Differential Expression of IgM and IgD Discriminates Two Subpopulations of Human Circulating IgM+IgD+CD27+ B Cells That Differ Phenotypically, Functionally, and Genetically

Abstract

The origin and function of blood IgM⁺IgD⁺CD27⁺ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM⁺IgD⁺CD27⁺: IgM^hi and IgM^lo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, <i>in vitro</i> function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgM^hi phenotypically resembling previously reported marginal zone B cells and the IgM^lo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgM^hi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (<i>p</i> < 0.05) higher frequencies (62.5%) of IgM^hi B cells proliferated, compared with IgM^lo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgM^hi and 7.19% for IgM^lo). IgM^hi B cells had significantly (<i>p</i> < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgM^lo B cells had higher usage of <i>IGHJ6</i> genes (<i>p</i> < 0.0001), and both subsets differed in their HCDR3 properties. IgM^hi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgM^lo B cells with IgM^hi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM⁺IgD⁺CD27⁺ B cells, with the IgM^hi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgM^lo B cells being the least differentiated amongst the IgM⁺CD27⁺ subsets.