HLA-DR限制性HBV表位介导慢加急性肝衰竭患者CD4+T细胞活化的机制研究

Acute-on-chronic liver failure (ACLF) is an acute severe exacerbation of chronic hepatitis B. Hepatitis B virus (HBV)-specific adaptive immune responses of CD4+ T cells play an important role during the immune-pathogenesis of ACLF, however, the detailed mechanisms of its triggering is unclear. Recently, we performed a genome-wide association study and identified the HLA-DRB1*12:02 as the top allele for HBV-related ACLF, which highlighted the importance for antigen-presentation via HLA-DR. This result had been published on Gut 2018. Furthermore, our group identified seven HLA-DRB1*12:02-specific restricted HBV epitope peptides and two draft epitope peptides in patients with chronic HBV infection, and found these peptides could stimulate CD4+ T cell proliferation and secretion of TNF-α and IFN-γ. It’s interesting that we found the TNF-α+ CD4+ T cells were associated with the hepatitis flare and injury in patients with severe hepatitis. All the results suggested that these new identified HBV epitope peptides may play a role as pro-inflammatory peptides in the activation of chronic hepatitis B, but how it works is not entirely clear. As the important role of antigen presentation by HLA class-II molecular in the CD4+ T cell adaptive immune response to the microbial pathogens, here we hypothesize the new identified HBV epitope peptides may promotes naïve CD4+ T cells activation and TNF-α-producing CD4 T cell subset differentiation by modulating the TCR-epitope-HLA-DRB1*12:02 signal, thus initiate start the adaptive immunity response and inflammatory in severe hepatitis. In this project, we plan to conduct serial experiments (the CD4+ T cell polarization via BLCL allele-specific presentation system, the affinity and stability of peptide-HLA-DR complex, inflammatory effects in HLA-DRB1*12:02 transgenic mouse models, etc.) and clarify the immune-pathogenesis of HLA-DRB1*12:02 restricted inflammatory epitopes. This study may provide new insights into the initiation of inflammatory in HBV-ACLF and new immunotherapy target of HBV-ACLF.

病毒特异性CD4+T细胞在慢加急性肝衰竭（ACLF）的免疫损伤中起重要作用。我们研究发现，HLA-DR是全基因组范围内影响HBV-ACLF免疫损伤的关键分子，提示HLA-DR抗原提呈信号的重要性。我们进一步鉴定出风险等位HLA-DRB1*12:02限制性的HBV表位肽，可刺激CD4+T细胞分泌产生大量的TNF-α，是可能的致炎表位肽，但功能效应及炎症启动机制尚不清楚。鉴于HLA-II类分子抗原提呈是CD4+T细胞活化的特异性第一信号，我们推测，新鉴定的表位肽可能通过DR抗原提呈信号促进CD4+T细胞活化及TNF-α+亚群分化，引发免疫损伤和肝炎活化。本研究拟从CD4+T细胞免疫效应和极化偏向、对抗原提呈细胞的反向调节、表位肽与DR的亲合力/稳定性、转基因小鼠模型等方面，明确HLA-DR限制性HBV表位介导CD4+T细胞活化的效应及机制，为ACLF免疫损伤关键始动环节提供新认识。

病毒特异性CD4 T细胞在慢加急性肝衰竭（ACLF）的免疫损伤中起重要作用。我们前期研究发现，HLA-DR是全基因组范围内影响HBV-ACLF免疫损伤的关键分子，提示HLA-DR抗原提呈信号的重要性。鉴于HLA-Ⅱ类分子抗原提呈是CD4 T细胞活化的特异性第一信号，我们推测，HBV表位肽可能通过DR抗原提呈信号促进CD4 T细胞活化及TNF-α亚群分化，引发ACLF免疫损伤和肝炎活化。本课题三年间围绕HBV再活化特征、HLA-DRB1限制性HBV致炎表位和保护性表位、表位肽介导的CD4 T细胞亚群分泌功能机制、小鼠模型研究等内容开展研究，取得以下成果：（1）认识到HBV再激活显著增加肝硬化患者发生肝损伤、肝功能衰竭、ACLF和短期死亡的风险，过程中伴随着HBV特异性免疫和天然免疫的激活及炎症因子释放；（2）鉴定到7条具有免疫原性的HBVS蛋白来源的表位肽，可诱导S抗原特异性CD4 T细胞应答，表现为IFN-γ单阳性、TNF-α单阳性以及IFN-γ/TNF-α双阳性三类CD4 T亚群；（3）发现HBs16-30、HBs46-60、HBs71-85、HBs76-90、HBs156-170和HBs166-180等6条肽均受到宿主HLA-DR限制性识别，其中HBs71-85和HBs166-180肽段的识别递呈同时还受到HLA-DQ的协同，并存在个体差异，协同作用的存在增加了抗原递呈的个体多样性和HBV抗原清除的复杂性；（4）证实参与HBs71-85短肽限制性识别的是HLA-DRB1*09:01等位和-DQB1*03:03等位；（5）构建了AE0-HLA-DRB1*1202小鼠模型和ConA诱导的肝损伤模型，观察了Ⅱ类分子在小鼠肝损伤中介导CD4 T细胞应答；（6）鉴定出新的与ACLF关联的拷贝数变异（HCG4B基因拷贝数丢失），可参与机体免疫炎症反应，并发现IGFs信号通路可能参与了慢加急性肝衰竭的肝细胞衰老和再生障碍。我们的研究首次鉴定出7条具有免疫原性的HBV S蛋白来源的表位肽，可诱导S抗原特异性CD4 T细胞应答，并证实这些表位肽受到HLA-DR限制性识别，并存在HLA-DQ协同。表位肽及其相应限制性等位的鉴定可为个体化的治疗性疫苗研发提供潜在的靶标分子及可获益人群。本课题成果已发表SCI论文5篇，中文论文1篇，参加学术报告4次，培养硕士生1名。