The proteasome generates the epitopes presented on human leukocyte antigen (HLA) class I molecules that elicit CD8+ T cell responses. Reports of proteasome-generated spliced epitopes exist, but they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one-third of the entire HLA class I immunopeptidome in terms of diversity and one-fourth in terms of abundance. This pool also represents a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, as well as multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events has implications for immunobiology and autoimmunity theories and may provide a previously untapped source of epitopes for use in vaccines and cancer immunotherapy.

RAW files C1Rmass spectrometry data for immunopeptidome and cell lysate o C1R cell line. File names include mass spectrometry method used.C1R.zipT2 mass spectrometry datamass spectrometry data for immunopeptidome and cell lysate o T2 cell line. File names include mass spectrometry method used.T2.zipGRLCL immunopeptidomemass spectrometry data for immunopeptidome of GRLCL cell line. File names include mass spectrometry method used.GRLCL_immunopeptidome.zipGRLCL lysate mass spectrometry datamass spectrometry data for cell lysate of GRLCL cell line. File names include mass spectrometry method used.GRLCL_lysate.zip