Hpd l1

hPD-L1-TCR-HEK293T and TCR-HEK293T: One day before transfection, seed HEK293T cells at a density of 2x10⁶ cells per ml, when cells reached 80% confluent at the time of transfection. The next day, transfect 1 μl TCR activator and hPD-L1 (Cat#79455, BPS Bioscience) or the only TCR activator (Cat#79455, Cat#79455) into cells following the manufacturer’s protocol. To sort the hPD-L1-TCR -HEK293T cells, we stain the cells with anti-human PD-L1 antibody (Cat #329706, Biolegend) by the FACSAria (BD) after 3 days transfection.
hPD-1-NFAT-Jurkat cells: Lentiviral packaging of the plasmid pLenti-NFAT-IRES-EGFP-PD-1 were performed by Gene Pharma. Lentivirus were infected into Jurkat cells at MOI 20 with 2µg/ml Polybrene, 7 days post infection, hPD-1-NFAT-Jurkat cells were sorted by FACSAria (BD), with staining of anti- human PD1 antibody (Cat #329906, Biolegend). Single clones were performed and selected by expression of PD-1.

Competitive ELISA was performed using a PD-1/PD-L1 or CTLA-4/CD80 inhibitor ELISA-screening kit (BPS Bioscience), according to the manufacturer’s instructions (25 (link)). Briefly, recombinant hPD-L1 (#71104, BPS Bioscience) or CTLA-4 (#71149, BPS Bioscience) was coated overnight at 1 μg/mL in PBS onto 96-well plates (Corning Inc., New York, NY, USA). The plates were washed with PBS containing 0.1% tween (PBS-T), blocked for 1 h at room temperature with PBS containing 2% (w/v) BSA, and washed again. Then, 5 µL of 0.5 μg/mL biotinylated hPD-1 (#71109, BPS Bioscience) or biotinylated hCD80 (#71114, BPS Bioscience) was added to the wells, and the plates were incubated for 2 h at room temperature. After three washes in PBS-T, 50 μL of 0.2 μg/mL HRP-conjugated streptavidin was added to each well, and the plates were incubated for 1 h. After incubation, plates were washed thrice with 0.1% PBS-T, and relative chemiluminescence was measured using a SpectraMax L Luminometer (Molecular Devices, San Jose, CA, USA).