肝脏驻留γδT细胞的区域免疫可视化与细胞亚群全景图谱研究

In the previous project we have found two novel subsets of liver-resident γδT cells and have established the innovative techniques of multicolor intravital microscopic imaging and in vivo optical spatio-temporal imaging of liver. In the present project, we will combine the single-cell sequence, bioinformatics, the multicolor intravital microscopic imaging and in vivo optical spatio-temporal imaging techniques to determine the clustering, phenotype and functional characteristics of intrahepatic γδT cells. The movement and migration characteristics of liver-resident γδT subsets will be dynamically monitored and compared with the traditional circulating immune cells in the liver. The distribution, adhesion and interactions of γδT subsets with the hepatic sinus endothelial cells, kupffer cells and other intrahepatic lymphocytes will be real time monitored by the multicolor intravital microscopic imaging technique. Further, the exact roles of liver-specific γδT cell subsets in maintaining liver immune tolerance and immune surveillance will be confirmed exquisitely in murine HBV- and MCMV-infected models respectively. The aims of this study are to comprehensively depict the panoramic pattern of the regional immunological characteristics and function of liver-specific γδT cells and its relationship with the development of liver-related diseases, and to determine the key functional molecules and potential intervention targets of γδT cell subsets. The panoramic description and deeply understanding of the liver-specialized immunologic features of γδT cells and their roles in sustaining liver immunotolerance or promoting immune surveillance and in the development of liver-related diseases will certainly lead to the development of novel therapeutic strategies for liver diseases.

本项目拟集成前期所发现的肝脏独特的组织驻留 γδT细胞亚群和所成功建立的肝脏区域免疫可视化研究新方法，通过进一步结合单细胞测序技术,确定肝内 γδT细胞的分群、表型和功能特点；通过多色标记的活体显微成像技术和活体肝脏的三色高分辨光声显微成像技术实时动态比较肝内肝脏驻留 γδT细胞与传统循环免疫细胞在肝内的运动轨迹、迁移、驻留等运动特征及肝脏 γδT细胞亚群与肝窦内皮细胞、Kupffer细胞及其他肝内淋巴细胞的分布、黏附和相互作用；通过建立不同肝脏疾病模型（HBV、MCMV感染）确定肝脏特有 γδT细胞亚群在维持肝脏免疫耐受和发挥免疫监视中的确切作用。力争全景描绘肝脏特有 γδT细胞的区域免疫特性以及与肝脏相关疾病发生发展的关系，确定 γδT细胞亚群发挥作用关键功能分子和潜在干预靶点，为肝脏相关疾病的治疗提供新的思路和治疗策略。

肝脏不仅是重要的代谢器官，也是重要的免疫器官和髓外造血器官。肝脏为γδ T细胞较为丰富的器官，但是肝脏γδ T细胞的亚群组成、发育来源及其区域免疫特性尚缺乏系统全面的研究。本项目结合单细胞测序技术、生物信息学分析、流式细胞术细胞表型鉴定和体内外生物学功能验证，系统描绘了小鼠肝脏γδ T细胞的分群、表型和功能特点，阐明了肝脏γδ T的区域免疫特性。探究和描绘了肝脏γδ T细胞的肝内发育特点及肝脏微环境对肝脏髓外造血的调控机制，为胸腺为γδ T细胞发育、分化与成熟的场所提供了依据。建立了适用于肝脏的长时程活体光学成像方法，为实时动态观察肝脏微环境不同细胞间的相互作用提供了可视化研究新技术。基于以上发现和技术，确定了肝脏特有γδ T细胞在肝脏肿瘤和感染性疾病中发挥的作用，全景描绘了肝脏特有γδ T细胞的区域免疫特性以及与肝脏相关疾病发生发展的关系，为肝脏相关疾病的治疗提供了新的思路和治疗策略。