DC exosome recruitment by T cells requires cellular activation and is dependent on active LFA-1. (A) p53 T cells (10⁶) were incubated for 24 hours with 2.5 mL control medium (medium) or exosome containing 10 000g supernatant derived from cocultures of p53p-pulsed DC and p53 T cells (exo), in the absence (resting) or presence (activated) of anti-CD3/anti-CD28. T cells were labeled for MHC class II and analyzed by flow cytometry. GeoMFI values are expressed as percentages of the values for activated T cells incubated with exosomes (mean ± SD of 3 independent experiments). (B) p53 T cells (■, 10⁶) or OVA T cells (, 10⁶) were cultured for 24 hours in 2.5 mL 10 000g supernatant from cognate DC-p53 T-cell cocultures (left) or cognate DC-OVA T-cell cocultures (right) during anti-CD3/anti-CD28 activation. T cells were labeled for MHC class II and analyzed by flow cytometry. GeoMFI values are expressed either as percentages of the values for p53 T cells incubated with exosomes from cognate DC-p53 T-cell cocultures (left) or as the percentages of the values for OVA T cells incubated with exosomes from cognate DC-OVA T-cell cocultures (right; mean ± SD of 3 independent experiments). (C) Exosomes present in 10 000g supernatant from 24-hour cognate DC-p53 T-cell cocultures were adsorbed onto anti-MHC class II or anti–ICAM-1–coated beads, stained for CD9 and MHC class II, and analyzed by flow cytometry. Histograms indicate staining with specific antibodies (open histograms) or isotype control antibodies (filled histograms). (D) p53 T cells (10⁶) were cultured for 5 hours with anti-CD3/anti-CD28 either in 2.5 mL control medium or in 10 000g supernatant from 24-hour cognate DC- p53 T-cell cocultures in the absence or presence of indicated amounts (μg/2.5 mL) of anti–LFA-1 or isotype control (i.c.) antibody. Cells were stained for acquired MHC class II (left) or the activation marker CD69 (right) and analyzed by flow cytometry. GeoMFI values are expressed as percentage of signal in the absence of anti–LFA-1 (mean ± SD of 3 independent experiments). (E) Resting p53 T cells were cultured with medium or exosomes as in panel D in the absence or presence of MnCl₂ and/or anti–LFA-1 as indicated. Cells were stained for MHC class II and analyzed by flow cytometry. For comparison, exosome binding by activated T cells is included (left bar in graph). The GeoMFI value for T cells incubated with exosomes in the absence of MnCl₂ was set to 1, and data are expressed as fold increase over this value (mean ± SD of 3 independent experiments).