title: T cell receptor signaling can directly enhance the avidity of CD28 ligand binding.
abstract: T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR) in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.
authors.affiliation: David H Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Mariano
authors.initials: M
authors.lastname: Sanchez-Lockhart
authors.affiliation: Department of Biostatistics and Computational Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Ana V
authors.initials: AV
authors.lastname: Rojas
authors.affiliation: David H Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Fettis
authors.affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.
authors.forename: Richard
authors.initials: R
authors.lastname: Bauserman
authors.affiliation: David H Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Trissha R
authors.initials: TR
authors.lastname: Higa
authors.affiliation: Department of Biostatistics and Computational Biology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Miao
authors.affiliation: Department of Biomedical Engineering, University of Rochester, Rochester, New York, United States of America.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Waugh
authors.affiliation: David H Smith Center for Vaccine Biology and Immunology and Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United States of America.
authors.forename: Jim
authors.initials: J
authors.lastname: Miller
mesh_terms: D000818:Animals; D018122:B7-1 Antigen / Q000276:immunology / Q000378:metabolism*; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism*; D008024:Ligands; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008822:Mice, Transgenic / Q000276:immunology / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
chemical_list: D018122:B7-1 Antigen; D018106:CD28 Antigens; D008024:Ligands; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins