The heterogeneous nature of Triple-Negative Breast Cancer (TNBC) has highlighted the need for novel approaches in treatment. Recent studies demonstrate the feasibility of a peptide vaccination approach to induce T-cell mediated killing of these cancerous cells.
To identify potential T-cell targets we examined how the pro-inflammatory cytokine interferon-γ (IFNγ), which is highly expressed within the microenvironment of infiltrated tumours, modulated the peptide repertoire of HLA-I and HLA-II alleles. We isolated native HLA-I and HLA-II-peptide complexes from a TNBC model cell line (MDA-MB-231) under mild lysis conditions using immunoaffinity chromatography, isolated their peptide cargo by RP-HPLC, prior to analysis of peptide containing fractions by high resolution mass spectrometry for peptide identification.
We identified 46,128 unique peptides presented across HLA-I and HLA-II. In general, IFNγ treatment caused a 2.2-fold increase in peptide abundance and increased the diversity of the peptide repertoire from 14,270 peptides (derived from 4892 source proteins) to 26,135 peptides (derived from 6668 source proteins) in the HLA-I immunopeptidome. We see a 66% overlap between peptides presented across HLA-I in IFNγ treated and untreated samples. In contrast, HLA-II was not expressed in untreated cells, yet was expressed upon IFNγ treatment and resulted in the detection of 25,441 peptides following isolation of HLA-II molecules from the treated cancer cells. In total, 8044 unique source proteins were represented via the TNBC peptidome presented by HLA-I and -II molecules, with 3152 being unique to IFNγ treatment and only 505 being unique to untreated cells.
These results highlight the high degree of plasticity of the TNBC immunopeptidome in response to changes in the tumour microenvironment. IFNg increased the diversity and abundance of the peptide repertoire suggesting that under pro-inflammatory conditions a greater variety of vaccine targets are unveiled to the immune system. This has important implications for the development of personalised cancer vaccination strategies.