**3.1. Cytokines produced by Th cells and polarization to Th1, Th2, Treg and Th17 cells**

Antigen stimulation, made by a complex of MHC class II and antigen peptide, allows naïve Th cells to proliferate and produce cytokines. Th cells produce various kinds of cytokines, which can be classified according to the role they have in immune functions. TNF-α and IFN-γ stimulate activation of NK cells and exert a promoting effect on the development of CTL, by which they support cell-mediated immunity for elimination of virus-infected or transformed/tumor cells. In contrast, IL-4 supports humoral immunity, in which it stimulates production of immunoglobulin, in particular IgE, by plasma cells differentiated from B cells, and is related to allergy and asthma. In addition, IFN-γ and IL-4 produced by Th cells show a mutual suppression in each other's production. As a result of such suppression, Th cells dominantly producing IFN-γ show less production of IL-4 and vice versa. Therefore, such development of Th cells exhibits a dominant production of cytokinesis, called "polarization", and the cells showing dominant production of IFN-γ and IL-4 are Th1 and Th2 cells, respectively (Liew, 2002). Moreover, part of the Th cells differentiate to regulatory T (Treg) cells, which dominantly produce IL-10 and TGF-β, immune-suppressive cytokines, and play a role in regulation of the immune response (Asseman et al., 1999; Dieckmann et al., 2002; Green et al., 2003; Jonuleit et al., 2002), although a part of Treg occurs naturally without antigen stimulation (Sakaguchi, 2005). It has also been reported that Th cells producing IL-17, called Th17, contribute to autoimmune diseases (Peck & Mellins, 2009). Thus, several cytokines produced by Th cells and subpopulations of polarized Th cells show a characteristic role in immune functions. Therefore, immune status can be estimated by examining the production of these cytokines.

#### **3.2. Chemokine receptors expressed on Th cells**

Although immune competent cells are spread over the body, they must move to the place where they are needed according to their functions, and this also applies to each subpopulation of Th cells. Naïve T cells, both CD4+ and CD8+ cells, have to reach secondary lymphoid organs to check an antigen processed by antigen-presenting cells. In contrast, Th1 and Th2 cells, as effector cells, move to the periphery where they must encounter appropriate partners. Therefore, the expression of receptors for chemokines differs among the subpopulations of Th cells (Sallusto & Lanzavecchia, 2000). Naïve T cells express the chemokine receptor CCR7, by which they acquire responsiveness to its ligands SLC and ELC and can move to secondary lymphoid organs. In contrast, Th1 and Th2 cells lose the expression of CCR7 and acquire the expression of CXCR3 and CCR5 or CCR3 and CCR4, respectively, although these expressions are a little flexible. CXCR3 is expressed on some NK cells, and CXCL10, the ligand of CXCR3, accumulates NK cells in tumors (Qin et al., 1998; Wendel et al., 2008). In addition, mice deficient for CXCL10 show impairment in T cell proliferation, IFN-γ production, contact hyper-sensitivity, a representative Th1 response, and recruitment of CD4+ and CD8+ cells in the periphery (Dufour et al., 2002). On the other hand, CCR3 is expressed at high levels on eosinophils, basophils, and mast cells, the population of cells related to allergy and asthma (Gerber et al., 1997). Thus, the subpopulations of Th cells differ in the expression of chemokine receptors, which is related to their different roles in immune functions, and the assay for expression of chemokine receptors on Th cells allows us to obtain information regarding the balance of the Th1/Th2 response as well as cell migration.

40 Malignant Mesothelioma

**cells** 

controlled by chemokines and chemokine receptors.

**3.2. Chemokine receptors expressed on Th cells** 

In addition, cytokines produced by Th cells can be transported by the blood stream, which allows them to exert an influence on distant cells as well as adjacent cells. Moreover, immune competent cells can migrate widely throughout the body, and Th cells are no exception to this rule. Chemokines, a group of cytokines, play a large role in such cell migration. For example, a chemokine produced in the periphery creates a concentration gradient, by which cells having the receptor for that chemokine can be attracted. Th cell migration is also controlled in a similar manner, in which the expression of receptors for chemokines influences the migration. As described below, our study discovered that the immunological effect of asbestos exposure involved a characteristic alteration in cytokine production and expression of chemokine receptor in Th cells chronically exposed to asbestos, and that Th cells in patients with malignant mesothelioma exhibited characteristics similar to those of asbestos-exposed cells. Before elaborating on these findings, in the next section we will detail the cytokines produced by Th cells and the Th cell migration

**3.1. Cytokines produced by Th cells and polarization to Th1, Th2, Treg and Th17** 

Antigen stimulation, made by a complex of MHC class II and antigen peptide, allows naïve Th cells to proliferate and produce cytokines. Th cells produce various kinds of cytokines, which can be classified according to the role they have in immune functions. TNF-α and IFN-γ stimulate activation of NK cells and exert a promoting effect on the development of CTL, by which they support cell-mediated immunity for elimination of virus-infected or transformed/tumor cells. In contrast, IL-4 supports humoral immunity, in which it stimulates production of immunoglobulin, in particular IgE, by plasma cells differentiated from B cells, and is related to allergy and asthma. In addition, IFN-γ and IL-4 produced by Th cells show a mutual suppression in each other's production. As a result of such suppression, Th cells dominantly producing IFN-γ show less production of IL-4 and vice versa. Therefore, such development of Th cells exhibits a dominant production of cytokinesis, called "polarization", and the cells showing dominant production of IFN-γ and IL-4 are Th1 and Th2 cells, respectively (Liew, 2002). Moreover, part of the Th cells differentiate to regulatory T (Treg) cells, which dominantly produce IL-10 and TGF-β, immune-suppressive cytokines, and play a role in regulation of the immune response (Asseman et al., 1999; Dieckmann et al., 2002; Green et al., 2003; Jonuleit et al., 2002), although a part of Treg occurs naturally without antigen stimulation (Sakaguchi, 2005). It has also been reported that Th cells producing IL-17, called Th17, contribute to autoimmune diseases (Peck & Mellins, 2009). Thus, several cytokines produced by Th cells and subpopulations of polarized Th cells show a characteristic role in immune functions. Therefore, immune status can be estimated by examining the production of these cytokines.

Although immune competent cells are spread over the body, they must move to the place where they are needed according to their functions, and this also applies to each

#### **3.3. Impaired Th1 function of the human T cell line continuously exposed to asbestos**

As described above, many immune functions are under the control of Th cells. Therefore, we examined the effect of asbestos exposure on Th cells. At the beginning of this study, we prepared an in vitro T-cell model of long-term and low-level exposure to chrysotile asbestos using MT-2 cells, a human adult T-cell leukemia virus (HTLV)-1-immortalized human polyclonal cell line, resulting in six sublines exposed to asbestos. These sublines were established from the independent cultures of MT-2 cells with chrysotile asbestos. All of the sublines acquired resistance to asbestos-induced apoptosis after more than eight months of continuous exposure, and were named MT-2Rsts. Those six MT-2Rsts were used for DNA microarray analysis, compared with the original MT-2 cells, named MT-2Org. The analysis clarified statistically significant alterations in expression of 139 genes in MT-2Rsts, greater than twofold changes. To identify genes related to the suppression of anti-tumor immunity, the expression data were processed by the MetaCore Analytical Suite (http://www.genego.com; GeneGo, St. Joseph, MI) to search for deregulated networks and pathways. The results obtained from pathway and network analysis showed downregulation of IFN-γ signalling and CXCR3 expression in MT-2Rsts. As mentioned above, both IFN-γ and CXCR3 are related to Th1 cells. Therefore, we focused on Th1 functions of MT-2Rsts. All MT-2Rsts showed reduction of cell-surface expression of CXCR3, the mRNA level of which also decreased in MT-2Rsts, as assayed by real-time PCR. In addition, MT-2Rsts showed a decrease in production of IFN-γ compared to MT-2Org, as assayed by ELISA. Moreover, the production of CXCL10 also decreased in MT-2Rsts (Maeda et al., 2010). These results indicate that continuous exposure of a human T cell line to asbestos impaired Th1 function, leading to decreases in cell-surface expression of CXCR3 and production of IFN-γ and CXCL10.

Effect of Asbestos on Anti-Tumor Immunity

and Immunological Alteration in Patients with Malignant Mesothelioma 43

attract anti-tumor T cells (Luster & Ravetch, 1987; Dufour et al., 2002; Homey et al., 2002). Moreover, the mesothelioma group showed a tendency for an inverse correlation between the percentage of CD4+CXCR3+ T cells and CXCL10 concentration, in comparison with the plaque and healthy groups. These results indicate that anti-tumor immune function in mesothelioma patients may be in the situation with less recruitment of Th1 cells using

We examined the effect of asbestos exposure on NK-cell and Th-cell functions using human NK- and T-cell lines continuously exposed to asbestos, primary cell cultures with asbestos, and analyses for peripheral blood NK and Th cells in plaque-positive individuals and patients with malignant mesothelioma. The results obtained from these studies indicate that asbestos exposure causes functional alterations in NK and Th cells, decreases in cytotoxicity and expression of NKG2D or NKp46, and decreases in production of IFN-γ and expression of CXCR3, respectively. It is known that NK cell-activating receptors transduce a signal to induce phosphorylation of ERK and JNK, causing degranulation of cytotoxic granules. The results of our study also showed the relationship between the expression level of NKG2D or NKp46, ERK phosphorylation, and cytotoxicity using the NK cell line and peripheral blood NK cells from healthy individuals. Therefore, the asbestos-induced decrease in expression of NKG2D or NKp46 is thought to cause impairment of NK cell-mediated anti-tumor immunity. In addition, IFN-γ is a key cytokine for the Th1 response and CXCR3 is one of the representative chemokine receptors expressed on Th1 cells. Therefore, the asbestos-induced decrease of IFN-γ and CXCR3 in Th cells indicates the decrease of the Th1 response, which contributes to impairment of the immune response to tumors. These findings concerning NK and Th cells indicate that asbestos fibers have the potential to cause impairment of anti-tumor immunity. This is the first demonstration that asbestos exerts an immune suppressive effect, as well as a tumorigenic effect. The immune-suppressive effect of asbestos might contribute to development of malignant mesothelioma in people exposed to asbestos. As described in the introduction, asbestos is known to accumulate in the lung-draining lymph nodes, as well as the lungs, where NK and Th cells might be exposed to asbestos. Furthermore, the results of our studies indicate that several kinds of functional impairment in NK and Th cells observed in experiments of in vitro or ex vivo exposure to asbestos can also be observed in the cells of patients with malignant mesothelioma, although the results include some inconsistencies. NK cells in patients with mesothelioma showed the same decrease in NKp46 as NK cells in the PBMC culture with chrysotile asbestos, and Th cells in plaquepositive individuals and mesothelioma patients showed the same decrease in CXCR3 as the Th cell line and primary Th cells continuously cultured with chrysotile. In addition, it is noteworthy that there is also the consistency of parameters showing no alteration in expression between data from patients and cultures of primary cells. Those parameters are NKG2D and 2B4 for NK cells and CCR5 for Th cells, the altered expressions of which were not found in primary cells either exposed to asbestos or derived from patients with

CXCR3, although the level of its ligand, CXCL10, is high.

**4. Conclusion and discussion** 

#### **3.4. Decreases in CXCR3 and IFN-γ in primary CD4+ T cells exposed to asbestos**

Following the results obtained from the experiment using the MT-2 cell line, we examined the effect of asbestos exposure on human primary CD4+ T cells (Maeda et al., 2011). CD4+ T cells freshly isolated from PBMCs were stimulated with antibodies to CD3 and CD28 and cultured in IL-2-supplemented media for 3 days, and the activated CD4+ T cells were transferred into a new culture plate and cultured with IL-2 for a week. These polyclonally expanded CD4+ T cells were used for culture with chrysotile B asbestos. After 40 days of culture, cell surface CXCR3 expression decreased in a dose-dependent manner. In contrast, the expression of CCR5 varied among all healthy volunteers, and there were no significant changes after culture with chrysotile. In addition, we examined intracellular expression and the mRNA level of IFN-γ in CD4+ T cells exposed to chrysotile B by flow cytometry and realtime PCR. The CD4+ T cells exposed to chrysotile B showed a decrease in IFN-γ mRNA level, for which there was no significant difference, whereas IFN-γ positive cells tended to be reduced in those asbestos-exposed cells. These results indicate that chronic exposure to asbestos caused the decrease in CXCR3 and IFN-γ in CD4+ T cells, in accordance with results obtained from the experiment using the cell line.

#### **3.5. Decrease in CD4+CXCR3+ T cells in patients with mesothelioma**

Finally, we determined whether CD4+ T cells in asbestos-exposed patients showed the same impairment as shown by MT-2Rst sublines and in vitro asbestos-exposed primary CD4+ T cells (Maeda et al., 2011). Individuals positive for pleural plaque and patients with malignant mesothelioma were examined for CXCR3 expression and IFN-γ production in peripheral blood CD4+ T cells. Both plaque-positive individuals and mesothelioma patients showed a significantly lower percentage of CXCR3+ cells in CD4+ T cells than healthy volunteers. In addition, the percentages of CD4+ CXCR3+ T cells in lymphocytes from the plaque and mesothelioma groups were also significantly lower than those of the healthy group, and the mesothelioma group showed the lowest percentage among the three groups. In contrast to CXCR3, the percentage of CCR5+ cells in CD4+ T cells and CD4+ CCR5+ T cells in lymphocytes was not low in the plaque and mesothelioma groups. To examine production of IFN-γ by CD4+ T cells from plaque-positive individuals and mesothelioma patients, CD4+ T cells were stimulated with antibodies to CD3 and CD28, and cells and culture supernatants were harvested and assayed for mRNA and secreted levels of IFN-γ. The CD4+ T cells of mesothelioma patients showed a significantly lower mRNA level of IFNγ than that of plaque-positive individuals or healthy volunteers, whereas the secreted level of IFN-γ did not differ among the three groups. In addition, the concentration of CXCL10 in plasma tended to be higher for the plaque and mesothelioma groups than the healthy group, although there were no significant differences. CXCL10 can be produced by a variety of cells including endothelial cells, fibroblasts and monocytes near a cancerous lesion, to attract anti-tumor T cells (Luster & Ravetch, 1987; Dufour et al., 2002; Homey et al., 2002). Moreover, the mesothelioma group showed a tendency for an inverse correlation between the percentage of CD4+CXCR3+ T cells and CXCL10 concentration, in comparison with the plaque and healthy groups. These results indicate that anti-tumor immune function in mesothelioma patients may be in the situation with less recruitment of Th1 cells using CXCR3, although the level of its ligand, CXCL10, is high.

#### **4. Conclusion and discussion**

42 Malignant Mesothelioma

production of IFN-γ and CXCL10.

obtained from the experiment using the cell line.

**3.5. Decrease in CD4+CXCR3+ T cells in patients with mesothelioma** 

Finally, we determined whether CD4+ T cells in asbestos-exposed patients showed the same impairment as shown by MT-2Rst sublines and in vitro asbestos-exposed primary CD4+ T cells (Maeda et al., 2011). Individuals positive for pleural plaque and patients with malignant mesothelioma were examined for CXCR3 expression and IFN-γ production in peripheral blood CD4+ T cells. Both plaque-positive individuals and mesothelioma patients showed a significantly lower percentage of CXCR3+ cells in CD4+ T cells than healthy volunteers. In addition, the percentages of CD4+ CXCR3+ T cells in lymphocytes from the plaque and mesothelioma groups were also significantly lower than those of the healthy group, and the mesothelioma group showed the lowest percentage among the three groups. In contrast to CXCR3, the percentage of CCR5+ cells in CD4+ T cells and CD4+ CCR5+ T cells in lymphocytes was not low in the plaque and mesothelioma groups. To examine production of IFN-γ by CD4+ T cells from plaque-positive individuals and mesothelioma patients, CD4+ T cells were stimulated with antibodies to CD3 and CD28, and cells and culture supernatants were harvested and assayed for mRNA and secreted levels of IFN-γ. The CD4+ T cells of mesothelioma patients showed a significantly lower mRNA level of IFNγ than that of plaque-positive individuals or healthy volunteers, whereas the secreted level of IFN-γ did not differ among the three groups. In addition, the concentration of CXCL10 in plasma tended to be higher for the plaque and mesothelioma groups than the healthy group, although there were no significant differences. CXCL10 can be produced by a variety of cells including endothelial cells, fibroblasts and monocytes near a cancerous lesion, to

2010). These results indicate that continuous exposure of a human T cell line to asbestos impaired Th1 function, leading to decreases in cell-surface expression of CXCR3 and

**3.4. Decreases in CXCR3 and IFN-γ in primary CD4+ T cells exposed to asbestos** 

Following the results obtained from the experiment using the MT-2 cell line, we examined the effect of asbestos exposure on human primary CD4+ T cells (Maeda et al., 2011). CD4+ T cells freshly isolated from PBMCs were stimulated with antibodies to CD3 and CD28 and cultured in IL-2-supplemented media for 3 days, and the activated CD4+ T cells were transferred into a new culture plate and cultured with IL-2 for a week. These polyclonally expanded CD4+ T cells were used for culture with chrysotile B asbestos. After 40 days of culture, cell surface CXCR3 expression decreased in a dose-dependent manner. In contrast, the expression of CCR5 varied among all healthy volunteers, and there were no significant changes after culture with chrysotile. In addition, we examined intracellular expression and the mRNA level of IFN-γ in CD4+ T cells exposed to chrysotile B by flow cytometry and realtime PCR. The CD4+ T cells exposed to chrysotile B showed a decrease in IFN-γ mRNA level, for which there was no significant difference, whereas IFN-γ positive cells tended to be reduced in those asbestos-exposed cells. These results indicate that chronic exposure to asbestos caused the decrease in CXCR3 and IFN-γ in CD4+ T cells, in accordance with results

We examined the effect of asbestos exposure on NK-cell and Th-cell functions using human NK- and T-cell lines continuously exposed to asbestos, primary cell cultures with asbestos, and analyses for peripheral blood NK and Th cells in plaque-positive individuals and patients with malignant mesothelioma. The results obtained from these studies indicate that asbestos exposure causes functional alterations in NK and Th cells, decreases in cytotoxicity and expression of NKG2D or NKp46, and decreases in production of IFN-γ and expression of CXCR3, respectively. It is known that NK cell-activating receptors transduce a signal to induce phosphorylation of ERK and JNK, causing degranulation of cytotoxic granules. The results of our study also showed the relationship between the expression level of NKG2D or NKp46, ERK phosphorylation, and cytotoxicity using the NK cell line and peripheral blood NK cells from healthy individuals. Therefore, the asbestos-induced decrease in expression of NKG2D or NKp46 is thought to cause impairment of NK cell-mediated anti-tumor immunity. In addition, IFN-γ is a key cytokine for the Th1 response and CXCR3 is one of the representative chemokine receptors expressed on Th1 cells. Therefore, the asbestos-induced decrease of IFN-γ and CXCR3 in Th cells indicates the decrease of the Th1 response, which contributes to impairment of the immune response to tumors. These findings concerning NK and Th cells indicate that asbestos fibers have the potential to cause impairment of anti-tumor immunity. This is the first demonstration that asbestos exerts an immune suppressive effect, as well as a tumorigenic effect. The immune-suppressive effect of asbestos might contribute to development of malignant mesothelioma in people exposed to asbestos. As described in the introduction, asbestos is known to accumulate in the lung-draining lymph nodes, as well as the lungs, where NK and Th cells might be exposed to asbestos. Furthermore, the results of our studies indicate that several kinds of functional impairment in NK and Th cells observed in experiments of in vitro or ex vivo exposure to asbestos can also be observed in the cells of patients with malignant mesothelioma, although the results include some inconsistencies. NK cells in patients with mesothelioma showed the same decrease in NKp46 as NK cells in the PBMC culture with chrysotile asbestos, and Th cells in plaquepositive individuals and mesothelioma patients showed the same decrease in CXCR3 as the Th cell line and primary Th cells continuously cultured with chrysotile. In addition, it is noteworthy that there is also the consistency of parameters showing no alteration in expression between data from patients and cultures of primary cells. Those parameters are NKG2D and 2B4 for NK cells and CCR5 for Th cells, the altered expressions of which were not found in primary cells either exposed to asbestos or derived from patients with mesothelioma. These findings suggest that those characteristic functional alterations of NK and Th cells shown in patients with malignant mesothelioma might be caused by inhaled and accumulated asbestos in the body. Furthermore, they also suggest the possibility that decreases in NKp46 on NK cells and CXCR3 on Th cells might contribute to early diagnosis of malignant mesothelioma as markers to monitor asbestos-related immune suppression. Today, the diagnosis of malignant mesothelioma is dependent on X-ray and CT image analyses, as well as pathohistological analysis, but these procedures involve several problems such as difficulty in obtaining a consistent diagnosis and a risk of radiation exposure or invasiveness. In contrast, the drawing of blood necessary for analysis of immunological markers is safe and easy, and can be performed frequently during a year. Therefore, imaging and pathohistological analyses of malignant mesothelioma combined with immunological analysis for expression of NKp46 and CXCR3 might provide more valuable information for people who are exposed to asbestos and worry about the development of malignant mesothelioma. Further studies regarding the immunological effect of asbestos exposure will contribute to the effective diagnosis and therapy of malignant mesothelioma.

Effect of Asbestos on Anti-Tumor Immunity

and Immunological Alteration in Patients with Malignant Mesothelioma 45

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