B-cell-intrinsic MHCII Signaling is a Diversifying Force of Selection on IgA Repertoires and the Gut Microbiota

项目摘要 T细胞依赖(TD)免疫球蛋白A(IgA)反应调节肠道微生物区系的组成。多么 B细胞固有的MHCII信号对TD IgA反应的发展至关重要，它影响宿主- 微生物区系之间的相互作用是未知的。MHCII被广泛认为促进效应淋巴细胞的克隆性多样性 人口，但这还没有经过测试。在这里，我们提出证据支持B细胞- 固有的MHCII信号控制GC动态、IgA谱系多样性和微生物区系组成。 此外，我们的实验证据还表明，B细胞对MHCII的固有消融和特异性 MHC基因型别与肠道细菌传播增强有关。基于这些 Kubinak博士的观察和以前的工作，这一R01试图解决B细胞固有的假设 MHCII信号是一种多样化的选择力量，促进了IgA浆细胞池的克隆多样性 和微生物区系组成上的个性。具体目标#1的目标是定义B细胞固有的 MHCII在肠道GC反应中的作用；特别关注其在形成浆细胞库中的作用 多样性。我们将使用五彩纸屑的小鼠模型来直观地演示MHCII消融对GC B细胞的影响 肠道中的克隆多样性。免疫球蛋白测序将用于确定MHCII多态和MHC的影响 肠道中IgA谱带多样性的杂合性。将使用scRNA测序来确定MHCII如何 影响粘膜浆细胞池和系统浆细胞池克隆多样性的重叠。RAG1-/-骨髓 (BM)嵌合体将被用来量化MHCII对肠道来源的浆细胞交叉种植到 BM。互易的BM嵌合体将用于确定B细胞固有缺陷的必要性/充分性 MHCII在调节肠道细菌传播中的作用。具体目标#2目标是测试微生物区系 成分是一种依赖于MHCII的表型。RAG1-/-和RAG1-/-IL7R-/-BM嵌合体将用于 确定肠道Peyer‘s补片在肠道内驱动MHCII介导的IgA选择中所起的作用。微生物 在无菌GF RAG1-/-BM嵌合体中的定植实验将确定B细胞固有的MHCII的作用 关于以IgA为靶点的共生菌。将使用RAG1-/-采用迁移模型来确定MHCII 表面密度影响IgA介导的共生体和微生物群组成的靶向。最后，一部小说 无菌MHC同源模型将被用来明确定义IgA在推动个性中所起的作用 微生物区系组成。这些研究的结果将解决B细胞在调节MHCII中的内在作用 粘膜免疫球蛋白A反应，微生物区系组成，和宿主健康。这是我们知识中的一个关键缺口，也就是 与人类健康高度相关。IgA缺乏症是人类最常见的抗体缺乏症 与MHCII基因的遗传变异密切相关，并已被证明导致与 患有慢性炎症。这里概述的实验是第一次解决MHC基因如何影响 微生物区系组成和肠道细菌渗漏，是慢性免疫激活的有力驱动因素。

Project Summary T-cell-dependent (TD) immunoglobulin A (IgA) responses regulate the composition of the gut microbiota. How B-cell-intrinsic MHCII signaling, which is central to the development of TD IgA responses, influences host- microbiota interactions is unknown. MHCII is widely assumed to promote clonal diversity in effector lymphocyte populations, but this has not been tested. Here, we present evidence in support of the argument that B cell- intrinsic MHCII signaling controls GC dynamics, IgA repertoire diversity, and microbiota composition. Additionally, evidence from our experiments also indicate that both B-cell-intrinsic ablation of MHCII and specific MHC genotypes are associated with enhanced bacterial dissemination from the gut. Based on these observations and previous work by Dr. Kubinak, this R01 seeks to address the hypothesis that B-cell-intrinsic MHCII signaling is a diversifying force of selection promoting clonal diversity in IgA plasma cell pools and individuality in microbiota composition. The objective of Specific Aim #1 is to define the B-cell-intrinsic role of MHCII during GC reactions in the gut; specifically focusing on its role in shaping plasma cell repertoire diversity. A 'Confetti' mouse model will be used to visually demonstrate the effect of MHCII ablation on GC B cell clonal diversity in the gut. IgH sequencing will be used to determine the effect of MHCII polymorphisms and MHC heterozygosity on IgA repertoire diversity in the gut. scRNA sequencing will be used to determine how MHCII influences overlap in clonal diversity between mucosal and systemic plasma cell pools. RAG1-/- bone marrow (BM) chimeras will be used to quantify the effect of MHCII on cross-seeding of gut-derived plasma cells into the BM. Reciprocal BM chimeras will be used to determine the necessity/sufficiency of defects in B-cell-intrinsic MHCII in regulating bacterial dissemination from the gut. The objective of Specific Aim #2 is to test that microbiota composition is an MHCII-dependent phenotype. RAG1-/- and RAG1-/-IL7R-/- BM chimeras will be used to determine the role gut peyer's patches play in driving MHCII-mediated IgA selection in the gut. Microbial colonization experiments in germfree GF RAG1-/- BM chimeras will determine the effect of B-cell-intrinsic MHCII on IgA-targeting of commensal bacteria. A RAG1-/- adoptive transfer model will be used to determine if MHCII surface density influences IgA-mediated targeting of commensals and microbiota composition. Finally, a novel germfree MHC congenic model will be used to explicitly define the role IgA plays in driving individuality in microbiota composition. Results from these studies will address the B-cell-intrinsic role of MHCII in regulating mucosal IgA responses, microbiota composition, and host health. This is a critical gap in our knowledge that is highly relevant to human health. IgA deficiency is the most common form of antibody-deficiency in humans, is strongly linked to genetic variation in MHCII genes, and has been shown to result in dysbiosis that is associated with chronic inflammation. Experiments outlined here are the first to address how MHC genotype influences microbiota composition and bacterial leakage from the gut, a potent driver of chronic immune activation.