Gut-associated lymphoid tissue (GALT) is organised lymphoid tissue that is chronically activated by the intestinal microbiota. It generates the IgA response that is critical for intestinal homeostasis. By iterative application of multiplexed technologies, we identify enrichment of double-negative 2 (DN2:CD27-IgD-CD21 lo CD11c hi) B cells in GALT, where they comprise the majority of intraepithelial and subepithelial B cells. We show that DN2 B cells in GALT interact with DC in the sub-epithelial dome that express DNASE1L3 and microbicides. Unlike in mice, DNASE1L3 in humans does not associate with apoptotic debris, but is located between sampled bacteria and host tissue where it is co-expressed with C1Q, consistent with management of bacterial debris. Thus we demonstrate that DN2 B cells that are otherwise associated with lupus nephritis, and DNASE1L3 and C1q that are lupus autoantigens, are microbiota-associated, interacting components of normal intestinal immunity.