Evaluation of Intrahepatic Immune and Virological Mechanisms of Immunopathology in Chronic Hepatitis B Patients using Fine Needle Aspiration Biopsy

T细胞耗尽是慢性病毒感染的一个重要特征。可以说，没有其他人类疾病比慢性乙肝病毒(乙肝病毒)感染更能导致抗原特异性T细胞耗尽。大多数患者在出生时就被感染了。乙肝病毒是一种DNA病毒，与丙型肝炎病毒(丙型肝炎病毒)、丙型肝炎病毒或人类免疫缺陷病毒(HIV)等其他病毒相比，变异率较低。再加上病毒和抗原的高负载量，慢性乙肝患者表现出严重的T细胞免疫衰竭。在患有肝癌的慢性乙肝患者中，这种极度疲惫的情况尤为突出，Gehring实验室表明，即使是肿瘤抗原特异性T细胞反应也强于乙肝病毒特异性反应。T细胞耗竭的机制已经在小鼠模型中定义，并被转化为检查点抑制剂治疗。抗PD-1治疗在体外部分恢复了HBV特异性T细胞免疫，并可能改善CHB患者的治疗。然而，检查点抑制剂治疗只能引起部分反应，因为抑制受体的表达只是控制T细胞功能的几个因素之一。对于慢性乙肝，慢性感染期间T细胞免疫的恢复意味着1)克服T细胞耗竭的特异性缺陷，2)打破耐受性肝脏环境--癌症免疫疗法熟悉的障碍。Gehring实验室的初步工作研究了来自不同患者队列的血液和肝脏样本，以确定肝脏炎症期间的不同免疫模式。利用肝脏活检的单细胞RNA测序，他们鉴定出一组CD8T细胞具有激活的组织驻留转录表型，这是肝脏炎症患者独有的。该人群表达高水平的抑制受体PD-1、TIM-3和LAG-3。与抑制表型相反，CD8T细胞群是唯一可检测到抗病毒细胞因子干扰素-γ和细胞毒效应分子Fas配体的T细胞群。这些数据表明，肝脏炎症期间的环境压倒了抑制性受体的表达，促进了T细胞的功能。因此，我们假设慢性乙肝患者肝脏红斑的免疫学定义将提供一种能够打破肝脏耐受性的免疫模式。明确炎症环境有可能确定免疫治疗靶点，从而恢复T细胞功能，用于慢性病毒感染或癌症的免疫治疗。这项拟议的研究将：1)验证CD8 T细胞与其遗传特征相对应的表型，并确定CD8 T细胞群是否是乙肝病毒特异性的；2)确定激活的CD8 T细胞群产生的静止T细胞群；3)确定驱动CD8 T细胞群功能的炎症环境。其目标是针对肝脏中特定的CD8T细胞进行免疫治疗，并确定有效的抗病毒免疫反应。

T cell exhaustion is a defining characteristic of chronic viral infection. There is arguably no other human disease that results in greater antigen-specific T cell exhaustion than chronic Hepatitis B virus (HBV) infection. A majority of patients are infected at birth. HBV is a DNA virus with a low mutation rate compared to other viruses such as hepatitis C virus (HCV) HCV or human immunodeficiency virus (HIV). Coupled with high virus and antigen load, chronic hepatitis B (CHB) patients display profound exhaustion of T cell immunity. The extreme exhaustion is highlighted in CHB patients with liver cancer, where the Gehring lab showed that even tumor antigen specific T cell responses were greater than HBV-specific responses . Mechanisms of T cell exhaustion have been defined in mouse models and translated to checkpoint inhibitor therapy. Anti-PD-1 therapy partially restores HBV-specific T cell immunity in vitro and may improve therapy in CHB patients. However, checkpoint inhibitor therapy elicits only a partial response because inhibitory receptor expression is only one of several factors governing T cell functionality. For CHB, restoration of T cell immunity during chronic infection means 1) overcoming T cell specific defects of exhaustion and 2) breaking the tolerogenic liver environment – obstacles familiar to cancer immunotherapy.Preliminary work in the Gehring lab investigated blood and liver samples from different patient cohorts to define distinct immune profiles during liver inflammation. Using single-cell RNA sequencing of liver biopsies, they identified a population of CD8 T cells with an activated tissue-resident transcriptional phenotype that was unique to patients with liver inflammation. The population expressed high levels of the inhibitory receptors PD-1, Tim-3 and Lag-3. In contrast to the inhibitory phenotype, this CD8 T cell population was the only one where the antiviral cytokine, IFN-γ, and cytotoxic effector, Fas ligand, were detectable. These data suggest that the environment during liver inflammation overrides inhibitory receptor expression, promoting T cell function. Therefore, we hypothesize that immunologic definition of hepatic flares in CHB patients will provide an immune profile capable of breaking liver tolerance. Defining the inflammatory environment has the potential to identify immunotherapeutic targets that could restore T cell function for immunotherapy of chronic viral infections or cancer. The proposed study will i) validate the phenotype of CD8 T cells corresponding to their genetic signature and determine if the CD8 T cell population is HBV-specific; ii) identify the resting population of T cells from which the activated CD8 T cell population arises and iii) define the inflammatory environment that drives the function of this CD8 T cell population. The goal is to target specific CD8 T cells for immunotherapy in the liver and to define an effective antiviral immune response.