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，胸腺细胞克隆性缺失和调节性T细胞(Treg)分化是如何协调的，这是一个基本的空白。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，对免疫调节和耐受至关重要的胸腺T细胞选择将在很大程度上仍然无法理解。本应用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，胸腺树突状细胞通过调节膜相关环指CH1(March1)E3泛素连接酶的表达来控制多肽/MHCII复合体的表面周转，从而协调Treg的诱导和克隆性删除。这一假设是根据申请人实验室提供的初步数据提出的。这项研究的基本原理是，一旦知道胸腺树突状细胞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树突状细胞进行免疫治疗，目的是诱导抗原特异性T细胞耐受。在强大的初步数据的指导下，这一假说将通过追求三个具体目标来检验：1)确定March1对胸腺Treg发育的作用和程度；2)确定March1介导Treg发育的特定分子机制；以及3)确定胸腺树突状细胞中March1异质性表达的机制和功能。在第一个目标下，将对March1基因完全缺陷或DC特异性方式缺陷的小鼠进行抗原特异性Treg的发育检查。在第二个目标下，将检测MHCII(K&GT；R)敲入小鼠，其中MHCII胞浆赖氨酸(K)被精氨酸(R)取代，因此对March1介导的MHCII泛素化具有抗药性，以确定March1介导的MHCII泛素化在Treg发育中的特定作用。在第三个目标下，胸腺DC的March1调控及其在克隆缺失中的功能作用将通过胸腺DC转移和March1去调控相结合的方法来确定。成功完成这些目标所需的老鼠品系和实验技能已经掌握在申请者手中。这种方法是创新的，因为它破坏了MHCII介导的DC抗原提呈的生理调节，并决定了其对T细胞选择的影响。这项拟议的研究具有重要意义，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，这种理解有可能在治疗上被利用来发展抗原特异性耐受性，用于治疗高免疫性疾病。

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.