Interaction with XIAP Prevents Full Caspase-3/-7 Activation in Proliferating Human T Lymphocytes

Eur. J. Immunol. 2008. 38: 1979–1987 DOI 10.1002/eji.200838211 Molecular immunology 1979

Interaction with XIAP prevents full caspase-3/-7 activation in proliferating human T lymphocytes

Maren Paulsen, Sandra Ussat, Marten Jakob, Gudrun Scherer, Inga Lepenies, Stefan Schtze, Dieter Kabelitz and Sabine Adam-Klages

Institute of Immunology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/ DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.

Key words: Caspases Á Proliferation Á T lymphocytes Á XIAP

Supporting Information available online

Introduction of death receptors) or intrinsic (activation of mitochondria) signaling pathways and cleave a limited number of substrates, Proteases belonging to the family of caspases are well character- most importantly other caspases. These effector caspases (cas- ized as essential enzymes regulating cytokine release and pase-3, -6, -7) then cleave a plethora of substrates distributed apoptosis [1, 2]. All caspases are expressed as zymogens consisting across the entire cell, leading to the regulated demise of the cell of a prodomain of various sizes, a large, and a small subunit (about [1–3]. 20 kDa and 10 kDa, respectively). Both subunits are liberated by Surprisingly, caspase activity has been found to be essential for proteolysis and form the active heterotetramer. The pro-apoptotic various differentiation and activation processes, most strikingly caspases are divided into two subgroups according to their for activation-induced proliferation of T lymphocytes [4–6]. function during the cell death process. Initiator caspases Several years ago, activation of caspase-3 has been observed in (caspase-2, -8, -9, and -10) are activated by extrinsic (activation living and proliferating T cells [7, 8]. Shortly thereafter, two independent groups described that broad-spectrum inhibitors of caspases prevent T cell proliferation [9, 10]. Independent in vivo

Correspondence: Prof. Dr. Sabine Adam-Klages observations corroborated these initial findings by convincingly e-mail: [email protected] proving that the initiator caspase-8 is indispensable for T cell

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activation and proliferation [11–13]. Whether the activation of detectable in apoptotic cells. Caspases-6 and -9 were cleaved in effector caspases like caspase-3 is as well a vital prerequisite for apoptotic cells only, while both most important effector caspases, T cell activation is less clear. namely caspase-3 and -7, were significantly cleaved in proliferating The initial studies reporting significant caspase-3 activation in cells as well. However, the cleavage patterns showed distinct living, proliferating T cells provoked the so far unsolved question differences between proliferating and apoptotic cells. The anti- as to how the cells are able to survive the presence of such a procaspase-3 antibody recognizes procaspase-3 (35 kDa) and clea- apoptotic enzyme. It has been speculated that active caspases, vage products thereof (p20, p19, and p17); the anti-caspase-7 especially caspase-3, might be restrained from cleaving substrates antibody recognizes procaspase-7 (35 kDa) and its cleavage that inevitably provoke apoptosis by a limited subcellular products (p30 and p20). The predominance of the p20 fragment compartmentalization [4, 5]. The localization of active caspases of caspase-3 visible in proliferating cells can be used as “footprint” during apoptosis has been investigated by several groups [14–18]. indicating that caspase-3 activity is inhibited, preventing auto- While caspase-8 appears to be exclusively restricted to the catalytic processing generating the fully mature p17 fragment cytoplasm of apoptotic cells, active caspase-3 displays a much [23]. This pattern of caspase-3 cleavage has first been observed in broader distribution. Notably, nuclear translocation of active Jurkat cells protected from Fas-induced apoptosis by overexpres-

caspase-3 depends on binding and cleavage of A-kinase-anchoring sion of Bcl-2 or Bcl-XL [24]. Therefore, we analyzed similar Jurkat protein 95 [19, 20]. The subcellular localization of active clones (successful Bcl-2 overexpression and protection is shown in caspase-3 in proliferating T cells has not been investigated. Supporting Information Fig. S2) for cleavage of the same caspases Another possibility to keep active caspase-3 in check might be by tested previously in T cells. In fact, the cleavage patterns detected blocking its activity through interaction with endogenous in vector-transfected Jurkat cells dying in response to Fas inhibitors like the inhibitor-of-apoptosis proteins (IAP) [21]. stimulation corresponded to the patterns seen in apoptotic In this study, we provide evidence that in proliferating human T cells. Vice versa, the patterns of Bcl-2-overexpressing cells T cells cleaved caspase-3 and -7 interact with X-linked inhibitor-of- corresponded to those detected in proliferating cells (Supporting apoptosis protein (XIAP). This interaction results in reduced Information Fig. S3 compared to Fig. 1A). autoprocessing of caspase-3, a largely restricted cytoplasmic Since the observed cleavage pattern especially of caspase-3 localization of both caspases, lack of pronounced substrate indicated an inhibited activity, we next wanted to determine the cleavage, and ultimately survival of the cells. activity of the effector caspases-3/-7. Since both caspases possess largely overlapping cleavage site specificity with the preferred site DEVD, all available activity assays are not able to distinguish Results between their activities. We thus measured DEVDase activity by three different methods, purely in vitro using DEVD-AMC or Attenuated activation of effector caspases-3 and -7 in DEVD-luciferase as substrates as well as intracellular activity with proliferating T cells PhiPhiLux-G2D2. As before, we compared the DEVDase activities of proliferating T cells with Bcl-2-overexpressing and anti-Fas To address the question how proliferating T lymphocytes survive antibody-stimulated Jurkat cells, and the activities of apoptotic despite caspase activation, we first wanted to identify which T cells with vector-transfected and Fas-stimulated Jurkat cells. caspases are cleaved in proliferating T cells compared to apoptotic Again, we found good correlations for both compared groups cells. To this end, human T lymphocytes were purified and left (Fig. 1B, C and Supporting Information Fig. S4A, B). Taken untreated (resting cells, r) or were stimulated with anti-CD3 and together, we concluded that in proliferating T cells initial cleavage anti-CD28 antibodies for 4 days (proliferating cells, p). To exclude of the effector caspases-3 and -7 takes place, but that their full cells dying from growth factor deprivation or by activation- activation is blocked. induced cell death, we purified living cells prior to the lysate preparation using Ficoll-Hypaque. An aliquot of the proliferating cells was additionally treated for the last 16 h with etoposide to Cytoplasmic localization of caspases-3/-7 without provoke apoptosis and was analyzed in parallel (apoptotic cells, a). detectable substrate cleavage Since it has been reported earlier that the detection of effector caspase cleavage might result from a lysis artifact [22], we first Since a limited subcellular localization of activated caspases would examined cleavage of caspase-3 in proliferating T cells using be one of the possibilities to assure the survival of proliferating different lysis conditions. As shown in Fig. S1 (see Supporting T cells, we prepared various fractions of resting, proliferating, and Information), caspase 3 cleavage was detected in lysates of apoptotic T cells and analyzed them for the presence of proliferating T cells as well as PHA blasts, regardless of the lysis caspase-3/-7 cleavage products. First, the purity of the subcellular buffer, confirming previous observations [9]. Therefore, whole fractions was verified by Western blotting using antibodies specific cell lysates were prepared using TNE buffer and analyzed by for calpain (cytosolic), lysosome-associated membrane protein Western blotting for cleavage of various caspases. As shown in (LAMP)-1 (lysosomal), histone (nuclear), and vimentin (cyto- Fig. 1A, cleavage of caspase-8 yielding the p43/41 form was visible skeletal; Fig. 2). The 35-kDa proform of caspase-3 was detected in in proliferating cells, but the mature p18 fragment was only the cytoplasmic fractions of all three T cell preparations, with

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Figure 1. Caspase cleavage and activity in proliferating and apoptotic human T lymphocytes. (A) Purified human T cells were left untreated (resting cells, r) or were stimulated for 4 days with immobilized anti-CD3/CD28 antibodies (proliferating cells, p). Immediately before lysis, the living cells were purified by centrifugation using Ficoll-Hypaque. One aliquot of the proliferating T cells was additionally treated with etoposide for the final 16 h (apoptotic cells, a). Cellular lysates were prepared and analyzed for the expression of various caspases by Western blotting. The proenzymes and the respective cleavage products are indicated. The positions of molecular weight markers in kDa are indicated at the left of each blot. Nonspecific bands are marked with an asterisk. (B) Cells stimulated as described for (A) were lysed and in vitro DEVDase activity was quantified using DEVD-luciferase as substrate. (C) Intracellular DEVDase activity was determined in cells stimulated as described for (A) using the cell-permeable substrate PhiPhiLux-G2D2 and flow cytometry. The results shown are representative of at least three experiments using different donors. fewer amounts in apoptotic cells due to excessive cleavage (Fig. 2). result of the destruction of cellular structures. In summary, our Cleavage products of caspase-3 were detected only in proliferating data indicate that active effector caspases might exhibit a broader and apoptotic T cells. Notably, in proliferating cells again only distribution in apoptotic T cells, while they are rather confined to incomplete cleavage of caspase-3 was detected, and these cleavage the cytoplasm of proliferating T cells. products were present predominantly in the cytoplasm. Only tiny The observed differences in caspase-3/-7 cleavage patterns amounts of cleavage products were observed also in the and in the localization of active fragments between proliferating membrane/organelle and nuclear fractions. A similar limited and apoptotic T lymphocytes prompted us to investigate the distribution was observed of caspase-7 cleavage products in cleavage of selective substrates in both cell populations. We chose proliferating cells (Fig. 2). In contrast, induction of apoptosis led to cytoplasmic (D4-GDI, vav), nuclear (PARP, p21Cip1/WAF1), and almost complete cleavage of caspase-3, indicated by the prominent membrane-associated (PLCc, TCRf) proteins, which have been detection of the p17 fragment which appeared broadly distributed described as substrates of effector caspases, and the characteristic throughout the entire cell including the nucleus (Fig. 2). However, caspase-8 substrate c-FLIP [3]. With the exception of c-FLIP, none since the marker protein histone was present also in the of the caspase substrates was cleaved in proliferating cells while all cytoplasmic fraction, most likely due to a breakdown of the of them were processed during apoptosis (Fig. 3 and data not nuclear envelope during apoptosis, we cannot rule out that the shown). broader distribution of caspase fragments in apoptotic cells is a

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caspase-7 are bound and thereby kept inactive by XIAP. Using lysates from resting, proliferating, and apoptotic T cells, we immunoprecipitated caspase-3 and tested for coprecipitating XIAP and vice versa. As shown in Fig. 4A, XIAP was detected in caspase-3 precipitates from proliferating cells, while no association was seen in resting or apoptotic cells. Immunoprecipitation of XIAP followed by immunoblotting with anti-caspase antibodies revealed association of the p19 and p20 fragments of caspase-3 as well as the p20 fragment of caspase-7, while both coprecipitations were detected again exclusively in proliferating cells (Fig. 4B and C). These results corresponded again well with results of similar

Figure 2. Subcellular localization of effector caspases-3/-7 in proliferating and apoptotic T lymphocytes. Purified human T cells were stimulated exactly as described for Fig. 1. Subcellular fractions were prepared (1, cytosolic; 2, membrane/organelle; 3, nuclear; 4, cytoskeletal) and analyzed for the presence of the marker proteins calpain, LAMP1, histone, and vimentin. Additionally, expression and cleavage pattern of caspase-3 and -7 were determined. Molecular weight markers are indicated in kDa. The results are from one experiment out of three with different donors.

Restriction of effector caspases-3/-7 activity by interaction with XIAP

The p20 cleavage product of caspase-3 is in vitro as active as the p17 fragment [25], but often kept inactive in living cells due to binding of XIAP [21, 24]. The p20 cleavage product of caspase-7 is also targeted for inhibition by XIAP [21]. Therefore, we examined whether in proliferating cells the p20 fragments of caspase-3 and

Figure 4. Interaction of XIAP with caspases-3/-7 in proliferating T cells. Purified T cells were stimulated as indicated and whole cell lysates (WCL) were generated. (A) IP were performed using the anti-caspase-3 rabbit antiserum and subjected to western blot analysis. The blot was stained with the mAb specific for XIAP and the peroxidase-labeled Figure 3. Cleavage of characteristic effector caspase substrates occurs Trueblot secondary antibody. For control, 20 lg WCL was subjected to only in apoptotic T lymphocytes. Purified human T cells were western blotting with anti-XIAP and anti-caspase-3 antibodies, respec- stimulated as indicated and cellular lysates were prepared. Equal tively. (B) IP were conducted using the anti-XIAP mAb and immuno- amounts of protein were analyzed for the specified proteins by western blotted using the anti-caspase-3 antiserum. WCL were run in parallel to blotting. The intact proteins are indicated, their respective cleavage identify coprecipitated caspase-3 fragments. (C) XIAP was immuno- products are marked by arrowheads, and nonspecific bands are precipitated and western blotting was conducted using the anti- indicated by an asterisk. caspase-7 antibody. WCL were again run in parallel.

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experiments performed with the previously used Jurkat cell model (Supporting Information Fig. S5A and B). Release of the mitochondrial protein second mitochondria- derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO) inhibits the binding of XIAP to caspase-3, allowing autoprocessing during death receptor-mediated apoptosis [23, 24]. Therefore, we analyzed cytosolic and membrane/organelle fractions of resting, proliferating, and apoptotic T cells using an anti-Smac/DIABLO antibody. As shown in Fig. 5, considerable amounts of Smac/ DIABLO in the cytosolic fraction were detected only in apoptotic cells. In conclusion, these data indicate that the inhibitory effect of XIAP on effector caspases limiting their full activation in proliferating T cells is abrogated after induction of apoptosis by the release of Smac/DIABLO, resulting in prominent substrate cleavage and cell death. To confirm the role of XIAP in proliferating T cells as inhibitor of effector caspase activity and therefore cell death, we first employed the pharmacological XIAP inhibitor embelin [26]. As shown in Fig. 6A, treatment with 20 lM embelin led to increased intracellular DEVDase activity in proliferating T cells, but not in resting T cells. In T cells already undergoing apoptosis due to stimulation with etoposide, intracellular DEVDase activity was unchanged (Fig. 6A). In addition, treatment with increasing amounts of embelin augmented cell death only in proliferating T cells but not in resting or etoposide-treated cells, arguing against a general cytotoxic effect of embelin (Fig. 6B). Embelin treatment also led to increased apoptosis in Bcl-2-overexpressing Jurkat cells treated with anti-Fas antibody previously used as model system for XIAP-mediated protection (Supporting Informa- tion Fig. S6). Figure 6. Pharmacological inhibition of XIAP leads to enhanced To definitely verify the important role of XIAP in proliferating DEVDase activity and cell death in proliferating T cells. (A) Purified T cells were left untreated or were stimulated with anti-CD3/28 mAb for T cells, we finally down-regulated XIAP expression by RNA 3 days and additionally treated with DMSO or 20 lM embelin. Where interference. We performed two different approaches to knock indicated, proliferating T cells were additionally incubated with 50 lg/ down XIAP in proliferating T cells. First, we transfected resting mL etoposide. After an additional 16 h of incubation, cells were analyzed for DEVDase activity by flow cytometry using the cell- T cells with a XIAP-specific siRNA or a scrambled control and permeable substrate PhiPhiLux-G2D2. Shown are results of one stimulated them after 2 days of incubation to allow for XIAP down- independent experiment out of three using different donors. modulation with anti-CD3/28 mAb for an additional 2 days. (B) T cells were stimulated as described for (A) for 3 days and additionally treated with DMSO or increasing amounts of embelin in Alternatively, we stimulated purified Tcells with anti-CD3/28 mAb the presence or absence of 5 lg/mL etoposide. After an additional 3 days for 2 days to initiate proliferation and then transfected the cells of incubation, cells were analyzed for cell death by PI staining and flow with the siRNA. Both approaches led to substantially reduced XIAP cytometry. Shown are average results of determinations in triplicate with the respective standard deviations. expression (Fig. 7A and E). As readout for induction of effector

caspase activation due to XIAP inhibition, we used quantification of DEVDase activity, [3H]-thymidine incorporation, and cell death. Only transfection with the XIAP-specific siRNA clearly resulted in increased DEVDase activity, reduced proliferation, and augmented cell death, regardless whether the transfection was performed before or after anti-CD3/28 stimulation (Fig. 7B–D, F–H). Although we detected enhanced caspase activity usually indicative Figure 5. Cytoplasmic detection of Smac/DIABLO in apoptotic T cells. for apoptosis, we cannot rule out that the observed cell death Purified T cells were stimulated as indicated and used for the occurred through non-apoptotic processes, since the PI staining preparation of cytosolic and membrane/organelle fractions. Equal amounts of protein were analyzed by western blotting for the release assay used to quantify cell death does not discriminate between of Smac/DIABLO from mitochondria. different modes of cell death. These data strongly support our

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hypothesis that interaction with XIAP inhibits full effector caspase Discussion activation, prominent substrate cleavage, and apoptosis in proliferating T cells. It is now widely accepted that caspases play an important role in activation-induced proliferation of T lymphocytes [4, 5]. While the requirement of caspase-8 has been undoubtedly proven by analysis of caspase-8-deficient patients and conditional knockout mice [11–13], the relevance of effector caspases for T cell proliferation is unclear. Moreover, it is still an open question how living cells facilitate their survival despite the presence of active, highly pro-apoptotic effector caspases. In this study, we analyzed which effector caspases are cleaved in proliferating human T lymphocytes, to which extent they are active in comparison to apoptotic cells, how subcellular distribution of active effector caspases and therefore the subset of substrates differ between proliferating and apoptotic cells, and what mechanism(s) might ensure the survival of the proliferating cells. At first, we observed that the effector caspases-3 and -7, but not caspase-6, are cleaved and therefore presumably activated in proliferating human T cells. Cleavage of caspase-8 was clearly detected in both cell populations, while caspase-9 processing and in vitro cleavage of a caspase-9-specific substrate (S.U. and S.A.-K., unpublished data) was measurable only in apoptotic cells. Therefore, it seems most likely that effector caspases-3/-7 are directly cleaved by caspase-8 without involvement of the mitochondrial pathway of caspase-9 activation. A possible mechanism that enables proliferating T cells to cope with active effector caspases could be that a limited localization within the cell allows access only to a subset of substrates whose cleavage is compatible with cell survival and/or required for proliferation [5]. Therefore, we analyzed the subcellular localization of active effector caspases in proliferating human T lymphocytes and compared it with the localization in apoptotic T cells. We found that the cleavage products of caspases-3/-7 were largely confined to the cytoplasm of proliferating T cells, while in apoptotic cells they seemed to display a rather general distribution. Thus, restricted localization could be responsible for a limited substrate cleavage in proliferating cells. However, when we tested cleavage of known substrates with varying intracellular localiza- tions, not even cytosolic substrates of effector caspases were cleaved in proliferating cells, although processing of all substrates was readily detected in apoptotic cells. Processing of the most renowned caspase-3 substrate PARP in proliferating T cells has been reported by one group [8], but was neither confirmed by others [9, 10] nor by us. Additionally, cleavage of the cell cycle- inhibitory kinase Wee1 [9], caspase-6 and caspase-7 [9], and the Figure 7. XIAP down-modulation by RNA interference. (A–D) Purified caspase-6 substrate lamin B [8, 9] was reported to occur in T cells were transfected with control or XIAP-specific siRNA and proliferating T cells. In our experiments using CD3/28-stimulated stimulated after 48 h of incubation as indicated for an additional 2 days. XIAP and actin expression were analyzed by Western blotting (A), T cells, none of the substrates we tested, among them caspase-6, proliferation was quantified by [3H]thymidine incorporation (B), DEV- was cleaved. This difference might be due to the fact that PBMC Dase activity was measured using DEVD-luciferase as substrate (C), and were used in the studies mentioned above [8–10] while we cell death was quantified by PI staining and flow cytometry (D). (E–H) Purified T cells were stimulated with anti-CD3/28 antibodies for employed purified T cells and we also excluded dead cells prior to 2 days followed by nucleofection with control or XIAP-specific siRNA. analysis. After an additional 48 h of incubation, cells were analyzed as described Interestingly, we noticed a different pattern of caspase-3 for (A–D). Shown are the average results of triplicate determinations with the respective standard deviations or of duplicates without cleavage products in proliferating T cells compared to apoptotic standard deviations, respectively. cells. The p17 fragment representing the fully mature large subunit

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predominated in apoptotic cells, while the partially cleaved p20 indicate that the functional importance of XIAP varies between fragment was the major product detected early during T cell mice and humans. proliferation. A similar cleavage pattern in proliferating T cells was Finally, our findings argue against caspase-3 activation as an observed in other studies, but was not compared with the same essential prerequisite for activation and proliferation of cells after induction of apoptosis [7, 9]. The p20 form of caspase-3 T lymphocytes. Interestingly, a kinetic analysis of anti-CD3/28 comprising the complete prodomain still attached to the large antibody-treated T cells showed that morphological changes subunit displays equal activity as the fully processed p17 product precede caspase-3 activation and the onset of proliferation when analyzed in vitro [25]. Using so-called type II cells, however, occurred simultaneously with it, while a broad-spectrum caspase it has been shown that overexpression of Bcl-2 or Bcl-XL protects inhibitor prevented already the activation-induced morphological the cells from Fas-induced apoptosis by keeping the p20 fragment changes (S.U. and S.A.-K., unpublished data). Therefore, cas- of caspase-3 inactive through binding of the endogenous caspase pase-3 activity is unlikely to be required for the initiation of T cell inhibitor XIAP [23, 24]. Therefore, we made use of the proliferation. This assumption is fostered by data derived from characteristic type II cell line Jurkat, either vector controls or knockout mice: T cells from caspase-3-deficient mice show cells overexpressing Bcl-2, and compared (1) Fas-stimulated increased growth due to reduced T cell apoptosis rather than vector-transfected Jurkat cells with etoposide-treated primary reduced proliferation [31]. In addition, possible compensation for T cells, both undergoing apoptosis, and (2) Fas-stimulated Bcl-2- the lack of caspase-3 by the closely related caspase-7 appears overexpressing and thereby protected Jurkat cells with proliferat- unlikely, because RAG-deficient mice reconstituted with cas- ing primary T cells. A detailed comparison revealed in both groups pase-3/7–/– fetal liver cells revealed a regular distribution of T cell similar cleavage patterns of all caspases investigated. In addition to populations in the thymus [32], while a complete lack of T cells the incomplete caspase-3 cleavage, we observed considerably was observed in mice reconstituted with caspase-8–/– cells [13]. lower DEVDase activity in lysates from proliferating T cells than in In conclusion, we propose the following model: caspase-8 apoptotic cells, confirming a previous study reporting lower activation induced either by death receptor ligation or by a TCR- DEVDase activity in proliferating CD4+ T cells than in the same dependent mechanism is essential for c-FLIP cleavage and thus cells after Fas stimulation [27]. Again, these findings resembled contributes to NF-jB activation, which is absolutely required for the results obtained with the Jurkat cell model. T cell activation. In addition, active caspase-8 cleaves caspase-3, Importantly, we provide evidence that caspases-3/-7 interact generating the p20 fragment. This p20 form of caspase-3 then is with XIAP in proliferating but not in apoptotic T cells, while the restrained by XIAP to prevent pronounced substrate cleavage and XIAP inhibitor Smac/DIABLO is released from mitochondria in untimely apoptosis. Since NF-jB activation up-regulates XIAP apoptotic but not in proliferating T cells. Pharmacological or expression [21], caspase-8 itself might be responsible for keeping siRNA-mediated inhibition of XIAP resulted in augmented the potentially deleterious activation of caspase-3 under control. DEVDase activity and cell death in proliferating but not in resting T cells. Thus, our data suggest that after T cell activation the effector caspases-3/-7 are cleaved most likely by caspase-8, but are restrained from pronounced substrate cleavage and thereby from inducing apoptosis by interaction with XIAP. Of note, nuclear translocation of active caspase-3 is achieved by active transport Isolation and stimulation of primary human T cells mediated through binding of its substrate A-kinase-anchoring protein 95 [20]. Therefore, the lack of substrate binding and Human PBMC were purified from buffy coats of healthy donors cleavage might also be responsible for the limited subcellular using Ficoll-Hypaque (the scientific use of buffy coat cells was distribution of caspase-3 in proliferating T cells. approved by the local Ethics Commission). A negative selection kit In view of our findings, it seems surprising that XIAP-deficient from Dynal (Hamburg, Germany) and magnetic separation were mice do not experience immunodeficiency due to decreased used to purify T lymphocytes from PBMC. The purification of the proliferative capacity of their T cells [28]. However, since the cells T cells was examined by anti-CD3 antibody staining and flow of these mice did not reveal any defects in apoptosis, it has been cytometry and reached routinely at least 97%. To induce suggested that a compensatory mechanism replaces the lack of proliferation, purified T cells were seeded at 5 Â 105 cells/mL XIAP with up-regulation of related molecules, thereby taking over on plastic dishes coated with 5 lg/mL anti-CD3 mAb (OKT3, XIAP's function. Similarly, T cells from mice lacking the adversary provided by Janssen Cilag, Neuss, Germany) and 1 lg/mL anti- of XIAP, namely Smac/DIABLO, show normal apoptotic as well as CD28 mAb (Coulter Immunotech, Krefeld, Germany). Immedi- proliferative responses [29]. Together, these studies argue that ately before harvesting resting and proliferating cells, living cells redundant molecules exist in mice as backup for XIAP as well as were separated from dead cells by additional centrifugation Smac/DIABLO. Recently, XIAP-deficient patients have been through a Ficoll-Hypaque gradient. To induce apoptosis, prolif- described whose lymphocytes are more vulnerable to apoptosis erating T cells (3 days after anti-CD3/28 antibody stimulation) induction in response to various stimuli. Most importantly, T cells were treated with 50 lg/mL etoposide (Sigma, Deisenhofen, are more sensitive towards stimulation through the TCR and this Germany) for 16 h. The inhibitor of XIAP, embelin, was purchased effect is reversed by retransfection of XIAP [30]. These data

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from Merck Biosciences (Darmstadt, Germany) and solubilized at Quantification of DEVDase activity 20 mM in DMSO. Intracellular DEVDase activity was determined using the cell- permeable fluorogenic substrate PhiPhiLux-G2D2 (Merck Bios- [3H]Thymidine incorporation assay ciences) as previously described [33]. Quantification of caspase activities in vitro using DEVD-luciferase as substrate was To measure T cell proliferation, [3H]thymidine incorporation was performed using the Caspase-Glo 3/7 Assay from Promega determined as described before [33]. (Madison, WI) following the instructions provided by the manufacturer.

Quantification of cell death RNA interference To quantify cell death, T cells stimulated with anti-CD3/CD28 antibody in the absence or presence of etoposide were analyzed for The validated XIAP-specific siRNA (ID #121292) and a control exclusion of PI using flow cytometry [34]. siRNA were purchased from Ambion (Huntingdon, UK). Purified resting T cells (5 Â 106) were transfected with 100 lM siRNA by nucleofection using the Human T cell Nucleofector kit (Amaxa Subcellular fractionation Biosystems, Cologne, Germany) and program U-014. After an incubation time of 48 h to allow for down-regulation of XIAP, cells To obtain different subcellular fractions of stimulated or non- were left untreated or were stimulated with anti-CD3/28 antibody stimulated T cells, a subcellular fractionation kit from Merck for an additional 2 days. Alternatively, Tcells were stimulated with Biosciences was employed following the instructions of the anti-CD3/28 antibody for 48 h followed by nucleofection using supplier. In parallel, whole cell lysates were prepared by lysing program X-001. Analyses were performed after incubating the cells cells in TNE buffer as previously described [34]. Protein for an additional 48 h. concentrations were determined using the BCA reagent kit from Perbio Science (Bonn, Germany).

Immunoprecipitation and Western blot analysis Acknowledgements: We thank S. Korsmeyer for the vector- From each TNE lysate, 20 lg of protein, and from each subcellular transfected and Bcl-2-overexpressing Jurkat cells. This work was fraction, 2–20 lg of protein were analyzed for the content of supported by the Faculty of Medicine, University of Kiel (to various proteins by Western blot analysis, as previously described S.A.-K.). [34]. The primary antibodies used were obtained from the following sources: anti-calpain and anti-histone H3: Abcam Conflict of interest: The authors declare no financial or (Cambridge, UK); anti-LAMP1, anti-PARP, anti-D4-GDI, and commercial conflict of interest. anti-XIAP: BD Biosciences (Heidelberg, Germany); anti-vimentin and anti-actin: Santa Cruz (Heidelberg, Germany); anti-caspase-3, -6, -7, -8, and -9, anti-PLCc, and anti-Smac/DIABLO: Cell Signaling References (Frankfurt, Germany); anti-c-FLIP: Qbiogene-Alexis (Grnberg,

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f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2008. 38: 1979–1987 Molecular immunology 1987

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18 Takemoto, K., Nagai, T., Miyawaki, A. and Miura, M., Spatio-temporal 34 Lschen, S., Ussat, S., Scherer, G., Kabelitz, D. and Adam-Klages, S., activation of caspases revealed by indicator that is sensitive to environmental Sensitization to death receptor cytotoxicity by inhibition of FADD/caspase effects. J. Cell Biol. 2003. 160: 235–243. signaling: Requirement of cell cycle progression. J. Biol. Chem. 2000. 275: 24670–24678. 19 Kamada, S., Kikkawa, U., Tsujimoto, Y. and Hunter, T., Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s). J. Biol. Chem. 2005. 280: Abbreviations: IAP: inhibitor-of-apoptosis protein Á LAMP: lysosome- 857–860. associated membrane protein Á Smac/DIABLO: second mitochondria- derived activator of caspases/direct inhibitor of apoptosis-binding 20 Kamada, S., Kikkawa, U., Tsujimoto, Y. and Hunter, T., A-kinase- anchoring protein 95 functions as a potential carrier for the nuclear protein with low isoelectric point Á WCL: whole cell lysate Á translocation of active caspase-3 through an enzyme-substrate-like associa- XIAP: X-linked inhibitor-of-apoptosis protein tion. Mol. Cell. Biol. 2005. 25: 9469–9477. Full correspondence: Prof. Dr. Sabine Adam-Klages, Institute of 21 Callus, B. A. and Vaux, D. L., Caspase inhibitors: Viral, cellular and Immunology, UK S-H Campus Kiel, Michaelisstrasse 5, D-24105 Kiel, chemical. Cell Death Differ. 2007. 14: 73–78. Germany 22 Zapata, J. M., Takahashi, R., Salvesen, G. S. and Reed, J. C., Granzyme Fax: +49-431-5973335 release and caspase activation in activated human T-lymphocytes. J. Biol. e-mail: [email protected] Chem. 1998. 273: 6916–6920. Supporting Information for this article is available at 23 Bratton, S. B. and Cohen, G. M., Death receptors leave a caspase footprint http://www.wiley-vch.de/contents/jc_2040/2008/38211_s.pdf that Smacs of XIAP. Cell Death Differ. 2003. 10: 4–6.

24 Sun, X., Bratton, S. B., Butterworth, M., MacFarlane, M. and Cohen, Received: 30/1/08

G. M., Bcl-2 and Bcl-XL inhibit CD95-mediated apoptosis by preventing Revised: 19/3/08 mitochondrial release of Smac/DIABLO and subsequent inactivation of Accepted: 18/4/08

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