of January 21, 2022.This information is current as ApoptosisTh2 Resistance from Fas-Mediated

Death-Inducing Complex: A Mechanism forTh2 Cells Inhibits Caspase-8 Cleavage at the

-Kinase Activity in′Phosphatidylinositol 3Selective Up-Regulation of

Peter H. Krammer and Padmini SalgameArun S. Varadhachary, Marcus E. Peter, Somia N. Perdow,

http://www.jimmunol.org/content/163/9/47721999; 163:4772-4779; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/9/4772.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Selective Up-Regulation of Phosphatidylinositol 3*-KinaseActivity in Th2 Cells Inhibits Caspase-8 Cleavage at theDeath-Inducing Complex: A Mechanism for Th2 Resistancefrom Fas-Mediated Apoptosis1

Arun S. Varadhachary,* Marcus E. Peter,†‡ Somia N. Perdow,* Peter H. Krammer,† andPadmini Salgame2*

In this study the mechanism of differential sensitivity of CD3-activated Th1- and Th2-type cells to Fas-mediated apoptosis wasexplored. We show that the Fas-associated death domain protein (FADD)/caspase-8 pathway is differentially regulated by CD3activation in the two subsets. The apoptosis resistance of activated Th2-type cells is due to an incomplete processing of caspase-8at the death-inducing signaling complex (DISC) whereas recruitment of caspase-8 to the DISC of Th1- and Th2-like cells iscomparable. Activation of phosphatidylinositol 3*-kinase upon ligation of CD3 in Th2-type cells blocked caspase-8 cleavage to itsactive fragments at the DISC, thereby preventing induction of apoptosis. This study offers a new pathway for phosphatidylinositol3*-kinase in mediating protection from Fas-induced apoptosis. The Journal of Immunology,1999, 163: 4772–4779.

M ature CD41 T cells can be separated into Th1- andTh2-type effector cells based on the pattern of cyto-kines they produce upon stimulation (1–3). The Th1

subset produces IFN-g and IL-2 and induces a cell-mediated im-mune response to Ag, whereas the Th2 subset produces IL-4, IL-5,and IL-10 and is necessary for generating a humoral immune re-sponse against Ag. The biological relevance of these two subsetsin infection is underscored by the ability of Th1 and Th2 cells toinfluence disease outcome (4). For example, protection againstmost intracellular pathogens requires cell-mediated immune re-sponses generated by Th1 cells, whereas biasing of the host im-mune response toward a Th2 type promotes humoral immunity,resulting in disease.

Although the impact on disease of a restricted cytokine produc-tion pattern is well established, a clear understanding of the mech-anisms leading to dominance of a particular T cell subset in diseaseis just emerging (5). Understanding both the factors that modulatethe decision of a naive CD41 T cell to develop into a Th1 or Th2cell in response to an Ag and the mechanisms regulating respon-siveness to Ag of differentiated subsets may lead to rational ap-proaches for generating appropriate immune responses to a patho-gen. Ag dose (6), APCs (7, 8) with their associated costimulatorysignals (9, 10) and cytokines (11, 12) all have varying effects onthe differentiation of T cells and the final outcome of a disease

state. Emerging evidence suggests that the Fas/Fas ligand (FasL)3

apoptotic pathway, required for maintenance of T cell homeostasisand self tolerance (13), in addition, may also contribute to certaindisease states through preferential induction of Th1 cell apoptosis(14–16).

Fas (APO-1, CD95) receptor is a cell death receptor that is ac-tivated by FasL, a trimeric transmembrane protein (17). Receptoroligomerization results in the recruitment of the death domain pro-tein FADD to the FasR (18–22), followed by the IL-1b-convertingenzyme (ICE)-like protease, caspase-8, to form a functional death-inducing signaling complex (DISC) (23). Upon triggering of theFasR, caspase-8 bound to the DISC gets proteolytically cleaved togenerate two active fragments (24). The active fragments are re-leased into the cytosol where they initiate activation of a cascadeof ICE-like proteases (25) and subsequent death. Following re-cruitment to the DISC, active caspase-8 that is generated can di-rectly mediate activation of caspase-3 and other downstreamcaspases, leading to apoptosis (26). Alternatively, in some celltypes, active caspase-8 cleaves BH3-interacting domain death ag-onist (Bid), which then acts on mitochondria to release cytochromec. Cytochromec, in association with Apaf-1 and procaspase-9,activates caspase-3 and other downstream caspases (26, 27).

There are two other pathways that may also be engaged follow-ing FasR ligation. Recruitment of an adaptor molecule Daxx to thereceptor activates a c-Jun amino-terminal kinase (JNK) pathwaythat leads to gene transcription and subsequent death (28, 29).FasR ligation also catalyzes the hydrolysis of membrane sphingo-myelin, which results in a rapid increase in endogenous levels ofceramides (30–32), which in turn can either directly activatecaspases or intersect with a ras-dependent death pathway. Recentstudies utilizing FADD (33), caspase-8, (34), caspase-9 (35, 36),and acid sphingomyelinase-knockout mice (37), however, indicate

*Department of Microbiology and Immunology, Temple University School of Med-icine, Philadelphia, PA 19140; and†Tumor Immunology Program, German CancerResearch Center, Heidelberg, Germany

‡Current address: Ben May Institute for Cancer Research, University of Chicago,Chicago, IL 60637.

Received for publication June 9, 1999. Accepted for publication August 16, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was partly supported by Grant HL 55972 from the National Institutes ofHealth (to P.S.).2 Address correspondence and reprint requests to Dr. Padmini Salgame, Departmentof Microbiology and Immunology, Temple University School of Medicine, Philadel-phia, PA 19140. E-mail address: salgame@astro.ocis.temple.edu

3 Abbreviations used in this paper: FasL, Fas ligand; DISC, death-inducing signalingcomplex; PI39-K, phosphatidylinositol 39-kinase; WT, wild-type; FADD, Fas-associ-ated death domain protein; ICE, IL-1b-converting enzyme; FLICE, FADD-like ICE;c-FLIP, cellular FLICE-inhibiting protein; LAT, linker for activation of T cells.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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that the FADD/caspase-8 signaling pathway is central and proba-bly nonredundant in inducing death by the FasR. Therefore, therole of other pathways in FasR signaling, besides FADD/caspase-8, remain debatable.

Previously, we demonstrated that mature Th1 and Th2 cells canbe differentially regulated to undergo Fas-mediated apoptosis uponCD3 ligation (14), implying that this form of apoptosis may playa significant role in suppressing Th1 cells. We showed that acti-vated Th1 cells are susceptible to Fas-mediated apoptosis uponCD3 ligation. In contrast, prior activation through CD3 of Th2cells induced resistance to Fas-mediated apoptosis. However, di-rect ligation of the FasR induced apoptosis of both Th1- and Th2-type cells. Thus, differences in sensitivity to apoptosis of Th1 andTh2 cells is not due to an intrinsic defect in the Fas signal trans-duction pathway in Th2 cells, as reported by others (16, 38, 39),but due to interception of this pathway from signals generatedthrough CD3.

In view of the role that apoptosis may play in the loss of Th1-dependent effector functions in disease, it becomes necessary toestablish the molecular mechanisms controlling T cell subset-spe-cific apoptosis. In this work, we therefore investigate the mecha-nism of differential susceptibility to apoptosis of Th1 and Th2 cellsand examine CD3-generated signals that regulate this apoptoticpathway. We report that the difference in Fas sensitivity of CD3-activated Th1 and Th2 cells is not at the level of recruitment ofcaspase-8 to the DISC, but at the level of caspase-8 cleavage to itsactive fragments. We identify phosphatidylinositol 39-kinase(PI39-K) as the key signaling molecule generated through CD3engagement on Th2 cells that regulates the resistance to Fas-in-duced apoptosis by preventing caspase-8 cleavage to its activefragments. These findings describe a novel mechanism that T cellsutilize to resist FasR-induced apoptosis. It also provides the firstevidence that PI39-K interferes with caspase-8 activation.

Materials and MethodsT cell clones

Tetanus-toxoid and purified protein derivative-reactive clones were estab-lished by limiting dilution technique, as previously described (3). Cytokineprofile was determined by stimulating cells with plate-bound anti-CD3 Absand by testing 24-h supernatants for the presence of TNF-a, IFN-g, andIL-4 by ELISA. To expand cell numbers, clones were maintained in IL-2,with biweekly stimulation of Ag and autologous APCs. Cytokine profile ofthe clones was routinely reconfirmed after several rounds of stimulation.We have observed that, even after several passages in vitro, the cytokineprofile and Ag reactivity are unchanged. As previously described, Th1 andTh0 type 1 clones undergo apoptosis when anti-CD3 stimulated; for sakeof clarity, herein Th1 and Th0 type 1 clones will be referred to as apop-tosis-susceptible clones. Conversely, Th2 and Th0 type 2 clones will bereferred to as apoptosis-resistant clones. A total of nine individual T cellclones of both the apoptosis-susceptible (CG2, JC2, HG17, and PS13) and-resistant (HG3, HG5, HG6, HG10, and SPU) phenotype were included inthis study.

Abs and reagents

Anti-CD3 mAb was obtained from Biosource International (Camarillo,CA). The anti-APO-1 mAb (IgG3,k) is an agonistic Ab recognizing anextracellular epitope of CD95 (40). The three anti-caspase-8 Abs utilizedrecognize the three functional domains of caspase-8. N2 recognizes anepitope adjoining the tandem death effector domain containing pro-domain.C15 detects the catalytically active p18 fragment, and C5 binds to anepitope within the p10 fragment (41). The NF6 anti-cellular FLICE-inhib-iting protein (c-FLIP) mAb (IgG1) recognizes the N-terminal part of hu-man c-FLIP (42). IETD-fmk is a cell permeable caspase-8-specific inhib-itor (Clontech, Palo Alto, CA) and was used at 25mM. Wild-type (WT)and dominant-negative phosphatidylinositol 39-kinase p85 regulatory sub-unit containing plasmids (43) were provided by Tomas Mustlin (La JollaInstitute of Allergy and Immunology, San Diego, CA). Successfully trans-fected T cells were selected on the basis of their expression of a murineMHC class I molecule encoded by the cotransfected plasmid, pMACS Kk

(Miltenyi Biotec, Gladbach, Germany). Wortmannin was purchased fromSigma (St. Louis, MO).

Apoptosis detection

Apoptosis of cells was determined using a sandwich ELISA (44). TheELISA relies on the detection of cytoplasmic nucleosomal fragments re-leased from the nucleus during the apoptotic process. To obtain cytoplas-mic nucleosomes, cells were lysed in ice cold buffer (1% Nonidet P-40, 20mM EDTA, 50 mM Tris-HCl (pH 7.5)), for 0.5 h, in a volume sufficient fora final concentration of 1.253 105 cell equivalents/ml. Nuclei were pel-leted by centrifugation at 13,0003 g for 10 min, following which thesupernatant was carefully removed and used for analysis. Lysates from 53103 cell equivalents were added to each well, with each sample tested intriplicate. Nucleosomal fragments were captured with an anti-histone mAb(LG11-2A) and detected with a biotinylated secondary mAb (PL2-3) thatrecognizes a DNA-histone complex. Bound Ab/Ag complexes were thenreacted with alkaline phosphatase-conjugated streptavidin (Southern Bio-technology, Birmingham, AL), to which substrate was then added. Colordevelopment was measured at 405 nm. The percentage of apoptosis isexpressed in arbitrary units, calculated as follows using cells maintained inIL-2 as control: 1002 [(absorbance values at OD405 of IL-2 culture/ab-sorbance value at OD405 of treated cells)3 100].

DISC analysis and Western blotting

Association of caspase-8 with the DISC of unactivated and CD3-activatedapoptosis-resistant and -susceptible clones was determined after directlyligating FasR. T cells (107) of the resistant and susceptible clone wereeither treated with CD3 Abs for 30 min or left untreated, following whichthe cells received 500 ng/ml anti-APO-1 Abs for 5 min at 37°C. Controlcells were stimulated with anti-CD3 Abs alone; however, the lysates ofthese cells received anti-APO-1 Abs. Cells were harvested, washed in coldbuffer, pelleted, and lysed in ice cold lysis buffer (30 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, and small peptideinhibitors, as described in Ref. 45, for 30 min. Cell debris was pelleted bycentrifugation for 10 min at 13,0003 g. To the lysates, 50ml of rabbitanti-mouse (Cappel, Cochranville, PA)-coated staphylococcus A wasadded. DISCs were allowed to immunoprecipitate in the cold for at least1 h, then were washed in cold TNE buffer. After washing 63, immunecomplexes and staphylococcus A were separated by 15 min incubation in2-ME containing sample buffer, staphylococcus A was spun out, and thesupernatants containing the immunoprecipitates were boiled. For Westernblotting, lysates were separated on 12% SDS-PAGE, transferred to sup-ported nitrocellulose membrane (Bio-Rad, Hercules, CA), and blockedwith nonfat milk-containing TTBS. Blots were incubated with primary Abdiluted in TTBS overnight at 4°C, washed 3 to 4 times for 10 min each withTTBS, then detected with HRP-conjugated secondary Ab diluted 1:3000 inTTBS 1 5% nonfat milk, and developed using the enhanced chemilumi-nescence method (ECL Plus, Amersham, Arlington Heights, IL) followingthe manufacturer’s protocol.

Transient transfection and positive selection of transfected T cellclones

Cells (103 106) of an apoptosis-resistant clone (SPU) were suspended in250ml serum free RPMI 1640 (Mediatech, Herndon, VA), and transferredto an electroporation cuvette specific for eukaryotic cells (Bio-Rad GenePulser Cuvette, 0.4-cm electrode, gap 50). To the cuvette, 20mg totalplasmid DNA was added, with the p85WT or p85 MUT DNA in a 1:1 ratiowith the cotransfected pMAX plasmid, and allowed to incubate for 10 minat room temperature. Electroporation was conducted at 250 V, 960mF.Immediately after electroporation, cells were placed at 37°C for 10 min,then cultured in IMDM (Life Technologies, Grand Island, NY) supple-mented with 10% AB serum (BioReclamation, Long Island, NY) and 20U/ml IL-2 (National Cancer Institute, Frederick, MD). Following 72 hincubation, dead cells were removed by separation using Ficoll-Paque(Pharmacia, Uppsala, Sweden) density gradient centrifugation, with theremaining cells resuspended in PBS1 0.5% BSA. To magnetically labelthe suspension, MACSelect Kk microbeads were added and allowed tobind the T cells at 4°C for 15 min, following which volume was adjustedwith PBS, and the cells were applied to the magnetic selection column.Cells not expressing the murine class I molecule were not retained in thecolumn, while transfectants were eluted and collected by removing thecolumn from the magnetic field and flushing the column with additionalbuffer. Recovery of cells after positive selection was 2% of the inputnumber.

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PI39-K activity

The activity of immunoprecipitated PI39-K was determined by the phos-phorylation of the substrate phosphatidylinositol to phosphatidylinositol3-phosphate with and without a 10-min anti-CD3 stimulation. Lipid sub-strate was chloroform extracted from the kinase reaction buffer, then sep-arated by TLC and analyzed by autoradiography. Quantitative assessmentof kinase activity was obtained using phosphorimaging (30).

ResultsApoptosis profile of T cell clones

Our previous data demonstrated that, upon CD3 stimulation, Th1cells undergo Fas-mediated apoptosis. In contrast, Th2 cells do notundergo apoptosis upon CD3 stimulation (14). We also showedthat Th0 cells consist of two subsets, one that behaves like Th1cells and undergoes Fas-mediated apoptosis following CD3 stim-ulation (Th0 type 1), and the other subset is similar to Th2 cellsand is resistant to apoptosis (Th0 type 2). Paradoxically, all cloneswere susceptible to apoptosis when Fas receptor was directly li-gated by anti-Fas Abs. However, prior activation of Th2 and Th0type 2 clones by mAbs to CD3/TCR complex induced resistanceagainst Fas-mediated apoptosis (14). In Table I, we have listed thesensitivity to both CD3 and direct FasR ligation-mediated apopto-sis of the clones utilized in the present study. As shown in TableI, and as previously reported (14), clones susceptible to CD3-me-diated apoptosis are Th1 and Th0 type 1, and clones resistant toCD3 stimulation are Th2 and Th0 type 2. Studies described in thisarticle are designed to provide a mechanism for our previous ob-servation that anti-CD3 rapidly protects Th2 cells from deathcaused by direct FasR ligation with agonistic Abs (14).

FADD/caspase-8 pathway is differentially regulated inapoptosis-resistant and -susceptible clones

To determine whether the FADD/caspase-8 pathway is being dif-ferentially regulated in the Th1 and Th2 clones, we utilized theinhibitor IETD-fmk, which interrupts the FADD/caspase-8 apo-ptotic pathway by functioning as a noncleavable substrate for ac-tive caspase-8 (20). CD3 ligation induces apoptosis in all threesusceptible clones (Fig. 1). However, addition of IETD-fmk duringCD3-activation fully protects these cells from Fas-mediated death(Fig. 1). We therefore further investigated the FADD/caspase-8-associated death pathway used in activated Th1- and Th2-typecells.

c-FLIP is recruited to the DISC of both apoptosis-resistant andsensitive clones.

Recently, a caspase-8-like protein without enzymatic activity, c-FLIP, was cloned (46). In stable transfectants, c-FLIP was shownto interfere with the activation of caspase-8 at the DISC (42). Thus,

we wished to determine whether preferential recruitment of c-FLIPto the DISC in Th2-type cells could be responsible for its resis-tance to Fas. An apoptosis-resistant and an apoptosis-susceptibleclone were treated with anti-CD3 Abs for 30 min before ligatingtheir Fas receptors with anti-APO-1 Abs. FasR was immunopre-cipitated, and the DISCs were Western blotted and probed withanti-c-FLIP Abs. As shown in Fig. 2, in anti-CD3-treated cells ofboth resistant (lane 1) and susceptible clones (lane 4), no recruit-ment of c-FLIP to the nonactivated Fas receptor is observed. How-ever, detectable c-FLIP recruitment to the DISC is observed inboth apoptosis-resistant and -susceptible clones, with (lanes 3and6) or without (lanes 2and4) prior CD3 treatment. This suggeststhat additional regulatory mechanisms, such as an improper DISCassembly or inappropriate caspase-8 cleavage at the DISC, areconferring Th2 resistance to Fas-induced apoptosis. Thus, we nexttested the ability of Th2 cells to recruit and process caspase-8.

CD3 activation does not prevent caspase-8 recruitment to theDISC of resistant Th2 clones

Caspase-8, as with all known caspases, exists in the cytoplasm ina pro-form (20). Previous studies have described that, on triggeringof the FasR, caspase-8 gets recruited to the DISC where it is pro-teolytically activated (24). Initial cleavage of DISC-associatedcaspase-8 generates a p43 and a p12 fragment. The latter getsfurther processed to a p10 fragment. Subsequently, the DISC-as-sociated p43 gets cleaved to release the active site-containing frag-ment p18 from the prodomain p26 (24) (Fig. 3,A and B). We

Table I. Characteristics of T cell clonesa

Clone Clone Type % Apoptosis-CD3 % Apoptosis-FasR Apoptosis Phenotype

CG2 Th1 77 67 SHG17 Th1 82 70 SJC2 Th0 type 1 70 75 SPS13 Th0 type 1 45 65 SHG3 Th2 1 36 RHG5 Th2 12 64 RSPU Th2 3 46 RHG6 Th2 1 76 RHG10 Th0 type 2 1 79 R

a Compilation of the apoptosis profiles of all the clones utilized in the present study. T cell clones (104) were stimulated witheither 2.5mg/ml plate bound anti-CD3 Abs (8 h) or with 500 ng/ml anti-APO-1 Abs (4 h). Eight-hour CD3 stimulation issufficient to upregulate FasL as determined in our previous study (14). Apoptosis was measured by previously described ELISA(44).

FIGURE 1. IETD-fmk blocks apoptosis in susceptible clones. CD3 Abswere made insoluble by coating 100ml of 2.5 mg/ml anti-CD3 Abs in96-well plates and allowing them to bind for 1 h atroom temperature. Thewells were rinsed thoroughly before addition of cells. Cells (13 105) weretreated with insoluble anti-CD3 Abs (2.5mg/ml) for 4 h in the presence orabsence of 25mM caspase-8 inhibitor (IETD-fmk), or the inhibitor alone.Apoptosis was determined by ELISA (1).

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therefore examined by immunoprecipitation and Western blottingthe DISCs of Th1 and Th2 cells activated under different condi-tions. FasR on a susceptible (JC-2) clone and a resistant (SP-U)clone was directly ligated with anti-APO-1 (anti-Fas) Abs for 5min, with or without prior stimulation with anti-CD3 Abs. FasRwas immunoprecipitated, separated on 12% SDS-PAGE gel, andprobed with anti-caspase-8 Ab C15, which recognizes full-lengthcaspase-8, and the first cleavage intermediate of caspase-8, thep43/41 fragment (Fig. 3,A and B). As seen in Fig. 3C, 56-kDapro-caspase-8 is associated with the FasR under conditions where

the cells are undergoing apoptosis (APO-1 Abs alone,lane 2) andalso under conditions where the cells are resistant to apoptosis(anti-CD31 anti-APO-1 Abs,lane 3). A 43-kDa band indicativeof the first cleavage fragment of caspase-8 is also observed in theresistant clone (Fig. 3C, lanes 2and3), suggesting that cleavagehas been initiated at the DISC of the apoptosis-resistant clone. Inthe susceptible JC-2 clone, as expected, both in the presence ofanti-APO-1 Abs alone (lane 5) and in the presence of anti-APO-11 anti-CD3 Abs (lane 6), caspase-8 is recruited to the DISC, andthe presence of p43/41 is observed (Fig. 3C, lanes 5and6). Bothapoptosis-resistant and -susceptible clones stimulated with CD3Abs alone do not demonstrate any caspase-8 recruitment (Fig. 3C,lanes 1and4).

In Fig. 3D, the blot was reprobed with the N2 Ab, which rec-ognizes p26 fragment of caspase-8, (Fig. 3,A andB). As observedpreviously with C15 Ab, p43/41 is found in the resistant clone,both under apoptosing and nonapoptosing conditions. p26, thecleavage product of p43/41, is observed in the resistant clone whentreated with anti-APO-1 Abs (Fig. 3D,lane 2). However, whencells are stimulated with anti-CD3 Abs before addition of anti-APO-1 Abs, conditions that protect these cells from apoptosis, nop26 is detected (Fig. 3D, lane 3). In the susceptible clone, again asexpected, p26 was observed both when cells are treated with anti-APO-1 Abs (Fig. 3D, lane 5) and also when pretreated with CD3before addition of anti-APO-1 Abs (Fig. 3D, lane 6). Inlane 4ofFig. 3D, the susceptible clones do not show any caspase-8 recruit-ment, since they are treated with CD3 for only 30 min, which isinsufficient length of time to induce endogenous FasL. However,30 min of CD3 engagement is sufficient to generate protectivesignals in resistant clones.

FIGURE 2. c-FLIP is recruited to the DISC of both apoptosis-resistantand -susceptible clones. Cells (107) of an apoptosis-resistant and an apop-tosis-susceptible clone were treated with either CD3 Abs (lanes 1and4),500 ng/ml anti-APO-1 Abs (lanes 2and5), or anti-CD31 anti-APO-1 Abs(lanes 3and6). Where appropriate, cells were treated with CD3 Abs for 30min before the addition of anti-APO-1 Abs for 5 min. Cells were lysed, andFasR with associated DISC components were immunoprecipitated. c-FLIPrecruitment to the DISC was determined by Western blotting analysis usingc-FLIP Abs (NF6) (42), and isotype-specific HRP-labeled secondary Ab.The blot was developed with an enhanced chemiluminescence system(Amersham). Levels of cellular c-FLIP expression were similar in Th1- andTh2-like cells (data not shown). Data presented here are representative oftwo individual experiments.

FIGURE 3. Recruitment of caspase-8 to the DISC in apoptosis-resistant and -susceptible cells.A, Schematic diagram of caspase-8 and the domainsrecognized by anti-caspase-8 Abs.B, Cleavage pattern of caspase-8.C and D, CD3 activation does not prevent caspase-8 recruitment to the DISC ofresistant Th2 cells. Cells (107) of a resistant and susceptible clone were treated with either CD3 Abs (lanes 1and4), 500 ng/ml anti-APO-1 Abs (lanes2 and5), or anti-CD31 anti-APO-1 Abs (lanes 3and6). Where appropriate, cells were treated with CD3 Abs for 30 min before the addition of anti-APO-1Abs for 5 min. Cells were lysed, and FasR with associated DISC components was immunoprecipitated. Caspase-8 recruitment to the DISC was determinedby Western blotting analysis using the C15 (C) and N2 (D) anti-caspase-8 Abs, and isotype-specific HRP-labeled secondary Abs. The blot was developedwith an enhanced chemiluminescence system (Amersham).E andF, Generation of catalytically active caspase-8 fragments is arrested in activated Th2 cells.Lysates fromC were subjected to Western blotting using the C15 (E) and C5 (F) mAbs, respectively, to monitor the appearance of active caspase-8fragments. Data presented in this figure are representative of one of three individual experiments.

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Generation of catalytically active caspase-8 fragments isarrested in Th2 clones

Lysates from Th1 and Th2 clones were further probed with C15and C5 Abs (Fig. 3,E and F) to analyze caspase-8 cleavage prod-ucts released from the DISC into the cytosol. C15 Ab detects thep18 fragment, and C5 Ab detects the p10 fragment (Fig. 3A). Thepresence of active site-containing p18 (Fig. 3E) and p10 (Fig. 3F)fragments is observed only in the apoptosis-sensitive clones (lanes5 and6). The presence of the p12 fragment, a cleavage interme-diate of full length caspase-8 (p56 cleavage giving rise to p43 andp12 products), suggests that caspase-8 cleavage is initiated in boththe resistant and susceptible clones. However, further processingof p12 to the active p10 fragment and cleavage of p43 to p26 andthe active p18 fragment do not occur in the resistant Th2 clone(Fig. 3, E andF). This cleavage process is being specifically pre-vented in the Th2 clone by signals generated upon CD3 ligation.

PI39-K inhibitor sensitizes activated Th2 cells to Fas-mediateddeath

The immunoprecipitation and Western blot results indicate thatTh2 cells utilize a novel strategy to protect themselves from Fas-mediated death, by actively disrupting the DISC. Since this pro-tection was only obvious after CD3 ligation, it was important touncover which signals generated by CD3 intersected with theFADD/caspase-8 pathway. Keeping in mind that PI39-K protectsfrom apoptosis in other systems, we used wortmannin, an inhibitorof PI39-K, and examined induction of apoptosis in five individualTh2 clones. As previously observed, anti-APO-1 (Fas) Abs induceapoptosis of Th2 cells that can be abrogated if the cells are stim-ulated with CD3 Abs before FasR ligation. However, CD3 stim-ulation of wortmannin-treated cells followed by FasR ligationleads to a dramatic increase in apoptosis of the normally resistantTh2 clones (Fig. 4A). Pretreatment of Th2 clones with wortmanninbefore CD3 stimulation does not induce death in the T cell clones,confirming that the wortmannin is not toxic to the cells.

Effect of PI39-K WT and dominant negative mutant onresistance to apoptosis of Th2-type cells

The wortmannin results suggest that, upon CD3 stimulation,PI39-K becomes active and, acting on downstream substrates, isable to interfere with an orderly apoptotic cascade. To further con-firm the role of PI39-K in preventing Fas-mediated apoptosis, WTand a dominant negative mutant of the p85 regulatory subunit ofPI39-K were transiently transfected into a typical apoptosis-resis-tant Th2-type clone (SPU). Successful transfectants were selectedon the basis of their expression of a murine MHC class I molecule,which had been cotransfected with the p85 subunit encoding plas-mid. Following 8 h of CD3stimulation, cells were harvested andprepared for apoptosis ELISA. Cells receiving the WT p85 be-haved as normal untransfected counterparts, i.e., resistant to Fas-mediated death (Fig. 4B). However, the cells transfected with thedominant-negative mutant become susceptible to apoptosis(Fig. 4B).

Differential ability of Th1 and Th2 cells to activate PI39-K

Pharmacological and gene transfer experiments imply that the ap-optosis-resistant and -susceptible clones differ in their ability toactivate PI39-K. To explore this possibility, we compared the ac-tivity of PI39-K in four resistant and four susceptible clones, beforeand after CD3 ligation. T cell clones were rested for 8 h in theabsence of IL-2 (this length of time does not affect the viability ofthe cells) to reduce background PI39-K activity. Cells (53 106)each were stimulated with anti-CD3 Abs, or the same number ofcells was left untreated. Cell lysates were prepared and subjected

to immunoprecipitation with Abs to the p85 regulatory subunit ofPI39-K. Kinase activity was measured using [g-32P]ATP and phos-phatidylinositol (PI) as the substrate. Radiolabeled lipids were sep-arated by TLC and quantitated by phosphorimaging. Radiolabeledlipids were identified by comparison ofrf to known standards (Fig.5). The four resistant clones SPU, HG3, HG5, and HG15 exhibitedpercentage increases in kinase activity of 156, 1000, 285, and251%, respectively. In contrast, the susceptible clones had signif-icantly lower activity. PS13, 59.5, JC6, and JC1 exhibited percent-age increases of kinase activity of only 79, 41, 26, and 16%, re-spectively (Fig. 5).

PI39-K mediates protection in Th2 cells from Fas-mediateddeath by blocking caspase-8 cleavage at the DISC

To test how PI39-K was regulating apoptosis in Th2 cells, we nextdetermined whether blockade of PI39-K permitted the generationof active caspase-8 fragments. We tested caspase-8 cleavage pat-tern in an apoptosis-resistant Th2-type clone (SPU) that was stim-ulated with anti-CD3 Abs in the presence or absence of wortman-nin, followed by FasR ligation. FasR was immunoprecipitated,separated on 12% SDS-PAGE gel, and probed with the anti-

FIGURE 4. A, PI39-K inhibitor sensitizes activated Th2 cells to Fas-mediated death. Cells (13 105) were treated for 4 h with either anti-APO-1Abs (500 ng/ml), anti-CD31 anti-APO-1 Abs, or wortmannin (1mM) 1anti-CD31 anti-APO-1 Abs. Where appropriate, anti-CD3 and wortman-nin treatment preceded anti-APO-1 addition by 30 min. Apoptosis wasdetermined by the previously described ELISA (1). Data from five indi-vidual Th2 clones is presented.ppp, p , 0.001 for anti-Fas-treated andanti-CD31 anti-Fas1 wortmannin-treated cells vs anti-CD3-treated cells.B, Effect of PI39-K WT and dominant negative mutant on resistance toapoptosis of Th2-type cells. A Th2 clone was transiently transfected withplasmids containing the WT and the mutant form of p85 subunit of PI39-Kalong with an expression vector for murine MHC class I. Transfected cellswere selected by the presence of surface expression of class I molecules asdescribed inMaterials and Methods. Transfectants (23 104) containingeither the WT or the mutant form of p85 were then cultured in the presenceof either IL-2 alone or insoluble anti-CD3 Abs for 8 h. The cells wereharvested, and apoptosis was determined by ELISA.

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caspase-8 Ab C15, which recognizes full-length caspase-8, and thefirst cleavage intermediate of caspase-8, the p43/41 fragment. Bothfull-length p56 caspase-8 and partially cleaved p43/p41 are de-tected with C15 Abs when cells are treated with anti-APO-1 alone(Fig. 6A,lane 3), Abs to APO-1 and CD3 (Fig. 6A, lane 4), or Absto APO-1 and CD3 in the presence of wortmannin (Fig. 6A, lane5). C15 Abs were also used to probe the lysates for the presence ofp18. Ligation of FasR alone leads to the generation of the catalyt-ically active p18 in the resistant clone (Fig. 6B, lane 3), which canbe blocked by CD3 stimulation (Fig. 6B, lane 4). However, in thepresence of wortmannin, complete cleavage of caspase-8 to p18fragment is observed (Fig. 6B, lane 5). These results demonstrate

that inhibiting PI39-K allows complete cleavage of caspase-8,which is inhibited in CD3-activated Th2 cells that resist apoptosis.

DiscussionIn this report we characterize the Fas signal transduction pathwayin activated Th1- and Th2-type cells and identify PI39-K as a crit-ical molecule in mediating protection from Fas-induced death inactivated Th2 cells. We present a novel mechanism of how Th2cells resist Fas-mediated apoptosis.

The search for mechanisms that permit T cells to resist apoptosishas led to the discovery of a number of molecules that block theFADD/caspase-8 apoptotic pathway (46–48). Previous work in-dicates that bringing caspase-8 into close proximity with anothercaspase molecule at the DISC is sufficient to permit autoproteoly-sis of the caspase to occur (24). Therefore, the most efficient wayto inhibit the FADD/caspase-8 apoptotic pathway is to preventrecruitment of caspase-8, resulting in a nonfunctional DISC. In-deed, recently such a protein, termed c-FLIP, has been cloned (46)that can compete with caspase-8 for binding to FADD, and therebyblocking recruitment of caspase-8 to the DISC (42). That this strat-egy may be utilized by cells that undergo IL-2-dependent Fas-mediated apoptosis has been suggested (49). However, recent stud-ies examining the inability of short-term activated peripheral Tcells to recruit caspase-8 (45) indicates that c-FLIP is not involvedin preventing caspase-8 recruitment to the DISC, unless it is over-expressed (42). The present data also support this finding by show-ing that resistance of Th2 cells is not at the level of c-FLIP. Theamount of endogenous c-FLIP present in T cells is insufficient toprevent caspase-8 recruitment and apoptosis induction (presentdata and Ref. 42). c-FLIP, only when overexpressed, can competewith caspase-8 for binding to FADD, and thereby halt caspase-8recruitment and activation.

Our results demonstrate that the differences in caspase-8 pro-cessing at the DISC of Th1 and Th2 cells is due to differentialPI39-K activation in the two subsets following CD3 stimulation.PI39-K has been shown previously to protect cells from a varietyof apoptotic stimuli (50–53). More recently, it has been clearlydemonstrated that serine threonine kinase AKT/protein kinase B, atarget of PI39-K, is an essential signaling intermediate in this an-tiapoptotic pathway. AKT can catalyze BAD phosphorylation andthereby sequester it from bcl-xL/bcl-2, thus preventing apoptosis

FIGURE 5. Differential ability of apoptosis-resistant and -susceptible clones to activate PI39-K. The activity of immunoprecipitated PI39-K was deter-mined by the phosphorylation of the substrate phosphatidylinositol to phosphatidylinositol 39-phosphate (PI39P) after either no treatment, or 10 minanti-CD3 stimulation. Lipid substrate was chloroform extracted from the kinase reaction buffer, then separated by TLC and analyzed by autoradiography.Quantitative assessment of kinase activity was obtained using phosphor-imaging.A, Autoradiogram of 1 representative susceptible clone and one resistantclone.B, Data are compiled from eight individual clones and presented as percentage increase in kinase activity.pp, p , 0.005 for Th2 vs Th1.

FIGURE 6. Inhibiting PI39-K in CD3-stimulated Th2 cells allows forcomplete cleavage of caspase-8. Cells (107) of an apoptosis-resistant clonewere treated with either CD3 Abs (lane 1), wortmannin (wort.) (1mM)alone (lane 2), 500 ng/ml anti-APO-1 Abs (lane 3), anti-CD31 anti-APO-1 Abs (lane 4), anti-CD31 anti-APO-1 Abs1 wortmannin (lane 5),or anti-CD3 1 wortmannin (lane 6). Where appropriate, anti-CD3 andwortmannin treatment preceded anti-APO-1 addition by 30 min. Cells werelysed, and FasR with associated DISC components were immunoprecipi-tated. Caspase-8 recruitment to the DISC was determined by Western blot-ting analysis using the C15 (A) anti-caspase-8 Abs, and the blot was de-veloped with an enhanced chemiluminescence system (Amersham).Lysates fromA were subjected to Western blotting using the C15 (B) mAbsto monitor the appearance of active p18 caspase-8 fragment. The bottom ofeach lane indicates percentage apoptosis of the T cell clone stimulatedunder each of those conditions. The cells were stimulated for 4 h withanti-CD3 Abs to detect apoptosis by ELISA (44). Data are representativeof one of three individual experiments.

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(51, 54). AKT can also phosphorylate caspase-9 and prevent itfrom activating caspase-3 (66). Both these targets of AKT arecomponents of the mitochondrial-dependent pathway of Fas-me-diated apoptosis. This pathway is characteristic of cells that recruitlow amounts of caspase-8 to the DISC and are therefore not effi-cient at directly activating caspase-3 (26). In contrast, cells thatrecruit large amounts of caspase-8 to the DISC can directly acti-vate caspase-3 and bypass the mitochondria. These cells thereforemay have developed other PI39-K/AKT targets whose phosphor-ylation would abrogate apoptosis. Cells described in these studiesrecruit large amounts of caspase-8 to the DISC and thus provide amodel system to address in the future how PI39-K directly orthrough AKT regulates caspase-8 cleavage and subsequentcaspase-3 activation in the mitochondrial-independent pathway ofFas signaling.

Engagement of the TCR/CD3 complex initiates the activation ofa number of protein tyrosine kinases, followed by recruitment andactivation of ZAP-70 (55, 56). One of the key substrates forZAP-70 is linker for activation of T cells (LAT). PhosphorylatedLAT associates with the p85 subunit of PI39-K directly and alsovia tyrosine phosphorylated cbl (57). TCR-interacting molecule(TRIM) (58) is another linker protein containing several tyrosine-based signaling motifs that, upon phosphorylation, can also asso-ciate with p85 (59). Thus, both TRIM and LAT function as linkermolecules to target PI39-K to the membrane where it can poten-tially interact with its lipid substrates and other activators, such asRas (60, 61). The data reported here reveal that Th1 and Th2 cellsregulate PI39-K activation differently upon CD3 activation leadingto differences in their ability to resist Fas-mediated apoptosis. Fu-ture studies will include a systematic comparative analysis of CD3signaling in the two subsets to gain a better insight into the mech-anisms of why CD3 signaling in Th1 cells results in poor activa-tion of PI39-K. Indeed, previous studies have indicated that Th1and Th2 cells signal differently upon engagement of their TCRs.Fitch and colleagues studying murine Th1 and Th2 clones haveshown that stimulation of Th1, but not Th2, clones with anti-TCRAbs leads to elevated [Ca21] (9, 62). Similar observations weremade in another study where calcium ion signaling was measuredin T cells as they differentiated from naive T cells to mature Th1and Th2 effector populations (63). Naive T cells engaged the Ca21

pathway, which was further enhanced if the cells were biased todifferentiate into Th1 cells. In contrast, under Th2-differentiatingconditions, the naive T cells selectively lost their ability to use thissignaling pathway. PKC-dependent signaling leading to preferen-tial up-regulation of FasL and subsequent susceptibility to apopto-sis occurring only in Th1 cells, is yet another instance of differ-ences between Th1 and Th2 cells in CD3 signaling (39). Thus,distinct signaling pathways are activated in Th1 and Th2 cellsupon ligation of their CD3/TCR that can lead to differences in theireffector functions and activities.

The biological significance of the differential susceptibility ofTh1 and Th2 cells to undergo apoptosis when activated in theabsence of costimulation become important during receptor trig-gering in response to Ag. During Ag presentation, and in a milieuthat provides T cells with necessary costimulatory signals, Th1cells will be capable of proliferating, and thus activating cellularimmune responses. However, if an intracellular pathogen has dis-abled much of the costimulatory machinery (64, 65), Ag triggeringof T cells will lead to skewing of the immune response by defaultto a Th2 type. In conclusion, this study provides a paradigm tofurther investigate how PI39-K regulates caspase-8 activation, andalso to examine whether preferential Fas-mediated apoptosis ofTh1 cells is an additional modality for immune deviation.

AcknowledgmentsWe thank Tomas Mustelin for his generous gift of the PI39-K constructs,Danny Dhanashekaran for help with the PI39-K assays, and the NationalCancer Institute for IL-2. We also thank Abul Abbas for helpfuldiscussions.

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