益气养阴解毒方联合抗CTLA-4单抗协同诱导抗肺癌免疫应答的作用及机制研究

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an important inhibitory molecule that mediates tumor immune escape. Treatment of lung cancer with anti-CTLA-4 monoclonal antibody has been proved to be effective, and the value of anti-CTLA-4 monoclonal antibody combined with other therapies of lung cancer should be paid more attention. The preliminary study shows that Yiqi Yangyin Jiedu decoction can improve the cellular immune function and down-regulate the expression of soluble CTLA-4 (sCTLA-4) sharing the same biological effects with CTLA-4 in peripheral blood of patients with lung cancer. In light of this, we put forward the hypothesis that “Yiqi Yangyin Jiedu decoction combined with anti-CTLA-4 monoclonal antibody enhances immune response against lung cancer”. Immunocyte co-culture and a system of orthotopic transplantation tumor model of lung cancer in mice will be used to study the impact of Yiqi Yangyin Jiedu decoction combined with anti-CTLA-4 monoclonal antibody on lung cancer , the immune function of T cell subsets and the expression of CTLA-4 on effector and regulatory T Cells. We will research into the molecular mechanism of synergistic enhancement of T cell immune response by anti-CTLA-4 monoclonal antibody combined with Yiqi Yangyin Jiedu decoction through PI3K/Akt pathway. This study will reveal the biological basis of the role of Yiqi Yangyin Jiedu decoction and the importance of its combination with immune targeted therapy from the perspective of immune checkpoint so as to advance the mechanism of “treating tumor by improving the general condition”.

细胞毒T淋巴细胞相关抗原4（CTLA-4）是介导肿瘤免疫逃逸的重要抑制性分子。抗CTLA-4单抗具有治疗肺癌的作用，其联合治疗的应用价值受到关注。课题组前期研究显示益气养阴解毒方改善机体细胞免疫功能，下调肺癌患者外周血中与CTLA-4生物学效应相同的可溶性CTLA-4（sCTLA-4）表达。基于此，提出“益气养阴解毒方联合抗CTLA-4单抗协同诱导抗肺癌免疫应答”的假说。拟用免疫细胞共培养、小鼠肺癌原位移植瘤模型研究益气养阴解毒方联合抗CTLA-4单抗的协同抗癌作用，对T细胞亚群免疫功能状态影响，对效应和调节性T细胞表面CTLA-4及sCTLA-4的下调作用；并通过PI3K/Akt通路探讨益气养阴解毒方联合抗CTLA-4单抗协同增强T细胞免疫应答的分子机制。从免疫检查点角度揭示益气养阴解毒方发挥作用的生物学基础及其与免疫靶向治疗联合应用的价值，丰富“扶正治癌”以提高自身抗癌能力的作用内涵。

肺癌的死亡率居我国恶性肿瘤首位，其主要治疗手段包括手术、化放疗、分子靶向治疗等。随着对肿瘤免疫调控机制的不断深入研究，肺癌免疫治疗得到了空前发展。细胞毒T淋巴细胞相关抗原4（CTLA-4）是介导肿瘤免疫逃逸的重要抑制性分子，靶向CTLA-4的肿瘤免疫治疗具有增强机体免疫应答的作用，其联合治疗的应用价值亦受到关注。课题组前期研究发现，益气养阴解毒方改善机体细胞免疫功能，下调肺癌患者外周血中与CTLA-4生物学效应相同的可溶性CTLA-4（sCTLA-4）表达。基于此，提出“益气养阴解毒方联合抗CTLA-4单抗协同诱导抗肺癌免疫应答”的假说。本课题采用肿瘤细胞免疫细胞共培养、小鼠肺癌原位模型，运用活体成像、免疫组化、免疫印迹、流式细胞术、ELISA等方法，研究益气养阴解毒方及其联合抗CTLA-4单抗的协同抗癌作用，对T细胞亚群的影响，对T细胞表面膜型CTLA-4（mCTLA-4）及sCTLA-4的下调作用，对肿瘤组织中免疫相关因子的调控作用，并通过PI3K/Akt通路探讨益气养阴解毒方及其联合抗CTLA-4单抗协同增效的分子机制。体内研究发现，益气养阴解毒方中高剂量、抗CTLA-4单抗和益气养阴解毒方联合抗CTLA-4单抗均可抑制肺癌原位模型小鼠的肿瘤生长，并延长小鼠生存期，且联合用药具有协同增效的作用，其机制可能与下调肿瘤组织中Foxp3和CTLA-4表达有关。黄芪甲苷联合抗CTLA-4单抗、益气养阴解毒方、益气养阴解毒方联合抗CTLA-4单抗抑制肺癌原位模型小鼠肿瘤生长，其机制可能与下调小鼠肿瘤组织中TGF-β表达、抑制肿瘤组织中PI3K/Akt通路激活有关；益气养阴解毒方联合抗CTLA-4单抗升高小鼠脾脏CD3+T细胞比例，下调CD3+T细胞表面CTLA-4表达，下调Treg细胞比例，并提高小鼠脾脏指数。体外研究发现，益气养阴解毒方及黄芪甲苷下调mCTLA-4及sCTLA-4表达，提高CD8+T细胞比例，以增强抗肺癌免疫应答、逆转CTLA-4介导的肺癌免疫逃逸，与抗CTLA-4单抗联合给药具有协同增效的作用。本课题从免疫检查点角度揭示益气养阴解毒方发挥作用的生物学基础及其与免疫靶向治疗联合应用的价值，体现了中医“扶正法”提高自身抗癌能力的治疗理念，为进一步探索“扶正治癌”如何调控CTLA-4表达的分子机制提供研究基础。