Liu Chen, Zhu Haizhen, Tu Zhengkun, Xu Yi-Ling, Nelson David R
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL, USA.
Hepatology. 2003 Jun;37(6):1335-42. doi: 10.1053/jhep.2003.50207.
The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.
宿主免疫反应与受感染肝细胞之间的相互作用在丙型肝炎病毒（HCV）发病机制中起核心作用。缺乏合适的动物或体外模型阻碍了我们对宿主T细胞/HCV相互作用的理解。我们的目的是建立一个体外模型来研究HCV特异性T细胞介导的抗病毒和细胞溶解功能机制。对HCV复制子进行HLA分型，并从一名HLA I类匹配的受试者获取淋巴细胞。用2种针对NS3的HCV特异性/HLA限制性肽扩增CD8(+) T细胞。将淋巴细胞制剂与用（51）Cr标记的HCV复制子（FCA1）细胞和对照（Huh7）细胞共培养。孵育48小时后，收获细胞用于RNA提取。在存在抗干扰素γ（IFN-γ）、抗肿瘤坏死因子α（TNF-α）和抗FasL的情况下进行标准阻断试验。通过（51）Cr释放测量细胞溶解活性。HCV复制子细胞表达纯合的HLA-A11等位基因并呈递HCV非结构蛋白。通过Northern印迹分析，CD8(+)细胞的HCV特异性扩增导致HCV复制下降10倍，FCA1细胞特异性裂解21%（与对照Huh7细胞的2%相比）。这种抗病毒活性的20%独立于T细胞结合，提示细胞因子介导的抗病毒活性。阻断IFN-γ或FasL可显著降低CD8(+)抗病毒作用，但阻断TNF-α则无影响。总之，HCV特异性CD8(+)细胞通过细胞因子介导的和直接的细胞溶解作用抑制病毒RNA复制。这个T细胞/HCV亚基因组复制子系统代表了一个研究CD8细胞与HCV感染肝细胞相互作用的模型。
