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

Abstract

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 ␣3 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 CD3 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.T lymphocytes recognize peptide antigen associated with major histocompatibility complex (MHC) 1 molecules on the surface of antigen-presenting cells (APCs). The antigen specificity of T lymphocytes is conferred by the T cell receptor (TCR), whose highly variable complementarity-determining regions interact with the ␣1/␣2 domain platform of the MHC and the bound peptide. Engagement of the TCR with peptide-MHC (pMHC) activates an intracellular signal transduction cascade via the TCR-associated chains of the CD3-2 complex. The signal from the TCR ultimately leads to activation of the various T cell effector functions.In addition to its role in presenting antigen to the TCR, pMHC also interacts with the T cell coreceptors CD4 and CD8, which bind to invariable regions of MHC class II and MHC class I molecules, respectively. These coreceptors are critical for the development and activation of most T cells. Three possible roles for the coreceptor in T cell activation can be envisaged (1). First, the coreceptors perform a role in TCR signal transduction. After T cells engage pMHC, the earliest intracellular events induce specific phosphorylation of tyrosine residues in the immunoreceptor tyrosine activation motifs within the cytoplasmic tails of the TCR-associated CD3 complex. The cytoplasmic tails of CD4 and the CD8 ␣-chain are associated with the protein tyrosine kinase p56 lck . Active p56 lck initiates TCR signal transduction by phosphorylating the immunoreceptor tyrosine activation motifs within the CD3 complex. However, functional T cell activation, including p56 lck activation, can be achieved without involving coreceptor-MHC contacts, for example with monoclonal antibodies against components of the TCR-CD3 complex (2, 3). Furth...

Availability of a Diversely Avid CD8+ T Cell Repertoire Specific for the Subdominant HLA-A2-Restricted HIV-1 Gag p2419–27 Epitope

“…Target cells were T2 cells, Jurkat cells cotransfected with HLA-A*0201 and the HIV-1 proviral clone R7hyg (JA2/R7/Hyg) (49), and C1R cells expressing full-length wild-type HLA-A*0201 (C1R-A2 wt ) or the mutant D227K/ T228A HLA-A*0201 (C1R-A2 CD8null ) that does not bind the CD8 coreceptor (50).…”

“…Plates were developed with Mabtech reagents (Mabtech). Half-maximal stimulatory Ag doses (SD 50 ) were determined as the peptide concentration needed to achieve a half-maximal number of spots in the ELISPOT (54). Thresholds for positive responses were determined as at least five spots (50 sfc/10 6 ) per well and responses exceeding a mean of negative wells plus 3 SDs.…”

High Avidity Antigen-Specific CTL Identified by CD8-Independent Tetramer Staining

“…Because a single amino acid substitution at position 245 in the ␣3 CD8 binding loop of A2 reduces, without completely abolishing, binding to CD8 molecules (35), it remains to be established whether CD8 binding to class I tetramers is an absolute requirement for staining all CD8 ϩ T cells with class I tetramers and whether tetramers lacking CD8 binding site can specifically identify high avidity CTL. Because murine CD8 molecules fail to bind to the ␣3 domain of human class I molecules (29), tetramer staining of A2-restricted responses in A2 transgenic mice provides a model to address whether high avidity CTL can be identified by A2 tetramers (i.e., tetramers lacking murine CD8 binding site). In contrast, A2K b tetramers (i.e., chimeric human murine tetramers containing ␣1/␣2 domain from A2 molecules and ␣3 domain from K b molecules), engineered to contain the murine CD8 binding site, should be able to stain both high and low avidity T cells.…”

Decreased Binding of Peptides-MHC Class I (pMHC) Multimeric Complexes to CD8 Affects Their Binding Avidity for the TCR But Does Not Significantly Impact on pMHC/TCR Dissociation Rate

“…Other mutations (D227K, or D227K/T228A) were then directly introduced in the 223-229 loop. The D227K/ T228A double mutation was previously shown to abrogate A2 binding to CD8 (10).…”

“…Multimers containing an A2 A245V single mutant, known to bind CD8 with decreased affinity compared with the wild-type molecule, showed a strongly decreased capacity to bind high-avidity tumor-Ag-specific CD8 ϩ T cells in different antigenic systems (9). Multimers containing the D227K or D227K/T228A mutations that abrogate A2 interaction with CD8 (10,11) showed low to undetectable binding, depending on the CD8 ϩ T cell clone analyzed. Interestingly, the association kinetics of A245V multimers containing the parental peptide was similar to that of wild-type multimers containing a weak agonist peptide.…”