Regulatory DCs induce the differentiation of splenic B cells into IL-10–producing B cells. (A) Freshly purified splenic naive B cells were cocultured with regulatory DCs at a ratio of 1:1 for 24 hours. Culture supernatants were collected and assayed for cytokines (IL-10, IP-10, IL-6, IL-12p70, PGE₂, TGF-β, and IFN-γ) by ELISA. (B) Splenic naive B cells were cocultured with various DCs at the indicated ratios (DC/B). Supernatants were collected at 24 hours and assayed for IL-10. (C) Splenic naive B cells were cocultured with various DCs at a ratio of 1:10 (DC/B), and supernatants were collected at different times for assay of IL-10. (D) Intracellular staining for IL-10 in splenic CD19⁺B220⁺CD11c⁻ B cells and B220⁻CD11c⁺ regulatory DCs in coculture system. The percentages of each population are indicated in the plots. (E) B220⁺CD11c⁻ B cells and B220⁻CD11c⁺ regulatory DCs were sorted after coculture, and IL-10 expression in the purified regulatory DCs and B cells was checked by quantitative RT-PCR. IL-10 expression in diffDCs was designated as 1 for the comparison of IL-10 expression between regulatory B cells and diffDCs. Results are representative of 3 independent experiments. Data are mean ± SD. **P < .01. NS indicates not significant.