肿瘤相关内源性新蛋白质配体hMSH2介导Vγ9δ2T细胞杀伤肺癌的作用及机制

Human MutS homologue 2 (hMSH2),a novel tumor-associated endogenous protein ligand recognized by human Vγ9δ2 T cells that has been identified and characterized in our previous study,can promote the cytotoxicity of Vγ9δ2 T cells against ectopic-membrane-hMSH2-expressed tumor cells.However, the underlying molecular mechanisms have not been elucidated so far.In previous work,we found that monoclonal antibody expanded-human Vγ9δ2 T cells could significantly kill human lung cancer cells in vitro.Furthermore,we observed an unusual ectopic membrane expression of hMSH2 on lung cancer cell line NCI-H520.Based on these results, we speculated that ectopic-expressed hMSH2 on lung cancer cells should contribute to the Vγ9δ2 T cell-mediated cytotoxicity towards targets.Data obtained from our pilot studies confirmed the unusual membrane location of hMSH2 in lung cancer cells and the involvement of hMSH2 in the cytolysis of lung cancer cells by Vγ9δ2 T cells.The present study aims to illustrate the specific pathways(i.e.granule exocytosis mediated by perforin/granzymes and the death receptor-mediated Fas/Fas ligand system) and critical factors(e.g.soluable molecules such as cytokines and intracellular adhension molecules,cell contact ,phosphoantigen costimulation,etc) which involve in the hMSH2-enhanced-killing by Vγ9δ2 T cells in anti-lung cancer immunity, by functional analyses on a RNAi-hMSH2 gene knockdown lung cancer cell model and oxidative stress-induced overexpression lung cancer cell model,combined with specific antibody blocking and use of chemical inhibitors.Our study will help to clarify the pattern and the molecular mechanisms by which hMSH2 enhances the cytotoxic effects of Vγ9δ2 T cells against lung cancer cells,to illustrate the relevance of its ectopic expression with lung cancer stages,prognosis, and γδT cell distribution,to reveal its role in Vγ9δ2 T cell-mediated innate lung cancer immune surveillance,and help to generate and optimize Vγ9δ2T cell-based lung cancer therapeutic immunotherapies in future.

Vγ9δ2 T细胞在体内外显著杀伤肺癌细胞，但杀伤机制不明。hMSH2是我们前期发现为Vγ9δ2T细胞识别的新肿瘤相关蛋白质配体，我们新近发现并证实：hMSH2在肺癌有较高水平膜异位表达，异位表达的膜hMSH2能显著增强Vγ9δ2T细胞对肺癌细胞杀伤效应。我们推测hMSH2介导Vγ9δ2T细胞对肺癌识别杀伤。以此为基础，本项目拟：结合hMSH2基因沉默及诱导过表达肺癌细胞模型，研究①hMSH2介导Vγ9δ2T细胞杀伤肺癌的分子效应(活化、增殖、CK分泌等)；②hMSH2介导杀伤的途径(Fas-FasL、穿孔素/颗粒酶、TRAIL等)、关键因子（IFN-r、TNF-a等）及作用机制；③异位表达hMSH2与肺癌分期预后及γδT细胞分布的相关性分析。所得结果将有助于揭示hMSH2调控Vγ9δ2 T 细胞杀伤肺癌的作用机制及肺癌特异γδT细胞的表型与功能，并为建立新型肺癌免疫诊断及预防策略提供依据

研究背景：肿瘤相关内源性新蛋白质配体hMSH2介导Vγ9Vδ2 T细胞在体内外识别杀伤肺癌的机制不明。.研究内容：结合hMSH2基因敲除及过表达肺癌细胞模型，研究了①hMSH2介导Vγ9Vδ2T细胞杀伤肺癌的分子效应(活化、增殖、CK分泌等)；②hMSH2介导杀伤的途径（Fas-FasL、穿孔素/颗粒酶、TRAIL等）、关键因子（IFN-γ、TNF-a等）及作用机制；③hMSH2与磷酸化抗原在Vγ9Vδ2 T细胞介导的肺癌免疫中的协同效应；④异位表达的hMSH2与肺癌类型、分期预后、外周血及肿瘤组织中γδ T细胞分布的相关性分析。.研究结果：①采用流式细胞术（FCM）、激光共聚焦显微镜术、免疫组化染色等方法证实了hMSH2在肺癌细胞系NCI-H520及临床肺癌组织标本中的异常亚细胞分布；②利用siRNA-hMSH2基因沉默肺癌细胞模型、Fugw-EGFP-hMSH2瞬时转染及慢病毒感染过表达肺癌细胞模型、hMSH2过表达裸鼠肺癌体内治疗模型在体内外证实肺癌细胞表面异位表达的膜hMSH2参与介导Vγ9Vδ2T细胞对肺癌的识别杀伤，并与磷酸化抗原存在协同效应；③明确了hMSH2活化Vγ9Vδ2 T细胞识别杀伤的主要效应分子，以及hMSH2过表达驱动的IFN-γ和促炎性细胞因子分泌为主的抑癌机制；④完成了hMSH2、hMSH3、hMSH6以及 γδ T细胞的抗原识别受体在肺癌组织中的表达分析，明确了其作为肺癌诊疗预后标志物的可能性；⑤比较分析了肺癌、肺结节患者及健康体检者外周血的TBNK亚群尤其是γδ T 细胞亚群的分布特征、细胞因子分泌谱及自身抗体谱，并首次证实了肺癌血清可溶性hMSH2、hMSH3、hMSH6的表达水平及形式；⑥阐明EBV感染诱导的异位表达的膜hMSH2可经TCR和NKG2D促进Vγ9Vδ 2T细胞对EBV转化B淋巴细胞的杀伤作用。.科学意义：研究结果有助于揭示hMSH2调控Vγ9Vδ2 T 细胞识别、杀伤肺癌细胞的分子效应与作用机制，明确肺癌患者外周血淋巴细胞亚群的分布以及肺癌特异γδ T细胞的表型，并为建立新型的肺癌免疫诊断、治疗预防策略提供线索和依据。