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MHC Class I Immunopeptidome: Past, Present, and Future

Open AccessPublished:April 04, 2022DOI:https://doi.org/10.1016/j.mcpro.2022.100230

      Highlights

      • Concise history of the discoveries leading to the molecular explanation for the phenomenon of the MHC class I–restricted nature of T-cell recognition.
      • Historical review of how MS became a critical technique for defining MHC class I–associated peptides and understanding how peptides are generated from proteins biosynthesized by the antigen-presenting cell.
      • Critical review of recent findings linking the translatome to the MHC class I immunopeptidome and the controversy regarding contribution of proteasome-mediated peptide splicing to the immunopeptidome.
      • Speculative discussion of the future contributions of MS to understanding the generation of the MHC class I immunopeptidome.

      Abstract

      In the 35 years since the revelation that short peptides bound to major histocompatibility complex class I and II molecules are the secret of the major histocompatibility complex–restricted nature of T-cell recognition, there has been enormous progress in characterizing the immunopeptidome, the repertoire of peptide presented for immunosurveillance. Here, the major milestones in the journey are marked, the contribution of proteasome-mediated splicing to the immunopeptidome is discussed, and exciting recent findings relating the immunopeptidome to the translatome revealed by ribosome profiling (RiboSeq) is detailed. Finally, what is needed for continued progress is opined about, which includes the infusion of talented young scientists into the antigen-processing field, currently undergoing a renaissance; thanks in part to the astounding success of T-cell–based cancer immunotherapy.

      Graphical Abstract

      Keywords

      Abbreviations:

      DRiP (generation of ribosomal product), ER (endoplasmic reticulum), HLA (human leukocyte antigen), IAV (influenza A virus), IFN (interferon), MAP (MHC-associated peptide), MHC (major histocompatibility complex), NP (nucleoprotein), nuORF (novel unannotated ORF), T Ag (tumor antigen), TCR (T-cell receptor)
      It is a great pleasure to contribute to this remarkable collection of reviews. As a card-carrying immunologist and one of the few non–mass spectrometrists among the August authors, I have tasked myself with succinctly recounting the origins of the immunopeptidome from when it was just a gleam in the eyes of Emil Unanue, Alain Townsend, Stan Nathenson, Hans-Georg Rammensee, Vic Engelhard, and Don Hunt, whose groups each made critical contributions to establishing the basic rules of antigen presentation. But first, a definition: the immunopeptidome (a term I may have coined (
      • Istrail S.
      • Florea L.
      • Halldorsson B.V.
      • Kohlbacher O.
      • Schwartz R.S.
      • Yap V.B.
      • Yewdell J.W.
      • Hoffman S.L.
      Comparative immunopeptidomics of humans and their pathogens.
      )) is the set of peptides presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells to enable T-cell immunosurveillance. I will also use this bully pulpit to highlight recent exciting findings linking the translatome to the class I immunopeptidome and finally to speculate about what the future might bring.

      Immunopeptidome Past

      The roots of the immunopeptidome lie in studying the genetics of tissue transplantation, which revealed the MHC as a principal locus governing transplant survival, first in mice (H-2) (
      • Gorer P.A.
      The genetic and antigenic basis of tumour transplantation.
      ), and then in man (human leukocyte antigen [HLA]) (
      • Dausset J.
      Iso-leuco-anticorps.
      ). Genetic loci led to gene products in the form of MHC class I and II proteins that could be studied first using serum alloantibodies that could be raised in inbred mice and recovered from multiparous women, and subsequently with monoclonal antibodies, which in simpler times, were generously shared gratis widely in the scientific community by their creators (
      • Ozato K.
      • Mayer N.
      • Sachs D.H.
      Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens.
      ,
      • Barnstable C.J.
      • Bodmer W.F.
      • Brown G.
      • Galfre G.
      • Milstein C.
      • Williams A.F.
      • Ziegler A.
      Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.
      )

      Setting the Stage: CD4+ T Cells Recognize MHC Class II–Bound Peptides

      T-helper cell studies performed using inbred guinea pigs (yes, guinea pigs) provided the first glimpse of the molecular function of MHC molecules (
      • Rosenthal A.S.
      • Shevach E.M.
      Function of macrophages in antigen recognition by Guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes.
      ). Activation of T cells specific for random synthetic peptides known to elicit antibody responses required genetic identity between the MHC of the antigen-presenting cell (macrophages) and the responding T cell, extending findings demonstrating that MHC class II genes controlled the T-helper cell response to synthetic peptides (
      • Benacerraf B.
      • McDevitt H.O.
      Histocompatibility-linked immune response genes.
      ). The concept of MHC restriction of T-cell responses fully flowered with Zinkernagel and Doherty's demonstration that T cells would only lyse virus-infected cells that genetically matched the class I genes of the host responding animal (
      • Zinkernagel R.M.
      • Doherty P.C.
      Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system.
      ). Antibodies specific for class II or class I molecules blocked, respectively, helper (
      • Shevach E.M.
      • Rosenthal A.S.
      Function of macrophages in antigen recognition by Guinea pig T lymphocytes. II. Role of the macrophage in the regulation of genetic control of the immune response.
      ) and killer T-cell (
      • Koszinowski U.
      • Ertl H.
      Lysis mediated by T cells and restricted by H-2 antigen of target cells infected with vaccinia virus.
      ) recognition of antigen-presenting cells, demonstrating the direct participation of these MHC gene products in the phenomenon of MHC restriction.
      The discovery of MHC control of antibody/helper T-cell responses using unstructured random and semirandom synthetic peptides provided a critical clue regarding the molecular explanation for MHC restriction, leading to the proposal that T cells recognize peptides bound to MHC class II molecules (
      • Benacerraf B.
      A hypothesis to relate the specificity of T lymphocytes and the activity of I region-specific Ir genes in macrophages and B lymphocytes.
      ). The first step in linking artificial synthetic peptides to natural antigens was the demonstration that peptides from “Sigma antigens,” that is, inexpensive purified proteins (that could be obtained from the Sigma Chemical Company) (e.g., hen egg lysozyme, myoglobin, ovalbumin) can induce antibody responses based on their abilities to activate both B cells and T-helper cells (
      • Mozes E.
      • Shearer G.M.
      • Maron E.
      • Arnon R.
      • Sela M.
      Cellular studies of the genetic control of immune response toward the loop region of lysozyme.
      ). Fine-mapping studies established that peptide fragments (
      • Solinger A.M.
      • Ultee M.E.
      • Margoliash E.
      • Schwartz R.H.
      T-lymphocyte response to cytochrome c. I. Demonstration of a T-cell heteroclitic proliferative response and identification of a topographic antigenic determinant on pigeon cytochrome c whose immune recognition requires two complementing major histocompatibility complex-linked immune response genes.
      ) and synthetic peptides as short as 10 residues could activate T cells induced by immunizing mice with a purified peptide fragment. This paved the way to the initial demonstration of peptide binding to class II molecules (
      • Babbitt B.P.
      • Allen P.M.
      • Matsueda G.R.
      • Haber E.
      • Unanue E.R.
      Binding of immunogenic peptides to Ia histocompatibility molecules.
      ).

      CD8+ T Cells—Class I in the Spotlight

      Because of the basic differences between class I and class II antigen presentation (
      • Yewdell J.W.
      • Bennink J.R.
      The binary logic of antigen processing and presentation to T cells.
      ), defining relevant class I–associated antigens for CD8+ T-cell recognition required cells expressing source proteins, precluding (though tricks (
      • Moore M.W.
      • Carbone F.R.
      • Bevan M.J.
      Introduction of soluble protein into the class I pathway of antigen processing and presentation.
      ) provide a workaround) the use of Sigma antigens. Because of the limited genetic engineering available, it was not possible to identify self-antigens (minor H or tumor antigens [T Ags]), limiting experimental approaches limited to viral proteins. The first viral gene product identified as a class I–restricted antigen was simian virus 40 T Ag, which was shown to be responsible for tumor rejection and T-cell killing of simian virus 40–transformed cells (
      • Lewis Jr., A.M.
      • Rowe W.P.
      Studies of nondefective Adenovirus 2-Simian virus 40 hybrid viruses. 8. Association of Simian virus 40 transplantation antigen with a specific region of the early viral genome.
      ,
      • Tevethia S.S.
      • Flyer D.C.
      • Tjian R.
      Biology of simian virus 40 (SV40) transplantation antigen (TrAg): VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein).
      ,
      • Tevethia S.S.
      • Greenfield R.S.
      • Flyer D.C.
      • Tevethia M.J.
      SV40 transplantation antigen: Relationship to SV40-specific proteins.
      ,
      • Trinchieri G.
      • Aden D.P.
      • Knowles B.B.
      Cell-mediated cytotoxicity to SV40-specific tumour-associated antigens.
      ). Indeed, the initial evidence (
      • Tevethia S.S.
      • Tevethia M.J.
      • Lewis A.J.
      • Reddy V.B.
      • Weissman S.M.
      Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen.
      ,
      • Reddy V.B.
      • Tevethia S.S.
      • Tevethia M.J.
      • Weissman S.M.
      Nonselective expression of simian virus 40 large tumor antigen fragments in mouse cells.
      ) regarding the involvement of intracellular degradation to the class I peptidome came from the finding that an unstable fragment of T Ag is fully antigenic. Notably, the same proteins were not immunogenic, which, retrospectively, was probably the first hint for the importance of metabolically stable proteins in T-cell “crosspriming” (
      • Bevan M.J.
      Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay.
      ,
      • Bevan M.J.
      Cross-priming.
      ), only firmly established 20 years later (
      • Norbury C.C.
      • Basta S.
      • Donohue K.B.
      • Tscharke D.C.
      • Princiotta M.F.
      • Berglund P.
      • Gibbs J.
      • Bennink J.R.
      • Yewdell J.W.
      CD8+ T cell cross-priming via transfer of proteasome substrates.
      ).
      T Ag turned out to be a harbinger for the general phenomenon of CD8+ T-cell recognition of internal viral proteins, which was first inferred from mapping the specificity of influenza A virus (IAV) strain–specific T-cell clones with reassortant IAV, which revealed recognition of a viral polymerase and nucleoprotein (NP) (
      • Bennink J.R.
      • Yewdell J.W.
      • Gerhard W.
      A viral polymerase involved in recognition of influenza virus-infected cells by a cytotoxic T-cell clone.
      ,
      • Townsend A.R.
      • Skehel J.J.
      • Taylor P.M.
      • Palese P.
      Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone.
      ). Expression of IAV genes in recombinant vaccinia viruses soon revealed that all IAV gene products can be recognized by CD8+ T cells (
      • Yewdell J.
      • Bennink J.
      • Smith G.
      • Moss B.
      Use of recombinant vaccinia viruses to examine cytotoxic T lymphocyte recognition of individual viral proteins.
      ). CD8+ T-cell recognition of cloned IAV NP fragments (
      • Townsend A.R.M.
      • Gotch F.M.
      • Davey J.
      Cytotoxic T cells recognize fragments of the influenza nucleoprotein.
      ) led to the 1986 discovery that peptides as short as 11 residues sensitized cells for T-cell recognition (
      • Townsend A.R.M.
      • Rothbard J.
      • Gotch F.M.
      • Bahadur G.
      • Wraith D.
      • McMichael A.J.
      The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides.
      ), thus unifying T-cell recognition of class I and class II molecules around the central principle of MHC molecules as peptide receptors that present the products of protein degradation. These seminal findings explained an otherwise puzzling series of earlier observations demonstrating that cyanogen bromide–generated peptide fragments and synthetic peptides as short as 21 residues from viral glycoproteins could stimulate antiviral CD8+ T cells in vitro (
      • Wabuke-Bunoti M.A.
      • Fan D.P.
      Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes.
      ,
      • Wabuke-Bunoti M.A.
      • Fan D.P.
      • Braciale T.J.
      Stimulation of anti-influenza cytolytic T lymphocytes by CNBr cleavage fragments of the viral hemagglutinin.
      ,
      • Guertin D.P.
      • Fan D.P.
      Stimulation of cytolytic T cells by isolated viral peptides and HN protein coupled to agarose beads.
      ,
      • Wabuke-Bunoti M.A.
      • Taku A.
      • Fan D.P.
      • Kent S.
      • Webster R.G.
      Cytolytic T lymphocyte and antibody responses to synthetic peptides of influenza virus hemagglutinin.
      ). Within a year (
      • Bjorkman P.J.
      • Saper M.A.
      • Samraoui B.
      • Bennett W.S.
      • Strominger J.L.
      • Wiley D.C.
      The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.
      ), the crystal structure of HLA-A2 firmly established the molecular basis for the phenomenon of MHC restriction: class I molecules possess a highly polymorphic-binding groove that binds short peptides in a manner that should enable T-cell receptor (TCR) contact with both the peptide and bounding alpha helices. Confirmation of this model came nearly a decade later with the publication of a TCR–MHC class I structure (
      • Garcia K.C.
      • Degano M.
      • Stanfield R.L.
      • Brunmark A.
      • Jackson M.R.
      • Peterson P.A.
      • Teyton L.
      • Wilson I.A.
      An àá T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex.
      ), fittingly in the same year that Doherty and Zinkernagel won the Nobel Prize in medicine and physiology for their 1974 landmark article.

      Milestones in Defining Natural MHC I Peptides

      The next major step toward defining the immunopeptidome was determining the actual foreign and self-peptides present in the class I–binding groove. A remarkable series of articles (
      • Falk K.
      • Rötzschke O.
      • Rammensee H.G.
      Cellular peptide composition governed by major histocompatibility complex class I molecules.
      ,
      • Rötzschke O.
      • Falk K.
      • Wallny H.J.
      • Faath S.
      • Rammensee H.G.
      Characterization of naturally occurring minor histocompatibility peptides including H-4 and H-Y.
      ,
      • Rötzschke O.
      • Falk K.
      • Deres K.
      • Schild H.
      • Norda M.
      • Metzger J.
      • Jung G.
      • Rammensee H.G.
      Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells.
      ,
      • Wallny H.J.
      • Rammensee H.G.
      Identification of classical minor histocompatibility antigen as cell-derived peptide.
      ) used classical biochemical methods to extract low–molecular weight peptides with trifluoroacetic acid and separate them by HPLC with organic solvents. Peptides in fractions were then used to sensitize target cells for CD8+ T-cell lysis or activation. By comparing natural peptide elution with a nested series of peptides containing a known T-cell determinant, or epitope, the natural sequence of the peptide could be inferred. Note that epitope was coined by Niels Jerne to describe antigen surface residues that contact antibodies (
      • Jerne N.K.
      Immunological speculations.
      ). Since most antigenic peptides are at least partially buried in proteins, endotope or cryptotope (as Jerne originally suggested (
      • Jerne N.K.
      Immunological speculations.
      )) is at least as accurate.
      This approach revealed that the original IAV NP peptide identified is presented as a 9-mer, which not coincidentally binds class I with the highest affinity among NP-derived peptides. Simultaneously, radiolabeling of vesicular stomatitis virus–infected cells with [3H]-amino acids was used in conjunction with HPLC and peptide sequencing to identify a natural viral 8-mer peptide bound to antibody-purified class I molecules (
      • Van Bleek G.M.
      • Nathenson S.G.
      Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule.
      ). Surprisingly, this appears to be the sole published example of using metabolic radiolabeling for peptide identification and characterization; it is well worth revisiting this technique to delineate the kinetics of peptide generation from new versus old proteins; though from bitter experience, this is easier written than done!.
      These initial studies established the critical point that recovering antigenic peptides requires the expression of class I molecules that bind the peptides with reasonably high affinity (KD in the neighborhood of 10–100 nM). Further work established that oligopeptides not bound to class I molecules are rapidly degraded (
      • Reits E.
      • Griekspoor A.
      • Neijssen J.
      • Groothuis T.
      • Jalink K.
      • van Veelen P.
      • Janssen H.
      • Calafat J.
      • Drijfhout J.W.
      • Neefjes J.
      Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I.
      ), though there are exceptions (
      • Lev A.
      • Takeda K.
      • Zanker D.
      • Maynard J.C.
      • Dimberu P.
      • Waffarn E.
      • Gibbs J.
      • Netzer N.
      • Princiotta M.F.
      • Neckers L.
      • Picard D.
      • Nicchitta C.V.
      • Chen W.
      • Reiter Y.
      • Bennink J.R.
      • et al.
      The exception that reinforces the rule: Crosspriming by cytosolic peptides that escape degradation.
      ). Pragmatically, the most important discovery in this set of articles was the revelation of simple class I allomorph-specific peptide-binding motifs (“anchor residues”) from sequencing of peptide pools recovered from antibody-collected class I molecules (
      • Falk K.
      • Rötzschke O.
      • Stevanović S.
      • Jung G.
      • Rammensee H.G.
      Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules.
      ). This was the first and most critical step to generate the ever-improving algorithms now used to predict class I–binding peptides, an essential step in filtering data to identify valid peptides in contemporary mass spectrometry (MS) characterization of the immunopeptidome.
      Just a year after the publication of peptide-binding motifs, the pioneering MS study to characterize host cell class I–bound (
      • Henderson R.A.
      • Michel H.
      • Sakaguchi K.
      • Shabanowitz J.
      • Appella E.
      • Hunt D.F.
      • Engelhard V.H.
      HLA-A2.1-associated peptides from a mutant cell line: A second pathway of antigen presentation.
      ) and class II–bound (
      • Hunt D.F.
      • Michel H.
      • Dickinson T.A.
      • Shabanowitz J.
      • Cox A.L.
      • Sakaguchi K.
      • Appella E.
      • Grey H.M.
      • Sette A.
      Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad.
      ) peptide ligands was published. This was followed by the first MS characterization of viral peptides (
      • Herr W.
      • Ranieri E.
      • Gambotto A.
      • Kierstead L.S.
      • Amoscato A.A.
      • Gesualdo L.
      • Storkus W.J.
      Identification of naturally processed and HLA-presented Epstein-Barr virus peptides recognized by CD4(+) or CD8(+) T lymphocytes from human blood.
      ,
      • Planz O.
      • Dumrese T.
      • Hülpüsch S.
      • Schirle M.
      • Stevanovic S.
      • Stitz L.
      A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.
      ,
      • van Els C.A.
      • Herberts C.A.
      • van der Heeft E.
      • Poelen M.C.
      • van Gaans-van den Brink J.A.
      • van der Kooi A.
      • Hoogerhout P.
      • Jan ten Hove G.
      • Meiring H.D.
      • de Jong A.P.
      A single naturally processed measles virus peptide fully dominates the HLA-A∗0201-associated peptide display and is mutated at its anchor position in persistent viral strains.
      ), then the initial description of the virus-infected cell immunopeptidome (
      • Hickman H.D.
      • Luis A.D.
      • Bardet W.
      • Buchli R.
      • Battson C.L.
      • Shearer M.H.
      • Jackson K.W.
      • Kennedy R.C.
      • Hildebrand W.H.
      Cutting edge: class I presentation of host peptides following HIV infection.
      ), which revealed major changes to the self-immunopeptidome. This phenomenon largely remains to be mechanistically dissected. It is likely related to findings (
      • Prasad S.
      • Starck S.R.
      • Shastri N.
      Presentation of cryptic peptides by MHC class I is enhanced by inflammatory stimuli.
      ,
      • Zanker D.J.
      • Oveissi S.
      • Tscharke D.C.
      • Duan M.
      • Wan S.
      • Zhang X.
      • Xiao K.
      • Mifsud N.A.
      • Gibbs J.
      • Izzard L.
      • Dlugolenski D.
      • Faou P.
      • Laurie K.L.
      • Vigneron N.
      • Barr I.G.
      • et al.
      Influenza A virus infection induces viral and cellular defective ribosomal products encoded by alternative reading frames.
      ) that viral infections alter translation to favor the generation of ribosomal products (DRiPs) from host mRNAs. In 1996, my colleagues and I proposed that DRiPs, rapidly degraded misfolded nascent proteins, account for the extremely rapid generation of peptides from ostensibly highly stable viral proteins (
      • Yewdell J.W.
      • Anton L.C.
      • Bennink J.R.
      Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules?.
      ). DRiPs appear to be the source of the vast majority of viral peptides, as elegantly shown by MS characterization of the immunopeptidomes of vaccinia and IAV-infected cells (
      • Wu T.
      • Guan J.
      • Handel A.
      • Tscharke D.C.
      • Sidney J.
      • Sette A.
      • Wakim L.M.
      • Sng X.Y.X.
      • Thomas P.G.
      • Croft N.P.
      • Purcell A.W.
      • La Gruta N.L.
      Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses.
      ,
      • Croft N.P.
      • Smith S.A.
      • Wong Y.C.
      • Tan C.T.
      • Dudek N.L.
      • Flesch I.E.
      • Lin L.C.
      • Tscharke D.C.
      • Purcell A.W.
      Kinetics of antigen expression and epitope presentation during virus infection.
      ,
      • Croft N.P.
      • Purcell A.W.
      • Tscharke D.C.
      Quantifying epitope presentation using mass spectrometry.
      ).
      MS played a key role in discovering the presentation of peptides with post-translational modifications (
      • Engelhard V.H.
      • Altrich-Vanlith M.
      • Ostankovitch M.
      • Zarling A.L.
      Post-translational modifications of naturally processed MHC-binding epitopes.
      ), which include Ser/Thr phosphorylation (recently shown to be bioinformatically predictable (
      • Solleder M.
      • Guillaume P.
      • Racle J.
      • Michaux J.
      • Pak H.S.
      • Müller M.
      • Coukos G.
      • Bassani-Sternberg M.
      • Gfeller D.
      Mass spectrometry based immunopeptidomics leads to robust predictions of phosphorylated HLA class I ligands.
      )), Asn deamidation during removal of N-linked glycans by peptide–N-glycanase (
      • Ostankovitch M.
      • Altrich-Vanlith M.
      • Robila V.
      • Engelhard V.H.
      N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase.
      ), N-terminal acetylation, Cys bound to Cys, glutathione (
      • Trujillo J.A.
      • Croft N.P.
      • Dudek N.L.
      • Channappanavar R.
      • Theodossis A.
      • Webb A.I.
      • Dunstone M.A.
      • Illing P.T.
      • Butler N.S.
      • Fett C.
      • Tscharke D.C.
      • Rossjohn J.
      • Perlman S.
      • Purcell A.W.
      The cellular redox environment alters antigen presentation.
      ), and other S-reactive compounds (
      • Trujillo J.A.
      • Croft N.P.
      • Dudek N.L.
      • Channappanavar R.
      • Theodossis A.
      • Webb A.I.
      • Dunstone M.A.
      • Illing P.T.
      • Butler N.S.
      • Fett C.
      • Tscharke D.C.
      • Rossjohn J.
      • Perlman S.
      • Purcell A.W.
      The cellular redox environment alters antigen presentation.
      ,
      • Chen W.
      • Yewdell J.W.
      • Levine R.L.
      • Bennink J.R.
      Modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class I-restricted viral determinants.
      ). Cys modification is probably the most prevalent and relevant modification, affecting up to a third of Cys-containing peptides, constituting 5 to 10% of the immunopeptidome (
      • Trujillo J.A.
      • Croft N.P.
      • Dudek N.L.
      • Channappanavar R.
      • Theodossis A.
      • Webb A.I.
      • Dunstone M.A.
      • Illing P.T.
      • Butler N.S.
      • Fett C.
      • Tscharke D.C.
      • Rossjohn J.
      • Perlman S.
      • Purcell A.W.
      The cellular redox environment alters antigen presentation.
      ). Any of these modifications can abrogate recognition by T cells and/or induce T cells specific for the modified peptide. Post-translational peptide modifications include covalent attachment to drugs (
      • Ostrov D.A.
      • Grant B.J.
      • Pompeu Y.A.
      • Sidney J.
      • Harndahl M.
      • Southwood S.
      • Oseroff C.
      • Lu S.
      • Jakoncic J.
      • de Oliveira C.A.
      • Yang L.
      • Mei H.
      • Shi L.
      • Shabanowitz J.
      • English A.M.
      • et al.
      Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire.
      ,
      • Norcross M.A.
      • Luo S.
      • Lu L.
      • Boyne M.T.
      • Gomarteli M.
      • Rennels A.D.
      • Woodcock J.
      • Margulies D.H.
      • McMurtrey C.
      • Vernon S.
      • Hildebrand W.H.
      • Buchli R.
      Abacavir induces loading of novel self-peptides into HLA-B∗57: 01: An autoimmune model for HLA-associated drug hypersensitivity.
      ,
      • Illing P.T.
      • Vivian J.P.
      • Dudek N.L.
      • Kostenko L.
      • Chen Z.
      • Bharadwaj M.
      • Miles J.J.
      • Kjer-Nielsen L.
      • Gras S.
      • Williamson N.A.
      • Burrows S.R.
      • Purcell A.W.
      • Rossjohn J.
      • McCluskey J.
      Immune self-reactivity triggered by drug-modified HLA-peptide repertoire.
      ) and other exogenous chemicals (including plant allergens (
      • López C.B.
      • Kalergis A.M.
      • Becker M.I.
      • Garbarino J.A.
      • De Ioannes A.E.
      CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells.
      )) that can lead to life-threatening autoimmune responses. Indeed, one of the earliest lines of evidence supporting MHC restriction came from studies of cellular modification with trinitrophenol, later shown to be based on peptide modification (
      • Ortmann B.
      • Martin S.
      • Von Bonin A.
      • Schiltz E.
      • Hoschützky H.
      • Weltzien H.
      Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.
      ).
      Doubtless, the most remarkable post-translational peptide modification discovered is peptide rearrangement via protease-catalyzed splicing. Peptide splicing was discovered while searching for a fibroblast growth factor 5 peptide recognized by human antitumor T cells. Puzzlingly, widely separated (40+ residues apart) single Ala substitutions abrogated antigenicity (
      • Hanada K.
      • Yewdell J.W.
      • Yang J.C.
      Immune recognition of a human renal cancer antigen through post-translational protein splicing.
      ). Taking inspiration from the well-characterized splicing of the plant lectin ConA to create the native protein (
      • Carrington D.M.
      • Auffret A.
      • Hanke D.E.
      Polypeptide ligation occurs during post-translational modification of concanavalin A.
      ), the peptide was revealed to be generated via intracellular protein splicing. Within a few months of publication, peptide splicing was reported to occur in the proteasome (
      • Vigneron N.
      • Stroobant V.
      • Chapiro J.
      • Ooms A.
      • Degiovanni G.
      • Morel S.
      • van der Bruggen P.
      • Boon T.
      • Van den Eynde B.J.
      An antigenic peptide produced by peptide splicing in the proteasome.
      ), which provides a ready container for preventing the diffusion of the initial cleavage products. Splicing can even lead to the reversal of the two fragments (
      • Warren E.H.
      • Vigneron N.J.
      • Gavin M.A.
      • Coulie P.G.
      • Stroobant V.
      • Dalet A.
      • Tykodi S.S.
      • Xuereb S.M.
      • Mito J.K.
      • Riddell S.R.
      • Van den Eynde B.J.
      An antigen produced by splicing of noncontiguous peptides in the reverse order.
      ,
      • Dalet A.
      • Robbins P.F.
      • Stroobant V.
      • Vigneron N.
      • Li Y.F.
      • El-Gamil M.
      • Hanada K.
      • Yang J.C.
      • Rosenberg S.A.
      • Van den Eynde B.J.
      An antigenic peptide produced by reverse splicing and double asparagine deamidation.
      ). Since yeast proteasomes splice peptides with similar efficiency as mammalian proteasomes (
      • Mishto M.
      • Goede A.
      • Taube K.T.
      • Keller C.
      • Janek K.
      • Henklein P.
      • Niewienda A.
      • Kloss A.
      • Gohlke S.
      • Dahlmann B.
      • Enenkel C.
      • Michael Kloetzel P.
      Driving forces of proteasome-catalyzed peptide splicing in yeast and humans.
      ), splicing is not evolutionarily adapted for immunosurveillance but likely results from the barrel-like nature of proteasomes. Confirmation from studies on peptide splicing by bacterial and mitochondrial barrel proteases remains to be established, but it is known since 1901 (!) (
      • Sawjalow W.W.
      Zur theorie der Eiweissverdauung.
      ) that endoproteases can splice reaction products if present at sufficiently high concentrations (
      • Goettig P.
      Reversed proteolysis—proteases as peptide ligases.
      ).

      Immunopeptidome Present: Pressing Issues

      Connecting the Translatome to the Immunopeptidome

      By definition, the endogenously presented peptides that constitute the immunopeptidome are synthesized by the antigen-presenting cell’s ribosomes. In 2011, the development of ribosome profiling (
      • Ingolia Nicholas T.
      • Lareau Liana F.
      • Weissman Jonathan S.
      Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.
      ) (RiboSeq) enabled global accounting of the translatome, which defines the potential nonspliced immunopeptidome. The translatome is the ideal database for matching peptides detected by MS since, by minimizing the search space, it optimizes the false discovery rate.
      RiboSeq is highly demanding technically and computationally, expensive in time, reagents, and sequencing. But the payback is exquisitely detailed information on exactly what ribosomes are synthesizing, where translation starts, slows, pauses, and stops. Combined with orthogonal techniques (
      • Jan C.H.
      • Williams C.C.
      • Weissman J.S.
      Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.
      ,
      • Reid D.W.
      • Nicchitta C.V.
      Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling.
      ), it determines the locality of translation of each mRNA (endoplasmic reticulum [ER], cytosol, etc).
      To date, only a few studies have related RiboSeq data to peptide generation. Smith et al. (
      • Smith C.C.
      • Beckermann K.E.
      • Bortone D.S.
      • De Cubas A.A.
      • Bixby L.M.
      • Lee S.J.
      • Panda A.
      • Ganesan S.
      • Bhanot G.
      • Wallen E.M.
      • Milowsky M.I.
      • Kim W.Y.
      • Rathmell W.K.
      • Swanstrom R.
      • Parker J.S.
      • et al.
      Endogenous retroviral signatures predict immunotherapy response in clear cell renal cell carcinoma.
      ) used a RiboSeq database to identify and validate peptides from endogenous human retroviruses as immunotherapy targets in kidney carcinoma. Erhard et al. (
      • Erhard F.
      • Halenius A.
      • Zimmermann C.
      • L'Hernault A.
      • Kowalewski D.J.
      • Weekes M.P.
      • Stevanovic S.
      • Zimmer R.
      • Dolken L.
      Improved Ribo-seq enables identification of cryptic translation events.
      ) developed a computational tool (PRICE) that they used to identify noncanonical translation products in a published RiboSeq dataset, some of which are the source of 112 peptides present in an MS immunopeptidome they characterized from the same cell type. This was the first generalization of late 80 s findings by Boon et al. that peptides can be generated from sequences lacking the canonical features thought to be necessary for translation (
      • Boon T.
      • Van Pel A.
      • De Plaen E.
      • Chomez P.
      • Lurquin C.
      • Szikora J.P.
      • Sibille C.
      • Mariamé B.
      • Van den Eynde B.
      • Lethé B.
      • Brichart V.
      Genes coding for T-cell-defined tum transplantation antigens: Point mutations, antigenic peptides, and subgenic expression.
      ). Although not included in the immunopeptidome analysis, Erhard et al. also found over 500 new noncanonical human cytomegalovirus proteins in a published RiboSeq dataset, all of which are potential peptide sources for antiviral immunosurveillance.
      Investigating the immunoribosome hypothesis, which posits a special class of DRiPs generated by a subset of ribosomes (
      • Wei J.
      • Yewdell J.W.
      Immunoribosomes: Where's there's fire, there's fire.
      ), Wei et al. (
      • Wei J.
      • Kishton R.J.
      • Angel M.
      • Conn C.S.
      • Dalla-Venezia N.
      • Marcel V.
      • Vincent A.
      • Catez F.
      • Ferre S.
      • Ayadi L.
      • Marchand V.
      • Dersh D.
      • Gibbs J.S.
      • Ivanov I.P.
      • Fridlyand N.
      • et al.
      Ribosomal proteins regulate MHC class I peptide generation for immunosurveillance.
      ) studied the effects of ribosomal protein knockdown on peptide generation. RiboSeq revealed that RPS28 knockdown increased noncanonical translation of proteins in 3′ and 5' UTRs and proteins initiating with non-AUG codon. Loss of RPS28 also increased HLA-A2 cell surface expression and export from the ER, consistent with an increase in peptide supply from the noncanonical proteins, many of which may represent DRiPs.
      Chong et al. (
      • Chong C.
      • Muller M.
      • Pak H.
      • Harnett D.
      • Huber F.
      • Grun D.
      • Leleu M.
      • Auger A.
      • Arnaud M.
      • Stevenson B.J.
      • Michaux J.
      • Bilic I.
      • Hirsekorn A.
      • Calviello L.
      • Simo-Riudalbas L.
      • et al.
      Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes.
      ) were the first to experimentally determine the RiboSeq translatome and the MS immunopeptidome on the same sample, a human melanoma cell line. RiboSeq identification of translated proteins expanded the MHC I immunopeptidome by 25%, though all but 56 of the 3606 extra peptides found derived from canonical proteins.
      Extending this finding, Ruiz Cuevas et al. (
      • Ruiz Cuevas M.V.
      • Hardy M.P.
      • Hollý J.
      • Bonneil É.
      • Durette C.
      • Courcelles M.
      • Lanoix J.
      • Côté C.
      • Staudt L.M.
      • Lemieux S.
      • Thibault P.
      • Perreault C.
      • Yewdell J.W.
      Most non-canonical proteins uniquely populate the proteome or immunopeptidome.
      ) were the first to correlate the RiboSeq translatome with the MS immunopeptidome and whole-cell proteome. The translatome was generated using a newly devised computational tool (RiboDB) that modestly surpassed PRICE in finding novel translation products. Extensive informatic analysis of three human B-cell lymphoma cell lines revealed that:
      • 1.
        Fully 85% of translation products (approximately 200,000 for each cell line) are not annotated in the standard human database and constitute the noncanonical translatome. Of these, only 0.08% and 0.36% were detected in the immunopeptidome and proteome, respectively. This is due, in part, to their sevenfold shorter length: the total noncanonical translatome encodes ∼11 million amino acids, compared with 24 million amino acids for the canonical translatome, because of a median length of 42 versus 280 residues per protein (this is consistent with RiboSeq-detected noncanonical proteins in other studies (
        • Ingolia N.T.
        • Hussmann J.A.
        • Weissman J.S.
        Ribosome profiling: Global views of translation.
        )). But even for canonical proteins, only 5.51% and 9.11% are detected, respectively, in the immunopeptidome and proteome, the latter emphasizing the current limitations of MS in completely cataloging the proteome.
      • 2.
        Of noncanonical translatome-defined proteins, half are novel isoforms, proteins that nearly exclusively use a nonannotated start codon, often a non-AUG codon downstream of the annotated start codon. The rest are “cryptic” proteins translated from alternative reading frames of canonical ORFs, “noncoding” ORFs, including introns, 3′ and 5' “UTRs,” intergenic regions, pseudogenes, and other “noncoding” RNAs. More than 70% of cryptic proteins initiate with non-AUG codons, most commonly CUG, but with all near-cognate codons (one base substitution from AUG) well represented.
      • 3.
        Of 14,498 proteins detected by MS, only 17% are noncanonical proteins, split, 60:28:12, respectively, among “UTRs,” novel isoforms, and frameshifted canonical ORFS. The absence of cryptic proteins from the proteome is based on several factors. Their short size limits the number of peptides that can be detected and likely increases their degradation rate. Their stability is probably also impaired by higher predicted disorder since proteins consisting of less than 80 residues typically need a binding partner for stability. Increased ribosome stalling detected by RiboSeq may also contribute to the decreased stability (
        • Brandman O.
        • Hegde R.S.
        Ribosome-associated protein quality control.
        ).
      • 4.
        Of 7045 MHC-associated peptides (MAPs) detected, 7.5% were derived from noncanonical proteins. Note that this is much greater fraction than detected by Chong et al., likely because of the ∼100-fold greater number of RiboSeq reads used to populate the translatome. Of noncanonical MAP source proteins, there is 50:50 split between novel isoforms and cryptic proteins. Canonical and noncanonical peptides had a similar length distribution and predicted class I–binding affinity. As expected, based simply on number of potential peptides in a given protein, longer proteins were a preferred source of both canonical and noncanonical peptides. Importantly, MAP source cryptic proteins were much shorter than canonical source proteins with a median of 49 versus 504 residues.
      • 5.
        For MAP source proteins, levels of canonical transcripts were only 1.4-fold and 2.1-fold higher, respectively, than novel isoform and cryptic protein transcripts. There were also surprisingly slight differences in the translation efficiency of these mRNAs (note that efficiency relates only to MAP detection, not MAP quantitation, an important limitation to nearly all global immunopeptidome studies, as discussed later). Critically, per translation event, mRNAs encoding cryptic proteins were fivefold more efficient at generating MAPs. Ribosome stalling, which is greater on such mRNA, may contribute to this increased efficiency, as elegantly shown by Trentini et al. (
        • Trentini D.B.
        • Pecoraro M.
        • Tiwary S.
        • Cox J.
        • Mann M.
        • Hipp M.S.
        • Hartl F.U.
        Role for ribosome-associated quality control in sampling proteins for MHC class I-mediated antigen presentation.
        ) using a model substrate.
      Ouspenskaia et al. (
      • Ouspenskaia T.
      • Law T.
      • Clauser K.R.
      • Klaeger S.
      • Sarkizova S.
      • Aguet F.
      • Li B.
      • Christian E.
      • Knisbacher B.A.
      • Le P.M.
      • Hartigan C.R.
      • Keshishian H.
      • Apffel A.
      • Oliveira G.
      • Zhang W.
      • et al.
      Thousands of novel unannotated proteins expand the MHC I immunopeptidome in cancer.
      ) determined the RiboSeq translatome for a remarkable 29 human primary and cancer cells. This generated 82,000 annotated and 237,000 novel unannotated ORFs (nuORFs), which were of similar brevity to the cryptic proteins detected by Ruiz Cuevas et al., and were also nearly undetected in the whole-cell proteome (0.1% of all tryptic peptides detected). About 6500 peptides from 3300 nuORFs were detected in the immunopeptidome, representing 3.3% of the large (198,000 peptides) 29 cell composite immunopeptidome. Though a tiny fraction of all peptides detected, it is still remarkable that 26 nuORFs encoded the exact peptide detected in the immunopeptidome: the genomic equivalent of minigenes (actually beyond minigenes, which typically possess the minimal peptide plus an initiating Met residue) used to study antigen processing in model systems. Looking specifically at cancer cells, nuORFs contributed to ∼2% of the immunopeptidome. Despite this, in given cancers, for example, melanoma, nuORF peptides augment the tumor-specific “neopeptide” repertoire by 25%, including peptides from tumor-specific gene products as well as tumor-specific mutations in antigenic peptides. This is consistent with nuORFs being a more efficient source of antigenic peptides per translation event, as reported by Ruiz Cuevas et al. (
      • Ruiz Cuevas M.V.
      • Hardy M.P.
      • Hollý J.
      • Bonneil É.
      • Durette C.
      • Courcelles M.
      • Lanoix J.
      • Côté C.
      • Staudt L.M.
      • Lemieux S.
      • Thibault P.
      • Perreault C.
      • Yewdell J.W.
      Most non-canonical proteins uniquely populate the proteome or immunopeptidome.
      ).
      Supporting the contribution of noncanonical translation to the immunopeptidome, Erhard et al. (
      • Erhard F.
      • Dölken L.
      • Schilling B.
      • Schlosser A.
      Identification of the cryptic HLA-I immunopeptidome.
      ) developed a new informatics pipeline to reanalyze published cancer cell immunopeptidome MS datasets from multiple laboratories consisting of over 400,000 identified peptides. For a selected single human melanoma line, 1563 peptides were derived from cryptic translation products, corresponding to a 4.5% increase in all peptides detected. For all tumors, 12,752 cryptic peptides detected constituted a 2.9% increase in total peptides. Careful analysis of these data led to several remarkable findings.
      • 1.
        About 18% of peptides from cryptic peptides derive from the COOH terminus of the predicted source protein, with more than half of these within just 10 amino acids from the predicted amino terminus. These peptides should be presented without proteasomal involvement, with amino-terminal residues removed either by cytosolic aminopeptidases or ER-associated aminopeptidases after transport into the ER. Based on this finding, it would be of great interest to reanalyze (
        • Milner E.
        • Gutter-Kapon L.
        • Bassani-Strenberg M.
        • Barnea E.
        • Beer I.
        • Admon A.
        The effect of proteasome inhibition on the generation of the human leukocyte antigen (HLA) peptidome.
        ) the peptide dataset tof Milner et al. to examine the contribution of cryptic translation to the immunopeptidome in proteasome inhibitor–treated cells.
      • 2.
        Certain HLA-A alleles demonstrated selective enhancement/reduction in presenting cryptic versus canonical peptides, ranging from 43% for HLA-A (136/316; 11:01) to negligible for HLA-B5 (1/7015; 8:01). Across all datasets, 10% of peptides assigned to A03 supertype allomorphs (supertypes are groups of closely related MHC genes) derive from noncanonical translation products compared with less than 2% for A02 supertypes. This could not be facilely attributed to differences in peptide composition and is consistent with potential allomorph-related differences in localized translation of class I molecules to facilitate channeled processing and presentation of cryptic translation products (
        • Yewdell J.W.
        • Dersh D.
        • Fahraeus R.
        Peptide channeling: The key to MHC class I immunosurveillance?.
        ).
      Bartok et al. (
      • Bartok O.
      • Pataskar A.
      • Nagel R.
      • Laos M.
      • Goldfarb E.
      • Hayoun D.
      • Levy R.
      • Körner P.R.
      • Kreuger I.Z.M.
      • Champagne J.
      • Zaal E.A.
      • Bleijerveld O.B.
      • Huang X.
      • Kenski J.
      • Wargo J.
      • et al.
      Anti-tumour immunity induces aberrant peptide presentation in melanoma.
      ) brilliantly combined RiboSeq and immunopeptidomics to explore how interferon gamma (IFN-γ) signaling–induced Trp deficiency affects the tumor cell immunopeptidome. The resulting reduction in Trp-charged tRNAs decreases protein synthesis because of selective ribosome stalling at Trp codons. Stalling is associated with translational frameshifting, which generates novel peptides, 94 of which were detected in the immunopeptidome of freshly isolated human melanoma cells. A number of these peptides were extensively validated and shown to be expressed in metastases from the same tumor. Several peptides were detected in a tumor from an another MHC-matched patient. One of the peptides was immunogenic for T cells ex vivo. This work establishes a new principle for how the tumor-specific peptide repertoire is naturally expanded during the course of an antitumor immune response.
      Taken together, the RiboSeq studies demonstrate that while noncanonical translation accounts for much less than one-half of total translation on a molar basis, it accounts for approximately two-thirds of the variety of translation products. Noncanonical proteins are mostly small metabolically unstable proteins, some of which are highly efficient sources of MAP that can be important targets for cancer immunotherapy and possibly autoimmunity as well (
      • Kracht M.J.
      • van Lummel M.
      • Nikolic T.
      • Joosten A.M.
      • Laban S.
      • van der Slik A.R.
      • van Veelen P.A.
      • Carlotti F.
      • de Koning E.J.
      • Hoeben R.C.
      • Zaldumbide A.
      • Roep B.O.
      Autoimmunity against a defective ribosomal insulin gene product in type 1 diabetes.
      ). Surely, some/many of these noncanonical proteins have other biological functions despite their small size and rapid turnover (
      • Anderson D.M.
      • Anderson K.M.
      • Chang C.L.
      • Makarewich C.A.
      • Nelson B.R.
      • McAnally J.R.
      • Kasaragod P.
      • Shelton J.M.
      • Liou J.
      • Bassel-Duby R.
      • Olson E.N.
      A micropeptide encoded by a putative long noncoding RNA regulates muscle performance.
      ,
      • Chen J.
      • Brunner A.-D.
      • Cogan J.Z.
      • Nuñez J.K.
      • Fields A.P.
      • Adamson B.
      • Itzhak D.N.
      • Li J.Y.
      • Mann M.
      • Leonetti M.D.
      • Weissman J.S.
      Pervasive functional translation of noncanonical human open reading frames.
      ). Likely, the noncanonical translatome and derived peptides are greatly altered under infectious stress, which is known to favor noncanonical translation initiation (
      • Starck S.R.
      • Tsai J.C.
      • Chen K.
      • Shodiya M.
      • Wang L.
      • Yahiro K.
      • Martins-Green M.
      • Shastri N.
      • Walter P.
      Translation from the 5' untranslated region shapes the integrated stress response.
      ). It will be critical to verify and extend findings currently limited to in vitro cultured cells to tissues in vivo.

      Peptide Splicing: Exception or Rule?

      Being the one to suggest splicing to Kenichi Hanada and Jim Yang as a potential mechanism to account for the effects of widely separated amino acid substitution on fibroblast growth factor 5 antigenicity (
      • Hanada K.
      • Yewdell J.W.
      • Yang J.C.
      Immune recognition of a human renal cancer antigen through post-translational protein splicing.
      ), I was still surprised at how rapidly other spliced peptides were reported. It was an even greater shock when Liepe et al. (
      • Liepe J.
      • Marino F.
      • Sidney J.
      • Jeko A.
      • Bunting D.E.
      • Sette A.
      • Kloetzel P.M.
      • Stumpf M.P.
      • Heck A.J.
      • Mishto M.
      A large fraction of HLA class I ligands are proteasome-generated spliced peptides.
      ) reported that spliced peptides account for a third of the immunopeptidome in diversity and a quarter in abundance. Supporting these findings, Faridi et al. (
      • Faridi P.
      • Li C.
      • Ramarathinam S.H.
      • Vivian J.P.
      • Illing P.T.
      • Mifsud N.A.
      • Ayala R.
      • Song J.
      • Gearing L.J.
      • Hertzog P.J.
      • Ternette N.
      • Rossjohn J.
      • Croft N.P.
      • Purcell A.W.
      A subset of HLA-I peptides are not genomically templated: Evidence for cis- and trans-spliced peptide ligands.
      ) reported that spliced peptides account for up to 44% of peptides associated with given HLA allomorphs, with a majority of spliced peptides derived from different proteins (trans-splicing).
      While proteasome-mediated splicing is a nonstochastic process that greatly favors ligating certain fragments, it is nonetheless an uncommon outcome in detailed studies of proteasome-mediated splicing of synthetic peptides (
      • Vigneron N.
      • Stroobant V.
      • Ferrari V.
      • Abi Habib J.
      • Van den Eynde B.J.
      Production of spliced peptides by the proteasome.
      ,
      • Paes W.
      • Leonov G.
      • Partridge T.
      • Nicastri A.
      • Ternette N.
      • Borrow P.
      Elucidation of the signatures of proteasome-catalyzed peptide splicing.
      ), undermining a major contribution of spliced peptides to the immunopeptidome. Further, could simultaneous or sequential proteasome degradation of two different proteins ever occur frequently enough to generate sufficient quantities of trans-spliced peptides for MS detection? This would be possibly plausible for two different DRiPs from highly translated viral proteins (which can each account for 5% or more of the total translation). However, for cellular proteins, it seems nearly impossible for proteins that are not physically associated when delivered to the proteasome for degradation (which, to my knowledge, has yet to be reported).
      But data are data, though the devil is always in the details. A critical factor in MS identification of peptides is the false discovery rate, which increases with the number of sequences in the database used to match peptide masses. Without this limitation, MS data could be queried against every possible n-mer peptide (i.e., 20 amino acids at each position [or more accurately 19 amino acids, since Leu and Ile can only be distinguished by special techniques (
      • Xiao Y.
      • Vecchi M.M.
      • Wen D.
      Distinguishing between leucine and isoleucine by integrated LC–MS analysis using an orbitrap fusion mass spectrometer.
      )). Indeed, minimizing the relevant peptide database is a major advantage of using RiboSeq in immunopeptidome studies.
      Several groups re-examined the reported spliced immunopeptidome and found that 1 to 6% of ostensibly spliced peptides fail to meet more stringent criteria (
      • Mylonas R.
      • Beer I.
      • Iseli C.
      • Chong C.
      • Pak H.S.
      • Gfeller D.
      • Coukos G.
      • Xenarios I.
      • Muller M.
      • Bassani-Sternberg M.
      Estimating the contribution of proteasomal spliced peptides to the HLA-I ligandome.
      ,
      • Rolfs Z.
      • Solntsev S.K.
      • Shortreed M.R.
      • Frey B.L.
      • Smith L.M.
      Global identification of post-translationally spliced peptides with neo-fusion.
      ). Further detailed informatic analysis, including matches with peptides from cryptic proteins and likelihood of binding to class I molecules, reduced the occurrence of spliced peptides to <1% of the immunopeptidome (
      • Erhard F.
      • Dölken L.
      • Schilling B.
      • Schlosser A.
      Identification of the cryptic HLA-I immunopeptidome.
      ,
      • Lichti C.F.
      Identification of spliced peptides in pancreatic islets uncovers errors leading to false assignments.
      ).
      The primary difficulty in studying spliced genomic-encoded peptides is the sheer size of the potential peptidome. This is obviated with studying viral peptides, which also provide a much easier method of verifying peptide identity by expressing individual viral proteins, mutating the corresponding gene, or by inducing T cells specific for the peptide. In a comprehensive study of HIV-spliced peptides, Paes et al. (
      • Paes W.
      • Leonov G.
      • Partridge T.
      • Chikata T.
      • Murakoshi H.
      • Frangou A.
      • Brackenridge S.
      • Nicastri A.
      • Smith A.G.
      • Learn G.H.
      • Li Y.
      • Parker R.
      • Oka S.
      • Pellegrino P.
      • Williams I.
      • et al.
      Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8(+) T cells in HIV-1 infection.
      ) determined that spliced peptides account for ∼2% of the viral peptidome and that while T cells from HIV-infected individuals can recognize spliced peptides, they do so based on TCR cross-reactivity with one of the unspliced parent peptides. The spliced peptide's lack of apparent intrinsic immunogenicity correlated with the relatively lower abundance of spliced versus unspliced peptides.
      While further studies are warranted, the present consensus is that spliced peptides constitute perhaps 1 to 3% of the immunopeptidome. Still, if the immunopeptidome is representative of the proteasome-generated peptidome, spliced peptides are likely to have roles beyond immunosurveillance, perhaps even as neurotransmitters, based on the remarkable findings of Margolis et al. that a large fraction of DRiPs are degraded by neuronal cell–surface proteasomes, with peptide products having signaling activity (
      • Ramachandran K.V.
      • Margolis S.S.
      A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function.
      ,
      • Ramachandran K.V.
      • Fu J.M.
      • Schaffer T.B.
      • Na C.H.
      • Delannoy M.
      • Margolis S.S.
      Activity-dependent degradation of the nascentome by the neuronal membrane proteasome.
      ).

      Immunopeptidome Future

      Sensitivity

      Progress in understanding the composition and origin of the immunopeptidome will be heavily dependent on advances in technology. Sooner or (probably) later, greatly increased sensitivity of MS (perhaps on the order of a billion-fold relative to standard experiments today, which routinely start with 107–108 cells) should enable characterizing the immunopeptidome at the level of single cells (
      • Kelly R.T.
      Single-cell proteomics: Progress and prospects.
      ), along with the proteome, degradome, and translatome (which is easier to envisage), with the ultimate goal of studying cells in their natural context in living vertebrates. All this is integral to the broader issue of protein synthesis and degradation at the level of individual cells. Superhigh sensitivity would also enable characterizing the complete immunopeptidomes of antigen-presenting cells engaged in T-cell activation/tolerance in lymph nodes, spleen, and thymus and dissect the contributions of endogenous and exogenous sources of peptides. Increased sensitivity of MS will also enable more precise stable isotope labeling by amino acids in cell culture to accurately measure the contribution of nascent versus retired proteins to the immunopeptidomes, where labeling intervals should be on the order of minutes rather than hours to enable detection of peptides from proteins degraded cotranslationally (
      • Wang F.
      • Durfee Larissa A.
      • Huibregtse Jon M.
      A cotranslational ubiquitination pathway for quality control of misfolded proteins.
      ) or within minutes of synthesis (
      • Schubert U.
      • Antón L.C.
      • Gibbs J.
      • Norbury C.C.
      • Yewdell J.W.
      • Bennink J.R.
      Rapid degradation of a large fraction of newly synthesized proteins by proteasomes.
      ,
      • Qian S.B.
      • Princiotta M.F.
      • Bennink J.R.
      • Yewdell J.W.
      Characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control.
      ).

      Quantitation

      A key to relating translatome and degradome to the immunopeptidome is quantitating the amounts of each peptide detected. At present, this requires using known amounts of synthetic peptides as standards (
      • Croft N.P.
      • Purcell A.W.
      • Tscharke D.C.
      Quantifying epitope presentation using mass spectrometry.
      ,
      • Hassan C.
      • Kester M.G.
      • Oudgenoeg G.
      • de Ru A.H.
      • Janssen G.M.
      • Drijfhout J.W.
      • Spaapen R.M.
      • Jiménez C.R.
      • Heemskerk M.H.
      • Falkenburg J.F.
      Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.
      ). In principle, it should be possible to obtain quantitative data directly during MS analysis. For peptides containing Trp (only ∼10% of the immunopeptidome, because of its being the least abundant amino acid), Trp intrinsic fluorescence (
      • Wisniewski J.R.
      • Gaugaz F.Z.
      Fast and sensitive total protein and Peptide assays for proteomic analysis.
      ) should enable direct quantitation after chromatographic separation with a sufficiently sensitive detector. Quantitating other peptides directly will require ingenuity but surely cannot be impossible!

      Cell Biology

      To date, the MS-defined immunopeptidome has almost exclusively been used to indirectly infer how peptides are generated. The Admon laboratory has pioneered using the MS immunopeptidome to directly characterize the contribution of cellular processes (e.g., proteasome participation, effects of IFN). Many more of these types of experiments would greatly advance the field. For example, it would be of great interest to examine the repopulation of the immunopeptidome after acid stripping cells to remove class I–bound peptides in the presence and absence of a protein synthesis inhibitor. This should reveal the contribution of DRiPs versus retirees at the level of the entire immunopeptidome. Incubation of cells with brefeldin A to characterize peptide class I dissociation would provide novel global insight into real versus in silico–predicted class I–binding affinity and how peptide affinity relates to the half-life of MHC I complex. Class I molecules purified from various intracellular compartments could be used to examine how the immunopeptidome matures with transport of class I molecules to the cell surface. Many more such experiments are possible.

      Viral Immunology

      To date, only a few studies have exploited the awesome power of MS to characterize the viral immunopeptidome. In a remarkable early MS viral immunopeptidome study, van Els et al. (
      • van Els C.A.
      • Herberts C.A.
      • van der Heeft E.
      • Poelen M.C.
      • van Gaans-van den Brink J.A.
      • van der Kooi A.
      • Hoogerhout P.
      • Jan ten Hove G.
      • Meiring H.D.
      • de Jong A.P.
      A single naturally processed measles virus peptide fully dominates the HLA-A∗0201-associated peptide display and is mutated at its anchor position in persistent viral strains.
      ) reported that HLA-A2 presented a single measles virus peptide in enormous numbers: greater than 105 complexes per virus-infected cell. Such levels are not even achieved by overexpressing preprocessed peptides (
      • Princiotta M.F.
      • Finzi D.
      • Qian S.B.
      • Gibbs J.
      • Schuchmann S.
      • Buttgereit F.
      • Bennink J.R.
      • Yewdell J.W.
      Quantitating protein synthesis, degradation, and endogenous antigen processing.
      ,
      • Anton L.C.
      • Yewdell J.W.
      • Bennink J.R.
      MHC class I-associated peptides produced from endogenous gene products with vastly different efficiencies.
      ), raising the possibility of virus-evolved peptide overexpression to modulate the CD8+ T-cell or NK cell response.
      Extending their prior studies on vaccinia virus, Purcell et al. (
      • Wu T.
      • Guan J.
      • Handel A.
      • Tscharke D.C.
      • Sidney J.
      • Sette A.
      • Wakim L.M.
      • Sng X.Y.X.
      • Thomas P.G.
      • Croft N.P.
      • Purcell A.W.
      • La Gruta N.L.
      Quantification of epitope abundance reveals the effect of direct and cross-presentation on influenza CTL responses.
      ) described the quantitative kinetics of IAV peptide generation, discovering seven new peptides in the process, despite the influenza system being intensively investigated previously. The study provides unique information quantitating peptides crosspresented by dendritic cells, explaining the strange immunodominance of the PA224–232 peptide, which is presented at extremely low levels on infected cells but is among the most abundant of cross-presented peptides. de Wit et al. (
      • de Wit J.
      • Emmelot M.E.
      • Meiring H.
      • van Gaans-van den Brink J.A.M.
      • van Els C.
      • Kaaijk P.
      Identification of naturally processed mumps virus epitopes by mass spectrometry: Confirmation of multiple CD8+ T-cell responses in mumps patients.
      ) provided a shining example of the power of MS discovery of viral peptides in reporting 41 mumps virus peptides presented by human class I molecules, six of which were recognized by CD8+ T cells from patients with mumps. If not at present, MS will likely soon become the most time and cost-effective method for identifying viral peptides (
      • Woon A.P.
      • Purcell A.W.
      The use of proteomics to understand antiviral immunity.
      ).
      Though I have focused this review on MHC class I, I would be remiss to omit class II from the discussion. It has long been known that TCD4+ can kill virus-infected cells (
      • Kaplan D.R.
      • Griffith R.
      • Braciale V.L.
      • Braciale T.J.
      Influenza virus-specific human cytotoxic T cell clones: Heterogeneity in antigenic specificity and restriction by class II MHC products.
      ) and that IFN-γ and other cytokines can induce class II expression on nearly all cell types. Class II is constitutively expressed not only by many bone marrow–derived cells but also by a surprising number of other cells, for example, type II pneumocytes, endothelial cells, and gut epithelial cells. In addition to classical endosomal loading of exogenous viral proteins, class II molecules also present endogenous viral peptides (
      • Veerappan Ganesan A.P.
      • Eisenlohr L.C.
      The elucidation of non-classical MHC class II antigen processing through the study of viral antigens.
      ). Class II–restricted responses to viruses have been greatly understudied generally; specifically, I am unaware of any published MS class II viral immunopeptidome studies. There are clearly opportunities here for basic discoveries in antigen processing/presentation and practical applications in monitoring class II responses, improving vaccines and dealing with viral immune escape.

      Tumor Immunology

      This is the one area of the immunopeptidomics that has attracted a reasonable amount of interest from the greater community, thanks to the success of T cell–based immunotherapy and the paucity of tumor-specific targets. Still, so many questions have yet to be addressed. For example, I cannot find a single published study on how radiation treatment alters the MS tumor immunopeptidome, as it certainly must do (
      • Reits E.A.
      • Hodge J.W.
      • Herberts C.A.
      • Groothuis T.A.
      • Chakraborty M.
      • Wansley E.K.
      • Camphausen K.
      • Luiten R.M.
      • de Ru A.H.
      • Neijssen J.
      • Griekspoor A.
      • Mesman E.
      • Verreck F.A.
      • Spits H.
      • Schlom J.
      • et al.
      Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy.
      ). Ditto for chemotherapeutic agents. Since most tumor antigens must be presented via crosspresentation to activate naïve T cells, an obvious issue is the cross-presented immunopeptidome, which is also a critical issue for peripheral tolerance to tissue-specific antigens.

      The Last Word

      Many of us generation 1 antigen processingologists have spent much of our careers studying how SIINFEKL and a few other model peptides are generated. This approach has been fruitful, but extending these findings to the greater immunopeptidome is essential. This can only be accomplished via MS-based immunopeptidomics. The field should aim at fostering interdisciplinary collaboration between laboratories focused on MS, cell biological aspects of antigen processing, and bioinformatics. Young scientists interested in T-cell immunosurveillance should be encouraged to receive training in at least two of these disciplines. All should attend the 11th and future Antigen Processing and Presentation workshops, where newcomers are not only welcome but cherished.
      See you there!

      Conflict of interest

      The authors declare no competing interests.

      Acknowledgments

      Devin Dersh provided excellent suggestions for improving the article. This article is dedicated to the memory of four outstanding scientists who laid the foundation for understanding the cell biological origins of the class I immunopeptidome: Enzo Cerundolo, Huib Ovaa, Nilabh Shastri, and Satvir Tevethia. While sadly no longer with us in body, their spirit lives on in their mentees, colleagues, and articles.

      Funding and additional information

      J. W. Y. is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases , National Institutes of Health . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Author contributions

      J. W. Y. conceptualization, writing–original draft, and writing–review & editing.

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      Linked Article

      • Immunopeptidomics: Reading the Immune Signal That Defines Self From Nonself
        Molecular & Cellular ProteomicsVol. 21Issue 6
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          Fifty years have passed since the study of Benacerraf and McDevitt (1) describing the exquisite regulation of the immune response by the major histocompatibility complex (MHC). The landmark discovery that T-cell activation requires corecognition of peptide antigens and self-MHC molecules (2) revealed not only a unique receptor–ligand interaction but also a delicate balance between autoimmune response and effective protection against infection. These seminal studies laid the foundation to our current understanding of how immune cells distinguish between self and nonself.
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