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

描述(申请人提供)：我是微生物学和免疫学系的博士后，血液科的讲师。我长期以来一直对免疫学感兴趣，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。在我的临床培训后，我决定继续学习免疫学，但由于人类T细胞生物学的临床潜力，我决定将重点转向人类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。强大的化学、工程和物理系的存在为斯坦福大学的研究人员提供了能够应用最新技术来解决生物和临床问题的优势。在我的初步研究中，我使用了多肽MHC四聚体浓缩的一种新方法：直接从献血者中分离前免疫CD8+T细胞。以前的研究依赖于体外扩增，这是一种可能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，我能够在高频下检测到具有自我反应性的CD8+T细胞--特别是识别胰岛素原的T细胞，这是一种与1型糖尿病(T1D)相关的表位。微流控定量聚合酶链式反应的基因转录分析表明，由于克隆性扩增能力受损，自身反应性CD8+T细胞仍保持耐受。这些结果表明，正常情况下健康人体内存在大量具有自我反应性的T细胞，对于这些细胞来说，细胞内在基因程序可能会加强耐受性，而不是克隆缺失。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。这项提案概述了一个研究T细胞耐受性和1型糖尿病患者的5年计划。主要的假设是，由于共同的细胞内在基因程序，人类体内正常存在大量具有自我反应性的T细胞，并保持耐受；在T1D中，该基因程序发生改变，增加了自身免疫的风险。这个项目的主要目标是定义一组对T细胞耐受性重要且与T1D相关的基因。我的长期目标是了解T1D是如何失去耐受性的，并将这一知识应用于这种疾病的治疗。具体的研究目标是：1)通过微阵列表达谱来确定定义健康人类捐赠者血液中自我反应性T细胞耐受性的基因特征；2)确定I型糖尿病患者自身免疫T细胞中耐受性基因特征的变化；以及3)开发一种系统来测试候选基因的功能。导师职业发展奖将使我成长为一名独立而成功的内科科学家。戴维斯实验室内部和外部合作的专业知识将提供一个关键的机会，以获得执行我的研究目标所需的技术技能，例如第二类MHC蛋白表达、微阵列表达谱和飞行时间质谱仪的细胞学。有组织的学习机会将使我能够改善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，以及由与我的目标相关的专业知识的教职员工组成的指导委员会一年两次的会议来监督。作为一名学术医学教授，我的长期目标是了解1型糖尿病的发病机制。我把90%的时间投入到研究和教学中，将10%的时间投入到临床工作中。

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.