Functional mapping of ex-regulatory T cell phenotypic diversity

项目摘要/摘要 调节性和效应性T细胞具有不同的免疫学作用，有助于免疫稳态。 虽然效应性T细胞具有保护宿主免受感染威胁和癌症的功能， 调节性T细胞(Tregs)本质上是抑制性的，可以防止失控的效应T细胞介导的炎症 和自身免疫力。尽管这些不同的细胞谱系主要是在胸腺选择过程中确定的，但它是 现在了解到，这两个可以相互转换的T细胞可以在外周呈现调节表型， 而Tregs可能会失去成为促炎“前调节”T细胞(ExTregs)的抑制能力。 因为exTregs可以颠覆传统的调节性T细胞来加剧炎症并促进 病理性自身免疫进展，人们对了解如何以及在什么条件下进行非常感兴趣 前树会发展成。在我们实验室的初步工作中，我们发现单细胞RNA测序揭示了广泛的 小鼠肠道、肺和淋巴结中初级Tregs和exTregs的表型异质性。我建议 使用单细胞基因组学方法全面定义管理exTreg的基因调控程序 不稳定的特雷格的发展和两极分化。在我的第一个目标中，我将深入分析单个细胞 确定不同组织中exTreg表型异质性的RNA测序数据集 它们被分离出来的微环境。对于我的第二个目标，我将利用我的赞助商的专业知识 在高通量遗传扰动的初级小鼠Tregs中执行正向遗传筛选以定位 维持Treg特性并将Treg破坏为ExTregs的基因调控程序 促炎细胞因子IL-4或IL-6。我的赞助人亚历克斯·马森在遗传学方面有丰富的专业知识 免疫细胞功能和自身免疫，以及使用CRISPR-Cas9进行原代T细胞的基因工程。我的 联合发起人吉米·叶是一位计算免疫学家和人类遗传学家，他的实验室使用 单细胞RNA测序等创新技术在分子上定义免疫细胞的过程 发展和功能。此外，我还获得了著名的科学和职业指导 免疫学家杰弗里·布鲁斯顿博士(ExTregs在自身免疫性炎症中的特征)和唐启智博士 (研究免疫耐受和自身免疫)。与此同时，我正在完成一项纵向临床试验 加州大学旧金山分校贝尼奥夫儿童医院院长陈丽思教授儿科免疫学 儿科免疫失调诊所和儿科免疫紊乱的基因诊断专家。总的来说， 这项拟议的工作将加深我们对exTregs的发育及其在自身免疫中的作用的理解 发病机制和进展。此外，这笔奖学金支持将促进我在免疫学和 计算生物学，支持我通过医学和博士相结合的培训走向职业生涯 学院派儿科免疫学家，研究免疫紊乱的遗传基础。

PROJECT SUMMARY/ABSTRACT Regulatory and effector T cells serve distinct immunological roles that contribute to immune homeostasis. Whereas effector T cells have the functional capacity to protect the host from infectious threats and cancer, regulatory T cells (Tregs) are suppressive in nature and prevent runaway effector T cell-mediated inflammation and autoimmunity. Although these are distinct cell lineages determined primarily during thymic selection, it is now understood that the two can interconvert – T cells can assume regulatory phenotypes in the periphery, and Tregs may lose suppressive capacity to become pro-inflammatory “ex-regulatory” T cells (exTregs). Because exTregs can subvert traditional regulatory T cells to exacerbate inflammation and contribute to pathologic autoimmune progression, there is great interest in understanding how and under what conditions exTregs develop. In preliminary work from our lab, we found that single cell RNA sequencing reveals extensive phenotypic heterogeneity of primary Tregs and exTregs from mouse gut, lungs, and lymph nodes. I propose using single cell genomics approaches to fully define the gene regulatory programs that govern exTreg development and polarization from destabilized Tregs. In my first aim, I will deeply analyze the single cell RNA sequencing data set to define the heterogeneity of exTreg phenotypes in distinct tissue microenvironments from which they were isolated. For my second aim, I will leverage my sponsors’ expertise in high-throughput genetic perturbation of primary mouse Tregs to perform a forward genetic screen to map the gene regulatory programs that maintain Treg identity and destabilize Tregs to exTregs induced by pro-inflammatory cytokines IL-4 or IL-6. My sponsor Alex Marson has extensive expertise in the genetics of immune cell function and autoimmunity, and in genetic engineering of primary T cells using CRISPR-Cas9. My co-sponsor Chun (Jimmie) Ye is a computational immunologist and human geneticist whose laboratory uses innovative technologies like single cell RNA sequencing to molecularly define processes of immune cell development and function. Additionally, I have secured scientific and career mentorship from renowned immunologists Dr. Jeffrey Bluestone (characterized exTregs in autoimmune inflammation) and Dr. Qizhi Tang (studies immune tolerance and autoimmunity). Concurrently, I am completing a longitudinal clinical preceptorship in pediatric immunology with Dr. Alice Chan, director of the UCSF Benioff Children’s Hospital Pediatric Immune Dysregulation Clinic and expert in genetic diagnosis of pediatric immune disorders. Overall, the proposed work will further our understanding of exTregs development and their role in autoimmune pathogenesis and progression. Moreover, this fellowship support will foster my training in immunology and computational biology, supporting me on my path through combined MD-PhD training towards a career as an academic pediatric immunologist studying the genetic bases of immune disorders.