Figure 3
Figure 3. The activation threshold for NKG2D-mediated F-actin polymerization and for symmetric spreading is altered in LAD-III NK cells. (A) Representative bright-field (top panels) and fluorescence images of F-actin (bottom panels) for healthy NK cells stimulated on surfaces coated with ICAM-1 (2.5 μg/mL) with increasing concentrations of MICA-Fc (0-2.5 μg/mL). (B) Representative bright-field (top panels) and fluorescence images of F-actin (bottom panels) for LAD-III NK cells stimulated as in panel A. White arrows indicate activated cells. Bars represent 10 μm. (C) The proportion 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 MICA-Fc (light blue and pink, respectively) or surfaces coated with ICAM-1 and indicated concentrations of MICA-Fc (blue and red, respectively). Data are mean ± SEM from 4 independent experiments; n = 50-90. ***P < .0005 (1-way ANOVA). *P < .05 (1-way ANOVA). (A-C) A complete ring of actin filaments is scored as a synapse, whereas an interrupted or patchy ring represents a kinapse. (D) The area of cells attached to the slide surface, as assessed by IRM imaging, was measured for healthy NK cells and LAD-III NK cells on surfaces coated with ICAM-1 and increasing concentrations of MICA-Fc (blue and red, respectively) and for healthy and LAD-III NK cells on surfaces coated with poly lysine and increasing concentrations of MICA-Fc (light blue and pink, respectively). Data are mean ± SEM from 4 independent experiments; n = 50. ***P < .0005 (1-way ANOVA). **P < .005 (1-way ANOVA). *P < .05 (1-way ANOVA). (E) The distance from the cell centroid of phalloidin-AlexaFluor488-stained healthy or LAD-III NK cells to the circumference was measured at 360 radii. The symmetry of the cells stimulated on ICAM-1–coated slides with or without MICA-Fc (2.5 μg/mL) is represented as the SD of the radial distances and the ratio of maximum and minimum lengths; n = 50-90. ***P < .0005. (F) The ratio of maximum and minimum lengths for healthy or LAD-III NK cells stimulated on surfaces coated with ICAM-1 and indicated concentrations of MICA-Fc; n = 50-90. *P < .05. NS indicates not significant.

The activation threshold for NKG2D-mediated F-actin polymerization and for symmetric spreading is altered in LAD-III NK cells. (A) Representative bright-field (top panels) and fluorescence images of F-actin (bottom panels) for healthy NK cells stimulated on surfaces coated with ICAM-1 (2.5 μg/mL) with increasing concentrations of MICA-Fc (0-2.5 μg/mL). (B) Representative bright-field (top panels) and fluorescence images of F-actin (bottom panels) for LAD-III NK cells stimulated as in panel A. White arrows indicate activated cells. Bars represent 10 μm. (C) The proportion 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 MICA-Fc (light blue and pink, respectively) or surfaces coated with ICAM-1 and indicated concentrations of MICA-Fc (blue and red, respectively). Data are mean ± SEM from 4 independent experiments; n = 50-90. ***P < .0005 (1-way ANOVA). *P < .05 (1-way ANOVA). (A-C) A complete ring of actin filaments is scored as a synapse, whereas an interrupted or patchy ring represents a kinapse. (D) The area of cells attached to the slide surface, as assessed by IRM imaging, was measured for healthy NK cells and LAD-III NK cells on surfaces coated with ICAM-1 and increasing concentrations of MICA-Fc (blue and red, respectively) and for healthy and LAD-III NK cells on surfaces coated with poly lysine and increasing concentrations of MICA-Fc (light blue and pink, respectively). Data are mean ± SEM from 4 independent experiments; n = 50. ***P < .0005 (1-way ANOVA). **P < .005 (1-way ANOVA). *P < .05 (1-way ANOVA). (E) The distance from the cell centroid of phalloidin-AlexaFluor488-stained healthy or LAD-III NK cells to the circumference was measured at 360 radii. The symmetry of the cells stimulated on ICAM-1–coated slides with or without MICA-Fc (2.5 μg/mL) is represented as the SD of the radial distances and the ratio of maximum and minimum lengths; n = 50-90. ***P < .0005. (F) The ratio of maximum and minimum lengths for healthy or LAD-III NK cells stimulated on surfaces coated with ICAM-1 and indicated concentrations of MICA-Fc; n = 50-90. *P < .05. NS indicates not significant.

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