Lenalidomide treatment augments opening of cortical actin mesh after CD16 stimulation. (A) Superresolution images obtained by STED microscopy of membrane proximal F-actin in pNK cells incubated for 120 minutes on coverslips coated with isotype-matched control antibody or anti-CD16 mAb (both 3 µg/mL) in the presence of DMSO vehicle control or 1.0 µM lenalidomide (+150 U/mL IL-2). Scale bars, 5 µm. Second column: Holes between actin filaments in the central region of the synapse shown as heat maps, with the smallest holes shown in blue (0.01 µm²) and largest holes shown in red (>3 µm²). Third column: Regions are shown within the actin mesh through which a particle (such as an IFN-γ vesicle) of diameter 200 nm (blue) to 800 nm (red) could fit. (B) Histogram of measured vesicle sizes from 10 cells from 2 independent donors. (C) Average size of holes in the actin mesh at the pNK synapse for cells stimulated as in panel A. Each data point represents a single cell; red lines shows mean from 3 donors, n = 18 to 59 per condition. (D) The proportion of the synapse area predicted to be penetrable by a vesicle of 200- to 500-nm diameter for same cells as in panel C. (E) Analysis of STED microscopy of membrane proximal actin in pNK cell stimulated as in panel A plus 5 µg/mL brefeldin A and costained with anti-IFN-γ conjugated to Alexa 647. The proportion of the synapse predicted to be penetrable by a particle of 200- to 500-nm diameter, stratified by whether or not the cells stained positive for IFN-γ. Graph shows mean from 3 donors, n = 50. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with Tukey posttest.