MHC II clustering and expression in pDCs exposed to various stimuli. pDCs were activated for the indicated periods of time with flu (20 μg/mL), IL-3 (10 ng/mL), CpG-C (10 μg/mL), or flu combined with IL-3 or CpG-C. In parallel, pDCs were activated with flu in presence of allogenic CD4⁺ T lymphocytes (ratio pDC/T 1:2) and of TSST1 at 10 ng/mL. (A) Cells were fixed and stained for HLA-DR and analyzed by 3-dimensional deconvolution microscopy. The percentage of cells exhibiting an intracellular pool of MHC II were estimated by 2 independent researchers (n = 75-150 cells per time point; 3 different donors). (B) Surface and total levels of HLA-DR expression were estimated by flow cytometry on fixed and permeabilized cells as in Figure 1B. (C,D) Surface expression of CD40 and CD86 were estimated by flow cytometry. MFI values presented are from a typical experiment representative of 3 independent experiments.