Lenalidomide increases the proportion of pNK cells expressing IFN-γ as well as the amount produced per cell. (A) Representative microscopy images of F-actin (red) and IFN-γ (shown in grayscale in middle column, and then green in merged image) in pNK cells stimulated for 120 minutes on surfaces coated with IgG1 isotype control mAb or anti-CD16 mAb in the presence of DMSO or 1 µM lenalidomide (+150 U/mL IL-2). Scale bars, 10 µm. (B) The proportion of cells expressing IFN-γ after stimulation on anti-CD16 mAb-coated surfaces in the presence of DMSO or 1 µM lenalidomide (+150 U/mL IL-2) for 30 to 240 minutes. Graph shows mean ± SD, n > 200 from 3 donors. (C) The proportion of cells expressing IFN-γ after stimulation on anti-CD16 mAb-coated surfaces in the presence of DMSO or 1 µM lenalidomide (+150 U/mL IL-2) for 30 to 240 minutes. All cells treated with DMSO or 1 µM lenalidomide for a total of 240 minutes and added to stimulating surfaces in the reverse order for the time course (for example, for 30-minute stimulation, pNK cells were treated with DMSO or 1 µM lenalidomide for 210 minutes before plating). Graph shows mean ± SD, n > 100 from 2 donors. (D) The proportion of pNK cells expressing IFN-γ after stimulation on anti-CD16 mAb-coated surfaces with 0.1 to 10 µM lenalidomide. Graph shows mean ± SD, n > 100 per donor from 3 donors. (E) Sum IFN-γ fluorescence per cell in pNK cells stimulated as in panel D. Each data point represents a single cell and red line shows the mean. n = 75 to 111 from 3 donors. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with Tukey posttest.