Single-cell RNA-seq of IgM⁺ B cells against bacterial challenge in vaccinated flounder (Paralichthys olivaceus)

B cells play a key role in vertebrate vaccinations induced protective immunity. However, teleost B cells still need to be investigated for their protective role mediated by vaccine immunization. To assess post-vaccination immune responses, flounder were intramuscularly immunized with a <i>Vibrio anguillarum</i> DNA vaccine and challenged 35 days later to assess protective immunity. IgM⁺ B cells from the head kidney were sorted using a previously developed monoclonal antibody and subjected to single-cell RNA sequencing at 7 days post-challenge. The sorted IgM⁺ B cells comprised 7 subsets: Innate B cells, CXCR4⁺ B cells, Mature B cells, Plasmablast, TIRAP⁺ B cells, CD22⁺ B cells, and CD44⁺B cells. Three differentiation trajectories were identified, with CXCR4⁺ B cells and Mature B cells defined as transitional states critical for B cell-mediated protective responses. Innate B cells and Plasmablast were significantly associated with phagocytosis and antibody secretion pathways, respectively. And after vaccination, both CXCR4⁺ and Mature B cell subsets expanded and were enriched in relevant pathways involved in T cell interactions, indicating their enhanced capacity to support T-cell responses. Notably, Plasmablast exhibited a transcriptional shift from positive response to immune homeostasis, suggesting that Plasmablast may play a sustained role in long-term protection. These findings demonstrate the functional diversity of teleost IgM⁺ B cells and identify T cell-dependent subsets as critical mediators of the vaccine-mediated immune response. This study reveals new insights into B cell differentiation and vaccine-induced immunity in teleost, advancing the understanding of vertebrate adaptive immunity.