Enumeration of human blood IL-10-competent B10 and B10pro cells. (A) Visualizing IL-10⁺ B cells. Purified blood mononuclear cells were cultured with BFA, LPS plus PMA, ionomycin and BFA (PIB), or LPS plus PMA, ionomycin, and monensin (PIM) for 5 hours and stained for cell viability, cell surface CD19 expression, and cytoplasmic IL-10. Representative cytoplasmic IL-10 staining by viable, single CD19⁺ B cells is shown in the flow cytometry dot-plots, with percentages indicating cytoplasmic IL-10⁺ B-cell frequencies within the indicated gates. Blood mononuclear cells that were cultured with BFA alone before immunofluorescence staining served as negative controls, with background staining similar to that obtained using isotype-matched control mAbs. Bar graphs represent mean (± SEM) B10-cell frequencies from 3 persons. (B) Representative IL-10 production by B cells from a person with relatively high B10-cell frequencies. B10 cells were identified after in vitro stimulation for 5 hours as in panel A. Alternatively, IL-10⁺ B-cell frequencies were determined after in vitro B10pro cell maturation by stimulation with LPS, CD40L + LPS, CpG, or CD40L + CpG, with PIB added during the final 5 hours of 48-hour cultures. As negative controls for IL-10 staining, only BFA was added to some cultures during the final 5 hours. Percentages indicate the frequencies of cytoplasmic IL-10⁺ B cells within the indicated gates among total CD19⁺ B cells. (C) B10-cell frequencies in persons after with TLR agonist stimulation as in panels A and B. Dots represent results from single persons after 5-hour culture with BFA alone, PIB, or the indicated TLR agonist + PIB. Horizontal bars represent means. (D) CD40L induced optimal B10 + B10pro cell maturation during 48 hours in vitro cultures with either recombinant CD40L or CD40 mAb, plus LPS for 48 hours, with PIB added during the final 5 hours. Bar graphs represent means (± SEM) from 5 persons. Similar results were obtained in 2 independent experiments. (E) Representative B10 + B10pro-cell frequencies after in vitro maturation and stimulation. Blood mononuclear cells were cultured for 48 hours with media alone or media containing CD40L, along with the indicated TLR agonists, with PIB added during the last 5 hours of culture. Significant differences between cultures with or without CD40L are indicated: #P < .05; ##P < .01. (C-E) Significant differences between means of controls and individual stimuli are indicated: *P < .05; **P < .01.