乙型肝炎患者体内滤泡辅助性T细胞的功能特点及临床意义研究

Functional characteristics and clinical significance of follicular helper T cells in patients with hepatitis B

目的 通过分析HBV感染者体内滤泡辅助性T细胞(follicular helper T cell,Tfh)的频率和功能,探讨其在HBV感染中的临床意义。方法 本研究入组HBV感染者36例,包括6例急性恢复期(acute recovery,AR)患者和30例慢性HBV感染者,其中慢性HBV感染者又包括免疫耐受期(immune tolerance,IT)患者10例、免疫活化期(immune activation,IA)患者20例。另外纳入8例健康对照(health control,HC)。通过流式细胞术和多色免疫组化技术比较HC组和HBV感染者外周血和肝内Tfh的表达差异,以及通过单细胞分泌蛋白组芯片检测Tfh的功能特点,并对HBV感染者Tfh的频率与HBV相关临床指标(HBV表面抗原、HBV载量、ALT、AST)的相关性进行分析。结果 IA组外周血中Tfh频率显著高于HC组(1.487%vs.0.876%,t=2.126,P=0.043)和IT组(1.487%vs.0.737%,t=3.144,P=0.004);与外周血一致,肝内Tfh也在IA组患者中富集。外周血中Tfh的频率与肝脏炎症指标ALT、AST水平呈正相关(ALT:r=0.488,P=0.000;AST:r=0.498,P=0.000)。单细胞分泌蛋白组芯片检测Tfh的多功能强度指数(polyfunctional strength index,PSI)在AR组患者中最高(AR组>IA组>IT组>HC组),其中AR组患者分泌TNF-、IL-2和IFN-能力较高、IA组分泌IL-10和IL-17A能力较高、IT期分泌粒细胞-巨噬细胞集落刺激因子和IFN-较高,HC组分泌IL-4较高。结论 肝内与外周血结果一致,Tfh在IA组CHB患者中增多,这可能提示Tfh与肝脏炎症的免疫状态密切相关。进一步功能分析表明Tfh在HBV感染不同阶段分泌的细胞因子不同,其可能倾向于不同的分化表型,其中IA期Tfh倾向于cTfh17类表型,而IT期和AR期Tfh倾向于cTfh1类表型,表明Tfh在HBV感染不同阶段表现出不同的CD4^(+)T细胞极化特征。

Objective By analyzing the frequency,phenotype,and function of follicular helper T cells (Tfh) in Hepatitis B Virus(HBV)-infected patients,we aim to explore the clinical significance of Tfh in HBV infection.Methods In this study,8 cases of the healthy control (HC) and 36 cases of HBV-infected patients were enrolled.The HBV infection group included 6 cases of acute recovery (Acute recovery,AR),10 of immune tolerance (IT),and 20 of immune activation (IA).Tfh were detected by flow cytometry and multicolor immunohistochemistry in peripheral blood and liver,respectively.The functional characteristics of Tfh were monitored by IsoPlexis single-cell highly multiplexed proteomic platform.Their correlations with clinical parameters (including HBV surface antigen,HBV viral load,ALT,AST,etc.) were examined.Results The proportion of peripheral Tfh from IA was significantly higher than that in healthy control (1.487% vs.0.876%,t=2.126,P=0.043),and (1.487%vs.0.737%,t=3.144,P=0.004).In line with peripheral blood,intrahepaticTfh were also enriched in IA patients.The proportion of Tfh was positively correlated with liver inflammatory indicators ALT and AST (ALT:r=0.488,P=0.000),(AST:r=0.498,P=0.000).Tfh from AR exhibited the highest polyfunctional strength index (AR>IA>IT>HC),in which TNF-,IL-2,and IFN-are high in AR patients,IL-10 and IL-17A are high in IA stage,while GM-CSF and IFN-are high in IT stage.Conclusions Tfh were increased in IA patients in both the blood and liver,which may suggest that Tfh are closely related to the immune state of liver inflammation.Further,Tfh were differently polarized among different stages:IA patients tend to have the cTfh17 phenotype,while IT and AR tend to have the cTfh1 phenotype.