DOI: 10.25345/c5dj58h15
title: MassIVE MSV000088876 - Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models
title: 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 naive 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. Methods pMHCs were isolated by affinity purification and purified prior to LC-MS/MS analysis. Exact methodological details can be found in the associated manuscript. Search All mass spectra were analyzed with Proteome Discoverer (PD, version 2.5) and searched using Mascot (version 2.4) against the mouse SwissProt database (2021_03; 2021_02 for label free quantification) supplemented with a list of murine ORFs previously identified by ribosome profiling (www.sorfs.org). Peptides were searched with no enzyme and variable methionine oxidation. Peptide spectrum matches (PSMs) were further filtered according to the following criteria: ions score &gt;= 15, search engine rank = 1 and results from technical replicates of each sample analysis were combined. Median retention time (RT) was calculated using the RT values of filtered PSMs from all replicates of a given sample. Label free quantitation was done using the Minora Feature Detector (precursor abundance values measured based on area under the curve) in PD with match between runs enabled and filtered for peptides with ions score &gt;= 15 and search engine rank = 1. Abundances were averaged across technical and biological replicates. The lot number used for TMT 6plex (Thermo fisher) was UG285371.