Enhanced cross-presentation of immune-complexes is not due to increased antigen uptake or DC maturation. (A) Day 5 human MoDCs were cultured in the presence of eGFP (gray bar) or eGFP-IC (black bar) for 10 minutes (pulse), washed 3 times, and cultured for 1 hour (chase) to assess uptake efficiency (mean + SEM, n = 3). Data shown are MFI values corrected for background MFI (DCs cultured without eGFP). (B) Day 4½ human MoDCs were cultured overnight in the presence of medium (white bars), pp65 antibody (light gray bars), pp65 alone (gray bars), pp65-IC (dark gray bars), or 100 ng LPS and 30 μg poly(I:C) (PIC) to assess maturation status. Data shown are MFI values (mean + SEM, n = 3) of CD40 (left graph), CD80 (middle graph), and CD86 (right graph). (C-D) MoDCs were cultured on confocal slides and incubated with eGFP (C) or eGFP-IC (D). (C) Nonopsonized eGFP (green) is internalized by human MoDCs (bright field, nucleus visualized using DAPI). (D-E) MoDCs were allowed to internalize eGFP-IC for1 hour and were chased for 1 and 4 hours (37°C, analysis of 10-25 slides containing multiple DCs for each condition in 2 separate experiments). Cells were fixed and stained for confocal microscopy. Distribution of internalized eGFP-IC was quantified as percentage of vesicles positive for EEA-1 (D) or LAMP-1 (E).