基于Exosome反应性B细胞过继免疫探讨边缘区B细胞在1型糖尿病发病机制中的作用

The early breach of B cell tolerance and sequential induction of autoreactive T cells are major contributors to type 1 diabetes (T1D). Our prior data found that marginal zone B (MZ B) cells，with innate like lymphocyte function, were effective in activating and expanding the autoreactive T cells. Their antigen receptors are cross-reactive to many different self antigens but with low affinity. The mechanism how MZ B cells contribute to autoimmunity remains unclear. Exosomes (EXOs) are nano-sized vesicles secreted by different kinds of cells, whose function is mainly to act as a cell-to-cell messenger, but also believed to mediate pathophysiology. Upon EXO stimulation, MZ B cells can release proinflammatory cytokines such as IL-6, TNFα. Moreover, autoantigens carried by the EXOs can stimulate EXO-specific autoreactive B and T cells. Therefore, EXO reactive B cells provide us a novel tool for the investigation of type 1 diabetes. In this study, we attempt to adopt cell culture, flow cytometry, and hybridoma techniques to analyze the phenotype of the EXO-induced B cells, and study their antigenic specificity.To confirm the T1D-causative role of the B cells, we will perform cell adoptive transferring experiments via B cell reconstitution in NOD.Ignull mice. Meanwhile, we will study EXO-induced innate response in the activation of MZ B via MDA5 pathway by using knockout mice, which may shed new light on the diagnosis, prevention and therapy of T1D.

因B细胞免疫耐受的破坏而激活自身反应性T细胞是1型糖尿病的重要发病机制之一。前期研究发现兼有固有免疫细胞样特征的边缘区B细胞具有选择和放大致病性T细胞的效应。目前对其自身活化机制仍然不明。Exosomes （外吐小体，EXOs)是一种新型的细胞分泌囊泡，参与细胞间的信号传递和病理生理过程。EXOs不但能够激活边缘区B细胞释放IL-6、TNFα等炎症细胞因子，还因为携带自身抗原而特异性的激活引起糖尿病的自身反应性B和CD4+ T细胞，所以EXO反应性B细胞为研究1型糖尿病提供了新的靶点。我们拟采用细胞培养、流式细胞术、杂交瘤技术等方法对EXO反应性B细胞进行表型和抗原特异性鉴定；利用B细胞重建NOD.Ignull小鼠糖尿病模型，体内验证EXO反应性B细胞在糖尿病中的作用；同时借助转基因动物明确MDA5固有免疫通路在活化边缘区B细胞中不可或缺的作用。为1型糖尿病的诊断、预防和治疗开辟新途径。

1型糖尿病（Type I diabetes, T1D）是以自身反应性B和T细胞克隆的激活和调控异常为发病机制核心环节的一种自身免疫性疾病，患者终身遭受身体病痛和经济压力，目前尚缺乏有效的治疗手段，开发安全有效的干预策略已十分迫切。B 细胞不但作为效应细胞还是抗原递呈细胞，直接激活自身反应性T 细胞，早期活化的边缘区B细胞（MZB细胞）可能为防治1型糖尿病提供新的靶点。我们的研究发现边缘区B细胞在STZ诱导T1DM小鼠模型中脾脏MZB细胞比例升高，且GC数量增加，表面积增大，脾脏TLR7的mRNA表达增加。由此论证MZB细胞很有可能对STZ诱导实验性T1DM产生了明显的作用。而糖尿病肾病小鼠胰腺组织来源外泌体可以明显刺激MZB细胞的增殖活化，提示MZB细胞在糖尿病发生发展中的重要作用。本研究系统阐明了MZB细胞以及外泌体在糖尿病发展过程中的变化规律，以及体内的功能，并对机制进行了初步的探索，研究成果不仅丰富了糖尿病研究的理论基础，也为糖尿病干预策略的研发提供新线索，有望为糖尿病的防治提供新的手段，具有潜在的经济和社会价值