KIR2DL2 does not affect the formation of a mature activating immune synapse. (A) Alexa488-conjugated CTXβ-labeled T cells and Alexa647-conjugated CTXβ target cells (721.221 [left panel] or 721.221/HLA-Cw3 [right panel]) were coincubated at a ratio of 1:2 in the absence or presence of the superantigen SEB (2 ng/mL) in the absence of IL-2. At the indicated time points, the percentage of T cells in conjugates was determined by FACS analysis. Results shown as means of triplicate measurements are representative of 3 experiments. (B) T cells labeled with Alexa555-conjugated CTXβ were coincubated with HLA-Cw3–positive and –negative target cells and SEB as described in panel A. Images of multiple fields were obtained by a blinded observer using a Zeiss LSM 510 confocal microscope equipped with a 63 ×/1.4 oil-immersion objective lens after 30 minutes of incubation, and conjugate formation and lipid raft clustering were analyzed by counting at least 100 T cells in different fields. Results are expressed as the percentage of T cells that were in conjugates with target cells (left panel) and the percentage of conjugates that showed lipid raft clustering at the T-cell–target-cell contact area (right panel). Results are representative of 3 experiments. (C) T cells were labeled with Alexa555-conjugated CTXβ (red; top panel) or with Alexa488-labeled CD3 mAbs (green; bottom panel). T cells and SEB-coated 721.221/HLA-Cw3 target cells were mixed at a 1:2 ratio and centrifuged for optimal cell-cell contact formation. Cells were examined at room temperature using a Zeiss LSM 510 confocal microscope equipped with a 100 ×/1.4 oil-immersion objective lens 15 minutes after initial cell contact. (D) HLA-Cw3 was visualized with Alexa488-labeled anti–HLA-ABC antibody (green), and CD3 was detected with Alexa555-labeled anti-CD3 mAb (red). Conjugates were formed and examined as described for panel C. (E) Lipid rafts (top panel) and CD3 (bottom panel) were visualized with Alexa555 (red). LFA-1 was detected by Alexa488-conjugated CD11a mAb (green). Conjugates were formed and examined as described in panel C. Taken together, the images demonstrate synapse formation with rafts and CD3 in the cSMAC; and KIR2DL2, HLA-Cw3, and LFA-1 in the pSMAC after 15 minutes. All confocal pictures are representative of at least 3 experiments with at least 5 contact areas analyzed in each experiment. Results are shown as mean ± SD of triplicate experiments.