**CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in Immune Deficiencies and Malignancies**

Christelle Freitas, Alexandre Bignon, Karl Balabanian and Ali Dalloul

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58699

#### **1. Introduction**

[142] Scharrer E. The histology of the meningeal myeloid tissue in the ganoids Amia and

[143] Dempster WT. The morphology of the amphibian endolymphatic organ. J Morphol.

[144] Sano Y, Imai C. Über ein blutbildendes Organ in der sella Turcica des Reisensala‐

[145] Horton JD. Ontogeny of the immune system in amphibians. Am Zool. 1971;11(1):

[146] Hadji-Azimi I, Schwager J, Thiebard CH, Perrenot N. B-lymphocyte differentitation

[147] Ardavin CF, Zapata A. Ultrastructure and changes during metamorphosis of the lympho-hemopoietic tissue of the larval anadromous sea lamprey Petromyzon mari‐

[148] O'Brien LE, Bilder D. Beyond the niche: tissue-level coordination of stem cell dynam‐

manders (*Megalobatrachus japonicus*). Z Zellfors. 1961;53(1):471-80.

Lepisosteus. Anat Rec. 1944;88(1):291-310.

in *Xenopus laevis larvae*. Dev Biol. 1982;90(1):253-8.

nus. Dev Comp Immunol. 1987 Winter;11(1):79-93.

ics. Annu Rev Cell Dev Biol. 2013;29:107-36.

1930;50(1):71-126.

219-28.

48 Adult Stem Cell Niches

The discovery of the cell-attracting chemokine Stromal cell Derived Factor 1 (SDF-1)/CXCL12 some twenty years ago and its ligand CXCR4 spurred tremendous research interest and generated an abundant literature reflecting the role of CXCR4 in various aspects of physiology and pathology, extending much beyond the role of CXCR4 as a co-receptor for Human Immunodeficiency Virus (HIV) on human T-cells [1-3].

Indeed, CXCL12 and CXCR4 are expressed in a complementary pattern during embryogenesis and are needed for endodermal migration [4, 5]. In addition, CXCL12 is induced by hypoxia through the transcription factor hypoxia-inducible factor 1 (HIF1) and accordingly stimulates CXCR4-positive stem cells that locate within cellular hypoxic niches [6, 7].

These features explain the lethal phenotype of *Cxcr4* null-mutated mice, hence the need for cell-specific conditional deletion models to be able to decipher its physiological functions [8]. This sharply contrasts with knockouts (KO) for most other chemokine/chemokine receptor pairs, which are almost viable throughout the adult life and point to the uniqueness of the CXCL12/CXCR4 pair in physiology. Note however that another CXCL12 receptor, ACKR3 (CXCR7) has been described, it may therefore overlap with the functions of CXCR4 [9-11]. As the physiological role of ACKR3 is not yet fully understood, its description is beyond the scope of this review.

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although CXCR4 signaling governs many aspects of stem cell development during embryonic and adult life, it also plays a role in the proper homing and migration of mature lymphocytes within the bone marrow and peripheral lymphoid organs. It also regulates B-cell differentia‐ tion as discussed later.

solid tumours (chapter 9); and on the other hand, pathologies with qualitative mutations of CXCR4 including the WHIM Syndrome (chapter 6) and Waldenström's Macroglobulinemia

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

The CXCL12/CXCR4 axis is pivotal in the survival of Hematopoietic Stem Cells (HSC) and early-committed progenitors, in their mobilization from the bone marrow to the peripheral blood and migration back from the blood to the bone marrow. This later process continuously seeds suitable niches from the bone marrow with functional HSC thereby allowing the maintenance of haematopoiesis. Hematopoietic stem and progenitors expressing CXCR4 interact with CXCL12-expressing stromal cells within niches thus delivering survival, and

The sequential appearance and disappearance of hematopoietic activity is notably governed by the CXCL12/CXCR4 axis and Very Late Antigen-4/Vascular cell adhesion molecule-1 (VLA4/VCAM-1) interactions [8, 22]. The role of CXCL12/CXCR4 interaction is needed for proper haematopoiesis throughout life as shown by induced deletion of *Cxcr4* in adult mice [23]. As mentioned above, the mechanisms of stem cell mobilization from the bone marrow are complex and regulated by factors of homing and adhesion to and within the niche. Adhesion is a multistep process that involves selectin-mediated tethering and rolling on stromal/endothelial cells followed by activation and ultimately firm adhesion mediated by

[25, 26]. Of interest in this respect, CXCL12 enhances integrin activation and at least *in vitro*,

their subsequent transmigration through endothelium [27, 28]. CXCL12 synergizes with Granulocyte colony-stimulating factor (GCSF) which activate the VLA4-ligand VCAM1and Vascular endothelial growth factor (VEGF) which activate both VCAM1 and E-selectin [28-30]. These findings were also supported by injection of antibodies and several KO mice models,

expression of which is unregulated by Stem cell Factor (SCF) [32]. Altogether, these findings demonstrated that CXCR4 signaling is required for stem cells to migrate, survive and differ‐ entiate into dedicated bone marrow niches. Studies dealing with effects of CXCR4 on the cell cycle on HSCs are contradictory. Some indicate that CXCR4 is required for the quiescence of primitive HSCs whereas others pointed a synergy between SCF/Kit-ligand and CXCL12 for

CXCL12 which protects them from apoptosis in an autocrine /paracrine manner [35, 36].

SCF and CXCL12 synergize for the maintenance of Kitand CXCR4 expressing HSC; this is highlighted by the finding that perivascular SCF expression pattern is very similar to that of

progenitors and CXCL12 induced CD34+

cells express both CD162 a sialomucin that binds all selectins and VLA4

progenitors [23, 33, 34]. Indeed, CXCR4 expression is higher in the

cells to the niche under flow, which allows

cells into severe combined immunodeficiency

cell engraftment on CXCR4, the

http://dx.doi.org/10.5772/58699

51

cell proliferation in

cells may produce

(chapter 8).

integrins [24]. CD34+

the proliferation of CXCR4+

most primitive CD34+

**2. CXCR4 and haematopoiesis**

mobilization signals to multipotent and committed progenitors.

E-selectin thereby promoting adhesion of CD34+

and most convincingly by grafting human CD34+

CD38-

mice [31]; this work demonstrated the dependency of CD34+

synergy with SCF or thrombopoietin TPO *in vitro*, further, cycling CD34+

From a paradigmatic point of view, upon engagement with its ligand, three processes namely desensitization (homologous and heterologous), internalization, and degradation regulate CXCR4. The homologous desensitization, or becoming refractory to continued stimulation, involves the recruitment of G-protein-coupled receptor kinases (GRK) that promote the recruitment of β-Arrestin which prevents coupling of receptor to new G protein and initiate endocytosis in clathrin-coated pits [12]. The receptor may then be recycled or degraded following ubiquitination by the E3 ubiquitin ligase AIP4 [13]. Another mechanism of heterol‐ ogous desensitization is mediated by the activation of second messenger dependent protein kinases such as protein kinase A and C (PKA and PKC) [14].

Abnormal expression and/or activity of CXCR4 due to an over stimulation or inhibition of transcription or internalization may therefore impact on several aspects of haematopoiesis and adaptive immune response, hence immune deficits and lymphoid neoplasms. A paradigmatic disease in this respect is the WHIM syndrome in which *CXCR4* is mutated and displays anomalous internalization and signaling properties [15]. More recently, one third of patients with Waldenström's macroglobilinemia, a disorder induced by an IgM-producing plasmacy‐ toma, were shown to harbour the WHIM syndrome mutation [16].

Altogether understanding the molecules that regulate CXCR4 expression and/or activity in leukocytes is a very active area of investigation inasmuch as CXCR4 expression can be regulated by the processing of CXCL12 either by cleavage or binding to glycosaminoglycans (GAGs) within extracellular matrix [17, 18]. Engagement of either the T-cell or the B-cell receptor (TCR or BCR) on lymphocytes can down regulate cell surface CXCR4 expression pointing to cross talks between CXCL12/CXCR4 axis and other chemokine independent pathways [19, 20].

These findings already translated into clinical applications with the invention of the first CXCR4 antagonist, AMD3100 and as our knowledge widens will be followed by other drugs aimed at directly or indirectly regulating CXCR4 expression and signaling, for multiple medical purposes [21].

The physiological role of chemokine receptors is still a matter of intense research and attempts to generate pharmacological antagonists of these receptors are numerous [205]. Among these receptors, CXCR4 is the one with the most successful implications in medicine, especially in the fields of immunology and haematology. Our goals were therefore to summarize or current knowledge on CXCR4 in these fields. This review describes our current knowledge on CXCR4 involvement in the development of lymphoid progenitors (chapter 2), B-cells (chapter 3), Tcells (chapter 4) and as recently described, in NK-cells (chapter 5). We describedon one hand, pathological lymphoid disorders with quantitative abnormalities of CXCR4 including Idiopathic T-cell lymphocytopenia (chapter 7), chronic lymphocytic leukaemia (chapter 8), and solid tumours (chapter 9); and on the other hand, pathologies with qualitative mutations of CXCR4 including the WHIM Syndrome (chapter 6) and Waldenström's Macroglobulinemia (chapter 8).

#### **2. CXCR4 and haematopoiesis**

Although CXCR4 signaling governs many aspects of stem cell development during embryonic and adult life, it also plays a role in the proper homing and migration of mature lymphocytes within the bone marrow and peripheral lymphoid organs. It also regulates B-cell differentia‐

From a paradigmatic point of view, upon engagement with its ligand, three processes namely desensitization (homologous and heterologous), internalization, and degradation regulate CXCR4. The homologous desensitization, or becoming refractory to continued stimulation, involves the recruitment of G-protein-coupled receptor kinases (GRK) that promote the recruitment of β-Arrestin which prevents coupling of receptor to new G protein and initiate endocytosis in clathrin-coated pits [12]. The receptor may then be recycled or degraded following ubiquitination by the E3 ubiquitin ligase AIP4 [13]. Another mechanism of heterol‐ ogous desensitization is mediated by the activation of second messenger dependent protein

Abnormal expression and/or activity of CXCR4 due to an over stimulation or inhibition of transcription or internalization may therefore impact on several aspects of haematopoiesis and adaptive immune response, hence immune deficits and lymphoid neoplasms. A paradigmatic disease in this respect is the WHIM syndrome in which *CXCR4* is mutated and displays anomalous internalization and signaling properties [15]. More recently, one third of patients with Waldenström's macroglobilinemia, a disorder induced by an IgM-producing plasmacy‐

Altogether understanding the molecules that regulate CXCR4 expression and/or activity in leukocytes is a very active area of investigation inasmuch as CXCR4 expression can be regulated by the processing of CXCL12 either by cleavage or binding to glycosaminoglycans (GAGs) within extracellular matrix [17, 18]. Engagement of either the T-cell or the B-cell receptor (TCR or BCR) on lymphocytes can down regulate cell surface CXCR4 expression pointing to cross talks between CXCL12/CXCR4 axis and other chemokine independent

These findings already translated into clinical applications with the invention of the first CXCR4 antagonist, AMD3100 and as our knowledge widens will be followed by other drugs aimed at directly or indirectly regulating CXCR4 expression and signaling, for multiple

The physiological role of chemokine receptors is still a matter of intense research and attempts to generate pharmacological antagonists of these receptors are numerous [205]. Among these receptors, CXCR4 is the one with the most successful implications in medicine, especially in the fields of immunology and haematology. Our goals were therefore to summarize or current knowledge on CXCR4 in these fields. This review describes our current knowledge on CXCR4 involvement in the development of lymphoid progenitors (chapter 2), B-cells (chapter 3), Tcells (chapter 4) and as recently described, in NK-cells (chapter 5). We describedon one hand, pathological lymphoid disorders with quantitative abnormalities of CXCR4 including Idiopathic T-cell lymphocytopenia (chapter 7), chronic lymphocytic leukaemia (chapter 8), and

kinases such as protein kinase A and C (PKA and PKC) [14].

toma, were shown to harbour the WHIM syndrome mutation [16].

tion as discussed later.

50 Adult Stem Cell Niches

pathways [19, 20].

medical purposes [21].

The CXCL12/CXCR4 axis is pivotal in the survival of Hematopoietic Stem Cells (HSC) and early-committed progenitors, in their mobilization from the bone marrow to the peripheral blood and migration back from the blood to the bone marrow. This later process continuously seeds suitable niches from the bone marrow with functional HSC thereby allowing the maintenance of haematopoiesis. Hematopoietic stem and progenitors expressing CXCR4 interact with CXCL12-expressing stromal cells within niches thus delivering survival, and mobilization signals to multipotent and committed progenitors.

The sequential appearance and disappearance of hematopoietic activity is notably governed by the CXCL12/CXCR4 axis and Very Late Antigen-4/Vascular cell adhesion molecule-1 (VLA4/VCAM-1) interactions [8, 22]. The role of CXCL12/CXCR4 interaction is needed for proper haematopoiesis throughout life as shown by induced deletion of *Cxcr4* in adult mice [23]. As mentioned above, the mechanisms of stem cell mobilization from the bone marrow are complex and regulated by factors of homing and adhesion to and within the niche. Adhesion is a multistep process that involves selectin-mediated tethering and rolling on stromal/endothelial cells followed by activation and ultimately firm adhesion mediated by integrins [24]. CD34+ cells express both CD162 a sialomucin that binds all selectins and VLA4 [25, 26]. Of interest in this respect, CXCL12 enhances integrin activation and at least *in vitro*, E-selectin thereby promoting adhesion of CD34+ cells to the niche under flow, which allows their subsequent transmigration through endothelium [27, 28]. CXCL12 synergizes with Granulocyte colony-stimulating factor (GCSF) which activate the VLA4-ligand VCAM1and Vascular endothelial growth factor (VEGF) which activate both VCAM1 and E-selectin [28-30]. These findings were also supported by injection of antibodies and several KO mice models, and most convincingly by grafting human CD34+ cells into severe combined immunodeficiency mice [31]; this work demonstrated the dependency of CD34+ cell engraftment on CXCR4, the expression of which is unregulated by Stem cell Factor (SCF) [32]. Altogether, these findings demonstrated that CXCR4 signaling is required for stem cells to migrate, survive and differ‐ entiate into dedicated bone marrow niches. Studies dealing with effects of CXCR4 on the cell cycle on HSCs are contradictory. Some indicate that CXCR4 is required for the quiescence of primitive HSCs whereas others pointed a synergy between SCF/Kit-ligand and CXCL12 for the proliferation of CXCR4+ progenitors [23, 33, 34]. Indeed, CXCR4 expression is higher in the most primitive CD34+ CD38 progenitors and CXCL12 induced CD34+ cell proliferation in synergy with SCF or thrombopoietin TPO *in vitro*, further, cycling CD34+ cells may produce CXCL12 which protects them from apoptosis in an autocrine /paracrine manner [35, 36].

SCF and CXCL12 synergize for the maintenance of Kitand CXCR4 expressing HSC; this is highlighted by the finding that perivascular SCF expression pattern is very similar to that of CXCL12 in bone marrow stromal cells especially in the perivascular niche [37]. This also is in agreement with the reported localization of the most primitive HSC adjacent to sinusoidal blood vessels [23, 38-40]. Most HSC are in contact with CXCL12-abundant reticular (CAR) cells mostly located around sinusoidal endothelial cells or near the endosteum [23]. Recent out‐ standing data from the groups of Morrison and Nagasawa using conditional deletion of *Cxcl12* in various stromal cells from the bone marrow redefined the HSC niches [37, 41]. These data demonstrate that the most primitive Cxcr4+ HSC reside in a perivascular region in close contact with endothelial cells and perivascular cells whereas early lymphoid progenitors occupy an endosteal niche in close contact with osteoblasts. This also implies that bone marrow progen‐ itors directly interact with Cxcl12-expressing stromal cells and do not respond to a Cxcl12 gradient, but that Cxcr4 expression and responsiveness of various progenitors to Cxcl12 dictate their fate within the bone marrow. It is indeed striking that CXCR4 signaling mediates quiescence in primitive HSC whereas it induces the proliferation of early B-cell progenitors [8, 33]. Thus genetic ablation of the *Cxcr4* transcript relieves HSC from quiescence and allows them to differentiate and migrate to the blood, as they fail to home back into their bone marrow niches, these cells ultimately loose their self renewal capacity and ability to restore blood lineages upon grafting into syngeneic recipient.

**3. CXCR4 and B-cell development**

Contrary to HSC, where CXCR4 signaling promotes survival and quiescence (see above), CXCR4 signaling in B-cell precursors promotes cell growth; this property led to the discovery of SDF1/CXCL12 [1]. The *Cxcl12*-null mice model established that Cxcl12 was necessary for Bcell lymphopoiesis in foetal liver and bone marrow, in addition to be necessary for bone marrow myelopoiesis [8]. This phenotype was close to that of *Cxcr4* null-mutated mice and is likely due to the inability of B-cells from the blood to home into bone marrow or foetal liver [48, 49]. Homing of B-cell precursors into specific bone marrow niches is indeed dependent on Cxcl12/Cxcr4 interactions [50]. Further experiments using adoptive transfer of cells from *Cxcr4-/-*animals, established the need for Cxcr4 expression by B-cell progenitors throughout adult life in order to home into the bone marrow [51]. Although myeloid lineages were also affected, the defect was predominant on the B-cell lineage further pointing to the dependency of B-cells towards the CXCL12/CXCR4 axis. As for HSC, B-cell precursors depend on VLA4/

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

http://dx.doi.org/10.5772/58699

53

VCAM1, and CXCR4/CXCL12 interactions for adhesion to stromal cells [52, 53].

Cxcl12-abundant reticular cells (CAR) are scattered throughout the bone marrow and in close contact with HSC and B-cell precursors [23, 54]. Recently, the redefinition of bone marrow niches established that early lymphoid precursors are in close contact with osteoblasts while committed B-lineage progenitors are in contact with the perivascular niche [37, 41]. Of interest, the ability of osteoblasts to support the differentiation of primitive HSC towards all stages of B-cells has been reported [55]. Furthermore, this differentiation appeared dependent on VCAM-1, CXCL12 and G protein αi subunit expression by osteoblasts [55, 56]. It should be mentioned that the expression of CXCR4 is sinusoidal in the B-cell lineage being highest in pre-B-cells and decreasing as cells develop in immature B-cells, and finally increases in mature B-cells; however the response of mature B-cells to CXCL12 remains poor despite their high CXCR4 expression [57]. Thus, as for HSC, B-cell precursors are highly sensitive to CXCL12; this is possibly linked to intrinsic properties of the cells such as sustained activation of the focal adhesion kinase pathway contrary to mature B-cells [53]. Altogether, the fine-tuning of CXCR4 response during B-cell maturation is complex and not only mediated by the regulation of CXCL12 production and proteolysis or by CXCR4 transcription and trafficking, but also by exogenous factors such as local production of CCR5 ligands that induce heterologous desen‐

As discussed below, mutant mice harbouring the WHIM Syndrome linked *Cxcr4* mutation display abnormal B-cell differentiation at the early B-cell committed progenitors stages combined to anomalous B-cell positioning within secondary lymphoid organs [59]. This points

to the role of CXCR4 in mature B-cells peripheral compartmentalization.

**3.1. Early steps of B-cell development**

sitization of CXCR4 to CXCL12 [58].

Interference with the CXCL12/CXCR4 axis, was initially thought to prevent the binding and entry of HIV in T-cells using CXCR4 antagonist AMD3100, but this approach was not used in clinics until now [42]. However, a clinical trial is underway with the CXCR4 allosteric antag‐ onist AMD11070 [43].

Nevertheless, interfering with CXCL12/CXCR4 axis by means of a pharmacological and reversible ligand of CXCR4, AMD3100, allows human HSC to leave their niche and migrate to the blood were they can easily be collected by apheresis in order to be grafted to a recipient (allograft) or to the donor himself (auto graft) following chemotherapy. AMD3100 is currently approved by the Food and Drug Administration (FDA) to induce in combination with GCSF the mobilization of human HSC [44]. Of interest, injection of GCSF to induce stem cell mobilization has long been used for clinical transplantation although its mechanism remained poorly understood [45]. Among the mechanisms evoked for GCSF action, is the degradation of CXCL12 by neutrophil elastase in parallel with up regulation of CXCR4 [46]. As CXCL12 has a very short half-life time (1 min), it is very sensitive to degradation by cleavage of its Nterminal domain [47].

In brief, CXCR4 is expressed on the most primitive and more committed hematopoietic progenitors and signals through interactions with CXCL12-expressing stromal cells in various bone marrow niches. CXCR4-mediated signaling results in distinct responses depending on the cell type (for instance quiescence *vs* proliferation), as also shown by predominant HSC and B-cell defects in *Cxcr4*-null mice, and neutrophil abnormalities in WHIM syndrome. It therefore remains to understand how CXCR4 is modulated and why progenitors behave differently following CXCR4 engagement. It is also unclear how progenitors move from one niche to another following CXCR4 engagement. Solving these challenges in the next years will undoubtedly teach us more in the fields of haematopoiesis and immunology.

### **3. CXCR4 and B-cell development**

#### **3.1. Early steps of B-cell development**

CXCL12 in bone marrow stromal cells especially in the perivascular niche [37]. This also is in agreement with the reported localization of the most primitive HSC adjacent to sinusoidal blood vessels [23, 38-40]. Most HSC are in contact with CXCL12-abundant reticular (CAR) cells mostly located around sinusoidal endothelial cells or near the endosteum [23]. Recent out‐ standing data from the groups of Morrison and Nagasawa using conditional deletion of *Cxcl12* in various stromal cells from the bone marrow redefined the HSC niches [37, 41]. These data

with endothelial cells and perivascular cells whereas early lymphoid progenitors occupy an endosteal niche in close contact with osteoblasts. This also implies that bone marrow progen‐ itors directly interact with Cxcl12-expressing stromal cells and do not respond to a Cxcl12 gradient, but that Cxcr4 expression and responsiveness of various progenitors to Cxcl12 dictate their fate within the bone marrow. It is indeed striking that CXCR4 signaling mediates quiescence in primitive HSC whereas it induces the proliferation of early B-cell progenitors [8, 33]. Thus genetic ablation of the *Cxcr4* transcript relieves HSC from quiescence and allows them to differentiate and migrate to the blood, as they fail to home back into their bone marrow niches, these cells ultimately loose their self renewal capacity and ability to restore blood

Interference with the CXCL12/CXCR4 axis, was initially thought to prevent the binding and entry of HIV in T-cells using CXCR4 antagonist AMD3100, but this approach was not used in clinics until now [42]. However, a clinical trial is underway with the CXCR4 allosteric antag‐

Nevertheless, interfering with CXCL12/CXCR4 axis by means of a pharmacological and reversible ligand of CXCR4, AMD3100, allows human HSC to leave their niche and migrate to the blood were they can easily be collected by apheresis in order to be grafted to a recipient (allograft) or to the donor himself (auto graft) following chemotherapy. AMD3100 is currently approved by the Food and Drug Administration (FDA) to induce in combination with GCSF the mobilization of human HSC [44]. Of interest, injection of GCSF to induce stem cell mobilization has long been used for clinical transplantation although its mechanism remained poorly understood [45]. Among the mechanisms evoked for GCSF action, is the degradation of CXCL12 by neutrophil elastase in parallel with up regulation of CXCR4 [46]. As CXCL12 has a very short half-life time (1 min), it is very sensitive to degradation by cleavage of its N-

In brief, CXCR4 is expressed on the most primitive and more committed hematopoietic progenitors and signals through interactions with CXCL12-expressing stromal cells in various bone marrow niches. CXCR4-mediated signaling results in distinct responses depending on the cell type (for instance quiescence *vs* proliferation), as also shown by predominant HSC and B-cell defects in *Cxcr4*-null mice, and neutrophil abnormalities in WHIM syndrome. It therefore remains to understand how CXCR4 is modulated and why progenitors behave differently following CXCR4 engagement. It is also unclear how progenitors move from one niche to another following CXCR4 engagement. Solving these challenges in the next years will

undoubtedly teach us more in the fields of haematopoiesis and immunology.

HSC reside in a perivascular region in close contact

demonstrate that the most primitive Cxcr4+

lineages upon grafting into syngeneic recipient.

onist AMD11070 [43].

52 Adult Stem Cell Niches

terminal domain [47].

Contrary to HSC, where CXCR4 signaling promotes survival and quiescence (see above), CXCR4 signaling in B-cell precursors promotes cell growth; this property led to the discovery of SDF1/CXCL12 [1]. The *Cxcl12*-null mice model established that Cxcl12 was necessary for Bcell lymphopoiesis in foetal liver and bone marrow, in addition to be necessary for bone marrow myelopoiesis [8]. This phenotype was close to that of *Cxcr4* null-mutated mice and is likely due to the inability of B-cells from the blood to home into bone marrow or foetal liver [48, 49]. Homing of B-cell precursors into specific bone marrow niches is indeed dependent on Cxcl12/Cxcr4 interactions [50]. Further experiments using adoptive transfer of cells from *Cxcr4-/-*animals, established the need for Cxcr4 expression by B-cell progenitors throughout adult life in order to home into the bone marrow [51]. Although myeloid lineages were also affected, the defect was predominant on the B-cell lineage further pointing to the dependency of B-cells towards the CXCL12/CXCR4 axis. As for HSC, B-cell precursors depend on VLA4/ VCAM1, and CXCR4/CXCL12 interactions for adhesion to stromal cells [52, 53].

Cxcl12-abundant reticular cells (CAR) are scattered throughout the bone marrow and in close contact with HSC and B-cell precursors [23, 54]. Recently, the redefinition of bone marrow niches established that early lymphoid precursors are in close contact with osteoblasts while committed B-lineage progenitors are in contact with the perivascular niche [37, 41]. Of interest, the ability of osteoblasts to support the differentiation of primitive HSC towards all stages of B-cells has been reported [55]. Furthermore, this differentiation appeared dependent on VCAM-1, CXCL12 and G protein αi subunit expression by osteoblasts [55, 56]. It should be mentioned that the expression of CXCR4 is sinusoidal in the B-cell lineage being highest in pre-B-cells and decreasing as cells develop in immature B-cells, and finally increases in mature B-cells; however the response of mature B-cells to CXCL12 remains poor despite their high CXCR4 expression [57]. Thus, as for HSC, B-cell precursors are highly sensitive to CXCL12; this is possibly linked to intrinsic properties of the cells such as sustained activation of the focal adhesion kinase pathway contrary to mature B-cells [53]. Altogether, the fine-tuning of CXCR4 response during B-cell maturation is complex and not only mediated by the regulation of CXCL12 production and proteolysis or by CXCR4 transcription and trafficking, but also by exogenous factors such as local production of CCR5 ligands that induce heterologous desen‐ sitization of CXCR4 to CXCL12 [58].

As discussed below, mutant mice harbouring the WHIM Syndrome linked *Cxcr4* mutation display abnormal B-cell differentiation at the early B-cell committed progenitors stages combined to anomalous B-cell positioning within secondary lymphoid organs [59]. This points to the role of CXCR4 in mature B-cells peripheral compartmentalization.

#### **3.2. B-cell homing and positioning within secondary lymphoid organs and regulation of CXCR4 expression by BCR signaling**

residues and recruit β-arrestins resulting in CXCR4 desensitization and finally its internaliza‐ tion [14]. CXCR4 is internalized and its surface expression down regulated following BCR activation or CXCL12 binding. Both signaling pathways induce CXCR4 phosphorylation on Ser residues albeit on distinct residues [20]. In fact, the migratory B-cell response to CXCL12 depends on both intrinsic factors and CXCR4 expression; indeed naive and memory B-cells are more sensitive to CXCL12 than germinal center B-cells in this respect [74]. The inhibition of CXCL12-mediated chemotaxis following BCR engagement depends on PKC but not on

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

In conclusion, CXCR4 expression in B-cell lineage cells depends on CXCL12 expression at the pro/pre B-cell stages and PC that lack functional BCR, whereas it also depends on the BCR

differentiate from common lymphoid progenitor and migrate from the bone marrow to the thymus through the blood and invade the epithelial rudiment at the cortico-medullary junction through post capillary venules where they loose progressively non-T-cell differentiated capacity and become fully T-cell committed as also described in human thymocytes [75-79]. These DN thymocytes make some 2% of thymocytes and are further divided in differentiation

CD25-

inner cortex [81]. Only two chemokines, CXCL12 and CCL25/thymus-expressed chemokine TECK are expressed in the thymic cortex namely by cortical epithelial thymic cells [82-84]. The CXCL12/CXCR4 axis is mandatory for retaining human double positive (DP) thymocytes in the cortex. In addition, in mice lacking Cxcr4 on thymocytes displayed defective DN migration to the cortex and defective DN to DP transition [82, 85-87]. DN migration can indeed be inhibited by AMD3100 [85, 88]. In the medulla, CCR7 promotes migration of mature thymo‐ cytes from the cortex. Thus, two opposing chemokine gradients regulate thymocyte migration

TCR βselection starts at the DN stage and DN3 cells express pre-TCR made of a pre-TCR α chain associated to TCR-β chain. These cells are selected positively by interactions with stromal cells in the cortex and CXCR4 is crucial for this process. Indeed, it is functionally associated with the pre-TCR and needed to activate phosphatidyl inositol 3-kinase (PI3K) and Notch pathway, the later which is mandatory for T-cell differentiation [86, 87, 89, 90]. In brief, DN thymocytes expand and differentiate in the cortex due to migration retention and survival signals dependent on CXCR4, and as for HSC, CXCR4 activation up regulate adhesion

DN2 cells following migration into the subcapsular zone, DN2 differentiate in

double positive DP thymocytes that acquire surface CD3 and move toward the

which reside in the thymic double negative DN1 fraction

CD25-

DN1 cells differentiate in

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55

cortical DN4 cells. In turn DN4 differentiate

Ca2+due to CXCR4 internalization [20).

signal strength at more mature stages.

Early thymic progenitors cKit+

CD44+

CD44-

in CD4+

CD25+

CD25+

CD8+

from the cortex to the medulla.

molecules integrin-α4β1/VLA4 [91, 92].

**4. CXCR4 and T-cell development**

stages based on CD44 and CD25 expression [80]. CD44+

DN3 cells and the later in CD44-

Following B-cell development in the bone marrow, transitional B-cells home into secondary lymphoid organs to become follicular naive B-cells that constitute a B-cell zone, called primary follicle, distinct from the T-cell one. B-cells use CCR7, CXCR4 and CXCR5 receptors to migrate from the blood into SLO [60, 61]. CXCR5+ B-cells are attracted and organize themselves around CXCL13-expressing follicular dendritic cells and marginal reticular cells at the periphery, whereas T-cells express CCR7 and are attracted by CCL19-and CCL21-producing reticular cells [62]. Following antigen-specific T-cell activation, T-cells down regulate CCR7 and move towards B-cell follicles. In parallel antigen-activated B-cells keep their CXCR5 expression unaltered while up regulating that of CCR7 thereby migrating towards the T-cell zone. Upon T-B encounter, antigen-specific B-cells then proliferate to constitute the dark zone of the germinal centre. These large cells so called centroblasts undergo Immunoglobulin (Ig) gene class-switch recombination, due to CD40/CD154 interactions on B-and T-cells respectively [63]. Class-switch recombination is also a prerequisite for B-cells to undergo somatic hyper mutation of the hyper variable regions of Ig genes, these random mutation process leads to antibody diversity [64]. Centroblasts constitute the dark zone and are CXCR4+/hi. As centro‐ blasts are promoted they down regulate CXCR4, enter a quiescent state and constitute the light zone made of CD23+ CD83+ CXCR4-/lo differentiated B-cells called centrocytes and are selected by antigen-presenting cells, so that B-cells with high affinity against antigen are positively selected for survival whereas low-affinity cells die in situ [65, 66]. Centrocytes ultimately give rise to plasma cells (PC) or to memory B-cells. B-cell differentiation in PC precursors also called plasmablasts, requires a synchronous up regulation of CXCR4 and down regulation of CXCR5, however migration of plasmablasts into splenic red pulp or lymph node medullary sinuses may not depend on CXCR4 expression inasmuch as mice with invalid *Cxcr4* in adult B-cells display PC in these respective anatomic sites [67-69]. A likely hypothesis is that CXCR4 is instead needed for long-lived PC survival in bone marrow niches and thatVLA4 interaction with VCAM1 is necessary in this respect [70, 71] whereas migration of antibody-producing cells into the bone marrow depends on the sphingosine-1 phosphate and its receptor S1P1 [68]. Finally, an unanswered and interesting question is whether long-lived PC and B-cell progen‐ itors compete for the same bone marrow niches.

Thus CXCR4 expression is sinusoidal in mature B-cells and tightly regulated within the secondary follicle intuitively suggesting that anomalous expression or signaling of CXCR4 may lead to B-cell defects or malignancies.

It is well known that CXCR4 expression is regulated directly by interactions with CXCL12 and indirectly by BCR signaling. CXCL12 interaction with CXCR4 induces CXCR4 inactivation on T-and B-cells and myeloid cells as well, so it is a general mechanism at least on mature cells that lead to CXCR4 phosphorylation on its intracytoplasmic C-terminal tail, leading to G protein uncoupling and CXCR4 internalization [20, 72, 73]. As for most chemokine receptor which are G-protein-coupled receptors, binding of CXCR4 with its ligand induces conforma‐ tional modifications and secondary activation of G-proteins and further intracellular recruit‐ ment of G-protein-coupled-receptor kinases (GRK) that phosphorylate CXCR4 on Ser/Thr residues and recruit β-arrestins resulting in CXCR4 desensitization and finally its internaliza‐ tion [14]. CXCR4 is internalized and its surface expression down regulated following BCR activation or CXCL12 binding. Both signaling pathways induce CXCR4 phosphorylation on Ser residues albeit on distinct residues [20]. In fact, the migratory B-cell response to CXCL12 depends on both intrinsic factors and CXCR4 expression; indeed naive and memory B-cells are more sensitive to CXCL12 than germinal center B-cells in this respect [74]. The inhibition of CXCL12-mediated chemotaxis following BCR engagement depends on PKC but not on Ca2+due to CXCR4 internalization [20).

In conclusion, CXCR4 expression in B-cell lineage cells depends on CXCL12 expression at the pro/pre B-cell stages and PC that lack functional BCR, whereas it also depends on the BCR signal strength at more mature stages.

#### **4. CXCR4 and T-cell development**

**3.2. B-cell homing and positioning within secondary lymphoid organs and regulation of**

Following B-cell development in the bone marrow, transitional B-cells home into secondary lymphoid organs to become follicular naive B-cells that constitute a B-cell zone, called primary follicle, distinct from the T-cell one. B-cells use CCR7, CXCR4 and CXCR5 receptors to migrate

CXCL13-expressing follicular dendritic cells and marginal reticular cells at the periphery, whereas T-cells express CCR7 and are attracted by CCL19-and CCL21-producing reticular cells [62]. Following antigen-specific T-cell activation, T-cells down regulate CCR7 and move towards B-cell follicles. In parallel antigen-activated B-cells keep their CXCR5 expression unaltered while up regulating that of CCR7 thereby migrating towards the T-cell zone. Upon T-B encounter, antigen-specific B-cells then proliferate to constitute the dark zone of the germinal centre. These large cells so called centroblasts undergo Immunoglobulin (Ig) gene class-switch recombination, due to CD40/CD154 interactions on B-and T-cells respectively [63]. Class-switch recombination is also a prerequisite for B-cells to undergo somatic hyper mutation of the hyper variable regions of Ig genes, these random mutation process leads to antibody diversity [64]. Centroblasts constitute the dark zone and are CXCR4+/hi. As centro‐ blasts are promoted they down regulate CXCR4, enter a quiescent state and constitute the light

by antigen-presenting cells, so that B-cells with high affinity against antigen are positively selected for survival whereas low-affinity cells die in situ [65, 66]. Centrocytes ultimately give rise to plasma cells (PC) or to memory B-cells. B-cell differentiation in PC precursors also called plasmablasts, requires a synchronous up regulation of CXCR4 and down regulation of CXCR5, however migration of plasmablasts into splenic red pulp or lymph node medullary sinuses may not depend on CXCR4 expression inasmuch as mice with invalid *Cxcr4* in adult B-cells display PC in these respective anatomic sites [67-69]. A likely hypothesis is that CXCR4 is instead needed for long-lived PC survival in bone marrow niches and thatVLA4 interaction with VCAM1 is necessary in this respect [70, 71] whereas migration of antibody-producing cells into the bone marrow depends on the sphingosine-1 phosphate and its receptor S1P1 [68]. Finally, an unanswered and interesting question is whether long-lived PC and B-cell progen‐

Thus CXCR4 expression is sinusoidal in mature B-cells and tightly regulated within the secondary follicle intuitively suggesting that anomalous expression or signaling of CXCR4

It is well known that CXCR4 expression is regulated directly by interactions with CXCL12 and indirectly by BCR signaling. CXCL12 interaction with CXCR4 induces CXCR4 inactivation on T-and B-cells and myeloid cells as well, so it is a general mechanism at least on mature cells that lead to CXCR4 phosphorylation on its intracytoplasmic C-terminal tail, leading to G protein uncoupling and CXCR4 internalization [20, 72, 73]. As for most chemokine receptor which are G-protein-coupled receptors, binding of CXCR4 with its ligand induces conforma‐ tional modifications and secondary activation of G-proteins and further intracellular recruit‐ ment of G-protein-coupled-receptor kinases (GRK) that phosphorylate CXCR4 on Ser/Thr

B-cells are attracted and organize themselves around

CXCR4-/lo differentiated B-cells called centrocytes and are selected

**CXCR4 expression by BCR signaling**

54 Adult Stem Cell Niches

from the blood into SLO [60, 61]. CXCR5+

CD83+

itors compete for the same bone marrow niches.

may lead to B-cell defects or malignancies.

zone made of CD23+

Early thymic progenitors cKit+ which reside in the thymic double negative DN1 fraction differentiate from common lymphoid progenitor and migrate from the bone marrow to the thymus through the blood and invade the epithelial rudiment at the cortico-medullary junction through post capillary venules where they loose progressively non-T-cell differentiated capacity and become fully T-cell committed as also described in human thymocytes [75-79]. These DN thymocytes make some 2% of thymocytes and are further divided in differentiation stages based on CD44 and CD25 expression [80]. CD44+ CD25- DN1 cells differentiate in CD44+ CD25+ DN2 cells following migration into the subcapsular zone, DN2 differentiate in CD44- CD25+ DN3 cells and the later in CD44- CD25 cortical DN4 cells. In turn DN4 differentiate in CD4+ CD8+ double positive DP thymocytes that acquire surface CD3 and move toward the inner cortex [81]. Only two chemokines, CXCL12 and CCL25/thymus-expressed chemokine TECK are expressed in the thymic cortex namely by cortical epithelial thymic cells [82-84]. The CXCL12/CXCR4 axis is mandatory for retaining human double positive (DP) thymocytes in the cortex. In addition, in mice lacking Cxcr4 on thymocytes displayed defective DN migration to the cortex and defective DN to DP transition [82, 85-87]. DN migration can indeed be inhibited by AMD3100 [85, 88]. In the medulla, CCR7 promotes migration of mature thymo‐ cytes from the cortex. Thus, two opposing chemokine gradients regulate thymocyte migration from the cortex to the medulla.

TCR βselection starts at the DN stage and DN3 cells express pre-TCR made of a pre-TCR α chain associated to TCR-β chain. These cells are selected positively by interactions with stromal cells in the cortex and CXCR4 is crucial for this process. Indeed, it is functionally associated with the pre-TCR and needed to activate phosphatidyl inositol 3-kinase (PI3K) and Notch pathway, the later which is mandatory for T-cell differentiation [86, 87, 89, 90]. In brief, DN thymocytes expand and differentiate in the cortex due to migration retention and survival signals dependent on CXCR4, and as for HSC, CXCR4 activation up regulate adhesion molecules integrin-α4β1/VLA4 [91, 92].

Thus CXCR4 plays a crucial and non-redundant role in thymic progenitors positioning in the cortex and in pre-TCR-mediated survival signals, the failure of which resulted in develop‐ mental arrest at the DN stage. Of interest, mice carrying a WHIM Syndrome heterozygous *Cxcr4* mutation display abnormal thymic maturation [59]. Once positive selection of DP thymocytes and negative selection of CD4+ and CD8+ single positive thymocytes has been achieved, these cells leave the thymus to seed secondary lymphoid organs. Naive T-cells do not rely on CXCR4 for thymic egress and to home into T-cell zones. However CXCR4 is needed for T-cell extravasation through high endothelial venules and entry into the lymph nodes [93].

marrow is associated with coordinated and tightly regulated changes of both expression and activity of distinct GPCRs including CXCR4. Indeed, CXCR4 is highly expressed on NK precursors but it gradually declines during maturation [103]. Conversely, expression of CXCR3, CCR1, CX3CR1 and S1P5 (Sphingosine-1-phosphate receptor 5) progressively increase with NK-cell maturation [104, 105]. CXCR4 and S1P5 appear as master regulators of NK-cell retention in, and egress from, the bone marrow. The exit process is obviously required for ensuring immunosurveillance. CXCR4 retains NK cells in the bone marrow parenchyma, whereas S1P5 promotes their exit from this organ through sinusoids. Using *S1p5*-null mice and knockin (KI) mouse model in which Cxcr4 cannot be desensitized, Mayol et al have recently showed that NK-cell exit from the bone marrow requires both Cxcr4 desensitization and S1p5 engagement by their corresponding ligands namely Cxcl12 and S1p, which are produced in the bone marrow and the bloodstream respectively [103]. In the bone marrow, CXCL12 is detected in different niches including the endosteal one and CAR cells as well [46, 106]. A recent study indicates that NK cells are found in close contact to CAR cells that also produce IL-15, another master regulator of NK-cell homeostasis [107]. Another work conduct‐ ed by Sciumè and collaborators has involved the integrin chain α4 and the Fractalkine/CX3CL1 receptor CX3CR1 in the positioning of mature NK cells in the sinusoidal compartment [104]. Once in the blood, the S1P concentration increases and S1P5 responsiveness decreases [108]. This responsiveness is recovered in the lymph nodes to allow NK-cell exit via lymphatics in a CXCR4-independent manner. The mechanism controlling NK-cell exit from the human bone marrow is likely to be similar to the one reported in the mouse counterpart. Several lines of evidence support this assertion. First, the absolute number of NK cells is deeply decreased in the peripheral blood of some WHIM syndrome patients that harbour a gain-of-*CXCR4* function mutation [109]. Second, CXCR4 has been shown to retain human NK cells in the bone marrow and spleen of immunodeficient mice reconstituted with human immune system [110]. Finally, S1P5 was reported to be unregulated during human NK-cell differentiation [105].

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57

The WHIM syndrome (WS) is a rare immunological disorder characterized by the presence of warts (W), hypogammaglobulinemia (H), bacterial infections (I) and myelokathexis (M) meaning an abnormal retention of pro-apoptotic neutrophils in the bone marrow [111]*.* WS is an inherited pathological disorder with an autosomal dominant transmission. The WS estimate incidence was of 0.23 per million births but the prevalence is < 1/1 000 000. In fact, there are

Inherited heterozygous autosomal dominant mutations of the *CXCR4* gene, which result in the truncation of the carboxyl-terminus of the receptor leading to a defect of CXCR4 inactiva‐ tion, were found to be associated with the WS [113]. The disorder is clinically and genetically heterogeneous, since hypogammaglobulinemia and verrucosis were absent in some cases, and individuals with isolated myelokathexis were found to be "wild type" for the *CXCR4* gene [114]. As described previously, CXCR4 is expressed in hematopoietic cells. Consequently, the

**6. CXCR4 in the WHIM syndrome**

less than 60 documented cases in the world [112].

As for the BCR, TCR engagement or signals that mimic PKC activation such as phorbol esters result in the phosphorylation and internalization of CXCR4 in T-cells, whereas infections with human herpes viruses HHV-6 or-7 down regulate*CXCR4* transcription [72, 94]. TCR-induced CXCR4 down modulation may therefore help T-cells stay at the site of self-antigen encounter by dendritic cells (DC) in the thymus in order to undergo negative selection in the medulla. Similar mechanism may operate in secondary lymphoid organs during presentation of antigens by DC. Of interest CXCR4 may increase the stability of the T-antigen-presenting cell (APC) immunologic synapses (IS) necessary for successful T-cell activation [95]. Interestingly, upon CXCL12 stimulation, a physical association between CXCR4 and TCR was described and promote TCR signal transduction. This association is responsible for prolonged extracellular signal regulated kinase activity, increased intracellular calcium ion concentrations, robust *activator protein-1* transcriptional activity, and SDF-1α costimulation of cytokine secretion. These pathways mediate costimulation of cytokine secretion by activated T-cells [96].

Following antigen encounter, naive T-cells differentiate in CD4+ or CD8+ memory T-cells that home into the bone marrow which is a survival site for memory T-cells [97, 98]. However CXCR4 does not seem to be necessary for the retention and survival of memory T-cells, as survival is mostly dependent on IL7 [99, 100].

In conclusion the physiological role of CXCR4 in T-cell development and functions predomi‐ nates on thymic progenitors although impaired peripheral T-cell responses in WHIM syn‐ drome and Idiopathic CD4+ T-cell Lymphocytopenia suggest a role of CXCR4 on peripheral Tcells.

#### **5. CXCR4 and NK-cell development**

Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in the early control of infections by viruses and other intracellular pathogens. NK cells can be identified by expression of the activating NK receptor NK1.1 associated with the absence of T-cell CD3 receptor complex. Based on the membrane expression of the tumour necrosis factor superfamily member CD27 and the integrin CD11b, four maturation stages can be identified: CD11blowCD27low ("double negative"), which likely encompass precursors, CD11blowCD27high ("CD11blow"), CD11bhighCD27high ("double positive"), and CD11bhighCD27low ("CD27low") [101, 102]. These NK-cell subsets display heterogeneous distribution in lymphoid organs. The bone marrow plays a pivotal role in NK-cell development. NK-cell differentiation in the bone marrow is associated with coordinated and tightly regulated changes of both expression and activity of distinct GPCRs including CXCR4. Indeed, CXCR4 is highly expressed on NK precursors but it gradually declines during maturation [103]. Conversely, expression of CXCR3, CCR1, CX3CR1 and S1P5 (Sphingosine-1-phosphate receptor 5) progressively increase with NK-cell maturation [104, 105]. CXCR4 and S1P5 appear as master regulators of NK-cell retention in, and egress from, the bone marrow. The exit process is obviously required for ensuring immunosurveillance. CXCR4 retains NK cells in the bone marrow parenchyma, whereas S1P5 promotes their exit from this organ through sinusoids. Using *S1p5*-null mice and knockin (KI) mouse model in which Cxcr4 cannot be desensitized, Mayol et al have recently showed that NK-cell exit from the bone marrow requires both Cxcr4 desensitization and S1p5 engagement by their corresponding ligands namely Cxcl12 and S1p, which are produced in the bone marrow and the bloodstream respectively [103]. In the bone marrow, CXCL12 is detected in different niches including the endosteal one and CAR cells as well [46, 106]. A recent study indicates that NK cells are found in close contact to CAR cells that also produce IL-15, another master regulator of NK-cell homeostasis [107]. Another work conduct‐ ed by Sciumè and collaborators has involved the integrin chain α4 and the Fractalkine/CX3CL1 receptor CX3CR1 in the positioning of mature NK cells in the sinusoidal compartment [104]. Once in the blood, the S1P concentration increases and S1P5 responsiveness decreases [108]. This responsiveness is recovered in the lymph nodes to allow NK-cell exit via lymphatics in a CXCR4-independent manner. The mechanism controlling NK-cell exit from the human bone marrow is likely to be similar to the one reported in the mouse counterpart. Several lines of evidence support this assertion. First, the absolute number of NK cells is deeply decreased in the peripheral blood of some WHIM syndrome patients that harbour a gain-of-*CXCR4* function mutation [109]. Second, CXCR4 has been shown to retain human NK cells in the bone marrow and spleen of immunodeficient mice reconstituted with human immune system [110]. Finally, S1P5 was reported to be unregulated during human NK-cell differentiation [105].

#### **6. CXCR4 in the WHIM syndrome**

Thus CXCR4 plays a crucial and non-redundant role in thymic progenitors positioning in the cortex and in pre-TCR-mediated survival signals, the failure of which resulted in develop‐ mental arrest at the DN stage. Of interest, mice carrying a WHIM Syndrome heterozygous *Cxcr4* mutation display abnormal thymic maturation [59]. Once positive selection of DP

achieved, these cells leave the thymus to seed secondary lymphoid organs. Naive T-cells do not rely on CXCR4 for thymic egress and to home into T-cell zones. However CXCR4 is needed for T-cell extravasation through high endothelial venules and entry into the lymph nodes [93]. As for the BCR, TCR engagement or signals that mimic PKC activation such as phorbol esters result in the phosphorylation and internalization of CXCR4 in T-cells, whereas infections with human herpes viruses HHV-6 or-7 down regulate*CXCR4* transcription [72, 94]. TCR-induced CXCR4 down modulation may therefore help T-cells stay at the site of self-antigen encounter by dendritic cells (DC) in the thymus in order to undergo negative selection in the medulla. Similar mechanism may operate in secondary lymphoid organs during presentation of antigens by DC. Of interest CXCR4 may increase the stability of the T-antigen-presenting cell (APC) immunologic synapses (IS) necessary for successful T-cell activation [95]. Interestingly, upon CXCL12 stimulation, a physical association between CXCR4 and TCR was described and promote TCR signal transduction. This association is responsible for prolonged extracellular signal regulated kinase activity, increased intracellular calcium ion concentrations, robust *activator protein-1* transcriptional activity, and SDF-1α costimulation of cytokine secretion.

These pathways mediate costimulation of cytokine secretion by activated T-cells [96].

home into the bone marrow which is a survival site for memory T-cells [97, 98]. However CXCR4 does not seem to be necessary for the retention and survival of memory T-cells, as

In conclusion the physiological role of CXCR4 in T-cell development and functions predomi‐ nates on thymic progenitors although impaired peripheral T-cell responses in WHIM syn‐

Natural killer (NK) cells are lymphocytes of the innate immune system that are involved in the early control of infections by viruses and other intracellular pathogens. NK cells can be identified by expression of the activating NK receptor NK1.1 associated with the absence of T-cell CD3 receptor complex. Based on the membrane expression of the tumour necrosis factor superfamily member CD27 and the integrin CD11b, four maturation stages can be identified: CD11blowCD27low ("double negative"), which likely encompass precursors, CD11blowCD27high ("CD11blow"), CD11bhighCD27high ("double positive"), and CD11bhighCD27low ("CD27low") [101, 102]. These NK-cell subsets display heterogeneous distribution in lymphoid organs. The bone marrow plays a pivotal role in NK-cell development. NK-cell differentiation in the bone

Following antigen encounter, naive T-cells differentiate in CD4+

survival is mostly dependent on IL7 [99, 100].

**5. CXCR4 and NK-cell development**

drome and Idiopathic CD4+

cells.

and CD8+

single positive thymocytes has been

or CD8+

T-cell Lymphocytopenia suggest a role of CXCR4 on peripheral T-

memory T-cells that

thymocytes and negative selection of CD4+

56 Adult Stem Cell Niches

The WHIM syndrome (WS) is a rare immunological disorder characterized by the presence of warts (W), hypogammaglobulinemia (H), bacterial infections (I) and myelokathexis (M) meaning an abnormal retention of pro-apoptotic neutrophils in the bone marrow [111]*.* WS is an inherited pathological disorder with an autosomal dominant transmission. The WS estimate incidence was of 0.23 per million births but the prevalence is < 1/1 000 000. In fact, there are less than 60 documented cases in the world [112].

Inherited heterozygous autosomal dominant mutations of the *CXCR4* gene, which result in the truncation of the carboxyl-terminus of the receptor leading to a defect of CXCR4 inactiva‐ tion, were found to be associated with the WS [113]. The disorder is clinically and genetically heterogeneous, since hypogammaglobulinemia and verrucosis were absent in some cases, and individuals with isolated myelokathexis were found to be "wild type" for the *CXCR4* gene [114]. As described previously, CXCR4 is expressed in hematopoietic cells. Consequently, the lack of CXCR4 inactivationis expected to generate significant immune and haematological disturbances [14].

lesser extent of immature B-cells, was reduced in *Cxcr4+/1013* mice. Therefore, the alteration of early B-cell development in *Cxcr4+/1013* mice was not associated with enhanced cell death. Regarding thymic differentiation, the absolute number of each thymic subset (from progen‐ itors to mature SP thymocytes) was significantly decreased in *Cxcr4+/1013* mice. Together, these findings reveal that altered Cxcr41013-driven development of B-and T-cells leads to chronic

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

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59

Regarding the periphery, the absolute number of each blood leukocyte subpopulation was significantly reduced in *Cxcr4+/1013* mice. Serum levels of natural IgA were similar in nonmanipulated *Cxcr4+/1013* and WT mice, whereas IgM and IgG concentrations increased in *Cxcr4+/1013* mice contrary to patients as mentioned above. In parallel, mature T-and B-cells were abnormally compartmentalized in the periphery, with fewer primary follicles in the spleen and absence in lymph nodes, mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B-and T-cells and to investigate which biological abnormalities of patients may be reversed by dampening the gain of CXCR4 function [59]. As mentioned above (chapter 4) CXCR4 may increase the stability of the T-APC interactions and is necessary for optimal T-cell activation [95]. Indeed, recent data demonstrated that WHIM-CXCR4–expressing retrogenic T-cells inhibits the formation of long-lasting T-APC interactions confirming the role of CXCR4 in the stability of T-APC Immune Synapse. Such anomalous synapse formation likely results in the failure to respond to vaccinations with the absence of specific antibody one year after vaccination observed in WS patient [117]. Thus, it will be interesting to investigate if there is an optimal adaptive immune response in these mice. In addition, the discovery of a mouse papillomavirus (MusPV) rendered possible to analyse HPV-infected *Cxcr4+/1013*mice [118], as done in nude mice [119].Finally our mice will also be useful to test new drugs including molecules that target

AMD3100 (plerixafor) a selective and competitive antagonist of CXCR4 appears to be a potential treatment for patients with WS, and clinical studies have been conducted accord‐ ingly. One study reported the effect of daily injections with increased concentrations of AMD3100 (0.02-0.24 mg/kg) in 3 adults with WS; an increase of white blood cells and of absolute lymphocyte, neutrophil and monocyte counts was observed in all patients. Further‐ more white blood cells mobilization is higher with AMD3100 than with GCSF [120]. Another study showed that treatment with AMD3100 every 2-4 days in 6 patients resulted in prompt leukocytosis [121]. Mice carrying the heterozygous *Cxcr4+/1013*mutation, were also treated with AMD3100 or chalcone 4 that binds Cxcl12 and prevents signaling through Cxcr4. After single intraperitoneal injection of either AMD3100 or chalcone 4, the absolute numbers of total leukocytes in the blood of WT mice, including neutrophils, B and T cells was increased within 3 hours [59].Therefore, AMD3100 is able to reverse the pan leukopenia in WS mice and patients albeit transiently because of its short half-life. A more recent trial involving 3 WHIM patients treated with plerixafor subcutaneously twice daily for 6 months in combination with the Interferon-αinducer imiquimod, showed improvement in warts and in infections, albeit with

partial restoration of Ig levels and vaccine responses [103].

circulating lymphopenia.

CXCR4.

Patients with WS exhibit a marked lymphopenia suggesting either a central defect of leukocyte differentiation in the bone marrow and the thymus or a peripheral defect such as an increase in apoptosis. Morphological analysis of bone marrow was performed in some patients; it evidenced an abnormal morphology of neutrophils called myelokathexis and an increase in neutrophil counts [111]. Furthermore the analysis of a bone marrow sample from one patient failed to detect abnormalities in lymphoid precursors and in immature and mature B-lym‐ phocytes [115]*.*

Biological features of WS include hypogammaglobulinemia, involving IgG. As expected, hypogammaglobulinemia combined to neutropenia in WS patients results in infections especially of encapsulated bacterias. Interestingly, infections transiently increase the number of neutrophil counts in the blood suggesting that neutropenia is caused by lack of neutrophils egress rather than to a defect in production. Patients also present recurrent pneumonias, sinusitis, urinary tract infection and skin infections among others. Strikingly, WS patients display high susceptibility to human papilloma virus (HPV) leading to skin lesions such as warts on hands, feet and trunk, genital and anal condylomas and mucosal lesions which often progress to carcinomas [116]. In two patients carrying the heterozygous *CXCR4* 1013 mutation, CXCR4 failed to internalize on lymphocytes upon stimulation. In PBMC, refractoriness of CXCR4 for desensitization and internalization led to an enhanced CXCL12-promoted chemo‐ taxis [114]*.* Thus the lack of CXCR4 inactivation is associated with a gain of function of the receptor at least based on migration capacity criteria.

Despite lymphopenia and hypogammaglobulinemia, WS patients immunized with tetanustoxoid produce normal amounts of antibodies against tetanus-toxoid 10 weeks after immuni‐ zation. However, no specific antibodies were found one year after immunization suggesting a defect in long-lived PC and/or in memory B-cell response in WS [115].

There are currently no specific treatments for WS patients, albeit symptomatic use of GCSF and intravenous immunoglobulins (Ig) combined to anti-infectious agents is of some help [116], however CXCR4-targeted therapy is promising as described below.

In order to better understand this pathology, to characterise lymphoid differentiation and haematopoiesis defects in WS, we sought to generate an animal model. Balabanian et *al* generated a KI mouse strain that harbours a WS-associated heterozygous mutation of the *Cxcr4* gene (*i.e.,Cxcr4+/1013)* to analyse the impact of Cxcr4 desensitization on leukocyte homeostasis [59]. These mice display a severe lymphoneutropenia. As in patients, CXCR4 failure to internalise doesn't lead to an increase in receptor expression in leukocytes in eitherbone marrow, thymus, spleen and blood. Moreover, all tested leukocyte subsets from *Cxcr4+/1013* mice, displayed increased sensitivity to Cxcl12-promoted chemotaxis compared to wild type mice as shown in blood T-and B-cells, and in DP, and SP thymocytes. Both frequency and absolute numbers of CD19+ B-cells were slightly, but significantly lower in the bone marrow from *Cxcr4+/1013* mice. In contrast to the myeloid series, leukocyte differentia‐ tion was altered in *Cxcr4+/1013* mice. Indeed, the absolute number of pro/pre-B cells, and to a lesser extent of immature B-cells, was reduced in *Cxcr4+/1013* mice. Therefore, the alteration of early B-cell development in *Cxcr4+/1013* mice was not associated with enhanced cell death. Regarding thymic differentiation, the absolute number of each thymic subset (from progen‐ itors to mature SP thymocytes) was significantly decreased in *Cxcr4+/1013* mice. Together, these findings reveal that altered Cxcr41013-driven development of B-and T-cells leads to chronic circulating lymphopenia.

lack of CXCR4 inactivationis expected to generate significant immune and haematological

Patients with WS exhibit a marked lymphopenia suggesting either a central defect of leukocyte differentiation in the bone marrow and the thymus or a peripheral defect such as an increase in apoptosis. Morphological analysis of bone marrow was performed in some patients; it evidenced an abnormal morphology of neutrophils called myelokathexis and an increase in neutrophil counts [111]. Furthermore the analysis of a bone marrow sample from one patient failed to detect abnormalities in lymphoid precursors and in immature and mature B-lym‐

Biological features of WS include hypogammaglobulinemia, involving IgG. As expected, hypogammaglobulinemia combined to neutropenia in WS patients results in infections especially of encapsulated bacterias. Interestingly, infections transiently increase the number of neutrophil counts in the blood suggesting that neutropenia is caused by lack of neutrophils egress rather than to a defect in production. Patients also present recurrent pneumonias, sinusitis, urinary tract infection and skin infections among others. Strikingly, WS patients display high susceptibility to human papilloma virus (HPV) leading to skin lesions such as warts on hands, feet and trunk, genital and anal condylomas and mucosal lesions which often progress to carcinomas [116]. In two patients carrying the heterozygous *CXCR4* 1013 mutation, CXCR4 failed to internalize on lymphocytes upon stimulation. In PBMC, refractoriness of CXCR4 for desensitization and internalization led to an enhanced CXCL12-promoted chemo‐ taxis [114]*.* Thus the lack of CXCR4 inactivation is associated with a gain of function of the

Despite lymphopenia and hypogammaglobulinemia, WS patients immunized with tetanustoxoid produce normal amounts of antibodies against tetanus-toxoid 10 weeks after immuni‐ zation. However, no specific antibodies were found one year after immunization suggesting

There are currently no specific treatments for WS patients, albeit symptomatic use of GCSF and intravenous immunoglobulins (Ig) combined to anti-infectious agents is of some help

In order to better understand this pathology, to characterise lymphoid differentiation and haematopoiesis defects in WS, we sought to generate an animal model. Balabanian et *al* generated a KI mouse strain that harbours a WS-associated heterozygous mutation of the *Cxcr4* gene (*i.e.,Cxcr4+/1013)* to analyse the impact of Cxcr4 desensitization on leukocyte homeostasis [59]. These mice display a severe lymphoneutropenia. As in patients, CXCR4 failure to internalise doesn't lead to an increase in receptor expression in leukocytes in eitherbone marrow, thymus, spleen and blood. Moreover, all tested leukocyte subsets from *Cxcr4+/1013* mice, displayed increased sensitivity to Cxcl12-promoted chemotaxis compared to wild type mice as shown in blood T-and B-cells, and in DP, and SP thymocytes. Both

bone marrow from *Cxcr4+/1013* mice. In contrast to the myeloid series, leukocyte differentia‐ tion was altered in *Cxcr4+/1013* mice. Indeed, the absolute number of pro/pre-B cells, and to a

B-cells were slightly, but significantly lower in the

receptor at least based on migration capacity criteria.

frequency and absolute numbers of CD19+

a defect in long-lived PC and/or in memory B-cell response in WS [115].

[116], however CXCR4-targeted therapy is promising as described below.

disturbances [14].

58 Adult Stem Cell Niches

phocytes [115]*.*

Regarding the periphery, the absolute number of each blood leukocyte subpopulation was significantly reduced in *Cxcr4+/1013* mice. Serum levels of natural IgA were similar in nonmanipulated *Cxcr4+/1013* and WT mice, whereas IgM and IgG concentrations increased in *Cxcr4+/1013* mice contrary to patients as mentioned above. In parallel, mature T-and B-cells were abnormally compartmentalized in the periphery, with fewer primary follicles in the spleen and absence in lymph nodes, mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B-and T-cells and to investigate which biological abnormalities of patients may be reversed by dampening the gain of CXCR4 function [59]. As mentioned above (chapter 4) CXCR4 may increase the stability of the T-APC interactions and is necessary for optimal T-cell activation [95]. Indeed, recent data demonstrated that WHIM-CXCR4–expressing retrogenic T-cells inhibits the formation of long-lasting T-APC interactions confirming the role of CXCR4 in the stability of T-APC Immune Synapse. Such anomalous synapse formation likely results in the failure to respond to vaccinations with the absence of specific antibody one year after vaccination observed in WS patient [117]. Thus, it will be interesting to investigate if there is an optimal adaptive immune response in these mice. In addition, the discovery of a mouse papillomavirus (MusPV) rendered possible to analyse HPV-infected *Cxcr4+/1013*mice [118], as done in nude mice [119].Finally our mice will also be useful to test new drugs including molecules that target CXCR4.

AMD3100 (plerixafor) a selective and competitive antagonist of CXCR4 appears to be a potential treatment for patients with WS, and clinical studies have been conducted accord‐ ingly. One study reported the effect of daily injections with increased concentrations of AMD3100 (0.02-0.24 mg/kg) in 3 adults with WS; an increase of white blood cells and of absolute lymphocyte, neutrophil and monocyte counts was observed in all patients. Further‐ more white blood cells mobilization is higher with AMD3100 than with GCSF [120]. Another study showed that treatment with AMD3100 every 2-4 days in 6 patients resulted in prompt leukocytosis [121]. Mice carrying the heterozygous *Cxcr4+/1013*mutation, were also treated with AMD3100 or chalcone 4 that binds Cxcl12 and prevents signaling through Cxcr4. After single intraperitoneal injection of either AMD3100 or chalcone 4, the absolute numbers of total leukocytes in the blood of WT mice, including neutrophils, B and T cells was increased within 3 hours [59].Therefore, AMD3100 is able to reverse the pan leukopenia in WS mice and patients albeit transiently because of its short half-life. A more recent trial involving 3 WHIM patients treated with plerixafor subcutaneously twice daily for 6 months in combination with the Interferon-αinducer imiquimod, showed improvement in warts and in infections, albeit with partial restoration of Ig levels and vaccine responses [103].

To conclude, patients with WS have a gain of CXCR4 function, which leads to severe disorders of lymphocytes and neutrophils and hence of immune responses. How it affects mature T-and B-cells is still unclear. Undoubtedly, studies on homozygous and heterozygous mice and on the hematopoietic compartment will help understand the pathologies associated with CXCR4 dysfunction.

ligand overexpression [137]. Markers for cell activation and turn-over, as indicated by HLA-

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

cells cycling was associated with levels of plasma lipopolysaccharide resulting from microbial translocation [139]. Therefore, immune activation and preferential loss of naive T-cells in ICL could result from chronic and persistent stimulation by an unidentified pathogen [122]. Mechanistic studies have also pointed out a defective TCR signal transduction in ICL raising the possibility that persistent T-cell activation leads to defective TCR signaling and may contribute to T-cell depletion [130-132]. Increased levels of the homeostatic cytokineIL7 in the

140]. This accumulation of IL7 likely results from both a diminished consumption of the cytokine and a decrease of IL7 receptor α chain expression (CD127) on the reduced T-cell pool. Moreover the induction of phospho-STAT5 after IL7 stimulation was decreased in residual

not necessarily represent a compensatory response but may be further accentuating T-cell apoptosis and lymphopenia [141, 142]. In addition, while one study reported a successful therapy with IL2, decreased IL2 responses correlated with impaired IL7 responses, which may

T-cells homeostasis in ICL [142-144].

Regarding the CXCR4/CXCL12 axis, some studies demonstrated it role for T-cell production, homing, positioning and activation within secondary lymphoid tissues. Furthermore, altera‐ tions of CXCR4 expression or activity are likely to severely impact T-cell differentiation and trafficking. Thus, we hypothesized that expression or function of CXCR4 could be altered in

intracellular accumulation of CXCR4 and CXCL12. This suggested a defective intracellular routing of the chemokines/chemokines receptors complex. Analyses of CXCR4 fate following CXCL12 stimulation indicated that CXCR4 preserved its ability to internalize, but thereafter poorly recycled back to the plasma membrane of ICL T-cells. Altered membrane CXCR4 recovery resulted in a loss of CXCR4 function, as illustrated by the impaired CXCL12-

newly ICL patients and suggest that impaired membrane CXCR4 expression may contribute

Altogether, ICL is a complex disorder with impaired membrane CXCR4 expression in peripheral lymphoid populations and in progenitors, possibly explaining the downstream lymphocytopenia. Deciphering the reasons for such dysfunction will help us discover new molecular targets for immune cell therapy. In this context, we derived induced pluripotent

T-cell homeostasis in the periphery but also in the thymus.

T-cells of some ICL patients. These data suggest that high serum IL7 levels do

T-cells alteration. Thus,ICL may mirror WHIM Syndrome as we observed

T-cells in ICL. We contributed to the identification of a

T-cells [144]. These results were recently extended to 20

T-cells of an ICL patient; this will offer unprecedented opportu‐

T-cells from 6 ICL patients with concomitant

T-cell counts [138]. In another report, abnormal CD4+

T-cells and this was

http://dx.doi.org/10.5772/58699

T-cells and may reflect

T-cell counts [133, 137,

T-

61

DR, Ki-67 expression and BrdU labelling are also increased in CD4+

sera of ICL patients inversely correlate with those of blood naive CD4+

the triggering of a homeostatic response in order to restore normal CD4+

inversely correlated with blood CD4+

memory CD4+

ICL causing CD4+

to the defective CD4+

stem cells iPSCs, from CD4+

account for the loss of CD4+

decreased CXCR4 expression on CD4+

promoted chemotaxis of ICL CD4+

defect in CXCR4 expression at the surface of CD4+

nities to study T-cell differentiation in this disease.

#### **7. CXCR4 in the Idiopathic CD4+ Lymphocytopenia syndrome**

Idiopathic CD4+ T-cell Lymphocytopenia (ICL) is a rare haematological disorder characterized by a profound and persistent CD4+ T-cell defect, defined by an absolute CD4+ T-cell count <300 cells/mm3 with lack of HIV infection or other known immune defect or therapy associated with lymphopenia [122]. This clinical entity was defined in 1992 by the Centers for Disease Control and then some 258 cases have been reviewed in the literature [122, 123]. ICL patients often present life threatening opportunistic infections similar to those observed in acquired immu‐ nodeficiency (AIDS) syndrome. The most common infections in ICL are cryptococcal (*Cryptococcus neoformans*), genital HPV, non-tuberculous mycobacterial infections (*Mycobacte‐ rium avium*),in addition to progressive multifocal leukoencephalopathy [122, 124]. Malignan‐ cies are also common in ICL, including EBV-related B-cell lymphomas, Kaposi's sarcoma and as seen in the WHIM Syndrome, cervical or perineal neoplasias in the setting of long-term HPV infections [125-129]. In addition, autoimmune diseases (e.g. Sjögren syndrome) were also frequently reported [122].

The aetiology of ICL is unlikely to involve an infectious or environmental agent but may have a genetic basis as loss-of-function mutations have been reported in genes encoding regulators of the TCR diversity and signaling (*i.e.Unc119*, *MAGT1* and *RAG1*) [130-132]. ICL is considered as a heterogeneous syndrome possibly encompassing different disorders sharing the common feature of reduced circulating CD4+ T-cell counts. One issue is to determine whether the ICL results from a defective bone marrow production or thymic output, or an exacerbated peripheral consumption of CD4+ T-cells, or any combination of central or peripheral defect.

The defective production of CD4+ T-cell hypothesis is supported by a study reporting a regenerative failure of HSCs and lymphoid precursors in the bone marrow of ICL patients [133]. A work dealing with T-cell maturation has shown apparent restriction of the α/β and γ/ δ TCR in ICL suggesting a disturbed thymic T-cell maturation in ICL [134]. In support of the central defect hypothesis, CD34+ HSCs derived from one ICL patient carrying a hypomorphic missense mutation in the *Recombination Activating Gene 1* (*RAG1*) were not able to repopulate humanized *Rag2-/-Il2Rgc-/-*mice, which is in favour for the lymphoid origin of the lymphopenia [132]. Finally, non-myeloablative allogeneic bone marrow transplantation in one patient was able to restore CD4+ T-cell numbers and functions [135].

In addition various works argue for an exacerbated peripheral consumption of CD4+ T-cells. Decreased T-cell responses as well as increased T-cell activation and apoptosis have been reported in ICL [136-138]. In terms of CD4+ T-cells activation, studies have reported enhanced propensity of ICL T-cells to undergo apoptosis, a process partially dependent on Fas and Fas ligand overexpression [137]. Markers for cell activation and turn-over, as indicated by HLA-DR, Ki-67 expression and BrdU labelling are also increased in CD4+ T-cells and this was inversely correlated with blood CD4+ T-cell counts [138]. In another report, abnormal CD4+ Tcells cycling was associated with levels of plasma lipopolysaccharide resulting from microbial translocation [139]. Therefore, immune activation and preferential loss of naive T-cells in ICL could result from chronic and persistent stimulation by an unidentified pathogen [122]. Mechanistic studies have also pointed out a defective TCR signal transduction in ICL raising the possibility that persistent T-cell activation leads to defective TCR signaling and may contribute to T-cell depletion [130-132]. Increased levels of the homeostatic cytokineIL7 in the sera of ICL patients inversely correlate with those of blood naive CD4+ T-cells and may reflect the triggering of a homeostatic response in order to restore normal CD4+ T-cell counts [133, 137, 140]. This accumulation of IL7 likely results from both a diminished consumption of the cytokine and a decrease of IL7 receptor α chain expression (CD127) on the reduced T-cell pool. Moreover the induction of phospho-STAT5 after IL7 stimulation was decreased in residual memory CD4+ T-cells of some ICL patients. These data suggest that high serum IL7 levels do not necessarily represent a compensatory response but may be further accentuating T-cell apoptosis and lymphopenia [141, 142]. In addition, while one study reported a successful therapy with IL2, decreased IL2 responses correlated with impaired IL7 responses, which may account for the loss of CD4+ T-cells homeostasis in ICL [142-144].

To conclude, patients with WS have a gain of CXCR4 function, which leads to severe disorders of lymphocytes and neutrophils and hence of immune responses. How it affects mature T-and B-cells is still unclear. Undoubtedly, studies on homozygous and heterozygous mice and on the hematopoietic compartment will help understand the pathologies associated with CXCR4

**Lymphocytopenia syndrome**

T-cell counts. One issue is to determine whether the ICL

T-cell hypothesis is supported by a study reporting a

HSCs derived from one ICL patient carrying a hypomorphic

T-cells activation, studies have reported enhanced

T-cells, or any combination of central or peripheral defect.

T-cell count <300

T-cells.

T-cell Lymphocytopenia (ICL) is a rare haematological disorder characterized

with lack of HIV infection or other known immune defect or therapy associated with

lymphopenia [122]. This clinical entity was defined in 1992 by the Centers for Disease Control and then some 258 cases have been reviewed in the literature [122, 123]. ICL patients often present life threatening opportunistic infections similar to those observed in acquired immu‐ nodeficiency (AIDS) syndrome. The most common infections in ICL are cryptococcal (*Cryptococcus neoformans*), genital HPV, non-tuberculous mycobacterial infections (*Mycobacte‐ rium avium*),in addition to progressive multifocal leukoencephalopathy [122, 124]. Malignan‐ cies are also common in ICL, including EBV-related B-cell lymphomas, Kaposi's sarcoma and as seen in the WHIM Syndrome, cervical or perineal neoplasias in the setting of long-term HPV infections [125-129]. In addition, autoimmune diseases (e.g. Sjögren syndrome) were also

The aetiology of ICL is unlikely to involve an infectious or environmental agent but may have a genetic basis as loss-of-function mutations have been reported in genes encoding regulators of the TCR diversity and signaling (*i.e.Unc119*, *MAGT1* and *RAG1*) [130-132]. ICL is considered as a heterogeneous syndrome possibly encompassing different disorders sharing the common

results from a defective bone marrow production or thymic output, or an exacerbated

regenerative failure of HSCs and lymphoid precursors in the bone marrow of ICL patients [133]. A work dealing with T-cell maturation has shown apparent restriction of the α/β and γ/ δ TCR in ICL suggesting a disturbed thymic T-cell maturation in ICL [134]. In support of the

missense mutation in the *Recombination Activating Gene 1* (*RAG1*) were not able to repopulate humanized *Rag2-/-Il2Rgc-/-*mice, which is in favour for the lymphoid origin of the lymphopenia [132]. Finally, non-myeloablative allogeneic bone marrow transplantation in one patient was

Decreased T-cell responses as well as increased T-cell activation and apoptosis have been

propensity of ICL T-cells to undergo apoptosis, a process partially dependent on Fas and Fas

T-cell numbers and functions [135].

In addition various works argue for an exacerbated peripheral consumption of CD4+

T-cell defect, defined by an absolute CD4+

dysfunction.

60 Adult Stem Cell Niches

Idiopathic CD4+

cells/mm3

**7. CXCR4 in the Idiopathic CD4+**

by a profound and persistent CD4+

frequently reported [122].

feature of reduced circulating CD4+

The defective production of CD4+

peripheral consumption of CD4+

central defect hypothesis, CD34+

reported in ICL [136-138]. In terms of CD4+

able to restore CD4+

Regarding the CXCR4/CXCL12 axis, some studies demonstrated it role for T-cell production, homing, positioning and activation within secondary lymphoid tissues. Furthermore, altera‐ tions of CXCR4 expression or activity are likely to severely impact T-cell differentiation and trafficking. Thus, we hypothesized that expression or function of CXCR4 could be altered in ICL causing CD4+ T-cells alteration. Thus,ICL may mirror WHIM Syndrome as we observed decreased CXCR4 expression on CD4+ T-cells in ICL. We contributed to the identification of a defect in CXCR4 expression at the surface of CD4+ T-cells from 6 ICL patients with concomitant intracellular accumulation of CXCR4 and CXCL12. This suggested a defective intracellular routing of the chemokines/chemokines receptors complex. Analyses of CXCR4 fate following CXCL12 stimulation indicated that CXCR4 preserved its ability to internalize, but thereafter poorly recycled back to the plasma membrane of ICL T-cells. Altered membrane CXCR4 recovery resulted in a loss of CXCR4 function, as illustrated by the impaired CXCL12 promoted chemotaxis of ICL CD4+ T-cells [144]. These results were recently extended to 20 newly ICL patients and suggest that impaired membrane CXCR4 expression may contribute to the defective CD4+ T-cell homeostasis in the periphery but also in the thymus.

Altogether, ICL is a complex disorder with impaired membrane CXCR4 expression in peripheral lymphoid populations and in progenitors, possibly explaining the downstream lymphocytopenia. Deciphering the reasons for such dysfunction will help us discover new molecular targets for immune cell therapy. In this context, we derived induced pluripotent stem cells iPSCs, from CD4+ T-cells of an ICL patient; this will offer unprecedented opportu‐ nities to study T-cell differentiation in this disease.

#### **8. CXCR4 in B-cell malignancies**

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia accounting for some 30% of adult leukaemia's in western countries It is due to an accumulation of small B-cells in the blood, the bone marrow and secondary lymphoid organs. Although this accumulation reflects lack of apoptosis, CLL cells do proliferate and their mitotic index and telomere length correlate with the degree of malignancy as reflected by the Binet [145] or Rai [146] clinical staging or the mutational status of Ig hyper variable regions, currently the gold biological standard for prognosis in this disease [147-149]. Somatically Ig hyper mutated (M-CLL) is typically indolent whereas Ig unmutated (U-CLL) is more aggressive [150, 151]. The cellular origin of CLL has been intensively debated, however contrary to the prevailing view that CLL originate from memory/activated B2 B-cells, we favour the recent hypothesis that CLL B-cells originate from malignant transformation of CD5+ B-cells [152]. Moreover both indolent and aggressive CLL originate from B-cells endowed with an auto reactive surface Ig which supports the hypothesis of a common molecular mechanism for both CLL types albeit occurring at two cellular differentiation stages [153].

PI3kinase-δ and CAL101 an inhibitor of this kinase was efficient against CLL [172]. Thus activating these pathways by molecules less toxic than phorbolmyristate acetate might prove useful to down regulate CXCR4, alternatively, pharmacological inhibitors of regulators, such

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

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63

In Waldenstom's Macroglobulinemia (WM), macroglobulinemia designs an increase in serum concentrations of IgM and causes much of the morbidity associated with the disease. WM is an indolent B-cell malignancy with a monoclonal proliferation of IgM-producing PCs that fail to undergo Ig isotype switching. WM is uncommon relative to plasma cells (PC) myeloma [173]. Although the pathogenesis of WM remained undetermined several data suggested that genetic factors contributed to the disease [174]. PCs from WM are CXCR4hiVLA4hi and as for normal PC, malignant PC continuously home into the bone marrow and trans endothelial migration of PC from WM depends on CXCL12/CXCR4 [175]. It is now clear that WM results

A recent study evidenced somatic*CXCR4* mutation in 28% of the patients PC [16]. Five distinct somatic mutations were located in the *CXCR4* C-terminal tail, each of which were identical or functionally similar to mutations associated with WHIM syndrome (WS), resulting in the loss of regulatory Ser/Thr residues likely leading to impaired inactivation. CXCR4 is the second most frequent somatic mutation in WM next to L265P-MYD88 mutation that was detected in 90% of cases. Most interestingly, 98% of patients with CXCR4 mutation har‐ boured the MYD88 mutation. These results imply that a gain of function CXCR4 is in‐ volved in the pathogeny of WM. It remains to understand how this helps malignant transformation to occur, and how to reconcile this observation with hypogammaglobuline‐ mia in the WS. An interesting perspective here is the potential useful treatment of WM patients mutated or not for CXCR4, with AMD3100. It will undoubtedly represent the second

CXCR4 plays a critical role in the promotion of several solid tumours. A recent study showed that non-small lung cancer cell lines did express high CXCR4 despite secreting CXCL12 [176]; this questions the inability of CXCL12 to down regulate CXCR4 in these cells. Moreover these cells had a high self-renewal and tumour promoting activity *in vivo*. Thus CXCR4 may behave as a growth stimulator and help cancer stem cells home into protective niches within the solid tumour or metastasize within the bone marrow where they may compete with normal HSC,

A common feature of WS and ICL is the occurrence of cancers that are often linked to HPV infections. In ICL, some 15% of the patients experienced HPV infections and / or squamous and basal cell carcinomas of the skin, Bowen's disease, vulvar, cervical or bladder carcinomas [122]. Interestingly, HPV has been detected in these pathologies [177-181]. In WS, clinical manifestations at diagnosis include cervical papillomatosis that can lead to invasive cancer [112]. This highlights the potential and still undetermined role of CXCR4 (over or impaired

as phosphatases of the PKC pathway might be useful as well.

from somatic mutations in PC precursors.

therapeutic indication of plerixafor after WS.

hence adverse effects on immunity and haematopoiesis.

**9. CXCR4 and solid tumours**

CLL B-cells overexpress functional CXCR4 that may help B-cell to survive/proliferate and is associated with increased response to CXCL12 [154]. Indeed it has been noticed that CLL cells survive longer *in vitro* when cocultured with bone marrow stromal cells [155-157]. Interestingly survival mechanisms were linked to stimulation of CXCL12/CXCR4 and VLA4/VCAM axis [158-160]. This suggested that CLL cells are stimulated in protective microenvironments in the bone marrow or in secondary lymphoid organs and indeed proliferation centres with stromal cells and T-cells associated with CLL cells so called pseudo follicles were observed in these tissues [147, 161-164]. CLL cells in the blood are likely more sensitive to drugs than they are in lymphoid tissues.

Thus a new therapeutic strategy in CLL would be to down regulate or desensitize CXCR4 or disrupt CXCL12/CXCR4 interactions by AMD3100 on malignant B-cells in order to force them to leave their protective environments and undergo apoptosis in the bloodstream [165, 166].

As CLL are endowed with auto reactive surface Ig, they may be triggered repeatedly *in vivo* by auto antigens and BCR signaling is expected to down regulate CXCR4. Thus increased CXCR4 expression likely reflects the poor BCR signaling *in vivo* compared to that in normal B-cells; in addition CLL cells are surface Iglo. Indeed, CLL cells are anergic to anti-IgM stimulation, although U-CLL respond better in this respect than M-CLL in terms of prolifera‐ tion and BCR signaling, and interestingly, U-CLL down regulate CXCR4 more efficiently than M-CLL upon sIg cross linking *in vitro* [167-169]. AMD3100 has been shown to potentiate Chemo/Immunotherapy in CLL *in vitro* [170]. This can be interpreted as either a blockade of the CXCR4 survival pathway or a help of CLL cells to detach from CXCL12+ stromal cells in SLO and move away from their protective microenvironment or both. These results need however to be reconciled with the finding that CXCR4 expression on CLL cells is lower in LO than it is in the blood [171].

As mentioned above (chapter 3), BCR-induced CXCR4 phosphorylation is PKC-but not Ca2+ dependent [20]. Either CXCL12 interaction with CXCR4, or BCR engagement activates PI3kinase-δ and CAL101 an inhibitor of this kinase was efficient against CLL [172]. Thus activating these pathways by molecules less toxic than phorbolmyristate acetate might prove useful to down regulate CXCR4, alternatively, pharmacological inhibitors of regulators, such as phosphatases of the PKC pathway might be useful as well.

In Waldenstom's Macroglobulinemia (WM), macroglobulinemia designs an increase in serum concentrations of IgM and causes much of the morbidity associated with the disease. WM is an indolent B-cell malignancy with a monoclonal proliferation of IgM-producing PCs that fail to undergo Ig isotype switching. WM is uncommon relative to plasma cells (PC) myeloma [173]. Although the pathogenesis of WM remained undetermined several data suggested that genetic factors contributed to the disease [174]. PCs from WM are CXCR4hiVLA4hi and as for normal PC, malignant PC continuously home into the bone marrow and trans endothelial migration of PC from WM depends on CXCL12/CXCR4 [175]. It is now clear that WM results from somatic mutations in PC precursors.

A recent study evidenced somatic*CXCR4* mutation in 28% of the patients PC [16]. Five distinct somatic mutations were located in the *CXCR4* C-terminal tail, each of which were identical or functionally similar to mutations associated with WHIM syndrome (WS), resulting in the loss of regulatory Ser/Thr residues likely leading to impaired inactivation. CXCR4 is the second most frequent somatic mutation in WM next to L265P-MYD88 mutation that was detected in 90% of cases. Most interestingly, 98% of patients with CXCR4 mutation har‐ boured the MYD88 mutation. These results imply that a gain of function CXCR4 is in‐ volved in the pathogeny of WM. It remains to understand how this helps malignant transformation to occur, and how to reconcile this observation with hypogammaglobuline‐ mia in the WS. An interesting perspective here is the potential useful treatment of WM patients mutated or not for CXCR4, with AMD3100. It will undoubtedly represent the second therapeutic indication of plerixafor after WS.

#### **9. CXCR4 and solid tumours**

**8. CXCR4 in B-cell malignancies**

62 Adult Stem Cell Niches

originate from malignant transformation of CD5+

occurring at two cellular differentiation stages [153].

in lymphoid tissues.

than it is in the blood [171].

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia accounting for some 30% of adult leukaemia's in western countries It is due to an accumulation of small B-cells in the blood, the bone marrow and secondary lymphoid organs. Although this accumulation reflects lack of apoptosis, CLL cells do proliferate and their mitotic index and telomere length correlate with the degree of malignancy as reflected by the Binet [145] or Rai [146] clinical staging or the mutational status of Ig hyper variable regions, currently the gold biological standard for prognosis in this disease [147-149]. Somatically Ig hyper mutated (M-CLL) is typically indolent whereas Ig unmutated (U-CLL) is more aggressive [150, 151]. The cellular origin of CLL has been intensively debated, however contrary to the prevailing view that CLL originate from memory/activated B2 B-cells, we favour the recent hypothesis that CLL B-cells

aggressive CLL originate from B-cells endowed with an auto reactive surface Ig which supports the hypothesis of a common molecular mechanism for both CLL types albeit

CLL B-cells overexpress functional CXCR4 that may help B-cell to survive/proliferate and is associated with increased response to CXCL12 [154]. Indeed it has been noticed that CLL cells survive longer *in vitro* when cocultured with bone marrow stromal cells [155-157]. Interestingly survival mechanisms were linked to stimulation of CXCL12/CXCR4 and VLA4/VCAM axis [158-160]. This suggested that CLL cells are stimulated in protective microenvironments in the bone marrow or in secondary lymphoid organs and indeed proliferation centres with stromal cells and T-cells associated with CLL cells so called pseudo follicles were observed in these tissues [147, 161-164]. CLL cells in the blood are likely more sensitive to drugs than they are

Thus a new therapeutic strategy in CLL would be to down regulate or desensitize CXCR4 or disrupt CXCL12/CXCR4 interactions by AMD3100 on malignant B-cells in order to force them to leave their protective environments and undergo apoptosis in the bloodstream [165, 166]. As CLL are endowed with auto reactive surface Ig, they may be triggered repeatedly *in vivo* by auto antigens and BCR signaling is expected to down regulate CXCR4. Thus increased CXCR4 expression likely reflects the poor BCR signaling *in vivo* compared to that in normal B-cells; in addition CLL cells are surface Iglo. Indeed, CLL cells are anergic to anti-IgM stimulation, although U-CLL respond better in this respect than M-CLL in terms of prolifera‐ tion and BCR signaling, and interestingly, U-CLL down regulate CXCR4 more efficiently than M-CLL upon sIg cross linking *in vitro* [167-169]. AMD3100 has been shown to potentiate Chemo/Immunotherapy in CLL *in vitro* [170]. This can be interpreted as either a blockade of

SLO and move away from their protective microenvironment or both. These results need however to be reconciled with the finding that CXCR4 expression on CLL cells is lower in LO

As mentioned above (chapter 3), BCR-induced CXCR4 phosphorylation is PKC-but not Ca2+ dependent [20]. Either CXCL12 interaction with CXCR4, or BCR engagement activates

the CXCR4 survival pathway or a help of CLL cells to detach from CXCL12+

B-cells [152]. Moreover both indolent and

stromal cells in

CXCR4 plays a critical role in the promotion of several solid tumours. A recent study showed that non-small lung cancer cell lines did express high CXCR4 despite secreting CXCL12 [176]; this questions the inability of CXCL12 to down regulate CXCR4 in these cells. Moreover these cells had a high self-renewal and tumour promoting activity *in vivo*. Thus CXCR4 may behave as a growth stimulator and help cancer stem cells home into protective niches within the solid tumour or metastasize within the bone marrow where they may compete with normal HSC, hence adverse effects on immunity and haematopoiesis.

A common feature of WS and ICL is the occurrence of cancers that are often linked to HPV infections. In ICL, some 15% of the patients experienced HPV infections and / or squamous and basal cell carcinomas of the skin, Bowen's disease, vulvar, cervical or bladder carcinomas [122]. Interestingly, HPV has been detected in these pathologies [177-181]. In WS, clinical manifestations at diagnosis include cervical papillomatosis that can lead to invasive cancer [112]. This highlights the potential and still undetermined role of CXCR4 (over or impaired membrane expression) in the control of immunity to HPV. It also points to the fact that the term "gain of function" for the *CXCR41013*mutation may be a misnomer as it points for an augmented capacity of the cells to migrate *in vitro* and most likely to stick to their niches *in vivo*. Of note, there is no correlation between HPV-associated cancers and Waldenstom's Macroglobulinemia, which only affects PC and leaves intact most immune cells with normal CXCR4. The germ line CXCR41013-mutation is clearly associated with a polyclonal loss of function of B-, T-and NK-cells *in vivo* and possibly of neutrophils as well and the respective contribution of these leukocytes to the adaptive immune response against HPV remains to be understood.

thelioma and the myelodysplastic syndromes [194-197]. In cancer, the balance between Tregsand T-effector cells is often deregulated and Tregs are recruited to the tumour, a process that suppress the anti-tumour immune response leading to tumour growth. In basal-like breast cancer, it was shown that there is a positive correlation between CXCL12 expression in the tumour and Tregs recruitment correlated with a poor survival in patients [198]. This was also shown in lung adenocarcinomas and malignant mesothelioma [195, 196]. In addition Yan et *al* showed that the recruitment of Tregs is correlated with hypoxia-induced CXCR4 expression in basal-like breast cancers. Furthermore, in lung adenocarcinoma, CXCR4 positive Tregs cells are able to regulate the immune response and to secrete tumour growth factor-β which upregulate CXCR4 on naive T-cells and contribute to their migration and retention in the tumour microenvironment and that contribute to increase pathogenesis [195]. Overall, these observa‐ tions suggest that CXCL12 expression may influence tumour progression by shaping the immune cell population infiltrating lung adenocarcinoma tumours. In human ovarian carcinoma, Tregs preferentially move to tumours and ascites and suppress tumour-specific Tcell immunity and contribute to growth of human tumours *in vivo.* The recruitment of Tregs represents a mechanism by which tumours may promote immune advantage. Altogether, these results suggest that AMD3100 or other drugs that target the CXCL12/CXCR4 axis may

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65

The contribution of the CXCL12/CXCR4 signaling axis to many aspects of physiology and pathology has been increasingly appreciated (Figure 1 & 2). Deregulations of CXCR4 signaling and/or expression are associated with several disorders including lymphoid and autoimmune diseases, solid tumours and immune defects [15, 200-203]. Therefore, interference with the CXCL12/CXCR4 interaction or modulation of CXCR4 expression and/or activity is potentially interesting in the treatment of diseases with loss or gain of function of CXCR4. In this context and for the last two decades, many pharmaceutical companies have been trying to develop specific drugs targeting this axis [204, 205]. In light of recent clinical investigations, the bicyclamplerixafor (AMD3100), has already been approved by the FDA for HSC mobilization in patients with non-Hodgkin's lymphoma and multiple myeloma [206]. In addition, Plerixafor is already under Phase I of clinical investigation in Glioma, Acute Myeloid leukaemia, CLL and WS [204, 207]. Furthermore, regarding the low half-life, the constraining frequency of administration of AMD3100, several promising CXCR4 antagonists have been developed to block CXCL12/CXCR4 interactions that are currently under different stages of clinical trials mainly for HSC transplantation in patients with multiple myeloma and non-Hodgkin's lymphoma [205]. Most of them neutralize the interaction of CXCL12 with CXCR4 by binding to the receptor. Another way is to directly target CXCL12 by developing molecules that interact directly with it thus diverting the chemokine from its receptor. For example blocking interac‐ tions of CXCL12 with the extracellular matrix and cell surface glycosaminoglycans may be an approach by developing CXCL12 binding heparansulphates. In addition other strategies will be to generate molecules interfering with intracellular trafficking and expression of CXCR4

be useful adjuncts for immune-chemotherapy in some cancers [199].

**10. Conclusion/therapeutic perspectives**

Within solid tumours or among malignant cells from leukaemia's, several lines of evidence suggest that there are cells called cancer stem cells or tumour initiating cells. They have been identified in many types of cancers including human gastric tumour, mammary gland, brain, prostate gland, colon pancreas, head and neck and liver [182-190]. On primary cultures of cells from a human gastric tumour, those cells are able to grow on extracellular matrix and to form spheroid, structures specific of stem cells. Furthermore those cells are more resistant to chemotherapy and capable of self-renewal [182]. This feature could explain the failure in the treatment of cancers. In a Gefitinib-resistant non-small cell lung cancer cells line (A549/GR), experiments show that these cells possess some features of stem cells such as the acquisition of epithelial mesenchymal transition property. More interestingly, there is a high proportion of CXCR4+ cells in A549/GR, with high self-renewal activity *in vitro*, high tumorigenic potential *in vivo*, a strong sphere-forming activity and a resistance to radiation. The use of si-RNA specific of CXCR4 or the AMD3100 are both able to suppressed sphere forming activity in those cell lines, demonstrating an important role of CXCR4 in maintaining cancer stem cells features in A549/GR [176]. Cancer stem cells are potential targets for therapies in order to eliminate malignant cells in solid tumours resistant to chemotherapy.

The CXCL12/CXCR4 axis is known to be involved in tumour growth and metastatic process. The microenvironment is essential for tumour development and stromal and malignant cells communicate *via* growth factors and cytokines [191, 192]. In order to understand the impact of Cxcr4 in the microenvironment on metastasis of tumour cells, D'Alterio et al injected murine melanoma B16 cells on C57BL/6 *Cxcr4+/+*or *Cxcr4+/-*mice in the presence of AMD3100. In *Cxcr4+/* mice nodule size were significantly smaller and bone marrow-derived cells recruitment was lower compared to *Cxcr4+/+*. Furthermore, the Cxcr4 inhibitor AMD3100 preserves the pulmo‐ nary architecture in Cxcr4+/-mice by reducing lung metastases. *Cxcr4+/-*mice also show a decrease in LY6G-positive myeloid/granulocytic cells and in p38 MAPK activation in lungs compared to *Cxcr4+/+*mice [193]. These experiments demonstrate the importance CXCL12/ CXCR4 axis in the microenvironment regarding metastases and tumour growth. Since CXCL12/CXCR4 axis may promote cancer cell survival, invasion, and tumour-initiating cell phenotype. Therefore blocking this axis may be a potential approach to target various components in solid tumours.

The CXCL12/CXCR4 axis is also involved in the recruitment of immune cells into the tumour as described for regulatory T-cells (Tregs). Indeed Tregs expressing CXCR4 are recruited in a number of tumours including ovarian cancer, adenocarcinoma of the lung, malignant meso‐ thelioma and the myelodysplastic syndromes [194-197]. In cancer, the balance between Tregsand T-effector cells is often deregulated and Tregs are recruited to the tumour, a process that suppress the anti-tumour immune response leading to tumour growth. In basal-like breast cancer, it was shown that there is a positive correlation between CXCL12 expression in the tumour and Tregs recruitment correlated with a poor survival in patients [198]. This was also shown in lung adenocarcinomas and malignant mesothelioma [195, 196]. In addition Yan et *al* showed that the recruitment of Tregs is correlated with hypoxia-induced CXCR4 expression in basal-like breast cancers. Furthermore, in lung adenocarcinoma, CXCR4 positive Tregs cells are able to regulate the immune response and to secrete tumour growth factor-β which upregulate CXCR4 on naive T-cells and contribute to their migration and retention in the tumour microenvironment and that contribute to increase pathogenesis [195]. Overall, these observa‐ tions suggest that CXCL12 expression may influence tumour progression by shaping the immune cell population infiltrating lung adenocarcinoma tumours. In human ovarian carcinoma, Tregs preferentially move to tumours and ascites and suppress tumour-specific Tcell immunity and contribute to growth of human tumours *in vivo.* The recruitment of Tregs represents a mechanism by which tumours may promote immune advantage. Altogether, these results suggest that AMD3100 or other drugs that target the CXCL12/CXCR4 axis may be useful adjuncts for immune-chemotherapy in some cancers [199].

#### **10. Conclusion/therapeutic perspectives**

membrane expression) in the control of immunity to HPV. It also points to the fact that the term "gain of function" for the *CXCR41013*mutation may be a misnomer as it points for an augmented capacity of the cells to migrate *in vitro* and most likely to stick to their niches *in vivo*. Of note, there is no correlation between HPV-associated cancers and Waldenstom's Macroglobulinemia, which only affects PC and leaves intact most immune cells with normal CXCR4. The germ line CXCR41013-mutation is clearly associated with a polyclonal loss of function of B-, T-and NK-cells *in vivo* and possibly of neutrophils as well and the respective contribution of these leukocytes to the adaptive immune response against HPV remains to be

Within solid tumours or among malignant cells from leukaemia's, several lines of evidence suggest that there are cells called cancer stem cells or tumour initiating cells. They have been identified in many types of cancers including human gastric tumour, mammary gland, brain, prostate gland, colon pancreas, head and neck and liver [182-190]. On primary cultures of cells from a human gastric tumour, those cells are able to grow on extracellular matrix and to form spheroid, structures specific of stem cells. Furthermore those cells are more resistant to chemotherapy and capable of self-renewal [182]. This feature could explain the failure in the treatment of cancers. In a Gefitinib-resistant non-small cell lung cancer cells line (A549/GR), experiments show that these cells possess some features of stem cells such as the acquisition of epithelial mesenchymal transition property. More interestingly, there is a high proportion

cells in A549/GR, with high self-renewal activity *in vitro*, high tumorigenic potential

*in vivo*, a strong sphere-forming activity and a resistance to radiation. The use of si-RNA specific of CXCR4 or the AMD3100 are both able to suppressed sphere forming activity in those cell lines, demonstrating an important role of CXCR4 in maintaining cancer stem cells features in A549/GR [176]. Cancer stem cells are potential targets for therapies in order to eliminate

The CXCL12/CXCR4 axis is known to be involved in tumour growth and metastatic process. The microenvironment is essential for tumour development and stromal and malignant cells communicate *via* growth factors and cytokines [191, 192]. In order to understand the impact of Cxcr4 in the microenvironment on metastasis of tumour cells, D'Alterio et al injected murine melanoma B16 cells on C57BL/6 *Cxcr4+/+*or *Cxcr4+/-*mice in the presence of AMD3100. In *Cxcr4+/* mice nodule size were significantly smaller and bone marrow-derived cells recruitment was lower compared to *Cxcr4+/+*. Furthermore, the Cxcr4 inhibitor AMD3100 preserves the pulmo‐ nary architecture in Cxcr4+/-mice by reducing lung metastases. *Cxcr4+/-*mice also show a decrease in LY6G-positive myeloid/granulocytic cells and in p38 MAPK activation in lungs compared to *Cxcr4+/+*mice [193]. These experiments demonstrate the importance CXCL12/ CXCR4 axis in the microenvironment regarding metastases and tumour growth. Since CXCL12/CXCR4 axis may promote cancer cell survival, invasion, and tumour-initiating cell phenotype. Therefore blocking this axis may be a potential approach to target various

The CXCL12/CXCR4 axis is also involved in the recruitment of immune cells into the tumour as described for regulatory T-cells (Tregs). Indeed Tregs expressing CXCR4 are recruited in a number of tumours including ovarian cancer, adenocarcinoma of the lung, malignant meso‐

malignant cells in solid tumours resistant to chemotherapy.

understood.

64 Adult Stem Cell Niches

of CXCR4+

components in solid tumours.

The contribution of the CXCL12/CXCR4 signaling axis to many aspects of physiology and pathology has been increasingly appreciated (Figure 1 & 2). Deregulations of CXCR4 signaling and/or expression are associated with several disorders including lymphoid and autoimmune diseases, solid tumours and immune defects [15, 200-203]. Therefore, interference with the CXCL12/CXCR4 interaction or modulation of CXCR4 expression and/or activity is potentially interesting in the treatment of diseases with loss or gain of function of CXCR4. In this context and for the last two decades, many pharmaceutical companies have been trying to develop specific drugs targeting this axis [204, 205]. In light of recent clinical investigations, the bicyclamplerixafor (AMD3100), has already been approved by the FDA for HSC mobilization in patients with non-Hodgkin's lymphoma and multiple myeloma [206]. In addition, Plerixafor is already under Phase I of clinical investigation in Glioma, Acute Myeloid leukaemia, CLL and WS [204, 207]. Furthermore, regarding the low half-life, the constraining frequency of administration of AMD3100, several promising CXCR4 antagonists have been developed to block CXCL12/CXCR4 interactions that are currently under different stages of clinical trials mainly for HSC transplantation in patients with multiple myeloma and non-Hodgkin's lymphoma [205]. Most of them neutralize the interaction of CXCL12 with CXCR4 by binding to the receptor. Another way is to directly target CXCL12 by developing molecules that interact directly with it thus diverting the chemokine from its receptor. For example blocking interac‐ tions of CXCL12 with the extracellular matrix and cell surface glycosaminoglycans may be an approach by developing CXCL12 binding heparansulphates. In addition other strategies will be to generate molecules interfering with intracellular trafficking and expression of CXCR4 which are two different ways to normalize CXCR4 expression and functioning in pathologies associated with CXCR4 anomalies as seen both in ICL and WS. As mentioned in the introduc‐ tion, ACKR3 may share overlapping functions with CXCR4 and is possibly involved in the same pathologies and need to be investigated in this respect [208, 209].

**Figure 1. CXCR4 in lymphopoiesis.** Most primitive hematopoietic stem cells (HSC) and downstream lymphoid pro‐ genitors, including common lymphoid (CLP) and committed B-cell precursors express CXCR4 and the differentiation of HSC and B-cell precursors is CXCR4-dependent. These cells interact with CXCL12-expressing stromal cells. These stro‐ mal cells comprise Nestin+Mesenchymal stem cells MSC, CXCL12-producing reticular cells (CAR), perivascular cells ex‐ pressing PDGFR and LeptinR, and osteoblasts. AMD3100 is already used to allow the migration of HSC in the peripheral blood of patients in order to be collected for grafting. Recent data suggest that B-cell progenitors locate near the perivascular niche. CLP locate in the osteoblastic niche. Downstream progenitors migrate through the blood and enter the thymus at the cortico-medullary junction where they constitute early thymic progenitors (ETP). ETP fur‐ ther move inside the cortex and differentiate in several double negative CD4- CD8 stages DN1 to DN4. This process de‐ pends on the production of CXCL12 by cortical thymic epithelial cells (cTEC). At the DN4 stage, thymocytes downregulate CXCR4, upregulate CCR7 and become CD4+CD8+(CD3+) cells double positive DP cells. DP thymocytes are attracted by CCL21-producing thymic medullary epithelial cells (mTEC). At the end of differentiation, and negative se‐ lection due to interactions with dendritic cells (DC) in the medulla, naive single positive (SP) CD4+or CD8+thymocytes egress from the thymus to the bloodstream and further migrate to the T-cell zones of secondary lymphoid organs (SLO). In parallel B-cell precursors differentiate within the bone marrow in naive B-cells that express CXCR4/5 and mi‐ grate to the SLO, where they organize themselves around CXCL12/13-producing follicular dendritic cells (FDC) and marginal central reticular cells (CRC). Following T-dependent response to antigen the germinal centre (GC)reaction is initiated (figure 2).

**Figure 2. CXCR4 in T-dependent antigen response, and in pathology.** Following antigen capture and processing, dendritic antigen-presenting cells (APC) move to the T-cell zone in the SLO to prime antigen-specific T-cells whereas follicular B-cells are stimulated by soluble antigen. In turn T-and B-cells move towards each other and antigen-specific B-cells expand to generate the dark zone DZ made mostly of centroblasts. These cells differentiate in centrocytes that constitute the light zone and downregulate CXCR4. Ultimately, centrocytes differentiate in memory B-cells or plasma cells (PC), the laterupregulate CXCR4 and migrate to the bone marrow. In the WS, one hypothesis is that the germ‐ line*CXCR4m* mutation impairs T-APC and T-B interactions leading to deficient GC formation. In Waldenstom's Macro‐ globulinemiaWM, the somatic *CXCR4m* mutation in B-cells confers a survival advantage and drives PC to migrate and home into the bone marrow were they become malignant due to additional gain of function mutations such as in the *MYD88* gene. In the Idiopathic CD4+T-cell LymphocytopeniaICL, downregulation of CXCR4 expression in T-cells may concur to the deficient adaptive immune response. In chronic lymphocytic leukaemia, PC differentiation is impaired and leukemic B-cells express CXCR4, which keeps them in a protective environment in contact with CXCL12-producing stromal cells thereby protecting them from immune/chemotherapeutic drugs. In this context, AMD3100 plerixafor may help sensitize chronic lymphocytic leukaemia CLL cells to drugs. Plerixafor is already used for the treatment of

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in...

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67

A new therapeutic era has begun, next years will witness the use of anti-chemokines receptors to prevent malignant cells from interacting with their protective stromal cells and of inflam‐ matory cells to migrate into tissues, these new families of drugs should clearly improve the efficacy of current anti-cancer, anti-autoimmune and anti-inflammatory molecules [182].

WHIM Syndrome WS, and may prove useful in the treatment of WM.

CXCR4 in Central and Peripheral Lymphoid Niches – Physiology, Pathology and Therapeutic Perspectives in... http://dx.doi.org/10.5772/58699 67

which are two different ways to normalize CXCR4 expression and functioning in pathologies associated with CXCR4 anomalies as seen both in ICL and WS. As mentioned in the introduc‐ tion, ACKR3 may share overlapping functions with CXCR4 and is possibly involved in the

**Figure 1. CXCR4 in lymphopoiesis.** Most primitive hematopoietic stem cells (HSC) and downstream lymphoid pro‐ genitors, including common lymphoid (CLP) and committed B-cell precursors express CXCR4 and the differentiation of HSC and B-cell precursors is CXCR4-dependent. These cells interact with CXCL12-expressing stromal cells. These stro‐ mal cells comprise Nestin+Mesenchymal stem cells MSC, CXCL12-producing reticular cells (CAR), perivascular cells ex‐ pressing PDGFR and LeptinR, and osteoblasts. AMD3100 is already used to allow the migration of HSC in the peripheral blood of patients in order to be collected for grafting. Recent data suggest that B-cell progenitors locate near the perivascular niche. CLP locate in the osteoblastic niche. Downstream progenitors migrate through the blood and enter the thymus at the cortico-medullary junction where they constitute early thymic progenitors (ETP). ETP fur‐

pends on the production of CXCL12 by cortical thymic epithelial cells (cTEC). At the DN4 stage, thymocytes downregulate CXCR4, upregulate CCR7 and become CD4+CD8+(CD3+) cells double positive DP cells. DP thymocytes are attracted by CCL21-producing thymic medullary epithelial cells (mTEC). At the end of differentiation, and negative se‐ lection due to interactions with dendritic cells (DC) in the medulla, naive single positive (SP) CD4+or CD8+thymocytes egress from the thymus to the bloodstream and further migrate to the T-cell zones of secondary lymphoid organs (SLO). In parallel B-cell precursors differentiate within the bone marrow in naive B-cells that express CXCR4/5 and mi‐ grate to the SLO, where they organize themselves around CXCL12/13-producing follicular dendritic cells (FDC) and marginal central reticular cells (CRC). Following T-dependent response to antigen the germinal centre (GC)reaction is

CD8-

stages DN1 to DN4. This process de‐

ther move inside the cortex and differentiate in several double negative CD4-

initiated (figure 2).

66 Adult Stem Cell Niches

same pathologies and need to be investigated in this respect [208, 209].

A new therapeutic era has begun, next years will witness the use of anti-chemokines receptors to prevent malignant cells from interacting with their protective stromal cells and of inflam‐ matory cells to migrate into tissues, these new families of drugs should clearly improve the efficacy of current anti-cancer, anti-autoimmune and anti-inflammatory molecules [182].

#### **Acknowledgements**

This work was supported by the Assistance Publique-Hôpitaux de Paris (Grant 07018) and the Agence Nationale de la Recherche (Grant 2010 JCJC 1104 01). C.F., A.B., K.B., and A.D. are membres of the Laboratory of Excellence in Research on Medication and Innovative Thera‐ peutics, supported by a grant from the Agence Nationale de la Recherche (ANR-10-LABX-33) under the program "Investissements d'Avenir" ANR-11-IDEX-0003-01. A.B., C.F. are fellow‐ ship recipients from the Fondation pour la Recherche Médicale (FDT20130928127), the French Ministry and the DIM Biothérapies, respectively.

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http://dx.doi.org/10.5772/58699

69

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## **Author details**

Christelle Freitas1,2, Alexandre Bignon1,2, Karl Balabanian1,2 and Ali Dalloul1,2\*

\*Address all correspondence to: ali.dalloul@u-psud.fr

1 Université Paris-Sud, Laboratoire "Cytokines, Chimiokines et Immunopathologie," Clamart, France

2 INSERM, Laboratoire d'Excellence en Recherche sur le Médicament et l'Innovation Thérapeutique, Clamart, France

Christelle Freitas and Alexandre Bignon contributed equally to this work.

#### **References**


[5] Nair S, Schilling TF. Chemokine signaling controls endodermal migration during ze‐ brafish gastrulation. Science. 2008 Oct 3;322 (5898):89-92.

**Acknowledgements**

68 Adult Stem Cell Niches

licenses/by/3.0/fr/.

**Author details**

Thérapeutique, Clamart, France

France

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Ministry and the DIM Biothérapies, respectively.

\*Address all correspondence to: ali.dalloul@u-psud.fr

This work was supported by the Assistance Publique-Hôpitaux de Paris (Grant 07018) and the Agence Nationale de la Recherche (Grant 2010 JCJC 1104 01). C.F., A.B., K.B., and A.D. are membres of the Laboratory of Excellence in Research on Medication and Innovative Thera‐ peutics, supported by a grant from the Agence Nationale de la Recherche (ANR-10-LABX-33) under the program "Investissements d'Avenir" ANR-11-IDEX-0003-01. A.B., C.F. are fellow‐ ship recipients from the Fondation pour la Recherche Médicale (FDT20130928127), the French

Figures were adapted from Servier Medical Art http://smart.servier.fr/servier-medicalartunder licence Creative Commons Attribution 3.0 France http://creativecommons.org/

1 Université Paris-Sud, Laboratoire "Cytokines, Chimiokines et Immunopathologie," Clamart,

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**Chapter 4**

**Hematopoietic Stem Cells, Tumor Cells and Lymphocytes**

During vertebrate embryogenesis, different anatomical sites are responsible for creating specific conditions to promote hematopoietic stem cells self-renewal, expansion, commitment, and differentiation of the hematopoietic stem cells (HSC) [1,2]. The first hematopoietic cells emerge within the blood islands of the yolk sac (YS), an extra-embryonic site. Most of the cells belong to the primitive erythroid lineage, but a few myeloid cells are also generated [3,4]. In a second wave, hematopoietic progenitors emerge from the mesoderm of the paraaorticsplanchnopleura (Sp), an intra-embryonic site, which later gives rise to the aorta, gonads and mesonephros, and has been named AGM region [1]. Data show that almost all long-term definitive progenitors derive from the AGM region, as those originated in the YS fail to properly reconstitute the adult bone marrow of a lethally irradiated animal [5]. However, when cultured under the right combination of cytokines, cells derived from the mesoderm of the YS, in which blood islands originate, can be instructed to become long-term hematopoietic progenitors [6]. In these conditions, even higher numbers of progenitors could be found in the YS compared to the AGM region. This shows that, in vivo, the YS niche does not hold proper

After the vascular system is established in the embryo, hematopoietic progenitors migrate and colonize the fetal liver. Fetal liver is the main hematopoietic organ during embryo's develop‐ ment [7,8] and its hematopoiesis requires exogenous colonization. So far, no data indicate that

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**— Party in the Bone Marrow**

Alex Balduíno

**1. Introduction**

http://dx.doi.org/10.5772/58843

**1.1. The hematopoietic stem cell niche**

Adriana Bonomo, Ana Carolina Monteiro and

Additional information is available at the end of the chapter

*1.1.1. Hematopoietic system development: distinct niches activities*

conditions to promote full commitment of the hematopoietic progenitors.

## **Hematopoietic Stem Cells, Tumor Cells and Lymphocytes — Party in the Bone Marrow**

Adriana Bonomo, Ana Carolina Monteiro and Alex Balduíno

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58843

### **1. Introduction**

#### **1.1. The hematopoietic stem cell niche**

#### *1.1.1. Hematopoietic system development: distinct niches activities*

During vertebrate embryogenesis, different anatomical sites are responsible for creating specific conditions to promote hematopoietic stem cells self-renewal, expansion, commitment, and differentiation of the hematopoietic stem cells (HSC) [1,2]. The first hematopoietic cells emerge within the blood islands of the yolk sac (YS), an extra-embryonic site. Most of the cells belong to the primitive erythroid lineage, but a few myeloid cells are also generated [3,4]. In a second wave, hematopoietic progenitors emerge from the mesoderm of the paraaorticsplanchnopleura (Sp), an intra-embryonic site, which later gives rise to the aorta, gonads and mesonephros, and has been named AGM region [1]. Data show that almost all long-term definitive progenitors derive from the AGM region, as those originated in the YS fail to properly reconstitute the adult bone marrow of a lethally irradiated animal [5]. However, when cultured under the right combination of cytokines, cells derived from the mesoderm of the YS, in which blood islands originate, can be instructed to become long-term hematopoietic progenitors [6]. In these conditions, even higher numbers of progenitors could be found in the YS compared to the AGM region. This shows that, in vivo, the YS niche does not hold proper conditions to promote full commitment of the hematopoietic progenitors.

After the vascular system is established in the embryo, hematopoietic progenitors migrate and colonize the fetal liver. Fetal liver is the main hematopoietic organ during embryo's develop‐ ment [7,8] and its hematopoiesis requires exogenous colonization. So far, no data indicate that

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

new progenitors emerge in the fetal liver. All hematopoietic cells are derived from the YS and AGM region [8–10].

*1.1.2. Inside the bone marrow*

based on technical issues arguments.

and behavior.

In spite of its high dynamic, the hematopoietic system, in the bone marrow cavity, is widely hierarchical and hematopoietic cells are not randomly distributed. As mentioned before, specific niches control HSC self-renewal and their engagement to a differentiation cascade.

Hematopoietic Stem Cells, Tumor Cells and Lymphocytes — Party in the Bone Marrow

http://dx.doi.org/10.5772/58843

89

The concept that different niches would compose the bone marrow microenvironment was envisioned already in the 70's [16]. Based on a stereological study, it was proposed that bone marrow microenvironment could be subdivided into, at least, four niches: endosteal, suben‐ dosteal, central, and perisinusoidal [13,16]. Histological and functional assays showed that HSC and primitive progenitors preferentially colonize the endosteal and subendosteal regions – close to the bone surface. Intermediate progenitors and differentiated cells are distributed in the central and perisinusoidal niches, respectively [13,14,16–21]. Due to their close range, endosteal and subendosteal regions are usually identified as one niche, named "endosteal niche". However, these two regions harbor very distinct stromal cells [15,22,23] and must then

be considered as two different niches, as they play distinct roles on HSC behavior.

Based on the expression of different surface markers [24,25] one can isolate the long-term HSC separately from other progenitors. Under physiological conditions, 20%-30% of the HSC are in a quiescent stage. Studies have shown that slow-cycling HSC are found in association with endosteal osteoblasts [19,21,26]. On the other hand, most of the fast-cycling HSC are found in close association with perivascular cells of the blood vessels distributed in the subendosteal zone [27,28]. This has been described *in vivo* in long-term BrdU retaining assays and myeloa‐ blation models. In experimental in vivo myelosuppressive models, HSC colonizing the vascular niches in the subendosteal region are mostly ablated. Almost all HSC in contact with endosteal osteoblasts are preserved [20,28]. By the time this chapter has been written, the existence of the two separate yet complimentary niches is still questioned by a few authors

The role of endosteal osteoblasts on the HSC maintenance and self-renewal was first proposed in vitro by Taichman and Emerson [14,29,30] and later evidenced in vivo by others [31–33]. In transgenic animals, increased numbers of osteoblasts results in an increased number of longterm HSC, without affecting any other hematopoietic subpopulation in the bone marrow [31, 32,34]. Furthermore, when osteoblasts are removed from the marrow cavity, HSC numbers reduces drastically [33]. This is evidence that osteoblasts play a crucial role in HSC maintenance

On the same study mentioned before, Lambertsen and Weiss [13] showed that most of the perivascular niches harboring HSC are distributed in the subendosteal zone. In the perivas‐ cular niche [19,20,27], HSC reside on the abluminal side of bone marrow sinusoids, and are supported by the endothelial and perivascular reticular cells. HSC residing in the perivascular niche are in close association with reticular cells, which express high levels of CXCL12, a chemokine required for HSC maintenance and lodging [17,27]. Most of the cells creating the proliferative niche express CXCL12. In situ observation demonstrated that most of hemato‐ poietic stem cells are concentrated in the trabecular zone of the marrow cavity, which also harbors high numbers of niche osteblasts, sinusoids, and CXCL12-positive reticular cells.

At this stage, fetal liver microenvironment is responsible for two very important tasks: full commitment of mesoderm derived progenitors to long-term HSC, and their increase in numbers (higher numbers). Although AGM derived progenitors are able to fully reconstitute a lethally irradiated animal in an experimental model, well-defined HSC can only be observed in the embryo a few days after fetal liver colonization. It has been shown that, to become adult long-term HSC, progenitors derived from the AGM region must go through the fetal liver microenvironment for proper instructions [11]. A few progenitors from the yolk sac become long-term HSC, but those originated from the AGM region are far more predominant. Longterm HSC in the fetal liver are highly proliferative and self-renewable. In mouse embryo, in five days, there could be an increase in 30 times the original number of HSC [7,9]. Different from what would be expected, not a huge number of progenitors colonize the fetal liver. Only a few are necessary. Commitment to HSC and their expansion require two distinct niches in the fetal liver microenvironment, at the same time or at different maturation stages of the liver. This requires further investigation.

The fetal liver remains hematopoietic until birth, or even a short period after. By the time the organ starts to acquire its metabolic properties, HSC are then progressively transferred to their final destination: the bone marrow [9]. In contrast to the fetal liver hematopoietic activity, bone marrow main assignment is blood production – not HSC expansion (only). All types of blood cells are produced in the bone marrow, except for the T lymphocytes, produced in the thymus. Despite its high dynamics, the bone marrow microenvironment is organized, in order to guarantee a finely tuned hierarchical differentiation cascade. Hematopoietic system organi‐ zation in the marrow cavity follows an also organized distribution of the stromal cells. Different stromal cell types – osteoblasts, reticular cells, perivascular cells, endothelial cells, macrophages – interact with different groups of hematopoietic cells, creating distinct niches in bone marrow microenvironment to harbor. This is the way the differentiation cascade is controlled as hematopoietic cells at different stages of differentiation demand distinct combi‐ nations of factors for their proliferation and differentiation [12–15].

Based on cells behavior, at least three niches can be identified in the marrow microenviron‐ ment: one responsible for HSC maintenance (self-renewal) throughout life; a second to induce intermediate progenitors expansion; and a third to guarantee hematopoietic cells full com‐ mitment and differentiation to the lineages.

In humans, during childhood, almost all bones in our body hold a "hematopoietically" active bone marrow (red bone marrow). After reaching maturity, active bone marrow is restricted to the sternum, ribs, vertebrae, ilium, and femurs' heads. The rest the bones are filled with "inactive" bone marrow, which is called yellow bone marrow, due to the high number of fat storing cells.

#### *1.1.2. Inside the bone marrow*

new progenitors emerge in the fetal liver. All hematopoietic cells are derived from the YS and

At this stage, fetal liver microenvironment is responsible for two very important tasks: full commitment of mesoderm derived progenitors to long-term HSC, and their increase in numbers (higher numbers). Although AGM derived progenitors are able to fully reconstitute a lethally irradiated animal in an experimental model, well-defined HSC can only be observed in the embryo a few days after fetal liver colonization. It has been shown that, to become adult long-term HSC, progenitors derived from the AGM region must go through the fetal liver microenvironment for proper instructions [11]. A few progenitors from the yolk sac become long-term HSC, but those originated from the AGM region are far more predominant. Longterm HSC in the fetal liver are highly proliferative and self-renewable. In mouse embryo, in five days, there could be an increase in 30 times the original number of HSC [7,9]. Different from what would be expected, not a huge number of progenitors colonize the fetal liver. Only a few are necessary. Commitment to HSC and their expansion require two distinct niches in the fetal liver microenvironment, at the same time or at different maturation stages of the liver.

The fetal liver remains hematopoietic until birth, or even a short period after. By the time the organ starts to acquire its metabolic properties, HSC are then progressively transferred to their final destination: the bone marrow [9]. In contrast to the fetal liver hematopoietic activity, bone marrow main assignment is blood production – not HSC expansion (only). All types of blood cells are produced in the bone marrow, except for the T lymphocytes, produced in the thymus. Despite its high dynamics, the bone marrow microenvironment is organized, in order to guarantee a finely tuned hierarchical differentiation cascade. Hematopoietic system organi‐ zation in the marrow cavity follows an also organized distribution of the stromal cells. Different stromal cell types – osteoblasts, reticular cells, perivascular cells, endothelial cells, macrophages – interact with different groups of hematopoietic cells, creating distinct niches in bone marrow microenvironment to harbor. This is the way the differentiation cascade is controlled as hematopoietic cells at different stages of differentiation demand distinct combi‐

Based on cells behavior, at least three niches can be identified in the marrow microenviron‐ ment: one responsible for HSC maintenance (self-renewal) throughout life; a second to induce intermediate progenitors expansion; and a third to guarantee hematopoietic cells full com‐

In humans, during childhood, almost all bones in our body hold a "hematopoietically" active bone marrow (red bone marrow). After reaching maturity, active bone marrow is restricted to the sternum, ribs, vertebrae, ilium, and femurs' heads. The rest the bones are filled with "inactive" bone marrow, which is called yellow bone marrow, due to the high number of fat

nations of factors for their proliferation and differentiation [12–15].

mitment and differentiation to the lineages.

storing cells.

AGM region [8–10].

88 Adult Stem Cell Niches

This requires further investigation.

In spite of its high dynamic, the hematopoietic system, in the bone marrow cavity, is widely hierarchical and hematopoietic cells are not randomly distributed. As mentioned before, specific niches control HSC self-renewal and their engagement to a differentiation cascade.

The concept that different niches would compose the bone marrow microenvironment was envisioned already in the 70's [16]. Based on a stereological study, it was proposed that bone marrow microenvironment could be subdivided into, at least, four niches: endosteal, suben‐ dosteal, central, and perisinusoidal [13,16]. Histological and functional assays showed that HSC and primitive progenitors preferentially colonize the endosteal and subendosteal regions – close to the bone surface. Intermediate progenitors and differentiated cells are distributed in the central and perisinusoidal niches, respectively [13,14,16–21]. Due to their close range, endosteal and subendosteal regions are usually identified as one niche, named "endosteal niche". However, these two regions harbor very distinct stromal cells [15,22,23] and must then be considered as two different niches, as they play distinct roles on HSC behavior.

Based on the expression of different surface markers [24,25] one can isolate the long-term HSC separately from other progenitors. Under physiological conditions, 20%-30% of the HSC are in a quiescent stage. Studies have shown that slow-cycling HSC are found in association with endosteal osteoblasts [19,21,26]. On the other hand, most of the fast-cycling HSC are found in close association with perivascular cells of the blood vessels distributed in the subendosteal zone [27,28]. This has been described *in vivo* in long-term BrdU retaining assays and myeloa‐ blation models. In experimental in vivo myelosuppressive models, HSC colonizing the vascular niches in the subendosteal region are mostly ablated. Almost all HSC in contact with endosteal osteoblasts are preserved [20,28]. By the time this chapter has been written, the existence of the two separate yet complimentary niches is still questioned by a few authors based on technical issues arguments.

The role of endosteal osteoblasts on the HSC maintenance and self-renewal was first proposed in vitro by Taichman and Emerson [14,29,30] and later evidenced in vivo by others [31–33]. In transgenic animals, increased numbers of osteoblasts results in an increased number of longterm HSC, without affecting any other hematopoietic subpopulation in the bone marrow [31, 32,34]. Furthermore, when osteoblasts are removed from the marrow cavity, HSC numbers reduces drastically [33]. This is evidence that osteoblasts play a crucial role in HSC maintenance and behavior.

On the same study mentioned before, Lambertsen and Weiss [13] showed that most of the perivascular niches harboring HSC are distributed in the subendosteal zone. In the perivas‐ cular niche [19,20,27], HSC reside on the abluminal side of bone marrow sinusoids, and are supported by the endothelial and perivascular reticular cells. HSC residing in the perivascular niche are in close association with reticular cells, which express high levels of CXCL12, a chemokine required for HSC maintenance and lodging [17,27]. Most of the cells creating the proliferative niche express CXCL12. In situ observation demonstrated that most of hemato‐ poietic stem cells are concentrated in the trabecular zone of the marrow cavity, which also harbors high numbers of niche osteblasts, sinusoids, and CXCL12-positive reticular cells. Nonetheless, HSC maintenance by both endosteal and perivascular niches are, at least in part, mediated by Jagged-Notch and angiopoietin-1-Tie2 interactions [20,27,31,32].

which are diverse at the population level and clonal and unique at individual cell level. These clonal receptors are not conserved and are generated by gene rearrangements during ontogeny

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Innate immune cells and cells from the adaptive immune system mostly differentiate within the adult bone marrow, except for T lymphocytes that differentiate inside the thymus but also arise from hematopoietic progenitors. Although we can didactically separate the immune system into two categories, an effective immune response depends on both innate and adaptive cells. For T cells to be activated, they depend to see antigen complexed to the Major Histo‐ compatibility Molecules (MHC) presented to them by antigen presenting cells (APC), having the dendritic cells (DC) as the most important cell type to initiate the response, or to prime the adaptive immune response. Also, the cytokines secreted by DC at the moment of antigen presentation, will define the fate of the T cell, meaning the cytokines these T cells will present or their specialization on Th1, Th2, Th17, etc for CD4+T cells or Tc1, Tc2, etc for CD8+cells. Also, the antibody class produced by B cells – IgG, IgA, IgE, etc – will be defined by cytokines produced by a given CD4T cell which will 'help' the given B cell at the moment of its activation. So, indirectly, it depends upon the APC which will modulate the CD4 fate. On the other hand, although the cells from the innate immune system can play their role independently, T helper cells can efficiently modulate it and, for example, optimize the microbicidal function of macrophages or even down modulate it. Also, through their role on dictating the immuno‐ globulin isotype to B cells, T cells will indirectly act on opsonization which will ultimately be effective through the innate system by optimizing phagocytosis and activation of phagoctyes

The important point to have in mind here, before getting into the T cells inside the bone marrow, is that the effective immune response depends on T cells and an important part of the immune effector mechanisms rely on the collaboration between adaptive and innate responses, with the innate response being in many cases, the main players at the effector phase.

After maturation inside the thymus, T cells gain the peripheral blood circulation and enter the secondary lymphoid organs (SLO) - lymph nodes and spleen - where they can be activated. Classically, these two SLO are considered the sites of naive T cell activation given their architecture, which allows concentration of antigen, DCs and naive T cells in the same neighborhood. This architecture is extremely important given the low frequency of antigen specific T cells making it difficult to meet with antigen, by chance, anywhere in the body.

Primed T cells will generate effectors cells, which will deal with the incoming antigen in the short-term response and will be vanished after antigen clearance. Primed T cells will also generate memory cells, which will be kept, even in the absence of antigen. Memory cells can be found in the SLO and in tissues as different memory cell subpopulations. Those in the tissues, are the effectors memory cells, which respond rather quickly after antigen exposure and those in the SLO are considered the central memory cells, responsible for keeping the memory pool and they take a little longer than effector memory cells to respond to antigen [39].

of T-and B-lymphocytes, the cellular components of the adaptive immune system.

and granulocytes, all actors of the so called innate response.

**2.2. Bone marrow : A hospitable environment for T cells**

So, all in one thought, in the bone marrow, there are two distinct niches to harbor HSC, referred as to "proliferative niche" and "quiescent niche", which are composed by perivascular cells and endosteal osteoblasts, respectively [15,22,31,32,35,36]. The real conversation between these niches, and how other elements, such as the immune system, would contribute to the niche formation, organization and dynamics are still to be understood.

The technique to isolate and culture separately endosteal osteoblasts and subendosteal reticular/perivascular cells from the marrow cavity of murine long bones was established [15] and global gene analyses data suggest that both endosteal and subendosteal stroma contribute to the formation of both niches in the marrow.

## **2. T cells as messengers from the periphery to the hematopoietic bone marrow**

#### **2.1. An overview of the immune system**

The immune system is composed of hematopoietic cells, which we can be characterized according to the way they recognize and respond to antigens.

The innate immune system, phylogenetically, arises before the adaptive immune system and is so called because its ability to respond to antigens is ready and immediate. Characteristically, the innate immune cells recognize antigen through Pathogen Recognition Receptors (PRR), which are evolutionary conserved and can be common to different cell types. PRRs recognize defined molecular patterns from a pathogen [37] or something that is 'dangerous' to the body [38]. These molecular patterns, which are named Pathogen or Danger Associated Molecular Patterns (PAMPs or DAMPs), are poorly present or even absent in healthy mammals and are rich in or characteristics of bacteria, fungi, virus and so on. The cellular composition of the innate immune system is represented by phagocytes (granulocytes, monocytes/macrophages and dendritic cells) which deals with antigen, ultimately, eliminating it by phagocytosis or secretion of the internal granules content, and some lymphocytes as Natural Killer (NK) cells, γδ T cells and B1 cells. In common, all these cellular types promptly respond to antigen and will do so in the same time frame and efficiency regardless their previous experience with the same antigen.

The adaptive immune system is so called because its components do not mount an immediate response to antigen. They need to be stimulated in order to mature their effectors functions and these take 3-5 days to happen, and will only be clinically effective after 7 days. Although it takes a while for the adaptive immune response to occur, it does so only once-on the first encounter with the antigen. On the following and subsequent encounters with the same antigen, the response will be fast occurring in less than 24h, revealing the existing memory response. Characteristically, the antigen recognition is done by antigen recognition receptors, which are diverse at the population level and clonal and unique at individual cell level. These clonal receptors are not conserved and are generated by gene rearrangements during ontogeny of T-and B-lymphocytes, the cellular components of the adaptive immune system.

Innate immune cells and cells from the adaptive immune system mostly differentiate within the adult bone marrow, except for T lymphocytes that differentiate inside the thymus but also arise from hematopoietic progenitors. Although we can didactically separate the immune system into two categories, an effective immune response depends on both innate and adaptive cells. For T cells to be activated, they depend to see antigen complexed to the Major Histo‐ compatibility Molecules (MHC) presented to them by antigen presenting cells (APC), having the dendritic cells (DC) as the most important cell type to initiate the response, or to prime the adaptive immune response. Also, the cytokines secreted by DC at the moment of antigen presentation, will define the fate of the T cell, meaning the cytokines these T cells will present or their specialization on Th1, Th2, Th17, etc for CD4+T cells or Tc1, Tc2, etc for CD8+cells. Also, the antibody class produced by B cells – IgG, IgA, IgE, etc – will be defined by cytokines produced by a given CD4T cell which will 'help' the given B cell at the moment of its activation. So, indirectly, it depends upon the APC which will modulate the CD4 fate. On the other hand, although the cells from the innate immune system can play their role independently, T helper cells can efficiently modulate it and, for example, optimize the microbicidal function of macrophages or even down modulate it. Also, through their role on dictating the immuno‐ globulin isotype to B cells, T cells will indirectly act on opsonization which will ultimately be effective through the innate system by optimizing phagocytosis and activation of phagoctyes and granulocytes, all actors of the so called innate response.

The important point to have in mind here, before getting into the T cells inside the bone marrow, is that the effective immune response depends on T cells and an important part of the immune effector mechanisms rely on the collaboration between adaptive and innate responses, with the innate response being in many cases, the main players at the effector phase.

#### **2.2. Bone marrow : A hospitable environment for T cells**

Nonetheless, HSC maintenance by both endosteal and perivascular niches are, at least in part,

So, all in one thought, in the bone marrow, there are two distinct niches to harbor HSC, referred as to "proliferative niche" and "quiescent niche", which are composed by perivascular cells and endosteal osteoblasts, respectively [15,22,31,32,35,36]. The real conversation between these niches, and how other elements, such as the immune system, would contribute to the niche

The technique to isolate and culture separately endosteal osteoblasts and subendosteal reticular/perivascular cells from the marrow cavity of murine long bones was established [15] and global gene analyses data suggest that both endosteal and subendosteal stroma contribute

**2. T cells as messengers from the periphery to the hematopoietic bone**

The immune system is composed of hematopoietic cells, which we can be characterized

The innate immune system, phylogenetically, arises before the adaptive immune system and is so called because its ability to respond to antigens is ready and immediate. Characteristically, the innate immune cells recognize antigen through Pathogen Recognition Receptors (PRR), which are evolutionary conserved and can be common to different cell types. PRRs recognize defined molecular patterns from a pathogen [37] or something that is 'dangerous' to the body [38]. These molecular patterns, which are named Pathogen or Danger Associated Molecular Patterns (PAMPs or DAMPs), are poorly present or even absent in healthy mammals and are rich in or characteristics of bacteria, fungi, virus and so on. The cellular composition of the innate immune system is represented by phagocytes (granulocytes, monocytes/macrophages and dendritic cells) which deals with antigen, ultimately, eliminating it by phagocytosis or secretion of the internal granules content, and some lymphocytes as Natural Killer (NK) cells, γδ T cells and B1 cells. In common, all these cellular types promptly respond to antigen and will do so in the same time frame and efficiency regardless their previous experience with the

The adaptive immune system is so called because its components do not mount an immediate response to antigen. They need to be stimulated in order to mature their effectors functions and these take 3-5 days to happen, and will only be clinically effective after 7 days. Although it takes a while for the adaptive immune response to occur, it does so only once-on the first encounter with the antigen. On the following and subsequent encounters with the same antigen, the response will be fast occurring in less than 24h, revealing the existing memory response. Characteristically, the antigen recognition is done by antigen recognition receptors,

mediated by Jagged-Notch and angiopoietin-1-Tie2 interactions [20,27,31,32].

formation, organization and dynamics are still to be understood.

according to the way they recognize and respond to antigens.

to the formation of both niches in the marrow.

**2.1. An overview of the immune system**

**marrow**

90 Adult Stem Cell Niches

same antigen.

After maturation inside the thymus, T cells gain the peripheral blood circulation and enter the secondary lymphoid organs (SLO) - lymph nodes and spleen - where they can be activated. Classically, these two SLO are considered the sites of naive T cell activation given their architecture, which allows concentration of antigen, DCs and naive T cells in the same neighborhood. This architecture is extremely important given the low frequency of antigen specific T cells making it difficult to meet with antigen, by chance, anywhere in the body.

Primed T cells will generate effectors cells, which will deal with the incoming antigen in the short-term response and will be vanished after antigen clearance. Primed T cells will also generate memory cells, which will be kept, even in the absence of antigen. Memory cells can be found in the SLO and in tissues as different memory cell subpopulations. Those in the tissues, are the effectors memory cells, which respond rather quickly after antigen exposure and those in the SLO are considered the central memory cells, responsible for keeping the memory pool and they take a little longer than effector memory cells to respond to antigen [39]. However, the above mentioned circulation pattern and activation sites of mature T cells had been challenged and revitalized by studies on BM T cells in the last decade.

**2.3. T cell help for hematopoiesis**

independent of antigen recognition.

corrected.

T cells (Figure 1).

BM is especialized in generating hematopoietic cells and T cells localize in the hematopoietic niche, on the perivascular regions where pericytes are present being one of the stem cell niches [50]. These data show that T cells are in close physical contact with the hematopoietic envi‐ ronment. In infectious situations, it appears that T cell amplification of hematopoiesis is required to clear pathogens [51–53]. These can be achieved by local secretion of cytokines by T cells (at the expense of antigen recognition *in situ*) including GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-13, IL17 and oncostatin M, which all contribute to amplify granulocyte generation inside the bone marrow. However, the role of T cells in "normal" hematopoiesis has not been extensively considered as normal hematopoiesis is considered an innate immune phenomena

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The relationship between hematopoiesis and T cells was first suggested almost 40 years ago when it was shown that 1-day thymectomized mice were anemic, showed arrested erythroid maturation and reduction in the number of spleen colony-forming units in the bone marrow and spleen [54,55]. In addition, intravenous injection of live thymocytes accelerated hemato‐ poietic reconstitution in sublethally irradiated mice [56]. In the 90's it was suggested that singeneic T cells could stimulate the growth of hematopoietic progenitors [57]. Much more recently, it was clearly shown that T cell deficient mice (nude and SCID mice) have a severe reduction in the number of granulocytes in peripheral blood, despite the high frequency of granulo-monocytic progenitors in the bone marrow. By injecting CD4 T cells into these animals, the peripheral cytopenia was corrected and the number of progenitors accumulated in the bone marrow diminished to levels similar to the ones found in normal euthymic animals [46]. Moreover, studies with TCR transgenic mice in the RAGKO background, i.e., in the absence of endogenous gene rearrangements to guarantee that the only T cell specificity in the animals was the one from the transgenic receptor, evidenced the same altered hematopoiesis present in the T cell deficient mice: accumulation of immature myeloid-monocytic progenitors in the BM and granulopenia in the peripheral blood. Also, their BM T cells did not show the characteristic activated phenotype found in regular animals. Strikingly after injection of the cognate antigen, BM T cells became activated and the abnormal hematopoietic phenotype was

All these results show that T cells, as antigen recognition entities present in the periphery, act as messengers to the hematopoietic bone marrow. They traffic to the bone mainly after activation, recognize antigen within the bone cavity and help hematopoiesis so that it acquire the so-called 'normal' configuration. Normal hematopoiesis is not a phenomenon independent of the adaptive immune response, but is a response to an immunological insult, instructed by

At the end, this makes a lot of sense since the optimal effector immune mechanisms relies on innate immune cells acting on its best with components of the adaptive responses. And if memory response need to be fast and precise, T cells need to be rapidly activated and find their way to the bone marrow to instruct hematopoiesis to produce more of effectors. In the absence of enough phagocytes, and these in the absence of T cell help and immunoglobulins, the

response will not be as efficient as necessary to counteract an invasive pathogen.

T cells account for only 3-8% of total BM cells, what seems a small number, but in fact it is estimated to be close to or even higher than the number of T cells in the spleen when all hematopoietic bones are considered [40]. Moreover, the CD4 to CD8 ratio is 1:2 instead of the 2:1 ratio found in peripheral blood, indicating a local microenvironmental regulation of these cell subsets. Interestingly, these cells do not seem to be BM resident cells nor depend on antigen presence for its location in the bone marrow. Naive as well as memory T cells carry CXCR4, a receptor for CXCL12 (SDF1) a critical chemokine produced by stromal cells in the bone marrow, which play a significant role on HSC migration into the BM and its specific niche. Although both, memory and naive cells can respond to CXCL12 in migration assays, memory or activated cells respond more efficiently [41]. Besides, parabiont studies had shown that activated/memory CD4 and CD8 T cells recirculate and distribute equally through SLO and BM between the two animals [42,43] indicating that T cells recirculate through the BM.

Antigen recognition in the bone marrow could be one important requirement to keep them there not only as spectators but as active cells influencing the microenvironment. Of note is the fact BM CD8 T cells are extremelly active, with a proliferation rate in vivo higher than the ones in spleen and lymph nodes [44,45] Similarly, BM CD4 T cells produce high amounts of cytokine in the absence of intentional stimulation [46,47]. However, in the case of CD8 cells, when taken out from the BM, their behavior in vitro is similar to the one from splenic cells, indicating that this is not an intrinsic characteristic of BM cells, but is a modulation imposed by the BM microenvironment [40]. The presence of antigen is actually possible, as bone marrow DC were shown to present blood born antigens to naive CD4 and CD8 T cells [48]. Moreover, not only DCs, but other myeloid cells can also present antigen to naive BM T cells, what is not observed in spleen where T cell primming depends mostly on DCs [49]. Another curious fact about the bone marrow environment and T cells is that antigen specific cells are found in several diseases but do not always relate to the presence of antigen, neither in the bone marrow nor in the periphery [40]. Memory CD8 T cells are maintained by IL-7 and IL-15 which are produced in copious amounts by stromal cells in the BM. On the other hand, memory CD4 cells do not need recognition of MHC with the cognate peptide, but depend on the presence of MHC and IL-7 to be maintained.

So, it seems that the BM environment have all the requirements to attract and eventually keep T cells active: BM DC and other myeloid cells can present antigen and prime T cells, the stroma produces IL-7 and IL-15 necessary for memory CD8 maintenance, and hematopoietic cells in the marrow express MHC molecules fulfilling the requirements to maintain memory CD4 cells.

But what are these cells doing in the bone marrow, since the majority of infections are not BM specific, and in pathological conditions such as cancer, antigen specific cells are found there in the absence of the pathogenic antigen? (although the antigen peptide might be presented by BM DCs as mentioned above)

#### **2.3. T cell help for hematopoiesis**

However, the above mentioned circulation pattern and activation sites of mature T cells had

T cells account for only 3-8% of total BM cells, what seems a small number, but in fact it is estimated to be close to or even higher than the number of T cells in the spleen when all hematopoietic bones are considered [40]. Moreover, the CD4 to CD8 ratio is 1:2 instead of the 2:1 ratio found in peripheral blood, indicating a local microenvironmental regulation of these cell subsets. Interestingly, these cells do not seem to be BM resident cells nor depend on antigen presence for its location in the bone marrow. Naive as well as memory T cells carry CXCR4, a receptor for CXCL12 (SDF1) a critical chemokine produced by stromal cells in the bone marrow, which play a significant role on HSC migration into the BM and its specific niche. Although both, memory and naive cells can respond to CXCL12 in migration assays, memory or activated cells respond more efficiently [41]. Besides, parabiont studies had shown that activated/memory CD4 and CD8 T cells recirculate and distribute equally through SLO and BM between the two animals [42,43] indicating that T cells recirculate through the BM.

Antigen recognition in the bone marrow could be one important requirement to keep them there not only as spectators but as active cells influencing the microenvironment. Of note is the fact BM CD8 T cells are extremelly active, with a proliferation rate in vivo higher than the ones in spleen and lymph nodes [44,45] Similarly, BM CD4 T cells produce high amounts of cytokine in the absence of intentional stimulation [46,47]. However, in the case of CD8 cells, when taken out from the BM, their behavior in vitro is similar to the one from splenic cells, indicating that this is not an intrinsic characteristic of BM cells, but is a modulation imposed by the BM microenvironment [40]. The presence of antigen is actually possible, as bone marrow DC were shown to present blood born antigens to naive CD4 and CD8 T cells [48]. Moreover, not only DCs, but other myeloid cells can also present antigen to naive BM T cells, what is not observed in spleen where T cell primming depends mostly on DCs [49]. Another curious fact about the bone marrow environment and T cells is that antigen specific cells are found in several diseases but do not always relate to the presence of antigen, neither in the bone marrow nor in the periphery [40]. Memory CD8 T cells are maintained by IL-7 and IL-15 which are produced in copious amounts by stromal cells in the BM. On the other hand, memory CD4 cells do not need recognition of MHC with the cognate peptide, but depend on the presence

So, it seems that the BM environment have all the requirements to attract and eventually keep T cells active: BM DC and other myeloid cells can present antigen and prime T cells, the stroma produces IL-7 and IL-15 necessary for memory CD8 maintenance, and hematopoietic cells in the marrow express MHC molecules fulfilling the requirements to maintain memory CD4 cells.

But what are these cells doing in the bone marrow, since the majority of infections are not BM specific, and in pathological conditions such as cancer, antigen specific cells are found there in the absence of the pathogenic antigen? (although the antigen peptide might be presented

of MHC and IL-7 to be maintained.

92 Adult Stem Cell Niches

by BM DCs as mentioned above)

been challenged and revitalized by studies on BM T cells in the last decade.

BM is especialized in generating hematopoietic cells and T cells localize in the hematopoietic niche, on the perivascular regions where pericytes are present being one of the stem cell niches [50]. These data show that T cells are in close physical contact with the hematopoietic envi‐ ronment. In infectious situations, it appears that T cell amplification of hematopoiesis is required to clear pathogens [51–53]. These can be achieved by local secretion of cytokines by T cells (at the expense of antigen recognition *in situ*) including GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-13, IL17 and oncostatin M, which all contribute to amplify granulocyte generation inside the bone marrow. However, the role of T cells in "normal" hematopoiesis has not been extensively considered as normal hematopoiesis is considered an innate immune phenomena independent of antigen recognition.

The relationship between hematopoiesis and T cells was first suggested almost 40 years ago when it was shown that 1-day thymectomized mice were anemic, showed arrested erythroid maturation and reduction in the number of spleen colony-forming units in the bone marrow and spleen [54,55]. In addition, intravenous injection of live thymocytes accelerated hemato‐ poietic reconstitution in sublethally irradiated mice [56]. In the 90's it was suggested that singeneic T cells could stimulate the growth of hematopoietic progenitors [57]. Much more recently, it was clearly shown that T cell deficient mice (nude and SCID mice) have a severe reduction in the number of granulocytes in peripheral blood, despite the high frequency of granulo-monocytic progenitors in the bone marrow. By injecting CD4 T cells into these animals, the peripheral cytopenia was corrected and the number of progenitors accumulated in the bone marrow diminished to levels similar to the ones found in normal euthymic animals [46]. Moreover, studies with TCR transgenic mice in the RAGKO background, i.e., in the absence of endogenous gene rearrangements to guarantee that the only T cell specificity in the animals was the one from the transgenic receptor, evidenced the same altered hematopoiesis present in the T cell deficient mice: accumulation of immature myeloid-monocytic progenitors in the BM and granulopenia in the peripheral blood. Also, their BM T cells did not show the characteristic activated phenotype found in regular animals. Strikingly after injection of the cognate antigen, BM T cells became activated and the abnormal hematopoietic phenotype was corrected.

All these results show that T cells, as antigen recognition entities present in the periphery, act as messengers to the hematopoietic bone marrow. They traffic to the bone mainly after activation, recognize antigen within the bone cavity and help hematopoiesis so that it acquire the so-called 'normal' configuration. Normal hematopoiesis is not a phenomenon independent of the adaptive immune response, but is a response to an immunological insult, instructed by T cells (Figure 1).

At the end, this makes a lot of sense since the optimal effector immune mechanisms relies on innate immune cells acting on its best with components of the adaptive responses. And if memory response need to be fast and precise, T cells need to be rapidly activated and find their way to the bone marrow to instruct hematopoiesis to produce more of effectors. In the absence of enough phagocytes, and these in the absence of T cell help and immunoglobulins, the response will not be as efficient as necessary to counteract an invasive pathogen.

16 T cells (Figure 1).

17 18 19 

27 

33 34 

36 

38 

*Bonomo , Monteiro, Balduino HSC, Tumor cells and T cells*

 accelerated hematopoietic reconstitution in sublethally irradiated mice [56]. In the 90's it was suggested that singeneic T cells could stimulate the growth of hematopoietic progenitors [57]. Much more recently, it was clearly shown that T cell deficient mice (nude and SCID mice) have a severe reduction in the number of granulocytes in peripheral blood, despite the high frequency of granulo-monocytic progenitors in the bone marrow. By injecting CD4 T cells into these animals, the peripheral cytopenia was corrected and the number of progenitors accumulated in the bone marrow diminished to levels similar to the ones found in normal euthymic animals [46]. Moreover, studies with TCR transgenic mice in the RAGKO background, i.e., in the absence of endogenous gene rearrangements to guarantee that the only T cell specificity in the animals was the one from the transgenic receptor, evidenced the same altered hematopoiesis present in the T cell deficient mice: accumulation of immature myeloid-monocytic progenitors in the BM and granulopenia in the peripheral blood. Also, their BM T cells did not show the characteristic activated phenotype found in regular animals. Strikingly after injection of the cognate antigen, BM T cells became activated and the abnormal hematopoietic phenotype was corrected. All these results show that T cells, as antigen recognition entities present in the periphery, act as messengers to

15 phenomenon independent of the adaptive immune response, but is a response to an immunological insult, instructed by

Once both immune and skeletal systems share many regulatory elements, including some which are key in bone remodelling, it seems reasonable to think that these two systems interact with each other. Indeed, the new and complex interdisciplinary field of osteoimmunology implies the concept that bone, and its cavity, crosstalk with the immune system. Osteoimmu‐ nology investigates the interactions between these two systems, since bone marrow stromal cells express surface molecules essential for hematopoiesis─ from which all cells of the mammalian immune system derive ─ and stimulate immune cells, which produce various

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It is clear that immune cells producing pro-inflammatory cytokines contribute to bone damage by potentiating the effects of RANK/RANKL/OPG pathway. The cytokines TNF-α, interleukin (IL)-1, IL-3, IL-6, IL-7, IL-11, IL-15 IL-17A/F, and prostaglandin-E2 (as well hormones and related peptides as parathyroid hormone, parathyroid hormone-related protein, and gluco‐ corticoids, besides 1,25(OH)2 vitamin D3) potentiate bone loss either by increasing osteoclast generation and activation or by inducing RANKL expression by the osteoblasts [60]. On the other hand, IL-4, IL-5, IL-10, IL-12, IL-13, IL-18 and interferon (IFN)-α, IFN-β and IFN-γ are inhibitors of osteoclastogenesis by blocking RANKL signalling, either directly or indirectly [58,59]. Interestingly, IL-1 is a stimulator of TRAF6 expression on the osteoclast, thereby potentiating RANK/RANKL signaling cascade, whereas IFN-γ is known to downregulate TRAF6 by proteossomal degradation aborting osteoclast formation [58,65]. In contrast with this effect, IFN-γ has also been implicated in osteoclast formation and bone resorption,

underlining the controversial role of IFN-γ in osteoclastogenesis [59,61,63,66].

The crosstalk mechanisms between T cells and osteoclasts have been extensively documented. The key role of Th 17 CD4 T cells, ─ an osteoclastogenic subclass of T cells expressing membrane and soluble RANKL, IL-17A/F and TNFa ─ on exacerbated and uncontrolled function of osteoclasts, has been investigated in models of inflammatory diseases, such as autoimmune rheumathoid arthritis [58,59], periodontitis [67], multiple myeloma [61] and breast tumor skeletal metastasis [68]. Nevertheless, the literature of this field has also been showing that these exacerbated osteoclast pattern, might also be controlled by other T cells subsets as regulatory CD4 T (Treg) cells. T reg cells produce anti-osteoclastogenic cytokines such as IL-4, IL-10 and TGF-β and express CTLA-4 inhibiting bone destruction [69,70]. Besides that, recently, it was showed that osteoclasts can present antigenic peptides to CD8 T cells, apart from CD4 T cells, resulting in FoxP3 expression. In this way, CD8 FoxP3+cells function as CD8 Treg cells, able to cause an inappropriate activation of the immune response through reciprocal interactions between CD137/CD137L and RANK/RANKL pathways. CD137, expressed on T cells, is a co-stimulatory molecule induced by TCR activation and its ligand, CD137L, is expressed on DCs and osteoclasts precursors. Once T cell CD137 binds to CD137L on osteoclasts precursors, multinucleation of osteoclasts is suppressed.. However, CD137/ CD137L will signal simultaneously with RANKL/RANK on the Tcell/Osteoclast pair and this might lead to increased apoptosis by T cells [71]. Therefore, in pathological conditions, the effects of T cells on osteoclastogenesis will depend on the balance between positive and

regulatory cytokines that influence the bone fate [58,64].

negative factors that they express.

26 **Figure 1**: *T cells help myeloid cell differentiation* - In A, in the absence of activated T cells, myeloid progenitor cells (light green) accumulate in the bone marrow. When T cells are activated and migrate to the bone cavity, as shown in B, they most probably interact with APC such as DCs, secrete cytokines which will help the terminal differentiation of myeloid cells (dark green cells) giving rise to the "normal' cells counts in the peripheral blood. **Figure 1.** T cells help myeloid cell differentiation-In A, in the absence of activated T cells, myeloid progenitor cells (light green) accumulate in the bone marrow. When T cells are activated and migrate to the bone cavity, as shown in B, they most probably interact with APC such as DCs, secrete cytokines which will help the terminal differentiation of myeloid cells (dark green cells) giving rise to the normal cells counts in the peripheral blood.

28 At the end, this makes a lot of sense since the optimal effector immune mechanisms relies on innate immune 29 cells acting on its best with components of the adaptive responses. And if memory response need to be fast and precise,

#### 30 T cells need to be rapidly activated and find their way to the bone marrow to instruct hematopoiesis to produce more of 31 effectors. In the absence of enough phagocytes, and these in the absence of T cell help and immunoglobulins, the 32 response will not be as efficient as necessary to counteract an invasive pathogen. **3. Roommates in the bone cavity: Tumor cells, HSC and T cells**

#### 35  **3. Room mates in the bone cavity: tumor cells, HSC and T cells 3.1. The crosstalk between T cells and bone: An overview of osteoimmunology**

5 37  *The crosstalk between T cells and bone system cell: An overview of "osteoimmunology"*  39 First of all, bone marrow is in close contact with bone tissue formed by the organized deposits of type I collagen 40 and hydroxyapatite, a calcium phosphate salt, in which bone cells are distributed. Rather than being an inert matrix, bone 41 undergoes a continuous turnover: osteoblast activity resulting in bone deposition is counteracted by osteoclast mediated 42 bone resorption. Osteoblasts are cells of mesenchymal origin, whereas osteoclasts are of hematopoietic origin ─ 43 multinucleated giant cells, derived from monocytes/macrophages progenitors expressing CD11b–c, CD14 and receptor 44 activator of nuclear factor, (RANK). Curiously, several factors regulating bone homeostasis are also molecular players of First of all, bone marrow is in close contact with bone tissue formed by the organized deposits of type I collagen and hydroxyapatite, a calcium phosphate salt, in which bone cells are distributed. Rather than being an inert matrix, bone undergoes a continuous turnover: osteoblast activity resulting in bone deposition is counteracted by osteoclast mediated boneresorption. Osteoblasts are cells of mesenchymal origin, whereas osteoclasts are of hemato‐ poietic origin ─ multinucleated giant cells, derived from monocytes/macrophages progenitors expressing CD11b–c, CD14 and receptor activator of nuclear factor, (RANK). Curiously, several factors regulating bone homeostasis are also molecular players of the immune response. For example, the TNF family member RANK ligand (RANKL) (also called TRANCE, OPGL, ODF), a potent regulator of osteoclast activation and differentiation, is expressed not only by osteoblasts, but also by monocytes, neutrophils, dendritic cells, B cells and activated CD4 and CD8 T cells [58–61]. RANKL mediates its biological effects by binding to RANK, expressed by osteoclast progenitors, mature osteoclasts, DCs and neutrophils [60,62]. RANKL can also bind to the soluble protein osteoprotegerin (OPG), which acts as an inhibitory decoy receptor and can be produced by osteoblasts, DCs and B cells [58–61]. By binding to RANK, RANKL strongly stimulates bone resorption, contributes to lymph node organogenesis, prolongs DC survival and augments DC adjuvant properties [63].

Once both immune and skeletal systems share many regulatory elements, including some which are key in bone remodelling, it seems reasonable to think that these two systems interact with each other. Indeed, the new and complex interdisciplinary field of osteoimmunology implies the concept that bone, and its cavity, crosstalk with the immune system. Osteoimmu‐ nology investigates the interactions between these two systems, since bone marrow stromal cells express surface molecules essential for hematopoiesis─ from which all cells of the mammalian immune system derive ─ and stimulate immune cells, which produce various regulatory cytokines that influence the bone fate [58,64].

*Bonomo , Monteiro, Balduino HSC, Tumor cells and T cells*

 accelerated hematopoietic reconstitution in sublethally irradiated mice [56]. In the 90's it was suggested that singeneic T cells could stimulate the growth of hematopoietic progenitors [57]. Much more recently, it was clearly shown that T cell deficient mice (nude and SCID mice) have a severe reduction in the number of granulocytes in peripheral blood, despite the high frequency of granulo-monocytic progenitors in the bone marrow. By injecting CD4 T cells into these animals, the peripheral cytopenia was corrected and the number of progenitors accumulated in the bone marrow diminished to levels similar to the ones found in normal euthymic animals [46]. Moreover, studies with TCR transgenic mice in the RAGKO background, i.e., in the absence of endogenous gene rearrangements to guarantee that the only T cell specificity in the animals was the one from the transgenic receptor, evidenced the same altered hematopoiesis present in the T cell deficient mice: accumulation of immature myeloid-monocytic progenitors in the BM and granulopenia in the peripheral blood. Also, their BM T cells did not show the characteristic activated phenotype found in regular animals. Strikingly after injection of the cognate antigen, BM T cells became activated and the abnormal hematopoietic phenotype was corrected. All these results show that T cells, as antigen recognition entities present in the periphery, act as messengers to the hematopoietic bone marrow. They traffic to the bone mainly after activation, recognize antigen within the bone cavity and help hematopoiesis so that it acquire the so-called 'normal' configuration. Normal hematopoiesis is not a phenomenon independent of the adaptive immune response, but is a response to an immunological insult, instructed by

 At the end, this makes a lot of sense since the optimal effector immune mechanisms relies on innate immune cells acting on its best with components of the adaptive responses. And if memory response need to be fast and precise, T cells need to be rapidly activated and find their way to the bone marrow to instruct hematopoiesis to produce more of effectors. In the absence of enough phagocytes, and these in the absence of T cell help and immunoglobulins, the

myeloid cells (dark green cells) giving rise to the "normal' cells counts in the peripheral blood.

myeloid cells (dark green cells) giving rise to the normal cells counts in the peripheral blood.

**Figure 1**: *T cells help myeloid cell differentiation* - In A, in the absence of activated T cells, myeloid progenitor cells (light green) accumulate in the bone marrow. When T cells are activated and migrate to the bone cavity, as shown in B, they most probably interact with APC such as DCs, secrete cytokines which will help the terminal differentiation of

**Figure 1.** T cells help myeloid cell differentiation-In A, in the absence of activated T cells, myeloid progenitor cells (light green) accumulate in the bone marrow. When T cells are activated and migrate to the bone cavity, as shown in B, they most probably interact with APC such as DCs, secrete cytokines which will help the terminal differentiation of

37  *The crosstalk between T cells and bone system cell: An overview of "osteoimmunology"* 

**3.1. The crosstalk between T cells and bone: An overview of osteoimmunology**

**3. Roommates in the bone cavity: Tumor cells, HSC and T cells**

 First of all, bone marrow is in close contact with bone tissue formed by the organized deposits of type I collagen and hydroxyapatite, a calcium phosphate salt, in which bone cells are distributed. Rather than being an inert matrix, bone undergoes a continuous turnover: osteoblast activity resulting in bone deposition is counteracted by osteoclast mediated bone resorption. Osteoblasts are cells of mesenchymal origin, whereas osteoclasts are of hematopoietic origin ─ multinucleated giant cells, derived from monocytes/macrophages progenitors expressing CD11b–c, CD14 and receptor activator of nuclear factor, (RANK). Curiously, several factors regulating bone homeostasis are also molecular players of

First of all, bone marrow is in close contact with bone tissue formed by the organized deposits of type I collagen and hydroxyapatite, a calcium phosphate salt, in which bone cells are distributed. Rather than being an inert matrix, bone undergoes a continuous turnover: osteoblast activity resulting in bone deposition is counteracted by osteoclast mediated boneresorption. Osteoblasts are cells of mesenchymal origin, whereas osteoclasts are of hemato‐ poietic origin ─ multinucleated giant cells, derived from monocytes/macrophages progenitors expressing CD11b–c, CD14 and receptor activator of nuclear factor, (RANK). Curiously, several factors regulating bone homeostasis are also molecular players of the immune response. For example, the TNF family member RANK ligand (RANKL) (also called TRANCE, OPGL, ODF), a potent regulator of osteoclast activation and differentiation, is expressed not only by osteoblasts, but also by monocytes, neutrophils, dendritic cells, B cells and activated CD4 and CD8 T cells [58–61]. RANKL mediates its biological effects by binding to RANK, expressed by osteoclast progenitors, mature osteoclasts, DCs and neutrophils [60,62]. RANKL can also bind to the soluble protein osteoprotegerin (OPG), which acts as an inhibitory decoy receptor and can be produced by osteoblasts, DCs and B cells [58–61]. By binding to RANK, RANKL strongly stimulates bone resorption, contributes to lymph node organogenesis,

32 response will not be as efficient as necessary to counteract an invasive pathogen.

prolongs DC survival and augments DC adjuvant properties [63].

35  **3. Room mates in the bone cavity: tumor cells, HSC and T cells** 

A B

16 T cells (Figure 1).

94 Adult Stem Cell Niches

17 18 19 

33 34 

36 

38 

5 

It is clear that immune cells producing pro-inflammatory cytokines contribute to bone damage by potentiating the effects of RANK/RANKL/OPG pathway. The cytokines TNF-α, interleukin (IL)-1, IL-3, IL-6, IL-7, IL-11, IL-15 IL-17A/F, and prostaglandin-E2 (as well hormones and related peptides as parathyroid hormone, parathyroid hormone-related protein, and gluco‐ corticoids, besides 1,25(OH)2 vitamin D3) potentiate bone loss either by increasing osteoclast generation and activation or by inducing RANKL expression by the osteoblasts [60]. On the other hand, IL-4, IL-5, IL-10, IL-12, IL-13, IL-18 and interferon (IFN)-α, IFN-β and IFN-γ are inhibitors of osteoclastogenesis by blocking RANKL signalling, either directly or indirectly [58,59]. Interestingly, IL-1 is a stimulator of TRAF6 expression on the osteoclast, thereby potentiating RANK/RANKL signaling cascade, whereas IFN-γ is known to downregulate TRAF6 by proteossomal degradation aborting osteoclast formation [58,65]. In contrast with this effect, IFN-γ has also been implicated in osteoclast formation and bone resorption, underlining the controversial role of IFN-γ in osteoclastogenesis [59,61,63,66].

The crosstalk mechanisms between T cells and osteoclasts have been extensively documented. The key role of Th 17 CD4 T cells, ─ an osteoclastogenic subclass of T cells expressing membrane and soluble RANKL, IL-17A/F and TNFa ─ on exacerbated and uncontrolled function of osteoclasts, has been investigated in models of inflammatory diseases, such as autoimmune rheumathoid arthritis [58,59], periodontitis [67], multiple myeloma [61] and breast tumor skeletal metastasis [68]. Nevertheless, the literature of this field has also been showing that these exacerbated osteoclast pattern, might also be controlled by other T cells subsets as regulatory CD4 T (Treg) cells. T reg cells produce anti-osteoclastogenic cytokines such as IL-4, IL-10 and TGF-β and express CTLA-4 inhibiting bone destruction [69,70]. Besides that, recently, it was showed that osteoclasts can present antigenic peptides to CD8 T cells, apart from CD4 T cells, resulting in FoxP3 expression. In this way, CD8 FoxP3+cells function as CD8 Treg cells, able to cause an inappropriate activation of the immune response through reciprocal interactions between CD137/CD137L and RANK/RANKL pathways. CD137, expressed on T cells, is a co-stimulatory molecule induced by TCR activation and its ligand, CD137L, is expressed on DCs and osteoclasts precursors. Once T cell CD137 binds to CD137L on osteoclasts precursors, multinucleation of osteoclasts is suppressed.. However, CD137/ CD137L will signal simultaneously with RANKL/RANK on the Tcell/Osteoclast pair and this might lead to increased apoptosis by T cells [71]. Therefore, in pathological conditions, the effects of T cells on osteoclastogenesis will depend on the balance between positive and negative factors that they express.

In addition to inflammatory pathological conditions, increasing evidence supports the notion that T cells are also involved in post-menopausal osteoporosis [66]. Experiments in mice showed that, in the absence of estrogens, higher numbers of TNF-α producing T cells were found in the bone marrow, stimulating directly osteoclasts activity and augmenting their response to RANKL. By comparing peripheral blood mononuclear cells from pre and postmenopausal women, it was observed that estrogen deficiency was associated with an increased production of TNF-α. The action of TNF-α is not limited to the induction of local inflammation, but is both directly and indirectly involved in the activation of osteoclasts. Although further work is necessary to clarify the complex changes leading to post-menopausal osteoporosis in women, a pro-osteoclastogenetic contribution of T cells has to be taken into account.

such model, it is not clear whether Th17 RANKL+ subset T cells exert a direct effect on osteoclastogenesis. It is more likely that Th17 RANKL+ subset T cells contribute indirectly through IL-17A activity over sinovial fibroblasts which produce RANKL and directly stimu‐ lates osteoclastogenesis. Another T cell shown to be potentially envolved is the CD4 Treg subset. In fact, an increase of Treg CD4 T cell number improves clinical signs of arthritis and suppressed local and systemic bone destruction. Synovial tissues of patients with RA also produce many factors regulating bone resorption, such as TNF-α, IL-1 and IL-6, which amplify osteoclast differentiation, activation and consequent bone destruction. Inhibitors that target TNF-α, IL-1 and IL-17A pro-inflammatory and osteoclastogenic cytokines have been approved

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More recently, investigators also demonstrated that RANKL plus macrophage colonystimulating factor can induce transdifferentiation of immature dendritic cells into the osteo‐ clastogenic lineage and that this process is significantly enhanced by RA synovial fluid [73]. Dendritic cells are antigen presenting cells, but they could function as osteoclasts precursors in inflammatory conditions. We can conclude that since dendritic cells modulate T cell activity through the RANK/RANKL pathway and other cytokines associated with osteoclastogenesis, as mentioned earlier, it can function as an osteoimmune interface, contributing to bone loss in

Although T cells clearly contribute to RA pathology, they do so in the outer face of the bone, outside the bone marrow cavity. By the same token, periodontal disease, which had been shown to be dependent of Th17 RANKL+ T cells activated by bacteria present in the oral cavity also lead to extra medullary lesions [67]. Similar mechanisms might act in pathological situations arising within the bone marrow cavity such as post-menopausal osteoporosis and cancers, as myeloma and solid tumor metastasis. In either case it is clear the potential for the

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of mature plasma cells in the bone marrow, leading to bone destruction and failure of normal hematopoiesis. However the cancer induced osteolytic disease in this case, may count on T cell activity inside the bone marrow, in addition to the presence of the tumor itself [61]. In multiple myeloma patients with lytic bone disease, it was observed an increase in bone marrow Th17 T cells expressing high levels of RANKL that can directly stimulate osteoclasts [49,74]; moreover, increased production of T cell derived IL-3, occurring in this disease, can inhibit

This is one the few, if not the only, malignant bone marrow disease associated with bone loss where T cell activity has been studied, and actually shown to be concordant with osteolytic activity. So, instead of having the osteolytic disease induced by cancer cells only, it is proposed the participation of Th17 cells in the pathogenesis of lytic lesions in bone marrow malignancies. In fact, as reported for other human malignacies [75] and in accordance to the phenotype of BM T cells, the number of memory/activated T cells in MM patients is increased, their activity is enhanced and they proliferate much more efficiently than blood derived T cells. These data

adaptive immune system to interact with the bone remodelling system.

osteoblast generation and facilitate hematopoiesis.

**3.3. Cancer: Multiple myeloma as an example of bone marrow derived tumors**

for the treatment of RA.

inflammatory diseases.

Finally, it was documented that T cells have a protective role on bone turnover under phys‐ iological conditions [66,72]. Hints that this modulation may occur came from in vitro studies showing that osteoclastogenesis was inhibited by CD8 T cells. Moreover, after CD3 and CD28 activation, mouse lymph node CD8 T cells showed a delayed kinetics of RANKL expression, as compared with corresponding CD4 T cells. Culture of bone marrow cells from CD4 and CD8 T cell depleted mice showed enhanced osteoclastogenesis in response to 1,25(OH)2 vitamin D3 stimulation, suggesting that T cells had a suppressive effect in this system [6]. Moreover, the protective role of T cells on bone metabolism was also documented by in vivo studies, showing that both B cell-and T cell-deficient mice have decreased bone mineral density. A detailed analysis demonstrated that osteoporosis was prevented by osteoprotegerin produced by bone marrow resident B cells stimulated by T cells through CD40L/CD40 interactions. In contrast, IL-17A does not play any relevant role in physiological bone homeo‐ stasis, as IL17A-deficient mice show normal bone mineral density and skeletal development [6]. Taken together, these findings support the notion that bone marrow derived CD4 and CD8 T cells play a protective role in physiological bone homeostasis, using pathways different from those associated with inflammatory bone diseases.

#### **3.2. Inflammatory bone diseases: Rheumatoid arthritis as a model of T cell involvement in bone diseases**

Rheumatoid arthritis (RA) is the prototype of chronic inflammatory joint diseases and is characterized by persistent inflammation and progressive bone erosions, leading to functional disability and high morbidity. In this disease it is clear that the pro-inflammatory cytokines IL-17A, TNF-α, IL-1 and IL-6 are involved in the perpetuation of the inflammatory condition. The RANK/RANKL/OPG pathway is also strongly involved in RA pathology and it was observed that the RANKL/OPG ratio is increased, leading to bone erosions [58,59]. This effect is mainly dependent on osteoclastic activity and is expressed by two main mechanisms: i) destruction of the organic matrix (type I collagen) by osteoclast cathepsin K, and ii) dissolution of the mineralized component (hydroxyapatite crystals) by the acidic microenvironment generated by the osteoclastic proton pump.

In RA, T cell derived RANKL was initially proposed to be the main contributor to exacerbated osteoclastogenesis, but Th17 RANKL+ subset T cells from RA joints also produce IFN-γ, an anti-osteoclastogenic cytokine, which counterbalance the action of RANKL [58–61]. Thus, in such model, it is not clear whether Th17 RANKL+ subset T cells exert a direct effect on osteoclastogenesis. It is more likely that Th17 RANKL+ subset T cells contribute indirectly through IL-17A activity over sinovial fibroblasts which produce RANKL and directly stimu‐ lates osteoclastogenesis. Another T cell shown to be potentially envolved is the CD4 Treg subset. In fact, an increase of Treg CD4 T cell number improves clinical signs of arthritis and suppressed local and systemic bone destruction. Synovial tissues of patients with RA also produce many factors regulating bone resorption, such as TNF-α, IL-1 and IL-6, which amplify osteoclast differentiation, activation and consequent bone destruction. Inhibitors that target TNF-α, IL-1 and IL-17A pro-inflammatory and osteoclastogenic cytokines have been approved for the treatment of RA.

In addition to inflammatory pathological conditions, increasing evidence supports the notion that T cells are also involved in post-menopausal osteoporosis [66]. Experiments in mice showed that, in the absence of estrogens, higher numbers of TNF-α producing T cells were found in the bone marrow, stimulating directly osteoclasts activity and augmenting their response to RANKL. By comparing peripheral blood mononuclear cells from pre and postmenopausal women, it was observed that estrogen deficiency was associated with an increased production of TNF-α. The action of TNF-α is not limited to the induction of local inflammation, but is both directly and indirectly involved in the activation of osteoclasts. Although further work is necessary to clarify the complex changes leading to post-menopausal osteoporosis in

women, a pro-osteoclastogenetic contribution of T cells has to be taken into account.

those associated with inflammatory bone diseases.

generated by the osteoclastic proton pump.

**bone diseases**

96 Adult Stem Cell Niches

Finally, it was documented that T cells have a protective role on bone turnover under phys‐ iological conditions [66,72]. Hints that this modulation may occur came from in vitro studies showing that osteoclastogenesis was inhibited by CD8 T cells. Moreover, after CD3 and CD28 activation, mouse lymph node CD8 T cells showed a delayed kinetics of RANKL expression, as compared with corresponding CD4 T cells. Culture of bone marrow cells from CD4 and CD8 T cell depleted mice showed enhanced osteoclastogenesis in response to 1,25(OH)2 vitamin D3 stimulation, suggesting that T cells had a suppressive effect in this system [6]. Moreover, the protective role of T cells on bone metabolism was also documented by in vivo studies, showing that both B cell-and T cell-deficient mice have decreased bone mineral density. A detailed analysis demonstrated that osteoporosis was prevented by osteoprotegerin produced by bone marrow resident B cells stimulated by T cells through CD40L/CD40 interactions. In contrast, IL-17A does not play any relevant role in physiological bone homeo‐ stasis, as IL17A-deficient mice show normal bone mineral density and skeletal development [6]. Taken together, these findings support the notion that bone marrow derived CD4 and CD8 T cells play a protective role in physiological bone homeostasis, using pathways different from

**3.2. Inflammatory bone diseases: Rheumatoid arthritis as a model of T cell involvement in**

Rheumatoid arthritis (RA) is the prototype of chronic inflammatory joint diseases and is characterized by persistent inflammation and progressive bone erosions, leading to functional disability and high morbidity. In this disease it is clear that the pro-inflammatory cytokines IL-17A, TNF-α, IL-1 and IL-6 are involved in the perpetuation of the inflammatory condition. The RANK/RANKL/OPG pathway is also strongly involved in RA pathology and it was observed that the RANKL/OPG ratio is increased, leading to bone erosions [58,59]. This effect is mainly dependent on osteoclastic activity and is expressed by two main mechanisms: i) destruction of the organic matrix (type I collagen) by osteoclast cathepsin K, and ii) dissolution of the mineralized component (hydroxyapatite crystals) by the acidic microenvironment

In RA, T cell derived RANKL was initially proposed to be the main contributor to exacerbated osteoclastogenesis, but Th17 RANKL+ subset T cells from RA joints also produce IFN-γ, an anti-osteoclastogenic cytokine, which counterbalance the action of RANKL [58–61]. Thus, in More recently, investigators also demonstrated that RANKL plus macrophage colonystimulating factor can induce transdifferentiation of immature dendritic cells into the osteo‐ clastogenic lineage and that this process is significantly enhanced by RA synovial fluid [73]. Dendritic cells are antigen presenting cells, but they could function as osteoclasts precursors in inflammatory conditions. We can conclude that since dendritic cells modulate T cell activity through the RANK/RANKL pathway and other cytokines associated with osteoclastogenesis, as mentioned earlier, it can function as an osteoimmune interface, contributing to bone loss in inflammatory diseases.

Although T cells clearly contribute to RA pathology, they do so in the outer face of the bone, outside the bone marrow cavity. By the same token, periodontal disease, which had been shown to be dependent of Th17 RANKL+ T cells activated by bacteria present in the oral cavity also lead to extra medullary lesions [67]. Similar mechanisms might act in pathological situations arising within the bone marrow cavity such as post-menopausal osteoporosis and cancers, as myeloma and solid tumor metastasis. In either case it is clear the potential for the adaptive immune system to interact with the bone remodelling system.

#### **3.3. Cancer: Multiple myeloma as an example of bone marrow derived tumors**

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of mature plasma cells in the bone marrow, leading to bone destruction and failure of normal hematopoiesis. However the cancer induced osteolytic disease in this case, may count on T cell activity inside the bone marrow, in addition to the presence of the tumor itself [61]. In multiple myeloma patients with lytic bone disease, it was observed an increase in bone marrow Th17 T cells expressing high levels of RANKL that can directly stimulate osteoclasts [49,74]; moreover, increased production of T cell derived IL-3, occurring in this disease, can inhibit osteoblast generation and facilitate hematopoiesis.

This is one the few, if not the only, malignant bone marrow disease associated with bone loss where T cell activity has been studied, and actually shown to be concordant with osteolytic activity. So, instead of having the osteolytic disease induced by cancer cells only, it is proposed the participation of Th17 cells in the pathogenesis of lytic lesions in bone marrow malignancies. In fact, as reported for other human malignacies [75] and in accordance to the phenotype of BM T cells, the number of memory/activated T cells in MM patients is increased, their activity is enhanced and they proliferate much more efficiently than blood derived T cells. These data suggest that BM T cells in pathological, non-infectious conditions such as cancer, can also migrate to the bone marrow and in addition to its effects over hematopoiesis, as discussed above, they can also influence bone remodelling.

*Bonomo , Monteiro, Balduino HSC, Tumor cells and T cells*

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 before metastasis started, to athymic mouse which never saw tumors, led to an intense osteolytic disease, similar in kinetics and intensity to the one observed in the tumor bearing donor animals. These indicate that T cells can induce osteolytic disease which precedes metastatic colonization. In vivo inhibition of RANKL production by Th17 CD4 T cells, but not of IL-17F, completely protects mice from osteolytic disease and, surprisingly, completely abolishes the development of bone metastases, suggesting that CD4 T cells prepare the metastatic niche for further establishment of tumor cells. In conclusion, these results unveil an unexpected role for RANKL derived from T cells in setting the pre-

 In summary, for tumor cells to first establish in the bone marrow niche, growth factors need to be available favoring its hostage, in other words, a pre-metastatic niche needs to be prepared. On that sense, again, T lymphocytes work as messengers from the periphery as they migrate to the BM after they get primed by tumor antigens. In the BM, they signal and alter bone homeostasis as to prepare the pre-metastatic niche instructed by the tumor modulation of T

selves in the marrow, and after that, they will progress by themselves, directly regulating bone remodeling.

cells, inside the BM, produce pro-osteolcastogenic cytokines (mainly IL-17F and RANKL) which promotes

in the marrow, and after that, they will progress by themselves ,directly regulating bone remodeling.

**Figure 2: T cells induce osteolytic disease which is critical for bone metastatsis establishment**. In A Tumor primed T

**Figure 2.** T cells induce osteolytic disease which is critical for bone metastatsis establishment. In A ; Tumor primed T cells, inside the BM, produce pro-osteolcastogenic cytokines (mainly IL-17F and RANKL) which promotes osteoclasto‐ genesis and activate osteoclasts. Osteoclast activity generates a pro-metastatic environment, with the release of growth factors from the bone matrix. In B, with the hospitable environment prepared, tumor cells can establish them‐

osteoclastogenesis and activate osteoclasts. Osteoclast activity generates a pró -metastatic environment, with the release of growth factors from the bone matrix. In B, with the hospitable environment prepared, tumor cells can establish themselves

In summary, for tumor cells to first establish in the bone marrow niche, growth factors need to be available favoring its hostage, in other words, a pre-metastatic niche needs to be prepared. On that sense, again, T lymphocytes work as messengers from the periphery as they migrate to the BM after they get primed by tumor antigens. In the BM, they signal and alter bone homeostasis as to prepare the pre-metastatic niche instructed by the tumor modulation of T

26 From all of the above it can be depicted that once the hematopoietic niche depends upon the bone lining 27 osteoblasts, it is reasonable to expect an interplay between bone and hematopoietic regulation. On the other hand, it is 28 known that T lymphocytes also communicate with the hematopoietic and bone tissues adding more complexity to the

 T cells in the bone marrow are compatible with memory cells and found in their activated state. In the absence of T cells, hematopoiesis is altered and a maturation arrest is observed in the bone marrow, were high numbers of immature myelo-monocytic progenitors are found accompanied by peripheral cytopenia. When T cells are replenished, the bone marrow arrested progenitors progress, differentiate and migrate to the periphery, correcting the myelogram and the peripheral cytopenia. This is conceptually important since what we use to understand as "normal" hematopoiesis, which should be an antigen independent activity, is already the result of the adaptive immune response, which, in fact,

37 The localization of active T cells within the marrow cavity coincides with the "proliferative niche" of adult HSC, or 38 the perivascular niche. In fact, the evidences favor a T cell function on the proliferative/differentiative phase of

From all of the above it can be depicted that once the hematopoietic niche depends upon the bone lining osteoblasts, it is reasonable to expect an interplay between bone and hematopoietic regulation. On the other hand, it is known that T lymphocytes also communicate with the hematopoietic and bone tissues adding more complexity to the whole balance of these systems.

T cells in the bone marrow are compatible with memory cells and found in their activated state. In the absence of T cells, hematopoiesis is altered and a maturation arrest is observed in the bone marrow, were high numbers of immature myelo-monocytic progenitors are found accompanied by peripheral cytopenia. When T cells are replenished, the bone marrow arrested progenitors progress, differentiate and migrate to the periphery, correcting the myelogram and the peripheral cytopenia. This is conceptually important since what we use to understand

40 Hematopoietic stem cells are supported and regulated by stromal cells covering the inner surface of bones, or 41 the endosteum. The endosteum also supports bone remodeling and osteoblasts are present there. The subendosteal

7 metastatic niche and promoting tumor spread,an extra role for T cells only recently explored (Figure 2).

A B 

8 9 

23 

25 

22 cell phenotype in the periphery [68].

29 whole balance of these systems.

24 **4.Conclusions and Perspectives** 

cell phenotype in the periphery [68].

**4. Conclusions and perspectives**

36 need the innate immune cells to operate!

39 myelopoiesis and not on stem cell maintenance, as stated above.

8 

#### **3.4. Breast tumor skeletal metastasis: the case of osteolytic bone disease in the absence of tumor cells**

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the meta‐ static foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodelling system and the tumor itself [76]. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear.

As already mentioned before, CD4 and CD8 T cells have been shown to unbalance the bone remodeling process in inflammatory osteolytic diseases, however, little is known about their role in cancer induced bone disease, a process that differs from inflammatory diseases as in the former it happens in the bone cavity and not on the periosteal surface.

It had been shown, in an experimental model, that tumor specific T cells have a pro-osteoclas‐ togenic phenotype, i.e., Th17 producers of IL-17F and RANKL among others, when tumors are highly metastatic. On the other hand, the T cell phenotype was not pro-osteoclastogenic, and even rich in anti-osteoclastogenic cytokines as IFN-γ and IL-10, if the tumor was localized to the breast and incapable of sending metastasis to any distant organ, including the BM. This suggest that T cells activity is modulated by the tumor since sibling cell lines, with different metastatic characteristics and sharing the same cognate T cell antigen, trigger different T cell phenotypes.

The pro-osteoclastogenic T cell phenotype observed with metastatic tumor was evident inside the BM, and preceded bone metastatic colonization. Also, osteolytic lesions were already present very early on disease evolution, and again, before metastatic colonization. By trans‐ fering BM T cells from animals bearing the highly aggressive tumors, before metastasis started, to athymic mouse which never saw tumors, led to an intense osteolytic disease, similar in kinetics and intensity to the one observed in the tumor bearing donor animals. These indicate that T cells can induce osteolytic disease which precedes metastatic colonization. In vivo inhibition of RANKL production by Th17 CD4 T cells, but not of IL-17F, completely protects mice from osteolytic disease and, surprisingly, completely abolishes the development of bone metastases, suggesting that CD4 T cells prepare the metastatic niche for further establishment of tumor cells. In conclusion, these results unveil an unexpected role for RANKL derived from T cells in setting the pre-metastatic niche and promoting tumor spread, an extra role for T cells only recently explored (Figure 2).

*Bonomo , Monteiro, Balduino HSC, Tumor cells and T cells*

1 before metastasis started, to athymic mouse which never saw tumors, led to an intense osteolytic disease, similar in 2 kinetics and intensity to the one observed in the tumor bearing donor animals. These indicate that T cells can induce

6 tumor cells. In conclusion, these results unveil an unexpected role for RANKL derived from T cells in setting the pre-

7 metastatic niche and promoting tumor spread,an extra role for T cells only recently explored (Figure 2).

8 9 

23 

25 

**Figure 2: T cells induce osteolytic disease which is critical for bone metastatsis establishment**. In A Tumor primed T cells, inside the BM, produce pro-osteolcastogenic cytokines (mainly IL-17F and RANKL) which promotes osteoclastogenesis and activate osteoclasts. Osteoclast activity generates a pró -metastatic environment, with the release of growth factors from the bone matrix. In B, with the hospitable environment prepared, tumor cells can establish themselves in the marrow, and after that, they will progress by themselves ,directly regulating bone remodeling. **Figure 2.** T cells induce osteolytic disease which is critical for bone metastatsis establishment. In A ; Tumor primed T cells, inside the BM, produce pro-osteolcastogenic cytokines (mainly IL-17F and RANKL) which promotes osteoclasto‐ genesis and activate osteoclasts. Osteoclast activity generates a pro-metastatic environment, with the release of growth factors from the bone matrix. In B, with the hospitable environment prepared, tumor cells can establish them‐ selves in the marrow, and after that, they will progress by themselves, directly regulating bone remodeling.

18 In summary, for tumor cells to first establish in the bone marrow niche, growth factors need to be available

30 T cells in the bone marrow are compatible with memory cells and found in their activated state. In the absence 31 of T cells, hematopoiesis is altered and a maturation arrest is observed in the bone marrow, were high numbers of

35 which should be an antigen independent activity, is already the result of the adaptive immune response, which, in fact,

19 favoring its hostage, in other words, a pre-metastatic niche needs to be prepared. On that sense, again, T lymphocytes 20 work as messengers from the periphery as they migrate to the BM after they get primed by tumor antigens. In the BM, 21 they signal and alter bone homeostasis as to prepare the pre-metastatic niche instructed by the tumor modulation of T 22 cell phenotype in the periphery [68]. 24 **4.Conclusions and Perspectives**  26 From all of the above it can be depicted that once the hematopoietic niche depends upon the bone lining 27 osteoblasts, it is reasonable to expect an interplay between bone and hematopoietic regulation. On the other hand, it is 28 known that T lymphocytes also communicate with the hematopoietic and bone tissues adding more complexity to the In summary, for tumor cells to first establish in the bone marrow niche, growth factors need to be available favoring its hostage, in other words, a pre-metastatic niche needs to be prepared. On that sense, again, T lymphocytes work as messengers from the periphery as they migrate to the BM after they get primed by tumor antigens. In the BM, they signal and alter bone homeostasis as to prepare the pre-metastatic niche instructed by the tumor modulation of T cell phenotype in the periphery [68].

#### 32 immature myelo-monocytic progenitors are found accompanied by peripheral cytopenia. When T cells are replenished, 33 the bone marrow arrested progenitors progress, differentiate and migrate to the periphery, correcting the myelogram and 34 the peripheral cytopenia. This is conceptually important since what we use to understand as "normal" hematopoiesis, **4. Conclusions and perspectives**

29 whole balance of these systems.

suggest that BM T cells in pathological, non-infectious conditions such as cancer, can also migrate to the bone marrow and in addition to its effects over hematopoiesis, as discussed

**3.4. Breast tumor skeletal metastasis: the case of osteolytic bone disease in the absence of**

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the meta‐ static foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodelling system and the tumor itself [76]. Yet, how the pre-metastatic niches arise in the

As already mentioned before, CD4 and CD8 T cells have been shown to unbalance the bone remodeling process in inflammatory osteolytic diseases, however, little is known about their role in cancer induced bone disease, a process that differs from inflammatory diseases as in

It had been shown, in an experimental model, that tumor specific T cells have a pro-osteoclas‐ togenic phenotype, i.e., Th17 producers of IL-17F and RANKL among others, when tumors are highly metastatic. On the other hand, the T cell phenotype was not pro-osteoclastogenic, and even rich in anti-osteoclastogenic cytokines as IFN-γ and IL-10, if the tumor was localized to the breast and incapable of sending metastasis to any distant organ, including the BM. This suggest that T cells activity is modulated by the tumor since sibling cell lines, with different metastatic characteristics and sharing the same cognate T cell antigen, trigger different T cell

The pro-osteoclastogenic T cell phenotype observed with metastatic tumor was evident inside the BM, and preceded bone metastatic colonization. Also, osteolytic lesions were already present very early on disease evolution, and again, before metastatic colonization. By trans‐ fering BM T cells from animals bearing the highly aggressive tumors, before metastasis started, to athymic mouse which never saw tumors, led to an intense osteolytic disease, similar in kinetics and intensity to the one observed in the tumor bearing donor animals. These indicate that T cells can induce osteolytic disease which precedes metastatic colonization. In vivo inhibition of RANKL production by Th17 CD4 T cells, but not of IL-17F, completely protects mice from osteolytic disease and, surprisingly, completely abolishes the development of bone metastases, suggesting that CD4 T cells prepare the metastatic niche for further establishment of tumor cells. In conclusion, these results unveil an unexpected role for RANKL derived from T cells in setting the pre-metastatic niche and promoting tumor spread, an extra role for T cells

the former it happens in the bone cavity and not on the periosteal surface.

above, they can also influence bone remodelling.

**tumor cells**

98 Adult Stem Cell Niches

phenotypes.

only recently explored (Figure 2).

bone tissue remains unclear.

36 need the innate immune cells to operate! 37 The localization of active T cells within the marrow cavity coincides with the "proliferative niche" of adult HSC, or 38 the perivascular niche. In fact, the evidences favor a T cell function on the proliferative/differentiative phase of 39 myelopoiesis and not on stem cell maintenance, as stated above. 40 Hematopoietic stem cells are supported and regulated by stromal cells covering the inner surface of bones, or 41 the endosteum. The endosteum also supports bone remodeling and osteoblasts are present there. The subendosteal From all of the above it can be depicted that once the hematopoietic niche depends upon the bone lining osteoblasts, it is reasonable to expect an interplay between bone and hematopoietic regulation. On the other hand, it is known that T lymphocytes also communicate with the hematopoietic and bone tissues adding more complexity to the whole balance of these systems.

8 T cells in the bone marrow are compatible with memory cells and found in their activated state. In the absence of T cells, hematopoiesis is altered and a maturation arrest is observed in the bone marrow, were high numbers of immature myelo-monocytic progenitors are found accompanied by peripheral cytopenia. When T cells are replenished, the bone marrow arrested progenitors progress, differentiate and migrate to the periphery, correcting the myelogram and the peripheral cytopenia. This is conceptually important since what we use to understand as "normal" hematopoiesis, which should be an antigen independent activity, is already the result of the adaptive immune response, which, in fact, need the innate immune cells to operate!

(figure 3). Whether or not, regulation of one system interferes with the other one, and most

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101

**Figure 3.** T cells modulate the bone and hematopoietic system after activation:. Antigen primed T cells, inside the BM, produce cytokines which can act in both, hematopoiesis and bone remodeling. Depending on the stimuli, each of the two systems can receive positive or negative signals. In the case exemplified here, positive signals for osteolytic disease and myeloid cell expansion and maturation is shown. On this case, T cell help metastasis establishment and boost hematopoiesis. (dark green : mature myeloid cells, light green: immature myeloid cells, Blue: T cells, brown: dendritic

We are deeply indebted with Romulo G. Galvani, who kindly did all the schematics presented

important, to which extend, is still a matter of debate.

cells, pink: osteoclas, red: tumor cells, pruple: osteoblast.

**Acknowledgements**

in this chapter

The localization of active T cells within the marrow cavity coincides with the "proliferative niche" of adult HSC, or the perivascular niche. In fact, the evidences favor a T cell function on the proliferative/differentiative phase of myelopoiesis and not on stem cell maintenance, as stated above.

Hematopoietic stem cells are supported and regulated by stromal cells covering the inner surface of bones, or the endosteum. The endosteum also supports bone remodeling and osteoblasts are present there. The subendosteal region harbors pre-osteoblast, reticular cells and mesenchymal stem cells, with higher hematopoietic supporting role, characterizing different niches involved in different activities that might be cross-regulated somehow.

Activated T cells are able to interact with hematopoietic system, apparently on the proliferative niche, and this is in close contact with the bone remodeling system. When activated, BM T cells can increase osteoclastogenesis and this will favor, in case of a bone metastatic disease, the establishment of bone colonization by the malignant cell. Curiously, metastatic cells in the BM use the same niche as hematopoietic progenitor cells [77] and might well be regulated by antigen specific activated T cells, which are able to prepare the pre-metastatic niche and interfere with the hematopoietic niche. At one side, increase in osteoclasts numbers might be the result of increased proliferation of myeloid progenitors, but it also result from direct T cell signaling to stimulate osteoclast differentiation (figure 3).

Whether or not alterations on bone remodeling are directly linked to alterations in hemato‐ poiesis and vice-versa, and the dependency on the T cell adaptive immune response is still a theme of debate.

Studies in germ free mice might elucidate the subject, since the absence of intentional stimu‐ lation is not synonymous of no stimulation making it difficult to address the immune regula‐ tion of blood and bone.

It is now believed that the commensal microbiota plays an important role on basically every system related to immune response. Very recently, it was shown that Germ Free mice are cytopenic not only in the peripheral blood, but also in the bone marrow. The cytopenia is reversed after intestinal colonization with commensal bacteria [78] and this provide an optimal tune to the immune system to fight infection. On the other hand, Germ Free mice were recently shown to be osteopetrotic and this is reversed by colonization with commensal microbiota [79]. Moreover, in this report, the number and activation state of immune cells was analyzed and in the BM, the number of T cells was diminished in GF mice and the frequency of osteoclast precursors was also deficient. These are in accordance with the view that in the steady state, recognition of antigen by T cells tune the bone remodelling system and this might be related to hematopoietic activity.

Altogether, we provide evidence that, inside the BM there are at least two co-existing systemsbone and hematopoietic-which can be regulated by T cells as they bring messages from the periphery to the BM, resulting in hematopoietic/cancer niche and bone remodeling regulation (figure 3). Whether or not, regulation of one system interferes with the other one, and most important, to which extend, is still a matter of debate.

**Figure 3.** T cells modulate the bone and hematopoietic system after activation:. Antigen primed T cells, inside the BM, produce cytokines which can act in both, hematopoiesis and bone remodeling. Depending on the stimuli, each of the two systems can receive positive or negative signals. In the case exemplified here, positive signals for osteolytic disease and myeloid cell expansion and maturation is shown. On this case, T cell help metastasis establishment and boost hematopoiesis. (dark green : mature myeloid cells, light green: immature myeloid cells, Blue: T cells, brown: dendritic cells, pink: osteoclas, red: tumor cells, pruple: osteoblast.

#### **Acknowledgements**

as "normal" hematopoiesis, which should be an antigen independent activity, is already the result of the adaptive immune response, which, in fact, need the innate immune cells to operate!

The localization of active T cells within the marrow cavity coincides with the "proliferative niche" of adult HSC, or the perivascular niche. In fact, the evidences favor a T cell function on the proliferative/differentiative phase of myelopoiesis and not on stem cell maintenance, as

Hematopoietic stem cells are supported and regulated by stromal cells covering the inner surface of bones, or the endosteum. The endosteum also supports bone remodeling and osteoblasts are present there. The subendosteal region harbors pre-osteoblast, reticular cells and mesenchymal stem cells, with higher hematopoietic supporting role, characterizing different niches involved in different activities that might be cross-regulated somehow.

Activated T cells are able to interact with hematopoietic system, apparently on the proliferative niche, and this is in close contact with the bone remodeling system. When activated, BM T cells can increase osteoclastogenesis and this will favor, in case of a bone metastatic disease, the establishment of bone colonization by the malignant cell. Curiously, metastatic cells in the BM use the same niche as hematopoietic progenitor cells [77] and might well be regulated by antigen specific activated T cells, which are able to prepare the pre-metastatic niche and interfere with the hematopoietic niche. At one side, increase in osteoclasts numbers might be the result of increased proliferation of myeloid progenitors, but it also result from direct T cell

Whether or not alterations on bone remodeling are directly linked to alterations in hemato‐ poiesis and vice-versa, and the dependency on the T cell adaptive immune response is still a

Studies in germ free mice might elucidate the subject, since the absence of intentional stimu‐ lation is not synonymous of no stimulation making it difficult to address the immune regula‐

It is now believed that the commensal microbiota plays an important role on basically every system related to immune response. Very recently, it was shown that Germ Free mice are cytopenic not only in the peripheral blood, but also in the bone marrow. The cytopenia is reversed after intestinal colonization with commensal bacteria [78] and this provide an optimal tune to the immune system to fight infection. On the other hand, Germ Free mice were recently shown to be osteopetrotic and this is reversed by colonization with commensal microbiota [79]. Moreover, in this report, the number and activation state of immune cells was analyzed and in the BM, the number of T cells was diminished in GF mice and the frequency of osteoclast precursors was also deficient. These are in accordance with the view that in the steady state, recognition of antigen by T cells tune the bone remodelling system and this might be related

Altogether, we provide evidence that, inside the BM there are at least two co-existing systemsbone and hematopoietic-which can be regulated by T cells as they bring messages from the periphery to the BM, resulting in hematopoietic/cancer niche and bone remodeling regulation

signaling to stimulate osteoclast differentiation (figure 3).

stated above.

100 Adult Stem Cell Niches

theme of debate.

tion of blood and bone.

to hematopoietic activity.

We are deeply indebted with Romulo G. Galvani, who kindly did all the schematics presented in this chapter

#### **Author details**

Adriana Bonomo1,2\*, Ana Carolina Monteiro2 and Alex Balduíno3,4

\*Address all correspondence to: acbonomo@gmail.com

1 Department of Immunology, Federal Universisty of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

72. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐

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103

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ble from: http://www.ncbi.nlm.nih.gov/pubmed/10733497

2 Laboratory of Thymus Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil

3 Technology and Research Center, Veiga de Almeida University, Rio de Janeiro, RJ, Brazil

4 Excellion Biomedical Services Lab, Rio de Janeiro, RJ, Brazil

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72. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?ar‐ tid=3986503&tool=pmcentrez&rendertype=abstract

[7] Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood [Internet]. 2000 Apr 1 [cited 2014 Jul 4];95(7):2284–8. Availa‐ ble from: http://www.ncbi.nlm.nih.gov/pubmed/10733497

**Author details**

102 Adult Stem Cell Niches

de Janeiro, RJ, Brazil

**References**

dertype=abstract

Adriana Bonomo1,2\*, Ana Carolina Monteiro2

\*Address all correspondence to: acbonomo@gmail.com

4 Excellion Biomedical Services Lab, Rio de Janeiro, RJ, Brazil

from: http://www.ncbi.nlm.nih.gov/pubmed/12154378

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and Alex Balduíno3,4

1 Department of Immunology, Federal Universisty of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

2 Laboratory of Thymus Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio

3 Technology and Research Center, Veiga de Almeida University, Rio de Janeiro, RJ, Brazil

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**Section 2**

**Stem Cell Niches and Reproductive Systems**


**Stem Cell Niches and Reproductive Systems**

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**Chapter 5**

**Male stem Cell Niche and Spermatogenesis in the**

A fundamental question in biology is how communication and exchange of short-range signals shape the microenvironment for setting up functional tissues. In all adult tissues and organs harboring stem cells, tissue homeostasis and repair relies on the proper communication of stem cells and their differentiating daughter cells with the local tissue microenvironment that homes them [1, 2]. Stem cell research has made outstanding contributions on the factors that maintain stem cells or drive them to generate differentiated daughter cells. The use of stem cells in the development of cell-based medicine and in repairing malformed, damaged or aging tissues demands a better understanding of stem cells at a molecular level and of how they behave in

The basic principles controlling stem cell self-renewal versus differentiation are strikingly conserved during evolution and their regulatory logic is often very similar among homologous stem cell niches. Since the signaling pathways and their regulatory circuits are highly complex in the mammalian system with significant molecular redundancy, they are often difficult to study. Therefore, using a simpler model system such as the *Drosophila* testis allows us to elucidate the underlying cellular and molecular mechanisms of stem cell maintenance and

The *Drosophila* testis provides an excellent system to study *in vivo* how two closely apposed cell types communicate and coordinate their reciprocal interaction. Recent advances in spermatogenesis have shown that testis morphogenesis is achieved through the physical contact and diffusible signals exchanged between the germline and the somatic cell popula‐ tions [3]. Moreover, the *Drosophila* testis provides a powerful system to study germline-soma

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Drosophila* **testis — A Tale of Germline-Soma**

Additional information is available at the end of the chapter

**Communication**

Fani Papagiannouli

**1. Introduction**

their physiological context.

differentiation in a straightforward way.

http://dx.doi.org/10.5772/58756
