Profiling SARS-CoV-2 HLA-I peptidome reveals T cell epitopes from out-of-frame ORFs.

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.

Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.

Within the tumour microenvironment, CD4+ T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules1,2, but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4+ T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4+ T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4+ T regulatory (TReg) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4+ TReg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4+ T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4+ TReg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

The peptide woods are lovely, dark and deep: Hunting for novel cancer antigens.

Harnessing the patient's immune system to control a tumor is a proven avenue for cancer therapy. T cell therapies as well as therapeutic vaccines, which target specific antigens of interest, are being explored as treatments in conjunction with immune checkpoint blockade. For these therapies, selecting the best suited antigens is crucial. Most of the focus has thus far been on neoantigens that arise from tumor-specific somatic mutations. Although there is clear evidence that T-cell responses against mutated neoantigens are protective, the large majority of these mutations are not immunogenic. In addition, most somatic mutations are unique to each individual patient and their targeting requires the development of individualized approaches. Therefore, novel antigen types are needed to broaden the scope of such treatments. We review high throughput approaches for discovering novel tumor antigens and some of the key challenges associated with their detection, and discuss considerations when selecting tumor antigens to target in the clinic.

The OTUD6B-LIN28B-MYC axis determines the proliferative state in multiple myeloma.

Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle-specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S-phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post-transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS-multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B-LIN28B axis.

Sensitive, High-Throughput HLA-I and HLA-II Immunopeptidomics Using Parallel Accumulation-Serial Fragmentation Mass Spectrometry.

Comprehensive and in-depth identification of the human leukocyte antigen class I (HLA-I) and class II (HLA-II) tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is a powerful technology for direct identification of HLA peptides from patient-derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare and clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high-throughput, sensitive, and single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight MS on the Bruker timsTOF single-cell proteomics system (SCP). We demonstrate greater than twofold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation, and reduces input requirements to as few as 1e6 A375 cells for >800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen and noncanonical proteins. We also apply our optimized single-shot SCP acquisition methods to tumor-derived samples, enabling sensitive, high-throughput, and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

MS-Based HLA-II Peptidomics Combined With Multiomics Will Aid the Development of Future Immunotherapies.

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.

Interactions between Transport Protein Particle (TRAPP) complexes and Rab GTPases in Arabidopsis.

Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little information is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab-A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP-bound) versus wild-type or constitutively active (GTP-bound) RAB-A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyzes of Rab-A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB-A2a, allowing us to propose that TRAPPII is likely to behave as a guanine nucleotide exchange factor (GEF) for the RAB-A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP-bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF-Rab interactions will be crucial to unravel the co-ordination of plant membrane traffic.

Evaluation of Advanced Precursor Determination for Tandem Mass Tag (TMT)-Based Quantitative Proteomics across Instrument Platforms.

Tandem mass tag (TMT)-based quantitation is a strong modality for quantitative proteomics, as samples can be multiplexed, creating large-scale data sets with high precision and minimal missing values. However, coisolation/cofragmentation of near isobaric, coeluting precursor peptide analytes has been well-documented to show ratio compression, compromising the accuracy of peptide/protein quantitation. Advanced peak determination (APD) is a new peak-picking algorithm that shows improved identification of peak detection in survey scans (MS1) to increase the number of precursors selected for unimolecular dissociation (MS2). To increase the number of these "features" selected for MS2 APD purposefully selects multiple peptide precursors of very similar m/ z that often derive from different proteins-a major source of ratio compression in TMT quantification. Here, we evaluate the effects of various data acquisition parameters combined with APD on ratio compression. We find that data acquisition with APD enabled results in more coisolated precursors, more mixed spectra, and in turn, fewer peptide spectral matches, especially at standard on-column loads. We conclude that APD should not be utilized for isobaric tagging, MS2-based experiments.

Pharmacoproteomic characterisation of human colon and rectal cancer.

Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.

Cell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis.

Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between cell cycle cues, membrane trafficking and microtubule dynamics. Plant cytokinesis occurs within a transient membrane compartment known as the cell plate, to which vesicles are delivered by a plant-specific microtubule array, the phragmoplast. While membrane proteins required for cytokinesis are known, how these are coordinated with microtubule dynamics and regulated by cell cycle cues remains unclear. Here, we document physical and genetic interactions between Transport Protein Particle II (TRAPPII) tethering factors and microtubule-associated proteins of the PLEIADE/AtMAP65 family. These interactions do not specifically affect the recruitment of either TRAPPII or MAP65 proteins to the cell plate or midzone. Rather, and based on single versus double mutant phenotypes, it appears that they are required to coordinate cytokinesis with the nuclear division cycle. As MAP65 family members are known to be targets of cell cycle-regulated kinases, our results provide a conceptual framework for how membrane and microtubule dynamics may be coordinated with each other and with the nuclear cycle during plant cytokinesis.

Phosphoproteome Profiling Reveals Molecular Mechanisms of Growth-Factor-Mediated Kinase Inhibitor Resistance in EGFR-Overexpressing Cancer Cells.

Although substantial progress has been made regarding the use of molecularly targeted cancer therapies, resistance almost invariably develops and presents a major clinical challenge. The tumor microenvironment can rescue cancer cells from kinase inhibitors by growth-factor-mediated induction of pro-survival pathways. Here we show that epidermal growth factor receptor (EGFR) inhibition by Gefitinib is counteracted by growth factors, notably FGF2, and we assessed the global molecular consequences of this resistance at the proteome and phosphoproteome level in A431 cells. Tandem mass tag peptide labeling and quantitative mass spectrometry allowed the identification and quantification of 22 000 phosphopeptides and 8800 proteins in biological triplicates without missing values. The data show that FGF2 protects the cells from the antiproliferative effect of Gefitinib and largely prevents reprogramming of the proteome and phosphoproteome. Simultaneous EGFR/FGFR or EGFR/GSG2 (Haspin) inhibition overcomes this resistance, and the phosphoproteomic experiments further prioritized the RAS/MEK/ERK as well as the PI3K/mTOR axis for combination treatment. Consequently, the MEK inhibitor Trametinib prevented FGF2-mediated survival of EGFR inhibitor-resistant cells when used in combination with Gefitinib. Surprisingly, the PI3K/mTOR inhibitor Omipalisib reversed resistance mediated by all four growth factors tested, making it an interesting candidate for mitigating the effects of the tumor microenvironment.

DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.

We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.

Optimized chemical proteomics assay for kinase inhibitor profiling.

Solid supported probes have proven to be an efficient tool for chemical proteomics. The kinobeads technology features kinase inhibitors covalently attached to Sepharose for affinity enrichment of kinomes from cell or tissue lysates. This technology, combined with quantitative mass spectrometry, is of particular interest for the profiling of kinase inhibitors. It often leads to the identification of new targets for medicinal chemistry campaigns where it allows a two-in-one binding and selectivity assay. The assay can also uncover resistance mechanisms and molecular sources of toxicity. Here we report on the optimization of the kinobead assay resulting in the combination of five chemical probes and four cell lines to cover half the human kinome in a single assay (∼ 260 kinases). We show the utility and large-scale applicability of the new version of kinobeads by reprofiling the small molecule kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil, Nilotinib, Ibrutinib, Imatinib, and Sunitinib.

Systematic identification of minor histocompatibility antigens predicts outcomes of allogeneic hematopoietic cell transplantation.

T cell alloreactivity against minor histocompatibility antigens (mHAgs)-polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms-is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D-R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D-R pairs demonstrated that total and organ-specific mHAg load could independently predict the occurrence of acute GvHD and chronic pulmonary GvHD, respectively, and defined promising GvL targets, confirmed in a validation cohort of 58 D-R pairs, for the prevention or treatment of post-transplant disease recurrence.

MScDB: a mass spectrometry-centric protein sequence database for proteomics.

Protein sequence databases are indispensable tools for life science research including mass spectrometry (MS)-based proteomics. In current database construction processes, sequence similarity clustering is used to reduce redundancies in the source data. Albeit powerful, it ignores the peptide-centric nature of proteomic data and the fact that MS is able to distinguish similar sequences. Therefore, we introduce an approach that structures the protein sequence space at the peptide level using theoretical and empirical information from large-scale proteomic data to generate a mass spectrometry-centric protein sequence database (MScDB). The core modules of MScDB are an in-silico proteolytic digest and a peptide-centric clustering algorithm that groups protein sequences that are indistinguishable by mass spectrometry. Analysis of various MScDB uses cases against five complex human proteomes, resulting in 69 peptide identifications not present in UniProtKB as well as 79 putative single amino acid polymorphisms. MScDB retains ~99% of the identifications in comparison to common databases despite a 3-48% increase in the theoretical peptide search space (but comparable protein sequence space). In addition, MScDB enables cross-species applications such as human/mouse graft models, and our results suggest that the uncertainty in protein assignments to one species can be smaller than 20%.

Pan-viral ORFs discovery using Massively Parallel Ribosome Profiling.

Unveiling the complete proteome of viruses is crucial to our understanding of the viral life cycle and interaction with the host. We developed Massively Parallel Ribosome Profiling (MPRP) to experimentally determine open reading frames (ORFs) in 20,170 designed oligonucleotides across 679 human-associated viral genomes. We identified 5,381 ORFs, including 4,208 non-canonical ORFs, and show successful detection of both annotated coding sequences (CDSs) and reported non-canonical ORFs. By examining immunopeptidome datasets of infected cells, we found class I human leukocyte antigen (HLA-I) peptides originating from non-canonical ORFs identified through MPRP. By inspecting ribosome occupancies on the 5'UTR and CDS regions of annotated viral genes, we identified hundreds of upstream ORFs (uORFs) that negatively regulate the synthesis of canonical viral proteins. The unprecedented source of viral ORFs across a wide range of viral families, including highly pathogenic viruses, expands the repertoire of vaccine targets and exposes new cis-regulatory sequences in viral genomes.

Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry.

Comprehensive, in-depth identification of the human leukocyte antigen HLA-I and HLA-II tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is powerful technology for direct identification of HLA peptides from patient derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare, clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high throughput, sensitive, single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight mass spectrometry on the Bruker timsTOF SCP. We demonstrate >2-fold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation and reduces input requirements to as few as 1e6 A375 cells for > 800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen, and novel/unannotated open reading frames. We also apply our optimized single-shot SCP acquisition methods to tumor derived samples, enabling sensitive, high throughput and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.