Discovery of perforin-expressing killer B cells in vertebrates

Abstract

Abstract Our previous work has shown the existence of distinct subsets of B cells in vertebrates that are highly phagocytic. To understand further the involvement of B cells in other potential innate immune functions, we performed the first comparative transcriptome analysis on FACS-sorted IgT+ and IgM+ B cells in rainbow trout, a primitive teleost fish species. To our surprise the gene that showed the highest differential expression between these two B cell subsets was perforin, a molecule that is not typically associated with B cells. The results obtained by transcriptome were expanded by further RT-PCR analysis confirming that unlike IgM+ B cells, IgT+ B cells expressed high transcript levels of several perforin isoforms, including, prf1-like-B, prf1-like-C and prf1-like-D. We also confirmed the unique expression of these perforin genes in IgT+ B cell by single cell transcriptome analysis. Moreover, we produced antibodies against these perforin molecules and demonstrated by immunohistochemistry the presence of several of these perforin isoforms in IgT+ B cells. Since perforin is a cytolytic protein we hypothesized that IgT+ B cells could possess cytotoxic activity similar to that of other perforin-expressing cells. To confirm this hypothesis, we tested the potential cytotoxic capacity of IgT+ B cells towards several mammalian cell lines, such as HL-60. Our results show that the killing activity of IgT+ B cells was significantly greater than that of IgM+ B cells. As IgT+ B cells and the IgT antibodies they produce play key roles in fish mucosal surfaces, our results strongly suggest a cytotoxic role for mucosal B cells in these species. Overall, our findings demonstrate a previously unrecognized new function for vertebrate B cells in immunity.