Defining the molecular determinants of the threshold for autoimmune arthritis

定义自身免疫性关节炎阈值的分子决定因素

• 批准号: 8431367
• 负责人: Lih-Yun Hsu
• 金额: $ 11.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431367
• 关键词: Address; Adoptive Transfer; Affect; Alanine; Antibodies; Arthritis; Autoantigens; Autoimmune Diseases; Autoimmunity; Award; Biochemistry; Bromodeoxyuridine; C-terminal; CD4 Positive T Lymphocytes; Cartilage; Cells; Collaborations; Data; Defect; Development; Disease; Environment; Event; Exhibits; Experimental Models; Faculty; Gene Mutation; Genes; Goals; Grant; Human; Immune; Immune Tolerance; Immune system; Immunology; In Vitro; Infection; Inflammatory Response; Interleukin-17; Intervention; Investigation; Joints; Journals; Kinetics; Knock-in Mouse; Knowledge; Label; Laboratories; Lead; Libraries; Mediating; Medicine; Mentors; Mentorship; Molecular; Mus; Mutant Strains Mice; Mutation; Nature; Occupations; Pathogenesis; Patients; Pattern; Peripheral; Persons; Phase; Phosphotransferases; Physiologic pulse; Play; Point Mutation; Positioning Attribute; Predisposition; Preparation; Prevalence; Property; Protein Tyrosine Kinase; Publications; Publishing; Reagent; Receptor Signaling; Regulatory T-Lymphocyte; Reporter; Research; Research Personnel; Research Project Grants; Rheumatoid Arthritis; Rheumatoid Factor; Role; Scientist; Signal Transduction; Specificity; Symptoms; Synovial Membrane; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Thymus Gland; Tissues; Training; Tyrosine; Work; ZAP-70 Gene; Zymosan; abstracting; autoimmune arthritis; autoreactive T cell; base; bone; cell type; experience; in vivo; interest; lymph nodes; member; migration; mouse model; mutant; new therapeutic target; research study; response; skills; src Homology Region 2 Domain; trafficking

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.

应聘者描述(申请人提供)：我的长期目标是在实验室研究中确立自己作为独立研究员的地位，重点研究免疫细胞中信号转导失调引起的自身免疫性疾病的机制。为了完成在加州大学旧金山分校的培训，并过渡到终身教职的职位，我正在寻求KO1奖。我在Mark Schlissel的指导下进行的研究生培训非常有成效，发表了五篇论文，其中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Weiss博士的实验室时，我启动了一个项目，旨在通过产生ZAP-70敲入小鼠YYAA小鼠，在体内定义ZAP-70的酪氨酸315和319在调节ZAP-70功能中的作用。有趣的是，YYAA小鼠的特征使我产生了新的研究兴趣--自身免疫和免疫耐受。通过比较SKG和YYAA小鼠中的两个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。在最近与我来自Weiss博士实验室的同事Byron Au-Yeung的合作中，我们发现了在T调节细胞中ZAP-70的一个以前被忽视的不依赖于激酶的功能。这项合作工作在我最初提交后于去年发表在《自然免疫学》杂志上。在获奖期间，我的近期目标是1)获得必要的技能来识别致病自身抗原和识别它们的T细胞；2)获得作为一名科学家的成熟指导，为我的学术求职做准备，以及获得领导研究小组所需的管理技能培训；3)开发对我在作为独立研究员的头两年提交竞争性RO1申请的目标至关重要的实验试剂。我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·马特鲁宾博士。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，结合我的说教培训计划，将帮助我实现我的目标，并成功过渡到一名独立的学术研究人员。环境：我相信加州大学旧金山分校为研究自身免疫提供了丰富的环境。利用Weiss实验室的专业知识，我将在生物化学方面获得丰富的实践经验，这对鉴定组织特异性自身抗原至关重要。此外，韦斯博士的实验室距离加州大学旧金山分校从事自身免疫研究的其他教职员工很近，比如阿巴斯博士和安德森博士。由于距离很近，合作和讨论频繁，很容易建立。虽然我将主要在韦斯实验室进行实验，但我将通过马特鲁宾、阿巴斯和安德森博士的实验室获得自身反应性T细胞贩运以及自身免疫/免疫耐受方面的专业知识。研究项目摘要：类风湿关节炎(RA)是人类最常见的自身免疫性疾病之一，主要累及关节滑膜。CD4+T细胞被认为在类风湿性关节炎的发展中起着至关重要的作用，在发炎的关节中它们是一种主要的细胞类型。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性，主要是由于患者出现症状之前的不同间隔。类风湿性关节炎的小鼠模型，如SKG小鼠，与人类类风湿性关节炎有许多相似之处，已成为研究疾病早期事件的工具。许博士的研究试图利用具有ZAP-70高形态突变的小鼠、SKG和YYAA小鼠来确定RA的病因和致病机制。许博士此前已经证明，YYAA和SKG小鼠都有T细胞发育障碍，对TCR刺激反应迟钝，阴性和阳性选择有缺陷。YYAA小鼠和SKG小鼠一样，会产生类风湿因子抗体，但不会患上自身免疫性关节炎。她的发现表明， TCR谱系可能是它们对关节炎的不同易感性的原因。拟议实验的中心假设是，在YYAA和SKG小鼠中选择的不同TCR谱系确定了导致关节炎的病理性自身反应T细胞的阈值。检验这一假说的具体目的如下：(AIM1)了解自身反应性T细胞在炎症关节中积聚的特性和动力学，(AIM2)确定在SKG小鼠中致炎的T细胞和一种主要的自身抗原。在目标1中，与YYAA或SKG小鼠杂交的IFN3和IL-17报告鼠将被用来确定IFN3和IL-17产生细胞在自身免疫性关节炎启动阶段的作用。BrdU脉冲标记实验将用于表征激活的自身反应性T细胞在YYAA和SKG小鼠体内的动力学和迁移模式。目的2比较YYAA和SKG小鼠关节内T细胞和外周淋巴结T细胞的TCR谱。从炎症关节中提纯的浸润性T细胞产生的TCR1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理解类风湿关节炎的细胞机制，为干预提供有用的靶点。