GLIS1介导TIGIT调控胃癌微环境中CD8+ T细胞增殖活化的机制研究

TIGIT is an immunological checkpoint receptor shared by NK and T cells, but its molecular mechanism of inhibiting CD8+ T cell function still needs further investigation. The applicants used single-cell sequencing to map the immune cells in tumor microenvironment of gastric cancer (GC). It was found that TIGIT was highly expressed in the CD8+T exhausted subpopulation and the transcription factor GLIS1 could regulate the transcription of TIGIT. The clinical investigation found that the high expression of TIGIT in GC was correlated with poor prognosis of patients. After knocking down the expression of GLIS1 in vitro, the proliferation of CD8+TIGIT+ T cells and the production of IFN-γ were significantly increased, and the phosphorylation level of VAV1 was up-regulated. Based on previous research, this project proposes a hypothesis that the transcription factor GLIS1 up-regulates TIGIT, inhibits VAV1 phosphorylation, and inhibits PI3K/AKT/NF-kB pathway-mediated activation and proliferation of gastric cancer CD8+TIL. This project will conduct a more comprehensive analysis and verification of the hypothesis from clinical samples, cells, tissues and animals with flow cytometry, ChIP, luciferase reporter assay, animal tumor model, etc. The aim of this project is to restore the proliferation and activation of exhausted CD8+ T cells by targeting TIGIT-based related signaling pathways, and provide a new theoretical basis and experimental basis for immunotherapy of gastric cancer.

TIGIT是NK细胞和T细胞共有的免疫检查点受体，但是其抑制CD8+T细胞功能的分子机制尚未阐述清楚。申请者前期采用单细胞测序绘制胃癌免疫细胞图谱，发现TIGIT在癌组织耗竭性CD8+T细胞亚群中高表达且转录因子GLIS1调控TIGIT转录，临床验证发现TIGIT在胃癌中高表达与患者预后负相关，敲低GLIS1后CD8+T细胞增殖和IFN-γ分泌增加，VAV1磷酸化水平上调。基于前期基础，本项目提出假设：转录因子GLIS1上调TIGIT表达，抑制VAV1磷酸化，调控PI3K/AKT/NF-κB通路介导胃癌微环境中CD8+T细胞的增殖及活化。本项目拟采用流式细胞术、ChIP、荧光素酶报告实验、小鼠成瘤等技术，从临床样本、细胞、组织、动物层面进行较全面的解析与验证。预期实现通过靶向以TIGIT为基础的相关信号通路，恢复耗竭性CD8+T细胞增殖与活化能力，为胃癌免疫治疗提供新的理论基础和实验依据。

目前随着PD1单抗和CTLA4单抗的应用，肿瘤免疫治疗虽然有了较大的进步，但是大多数实体肿瘤患者仍未能临床获益。因此寻找新的有效免疫检查点及导致CD8+TIL耗竭的新机制对肿瘤治疗有重要的临床意义。免疫检查点TIGIT 和PD1在不同免疫细胞中包括 T 细胞， NK 细胞中发挥免疫抑制功能的现象，为阐明TIGIT 和PD1介导肿瘤微环境中 CD8+ T细胞耗竭的具体机制。本课题组通过采用多种肿瘤模型研究TIGIT和PD1的转录调控及促进CD8+TIL耗竭机制。结果表明：1）TIGIT,PD1和转录因子GLIS1在癌组织耗竭性CD8+T细胞亚群中高表达。2）转录因子GLIS1参与TIGIT和PD1转录调控，3）敲低GLIS1后CD8+T细胞增殖和促炎因子分泌增加，4）TIGIT通过PI3K/AKT/NF-κB 通路抑制CD8+TIL的抗肿瘤功能，5）GLIS1通过转录调节SGK1-STAT1-PD1途径促进肿瘤微环境中CD8+TIL细胞耗竭，6）PDX模型前瞻性验证了在CD8+T细胞中敲低GLIS1协同免疫检查点阻滞对起到明显抑制肿瘤的作用。