Dissecting the interactions between TCR signaling strength and inhibitory pathways

项目总结/摘要 这个为期5年的K 08培训计划旨在促进我（沈林博士）的职业发展， 为我作为一名医生兼科学家的独立研究生涯做准备。我的长期目标是研究 发病机制和潜在的自身免疫性疾病的治疗。该提案的重点是如何抑制 途径抑制异常TCR信号强度以实现外周耐受。中央指导和 培训环境将由我的主要导师亚瑟韦斯博士提供，他是TCR信号传导方面的专家。 进一步的专业知识将提供一个强大的咨询小组的专家在淋巴细胞信号，免疫 耐受性、自身免疫和转录组学分析。一个包含实验研究、教学法和 研讨会已经开发，以促进我对我的职业目标。广泛的资源和支持 在加州大学旧金山分校的工作将有助于我实现独立和长期目标。 TCR信号转导的失调长期以来被认为在肿瘤的发病机制中起重要作用。 自身免疫性疾病然而，抑制途径如何通过增强的TCR信号强度参与， 调节外周耐受和预防自身免疫仍不完全清楚。细胞质 酪氨酸激酶ZAP 70在TCR信号传导中起必要的作用。两个ZAP 70超型突变体，越强 W131 A和较弱的R360 P干扰自身抑制，并对TCR信号传导具有不同的影响。作为 作为增强的基础信号传导的结果，OTII-TCR转基因W131 A T细胞表现出显著的上调， 抑制性受体和无反应性表型的获得。ZAP 70 R360 P小鼠突变体， 是家族性严重自身免疫综合征R360 P小鼠未能出现明显的 在C57 BL/6背景下，该患者患有自身免疫性疾病，但表现出调节性和无反应性T细胞的扩增。 引人注目的是，OTI-TCR转基因R360 P T细胞显示出对弱肽和自身肽的增强的应答。 引入TCR信号负调节因子Cbl-b的缺陷导致R360 P-OTI T细胞的增强， 在W131 A-OTII T细胞中对弱激动剂肽的应答和逆转功能性无能。所以我 假设增加的TCR信号强度使T细胞受到抑制性T细胞的更大控制， 但是可能赋予它们对这些抑制途径的破坏更大的敏感性。在这 建议，我将利用R360 P和W131 A小鼠模型来研究如何增强TCR信号传导 强度和抑制途径耦合在一起以维持或破坏外周耐受性。在Aim中 1，使用一系列的生物化学技术和转录组学分析，我将剖析TCR信号如何 强度与抑制性程序动态耦合。在目标2中，我将研究增强的TCR信号传导是否 强度促进对负调节途径的破坏的敏感性。这项研究将提供新的 了解这些相反的信号通路如何整合以影响T细胞抗原敏感性的机制 并调节外周耐受性和免疫应答的质量。

PROJECT SUMMARY/ABSTRACT This 5-year K08 training program is designed to facilitate my (Dr. Lin Shen) career development in preparation of my independent research career as a physician-scientist. My long-term goal is to study the pathogenesis and, potentially, the therapy of autoimmune diseases. This proposal focuses on how inhibitory pathways restrain abnormal TCR signaling strength to achieve peripheral tolerance. Central guidance and the training environment will be provided by my primary mentor, Dr. Arthur Weiss, an expert in TCR signaling. Further expertise will be provided by a strong advisory panel of experts in lymphocyte signaling, immune tolerance, autoimmunity and transcriptomic profiling. A training plan with experimental research, didactics, and seminars has been developed to advance me towards my career goals. The extensive resources and support at the UCSF will facilitate my pathway to independence and my long-term goals. Dysregulation of TCR signaling has long been recognized to play important roles in the pathogenesis of autoimmune diseases. However, how inhibitory pathways are engaged by enhanced TCR signaling strength to regulate peripheral tolerance and prevent autoimmunity remains incompletely understood. The cytoplasmic tyrosine kinase ZAP70 plays a requisite role in TCR signaling. Two ZAP70 hypermorphic mutants, the stronger W131A and the weaker R360P, interfere with autoinhibition and have varying effects on TCR signaling. As a consequence of enhanced basal signaling, OTII-TCR transgenic W131A T cells exhibit marked upregulation of inhibitory receptors and acquisition of an anergic phenotype. The ZAP70 R360P mouse mutant that I generated derives from a familial severe autoimmune syndrome. R360P mice failed to develop overt autoimmune disease on a C57BL/6 background but exhibited expansion of regulatory and anergic T cells. Strikingly, OTI-TCR transgenic R360P T cells showed enhanced responses to weak and self-peptides. Introducing deficiency in Cbl-b, a negative regulator for TCR signaling, resulted in enhanced R360P-OTI T cell responses to a weak agonist peptide and reversed functional anergy in W131A-OTII T cells. Therefore, I hypothesize that increased TCR signaling strength renders T cells subject to greater control by inhibitory pathways but may confer upon them greater sensitivity to disruption of these inhibitory pathways. In this proposal, I will take advantage of the R360P and W131A mouse models to study how enhanced TCR signaling strength and inhibitory pathways are coupled together to either maintain or subvert peripheral tolerance. In Aim 1, using a range of biochemical techniques and transcriptomic profiling, I will dissect how TCR signaling strength is dynamically coupled to inhibitory programs. In Aim 2, I will study whether augmented TCR signaling strength promotes sensitivity to the disruption of negative regulatory pathways. This study will provide new mechanistic insights into how these opposing signaling pathways integrate to impact T cell antigen sensitivity and to regulate peripheral tolerance and the quality of immune responsiveness.