Fig. 6.

MARCH I depletion promotes MHC class II early-endosomal retention in MoDCs. (A) Confocal microscopy analysis of MARCH I-depleted cells. MHC II (ISCR3 mAb, green) fails to reach lysosomal compartments (blue), being retained in early endosomes (red) and at cell surface, whereas it accumulates in lysosomes in nonrelevant (NR) siRNA-treated cells. (B) Quantification of molecules colocalization using the ImageJ image analysis software and Pearson coefficient determination. A low Pearson coefficient is indicative of the absence of staining overlap. Histograms represent means ± SEM of Pearson coefficient between MHC II and HLA-DM (black) or MHC II and EEA1 (gray) on three independent experiments. (C) FACS quantification of L243 antibody uptake by immature and mature (24-h LPS-activated) MoDCs or by nonrelevant and MARCH I siRNA-transfected MoDCs (lower line). MoDCs were stained with L243 antibody for 30 min at 4°C, warmed at 37°C for indicated times, and stained with a secondary anti-mouse antibody at 4°C before FACS analysis. The kinetics of surface MHC II internalization in MARCH I siRNA-transfected cells is strikingly similar to the one observed in LPS stimulated-MoDCs. Results for two representative blood donors are shown (C and D).