Tumour‐associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles

The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer‐related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA‐I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA‐I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA‐MB‐231, MDA‐MB‐361, MDA‐MB‐415, MDA‐MB‐453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB‐056 and the multiple myeloma line RPMI 8226. Utilizing HLA‐I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA‐I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T‐cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour‐associated antigenic peptides in the HLA‐I ligandome presented on EV. The HLA‐I peptidomes of both EV and cell were identified from 10 cancer cell lines and 150 peptides derived from tumour‐associated antigens were identified in HLA‐I ligandome of EVs. Our data, show, for the first time the prevalence of clinically relevant tumour‐associated antigenic peptides in the HLA‐I ligandome presented on EV and the potential of EV ligandomes for identification of CD8+ T‐cell epitopes.