We do not yet know whether DM physically blocks antigen re-binding or stabilizes a confirmation of Ig that prevents rebinding

By | July 17, 2022

We do not yet know whether DM physically blocks antigen re-binding or stabilizes a confirmation of Ig that prevents rebinding. 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 name, 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. Introduction B cells, dendritic cells, and macrophages – collectively called professional antigen presenting cells (APCs) – play a central role in the initiation of adaptive immune responses. These cells internalize antigen into endosomes, process antigen into peptide fragments that bind to MHC class II proteins (MHC-II) and present peptide/MHC-II complexes at the cell surface (1, 2). Recognition of peptide/MHC-II complexes activates CD4+ T cells (3). Activated CD4+ T cells in turn can license dendritic cells (DCs) to activate CD8+ T cells (4) and provide help to B cells for immunoglobulin (Ig) production (5). MHC-II antigen presentation initiates protective adaptive immune responses in host defense and vaccination, as well as pathogenic responses, CPI-0610 carboxylic acid in autoimmune disease (6) and transplant rejection (7). Human MHC-II proteins (HLA-DP, HLA-DQ, and HLA-DR) are / heterodimers. Actions in their biosynthesis and peptide loading have been extensively studied (1). In the endoplasmic reticulum (ER), the invariant chain (Ii) binds and stabilizes newly synthesized MHC-II proteins. Nascent MHC-II/Ii complexes leave the ER, traverse the Golgi, and traffic to the MHC-II loading compartment(s) (MIIC), which are typically late endosomal, multivesicular bodies made up of the protein cofactors required for antigen presentation. In the MIIC, acid-activated proteases degrade Ii into a nested set of short peptides termed CLIP (class II associated invariant chain peptide), which remains associated with the peptide-binding groove of MHC-II (8). CLIP serves as a placeholder peptide and stabilizes the MHC-II dimer until HLA-DM (DM), an MHC-II-like heterodimer, catalyzes its release. Internalized antigen also localizes to the MIIC, and after CLIP dissociation, DM influences peptide loading by chaperoning vacant MHC-II and editing the peptides bound to MHC-II in favor of peptides that form stable peptide/MHC class II complexes (9). The conversation between CPI-0610 carboxylic acid DM and MHC-II is usually mediated by parallel alignment of the luminal domains of each protein (9). The endosomal protein HLA-DO (DO) interacts with DM and inhibits the DM/MHC-II conversation by steric competition (10). The peptides presented by MHC-II are typically 12-18 amino acids long, but the open-ended geometry of the binding groove can accommodate antigen fragments of larger sizes (11) . In B cells, transmembrane Ig associates with the signaling chains Igand Igat the cell surface, forming the B cell receptor (BCR). The Ig component of the BCR captures antigen, which results in activation of an intracellular signaling pathway that emanates from phosphorylated BCR. Unphosphorylated BCR/antigen complexes are internalized and traffic to early endosomes, where they initially co-localize with MHC-II prior to migration of both proteins to the MIIC (12). Although the Fc and Fab domains of Ig remain largely intact in the MIIC of B cells (13, 14), the two domains can be immunoprecipitated separately, and co-precipitation of antigen with the Fab domain name can be detected (14). This result implies that proteolytic enzyme(s) resident in the MIIC cleave intact Ig and provides evidence that at least some antigens remain bound to intact Fab in late endosomes. The pathway that bridges antigen/Ig delivery to MIIC compartments with peptide/MHC-II association remains unclear (15). It is known that antigen capture by the BCR and its trafficking to late endocytic compartments contribute to the significant enhancement of Ig-mediated antigen presentation compared to presentation after fluid-phase antigen uptake (16). We investigated whether HLA-DM, a known catalyst of peptide/MHC-II association, directly interacts with Ig in B cells. Materials and Methods Cells The non-Hodgkin’s human B cell lymphoma cell line, DHL-4 (IgG-) (17), and the human B cell lymphoma cell line, OCI-LY8 (IgM/IgD-) (18), (provided CPI-0610 carboxylic acid by Dr. Ron Levy, Stanford University, Stanford, CA) were produced in Rabbit Polyclonal to REN RPMI 1640 supplemented with 10% FBS and 2 mM L-Glutamine (cRPMI). Epstein-Barr computer virus transformed human B cell lines DPA (IgG1-) and DPD (IgG1-) (19) (provided by Christiane Hampe, University of Washington, Seattle, WA) were produced in cRPMI. Epstein-Barr transformed human.