A subset of HLA-I peptides are not genomically templated: Evidence for cis- and trans-spliced peptide ligands

Abstract

The diversity of peptides displayed by class I human leukocyte antigen (HLA) plays an essential role in T cell immunity. The peptide repertoire is extended by various posttranslational modifications, including proteasomal splicing of peptide fragments from distinct regions of an antigen to form nongenomically templated cis-spliced sequences. Previously, it has been suggested that a fraction of the immunopeptidome constitutes such cis-spliced peptides; however, because of computational limitations, it has not been possible to assess whether trans-spliced peptides (i.e., the fusion of peptide segments from distinct antigens) are also bound and presented by HLA molecules, and if so, in what proportion. Here, we have developed and applied a bioinformatic workflow and demonstrated that trans-spliced peptides are presented by HLA-I, and their abundance challenges current models of proteasomal splicing that predict cis-splicing as the most probable outcome. These trans-spliced peptides display canonical HLA-binding sequence features and are as frequently identified as cis-spliced peptides found bound to a number of different HLA-A and HLA-B allotypes. Structural analysis reveals that the junction between spliced peptides is highly solvent exposed and likely to participate in T cell receptor interactions. These results highlight the unanticipated diversity of the immunopeptidome and have important implications for autoimmunity, vaccine design, and immunotherapy.

Proteogenomic discovery of cancer antigens: Neoantigens and beyond

“…have leveraged proteogenomics and reported that, in hematological and fibroblast cell lines, spliced peptides accounted for up to one‐third of the entire HLA class I peptide repertoire . A following study showed that both cis‐ and trans‐splicing give rise to spliced HLA ligands . These results imply that antigen processing contributes to the creation of new peptides using a limited gene resource, significantly expanding the range of the immunopeptidome for T‐cell surveillance.…”

“…39 A following study showed that both cis-and trans-splicing give rise to spliced HLA ligands. 40 These results imply that antigen processing contributes to the creation of new peptides using a limited gene resource, significantly expanding the range of the immunopeptidome for T-cell surveillance. Lastly, such a large amount of spliced peptide presentation raises a question: do they give rise to neoantigens along with corresponding somatic mutations?…”

Mass Spectrometry-Based Identification of MHC-Associated Peptides

“…The exact source of targeted tissue-specific antigens and MHC expression pathways is still unclear. Moreover, there are many potential sources of MHC-associated antigens such as classical MHC pathways, pioneer translation products (PTPs) [41], defective ribosomal products (DRiPs) [42], cisand trans-spliced peptide [43], non-canonical reading frames [44], etc. Therefore, the combination of high-resolution MS and MHC peptide isolation methods (MAE and IP) is not only convenient for accurate identification of MHC peptide neoantigens; it also indirectly contributes to detecting the source of origin at a genomic location and to tracing the exact mechanism of MHC peptide expression, which is a pressing issue [40].…”

HIPs and HIP-reactive T cells

“…Candidate spliced peptide sequences were added to a custom database, and data were then searched against this database using a standard search algorithm. Faridi et al used a similar workflow to identify both cis‐ and trans‐spliced peptides .…”