ABSTRACT: The majority of peptides presented by MHC class I results from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides for MHC class I loading non-canonically. We used an im-munopeptidomics workflow combined with a prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect proteasomal processing of immune epitopes presented by A549 cells. Treatment of A549 cells with IFNγ enhanced proteasomal cleavage probability of MHC class I ligands for both, the constitutive proteasome and the im-munoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated HLA allotype-specific proteasomal cleavage probabili-ties for MHC class I ligands, peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. Peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases. The mass spectrometry raw files used in this study were originally produced for a previous publication, where they were utilized to address the influence of cigarette smoke on the antiviral T-cell immune response. Here, we have reanalyzed the data to investigate processing of MHC class I ligands by c20S or i20S, respectively. Original publication: Chen, J.; Wang, X.; Schmalen, A.; Haines, S.; Wolff, M.; Ma, H.; Zhang, H.; Stoleriu, M.G.; Nowak, J.; Nakayama, M.; et al. Antiviral CD8(+) T cell immune responses are impaired by cigarette smoke and in COPD. Eur Respir J 2023, doi:10.1183/13993003.01374-2022.
Adrian Schmalen 
