Specificity of the signaling required for acquisition of a dendritic phenotype. pDCs were exposed for 24 hours to the indicated stimulus (A-E) or were preincubated for 1 hour with the IKKγ NBD peptide (IKKγ) or LY294002 (LY), and exposed to flu for 12 hours (F-H). Cells were then stained for HLA-DR and analyzed by 3-dimensional deconvolution microscopy. (A) Typical images (equatorial zone of 3-dimensional reconstruction) for each condition are presented. Scale bar represents 2 μm. (B,F) Quantification of pDCs displaying an intracellular MHC II pool on stimulation. Percentages of pDCs exhibiting an intracellular pool of MHC II were blindly estimated by 2 independent researchers (n = 75-150 cells per time point; 3 different donors). Error bars represent SD. (C,G) Levels of IFN-α produced in the culture supernatants were determined by ELISA. (D,E,H) Surface expression of the indicated markers was estimated by flow cytometry. (H) Surface (■) and total (▩) levels of peptide-loaded MHC II were determined as in Figure 1B. CD86 surface expression in the various cell populations was very similar to the CD80 pattern and therefore is not shown. MFI values of a typical experiment representative of 3 to 4 independent experiments are presented.