HBV致肝细胞PD-L1表达及其T细胞免疫耗竭的miRNA调控机制探索

Accumulating clinical data has shown that the immune exhaustion of T cells in chronic HBV infection (CHB) patients is usually accompanied by the over-expression of PD-1, indicating a possible incentive of its ligand, PD-L1, which highly expresses on the hepatocytes, in the induction of such exhaustion. However, the mechanism underlying the induction of PD-L1 by HBV is still unclear. In our present study, we found that PD-L1 and miR-200c are negatively correlated in HBV-infected hepatocellular carcinoma (HCC) patients, meanwhile, it was preliminarily observed in persistent HBV-infected mice that miR-200c may repress the expression of PD-L1 on hepatocytes and reverse the expression of PD-1 on T cells. We are planning to further demonstrate the interactive relations between HBV (or its components), PD-L1, and miR-200c, and the possible mechanisms underlying their interactions that may contribute to the reversion of PD-1 expression on T cells. HBV-infected liver cell lines and hepatocytes of persistent HBV-infected mice will be used to re-confirm the negative regulation of PD-L1 by miR-200c; the negative regulation of miR-200c by HBV (or its components); the inhibition of HBV replication by miR-200c; and the synthetic effect on the exhaustion of T cells in HBV-infected mice, respectively. The results would possibly reveal the epigenetic negative regulatory mechanisms of miR-200c on HBV-induced PD-L1 expression that may suggest a promising role of miR-200c as a new therapeutic treatment to reverse the immune escaping of HBV.

临床研究提示HBV感染患者体内T细胞PD-1高表达而导致免疫耗竭, 推测肝细胞高表达PD-L1是主要诱因，目前尚不知HBV导致PD-L1表达的分子机制。我们在HBV肝细胞癌病人队列发现PD-L1与miR-200c呈明显负相关，同时在HBV持留感染小鼠初步观察到miR-200c可阻遏肝细胞PD-L1表达并逆转T细胞PD-1表达。拟进一步论证HBV或组分、PD-L1、miR-200c三者之间互作机制及其与T细胞耗竭关系，将通过人HBV感染肝细胞系、HBV持留感染小鼠肝细胞，分别确认miR-200c对PD-L1的负调效应、HBV或组分对miR-200c的负调效应、miR-200c对HBV复制的负调效应、以及对HBV持留小鼠体内T细胞耗竭的综合作用，以揭示miRNA对HBV导致肝细胞PD-L1表达的表观遗传负调机制以及逆转体内HBV免疫逃逸的基因治疗新途径。

慢性病毒感染和肿瘤微环境可以诱使机体抗病毒或抗肿瘤特异性T细胞衰竭，使其增殖能力和效应功能严重受损，致使机体免疫应答无法抵抗病毒感染或肿瘤发生。T细胞共抑制受/配体被认为是慢性病毒感染和肿瘤中T细胞耗竭的关键调节因素，据此原理诞生了卡控点免疫疗法（Checkpoint Immunotherapy），成为2013年度世界十大科技进展榜首和美国2016年启动的“癌症登月计划（Cancer MoonShot）”的核心手段。然而，慢性病毒感染、肿瘤发生和介导T细胞耗竭的共抑制受/配体三者之间相互影响的分子机制尚不清楚。我们通过HBV+肝癌患者队列进行肝癌组织的分子病理学研究，发现肝癌细胞的锌指转录因子SALL4（一种肝脏胚胎蛋白）和T细胞共抑制配体PD-L1的表达水平均与miR-200c水平呈明显的负相关；通过大样本肝癌病人生存期回顾分析，发现低表达SALL4或PD-L1及高表达miR-200c的患者具有更长的生存期。以HBV+肝癌细胞为模型探讨其分子机制，发现miR-200c可直接靶向PD-L1的3'-UTR以控制其表达，过表达miR-200c能直接抑制HBV诱导的肝细胞PD-L1表达，证实miR-200c是PD-L1表达的阻遏因子；进一步发现HBV感染可以再激活正常成年肝脏不表达的癌胚蛋白SALL4，而SALL4可特异性负向调控miR-200c的转录，使miR-200c失去对PD-L1表达的阻遏作用，导致PD-L1高表达。应用HBV携带小鼠模型进一步确证，HBV可以导致小鼠肝细胞PD-L1高表达，并伴随抗HBV特异性CD8+ T细胞的耗竭；抑制SALL4表达或增强miR-200c表达或用抗体阻断PD-L1均能明显削弱PD-L1介导的T细胞耗竭。我们揭示了在HBV感染和肝癌发展期间存在调节PD-L1表达的HBV-pSTAT3-SALL4-miR-200c-PDL1分子轴，基于该分子轴的干预策略对于逆转病毒或肿瘤诱导的免疫细胞功能衰竭具有重要意义，为Checkpoint免疫治疗提供了新思路。同时还揭示miR-200c和SALL4可能对HBV+肝癌患者的预后具有预测价值，具有重要的分子病理学意义；本项目如期按照计划完成，共发表标注该基金的SCI论文10篇，其中第一标注论文7篇，影响因子大于10分论文2篇，中科院一区论文5篇。