Abstract
Peptides bound to class I major histocompatibility complexes (MHC) 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’s response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking normalization controls. We describe an experimental platform 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 immunotherapy design. Applying this platform in melanoma cell lines to profile the immunopeptidome response to CDK4/6 inhibition and interferon-γ — known modulators of antigen presentation — uncovers treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.
Highlights
Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer
To control for loading efficiency of synthetic peptides into recombinant MHC proteins, the concentration of stable heavy isotope-coded peptide MHCs (hipMHCs) complexes was determined by an enzyme-linked immunosorbent assay (ELISA)
Stable hipMHC complexes were used in two ways: selected hipMHC complexes were spiked at the same concentration into the whole-cell lysate from each sample to provide a normalization correction for relative quantification across samples, while other hipMHC complexes were titrated at different concentrations into each sample to verify correction parameters, estimate dynamic range suppression for quantification, and/or create an internal standard curve for absolute quantification of a specific peptide
Summary
Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. 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 immunotherapy design Applying this platform in melanoma cell lines to profile the immunopeptidome response to CDK4/6 inhibition and interferon-γ — known modulators of antigen presentation — uncovers treatment-specific alterations, connecting the intracellular response to extracellular immune presentation. We utilize hipMHC multipoint-embedded standard curves coupled with isobaric mass tags to accurately quantify the absolute number of copies per cell of target antigens within a single analysis We apply this platform to profile immunopeptidomic changes in melanoma cell lines, comparing treatment with palbociclib (a small-molecule CDK4/6 inhibitor) and interferon-γ (IFN-γ), both known modulators of antigen presentation[7,26]. We propose this platform can be broadly applied to profile immunopeptidomic changes in a high-throughput, lowinput format across sample types and treatments to inform combination therapy strategies and can be used to identify and quantify treatment-modulated antigen targets for targeted immunotherapy
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