鱼类γδ T细胞分离鉴定及其免疫学功能与机制研究

γδ T cells represent a distinct subset of T cells characterized by special γδ T cell receptors (TCRs), which considered to play a vital role in protective immunity. However, the functions and regulatory mechanisms of γδ T cells involved in the interface between innate and adaptive immunity remains poorly understood. Besides, data on their occurrence and functions in fish are still limited. To this end, this project contributes to the cellular and molecular identification of fish γδ T cells and their antigen markers. The structural features, tissue distribution patterns and the possible evolutionary history of the marker genes, as well as the existence and functions of γδ T cells are to be elucidated. To reveal the special evolutionary position of γδ T cells on the transitional state from innate to adaptive immunity, both the innate and primitive adaptive immune functions of this cell subset are sought to be analyzed. Moreover, we attempt to propose that the mutual regulatory mechanisms exist between innate and adaptive immunity, which means not only the innate immunity can promote the adaptive immunity, but also the adaptive immunity exert feedback inhibition to the innate immunity. To define this hypothesis, γδ T cells bridging innate and adaptive immunity are chosen as the research candidate. The professional antigen presenting function of γδ T cells and the related regulatory mechanisms, including costimulatory and TCR signaling, will be first identified. Furthermore, it is expected to confirm that the stress signals delivered by the innate immune cells, such as the macrophages and DCs, will be recognized by γδ T cells and further induce CD8+ CTL activation. In contrast, to certify that γδ T cells mediate the negative feedback regulation of the adaptive immunity to the innate immunity, we sought to analyze whether the IL-2 secreted by αβ T cells could induce the FasL expression on γδ T cells, which finally induce the macrophage apoptosis. Through these investigations, we hope to enrich the knowledge of T cell subsets in fish, certify the γδ T cells as a primitive cell population bridging the innate and adaptive immunity, and put forward to the theory that the γδ T cell-mediated intrinsic relationships and mutual regulations between innate and adaptive immunity are actually exist. Finally, we wish that it will not only be beneficial for better recognition of the evolutionary relationship and regulatory mechanism between innate and adaptive immunity, but also has important application potential in the development of vaccines and immune adjuvants in the prevention and control of fish diseases.

本项目拟首次在鱼类中开展γδT细胞分离鉴定及其功能机制的研究。通过揭示γδT细胞兼具先天（APC）和适应性（αβT）免疫细胞的双重功能，阐明其介于先天和适应性免疫中间进化状态的特殊地位。以此为模型，研究先天免疫通过何种方式与机制激活适应性免疫，反之适应性免疫又通过何种机制实现对先天免疫的反馈调节。前者重点阐明γδT细胞的专职抗原递呈功能及其通过γδTCR/NKG2D信号通路促进共刺激分子表达并增强对应激信号识别从而启动适应性免疫的调节机制；后者重点阐明适应性免疫细胞通过γδTCR-应激信号（γδTCR-HSP60/MIC）促进γδT细胞表达FasL诱导天然免疫细胞凋亡的调节机制。通过项目实施，揭示先天与适应性免疫间的内在联系与相互作用规律，丰富鱼类免疫学内容，为鱼类疫苗和分子佐剂设计提供新的科学依据，也为深入阐明脊椎动物免疫系统及其细胞与分子调节机制的起源进化规律提供理论基础。

γδ T细胞是一类重要而特殊的免疫细胞类群，因其在免疫防御、监视与调节以及多种疾病发生中的突出地位和作用而受到学术界的关注，已成为当前免疫学研究的前沿和热点领域。γδ T细胞被认为是一类起源较早的原始淋巴细胞，起到连接先天和适应性免疫的功能，但对于该假设尚缺乏比较与发育免疫学的直接证据。由于鱼类是早期脊椎动物的代表物种，可以成为相关研究的重要环节。另一方面，当前对于鱼类免疫细胞的研究尚比较薄弱，这是鱼类免疫学亟待加强的重要研究方向。本项目针对上述问题，在鱼类中系统分离鉴定了 T细胞的主要表型分子和功能调节因子如TCR-γ、TCR-δ、CD8α和NKG2D/Illr3b等。构建了其真核或原核表达质粒，开展了蛋白的重组表达和特异性抗体制备，并构建了慢病毒介导的RNA干扰载体系统。在此基础上，成功地从斑马鱼中分离鉴定出 T细胞，深入研究了其组织分布规律、形态结构和表型特征。发现鱼类 T细胞具有吞噬和加工递呈抗原的功能，是一类具有活化初始CD4+ T细胞功能的抗原递呈细胞，在系统性IgM抗体以及肠道粘膜抗体IgZ的产生中发挥重要作用。 T细胞可分别通过αβ T细胞依赖和非依赖的途径介导IgM和IgZ的产生，共刺激信号分子CD40以及共抑制信号分子TIM-3在γδ T介导的适应性体液免疫活化中发挥调节功能。此外，还发现 T细胞具有CD8+ CTL样杀伤功能，能通过诱导细胞凋亡的方式发挥对感染细胞的杀伤作用，其中NKG2D/Illr3b分子对 T细胞的CTL杀伤活性具有调节功能。研究揭示了γδT细胞兼具先天和适应性免疫细胞的双重功能，阐明了其介于先天和适应性免疫中间进化状态的特殊地位。项目研究成果不仅丰富了鱼类免疫学内容，为深入阐释脊椎动物原始淋巴细胞以及适应性免疫系统的起源进化规律提供了理论依据，也为鱼类病害免疫防治中针对基于 T细胞的疫苗开发以及分子佐剂的设计和研制提供了新的思路，具有重要的潜在转化与应用价值。