**3. Immunological risks caused by asbestos fibers**

As shown in **Figure 1**, the most important and critical complications that arise in asbestosexposed patients concern the development of malignancies, such as lung cancer and MM [25– 29]. Of course, asbestos fibers possess carcinogenic-related activities, such as oxygen stress caused by iron in the asbestos fibers, frustrated macrophages incapable of phagocytosing asbestos fibers, chromosome tangling, and the absorption of other carcinogenic substances inhaled in the lung, such as materials from tobacco smoke and other air pollutants [34–36]. However, given the long latency period that precedes the onset of MM following initial exposure to asbestos, it was considered that asbestos fibers cause alterations in antitumor immunity by recurrent and chronic encounters with various immune cells at the lung and related lymph nodes.

As shown in **Table 2**, our findings show altered immune cell function and manifestations from experimental settings as well as PBMC derived from pleural plaque and MM [11, 12, 15, 16, 80, 81].



CXCR3, CXC chemokine receptor 3; HD, healthy donor; IFN, interferon; IL, interleukin; MLR, mixed lymphocyte reaction; MM, malignant mesothelioma; NK, natural killer; PP, pleural plaque; TGF, transforming growth factor; SMAD, vertebrate homologues of *Sma* and *Mad* [Drosophila protein; mothers against decapentaplegic (MAD) and the Caenorhabditis elegans protein SMA (from the sma gene for small body size)]; and Th1, T helper.

**Table 2.** Immunological risks caused by asbestos fibers.

#### **3.1. NK cells**

Regarding NK cells, cytotoxicity was reduced in peripheral NK cells from pleural plaque (PP) and MM, in NK cells from HD cultured in vitro with asbestos fiber, and in a human NK cell line continuously exposed to asbestos [82]. Additionally, the expression of various NK cell activating receptors, such as NKG2D, 2B4, and NKp46, was reduced in a human NK cell line cultured continuously with asbestos, in freshly isolated NK cells from HD cultured in vitro with asbestos, and in fresh NK cells from PP and MM [82, 83]. Among these receptors, NKp46 was thought to be an important marker for impaired function of NK cells exposed to asbestos. Moreover, reduced cytotoxicity in NK cells exposed to asbestos was accompanied with reduced phosphorylation of extracellularly regulated kinases (ERK) 1 and 2 and reduced degranulation of perforin and granzyme B, which are the killing small molecules secreted from NK cells [82, 83].

#### **3.2. Cytotoxic T lymphocytes**

Other types of cytokilling immune cells, CTLs, are also involved and have their functional and cellular properties altered by asbestos exposure. From in vitro analyses using peripheral CTLs in a mixed lymphocyte reaction (MLR), it was found that differentiation and proliferation of CD8+ naïve T cells were disturbed by the presence of cocultured asbestos fibers with decreased expression of killing small molecules, such as granzyme B and interferon γ (IFNγ) [84]. Moreover, alteration of killing molecules, as well as the phenotype of CD8+ cells, was mani‐ fested by CD45RA as the marker of effector/memory T cells. Freshly isolated CD8+ cells derived from asbestos-exposed patients, such as PP and MM, showed a higher predominance of CD45RA-negative cells compared with HD [85]. However, the cytokilling activity differed between isolated and in vitro*-*stimulated CD8+ cells. CD8+ cells from PP and MM revealed an increase in the number of perforin-positive cells; however, after in vitro stimulation, only CD8+ cells from MM showed a decrease in the perforin-positive cell population when subtracted from the unstimulated base line [85].

These findings indicated that asbestos exposure caused dysfunction of CTLs, while specific cell functions differed depending on disease status, for example, PP patients do not carry any malignant tumors, whereas MM patients suffer from mesothelioma. However, the impact of asbestos fibers on CTLs is considered to involve a reduction of tumor immunity, as we showed in NK cells mentioned above [84, 85].

#### **3.3. Th 1 cells**

Asbestos fibers are also known to modify Th1 cells. We developed continuously exposed sublines using a cell line model. The cDNA microarray data were examined of the original cell line, which has had no contact with asbestos fibers, and six independently established sublines, which were continuously exposed to asbestos fibers for more than 8 months using an asbestos concentration that did not induce apoptosis in more than half of the cells by transient exposure. The microarray showed a decrease in IFNγ and related molecules, such as IFN regulatory factor 9 (IRF9) and IFN-stimulating gene factor-3 (ISGF3), in addition to a decrease in CXC chemokine receptor 3 (CXCR3), which is regulated by IRF9 [86].

CXCR3 is important in antitumor immunity to summon IFNγ-positive tumor antigen recog‐ nizing Th1 cells to the tumor. Thus, the asbestos-induced reduction of CXCR3 and IFNγ seems to cause a reduction of antitumor immunity in asbestos-exposed patients. As we assumed, examination of freshly isolated CD4+ cells from HD stimulated in vitro and cocultured with asbestos fibers as well as peripheral CD4+ cells from PP and MM revealed a decrease in the cell surface expression of CXCR3 in addition to a decrease in the number of intracellular IFNγpositive cells [87].

Taken together, one of the immunological risks resulting from asbestos exposure concerns a reduction of Th1-type T cell–derived antitumor immunity.

### **3.4. Treg cells**

Treg cells are important in antitumor immunity. If the function and number of Treg cells are enhanced, immune cells responding to tumor antigen show suppressed function, which causes a reduction of antitumor immunity [75, 76].

Our cell line model continuously exposed to asbestos fibers using MT-2, a human T-lympho‐ tropic virus type 1, which causes adult T cell leukemia/lymphoma, showed excess production of transforming growth factor (TGF) β and IL-10, typical soluble factors examined to reveal the function of Treg cells [88, 89]. Overproduction of IL-10 is regulated by the Src-family receptor and is used by the IL-10 receptor via autocrine mechanisms, which then causes activation of the signal transducer and activator of transcription 3 (STAT 3) and upregulation of antiapoptotic molecule Bcl-2 located downstream of STAT3 [88]. Continuously exposed sublines acquire resistance to apoptosis induced via transient exposure to asbestos [88]. Furthermore, overproduction of TGFβ induces resistance to TGFβ-induced growth inhibition in continuously exposed sublines with phosphorylation of p38, one of the signaling molecules in the mitogen-activated protein kinase (MAPK) signaling pathway, as well as phosphoryla‐ tion of SMAD3 [SMAD; vertebrate homologues of *Sma* and *Mad* [Drosophila protein, mothers against decapentaplegic (MAD) and the Caenorhabditis elegans protein SMA (from the sma gene for small body size)] [89].

In addition to the two aforementioned typical soluble factors, continuous exposure of MT-2 sublines to asbestos resulted in markedly higher suppressive activity when mixed with cultures of CD4+ responder cells activated with anti-CD3 antibody and autologous peripheral blood monocyte-derived dendritic cells compared with the original MT-2 cell line, which has had no contact with asbestos [90].

Taken together, exposure to asbestos results in enhanced Treg function, which is manifested by a reduction of antitumor immunity [11, 12, 15, 16, 80, 81].

#### **3.5. Risks of asbestos on antitumor immunity**

As mentioned above and shown in **Table 2**, all of the examined effects of asbestos on NK cells, CTLs, Th1, and Treg cells indicate that asbestos exposure can cause a reduction of antitumor immunity. These findings are considerable and the risks associated with asbestos exposure may be used as early detection markers for the occurrence of asbestos-induced malignancies. Additionally, the ability to mitigate the observed reduction of antitumor immunity through the use of chemopreventive substances derived from foods or plants may be an important strategy in the treatment of high-risk groups exposed to asbestos, such as residents who have a history of living near factories handling asbestos and workers in the building demolition and rubble processing fields.
