Analysis of the PD-L1 protein with in-frame deletion in an overexpression system. (A and B) PD-L1 protein levels. Raji B-lymphoma cells were lentivirally transduced with cDNA encoding the WT or a mutant PD-L1 isoform, or with EV, and were then subjected to selection on puromycin. PD-L1 protein levels were determined by (A) immunoblotting and (B) flow cytometry with monoclonal antibodies (mAb) against PD-L1. In B, a vertical dotted line within a histogram indicates the median. Representative results from two experiments are shown. (C and D) PD-1:PD-L1-mediated suppression assay. (C) Schematic diagram. HuT78 T-lymphoma cells lentivirally transduced with EV or with WT PD-1 were cocultured with Raji cells transduced with EV or a WT or mutant PD-L1 isoform for 24 h without stimulation or with blinatumomab (CD3-CD19 bispecific antibody, BiTE). Secretion inhibitors were added for the last 6 h. IFN-γ production was quantified by intracellular flow cytometry. The effect of anti-PD-L1 neutralizing mAb (equivalent to atezolizumab) or its isotype control was also assessed in this system. (D) Summary plot. The readout (percentage of IFN-γ⁺ HuT78 cells) was normalized against the mean in the “BiTE plus anti-PD-L1 antibody” group. Results from two independent experiments with 12 technical replicates in total were compiled. Statistical significance was determined for differences between EV and each PD-L1 construct in BiTE-stimulated conditions by two-tailed Wilcoxon’s rank sum tests with FDR adjustment. n.s., not significant. ****, P < 0.0001. Source data are available for this figure: SourceData F3.