胃癌中巨噬细胞通过PVR信号抑制NK细胞杀伤功能的机制研究

The progression of gastric cancer is influenced by the regulation of immune cells in the tumor microenvironment. Understanding the infiltration and regulation of immune cells in the tumor microenvironment will provide a new strategy for immunotherapy of gastric cancer. Our study showed that compared with non-tumor tissues, the infiltration and CD107a and IFN-γ expression of NK cells in gastric cancer tissues were decreased, and the frequency of tumor-infiltrating macrophages was negatively correlated with the proportion of CD107-positive NK cells. In addition, the level of PVR, a ligand of NK-cell immunosuppressive receptor TIGIT, was up-regulated on tumor-infiltrating macrophages. These data suggested that the decreased cytotoxic function of NK cells may be mediated by macrophages in the gastric cancer microenvironment and PVR signal pathway may be involved in this process. Therefore, we collected clinical specimens from patients of gastric cancer and analyzed the immune response characteristics of NK cells and macrophages. Then macrophages were sorted from tissues and co-cultured with purified peripheral NK cells, and anti-PVR or anti-TIGIT blocking antibody was also added in the co-cultured system. Moreover, macrophages induced in vitro were stimulated by low oxygen pressure for investigating the mechanism of PVR up-regulation. Understanding the mechanism of the decreased cytotoxic function of NK cells induced by tumor microenvironment-mediated macrophages will add new evidence for elucidating the forming of immunosuppressive environment, which will provide an important guidance for the immunotherapy of gastric cancer.

胃癌的发生发展受到胃癌微环境中免疫细胞调控的影响，明确参与其中的免疫细胞及其功能调控可为胃癌的免疫治疗提供新思路。课题组研究发现：在胃癌组织中，NK细胞的浸润及杀伤效应分子CD107a和IFN-γ的表达显著下降，而巨噬细胞则与表达CD107a的NK细胞成反比并上调表达NK细胞抑制型受体TIGIT的配体PVR，提示胃癌组织中的巨噬细胞可能通过PVR信号抑制了NK细胞的杀伤功能。因此，本项目拟通过收集临床胃癌病人的标本，检测NK细胞与巨噬细胞在组织中的应答特征；同时分选组织中的巨噬细胞与外周NK细胞共培养，并加入抗PVR或抗TIGIT抗体进行阻断，阐明巨噬细胞抑制NK细胞杀伤功能的机制；随后利用低氧分压刺激诱导分化的巨噬细胞，探讨缺氧对巨噬细胞上调表达PVR的影响。明确胃癌组织中巨噬细胞抑制NK细胞杀伤功能的机制，将为阐明胃癌免疫抑制的机制提供新的实验证据，对胃癌的免疫治疗具有重要的指导意义。

项目背景：胃癌的发生发展受到胃癌微环境中免疫细胞相互作用的影响，阐明参与胃癌微环境的免疫细胞及其功能的调控可为胃癌的免疫治疗提供理论依据。NK细胞是机体抵抗肿瘤发生发展的一类重要免疫细胞。然而，NK细胞在人胃癌中的应答特性、功能调控及其临床意义尚未完全阐明。.研究内容：本项目基于课题组前期关于NK细胞在胃癌组织中的浸润和功能下降及其功能的下降与巨噬细胞密切相关这一发现，拟进一步通过流式细胞术和免疫组化技术分析NK细胞与巨噬细胞在胃癌组织中的应答特征，Kaplan-Meier plot方法评估NK细胞应答水平与胃癌病人总体生存率的关系，细胞共培养技术探讨胃癌组织中的巨噬细胞抑制NK细胞抗肿瘤功能的具体机制。.研究结果：第一，在胃癌组织中，NK细胞的比例和数目显著低于正常组织和外周血，且胃癌浸润的NK细胞比例与肿瘤进展、肿瘤大小、组织侵袭、淋巴结和远端转移成负相关，但与患者的生存时间成正相关。第二，表型分析显示与正常组织中的NK细胞相比，胃癌组织中的NK细胞表达活化型受体与抑制型受体的能力并无显著统计学差异，但功能学鉴定发现胃癌组织中表达IFN-γ和TNF-α的NK细胞水平显著下降，且其下降水平与肿瘤中的巨噬细胞浸润密切相关。第三，胃癌组织中的巨噬细胞可分泌免疫抑制性的细胞因子TGF-β1，进而能够有效抑制NK细胞的抗肿瘤功能，阻断TGF-β1信号能够解除胃癌相关巨噬细胞对NK细胞功能的抑制效果。.结论：该项目阐明了胃癌组织中的巨噬细胞抑制NK细胞抗肿瘤功能的新机制，为临床基于阻断TGF-β1信号的胃癌免疫治疗提供了新思路。