The CD16-mediated symmetric spreading is LFA-1– and kindlin-3–dependent. (A) Fluorescence images of F-actin in healthy donor NK cells or LAD-III NK cells stimulated on surfaces coated with anti-NKG2D (3.0 μg/mL) or anti-CD16 (5.0 μg/mL). (B) The percentage of healthy or LAD-III NK cells forming a peripheral ring of F-actin in response to surfaces coated with poly lysine and indicated concentrations of anti-CD16 (light blue and pink, respectively) or surfaces coated with ICAM-1 and indicated concentrations of anti-CD16 (blue and red, respectively). Graphs represent mean ± SEM; n = 60-90. (C) The area of cells attached to the slide surface, as assessed by IRM imaging, was measured for healthy and LAD-III NK cells on surfaces coated with ICAM-1 and increasing concentrations of anti-CD16 (blue and red, respectively) and for healthy and LAD-III NK cells on surfaces coated with poly lysine and increasing concentrations of anti-CD16 (light blue and pink, respectively). Graphs represent mean ± SEM; n = 60-90. (D) The distance from the centroid of healthy or LAD-III NK cells (cell activated using anti-CD16 and anti-NKG2D at 5.0 μg/mL) stained with phalloidin-ATTO-647N to the circumference was measured at 360 radii, and the symmetry of the cells was assessed by the SD of the radii (left) and the ratio of maximum and minimum lengths (right, n = 40-70). ***P < .0005 (ANOVA). **P < .005 (ANOVA). *P < .05 (ANOVA). NS indicates not significant.