Competition experiments suggest that different particles derived from cholesterol-enriched domains use the same entry pathway into mDCs. (A) Capture of VLP_HIV-Gag-eGFP by mDCs previously exposed to increasing amounts of Jurkat-derived Exosomes_DiI. Cells were preincubated for 30 minutes with increasing amounts of Exosomes_DiI and then pulsed with 625 pg of VLP_HIV-Gag-eGFP p24^Gag for 1 hour at 37°C, washed with PBS, fixed, and analyzed by FACS to determine the percentage of eGFP- and DiI-positive cells. mDCs captured fewer VLP_HIV-Gag-eGFP in the presence of increasing amounts of Exosomes_DiI (P = .0078, paired t test). (B) Capture of HIV_Δenv-NL43 by mDCs previously exposed to increasing amounts of yellow carboxylated 100-nm beads. A total of 5 × 10⁵ mDCs were preincubated for 30 minutes with the beads and then pulsed for 1 hour at 37°C with 130 ng HIV_Δenv-NL43 p24^Gag in 0.5 mL and extensively washed with PBS. Each sample was then divided and either fixed for analysis by FACS for bead capture or lysed with 0.5% Triton (at a final concentration of 5 × 10⁵ cells per milliliter) to measure p24^Gag content in the cell lysate by an ELISA. Results represent the percentage of yellow positive mDCs (○) and the amount of pg of p24^Gag bound per mL of cell lysate (♦). (C,D) Capture of VLP_HIV-Gag-eGFP by mDCs previously exposed to increasing amounts of HIV_Δenv-Bru (C) and VLP_MLV-Gag (D). Cells were preincubated for 30 minutes with increasing amounts of HIV_Δenv-Bru or VLP_MLV-Gag and then pulsed with 625 pg of VLP_HIV-Gag-eGFP p24^Gag for 1 hour at 37°C, washed with PBS, and fixed to analyze the percentage of eGFP-positive cells by FACS. mDCs capture less VLP_HIV-Gag-eGFP in the presence of increasing concentrations of particles derived from cholesterol-enriched membrane microdomains (P values on the graphs, paired t test). (E) The data represent the relative VLP_HIV-Gag-eGFP capture by mDCs that had been preincubated with 200 ng of p24^Gag of HIV_NL43 obtained from either MT4, MOLT, or PHA-stimulated PBMCs and normalized to the level of VLP_HIV-Gag-eGFP capture by mock-treated mDCs (set at 100%). mDCs captured less VLP_HIV-Gag-eGFP in the presence of these different viral stocks. (F) Capture of VLP_HIV-Gag-eGFP by mDCs that had been preincubated with increasing amounts of pronase-treated VSV particles. Cells were preincubated for 30 minutes in the presence of pronase-treated VSV particles and then pulsed with the 625 pg of VLP_HIV-Gag-eGFP p24^Gag for 1 hour at 37°C, washed with PBS, and fixed to analyze the percentage of eGFP-positive cells by FACS. mDCs captured similar amounts of VLP_HIV-Gag-eGFP in the presence of pronase-treated VSV particles. Panels A through F show mean values and SEM from 3 independent experiments, including cells from at least 4 different donors.