Blinatumomab-mediated cytotoxicity assay

The blinatumomab-mediated cytotoxicity assay was developed upon, and modified from, previous methods [23, 33, 34]. Briefly, we used frozen healthy Peripheral blood mononuclear cells (PBMCs, HemaCare, #PB009C-2) and the B-ALL cell lines RS4;11 (ATCC, CRL-1873) and SUP-B15 (ATCC, CRL-1929) as effector and target cells, respectively. The frozen PBMCs were commercial products and collected by vendor with a limited cell number from each donor. Thus, the PBMCs that were used in the different assays came from different donors. The target to effector cell ratio and blinatumomab concentration were titrated for different experiments. For the cell line cytotoxicity assay, the target cells were stained using the CellTrace™ Far Red Cell Proliferation Kit (Invitrogen, # {"type":"entrez-nucleotide","attrs":{"text":"C34564","term_id":"2370705","term_text":"C34564"}}C34564) and then co-cultured with PBMCs for 16 or 48 h at an effector-to-target cell ratio of 10:1 and in the presence of blinatumomab. For the TNFSF4 assay, titration started from an E:T ratio of 10:1 for 16 and 48 h. In order to observe a significant dose-response effect of TNFSF4, the PBMCs were co-cultured with target cells for 5 days at a ratio of 1:2 in the presence of recombinant human TNFSF4 protein (R&D system, #1054-OX-010). The cell mixture was then stained using a L/D staining solution (Zombie Aqua Fixable viability kit, Biolegend, #423101) for 30 min, resuspended in a flow cytometry staining buffer (eBioscience, #00422226), and then 10000 target cells were analyzed on a BD LSR Fortessa™ X-20. Specific lysis was calculated according to the following equation: % specific lysis = (proportion of live target cells in sample - proportion of live target cells in control sample) / (1 - proportion of live target cells in control sample) × 100.