IFN-γ/IL-4–primed PCMCs and freshly isolated mast cells process whole ovalbumin and form functional ISs. IFN-γ/IL-4–primed PCMCs (A) or IFN-γ/IL-4–primed peritoneal mast cells (B) were pulsed or not for 16 hours with 400 μg/mL ovalbumin. After washing, APCs were cocultured with effector OT-II T cells for 2.5 hours. Cells were fixed, permeabilized, and stained for mast cell granules with avidin-sulforhodamine 101 (red), PKCθ (blue), and IFN-γ (green) and analyzed by confocal laser scanning microscopy. (Top) IFN-γ (green) and avidin-sulforhodamine 101 (red) are merged with DIC images; (middle) avidin-sulforhodamine 101 (red) and PKCθ staining (blue) are shown; (bottom) PKCθ staining is shown as pseudocolor intensity merged with DIC images. (C-D) For each condition, OT-II T-cell/APC conjugates were randomly selected and scored for the recruitment of PKCθ to the cell–cell contact site either alone or in combination with IFN-γ polarization. indicates conjugates exhibiting recruitment of PKCθ alone; ■, conjugates exhibiting polarization of both PKCθ and IFN-γ; □, conjugates exhibiting neither PKCθ nor IFN-γ polarization. Approximately 50 conjugates were analyzed per experiment. Histograms represent means ± SEM of 3 independent experiments. Bar = 5μm.