环孢素A增强黑色素瘤CD8+T细胞抗肿瘤活性机制研究

T cell exhaustion, which causes T cell dysfunction, is the main reason for the failure of cancer immunotherapy. It is synthetically caused by signals generated from multiple inhibitory receptors. In order to retard T cell exhaustion, intervention during the onset of exhaustion to suppress signals that initiate exhaustion may provide an effective approach to simultaneously down-regulate the expression of multiple inhibitory receptors and improve T cell function. Considering that nuclear factor of activated T cells (NFAT) family are the essential molecules for the development of T cell exhaustion and the main targets of cyclosporine A, we have successfully applied cyclic di-guanylate to induce anti-melanoma immunity by mice CD8+T cells and confirmed that low dose cyclosporine A enhanced the immunity against melanoma when it was administrated during the early stage of immunotherapy. However, the underlying mechanism was not clear. We speculate that low dose cyclosporine A may regulate the expression of NFAT family proteins and dampen the signal transduction which triggers exhaustion, resulting in enhanced anti-tumor immunity. This study will further take advantage of this mouse melanoma model and methods such as immune cell sorting and small interference RNA, to investigate the effects of low dose cyclosporine A on the exhaustion process of melanoma specific CD8+T cells activated by cyclic di-guanylate, as well as the expression of NFAT family members. This study may gain the molecular mechanism of the immune boosting effects induced by cyclosporine A and extend clinical application of NFAT inhibitors in cancer immunotherapy.

T细胞衰竭引起T细胞功能障碍，是肿瘤免疫治疗失败的重要原因；它由多个抑制性受体介导信号综合作用所致。为延缓T细胞衰竭，在衰竭启动阶段抑制诱发衰竭的信号传导，同时降低多种抑制性受体表达，有望改善T细胞功能。考虑到活化T细胞核因子（NFAT）家族是诱发T细胞衰竭的关键节点，且为环孢素A的主要靶点；我们前期以环二鸟苷酸成功激活小鼠CD8+T细胞抗黑色素瘤免疫，并证实在免疫活化初期应用低剂量环孢素A可以增强黑色素瘤免疫治疗效果，但具体机制不明确。我们假设：低剂量环孢素A可能通过调节NFAT蛋白各亚型表达，削弱诱发衰竭的信号传导，从而增强抗肿瘤免疫。本项目将继续采用黑色素瘤小鼠模型，通过免疫细胞分选、小干扰RNA等手段，研究低剂量环孢素A对环二鸟苷酸活化的黑色素瘤特异性CD8+T细胞衰竭发展及NFAT各亚型的影响；有望揭示环孢素A免疫增强作用的分子机制，拓展NFAT抑制剂在肿瘤免疫治疗领域的新应用。

持续性抗原刺激是CD8+T细胞衰竭的重要原因之一，由多个抑制性受体综合作用引起，抑制性受体表达是抗原活化信号介导并维持的，其中活化T细胞核因子（NFAT）信号是诱导CD8+T细胞向记忆或衰竭分化的关键节点；为延缓CD8+T细胞衰竭，利用NFAT抑制剂削弱诱发抑制性受体表达的信号传导，有望改善CD8+肿瘤浸润T细胞（CD8+TILs）功能。我们利用环二鸟苷酸激活小鼠CD8+T细胞介导的抗黑色素瘤免疫，发现：低剂量NFAT抑制剂环孢素A（CsA）可以增强环二鸟苷酸的抗黑色素瘤效果。考虑到另一种NFAT抑制剂他克莫司免疫抑制活性比CsA强且可以抑制辅助刺激信号诱导的CsA抵抗的T细胞活化信号，我们对比他克莫司和CsA单药对抗肿瘤免疫的影响。我们发现单独间断性利用他克莫司治疗（每5天1次）显著抑制小鼠黑色素瘤生长，其单药抗肿瘤作用比CsA更稳定；通过CD8+细胞敲除实验证实间断性他克莫治疗的抗肿瘤效应依赖于CD8+T细胞。间断性他克莫司治疗显著改善黑色素瘤荷瘤小鼠CD8+TILs功能，增加表面活性标志物CD107a表达，促进其合成IFN-γ，而主要抑制性受体（如PD-1、LAG-3、TIM-3）表达无明显变化。分选荷瘤小鼠的CD8+TILs，通过转录组分析（RNA-seq）及qRT-PCR证实：间断性他克莫司治疗组Nr4a1（衰竭相关转录因子）及Ctla4（抑制性受体）转录显著下降。接着，分选小鼠CD8+T细胞并在体外诱导衰竭，间断性他克莫司干预显著促进衰竭型CD8+T细胞合成IFN-γ，抑制其表达NFATc1，但NFATc2表达无明显差异。为进一步明确NFATc1与NR4A1、CTLA-4的调控关系，我们分选小鼠CD8+T细胞，通过Crispr-cas9技术敲除NFATc1并在体外刺激活化，其NR4A1、CTLA-4表达也下降。本项目目前研究结果证实，间断性他克莫司治疗可以显著延缓黑色素瘤CD8+TILs衰竭并增强抗肿瘤免疫，其机制可能与抑制NR4A1、CTLA-4表达有关，需要后续研究进一步证实。