Transfer of DC proteins to T cells and bystander DCs during cognate T cell–DC interactions. (A) DCs were pulsed for 2 hours with 5 μM p53p before coculture with T cells. Pulsed DCs (5 × 10⁶) were cultured for 24 hours with CFSE-labeled p53 T cells (10⁷). Cells were stained for MHC class II, CD9, CD3, CD69, or IFN-γ and analyzed by flow cytometry. Histograms indicate staining of gated CFSE-positive T cells derived from either T-cell cultures (solid histograms) or from cognate cocultures with DCs (open histograms) and were corrected for nonspecific staining as determined by isotype control antibodies. Reduction of CD3 and up-regulation of CD69 expression and IFN-γ production are indicative of T-cell activation. (B) Nonlabeled nontransduced (bystander) DCs (8 × 10⁵) were cultured for 24 hours in the presence of CFSE-labeled DCs transduced with I-A^kβ (8 × 10⁵), PKH26-labeled p53 T cells (2 × 10⁶), and 5 μM p53p in 4 mL of culture medium. Cocultured p53 T cells (left) and CFSE-negative bystander DCs (right) were gated on FL2 or FL1 and FSC levels as indicated (top panels), to exclude T-cell–DC clusters. The indicated gated populations were analyzed for I-A^kβ (OX6) staining (bottom panels). Indicated are geometric mean fluorescence intensity (GeoMFI) values of cells derived from the cognate 3-cell culture system (cognate) or control DCs and T-cell cultures. The data shown are representative for 3 independent experiments. (C) Exosomes produced during 24-hour cognate DC–T-cell cocultures were collected by centrifuging the 10 000g supernatant for 1 hour at 100 000g, resuspended in 2.5 M sucrose, 20 mM Tris-HCl, pH 7.2, and floated by centrifugation to equilibrium into a 2.0- to 0.4-M sucrose gradient.¹⁹  The presence of MHC class II-β in gradient fractions was analyzed by Western blotting using rabbit polyclonal anti–MHCII-β obtained from Dr N. Barois (University of Oslo, Oslo, Norway). The density of the gradient fractions is indicated at the bottom of the blot.