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To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-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 Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck 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 p56lck. 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 p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic disorder characterized by the presence of abnormal cells of various hematopoietic cell lineages deficient in surface expression of glycosylphosphatidylinositol (GPI)-anchored molecules. By analyzing T cells isolated from patients affected with PNH, it was found that ex vivo GPI-deficient CD4(+) and CD8(+) peripheral T cells display a more naive phenotype as compared to wild-type cells. In addition, in vitro proliferative responses to allogeneic antigen-presenting cells were shown to be reduced in mutant T cells. To investigate the molecular basis responsible for defective T cell activation in GPI-deficient T cells, T cell lines and T cell clones were generated from patients affected with PNH. When stimulated with anti-CD3epsilon mAb, mutant cells displayed a significantly decreased activation of protein tyrosine kinase p56(lck). The decreased kinase activity was accompanied by a delayed TCR capping and internalization. Interestingly, protein tyrosine phosphorylation is not only quantitatively but also qualitatively affected, with one substrate being more intensively phosphorylated in mutant than in wild-type cells. These observations suggest that a defective activation of p56(lck) contributes to the depressed immune responses observed in GPI-deficient T cells derived from PNH patients.

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.