TCR-pMHC 亲和力调控的CD8+T细胞功能分化异常与慢性乙肝病毒感染

Our previous work shows that functional subsets of effector, low responsiveness and inhibitor are induced to proliferate when polyclonal T cells respond against a single pMHC epitope, and the low-responsive and inhibitory subsets are more frequent in the samples of patients with chronic HBV infection. Based on the above observations, we choose HBcAg18-27/HLA-A2 as a single pMHC in this proposal, setting up an in vitro co-culture which allows polyclonal T cells respond to the pMHC, to expand and sort the above 3 functional subsets of the pMHC-specific CD8+T cells in the samples of normal controls, HBV infected patients and a chronic HBV infection mouse model (the mouse is HLA-A2 transgenic). The CD8+T cell subsets will be tested for their frequency and ratio, TCR-pMHC affinity, metabolic pathway and relevant transcript factors, and their metabolic pathway will be alternated with metabolic interfering drugs. This proposed study would provide experimental evidences to show that (1) T-cell repertoire has got a series of clones able to bind to a given pMHC with gradual affinities; the TCR-pMHC affinity would determine which metabolic pathway the clone is to take, and then which functional subset the clone is to differentiate into. (2) The TCR-pMHC affinities of pMHC-specific CD8+T cells in the patients with chronic HBV infection would be lower than those of normal controls, which would make the clones more likely to take fatty acid metabolic pathway, and differentiate into the low-responsive and inhibitory subsets, delaying the virus clearance. (3) Inhibition of fatty acid metabolic pathway would be hopeful for the lower TCR-pMHC affinity clones to differentiate into effector subset, promoting the virus clearance. This proposed study would help to understand immune regulation and its association with diseases, and to develop immunotherapy method.

前期工作显示：多克隆T细胞针对单一pMHC应答时，可分化为效应性、低反应性和抑制性亚群；慢性乙肝患者的低反应/抑制性亚群增多。在此基础上，本项目选择HBcAg18-27/HLA-A2作为pMHC，用多克隆T细胞对单一pMHC应答的体外共培养方法，在正常人、慢性乙肝患者和慢性乙肝模型小鼠（HLA-A2转基因小鼠）样本中，扩增该pMHC特异性T细胞的上述三个亚群。通过检测各亚群的数量、比值、TCR-pMHC亲和力和代谢途径，并在体内外干预代谢途径，结果将显示：①不同T细胞克隆的TCR-pMHC亲和力存在差异，使之采用不同的能量代谢途径，分化为不同的亚群；②慢性乙肝患者T细胞的TCR-pMHC亲和力较低，易采用脂肪酸代谢、分化为低反应/抑制性亚群，延缓病毒清除；③通过抑制脂肪酸代谢，促进亲和力低的T细胞分化为效应性亚群，加速病毒清除。目的是认识免疫调节机制及其异常与疾病的关系，发展免疫干预手段。

利用多克隆CD8+T细胞对单一pMHC应答的“单核细胞-自身淋巴细胞”共培养方法，我们将在单一pMHC刺激下发生增殖的CD8+T细胞视为该pMHC特异性CD8+T细胞。本项目选用的pMHC为HBcAg抗原肽/MHC复合物（HBC18-27/HLA-A2），在正常个体、慢性乙肝患者和急性乙肝康复者的样本中，扩增和分选出该pMHC特异性CD8+T细胞，并证实其中存在着效应性、抑制性和低反应性亚群。这些亚群能够通过检测IFN-gamma和mTGF-beta的表达进行区分，效应性亚群表达IFN-gamma；抑制性亚群表达mTGF-beta；低反应性亚群既不表达IFN-gamma，又不表达mTGF-beta。通过检测上述样本中pMHC特异性CD8+T细胞各亚群的数量和比值，我们发现效应性/抑制性亚群的比值在急性乙肝康复者的样本中高于慢性乙肝患者，且该比值与样本中乙肝病毒DNA浓度成反比，显示pMHC特异性CD8+T细胞中效应性/抑制性亚群的比值决定了清除病毒的能力。采用pMHC四聚体染色法评估各亚群的TCR-pMHC亲和力，结果显示效应性亚群TCR-pMHC亲和力最高，抑制性亚群其次，而低反应性亚群最低。提示个体T细胞库对单一pMHC应答时，该pMHC特异性CD8+T细胞克隆依据TCR-pMHC亲和力的高低分别分化为效应性、抑制性和低反应性亚群。免疫抑制剂（环孢霉素）和免疫增强剂（PD-1抗体）分别能够减低和升高pMHC特异性CD8+T细胞中效应性/抑制性亚群的比值。