α-GalCer-induced cytokine production by splenocytes from IL-4C-pretreated mice. (A) IL-4C, anti-IL-4 mAb, or PBS (cont.) was intravenously injected into mice. After 3 days, each spleen was separated, and a single cell suspension was prepared. The splenocytes were stimulated with various concentrations of α-GalCer for 48 hours. The culture supernatants were subjected to ELISA to analyze IFN-γ and IL-4 production. (B,C) α-GalCer was injected intravenously into WT or CD1d^-/- mice 3 days after administration of IL-4C or PBS (cont.). The amount of IFN-γ, IL-4, and IL-12 (at 6 hours) in the sera was measured by ELISA. Intracellular IFN-γ production of splenic NKT cells and NK cells was examined by flow cytometry 2 hours after α-GalCer or vehicle injection into BALB/c (D) or B6 mice (E) pretreated with PBS (cont.) or IL-4C. The intracellular IFN-γ production was analyzed on CD1d dimer⁺ TCRβ⁺ NKT cells or DX5⁺ TCRβ^- NK cells in BALB/c strain and on NK1.1⁺ TCRβ⁺ NKT cells or NK1.1⁺ TCRβ^- NK cells in B6 strain (middle). The percentage of marker-positive cells was determined based on the staining with α-GalCer-unloaded CD1d-dimer or isotype-matched Ig. Each symbol or column represents the mean MFI of IFN-γ ± SE of 3 independent experiments. Statistical significance was calculated by Student t test (^*P < .05; ^**P < .01 vs. cont.).