细胞外Calpain激活PD-1/PDL-1通路促进肿瘤细胞免疫逃逸的功能机制

How to activate the immune system and block the negative signal transduction is a currently needed to be solved problem in the study of tumor immunotherapy. Calpain is a family of calcium-dependent intracellular cysteine proteases with a wide range of substrates including various cell skeletal proteins and enzymes involved in intracellular signal transduction. Previous studies showed that calpain mainly plays physiological roles in cytoplasm, while its extracellular function is poorly understood. Our previous study showed a significant inhibitory effect of extracellular calpain1 on proliferation of T lymphocytes and activation. Calpain1 increased the expression of inhibitory molecules PD-1 on the surface of T cells. Accordingly, we put forward the scientific question: whether the calpain is an independent inhibitory molecule in tumor microenvironment? Does its inhibitory effect play an important role in the activation of the downstream PD-1/PDL-1 pathway? In this study, we will analyze the relationship between extracellular expression of calpain and clinical prognosis; we will study the mechanism of calpain on T cell proliferation and killing efficiency with Adoptive transfer model and mouse melanoma model in vivo; through detection of PD-1 methylation in T cell surface in vitro and PD-1KO mice study in vivo, we will investigate the effect of calpain on the PD-1/PDL-1 pathway. All these works would be helpful for providing theoretical basis for the development of tumor immunotherapy.

如何激活机体的免疫系统，阻断负向免疫调控分子的信号转导是肿瘤免疫研究的热点。Calpain作为细胞内的蛋白酶，以往的研究认为其主要在胞内中发挥生理性调节作用，而对胞外Calpain的功能知之甚少。我们前期研究首次显示胞外Calpain1对T细胞的增殖和杀伤效能具有显著抑制作用，并且能促进T细胞表面抑制性分子PD-1的表达。据此，我们提出科学问题： Calpain是否为肿瘤微环境中独立的T细胞抑制分子？其抑制效应是否通过激活下游PD-1/PDL-1通路起作用？本研究将通过分析Calpain胞外表达与临床预后的关系；利用体内过继免疫模型和小鼠黑色素瘤模型确立Calpain抑制T细胞效能的作用；通过检测PD-1甲基化位点的变化和PD-1KO鼠模型研究Calpain促进PD-1/PDL-1通路活化的作用机制，为开发以该分子为核心的肿瘤免疫治疗提供理论依据。

在肿瘤微环境中，肿瘤细胞可通过表达或分泌某些信号分子逃避免疫系统的识别和攻击，而寻找这些信号分子并阻断其通路，促进更多的效应性淋巴细胞参与肿瘤的免疫应答是提高肿瘤免疫治疗的有效手段。本项目以钙激活中性蛋白酶Calpain为核心，以口腔鳞癌为研究模型，首先研究了Calpain2和Calpastatin在肿瘤上皮中的表达情况及与患者临床预后的相关性，结果发现Calpain2和Calpastatin在口腔癌组织中表达升高，其高表达与患者的临床不良预后相关；其次我们发现Calpain1，2体外可抑制T淋巴细胞的增殖效能，Calpain2体内可通过抑制T淋巴细胞的活性促进肿瘤的进展；另外，我们对可能影响Calpain活性的钙连蛋白（Calnexin）、血栓调节蛋白（TM/CD141）也进行了相关的研究，发现TM/CD141在肿瘤组织中的高表达与其更高的分化程度相关，而Calnexin的膜表达与患者的临床预后和肿瘤周围淋巴细胞浸润相关。本研究通过探讨Calpain及其内源性抑制剂Calpastatin在口腔鳞癌中的表达验证以及Calpain与T细胞的相互作用，初步揭示了Calpain在肿瘤微环境中调节T细胞免疫应答的机制，获得相关的应用成果，发表标注SCI 收录论文 5 篇，总 IF 值 31.816 分。