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 choice of binding register along 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.
Highlights
II peptidome subsets based on their apparent sensitivity to DM activity and the corresponding tuning property of DOnullDMnull and (DO):DM
In B cells and several other antigen presenting cell (APC) described above, this process is further modulated by DO, likely via the competition between DO and Major histocompatibility complex class II (MHC-II) for binding to DM [19]
To further examine how tuning DO:DM can Journal Pre-proof affect different subsets of peptides presented by DR4, we compared the relative abundances of each core type across all cell lines (Fig. 5A)
Summary
Cell line construction The previously described cell lines included in this work are T2 (MHC-IInullDOnullDMnull TxB hybrid), T2DR4 (DOnullDMnull), T2DR4DM and two clonal cell lines, T2DR4DMDO++ (1C3) and. 2. To assess labelling efficiency, a ratio-check was performed: 5 μl of TMT-labeled peptides from each cell sample or replicate were combined, desalted by StageTip [34] and analyzed on the liquid chromatography–mass spectrometry (LC/MS)-instrument (detailed methods described below). Mass spectrometry-facilitated proteomic analysis Proteome-wide MS analyses were carried out as follows: TMT-labeled and Hp-RP-fractionated peptides described above were resuspended in 20 μl 0.1% formic acid, of which 10 % of the material was injected onto the Dionex Ultimate 3000 autosampler and LC-system (Thermo Fisher Scientific). Proteomic data were measured as preparative triplicates, such that each cell culture was divided into three aliquots, and each was lysed, digested, and labeled with TMT reagents in parallel as described above. Carry-over between different samples was minimized by acquiring blank analyses between each
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