The EBNA3A-specific TCR can be matched to its cognate peptide. (A) The EBNA3A-specific TCR (supplemental Figure 1B-E) was cloned from the plasmid library, and expressed in Jurkat∆αβ reporter cells by lentiviral transduction. Expression of the TCR on the reporter cells was verified by staining with EBNA3A tetramer, anti-Vβ5-1, and anti-CD3 antibodies. (B) Specificity of the EBNA3A-specific TCR for cognate antigen was confirmed by detection of IL-2 secretion selectively after coculture of reporter cells with B721.221-A*24:02 cells presenting EBNA3A peptide compared with control HLA-A*24:02-restricted peptide BRLF1 (P = .014; 2-sample t-test with Welch’s correction from 2 technical replicates). Phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation was used as a positive control.