Anti cd19 anti cd3 bispecific antibody

HuT78 cells (1 × 10⁵ cells per well) and Raji cells (1 × 10⁵ cells per well) were cocultured in lymphocyte medium for 24 h with or without anti-CD19-anti-CD3 bispecific antibody (equivalent to blinatumomab) (10 ng/ml; BPS Bioscience) and anti-PD-L1-hIgG1 antibody (N298A; equivalent to atezolizumab) (Cat: hpdl1-mab12, 5 μg/ml; InvivoGen) or isotype control (Cat: bgal-mab12; InvivoGen). Monensin and brefeldin A (1:1,000 each; Cytek) were added for the last 6 h. Cells were stained with Zombie NIR Fixable Viability dye (1:1,000 in PBS; BioLegend) for 15 min at 4°C in the dark, washed with FACS buffer, and fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (Cytek). Cells were then stained by incubation overnight at 4°C in the dark with the following reagents in permeabilization buffer: FcR blocking reagent (1:50; Miltenyi Biotec), anti-CD3-APC (Clone: UCHT1, 1:100; Cytek), anti-IFN-γ-PE-Dazzle 594 (Clone: 4S.B3, 1:500; BioLegend), and anti-TNF-BV711 (Clone: MAb11, 1:500; BioLegend) mAbs. The cells were washed with FACS buffer and acquired with an Attune NxT Flow Cytometer with the CytKick MAX Autosampler (Invitrogen). Data were analyzed with FlowJo and R software. The percentage of IFN-γ⁺ cells was used as a readout. The data were normalized against the mean for the blinatumomab plus atezolizumab group for each combination of HuT78 and Raji cells.