CT-011 enhances NK-cell GrB and IFN-γ production against PD-L1–bearing tumor cell targets. (A) By using an ELISPOT effector-based assay, we studied the effects of CT-011 on GrB production by primary, human NK cells against the K562 and RPMI8226 cell lines (E:T = 10:1). After 72 hours of pretreatment with 150 IU/mL IL-2 and control or CT-011 mAb, the latter led to statistically significant increases in NK-cell degranulation of GrB against K562 (*P < .05) and RPMI8226 (**P < .05; data shown are representative of 3 independent experiments against each line). (B) By using the same effector-based ELISPOT cytotoxicity assay, we found that CT-011 also enhanced IFN-γ production by NK cells (pretreated for 72 hours in IL-2 150 IU/mL and control or CT-011 mAb) against primary MM tumor cell targets (*P = .01; data shown from 3 independent experiments). (C) By using a target-based cytotoxicity assay, primary, we pretreated human NK cells for 72 hours in IL-2 150 IU/mL and control, lenalidomide (5nM), CT-011, or the combination. Lenalidomide and CT-011 statistically significantly increased cytotoxicity against RPMI8226 cell line targets (E:T 50:1, *P = .03, ET:100:1, **P = .02; data shown are from 3 independent experiments at both E:T ratios). (D) PBMCs and marrow aspirates were obtained from patients with MM (n = 3) and CD138⁺ tumor cells were isolated from the whole marrow aspirate. Effector cells were cultured in IL-2 and control or CT-011 for 48 hours and CD138⁺PD-L1⁺ MM tumor cells and CD138⁻PD-L1⁻ cellular marrow fraction served as target populations. On the left, cytotoxicity is enhanced against autologous CD138⁺PD-L1⁺ MM tumor cells (*P = .005; data shown from n = 3 independent experiments), yet no increase in cytotoxicity is conferred against autologous CD138⁻PD-L1⁻ cellular marrow elements (right; P = ns).