Antitumor efficacy of anti–CD20-mIFNα requires IFNAR expression on tumor cells. (A) Flow cytometric analysis of IFNAR expression on indicated cell lines. 38C13-huCD20 transduced with IFNAR-specific shRNA (38C13-huCD20 IFNAR KD), 38C13-huCD20 transduced with nonspecific shRNA (38C13-huCD20 control), and 38C13-CD20 parental cells were stained with anti–IFNAR-biotin primary antibody (clone MAR1-5A3) and detected with streptavidin-PE. Biotinylated IgG1 isotype–stained control is also shown. (B) Apoptosis assay using parental 38C13-huCD20 and 38C13-huCD20 IFNAR KD. Cells were treated with 1000pM anti–CD20-mIFNα and stained with annexinV-FITC and PI 48 hours later. Flow cytometry patterns and gate frequencies in percentages are shown in the upper right hand corner of the annexin⁺ gates. (C) Effective in vivo treatment of 38C13-huCD20 with anti–CD20-mIFNα requires IFNAR expression on tumor cells. Mice (n = 8 per group) were treated 5, 6, and 7 days after tumor inoculation with 10 μg of anti–CD20-mIFNα or the molar equivalent of the indicated proteins. Mice were followed for survival and killed when tumors reached 1.4 cm in diameter as per institutional guidelines. Mice treated with HBSS were used as control.