The MHC Class II Cofactor HLA-DM Interacts with Ig in B Cells

Abstract

B cells internalize extracellular antigen into endosomes using the immunoglobulin (Ig) component of the B cell receptor. In endosomes, antigen-derived peptides are loaded onto MHC class II proteins (MHC-II). How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC-II peptide loading, co-precipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes co-localize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble HLA-DM (sDM) directly binds the Ig Fab domain, and increases levels of free antigen released from immune complexes. Together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences.

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

“…Our next goal was to evaluate the hypothesis that cross-linking biologically complementary phospholipids and proteins in a chemically efficient way would decrease unspecific binding of the resulting antigens. To do this, we carried out series of IgG ELISA assays using human monoclonal antibodies against HIV-1 antigens [ 14 ] and polyclonal healthy control sera obtained at Stanford University Hospital (n = 14) and from Immunovision (HNP) [ 15 ].…”

“…Human monoclonal antibodies used as controls for unspecific binding were purchased from NIH AIDS Reagent Programme (a-p24, a-gp41) or provided by Stanford University (B12; produced in Mellins lab) [ 14 , 15 ]. Catalogue numbers: a-p24, 530; a-gp41, 531.…”

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

“…Therefore, any complexes possessing significant levels of DM molecules may not be recovered because the DM would have catalyzed the release of the biotin-labeled antigen from the complex. Taken together, our work and the work of Macmillan et al (27) suggest that internalized Ag-BCR complexes may interact with intracellular class II or class II-DM complexes, where DM catalyzes Ag-BCR dissociation as a potential prelude to proteolytic antigen processing and class II association. This hypothesis will require further investigation.…”

“…However, Macmillan et al (27) have reported recently that DM interacts with the Fab region of internalized Ag-BCR complexes to catalyze the release of BCR-bound antigen. In this report, we isolate Ag-BCR-MHC class II complexes by pulldown of biotin-labeled antigen.…”

Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning

“…These ligands become locally available in MIIC likely due to the delivery and protection of antigen from degradation by high affinity BCR 51 and the intersection of the intracellular trafficking paths of BCR-bound antigen and nascent MHCII 1,50 . Further, in findings that imply facilitated hand-off of Ig-associated antigen for MHCII binding, we have previously shown that cross-linking of surface BCR increases the subsequent co-precipitation of Ig and free DM 52 . As DO-associated DM does not co-precipitate with Ig, this is likely another benefit of synchronized antigen binding and DO downregulation.…”