Lenalidomide lowers the threshold for NK-cell activation through NKG2D and CD16. (A-B) pNK cells were treated with DMSO or 1.0 µM lenalidomide (+150 U/mL IL-2) for 24 hours in wells coated with (A) 0.1 to 10 µg/mL MICA plus 2.5 µg/mL ICAM-1 or (B) 1 to 500 µg/mL human IgG. IFN-γ release was measured by ELISA. Data show mean ± SD from 3 donors. Nonlinear regression fit was applied to data. EC₅₀ values were calculated to be: 2.1 µg/mL (MICA DMSO), 1.3 µg/mL (MICA 1.0µM Len), 62 µg/mL (hIgG DMSO), 21 µg/mL (hIgG 1.0µM Len). (C) 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 90 minutes on surfaces coated with 1 µg/mL or 2.5 µg/mL MICA (+2.5 µg/mL ICAM-1) in the presence of DMSO or 1.0 µM lenalidomide (+150 U/mL IL-2). Scale bars, 10 µm. (D) The proportion of pNK cells expressing IFN-γ after stimulation with 3 different concentrations of MICA. Graph shows mean ± SD, n > 100 per donor from 3 donors. (E) Sum fluorescence staining for IFN-γ per cell in the same cells as in panel D. Each data point represents a single cell and red line shows the mean. *P < .05, **P < .01, ****P < .0001, 1-way ANOVA with Tukey posttest.