Bioinformatic identification of HLA-I peptides. (A,B) The distribution of identified canonical and cryptic peptide sequences restricted by HLA-A∗03 (A) and HLA-B∗15 (B) including an overlap of HLA-A∗03 restricted, canonical peptides published in the report by Lübke et al,¹³ which relies on 1 of the data sets examined in our study (A). (C) Identified and selected canonical and cryptic peptides for the haplotypes HLA-A∗03:01 and HLA-B∗15:01. (D,E) Box plot showing the distributions of netMHC ranks and translational activity measured by Ribo-seq of all peptides selected for testing (selected) and all other peptides identified by MHC-I immunopeptidomics (filtered). (D) The default cutoffs for strong binders (cutoff = 0.5) and binders (cutoff = 2) of the netMHC score are indicated. (F) Validation of immunogenicity of the top ranked 14 peptides vs the bottom ranked 14 peptides used for pool testing. Background-corrected peptide-specific T-cell frequencies of PBMCs from healthy donors with HCMV-seropositive HLA-B∗15⁺ stimulated with peptide pools A, B, F, and G (as described in Figure 3C, HLA-B∗15 canonical) are shown. Each symbol represents 1 donor. Mann-Whitney U test was used for statistical analysis (∗∗P < .01). can, canonical; cry, cryptic.