Objective. The association of HLA-B27 with spondyloarthropathy is one of the strongest documented for any autoimmune disease. A common hypothesis for this association is the arthritogenic peptide concept. This dictates that differences in the peptide binding preferences of disease-associated and non-diseaseassociated HLA-B27 allotypes underlie the presentation of bacterial and self-peptides, leading to crossreactive T cell immunity and subsequent autoimmune attack of affected tissues. The aim of this study was to analyze and compare self-peptides from 8 HLA-B27 allotypes, to increase existing data sets of HLA-B27 ligands, to refine and compare their consensus-binding motifs, and to reveal similarities and differences in the peptide repertoire of the HLA-B27 subtypes.Methods. Qualitative differences in the peptides bound to the 8 most frequent HLA-B27 subtypes were determined by tandem mass spectrometry, and quantitative changes in allelic binding specificities were determined by highly sensitive and targeted multiple reaction monitoring mass spectrometry.Results. We identified >7,500 major histocompatibility complex class I peptides derived from the 8 most common HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09). We describe individual binding motifs for these alleles for the 9-12-mer ligands. The peptide repertoires of these closely related alleles showed significant overlap. Allelic polymorphisms resulting in changes in the amino acid composition of the antigenbinding cleft manifested largely as quantitative changes in the peptide cargo of these molecules.Conclusion. Absolute binding preferences of HLA-B27 allotypes do not explain disease association. The arthritogenic peptide theory needs to be reassessed in terms of quantitative changes in self-peptide presentation, T cell selection, and altered conformation of bound peptides.

SummaryAntigen-recognition by CD8+ T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Recent studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasome-mediated splicing of non-contiguous regions of proteins to form novel peptide antigens. Here we used high-resolution mass-spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of interferon-γ. In total, we identified more than 60,000 peptides from a single patient derived cell line (LM-MEL-44) and demonstrated that interferon-γ induced marked changes in the peptidome with an overlap of only ∼50% between basal and treated cells. Around 6-8% of the peptides were identified as cis-spliced peptides, and 2213 peptides (1827 linear, 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive and interferon-γ induced peptidome. We next examined additional HLA-matched patient derived cell lines to investigate how frequently these peptides were identified and found that a high proportion of both linear and spliced peptides were conserved between individual patient tumors, drawing on data amassing to over 100,000 peptide sequences from these extended data sets. Moreover, several of these peptides showed in vitro immunogenicity across multiple melanoma patients. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that can be exploited therapeutically and suggest that spliced peptides are a major new class of tumor antigens.

Antigen-recognition by CD8 + T cells is governed by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Recent studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides is generated through proteasomemediated splicing of non-contiguous regions of proteins to form novel peptide antigens. Here we used high-resolution mass-spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of interferon-γ. In total, we identified more than 60,000 peptides from a single patient derived cell line (LM-MEL-44) and demonstrated that interferon-γ induced marked changes in the peptidome with an overlap of only ~50% between basal and treated cells. Around 6-8% of the peptides were identified as cisspliced peptides, and 2213 peptides (1827 linear, 386 cis-spliced peptides) were derived from known melanoma-associated antigens. These peptide antigens were equally distributed between the constitutive and interferon-γ induced peptidome. We .