Regulatory DCs program generation of IL-10–producing B population with CD19^hiFcγRIIb^hi phenotype. (A) Purified splenic naive B cells cocultured with DCs at a ratio of 1:10 (DC/B) for 48 hours were stained with antibodies to CD19, CD62L, or CD16/CD32 and analyzed by flow cytometry. Numbers in plots indicate percentage of CD19^hi cells or CD19^hiCD62L⁺ cells or CD19^hiCD16/CD32^hi in the gated CD19⁺ B cells. (B) FcγIIb and FcγRIII mRNA expression by conventional splenic CD19⁺ B cells or sorted CD19^hiCD16/CD32^hi B cells was assessed by RT-PCR. The transcript of mouse GAPDH gene was used as the amplification control. (C) Purified splenic naive B cells were cocultured with regulatory DCs at a ratio of 1:10 (DC/B) for 48 hours. Cells were stained with antibodies to CD19, B220, CD11c, and CD16/CD32 and analyzed by flow cytometry. Numbers in plots indicate percentage of CD19^hiFcγRIIb^hi in the gated B220⁻CD11c⁺ regulatory DCs or B220⁺CD11c⁻ B cells. (D) Purified splenic B cells were cocultured with regulatory DCs. CD19⁺IL-10⁺ B cells or CD19⁺IL-10⁻ B cells were gated and assessed for FcγIIb expression. (E) Twenty-four hours after conventional splenic B cells or sorted CD19^hiFcγRIIb^hi B cells were stimulated with 20 μg/mL anti-IgM Ab, 2 μg/mL sCD40L, 200 ng/mL LPS, 2 μg/mL CpG ODN, or 2 μg/mL poly I:C, respectively, IL-10 production was measured by ELISA. (F) Conventional splenic B cells or sorted CD19^hiFcγRIIb^hi B cells were labeled with CFSE and cultured with anti-IgM Ab, sCD40L, LPS, or CpG ODN for 5 days. Histograms represent CFSE expression by the CD19⁺ or CD19^hiFcγRIIb^hi B cells. Gray lines represent CFSE staining of the unstimulated CD19⁺ or CD19^hiFcγRIIb^hi B cells. Results are representative of 3 independent experiments. Data are mean ± SD. **P < .01. NS indicates not significant.