MAGE-A4 TCR-like antibody IP in human lung and colon tissue
Ontology highlight
ABSTRACT: We chose to perform immune-enrichment from primary lung and colon tissue resections using both, anti-MAGE-A4 in IgG format and control (anti-HLA-DQ, clone SPVL3) antibodies.
Project description:To demonstrate the on-target binding of the MAGE-A4 antibody to the target HLAp GVYDGREHTV, we developed a reverse immunopeptidomics strategy that uses the HLAp-targeting antibody as a bait to enrich interacting HLAp from A375 xenografts. For this purpose, we immobilized the IgG-format of the MAGE-A4 TCR-like antibody on Protein A beads by cross-linking, and exposed the molecule to the solubilized proteome, including membrane bound HLAp, of the A375 xenografts. We subsequently eluted the interacting HLAp bound to the MAGE-A4 antibody and purified the HLA-associated peptides through a 5 kDa molecular weight filter before liquid chromatography tandem mass spectrometry analysis for sequence determination.
Project description:We chose to perform a head-to-head immune-enrichment from primary liver tissue resections using both, MAGE-A4 and ESK1 TCR-like antibodies. Since both target antigens MAGE-A4 and WT-1 are not expressed in hepatocytes (Human Protein Atlas, www.proteinatlas.org), the ESK1 enrichment could serve as isotype control for the MAGE-A4 antibody, and vice versa.
Project description:To demonstrate the on-target binding of the MAGE-A4 antibody to the target HLAp GVYDGREHTV, we developed a reverse immunopeptidomics strategy that uses the HLAp-targeting antibody as a bait to enrich interacting HLAp from A375 xenografts. For this purpose, we immobilized the IgG-format of the MAGE-A4 TCR-like antibody on Protein A beads by cross-linking, and exposed the molecule to the solubilized proteome, including membrane bound HLAp, of the A375 xenografts. We subsequently eluted the interacting HLAp bound to the MAGE-A4 antibody and purified the HLA-associated peptides through a 5 kDa molecular weight filter before liquid chromatography tandem mass spectrometry analysis for sequence determination.
Project description: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 β-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed β-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 β-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 islet-infiltrating CD8+ T-cells from T1D donors were reactive to some 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.
Project description: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 β-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed β-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 β-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 islet-infiltrating CD8+ T-cells from T1D donors were reactive to some 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.
Project description:Our extended analysis of the HLA-presented antigen landscape in cervical cancer cells using an integrative proteogenomics approach identifies, next to tumour-associated antigens and tumour-specific neoantigens, presentation of viral canonical, and alternative reading frame (ARF)-derived HLA-presented sequences, including peptides derived from HPV-E1.
Project description:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the presentation and immunogenicity of nonC antigens across different cancer types to better understand their contribution to cancer immunosurveillance and to address whether they could be exploited therapeutically. To this end, we employed a proteogenomics pipeline to identify nonC HLA-I ligands derived from off-frame translation of coding sequences and non-coding regions (UTR, ncRNA, intronic and intergenic) in patient-derived tumor cell lines (TCL) of different histological types (4 gynecological cancer, 3 melanoma and 2 head and neck cancer patients). First, peptides bound to HLA-I were isolated and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using state-of-the-art procedures. Amino acid (Aa) sequences were identified through the previously described pipeline Peptide-PRISM, with some modifications. Briefly, for each MS spectrum, the top 10 candidates were first identified by de novo sequencing and later mapped to a database including the 3-frame transcriptome and 6-frame genome. Additionally, whole-exome sequencing (WES) information of each TCL was included to interrogate the presentation of mutated peptides derived cancer-specific NSM. The false-discovery rate (FDR) was calculated independently for each category considering the search space and peptide length in a stratified mixture model as previously described. Next, in order to select nonC peptides preferentially presented by tumor cells, immunopeptidomics data from several healthy tissues and samples available from the HLA ligand atlas was used to filter out peptides presented in healthy tissues. To overcome a potential bias toward frequent alleles, the peptides were excluded at the ORF level rather than the Aa sequence. As a result, we found that from a total of 839 unique nonC peptides detected in our tumor samples, 38.5% were predicted to derive from ORFs also present in healthy tissue (nonC-HL). Hence, 61.6% (n=517) were considered preferentially presented on tumor HLA-I, referred to as non-canonical tumor ligands (nonC-TL). Out results showed that, although nonC-TL constitued the most abundant source of candidate tumor antigens, as compared to neoantigens, cancer-germline or melanoma-associated antigens, pre-existing antitumor T cells in cancer patients preferentially recognized neoantigens rather than nonC-TL. Nonetehless, nonC-TL elicited de novo T-cell responses via in vitro sensitization of donor lymphocytes. We identified TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of the C5orf22C. These immunogenic nonC-TL were expressed across tumor types but were barely or not detected in healthy cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they are attractive novel targets for widely applicable immunotherapies.