Table 2.

Most commonly used functional assays to monitor the impact of small molecule agents on PD-1 pathway.

Type of assay	Source for PD-1	Source for PD-L1	Consequence of interfering in the PD-1:PD-L1 interaction by small molecules	Detection system	Comments
NFAT reporter	Jurkat (immortalized line of human T cells) overexpressing PD-1 and luciferase gene controlled by the NFAT-response element	CHO-K1 (Chinese hamster ovary cells) cell line overexpressing PD-L1 and T-cell receptor activator	Activation of TCR signaling leading to greater luciferase expression	Chemiluminescence	Engineered cell line as T cells in the assay. Assay commercially available
SHP-1	Jurkat cells expressing PD-1 and SHP-1 each fused with different individually inactive fragments of the β-galactosidase	U-2 OS osteosarcoma cell line expressing PD-L1	Decrease in SHP-1 recruitment leading to lower β-galactosidase activity due to reduced enzyme fragment complementation	Chemiluminescence	Engineered cell line as T cells in the assay. Assay commercially available
PBMC	T cells activated by anti-CD3/anti-CD28	Recombinant protein	Activation of TCR signaling leading to the rescue of proliferation or cytokine release	FACS or ELISA	Primary T cells in the assay—closer to physiological context
PBMC/whole blood	T cells activated by staphylococcal enterotoxin B (SEB) or CMV antigen	Other cells in PBMCs or blood; not controlled likely leading to greater variability in the assay	Activation of TCR signaling leading to the rescue of proliferation or cytokine release	FACS or ELISA	Primary T cells in the assay—closer to physiological context