Defining the molecular determinants of the threshold for autoimmune arthritis

描述（由申请人提供）：候选人：我的长期目标是建立自己作为一个独立的研究人员在实验室为基础的研究，重点是在免疫细胞信号转导失调引起的自身免疫性疾病的机制。我正在寻求一个KO 1奖，以完成我在加州大学旧金山分校的培训和过渡到终身职位。在Mark Schlissel的指导下，我的研究生培训非常富有成效，发表了五篇论文，包括两篇第一作者文章和三篇第二作者文章。当我作为博士后学者加入韦斯博士的实验室时，我发起了一个项目，旨在通过产生ZAP-70基因敲入小鼠，YYAA老鼠有趣的是，YYAA小鼠的特征使我产生了一个新的研究兴趣--自身免疫和免疫耐受。通过比较SKG和YYAA小鼠中的两种ZAP-70突变体，我定义了自身免疫性关节炎与自身免疫性关节炎的阈值。这些发现发表在2009年的《实验医学杂志》上。在最近的一次合作中，我与韦斯博士实验室的同事Byron Au-Yeung发现了ZAP-70在T调节细胞中的一种以前被忽视的激酶非依赖性功能。这项合作工作在我首次提交后于去年发表在《自然免疫学》上。我在获奖期间的直接目标是：1）获得识别致病性自身抗原和识别它们的T细胞的必要技能; 2）获得作为科学家成熟的指导，为学术求职做好准备，以及获得领导研究小组所需的管理技能培训; 3）开发实验试剂，这对我在作为独立研究者的头两年提交竞争性RO 1申请的目标至关重要。我已经组建了一个指导委员会，除了我的导师韦斯博士外，还包括马克安德森博士、阿布·阿巴斯博士和迈赫达德·马特卢比安博士。每个人都带来了与我的研究计划直接相关的不同水平的专业知识，结合我的教学培训计划，将帮助我实现我的目标，并成功地过渡到一个独立的学术研究者。环境：我相信UCSF为研究自身免疫提供了丰富的环境。利用韦斯实验室的专业知识，我将获得广泛的生物化学实践经验，这对组织特异性自身抗原的鉴定至关重要。此外，韦斯博士的实验室位于加州大学旧金山分校的其他教师，如阿巴斯博士和安德森博士在自身免疫工作。由于距离很近，合作和讨论很频繁，很容易建立。虽然我将主要在韦斯实验室进行实验，但我将通过Matloubian、Abbas和安德森博士的实验室获得自身反应性T细胞运输以及自身免疫/免疫耐受方面的专业知识。研究项目摘要：类风湿性关节炎（RA）是人类最常见的自身免疫性疾病之一，主要影响关节的滑膜。CD 4 + T细胞被认为在RA的发展中起重要作用，它们是在发炎关节中发现的主要细胞类型。尽管进行了深入的调查，这种疾病的初始阶段仍然没有特征，主要是由于患者症状发作前的时间间隔不同。RA小鼠模型（例如SKG小鼠）与人类RA有许多共同特征，已成为研究该疾病早期事件的工具。徐博士的研究旨在确定RA的病因和致病机制，使用具有ZAP-70的低形态突变体的小鼠，SKG和YYAA小鼠。Hsu博士先前已经证明，YYAA和SKG小鼠的T细胞发育受损，对TCR刺激的低反应性，以及有缺陷的阴性和阳性选择。YYAA小鼠，像SKG小鼠一样，产生类风湿因子抗体，但不能发展自身免疫性关节炎。她的研究结果表明， TCR库可能是其对关节炎的不同易感性的原因。所提出的实验的中心假设是，在YYAA和SKG小鼠中选择的不同TCR库决定了导致关节炎的致病性自身反应性T细胞的阈值。检验这一假设的具体目的如下：（AIM 1）了解自身反应性T细胞在发炎关节中积累的性质和动力学，（AIM 2）鉴定SKG小鼠中的致关节炎T细胞和显性自身抗原。在目标1中，IFN 3和IL-17报告小鼠与YYAA或SKG小鼠杂交，将用于确定IFN 3和IL-17产生细胞在自身免疫性关节炎起始阶段的作用。BrdU脉冲标记实验将用于表征YYAA和SKG小鼠中活化的自身反应性T细胞的动力学和迁移模式。在目的2中，将进行实验以比较YYAA和SKG小鼠关节中浸润T细胞和来自淋巴结的外周T细胞的TCR库。从发炎关节中纯化的浸润T细胞产生的TCR 1文库的逆转录病毒感染将与过继转移实验相结合，以鉴定和克隆SKG小鼠的致关节炎T细胞。这些实验将有助于从根本上了解RA的细胞机制，这可能为干预提供有用的靶点。

DESCRIPTION (provided by applicant): The Candidate: My long-term goal is to establish myself as an independent investigator in laboratory based research focused on the mechanisms of autoimmune diseases caused by dysregulated signal transduction in immune cells. I am seeking a KO1 Award in order to complete my training at UCSF and transition to a tenure- track position. My graduate training under the mentorship of Mark Schlissel was highly productive, resulting in five publications including two first-author articles and three second-author articles As I joined Dr. Weiss' laboratory as a postdoctoral scholar, I initiated a project aimed at definin the in vivo role of tyrosine 315 and 319 of ZAP-70 in regulating ZAP-70 function by generating a ZAP-70 knock-in mouse, the YYAA mouse. Interestingly, characterization of YYAA mice has led me to a new research interest --autoimmunity and immune tolerance. By comparing two ZAP-70 mutants in the SKG and YYAA mice, I have defined a threshold for autoimmunity versus autoimmune arthritis. These findings were published in Journal of Experimental Medicine in 2009. In a recent collaboration with my co-worker from Dr. Weiss lab, Byron Au-Yeung, we uncovered a previously overlooked kinase-independent function of ZAP-70 in the T regulatory cells. This collaborative work was published last year in Nature Immunology after my initial submission. My immediate goals for the duration of the award are 1) to acquire the necessary skills to identify pathogenic autoantigens and the T cells that recognize them; 2) to obtain guidance for maturation as a scientist and my preparation for an academic job search as well as to acquire training in management skills needed to lead a research group; 3) to develop experimental reagents that are critical in my goal of submitting a competitive RO1 application during my first two years as an independent investigator. I have assembled a mentoring committee that, in addition to my mentor Dr. Weiss, includes Drs. Mark Anderson, Abul Abbas and Mehrdad Matloubian. Each person brings a different level of expertise that is directly relevant to my research plan and, combined with my didactic training plan, will help me achieve my goals and successfully transition to an independent academic investigator. Environment: I believe UCSF provides the rich environment in studying autoimmunity. Using the expertise in the Weiss lab, I will gain extensive hands-on experience in biochemistry which is essential for the identification of tissue-specific autoantigens. Additionally, Dr. Weiss' lab is located in clos proximity to other faculty members at UCSF working on autoimmunity such as Dr. Abbas, and Dr. Anderson. Because of the close proximity, the collaborations and discussions are frequent and easily established. Although I will conduct my experiments mainly in the Weiss lab, I will gain expertise in trafficking of autoreactive T cells as well as autoimmunity/immune tolerance through the laboratories of Drs. Matloubian, Abbas, and Anderson. Abstract of Research Project: Rheumatoid arthritis (RA), one of the most common human autoimmune diseases, primarily affects the synovial membranes of joints. CD4+ T cells are thought to play an essential role in the development of RA, where they are a predominant cell type found in the inflamed joint. Despite intensive investigation, the initial phase of this disease remains uncharacterized, largely due to varied intervals before the onset of symptoms in patients. Mouse models of RA such as the SKG mouse, which shares many features with human RA, have become instrumental to study early events in the disease. Dr. Hsu's research seeks to define the cause and pathogenic mechanisms of RA using mice possessing hypormorphic mutants of ZAP-70, the SKG and YYAA mice. Dr. Hsu has previously demonstrated that both YYAA and SKG mice have impaired T cell development, hyporesponsiveness to TCR stimulation, and defective negative and positive selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Her findings suggested that the difference in TCR repertoires may be responsible for their differential susceptibility to arthritis. The central hypothesis of the proposed experiments is that distinct TCR repertoires selected in the YYAA and SKG mice determine a threshold for pathogenic autoreactive T cells leading to arthritis. The specific aims testing this hypothesis are as follows: (AIM1) understand the properties and kinetics of the accumulation of autoreactive T cells into the inflamed joints, (AIM2) identify arthritogenic T cells and a dominant autoantigen in SKG mice. In Aim 1, IFN3 and IL-17 reporter mice crossed to either YYAA or SKG mice will be used to define the roles of IFN3 and IL-17-producing cells in the initiation phase of autoimmune arthritis. BrdU pulse labeling experiments will be used to characterize the kinetics and migration patterns of activated autoreactive T cells in YYAA and SKG mice. In Aim 2, experiments will be conducted to compare YYAA and SKG mouse TCR repertoires of the infiltrating T cells in the joints and peripheral T cells from lymph nodes. Retroviral infection of a TCR1 library generated from purified infiltrating T cells in the inflamed joints will be combined with adoptive transfer experiments to identify and clone the arthritogenic T cells from SKG mice. These experiments will contribute to the fundamental understanding of the cellular mechanisms of RA which may provide useful targets for intervention.