T Lymphocyte Apoptosis in Hepatitis C Persistence

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染已日益被认为是慢性肝炎、肝硬变和肝细胞癌的主要原因。预计在未来10到20年中，面临最高并发症发生率的20年以上感染者的比例将会增加。大约85%的感染者未能清除病毒。丙型肝炎病毒感染的一个核心悬而未决的问题是，病毒如何在大多数感染者中建立持续感染。最近的一些研究表明，丙型肝炎病毒清除失败与针对该病毒的1细胞反应较弱有关。虽然在大多数慢性感染者中很容易发现丙型肝炎病毒特异的T细胞，但与其他病毒感染相比，它们在肝脏和外周血中的出现频率非常低。目前尚不清楚这是由于未能产生病毒特异性T细胞反应，还是由于激活的效应器1细胞凋亡增加所致。越来越多的证据表明，肝脏可能通过捕获和清除活化的T细胞，特别是CD8+1细胞，在T细胞动态平衡中发挥重要作用。这一建议的中心假设是，丙型肝炎病毒感染的肝细胞会导致肝脏中丙型肝炎病毒特异性T细胞的过早凋亡，导致T细胞反应的减弱。为了支持这一假说，我们的初步研究表明，无论是在体外还是在体内，表达丙型肝炎病毒核心、EL和E2转基因的肝细胞都能加速活化的CD4+和CD8+T细胞的凋亡。与此同时，转基因肝细胞中死亡诱导分子之一Fas配体(FasL或Apo-I配体)的表达上调，并可在体内外被抗Fas抗体阻断。这里提出的实验是专门为扩大我们的观察范围而设计的，并在体内验证我们的假设。具体地说，我们建议：1)确定丙型肝炎病毒转基因小鼠T淋巴细胞凋亡增加的体内意义；2)确定Fas：FasL相互作用是否对该系统中激活的T细胞的凋亡起关键作用；以及3)利用新型腺相关病毒载体在小鼠肝细胞中表达丙型肝炎病毒蛋白，以表征调控激活的T淋巴细胞凋亡的丙型肝炎病毒蛋白。这些数据将证实，丙型肝炎病毒持续存在的一个机制可能是病毒复制主要部位的免疫反应减弱，并可能提出新的治疗策略来增强免疫反应的有效性。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection has been increasingly recognized as a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). The proportion of individuals infected more than twenty years, who face the highest rate of complications, is expected to increase over the next ten to twenty years. Approximately 85 percent of infected individuals fail to clear the virus. A central unresolved issue in HCV infection is how the virus establishes persistent infection in most infected individuals. A number of recent studies have suggested that failure of HCV clearance is associated with weakness in 1-cell responses against the virus. Although HCV-specific T cells are readily demonstrable in most chronically infected individuals, their frequency in the liver and peripheral blood is unusually low, when compared with other viral infections. It is not clear whether this is due to failure of generating virus-specific T-cell response or due to increased apoptosis of activated effector 1 cells. Accumulating evidence has shown that the liver may play an important role in T cell homeostasis by trapping and eliminating activated T cells, especially CD8+ 1 cells. The central hypothesis of this proposal is that hepatocytes infected by HCV causes premature apoptosis of HCV-specific T cells in the liver, leading to attenuation of T-cell response. In support of this hypothesis, our preliminary studies have shown that apoptosis of activated CD4+ and CD8+ T cells was accelerated after exposure to hepatocytes expressing HCV core, El and E2 transgenes either in vitro or in vivo. This was accompanied by up-regulation of the expression of Fas ligand (FasL or Apo- I ligand), one of the death-inducing molecules, in transgenic hepatocytes, and could be blocked by antibodies against Fas in vitro and in vivo. The experiments proposed here are specifically designed to extend our observations, and to validate our hypothesis in vivo. Specifically, we propose to: 1) Determine the in vivo significance of increased T lymphocyte apoptosis in HCV transgenic mice; 2) Determine whether Fas: FasL interactions are critical to apoptosis of activated T cells in this system; and 3) Characterize the HCV proteins regulating apoptosis of activated T lymphocytes using novel adeno-associated virus vectors to express HCV proteins in murine hepatocytes. These data would confirm one mechanism of HCV persistence may be attenuation of the immune response in the principal site of viral replication, and might suggest new therapeutic strategies to augment the effectiveness of the immune response.