Lenalidomide treatment increases NK-cell activation through rituximab. (A) pNK cells were treated with DMSO or 1.0 µM lenalidomide (+150 U/mL IL-2) for 24 hours in wells coated with 0.0001 to 100 µg/mL rituximab. IFN-γ release was measured by ELISA. Data shows mean ± SD from 3 donors. Nonlinear regression fit was applied to data. EC₅₀ values calculated to be: 1.6 µg/mL without lenalidomide and 0.4 µg/mL with lenalidomide. (B) Proportion of DMSO-treated cells expressing IFN-γ after stimulation on rituximab-coated surfaces in the presence of DMSO or 1 µM lenalidomide for 30 to 120 minutes. Graph shows mean ± SD, n > 200 from 3 donors. (C) Superresolution images obtained by STED microscopy of membrane proximal F-actin in pNK cells incubated for 60 or 120 minutes on coverslips coated with rituximab (10 µg/mL) in the presence of DMSO or 1.0 µM lenalidomide (+150 U/mL IL-2). Scale bars, 5 µm. Second column: Holes between actin filaments 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 through which a particle (such as an IFN-γ vesicle) of diameter 200 nm (blue) to 800 nm (red) could fit. (D) Proportion of the synapse area predicted to be penetrable by a vesicle of 200- to 500-nm diameter for cells stimulated on 10 µg/mL rituximab for 60 minutes. (E) Proportion of the synapse area predicted to be penetrable by a vesicle of 200- to 500-nm diameter for cells stimulated on 10 µg/mL rituximab for 120 minutes. Graph shows mean ± SD, n = 45 from 3 donors. *P < .05, **P < .01, ***P < .001, 1-way ANOVA with Tukey posttest.