B cell clonal selection in gut-associated germinal centers

除了在诸如食物消化等生理过程中发挥关键作用外， 微生物还提供了一个低级别的刺激肠道免疫系统，其中含有 大多数的淋巴细胞和抗体在体内。肠道菌群定植 在很大程度上通过肠道相关的次级淋巴细胞影响适应性免疫 包括肠引流肠系膜淋巴结（mLN）和派尔集合淋巴结（PP）的结构。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， 部分通过分泌抗体。然而，肠道中抗体的形成和分泌是如何 受微生物组调节和调节微生物组的机制还不完全清楚。特别是，它仍然 目前尚不清楚天然存在的肠道相关生发中心（gaGCs）， 对于B细胞受体亲和力成熟和类别转换， 管腔抗原或反过来，在它们不存在的情况下发展。在我们以前的工作和初步的 在这里，我们展示了支持的相关性和可行性的证据线， 建议的研究。首先，我们使用“Brainbow”等位基因开发了一种抗抑郁剂-映射， 作为一个系统来测量阳性GC选择的程度（从而亲和力成熟）， 具有高通量的单个GC。第二，我们提供的数据支持证据表明， 稳定状态下的gaGC中的单克隆选择以及基于亲和力的选择。 第三，通过将我们的Brainbow小鼠重新衍生到我们现有的GF设施中，并通过分析克隆 在没有肠道微生物群的情况下，我们发现gaGCs仍然是 在GF条件下大量观察到，令人惊讶地由高度公开的克隆型组成 它们经历了异常快速的正向选择。因此，我们假设， 肠道的巨大抗原多样性，对大肠杆菌的亲和力成熟确实发生在 生理学在目标1&2中，我们建议利用容易识别“赢家”B的能力 由Brainbow系统提供的细胞克隆（目的1），以分离具有强亲和力的B细胞克隆 成熟到稳定状态的生物学，作为一种工具，以深入了解基本的生物学基础 gaGCs的克隆动力学和一般的粘膜抗体应答（目的2）。在目标3中， 我建议调查在GF小鼠中观察到的不寻常GC的生物学，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），与gnotobiotic和最先进的成像，单细胞 测序，生物化学和微生物学方法，我们试图确定的影响， 微生物组对B细胞选择、抗体亲和力成熟和 肠;以及在没有微生物的情况下控制这些过程的机制 刺激.

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.