肥胖新生抗原特异性CD8+T细胞在肥胖相关慢性炎症中的作用研究

Obesity-induced adipose tissue chronic inflammation is now considered to have a pivotal role in the development of metabolic diseases including insulin resistance, type 2 diabetes and atherosclerosis. A great deal of evidence has pointed to the role of innate immune cells, in particular,adipose tissue macrophages(ATMs), in the regulation of fat inflammation. However, more recently, cells of the adaptive immune system, specifically T lymphocytes, have emerged as unexpected promoters and controllers of fat inflammation and insulin resistance. These adaptive immune cells infiltrate obesity expanded visceral adipose tissue(VAT) and through cytokine secretion and macrophage modulation dictate the extent of the local inflammatory response, thereby directly impacting insulin resistance.Interestingly,evidence suggests that obesity-induced inflammation is initiated by CD8+ T cells: the adipose tissue attracts and activates CD8+ T cells that in turn initiate and maintain adipose tissue inflammation and systemic insulin resistance. Adaptive CD8+ T cells recognizes MHC I-associated peptides.These MHC I peptides are collectively referred to as the MHC-I associated immunopeptidome (MIP).The composition of the MIPs varies from one cell type to another and can be perturbed by cell-intrinsic and -extrinsic factors including dysregulation of cellular metabolism and infection.Over the past years, the ability of mass spectrometry to analyze complex peptide mixtures has provided unprecedented insights into the repertoire of peptide antigens displayed by MHC molecules and recognized by T lymphocytes.Here we will applicate mass spectrometry to analysis of self-MIPs distinction between obese and lean mice adipocytes. Because excessive amounts of nutrients induce endoplasmic reticulum stress in adipocytes,it would be interesting for us to identify some neo-self antigenic peptide ligands on overloaded adipocytes, which be specifically recognized by CD8+ T cells that initiate obesity-induced inflammation.We also want to know whether the tolerogenic treatment with neoantigen can protect C57BL/6 mice from obese-induced inflammation and metabolic abnormalitiesinduction.

肥胖诱导的内脏脂肪组织慢性炎症与各种代谢综合征密切相关。脂肪组织巨噬细胞（ATMs）在内脏脂肪组织慢性炎症中发挥主要作用。近来发现适应性免疫应答参与ATMs调控，其中未知抗原驱动的效应性CD8+T细胞最早参与ATMs招募及内脏脂肪组织炎症启动。CD8+T细胞特异性识别有核细胞表面MHC-Ⅰ类分子结合的肽而活化，其肽总和称为MHC-Ⅰ免疫肽组（MIP）。最新研究显示多种刺激因素导致的细胞代谢异常可重塑细胞表面MIPs组成。本课题拟利用高分辨率质谱技术，鉴定高脂饮食诱导的肥胖小鼠和正常小鼠内脏脂肪细胞MIPs差异，结合细胞免疫学功能实验明确:肥胖时由脂肪代谢紊乱所产生的脂肪细胞新生抗原是否成为驱动脂肪组织中CD8+T细胞激活的关键抗原因素，并尝试通过诱导小鼠对肥胖相关新生抗原的免疫耐受而实现对高脂饮食诱导的系统性炎症及胰岛素抵抗的防治，为肥胖相关炎症性疾病提供新的特异性干预靶点和策略。

肥胖诱导的内脏脂肪组织慢性炎症与各种代谢综合征密切相关。脂肪组织巨噬细胞（ATMs）在内脏脂肪组织慢性炎症中发挥主要作用。近来发现适应性免疫应答参与ATMs调控，其中未知抗原驱动的效应性CD8+T 细胞最早参与ATMs 招募及内脏脂肪组织炎症启动。CD8+T 细胞特异性识别有核细胞表面MHC-Ⅰ类分子结合的肽而活化，其肽总和称为MHC-Ⅰ免疫肽组（MIP）。最新研究显示多种刺激因素导致的细胞代谢异常可重塑细胞表面MIPs 组成。本课首先成功建立高脂饮食诱导的肥胖及代谢异常的小鼠模型，并证实在高脂饮食第二周，内脏脂肪组织（VAT）中浸润的CD8+T细胞比例和绝对数量均明显增多；利用免疫亲和层析技术成功分离获得来源于VAT的MIP，并联合高分辨率质谱技术，成功鉴定出分别来自正常饮食的正常对照小鼠及高脂饮食肥胖小鼠的VAT中MIPs。我们根据三次质谱结果重现性，并比对Uniprot数据库中小鼠proteome，对三次质谱结果均为高可信度的多肽进行多肽来源分析，我们发现MIPs中约有三分之一的高重现性多肽为剪切多肽，无法追索其蛋白来源，因此在后续分析时被忽略。后续分析仅保留来源蛋白属于小鼠蛋白质组，且同时满足与H2-Kb结合亲和力IC50<1000nM的多肽，筛选获得肥胖小鼠 VAT MIP中存在的，而在正常小鼠VAT MIPs 中不存在的若干新生肽。进一步结合细胞免疫学功能实验明确：肥胖时由脂肪代谢紊乱所产生的VAT 新生抗原肽具有明显的免疫原性，可有效激活脾初始CD8+T 细胞激活并分泌促炎因子，而两者共同存在的多肽则无免疫原性。尝试通过口服脂肪组织来源的混合抗原，诱导小鼠免疫耐受而实现对高脂饮食诱导的系统性炎症及胰岛素抵抗的防治，为肥胖相关炎症性疾病提供新的特异性干预靶点和策略。