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Multiplexed relative and absolute quantitative immunpeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition [I]

ABSTRACT: Major histocompatibility complex (MHC) class I peptides play a critical role in immune cell recognition. Cancer cells modulate surface MHC levels in response to therapy, thereby affecting antitumor immunity. However, understanding the peptide repertoire response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging for quantitation of peptide repertoires using low sample input. HipMHCs improve quantitative accuracy by normalizing for variation across analyses, and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens. Application of this platform to profile the immunopeptidome response to CDK4/6 inhibition and Interferon gamma, known modulators of antigen presentation, uncovered treatment-specific alterations that connect the intracellular response to extracellular immune presentation. This method quantifies repertoire changes that can inform targeted and combination immunotherapy design.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144373 | GEO | 2020/04/29

REPOSITORIES: GEO

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Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

Project description:Major histocompatibility complex (MHC) class I peptides play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be modulated by anticancer agents, altering immunity. However, understanding the peptide repertoire response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform targeted and combination immunotherapy design. Applying this platform to profile the immunopeptidome response to CDK4/6 inhibition and Interferon gamma, known modulators of antigen presentation, uncovered treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.

2020-06-04 | PXD017407 | Pride

Editing the immunopeptidome of melanoma cells using a potent inhibitor of ER aminopeptidase 1

Project description:The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, is often limited by ineffective presentation of antigenic peptides that can elicit T-cell mediated anti-tumor cytotoxic responses. Therefore, manipulating antigen presentation is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides that can bind onto MHC class I molecules (MHC-I). We hypothesized that pharmacologically inhibiting ERAP1 in cells can regulate the global cellular immunopeptidome. To test this hypothesis, we treated the A375 melanoma cell line with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting the bound peptides and identifying them using capillary chromatography and tandem mass spectrometry. Although the inhibitor did not negatively affect overall MHC-I presence on the cell surface, it induced significant changes on the presented peptidomes, both at the qualitative and quantitative levels. Specifically, inhibitor treatment altered about half of the total 3204 identified peptides and about one third of the peptides predicted to be good ligands for MHC-I, affected length and sequence without however interfering with basic binding motifs. Strikingly, the inhibitor enhanced overall MHC-I binding affinity by reducing presentation of sub-optimal long peptides and generating many high-affinity 9-12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 is a valid approach for manipulating the immunopeptidome of cancer and autoimmunity.

2019-06-24 | PXD011766 | Pride

Integrative MHC-I peptidome quantitation with isobaric tagging mass spectrometry

Project description:Despite the appreciation of CD8+ T cell anti-tumor immune responses towards improvement patient outcomes, the MHC-I peptides that facilitate the response are poorly described. Alternatively, whether cancer therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. In this regard, anthracyclines and ionizing radiation both increase the infiltration of CD8+ T cells into tumors but whether they do so by altering the MHC-I peptidome repertoire is not known. To investigate this, we developed a platform for screening therapy-induced MHC-I peptides by quantitative, multiplexed measurement of MHC-I peptides with tandem mass tags (TMT). We show that both ionizing radiation and the anthracycline doxorubicin induce MHC-I peptides that are largely derived from mitotic progression and cell cycle proteins. Our approach enables further strategies to understand how small molecules and other therapies alter MHC-I peptide presentation that may be harnessed for CD8+ T cell-based immunotherapies.

2019-03-05 | PXD011464 | Pride

Integrative MHC-I peptidome quantitation with isobaric tagging mass spectrometry

2019-03-05 | PXD011444 | Pride

Deciphering the immunopeptidome in vivo reveals novel tumor antigens

Project description:Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the “immunopeptidome”. Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of cancer cell-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP/KbStrep). This novel approach has allowed us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome from the alveolar type 2 cell-of-origin through late-stage disease. Differential peptide presentation in LUAD is not driven by increased mRNA expression or translation rate and is likely driven by post-translational mechanisms. Vaccination of mice with peptides presented by LUAD in vivo provoked CD8 T-cell responses in naïve and tumor bearing mice. Many peptides unique to LUAD, including immunogenic peptides, exhibited very low expression of the cognate mRNA provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

2022-06-20 | PXD033232 | Pride

Mapping of immunopeptidome of cancer immunotherapy with oncolytic virus and checkpoint blockade

Project description:The elucidation of therapy-induced changes to the class I major histocompatibility complex (MHC-I)-bound tumor antigens is crucial for understanding immune-mediated tumor eradication and identifying potential targets for peptide vaccines to enhance the efficacy of immunotherapies. Here, we investigated how oncolytic reovirus therapy with and without immune checkpoint blockade (ICB) alters the tumor peptide-MHC repertoire. Using mass spectrometry analysis of immunoaffinity purified MHC peptides, we first showed that changes to the MHC immunopeptidome following reovirus treatment is cancer type-dependent, where a murine fibrosarcoma model displayed quantitative and qualitative variance in differentially expressed peptides (DEPs) as compared to those identified in a murine ovarian cancer model. We then determined that the combination therapy of reovirus and ICB in the fibrosarcoma model resulted in higher numbers of DEPs relative to either monotherapy alone. Most importantly, we identified reovirus and ICB-induced MHC peptides that are biologically active in stimulating interferon-gamma response in cognate CD8+ T cells, which likely contribute to cancer immunoediting. These findings highlight the importance of therapy-induced changes to the MHC immunopeptidome in shaping the underlying anti-tumor immune responses during reovirus and ICB combination therapy.

2021-12-06 | PXD024369 | Pride

Transcription profiling and proteomic profiling of human B-LCL cell lines (TAP1/2 +/+ and TAP1/2 -/-) to analyse HLA peptide repertoire

Project description:The TAP transporter is responsible for transferring cytosolic peptides into the ER where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC surface expression and alters the presented peptide pattern. Using the TAP deficient cell line LCL721.174 and its TAP expressing progenitor cell line LCL721.45, we have identified and quantified more than 160 HLA ligands, 50 out of which were presented TAP independently. Peptides which were predominantly presented on the TAP deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded that different HLA presentation ratios were due to varying expression of the respective protein or due to changes in the antigen loading complex. Features of TAP-independently presented peptides as well as proteasomal contribution to their generation provides an insight into basic immunological mechanisms. Experiment Overall Design: Two B-LCL cell lines (TAP1/2 +/+ and TAP1/2 -/-) were compared in their gene and protein expression as well as in their HLA peptide repertoire

2008-06-16 | E-GEOD-9437 | biostudies-arrayexpress

ERAP2 Increases the Abundance of a Peptide Submotif Highly Selective for the Birdshot Uveitis-Associated HLA-A29

Project description:Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patientderived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU.

2021-09-10 | PXD024109 | Pride

Distinct modulation of cellular immunopeptidome by the allosteric regulatory site of ER aminopeptidase 1

Project description:ER aminopeptidase 1 (ERAP1) is an ER-resident aminopeptidase that excises N-terminal residues off peptides that then bind onto Major Histocompatibility Complex I molecules (MHC-I) and indirectly modulates adaptive immune responses. ERAP1 contains an allosteric regulatory site that accommodates the C-terminus of only some peptide substrates, raising questions about its exact influence on antigen presentation and the potential of allosteric inhibition for cancer immunotherapy. We used an inhibitor that targets this regulatory site to study its effect on the immunopeptidome of a human cancer cell line. The immunopeptidomes of allosterically inhibited and ERAP1 knockout cells contain high-affinity peptides with sequence motifs consistent with the cellular HLA class I haplotypes, but were strikingly different in peptide composition. Compared to knockout cells, allosteric inhibition did not affect the length distribution of peptides and skewed the peptide repertoire both in terms of sequence motifs and HLA allele utilization, indicating significant mechanistic differences between the two ways of disrupting ERAP1 function. These findings suggest that the regulatory site of ERAP1 plays distinct roles in antigenic peptide selection, which should be taken into consideration when designing therapeutic interventions targeting the cancer immunopeptidome.

2023-11-21 | PXD039584 | Pride

Induced pluripotent stem cells display a unique set of MHC I-associated peptides shared by cancer cells

Project description:We used a proteogenomic approach combining RNA-sequencing and mass spectrometry to study the MHC I-immunopeptidome specific to human iPSC samples. iPSCs were also analyzed after a 72h treatment with IFN-g (IFN-gamma) to boost MHC I levels and increase our chance for pluripotency-associated MHC I-associated peptide (paMAP) discovery. We identified 46 paMAPs which were absent from the RNA-seq of healthy somatic tissues and adult stem cells, but 40 of them were expressed in multiple cancer types from The Cancer Genome Atlas (TCGA). The antigens shared by iPSCs and tumors represent attractive targets for immunotherapy of poorly differentiated cancers.

2022-07-20 | GSE171226 | GEO

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