Human blood B-cell stimulation induces IL-10 transcription and secretion in vitro. (A) Time course of IL10 transcript induction. Purified CD19⁺ B cells were cultured with media alone or CD40L + CpG for the times indicated, with IL10 transcripts quantified by real-time reverse-transcribed polymerase chain reaction analysis. Bar graphs indicate mean relative IL10 transcript (± SEM) levels in 6 persons. (B) B cells secreting IL-10 express IL10 transcripts. Purified blood B cells were cultured with PMA and ionomycin for 4 hours before CD19 staining and secreted IL-10 capture (left panel). Cell surface IL-10⁺ and IL-10⁻ B cells were isolated using the indicated gates and subsequently reassessed for IL-10 secretion (right panels) before relative IL10 transcript levels were quantified by real-time reverse-transcribed polymerase chain reaction analysis. Mean fold differences (± SEM) for IL10 transcript levels from 3 different persons are shown, with transcript levels normalized so that the relative mean IL-10⁻ B-cell value is 1.0. (C) Cell surface signals that regulate cytoplasmic IL-10 expression. Blood B cells were cultured with CpG, CD40L, and antiIgM Ab (IgM) as indicated for 48 hours with PIB added during the final 5 hours of culture. Representative frequencies of IL-10-producing cells are shown, with bar graphs representing mean (± SEM) percentages in 5 persons. (D) TLR agonists that induce IL-10 secretion. Purified CD19⁺ B cells were cultured with media alone, CD40L, or with TLR agonists and CD40L as indicated for 48 or 72 hours. IL-10 secreted into the culture supernatant fluid was quantified by ELISA. Bar graphs represent mean IL-10 (± SEM) concentrations from more than or equal to 4 different persons. (A-D) Similar results were obtained in 2 independent experiments. Significant differences between means of cells cultured in media alone and stimulated cultures are indicated: *P < .05; **P < .01.