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: 10684881
• 负责人: Daniel S Mucida
• 金额: $ 81.16万
• 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/10684881
• 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; Color; 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; Maps; 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; 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.

除了在消化食物等生理过程中发挥关键作用外，肠道 微生物还对肠道免疫系统提供低级刺激，其中包含 体内的大多数淋巴细胞和抗体。肠道微生物区系定植 影响获得性免疫，在很大程度上是通过肠道相关的次级淋巴 结构包括引流性肠系膜淋巴结(MLN)和Peyer补片(PPS)。在……里面 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，但也代表了微生物区系组成本身的主要调节因素，在 部分通过抗体的分泌。然而，肠道中抗体的形成和分泌是如何 微生物群的调节和调节还没有完全被理解。特别是，它仍然 尚不清楚自然发生的肠道相关生发中心(GaGC)、基本结构 对于B细胞受体亲和力成熟和类别转换，处理过多的 管腔抗原，或者反过来，在没有它们的情况下产生。在我们之前的工作和初步的 在这里，我们展示了一系列证据，支持 建议进行的研究。首先，我们开发了一种使用“脑弓”等位基因的多色命运图 作为一个系统来衡量积极GC选择的程度(因此亲和力成熟) 具有高吞吐量的单GC。其次，我们给出的数据支持了强劲的 向单克隆性的选择，以及稳定状态下的基于亲和力的选择。 第三，通过将我们的Brain弓小鼠重新派生到我们现有的GF设备中，并通过分析克隆 在没有肠道微生物区系的情况下，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类型组成 它们经历了非常快速的正选择。因此，我们假设，尽管 肠道的巨大抗原性多样性，亲和力成熟的共生体确实发生在 生理学。在目标1和目标2中，我们建议利用易于识别“赢家”B的能力 脑弓系统(AIM 1)提供的细胞克隆以分离亲和力强的B细胞克隆 成熟到稳定的共生体，作为洞察潜在的基本生物学基础的工具 GaGCs的克隆动力学和一般的粘膜抗体反应(目标2)。在目标3中，我们 建议对在GF小鼠中观察到的异常GC的生物学进行研究，以确定 支配肠道的公共B细胞克隆的起源、特异性和功能 在没有微生物区系的情况下的反应。通过结合我们在GUT方面的互补专业知识(Mucida实验室) 和B细胞生物学(Victora Lab)，拥有诺维菌素和最先进的成像技术，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转换的影响 肠道；以及在没有微生物的情况下管理这些过程的机制 刺激。