iTR35诱导HBV特异性CTL耗竭的分子机制及其靶向干预研究

Hepatitis B virus (HBV) specific cytotoxic T lymphocytes (CTL) is a key factor in determining the outcome of HBV infection, its functional impairment or exhaustion are closely related to chronic hepatitis. Interleukin-35 (IL-35) induced regulatory T cells (iTR35) are new members of regulatory T cells, which exert immunosuppressive function by secretion of IL-35, meanwhile, special AT rich sequence binding protein 1 (SATB1) can induce regulatory T cell transformation and therefore lose their immunosuppressive effect. Our previous study demonstrated that IL-35 can inhibit the function of HBV-specific CTL and induce their exhaustion, however, the mechanism is unknown. This project aims to investigate from the aspects including the secretion of IL-35 by iTR35 via JAK/STAT pathway to trigger HBV-specific CTL exhaustion, SATB1 induced iTR35 conversion and block CTL exhaustion. Our expected results will firstly understand the function of iTR35 and HBV-specific CTL of chronic hepatitis B patients as well as their relationship, and then explore the mechanism of how iTR35 cells inhibit CTL proliferation, up-regulate the expression of exhaustion molecules and down-regulate effector molecules through this way. SATB1 intervention can inhibit iTR35 cells to express IL-35 and lose their inhibition function to CTL, and the transfer of SATB1-overexpressed iTR35 in mice are able to terminate the development of HBV persistent infection, which provides research foundation for new drugs targeting SATB1 to treat hepatitis B patients.

HBV特异性细胞毒T细胞（CTL）是决定HBV感染转归的关键因素，其功能受损或耗竭与乙肝慢性化密切相关。iTR35是调节性T细胞新成员，通过分泌IL-35发挥免疫抑制功能，而核基质蛋白SATB1可诱导调节性T细胞转化使其失去免疫抑制效应。我们前期研究发现IL-35可明显抑制HBV特异性CTL功能而致其耗竭，但机制不明。本项目从iTR35分泌IL-35并经JAK/STAT通路引发HBV特异性CTL耗竭、SATB1诱导iTR35转化而阻断CTL耗竭的思路出发，首先了解慢性乙肝患者iTR35和HBV特异性CTL功能及其相互关系，进而探明iTR35经上述通路抑制CTL增殖及上调耗竭分子或下调效应分子表达、SATB1干预iTR35表达IL-35而使其丧失抑制CTL功能、输入SATB1过表达iTR35能终止小鼠HBV持续感染的作用及其分子机制，为未来进一步发展以SATB1为靶点的乙肝治疗新药奠定基础。

HBV特异性T细胞免疫弱或无应答是HBV难以彻底清除的关键。本项目以新型调节性T细胞（Treg）-iTR35为突破口，探索导致HBV特异性细胞毒T细胞（CTL）免疫应答失调的可能机制，设计并完成了本项目研究，取得以下结果：.1）发现iTR35功能效应分子IL-35通过激活JAK/STAT信号通路，促进HBV特异性CTL细胞表达耗竭分子，导致功能下调；通过阻断该通路后，HBV特异性CTL细胞分泌抗病毒因子INF-γ及TNF-α能力增强，细胞表面耗竭分子表达下降，清除病毒的功能恢复。2）利用蛋白质组学技术发现，IL-35作用CTL细胞后，在生物过程中负调控蛋白占比增加，其中细胞免疫抑制调节及外界刺激调节蛋白受IL-35影响最明显；通过蛋白互作分析显示，差异蛋白功能基本围绕在CTL细胞信号传导，细胞迁移、细胞代谢、细胞抑制等功能范围，这些发现有助于进一步明确IL-35对CTL细胞的抑制作用途径。3）发现处于耗竭状态CTL、CD4+T细胞表面高表达PD-1、LAG-3等耗竭分子，且耗竭程度与肝脏炎症程度（ALT水平）呈正相关；在体外细胞培养研究中发现，用特异性抗体封闭细胞表面耗竭分子能恢复效应T细胞功能。4）发现慢性乙肝患者外周血Treg细胞的核转录因子SATB1表达明显下降，且与HBV DNA载量、肝脏炎症程度呈负相关；发现经转染过表达核转录因子SATB1，可促使Treg细胞向常规T细胞（Tconv）转换，解除对HBV特异性CTL应答的抑制作用。课题研究成果揭示了病毒持续感染导致效应细胞失活的新分子机制，为恢复HBV特异性T细胞功能，实现HBV清除的干预治疗策略提供新的切入点。通过本项目研究，已发表论文7篇，包括6篇SCI收录论文，培养博士研究生1名，硕士研究生3名，专业技术人员数名，研究成果获得2019年度浙江省自然科学奖一等奖，圆满完成原计划的研究目标和预期指标。