VLP and exosome uptake in mDCs is a dose-dependent mechanism that increases over time, allowing efficient transfer to target T cells. (A) Time course of mDCs (n = 4) exposed to 2 different concentrations of Exosomes_DiI and fixed at each of the indicated time points and analyzed by FACS. Exosome capture by mDCs increases over time in a dose dependent manner. (B) Time course of mDCs (n = 4) exposed to 2 different concentrations of VLP_HIV-Gag-eGFP and fixed at each of the indicated time points and analyzed by FACS. VLP_HIV-Gag-eGFP capture by mDCs increases over time in a dose-dependent manner. (C) Fate of VLP_HIV-Gag-eGFP captured by mDCs and followed by flow cytometry for 2 days. Graph shows the percentage of Gag-eGFP-positive cells measured by FACS at the indicated time points. P values on the graph reveal that, at 48 hours after pulse with VLP_HIV-Gag-eGFP, a significant percentage of mDCs still retained VLPs (one sample t test). Data (mean and SEM from 3 independent experiments) include cells from 4 different donors. (D) Orange cell tracker dye-labeled Jurkat T cells were analyzed by deconvolution microscopy after 4 hours of coculture with mDCs previously pulsed with VLP_HIV-Gag-eGFP and extensively washed before coculture. The cells shown in the panels are projections of stack images obtained by merging the red and green fluorescence. Arrows indicate Gag-eGFP dots associated to Jurkat T cells, magnified in the nearby marked boxes (E). Viral synapse could also be observed in these cocultures, where mDCs pulsed with VLP_HIV-Gag-eGFP were stained with DAPI. Images shown, from left to right, depict the red and green fluorescence channels merged with DAPI, the bright-field cellular shape and the combination of both. (F) Jurkat T cells labeled with a green cell tracker dye were analyzed by confocal microscopy after 4 hours of coculture with mDCs previously pulsed with Exosomes_DiI and extensively washed. Images were obtained by merging the red and green fluorescence. Arrows indicate DiI dots associated with Jurkat T cells, magnified in the nearby marked boxes. Bright-field cellular shape merged with the red and green fluorescence is also shown. (G) Exosome polarization to the site of DC-T cell–contact, where mDCs pulsed with Exosomes_DiI were stained with DAPI. Images shown, from left to right, depict the red and green fluorescence channels merged with DAPI, the bright-field cellular shape and the combination of both. (H) Quantification of mDCs forming synapses like those shown in panels E and G. Polarization of particles toward the synapse was considered when VLPs_HIV-Gag-eGFP (green) or Exosomes_DiI (red) were found within one-third of the cell proximal to the contact zone (as represented in the illustration by the blue colored area). Mean values and SEM of 50 synapses from 2 donors counted by 3 distinct observers.