免疫重建窗口期过继转输CD8+Tcm细胞联合阻断PD-1/PD-L1通路抗卵巢癌的作用及机制

In our previous project funded by The National Natural Science Foundation of China, it was found that, compared to other time points, adoptive transfer of T cells (ACT) when lymphocytes decreased to the lowest after chemotherapy induces strongest anti-tumor response. However, there are two big obstacles severely restricting the efficacy of ACT: (1) the transferred T cells in most clinical trials are terminally differentiated effector T (Tte) cells, which, having been stimulated multiple times with their specific antigen and high dose IL-2, have weak survival and proliferative capacities and are unable to home to lymph nodes; (2) there is an inhibitory pathway PD-1/PD-L1 in tumor microenvironment as an important mediator of the evasion of tumor immunity. PD-1/PD-L1 is a chief pathway that regulates tumor specific T cell exhaustion and inhibits the migration of T cells to target tumor cells. Since central memory T (Tcm) cells exhibit far greater proliferative, survival and lymph node homing capacities in vivo than Tte cells, we intend to transfer tumor antigen specific Tcm cells （instead of Tte cells） combined with blocking PD-1/PD-L1 pathway in the treatment of ovarian cancer. We have found, in an early preparatory work, that the high expression of PD-L1 by ovarian cancer cells is associated with poor prognosis, and that blocking PD-1/PD-L1 pathway enhanced the effector function of CD8+ cytotoxic T lymphocytes against PD-L1+ ovarian cancer cells. In this project we take the lead in exploring the procedure to obtain the polyclonal and ovarian caner antigen specific CD8+Tcm cells, and in detecting the impact of blocking PD-1/PD-L1 pathway in the functioning of CD8+Tcm cells. Meanwhile, we will study the antitumor effects of adoptive transfer of tumor antigen specific CD8+Tcm cells during the window period of immune reconstruction after chemotherapy combind with blocking PD-1/PD-L1 pathway in mice bearing ovarian cancer. Furthermore, we will evaluate the effect of blocking PD-1/PD-L1 pathway in the proliferation, migration, functioning of transfered T cells and in the tumor microenvironment, so as to reveal the mechanism of the synergistic antitumor effects of combination therapy. This pioneering study will greatly help to enhance the efficacy of comprehensive treatment of ovarian cancer.

此前我们已发现化疗后免疫重建期是实施过继转输T细胞（ACT）治疗的最佳时机，然而两大问题严重制约ACT疗效：一是目前普遍使用的转输细胞为终末分化效应Tte细胞，其增殖及归巢能力差，存活时间短；二是肿瘤微环境存在的PD-1/PD-L1（P/P）通路可致T细胞耗竭并抑制其募集。鉴于中枢型记忆Tcm细胞的增殖及归巢能力显著强于Tte细胞，我们设计以Tcm作为转输细胞，并联合阻断P/P通路以期显著提高ACT疗效。前期我们已发现卵巢癌组织PD-L1高表达与预后不良密切相关，且阻断P/P通路可增强CTLs杀伤功能。本研究将首次探索体外诱导卵巢癌特异性Tcm细胞的培养体系，检测阻断P/P通路对Tcm细胞功能的影响，进而研究免疫重建期转输Tcm细胞联合阻断P/P通路的抗瘤效应，分析阻断该通路对转输细胞增殖、迁移、功能及肿瘤微环境影响，揭示两者协同抗瘤的内在机制，为大幅提升卵巢癌综合治疗效果作出开拓性探索。

此前我们已发现化疗后免疫重建期是实施过继转输T细胞（ACT）治疗的最佳时机，然而两大问题严重制约ACT疗效：一是目前普遍使用的转输细胞为终末分化效应Tte细胞，其增殖及归巢能力差，存活时间短；二是肿瘤微环境存在的PD-1/PD-L1通路可致T细胞耗竭并抑制其募集。鉴于中枢型记忆Tcm细胞的增殖及归巢能力显著强于Tte细胞，因此本研究设计以Tcm作为转输细胞，并通过联合阻断PD-1/PD-L1通路以期显著提高ACT疗效。T细胞的分化呈线性模式，即初始T（TN）细胞→中枢型记忆Tcm细胞→效应型记忆Tem细胞→终末分化的Tte细胞，本研究通过将CD8+TN细胞与负载卵巢癌抗原的DCs共培养，并通过添加IL-7、IL-15及IL-21等细胞因子,成功诱导及构建了多克隆的卵巢癌特异性CD8+Tcm细胞培养体系，体外研究显示卵巢癌特异性CD8+Tcm细胞有较强的增殖及特异性杀伤功能；在CD8+Tcm细胞与PD-L1+同源人卵巢癌细胞共培养体系中，阻断PD-1/PD-L1通路显著增强了CD8+Tcm细胞的特异性杀伤功能。在ID8小鼠卵巢癌模型的体内研究中，我们于紫杉醇诱导的免疫重建期过继转输卵巢癌特异性CD8+Tcm细胞，并联合阻断PD-1/PD-L1通路，较之对照组（未阻断PD-1/PD-L1通路），募集至淋巴组织及肿瘤局部的卵巢癌特异性CD8+Tcm细胞数量显著增加，其增殖及特异性杀伤能力显著增强；此外与对照组相比，联合治疗组受体小鼠肿瘤生长显著减缓，小鼠生存时间显著延长。由于PD-1或PD-L1单抗存在原发或继发耐药，基于动物模型本研究进一步揭示阻断PD-1/PD-L1通路联合小分子抗血管生成药物阿帕替尼具有协同抗肿瘤作用，为逆转PD-1或PD-L1单抗原发或继发耐药、提高PD-1或PD-L1单抗的客观有效率提供重要的借鉴信息。本研究结果为临床上制定有效的卵巢癌化疗联合免疫治疗方案提供不可或缺的信息，为完善卵巢癌综合治疗模式作出开拓性探索。此外，本研究中建立的肿瘤特异性CD8+Tcm细胞培养体系、ACT治疗与阻断PD-1/PD-L1通路可能产生的协同效应等对于其它肿瘤的免疫治疗也具有重要借鉴价值。