Role of MARCH1 E3 ubiquitin ligase in thymic dendritic cell function

描述（由申请人提供）：在胸腺T细胞选择过程中，胸腺细胞克隆缺失和调节性T细胞（Treg）分化是如何协调的，这是一个根本性的空白。这个缺口的持续存在代表了一个重要的问题，因为直到它被填补，胸腺T细胞的选择，这是免疫调节和耐受性的核心，将在很大程度上仍然是不可理解的。本应用程序的目的是确定树突状细胞（dc）介导胸腺克隆缺失和Treg分化的特定分子机制。我们的中心假设是胸腺dc通过调节膜相关环指CH1 (MARCH1) E3泛素连接酶的表达来控制肽/MHCII复合物的表面周转，从而协调Treg的诱导和克隆缺失。这一假设是根据申请人实验室产生的初步数据制定的。这项研究的基本原理是，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，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，这一假设将通过追求三个具体目标来验证：1)确定MARCH1在胸腺中促进Treg发育的作用和程度；2)明确MARCH1介导Treg发育的具体分子机制；3)明确胸腺dc中MARCH1异质表达的机制和功能。在第一个目标下，将对完全缺乏MARCH1或dc特异性缺乏的小鼠进行检查，以研究抗原特异性Treg的发育。在第二个目标下，MHCII （K>R）敲入小鼠中，MHCII细胞质赖氨酸(K)被精氨酸(R)取代，从而抵抗MARCH1的泛素化，将被检查以确定MARCH1介导的MHCII泛素化在Treg发育中的具体作用。在第三个目标下，将通过胸腺DC转移结合MARCH1解除调控的方法来确定胸腺DC中的MARCH1调控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。该方法具有创新性，因为它消除了dc中mhcii介导的抗原呈递的生理调节，并确定了其对T细胞选择的影响。这项提议的研究是重要的，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的理解。最终，这种理解有可能被用于治疗开发抗原特异性耐受性，以治疗超免疫疾病。

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.