CD38 upregulation through lenalidomide-induced loss of Ikaros or interferon treatment enhances daratumumab-stimulated NK-cell mediated ADCC. (A) CD38 expression (mean RPKM ± SD) from RNA sequencing in OPM2 cells after deletion of Ikaros, Aiolos, or lenalidomide (Len) 10 μM, and MM1S and H929 cells after deletion of Ikaros, vs control. Data are described in Figure 3. (B) CD38 mean fluorescence intensity (MFI) ± SD determined (relative to control) by flow cytometry from 3 experiments in H929, OPM2, AMO-1, and MM1S cells 48 hours after 0, 1, or 10 μM Len. ****P < .0001 using an unpaired Student t test. Refer to supplemental Figure 6 for corresponding flow cytometry histograms. (C) Histogram of CD38 surface expression by flow cytometry in H929 cells at baseline, or 96 hours after dox IKZF1 gRNA induction, 72 hours after treatment with 1μ M Len, and/or 48 hours after 50 IU β-IFN. (D) CD38 MFI ± SD determined by flow cytometry in 5 newly diagnosed treatment-naïve MM patient samples isolated from bone marrow aspirate by flow cytometry sorting and treated for 60-72 hours with 1 μM Len, 100 IU β-IFN, combination Len + β-IFN, or control. ***P < .001 using an unpaired Student t test. (E) Calcein-AM cell lysis assay evaluating human NK cell (effector)-mediated antibody-dependent cellular cytotoxicity of H929 (target) cells after a dose titration of daratumumab. Effector to target (E:T) ratio 5:1. H929 IKZF1 gRNA cells pretreated with dox (day, −4), lenalidomide 1 μM (day −3), and/or β-IFN 50 IU (day −2) before assay (D0). Percentage lysis determined relative to internal “spontaneous” (no NK cells or daratumumab) and “max” (media with 2% Triton-X) lysis controls for each sample to exclude effect of differing pretreatments. Data are the mean % lysis ± SD and are representative of 4 experiments. The untreated H929 control data are common to both data sets.