The EBNA1-specific CD4⁺ T-cell clone A4. E116 responds to EBNA1 peptide pools containing the EBNA1 peptide sequence 513-527. (A) Scheme of EBNA1 amino acid sequence represented by overlapping peptide pools (EBNA1, all EBNA1 peptides; pool I, aa's 400-461; pool II, aa's 452-508; pool III, aa's 499-548; pool IV, aa's 539-593; and pool V, aa's 584-641). (B) ELISPOT assay indicating spots per 10⁴ cells in response to dilutions of peptide 513-527, and a noncognate peptide sequence as a negative control. Error bars indicate standard deviations of duplicates. (C) Intracellular cytokine staining assay for IFNγ in response to EBNA1 peptide pools, known CD8⁺ T-cell epitopes from CMV and EBV (Table S1), medium alone, and Staphylococcus enterotoxin B (SEB); note significantly positive response to the pool of all EBNA1 peptides and pool III, which both contain peptide 515-529. (D) CFSE proliferation assay in response to EBNA1 peptide pools. The data are representative of 3 independent experiments.