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.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Neopeptide-based immunotherapy has been recognised as a promising approach for the treatment of cancers. For neopeptides to be recognised by CD8+ T cells and induce an immune response, their binding to human leukocyte antigen class I (HLA-I) molecules is a necessary first step. Most epitope prediction tools thus rely on the prediction of such binding. With the use of mass spectrometry, the scale of naturally presented HLA ligands that could be used to develop such predictors has been expanded. However, there are rarely efforts that focus on the integration of these experimental data with computational algorithms to efficiently develop up-to-date predictors. Here, we present Anthem for accurate HLA-I binding prediction. In particular, we have developed a user-friendly framework to support the development of customisable HLA-I binding prediction models to meet challenges associated with the rapidly increasing availability of large amounts of immunopeptidomic data. Our extensive evaluation, using both independent and experimental datasets shows that Anthem achieves an overall similar or higher area under curve value compared with other contemporary tools. It is anticipated that Anthem will provide a unique opportunity for the non-expert user to analyse and interpret their own in-house or publicly deposited datasets.

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 .

Human islets are subjected to a number of stresses before and during their isolation that may influence their survival and engraftment after transplantation. Apoptosis is likely to be activated in response to these stresses. Apoptosis due to intrinsic stresses is regulated by pro-and antiapoptotic members of the Bcl-2 family. While the role of the Bcl-2 family in apoptosis of rodent islets is becoming increasingly understood, little is known about which of these molecules are expressed or required for apoptosis of human islets. This study investigated the expression of the Bcl-2 family of molecules in isolated human islets. RNA and protein lysates were extracted from human islets immediately postisolation. At the same time, standard quality control assays including viability staining and β-cell content were performed on each islet preparation. Microarrays, RT-PCR, and Western blotting were performed on islet RNA and protein. The prosurvival molecules Bcl-xl and Mcl-1, but not Bcl-2, were highly expressed. The multidomain proapoptotic effector molecule Bax was expressed at higher levels than Bak. Proapoptotic BH3-only molecules were expressed at low levels, with Bid being the most abundant. The proapoptotic molecules BNIP3, BNIP3L, and Beclin-1 were all highly expressed, indicating exposure of islets to oxygen and nutrient deprivation during isolation. Our data provide a comprehensive analysis of expression levels of pro-and antiapoptotic Bcl-2 family members in isolated human islets. Knowledge of which molecules are expressed will guide future research to understand the apoptotic pathways activated during isolation or after transplantation. This is crucial for the design of methods to achieve improved transplantation outcomes.Key words: Human islet transplantation; Bcl-2; Apoptosis INTRODUCTIONThe requirement for islets from more than one donor to reverse diabetes in most recipients is one limitation to the wider application of the procedure. This is due to Type 1 diabetes (T1D) is the result of autoimmune destruction of the insulin-producing pancreatic β-cells losses of islets in the isolation process and following transplantation, when many islets die prior to engraftby cells of the immune system, resulting in lifelong dependence on insulin injections. One approach to corment even in syngeneic models (6). Islets are exposed to many insults during their isolation, including mechanrecting blood glucose levels in T1D is allogeneic human islet transplantation. In this procedure pancreases obtained ical stress, hypoxia, and variations in temperature and collagenase blends (3,5,31,43). After transplantation from organ donors are processed to yield isolated islets by a combination of mechanical and enzymatic digestion other factors have been identified that may influence the viability and function of transplanted islets over followed by density gradient purification. Islets are transplanted into the portal circulation of recipients who the short and long term. These include an instant blood-mediated inflammatory reac...

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This is our response to the Technical Comment by Rolfset al.where we point out errors in their reanalysis of our data.