Viral degradation in both immature and mature DCs. (A) Quantitative decay of intracellular HIV p24. (Ai) Purified AT-2 HIV-1_BaL was incubated with immature and mature MDDCs (1 × 10⁶ cells/mL, 2 μg/mL HIV-1 p24) for 2 hours. Cells were washed 3 times and cultured at 0.5 × 10⁶ cells/mL in IL-4/GM-CSF media. At the indicated times, cells were pelleted for cell-associated p24 quantified as for Figure 1. At time 0 hours, immediately following the pulse, cell-associated p24 was 18 ng p24/10⁶ for immature DCs and 5 ng p24/10⁶ for mature DCs. The p24 amounts measured at each time point are expressed as a percentage relative to the 100% starting points and are representative of 3 experiments. (Aii) Alternatively, cytospin preparations were made at the indicated time points and acetone-fixed on glass. AT-2 HIV-1 was detected using a murine anti–HIV-1 p24 monoclonal antibody followed by Alexa 488–conjugated goat anti–mouse IgG. At the 6- and 24-hour time points, exposure was enhanced 3-fold to demonstrate the presence of fluorescent granules. Original magnification, × 100. (B) Degradation of SIV (envelope) in DCs over 24 hours. (Bi) AT-2 SIV CP-mac–loaded immature or mature DCs were sampled immediately (0 d) or after being recultured for 1 day (1 d) (10⁶ cells/well, 24-well plate; immature in GM-CSF/IL-4 and mature in medium alone) adhered to slides. Cells were stained for SIV envelope and nuclei using biotinylated CD4-IgG₂ and DAPI, respectively, as outlined in Figure 3. A low-power image (i, upper panels; original magnification, × 10) and a higher-power deconvoluted Z series image (i, lower panels; original magnification, × 100) are shown. Data are representative of DCs from 7 donors. (Bii) Immature DCs and cells matured for one day in cocktail (1d Mature) were pulsed with AT-2 SIV E11S (30 ng p27/10⁵ DCs, 2 hours at 37°C) and sampled immediately (0 d) or recultured in the presence of medium, GM-CSF/IL-4, or the maturation cocktail for a day (1d). Cells were adhered to slides and stained for envelope expression. Immature and 1-day matured DCs yielded the same results from 5 different individuals. Original magnification, × 100. Comparative analyses confirmed the similarities between the different SIV isolates (as apparent in panels Bi and Bii).