Diversification of immune responses through degenerate and reconstructive proteolysis

The cellular immune response relies upon T cell recognition of peptides presented on the cell surface in complex with HLA molecules. As such, it is the peptide cargo of these HLA molecules that dictates the quality of the immune response and ultimately the efficacy of protective immunity. Relatively simplistic models have been used to explain how these peptide antigens are generated and selected for presentation, however, such models fail to predict and explain the diversity and complexity of the immune response. Much of this unexplained complexity resides in degenerate and reconstructive proteolysis. We have recently highlighted the complex role of proteolysis in the generation and diversification of peptide antigens displayed for T cell recognition. For example, functional heterogeneity is observed during degenerate peptidase trimming of T cell epitopes such that peptides with ragged N- or C-termini (nested peptides) can be presented with different immune outcomes. Moreover, we have generated evidence that reinforces recent surprising studies that up to 30% of peptides presented by class I HLA molecules are generated by post-translational proteasomal splicing (i.e. the ligation of peptide fragments within the proteasome rather than peptide destruction). Using a novel bioinformatic workflow we show that these spliced peptides can be generated either from two regions of the same antigen (cis-splicing) or two distinct antigens (trans-splicing) by retrospectively interrogating datasets for several common HLA allotypes. This is the first demonstration that trans-spliced peptides are abundantly represented in the immunopeptidome. However, the role of spliced peptides in immune responses and the precise mechanism of their generation remains poorly understood and will be discuss in the context of Type 1 Diabetes and other immune disorders.