Mass spectrometry validation of predicted GVL mHAs for HLA-A∗02:01, B∗35:01, and C∗07:02. (A) Shows representative spectra for heavy-labeled peptide standard for HLA-C∗07:02–binding mHA LPAAYHHH. (B) Shows endogenous LPAAYHHH peptide identified from immunoprecipitated peptide sample from cell line MONOMAC1. (C) Shows all novel identified peptides from cell line U937A2 sample. “Noncumulative population coverage” shows the percentage of DRPs expressing HLA-A∗02:01 within the DISCOVeRY-BMT data set where the recipient expresses the mHA allele and the donor does not. “Cumulative population coverage” shows the output from the greedy algorithm calculating total population coverage by each peptide and the ones preceding it, with a total of 98.8% population coverage by the 10 peptides. (D) Shows all identified peptides from cell line NB4 sample, with a 60.7% cumulative coverage of DRPs expressing HLA-B∗35:01 within the data set by the 3 peptides. (E) Shows all identified peptides from cell line MONOMAC1, with a 78.9% cumulative coverage of HLA-C∗07:02–expressing DRPs within the data set. (F) Shows cumulative coverage by the 16 novel confirmed HLA-A∗02:01–binding mHAs and 1000 simulated sets of 16 peptides from the set of mHAs searched by mass spectrometry. Cumulative coverage by confirmed peptides is shown in blue, whereas each simulated run is shown as an individual gray line. (G) Shows cumulative coverage for the 3 confirmed HLA-B∗35:01–binding mHAs and 1000 simulated sets of 3 peptides. (H) Shows cumulative coverage for the 5 confirmed HLA-C∗07:02–binding mHAs and 1000 simulated sets of 5 peptides. (I) Shows flow cytometry staining from the mHA immunogenicity experiment. “UV only” shows negative control stained with tetramer exposed to UV light with no peptide. “Flu-M15_8-66” shows CD8 T cells cocultured with M1_58-66 pulsed DCs, stained with M1_58-66 tetramer. “UNC-HEXDC-V” shows CD8 T cells cocultured with novel mHA UNC-HEXDC-V stained with UNC-HEXDC-V tetramer.