Figure 1.

T cell responsiveness to synthetic EBV peptide antigens in healthy, EBV-seropositive donor IP1. CD8⁺ T cells were isolated from PBMC and were analyzed in IFN-γ ELISPOT assays for reactivity against EBV peptide epitopes known to bind to HLA-A2.1 (BMLF-1 280–288, LMP-2 329–337, LMP-2A 426–434, EBNA-2 67–76, EBNA-3 596–604, EBNA-6 284–293) or HLA-B7 (EBNA-3A 379–387, EBNA-3C 881–889). Autologous immature DC served as APC. Control wells contained CD8⁺ T cells with nonpeptide-pulsed DC or CD8⁺ T cells with DC loaded with the HLA-A2.1-restricted CTL epitopes Flu MP 58–66 (positive control) or HIV nef 180–189 (negative control). For these latter controls and for experimental EBV-derived epitopes, DC were loaded with 10 μg/ml concentrations of the indicated peptides. After a culture period of 20 h, IFN-γ spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD per 10⁵ CD8⁺ T lymphocytes initially seeded per well. The numbers of peptide-responsive T cells per 10⁵ CD8⁺ T lymphocytes are calculated by subtraction of mean spot numbers induced by DC alone from mean spot numbers induced by peptide-loaded DC (∗ indicate significant results, i.e., P < 0.05). Results were confirmed in three independent experiments.