Abstract
Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a nonclassical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by MHC-II molecules expressed by DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro using quantitative mass spectrometry approaches. We found that DO controlled the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. Antigen-presenting cells express another nonclassical MHC-II protein, DM, which acts as a peptide editor by preferentially catalyzing the exchange of less stable MHC-II peptide complexes, and which is inhibited when bound to DO. Peptides presented uniquely in the presence of DO were sensitive to DM-mediated exchange, suggesting that decreased DM editing was responsible for the increased diversity. DO-deficient mice mounted CD4 T cell responses against wild-type antigen-presenting cells, but not vice versa, indicating that DO-dependent alterations in the MHC-II peptidome could be recognized by circulating T cells. These data suggest that cell-specific and regulated expression of HLA-DO serves to fine-tune MHC-II peptidomes, in order to enhance self-tolerance to a wide spectrum of epitopes while allowing focused presentation of immunodominant epitopes during an immune response.
Highlights
Antigen presentation by MHC-II molecules is required for development of CD4 T cells and regulation of CD4-mediated cellular and humoral immune responses
Generation and validation of DO knockout (DO-KO) and WT clones In order to study the effect of DO on the self-peptide repertoire, we used CRISPR/Cas9 gene editing to delete HLA-DO from the HLA-DR-expressing lymphoblastoid cell line LG-2
The role of DO in MHC-II antigen presentation has been the subject of numerous biochemical and immunological studies, but understanding of the biological function of DO has been complicated by conflicting reports, in which differing effects have been described depending on the epitope(s) examined [9, 11, 13, 24, 25, 28, 30,31,32,33, 72, 73]
Summary
Antigen presentation by MHC-II molecules is required for development of CD4 T cells and regulation of CD4-mediated cellular and humoral immune responses. Isolation and characterization of I-Ab-bound peptides Mouse B cells (~3x108) were solubilized in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, containing protease inhibitors and 5% β-octylglucoside and were processed as for LG-2 membrane fractions described above, except that whole cell lysates instead of solubilized membrane fractions were used, before loading on an affinity column of I-Ab-specific mAb M5114 coupled to CNBr-activated Sepharose, with elution and analysis as described above for isolation of HLA-DR1-bound peptides.
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