Lenalidomide treatment increases IFN-γ secretion from NK cells. (A) Healthy donor pNK cells and Daudi target cells were pretreated with DMSO or 0.001 to 10 µM lenalidomide for 24 hours then the Daudi cells were tested for susceptibility to NK-cell–mediated lysis. Graph shows mean ± SD from 3 independent donors. E:T ratio was 10:1. (B-C) pNK cells and (B) Daudi cells or (C) Raji cells were cocultured for 24 hours with DMSO or 0.001 to 10 µM lenalidomide (±150 U/mL IL-2). IFN-γ release was measured by ELISA. E:T ratio was 10:1 in all experiments. Data shows mean ± SD from 3 donors. Significance compared with DMSO (‘0 µM’) condition. (D-G) Primary NK cells were treated with DMSO or 0.1 to 10 µM lenalidomide (+150 U/mL IL-2) for 24 hours in wells coated with (D) anti-CD16 mAb or IgG1 isotype control mAb, (E) recombinant ICAM-1, anti-NKG2D mAb, or both, (F) recombinant ICAM-1, recombinant MICA, or both, (G) anti-NKG2D mAb, anti-2B4, or both. IFN-γ release was assessed by ELISA. Data show mean ± SD from 3 donors. (H-I) Primary NK cells were treated with DMSO or 1.0 µM lenalidomide (+150 U/mL IL-2) for 4 hours in wells coated with (H) anti-CD16 mAb or IgG1 isotype control mAb, or (I) recombinant ICAM-1 or recombinant MICA and ICAM-1. IFN-γ mRNA was assessed by qRT-PCR and is normalized to GAPDH. Data shows triplicate data from (H) 3 donors and (I) 2 donors. Data points for each donor are shaded the same. Red line shows the mean. ns, not significant; qRT-PCR, quantitative reverse transcription polymerase chain reaction. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with Tukey posttest.