DC maturation alters IFN-α/β signaling by modulating STAT expression. (A) iDCs and mDCs were stained with anti-CD83-Cy5 and mAbs specific for IFNAR1 (AA3, Biogen, Cambridge, MA) or IFNAR2 (MMHAR-2, Calbiochem, San Diego, CA). Anti–mouse IgG PE (Jackson Immunochemicals, Raritan, NJ) was used to visualize anti-IFNAR binding. Dot plots representative of a single donor are shown. Three individuals were evaluated and revealed similar results. (B) Transcriptional analysis of iDCs and mDCs was performed using Affymetrix microarray U133A. Data were analyzed and normalized using MAS5.0. The relative signal intensity of 4 biologic replicates is displayed for STAT1 and STAT4 expression in iDCs and mDCs. (C) Quantitative PCR was performed as described in “Materials and methods.” Fold increase in mRNA expression of mDCs/iDCs is displayed graphically for STAT1 and STAT4 as indicated. (D) iDCs and mDCs were stimulated with media alone or indicated concentrations of IFN-α/β for 30 minutes. Cells were stained with anti-CD83–FITC to identify CD83⁻ iDCs and CD83⁺ mDCs. Cells were fixed and permeabilized as described in “Materials and methods” and stained for p-STAT1-PE and p-STAT4-APC. Percentage of events in each quadrant is indicated on the respective plot. Data are representative of 3 independent experiments. (E) iDCs and mDCs were stimulated with media alone, 60 IU/mL IFN-α/β, or 10 ng/mL IFN-γ for 30 minutes as indicated. Cells were fixed, permeabilized, and stained for p-STAT1, as described. (F-G) iDCs and mDCs were exposed to 60 IU/mL IFN-α/β for 30 minutes. Protein (20 μg) from the total-cell lysates were run on an 8% SDS-PAGE, transferred to PVDF membranes, and blotted with indicated antibodies. Blots were stripped and probed for total STAT1/2 or β-actin as a control for loading the gel.