Tolerance mechanisms in self reactive T cells in healthy people and type 1 diabet

简介（申请人提供）：我是微生物与免疫学系博士后，血液学系讲师。我对免疫学有着长期的兴趣，并完成了利用转基因动物模型进行B细胞发育的研究生工作。在我的临床培训之后，我决定继续免疫学，但由于其临床潜力，我将重点转向人类T细胞生物学。为了实现这一目标，我加入了国际知名免疫学家、斯坦福大学免疫、移植和感染研究所（ITI）所长马克·戴维斯博士的实验室。斯坦福大学拥有强大的化学系、工程系和物理系，为研究人员提供了能够应用最新技术解决生物学和临床问题的优势。在我的初步研究中，我使用肽MHC四聚体富集的新方法：直接从献血者中分离免疫前CD8+ T细胞。以前的研究依赖于体外扩增，这是一种可以改变细胞表型并选择具有增殖优势的细胞的操作。出乎意料的是，我能够高频率地检测到自我反应性CD8+ T细胞，特别是识别胰岛素前原的T细胞，1型糖尿病（T1D）相关的表位。基于微流体的qPCR基因转录分析表明，由于克隆扩增能力受损，自反应性CD8+ T细胞仍然具有耐受性。这些结果表明，健康人群中通常存在大量的自我反应性T细胞，对于这些细胞，而不是克隆缺失，细胞内在的基因程序可能会增强耐受性。这为在T1D的背景下检查人类T细胞耐受性和自身免疫提供了机会。该提案概述了研究人类T细胞耐受性和1型糖尿病的5年计划。主要的假设是，大量的自我反应性T细胞正常存在于人体内，并且由于共同的细胞内在基因程序而保持耐受性；在T1D中，这种基因程序发生改变，从而增加自身免疫的风险。该项目的主要目标是确定一组对T细胞耐受性和T1D相关的重要基因。我的长期目标是了解T1D患者的耐受性是如何丧失的，并将这些知识应用于这种疾病的治疗。具体的研究目标是：1)通过微阵列表达谱确定基因标记，定义来自健康人类供者血液的自反应性T细胞的耐受性；2)确定1型糖尿病患者自身免疫T细胞耐受基因信号的变化；3)开发候选基因功能测试系统。导师职业发展奖将使我成为一名独立成功的内科科学家。戴维斯实验室的专业知识和外部合作将为获得执行我的研究目标所需的技术技能提供关键机会，例如II类MHC蛋白表达，微阵列表达谱和飞行时间质谱的细胞测定。结构化的学习机会将使我能够提高我在生物信息学方面的背景知识，并扩大我对人类免疫学进展的了解。我的研究和/或专业发展将通过与导师的讨论，定期向科学界展示我的成果，以及由具有与我的目标相关的专业知识的教师组成的指导委员会的两年一次的会议来监督。我的长期目标是了解1型糖尿病的发病机制，作为一名学术医学教授，将90%的时间用于研究和教学，10%的时间用于临床工作。

DESCRIPTION (provided by applicant): I am a postdoc in the Department of Microbiology and Immunology, and an Instructor in the Division of Hematology. I have a long-standing interest in immunology and completed graduate work in B cell development using transgenic animal models. After my clinical training, I decided to continue in immunology, but with a change of focus to human T cell biology because of its clinical potential. To realize this goal, I joined the laboratory of Dr. Mark Davis, an internationally renowned immunologist and Director of the Stanford Institute for Immunity, Transplantation, and Infection (ITI). The presence of strong departments in chemistry, engineering and physics affords researchers at Stanford University the advantage of being able to apply the newest technologies to address biological and clinical problems. In my preliminary studies, I used peptide MHC tetramer enrichment for a novel approach: to directly isolate preimmune CD8+ T cells from blood donors. Previous studies have relied on in vitro expansion, a manipulation that may alter the cell phenotype and select for cells with a proliferative advantage. Unexpectedly, I was able to detect self-reactive CD8+ T cells at high frequency - in particular T cells recognizing preproinsulin, a type 1 diabetes (T1D) associated epitope. A gene transcript analysis by microfluidics-based qPCR suggested that self-reactive CD8+ T cells remain tolerant due to an impaired ability to clonally expand. These results show that a large pool of self-reactive T cells is normally present healthy people, and that for these cells, rather than clonal deletion, a cell intrinsic gene program may enforce tolerance. This presented an opportunity to examine human T cell tolerance and autoimmunity in the context of T1D. This proposal outlines a 5-year plan to study T cell tolerance and type 1 diabetes in people. The main hypothesis is that a large pool of self-reactive T cells exists normally in people and remains tolerant due to a common cell intrinsic gene program; in T1D, this gene program is altered in a way that increases the risk of autoimmunity. The primary goal of this project is to define a set of genes important for T cell tolerance and relevant to T1D. My long term goal is to understand how tolerance is lost in T1D and apply that knowledge to the treatment of this disease. The specific research aims are: 1) To determine a gene signature by microarray expression profiling that defines tolerance in self-reactive T cells from the blood of healthy human donors; 2) To determine the changes to a tolerance gene signature in autoimmune T cells from individuals with type I diabetes mellitus; and 3) To develop a system to test the function of candidate genes. A Mentored Career Development Award will enable me to develop into an independent and successful physician scientist. Expertise within the Davis laboratory and from external collaborations will provide a critical opportunity to acquire the technical skills necessary to execute my research aims, such as class II MHC protein expression, microarray expression profiling, and cytometry by time of flight mass spectrometry. Structured learning opportunities will allow me to improve my background in bioinformatics and expand my knowledge of advancements in human immunology. My research and/or professional development will be monitored through discussions with my mentor, regular presentations of my results to the scientific community, and a biannual meeting of a mentoring committee composed of faculty with expertise related to my aims. My long-term goal is to understand the pathogenesis of type 1 diabetes mellitus as a professor in academic medicine devoting ~90% of time to research and teaching and ~10% of time to clinical work. PUBLIC HEALTH RELEVANCE: All people have T cells that recognize molecules found normally in the body. These self-reactive T cells do not attack because of a biological state called tolerance. We will use new technologies to understand how T cell tolerance is maintained in healthy people, and how T cells tolerance is lost and contributes to autoimmunity in type 1 diabetes mellitus.