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Summary: The non-classical Major Histocompatibility Complex class II (MHCII) protein, H2-M, edits peptides bound to conventional MHCII in favor of stable peptide/MHCII (p/MHCII) complexes. Here, we show that H2-M deficiency affects B-1 cell survival, reduces cell renewal capacity, and alters immunoglobulin repertoire, allowing for the selection of cells specific for highly abundant epitopes, but not low-frequency epitopes. H2-M-deficient B-1 cells have shorter CDR3 length, higher content of positively charged amino acids, shorter junctional regions, less mutation frequency, and a skewed clonal distribution. Mechanistically, H2-M loss reduces plasma membrane p/MHCII association with B cell receptors (BCR) on B-1 cells and diminishes integrated BCR signal strength, a key determinant of B-1 cell selection, maturation, and maintenance. Thus, H2-M:MHCII interaction serves as a cell-intrinsic regulator of BCR signaling and influences the selection of the B-1 cell clonal repertoire.

B-1a cells play an important role in mediating tissue homeostasis and protecting against infections. They are the main producers of ‘natural’ IgM, spontaneously secreted serum antibodies predominately reactive to self antigens, like phosphatidylcholine (PtC), or antigens expressed by the intestinal microbiota. The mechanisms that regulate the B-1a immunoglobulin (Ig) repertoire and their antibody secretion remain poorly understood. Here, we use a novel reporter mouse to demonstrate that production of self- and microbiota-reactive antibodies is linked to BCR signaling in B-1a cells. Moreover, we show that Toll-like receptors (TLRs) are critical for shaping the Ig repertoire of B-1a cells as well as regulating their antibody production. Strikingly, we find that both the colonization of a microbiota as well as microbial-sensing TLRs are required for anti-microbiota B-1a responses, whereas nucleic-acid sensing TLRs are required for anti-PtC responses, demonstrating that linked activation of BCR and TLRs controls steady state B-1a responses to both self and microbiota-derived antigens.

Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.