MHC II synthesis and redistribution in pDCs stimulated by flu. (A) Freshly purified (2 × 105) pDCs were stimulated or not with 20 μg/mL flu for different periods of time, as indicated. Levels of expression of αβIi complexes were analyzed by flow cytometry using an Ii chain-specific mAb for surface staining (Surf), or for surface and intracellular staining of permeabilized cells (Tot; “Methods”). (B) Similarly, total and surface levels of expression of peptide-loaded MHC II were estimated by flow cytometry using a mAb specific for peptide-bound MHC II. (A,B) Results shown were obtained from 3 independent donors and normalized taking the “Tot” MFI values obtained at 48 hours as 100%. See Figure S1 for raw data expressed in MFI from a typical experiment. Error bars represent SD. (C) Purified pDCs (105) were stimulated with 20 μg/mL flu for 0, 24, or 48 hours. Cells were fixed and stained for HLA-DR and analyzed by 3-dimensional deconvolution microscopy. Equatorial zones of 3-dimensional reconstructions are presented on the left panels. When present, white crosses delimit the zone of the captured images, which was subjected to the deconvolution process. Representative images of 6 individual donors are presented. On the right panels are displayed the quantification of the fluorescence means by a line scanning the corresponding cell on the left. Scale bar represents 3 μm. PM indicates plasma membrane.
