br Acknowledgments br Introduction Human innate immune syste

Acknowledgments
Introduction Human innate immune system provides first line of defense against multiple viral or bacterial attacks, and provides critical surveillance against oncogenic development. Evidences show that individuals with primary immunodeficiency or induced immunosuppression during organ or cell transplantation showed higher risk for tumor development [1]. Alternately, impaired function of innate immune cells such as natural killer (NK) cells, subset of αβ T cells, and γδ T cells leads to increased susceptibility of the host for tumor growth [2], [3]. Among these innate immune cells, γδ T cells (Vγ2Vδ2 subset) are of particular interest due to their dual role in the immune system for bridging the gap between the innate and adaptive immunity, and have been demonstrated critical anti-tumor activities [4], [5], [6]. Due to the robust metabolic activities in tumor cells, specific diltiazem hydrochloride sale such as MHC class-I chain-related molecules MICA/B were typically found to be highly expressed in most tumor cells compared to normal healthy cells [7]. These antigens were specifically recognized by the Vγ2Vδ2 T cells and were proposed to enhance the cytotoxic activity of Vγ2Vδ2 T cells towards a variety of tumor cells [8], [9], [10], [11]. The activating receptors, NKG2D, serves as one of the most important receptors present in Vγ2Vδ2 T cells and mediate the recognition process to eliminate the tumor cells. This NKG2D molecule not only interacts to the receptor MICA/B, but also interacts with molecules such as ULBPs [12]. Emerging T cell-based adaptive immunotherapy is under consideration for various cancers as a therapeutic regimen [13]. In the adoptive therapy, T cells are activated, expanded ex vivo and reinjected into the patients with tumors [14], [15]. Adoptive T-cell therapy in renal cancer patients showed no adverse events, and 3 of 5 patients showed slower tumor progression. Patients documented with a positive response showed an increased number of Vγ2Vδ2 T cells in the peripheral blood and a strong in vitro response to phosphoantigen stimulation [14]. Various trials show promise for development of autologous Vγ2Vδ2 T cell therapies in eligible patients. However, for ovarian cancer, there is currently no effective immunotherapy. Interestingly, chemotherapeutic agents were shown to induce immunogenic tumor cell death, which is crucial for tumor eradication and long-term protection against relapse. Moreover, Vγ2Vδ2 T cells were recruited to the tumor bed after immunogenic chemotherapy and appear to be contributors to the efficacy of chemotherapy [16]. So, developing a combination therapy using chemotherapeutic reagent and Vγ2Vδ2 T cells will be a valuable option to be tested. The Vγ2Vδ2 T cells induce cytotoxicity in many ovarian tumor cells via induction of apoptosis [17]. However, some of the ovarian tumor cells evade the apoptosis process and became resistant towards Vγ2Vδ2 T cell-mediated cytotoxicity. These resistant cell lines (such as A2780) showed slower proliferation compared to the sensitive cell line (such as OV4); interestingly, we found that the resistant cell line has reduced expression of MICA [17]. We proposed that the tumor cells may evade the Vγ2Vδ2 T cell cytotoxicity by down-regulating their MICA expression and at the same time enter into a dormancy stage, in which their proliferation was slowed down. In the current study, we further investigated the molecular mechanisms involved in the immune escape process. It has been shown that genotoxic stress or inhibitors of DNA-replication could up-regulate the expression of NKG2D ligand through activation of ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) protein kinase pathway in human fibroblast and in mouse tumor cell lines, which led to enhance cytotoxic lysis by NK cells [18], [19]. ATM and ATR are activated in response to DNA damage, oxidative stress, and replication stress resulting in apoptosis or cell cycle arrest. After activation ATM phosphorylates Chk2, and ATR phosphorylates Chk1 to start a cascade of downstream signaling events [20]. Activated Chk1 and Chk2 phosphorylate Cdc25 phosphatases, to inhibit their function, and the cells delay progression though the cell cycle [20]. After activation ATR and ATM also phosphorylate H2A variant H2AX at Ser-139 (γH2AX) at the damage sites, or where chromosomes are fragmented by oxidative stress [21]. The γH2AX has been used as a marker for DNA damage, oxidative stress, and replication stress. It was also shown that inhibition of ATM pathway by using synthetic inhibitor such as KU-55933 suppressed cell proliferation and induced apoptosis [22]. In this study, we examined whether the ATM and ATR protein kinases play a role in Vγ2Vδ2 T cell-mediated recognition of ovarian cancer cells. We found that treatment of ovarian cancer cells with Vγ2Vδ2 T cells results in down regulation of ATR and ATM signal transduction in resistant cells, but remain unchanged in sensitive cells. When we treated the cells with Vγ2Vδ2 T cells along with drugs activating ATM pathway, it resulted a significant increase in cytotoxicity of tumor cells. Thus, ATM–Chk2 signal transduction plays a critical role in regulating tumor survival in ovarian cancer upon Vγ2Vδ2 T cell treatment.