Understanding the Impacts of HLA-DO in vivo

项目标题：了解人类白细胞抗原-DO在体内的影响 在此R01申请中描述的项目解决了旨在验证机制的细胞研究 对抗原辅助分子HLADO(小鼠体内的H2-O)(DO)调节作用的认识 依赖于细胞表达的人类白细胞抗原-DM(小鼠为H2-M)的加工和提呈途径 并限制了组织的分布。尽管DO在二十多年前就被发现了，但人们对 它在调节抗原处理和表位选择方面的影响一直缺乏。海流 对DO功能的理解是它抑制DM。最近，我们已经演示了DO并不是 抑制糖尿病：我们已经证明DO与人类MHC II类分子，人类白细胞抗原-DR1(DR1)分子直接相互作用 并与DM合作优化表位选择。我们证明了DO对 不同多肽与DR1分子的结合。这些发现需要在体内进行验证。关键问题 这里讨论的是DO是否在胸腺中导致改变的表位选择的调节中起作用 表达的T细胞谱系，以及对自身免疫发展易感性的可能调节 疾病。在目标I中，我们将研究DO在表达DR1的转基因中改变T细胞谱系中的作用 表达或不表达H2-O的小鼠。使用表达突变DR1(DR1bG86Y)的独特小鼠模型 与DO相互作用，但不与DM相互作用，有或没有H2-O。在目标2中，我们将解决DO是否抑制 DM，或其功能不同于体内抑制DM。在目标3a中，我们将检查前兆频率 胶原性关节炎的致病抗原II型胶原的主要疾病相关表位 (CIA)，在DR1+DO基因敲除小鼠中。在Aim3b中，我们将发现DO-KO小鼠对CIA的易感性，使用 JHU同事开发的新型非侵入性荧光成像技术。在Aim 3c中，我们将 检查类风湿性关节炎患者的样本，确认为具有单核苷酸 细胞内流式细胞仪检测人类白细胞抗原DOA 3‘非编码区基因SNP对人类白细胞抗原DO表达的影响 染色。人类白细胞抗原DO对抗原加工和表位调控的体内验证 选择将是填补了解人类白细胞抗原DO生物学意义的空白的重大飞跃， 这对今后设计有效的免疫治疗药物具有指导意义。

Project Title: Understanding the impacts of HLA-DO in vivo The project described in this R01 application addresses a cellular study aimed at verification of mechanistic understanding of the regulatory role HLA-DO (H2-O in mice) (DO), an accessory molecule of antigen processing and presentation pathway that is dependent on HLA-DM (H2-M in mice) on its cellular expression and has restricted tissue distribution. Despite the discovery of DO for over two decades ago, an understanding of its impact in regulation of antigen processing and epitope selection has been lacking. The current understanding of the DO function is that it inhibits DM. Recently, we have demonstrated that DO does not inhibit DM: we have shown that DO interacts with human MHC class II, HLA-DR1 (DR1), molecules directly and in collaboration with DM optimizes epitope selection. We demonstrated that DO has differential effects on binding of different peptides to DR1 molecules. Those findings need in vivo verification. The key question addressed here is whether DO plays a role in regulation of epitope selection in the thymus leading to changing the expressed T cell repertoire, and possibly regulation of susceptibility to the development of autoimmune diseases. In aim I, we would examine the role of DO in altering T cell repertoire in DR1 expressing transgenic mice that do, or do not express H2-O. Using unique mouse models expressing a mutant DR1 (DR1bG86Y) that interacts with DO but not with DM, with or without H2-O. In Aim 2, we would address whether DO inhibits DM, or its function is different from inhibiting DM in vivo. In Aim 3a, we would examine precursor frequency for the dominant disease associated epitope of collagen II, the causative antigen in Collagen Induced Arthritis (CIA), in in DR1+ DO-knockout mice. In Aim3b we would find out susceptibility of DO-KO mice to CIA, using novel non-invasive fluorescent imaging technology developed by our colleagues at JHU. In Aim 3c we would examine samples from Rheumatoid Arthritis patients that are identified as having a single nucleotide polymorphism (SNP) in their HLA-DOA 3'UTR gene for the expression of HLA-DO by intracellular FACS staining. The in vivo verification of HLA-DO contribution to the regulation of antigen processing and epitope selection would be a major leap towards filling the gap in understanding the biological significance of HLA-DO, which can guide the design of effective immunotherapeutics in future.