The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor sigma chain

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by in teraction of both the T cell receptor and its CD8 coreceptor with peptide-m ajor histocompatibility complex (pMHC) class I molecules. We examine the re lative roles of these receptors in the activation of human CTL using mutati ons in MHC class I designed to diminish or abrogate the CD8/pMHC interactio n. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting c ells expressing HLA A2 which does not bind to CD8 fail to activate CTL at a ny peptide concentration. Comparison of CTL activation by targets expressin g HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show th at the CD8/pMHC interaction enhances sensitivity to antigen. We determine t hat the biochemical basis for coreceptor dependence is the activation of th e 23-kDa phosphoform of the CD3 zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. Thes e reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to ph enotype antigen-specific CD8+ lymphocytes.