Confocal microscopic analysis of the distribution of p-Tyr, p-ZAP70, and KIR2DL1 in EV and JK32 cells conjugated with SEE-pulsed 721.221/HLA-Cw7 or 721.221/HLA-Cw4 cells. (A) JK32 and EV cells were incubated in a 1:1 ratio with 721.221/HLA-Cw7 or 721.221/HLA-Cw4 cells for 20 minutes to ensure optimal conjugate formation. Conjugates were fixed and processed for immunofluorescence staining of p-Tyr with 4G10 or p-ZAP70 (red fluorescence) and nuclei with TO-PRO-3 iodide (blue fluorescence). KIR2DL1 and GFP appear as green fluorescence. All confocal pictures are representative of 3 experiments with at least 50 contact areas analyzed in each experiment. Bars represent 5 μm. (B,C) Cells were prepared and treated as described in panel A, and images of 10 fields were visualized with a 63×/1.4 oil-immersion objective. Signal intensity for p-Tyr and p-Zap70 was quantified with ImageJ software. (D) Efficiency of conjugate formation between 721.221/HLA-Cw7 or 721.221/HLA-Cw4 and EV or JK32 cells was calculated by determining the ET ratio × 100, as described in “Confocal microscopy.” Data are expressed as the mean plus or minus SD of 10 fields.