Christopher Wichmann

• The gut is an extremely complex environment. Many sources of foreign proteins, especially the resident microbiota, are separated from the host’s immune system by merely a single layer of epithelial cells. B cells, as part of the adaptive immune system, are important for the regulation of the microbial community. B cells can change the sequence of the immunoglobulin (Ig) they produce through somatic hypermutation (SHM) in germinal centres (GC). Within these GCs, B cells undergo iterative rounds of selection guided by T cells. In some cases, one B cell clone can out-compete the other clones in a given germinal centre and massively expand whilst creating large numbers of progeny in what is called a clonal burst. GCs form upon infection or vaccination but are constantly present in Peyer’s patches (PP) and mesenteric lymph nodes (mLN). The functional mechanisms of gut-associated GCs (gaGC) are still under active investigation. Here, the dynamics of gaGCs in settings of different antigenic diversity were investigated. Furthermore, the rules underlying selection processes in gaGCs, especially their potential to create affinity-matured B cells were explored. Lastly, the occurrence of public B cell clonotypes across different mice in settings of reduced antigenic diversity were analysed. To address these questions, fate-mapping mouse models like the AID-confetti model were used, which allowed for visual identification of selection in gaGCs and the isolation of individual GCs. The diversity, turnover and clonal composition of GC B cells, was analysed, and Ig sequences were produced as monoclonal antibodies (mAb) in vitro. The mAb were tested for binding to commensal bacteria by bacterial flow cytometry and enzyme-linked immunosorbent assay (ELISA), and to markers for self-reactivity, polyactivity and reactivity to mouse chow protein extract by ELISA. To test settings of different antigenic diversity mice from specific pathogen free (SPF) settings, germ free (GF) settings and a minimal bacterial community (Oligo-MM12) were analysed. In SPF mice gaGCs are constantly present. The half-life of GCs is approximately two weeks in mLNs and 1.5 weeks in PPs. Despite the rapid turnover selection events occur in these gaGCs. After 23 days six out of 57 GCs in mLN showed dominance by a highly expanded B cell clone. When producing mAbs from those highly expanded B cell clones, binding to faecal bacteria was detected. There was no detectable self-reactivity or reactivity to mouse chow protein and only low levels of polyreactivity. Reversion of mutations to the inferred germline unmutated common ancestor (UCA) led to a decrease in bacterial binding indicative of affinity maturation. In GF mice, enhanced numbers of highly selected gaGCs were found, with over 50 % of mLN GCs showing a dominant B cell clone as opposed to 11 % in SPF mice. Furthermore, the isotype usage in gaGCs deviated from IgG2b and IgA to predominantly IgG1. Oligo-MM12 mice displayed an intermediate phenotype for selection and isotype usage. When assaying mAbs derived from highly selected GF GCs, no binding was detected in any of the assays. For the mAbs derived from Oligo-MM12 mice, binding to faecal Oligo-MM12 bacteria was detected, which diminished upon reversion of mutations to the respective UCA. One mAb displayed binding to the Oligo-MM12 strain Enterococcus faecalis KB1. Lastly, the use of common clonotypes in GF and Oligo-MM12 mice was detected. In a broader analysis of gaGC B cells from GF, SPF, and Oligo-MM12 mice, increased usage of public clones in GF mice was found, with a specific enrichment of two clonotypes relying on VH1-47 and VH1-12. The usage of public clonotypes was reduced in Oligo-MM12 mice, as well as SPF mice and public clonotypes specifically relying on VH1-47 and VH1-12 were greatly reduced in SPF mice. Here, the dynamics of gaGCs in settings of different antigenic diversities are described. Whilst gaGCs are diverse and have short half-lives, selection events occur in a clonal burst fashion as described in peripheral immunisation. Those selection processes are tuned towards microbial diversity with markedly increased selection in GF mice. Mice colonised with a minimal bacterial community display an intermediate phenotype, showing the responsiveness of the system. Furthermore, in GF mice, B cell responses converged on recurrent public clonotypes between individual mice. The use of these clonotypes was drastically reduced in SPF mice. The binding antigen(s) of these public clonotypes remain elusive, although bacterial reactivity, mouse chow protein reactivity, self-reactivity, or polyreactivity were undetectable. For mAbs produced from SPF and Oligo-MM12 mice, it was possible to reveal bacterial binding which diminished upon reversion of mutations to the UCA, showing antigen-driven selection towards commensal binding under homeostatic conditions in these chronic gaGCs. The different responses to the microbiota might have clinical relevance, as bacteria highly coated in IgA drive disease in mouse models of colitis and malnutrition. Furthermore, differences in mutations of Ig sequences are found in B cells reacting to transglutaminase 2, a major serological marker of celiac disease. Even under non-pathological circumstances, the interaction with the microbiota seems important for the maturation of the mucosal immune system during early life. It is even more surprising that people with selective IgA deficiency show a variety of clinical outcomes with many asymptomatic cases. A deeper understanding of the adaptive mucosal immune system could aid the diagnosis and therapy of those clinical conditions.