Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas -Induced

This information is current as Peter Küenzi, Pascal Schneider and Dirk A. E. Dobbelaere of October 2, 2021. J Immunol 2003; 171:1224-1231; ; doi: 10.4049/jimmunol.171.3.1224 http://www.jimmunol.org/content/171/3/1224 Downloaded from References This article cites 69 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/171/3/1224.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/-Induced Apoptosis1

Peter Ku¨enzi,2* Pascal Schneider,† and Dirk A. E. Dobbelaere3*

Lymphocyte homeostasis is regulated by mechanisms that control lymphocyte proliferation and apoptosis. Activation-induced cell death is mediated by the expression of death ligands and receptors, which, when triggered, activate an apoptotic cascade. Bovine T cells transformed by the intracellular parasite Theileria parva proliferate in an uncontrolled manner and undergo clonal expansion. They constitutively express the death Fas and its ligand, FasL but do not undergo apoptosis. Upon elimination of the parasite from the host cell by treatment with a theilericidal drug, cells become increasingly sensitive to Fas/FasL-induced apoptosis. In normal T cells, the sensitivity to death receptor killing is regulated by specific inhibitor . We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of -8, and X-- linked (IAP) as well as c-IAP, which can block downstream executioner , are constitutively expressed in T. parva-transformed T cells. Expression of these proteins is rapidly down-regulated upon parasite elimination. Downloaded from

Antiapoptotic proteins of the Bcl-2 family such as Bcl-2 and Bcl-xL are also expressed but, in contrast to c-FLIP, c-IAP, and X-chromosome-linked IAP, do not appear to be tightly regulated by the presence of the parasite. Finally, we show that, in contrast to the situation in tumor cells, the phosphoinositide 3-kinase/Akt pathway is not essential for c-FLIP expression. Our findings indicate that by inducing the expression of antiapoptotic proteins, T. parva allows the host cell to escape destruction by homeostatic mechanisms that would normally be activated to limit the continuous expansion of a T cell population. The Journal of Immu- nology, 2003, 171: 1224–1231. http://www.jimmunol.org/

ast Coast fever is a tick-transmitted disease of cattle, cells have fulfilled their immune function, homeostatic mecha- caused by infection of lymphocytes by the intracellular nisms are activated to reduce the pool of cells that was generated. E parasite Theileria parva. In its early stages, the pathogen- This relies on the down-regulation of growth factor and growth esis of East Coast fever is dominated by massive lymphoprolif- factor receptor expression to halt proliferation, as well as on eration, which accompanies the clonal expansion of parasitized T the active elimination of cells. Thus, in a final deletion phase, most cells (reviewed in Ref. 1). T. parva is unique among parasites in its activated T cells, with the exception of memory T cells, are even- capacity to stimulate uncontrolled proliferation of the cells it in- tually eliminated in a process called activation-induced cell death by guest on October 2, 2021 fects. To induce transformation, the parasite directly or indirectly (AICD) (11). Failure to do this increases the risk of malignancy activates a number of host cell signaling pathways including the (12) or the development of autoimmune disorders triggered by Jun-N-terminal kinase (2), NF-␬B (3–5) and phosphoinositide unchecked proliferation (13–15). 3-kinase (PI3-K)4 pathways (6, 7). Additionally, enhanced activity Fas (CD95), a cell surface death receptor belonging to the tumor of casein kinase II (8) and Src-related kinases (9, 10) has been necrosis factor/nerve growth factor receptor superfamily and its demonstrated. ligand (FasL, CD95L) are both expressed on activated T cells (14) Under physiological conditions, an acquired immune response in a process which is in part regulated by c- (16) and NF-␬B results in the generation of an expanded pool of effector T cells. (17–21). They are important contributors to AICD (22–24), given This process involves a series of defined steps that lead to activa- that the engagement of Fas by FasL triggers downstream pathways tion, proliferation, and differentiation of the recruited cells. Once T that lead to caspase activation and apoptosis. The process is tightly regulated and can be blocked at different levels by a range of cellular inhibitors. Caspases can be divided into upstream or ini- *Division of Molecular Pathology, Institute of Animal Pathology, University of Bern, tiator caspases (such as caspase-8 and caspase-10), which are Bern, Switzerland; and †Institute of Biochemistry, Faculty of Medicine, University of Lausanne, Lausanne, Switzerland linked to apoptotic signaling through death receptors, and down- Received for publication January 27, 2003. Accepted for publication May 22, 2003. stream effector/executioner caspases, which can become activated by initiator caspases and trigger the final steps of apoptosis (re- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance viewed in Refs. 25 and 26). Ligation of Fas induces recruitment of with 18 U.S.C. Section 1734 solely to indicate this fact. caspase-8 via adaptor molecules to the cytoplasmic portion of Fas 1 This research was funded by grants from the Swiss League (KFS to form a death-inducing signaling complex called DISC. There, 625.2.1998) and the Swiss National Science Foundation (31.61340) to D.A.E.D. caspase-8 is autoproteolytically cleaved, resulting in the release of 2 Current address: Division of Biochemistry Department of Medicine, University of catalytically active caspase fragments. Apoptosis can also be elic- Fribourg, Rue de la Muse´e 5, CH-1700 Fribourg, Switzerland. ited by the release of mitochondrial in response to 3 Address correspondence and reprint requests to Dr. Dirk Dobbelaere, Division of Mo- lecular Pathology, Institute of Animal Pathology, University of Bern, La¨nggass-Strasse cellular stress and internal cellular damage, stimulating the acti- 122, CH-3012 Bern, Switzerland. E-mail address: [email protected] vation of a complex consisting of apoptotic protease-activating 4 Abbreviations used in this paper: PI3-K, phosphoinositide 3-kinase; AICD, activa- factor 1 and caspase-9. Caspase-9 activates further downstream tion-induced cell death; FasL, Fas ligand; IAP, inhibitor of apoptosis protein; c-, caspases, resulting in apoptosis. Whereas members of the Bcl-2 cellular; rhs, recombinant human soluble; PARP, poly(ADP-ribose) polymerase; VAD-fmk, valylalanylaspartic acid fluoromethyl ketone; XIAP, X-chromosome- family regulate apoptosis induced through the mitochondrial path- linked IAP; ACAD, activated T cell autonomous death. way, proteins of the inhibitor of apoptosis protein (IAP) family

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 1225 bind to and inhibit caspases such as caspase-3, -6, -7, or -9. More preincubated in the absence or presence of recombinant Fc-FasL followed recently, a family of inhibitors called FLIP (FLICE-inhibitory pro- by incubation with 1 ␮g of rhsFasL in a total volume of 25 ␮l of PBS, teins) has been described that blocks caspase-8 activation at the containing 10% heat-inactivated FCS and 0.02% sodium azide. FasL bind- ing was detected by incubation with the anti-FLAG mAb M2 (0.16 mg/ml) level of the death receptor (27, 28). Whereas T cells are initially and a FITC-conjugated anti-mouse IgG (diluted 1/25; Jackson ImmunoRe- resistant to Fas/FasL-induced apoptosis, with time they become search Laboratories, West Grove, PA). Staining with recombinant annexin increasingly sensitive, and it has been shown that reduced resis- V-FITC (Boehringer Mannheim, Mannheim, Germany) and propidium io- tance to apoptosis is paralleled by gradually waning levels of FLIP dide was performed according to the manufacturer’s instructions. All cells were analyzed in PBS containing 0.02% sodium azide using a FACScan (reviewed in Ref. 27). (BD Biosciences). Continuous proliferation and survival of the T. parva-infected T Alternatively, cells were seeded in 96-well microtiter plates and treated cell is dependent on the presence of the parasite in the host cell as described above. FITC-VAD-fmk (final concentration 5 ␮M; Promega) cytoplasm. Addition of the naphthoquinone derivative BW720c to was added directly to the cell suspension (4 ϫ 105 cells/ml), and cells were cultures of T. parva-transformed cells specifically kills the parasite incubated at 37°C for 20 min, washed with PBS, and analyzed in PBS containing 0.02% sodium azide using a FLX800 Microplate Fluorescence (29). BW720c does not affect the expression of housekeeping Reader (Bio-Tek Instruments, Winooski, VT). (5, 30, 31), shows no toxicity for the host cell, and does not interfere with the capacity of T cells to become activated and pro- Western blot liferate in response to mitogens (32). When the parasite is elimi- Immunoblot analysis was performed according to standard procedures. nated, cells lose their activated phenotype, cease to proliferate after TpM(803) T cells were harvested and washed in PBS. Cell pellets were 3–4 days, and finally undergo apoptosis (10, 33). Despite the fact resuspended in lysis buffer (50 mM Tris (pH 7.4), 100 mM NaCl, 30 mM sodium pyrophosphate; 50 mM NaF, 0.5% Nonidet P-40, 1ϫ protease that T. parva-infected T cells show many characteristics of acti-

inhibitors complete; Roche Diagnostics, Rotkreuz, Switzerland) and incu- Downloaded from vated T cells (reviewed in Ref. 1) (30), they apparently escape bated on ice for 15 min, followed by a centrifugation step (4°C, 17,500 ϫ AICD. To what extent molecular pathways that govern AICD are g, 15 min). Cell lysates equivalent to 20 ␮g of protein were separated using activated in T. parva-infected T cells and how parasitized cells 12% SDS-PAGE and blotted onto nitrocellulose membranes using standard acquire protection against this powerful homeostatic process have procedures. After a 2-h incubation at 37°C in a blocking mixture (5% milk powder, 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.05% Tween), mem- not yet been investigated. In the present study, we exploited the branes were incubated overnight at 4°C (mAb) or for1hatroom temper- reversibility of Theileria-induced transformation to tackle these ature (polyclonal Ab) with specific primary Ab diluted in blocking buffer: questions. We investigated Fas and FasL expression and compared anti-Fas, 1/500, anti-caspase-8, 1/200; anti-caspase-9, 1/200; anti-FLIP, http://www.jimmunol.org/ the sensitivity of T. parva-transformed T cells to Fas/FasL-medi- 1/1000; anti-XIAP, 1/1000; anti-Bcl-2, 1/500; anti-Bcl-x(L/S), 1/500; anti- Bax, 1/500. Secondary Abs used were affinity-purified goat anti-rabbit IgG ated apoptosis with that of BW720c-cured cells. In addition, we (H ϩ L) Ab conjugated to HRP (Cell Signaling Technology, Beverly, MA) monitored the potential involvement of members of the different used 1/5000, or affinity-purified rabbit anti-mouse IgG (H ϩ L) Ab con- antiapoptotic protein families. jugated to HRP (DAKO, Glostrup, Denmark) used 1/2000 in blocking buffer for 45 min at room temperature. Specific bands were detected using Materials and Methods the ECL System (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Cell culture Immunofluorescence microscopy ϩ Ϫ ϩ TpM(803) T cells are CD4 CD8 ␣␤ TCR T cells of bovine origin, trans- Cytospins were prepared using 100 ␮l of cell suspension (1 ϫ 106 cells/ by guest on October 2, 2021 formed by T. parva; the characteristics of this cloned cell line have been ml). Cells were fixed either in absolute methanol (Ϫ20°C) for 5 min (c- described elsewhere (33). Cells were cultured at 37°C in Leibovitz L-15 IAP-1; FasL) or in 3.7% paraformaldehyde (prepared in PBS containing medium (Life Technologies, Paisley, U.K.) containing 10% (v/v) heat- 10% heat-inactivated FCS) for 20 min at room temperature (PARP p85) inactivated FCS, 20 mM HEPES (pH 7.1), 2 mM L-glutamine, and 1ϫ and permeabilized with 0.2% Triton X-100 (prepared in PBS containing antibiotic/antimycotic solution (Life Technologies). To eliminate the par- 10% heat-inactivated FCS) for 10 min at room temperature. The Abs were asite, BW720c (Buparvaquone; Pitman-Moore, Mundelein, IL) was added diluted in PBS containing 10% heat-inactivated FCS and applied for1hat at 50 ng/ml to the culture medium. the following dilutions: rabbit anti-c-IAP-1 (1/50); rabbit anti-PARP p85 (1/200); and mouse anti-FasL (1/50). The cells were subsequently washed Ligands and Abs twice with PBS and incubated for 1 h with affinity-purified Texas Red- conjugated goat anti-rabbit IgG (H ϩ L; Vector Laboratories, Burlingame, Trimerized recombinant FasL (SuperFasL) was purchased from Apotech CA) diluted 1/500 or affinity-purified Alexa-Fluor 488 goat anti-mouse IgG (Epalinges, Switzerland). The recombinant human soluble FasL fused to a (H ϩ L; Molecular Probes, Leiden, The Netherlands) diluted 1/1500 in linker peptide (26 aa) and a FLAG tag at the N terminus (rhsFasL; Alexis, PBS containing 10% heat-inactivated FCS. Nuclei were stained with San Diego, CA) was used together with an enhancer Ab for multimeriza- Hoechst 33258 dye (diluted 1/2000 in PBS) for 1 min. tion (M2 mouse mAb anti-FLAG; Sigma-Aldrich, Buchs, Switzerland). The mAb directed against FasL was purchased from Transduction Labo- ratories (BD Biosciences, Heidelberg, Germany). mAb raised against hu- Results man caspase-8 (AM46) and caspase-9 (AM47) were obtained from Cal- Caspases are activated in T cells cured of the parasite biochem-Novabiochem (San Diego, CA). Ab specific for Fas (sc715), Bcl- It has previously been reported that the proliferation of T. parva- x(L/S) (sc1041), Bax (sc493), and cellular (c-) IAP-1 (sc7943) and mAb for Bcl-2 (sc7382), which detect the corresponding proteins in multiple infected T cells ceases when the parasite is removed from the host species such as mouse, rat, and human, were purchased from Santa Cruz cell cytoplasm by treatment with BW720c and that cells displaying Biotechnology (Santa Cruz, CA). An Ab directed against human FLIP the morphological features of apoptosis appear after ϳ4 days in (I-Flice; 67071N) was obtained from BD PharMingen (Heidelberg, Ger- cultures of BW720c-cured cells (10, 33). We first monitored many). A mAb directed against human XIAP (M044-3) was purchased from Medical and Biological Laboratories (Nagoya, Japan). An Ab that whether caspases become activated upon killing of the parasite. detects human, rat, and bovine poly(ADP-ribose) polymerase (PARP) p85 The caspase inhibitor VAD-fmk binds specifically to caspases that (G7341) was obtained from Promega (Catalyz, Wallisellen, Switzerland). have become activated by proteolytic cleavage. Flow cytometric Abs other than the mAbs were all rabbit polyclonal Abs. analysis using FITC-labeled VAD-fmk showed that activated Flow cytometry caspases can be detected in a distinct population of T cells treated with BW720c for 3 days (Fig. 1A). To detect caspase activation, FITC-valylalanylaspartic acid fluoromethyl Caspases are normally present in the cell as proenzymes that are ketone (VAD-fmk; final concentration, 5 ␮M; Promega) was added di- rectly to the cell suspension (4 ϫ 105 cells/ml), and cells were incubated at processed into activated fragments in response to an apoptotic 37°C for 20 min followed by a 15-min fixation in PBS (pH 7.4) containing stimulus. To investigate the processing of procaspase-8 and -9, 0.5% paraformaldehyde. For detection of FasL binding, 5 ϫ 105 cells were immunoblot analysis was made of protein extracts prepared from 1226 T. parva-INFECTED T CELLS RESIST Fas-INDUCED APOPTOSIS

obvious only once the cells had been cultured for 3 days in the presence of the drug.

T. parva-transformed T cells express Fas and FasL Caspase-8 activation has been implicated in apoptosis induced through the stimulation of surface death receptors such as Fas. Immunoblot analysis was conducted to determine whether the death receptor Fas is expressed by T. parva-transformed cells. Fas could readily be detected in T. parva-infected cells, and levels did not differ significantly when the parasite was killed by treatment with BW720c (Fig. 2A). To demonstrate Fas expression on the cell surface, FasL binding to Fas was monitored by flow cytometry. In agreement with the immunoblot data, the pattern of FasL binding to Fas was almost identical for T. parva-infected cells and cells FIGURE 1. Caspase activity can be detected in T cells upon elimination cured of the parasite, although the basic fluorescence was slightly of the parasite. A, T. parva-transformed cells were cultured for 3 days in the lower (Fig. 2B). Specificity of the binding was ensured by prein- absence or presence of the theilericidal drug BW720c. To monitor caspase cubation of cells with Fc-FasL, which completely blocked the activity, cells were incubated with FITC-VAD-fmk for 20 min, fixed in binding of rhsFasL. paraformaldehyde and analyzed by flow cytometry. Shaded profile repre- Downloaded from Immunofluorescence microscopy was conducted to determine sents T. parva-infected cells; open profile represents BW720c-cured cells. B, Immunoblot analysis of protein extracts prepared from T. parva-trans- whether FasL is expressed by T. parva-transformed cells. FasL formed T cells and cured T cells cultured in the presence of BW720c for could be detected in T. parva-infected cells, and staining intensity up to 3 days. Caspase-8 and -9 activation is reflected by the proteolytic did not significantly change after removal of the parasite by culturing processing of the respective proenzymes (indicated as Pro-Casp 8 and 9). the cells for up to 3 days in the presence of BW720c (Fig. 2C). The size of the cleavage products is indicated. FL1-H, Fluorescence. Despite the fact that both Fas and FasL are expressed by T. http://www.jimmunol.org/ parva-infected cells, they do not undergo apoptosis. This could be due to the fact that Fas is incapable of transmitting death signals in T. parva-transformed T cells and cells cured by treatment with T. parva-infected T cells. Alternatively, it is conceivable that ap- BW720c for up to 3 days. Low levels of processing of the initiator optosis occurs only provided a threshold level of stimulation is caspase-8 could be observed in T. parva-transformed T cells and exceeded. To establish whether the Fas/FasL pathway is functional also in cells treated with BW720c for 1 day. This is reflected by the in T. parva-infected T cells, we tested whether a strong stimulus presence of the p44 and p18 caspase-8 fragments. Complete pro- delivered to Fas could induce apoptosis in T. parva-infected cells. cessing of caspase-8 could be observed after 2 days of BW720c Cells were incubated with 250 ng/ml SuperFasL, a recombinant,

treatment (Fig. 1B). Weak caspase-9 processing, indicated by the trimerized form of FasL which, by inducing Fas oligomerization, by guest on October 2, 2021 appearance of the p32 and p20 fragments, could be observed provides a potent apoptotic trigger (34). Flow cytometric analysis within 1 day of BW720c treatment, but complete processing was using FITC-labeled annexin V showed that the majority of T.

FIGURE 2. Fas and FasL are expressed by T. parva-transformed (tr.) T cells and BW720c-cured T cells. A, Immunoblot analysis of T. parva-transformed T cells (labeled 0) and cells from which the parasite was eliminated by BW720c treatment for 1, 2, or 3 days (d). Protein extracts were analyzed by immunoblot analysis, using an Ab directed against Fas. B, Flow cytometric analysis of Fas expression. T. parva-transformed T cells and T cells cured of the parasite by treatment with BW720c for 3 days were first incubated with FLAG-tagged rhsFasL, followed by M2-anti-FLAG mAb and FITC-labeled anti-mouse IgG. Binding was monitored by flow cytometry. The dotted line shows the specific binding of rhsFasL to T. parva-transformed cells; the gray profile represents background fluorescence in the absence of rhsFasL. As control, cells were incubated with a 10-fold excess of Fc-FasL before the addition of FLAG-tagged rhsFasL; this blocked the available binding sites for Flag-tagged rhsFasL (black line), thus confirming the specificity. FL1-H, Fluores- cence. C, Immunofluorescence microscopy of T. parva-transformed T cells and cells from which the parasite was eliminated by BW720c treatment for up to 3 days. Cells were stained using an Ab directed against FasL (white). Nuclei were visualized by counterstaining with 4,6-diamidino-2-phenylindole (Hoechst 33258; gray). The Journal of Immunology 1227 parva-transformed T cells exposed to SuperFasL for 16 h ex- To further demonstrate the enhanced sensitivity of cured cells to pressed phosphatidylserine, a marker for apoptosis, on their sur- Fas-mediated apoptosis, we subjected cells that had been treated face, and cells displayed all the classical morphological features of with BW720c for 4 days to Fas stimulation and monitored the apoptosis (not shown). These findings suggest that the expression specific cleavage of PARP, which occurs as one of the last steps in of Fas and FasL alone does not suffice to induce apoptosis in T. the apoptotic cascade. Using Abs that specifically detect p85, a parva-transformed T cells, whereas a strong trigger, delivered by cleavage product of PARP, advanced apoptosis could be demon- the artificial oligomerization of Fas, can initiate cell death. strated in cured cells within2hofFastriggering, whereas only little PARP activation could be detected in T. parva-transformed T cells cured of the parasite become increasingly sensitive to cells (Fig. 4B). Although BW720c is strictly parasite specific, we Fas-induced apoptosis also tested its effects on a control transformed T cell line. Neither We next investigated whether cells from which the parasite has the proliferation rate nor the sensitivity of Jurkat T cells to Fas- been eliminated differ from T. parva-infected cells in their sensi- induced apoptosis was affected by culturing the cells for 3 days in tivity to Fas-mediated apoptosis. For this purpose, T. parva-in- the presence of BW720c (data not shown). Taken together, these fected T cells and cells cured by treatment with BW720c for 3 days findings indicate that although Fas and FasL are expressed on T. were incubated with increasing doses of soluble, epitope-tagged parva-infected T cells, they display a high level of resistance FasL, which binds to Fas without activating it. Fas oligomerization against Fas-mediated apoptosis relative to cured cells and that pro- and activation were triggered by Ab-mediated cross-linking of tection appears to be tightly associated with the presence of the epitope-tagged FasL, and caspase activation was monitored by parasite in the host cell cytoplasm. flow cytometry 4 h after induction. The results show that elimina- c-FLIP, c-IAP, and XIAP are expressed in a parasite-dependent Downloaded from tion of the parasite leads to a pronounced increase in sensitivity to manner in T. parva-transformed T cells Fas-mediated caspase activation (Fig. 3). Additional experiments were also conducted to determine how soon after parasite elimi- Fas stimulation involves the activation of the initiator caspase-8 nation the cells became sensitive to Fas/FasL-induced apoptosis. In and the subsequent activation of effector/executioner caspases such cells treated with BW720c for only 1 day, Fas-dependent caspase as caspase-3, -6, and -7. Caspase-8 activity is inhibited by FLIP activation could be detected within 6 h after induction (Fig. 4A). In and effector/executioner caspases can be blocked by the binding of cells treated for 3 days, a robust induction of caspase activity could IAPs such as c-IAP1/2 and XIAP. In primary T cells, high levels http://www.jimmunol.org/ be measured within3hofFastriggering. Under these conditions, of FLIP are transiently expressed upon activation; down-regulation T. parva-transformed T cells did not respond to Fas stimulation. of FLIP expression coincides with the onset of sensitivity to Fas/ FasL-mediated apoptosis. We examined whether FLIP and other IAP proteins are expressed in T. parva-infected T cells. Immuno- blot analysis shows that c-FLIP and XIAP are expressed in T. parva-infected cells (Fig. 5A). Levels of both proteins start to de- crease, however, within 1 day of BW720c treatment; after 3 days, no protein could be detected. Importantly, in transformed Jurkat by guest on October 2, 2021 T-cells, treatment for 3 days with BW720c did not affect the steady state levels of c-FLIP and X-IAP expression (Fig. 5A, lower pan- el), indicating that the drug per se does not block the expression of these antiapoptotic proteins. Together, our findings strongly indi- cate that in T. parva-transformed T-cells expression of antiapop- totic proteins is tightly linked to the parasitized state of the cell When analyzed by immunofluorescence microscopy, cytoplas- mic c-IAP expression could readily be demonstrated in T. parva- infected cells, whereas only weak or no signals could be detected in BW720c-cured cells (Fig. 5B). The low level of c-IAP expres- sion that can be detected in control Jurkat T-cells, in contrast, was not affected by culturing the cells for three days in the presence of BW720c (data not shown). Bcl-2 family members regulate apo- ptosis induced through the mitochondrial pathway. Interestingly, in cured cells, members of the Bcl-2 family are still expressed at similar levels as in T. parva-transformed T cells, independently of

whether they belong to the prosurvival (e.g., Bcl-2 and Bcl-xL)or

proapoptotic (e.g., Bax and Bcl-xS) family of Bcl-2 proteins. Finally, it has recently been reported that the PI3-K/Akt path- way regulates c-FLIP expression in tumor cells (35). T. parva- transformed T cells show many properties of tumor cells (1), and FIGURE 3. Increased sensitivity to Fas-induced apoptosis in BW720c- we have demonstrated that the PI3-K/Akt pathway is constitutively cured cells (BW). Upon elimination of the parasite, T cells become more activated (6). We therefore tested whether blocking PI3-K by treat- sensitive to Fas-induced apoptosis. T. parva-transformed (Tp-transf.) T ment of the cells with the inhibitor LY294002 could down-regulate cells (gray profile) and cells cured of the parasite by treatment with c-FLIP expression, as has been shown to be the case in tumor cells BW720c for 3 days (open profile) were incubated with increasing doses of FLAG-tagged recombinant soluble FasL. Fas activation was induced by (35). Fig. 5C shows that treatment with LY294002 for up to 3 days cross-linking FasL using an anti-FLAG mAb. Caspase activation was mon- did not adversely affect the expression of c-FLIP, c-IAP, or XIAP. itored 4 h after induction, using FITC-VAD-fmk and flow cytometry. The On the contrary, treatment with LY294002 resulted in a clear in- percent of T. parva-transformed cells or BW720c-cured cells that were posi- crease in the levels of c-IAP and also in a modest increase in tive for FITC-VAD-Fmk are indicated on the right. FL1-H, Fluorescence. XIAP. Levels of procaspase-8 remained constant, indicating that 1228 T. parva-INFECTED T CELLS RESIST Fas-INDUCED APOPTOSIS

FIGURE 4. Increased sensitivity to Fas/FasL-induced apoptosis occurs early upon parasite elimination and becomes more pronounced with time. A, T. parva-transformed (Tp) T cells and cells cured for 1 or 3 days (d) were incubated with 100 ng/ml recombinant soluble FasL followed by FasL cross-linking to trigger Fas activation. Relative caspase activity was monitored 3 and 6 h after Fas activation by measuring FITC-VAD-fmk binding in a microplate fluorescence reader. B, The sensitivity of T. parva-transformed T cells and cells treated with BW720c (BW) for 4 days was compared by monitoring the

cleavage of PARP upon Fas triggering. Fas signaling was induced by cross-linking recombinant soluble FasL (added at 200 ng/ml) as described above; after Downloaded from 2 h, the processing of PARP was monitored by immunofluorescence microscopy using an Ab specific for p85 fragment of PARP which results from caspase-3-mediated cleavage of the intact PARP protein. caspase-8 does not become activated. Taken together, these data mechanism and is not absolute. This is reflected by the fact that demonstrate that c-FLIP, which interferes with apoptotic signaling crossing the threshold level of protection by artificially over-stim- through death receptors, and c-IAP or XIAP, which block the ac- ulating Fas with SuperFasL results in apoptosis of T. parva-in- http://www.jimmunol.org/ tivation of downstream effector/executioner caspases, are all up- fected T cells. regulated in a parasite-dependent manner in T. parva-transformed In previous studies, we have shown that the transcription factor T cells. NF-␬B is constitutively activated in a parasite-dependent manner (3, 4). NF-␬B plays a dual, apparently contradictory, role in the Discussion regulation of apoptosis (reviewed in Refs. 36 and 37). Its anti- T cells transformed by the intracellular parasite T. parva express apoptotic function is well documented and has been linked to the both the death receptor Fas and its ligand, a combination that is fact that the expression of several antiapoptotic proteins, including potentially lethal to the cell. Parasite-transformed cells appear to c-FLIP (38–40), c-IAP1/2 (41–44), and XIAP (45, 46), is regu- be protected, however, through the concomitant expression of sev- lated, at least in part, by this transcription factor. In contrast, by guest on October 2, 2021 eral antiapoptotic proteins known to block apoptotic signaling at NF-␬B also appears to be involved in regulating the expression of the level of initiator as well as effector/executioner caspases. This both the fas (17, 18) and fasl (19–21) genes, thus potentially prim- way, parasite-transformed T cells maintain a delicate balance be- ing the T cell for AICD. The complex role of NF-␬B in apoptotic tween cell death and cellular proliferation. regulation is further highlighted by the finding that c-FLIP, XIAP, When the parasite is eliminated from the host cell cytoplasm, and c-IAP can themselves, in turn, contribute to the regulation of expression of antiapoptotic proteins is down-regulated and cured NF-␬B (47–49), potentially providing regulatory feedback loops. cells become increasingly sensitive to Fas-mediated apoptosis. The To what extent such feedback loops participate in regulating protection of parasitized cells appears to depend on a threshold NF-␬BinT. parva-infected cells is not known, but the fact that

FIGURE 5. The expression of the antiapoptotic proteins c-FLIP, c-IAP-1, and XIAP in T. parva-trans- formed cells is down-regulated upon elimination of the parasite. A, Top panel, Immunoblot analysis of protein extracts prepared from parasitized cells and cells cured by treatment with BW720c for up to 3 days. Lower panel, Control experiment demonstrating that treatment of Jurkat T-cells for 3 days with BW720c does not affect the expression of c-FLIP and XIAP. B, Immunofluorescence picture of T. parva- transformed cells expressing c-IAP-1 (Tp) and a cell from which the parasite has been eliminated by treat- ment with BW720c for 3 days (BW). C, Immunoblot analysis demonstrating that treatment of T. parva- transformed cells with the PI3-K inhibitor LY294002 (50 ␮M) for up to 3 days does not adversely affect the expression of antiapoptotic proteins and does not lead to caspase-8 activation. The Journal of Immunology 1229

NF-␬B is activated in a constitutive rather than transient manner Kuenzi, unpublished observation). These receptors are functional may be a critical factor in guaranteeing protection against Fas- because they are capable of enhancing I␬B-␣ degradation upon mediated apoptosis. Increased resistance to Fas-induced apoptosis stimulation with recombinant TNF. As has been demonstrated be- might also contribute to the pathogenesis of East Coast fever. Thei- fore (62), however, TNF-receptor triggering did not induce apo- leria-transformed cells have been shown to induce a pronounced ptosis and a logical explanation would be that parasite-dependent autologous mixed lymphocyte reaction in vitro (50, 51), a process up-regulation of antiapoptotic proteins also provides protection that, in vivo, is thought to contribute to the accumulation of large against TNF-induced apoptosis. numbers of uninfected lymphoblastoid cells in the animal’s lym- Whereas c-FLIP, c-IAP, and XIAP were down-regulated within phoid tissues (52, 53). As it can be expected that Fas and FasL are 1–2 days of BW720c treatment, levels of Bcl-2 family proteins up-regulated as a consequence of this activation, enhanced resis- remained constant, even after 3 days of treatment. Several obser- tance against Fas-induced apoptosis might impart a selective ad- vations support the notion that in activated T cells Bcl-2 controls vantage to parasitized cells over uninfected cells, thus contributing a death pathway that is distinct from that induced by Fas (63, 64) to the clonal expansion and spread of the infected cells through the because Bcl-2 overexpression does not protect activated T cells body. East Coast fever is characterized by an initial phase of lym- from Fas-driven death. Bcl-2 protects T cells from activated T cell phoproliferation, which is usually followed by massive lympho- autonomous death (ACAD), a form of T cell death in response to cytolysis involving uninfected as well as T. parva-infected cells activation by foreign Ags, which is distinguishable from that (53). At this stage, the molecular basis for this lytic response is not driven by death receptors (see review in Ref. 13). ACAD is typ- known, but it will be of interest to determine to what extent Fas/ ically accompanied by a pronounced decrease in Bcl-2 levels. The FasL expression plays a role in this process. fact that Bcl-2 is not down-regulated indicates that ACAD is prob- Recent reports point toward an important role for c-FLIP in the ably not involved in the apoptotic death of BW720c-cured cells. Downloaded from control of AICD (54). It is thought that upon antigenic stimulation In addition to ACAD, Bcl-2 also counters other apoptotic path- of primary T cells, c-FLIP is first up-regulated and then down- ways. Effector caspases can be induced by both caspase-8 and -9. regulated. Down-regulation requires IL-2, which may explain the Caspase-9 is predominantly triggered through the mitochondrial role of IL-2 in sensitizing T cells to cell death (55, 56). A survey pathway, which is induced by cellular stress and lymphokine/cy- of different cell lines has shown that T. parva-transformed cells tokine withdrawal. This process involves the activity of proapop- express only low levels of (31) or no (57, 58) IL-2. This might be totic BH3-only proteins (65) which, in turn, require the presence of http://www.jimmunol.org/ explained by the fact that signaling through the TCR does not proapoptotic Bax and Bak to exert their proapoptotic function (66). occur in T. parva-transformed T cells (2). It could be argued that Antiapoptotic Bcl-2 family members protect cells against apopto- suppressed levels of IL-2 would help protect T. parva-transformed sis by antagonizing proapoptotic Bcl-2 proteins. Thus, whereas T cells against AICD. In contrast, it has been observed that long c-FLIP may protect T. parva-transformed T cells against Fas and term propagation of T. parva-infected T cells in the presence of TNF-R-mediated apoptosis, Bcl-2 may contribute to protection IL-2 enhances proliferation without inducing cell death (59). against ACAD, lymphokine/cytokine withdrawal, and other forms Several lines of evidence link FLIP to tumor progression. FLIP of cellular stress. expression correlates with resistance to Fas-induced apoptosis in Viruses have evolved a range of efficient strategies to avoid by guest on October 2, 2021 cell lines derived from B cell and is also linked to the destruction by delaying death of the infected cell (67). In fact, the escape of tumors from T cell immunity (see review in Ref. 27 and search for apoptosis-regulatory proteins first led to the discovery of references therein). Our findings indicate that this might also apply v-FLIP in ␥-herpesviruses, which in turn led to the identification of to T. parva-transformed T cells. Despite the many similarities, c-FLIP (27). There is increasing evidence that, at one stage in differences between established tumor cell lines and T. parva-in- evolution, intracellular eukaryotic parasites joined the ranks of fected cells do exist. Studies on a number of tumor cell lines have pathogens that developed strategies to prevent death of the cell shown that c-FLIP expression is dependent on the activity of the they seek refuge in (68, 69). Our findings indicate that the parasite PI3-K/Akt pathway (35) and is down-regulated by inhibitors of T. parva also engages apoptosis-regulating proteins such as FLIP,

PI3-K. We and others (6, 7) have recently demonstrated that the IAP, Bcl-2, and Bcl-xL to mediate resistance against apoptosis. In PI3-K/Akt pathway is activated in T. parva-transformed T cells. contrast to viruses that directly appropriate host cell genes, this is Culturing cells in the presence of the PI3-K inhibitor LY294002 achieved indirectly by selectively scavenging those host cell sig- resulted in a rapid arrest of proliferation rather than apoptosis (6, naling pathways such as NF-␬B that control the expression of 60). Furthermore, in contrast to observations made in tumor cells these genes. (35), we observed that LY294002 treatment over a period of 3 days did not result in down-regulation of c-FLIP, c-IAP, or XIAP ex- Acknowledgments pression. Instead, a modest increase in the protein levels of c-FLIP We thank Thomi Brunner and Ju¨ng Tschopp for helpful advice and support and c-IAP1 could be observed that is consistent with the increased and Isabel Roditi for reading the manuscript and useful comments. NF-␬B activity observed in T. parva-infected T cells upon LY294002 treatment (6). References Upon elimination of the parasite, c-FLIP, c-IAP, and XIAP were 1. Dobbelaere, D., and V. Heussler. 1999. Transformation of leukocytes by Thei- down-regulated within 1–2 days of BW720c treatment, whereas leria parva and T. annulata. Annu. Rev. Microbiol. 53:1. the expression of Fas and FasL remained constant for at least 3 2. Galley, Y., G. Hagens, I. Glaser, W. C. Davis, M. Eichhorn, and days. The down-regulation correlated with the appearance of ac- D. A. E. Dobbelaere. 1997. Jun NH2-terminal kinase is constitutively activated in T-cells transformed by the intracellular parasite Theileria parva. Proc. Natl. tivated caspases. The rapid disappearance of c-FLIP might point Acad. Sci. USA 94:5119. toward a short half-life of the protein. Moreover, it has been shown 3. Palmer, G. H., J. J. Machado, P. Fernandez, V. Heussler, T. Perinat, and D. A. Dobbelaere. 1997. Parasite-mediated nuclear factor ␬B regulation in lym- that c-FLIP mRNA is unstable, given that inhibitors that act either on phoproliferation caused by Theileria parva infection. Proc. Natl. Acad. Sci. USA transcription or translation rapidly abolish c-FLIP expression (61). 94:12527. In addition to Fas, AICD can also be triggered through TNF 4. Ivanov, V., B. Stein, I. Baumann, D. A. Dobbelaere, P. Herrlich, and R. O. Williams. 1989. Infection with the intracellular protozoan parasite Theileria receptors. We have found in binding studies that receptors for parva induces constitutively high levels of NF-␬B in bovine T lymphocytes. Mol. TNF-␣ are expressed on the surface of T. parva-infected cells (P. Cell. Biol. 9:4677. 1230 T. parva-INFECTED T CELLS RESIST Fas-INDUCED APOPTOSIS

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