The pathways of MHC class I (MHCI) antigen processing and presentation play a crucial role during the recognition and elimination of virus-infected cells by CD8+ T cells. Two major pathways of MHCI processing exist, that of direct presentation whereby endogenous proteins are cleaved into short peptides for MHC-binding, or cross-presentation whereby exogenous antigens are taken up by professional antigen presenting cells and subsequently processed. Both pathways result in MHC-bound peptides being displayed on the cell surface, but the relative contribution of each pathway to T cell immunity, especially at the level of epitope abundance, remains unclear.
In the present study we used MHCI-peptide elution and LC-MRM to monitor the absolute and relative levels of influenza A virus (IAV) peptides during in vitro models of direct and cross-presentation. A total of 22 virus-derived MHCI-peptides were identified and quantified, including the immunodominant epitopes NP366-374 and PA224-233. Direct presentation revealed a diverse range of peptide abundance, with NP366-374 being present at more than a thousand copies per cell, whereas PA224-233 was the lowest at just 10 copies per cell. In stark contrast, cross-presentation resulted in a much narrower focus of peptide abundance at relatively low levels, with highly diminished presentation of NP366-374 yet sustained abundance of PA224-233 compared to direct presentation. Given that ex vivo CD8+ T cell responses were monitored towards all peptides, these data allow correlations between epitope presentation pathways, peptide abundance and resultant T cell immunity to be explored. These novel insights into the quantitative nature of antigen presentation hold promise for the future development of antiviral vaccine strategies.