4.9. PD-1/PD-L1 Blockade Bioassay

The PD-1/PD-L1 immune checkpoint bioassay (PD-1/PD-L1 Bioassay, Promega) was performed according to the manufacturer’s manual. PD-L1 + aAPC/CHO-K1 cells were plated in 96-well, white, flat bottom assay plates at 40 × 10⁴ cells in 100 µL of medium (Ham’s F12, 10% FBS) and incubated overnight at 37 °C, 5% CO₂. The next day, the medium was removed from the assay plate, and serially diluted antibodies were added at 40 µL per well in the assay buffer (RPMI1640 + 1% FBS + 1% DMSO). Next, PD-1 Effector Jurkat cells (included in the assay kit) were resuspended in assay buffer (RPMI 1640 + 1% FBS) at a concentration of 1.25 × 10⁶ cells/mL and added to the assay plate at 40 µL per well (total of 50 × 10⁴ cells). The cells were co-cultured for 6 h (37 °C, 5% CO₂) and then removed from the incubator and equilibrated at room temperature for 5 min. Bio-GloTM Reagent (Promega) was prepared according to the manufacturer’s manual and added to each well at 80 µL per well. Assay plates were incubated in room temperature for 15 min; luminescence was measured on the Tecan Spark microplate reader. Data were analyzed in GraphPad Prism software (v.9.5.1) using a log(inhibitor) vs. response—Variable slope (four parameters) model.