Investigating the role of the Non-Classical MHCII Molecule HLA-DO in Regulating Exogenous Antigen Presentation in B Cells

Abstract

In B cells, exogenous antigens internalized through the BCR are processed in the endocytic pathway and loaded onto class II MHC protein (MHCII) for presentation to CD4+ T cells. The non-classical MHCII molecule HLA-DM (DM) catalyzes peptide exchange, selecting peptides with high affinity to MHCII, while HLA-DO (DO) binds to and inhibits DM in a pH-dependent manner. DO expression increases the diversity of the MHCII self-peptide repertoire and, through its inhibition of DM, allows for the presentation of low-affinity self-peptides, but the effect on exogenous antigens is unclear. We wanted to understand if DO expression in B cells affects exogenous antigen presentation and determine whether DO promotes peptide loading in particular intracellular compartments. We used an immunopeptidome workflow to identify naturally processed endogenous, exogenous, and virus-derived peptides in DO-sufficient and DO-deficient B cells infected with influenza virus, and we used a T cell assay to track the presentation of a single DR1-bound epitope derived from influenza HA with altered pH of fusion. We found that DO increases the diversity of peptides presented in infected cells favoring presentation of DM-susceptible viral epitopes. Tracking a single epitope, we show that the absence of DO favors exogenous peptide presentation, driving loading to later endocytic compartments. Our study expands on the role of DO in modulating the MHCII peptidome via a DM-dependent peptide-selection mechanism that regulates presentation of exogenous antigens and helps to illuminate its role in humoral responses to infections.