Ectopic HMSH2 Molecule Promotes In Vitro Cytotoxicity Of ??T Cells Against Lung Cancer Cells

Human ??T cells,which have many characteristics of natural immune cells,are a special subgroup of T lymphocytes.??T cells are considered as a bridge that links innate immunity and adaptive immunity and they play an important role in the process of immune surveillance and anti-tumor immune response.Because of their major histocompatibility complex(MHC)-unrestricted manner to recognize broad tumor associated antigens,mechanism of prompt activation effect,potent cytotoxic effect and secretion of multiple anti-tumor cytokines,??T cells attract much attention in immunotherapy for tumors.??T cells can directly recognize a variety of antigen molecules in which the most studied is the small molecule phosphoantigens.Adoptive cell therapy using Vy9V82 T cells which are expanded by phosphoantigens had long been entered into phase I/II clinical trials.Besides,y8T cells can also recognize protein ligands that are stress-induced,abnormally expressed on the cellular surface.The preliminary results of our research group showed that human MutS homologue 2(hMSH2)was a tumor-associated protein antigen recognized by y8T cells.hMSH2 molecule,a critical component of DNA mismatch repair system,is mainly synthesized in the cytoplasm and transported into the nucleus via the formation of heterodimer with hMSH3 or hMSH6 molecule,the heterodimer that contains hMSH2 molecule contributes to the restoration of impaired or mismatched DNA.Defects of hMSH2 or functional abnormalities of hMSH2 protein are often detected in the process of tumorigenesis.Previously,with a complementary determining region 3 of T cell receptor ? chain(CDR3?)peptide-based affinity screening system,our research group identified hMSH2 as a putative tumor-associated antigen recognized by V?2 TCR.Further research showed that hMSH2 molecule exhibited extensive,varying level of ectopic expression on the membrane surface of many tumor cells.Besides of the condition of tumorigenesis,ectopic expression of hMSH2 molecule on the cellular membrane could also be induced by virus infection or oxidative stress.Ectopic expression of hMSH2 molecule could be dually recognized by TCRy5 and natural killer group 2 member D(NKG2D),which was involved in mediating cytotoxicity or clearance of target cells by y8T cells.However,currently the related mechanism of ??T cells killing tumor cells mediated by ectopically expressed hMSH2 molecule remains unclear.Clarifying the mechanism on cytotoxicity of ??T cells against tumor cells mediated by hMSH2 molecule may contribute to furtherly revealing the important role of ??T cells in immune surveillance of tumors and in anti-tumor immunity.Moreover,investigating the effect of hMSH2 molecule on promoting cytotoxicity of ??T cells is of great significance for optimizing the adoptive immunotherapy strategy based on ??T cells.In this study,we explored the mechanism of hMSH2 molecule that ectopically expressed on the cellular membrane on mediating cytotoxicity of ??T cells against lung cancer cells and the effect of hMSH2 in promoting cytotoxicity of ??T cells through construction of a lung cancer cell model that overexpressed hMSH2 molecule.First of all,we detected the level of ectopically expressed hMSH2 molecule on the cellular membrane of four commonly used lung cancer cell lines(NCI-H520,GLC-82,NCI-1703 and NCI-H446)and human embryonic lung fibroblast(MRC-5)as a control.The result showed that the expression of hMSH2 molecule on the cellular membrane of different lung cancer cell lines varied a lot.NCI-H520 cells which had lower level of hMSH2 molecule on the cellular membrane were selected as the lung cancer cell model to overexpress hMSH2 molecule.The constructed recombinant vector cFugw-hMSH2 was transfected into NCI-H520 cells to transiently express hMSH2-EGFP fusion protein.The results showed that the level of hMSH2 molecule on the surface of NCI-H520 cells was significantly increased after transfection,and the proportion of positive cells increased from 12.67±0.80%to 62.53±8.22%.Results of cytotoxicity assay in vitro showed that increased level of ectopic hMSH2 molecule on the cellular membrane of NCI-H520 cells could effectively enhance the cytotoxic activity of ??T cells.While detecting the level of activation and cytotoxicity associated effector molecules of ??T cells,we found that the level of CD69 and CD 107a which are activation associated effector molecules on ??T cells were significantly increased and the secretion of anti-tumor cytokines:IFN-y and TNF-a were enhanced after interaction with NCI-H520 cells overexpressing hMSH2 molecule on the cellular membrene.These results suggested that hMSH2 molecule might promote the cytotoxic activity of ??T cells against lung cancer cell NCI-H520 in vitro through the path of promoting the activation and degranulation of ??T cells and the secretion of anti-tumor cytokines:IFN-y and TNF-a.In addition,we also found that the cytotoxic activity of ??T cells against NCI-H520 cells was enhanced after pretreating NCI-H520 cells with zoledronic acid(ZOL,a small phosphoantigen molecule)in a dose-dependent manner.Meanwhile,in those groups in which NCI-H520 cells were pretreated with identical concentration of zoledronic acid,the cytotoxicity efficiency of y8T cells against NCI-H520 cells with up-regulated ectopic expression of hMSH2 molecule was significantly higher than those against wild type NCI-H520 cells.Thus,there existed a synergistic effect between hMSH2 molecule and zoledronic acid on the aspect of promoting cytotoxicity of ??T cells against lung cancer cell line NCI-H520 cells,and they might be synergistically used in anti-tumor immunotherapy.In summary,our main conclusions in this study include:1.Ectopic expression of hMSH2 molecule on the cellular membrane contributed to mediating the cytotoxicity of y8T cells against lung cancer cells and the ectopic expression of hMSH2 molecule was positively correlated with the cytotoxic activity of ??T cells;2.The mechanism of hMSH2 molecule-mediated cytotoxicity of y8T cells against lung cancer cells was probably related to promoting activation of y8T cells and secretion of anti-tumor cytokines IFN-y and TNF-a;3.There existed a synergistic effect between hMSH2 molecule and zoledronic acid on the aspect of promoting cytotoxicity of y8T cells against lung cancer cells in vitro.