Highlights d Germline transcripts peak prior to GC formation and rapidly decline in GCs d IgM-dominated clones are found in late GCs, arguing against ongoing Ig switching d CSR largely ceases upon the onset of somatic hypermutation d CSR decline due to low GLT and APE1 expression is possibly orchestrated by BCL6

Highlights d Memory B cell reentry into germinal centers is rare under typical boost regimens d Most (>90%) B cells in secondary GCs have no prior GC experience d A clonality bottleneck restricts the diversity of recall antibody-producing cells d Most primary diversity is found in an MBC compartment not accessed by boosting

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Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton-Valentine Leukocidin (PVL) and γ-Hemolysin CB (HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the apparent redundancy of both toxins cannot be adequately addressed in experimental models of infection since mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity towards neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Over-expression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity towards neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that toxicity of PVL, but not HlgCB, is neutralized by various C5aR1-antagonists. This study offers directions for the development of improved preclinical models for infection, and the design of drugs antagonizing leukocidin toxicity.

Germinal centers (GCs), structures normally associated with B cell immunoglobulin (Ig) hypermutation and development of high-affinity antibodies upon infection or immunization, are present in gut-associated lymphoid organs of humans and mice under steady state. Gut-associated (ga)GCs can support antibody responses to enteric infections and immunization 1 . However, whether B cell selection and antibody affinity maturation can take place in face of the chronic and diverse antigenic stimulation characteristic of steady-state gaGCs is less clear 2 – 8 . Combining multicolor “Brainbow” fate-mapping and single-cell Ig sequencing, we find that 5–10% of gaGCs from specific pathogen-free (SPF) mice contained highly-dominant “winner” clones at steady state, despite rapid turnover of GC B cells. Monoclonal antibodies (mAbs) derived from these clones showed increased binding to commensal bacteria compared to their unmutated ancestors, consistent with antigen-driven selection and affinity maturation. Frequency of highly-selected gaGCs was markedly higher in germ-free (GF) than in SPF mice, and winner B cells in GF gaGCs were enriched in public IgH clonotypes found across multiple individuals, indicating strong B cell receptor (BCR)-driven selection in the absence of microbiota. Vertical colonization of GF mice with a defined microbial consortium (Oligo-MM 12 ) did not eliminate GF-associated clonotypes, yet induced a concomitant commensal-specific, affinity-matured B cell response. Thus, positive selection can take place in steady-state gaGCs, at a rate that is tunable over a wide range by the presence and composition of the microbiota.