TAK-981 treatment augments rituximab-mediated macrophage phagocytosis and NK cell cytotoxicity in an IFN1-dependent manner. (A) Representative flow cytometry scatter plots. Macrophages are defined as CellTrace Red⁺ events and Daudi cells as CellTrace Violet⁺ events. Percent of CellTrace Red⁺Violet⁺ in CellTrace Violet⁺ events corresponds to percent of phagocytosed Daudi cells in total Daudi cells. (B) Phagocytic activity of 1 μM TAK-981–treated hMDM against Daudi cells in the presence of 1 μg/mL human IgG1 (hIgG1) or rituximab at an effector to target (E:T) ratio of 1:1 (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (C) Phagocytic activity of 50 ng/mL IFNγ-, 20 ng/mL IL-4-, and/or 1 μM TAK-981–treated hMDM against Daudi-KILR cells in the presence of hIgG1 or rituximab at an E:T ratio of 4:1 (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (D) Phagocytic activity of 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated hMDM against Daudi-KILR cells in the presence of human IgG1 or rituximab at an E:T ratio of 4:1 (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (E) Phagocytic activity of 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated hMDM against Daud-KILR cells in the presence of 1 μg/mL human IgG1, daratumumab, or 1 μg/mL rituximab at an E:T ratio of 4:1 (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (F) Cytotoxicity of 20 μg/ml aIFNAR2-, 1 μM TAK-981-, and/or 10 kU/mL IFNα2-treated human NK cells against Daudi-KILR cells in the presence of 1 μg/mL human IgG1 or 1 μg/mL rituximab at an E:T ratio of 4:1 (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). *P < .05, **P < .01, ***P < .001. Experiment was repeated using hMDM or human NK cells generated from at least 2 healthy PBMC donors. SD, standard deviation; N.S., not significant (P > .05).