Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Mass spectrometry-based thermostability measurements of virus-derived MHC peptides correlate with immunogenicity (#177)

Mohammad Shahbazy 1 , Pouya Faridi 2 , David C Tscharke 3 , Sri H Ramarathinam 1 , Nathan P Croft 1 , Anthony W Purcell 1
  1. Department of Biochemistry and Molecular Biology and Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia
  2. Department of Medicine, School of Clinical Sciences, Monash University, Melbourne, Victoria, Australia
  3. John Curtin School of Medical Research, The Australian National University, Canberra, Australia

The major histocompatibility complex (MHC) encodes molecules that bind and present peptides on the surface of cells to stimulate T-cell responses. Although the stability of peptide-bound MHC (pMHC) complexes can influence their immunogenicity, peptidome-wide analysis of pMHC stability has only recently been achieved (1). Here, we refined a wide-scale immunopeptidomics workflow to improve accurate thermostability profiling of thousands of individual pMHC complexes and then assessed the extent that thermostability correlates with viral-pMHC immunogenicity. We utilized a C57/BL6 mouse DC2.4 cell line (MHC class I H2-Db/Kb) to assess the thermostability of a set of vaccinia virus-derived peptides with known CD8+ T cell response profiles (2). Cells were loaded with an exogenous viral peptide mixture for 1hr before being washed extensively. After mild cell lysis, the resultant lysate was aliquoted equally and then subjected to a thermal gradient from 37℃ to 73℃. Subsequently, immunoprecipitation was carried out to capture pMHC complexes, before peptide elution and quantitation by data-independent mass spectrometry on a SCIEX ZenoTOF 7600 LC-MS/MS using an in-house spectral library of viral and endogenous peptides. ZenoSWATH DIA analysis, incorporating computational mass spectrometry and bioinformatics, allowed for thermostability profiling of >3500 pMHC, including 118 vaccinia peptides. This methodology allowed the calculation of Tm values for each peptide and provides insights into the diversity of pMHC stability across many peptide ligands. Importantly, we showed a positive correlation between virus pMHC stability and T-cell immunogenicity. The inclusion of pMHC stability information is therefore an important feature to consider when choosing potential targets for T cell immunotherapy in infection and cancer.

1. Jappe, E. C. et al. Thermostability profiling of MHC-bound peptides: a new dimension in immunopeptidomics and aid for immunotherapy design. Nat Commun 11, 6305 (2020). https://doi.org:10.1038/s41467-020-20166-4
2. Croft, N. P. et al. Most viral peptides displayed by class I MHC on infected cells are immunogenic. Proc Natl Acad Sci U S A 116, 3112-3117 (2019). https://doi.org:10.1073/pnas.1815239116