3. Workflow Overview

This Jurkat-K562 system enables efficient screening of synthetic peptide libraries or minigene-encoded epitopes in the context of specific HLA alleles. The platform is particularly well-suited for validating TCR specificity by co-expressing target TCRs in reporter Jurkat cells and peptide libraries in K562-HLA APCs. Below is an outline of the system’s standard workflow:

1. Generate a TCR-Specific Reporter Cell Line
   Use the DriverMap scAIR TCR Chain Pairing Assay to obtain TRA and TRB full-length paired receptor sequences. Clone the target TCR gene (TRA and TRB) into a cDNA expression vector and transduce it into Cellecta’s Jurkat NFAT-GFP Reporter Cell Line (Cat# ZJUR-T-Ble).

2. Prepare Antigen-Presenting K562-HLA Cells:

• Option 1: Use pre-made K562-HLA-A cells—such as Cat# YK562-MH-A0201-P, YK562-MH-A0201-BFP, or YK562-CMV-A0201-RFP—and load them (pulse) with any synthetic peptide set.
• Option 2: Clone the sequences encoding desired peptides into Cellecta’s cDNA expression vectors and transduce them into K562-HLA cells to express peptide minigenes endogenously.

3. Co-Culture Jurkat Reporter and K562-HLA Cells
   Mix the TCR-expressing Jurkat NFAT-GFP reporter cells (from Step 1) with the antigen-presenting K562-HLA cells (from Step 2).

4. Detect TCR–Epitope Interactions
   Specific TCR–peptide–MHC interactions trigger GFP expression in Jurkat reporter cells, which can be detected via flow cytometry. Alternatively, the TCR-expressing Jurkat cells can be validated by staining with labeled peptide–MHC tetramers or dextramers to assess antigen specificity.