Stitchr TCR validation | KFJ5 | rep2

Validate the antigenic specificity of TCRs produced by Stitchr from published V/J/CDR3 information, by cloning the sequences produced into CD8+ TCR-deficient Jurkat cells. Target cells either positive or negative for the appropriate HLA allele were stained with CFSE, peptide pulsed with the known epitope of the TCR, then co-cultured overnight. TCR activity was assessed the following day by gating on live Jurkats (CFSE-negative, 7AAD-negative) and measuring Jurkat activation by taking the % of cells CD69+ (APC), CD62L negative (PE). The KFJ5 TCR recognises the peptide APRGPHGGAASGL restricted by HLA-B*07:02. The HLA-matched line used here was Karpas-299, the HLA-mismatched line was SK-N-SH.