High-throughput discovery of MART1-specific TCR-peptide interactions using a Jurkat NFAT Reporter-K562 antigen-presenting cell system and single-cell adaptive immune receptor profiling

In this study, we developed a high-throughput workflow for identifying MART1-specific T cell receptor (TCR)-peptide interactions relevant to melanoma immunotherapy. Jurkat NFAT-GFP reporter cells expressing individual candidate MART1-TCRs (n=10 to 20) were co-cultured with K562 artificial antigen-presenting cells (APCs) engineered to express a MART1 120-peptide, tumor-associated library in a single-chain trimer (peptide-B2M-HLA-A) format. TCR activation was quantified using the CellDom Microwell EliSpot platform, where GFP expression and IL-2 secretion signaled productive antigen engagement. Cells from positive wells were harvested, and DriverMap adaptive immune receptor (AIR) bulk and single-cell TCR-Seq assays and subsequent NGS analysis were performed to identify the cognate TCR epitopes.To improve epitope presentation, we introduced a G2C substitution in the G4S linker of the single-chain trimer, which enhanced HLA surface expression and increased Jurkat activation compared with the wild-type construct. MART1-TCRs showed differential reactivity across the MART peptide panel, enabling ranking of functional affinity and identification of cross-reactive peptide candidates. Both the bulk AIR DNA and bulk AIR RNA assays show clonotypes that are expanded in MART1 vs Day 0 (control) or DMSO control. On Day 9, approximately 12 clonotypes are expanded at 250x compared to Day 0 in the MART1-stimulated sample. With the single-cell AIR assay, the MART1-stimulated samples showed overrepresentation of clones, whereas there was no clonal overrepresentation in the DMSO control plate.This end-to-end platform shows TCR-peptide interactions with bulk and & single-cell NGS readout, which could enable identification of therapeutic TCR candidates, and provide a scalable method for characterizing antigen specificity for cancer immunotherapy applications.