title: The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.
abstract: Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, USA.
authors.forename: Hugo G
authors.initials: HG
authors.lastname: Hilton
authors.affiliation: Discovery Research, Amgen Inc., South San Francisco, CA, USA.
authors.forename: Achim K
authors.initials: AK
authors.lastname: Moesta
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, USA.
authors.forename: Lisbeth A
authors.initials: LA
authors.lastname: Guethlein
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, USA.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Blokhuis
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Parham
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, USA; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, USA. Electronic address: paul.norman@stanford.edu.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Norman
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000276:immunology; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology*; D007313:Insecta; D007694:Killer Cells, Natural / Q000276:immunology; D008024:Ligands; D011485:Protein Binding / Q000276:immunology*; D017510:Protein Folding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D054340:Receptors, KIR / Q000276:immunology*; D055607:Receptors, Natural Killer Cell / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology*; D061987:Sf9 Cells; D018411:Spodoptera / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D008024:Ligands; D011948:Receptors, Antigen, T-Cell; D054340:Receptors, KIR; D055607:Receptors, Natural Killer Cell; D011994:Recombinant Proteins
keywords: Antigens/peptides/epitopes; Comparative immunology/evolution; MHC; Natural killer cells