PO-406 Investigation of the repertoire of peptides bound to MHC class I molecules in tasmanian devil transmissible cancers for the development of a peptide vaccine

Abstract

IntroductionThe marsupial species Tasmanian devil harbours two contagious cancers, Devil Facial Tumour 1 and 2 (DFT1 and DFT2); both are passed between individuals as an allogeneic graft, initiating tumours around the neck and face. DFT1 emerged 20 years ago, causes 100% mortality and has decimated the devil population. In contrast, DFT2 emerged only recently and is still being characterised. As allografts, both tumours should be recognised by T cells interacting with non-self Major Histocompatibility Complex (MHC) molecules and associated peptides. We aim to isolate tumour-specific MHC-bound peptides in order to determine MHC/peptide complexes breaking or inducing tolerance and design a vaccine.Material and methodsWe have characterised the immunopeptidomes of DFT1-IFNγ, DFT2 and devil fibroblast cell lines. The DFT1 cell line was stimulated with devil interferon-gamma to up-regulate MHC class I expression. MHC class I molecule-peptide complexes were isolated by immunoaffinity purification using an antibody against Tasmanian devil β2-microblobulin. Eluted peptide/MHC complexes were fractionated by reversed phase HPLC and analysed by Liquid Chromatography tandem mass spectrometry (LC-MS/MS). All experiments were performed in triplicate. Peptides were identified by searching spectra against custom Tasmanian devil databases using the PEAKS software and data visualised in R studio and GraphPad Prism.Results and discussionsBetween 6373 and 2243 peptides were identified for each cell line, but only peptides found in all replicates for each cell line analysed further. 1806 peptides were identified in all three replicates of the fibroblast cell line, while 455 and 379 peptides were common to all three replicates for the DFT1-IFNγ and DFT2 cell lines respectively. Analysis of peptide length shows a preference for 8mers and 9mers in devil fibroblasts and DFT2 cells and a preference for 9mers in DFT1-IFNγ. We next searched for binding motifs for the 8mers and 9mers across all cell lines and found potential anchor residues at position 3 and position 8/9, where there was a preference for hydrophobic amino acids (in particular Leucine). We then identified 61 and 55 peptides unique to DFT1-IFNγ and DFT2 respectively.ConclusionThis is the first study to characterise the repertoire of peptides bound to MHC molecules in contagious cancers and represents a pivotal step for understanding the immunological features of transmissible cancers.