应用人源化小鼠研究皮肤区域免疫耐受机制

Regional immune system in skin is composed of a variety of specialized immune cells and immunoregulatory molecules, which possesses unique regional immune features. In human, it has been indicated that there is usually a significant population of T cells that specifically recognize the melanocyte associated antigen MART1 expressed by human melanocytes in skin. However, these MART1-specific T cells maintain tolerant in normal individuals and do not attack self-cells which express MART1. The maintenance and breaking of this immune tolerance involve co-participation and co-regulation of various immune cells and molecules in skin region，of which the mechanisms have not been well explored thus far. In this project, we will clarify the mechanisms for tolerance of these MART1-specific T cells in skin region as a starting point of the study. We will establish an animal model of the maintenance and breaking of the immune tolerance in human skin by transplanting homologous human skin tissue to MART1-specific TCR-transgenic humanized mice (MART1-TCR-Tg hu-mice). We wil also examine the distribution, phenotype and tissue resident characteristics of the MART1-specific T cells in the skin. Moreover, we will apply the two-photon confocal microscopy technique and single-cell sequencing-based analysis to study the dynamic population formation, function and gene spectrum characteristics of the tissue resident T cells in the skin. Based on the above analysis, we could explore the functions and mechanisms of the multiple skin immune cells, immunoregulatory and signal transduction molecules in the immune tolerance and the activation of the MART1-specific T cells. Combining with the systematic analysis on human skin samples, the proposed studies are expected to provide comprehensive insight into the basic immunological characteristics of human skin, and help to develop new effective therapies for patients with skin autoimmune disorders.

皮肤区域免疫系统由多种免疫细胞和功能分子组成，具有独特的区域免疫特性。研究表明部分人体内有大量皮肤组织抗原-黑色素抗原（MART1）特异性T细胞，但不发生对靶细胞的攻击。这种免疫耐受的维持涉及到皮肤区域多种免疫细胞与分子的共同参与和调控，机制尚不清楚。本研究将以MART1特异性T细胞在皮肤区域的免疫耐受机制为切入点，通过移植同源人皮肤给MART1-TCR转基因人源化小鼠，建立人皮肤免疫耐受维持及打破的动物模型；检测MART1特异性T细胞在皮肤组织中的分布、表型及定居特征；应用双光子共聚焦显微镜及单细胞测序等技术解析人皮肤定居T细胞的动态形成过程、功能特点和基因谱特征；以此为基础研究皮肤区域多种免疫细胞、功能分子和信号通路分子在MART1特异性T细胞免疫耐受中的作用及机制；结合对人皮肤样本的系统分析，全面解析人皮肤区域基础免疫学特性，为皮肤区域免疫紊乱相关疾病机制的阐明提供新的研究思路。

皮肤区域免疫系统由多种免疫细胞和功能分子组成，具有独特的区域免疫特性。部分人体内有大量皮肤组织抗原-黑色素抗原（MART1）特异性T细胞，但不发生对靶细胞的攻击。这种免疫耐受的维持涉及到皮肤区域多种免疫细胞与分子的共同参与和调控，机制尚不清楚。本研究以MART1特异性T细胞在皮肤区域的免疫耐受机制为切入点，首先建立了造血细胞表达MART-1抗原的MART-1-TCR转基因人源化小鼠，在活体条件下揭示了人体中枢免疫耐受现象。进而成功实现了人胎皮肤在NOD/SCID-IL2Rgamma-/-小鼠上的移植，并结合免疫系统人源化小鼠模型，建立了具有人皮肤和同源人免疫系统重建的“皮肤-免疫”双人源化小鼠模型，为研究人皮肤区域免疫学问题提供强大的工具。在“皮肤-免疫”双人源化小鼠模型的基础上，建立了MART-1-TCR转基因的皮肤-免疫双人源化小鼠模型；通过区域炎症诱导首次建立人源化小鼠白癜风模型（即由人免疫系统导致的人类皮肤黑色素细胞损伤的人源化小鼠模型），表现为移植在人源化小鼠上的人皮肤内人黑色素抗原特异性T细胞（即，MART-1-TCR+CD8+T细胞）浸润，皮肤毛发由黑色转为白色；在人MART-1-TCR+CD8+T细胞大量浸润的皮肤基底层，人黑色素细胞破坏显著。人源化小鼠白癜风模型的成功建立将为深入研究人皮肤组织定居T细胞的动态形成过程、功能特点和基因谱的特征提供有力平台和手段。另外，本项目在免疫系统人源化小鼠研究领域取得多项原创性结果，主要包括：（1）成功构建由人胰岛抗原特异性T细胞在同源人免疫系统监视下导致胰岛beta细胞免疫损伤的I型糖尿病（T1D）人源化小鼠模型，揭示了人T1D发病中自身反应性CD4+T细胞的重要作用。（2）建立了同时具有人肝细胞嵌合和同源人免疫系统重建的人源化小鼠模型，阐明人的肝脏细胞在肝脏免疫细胞谱的发育和维持中的重要作用。（3）成功构建人急性淋巴细胞白血病人源化小鼠模型，发现骨髓部位的免疫豁免状态是白血病患者免疫治疗后复发的重要原因。