Tuning DO:DM ratios modulates MHC class II immunopeptidomes

ABSTRACT Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM-sensitivity, peptide abundance, binding affinity and motif, peptide length and register positioning on the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body. IN BRIEF Peptides presented by MHC-II are critical to adaptive immune function. The non-canonical MHC molecules HLA-DM and HLA-DO cooperatively regulate MHC-II function, but how varied DO-to-DM ratios across different APCs and cellular contexts might influence their immunopeptide repertoires is unclear. We address this by measuring cell lines expressing these two proteins spanning a range of relative abundances. We found that peptides could be categorized according to how robustly they were presented at different DO:DM ratios. Importantly, this presentation was only partially linked to predicted affinity to the MHC-II molecule. HIGHLIGHTS Describe MHC-class II peptide repertoires from a unique HLA-DR4 cell line panel with increasing DO:DM ratios. Demonstrate striking and divergent changes in MHC-II immunopeptidomes that result from the tuning function of DO:DM. These findings bridge gap in understanding and predicting MHC-II antigen presentation. GRAPHICAL ABSTRACT