The Human CD8 Coreceptor Effects Cytotoxic T Cell Activation and Antigen Sensitivity Primarily by Mediating Complete Phosphorylation of the T Cell Receptor ζ Chain*

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the α3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3ζ chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.

[1]  Philip J. R. Goulder,et al.  Phenotypic Analysis of Antigen-Specific T Lymphocytes , 1996, Science.

[2]  A. O'Rourke,et al.  Cytotoxic T-lymphocyte activation involves a cascade of signalling and adhesion events , 1992, Nature.

[3]  B M Baker,et al.  Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical. , 1999, Immunity.

[4]  P. Allen,et al.  Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation. , 1998, Science.

[5]  P. Marrack,et al.  Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. , 1998, Immunity.

[6]  Y. Chien,et al.  CD4 augments the response of a T cell to agonist but not to antagonist ligands. , 1997, Immunity.

[7]  A. Smolyar,et al.  Structural Basis of CD8 Coreceptor Function Revealed by Crystallographic Analysis of a Murine CD8αα Ectodomain Fragment in Complex with H-2Kb , 1998 .

[8]  T. Greten,et al.  MHC-based diagnostics and therapeutics - clinical applications for disease-linked genes. , 1999, Immunology today.

[9]  P. A. Peterson,et al.  CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes , 1996, Nature.

[10]  W. Fung-Leung,et al.  Thymic selection of cytotoxic T cells independent of CD8 alpha-Lck association. , 1993, Science.

[11]  R. Phillips,et al.  Antagonism of cytotoxic T lymphocyte‐mediated lysis by natural HIV‐1 altered peptide ligands requires simultaneous presentation of agonist and antagonist peptides , 1997, European journal of immunology.

[12]  J. Altman,et al.  Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected]. , 1998, Immunity.

[13]  J. Yewdell,et al.  Quantitating presentation of MHC class I-restricted antigens. , 2001, Methods in molecular biology.

[14]  P. Parham,et al.  The antigenic structure of HLA-A2: an analysis with competitive binding assays and monoclonal antibodies. , 1983, Journal of immunology.

[15]  Z Reich,et al.  Ligand recognition by alpha beta T cell receptors. , 1998, Annual review of immunology.

[16]  P. Parham,et al.  Cell-cell adhesion mediated by CD8 and MHC class I molecules , 1988, Nature.

[17]  C. Tournay,et al.  Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding , 2000, Nature Medicine.

[18]  B K Jakobsen,et al.  TCR binding to peptide-MHC stabilizes a flexible recognition interface. , 1999, Immunity.

[19]  D. Fremont,et al.  High- and low-potency ligands with similar affinities for the TCR: the importance of kinetics in TCR signaling. , 1998, Immunity.

[20]  P. Allen,et al.  Partially Phosphorylated T Cell Receptor ζ Molecules Can Inhibit T Cell Activation , 1999, The Journal of experimental medicine.

[21]  S. Rowland-Jones,et al.  Antigen–specific release of β-chemokines by anti-HIV-1 cytotoxic T lymphocytes , 1998, Current Biology.

[22]  J. Bell,et al.  BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. , 1999, Analytical biochemistry.

[23]  P. Romero,et al.  CD8 modulation of T-cell antigen receptor–ligand interactions on living cytotoxic T lymphocytes , 1995, Nature.

[24]  P. Klenerman,et al.  Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  B K Jakobsen,et al.  Molecular interactions of coreceptor CD8 and MHC class I: the molecular basis for functional coordination with the T-cell receptor. , 2000, Immunology today.

[26]  Paul M. Allen,et al.  Partial T cell signaling: Altered phospho-ζ and lack of zap70 recruitment in APL-induced T cell anergy , 1994, Cell.

[27]  B. Walker,et al.  β-Chemokines are released from HIV-1-specific cytolytic T-cell granules complexed to proteoglycans , 1998, Nature.

[28]  R. Germain,et al.  Partial signaling by CD8+ T cells in response to antagonist ligands , 1996, The Journal of experimental medicine.

[29]  B K Jakobsen,et al.  T cell receptor and coreceptor CD8 alphaalpha bind peptide-MHC independently and with distinct kinetics. , 1999, Immunity.

[30]  G. Ogg,et al.  Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma. , 1999, Journal of immunology.

[31]  R. Phillips,et al.  Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation , 1997, The Journal of experimental medicine.

[32]  C. Janeway,et al.  Both high and low avidity antibodies to the T cell receptor can have agonist or antagonist activity. , 1994, Immunity.

[33]  N. Shastri,et al.  Requirement for association of p56 lck with CD4 in antigen-specific signal transduction in T cells , 1991, Cell.

[34]  P. Cresswell,et al.  NK susceptibility varies inversely with target cell class I HLA antigen expression. , 1987, Journal of immunology.

[35]  A. Hamad,et al.  Potent T Cell Activation with Dimeric Peptide–Major Histocompatibility Complex Class II Ligand: The Role of CD4 Coreceptor , 1998, The Journal of experimental medicine.

[36]  R. Zamoyska,et al.  Is CD8 dependence a true reflection of TCR affinity for antigen? , 1993, International immunology.

[37]  L. Samelson,et al.  Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists , 1995, Science.

[38]  V. Appay,et al.  A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha3 domain mutants of MHC class I/peptide complex. , 2001, Immunity.

[39]  S. Jameson,et al.  Critical Role for Cd8 in T Cell Receptor Binding and Activation by Peptide/Major Histocompatibility Complex Multimers , 2000, The Journal of experimental medicine.

[40]  David I. Stuart,et al.  Classical and Nonclassical Class I Major Histocompatibility Complex Molecules Exhibit Subtle Conformational Differences That Affect Binding to CD8αα* , 2000, The Journal of Biological Chemistry.

[41]  G. Gao,et al.  Antagonism of cytotoxic T-lymphocyte activation by soluble CD8 , 1999, Nature Medicine.

[42]  J. Tschopp,et al.  Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production. , 1998, Journal of immunology.