Photostimulation of KIR2DL2 induces receptor microclusters and cellular retraction. (A–D) NKL cells expressing KIR2DL2-GFP were imaged using TIRF microscopy and UV irradiated on bilayers containing ULBP3, ICAM, and either HLA-Cw3(Tyr) or HLA-Cw3(cage). (A and C) Representative time-lapse montages (∼25-s intervals) showing NKL cells responding to photostimulation on surfaces containing the indicated proteins. UV irradiation is denoted in magenta. (B) Graph showing the change in KIR2DL2 microclusters and cell contact area after photostimulation on bilayers containing HLA-Cw3(cage). Data were derived from seven cells. (D) Graph showing mean cell contact area before and after photostimulation on bilayers containing the indicated HLA-Cw3 proteins. Each curve was derived from at least nine cells. (E) PKH26-stained NKL cells expressing KIR2DL2 were imaged on bilayers containing ULBP3, ICAM, and the indicated HLA-Cw3 proteins for up to 15 min before UV irradiation. Cells on the HLA-Cw3(cage) bilayer were grouped based on when they formed stable contacts with the bilayer (early, first 5 min; late, between 5 and 15 min) and how quickly they collapsed after UV (within 5 or 17.5 min). A timeline for the experiment is shown on the right in a gray box. Cells were defined as collapsed once their footprint on the bilayer shrank to <50% of its value before UV irradiation. Error bars represent SEM. Purple lines in graphs denote UV irradiation. All data are representative of at least two independent experiments. norm., normalized. Bars, 5 µm.