Cytokine production by splenocytes stimulated with α-GalCer-loaded DCs in vitro. SDDCs (prepared from WT or IL-12^-/- mice) or BC1 cells were unstimulated (iDCs) or stimulated with IFN-γ (IFN/DCs) or IL-4 (IL-4/DCs). α-GalCer or vehicle-loaded iDCs, IFN/DCs, or IL-4/DCs were cocultured with nylon nonadherent splenocytes (prepared from WT or CD1d^-/- mice) for 48 hours at various DC/splenocyte ratios (0.001 to 0.1). The amount of cytokines in the culture supernatants was measured by ELISA. (A) IFN-γ and IL-4 production in cultures with SDDCs. Each symbol represents the mean ± SE of 3 independent experiments (WT SDDC or IL-12^-/- SDDC vs. WT splenocytes). Data are representative of 2 independent experiments (WT SDDC vs. CD1d^-/- splenocytes). (B) IFN-γ and IL-4 production in culture with BC1 cells. Each symbol represents the mean ± SE of 3 independent experiments. (C) IFN-γ production in cultures of sorted splenic NKT cells and BC1 cells. Each column represents the mean ± SE of triplicate wells. The analysis was repeated twice with similar results. (D) CD40-mediated IL-12 production by DCs. iDCs, IFN/DCs, or IL-4/DCs from BC1 cells were stimulated with anti-CD40 mAb for 48 hours. Each column represents the mean ± SE of 3 independent experiments. (E,F) IL-12 production in DC cultures with nylon nonadherent splenocytes. α-GalCer-loaded iDCs, IFN/DCs, or IL-4/DCs from SDDCs (E) or BC1 cells (F) were cocultured with nylon nonadherent splenocytes for 48 hours at various DC/splenocyte ratios (0.001 to 0.1). Each symbol represents the mean ± SE of 3 independent experiments. Statistical significance was calculated by Student t test (^*P < .05; ^**P < .01; and ^***P < .001 vs iDCs).