Apoptosis Signaling in T Lymphocyte Leukemia Cells Induced by the TCR/CD3 Complex

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TCR variable heterodimers of a/b and g/d are associated with invariable CD3 g,d,e and z proteins, thus forming the TCR/CD3 complex. The activation signals of TCR are transduced by immunoglobulin family tyrosine-based activation motifs (ITAMs), which couple the TCR to cellular proteins and adapter molecules. CD3g,d and e each contain a single ITAM whereas CD3z chain contains three in its cytoplasmic tail. The tyrosine residues in the ITAMs of CD3g,d and e are phosphorylated during activation. The CD3e subunit plays an important role in the initiation of TCR signal transduction. Stimulation by anti-CD3e monoclonal antibodies causes cell death by apoptosis in immature thymocytes, but activation, anergy and apoptosis in mature T cells. Published work suggests that there might be two different death-signaling pathways: one triggered by CD3e and another by CD3z. The apoptosis signaling pathway initiated by CD3e is poorly understood. To address the role of aberrant signaling through CD3e in T cell apoptosis a chimeric molecule (termed CD8e) that fuses the extracellular and transmembrane domains of human CD8 to the cytoplasmic domain of CD3e was constructed and the mutants Y171F, Y180F and Y171F/Y180F were generated. These DNA contructs were stably introduced into Jurkat leukemia CD8 cells. Stimulation with anti-CD8 antibody showed that apoptosis occurred in the wild type CD3e expressing cells but not in the mutant cells, suggesting that the two tyrosine residues are required for CD3e-induced apoptotic signaling in leukemia cells. Tyrosine phosphorylation of CD3e resulted in the recruitment of the phosphatidylinositol 3'-kinase (PI3-kinase) p85a subunit iundicating a role for PI3K/AKT dependent signaling pathways in apoptosis regulation. Stimulation of the TCR/CD3 complex also results in the activation of the Ras-Raf-Mek-Erk cascade. We have previously demonstrated that Raf-1 functions as effector kinase for Cl1-2 in preventing apoptosis. To further define the role of these pathways for apoptosis signaling by CD3e we will utilize Jurkat cell lines expressing wild type (competent for apoptosis induction) or mutant forms of the CD3e receptor (defective for apoptosis induction) and analyze them for signaling pathway utilization. Understanding of the mechanisms governing death or survival after CD3e stimulation will allow for the design of new therapeutic approaches for the treatment of leukemia.