Figure 4.
Viral degradation in both immature and mature DCs. (A) Quantitative decay of intracellular HIV p24. (Ai) Purified AT-2 HIV-1BaL 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).

Viral degradation in both immature and mature DCs. (A) Quantitative decay of intracellular HIV p24. (Ai) Purified AT-2 HIV-1BaL 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).

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