Stitchr TCR validation | LC13 | 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 LC13 TCR recognises the peptide FLRGRAYGL restricted by HLA-B*08:01. The HLA-matched line used here was MDA-MB-463, the HLA-mismatched line was Karpas-299.