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A PD-1/PD-L1-Sensitive Co-culture-Based Primary T-Cell Activation Assay
Justyna Kocik-Krol1, Malgorzata Stec2, Maciej Siedlar2
1Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Krakow, Poland.
Insights
This study introduces a new in vitro assay using co-cultured cells to measure T-cell activation. The assay assesses the potency of molecules targeting the Programmed cell death protein 1 (PD-1)/PD-L1 immune checkpoint.
Area of Science:
- Immunology
- Cell Biology
- Assay Development
Background:
- Programmed cell death protein 1 (PD-1) regulates T-cell activity and immune responses.
- The PD-1/PD-L1 pathway is a key target for immunotherapies.
- Existing methods for assessing PD-1/PD-L1 interactions require refinement.
Purpose of the Study:
- To develop and validate a novel in vitro co-culture assay for evaluating PD-1/PD-L1 immune checkpoint inhibitors.
- To quantify T-cell activation in the presence of PD-1/PD-L1 signaling.
- To assess the potency of molecules targeting the PD-1/PD-L1 pathway.
Main Methods:
- Co-culture of human peripheral blood mononuclear cells (PBMCs) with Chinese hamster ovary (CHO-K1) cells engineered to express PD-L1 and a T-cell receptor activator (TCRAct).
- Mimicking antigen-presenting cells (APCs) with CHO-K1/TCRAct/hPD-L1 cells to activate T cells via TCR.
- Monitoring T-cell activation using flow cytometry, analyzing surface markers like CD69, CD25, HLA-DR, and PD-1.
Main Results:
- The assay successfully demonstrates T-cell activation (TCA) via TCR signaling.
- PD-1/PD-L1 immune checkpoint signaling diminishes T-cell activation in the co-culture system.
- Blocking the PD-1/PD-L1 interaction leads to increased expression of T-cell activation markers.
Conclusions:
- The developed in vitro co-culture assay provides a robust method for testing PD-1/PD-L1 checkpoint inhibitor potency.
- This assay enables the study of T-cell modulation by the PD-1/PD-L1 pathway.
- The findings support the utility of this assay in preclinical drug development for immunotherapies.
Abstract:
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.
