Confirmation of identified immunogenic epitopes at the single peptide level. (A-B) PBMCs from healthy donors were stimulated with HCMV-derived single peptides. Peptide-specific T-cell frequencies (IFN-γ⁺/CD8⁺) were quantified by flow cytometry. (A) Representative flow cytometric density plots of IFN-γ⁺/CD8⁺ T-cell frequencies after stimulation with a background control (HIV_Gag HLA-A∗03 or HIV-1 p17 HLA-B∗15) or HCMV-derived peptides are shown. (B) Background-corrected peptide-specific T-cell frequencies of PBMCs from healthy donors with HCMV seropositivity (red circles) or seronegativity (blue triangles). PBMCs were also stimulated with pp65 peptides that were presented on the donors' alternative HLA allotype for comparative analysis. Each symbol represents 1 donor. The Mann-Whitney U test and Benjamini-Hochberg procedure for a false-positive discovery rate of <0.2 were used for statistical analysis (∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001). (C) Volcano plot showing the P value (Mann-Whitney U test) and respective log₂-transformed mean-to-mean ratios between healthy donors with HCMV seropositivity and HCMV seronegativity from Figure 4B. Peptides were classified into nonimmunogenic (●; log₂-transformed mean-to-mean ratio < 1.5; P > .05) or immunogenic (▲; log₂-transformed mean-to-mean ratio > 1.5; P < .05) peptides.