IL-10–producing B cells from healthy donors are enriched within the IgM memory and transitional B-cell subsets. PBMCs were stimulated with irradiated CD40L-transfected fibroblasts (L cells) for 12 to 15 hours. PMA (50 ng/mL), ionomycin (250 ng/mL) (Sigma Aldrich), and Brefeldin A (5 µg/mL) (Sigma Aldrich) were added for the last 6 to 8 hours of the culture. Cells were harvested, washed in staining buffer (1× phosphate-buffered saline, 2% heat-inactivated fetal calf serum, 0.1% sodium azide), and incubated for 20 minutes at room temperature with a cocktail of CD19-PE, CD3 PerCP (BD Biosciences); CD22 FITC, CD24 FITC, CD5 FITC or BAFFR FITC, CD1d PE, CD27 PE, IgD PE or CD40 PE, and IgM PerCP Cy5.5 (all from BD Pharmingen); CD21 FITC (BioLegend); and CD38 PE Cy7 (eBioscience). Cells were fixed/permeabilized (eBioscience) and stained with APC-conjugated IL-10 or IgG2a-К isotype antibodies. All data were analyzed with FlowJo software. (A) Representative flow cytometry plots are shown for IL-10⁺CD19⁺ B cells vs IL-10-CD19⁺ B cells. (B) Extended phenotyping of IL-10⁺ vs IL-10^– B cells vs isotype control. (C) IL-10 production by sort-purified B-cell subsets cultured with irradiated CD40L-expressing fibroblasts (no Brefeldin A was added). (D) IL-10–producing B cells are enriched within the transitional and IgM memory B-cell compartments. The data in (C) and (D) are from 3 independent experiments using PB samples from 5 healthy controls. Bars represent medians, and whiskers indicate the upper range. P < .001 by nonparametric ANOVA.