First Online: 1.10.2025

Programmed cell death protein 1 (PD-1) is crucial in inhibiting immune responses by modulating the activity of T cells. We present an in vitro assay that is based on a co-culture of primary immune cells represented by human peripheral blood mononuclear cells (PBMCs), isolated from healthy donors with Chinese hamster ovary-derived cell line (CHO-K1) overexpressing human PD-L1 protein (hPD-L1) and an artificial TCR-activator construct (TCRAct). CHO-K1/TCRAct/hPD-L1 cells mimic antigen-presenting cells by activating T cells via the T-cell receptor (TCR) and providing a ligand for the negative immune checkpoint (PD-L1 protein). The two components, PBMCs and CHO-K1/TCRAct/hPD-L1 cells, when in co-culture, provide a T-cell activation (TCA) assay, which may be used to test the potency of molecules targeting the PD-1/PD-L1 immune checkpoint. This method relies on monitoring the activation of helper CD4+ and cytotoxic CD8+ T cells using flow cytometry by analyzing the expression levels of early (CD69), intermediate (CD25 and HLA-DR), and late (PD-1) activation/exhaustion markers. This is the well-established in vitro co-culture assay in which primary T-cell activation via the TCR is diminished by the concurrent presence of PD-1/PD-L1 immune checkpoint, which can be blocked resulting in increased expression of T-cell surface markers.