DYNAMICS AND TUNING OF THE MHC II PRESENTED PEPTIDOME

本申请解决了关于MHC-II多肽选择和组合物的基本开放问题， 即常规树突状细胞(CDC)在不同时间表现出的MHC-II多肽的可塑性。 器官位置以及在生理和病理条件下。 总体而言，我们的分析将生成以下定量图谱：(I)MHC-II多肽如何反映“器官-- 特定的蛋白质组特征“来自CDC和(Ii)呈现的MHC II之间的关系 对于自身表位和非自身表位，在静息和炎症条件下的多肽。 目的1是了解MHC II表位拷贝数的定性和定量变化 在局部淋巴中的表达有助于耐受和自身免疫之间的平衡。这个 将比较从不同器官和器官引流的淋巴结中获得的CDC的MHC II肽段。 定量质谱法将被用来监测免疫多肽和多肽的变化。 跨组织疾病预防控制中心的丰度。特别是，我们将量化局部CDC上组织的多肽拷贝数 特定的自身抗原，已知是自身免疫性疾病的靶标，其自身反应性T细胞被发现 在外周，包括MBP、PLP、MOG、(多发性硬化症)D_1-抗胰蛋白酶(克罗恩病)、胰岛素、 GAD65，嗜铬粒蛋白，(I型糖尿病)，组蛋白，(系统性红斑狼疮)和II型胶原 (类风湿性关节炎)和促甲状腺激素(类风湿关节炎和幼年特发性关节炎)。此外，我们还将使用这口井- NOD小鼠1型糖尿病模型的特征，以回答小鼠是否跨越 生理条件和糖尿病前期胰岛素炎之间的阈值在数量和质量上的变化 观察到与疾病发展相关的I-Ag7-肽段(胰岛素、嗜铬粒蛋白、GAD65、S100)。 目的2是了解感染和炎症对MHC II自体多肽的影响。 使用定量同位素标记和无标记蛋白质组学方法，我们将在体内监测呈现的 自身和非自身多肽在静息小鼠和感染结核分枝杆菌后呈现CDC 或鼠伤寒沙门氏菌，以确定CDC成熟过程如何改变呈现的MHC-II自身 多肽。我们将确定MHC II亲和力和人类白细胞抗原-DM敏感性在MHC II自身 感染诱导的多肽，以及未成熟和未成熟的MHC II抗原处理途径的差异 病原体成熟的CDC。最后，我们评估了翻译后修饰加工过程中的变化 树突状细胞成熟过程中的诱导会影响所呈现的多肽。 总体而言，这些研究将确定局部淋巴中出现的MHC II表位拷贝数如何 有助于在耐受性和自身免疫之间取得平衡，感染的后果是什么？ 显示的MHC II自体多肽上的炎症。

This application addresses fundamental open questions on MHC-II peptidome selection and composition, namely, the plasticity of the MHC-II peptidome displayed by conventional dendritic cells (cDC) at different organ locations and under physiological and pathological conditions. Overall our analysis will generate a quantitative mapping of (i) how the MHC-II peptidome reflects the “organ- specific proteomic signature” from where the cDC originates and (ii) the relationship between presented MHC II peptidome in resting and inflammatory conditions, for both self and non-self-epitopes. Aim 1 is to understand how qualitative and quantitative changes in MHC II epitope copy number presented in local lymph node contribute to the balance between tolerance and autoimmunity. The MHC II peptidome will be compared among cDC harvested from lymph nodes draining different organs and quantitative mass spectrometry will be utilized to monitor changes in the immune-peptidome and peptide abundance across tissue cDC. In particular we will quantify the peptide copy numbers on local cDC for tissue specific self antigens, known to be targets in autoimmune diseases and whose auto-reactive T cells are found in the periphery, including MBP, PLP, MOG, (multiple sclerosis) D1-antitrypsin (Crohn's disease), Insulin, GAD65, Chromogranin, (diabetes type I), histones, (systemic lupus erythematosus) and collagen II (rheumatoid arthritis) and Transthyretin (RA and juvenile idiopathic arthritis). Additionally we will use the well- characterized NOD mice Type 1 Diabetes model to answer the question of whether when mice cross the threshold between physiological conditions and pre-diabetic insulitis quantitative and qualitative changes in the I-Ag7-peptidome associated with disease development (insulin, chromogranin, GAD65, S100), are observed. Aim 2 is to understand the consequences of infection and inflammation on the MHC II-self peptidome . Using quantitative isotope labeling and label-free proteomic approaches we will monitor the in vivo presented self- and noself-peptidomes presented cDC in resting mice and after infection with Mycobaterium tuberculosis or Salmonella typhimurium, to determine how the cDC maturation process changes the presented MHC-II self peptidome. We will determine any changes in MHC II affinity and HLA-DM sensitivity in the MHC II self- peptidome induced by infection, and differences in the MHC II antigen processing pathways in immature and pathogen-matured cDC. Finally, we evaluate how changes in post-translational modification processing induced during DC maturation impact the presented peptidome. Overall these studies will determine how MHC II epitope copy number presented in local lymph node can contributes to the balance between tolerance and autoimmunity and what are the consequences of infection and inflammation on the displayed MHC II self peptidome.