B cell clonal selection in gut-associated germinal centers

肠道相关生发中心的 B 细胞克隆选择

• 批准号: 10265570
• 负责人: Daniel S Mucida
• 金额: $ 76.86万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-17 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10265570
• 关键词: Acceleration; Adaptive Immune System; Address; Affinity; Alleles; Antibodies; Antibody Affinity; Antibody Formation; Antibody Response; Antigenic Diversity; Antigens; Autoimmunity; Automobile Driving; B-Cell Antigen Receptor; B-Lymphocytes; Biochemical; Biology; Bone Marrow; Cellular biology; Clone Cells; Complex; Data; Digestion; Disease; Food; Food Hypersensitivity; Gastrointestinal tract structure; Germ-Free; Gnotobiotic; Image; Immune response; Immune system; Immunization; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunology; Infection; Inflammatory Bowel Diseases; Intestines; Knowledge; Lamina Propria; Lymphocyte; Malignant Neoplasms; Measures; Microbe; Microbiology; Mucous Membrane; Mus; Nature; Outcome; Peyer' s Patches; Physiological; Physiological Processes; Physiology; Plasma Cells; Play; Process; Reagent; Recombinants; Reporting; Resistance; Role; Secretory Immunoglobulin A; Series; Specificity; Stimulus; Structure; Structure of germinal center of lymph node; System; Techniques; Work; adaptive immunity; base; commensal microbes; dimer; enteric infection; experimental study; germ free condition; gut colonization; gut microbes; gut microbiome; gut microbiota; insight; lymphoid structures; mesenteric lymph node; microbial; microbiome; microbiota; mouse model; plasma cell differentiation; resistance mechanism; response; single cell sequencing; tool

In addition to playing a crucial role in physiological processes such as digestion of food, gut microbes also provide a low-grade stimulation of the intestinal immune system, which contains the majority of the lymphocytes and antibodies in the body. Colonization by gut microbiota influences adaptive immunity, in large part via gut- associated secondary lymphoid structures including gut-draining mesenteric lymph nodes (mLN) and Peyer patches (PPs). In turn, the gut-associated adaptive immune system provides crucial resistance mechanisms against enteric infections, but also represents a major regulator of the microbiota composition itself, in part via secretion of antibodies. However, how antibody formation and secretion in the intestine is regulated by, and regulate the microbiome is incompletely understood. Particularly, it remains unclear how naturally-occurring gut- associated germinal centers (gaGCs), essential structures for B cell receptor affinity maturation and class switching, deal with the plethora of luminal antigens or in turn, develop in their absence. In our previous work and preliminary presented here, we demonstrate lines of evidence supporting the relevance and feasibility of the proposed studies. First, we developed a multicolor fate-mapping using "Brainbow" alleles as a system to measure the extent of positive GC selection (and thus of affinity maturation) in single GCs with high throughput. Second, we present data with supportive evidence of strong selection towards monoclonality, as well as affinity- based selection in gaGCs in the steady state. Third, by re-deriving our Brainbow mice into our existing GF facility, and by analyzing clonal dynamics and winner clones under absence of gut microbiota, we found that gaGCs are still abundantly observed in GF conditions, surprisingly consisting of highly public clonotypes that undergo extraordinarily fast positive selection. We thus hypothesize that, despite the enormous antigenic diversity of the gut, affinity maturation towards commensals does take place in the physiology. In Aims 1&2, we propose to leverage the ability to readily identify "winner" B cell clones afforded by the Brainbow system (Aim 1) to isolate B cell clones with strong affinity maturation to steady state commensals, as a tool to gain insight into the basic biology underlying the clonal dynamics of gaGCs and the mucosal antibody response in general (Aim 2). In Aim 3, we propose to investigate the biology of the unusual GCs observed in GF mice to determine the origin, specificity, and function of public B cell clones that dominate the intestinal response in the absence of microbiota. By combining our complementary expertise in gut (Mucida lab) and B cell biology (Victora lab), with gnotobiotic and state-of-the-art imaging, single-cell sequencing, biochemical and microbiology approaches, we seek to determine the influence of the microbiome on B cell selection, antibody affinity maturation and class switching in the intestine; as well as the mechanisms that govern these processes in the absence of microbial stimulation.

除了在食物消化等生理过程中发挥至关重要的作用外，肠道 微生物还对肠道免疫系统提供低度刺激，其中包含 体内大部分淋巴细胞和抗体。肠道微生物群的定植 影响适应性免疫，很大程度上是通过肠道相关的次级淋巴 结构包括肠道引流肠系膜淋巴结（mLN）和派尔淋巴结（PP）。在 反过来，肠道相关的适应性免疫系统提供了关键的抵抗机制 肠道感染，但也代表了微生物群组成本身的主要调节剂， 部分通过抗体的分泌。然而，抗体在肠道中的形成和分泌是如何进行的？ 微生物组的调节和调节尚不完全清楚。特别是，它仍然 不清楚天然存在的肠道相关生发中心 (gaGC)、基本结构是如何产生的 对于 B 细胞受体亲和力成熟和类别转换，需要处理过多的 管腔抗原，或者反过来，在没有管腔抗原的情况下发育。在我们之前的工作和前期工作中 在这里，我们展示了支持该计划的相关性和可行性的证据 拟议的研究。首先，我们使用“Brainbow”等位基因开发了多色命运图谱 作为测量阳性 GC 选择程度（以及亲和力成熟程度）的系统 具有高通量的单 GC。其次，我们提供的数据具有强有力的支持证据 针对单克隆性的选择，以及稳定状态下 gaGC 中基于亲和力的选择。 第三，通过将我们的 Brainbow 小鼠重新派生到我们现有的 GF 设施中，并通过分析克隆 在缺乏肠道微生物群的情况下的动态和获胜克隆，我们发现 gaGC 仍然是 在 GF 条件下大量观察到，令人惊讶的是由高度公开的克隆型组成 经历非常快的正选择。因此我们假设，尽管 肠道具有巨大的抗原多样性，对共生体的亲和力成熟确实发生在 生理学。在目标 1 和 2 中，我们建议利用轻松识别“获胜者”B 的能力 Brainbow 系统提供的细胞克隆（目标 1），用于分离具有强亲和力的 B 细胞克隆 成熟到稳态共生体，作为深入了解基础生物学的工具 gaGC 的克隆动力学和一般粘膜抗体反应（目标 2）。在目标 3 中，我们 提议研究 GF 小鼠中观察到的异常 GC 的生物学以确定 主宰肠道的公共 B 细胞克隆的起源、特异性和功能 在没有微生物群的情况下的反应。通过结合我们在肠道方面的互补专业知识（Mucida 实验室） 和 B 细胞生物学（Victora 实验室），具有 gnotobiotic 和最先进的成像、单细胞 测序、生化和微生物学方法，我们试图确定 微生物组对 B 细胞选择、抗体亲和力成熟和类别转换的影响 肠;以及在没有微生物的情况下控制这些过程的机制 刺激。