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

描述（由申请人提供）：我是微生物学和免疫学系的博士后，也是血液学系的讲师。我对免疫学有着长期的兴趣，并完成了使用转基因动物模型进行B细胞开发的研究生工作。在我的临床培训之后，我决定继续从事免疫学，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）主任马克·戴维斯博士的实验室。在化学，工程和物理强大的部门的存在提供了研究人员在斯坦福大学的优势，能够应用最新的技术来解决生物和临床问题。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CD 8 + 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，我能够以高频率检测到自身反应性CD 8 + T细胞-特别是识别前胰岛素原（1型糖尿病（T1 D）相关表位）的T细胞。通过基于微流体的qPCR的基因转录物分析表明，由于克隆扩增能力受损，自身反应性CD 8 + T细胞保持耐受性。这些结果表明，健康人通常存在大量的自身反应性T细胞，并且对于这些细胞，而不是克隆缺失，细胞内在基因程序可能会增强耐受性。这提供了在T1 D背景下检查人T细胞耐受性和自身免疫性的机会。该提案概述了一项研究T细胞耐受性和1型糖尿病的5年计划。主要假设是，大量的自身反应性T细胞正常存在于人体中，并且由于共同的细胞内在基因程序而保持耐受性;在T1 D中，这种基因程序以增加自身免疫风险的方式改变。该项目的主要目标是确定一组对T细胞耐受性重要且与T1 D相关的基因。我的长期目标是了解T1 D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签名，其定义来自健康人类供体血液的自身反应性T细胞中的耐受性; 2）确定来自I型糖尿病个体的自身免疫性T细胞中的耐受性基因签名的变化;以及3）开发测试候选基因功能的系统。一个指导职业发展奖将使我能够发展成为一个独立的和成功的医生科学家。戴维斯实验室内的专业知识和外部合作将提供一个关键的机会，以获得必要的技术技能，以执行我的研究目标，如II类MHC蛋白表达，微阵列表达谱，并通过飞行时间质谱细胞计数。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，并扩大我对人类免疫学进步的知识。我的研究和/或专业发展将通过与我的导师讨论，定期向科学界介绍我的成果，以及由与我的目标相关的专业知识的教师组成的指导委员会的一年两次会议进行监测。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，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.