A novel signaling mechanism for soluble CD95 ligand. Synergy with anti-CD95 monoclonal antibodies for apoptosis and NF-kappaB nuclear translocation

Soluble CD95 (Fas) ligand (sFasL) is known to be deficient in transducing signals upon engagement with membrane Fas. Here we report that sFasL tranduces, in synergy with non-cytotoxic anti-Fas monoclonal antibody (mAb), signals for apoptosis and nuclear translocation of the NF-kappaB (p65/p50) heterodimer. Activation of the specific signaling pathways correlates with target Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein expression. Synergy with anti-Fas mAb was demonstrated with a trimeric unit of sFasL bearing a single binding site for Fas trimer. In contrast, membrane-bound FasL as expressed on cell-derived vesicles was fully competent in transducing Fas-mediated signals for apoptosis and NF-kappaB nuclear translocation. We propose a model in which the trimeric sFasL signaling requires target expression of a high focal density of Fas, which is induced by the signaling-incompetent anti-Fas mAb. Membrane-bound FasL induces powerful Fas-mediated signals because it possesses both Fas-focusing and signal-transducing functions.     

Fas aggregation does not correlate with Fas-mediated apoptosis.

Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-alpha or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and beta-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Fas cross-linking induces apoptosis in human airway smooth muscle cells

Hypertrophy and hyperplasia lead to excess accumulation of smooth muscle in the airways of human asthmatic subjects. However, little is known about mechanisms that might counterbalance these processes, thereby limiting the quantity of smooth muscle in airways. Ligation of Fas on the surface of vascular smooth muscle cells and nonmuscle airway cells can lead to apoptotic cell death. We therefore tested the hypotheses that 1) human airway smooth muscle (HASM) expresses Fas, 2) Fas cross-linking induces apoptosis in these cells, and 3) tumor necrosis factor (TNF)-alpha potentiates Fas-mediated airway myocyte killing. Immunohistochemistry using CH-11 anti-Fas monoclonal IgM antibody revealed Fas expression in normal human bronchial smooth muscle in vivo. Flow cytometry using DX2 anti-Fas monoclonal IgG antibody revealed that passage 4 cultured HASM cells express surface Fas. Surface Fas decreased partially during prolonged serum deprivation of cultured HASM cells and was upregulated by TNF-alpha stimulation. Fas cross-linking with CH-11 antibody induced apoptosis in cultured HASM cells, and this effect was reduced by long-term serum deprivation and synergistically potentiated by concomitant TNF-alpha exposure. TNF-alpha did not induce substantial apoptosis in the absence of Fas cross-linking. These data represent the first demonstration that Fas is expressed on HASM and suggest a mechanism by which Fas-mediated apoptosis could act to oppose excess smooth muscle accumulation during airway remodeling in asthma.     

The Fas death signaling pathway connecting reactive oxygen species generation and FLICE inhibitory protein down-regulation

Fas-mediated apoptosis plays an important role in normal tissue homeostasis, and disruption of this death pathway contributes to many human diseases. Induction of apoptosis via Fas activation has been associated with reactive oxygen species (ROS) generation and down-regulation of FLICE inhibitory protein (FLIP); however, the relationship between these two events and their role in Fas-mediated apoptosis are unclear. We show herein that ROS are required for FLIP down-regulation and apoptosis induction by Fas ligand (FasL) in primary lung epithelial cells. ROS mediate the down-regulation of FLIP by ubiquitination and subsequent degradation by proteasome. Inhibition of ROS by antioxidants or by ectopic expression of ROS-scavenging enzymes glutathione peroxidase and superoxide dismutase effectively inhibited FLIP down-regulation and apoptosis induction by FasL. Hydrogen peroxide is a primary oxidative species responsible for FLIP down-regulation, whereas superoxide serves as a source of peroxide and a scavenger of NO, which positively regulates FLIP via S-nitrosylation. NADPH oxidase is a key source of ROS generation induced by FasL, and its inhibition by dominant-negative Rac1 expression or by chemical inhibitor decreased the cell death response to FasL. Taken together, our results indicate a novel pathway of FLIP regulation by an interactive network of reactive oxygen and nitrogen species that provides a key mechanism of apoptosis regulation in Fas-induced cell death and related apoptosis disorders.     

Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.     

IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells.

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcgamma RII/RIII receptor (FcgammaR) antibody, and defective in FcgammaR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcgammaR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcgammaR. Although cytochalasin D affected FcgammaR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.     

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.     

The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.     

Inhibiting apoptosis of CTLL-2 cells to enhance their GVL effects via anti-Fas ribozyme

Donor lymphocyte infusion (DLI) is an adoptiveimmunotherapy to achieve particular therapy aims forpatients accepting allogenetic hemopoietic stem celltransplantation [1–3]. Recently, many researches havetestified that the graft-versus-leukemia effect (GV…     

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined.     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells.

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.     

Fas, ceramide and serum withdrawal induce apoptosis via a common pathway in a type II Jurkat cell line

Ceramide is a key mediator of apoptosis, yet its role in Fas-mediated apoptosis is controversial. Some reports have indicated that ceramide is either a primary signaling molecule in Fas-induced cell death, or that it functions upstream of Fas by increasing FasL expression. Other studies have suggested that ceramide is not relevant to Fas-induced cell death. We have approached this problem by studying ceramide-induced apoptosis in unique Jurkat cell clones selected for resistance to membrane-bound FasL-induced death. Resistance of the mutant Jurkat cells was specific for FasL killing, since the mutant clones were sensitive to other apoptotic stimuli such as cycloheximide and staurosporine. We tested the effects of serum withdrawal, one of the strongest inducers of ceramide, and of exogenous ceramide on apoptosis of both wild-type and FasL-resistant clones. Wild-type Jurkat cells were remarkably sensitive to serum withdrawal and to exogenous ceramide. In contrast all FasL-resistant mutant clones were resistant to these apoptosis-inducing conditions. In contrast to previous work, we did not detect an increase in FasL in either wild-type or mutant clones. Moreover activation of stress-activated protein kinases (JNK/SAPKs) after serum withdrawal and exogenous ceramide treatment was detected only in the wild-type and not in the resistant clones. Because of the parallel resistance of the mutant clones to Fas and to ceramide-induced apoptosis, our data support the notion that ceramide is a second messenger for the Fas/FasL pathway and that serum withdrawal, through production of ceramide, shares a common step with the Fas-mediated apoptotic pathway. Finally, our data suggest that activation of JNK/SAPKs is a common mediator of the three pathways tested.     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.