show Abstracthide AbstractMouse B-1 B lymphocytes play essential roles in defense against infectious microorganisms and maintenance of self-tolerance by producing natural antibodies. However, whether exogenous factors influence B-1 B cell repertoire formation remains poorly understood. Herein, we demonstrate that early immunization with N-acetyl-D-glucosamine (GlcNAc)-bearing Streptococcus pyogenes elicits GlcNAc-reactive B-1 B cells in C57BL/6 mice expressing immunoglobulin heavy chains distinct from those of naïve mice and mice immunized with S. pyogenes in adulthood. Moreover, germ-free mice exhibit greatly reduced GlcNAc-reactive serum antibodies and deficiencies in oligoclonal GlcNAc-reactive B-1 cell clonotypes dominant in conventionally raised mice. These phenotypes are observed in Myd88-deficient mice and reversed following conventionalization of adult germ-free mice. Microbiota colonization promotes development of GlcNAc-reactive B cells and IgA-secreting plasma cells in the small intestine that are clonally related to PerC and splenic B-1 B cells. These results indicate that environmental and microbiota-derived antigens influence B-1 clonotype selection into the mature repertoire.