Role of MARCH1 E3 ubiquitin ligase in thymic dendritic cell function

描述（由申请人提供）：在理解胸腺T细胞选择过程中胸腺细胞克隆缺失和调节性T细胞（Treg）分化如何协调方面存在根本性差距。这个缺口的持续存在是一个重要的问题，因为在它被填补之前，胸腺T细胞的选择，这是免疫调节和耐受的核心，将在很大程度上仍然是不可理解的。本申请的目的是鉴定树突状细胞（DC）介导胸腺中的克隆缺失和Treg分化的特异性分子机制。我们的中心假设是，胸腺DC协调Treg诱导和克隆删除通过控制表面营业额的肽/MHCII复合物通过调节表达的膜相关的环指CH 1（MARCH 1）E3泛素连接酶。这一假设是根据申请人实验室提供的初步数据提出的。提出的研究的基本原理是，一旦知道胸腺DC如何介导克隆缺失和Treg发育，就可以开发新的策略来利用胸腺DC进行免疫治疗，旨在诱导抗原特异性T细胞耐受。在强有力的初步数据的指导下，该假设将通过追求三个特定目标进行测试：1）确定MARCH 1在胸腺中促进Treg发育的作用和程度; 2）确定MARCH 1介导Treg发育的特定分子机制;和3）定义胸腺DC中异质性MARCH 1表达的机制和功能。在第一个目标下，将检查MARCH 1完全或DC特异性方式缺陷的小鼠的抗原特异性Treg的发育。在第二个目标下，将检查MHCII（K>R）敲入小鼠，其中MHCII细胞质赖氨酸（K）被精氨酸（R）取代，从而抵抗MARCH 1的泛素化，以确定MARCH 1介导的MHCII泛素化在Treg发育中的特定作用。在第三个目标下，将通过胸腺DC转移结合MARCH 1去调节的方法来确定胸腺DC中MARCH 1的调节及其在克隆缺失中的功能作用。成功完成这些目标所需的小鼠品系和实验技能已经掌握在申请人手中。该方法是创新的，因为它消除了MHCII介导的抗原呈递在DC中的生理调节，并确定了其对T细胞选择的结果。这项研究意义重大，因为它有望改善和垂直推进我们对T细胞选择的理解，从概念理解到机械定义。最终，这样的理解有可能被治疗性地利用，以开发用于治疗超免疫性疾病的抗原特异性耐受。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how thymocyte clonal deletion and regulatory T cell (Treg) differentiation are coordinated during thymic T cell selection. Continued existence of this gap represents an important problem because, until it is filled, thymic T cell selection, which is central to immune regulation and tolerance, will remain largely incomprehensible. The objective of this application is to identify te specific molecular mechanisms by which dendritic cells (DCs) mediate clonal deletion and Treg differentiation in the thymus. Our central hypothesis is that thymic DCs coordinate Treg induction and clonal deletion by controlling surface turnover of peptide/MHCII complexes via regulated expression of membrane-associated ring finger CH1 (MARCH1) E3 ubiquitin ligase. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that once it is known how thymic DCs mediate clonal deletion and Treg development, novel strategies could be developed to exploit thymic DCs for immunotherapy aiming for the induction of antigen-specific T cell tolerance. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the role and extent to which MARCH1 contributes to Treg development in the thymus; 2) Identify the specific molecular mechanism by which MARCH1 mediates Treg development; and 3) Define the mechanism and function of heterogeneous MARCH1 expression among thymic DCs. Under the first aim, mice that are deficient in MARCH1 either entirely or DC-specific fashion will be examined for the development of antigen-specific Treg. Under the second aim, MHCII (K>R) knock-in mice in which MHCII cytoplasmic lysine (K) is replaced with arginine (R) and thus resistant to ubiquitination by MARCH1, will be examined to determine the specific role of MARCH1-mediated MHCII ubiquitination in Treg development. Under the third aim, MARCH1 regulation in thymic DCs and its functional role in clonal deletion will be determined by the approach of thymic DC transfer in combination with MARCH1 deregulation. Mouse strains and experimental skills required for successful completion of these aims are already on the applicants' hand. The approach is innovative, because it ablates physiologic regulation of MHCII-mediated antigen presentation in DCs and determines its consequence to T cell selection. The proposed research is significant, because it is expected to improve and vertically advance our understanding of T cell selection from conceptual understanding to mechanistically defined one. Ultimately, such understanding has the potential to be therapeutically exploited to develop antigen-specific tolerance for the treatment of hyper-immune disorders.