High-throughput TCR-epitope screening using the Jurkat NFAT Reporter-K562 APC cell system on the Microwell ELISpot Platform

Introduction/Rationale: Despite the critical role of T-cell receptors (TCRs) in adaptive immunity, experimental validation of predicted cognate antigens remains a major challenge.

Methods: . For screening TCR-epitope interaction, we developed Jurkat-NFAT-GFP reporter cells expressing TCRs of interest and co-cultured them with K562 antigen-presenting cells (APCs) expressing peptide libraries in a single-chain trimer (peptide–B2M–HLA-A) format. To enhance HLA surface expression, we introduced a G2C mutation in the G4S linker of the single-chain trimer. TCR activation, cell proliferation, and cytotoxicity were monitored using a novel microwell ELISpot assay, where GFP expression signaled functional TCR–peptide–MHC interactions. Cells from GFP+ wells were isolated using CloneXplorer colony picker for NGS to identify cognate epitopes and TCRs. For proof of concept, a library of ~100 peptides (cancer-associated and viral peptides) were short-listed using the DETECT algorithm and expressed in K562 cells, and tested against Jurkat cells expressing CMV, MART1, p53, and Flu TCRs. Microarrays were used to establish tens of thousands of Jurkat/K562 co-cultures for each TCR construct. Peptide identity was determined by sequencing isolated cells to match the expected TCR specificity.

Results: For proof of concept, a library of ~100 peptides (cancer-associated and viral peptides) were short-listed using the DETECT algorithm and expressed in K562 cells, and tested against Jurkat cells expressing CMV, MART1, p53, and Flu TCRs. Microarrays were used to establish tens of thousands of Jurkat/K562 co-cultures for each TCR construct. Peptide identity was determined by sequencing isolated cells to match the expected TCR specificity.

Conclusion: This end-to-end platform enables high-throughput discovery to screen 1000s of peptides, and functional characterization of TCR–peptide–MHC interactions. It has broad applications in cancer immunotherapy and vaccine development.