T cells can recognise transformed cells by virtue of the presentation of tumour-associated antigens at their cell surface. This critical immune recognition occurs through the detection of human leukocyte antigen (HLA) bound peptide antigens by T cells. Thus, HLA molecules display peptide cargo that acts as a molecular snapshot of events in the cell including tell-tale signs of malignancy. T cell recognition of these HLA-peptide complexes (epitopes) can lead to eradication of tumours and is the main mediator of immunotherapy. However, the antigenic peptides recognized by the vast majority of anti-tumour T cells are not known.
While the peptides that are recognized by T cells were thought to arise in a linear fashion from proteins expressed in cancer cells, it has recently been shown that a prominent subset is generated by splicing of two different pieces of an antigen(s) together. The role of these novel spliced peptides in tumour immunity remains unknown. In studies utilizing mass-spectrometry to identify HLA-eluted peptides together with a novel bioinformatics approach we have shown a high proportion of HLA-bound peptides from melanoma cells are generated by this splicing mechanism. We identified 38766 peptides of which 6% were cis-spliced and 21% trans-spliced. Of note, more than 100 spliced peptides were derived from melanoma-associated antigens (MAA). Immunogenicity studies of a subset of the MAA utilizing T cell stimulation with subsequent ICS have shown that multiple of the tested cis-spliced peptides were highly immunogenic, compared to a smaller fraction of linear peptides. Moreover, ~40% of known MAA were only represented by spliced peptides.
Understanding the nature and abundance of spliced peptides has a high relevance for our understanding of cancer antigenicity and immunotherapy: firstly, spliced peptides may yield more immunogenic epitopes than are available from the cancer genome, especially when mutational burden is low. Secondly, some antigens lack high affinity HLA-ligands and the peptide splicing mechanism can generate higher affinity neoepitopes for interaction with host HLA allomorphs.