IFN-β and CD40L/CD40 interaction is required to induce regulatory B-cell generation driven by regulatory DCs. (A) Splenic naive B cells were cocultured with regulatory DCs either directly or separated from regulatory DCs by transwell system. (B) Splenic naive B cells were cocultured with regulatory DCs in the presence of the indicated neutralizing antibodies (5 μg/mL). (C) Neutralizing anti–IFN-β antibody was added to the B-cell culture system in the presence of regulatory DC supernatants (regulatory DC-SN, collected after 24 hours of culture). And the final concentration of supernatants used was 50%. (D) CD40L expression on regulatory DCs and CD40L^−/− regulatory DCs was tested by flow cytometry. (E) Wild-type B cells or CD40^−/− B cells were incubated with regulatory DCs or CD40L^−/− regulatory DCs. Culture supernatants were collected at 48 hours and assayed for IL-10. (F-G) imDCs or maDCs were cocultured with purified splenic naive B cells in the presence of IFN-β (2 μg/mL) and sCD40L (2 μg/mL) for 48 hours. Culture supernatants were collected and assayed for IL-10 (F). Cells were stained with antibodies to CD19 and CD16/CD32 and analyzed by flow cytometry (G). The DC/B ratio in all these experiments was 1:10. Data are mean ± SD of triplicate wells. *P < .05. **P < .01.