Commensal-dependent maturation of the natural IgM repertoire

 描述(由申请人提供)：这项提案将确定肠道微生物区系如何塑造先天类B细胞的发育，以及天然IgM(NIGM)谱系中所代表的抗体的特异性。NIGM在维持免疫稳态和清除凋亡细胞上潜在的免疫原性自身抗原，抑制自身免疫性和变态反应性疾病的发展方面发挥着重要作用。先天类B细胞是NIGM的来源，具有多糖和磷脂表位的特异性，这些表位在哺乳动物抗原和肠道内共生细菌之间保守，但关于微生物来源的抗原对这一抗体亚群发展的影响知之甚少。观察到先天的类B细胞在小肠固有层经历抗原选择导致这些组织中分泌IgA的浆细胞的分化，以及观察到在无菌条件下维持的小鼠在过敏模型和几种自身免疫性疾病模型中经历了疾病的恶化，这使我们假设微生物区系在塑造系统B细胞库和生成NIGM中发挥了以前未被认识到的作用。我们研究了非T细胞(TI)抗原N-乙酰-D-氨基葡萄糖胺(GlcNAc)特异性B细胞在无特定病原体和无菌小鼠之间的分布，发现大量证据表明，肠道定植增加了B细胞的数量和血清中针对GlcNAc的抗体水平，同时诱导了用于产生这种特异性的显性免疫球蛋白V基因的改变。为了进一步分析这些相互作用，我们开发了一种新的荧光激活细胞分类方法来研究TI抗原在共生细菌物种上的分布。通过同时评估这些抗原的IgA调理状态，我们期望(I)鉴定出免疫相关的微生物，这些微生物携带与肠道中的体液免疫相互作用的特异性TI抗原。利用灵知生菌动物模型，在上述平台上，无菌小鼠与富含特定抗原决定簇的菌群重组，我们计划验证这样的假设，即来自共生微生物群的抗原导致B细胞克隆型选择进入产生Nigm的生态位。我们计划通过(Ii)评估脾GlcNAc和Lewis X抗原特异性B细胞在与富含这些抗原的细菌共生分离株进行微生物重组后的频率和表型，(Iii)监测LP、脾和骨髓局部浆细胞的产生，以及(Iv)监测组成这些特异性的B细胞克隆型的变化来检验这一假设。这些实验将首次证明，在外周组织中与外源抗原的相互作用是发展完全合格的NIGM谱系所必需的。这项工作将导致开发对肠道微生物区系多样性进行分类的新方法，并为开发NIGM谱系在设计旨在提高自然免疫力的免疫疗法方面的价值提供有价值的见解。

 DESCRIPTION (provided by applicant): This proposal will determine how the intestinal microbiota shapes the development of innate-like B cells and the specificities of antibodies represented in the natural IgM (nIgM) repertoire. nIgM plays significant roles in maintenance of immunological homeostasis and the clearance of potentially immunogenic autoantigens represented on apoptotic cells, suppressing the development of autoimmune and allergic diseases. Innate-like B cells are the source of nIgM, and possess specificities for polysaccharide and phospholipid epitopes that are conserved between mammalian antigens and commensal bacteria harbored within the gut, however little is known regarding the impact of microbiota-derived antigens on the development of this subset of antibodies. Observations that innate-like B cells undergo antigen selection in the small intestine lamina propria leading to the differentiation of IgA secreting plasma cells in those tissues, and observations that mice maintained under germ free conditions experience exacerbated diseases in allergy models and in several autoimmune disease models led us to hypothesize that the microbiota plays a previously unappreciated role in shaping the systemic B cell repertoire and the generation of nIgM. We investigated the distribution of B cells specific for the T cell-independent (TI) antigen N-acetyl-D-Glucosamine (GlcNAc) between specific pathogen free and germ free mice and have found substantial evidence that intestinal colonization increases the numbers of B cells and levels of serum antibody specific for GlcNAc, while inducing shifts in the dominant immunoglobulin V-gene used to generate this specificity. To further dissect these interactions, we developed a novel fluorescence-activated cell sorting approach to study the distribution of TI antigens on species of commensal bacteria. Through simultaneous evaluation of the IgA opsonization status of these antigens, we expect to (i) identify immunologically relevant microorganisms bearing specific TI- antigens that interface with humoral immunity in the gut. Using gnotobiotic animal models, wherein germ free mice are reconstituted with flora enriched for particular antigenic determinants with the platform described above, we plan to test the hypothesis that antigen derived from the commensal micoflora leads to selection of B cell clonotypes into the nIgM-producing niche. We plan to test this hypothesis by (ii) evaluating the frequency and phenotype of splenic GlcNAc and Lewis X antigen-specific B cells following microbial reconstitution with bacteria commensal isolates enriched for these antigens, (iii) monitoring the generation of LP, spleen and bone marrow-localized plasma cells, and (iv) monitoring shifts in the B cell clonotypes composing those specificities. These experiments will demonstrate for the first time that interaction with exogenous antigen at peripheral tissues is required for development of a fully competent nIgM repertoire. This work will lead to the development of novel means of classifying the diversity of the intestinal microbiota, and offer valuable insight to the development of the nIgM repertoire value in designing immunotherapies aimed at boosting natural immunity.