Recruitment of KIR2DL2 to the cSMAC is delayed. (A) KIR2DL2 was visualized by Alexa555-labeled CH-L, and HLA-Cw3 was detected by Alexa488-labeled anti–MHC class I mAb. KIR2DL2⁺ CD4⁺ T cells were pelleted with 721.221/HLA-Cw3 target cells in the presence of 2 ng/mL SEB. The cells were transferred to optical dishes, and the movement of KIR2DL2 and HLA-Cw3 was observed using a confocal microscope at room temperature. Representative x-y sections for KIR2DL2 (red) and HLA-Cw3 (green) are shown for 10, 20, and 30 minutes (left 2 columns). The middle 3 columns show z-axis image reconstruction in the plane of the contact site. Line plots show a quantitative image analysis in this plane, demonstrating that the green and red fluorescence colocalize in the pSMAC after 10 minutes and in the cSMAC after 20 minutes. (B) T cells were stained with Alexa488-labeled anti-CD3 mAb and Alexa555-labeled anti-KIR2DS2/2DL2/2DL3 mAb and then coincubated with HLA-Cw3–expressing target cells as described in panel A. Representative sections at 2 time points are shown. At 10 minutes, CD3 (green) localized to the cSMAC, while KIR2DL2 (red) was found in the pSMAC. At 30 minutes, both molecules were in the cSMAC. Representative x-y section images and z-axis–reconstructed images in the plane of contact site are shown for both time points. (C) T cells were stained with Alexa555-labeled anti-CD3 mAb, and target cells (721.221/HLA-Cw3) were stained with Alexa488-labeled anti–MHC class I mAb. At 10 minutes, CD3 (red) localized to the cSMAC, while HLA-Cw3 (green) was found in the pSMAC. At 30 minutes, both molecules were in the cSMAC. Representative x-y section images and z-axis–reconstructed images in the plane of contact site are shown for both timepoints. For all panels, conjugates were formed and examined as described for Figure 2C.