【作者】 李洋；

【导师】 杨永广；

【作者基本信息】 吉林大学 ， 免疫学， 2019， 博士

【摘要】 实验背景:人类自身免疫性疾病越来越普遍,一方面原因是由于在整个基因库中保留了许多易感基因,另一方面原因是在过去一个世纪中出现了新的环境因素变化。这些疾病是由不受控制的自身反应性T细胞引起的,其存在于患者和许多无症状个体的外周血液循环中,可归因于不完全的胸腺选择过程。T细胞在胸腺发育期间获得T细胞受体(TCR),于皮质中与自身MHC结合进行阳性选择,而后于髓质中与MHC/自身肽复合物结合进行阴性选择,导致清除大多数自身反应性T细胞,其余成熟T细胞可以释放到外周。我们对胸腺选择机制的了解大多来自于动物模型。一些对人类在心脏手术期间被摘除的胸腺组织进行的研究,提供了关于人胸腺细胞表型和分布的一些信息,对其功能研究的见解有限。然而,除了血液样本之外,人体免疫细胞和组织的纵向研究是不可行的,并且使用人胸腺组织来评估与其抗原特异性相关的胸腺细胞的发育和命运是具有挑战性的。现在有可能从单个人供体获取胸腺组织和造血干细胞(HSC),产生多个“相同”的人源化小鼠,这些小鼠允许纵向研究以及各种操作,例如将特定TCR和/或抗原引入造血干细胞中,这将在小鼠上重建人类免疫系统。这种体内模型为研究人类自身反应性T细胞的胸腺发育,APC特定亚群对中枢耐受的贡献以及双重TCR表达在自身免疫和肿瘤免疫中的作用开辟了新的可能性。实验目的:本研究目的为建立T细胞胸腺选择的免疫系统人源化小鼠模型,以充分地了解人体自身反应性T细胞在胸腺中的发育过程及其表型的变化,更好地了解各种类型的抗原呈递细胞在胸腺选择中的作用。实验方法:(1)用基因工程方法制备带有MART1 TCR基因和MART1抗原肽-GFP的慢病毒载体。包装慢病毒感染HLA-A2+MART1~-肿瘤细胞,以鉴定MART1抗原肽功能。(2)构建免疫系统人源化小鼠模型:NSG小鼠先接受亚致死剂量1.5Gy全身照射,肾被膜下移植胚胎胸腺组织。MART1抗原肽实验组小鼠尾静脉同时输注1×10~5转导MART1 TCR基因的造血干细胞和1×10~5转导MART1抗原肽基因的造血干细胞。另一组为对照组小鼠将同时输注1×10~5转导MART1 TCR基因的造血干细胞和1×10~5转导对照慢病毒的造血干细胞。用流式细胞术(FCM)测定人源化小鼠中人的造血细胞重建的水平。(3)检测人源化小鼠模型各组织中人抗原呈递细胞亚群。(4)检测分析抗原肽存在与否对MART1 TCR特异性T细胞表型变化、辅助分子及Treg细胞分化的影响。实验结果:我们建立了一种免疫系统人源化小鼠模型,通过转染造血干细胞引入了自身反应性TCR和同源自身抗原,其使在体内分析人T细胞的胸腺选择成为可能。本研究发现造血干细胞进入胸腺后发育为T细胞、及多种抗原呈递细胞(APC)亚群,胸腺对自身反应性T细胞克隆清除效果显著,通过胸腺选择的TCR特异性T细胞表现为CCR7~+PD-1~-,而被克隆清除的T细胞表现为CCR7~-PD-1~+。MART1 TCR特异性T细胞在胸腺中接触自身抗原后其TCR,CD3,CD8和CD4的水平显著降低。本研究另发现T细胞上第二条TCRα链的生成影响MHC I类限制性T细胞发育为CD4+,特别是调节性T细胞。结论:本论文的人源化小鼠模型是可用于研究人胸腺抗原特异性T细胞与一般多克隆胸腺细胞群发育的有力工具,其允许在有无自身反应性抗原肽存在的情况下更准确地分析T细胞表型。可以利用它来鉴定在人类自身免疫疾病哪些特定自身反应性T细胞克隆经历中枢耐受并且是否在胸腺选择后彻底清除。该模型的建立对于研究T细胞免疫耐受、自身反应性T细胞导致的自身免疫疾病的发生,以及胸腺选择逃逸机制的研究具有重要的应用价值。更多还原

【Abstract】 Background:Human autoimmune diseases are increasingly prevalent,partly because of retention of many susceptibility genes in the overall gene pool and partly because of new environmental factors that have surged in the past century.These diseases are contributed by uncontrolled autoreactive T cells,whose presence in the blood circulation of patients as well as many asymptomatic individuals,can be ascribed to an imperfect thymic selection process.T cells acquire a T cell receptor(TCR)during their development in the thymus,and then become exposed to self-MHC for positive selection,and to MHC/self-peptide complexes for negative selection,leading to a purge of most autoreactive T cells before the remaining mature T cells can be released into the periphery.Most of what we know regarding mechanisms of thymic selection comes from animal models.Some studies performed on human thymic tissue,usually removed during cardiac surgery,have offered snapshots on the phenotype and distribution of human thymocytes,with limited insights on their function.However,longitudinal studies of human immune cells and tissues are not feasible beyond blood samples,and using human thymic tissue to assess the development and fate of thymocytes in relation to their antigen specificity is challenging.It is now possible to generate multiple‘identical’humanized mice from a single human donor that allow longitudinal studies as well as various manipulations such as introduction of specific TCRs and/or antigens into the hematopoietic stem cells(HSCs)that will reconstitute the human immune system.This in vivo model opens new possibilities for studying the thymic development of human autoreactive T cells,the contribution of specific subsets of APCs to central tolerance and the implications of dual TCR expression in autoimmunity and tumor immunity.Objective:The purpose of this study was to establish a humanized mouse model of T cells thymic selection,to fully understand the developmental process and phenotypic changes of human autoreactive T cells in the thymus,and to better understand the function of various antigen presenting cells in thymus selection.Methods:(1)A lentiviral vector carrying the MART1 TCR gene and the MART1antigen peptide-GFP was prepared by a genetic engineering method.The lentivirus was packaged to transduce HLA-A2+MART1~-tumor cells to identify the function of the MART1 peptide.(2)Establishment of humanized mouse model with human immune system:NSG mice were conditioned with sublethal(1.5 Gy)toatal body irradiation,and transplanted fetal thymus tissue graft under the kidney capsule.MART1 peptide group mice were simultaneously infused with1×10~5 MART1 TCR transduced hematopoietic stem cells and 1×10~5MART1 peptide transduced hematopoietic stem cells.Another group of control mice were simultaneously infused with 1×10~5 Mart-1 TCR transduced hematopoietic stem cells and 1×10~5 control lentivirus transduced hematopoietic stem cells.The level of human hematopoietic cell reconstitution in humanized mice was determined by flow cytometry(FCM).(3)Detection of human antigen presenting cell subsets in each tissue of a humanized mouse model.(4)Analyzing the phenotypic changes and costimulatory molecule alteration of MART1 reactive TCR T cells and Treg cells development influenced by absent or present autoantigen.Results:We developed a humanized mouse model involving transduction of autoreactive TCRs and cognate autoantigens on human hematopoietic stem cells,which allows analysis of thymic selection of human T cells in vivo.This study found that hematopoietic stem cells developed into T cells and a variety of antigen-presenting cells(APC)subpopulations after entering the thymus.The effect of thymus deletion of autoreactive T cell clones was significant.TCR-specific T cells pass through thymic selection behaved as CCR7~+PD-1~-,while the thymic deleted T cells showed CCR7~-PD-1~+.MART1 TCR T cells down-regulated TCR,CD3,CD8 and CD4 after they encountered autoantigen in the thymus.This study also provide evidence that secondary TCRαinfluence MHC class I-restricted T cells to develop as CD4+,particularly regulatory T cells.Conclusions:The humanized mouse model described created a powerful tool to study the thymic development of human antigen-specific T cells side-by-side with the general polyclonal thymocyte population,allowing a more accurate dissection of T cell phenotypes in the presence or absence of a specific self-antigens.It may be leveraged to address the extent to which specific autoreactive T cell clones identified in human autoimmune diseases undergo central tolerance,or absent complete thymic deletion.The establishment of this model has important application value for the study of T cell immune tolerance,the occurrence of autoimmune diseases caused by autoreactive T cells,and the study of thymus selection escape mechanism.更多还原