Sven Baumann

Cellular FLICE-inhibitory Protein Splice Variants Inhibit Different Steps of Caspase-8 Activation at the CD95 Death-inducing Signaling Complex

Upon stimulation, CD95 (APO-1/Fas) recruits the adapter molecule FADD/MORT1, procaspase-8, and the cellular FLICE-inhibitory proteins (c-FLIP) into the death-inducing signaling complex (DISC). According to the induced proximity model, procaspase-8 is activated in the DISC in an autoproteolytic manner by two subsequent cleavage steps. c-FLIP proteins exist as a long (c-FLIPL) and a short (c-FLIPS) splice variant, both of them capable of protecting cells from death receptor-mediated apoptosis. In stably transfected BJAB cells, both c-FLIPS and c-FLIPL block procaspase-8 activation at the DISC. However, cleavage is blocked at different steps. c-FLIPL allows the first cleavage step of procaspase-8, leading to the generation of the p10 subunit. In contrast, c-FLIPS completely inhibits cleavage of procaspase-8. Interestingly, p43-c-FLIPL lacking the p12 subunit also prevents cleavage of procaspase-8. In contrast, a nonprocessable mutant of c-FLIPL allows the first cleavage of procaspase-8. In conclusion, both c-FLIP proteins prevent caspase-8 activation at different levels of procaspase-8 processing at the DISC. Our results indicate that c-FLIPL induces a conformation of procaspase-8 that allows partial but not complete proteolytical processing, whereas in contrast c-FLIPS even prevents partial procaspase-8 activation at the DISC.

FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis.

Cell death is executed along several pathways. Apart from necrotic cell death occurring upon tissue injury, several distinct types of apoptosis have been observed. Apoptosis, or programmed cell death, is critical for tissue homeostasis in multicellular organisms. It plays an important role in many physiological processes, especially in development and in the immune system (39, 80). Many diseases are associated with either too much or too little apoptosis, such as AIDS, cancer, and autoimmunity (39).

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

The role of CD95 in the regulation of peripheral T‐cell apoptosis

Summary:  Apoptosis of activated peripheral T cells during the termination phase of an immune response is critical to maintain T‐cell homeostasis. Activated T cells can be removed by two mechanisms: activation‐induced cell death (AICD) and death by neglect. AICD is triggered by death receptors, whereas death by neglect is induced by cytokine withdrawal. CD95 (APO‐1/Fas) belongs to the subfamily of death receptors and plays a major role in AICD. In this review, we focus on the molecular mechanisms of AICD, in particular those involving the CD95 system. Moreover, we discuss the relative contribution of AICD and death by neglect to terminate a T‐cell immune response. In order to become fully activated, T cells require a second signal provided by antigen‐presenting cells. We discuss how these costimulatory signals counteract pro‐apoptotic signals and, finally, which signals might protect T cells from death to generate a pool of memory T cells.

Resistance of Short Term Activated T Cells to CD95-Mediated Apoptosis Correlates with De Novo Protein Synthesis of c-FLIPshort

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIPlong was only slightly down-regulated in sensitized T cells, c-FLIPshort became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIPshort, rather than c-FLIPlong, confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.

Regulation of T cell apoptosis during the immune response

Apoptosis of T-lymphocytes is a fundamental process regulating antigen receptor repertoire selection during T cell maturation and homeostasis of the immune system. It also plays a key role in elimination of autoreactive lymphocytes. Resting mature T cells are activated by antigen to elicit an appropriate immune response. In contrast, preactivated T cells undergo activation-induced cell death (AICD) in response to TCR triggering alone. Thus, death by apoptosis is essential for function, growth and differentiation of T-lymphocytes. This review focuses on apoptosis mechanisms involved in T cell development and during the course of an immune response.

The active caspase-8 heterotetramer is formed at the CD95 DISC.

Caspase-8 plays an important role in the CD95 (APO-1/ Fas) signalling pathway. It is activated at the CD95 DISC. The mechanism of caspase-8 activation at the DISC is in the focus of intensive studies. It is well established that binding of procaspases-8/a and -8/b to the DISC results in autocatalytic cleavage via a two-step mechanism (Figure 1a). The initial cleavage at Asp generates two cleavage intermediates p43/p41 and p12. Subsequently, p12 is rapidly converted to p10. Afterwards, an additional cleavage occurs at Asp, producing the active enzyme subunit p18 and the inactive p26/p24 prodomain. The DED-containing cleavage products p43/p41 and p26/p24 were reported to be present in the DISC in previous biochemical studies. We analysed in more detail all cleavage products of procaspase-8 in the DISC formed upon stimulation of the Blymphoblastoid cell line SKW6.4. Cells were treated with LZCD95L for different time points, followed by immunoprecipitation of the DISC components with the agonistic anti-APO-1 and by immunoblotting with a panel of mAbs. The epitopes recognised by these antibodies are shown in Figure 1a. In addition to the established components of the DISC, including full-length procaspase-8 (p55/p53) and its cleavage product p43/p41, we clearly detected the p18 and p10 subunits of procaspase-8 in the DISC (Figure 1b). The presence of p10 and p18 at the DISC was also observed using other T-cell lines Hut78, CEM, H9 as well as primary T cells (data not shown). The detection of these products became possible because we used different conditions for electrophoresis, focusing on lower molecular weight products in comparison to previous studies. In addition, the presence of the p10 subunit was detected using the C5 antibody that had not been used in the analysis of the lower molecular weight procaspase-8 subunits in the CD95 DISC before. The kinetic analysis in Figure 1b demonstrated that p10 and p18 are detectable at the CD95 DISC within 30 s after stimulation. The amount of both p10 and p18 at the DISC reached its maximum 1–5 min after receptor engagement. After 10 min, the intensity of the bands corresponding to p10 and p18 started to decrease and after 20 min, the intensity of bands was markedly reduced. The kinetic analysis performed with lysates of SKW6.4 cells (Figure 1b) showed the same main features as reported earlier. P10 and p18 were detected in the cytosol 10 min after the receptor stimulation, and their presence reached a maximum after 20 min, exactly at the time when their amount in the DISC was found to be decreased. To ensure that the p10 and p18 subunits are components of the DISC under different stimulation conditions, SKW6.4 were stimulated in parallel with LZ-CD95L and anti-APO-1 antibody. We observed the presence of p10 and p18 in the DISC in both cases (data not shown). To exclude the possibility that p10 and p18 subunits are generated during the washing steps, DISC formation and consecutive immunoprecipitation experiments were performed in the presence of the caspase inhibitor zVAD-fmk. Under these conditions, we could also detect active caspase-8 subunits at the DISC (data not shown). Additionally, we addressed the question of whether the caspase-8 subunits generated at the DISC were functionally active and did not require additional cytosolic processing and can cleave caspase-8 substrates. Thus, in vitro translated, [S]-labelled procaspase-3 was added to protein-A sepharose containing CD95 immunoprecipitates from unstimulated and stimulated cells. Incubation of protein-A sepharose beads with caspase-3 resulted in the cleavage of procaspase-3 to p20, p19, p17 and p12 subunits only in the stimulated case (Figure 1c, lane 3). Cleavage was blocked in the presence of the specific inhibitor of caspase-8 IETD-fmk (Figure 1c, lane 4). To ensure that autocatalytic processing of procaspase-3 does not blur our results in these experiments, we performed a similar assay with procaspase-3 carrying a mutation in the catalytic site (C163S). This mutant was supposed to be a substrate of caspase-8, but it excludes the formation of any cleavage product as a result of genuine caspase-3 proteolytic activity. In the presence of immunoprecipitates from the CD95 DISC, we observed only the first cleavage step with the formation of the p20 subunit (Figure 1c, lane 7). No further processing to the p17 subunit was detected. This observation is in accordance with published data indicating that the generation of the mature p17–p12 enzyme of caspase-3 only occurs autocatalytically. Thus, the caspase-3 mutant C163S is catalytically inactive in our in vitro system. Cleavage was not observed in the presence of IETD-fmk (Figure 1c, lane 8), as well as in the case of immunoprecipitates from unstimulated cells (Figure 1c, lane 6). These data point out that the cleavage of caspase-3 results from the catalytic activity of the caspase-8 heterotetramer formed at the DISC. In previous studies, it has been suggested that formation of the tetramer takes place in the cytosol. In our view, this is a less likely event than the interaction between subunits remaining bound to the DISC. Thus, the DISC not only brings the molecules of procaspase-8 into close proximity to each other, but it also places them in a spatial orientation favourable for their activation. A number of biochemical studies provide evidence that the activation of procaspase-8 occurs via its dimerisation or oligomerisation. The conditions for the dimer formation and the following cleavage steps must be provided by the spatial structure of the DISC. However, further Cell Death and Differentiation (2003) 10, 144–145 & 2003 Nature Publishing Group All rights reserved 1350-9047/03

An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death

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Viral IFN-Regulatory Factors Inhibit Activation-Induced Cell Death Via Two Positive Regulatory IFN-Regulatory Factor 1-Dependent Domains in the CD95 Ligand Promoter

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-xL and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.

Vitamin E inhibits CD95 ligand expression and protects T cells from activation-induced cell death

The CD95 (also called APO-1/Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 ligand (CD95L) is induced in response to a variety of signals, including IFN-γ and TCR/CD3 stimulation. Here we report the identification of two positive regulatory IFN-regulatory factor-dependent domains (PRIDDs) in the CD95L promoter and its 5′ untranslated region, respectively. EMSAs demonstrate specific binding of IFN-γ-induced IFN-regulatory factor 1 (IRF-1) to the PRIDD sequences. Ectopic IRF-1 expression induces CD95L promoter activity. Furthermore, we demonstrate that PRIDDs play an important role in TCR/CD3-mediated CD95L induction. Most interestingly, viral IRFs of human herpes virus 8 (HHV8) totally abolish IRF-1-mediated and strongly reduce TCR/CD3-mediated CD95L induction. We demonstrate here for the first time that viral IRFs inhibit activation-induced cell death. Thus, these results demonstrate an important mechanism of HHV8 to modulate the immune response by down-regulation of CD95L expression. Inhibition of CD95-dependent T cell function might contribute to the immune escape of HHV8.