**3. CD4+ T cells**

38 Malignant Mesothelioma

cytotoxicity.

**asbestos** 

asbestos caused an impairment in cytotoxicity of NK cells with decreases in NK cellactivating receptors. The decrease in NKG2D caused the low level of signal transduction,

**2.4. Low cytotoxicity and low NKp46 level in NK cells of mesothelioma patients** 

The result of the experiment using the human NK cell line described above suggested that inhaled asbestos might cause impairment in cytotoxicity with altered expression of NK cellactivating receptors, and that PB-NK cells in patients with malignant mesothelioma might show a similar impairment in cytotoxicity to YT-CB cells. Therefore, peripheral blood mononuclear cells (PBMCs) prepared from the blood of patients with mesothelioma were examined for cytotoxicity against K562 cells and expression levels of NKG2D, 2B4, and NKp46 in NK cells. The cytotoxicity of PBMCs against K562 in the tree different ratios of effector and targets was measured for each individual, and expressed as the percentage of specific lysis. To evaluate cytotoxicity per given number of NK cells, The number of NK

PBMCs and the number of PBMCs dispensed to each well of the culture plate for cytotoxic reaction. A linear regression line with the percentage of specific lysis and number of NK cells, in which the formula is *[percentage of specific lysis] = A [number of NK cells] + B,* was determined by using the three sets of percentage of specific lysis and number of NK cells for each individual. Finally, the percentages of specific lysis per 5000 NK cells from each individual were calculated from these regression lines and compared with those of healthy volunteers. Mesothelioma patients showed significantly lower cytotoxicity than healthy volunteers (Nishimura et al., 2009b). However, unlike YT-CB5, NK cells in patients with mesothelioma did not show a decrease in expression level of NKG2D or 2B4, whereas a decrease in NKp46 was observed in those NK cells. Thus, although NK cells in the peripheral blood of patients with malignant mesothelioma were not of the same character as YT-CB5, they also showed alteration in the expression of one of the NK-cell activating receptors, albeit a different one namely NKp46, with low expression of NKp46, and low

**2.5. Low cytotoxicity of NK cells with low NKp46 in the culture of PBMCs with** 

As described above, NK cells in patients with malignant mesothelioma showed low cytotoxicity with low expression of NKp46, which was not found in YT-CB5. Therefore, to examine whether asbestos exposure causes such a decrease in NKp46 on NK cells, we performed a different experiment in which PBMCs from healthy volunteers were cultured in IL-2-supplemented media with chrysotile B at 5 µg/ml, and after 7 days cells were harvested and examined for expression levels of NKG2D, 2B4, and NKp46 in CD3-

NK cells by flow cytometry. IL-2 is a representative cytokine that induces proliferation and activation of NK cells and is commonly used to culture these cells. The results showed no decreases in cell-surface expression of NKG2D and 2B4 in NK cells derived from the culture

CD56+ NK cells in

CD56+

followed by a decrease in degranulation, in the asbestos-exposed subline of cells.

cells in the cytotoxic reaction was calculated from the percentage of CD3-

What is the function of CD4+ T cells in regard to an appropriate immune response? CD4+ T cells, Th, contribute to various kinds of responses in innate and acquired immunity, including activation of NK cells, macrophages and dendritic cells (DC), as well as induction of antibody production and CTL development (Monney et al., 2002; Parker, 1993; Smith et al., 2004; Vivier et al., 2008). The production of various cytokines, including IL-2, IL-4, IL-5, IL-10, IL-17, IFN-γ, and TNF-α, by Th cells is one of the reasons for the multiple contributions of those cells to immune function (Fig. 5).

**Figure 5.** Cytokine-mediated control of immune functions and chemokine-mediated control of cell migration. The various kinds of cytokines produced by Th cells allow them to contribute widely to functions of immune competent cells (top). Concentration gradient of chemokines and expression of chemokine receptors play a key role in appropriate cell migration (bottom).

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 controlled by chemokines and chemokine receptors.

Effect of Asbestos on Anti-Tumor Immunity

and Immunological Alteration in Patients with Malignant Mesothelioma 41

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

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

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.,

response as well as cell migration.

**asbestos** 
