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Summary Antigen-recognition by CD8 + T cells is governed largely by the pool of peptide antigens presented on the cell surface in the context of MHC class I complexes. Recent studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides are generated through proteasome-mediated splicing of non-contiguous regions of proteins to form novel peptide antigens. Here we used high-resolution mass-spectrometry combined with new bioinformatic approaches to characterize the immunopeptidome of melanoma cells in the presence or absence of interferon-γ. In total, we identified 30,120 peptides and demonstrate that interferon-γ induces marked changes in the peptidome (with an overlap of only 49.3% between conditions as revealed by data independent acquisition mass spectrometry). Moreover, around 6% (1,821) of the peptides were identified as cis -spliced peptides with 712 derived from known melanoma-associated antigens. Several of these peptides were shown to be immunogenic in unrelated melanoma patients. These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that may be exploited therapeutically and suggest that spliced peptides may be a major class of tumour antigens.

Murine models of Salmonella enterica serovar Typhimurium infection are one of the commonest tools to study host-pathogen interactions during bacterial infections. Critically, the outcome of S. Typhimurium infection is impacted by the genetic background of the mouse strain used, with macrophages from C57BL/6 and BALB/c mice lacking the capacity to control intracellular bacterial replication. For this reason, the use of congenic strains, which mix the genetic backgrounds of naturally protected mouse strains with those of susceptible strains, has the capacity to significantly alter results and interpretation of S. Typhimurium infection studies. Here, we describe how macrophage knockout cell lines generated by CRISPR/Cas9 gene editing can help determine the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice, on the outcome of S. Typhimurium infection studies. Our own experience illustrates how the CRISPR/Cas9 technology can be used to complement pre-existing knockout models, and shows that there is great merit in performing concurrent studies with both genetic models, to exclude unanticipated side-effects on host-pathogen interactions. Refereed/Peer-reviewed