Induction of Th phenotype by IL-18–DC2's. (A) Th phenotype determined by intracellular FACS. DC2s were obtained by culture of pre-DC2s with IL-3 (DC2) or IL-3 plus IL-18 (IL-18–DC2). After washing, DC2/IL-18–DC2s were cocultured with allogeneic naive Th cells for 6 days. Subsequently, Th cells were harvested and restimulated with anti-CD3 and anti-CD28 for 5 hours. Intracellular FACS staining was performed as described in “Materials and methods.” The percentage of IFN-γ⁺ and IL-4⁺ cells is given for each quadrant. The experiment shown is representative of 5 performed. (B) IFN-γ and IL-4 secretion by restimulated Th cells. Experiments were performed as described in the legend for panel A, with restimulation of Th cells for 24 hours instead of 5 hours, and cytokine secretion determined by ELISA. IFN-γ and IL-4 secretion induced in Th cells by DC2 was set at 1 in each case for further analysis owing to the large interindividual variation (medium baseline IFN-γ, 23 ± 9 ng/mL; baseline IL-4, 661 ± 324 pg/mL). The y-axis shows the ratio of Th cytokine secretion induced by IL-18–DC2 per DC2. Differentiation of allogeneic naive Th cells with IL-18–DC2 significantly increased IFN-γ release upon restimulation as compared with DC2 (n = 6). (C) Th phenotype and effect of IL-12 neutralization after differentiation with CD40L-matured DC2. Experiments were performed as described for panel A, except that CD40L-activated DC2 and IL-18–DC2 were added for 24 hours before the onset of coculture with allogeneic naive Th cells. Anti–IL-12 mAb or isotype-matched control immunoglobulin was present during the coculture as indicated. The percentage of IFN-γ⁺ and IL-4⁺ cells is given for each quadrant. The experiment shown is representative of 3 performed with respect to CD40L-matured DC2/IL-18–DC2, and is representative of an additional 2 with respect to the effect of anti–IL-12. Error bars indicate SEM.