MHC class II peptide loading regulates selection and function of regulatory T cells

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 non-classical MHC-II protein implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role for DO in selecting MHC-II epitopes. We previously characterized the immunopeptidome of DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro, and found that DO controls the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. DO expression is regulated differently from the coordinate regulation of other MHC-II antigen processing components, with prominent expression in medullary thymic epithelial cells suggesting a potential role in shaping the repertoire of developing T cells. To evaluate the role of DO in regulating T cell development, we compared the TCR sequences and functional characteristics of T cells from h2-ob-/-and wild-type littermate controls. Conventional CD8 and CD4 populations were not significantly altered, but Treg populations were increased and more activated in h2-ob-/-mice than in littermate controls, and exhibited greater suppression activity in vivo. We found reduced restraint of germinal center subsets and increased ANAs in the absence of DO, consistent with a reduced ability of activated Tregs to enter germinal centers. To follow T cell selection at the clonotype level, we used mice carrying a self-reactive TCRβ transgene on a TCRα haploinsufficient background. We found decreased diversity in the TCR repertoire of Tregs but not conventional CD4 or CD8 T cells in h2-ob-/-mice, with higher CDR3 hydrophobicity. These results suggest that Treg selection in the thymus is highly sensitive to antigen density, and highlight an unexpected for MHC class II antigen presentation in controlling regulatory T cell populations.