Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis.
Antigenic peptides are presented to CD8+T lymphocytes by class I major histocompatibility complex (MHC) molecules. Peptides specifically bind to purified class I molecules in vitro, and to class I molecules on cells at nonphysiological temperatures. We report here the kinetic and equilibrium parameters for the binding of radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter homologues) to the murine H-2Db molecule on intact, viable cells at 37 degrees C. In contrast to earlier reports, we show that peptide binding is rapid and reversible, with dissociation constants ranging from nanomolar to micromolar, suggestive of typical ligand-receptor interactions. Only 10% of cell-surface Db molecules can bind these peptides. To address the relationship between peptide binding and T-cell recognition of the antigen-MHC complex, we determined the minimum number of complexes required to sensitize a target cell for lysis by class I-restricted cytotoxic T-lymphocytes. Our data indicate that EL4 thymoma cells (H-2b) can be sensitized for lysis by cytotoxic T-lymphocytes when as few as 200 class I-peptide complexes (less than 0.08% of surface Db molecules) are present per cell.
Christinck
E R
ER
Department of Biochemistry, University of Toronto, Canada.
Luscher
M A
MA
Barber
B H
BH
Williams
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D006183:H-2 Antigens / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014758:Viral Core Proteins / Q000378:metabolism*
D006183:H-2 Antigens; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins