A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches.

Blockade of programmed cell death protein 1 (PD-1) immune checkpoint receptor signaling is an established standard treatment for many types of cancer and indications are expanding. Successful clinical trials using monoclonal antibodies targeting PD-1 signaling have boosted preclinical research, encouraging development of novel therapeutics. Standardized assays to evaluate their bioactivity, however, remain restricted. The robust bioassays available all lack antigen-specificity. Here, we developed an antigen-specific, short-term and high-throughput T cell assay with versatile readout possibilities. A genetically modified T cell receptor (TCR)-deficient T cell line was stably transduced with PD-1. Transfection with messenger RNA encoding a TCR of interest and subsequent overnight stimulation with antigen-presenting cells, results in eGFP-positive and granzyme B-producing T cells for single cell or bulk analysis. Control antigen-presenting cells induced reproducible high antigen-specific eGFP and granzyme B expression. Upon PD-1 interaction, ligand-positive antigen-presenting immune or tumor cells elicited significantly lower eGFP and granzyme B expression, which could be restored by anti-PD-(L)1 blocking antibodies. This convenient cell-based assay shows a valuable tool for translational and clinical research on antigen-specific checkpoint-targeted therapy approaches.