DYNAMICS AND TUNING OF THE MHC II PRESENTED PEPTIDOME

本申请解决了关于MHC-II肽组选择和组成的基本开放问题， 也就是说，在不同温度下，由常规树突状细胞（cDC）显示的MHC-II肽组的可塑性， 器官位置以及生理和病理条件下。 总体而言，我们的分析将产生以下的定量映射：（i）MHC-II肽组如何反映"器官-细胞"， 特异性蛋白质组学标记"从哪里cDC起源和（ii）之间的关系提出的MHC II 在静息和炎症条件下，对于自身和非自身表位， 目的1是了解MHCII表位拷贝数的定性和定量变化 在局部淋巴结中的存在有助于耐受和自身免疫之间的平衡。的 将在从引流不同器官的淋巴结收获的cDC之间比较MHC II肽组， 定量质谱法将用于监测免疫肽组和肽 跨组织cDC的丰度。特别地，我们将定量组织的局部cDC上的肽拷贝数， 特异性自身抗原，已知是自身免疫性疾病的靶点，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）D1-抗胰蛋白酶（克罗恩病）、胰岛素， GAD 65、嗜铬粒蛋白（I型糖尿病）、组蛋白（系统性红斑狼疮）和胶原蛋白II （类风湿性关节炎）和甲状腺素运载蛋白（RA和幼年特发性关节炎）。另外，我们将使用井- 特征NOD小鼠1型糖尿病模型，以回答当小鼠穿过 阈值之间的生理条件和糖尿病前期胰岛炎定量和定性的变化， 观察到与疾病发展相关的I-Ag 7-肽组（胰岛素、嗜铬粒蛋白、GAD 65、S100）。 目的2是了解感染和炎症对MHC II自身肽组的影响。 使用定量同位素标记和无标记蛋白质组学方法，我们将监测体内呈现的 自身和非自身肽段在静息小鼠和结核分枝杆菌感染后呈递cDC 或鼠伤寒沙门氏菌，以确定cDC成熟过程如何改变呈递的MHC-II自身 肽组。我们将确定MHC II自身免疫细胞中MHC II亲和力和HLA-DM敏感性的任何变化。 肽组诱导的感染，以及在MHC II抗原加工途径的差异，在未成熟的， 病原体成熟的cDC。最后，我们评估了翻译后修饰过程中的变化 在DC成熟过程中诱导的表达影响呈递的肽组。 总的来说，这些研究将确定局部淋巴结中的MHC II表位拷贝数如何能够 有助于耐受性和自身免疫之间的平衡，感染的后果是什么？ 和炎症反应。

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.