IL-12 and IL-18 stimulation induces IFN-γ mRNA transcription. (A) Real-time PCR analysis of IFN-γ or GM-CSF mRNA from cultured iNKT cells that were transiently exposed to CD1d⁻ or CD1d⁺ APCs and then stimulated with IL-12 and IL-18 or incubated in medium alone. Results are expressed as the signal obtained using IFN-γ or GM-CSF primers, divided by the signal from the same cDNA sample using β-actin primers. The plot shows one representative experiment out of three for a single iNKT cell clone; similar results were also obtained in independent experiments using two additional iNKT cell clones. (B) Kinetics of IFN-γ mRNA and protein production after IL-12 and IL-18 stimulation of cultured iNKT cells that were transiently exposed to CD1d⁺ APCs. Results are expressed as fold increase compared with control iNKT cells that were not stimulated with IL-12 and IL-18. The plot shows one representative experiment out of two; similar results were also obtained in two independent experiments using a different iNKT cell clone. (C) Real-time PCR analysis of IFN-γ mRNA in cultured iNKT cells that were first transiently exposed to CD1d⁺ APCs, then rested for the indicated times, and then stimulated with IL-12 and IL-18. Corresponding measurements of IFN-γ protein secretion from Fig. 2 A are shown for comparison. Results are expressed as fold increase over iNKT cells that were exposed to CD1d⁻ APCs in the first step. The plot shows one representative experiment out of two; similar results were also obtained in two independent experiments using a different iNKT cell clone.