谷胱甘肽联合PD-1/PD-L1阻断剂增强CD8+T细胞抗肿瘤效应的机制研究

Targeting immune checkpoints PD-1 in the treatment of various malignant tumors have been obtaining curative effects，however， some patients still have no response due to the resistance to PD-1 therapy. In preliminary study, the level of PD-1 expression was higher on the surface of nonresponsive MAGE-A3 specific T cells, but the proliferation and function of PD-1 positive CD8+ T cells were partially reinvigorated by PD-1/PD-L1 blockade. Thus, it is a hot area to explore novel strategy to enhance the effect of targeting PD-1 immunotherapy. Database results showed that PD-1 was positively associated the lipid metabolism that was closed to reactive oxygen species. It was reported that the unbalance of ROS could influence the activation and function of T cells. Preliminary test results showed that ROS scavenger glutathione (GSH) could reduce the apoptosis and improve the function of PD-1+ CD8+T cells. Therefore, we propose the hypothesis that ROS level is higher and could not be affected by PD-1/PD-L1 blockade in PD-1+CD8+T cells , we further explore the mechanism that how combing GSH with PD-1/PD-L1 blockade improve the antitumor ability of CD8+T cells.

靶向免疫检查点PD-1的治疗在多种恶性肿瘤中显示了惊人的疗效，但仍有部分患者对靶向PD-1的免疫治疗无反应。我们前期研究结果显示：PD-1在低反应性MAGE-A3特异性CD8+T细胞表面高表达；而应用PD-1/PD-L1阻断剂后仅能轻度增强CD8+T细胞的增殖能力和功能，因而寻求新的策略以增强靶向PD-1免疫治疗的疗效是肿瘤免疫治疗领域的研究热点。数据库分析发现PD-1正向调控脂类代谢，而脂类代谢同活性氧密切相关；活性氧的失衡将影响T细胞的活化和功能，预实验结果显示活性氧清除剂谷胱甘肽能够减少 PD-1+ CD8+T细胞的凋亡并增强其分泌IFN-r的能力；因而我们提出假说，PD-1+CD8+T细胞表达更高水平的活性氧，PD-1/PD-L1阻断剂对活性氧的水平无影响；我们将进一步探讨谷胱甘肽联合PD-1/PD-L1阻断剂增强 CD8+T细胞抗肿瘤作用的机制。

免疫治疗已成为肿瘤治疗的第四大手段，以嵌合抗原受体T细胞为代表的过继细胞治疗能够显著改善B细胞恶性肿瘤的预后，但在实体瘤中的治疗效果欠佳；靶向免疫检查点PD-1的单克隆抗体已被批准应用于多种恶性肿瘤，但部分患者对PD-1单克隆抗体治疗无反应。探讨对PD-1/PD-L1阻断剂无反应的机制将为解决PD-1单抗治疗抵抗提供新的靶点，新的联合策略以提高PD-1为靶点的治疗疗效是免疫治疗领域的热点。我们以MAGE-A3特异性CD8+T细胞为食管癌治疗的研究模型，探明食管癌肿瘤微环境中影响其功能的因素。主要研究内容为：①以食管癌患者为研究模型，对比不同表达水平PD-1的CD8+T细胞对PD-1/PD-L1阻断剂的敏感性; ②从PD-1的表达、ROS的分泌、T细胞的凋亡及之间的内在联系为纽带，探讨谷胱甘肽（GSH）增强CD8+T细胞增殖能力和功能的机制;③明确GSH联合PD-1/PD-L1阻断剂增强CD8+T细胞功能的作用机制。结果显示：通过检测食管癌患者PBMCs中抗原特异性CD8+T细胞的频率和对MAGE-A3肽的反应发现，来自PBMCs的功能性MAGE-A3特异性T细胞的比例同食管癌的预后正相关；PD-1+ MAGE-A3 特异性CD8+T细胞功能受限，PD-1高表达CD8+T细胞对PD-1/PD-L1阻断剂反应性较低；PD-1调控T 细胞活性氧表达水平，通过CD95-Caspase 8 调控T细胞凋亡，GSH能够降低PD-1+CD8+T细胞的ROS、CD95、Caspase-8和凋亡水平； GSH联合PD-1/PD-L1阻断剂（DPPA-1）减少PD-1+CD8+T细胞的凋亡、增强CD8+T细胞的内因子释放功能；动物实验证实了 GSH联合PD-1/PD-L1阻断剂协同增强MAGE-A3特异性CD8+T细胞的抗肿瘤作用。本项目将为开发新的联合策略增强T细胞免疫治疗疗效提供理论基础。