CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2K(d)-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric K-d-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric K-d-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56(ick). Dimerization of these adducts results in activation of p56(ick) in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56(ick) kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56(ick). They are also inhibited by methyl-beta -cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56(ick) takes place in rafts, both reagents, though in different ways, impair coupling of p56(ick) to TCR, thereby inhibiting the initial and essential activation of p56(ick) induced by cross-linking of engaged TCR.