草鱼CD8+记忆型T细胞反应的特征和分子机制研究

Memory immunity is one of the most important characteristics of the adaptive immune system, and is the foundation of vaccine development. In recent years, in mammals, the kinetics of the CD8+ memory T cell response and their functions and phenotypes and the molecular regulations by inflammatory cytokines and transcription factors have been illustrated. However, the cellular memory immunity of fish, in which the classical adaptive immune system first emerged, have been known little till now. In this study, we will first determine the kinetics of grass carp CD8+ memory T response by monitoring the cell number of the specific CD8 and MHC I-tetramer double positive subgroup. The naïve, effector and memory CD8+ T cells will be selected at different time points and their cytotoxicities and proliferative protentials will be tested in vitro. The genetic expressions of these three cell types will be profiled by RNA–seq, and the differential genes in them will be screened out to distinguish each other. According to the research background in mammals, the molecules playing a key role in CD8+ memory T response will be cloned and expressed, and their monoclonal antibodies will be produced to further identify the subtypes of the memory cells. At last, the molecular regulation of the memory response by candidate inflammatory factors and transcription factors will be explored. This study will systemically elucidate the molecular features and mechanisms of CD8+ memory T response in grass carp, and will be benefit for the further studies of fish and comparative immunology, and guide the fish vaccine development

免疫记忆是获得性免疫系统的最重要特征之一，也是疫苗设计的基础。近年来，哺乳动物免疫记忆反应的分子机制研究进展显著，阐释了CD8+记忆型T细胞形成的周期、细胞功能与表型特征和胞内外信号的调控机制。然而，在获得性免疫系统最早出现的鱼类中，细胞性免疫记忆的研究还很欠缺。本研究利用草鱼MHC I四聚体和CD8单抗，通过流式细胞术监测特异性CD8+T细胞数目的变化，确定记忆型细胞产生的周期；在不同节点上分离幼稚型、效应型和记忆型CD8+T细胞，鉴定其杀伤活性和增殖能力，并进行比较转录组测序。结合哺乳动物研究基础，寻找关键的差异表达基因，确定记忆型细胞表型和转录因子表达特征。克隆、表达关键性分子并制备单抗，探索草鱼记忆型细胞亚型的分类，以及胞外炎症因子和胞内转录因子的影响。本研究将系统的阐释草鱼CD8+T细胞反应的特征和分子机制，推动鱼类免疫学和比较免疫学的研究，并为鱼类疫苗的开发设计提供理论基础。

关于鱼类效应性和记忆性细胞性免疫的研究十分欠缺，本项目从草鱼MHC I和CD8分子入手，克隆了大量草鱼MHC-I等位基因，对草鱼MHC-I分子的多态性进行了系统进化分析，解析了草鱼MHC-I（Ctid-UAA）的晶体结构，对鱼类特有的beta微球蛋白（β2m）多态性进行了分析，解析了两种不同的草鱼β2m结构及形成的两种不同MHC-I复合体结构，阐释了草鱼β2m对于多肽结合的影响及其结构基础，制备了草鱼CD8分子的单克隆抗体，为解决缺乏纯系草鱼的问题，绘制了模式动物斑马鱼的MHC-I分子的表达谱，测定了主要表达的UBA分子的多肽结合基序，筛选了鲤春病毒和草鱼出血症病毒的表位，构建了草鱼出血症病毒弱毒苗感染斑马鱼的模型，以上结果为检测鱼类幼稚型、效应型和记忆型CD8+T细胞，鉴定鱼类记忆型细胞亚型的分类，探索鱼类CD8+T记忆型T细胞反应的特征和分子机制奠定了基础。