丙肝病毒感染者T细胞免疫反应及其亲和性的研究

Hepatitis C virus (HCV) infection is a major public health concern with approximately 3% of the worlds population being infected. Twenty to forty percent of HCV patients naturally clear their infections, presumably through immune mechanisms. Of those patients that fail to naturally clear their infections due to the defect of T cells responses, approximately half respond to fail anti-viral therapy. However, this leaves a large number of patients with persistent infection that eventually leads to cirrhosis of the liver or hepatocellular carcinoma which are the leading indications for liver transplantation. Therefore, any new therapy capable of treating patients with chronic HCV infections or HCV-related malignancies would be a significant public health benefit. T cells responses is mainly dependent on the T cell affinity and T cell receptor is a key role to define the T cell specificity and affinity. We have previously shown that it is possible to use retroviral vectors encoding TCR genes isolated from HCV NS3:1406-1415 and HCV NS3:1073-1081 reactive T cell clones to engineer normal peripheral blood derived T cells to recognize HCV peptide loaded cells and cells transfected to express HCV sequences. These HCV TCRs has sufficient affinity to enable both CD8+ and CD4+ T cells to recognize HCV antigen presented in the context of MHC class I molecules. Furthermore, the HCV TCR can recognize seven of eight naturally occurring muatnt variants of NS3:1406-1415 and HCV NS3:1073-1081 suggesting that T cells expressing these TCRs may be effective in combating mutant escape variants of HCV and is highly promising procedure for treatment of HCV chronic infection and HCV-related diseases. In this application, we propose to test the following major hypotheses. First, we hypothesize that HCV-infected individuals that have resolved their infections have T cells with higher avidity, higher TCR affinity, and broader crossreactivity with mutant HCV peptides than patients with chronic infections. Second, we hypothesize that HCV TCR gene modified T cells can well recognize HCV and its mutations in vivo and the TCR tranduced CD4+ T cells can help the TCR transduced CD8+ T cell function. We will use a combination of in vitro assays and the Nude mouse model to test these hypotheses. Sucessful completion of these studies outlined in this application will provide useful information related to the genetics of the anti-HCV immune response as well as the necessary preclinical data to support phase I clinical trials of HCV TCR gene modified T cells for patients with chronic HCV and HCV-related malignancies.

丙肝病毒（HCV）感染者体内T细胞免疫反应降低是HCV感染慢性化的主要原因，T细胞的亲和性是决定T细胞免疫反应的关键因素，而T细胞的特异性和亲和性主要是由T细胞受体（TCR）决定的，从HCV特异性T细胞中克隆高亲和性的HCV特异性TCR基因并把其导入患者的T细胞中，使这些T细胞成为识别并清除HCV感染的特异性T细胞，研究这些T细胞的功能和特性以及探讨其成为丙肝治疗效应T细胞的可能性是国际上研究的重点领域。我们前期工作已经克隆到了两个HCV NS3区域的特异性TCR。本课题将在前期的研究基础上进一步研究HCV感染者体内HCV特异性T细胞免疫反应及TCR亲和力的多样性；分析不同亲和力TCR转染的T细胞对基因变异HCV的杀伤能力，然后在动物模型体内研究TCR转染的T细胞对HCV抗原的识别能力和CD4+T细胞对CD8+T细胞清除HCV抗原的辅助作用。为T细胞在HCV感染及相关疾病应用提供实验证据。

HCV在机体内的持续存在是引起慢性丙型肝炎的主要原因。在病毒复制及感染的过程中CD8+T细胞发挥着重要的作用，然而在慢性感染的过程中大部分CD4+T细胞和CD8+T细胞都出现功能失调的现象，体外培养并克隆出HCV特异性的TCR，为慢性丙型肝炎治疗提供新的方法。项目开展的过程中，首先我们研究了慢性丙型肝炎患者中CD4+T细胞和CD8+T细胞的功能，研究发现Treg细胞在慢性丙型肝炎患者中的比例升高，且高表达CD39，CD73等抑制性标志物；CD8+T细胞的杀伤功能在不进行抗病毒治疗的过程中是减弱的。其次我们研究HLA-A2在我国慢性丙型肝炎患者中的表达，并探讨HLA-A2的表达与慢性丙型肝炎患者抗病毒治疗的相关性，研究发现HLA-A2+的患者通过抗病毒治疗更容易获得快速病毒学应答和稳定病毒学应答。最后我们分析了慢性丙型肝炎患者外周血中CD8+T细胞TCR的多样性，研究发现TCR多样性与患者的抗病毒治疗周期具有一定的联系。同时我们也培养了HCV特异性的克隆，并对这些克隆做了亲和性的分析，将于今年年底完成所有的标书规定的研究方向，为TCR转基因T细胞在临床上的应用提供理论基础，同时我们也对食管癌和肝癌进行了同样的分析，试图研究TCR转基因T细胞对实体瘤的作用。