沉默SOCS1的TIL联合Tim-3单抗重塑免疫微环境提高肺癌疗效的机制研究

The incidence and mortality of lung cancer in Yunnan keep up at a high level, it is urgently needed to develop more effective treatment methods or drugs, so as to reduce its fatality rate. Recent studies have found that the immunotherapy based on T cells is a new breakthrough among the treatment strategies in lung cancer. In the previous study, we introduced TIL cell therapy from NIH (Results 1). It was found that the expression of SOCS1 in TIL cells was in direct proportion to Treg cells, while TIL cells with silencing SOCS1 were overexpressing IFN-γ, low expression of IL-10 (Results 2~3), the results suggested that silencing SOCS1 has a reversal effect of immunosuppressive. Further studies found that Tim-3 was highly expressed in TIL of lung cancer, treating lung cancer using TIL with silencing SOCS1 combined with Tim-3 monoclonal antibody, increased IFN-γsecretion, and increased overall cytotoxic activity (Results 4 to 5), supporting the viewpoint that TIL withsilencing SOCS1 combined with Tim-3 monoclonal antibody enhancing efficacy synergistically by remodeling immuno-microenvironment. The aim of this study is to clarify the mechanism of TIL with silencing SOCS1 combined with Tim-3 monoclonal antibody, activating the third signal of cytokines, remodeling immune microenvironment, and then improving the efficacy of lung cancer using flow cytometry, animal experiments and so on., so as to provide experimental basis for promoting the clinical transformation of new TIL immunotherapy.

云南肺癌发病率和死亡率居高不下，亟需研发更有效的治疗方法或药物，降低其病死率。新近研究发现，以T细胞为基础的免疫治疗是肺癌治疗策略的新突破点。前期研究中，我们引进NIH的TIL细胞疗法（结果一），检测发现，TIL细胞SOCS1表达量与Treg细胞呈正比，而沉默SOCS1的TIL细胞，高表达IFN-γ、低表达IL-10（结果二~三），提示：沉默SOCS1具有逆转免疫抑制作用；进一步研究发现，肺癌TIL的Tim-3高表达，沉默SOCS1的TIL联合Tim-3单抗治疗肺癌，IFN-γ分泌增加，并提高总杀伤率（结果四~五），支持沉默SOCS1的TIL联合Tim-3单抗重塑免疫微环境，协同增强疗效的观点。本课题拟通过流式细胞术、动物实验等，阐明沉默SOCS1的TIL联合Tim-3单抗，激活细胞因子第三信号，重塑免疫微环境，提高肺癌疗效的机制，为促进TIL新疗法的临床转化应用提供实验依据。

背景：在所有恶性肿瘤中肺癌的发病率和死亡率一直以来均居全球首位，因此目前亟需根据肺癌的分子特征和免疫特征，研发更有效的治疗方法或药物，降低肺癌的死亡率。而T细胞免疫疗法近年来逐渐成为研究热点，其中肿瘤浸润淋巴细胞（Tumor-Infiltrating Lymphocytes,TIL）是当前最有研发前景的细胞免疫治疗方法之一。如何在TIL治疗的同时克服肿瘤局部和整体免疫微环境的免疫抑制，是当前肿瘤免疫治疗研究的热点领域。本项目主要通过沉默SOCS1的TIL联合Tim-3单抗解除肺癌患者体内局部和整体的免疫抑制状态从而提高其治疗肺癌的疗效。方法：通过分离和提取肺癌组织及外周血中的TIL在体外进行培养，随后通过慢病毒转染TIL建立沉默SOCS1的TIL细胞株，在细胞层面探究沉默SOCS1的TIL联合Tim-3单抗提高对NSCLC细胞株杀瘤活性的机制及其免疫微环境的变化情况；最后通过动物实验验证沉默SOCS1的TIL联合Tim-3单抗通过解除小鼠移植瘤模型局部和整体的免疫抑制重塑免疫微环境提高其免疫活性及杀瘤活性。结果：（1）在人肺癌组织中可分离并提取出TIL细胞；通过体外活化增殖后的TIL较培养前其免疫活性被激活。（2） SOCS1在肺腺癌癌组织中高表达，SOCS1在肺腺癌中高表达提示肺癌患者预后更差。（3） SOCS1在肺癌组织TIL表达量显著高于外周血T细胞，并且SOCS1高表达患者的Treg细胞比例显著高于SOCS1低表达患者，提示SOCS1与Treg细胞介导的免疫负性调控作用密切相关。（4） 慢病毒可成功转染TIL细胞，其中2号慢病毒转染效率最高，经过慢病毒转染后的TIL细胞其SOCS1表达降低。（5） 沉默SOCS1的TIL不仅可以进一步增强其免疫活性，同时也可以增强其对NSCLC的杀瘤活性，说明通过沉默SOCS1能有效的解除肿瘤局部免疫抑制状态。（6） 沉默SOCS1的TIL细胞联合Tim-3单抗可以通过重塑免疫微环境提高TIL的免疫活性及杀瘤活性。（7）动物实验结果提示沉默SOCS1的TIL联合Tim-3单抗可以有效抑制肺癌细胞在小鼠体内的增殖，其主要机制是通过解除小鼠体内局部及整体免疫抑制并重塑免疫微环境提高TIL对肺癌细胞的杀伤作用。本研究的意义在于为未来NSCLC的免疫治疗提供一个新的可行方向。