Theileria parva-Transformed T Cells Show Enhanced Resistance to Fas/Fas Ligand-Induced Apoptosis 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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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/Fas Ligand-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 receptor 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 proteins. We found that anti-apoptotic proteins such as cellular (c)-FLIP, which functions as a catalytically inactive form of caspase-8, and X-chromosome- linked inhibitor of apoptosis protein (IAP) as well as c-IAP, which can block downstream executioner caspases, 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 gene 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-Myc (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 Cancer 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 cytochrome c 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).