Are There Indeed Spliced Peptides in the Immunopeptidome?

The claims that a large fraction of the immunopeptidome is composed of spliced major histocompatibility complex (MHC) peptides have stirred significant excitement and raised controversy. Here, I suggest that there are likely no spliced peptides in the immunopeptidome, and if they exist at all, they are extremely rare. I base this claim on both biochemical and bioinformatics considerations. First, as a reactant in normal proteolytic reactions, water will compete with transpeptidation, which has been suggested as the mechanism of peptide splicing. The high mobility and abundance of water in aqueous solutions renders transpeptidation very inefficient and therefore unlikely to occur. Second, new studies have refuted the bioinformatics assignments to spliced peptides of most of the immunopeptidome MS data, suggesting that the correct assignments are likely other canonical, noncanonical, and post-translationally modified peptides. Therefore, I call for rigorous experimental methodology using heavy stable isotope peptides spiking into the immunoaffinity-purified mixtures of natural MHC peptides and analysis by the highly reliable targeted MS, to claim that MHC peptides are indeed spliced.

Commentary: Are There Indeed Spliced Peptides in the Immunopeptidome?

Proteasome-generated spliced epitopes presented by HLA class I complexes are emerging targets for T cell targeted immunotherapies. Their identification by mass spectrometry triggered heated debates, which find a representative opinion in one of the two fronts in the recent perspective article by Arie Admon. Briefly, he suggests that proteasomes cannot efficiently catalyze such a reaction, and, thus, that all spliced peptides identified in HLA class I immunopeptidomes and other specimens are artifacts. This hypothesis is in contrast with in vitro, in cellula, and in vivo results published since the discovery of proteasome-catalyzed peptide splicing in 2004.

Database search engines and target database features impinge upon the identification of post-translationally cis-spliced peptides in HLA class I immunopeptidomes.

Unconventional epitopes presented by HLA class I complexes are emerging targets for T cell targeted immunotherapies. Their identification by mass spectrometry (MS) required development of novel methods to cope with the large number of theoretical candidates. Methods to identify post-translationally spliced peptides led to a broad range of outcomes. We here investigated the impact of three common database search engines - that is, Mascot, Mascot+Percolator, and PEAKS DB - as final identification step, as well as the features of target database on the ability to correctly identify non-spliced and cis-spliced peptides. We used ground truth datasets measured by MS to benchmark methods' performance and extended the analysis to HLA class I immunopeptidomes. PEAKS DB showed better precision and recall of cis-spliced peptides and larger number of identified peptides in HLA class I immunopeptidomes than the other search engine strategies. The better performance of PEAKS DB appears to result from better discrimination between target and decoy hits and hence a more robust FDR estimation, and seems independent to peptide and spectrum features here investigated.

Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8+ T cells in HIV-1 infection.

Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8+ T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I-bound peptides on HIV-infected cells. We demonstrate that HIV-1-derived spliced peptides comprise a relatively minor component of the HLA-I-bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8+ T cell responses relatively infrequently during infection, CD8+ T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection.

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this approach, the fragmentation spectrum and chromatographic retention time of a peptide within a biological sample are compared with those of a synthetic version of the putative peptide sequence match. Similarity measurements obtained for a panel of internal standard peptides are then used to calculate a prediction interval for valid matches. If the observed degree of similarity between the biological and the synthetic peptide falls within this prediction interval, then the match is considered valid. P-VIS enables systematic and objective assessment of the validity of individual PSMs, providing a measurable degree of confidence when identifying peptides by mass spectrometry.

Spliced HLA-bound peptides: a Black Swan event in immunology.

Peptides that bind to and are presented on the cell surface by human leucocyte antigen (HLA) molecules play a critical role in adaptive immunity. For a long time it was believed that all the HLA-bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated during the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA-bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will evaluate recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing and the potential role of these spliced peptides in immune recognition.

What We See, What We Do Not See, and What We Do Not Want to See in HLA Class I Immunopeptidomes.

The identification of peptides bound to human leukocyte antigen class I (HLA-I) molecules-that is, the HLA-I immunopeptidome-is a useful tool in the hunt for epitopes suitable for vaccinations and immunotherapies. These peptides are mainly generated by proteasomes through peptide hydrolysis and peptide splicing. In this issue, Nicastri and colleagues compared different methods for the elution of HLA class I-associated peptides. It is demonstrated that the choice of HLA-associated peptide enrichment and purification strategy affects peptide yields and creates a bias in detected sequence repertoire. The author carried out this technical brief through the analysis of canonical non-spliced peptides. However, their study left out any analysis of post-translationally spliced peptides, thereby missing an opportunity to shed light on the persistent debate of the frequency of these unconventional peptides.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.

Spliced analogues of trypsin inhibitor SFTI-1 and their application for tracing proteolysis and delivery of cargos inside the cells.

A series of analogues of trypsin inhibitor SFTI-1 were designed and synthesized to monitor peptide splicing. In the middle part of the SFTI-1 analogues, which is released upon incubation with proteinase, the RGD sequence or an acceptor of fluorescence for FRET was introduced. The results of studies with trypsin confirmed that the designed analogues underwent peptide splicing. Furthermore, we showed that a FRET displaying SFTI-1 analogue was internalized into the HaCaT keratinocytes, where it was degraded. Therefore, both proteolysis and the reduction of the disulfide bridge of the peptide took place. As a result, such analogues are a convenient tool to trace the proteolytic activity inside the cell. However, the cytotoxicity of SFTI-1 analogues grafted with the RGD sequence did not correlate with their susceptibility to peptide splicing. Nevertheless, these peptides were slightly more active than the reference peptide (GRGDNP). Interestingly, one of the analogues assigned as [desSer6 ]VI, under experimental conditions, appeared significantly more cytotoxic towards cancer cells U87-MG in contrast to the reference peptide.

Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor†1 (SFTI-1).

Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.

Peptide splicing in a double-sequence analogue of trypsin inhibitor SFTI-1 substituted in the P1 positions by peptoid monomers.

Recently, we described a process of trypsin-assisted peptide splicing of analogs of trypsin inhibitor SFTI-1, that seems to be very similar to proteasome-catalyzed peptide splicing. Here, we show, for the first time, that a peptide-peptoid hybrid (peptomer) can also be spliced by trypsin. Incubation of a double sequence SFTI-1 analog, containing two peptoid monomers, with equimolar amount of trypsin leads to formation of monocyclic peptomer as the main product. We proved that the peptide bond formed by a peptoid monomer is not only digested by trypsin but also participates in the enzyme-assisted splicing process.

Neo-Splicetopes in Tumor Therapy: A Lost Case?

Proteasome generates spliced peptides by ligating two distant cleavage products in a reverse proteolysis reaction. The observation that CD8+ T cells recognizing a spliced peptide induced T cell rejection in a melanoma patient following adoptive T cell transfer (ATT), raised some hopes with regard to the general therapeutic and immune relevance of spliced peptides. Concomitantly, the identification of spliced peptides was also the start of a controversy with respect to their frequency, abundancy and their therapeutic applicability. Here I review some of the recent evidence favoring or disfavoring an immune relevance of splicetopes and discuss from a theoretical point of view the potential usefulness of tumor specific splicetopes and why against all odds it still may seem worth trying to identify such tumor and patient-specific neosplicetopes for application in ATT.

Corrigendum: Elucidation of the Signatures of Proteasome-Catalysed Peptide Splicing.

[This corrects the article DOI: 10.3389/fimmu.2020.563800.].

Proteasome-Generated cis-Spliced Peptides and Their Potential Role in CD8+ T Cell Tolerance.

The human immune system relies on the capability of CD8+ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8+ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes-i.e., HLA-I immunopeptidomes-of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8+ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8+ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8+ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8+ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8+ cytotoxic T cells (CTLs) involved in anti-viral response.

Elucidation of the Signatures of Proteasome-Catalyzed Peptide Splicing.

Proteasomes catalyze the degradation of endogenous proteins into oligopeptides, but can concurrently create spliced oligopeptides through ligation of previously non-contiguous peptide fragments. Recent studies have uncovered a formerly unappreciated role for proteasome-catalyzed peptide splicing (PCPS) in the generation of non-genomically templated human leukocyte antigen class I (HLA-I)-bound cis-spliced peptides that can be targeted by CD8+ T cells in cancer and infection. However, the mechanisms defining PCPS reactions are poorly understood. Here, we experimentally define the biochemical constraints of proteasome-catalyzed cis-splicing reactions by examination of in vitro proteasomal digests of a panel of viral- and self-derived polypeptide substrates using a tailored mass-spectrometry-based de novo sequencing workflow. We show that forward and reverse PCPS reactions display unique splicing signatures, defined by preferential fusion of distinct amino acid residues with stringent peptide length distributions, suggesting sequence- and size-dependent accessibility of splice reactants for proteasomal substrate binding pockets. Our data provide the basis for a more informed mechanistic understanding of PCPS that will facilitate future prediction of spliced peptides from protein sequences.

Production of spliced peptides by the proteasome.

CD8+ cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.

An in silico-in vitro Pipeline Identifying an HLA-A*02:01+ KRAS G12V+ Spliced Epitope Candidate for a Broad Tumor-Immune Response in Cancer Patients.

Targeting CD8+ T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes.

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection.

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

An Unexpected Major Role for Proteasome-Catalyzed Peptide Splicing in Generation of T Cell Epitopes: Is There Relevance for Vaccine Development?

Efficient and safe induction of CD8+ T cell responses is a desired characteristic of vaccines against intracellular pathogens. To achieve this, a new generation of safe vaccines is being developed accommodating single, dominant antigens of pathogens of interest. In particular, the selection of such antigens is challenging, since due to HLA polymorphism the ligand specificities and immunodominance hierarchies of pathogen-specific CD8+ T cell responses differ throughout the human population. A recently discovered mechanism of proteasome-mediated CD8+ T cell epitope generation, i.e., by proteasome-catalyzed peptide splicing (PCPS), expands the pool of peptides and antigens, presented by MHC class I HLA molecules. On the cell surface, one-third of the presented self-peptides are generated by PCPS, which coincides with one-fourth in terms of abundance. Spliced epitopes are targeted by CD8+ T cell responses during infection and, like non-spliced epitopes, can be identified within antigen sequences using a novel in silico strategy. The existence of spliced epitopes, by enlarging the pool of peptides available for presentation by different HLA variants, opens new opportunities for immunotherapies and vaccine design.