CD40L/IFN-γ induces IL-12 production specifically from CD80-bright and CD83-bright dendritic cells.

Monocyte-derived DCs were thawed and plated in 1 mL AIM V medium with no added cytokine, or with 1 μg/mL CD40 ligand (CD40L) alone, 1000 U/mL IFN-γ alone, or both 1 μg/mL CD40L and 1000 U/mL IFN-γ. Cells were incubated for 12 hours, 10 μg/mL brefeldin A was added, and incubation was continued for another 7 hours before cell harvest, fixation, and permeabilization. Cells were stained with α-CD11c-FITC, α-CD80 or α-83-PE, α-CD14-PerCP, and α-IL-12-APC. The fluorescence intensity of DCs stained with isotype control antibodies (used to construct regions) is shown in the upper panel. For the remaining scatter diagrams, 25 000 large, CD11c⁺/CD14⁻ cells analyzed by flow cytometry are shown, and the indicated percentage represents the frequency of CD83⁺IL-12⁺ DCs. R3 and R4 denote the regions containing IL-12⁻ and IL-12⁺ DCs; they are emboldened in the first graph of each set for the sake of clarity. The CD80 and CD83 data are representative of 4 and 11 experiments with similar results, respectively.