Ewing Sarcoma (ES) is the second most prevalent primary bone tumour in children, adolescents, and young adults with less than 30% to survive after five years if the tumour relapses or spreads to other parts of the body. Current treatment is rapidly losing effectiveness in treating the tumour. A potential novel therapy for ES is HLA-dependent T cell-based immunotherapy, which uses the body’s immune system to target peptide antigens displayed on human leucocyte antigens (HLA) and destroy the tumour cells. In this study, we present an in-depth immunopeptidomics analysis to identify peptide antigens present on HLA class I in a cohort of ES cell lines.
We analysed the immunopeptidome of four ES cell lines under basal condition and 100IU/ml interferon-gamma (IFNγ) treatment for 72hr. Our optimised immunopeptidomics protocol allowed us to identify 35,132 peptides, matched against the human proteome database across all cell lines by processing 100 million cells per cell line and condition (n=1). We found that IFNγ treatment increased HLA class I peptide antigens by 4-fold, with over 78% of unique peptides in each cell line after treatment. Identified peptides were found to be sourced from 8657 proteins. Similarly, IFNγ also increased the diversity of sourced proteins and cancer antigens (CAs), with over 65% and 60% found after IFNγ treatment in each cell line, respectively. Moreover, 35 CAs were shared across our cohort. Our findings suggest that an antigen discovery approach using in-depth immunopeptidomics analysis and IFNγ stimulation can help identify potential peptide antigen targets for ES immunotherapy.