(A) DCs from age- and gender-matched healthy donors (H) and melanoma patients (M) were activated with CD40L/IFN-γ for 24 hours, and supernatants were harvested and assayed for IL-12p70 production by ELISA. Horizontal lines and error bars denote median and interquartile range. P value (Wilcoxon matched-pairs test) is indicated. (B) Patient DCs were activated with CD40L/IFN-γ for 24 hours, supernatants were collected, and IL-12p40 and IL-12p70 production was measured by ELISA. Results are shown for 10 melanoma patients. Horizontal lines and error bars denote median and interquartile range. (C) To examine IL-12p35 gene activation, DCs were activated with CD40L/IFN-γ for 6 hours, cells were harvested and washed, and total RNA was prepared. Total RNA was also prepared from iDCs. Using primers specific for IL-12p35 and CD11c (DC lineage marker), qRT-PCR was performed and analyzed using the relative standard method. Results were normalized to CD11c, and values are expressed as fold IL-12p35 induction in mDCs relative to iDCs. Results are representative of 2 experiments.