Polarization of IFN-γ in NK cells stimulated through NKG2D and LFA-1. (A) Images of conjugates between cultured pNK cells and Daudi/MICA or Daudi/β2M. Cells were coincubated for 30 or 90 minutes and stained for F-actin (red) and IFN-γ (green). Bars represent 10 μm. (B) The proportion of pNK cells expressing IFN-γ in conjugates formed between fresh or cultured pNK cells and Daudi/MICA for the times indicated. Graph represents mean ± SEM from 3 experiments; n = 76-170. (C) Images of F-actin (green) and IFN-γ (red) in cultured pNK cells stimulated for 90 minutes on surfaces coated with MICA-Fc (2.5 μg/mL) and ICAM-1 (2.5 μg/mL). Bar represents 20 μm. (D) The proportion of pNK cells expressing IFN-γ after stimulation as in panel D for 5-180 minutes. Graph represents mean ± SEM from 5 donors; n > 200 cells. (E) Images of cultured pNK cells stimulated as in panel E. Right panels: Orthogonal (XZ, YZ) views, perpendicular to the planes marked with dotted lines in the XY image. (F) The proportion of IFN-γ staining in 4 quadrants of the cell, denoted by their relative distance from the coverslip (0 at the coverslip, 1 the top of the cell). Graph represents data for cells incubated on surfaces coated as in panel C for 1.5 or 3 hours as mean ± SEM (n = 60 or 30). (G) The surface area of pNK cells that express IFN-γ (IFN-γ⁺), or are IFN-γ negative (IFN-γ⁻), after incubation on surfaces coated as in panel C. (H) The symmetry of pNK cells that either express IFN-γ or are IFN-γ⁻ after stimulation on slides coated with MICA-Fc and ICAM-1 for 90 minutes is represented as the SD of the radial distances for n = 50 or 80 cells. Graphs represent mean ± SEM; n = 50-100. ***P < .0001 (t test). **P < .001 (t test).