Various cancer immunotherapies rely on the T cell recognition of peptide antigens presented on human leukocyte antigens (HLA). However, the identification and selection of naturally presented peptide targets for the development of personalized as well as off-the-shelf immunotherapy approaches remains challenging. Here, we introduce the open-access Peptides for Cancer Immunotherapy Database (PCI-DB, https://pci-db.org/), a comprehensive resource of immunopeptidome data originating from various malignant and benign primary tissues that provides the research community with a convenient tool to facilitate the identification of peptide targets for immunotherapy development. The PCI-DB includes > 6.6 million HLA class I and > 3.4 million HLA class II peptides from over 40 tissue types and cancer entities analyzed uniformly using high-sensitive nf-core bioinformatics pipelines and applying a global peptide false discovery rate (FDR) approach. First application of the database provided insights into the representation of cancer-testis antigens (CTA) across malignant and benign tissues and enabled the identification and characterization of the cross-tumor entity and entity-specific tumor-associated antigens as well as naturally presented neoepitopes from frequent cancer mutations. Further, we used the PCI-DB to design personalized peptide vaccines for two patients suffering from metastatic cancer. PCI-DB enabled the composition of both a multi-peptide vaccine comprising non-mutated, highly frequent tumor-associated antigens matching the immunopeptidome of the individual patient´s tumor and a neoepitope-based vaccine matching the mutational profile of the cancer patient. Both vaccine approaches induced potent and long-lasting T-cell responses, accompanied by long-term survival of these advanced cancer patients. In conclusion, the PCI-DB provides a highly versatile tool to broaden the understanding of cancer-related antigen presentation and, ultimately, supports the development of novel immunotherapies.

Additional file 8: Detailed data of peptides binding affinity to HLA-I molecules.

Defining the repertoire of peptides presented by the major histocompatibility complex class I (MHC I) is a key step toward the identification of relevant antigens for cancer immunotherapy. However, the identification of cancer-specific antigens is a significant analytical challenge in view of their low abundance and low mutational load found in most primary cancer specimens. Here, we describe the application of isobaric peptide labeling with tandem mass tag (TMT) to improve the detection of the MHC I peptides. Isobaric peptide labeling was found to promote the formation of multiply charged ions and to enhance the formation of b-type fragment ions, thus resulting in a 50% improvement of MHC I peptide identification. The gain in sensitivity obtained using TMT labeling enabled the detection of low-abundance MHC I peptides including tumor-specific antigens (TSAs) and minor histocompatibility antigens (MiHAs). We further demonstrate the application of this approach to quantify MiHAs presented by B-cell lymphocytes and determined their expression levels by LC-MS/MS using both synchronous precursor selection (SPS) and high-field asymmetric waveform ion mobility spectrometry (FAIMS).

Additional file 5: Detailed data of HLA-I class I peptides Gibbs cluster.

We propose a new pipeline for the refinement of spectrum-peptide assignment, designed specifically for MHC ligand identification. By modeling the peptidome as a collection of a limited number of specificities, corresponding to the MHC alleles of the cell line, our method achieves increased sequencing depth, while at the same time removing potential experimental outliers and contaminants.

In Dendritic cells (DC), the MHC II eluted immunopeptidome reflects the antigenic composition of the microenvironment. Proteins are transported and processed into peptides in endosomal MHC II compartments through autophagy or phagocytosis; extracellular peptides can also directly bind MHC II proteins at the cell surface. Altogether, these mechanisms allow DC to sample both the intra and extracellular environment. With an increase in mass spectrometry sensitivity and accuracy, we can now finally tackle important questions on the nature and plasticity of the MHC-II immunopeptidome in health and disease. Presented epitopes, neoepitopes, and PTM-modified epitopes can be quantitatively and qualitatively analyzed to provide a comprehensive picture of DC role in immunosurveillance. To determine whether the redox metabolic conditions induce an altered spectrum of presented peptides, we eluted immunoaffinity-purified I-Ab from conventional dendritic cells isolated from control B6 or obese Ob/Ob mice, and analyzed MHC-II-associated peptides by LC/MS/MS using combined data-dependent (DDA) and data-independent acquisition (DIA) approaches. We analyzed the DIA data by employing a reference spectral library consisting of all peptides identified by database matching in the pool of spectra from combined DDA dataset, thus allowing a direct label-free quantitation of relative abundances between the two sample categories. The quantitative analysis of the I-Ab-eluted immunopeptidomes pinpoint important differences in peptide presentation and epitope selection in obese mice.

epitope description:SNTYTFPNPAHPPG,antigen name:Probable G-protein coupled receptor 132,host organism:Mus musculus C57BL/6,mhc allele name:H2-IAb

Additional file 8: Detailed data of peptides binding affinity to HLA-I molecules.

epitope description:SLLSSVFKL,antigen name:Uncharacterized aarF domain-containing protein kinase 2,host organism:Homo sapiens,mhc allele name:HLA-A*02:01

Immunotherapy has shown great therapeutic potential for cancers with high tumor mutational burden (TMB), but much less promise for cancers with low TMB. One primary approach for adoptive lymphocyte transfer-based immunotherapy is to target the somatic mutated peptide neoantigens and cancer testis (CT) antigens recognized by cytotoxic T cells. Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class ǀ- associated peptides in both melanoma, a “hot tumor”, and EGFR mutant lung adenocarcinoma, a “cold tumor”. We uncovered 19 common driver oncogene-derived peptides and more than 1000 post-translationally modified peptides (PTM) representing 58 different PTMs. We constructed a CT antigen database with 286 antigens by compiling reputed CT antigen resources and “in-house” genomic data and used this to identify 45 CT antigen-derived peptides from the identified HLA peptidome. Using integrated next generation sequencing data, we discovered 12 neopeptides in EGFR mutant lung cancer cell lines. Finally, we report a novel approach for non-canonical peptide discovery, whereby we leveraged a deep learning-based de novo search and a high confidence annotated long noncoding RNA (LncRNA) database to identify 44 lncRNA-derived peptides. Findings of this study, for the first time, provide evidence for a large pool of actionable cancer antigen-derived peptides for use in mutant EGFR lung cancer immunotherapy.

WES sequencing of multiple regions per tumor from 8 lung cancer patients (LUSC, LCNEC and LUAD) and adjacent healthy lung tissue for each patient.

HLA-DQ molecules can be formed as both cis and trans variants. So far, the progress for predicting HLA-DQ antigen presentation has been limited. In addition, the contribution of trans-only variants in shaping the HLA-DQ immunopeptidome remains largely unresolved. Here, we address these issues by integrating state-of-the-art immunoinformatics data mining models with large volumes of high-quality HLA-DQ specific immunopeptidomics data. The analysis demonstrated a highly improved predictive power and molecular coverage for models trained with these novel HLA-DQ data and a limited to no contribution for trans-only HLA-DQ variants to the overall HLA-DQ immunopeptidome.

The proteasome generates the epitopes presented on human leukocyte antigen (HLA) class I molecules that elicit CD8+ T cell responses. Reports of proteasome-generated spliced epitopes exist, but they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one-third of the entire HLA class I immunopeptidome in terms of diversity and one-fourth in terms of abundance. This pool also represents a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, as well as multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events has implications for immunobiology and autoimmunity theories and may provide a previously untapped source of epitopes for use in vaccines and cancer immunotherapy.

Biomaterials play an increasing role in clinical applications and regenerative medicine. Modern biomaterials should imitate the function of damaged tissue without triggering an immune response, initiate self-regeneration of the body and gradually degrade after implantation. The immune system is now well recognized to play a major role in influencing the biocompatibility of implanted medical devices. To develop a better understanding of the effects of biomaterials on the immune response, we have developed a highly sensitive novel test system capable of examining changes in the immune system by biomaterial. Here, we evaluate for the first time the immunopeptidome, a highly sensitive system that reflects cancer transformation, virus or drug influences and passes these changes directly to T cells, as a test system to examine the effects of contact with materials. Since monocytes are one of the first immune cells reacting to biomaterials, we have tested the influence of different materials - stainless steel, aluminum, zinc, high-density polyethylene, polyurethane films containing zinc diethyldithiocarbamate, copper, and zinc sulphate - on the monocytic THP-1 cell line. Material-associated peptides were identified after stimulation with all material types examined. The magnitude of induced changes in the immunopeptidome after the stimulation appears comparable to that of bacterial lipopolysaccharides (LPS). The source proteins of many detected peptides are associated with cytotoxicity, fibrosis, autoimmunity, inflammation and cellular stress. Considering all tested materials, it was found that the LPS-induced cytotoxicity-, inflammation- and cellular stress-associated HLA class I peptides were mainly induced by aluminum and HLA class II peptides mainly by stainless steel. These findings provide some of the first insights into the effects on the immunopeptidome by biomaterials. A more thorough understanding of these effects may enable the design of more biocompatible implant materials using in vitro models in future. This may be achieved through developing a deeper understanding of possible immune responses induced by biomaterials such as fibrosis, inflammation, cytotoxicity, and autoimmune reactions.

Characterisation of peptide ligands of Major histocompatibility class (MHC) I isolated by immunoaffinity purification from the C1R (Class I reduced) B-lymphoblastoid cell line, transfected with the MHC class I allele HLA-B*57:03.

Additional file 3: Observed and predicted retention time of HLA-I class I peptides.

MHC I-associated peptides (MAPs) presented at the surface of nucleated cells play a central role in CD8 T-cell immunosurveillance. MAPs presented by mature (i.e. MHC IIhi) medullary thymic epithelial cells (mTEChi) are essential to eliminate self-reactive CD8 T cells in a process called central tolerance. On tumor cells, MAPs that do not induce tolerance (i.e. non-tolerogenic MAPs), because absent from mTEChi or any other normal cells, are referred to as tumor-specific antigens (TSAs). Despite their clinical relevance, very few have been identified, even in highly mutated tumor types. Thus, we developed a novel proteogenomic workflow able to characterize the full TSA landscape of any tumor. Briefly, using RNA-seq data, we subtracted the mTEChi from the tumor signal to generate tumor-specific protein databases enriched in non-tolerogenic sequences. Using these databases to analyze the MAP repertoire of two murine cell lines (CT26 and EL4) sequenced by mass spectrometry, we identified a total of 21 TSAs, 90% of which derived from allegedly non-coding regions. Interestingly, our results highlighted that 70% of those TSAs derived from non-mutated yet tumor-restricted sequences, e.g. endogenous retroelements. Moreover, we showed that our approach is easily amenable to analyze human primary samples as we were able to identify TSAs in three lung tumor biopsies and four B-ALL specimens. Focusing on 5 TSAs, we demonstrated that both TSA expression and TSA-specific T-cell frequency in the pre-immune repertoire influenced the overall survival of pre-immunized tumor-bearing mice. In conclusion, this proof-of-concept study demonstrates that non-coding-derived TSAs are frequent and protective in vivo, while they could be shared by several individuals. Altogether, our findings will help expand the repertoire of human TSAs and facilitate their prioritization in the clinic.

Here, we describe a secreted HLA (sHLA) Fc-fusion construct for simple single HLA allele profiling in hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. This method streamlines sample preparation, enables temporal control, and provides allele-restricted target identification. Over 30,000 unique HLA-associated peptides were identified across two different HLA alleles and seven cell lines, with ~9,300 peptides newly discovered. The sHLA Fc-fusion capture technology holds potential to expedite immunopeptidomics and advance therapeutic interest in peptide-HLA complexes.

Human leukocyte antigen (HLA) molecules play a crucial role in the development of adaptive immune responses and therefore have been of major interest in the development of immunotherapies such as cancer vaccines and chimeric antigen receptor (CAR) T cells. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster growth for these personalised solutions. We herein describe a novel immunopeptidomics workflow involving the Kingfisher platform (Thermo Scientific) to isolate immunopeptidomes with anti-HLA antibodies coupled to a proprietary hyper-porous protein A microparticle (MagReSyn®), and a modified variable window data independent acquisition (DIA) method. Using the workflow, we were able to identify ~1500 peptides in as low as 5e6 cells. Overall, the robustness of this workflow will be very important for the future of the immunopeptidome profiling, especially for smaller sample types, and will be a useful tool for the development of immunotherapies and other precision medicine approaches.

In Dendritic cells (DC), the MHC II eluted immunopeptidome reflects the antigenic composition of the microenvironment. Proteins are transported and processed into peptides in endosomal MHC II compartments through autophagy or phagocytosis; extracellular peptides can also directly bind MHC II proteins at the cell surface. Altogether, these mechanisms allow DC to sample both the intra and extracellular environment. With an increase in mass spectrometry sensitivity and accuracy, we can now finally tackle important questions on the nature and plasticity of the MHC-II immunopeptidome in health and disease. Presented epitopes, neoepitopes, and PTM-modified epitopes can be quantitatively and qualitatively analyzed to provide a comprehensive picture of DC role in immunosurveillance. To determine whether the redox metabolic conditions induce an altered spectrum of presented peptides, we eluted immunoaffinity-purified I-Ab from conventional dendritic cells isolated from B6 mice on normal or high fat/high fructose (HFHF) diet and analyzed MHC-II-associated peptides by nano LC-MS/MS using data-dependent (DDA) acquisition approaches. Our results show that the environment-driven lipotoxicity and glucotoxicity induced an MHC-class-II immunopeptidome enriched in peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and responses to redox mediated cellular stress. The quantitative analysis of the I-Ab-eluted immunopeptidomes pinpoint important differences in peptide presentation and epitope selection in mice subjected to a diet rich in saturated fat and carbohydrates, which in turn, could be responsible for inducing a low-grade chronic inflammation in several organs including nonalcoholic steatohepatitis.