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.
The gut-associated adaptive immune system provides crucial resistance mechanisms against enteric infections, but also represents a major regulator of the microbiota composition itself. This proposal seeks to define how antibody formation and secretion in the intestine are regulated, including the influence of the microbiome. This knowledge is relevant for a better understanding of several diseases process, ranging from inflammatory bowel diseases (IBD) to food allergy, autoimmunity and cancer.
