鱼类Tim基因介导的适应性免疫活化调控新机制

The T cell Ig and mucin (Tim) molecules are a newly identified immune-relevant family of proteins involved in various cellular activities and diseases. However, the role of this family member plays in adaptive immunity remains poorly understood, and data on their occurrence and functions in fish are limited. To this end, this project focuses on the molecular identification and functional characterization of Tim family in teleost fish. The structural features, tissue distribution patterns, subcellular localizations and the possible evolutionary history of this family are to be elucidateed. To understand the signaling pathways and mechanisms underlying the initiation of adaptive immunity, the role of Tim-1 and Tim-4, two critical hallmark molecules distributes on CD4+Th and dentritic cell (DC) surfaces, plays in the activation of adaptive immunity will be particularly investigated. It is hoped that Tim-1 and Tim-4 may serve as reciprocal molecules and function as a novel costimulatory signal, which drives the adaptive immunity together with the known costimulatory molecules such as B7 and CD40. Furthermore, we also attempt to propose that the pathogen-induced apoptosis signal, such as the phosphatidylserine (PtdSer) exposed on the surfaces of apoptotic bodies, is a novel initial signal, which is essential to the activation of DC and CD4+Th in adaptive immunity. To define this hypothesis, interaction between Tim-4 and PtdSer, and its role in DC and CD4+Th activation will be characterized. It is expected to demonstrate that Tim-4, as a receptor of PtdSer, mediates apoptotic-signal transduction by receptor tyrosine phosphorylation signaling pathway. For this, the key adaptors and protein components in this Tim-4 mediated signaling pathway will be in-depth identified. Through these researches, we hope to put forward a new theory that the pathogen-induced apoptotic signal serves as a novel signal, which cooperates with the signals from pathogens and costimulatory factors to constitute a "tri-signal system" in adaptive immunity. We hope that it will not only improve the traditional "bi-signal system" of the activation of adaptive immunity, but also has important application potential in the development of vaccines and immune adjuvants in the prevention and control of fish diseases.

本项目拟在鱼类中系统开展Tim家族的基因克隆与鉴定，阐明其分子结构、细胞定位和分子进化等基本特征和规律；针对适应性免疫通过何种信号途径和机制被激活这一重大科学命题，探索Tim-1和Tim-4家族成员在鱼类适应性免疫活化中的共刺激信号功能与作用机制，揭示基于Tim分子调控的新型共刺激信号系统，丰富共刺激信号学说的内容；同时探索病原诱导的细胞凋亡信号在适应性免疫活化中的作用，阐明凋亡信号是适应性免疫活化必需的重要起始信号；证明Tim-4是凋亡信号分子磷脂酰丝氨酸的受体并介导了凋亡信号对适应性免疫的调节，阐明Tim-4的受体酪氨酸磷酸化信号转导途径；提出凋亡信号、病原信号和共刺激信号构成适应性免疫活化"三信号"系统的新观点。研究成果不仅将修正和完善传统的适应性免疫双信号活化理论，具有重要的原创性理论意义，而且可为鱼类病害防治中疫苗及免疫佐剂的开发与应用提供新的思路和策略，具有重要的潜在应用价值。

天然免疫调控适应性免疫起始活化的分子机制是长期来备受关注的免疫学前沿科学问题。适应性免疫起始活化是指抗原递呈细胞（APC）激活初始T细胞增殖并分化成不同效应细胞进而引发抗原特异性细胞和体液免疫的过程，其中APC递呈的抗原肽及其表达的共刺激信号分子发挥了重要作用。目前已分离鉴定了若干关键的共刺激信号分子如CD80、CD86、CD83和CD40等，但对于是否存在其他的共刺激信号分子仍受到高度关注。T细胞免疫球蛋白粘蛋白（TIM）是新近发现的一类Ⅰ型跨膜糖蛋白家族成员，参与多种感染和自身免疫性疾病的发生，但对于其在适应性免疫中的功能尚缺乏深入研究。本项目首次在鱼类中系统开展了Tim家族的分子鉴定与免疫学功能研究。揭示了TIM-1和TIM-4分布于CD4+ T和MHCII+ APC细胞并受抗原诱导表达；TIM-1和TIM-4作为一对相互结合的互作分子，对抗原特异性CD4+ T细胞和IgM+ B的增殖活化以及抗体IgM的产生起到关键的促进作用，表明TIM-1和TIM-4是一对重要的共刺激信号分子。发现TIM-1在静息CD4+ T细胞中分布于胞内高尔基体，在TIM-4和Ca2+ 激动剂刺激下从细胞质迁移至细胞膜表面，从而启动CD4+ T细胞增殖活化，这代表了一种通过分子转位效应调节适应性免疫的新机制。还发现了一种新的可溶性sTIM-4可变剪接体，它通过竞争性结合CD4+ T细胞表面的TIM-1分子，从而阻断APC表面TIM-4与TIM-1的结合而抑制抗原特异性CD4+ T细胞的增殖活化，在Th2型体液免疫中发挥负调节作用。发现磷酯酰丝氨酸PtdSer能促进白细胞表达TIM-1、TIM-4、MHC-II以及共刺激分子CD80/86和CD40，同时伴随抗原特异性CD4+ T细胞增殖活化以及IgM抗体产生，表明PtdSer介导的凋亡信号通路在TIM-1-TIM-4介导的适应性免疫起始活化中发挥调节作用。此外，还发现TIM-3在γδ T细胞介导的CD4+ T 细胞增殖活化以及适应性体液免疫活化中发挥抑制性调节作用，表明TIM-3是一种共抑制信号分子。研究成果不仅丰富了适应性免疫活化的共刺激信号学说，充实了鱼类免疫学理论，而且对于我国渔业生产中的病害防治具有重要潜在应用价值，可为鱼类病害防治中疫苗和分子佐剂的设计提供新思路、开辟新途径。