IL-12 and IL-18 receptor expression and STAT-4 signaling. (A) Cultured iNKT cells were rested by incubation in medium lacking IL-2, then exposed to CD1d⁻ or CD1d⁺ APCs, and stained with antibodies against IL-12Rβ1 or IL-18R1 (solid gray histograms), compared with isotype-matched negative control antibodies (dotted lines). Results are from one representative experiment out of two; similar results were also obtained in two independent experiments using a different iNKT cell clone. (B) Rested iNKT cells were preexposed to CD1d⁻ or CD1d⁺ APCs, then the APCs were removed, and the iNKT cells were stimulated with the cytokine mixtures shown on the x axis or left unstimulated. The iNKT cells were fixed, permeabilized, and analyzed by flow cytometry for intracellular levels of phospho–STAT-4. The plot shows the increase in the median phospho–STAT-4 signal in cytokine-treated iNKT cells compared with those incubated in medium alone. The bars show the means of four independent analyses on two different iNKT cell clones, with error bars showing the standard deviations. mfi, mean fluorescence intensity.