Mab139

DCA was from Aldrich. Fluorescence coupled antibodies against human ULBP1-488 and the blocking antibody against NKG2D (MAB139) were from R&D Systems; ICAM-1, MICA/B, CD155, CD33, CD138 and HLA were from Miltenyi; DR4 (12-6644-73), DR5 (12-9908-42) and Fas (BMS140FI) were from eBiosciences and 7AAD from BD Biosciences. Recombinant human Fas-Fc chimera (AFA0114121) was purchased from R&D Systems and human TRAIL-blocking antibody RIK2 (550912) from BD Biosciences. For in vivo experiments we used the anti-CD20 rituximab. The D1D2 construct that binds to LFA-1 has been described³¹ . Blocking anti-ULBP1 antibody MAB1380, anti-MICA/B antibody MAB13001 and anti-β2 integrin AF1730 were from R&D systems.

3x10⁵ MNT-1 cells were infected with 3x10⁷ PFU of reovirus for 1 hr at room temperature, then incubated at 37˚C. At 48 hrs of infection, the cells were harvested and labelled with CFSE (ThermoFisher Scientific, C34570) according to the manufacturer’s instructions. They were then cocultured with purified primary NK cells for 4 hrs at effector-to-target (E:T) ratios of: 10:1, 5:1, 2.5:1, or 1:1. NK cells were adjusted to the number of target cells. To assess spontaneous lysis of cells, samples containing target cells only were included for both the infected and uninfected groups. The lysis of target cells was assessed by staining each sample with 0.01% fixable viability dye (FVD), and was measured by flow cytometer. Following doublets discrimination, the percentage of the specific lysed population was gated as %CFSE⁺FVD⁺ out of the CFSE⁺ population. NK cell specific cytotoxicity was calculated by following formula . All tested groups, including different E:T ratios, were done in triplicate. In assays with NKG2D receptor blocking, prior to coculturing with target-cells, NK cells were washed twice in PBSx1 and incubated on ice for 30 min with anti-NKG2D (R&D, MAB139) at the concentration of 10µg/mL. The cells were then washed twice in PBSx1 and cocultured as described above.