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:Ewing Sarcoma (EwS) is a rare pediatric malignancy characterized by a unique t(11:22) (q24;q12) translocation resulting in the EWSR1::FLI1 fusion. Recent reports indicate the EWSR1::FLI1 fusion onco-protein drives aberrant expression of numerous transcripts, including Lipoxygenase Homology Domains 1 (LOX¬HD1). Given its highly restricted protein expression pattern and role in EwS tumorigenesis and metastasis, LOXHD1 may serve as a novel immunotherapeutic target in this malignancy. LOXHD1 immunogenic epitopes restricted to HLA-A*02:01 allowed for the isolation of high avidity TCRs. LOXHD1-specific TCR engineered CD8+ T cells confer cytotoxic activity against a panel of HLA-A*02:01+ EwS tumor cell lines and adoptive transfer leads to tumor eradication in a mouse xenograft model of EwS. This study nominates LOXHD1 as an onco-fusion regulated / non-mutated EwS tumor associated antigen with expression limited to inner hair cells of the cochlea, adult testis, and EwS