Oral Presentation 29th Annual Lorne Proteomics Symposium 2024

Mass spectrometry-based thermostability profiling of virus-derived MHC peptide complexes serves as an effective predictor of immunogenicity (#18)

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-mediated immune responses. A comprehensive snapshot of immune specificity can be achieved by studying the interaction between peptide-MHC class I complexes (pMHCI) and CD8+ T cells. Since the stability of pMHCI has been postulated to influence the immunogenicity of virus-derived epitopes and cancer neoepitopes, we sought to further establish the correlation between stability and T cell immunogenicity. A peptidome-wide analysis of pMHC stability has recently been achieved by mass spectrometry-based quantitative thermal profiling1. Here, we refined this workflow for accurate thermostability profiling of pMHCI isolated from a murine dendritic cell line presenting vaccinia virus (VACV)-derived peptides with known CD8+ T cell response profiles2. Cells were loaded with an exogenous mixture of viral peptides for 1hr, washed extensively, lysed under mild conditions, and aliquots from the resultant lysate were subjected to thermal treatments between 37℃ to 73℃. Following immunoprecipitation to capture remaining thermostable pMHC complexes, peptides were eluted and quantified by data-independent and targeted mass spectrometry on a SCIEX ZenoTOF 7600 LC-MS/MS using an in-house spectral library of viral and endogenous murine peptides. ZenoSWATH DIA and MRMHR analyses allowed thermostability profiling of >3,600 pMHCI, including 110 vaccinia virus-derived peptides. This methodology enabled the calculation of Tm values for each peptide and insights into the diversity of pMHC stability, with Tm ranges spanning 41.2°C to 65.1°C. A positive correlation between virus pMHC stability and the magnitude of T-cell response in infected mice was established. From these data, we successfully developed two machine learning-based quantitative and qualitative models to predict VACV peptide immunogenicity, and we extended the model to distinguish immunogenic epitopes derived from other infectious pathogens, including influenza A and SARS-CoV-2 viruses. Using these models, we further demonstrated that the thermostability of pMHCI is an effective predictor of immunogenic CD8+ T cell epitopes and is thus an important feature in selecting optimal targets for T cell-mediated immunotherapy.

  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