title: A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.
abstract: HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.
authors.affiliation: Program in Immunology and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, United States.
authors.forename: Liusong
authors.initials: L
authors.lastname: Yin
authors.affiliation: Program in Immunology and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, United States; Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01605, United States; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, United States. Electronic address: Lawrence.Stern@umassmed.edu.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Stern
mesh_terms: D000915:Antibody Affinity / Q000276:immunology; D017951:Antigen Presentation / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005454:Fluorescence Polarization / Q000379:methods*; D006681:HLA-D Antigens / Q000032:analysis / Q000276:immunology*; D059805:HLA-DR alpha-Chains / Q000276:immunology; D059811:HLA-DRB1 Chains / Q000276:immunology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D020128:Inhibitory Concentration 50; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D059805:HLA-DR alpha-Chains; D059811:HLA-DRB1 Chains; C487961:HLA-DRB1*01:01 antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D000949:Histocompatibility Antigens Class II; D010446:Peptide Fragments
keywords: Binding competition; DM-susceptibility; Fluorescence polarization; IC(50); MHCII–peptide complex; Off-rate