Titrating CD45 expression in mice to study development of T cell tolerance

描述（由申请人提供）：该提案描述了一个为期五年的职业发展计划，其目标是让 Julie Zikherman 博士为独立研究者的职业生涯做好准备。该项目将以 Zikherman 博士在临床风湿病学、基础发育生物学和基础免疫学方面的经验为基础，扩大新兴研究项目，并为她提供设计和进行细胞和分子免疫学研究方面的进一步专业知识。长期目标是建立一个独立的实验室，致力于研究自身免疫性疾病发病机制的基本机制，重点研究抗原受体信号传导如何建立和颠覆免疫耐受。该项目的核心指导和培训环境将由导师 Art Weiss 博士提供，他是加州大学旧金山分校 Engleman 杰出医学教授、风湿病科主任，也是 T 细胞受体信号传导方面的专家。培训计划包括通过参加研讨会系列、期刊俱乐部、加州大学旧金山分校务虚会和科学会议进行的正式课程和教学培训，以及由知名科学家组成的咨询委员会的指导，以及一项研究计划，该计划将提供额外的技术培训以及独立调查的新方向。本次资助中提出的研究旨在阐明改变的 T 细胞受体信号传导如何颠覆中枢耐受机制并在人类中产生自身免疫。 Zikherman 博士在她的初步数据中培育了一系列表达不同水平 CD45 的等位基因小鼠，并证明不同的 CD45 剂量可产生 TCR 信号强度的稳定滴定以及胸腺输出的定量变化。在该研究计划中，Zikherman 博士将利用这一新颖的系统来研究 TCR 信号强度的滴定如何调节胸腺细胞的命运、转录机制以及最终的功能性 T 细胞库。在该提案的第一部分，Zikherman 博士将使用遗传方法和骨髓嵌合体来定义等位基因系列中正向和负向选择的调节。在第二部分中，她将通过定义正选择的关键转录调节因子的表达来表征等位基因系列胸腺细胞的分子表型。在提案的最后部分，Zikherman 博士将利用在器官特异性自身免疫的 Aire-/- 模型背景下追踪个体自身反应细胞的能力来研究 TCR 信号传导如何影响功能性 T 细胞库。总之，这些实验将有助于表征 T 细胞发育过程中 TCR 信号传导改变的细胞、分子和功能后果。这些研究将阐明改变 TCR 信号传导破坏中枢耐受性以产生疾病的机制。除了解决本研究提案中提出的问题之外，上述研究还将作为未来工作的基础，使 Zikherman 博士能够成为一名独立研究者。

DESCRIPTION (provided by applicant): This proposal describes a five year career development program whose goal is to prepare Dr. Julie Zikherman for a career as an independent investigator. This program will build on Dr. Zikherman's experience in clinical rheumatology, basic developmental biology and basic immunology in order to expand an emerging research program and provide her with further expertise in the design and conduct of cellular and molecular immunology research. The long-term goal is to establish an independent laboratory dedicated to the study of basic mechanisms of autoimmune disease pathogenesis, with a focus on how antigen receptor signaling can establish and subvert immune tolerance. The central guidance and training environment for this project will be provided by the mentor, Dr. Art Weiss, Engleman Distinguished Professor of Medicine, and Division Chief of Rheumatology at UCSF, as well as an expert in T cell receptor signaling. The training plan includes formal course work and didactic training through attendance of seminar series, journal clubs, UCSF retreats and scientific meetings, as well as guidance by an advisory committee of established scientists, and a research program that will provide additional technical training along with new directions for independent investigation. The studies proposed in this grant are intended to elucidate how altered T cell receptor signaling can subvert mechanisms of central tolerance and produce autoimmunity in humans. In her preliminary data, Dr. Zikherman has generated an allelic series of mice expressing distinct levels of CD45, and demonstrated that varying CD45 dose produces stable titration of TCR signaling intensity as well as quantitative changes in thymic output. In the research proposal, Dr. Zikherman will take advantage of this novel system to study how titration of TCR signaling intensity regulates thymic cell fate, transcriptional machinery and ultimately functional T cell repertoire. In the first part of the proposal, Dr. Zikherman will use genetic approaches and bone marrow chimeras to define the regulation of positive and negative selection in the allelic series. In the second part she will characterize the molecular phenotype of allelic series thymocytes by defining expression of key transcriptional regulators of positive selection. In the final part of the proposal, Dr. Zikherman will capitalize on the ability to track individual autoreactive cells in the context of the Aire-/- model of organ-specific autoimmunity to study how TCR signaling affects functional T cell repertoire. Together, these experiments will serve to characterize the cellular, molecular, and functional consequences of altered TCR signaling during T cell development. These studies will illuminate the mechanisms by which altered TCR signaling undermines central tolerance to produce disease. In addition to addressing the questions posed in this research proposal, the studies outlined above will serve as a foundation for future work that will enable Dr. Zikherman to become an independent investigator.