and to upregulate production of IFN-, IL-8 and IL-10, but not IL-4, in human CD4+ T-cells (Caron 2005). In particular, engagement of TLR-5 with flagellin enhances the suppressive capacity and FOXP3 expression in Treg cells (Crellin 2005). Direct modulation of Treg function by TLR2 ligands has been also reported (Oberg 2010). On the other hand, IRF-5 (P68) with a mutation of Ala to Pro at amino acid 68 (G202C; position relative to translation start codon) suppresses TLR-mediated IL-6 and IL-12p40 induction.

A more recent report has emphasized the significance of MyD88 in the pathogenesis and therapeutic approach for lymphoma. Ngo *et al*. identified a single leucine-to-proline substitution at amino acid position 265 of MyD88 protein (L265P) in 29% of activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) biopsy samples. This mutation occurs at an evolutionally invariant residue in the hydrophobic core and is rare or absent in the other DLBCL subtypes. They further demonstrated that in ABC DLBCL with L265P mutation, MyD88 L265P rescued the cell after MyD88 knockdown, but wild-type MyD88 was ineffective, showing that the L265P is a gain-of-function mutation and ABC DLBCL with L265P mutation depends upon the MyD88 signaling pathway. A selective small molecule inhibitor of IRAK1 and IRAK4 killed the ABC DLBCL cells. Moreover, in ABC DLBCL cell lines, MyD88 knockdown diminishes the secretion of IL-6 and IL-10 and

Investigations have led to the demonstration of the several regulatory mechanisms presented in this review. Recent reports have provided detailed insight into the crucial functional roles of JAK-STAT and MyD88-TLR in ATLL. An important goal of such approaches would be the identification of unique targets for clinical intervention. The fact that the two signaling pathway are attractive targets for leukemia therapies further argues

Akira, S (1997) IL-6-regulated transcription factors. *Int J Biochem Cell Biol,* 29, 12, 1401-18. Akira, S (2003) Toll-like receptor signaling. *J Biol Chem,* 278, 40, 38105-8, 0021-9258.

Akira, S, Isshiki, H, Sugita, T, Tanabe, O, Kinoshita, S, Nishio, Y, Nakajima, T, Hirano, T &

Akira, S, Takeda, K & Kaisho, T (2001) Toll-like receptors: critical proteins linking innate and

Akira, S, Yamamoto, M & Takeda, K (2003) Role of adapters in Toll-like receptor signalling.

Arima, N, Hidaka, S, Fujiwara, H, Matsushita, K, Ohtsubo, H, Arimura, K, Kukita, T,

Arima, N, Matsushita, K, Obata, H, Ohtsubo, H, Fujiwara, H, Arimura, K, Kukita, T, Suruga,

Kishimoto, T (1990) A nuclear factor for IL-6 expression (NF-IL6) is a member of a

Fukumori, J & Tanaka, H (1996) Relation of autonomous and interleukin-2 responsive growth of leukemic cells to survival in adult T-cell leukemia. *Blood,* 87,

Y, Wakamatsu, S, Hidaka, S & Tei, C (1999) NF-B involvement in the activation of

the importance of constitutive activation of these factors in ATLL cells.

C/EBP family. *EMBO J,* 9, 6, 1897-906, 0261-4189.

*Biochem Soc Trans,* 31, Pt 3, 637-42, 0300-5127.

7, 2900-4, 0006-4971.

acquired immunity. *Nat Immunol,* 2, 8, 675-80, 1529-2908.

The mutation has been identified in peripheral blood of ATLL patients (Yang 2009).

phosphorylation of STAT3 (Ngo 2011).

**3. Conclusion** 

**4. References** 

primary adult T-cell leukemia cells and its clinical implications. *Exp Hematol,* 27, 7, 1168-75, 0301-472X.


Constitutive Activation of the JAK/STAT

11, 1724-30.

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 91

Frank, DA & Varticovski, L (1996) BCR/abl leads to the constitutive activation of Stat

Geleziunas, R, Ferrell, S, Lin, X, Mu, Y, Cunningham, ET, Jr., Grant, M, Connelly, MA,

Gelman, AE, Zhang, J, Choi, Y & Turka, LA (2004) Toll-like receptor ligands directly promote activated CD4+ T cell survival. *J Immunol,* 172, 10, 6065-73, 0022-1767. Gouilleux-Gruart, V, Gouilleux, F, Desaint, C, Claisse, JF, Capiod, JC, Delobel, J, Weber-

Grossman, WJ & Ratner, L (1997) Cytokine expression and tumorigenicity of large granular

Harhaj, EW & Sun, SC (1999) IKK serves as a docking subunit of the IB kinase (IKK) and

Hayakawa, F, Towatari, M, Iida, H, Wakao, H, Kiyoi, H, Naoe, T & Saito, H (1998)

Higuchi, M, Matsuda, T, Mori, N, Yamada, Y, Horie, R, Watanabe, T, Takahashi, M, Oie, M

possible role in constitutive NF-B activation. *Retrovirology,* 2, 29, 1742-4690. Hironaka, N, Mochida, K, Mori, N, Maeda, M, Yamamoto, N & Yamaoka, S (2004) Tax-

Huang, GJ, Zhang, ZQ & Jin, DY (2002) Stimulation of IKK- oligomerization by the human T-cell leukemia virus oncoprotein Tax. *FEBS Lett,* 531, 3, 494-8, 0014-5793. Ihle, JN (2001) The Stat family in cytokine signaling. *Curr Opin Cell Biol,* 13, 2, 211-7.

Ito, S, Oyake, T, Murai, K & Ishida, Y (2010) Deguelin suppresses cell proliferation via the

Jacobson, NG, Szabo, SJ, Weber-Nordt, RM, Zhong, Z, Schreiber, RD, Darnell, JE, Jr. &

Jefferies, C, Bowie, A, Brady, G, Cooke, EL, Li, X & O'Neill, LA (2001) Transactivation by the

in primary acute myelogenous leukaemia. *Br J Haematol,* 101, 3, 521-8. Higashi, T, Tsukada, J, Yoshida, Y, Mizobe, T, Mouri, F, Minami, Y, Morimoto, H & Tanaka,

transformed with HTLV-I. *Genes Cells,* 10, 12, 1153-62, 1356-9597.

cellular kinases. *Mol Cell Biol,* 18, 9, 5157-65, 0270-7306.

acute leukemia patients. *Blood,* 87, 5, 1692-7.

*Biol Chem,* 274, 33, 22911-4, 0021-9258.

*Neoplasia,* 6, 3, 266-78, 1522-8002.

and Stat4. *J Exp Med,* 181, 5, 1755-62.

0270-7306.

leukemia virus type I. *Blood,* 90, 2, 783-94, 0006-4971.

transformed T cells. *Leuk Res,* 34, 3, 352-7, 1873-5835.

proteins, and shares an epitope with tyrosine phosphorylated Stats. *Leukemia,* 10,

Hambor, JE, Marcu, KB & Greene, WC (1998) Human T-cell leukemia virus type 1 Tax induction of NF-B involves activation of the IB kinase (IKK) and IKK

Nordt, R, Dusanter-Fourt, I, Dreyfus, F, Groner, B & Prin, L (1996) STAT-related transcription factors are constitutively activated in peripheral blood cells from

lymphocytic leukemia cells from mice transgenic for the tax gene of human T-cell

mediates interaction of IKK with the human T-cell leukemia virus Tax protein. *J* 

Differential constitutive activation between STAT-related proteins and MAP kinase

Y (2005) Constitutive tyrosine and serine phosphorylation of STAT4 in T-cells

& Fujii, M (2005) Elevated expression of CD30 in adult T-cell leukemia cell lines:

independent constitutive IB kinase activation in adult T-cell leukemia cells.

inhibition of survivin expression and STAT3 phosphorylation in HTLV-1-

Murphy, KM (1995) Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3

p65 subunit of NF-B in response to interleukin-1 (IL-1) involves MyD88, IL-1 receptor-associated kinase 1, TRAF-6, and Rac1. *Mol Cell Biol,* 21, 14, 4544-52,


Claudio, E, Brown, K, Park, S, Wang, H & Siebenlist, U (2002) BAFF-induced NEMO-

Coffer, PJ, Koenderman, L & de Groot, RP (2000) The role of STATs in myeloid

Coope, HJ, Atkinson, PG, Huhse, B, Belich, M, Janzen, J, Holman, MJ, Klaus, GG, Johnston,

Crellin, NK, Garcia, RV, Hadisfar, O, Allan, SE, Steiner, TS & Levings, MK (2005) Human

Danial, NN, Pernis, A & Rothman, PB (1995) Jak-STAT signaling induced by the v-abl

Dejardin, E, Droin, NM, Delhase, M, Haas, E, Cao, Y, Makris, C, Li, ZW, Karin, M, Ware, CF

Faggioli, L, Costanzo, C, Merola, M, Bianchini, E, Furia, A, Carsana, A & Palmieri, M (1996)

Feng, X & Ratner, L (2008) Human T-cell leukemia virus type 1 blunts signaling by

Flex, E, Petrangeli, V, Stella, L, Chiaretti, S, Hornakova, T, Knoops, L, Ariola, C, Fodale, V,

Franchini, G (1995) Molecular mechanisms of human T-cell leukemia/lymphotropic virus

Franchini, G, Wong-Staal, F & Gallo, RC (1984) Human T-cell leukemia virus (HTLV-I)

Frank, DA, Mahajan, S & Ritz, J (1997) B lymphocytes from patients with chronic

breast carcinoma cell line. *Eur J Biochem,* 239, 3, 624-31, 0014-2956.

interferon . *Virology,* 374, 1, 210-6, 0042-6822.

leukemia. *J Exp Med,* 205, 4, 751-8, 1540-9538.

type I infection. *Blood,* 86, 10, 3619-39, 0006-4971.

*Natl Acad Sci U S A,* 81, 19, 6207-11, 0027-8424.

differentiation and leukemia. *Oncogene,* 19, 21, 2511-22.

Darnell, JE, Jr. (1997) STATs and gene regulation. *Science,* 277, 5332, 1630-5.

1529-2908.

1525-8882.

100, 12, 3140-8.

21, 20, 5375-85, 0261-4189.

175, 12, 8051-9, 0022-1767.

oncogene. *Science,* 269, 5232, 1875-7.

independent processing of NF-B2 in maturing B cells. *Nat Immunol,* 3, 10, 958-65,

LH & Ley, SC (2002) CD40 regulates the processing of NF-B2 p100 to p52. *EMBO J,*

CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells. *J Immunol,*

& Green, DR (2002) The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-B pathways. *Immunity,* 17, 4, 525-35, 1074-7613. Dunne, A & O'Neill, LA (2003) The interleukin-1 receptor/Toll-like receptor superfamily:

signal transduction during inflammation and host defense. *Sci STKE,* 2003, 171, re3,

Nuclear factor B (NF-B), nuclear factor interleukin-6 (NFIL-6 or C/EBP) and nuclear factor interleukin-6(NFIL6- or C/EBP) are not sufficient to activate the endogenous interleukin-6 gene in the human breast carcinoma cell line MCF-7. Comparative analysis with MDA-MB-231 cells, an interleukin-6-expressing human

Clappier, E, Paoloni, F, Martinelli, S, Fragale, A, Sanchez, M, Tavolaro, S, Messina, M, Cazzaniga, G, Camera, A, Pizzolo, G, Tornesello, A, Vignetti, M, Battistini, A, Cave, H, Gelb, BD, Renauld, JC, Biondi, A, Constantinescu, SN, Foa, R & Tartaglia, M (2008) Somatically acquired JAK1 mutations in adult acute lymphoblastic

transcripts in fresh and cultured cells of patients with adult T-cell leukemia. *Proc* 

lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues. *J Clin Invest,*


Constitutive Activation of the JAK/STAT

0008-5472.

2360-8, 0006-4971.

99, 19, 12281-6.

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 93

Mizobe, T, Tsukada, J, Higashi, T, Mouri, F, Matsuura, A, Tanikawa, R, Minami, Y, Yoshida,

Mohapatra, S, Chu, B, Wei, S, Djeu, J, Epling-Burnette, PK, Loughran, T, Jove, R & Pledger,

Mori, N, Fujii, M, Ikeda, S, Yamada, Y, Tomonaga, M, Ballard, DW & Yamamoto, N (1999)

Mori, N, Gill, PS, Mougdil, T, Murakami, S, Eto, S & Prager, D (1996) Interleukin-10 gene

Morinobu, A, Gadina, M, Strober, W, Visconti, R, Fornace, A, Montagna, C, Feldman, GM,

Muzio, M, Polentarutti, N, Bosisio, D, Manoj Kumar, PP & Mantovani, A (2000) Toll-like receptor family and signalling pathway. *Biochem Soc Trans,* 28, 5, 563-6, 0300-5127. Nakama, S, Ishikawa, C, Nakachi, S & Mori, N (2011) Anti-adult T-cell leukemia effects of

Ngo, VN, Young, RM, Schmitz, R, Jhavar, S, Xiao, W, Lim, KH, Kohlhammer, H, Xu, W,

mutations in human lymphoma. *Nature,* 470, 7332, 115-9, 1476-4687 . Nicot, C, Mulloy, JC, Ferrari, MG, Johnson, JM, Fu, K, Fukumoto, R, Trovato, R, Fullen, J,

human peripheral blood mononuclear cells. *Blood,* 98, 3, 823-9, 0006-4971. O'Neill, LA (2003) The role of MyD88-like adapters in Toll-like receptor signal transduction.

Oberg, HH, Ly, TT, Ussat, S, Meyer, T, Kabelitz, D & Wesch, D (2010) Differential but direct

Ohashi, T, Masuda, M & Ruscetti, SK (1995) Induction of sequence-specific DNA-binding factors by erythropoietin and the spleen focus-forming virus. *Blood,* 85, 6, 1454-62. Oliere, S, Hernandez, E, Lezin, A, Arguello, M, Douville, R, Nguyen, TL, Olindo, S,

expression in adult T-cell leukemia. *Blood,* 88, 3, 1035-45, 0006-4971. Mori, N & Prager, D (1996) Transactivation of the interleukin-1alpha promoter by human Tcell leukemia virus type I and type II Tax proteins. *Blood,* 87, 8, 3410-7, 0006-4971. Mori, N, Yamada, Y, Ikeda, S, Yamasaki, Y, Tsukasaki, K, Tanaka, Y, Tomonaga, M,

leukemia cells. *Blood,* 100, 5, 1828-34, 0006-4971.

Bidens pilosa. *Int J Oncol,* 38, 4, 1163-73, 1791-2423.

*Biochem Soc Trans,* 31, Pt 3, 643-7, 0300-5127.

ligands. *J Immunol,* 184, 9, 4733-40, 1550-6606.

transformed T cells. *Exp Hematol,* 35, 12, 1812-22, 0301-472X.

Y & Tanaka, Y (2007) Constitutive association of MyD88 to IRAK in HTLV-I-

WJ (2003) Roscovitine inhibits STAT5 activity and induces apoptosis in the human leukemia virus type 1-transformed cell line MT-2. *Cancer Res,* 63, 23, 8523-30,

Constitutive activation of NF-B in primary adult T-cell leukemia cells. *Blood,* 93, 7,

Yamamoto, N & Fujii, M (2002) Bay 11-7082 inhibits transcription factor NF-B and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell

Nishikomori, R & O'Shea, JJ (2002) STAT4 serine phosphorylation is critical for IL-12-induced IFN- production but not for cell proliferation. *Proc Natl Acad Sci U S A,*

Yang, Y, Zhao, H, Shaffer, AL, Romesser, P, Wright, G, Powell, J, Rosenwald, A, Muller-Hermelink, HK, Ott, G, Gascoyne, RD, Connors, JM, Rimsza, LM, Campo, E, Jaffe, ES, Delabie, J, Smeland, EB, Fisher, RI, Braziel, RM, Tubbs, RR, Cook, JR, Weisenburger, DD, Chan, WC & Staudt, LM (2011) Oncogenically active MYD88

Leonard, WJ & Franchini, G (2001) HTLV-1 p12(I) protein enhances STAT5 activation and decreases the interleukin-2 requirement for proliferation of primary

abolishment of human regulatory T cell suppressive capacity by various TLR2

Panelatti, G, Kazanji, M, Wilkinson, P, Sekaly, RP, Cesaire, R & Hiscott, J (2010)


Jin, DY, Giordano, V, Kibler, KV, Nakano, H & Jeang, KT (1999) Role of adapter function in

interacts directly with IB kinase . *J Biol Chem,* 274, 25, 17402-5, 0021-9258. Ju, W, Zhang, M, Jiang, JK, Thomas, CJ, Oh, U, Bryant, BR, Chen, J, Sato, N, Tagaya, Y,

Kanno, T, Brown, K, Franzoso, G & Siebenlist, U (1994) Kinetic analysis of human T-cell

Kaplan, MH, Sun, YL, Hoey, T & Grusby, MJ (1996) Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. *Nature,* 382, 6587, 174-7. Kayagaki, N, Yan, M, Seshasayee, D, Wang, H, Lee, W, French, DM, Grewal, IS, Cochran,

Kitajima, I, Shinohara, T, Bilakovics, J, Brown, DA, Xu, X & Nerenberg, M (1992) Ablation of

Leonard, WJ & O'Shea, JJ (1998) Jaks and STATs: biological implications. *Annu Rev Immunol,*

Levy, DE & Gilliland, DG (2000) Divergent roles of STAT1 and STAT5 in malignancy as

Lin, TS, Mahajan, S & Frank, DA (2000) STAT signaling in the pathogenesis and treatment of

Maeda, M, Arima, N, Daitoku, Y, Kashihara, M, Okamoto, H, Uchiyama, T, Shirono, K,

Matsushima, N, Tanaka, T, Enkhbayar, P, Mikami, T, Taga, M, Yamada, K & Kuroki, Y

McGettrick, AF & O'Neill, LA (2007) Toll-like receptors: key activators of leucocytes and regulator of haematopoiesis. *Br J Haematol,* 139, 2, 185-93, 0007-1048. Mercurio, F, Zhu, H, Murray, BW, Shevchenko, A, Bennett, BL, Li, J, Young, DB, Barbosa, M,

kinases essential for NF-B activation. *Science,* 278, 5339, 860-6, 0036-8075. Migone, TS, Lin, JX, Cereseto, A, Mulloy, JC, O'Shea, JJ, Franchini, G & Leonard, WJ (1995)

Matsuoka, M, Hattori, T, Takatsuki, K & et al. (1987) Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia. *Blood,* 70, 5, 1407-

(2007) Comparative sequence analysis of leucine-rich repeats (LRRs) within

Mann, M, Manning, A & Rao, A (1997) IKK-1 and IKK-2: cytokine-activated IB

Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I.

promotes processing of NF-B2. *Immunity,* 17, 4, 515-24, 1074-7613.

revealed by gene disruptions in mice. *Oncogene,* 19, 21, 2505-10.

vertebrate toll-like receptors. *BMC Genomics,* 8, 124, 1471-2164.

NF-B. *Science,* 258, 5089, 1792-5, 0036-8075.

leukemias. *Oncogene,* 19, 21, 2496-504.

patients with ATL and HAM/TSP. *Blood,* 117, 6, 1938-46, 1528-0020. Kameda, T, Shide, K, Shimoda, HK, Hidaka, T, Kubuki, Y, Katayose, K, Taniguchi, Y,

leukemia/lymphoma. *Int J Hematol,* 92, 2, 320-5, 1865-3774.

51, 0270-7306.

16, 293-322.

11, 0006-4971.

*Science,* 269, 5220, 79-81.

oncoprotein-mediated activation of NF-B. Human T-cell leukemia virus type I Tax

Morris, JC, Janik, JE, Jacobson, S & Waldmann, TA (2011) CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3 activation and proliferation of T cells from

Sekine, M, Kamiunntenn, A, Maeda, K, Nagata, K, Matsunaga, T & Shimoda, K (2010) Absence of gain-of-function JAK1 and JAK3 mutations in adult T cell

leukemia virus type I Tax-mediated activation of NF-B. *Mol Cell Biol,* 14, 10, 6443-

AG, Gordon, NC, Yin, J, Starovasnik, MA & Dixit, VM (2002) BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and

transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of


Constitutive Activation of the JAK/STAT

cells. *Nature,* 382, 6587, 171-4.

*Res,* 30, 3, 313-21, 0145-2126.

127, 7, 1584-94, 1097-0215 .

cells. *Int J Cancer,* 118, 3, 765-72, 0020-7136.

activity. *Int J Cancer,* 124, 11, 2607-15, 1097-0215.

*Pharmacol,* 81, 6, 713-22, 1873-2968.

*Immunogenetics,* 48, 1, 1-7, 0093-7711 .

2, 169-76, 0007-1048 .

lines. *Blood,* 88, 3, 809-16.

of LIL-Stat in adult T-cell leukemia cells. *Blood,* 95, 8, 2715-8.

And Toll-Like Receptor Signaling Pathways in Adult T-Cell Leukemia/Lymphoma 95

Tomita, M, Kawakami, H, Uchihara, JN, Okudaira, T, Masuda, M, Takasu, N, Matsuda, T,

Tomita, M, Kawakami, H, Uchihara, JN, Okudaira, T, Masuda, M, Takasu, N, Matsuda, T,

Tomita, M, Tanaka, Y & Mori, N (2010) Aurora kinase inhibitor AZD1152 negatively affects

Tomita, M, Toyota, M, Ishikawa, C, Nakazato, T, Okudaira, T, Matsuda, T, Uchihara, JN,

Tsukada, J, Toda, Y, Misago, M, Tanaka, Y, Auron, PE & Eto, S (2000) Constitutive activation

Tsukada, J, Yoshida, Y, Kominato, Y & Auron, PE (2011) The CCAAT/enhancer (C/EBP)

Ueda, M, Imada, K, Imura, A, Koga, H, Hishizawa, M & Uchiyama, T (2005) Expression of

Visconti, R, Gadina, M, Chiariello, M, Chen, EH, Stancato, LF, Gutkind, JS & O'Shea, JJ

Watanabe, M, Ohsugi, T, Shoda, M, Ishida, T, Aizawa, S, Maruyama-Nagai, M, Utsunomiya,

Weber-Nordt, RM, Egen, C, Wehinger, J, Ludwig, W, Gouilleux-Gruart, V, Mertelsmann, R

therapy of adult T-cell leukemia. *Blood,* 106, 7, 2462-71, 0006-4971.

serine phosphorylation and transcriptional activity. *Blood,* 96, 5, 1844-52. Washizu, J, Nishimura, H, Nakamura, N, Nimura, Y & Yoshikai, Y (1998) The NF-B

regulated system for gene regulation. *Cytokine,* 54, 1, 6-19, 1096-0023. Tsumuraya, T, Ishikawa, C, Machijima, Y, Nakachi, S, Senba, M, Tanaka, J & Mori, N (2011)

Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T

Ohta, T, Tanaka, Y & Mori, N (2006a) Curcumin suppresses constitutive activation of AP-1 by downregulation of JunD protein in HTLV-1-infected T-cell lines. *Leuk* 

Ohta, T, Tanaka, Y, Ohshiro, K & Mori, N (2006b) Curcumin (diferuloylmethane) inhibits constitutive active NF-B, leading to suppression of cell growth of human T-cell leukemia virus type I-infected T-cell lines and primary adult T-cell leukemia

the growth and survival of HTLV-1-infected T lymphocytes in vitro. *Int J Cancer,*

Taira, N, Ohshiro, K, Senba, M, Tanaka, Y, Ohshima, K, Saya, H, Tokino, T & Mori, N (2009) Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-B

family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-

Effects of hippuristanol, an inhibitor of eIF4A, on adult T-cell leukemia. *Biochem* 

functional interleukin-21 receptor on adult T-cell leukaemia cells. *Br J Haematol,* 128,

(2000) Importance of the MKK6/p38 pathway for interleukin-12-induced STAT4

binding site is essential for transcriptional activation of the IL-15 gene.

A, Koga, S, Yamada, Y, Kamihira, S, Okayama, A, Kikuchi, H, Uozumi, K, Yamaguchi, K, Higashihara, M, Umezawa, K, Watanabe, T & Horie, R (2005) Dual targeting of transformed and untransformed HTLV-1-infected T cells by DHMEQ, a potent and selective inhibitor of NF-B, as a strategy for chemoprevention and

& Finke, J (1996) Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell

HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1). *PLoS Pathog,* 6, 11, e1001177, 1553-7374.


Portis, T, Harding, JC & Ratner, L (2001) The contribution of NF-B activity to spontaneous

Rincon, M, Enslen, H, Raingeaud, J, Recht, M, Zapton, T, Su, MS, Penix, LA, Davis, RJ &

Saitoh, T, Nakano, H, Yamamoto, N & Yamaoka, S (2002) Lymphotoxin-beta receptor

Saitoh, T, Nakayama, M, Nakano, H, Yagita, H, Yamamoto, N & Yamaoka, S (2003) TWEAK

Schmitz, ML & Baeuerle, PA (1995) Multi-step activation of NF-B/Rel transcription factors.

Shuai, K, Halpern, J, ten Hoeve, J, Rao, X & Sawyers, CL (1996) Constitutive activation of

Shuh, M, Morse, BA, Heidecker, G & Derse, D (2011) Association of SRC-related kinase lyn

Siekevitz, M, Feinberg, MB, Holbrook, N, Wong-Staal, F & Greene, WC (1987) Activation of

Spiekermann, K, Biethahn, S, Wilde, S, Hiddemann, W & Alves, F (2001) Constitutive

Takeda, K, Kaisho, T & Akira, S (2003) Toll-like receptors. *Annu Rev Immunol,* 21, 335-76,

Takemoto, S, Mulloy, JC, Cereseto, A, Migone, TS, Patel, BK, Matsuoka, M, Yamaguchi, K,

Takeuchi, O & Akira, S (2007) Signaling pathways activated by microorganisms. *Curr Opin* 

Taylor, JM, Brown, M, Nejmeddine, M, Kim, KJ, Ratner, L, Lairmore, M & Nicot, C (2009)

Thierfelder, WE, van Deursen, JM, Yamamoto, K, Tripp, RA, Sarawar, SR, Carson, RT,

cytokine signaling 1 (SOCS1). *PLoS Pathog,* 6, 11, e1001177, 1553-7374. Perkins, ND (1997) Achieving transcriptional specificity with NF-B. *Int J Biochem Cell Biol,*

29, 12, 1433-48, 1357-2725.

278, 38, 36005-12, 0021-9258.

*Immunobiology,* 193, 2-4, 116-27, 0171-2985.

cells. *J Virol,* 85, 9, 4623-7, 1098-5514 .

*Sci U S A,* 84, 15, 5389-93, 0027-8424.

*Cell Biol,* 19, 2, 185-91, 0955-0674.

*Virol,* 83, 22, 11467-76, 1098-5514 .

*Haematol,* 67, 2, 63-71.

0732-0582.

13897-902.

5793.

2, 247-54.

induced tumors. *Blood,* 98, 4, 1200-8, 0006-4971.

p38 MAP kinase signaling pathway. *Embo J,* 17, 10, 2817-29.

HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of

proliferation and resistance to apoptosis in human T-cell leukemia virus type 1 Tax-

Flavell, RA (1998) Interferon- expression by Th1 effector T cells mediated by the

mediates NEMO-independent NF-B activation. *FEBS Lett,* 532, 1-2, 45-51, 0014-

induces NF-B2 p100 processing and long lasting NF-B activation. *J Biol Chem,*

STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. *Oncogene,* 13,

with the interleukin-2 receptor and its role in maintaining constitutive phosphorylation of Jak/STAT in human T-cell leukemia virus type 1-transformed T

interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the transactivator (tat) gene product of human T-cell leukemia virus, type I. *Proc Natl Acad* 

activation of STAT transcription factors in acute myelogenous leukemia. *Eur J* 

Takatsuki, K, Kamihira, S, White, JD, Leonard, WJ, Waldmann, T & Franchini, G (1997) Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins. *Proc Natl Acad Sci U S A,* 94, 25,

Novel role for interleukin-2 receptor-Jak signaling in retrovirus transmission. *J* 

Sangster, MY, Vignali, DA, Doherty, PC, Grosveld, GC & Ihle, JN (1996)

Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. *Nature,* 382, 6587, 171-4.


**1. Introduction** 

shed light on the role of Ikaros in T-cell leukemia.

**2. Structure and function of Ikaros** 

**2.1 Molecular structure of Ikaros 2.1.1 DNA-binding domain** 

**6** 

**Ikaros in T-Cell Leukemia** 

Sinisa Dovat1 and Kimberly J. Payne2

*2Loma Linda University United States of America* 

*1Pennsylvania State University College of Medicine* 

Although extensive clinical data have established that the loss of Ikaros tumor suppressor activity *via* genetic inactivation is a major contributor to leukemogenesis leading to B-cell ALL, less is known about the role of Ikaros in T-cell leukemia. The T-cell malignancies observed in Ikaros-deficient mice suggest that Ikaros is likely to function as a tumor suppressor in T-cells. In human, multiple studies have identified genetic inactivation of Ikaros (deletion or mutation) to be associated with ~5% of T-ALL cases. Additional studies provide evidence that the functional inactivation of Ikaros due to defects in signal pathways that normally regulate Ikaros activity is likely to be a critical factor in the development of Tcell malignancies. These studies provide a rationale for new chemotherapeutic strategies in the treatment of T-cell leukemia. In this chapter, we summarize the newest advances that

The *Ikaros* gene, discovered independently by K. Georgopoulos and S. Smale, encodes multiple Ikaros protein isoforms *via* alternate splicing (Lo et al. 1991; Georgopoulos et al. 1992; Hahm et al. 1994). The structure of Ikaros protein reveals several known structural

The N-terminal region of the Ikaros protein contains four zinc finger motifs (Fig. 1). Three of these exhibit a typical C2H2 structure with two cysteines and two histidines covalently bound to a zinc atom, while the fourth zinc finger has a CCHC structure. The four N terminal zinc fingers in the Ikaros protein function in DNA binding. Point mutational analysis revealed that zinc fingers #2 and #3 are essential for the DNA-binding function of Ikaros, as well as its localization to pericentromeric heterochromatin (see 2.2.1). Based on DNA-se footprint analysis, it has been suggested that the first zinc finger contributes to DNA binding specificity. The role of the fourth zinc finger in DNA-binding remains unknown, although it has been noted that Ikaros isoforms that lack the fourth zinc finger exhibit a unique expression pattern during hematopoiesis (Payne et al. 2003). Recently, a point mutation in a single allele of the fourth zinc finger has been associated with primary

motifs corresponding to distinct functional domains as described below.

immunodeficiency and pancytopenia in humans (Goldman, et al. In press*.).*

