Commensal-dependent maturation of the natural IgM repertoire

 描述（由申请人提供）：该提案将确定肠道微生物群如何塑造先天性B细胞的发育以及天然IgM（nIgM）库中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代表的潜在免疫原性自身抗原，抑制自身免疫性和过敏性疾病的发展中起重要作用。天然样B细胞是nIgM的来源，并且对多糖和磷脂表位具有特异性，所述多糖和磷脂表位在哺乳动物抗原和肠道内窝藏的肠道细菌之间是保守的，然而关于微生物来源的抗原对该抗体子集的发展的影响知之甚少。观察到先天样B细胞在小肠固有层中经历抗原选择，导致这些组织中分泌伊加的浆细胞分化，并且观察到保持在无菌条件下的小鼠在变态反应模型和几种自身免疫性疾病模型中经历了恶化的疾病，这使我们假设微生物群在形成系统性B细胞库和免疫性疾病中起着以前未被认识到的作用。nIgM的生成。我们研究了特异性针对T细胞非依赖性（TI）抗原N-乙酰基-D-氨基葡萄糖（GlcNAc）的B细胞在无特定病原体和无菌小鼠之间的分布，发现了大量证据表明肠道定植增加了B细胞数量和GlcNAc特异性血清抗体水平，同时诱导了用于产生这种特异性的显性免疫球蛋白V基因的变化。为了进一步剖析这些相互作用，我们开发了一种新的荧光激活细胞分选方法来研究TI抗原在细菌物种上的分布。通过同时评价这些抗原的伊加调理作用状态，我们期望（i）鉴定携带特异性TI-抗原的免疫相关微生物，所述TI-抗原与肠道中的体液免疫相互作用。使用无菌动物模型，其中无菌小鼠用上述平台用富集特定抗原决定簇的植物群重建，我们计划测试来源于肠道微生物植物群的抗原导致B细胞克隆型选择进入产生nIgM的小生境的假设。我们计划通过（ii）用富含这些抗原的细菌分离株进行微生物重建后，评价脾脏GlcNAc和刘易斯X抗原特异性B细胞的频率和表型，（iii）监测LP、脾脏和骨髓定位浆细胞的生成，以及（iv）监测构成这些特异性的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，在外周组织与外源性抗原的相互作用是开发完全胜任的nIgM库所必需的。这项工作将导致开发对肠道微生物群多样性进行分类的新方法，并为nIgM库的开发提供有价值的见解，以设计旨在增强天然免疫力的免疫疗法。

 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.