IL-10 production by CB-derived B cells after stimulation with CD40L, CpG, or BCR ligation. (A) Phenotypic characterization of cord blood B-cell subsets, as shown in representative fluorescence-activated cell sorter plots illustrating gating strategy on lymphocyte population, total CD19⁺ B cells and CD19⁺CD38^hiCD24^hi transitional B cells and CD19⁺CD38^intCD24^int naive B cells. (B) Bar graphs showing cumulative time-dependent IL-10 production by CB-derived CD19⁺ B cells in response to stimulation with CD40L, CpG, or BCR ligation (n = 10). CBMCs were stimulated with irradiated CD40L-transfected fibroblasts (L cells), CpG, or BCR ligation for 24, 48, or 72 hours. PMA (50 ng/mL) and ionomycin (250 ng/mL; Sigma-Aldrich) were added for the last 6 to 8 hours of the culture. Supernatants were harvested and assayed for IL-10 secretion by ELISA. (C) Cumulative IL-10 production by total CD19⁺ B cells vs sort-purified naive and transitional B-cell subsets after stimulation with CD40L, CpG, BCR ligation or their combination. Resting B cells (unstimulated) were used in each experiment as a negative control (n = 10). The bars in panels B and C represent the means and ranges. (D-E) CBMCs were stimulated with irradiated CD40L-transfected fibroblasts (L cells), BCR ligation, and CpG for 24 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, and surface stained with a cocktail of CD38 PEcy7 (eBioscience), HLA-DR FITC, CD40 PEcy7BAFFR FITC, CD22 FITC, CD23 PEcy7, CD21 FITC, CD25 PE, CD1d PE (all from Biolegend), CD24 FITC, CD27 PE, IgM PerCPcy5.5, IgD BV605, CD10 BV605, CD5 FITC, and CD43 FITC (all from BD Biosciences). Cells were fixed/permeabilized (eBioscience) and stained with APC-conjugated IL-10 or IgG2a-К isotype antibodies. Unstimulated B cells were also included as a negative control to validate IL-10 detection. All data were analyzed with FlowJo software. (D) Representative flow cytometry plots are shown for the distribution of IL-10⁺CD19⁺ B cells (black) and IL-10⁻CD19⁺ B cells (gray) on the CD24 v CD38 axis. lL-10⁺CD19⁺ B cells were enriched in both transitional (45%) and naive (55%) subsets. (E) Extended phenotyping of IL-10⁺ B cells (dark gray) vs IL-10⁻ B cells (light gray) vs isotype control (white). APC, allophycocyanin; FITC, fluorescein isothiocyanate; FSC-A, forward scatter area; ISO, isotype control; PE, phycoerythrin; PerCP, peridinin chlorophyll; PMA, phorbol 12-myristate 13-acetate; SSC-A, side scatter area.