Resistance of mature DCs to negative regulation mediated by prereceptor amplification of GCs. (A) Monocytes were cultured in the presence of IL-4/GM-CSF for 7 days and then exposed to TNF-α for 48 hours. In parallel cultures, 10^-7 M cortisone ± GA were added for 72 hours from days 1 to 3, days 4 to 6, or days 7 to 9. Every 72 hours cultured cells in all groups were washed extensively and resuspended in fresh media. On day 9, output cells were used as stimulators in an allogeneic-mixed leukocyte reaction as outlined in Figure 4B using 0.05 to 1.5 × 10⁴ irradiated DCs. Data are representative of 3 independent experiments. Symbols represent mean ± SEM of triplicate samples. (B-C) Vehicle or 10^-7 M cortisone ± GA were added (B) on day 0 at the initiation of DC culture in IL-4/GM-CSF for 7 days followed by the addition of TNF-α for 48 hours or (C) on day 9 for 48 hours to TNF-α–matured DCs. Flow cytometric histograms show log fluorescence staining of CD83, CD86, and HLA-DR expression (bold line) versus that of isotype control (dotted line). Figures at the top right of each subpanel indicate mean cellular fluorescence. Data are representative of 4 independent experiments.