The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptide sequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer.

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

Interferon-α promotes neo-antigen formation and preferential HLA-B-restricted antigen presentation in pancreatic ß-cells.

Interferon (IFN)-α is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic ß-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed ß-cells. IFN-α increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting ß-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN-α only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and we identified islet-infiltrating CD8+ T-cells from T1D donors reactive to HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.

Novel canonical and non-canonical viral antigens extend current targets for immunotherapy of HPV-driven cervical cancer.

Current immunotherapeutic approaches for human papillomavirus (HPV)-driven cervical cancer target the viral oncogenes E6 and E7. We report viral canonical and alternative reading frame (ARF)-derived sequences presented on cervical tumor cells, including antigens encoded by the conserved viral gene E1. We confirm immunogenicity of the identified viral peptides in HPV-positive women, and women with cervical intraepithelial neoplasia. We observe consistent transcription of the E1, E6, and E7 genes in 10 primary cervical tumor resections from the four most common high-risk HPV subtypes (HPV16, 18, 31, and 45), suggesting the suitability of E1 as therapeutic target. We finally confirm HLA presentation of canonical peptides derived from E6 and E7, and ARF-derived viral peptides from a reverse-strand transcript spanning the HPV E1 and E2 genes in primary human cervical tumor tissue. Our results extend currently known viral immunotherapeutic targets in cervical cancer and highlight E1 as an important cervical cancer antigen.

Large-Scale Immunopeptidome Analysis Reveals Recurrent Posttranslational Splicing of Cancer- and Immune-Associated Genes.

Posttranslational spliced peptides (PTSPs) are a unique class of peptides that have been found to be presented by HLA class-I molecules in cancer. Thus far, no consensus has been reached on the proportion of PTSPs in the immunopeptidome, with estimates ranging from 2% to as high as 45% and stirring significant debate. Furthermore, the role of the HLA class-II pathway in PTSP presentation has been studied only in diabetes. Here, we exploit our large-scale cancer peptidomics database and our newly devised pipeline for filtering spliced peptide predictions to identify recurring spliced peptides, both for HLA class-I and class-II complexes. Our results indicate that HLA class-I-spliced peptides account for a low percentage of the immunopeptidome (less than 3.1%) yet are larger in number relative to other types of identified aberrant peptides. Therefore, spliced peptides significantly contribute to the repertoire of presented peptides in cancer cells. In addition, we identified HLA class-II-bound spliced peptides, but to a lower extent (less than 0.5%). The identified spliced peptides include cancer- and immune-associated genes, such as the MITF oncogene, DAPK1 tumor suppressor, and HLA-E, which were validated using synthetic peptides. The potential immunogenicity of the DAPK1- and HLA-E-derived PTSPs was also confirmed. In addition, a reanalysis of our published mouse single-cell clone immunopeptidome dataset showed that most of the spliced peptides were found repeatedly in a large number of the single-cell clones. Establishing a novel search-scheme for the discovery and evaluation of recurring PTSPs among cancer patients may assist in identifying potential novel targets for immunotherapy.

Long non-coding RNA-derived peptides are immunogenic and drive a potent anti-tumour response.

Protein arginine methyltransferase (PRMT) 5 is over-expressed in a variety of cancers and the master transcription regulator E2F1 is an important methylation target. We have explored the role of PRMT5 and E2F1 in regulating the non-coding genome and report here a striking effect on long non-coding (lnc) RNA gene expression. Moreover, many MHC class I protein-associated peptides were derived from small open reading frames in the lncRNA genes. Pharmacological inhibition of PRMT5 or adjusting E2F1 levels qualitatively altered the repertoire of lncRNA-derived peptide antigens displayed by tumour cells. When presented to the immune system as either ex vivo-loaded dendritic cells or expressed from a viral vector, lncRNA-derived peptides drove a potent antigen-specific CD8 T lymphocyte response, which translated into a significant delay in tumour growth. Thus, lncRNA genes encode immunogenic peptides that can be deployed as a cancer vaccine.

A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek's Disease.

The chicken MHC is known to confer decisive resistance or susceptibility to various economically important pathogens, including the iconic oncogenic herpesvirus that causes Marek's disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The class I molecule BF2*1901 is better expressed and more thermostable than the closely related BF2*1501, but the peptide motif was not simpler as expected. In this study, we confirm for newly developed chicken lines that the chicken MHC haplotype B15 confers resistance to MD compared with B19. Using gas phase sequencing and immunopeptidomics, we find that BF2*1901 binds a greater variety of amino acids in some anchor positions than does BF2*1501. However, by x-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Although the self-peptides that bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows stronger binding and thus more peptides overall, correlating with the expected hierarchies for expression level, thermostability, and MD resistance. Our study provides a reasonable explanation for greater promiscuity for BF2*1501 compared with BF2*1901, corresponding to the difference in resistance to MD.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

Immunopeptidomic Analysis of BoLA-I and BoLA-DR Presented Peptides from Theileria parva Infected Cells.

The apicomplexan parasite Theileria parva is the causative agent of East Coast fever, usually a fatal disease for cattle, which is prevalent in large areas of eastern, central, and southern Africa. Protective immunity against T. parva is mediated by CD8+ T cells, with CD4+ T-cells thought to be important in facilitating the full maturation and development of the CD8+ T-cell response. T. parva has a large proteome, with >4000 protein-coding genes, making T-cell antigen identification using conventional screening approaches laborious and expensive. To date, only a limited number of T-cell antigens have been described. Novel approaches for identifying candidate antigens for T. parva are required to replace and/or complement those currently employed. In this study, we report on the use of immunopeptidomics to study the repertoire of T. parva peptides presented by both BoLA-I and BoLA-DR molecules on infected cells. The study reports on peptides identified from the analysis of 13 BoLA-I and 6 BoLA-DR datasets covering a range of different BoLA genotypes. This represents the most comprehensive immunopeptidomic dataset available for any eukaryotic pathogen to date. Examination of the immunopeptidome data suggested the presence of a large number of coprecipitated and non-MHC-binding peptides. As part of the work, a pipeline to curate the datasets to remove these peptides was developed and used to generate a final list of 74 BoLA-I and 15 BoLA-DR-presented peptides. Together, the data demonstrated the utility of immunopeptidomics as a method to identify novel T-cell antigens for T. parva and the importance of careful curation and the application of high-quality immunoinformatics to parse the data generated.

Persistent but dysfunctional mucosal SARS-CoV-2-specific IgA and low lung IL-1ß associate with COVID-19 fatal outcome: A cross-sectional analysis.

The role of the mucosal pulmonary antibody response in coronavirus disease 2019 (COVID-19) outcome remains unclear. Here, we found that in bronchoalveolar lavage (BAL) samples from 48 patients with severe COVID-19-infected with the ancestral Wuhan virus, mucosal IgG and IgA specific for S1, receptor-binding domain (RBD), S2, and nucleocapsid protein (NP) emerged in BAL containing viruses early in infection and persist after virus elimination, with more IgA than IgG for all antigens tested. Furthermore, spike-IgA and spike-IgG immune complexes were detected in BAL, especially when the lung virus has been cleared. BAL IgG and IgA recognized the four main RBD variants. BAL neutralizing titers were higher early in COVID-19 when virus replicates in the lung than later in infection after viral clearance. Patients with fatal COVID-19, in contrast to survivors, developed higher levels of mucosal spike-specific IgA than IgG but lost neutralizing activities over time and had reduced IL-1ß in the lung. Altogether, mucosal spike and NP-specific IgG and S1-specific IgA persisting after lung severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance and low pulmonary IL-1ß correlate with COVID-19 fatal outcome. Thus, mucosal SARS-CoV-2-specific antibodies may have adverse functions in addition to protective neutralization. Highlights: Mucosal pulmonary antibody response in COVID-19 outcome remains unclear. We show that in severe COVID-19 patients, mucosal pulmonary non-neutralizing SARS-CoV-2 IgA persit after viral clearance in the lung. Furthermore, low lung IL-1ß correlate with fatal COVID-19. Altogether, mucosal IgA may exert harmful functions beside protective neutralization.

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome.

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.

Decitabine increases neoantigen and cancer testis antigen expression to enhance T-cell-mediated toxicity against glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Despite maximal treatment, median survival remains dismal at 14-24 months. Immunotherapies, such as checkpoint inhibition, have revolutionized management of some cancers but have little benefit for GBM patients. This is, in part, due to the low mutational and neoantigen burden in this immunogenically "cold" tumor. METHODS: U87MG and patient-derived cell lines were treated with 5-aza-2'-deoxycytidine (DAC) and underwent whole-exome and transcriptome sequencing. Cell lines were then subjected to cellular assays with neoantigen and cancer testis antigen (CTA) specific T cells. RESULTS: We demonstrate that DAC increases neoantigen and CTA mRNA expression through DNA hypomethylation. This results in increased neoantigen presentation by MHC class I in tumor cells, leading to increased neoantigen- and CTA-specific T-cell activation and killing of DAC-treated cancer cells. In addition, we show that patients have endogenous cancer-specific T cells in both tumor and blood, which show increased tumor-specific activation in the presence of DAC-treated cells. CONCLUSIONS: Our work shows that DAC increases GBM immunogenicity and consequent susceptibility to T-cell responses in vitro. Our results support a potential use of DAC as a sensitizing agent for immunotherapy.

The interdependence of machine learning and LC-MS approaches for an unbiased understanding of the cellular immunopeptidome.

INTRODUCTION: The comprehensive collection of peptides presented by major histocompatibility complex (MHC) molecules on the cell surface is collectively known as the immunopeptidome. The analysis and interpretation of such data sets holds great promise for furthering our understanding of basic immunology and adaptive immune activation and regulation, and for direct rational discovery of T cell antigens and the design of T-cell-based therapeutics and vaccines. These applications are, however, challenged by the complex nature of immunopeptidome data. AREAS COVERED: Here, we describe the benefits and shortcomings of applying liquid chromatography-tandem mass spectrometry (MS) to obtain large-scale immunopeptidome data sets and illustrate how the accurate analysis and optimal interpretation of such data is reliant on the availability of refined and highly optimized machine learning approaches. EXPERT OPINION: Further, we demonstrate how the accuracy of immunoinformatics prediction methods within the field of MHC antigen presentation has benefited greatly from the availability of MS-immunopeptidomics data, and exemplify how optimal antigen discovery is best performed in a synergistic combination of MS experiments and such in silico models trained on large-scale immunopeptidomics data.

Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection.

There is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells. IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.

HLA variants have different preferences to present proteins with specific molecular functions which are complemented in frequent haplotypes.

Human leukocyte antigen (HLA) genes are the most polymorphic loci in the human genome and code for proteins that play a key role in guiding adaptive immune responses by presenting foreign and self peptides (ligands) to T cells. Each person carries up to 6 HLA class I variants (maternal and paternal copies of HLA-A, HLA-B and HLA-C genes) and also multiple HLA class II variants, which cumulatively define the landscape of peptides presented to T cells. Each HLA variant has its own repertoire of presented peptides with a certain sequence motif which is mainly defined by peptide anchor residues (typically the second and the last positions for HLA class I ligands) forming key interactions with the peptide-binding groove of HLA. In this study, we aimed to characterize HLA binding preferences in terms of molecular functions of presented proteins. To focus on the ligand presentation bias introduced specifically by HLA-peptide interaction we performed large-scale in silico predictions of binding of all peptides from human proteome for a wide range of HLA variants and established which functions are characteristic for proteins that are more or less preferentially presented by different HLA variants using statistical calculations and gene ontology (GO) analysis. We demonstrated marked distinctions between HLA variants in molecular functions of preferentially presented proteins (e.g. some HLA variants preferentially present membrane and receptor proteins, while others - ribosomal and DNA-binding proteins) and reduced presentation of extracellular matrix and collagen proteins by the majority of HLA variants. To explain these observations we demonstrated that HLA preferentially presents proteins enriched in amino acids which are required as anchor residues for the particular HLA variant. Our observations can be extrapolated to explain the protective effect of certain HLA alleles in infectious diseases, and we hypothesize that they can also explain susceptibility to certain autoimmune diseases and cancers. We demonstrate that these differences lead to differential presentation of HIV, influenza virus, SARS-CoV-1 and SARS-CoV-2 proteins by various HLA alleles. Taking into consideration that HLA alleles are inherited in haplotypes, we hypothesized that haplotypes composed of a combination of HLA variants with different presentation preferences should be more advantageous as they allow presenting a larger repertoire of peptides and avoiding holes in immunopeptidome. Indeed, we demonstrated that HLA-A/HLA-B and HLA-A/HLA-C haplotypes which have a high frequency in the human population are comprised of HLA variants that are more distinct in terms of functions of preferentially presented proteins than the control pairs.

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

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

Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

The Choice of Search Engine Affects Sequencing Depth and HLA Class I Allele-Specific Peptide Repertoires.

Standardization of immunopeptidomics experiments across laboratories is a pressing issue within the field, and currently a variety of different methods for sample preparation and data analysis tools are applied. Here, we compared different software packages to interrogate immunopeptidomics datasets and found that Peaks reproducibly reports substantially more peptide sequences (~30-70%) compared with Maxquant, Comet, and MS-GF+ at a global false discovery rate (FDR) of <1%. We noted that these differences are driven by search space and spectral ranking. Furthermore, we observed differences in the proportion of peptides binding the human leukocyte antigen (HLA) alleles present in the samples, indicating that sequence-related differences affected the performance of each tested engine. Utilizing data from single HLA allele expressing cell lines, we observed significant differences in amino acid frequency among the peptides reported, with a broadly higher representation of hydrophobic amino acids L, I, P, and V reported by Peaks. We validated these results using data generated with a synthetic library of 2000 HLA-associated peptides from four common HLA alleles with distinct anchor residues. Our investigation highlights that search engines create a bias in peptide sequence depth and peptide amino acid composition, and resulting data should be interpreted with caution.