Introduction: B cells contribute to immune tolerance mainly by producing IL-10, IgG4, TGFβ, IL-35, among others, yet additional regulatory mechanisms remain unclear. Recent studies suggest a role for immunoglobulin D (IgD) in immune homeostasis during high-dose allergen tolerance. Here, we characterize IgD-expressing memory B cells and investigate their functional relevance as a proxy for secreted IgD.
Methods: Model of high-dose allergen exposure (beekeepers; n = 10) and tonsillar mononuclear cells (TMC) from allergic and non-allergic individuals(n = 17) were used to analyze the frequency of memory (CD27⁺) IgD⁺ B cells by flow cytometry. Plasma Api m 1–specific IgD levels were quantified in beekeepers by ELISA. PBMC-purified B cells were stimulated in conditions such as CpG, IL-4, and IL-10, and total Ig(tIg) isotypes were measured in supernatants by multiplex assay. Transcriptomic and proteomic profiling were performed using bulk-RNAseq and LC–MS/MS on fluorescence-activated cell-sorted B cell subsets based on IgD expression.
Results: Api m 1–specific IgD levels were increased during the high-dose exposure season in individuals occupationally exposed to bee venom compared with the pre-season period. Frequencies of memory IgD⁺ B cells were significantly elevated in long-term exposed beekeepers and in tonsillar mononuclear cells (TMCs). Notably, allergic TMCs exhibited lower frequencies of IgD⁺ memory B cells compared with non-allergic TMCs. In vitro stimulation of purified B cells with CpG (± IL-10), induced increased IgD production after 10 days compared with unstimulated cells, along with concomitant IgA and IgM secretion. Transcriptomic profiling of sorted tonsillar CD27⁺IgD⁺ memory B cells, compared with CD27⁺IgD⁻, revealed a distinct signature comprising 279 upregulated and 51 downregulated genes. Upregulated genes included several with immunoregulatory and anti-inflammatory functions, such as IGHD, TNFRSF13B, TIMP1, IL18BP, TNFRSF1B, PRAL, SIGLEC6, FCGR2A, RORA, HPGD, VSIR, CD70, FCGR2C, and XAF1. Galectins (LGALS3 and LGALS1) and molecules previously implicated in basophil–IgD interactions (SRGN) were also enriched in IgD⁺ memory B cells. Pathway analysis demonstrated downregulation of IL-4 signaling (IL4R) and leukotriene biosynthesis (ALOX5AP), alongside upregulation of pathways involved in negative regulation of T-cell activation. Proteomic analysis reveals reduced expression of leukotriene synthesis–related proteins (ALOX5AP, ALOX5), RAC2, and CD79B, and increased expression of LGALS3, MZB1, and IGLL5 in IgD⁺ memory B cells compared with other memory B-cell subsets.
Conclusions: IgD-expressing memory B cells are enriched during sustained high-dose antigen exposure and display transcriptional and proteomic features consistent with immune regulation. These findings support a role for IgD⁺ memory B cells in immune tolerance and homeostasis during high-dose exposure.