Fasbased cytotoxicity

1.1.1 Induction of FasL expression in CTL

The expression of FasL in CTL is not constitutive and a strong activation signal through the T cell antigen receptor (TCR) is needed (13). This can also be achieved using a combination of the phorbol ester PMA, which activates efficiently protein kinase C (PKC), and the calcium ionophore ionomycin (2). The induction of FasL expression in CTL can be prevented by protein tyrosine kinase inhibitors, and by cyclosporin A, presumably through the inhibition of calcineurin phosphatase (13). Hence, although the execution of Fas-based cytotoxicity occurs in the absence of extracellular Ca2+, the TCR-induced FasL expression is dependent on Ca2+ (14). It was later demonstrated that TCR-induced, but not PMA/ionomycin-induced, FasL expression in CTL was dependent on phosphatidylinositol 3-kinase activity (15), and that the transcriptional regulation of FasL expression was dependent on the activation of the transcription factor NF-AT (16), according to the observed inhibition by cyclosporin A.

1.1.2 Mechanism for Fas-induced apoptosis

Fas (Apo-l/CD95) is a type I transmembrane glycoprotein belonging to the TNF receptor superfamily. Fas is expressed in some thymocyte subsets, activated and tumour T and B cells, and in several non-immune tissues. Fas induces apoptosis when cross-linked with agonist mAbs or by its natural ligand, FasL (17). Fas and the TNF receptor share a cytoplasmic domain needed to induce apoptosis that has been termed the 'death domain' (18).

A great deal of research effort has demonstrated the implication of intracellular cysteine-proteases with Asp specificity of the interleukin-l(3-converting enzyme (ICE) family, later named as caspases, in apoptosis (see Chapter 10), and in particular in Fas-induced cell death (reviewed in refs 19 and 20)). Using the peptide inhibitor, Ac-DEVD-CHO, which inhibits, preferentially, caspases of the CPP32 (caspase-3) subfamily (21), it has been shown that these caspases play a key role in cytotoxicity exerted by anti-Fas mAbs (22), and by FasL-expressing T cell effectors (23). The finding that activation of CPP32-like caspases plays a pivotal role in many types of apoptotic process has led to the proposal that these caspases constitute the apoptotic executioner (24).

The connection between Fas ligation and the activation of the apoptotic executioner has recently been unveiled. Aggregation of Fas through agonist antibodies or by its physiological ligand induces the recruitment to the cross-linked receptors and, through their respective death domains, of a molecule termed FADD (Fas-associated death domain) (25). In addition to the death domain, which allows its interaction with the aggregated receptors, FADD also contains a 'death effector domain' that mediates the recruitment of another molecule composed of two death effector domains and a caspase domain, named FLICE, MACH, Mch5, or caspase-8 (26-28). The recruitment of this molecule induces activation of its protease activity, resulting in the cleavage and activation of CPP32-like proteases, the apoptotic executioner. An alternative pathway implicated in the activation of CPP32-like caspases is the release of pro-apoptotic proteins (cytochrome c and the apoptosis-inducing factor, AIF) from mitochondria in the first stages of apoptosis (29, 30). This process takes place through the assembly of a complex that also includes Apaf-1, the mammalian homologue of Ced-4, and caspase-9, which in turn, activates caspase-3 (31) (see also Chapter 1, Fig. 6). Both processes, 'direct' activation of caspase-3 through caspase-8 recruitment to the aggregated receptor and 'indirect' activation of caspase-3 through mitochondrial activation of caspase-9, are not mutually exclusive. In fact, it has recently been demonstrated that the relative importance of one over the other depends on the nature of the cells undergoing apoptosis (32).

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