The PD-1:PD-L1/PD-L2 Cell-Based Inhibitor Screening Assay is a bioluminescent cell-based assay for screening and profiling inhibitors of the PD-1:PD-L1 or PD-1:PD-L2 interaction. The assay consists of two main components: effector cells and expression vectors encoding TCR activator, human PD-L1, and human PD-L2. Growth-Arrested PD-1 Effector Cells (PD-1/NFAT Reporter Jurkat cells) are Jurkat cells expressing firefly luciferase, under the control of NFAT response elements, and human PD-1. These cryopreserved cells are provided in a thaw-and-use format that does not require cell propagation. These cells cannot be expanded and are intended to be single use. The expression vectors for TCR Activator, human PD-L1, and human PD-L2 are transfection-ready vectors that can be used to transfect cells and create target cells that overexpress PD-L1 or PD-L2 and an engineered cell surface T cell receptor (TCR) activator. The kit contains enough cells and plasmids for 100 assays if using a 96-well plate.

Figure 1: Illustration of the principle of the PD-1:PD-L1/PD-L2 Cell-Based Inhibitor Screening Assay. Jurkat T cells expressing NFAT reporter with constitutive expression of PD-1 (PD-1/NFAT Reporter Jurkat Cells) act as effector cells. When co-c*ted, TCR complexes on PD-1/NFAT Reporter Jurkat cells are activated by the TCR activator expressed in the transfected cell line of interest, resulting in expression of the NFAT luciferase reporter. However, PD-1 and PD-L1/ PD-L2 ligation prevents TCR activation and suppresses the NFAT-responsive luciferase activity. This inhibition can be specifically reversed by anti- PD-1 or anti- PD-L1/PD-L2 antibodies. Neutralizing antibodies block PD-1:PD-L1/PD-L2 interaction and result in reactivation of the NFAT responsive luciferase reporter.

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