Effector CD4+ T Cells Generate Intermediate Activity and Cleavage of Caspase-8 Substrates

This information is current as Ravi S. Misra, Dawn M. Jelley-Gibbs, Jennifer Q. Russell, of October 1, 2021. Gail Huston, Susan L. Swain and Ralph C. Budd J Immunol 2005; 174:3999-4009; ; doi: 10.4049/jimmunol.174.7.3999 http://www.jimmunol.org/content/174/7/3999 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Effector CD4؉ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates1

Ravi S. Misra,* Dawn M. Jelley-Gibbs,† Jennifer Q. Russell,* Gail Huston,† Susan L. Swain,† and Ralph C. Budd2*

Caspase-8 activation promotes cell but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4؉ T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIPL, interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4؉ T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling. The Journal of Immunology, 2005, 174: 3999–4009. Downloaded from

t is becoming increasingly evident that ligation of death re- The earlier view that caspase activation occurs only through its ceptors and activation of cellular does not necessar- cleavage has been revised for certain upstream caspases. An alter- I ily always result in apoptosis. Involvement of the death re- native method of caspase activation is through a conformational ceptor, Fas, in processes other than cell death has been revealed by change. For example, when caspase-9 interacts with the ability of Fas ligation to enhance neural outgrowth (1), cardiac complexed to Apaf-1, a conformational change of caspase-9 ex- hypertrophy (2), liver regeneration (3), fibroblast proliferation (4), poses its (16, 17). In a similar manner, procaspase-8 can http://www.jimmunol.org/ dendritic cell maturation (5), and T cell activation (6). More di- induce a structural change in a neighboring procaspase-8 molecule, rectly, caspase-8 plays an integral role in T cell function, as hu- resulting in active full-length caspase-8 (10). In addition, the en- mans bearing nonfunctional caspase-8, as well as caspase-8 con- zymatically inactive casapase-8-like molecule c-FLIPL can also ditional knockout mice, exhibit severe defects in T cell activation associate with and activate full-length caspase-8 (18). As c-FLIPL (7, 8). However, there is currently no clear picture of which also contains a known caspase cleavage site, it represents poten- caspases or which substrates are involved with T cell activation tially one of the earliest substrates of caspase-8 (19). and how far down the caspase cascade these signals propagate. The significance of caspase activity in nonapoptotic effector T Traditionally, activation of the upstream initiator caspases that cells is still poorly understood. It is not known how the levels of by guest on October 1, 2021 possess large prodomains, such as procaspases-8, -9, and -10, had active caspases or cleavage of their substrates in viable effector T been thought to require proteolytic cleavage of the zymogen, resulting cells compare with those in resting T cells or those undergoing in the release of large and small subunits (9). These fragments then apoptosis after Fas ligation. We observe that intermediate levels of heterodimerize to form an active caspase molecule. Typically, the caspase activity are generated at the effector stage of T cell acti- cleavage occurs by auto- and cross- in response to the li- vation, which are proportional to cell cycling, and that blockade of gation of death receptors, which increases the local concentrations of caspase activity inhibits cell cycling. Proximal caspase-8 sub- caspases (9–11). The upstream initiator caspase signal is propagated strates such as c-FLIPL and receptor interacting protein 1 (RIP1) to the effector procaspases, such as procaspase-3, by proteolytic cleav- are cleaved in effector T cells, but not the downstream caspase-3 age of the procaspase. As part of this caspase cascade, various caspase substrate ICAD. Furthermore, the caspase activity and cleavage of substrates are cleaved, including the DNase inhibitor inhibitor of substrates is more extensive in Th2 effector cells than in Th1 ef- caspase-activated DNase (ICAD)3 (12) and structural proteins, such fectors. Collectively, these results define the caspase activity pro- as actin (13), fodrin (14), and lamin (15). file in viable T cells and underscore the functional requirements of such activity.

*University of Vermont, Immunobiology Program, College of Medicine, Burlington, Materials and Methods VT 05405; and †Trudeau Institute, Saranac Lake, NY 12983 Mice Received for publication July 14, 2004. Accepted for publication January 11, 2005. C57BL/6 mice were housed and bred in the University of Vermont animal The costs of publication of this article were defrayed in part by the payment of page facility and were used at 2–6 mo of age. B10.Br V␣11/V␤3 AND TCR charges. This article must therefore be hereby marked advertisement in accordance transgenic (pigeon cytochrome c/I-Ek responsive) mice were used at 2–3 with 18 U.S.C. Section 1734 solely to indicate this fact. mo of age and were bred in the animal facilities at the Trudeau Institute. 1 This work was supported by grants from the National Institutes of Health (AI36333 Both facilities are American Association of Laboratory Animal Care ap- and AI45666 to R.C.B.; AI26887 and AI46530 to S.L.S.). R.S.M. is supported by proved and protocols were approved by the Institutional Animal Care and National Institutes of Health Grant No. 5T32ES007122. Use Committee at the respective institutions. 2 Address correspondence and reprint requests to Dr. Ralph C. Budd, University of Vermont, Immunobiology Program, College of Medicine, 89 Beaumont Avenue, T cell purification Given Building D305, Burlington, VT 05405. E-mail address: [email protected] 3 Abbreviations used in this paper: ICAD, inhibitor of caspase-activated DNase; C57BL/6 spleens and lymph nodes were isolated and disrupted through RIP1, receptor interacting protein 1; PCCF, pigeon cytochrome c fragment; z-VAD, nylon mesh in RPMI 1640 (MediaTech) containing 5% (v/v) FCS (Hy- z-Val-Ala-Asp-fluoromethylketone; FasL, Fas ; WCL, whole cell lysate; XIAP, Clone). Erythrocyte lysis of splenocytes was performed using Gey’s solu- ϩ X-linked protein. tion. and splenocytes were combined and CD4 T cells were

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4000 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS

isolated by negative selection. C57BL/6 cells were incubated with anti- Immunoblot analysis CD8 (Tib 105), anti-MHC class II (M5/114/15/2; a kind gift of M. Rinco´n, University of Vermont, Burlington, VT), anti-CD11b (M1/70), anti-NK1.1 Viable cells were lysed in buffer containing 0.2% Nonidet P-40, 20 mM (PK136), and anti-B220 (RA3-6B2) on ice for 45 min. For total T cell Tris-HCl (pH 7.4), 2 mM sodium orthovanadate, 10% glycerol, 150 mM ␮ isolation, anti-CD8 was omitted from Ab mix. Cells were washed three NaCl, complete inhibitor (Roche Diagnostics), and 10 M z-VAD times and rocked with goat anti-mouse and goat anti-rat conjugated mag- ( System Products). Protein concentration was determined by netic beads at a 10:1 ratio of beads to cell (Qiagen) at 4°C for 45 min. Bradford assay (Bio-Rad). Protein lysates were boiled for 5 min in loading Magnetic depletion was used to remove bead-bound cells, routinely yield- buffer containing 2-ME and were separated using SDS-PAGE on 12.5% ing Ͼ90% CD4ϩ T cells. Cells were washed and resuspended in culture gels. Proteins were transferred onto a polyvinylidene difluoride membrane medium (RPMI 1640 supplemented with 25 mM HEPES, 2.5 mg/ml glu- (Bio-Rad) and blocked using 4% milk in TBS plus 0.1% Tween 20 at room cose (Sigma-Aldrich), 10 ␮g/ml folate (Invitrogen Life Technologies), 110 temperature for 1 h. Membranes were incubated at 4°C overnight in milk Ϫ ␮ ␮g/ml pyruvate (Invitrogen Life Technologies), 5 ϫ 10 5 M 2-ME (Sig- containing one of the following primary detection Abs: 1 g/ml actin (I-19; ma-Aldrich), 292.3 ␮g/ml glutamine (Invitrogen Life Technologies), 100 Santa Cruz Biotechnology), Bid (R&D Systems), caspase-3 (a kind gift of U/ml penicillin-streptomycin (Invitrogen Life Technologies), and Y. Lazebnik, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY), 5% FBS). caspase-8 (a kind gift of A. Stasser, The Walter and Eliza Hall Institute of Naive AND CD4ϩ T cells were enriched from pooled spleen and lymph Medical Research, Melbourne, Australia), ICAD (BD Biosciences), RIP1 ␮ node cells as previously described (20). In brief, spleen and lymph node (BD Biosciences), or 2 g/ml FLIP (Dave-2; Apotech). Blots were washed ␮ cells were passed through a nylon wool column, and nonadherent cells three times for 15 min each and incubated for 90 min with 0.5 g/ml ␮ were treated with a panel of CD8, HSA, and MHC class II depleting Ab (caspase blots) or 1 g/ml (all others) of appropriate secondary Ab con- and complement followed by Percoll (Amersham Biosciences) centrifuga- jugated to HRP (Santa Cruz Biotechnology). Blots were washed again and tion. CD4ϩ purity was routinely Ͼ95%, 90–95% of which had a naive developed using LumiGLO (Kirkegaard & Perry Laboratories). high high low low phenotype (CD45RB , CD62 ligand , CD44 , CD25 ) and ex- Biotin-VAD-fmk caspase precipitation assay pressed the TCR transgene. AND transgenic cells were cultured in RPMI Downloaded from 1640 plus penicillin (200 ␮g/ml; Sigma-Aldrich), streptomycin (200 ␮g/ Viable T cells were incubated with z-FA-fmk control peptide or 100 ␮M ml; Sigma-Aldrich), glutamine (4 mM; Sigma-Aldrich), 2-ME (50 ␮M; nonbiotinylated z-VAD at 37°C for 15 min followed by another incubation Sigma-Aldrich), HEPES (10 mM; Sigma-Aldrich), and 8% FBS (Intergen). with 10 ␮M biotin-VAD (Enzyme System Products). Cells were lysed in buffer containing 20 ␮M biotin-VAD. A total of 600 ␮g of lysate was then precleared by rocking with 40 ␮l of Sepharose 6B agarose beads (Sigma- T cell culture Aldrich) at 4°C for 2 h. Supernatant was then rocked with 30 ␮l of Strepa- ϩ vidin-Sepharose beads (Zymed Laboratories) at 4°C overnight. Beads were C57BL/6 CD4 T cells were activated in culture medium by plate-bound washed five times in lysis buffer without protease inhibitor, and then were http://www.jimmunol.org/ anti-CD3 (5 ␮g/ml; 145-2C11), anti-CD28 ascites (1:500), and recombi- boiled in loading buffer. Beads were removed by centrifugation, and im- nant human IL-2 (50 U/ml; Cetus) for 2 days. Cells were then removed munoblot analysis was then performed on supernatants. from anti-CD3 and fed with fresh medium plus IL-2. AND effectors were ϩ generated by culturing transgenic CD4 cells as previously described (20). CFSE dye labeling of cells Alternatively, AND CD4ϩ T cells were stimulated with DCEK-ICAM APCs (1.5 ϫ 105 cells/ml) pulsed with 5 ␮M pigeon cytochrome c frag- Cells were washed with PBS plus 0.1% BSA and then were mixed with 5 ment (PCCF; KAERADLIAYLKQATAK) and IL-2 (11 ng/ml). The ␮M CFSE in PBS plus 0.1% BSA (Molecular Probes). Samples were di- DCEK-ICAM cell line was originally generated by R. Germain (National vided into 1-ml aliquots and incubated for 10 min at 37°C. Cells were then Institutes of Health, Bethesda, MD) and expresses B7.1, ICAM-1, and class washed three times. A portion of cells was incubated overnight at 37°C to II MHC (I-Ek). APCs were treated with 100 ␮g/ml mitomycin C (Sigma- serve as a noncycling control for flow cytometry, and the remaining cells

Aldrich) for 45 min at 37°C, extensively washed, and then used at a 2:1 T were stimulated. by guest on October 1, 2021 cell/APC ratio. Murine IL-2 and IL-4 were obtained from culture supernatant of Flow cytometry X63.Ag8-653 cells transfected with cDNA for the respective cytokines. For surface staining, cells were washed in PBS and then incubated with 1 Th1 culture conditions included 10 ␮g/ml anti-IL-4 neutralizing Ab ␮g/ml FITC-conjugated anti-CD4 Ab diluted in PBS/1% BSA for 30 min. (11b11; BD Biosciences) and 4 ng/ml IL-12 (R&D Systems). Th2 cells Cells were then washed with PBS/BSA and fixed using 1% formaldehyde were stimulated in the presence of 10 ␮g/ml anti-IFN-␥ neutralizing Ab in PBS. Samples were analyzed using an LSRII flow cytometer (BD (XMG1.2; BD Biosciences) and 30 ng/ml IL-4 (R&D Systems). Recom- Biosciences). binant murine IL-12 used in AND T cell experiments was a gift of S. Wolf For TUNEL and Bcl-2 staining, surface staining was first completed as (Genetics Institute, Cambridge, MA). Where indicated, culture conditions above, with the exception that cells were fixed on ice for 15 min using 2% included the pan-caspase blocker z-Val-Ala-Asp-fluoromethylketone (z- formaldehyde in PBS. Cells were then washed twice with PBS, fixed in VAD; Enzyme System Products; 100 ␮M) or comparable DMSO (Sigma- 70% ice-cold ethanol for 15 min, and then washed twice with PBS. Nicked Aldrich) vehicle control. Viable cells were isolated by layering cell sus- DNA was labeled by incubating cells with TdT buffer (2.5 mM CoCl ,1U pensions over Lympholyte-M (Cedarlane Laboratories) and centrifugation 2 TdT, and 0.5 nmol biotin-dUTP (Roche Diagnostics) in a total volume of at 850 ϫ g for 20 min. Cells were washed three times and cell counts 50 ␮l) at 37°C for 1 h. Cells were then washed twice with 1% BSA in PBS performed. and subsequently incubated with Strepavidin-Tricolor for 30 min. Samples were then washed twice with 1% BSA in PBS and fixed using 1% form- RIP1-deficient 3T3 cell culture aldehyde (Ted Pella) in PBS. For further Bcl-2 staining, cells were per- meabilized with 1% BSA and 0.03% saponin (Sigma-Aldrich) in PBS on RIP1-deficient 3T3 cells were a kind gift of M. Kelliher (University of ice for 10 min. Cells were then washed and incubated with FITC-conju- Massachusetts, Worcester, MA) and were grown in DMEM supplemented gated anti-Bcl-2 (BD Biosciences) on ice for 30 min. Samples were washed with Hams 3F12 (Invitrogen Life Technologies), 5 ϫ 10Ϫ5 M 2-ME (Sig- three times and fixed using 2% formaldehyde (Ted Pella) in 1% BSA in ma-Aldrich), 292.3 ␮g/ml glutamine (Invitrogen Life Technologies), 100 PBS. For staining of active caspase-3 intracellular staining, surface staining U/ml penicillin-streptomycin (Invitrogen Life Technologies), and 10% was first completed and cells were fixed. Cells were washed twice after FBS (HyClone). fixation and the Anti-Active Caspase-3 Apoptosis kit 1 (BD Biosciences) was used to detect intracellular active caspase-3, with PBS/0.03% saponin used in place of Perm/Wash buffer. Cells were washed twice with PBS Caspase activity assays before TUNEL staining. Relative caspase activities were determined per vendor recommendation Results using the Apo-ONE Caspase-3/7 Assay (Promega). Viable cells were iso- ϩ lated and resuspended in complete medium at 10 ϫ 106/ml. Cells were Effector CD4 T cells possess active caspases serially diluted in complete medium and then mixed with 100 ␮lof Although caspase activity is essential for T cell activation (7, 21, Caspase-3/7 Reagent (DEVD-rhodamine) per the manufacturer’s protocol. Competition experiments included 100 ␮M z-VAD in the Caspase-3/7 Re- 22), the extent of caspase activation and their substrate cleavage agent mix. Spectrophotomeric readings were taken over a range of times have not been defined. Caspase activation and cleavage of known ϩ and data shown represent readings at 90 or 150 min. substrates were examined during the progression of naive CD4 T The Journal of Immunology 4001 cells to the effector stage. Fresh polyclonal CD4ϩ T cells were of upstream caspase-8 and downstream caspase-3 as defined by purified from C57BL/6 mice, a portion were stimulated with anti- their cleavage at specific aspartate residues. We also determined

CD3/CD28 in the presence of IL-2, and nonviable cells were re- cleavage of the known caspase-8 substrates c-FLIPL and Bid as moved at all time points by Ficoll centrifugation. Caspase activity well as the caspase-3 substrate ICAD by immunoblot using lysates was then determined using a DEVD-rhodamine substrate (Fig. from purified CD4ϩ T cells that were either unstimulated or acti- 1A). Although DEVD has a high affinity for active caspase-3, it vated by anti-CD3/CD28 (Fig. 2). Lysates from FasL-treated cells also has an appreciable affinity for the other caspases, including were included as a positive control for substrate cleavage products. caspase-8 (23). This assay is therefore best viewed as an indication One means of caspase-8 activation is by cleavage of its full-length ϩ of overall caspase activity (9, 24). Fresh CD4 T cells had neg- 55-kDa form, resulting in p43/41 caspase-8, through the liberation ligible levels of caspase activity, whereas viable day 4 effectors of a C-terminal p10/12 fragment. Further cleavage of p43/41 manifested considerably increased caspase activity (Fig. 1A). ϩ caspase-8 leads to the generation of a p18 fragment that het- Caspase activity in effector CD4 T cells was greatly diminished erodimerizes with the p10 fragment to form a fully active protease in the presence of the pan-caspase blocker z-VAD-fmk (Fig. 1B), (25). Caspase-8 was largely present in its full-length 55-kDa form a competitive inhibitor of DEVD, confirming the specificity of the in freshly isolated CD4ϩ T cells (Fig. 2A). Of note, p18 caspase-8 assay. In contrast, apoptotic T cells generated by incubating T cell was absent in effector lysates, but was present in lysates from the blasts with soluble (FasL) manifested a 4-fold increase FasL-treated population (Fig. 2A). Cleavage of caspase-8 to in caspase activity compared with viable effector T cells (Fig. 1C). p43/41 caspase-8 was seen 2 days after activation. Evidence of Intracellular levels of active caspase-3 were assessed in fresh day caspase-8 cleavage was apparent as early as 4 h after T cell acti- 4 effectors, and day 4 effectors were treated with FasL (Fig. 1D). vation (21). The intensity of the p43/41 complex increased grad- Downloaded from This demonstrated monophasic peaks of active caspase-3, whose ually through day 4 and then decreased slightly by day 6 (Fig. 2A). mean fluorescence intensity increased from 82 in fresh cells to 825 in day 4 effectors and 1717 in effectors treated with FasL. Cells The kinetics of the cleavage pattern closely paralleled CD25 ex- were simultaneously stained for TUNEL, which revealed 3% of pression and cell cycle rates (Ref. 21 and data not shown). fresh cells, 9% of effectors, and 63% of FasL-treated cells were c-FLIPL heterodimerizes with caspase-8 and is a caspase-8 sub- TUNELϩ. This demonstrated that the level of caspase activity in strate (19, 26). In a manner similar to caspase-8, cleavage of c-

ϩ http://www.jimmunol.org/ effector CD4 T cells was intermediate between that of resting T FLIPL from its full-length 55-kDa form to p43FLIP was consid- cells and of those undergoing . To deter- erably more apparent by day 2 (Fig. 2B). This corresponds with a Ϫ 376 mine the levels of active caspase-3 in truly nonapoptotic TUNEL known caspase-8 cleavage site in c-FLIPL at Asp (27, 28). Bid effectors, TUNELϪ and TUNELϩ populations of effector cells was only weakly expressed in fresh T cells, but was considerably were gated separately and caspase-3 activity was measured. Active up-regulated by day 2 after activation (Fig. 2C). Cleavage of Bid caspase-3 levels were clearly higher in TUNELϩ cells, but was only slight but mirrored that of caspase-8 activation, becoming TUNELϪ cells had levels of active caspase-3 that were still sub- apparent at day 2, peaking at day 4, followed by a gradual decrease ϩ stantially above those of fresh T cells (Fig. 1E). by day 6 (Fig. 2C). Thus, caspase activation in effector CD4 T To further define which caspases contributed to the caspase ac- cells led to the cleavage of the known caspase-8 substrate, ϩ by guest on October 1, 2021 tivity of day 4 effector CD4 T cells, we examined the activation c-FLIPL, and less extensively downstream, Bid.

FIGURE 1. Intermediate levels of caspase activity are present in effector CD4ϩ T cells. Purified poly- clonal CD4ϩ T cells were stimulated for 2 days with immobilized anti-CD3, soluble anti-CD28, and IL-2, and then were expanded in fresh medium supplemented with IL-2 for an additional 2 days. A, Viable unstimu- lated (Fresh) and day 4 effector CD4ϩ T cells were purified by ficoll centrifugation and caspase activity was determined using a DEVD-rhodamine fluorogenic assay. B, To confirm specificity of the assay, the broad spectrum caspase inhibitor z-VAD-fmk (100 ␮M) was used as a competitive inhibitor of DEVD-rhodamine using day 4 effector cells. Results are representative of five experiments. C, As a positive control for caspase activation and as a point of reference, a portion of the same effector T cells was treated with FLAG-tagged FasL cross-linked with anti-FLAG Ab for 4 h before the caspase assay was performed. D, Fresh CD4ϩ T cells (peak 1), day 4 effector cells (peak 2), and 2-h FasL- treated effector cells (peak 3) were simultaneously an- alyzed for intracellular active caspase-3 and TUNEL. E, The day 4 effector cells were further subdivided into TUNELϪ and TUNELϩ subsets, and levels of active caspase-3 were displayed for each. Note that the non- apoptotic TUNELϪ effector cells nonetheless have higher levels of active caspase-3 than do fresh cells. Results in D and E are representative of two indepen- dent experiments. 4002 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS

enriched after the precipitation of active caspases, which provides support for p17 caspase-3 being catalytically active in effector T cells (see Fig. 3, and data not shown). Despite the degree of caspase-3 cleavage, only minimal cleavage of ICAD was detect- able (Fig. 2E). Cleavage of each of these caspase substrates was considerably less than that observed in the same cells treated with FasL (Fig. 2).

A conformational change in procaspase-8 induced by c-FLIPL interaction results in an active full-length protease (10, 18, 27). Distinguishing active full-length caspase-8 from inactive pro- caspase-8 is thus not possible by standard immunoblot analysis of whole cell lysates. To better identify the degree of caspase acti- vation and the size of active caspases, we used biotin-VAD-fmk to selectively bind enzymatically active caspase catalytic pockets. Fresh day 0 or day 4 effector CD4ϩ T cells were incubated with biotin-VAD. Lysates were then incubated with avidin-Sepharose beads to affinity purify active caspases. Active caspase-8 and caspase-3 levels were identified using specific Abs in immunoblot analysis of precipitates (Fig. 3A). Whole cell lysates were included to positively identify each caspase band and assess their overall Downloaded from expression (Fig. 3A, lanes 1 and 2). This analysis revealed that full-length caspase-8 bound biotin-VAD in effector T cells (Fig. 3A, top panel, lane 4), but not in unstimulated cells (Fig. 3A, lane 3). A longer exposure of the caspase-8 immunoblot revealed ad- ditional p43/41 caspase-8 bands, indicating that this species is also enzymatically active (Fig. 3B, right panel). The binding of biotin- http://www.jimmunol.org/ VAD to caspases in effector cell lysates was effectively competed by the addition of 10-fold excess nonbiotinylated z-VAD before incubation of cultures with biotin-VAD (Fig. 3A, lane 5). In ad- dition, no detectable caspase-8 was precipitated using only nonbi- otinylated z-VAD (data not shown). As with caspase-8, more active caspase-3 was present in effector cell lysates than in fresh lysates (Fig. 3A, middle panel, lanes 3 and 4). In contrast with caspase-8, however, there was negligible bind- by guest on October 1, 2021 FIGURE 2. Caspase activation and substrate cleavage are concomitant ing of biotin-VAD to full-length caspase-3, but considerable bind- with CD4ϩ T cell activation. Day 4 polyclonal effector CD4ϩ T cells were ing to the small amount of the cleaved p12/10 fragments present in generated as in Fig. 1. Lysates were prepared from viable cells at the times day 4 effector T cell lysates (Fig. 3A, middle panel, lane 4). As indicated. Immunoblot analysis was performed for caspase-8 (A), c-FLIPL with caspase-8, competition with nonbiotinylated z-VAD de- (B), Bid (C), caspase-3 (D), and ICAD (E). Caspase-8 represents non- creased the amount of precipitated active caspase-3 (Fig. 3A, mid- cleaved procaspase-8, whereas p43/41caspase-8 bands represent fragments dle panel, lane 5). These findings are consistent with the accepted generated by cleavage at Asp374 or Asp384. c-FLIP represents full-length L model that upstream caspases (e.g., caspase-8) can be active in protein, p43FLIP represents a fragment generated by cleavage at Asp376, their full-length form, whereas downstream effector caspases (e.g., and p27FLIP represents a further cleavage product from cleavage at Asp258. t-Bid is generated by cleavage of full-length Bid at Asp75. caspase-3) require cleavage and dimerization to be active (9, 32). The results demonstrate that activated nonapoptotic CD4ϩ T cells Caspase-3 represents noncleaved procaspase-3, whereas p20 caspase-3 and ϩ p12/10 caspase-3 represent fragments generated by cleavage at Asp169 or possess more caspase activity than do unstimulated CD4 T cells. Asp175. ICAD represents noncleaved ICAD, and p18ICAD is generated by Of additional interest was that death domain RIP1 (Fig. cleavage at Asp117. Lysates from FasL-treated populations were included 3A, bottom panel, lane 4), but not Fas-associated death domain in the analysis to illustrate cleavage products. NS, Nonspecific band protein (data not shown), was found to be associated with active caspases precipitated from effector cells. RIP1 is involved with the generation of apoptotic signals by TNFR2 (33) and Fas (34, 35), as Caspase-3 is inactive in its full-length 33-kDa form and is well as with the activation of NF-␬B in response to TNF-␣ (36, cleaved by upstream caspases, including caspase-8, which liber- 37), and thus acts as a link between NF-␬B activation and apo- ates a large p20 and a small p12/10 fragment (25, 29). The large ptosis. RIP1 both associates with c-FLIP and is a known caspase-8 and small fragments then dimerize to form the active protease (25). substrate (33, 38, 39). Reinforcing the observation that RIP1 was p20 caspase-3 may then be autoproteolytically processed to a p17 associating with active caspases, RIP1 was not seen in precipitates fragment, which is considered the mature large caspase-3 subunit from fresh T cells (Fig. 3A, bottom panel, lane 1). RIP1 association (29–31). In fresh CD4ϩ T cells, full-length caspase-3 and p20 was specifically reduced upon the addition of excess nonbiotiny- caspase-3 were present, whereas active p12/10 caspase-3 was not lated z-VAD (Fig. 3A, bottom panel, lane 5). In addition to RIP1, apparent until day 2 (Fig. 2D). p17 caspase-3 was never observed both full-length c-FLIPL and p43FLIP were also precipitated with in the immunoblot of whole cell lysates, even those treated with the active caspase complex (Fig. 3B). Thus, RIP1 and c-FLIP be- FasL. This may be explained by the lack of ability of the anti- come complexed with active caspases. caspase-3 Ab to strongly detect p17 caspase-3. However, in lysates To illustrate that p55 caspase-8 is in fact active, effector cells made from cells treated with biotin-VAD, a 17-kDa band was re- were treated with FasL for 1 or 2 h, and active caspases were vealed with HRP-conjugated Strepavidin or anti-biotin Ab and was labeled, precipitated, and analyzed by immunoblot (Fig. 3C). The Journal of Immunology 4003 Downloaded from

FIGURE 3. Activated T cells possess active full-length caspase-8, active cleaved caspase-3, full-length FLIP , p43FLIP, and RIP1 complexed with

L http://www.jimmunol.org/ active caspases. Freshly isolated polyclonal T cells (lanes 1 and 3) or day 4 effectors (lanes 2, 4, and 5) were incubated with control peptide z-FA-fmk (lanes 1-4)or100␮M nonbiotinylated z-VAD (lane 5) at 37°C for 15 min. All cells were then incubated with 10 ␮M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 ␮M biotin-VAD (lanes 1-5). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 (top panel), caspase-3 (middle panel), or RIP1 (bottom panel) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh (lane 1) or day 4 effectors (lane 2). B, Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C, Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab (top panel)or by guest on October 1, 2021 HRP-conjugated Strepavidin (middle and bottom panels). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots (top and middle panels). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 (bottom panel).

Whole cell lysates and the portion of cell lysates that did not bind cycling. CD4ϩ T cells were labeled with the membrane dye CFSE to avidin-Sepharose (“Flow Through”) were also included in the and stimulated in vitro with anti-CD3/CD28 plus IL-2 in the pres- immunoblot. As expected, anti-caspase-8 Ab revealed that p55 and ence of z-VAD or DMSO vehicle control added at the time of p43 caspase-8 were precipitated with biotin-VAD (Fig. 3C, top activation. As shown in Fig. 4, both medium control and DMSO- panel). It is important to note that the proportion of total caspase-8 treated cells cycled to a similar degree (36%) by day 2, whereas that was precipitated as active caspase-8 was relatively low, as only 20% of z-VAD-treated cells underwent cell cycling. By day most caspase-8 in effector T cells did not bind avidin-Sepharose (Fig. 3C, Flow Through). To confirm that biotin-VAD directly bound a 55-kDa species rather than merely coimmunoprecipitating p55 caspase-8, we visualized protein species that bound biotin- VAD using Strepavidin-HRP (Fig. 3C, middle panel) and anti- biotin (data not shown). This also identified bands at 55 kDa and 43/41 kDa, corresponding with full-length caspase-8 and p43/41 caspase-8. In addition, the vast majority of biotin-labeled caspase-8 was precipitated by avidin-Sepharose, as only a very weak band can be visualized in the flow-through fraction that did not bind avidin-Sepharose. An additional 17-kDa band was visu- alized in whole cell lysates, was enriched with Strepavidin-Sepha- rose beads, and was absent in the flow-through fractions (Fig. 3C, bottom panel). This band likely corresponds with p17 caspase-3. Thus, biotin-VAD bound directly to full-length p55 caspase-8 in ϩ FIGURE 4. z-VAD inhibits CD4 T cell proliferation. z-VAD (100 effector but not fresh naive T cells, and this represented only a ␮M) or a comparable volume of DMSO vehicle control was added to small fraction of the total caspase-8 in the effector cells. CFSE-labeled polyclonal CD4ϩ T cell cultures at day 0 and stimulated ϩ Caspase blockade inhibits CD4ϩ T cell proliferation with anti-CD3/CD28 in the presence of IL-2. CD4 T cells were analyzed for proliferation as measured by CFSE dye loss on day 2 (top panel)or3 ϩ Given the caspase activity of cycling effector CD4 T cells, we (bottom panel). Number insets represent the percentage of cells that di- investigated whether this activity was necessary to initiate cell vided at least one time. 4004 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS

3, 60% of medium control cells cycled and 58% of the DMSO cycling (43), and viable rested effectors were then purified. Con- control cells, compared with only 32% of cells that received z- comitant with their reduced cell cycling, rested effectors mani- VAD. These results indicate that a blockade in caspase activation fested considerably diminished caspase activity (Fig. 5B). None- prevents cycling of murine T cells. theless, Th2 rested effector cells possessed higher caspase activity than did Th1 rested effector cells (Fig. 5B, inset). Thus, caspase Th2 effectors manifest more caspase activity than do Th1 activity closely paralleled cell cycling. effectors To better define which caspases were active, and to what degree, Given the role of caspase activity for both cell growth and cell in Th1 and Th2 effectors, biotin-VAD was again used to purify death, the level of caspase activity in effector T cells could pro- active caspases. In agreement with the DEVD-rhodamine caspase foundly influence the proportion of various effector subsets. As activity assay, Th2 effectors possessed more active full-length Th1 and Th2 cells are differentially responsive to Fas-mediated caspase-8 than did Th1 effectors (Fig. 6, top panel, lanes 3 and 4), cell death, we characterized caspase activity and cleavage of despite having identical overall levels and cleavage patterns of caspase substrates in these cells (40, 41). We also wished to de- caspase-8 in their whole cell lysates. Comparable results were seen termine whether our findings using anti-CD3/CD28 stimulation of with analysis of active caspase-3 (Fig. 6, middle panel). In addi- polyclonal CD4ϩ T cells extended to Ag-specific CD4ϩ T cells. tion, more RIP1 was seen in complex with active caspases in Th2 For this purpose we used CD4ϩ T cells from AND TCR transgenic cells than in Th1 cells (Fig. 6, bottom panel). mice, which recognize PCCF presented in the context of I-EK (42). To determine the downstream targets of increased caspase ac- AND Th1 and Th2 effector CD4ϩ T cells were produced in tivity in Th2 effectors, we examined cleavage patterns of candidate vitro, as described in Materials and Methods. Intracellular cyto- caspase substrates in viable AND naive cells as well as in viable ϩ Downloaded from kine staining and ELISA were used to verify correct polarization polarized AND CD4 effector and rested effector T cells stimu- ϩ of the resulting day 4 effectors (data not shown). Viable day 4 lated with PCCF. AND naive CD4 T cells contained predomi- effector T cells were purified by Percoll centrifugation, and a por- nantly full-length c-FLIPL, whereas activated day 4 Th1 and Th2 tion was then used in the DEVD-rhodamine release assay to de- effectors expressed cleaved p43FLIP in addition to full-length c- termine overall caspase activity (Fig. 5A). AND Th2 effectors had FLIPL (Fig. 7A). No difference was observed in FLIP RNA levels significantly higher overall caspase activity than did Th1 effectors. between Th1 and Th2 effectors (data not shown). However, Th2 The remaining day 4 effectors were extensively washed and rested cells possessed an additional p27FLIP product with proportionally http://www.jimmunol.org/ for 3 days in the absence of exogenous cytokines to reduce cell less full-length c-FLIPL and p43FLIP. The p27FLIP fragment that we observed was of a molecular mass similar to that of an alter-

native splice product of FLIP message (FLIPshort), which has been reported to be up-regulated in activated T cells and during restimu- lation events (44, 45). The p27FLIP fragment that we observed corresponds with a second putative caspase cleavage site at Asp258. In this instance, it would appear that p27FLIP was more likely a further cleavage product than an alternative splice form. by guest on October 1, 2021 Evidence for this was observed in CD4ϩ T cell lysates from full-

length c-FLIPL transgenic mice. In the case of the transgenic an- imal, the transgene cannot be alternatively spliced (Fig. 7A). This finding is also consistent with the observation of elevated caspase

FIGURE 5. AND Th2 CD4ϩ T cells possess higher levels of caspase activity than do Th1 cells at the effector and rested effector stages. AND naive CD4ϩ T cells were stimulated in vitro under polarizing conditions for 4 days using DCEK-ICAM APCs pulsed with 5 ␮M PCCF. Viable cells were used to perform caspase activity assays. Shown is a representative FIGURE 6. Th2 effector CD4ϩ T cells possess more active full-length example of three experiments. A, Caspase activity of day 4 effectors. Re- caspase-8, active cleaved caspase-3, and RIP1 in complex with active sults are representative of three experiments (p ϭ 0.0044, two-way caspases than do Th1 effectors. Day 4 Th1 and Th2 effector CD4ϩ T cells ANOVA column factor). B, Portions of Th1 and Th2 effector populations were generated using anti-CD3/CD28 stimulation and then were incubated were washed three times to remove exogenous cytokines and were cultured at 37°C for 15 min with biotin-VAD followed by lysis in buffer containing in the absence of exogenous cytokines for an additional 3 days to generate 20 ␮M biotin-VAD. A portion of each lysate was incubated with avidin- rested effector populations (43). Results are displayed using the same scale Sepharose beads to isolate active caspases. Immunoblot analysis was per- as in A. Figure inset in B represents results from the same rested effectors formed on nonprecipitated WCLs (lanes 1 and 2) and precipitates (lanes 3 using a more sensitive scale that shows residual persistent caspase activity and 4) for caspase-8 (top panel), caspase-3 (middle panel), or RIP1 (bottom to be more prominent in Th2 rested effectors. panel). NS, Nonspecific band. The Journal of Immunology 4005

FIGURE 7. AND Th2 effector CD4ϩ T cells show more extensive cleavage of selective upstream caspase substrates than do Th1 effectors. Viable naive (Fresh) day 4 Ag-activated Th1 and Th2 effectors and Th1 and Th2 rested effector populations were purified, and im- munoblot analysis of lysates was performed for c-FLIPL (A), RIP1 (B), Bid (C), and ICAD (D). Lysates ϩ from c-FLIPL transgenic CD4 T cells were included in A to demonstrate the p43FLIP and p27FLIP cleavage fragments of overexpressed full-length c-FLIPL. B, RIP1 represents noncleaved protein and p38RIP1 rep- resents a cleavage product generated by cleavage at Asp324. NS, Nonspecific band. C, t-BID is generated by caspase-mediated cleavage at Asp75. D, Cleavage of ICAD at Asp224 results in p18ICAD. Lysates of RIP1Ϫ/Ϫ 3T3 fibroblasts were included as negative con- trols. Bid (C) and ICAD (D) remained as mostly non- cleaved proteins under these culture conditions. Results are representative of four experiments. Downloaded from

activity in T cells from c-FLIPL transgenic mice (R. C. Budd, Th2 effectors may better tolerate elevated caspase activity due unpublished observations). Consistent with the concept that to higher levels of Bcl-2

ϩ http://www.jimmunol.org/ p27FLIP can be generated by active caspases, wild-type CD4 T Conceivably, the higher levels of caspase activity of Th2 cells cells treated with FasL also manifest a p27FLIP fragment (Fig. might result in greater cell death over Th1 cells. However, TUNEL 2B). Rested effectors showed a return of full-length c-FLIP (Fig. L analysis of polarized Th1 and Th2 effector cells actually revealed 7A), consistent with the observed decrease in the caspase activity higher levels of apoptosis in Th1 effectors on days 3 and 4 (Fig. of these cells (Fig. 5). Nonetheless, the residual higher level of ϩ 8A). Similar results were found in polarized polyclonal CD4 T caspase activity in Th2 rested effectors (Fig. 5) was reflected in an cell populations (data not shown). The differences between increased amount of p27FLIP compared with that seen in Th1 ϩ TUNEL cells in Th1 and Th2 populations on day 4, but not day rested effector cells. 3, reached statistical significance ( p Ͻ 0.05, n ϭ 2). This finding To assess the extent of propagation of the caspase cascade in is consistent with an earlier report that a higher proportion of Th1 by guest on October 1, 2021 polarized effector T cells, we examined cleavage of the caspase-8 cells die compared with Th2 effectors (40). substrates RIP1 and Bid and the downstream caspase-3 substrate Because the increased caspase activity in Th2 effectors did not ICAD (Fig. 7, B–D). Given our findings with c-FLIP cleavage, result in increased apoptosis, we considered that there might be a RIP1 was a particularly attractive candidate as it both associates with c-FLIP and is a known caspase-8 substrate, yielding a mechanism by which Th2 effectors better tolerate elevated caspase p38RIP1 fragment (33, 38). We observed that RIP1 cleavage pat- activity. To explore this possibility, we examined expression of a major protector against caspase-driven death, Bcl-2, which func- terns in Th1 and Th2 effectors mimicked that of c-FLIPL. AND naive CD4ϩ T cells expressed mainly full-length RIP1, whereas tions to protect the outer mitochondrial membrane from caspase- effector cells manifested the p38RIP1 cleavage product with con- initiated cell death (47). Day 4 Th1 and Th2 effectors were ana- comitant reduction of full-length RIP1 (Fig. 7B). In each sample, lyzed by simultaneous staining for apoptotic cells by TUNEL assay and for intracellular Bcl-2. Th1 effectors expressed a higher full-length RIP1 appeared as three distinct bands, consistent with Ϫ recent reports that RIP1 can be ubiquitylated (46). Th2 effectors proportion of Bcl-2 cells (23%) than did Th2 effectors (12%) possessed less full-length RIP1 than did Th1 effectors (Fig. 7B). As (Fig. 8B). Th2 AND effectors manifest a statistically higher per- high Ϫ with c-FLIP cleavage products, levels of p38RIP1 diminished in centage of cells of the phenotype Bcl-2 /TUNEL and lower low Ϫ Ͻ ϭ both Th1 and Th2 rested effector cells, but Th2 rested effectors levels of Bcl-2 /TUNEL cells ( p 0.05, n 2). In addition, Ϫ manifested less full-length RIP1 (Fig. 7B). The specificity of the the apoptotic cells were almost entirely confined to the Bcl-2 full-length RIP1 and p38RIP1 bands was confirmed using lysates subpopulation, suggesting that the presence of Bcl-2 was protec- ϩ from both RIP1-deficient 3T3 cells as a negative control (Fig. 7B). tive against the caspase activity in CD4 effector T cells. This was Unlike c-FLIP and RIP1, Bid is not known to form a complex reinforced by the addition of z-VAD to cultures at day 0, which L Ϫ with caspase-8, although Bid can be cleaved by caspase-8. Inter- reduced the proportion of TUNEL-positive cells in the Bcl-2 population from 22% to 15% in Th1 cultures and from 17% to 8% estingly, in contrast with c-FLIPL and RIP1, Bid showed only min- imal levels of cleavage in either polarized effector or rested effector in Th2 cultures (Fig. 8B). Caspase blockade also yielded an overall Ϫ populations (Fig. 7C). This finding closely paralleled the earlier increase in the proportion of Bcl-2 cells. Th2 effector cells are observations with nonpolarized CD3/CD28-stimulated effector thus potentially better adapted to sustain higher levels of caspase cells (Fig. 2C). In addition, no observed cleavage of the caspase-3 activity, in part through maintenance of higher levels of Bcl-2 substrate ICAD was detected in either Th1 or Th2 effector or rested expression. effector populations. Collectively, these data show that Th2 cells An additional, though not mutually exclusive, possibility is that manifest greater caspase activity and cleavage of upstream caspase Th2 effector cells may have a greater requirement for caspase ac- substrates than do Th1 cells at the effector and rested effector tivity for cell cycling. To examine this, CFSE-labeled AND CD4ϩ stages. T cells were stimulated with PCCF under Th1 and Th2 polarizing 4006 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS Downloaded from

FIGURE 8. AND Th1 effector CD4ϩ T cells exhibit a greater popula- http://www.jimmunol.org/ tion of Bcl-2Ϫ and dead cells than do Th2 cells. A, TUNEL analysis of day 3 and 4 AND effector CD4ϩ T cells stimulated with APCs and PCCF. Data represent mean Ϯ SD from triplicate cultures. B, Cell death (nicked DNA) was assessed by TUNEL assay and intracellular Bcl-2 levels were deter- FIGURE 9. Addition of z-VAD inhibits T cell proliferation under either mined on day 4 Th1 and Th2 effectors in which 100 ␮M z-VAD, or equiv- Th1 or Th2 polarization conditions. CFSE-labeled AND CD4ϩ T cells alent volume of DMSO, was added at day 0. Percentage insets represent the were cultured under Th1 or Th2 polarizing conditions in the presence of proportion of total cells. Numbers in parentheses represent the proportion 100 ␮M z-VAD or appropriate DMSO control added at day 0 and then Ϫ of Bcl-2 cells containing nicked DNA. Data are representative of five were analyzed for cell cycling on days 2 (top panel)and4(bottom panel). independent experiments. Number insets represent the percentage of divided cells. by guest on October 1, 2021 conditions with or without z-VAD added at day 0 of culture. sufficient caspase activity to promote cell cycling, but not so much Equivalent fractions of Th1 (10%) and Th2 (7%) cells proliferated as to induce cell death. The levels of caspase activity may pro- by day 2 in the absence of caspase blockade (Fig. 9, DMSO). foundly affect the population of T cell effector subsets. However, caspase blockade reduced the percentage of proliferating Because there is a fine balance of caspase activity between pro- cells to 1% in both the Th1 and Th2 populations. By day 4, 94% liferation and cell death, it is important to restrain and target of Th1 and 95% of Th2 cells had proliferated, whereas caspase caspase activity to specific substrates in living cells. The recent blockade at day 0 decreased proliferation to 58% in Th1 and 55% concept that noncleaved caspase-8 can manifest intermediate ac- in Th2 cells. Furthermore, a delay in the addition of z-VAD until tivity is fully consistent with our results (18). Our findings further ϩ day 2 had no effect on subsequent proliferation by AND CD4 T suggest that this form of active caspase-8 leads to the cleavage of cells (data not shown). These findings suggest that caspase acti- molecules known to associate with it, including c-FLIPL and RIP1 vation is equally necessary for initial activation of the majority of (39), with less extensive cleavage of more distal substrates such as Th1 and Th2 populations, but that it is not required for continued Bid. This would suggest that molecules that complex with proliferation during the Ag-independent, cytokine-driven phase of caspase-8 are most efficiently cleaved in effector cells. Although ϩ CD4 T cell growth (20). we speculate that RIP1 is binding to caspase-8, it remains formally possible that RIP1 is binding to and cleaved by active caspases Discussion other than caspase-8. Furthermore, the limited degree of caspase-3 Our results show that viable effector CD4ϩ T cells generate activity in effector T cells does not lead to any appreciable ICAD caspase activity levels that are between those of fresh CD4ϩ T cleavage. cells and FasL-treated effector T cells. This is reflected in the Recent reports show that caspase-3 and caspase-8 can localize in cleavage of caspase-8 substrates c-FLIPL, RIP1, and to a lesser lipid rafts of nonactivated peripheral blood lymphocytes and that extent Bid, but not the downstream caspase-3 substrate ICAD. In caspase-3 levels increase in lipid rafts upon activation (48). In addition, RIP1, full-length c-FLIPL, and p43FLIP were precipi- addition, these and other investigators have shown that p20 tated with active caspases in effector T cells. Furthermore, this caspase-3 is kept inactive by X-linked inhibitor of apoptosis pro- intermediate caspase activity is necessary for the initiation of cell tein (XIAP) and is activated upon further cleavage in which the cycling by the majority of T cells. Caspase activity is also higher prodomain of capase-3 is released, thereby generating p17 in Th2 vs Th1 viable effector CD4ϩ T cells and subsides in each caspase-3 (29, 48). If second mitochondria-derived activator of population as they decrease cell cycling and become rested effector caspase/direct inhibitor of apoptosis-binding protein with low iso- cells. Activated T cells thus run a delicate balance of generating electric point protein is released from the mitochondria, then XIAP The Journal of Immunology 4007 is inactivated, thereby enabling the autoproteolytic processing of of wild-type caspase-8 in these cells promoted normal induction of p20 caspase-3 to p17 caspase-3. Bcl-2 protein present in the mi- CD25 upon TCR stimulation (7). Consistent with these findings, tochondria inhibits release of second mitochondria-derived activa- inhibition of caspase-8 expression by siRNA decreased activation tor of caspase, thereby maintaining the association of XIAP and of T cells (7). In contrast, caspase-8 conditional knockout mice did p20 caspase-3 and preventing further processing of caspase-3 (29– not show a defect in IL-2 production, but did exhibit a defect in 31). Studies have suggested that other active caspases, such as response to IL-2, which could not be rescued by PMA and iono- caspase-8 or caspase-9, can process caspase-3 to its fully mature mycin (8). Thus, multiple studies highlight the importance of 17-kDa fragment (49). Thus, the likely need to maintain mitochon- caspases in T cell effector function. Yet, it has been uncertain just drial integrity to prevent excessive caspase activity at the effector how much caspase activity is present in cycling vs apoptotic T T cell stage is reflected in the high percentage of apoptotic cells in cells, what the substrates are, and how extensive is the progression Ϫ the Bcl-2 subset. In agreement with this concept, caspase block- of the caspase cascade. Ϫ ade allowed greater survival of Bcl-2 cells. It is not presently clear why Th2 cells exhibit higher caspase Further support for the notion that p20 caspase-3 is inactive activity than do Th1 cells. One potential explanation is that Th2 derives from its presence in freshly isolated T cells, in which neg- effectors are better equipped to survive caspase activation. This ligible levels of caspase activity were detected. However, in day 4 possibility is supported by the findings of a higher percentage of effector T cells, where caspase activity was detected, the intensity Bcl-2ϩ cells among Th2 cells compared with Th1 effectors. These of the p20 caspase-3 band decreased concomitant with an increase results may offer a partial explanation for two reports that find that of detectable p12/10 caspase-3. By day 6, there was a decrease in Th1 cells are more susceptible to Fas-mediated cell death (40, 41). p12/10 caspase-3 with a proportional increase in p20 caspase-3. Alternatively, though not mutually exclusively, Th2 cells may re- Downloaded from Taken together, these results suggest that p20 caspase-3 is likely quire higher levels of caspase activity to obtain their fully polar- inactive, consistent with it being complexed with XIAP (29). Fur- ized cytokine phenotype by cleavage of selective substrates im- thermore, our results showing negligible binding of biotin-VAD to portant in pathways. Studies to explore the either full-length pro-caspase-3 or p20 caspase-3, but binding to a latter possibility are currently underway. 17-kDa fragment, are consistent with this view and with other The exact contribution of caspase activity and their substrates in studies (29, 48). T cell activation remains undefined, though the current findings http://www.jimmunol.org/ It was recently reported that c-FLIP , a 27-kDa alternate splice S identify logical candidate substrates. One role for caspases could variant of c-FLIP, blocks caspase-8 activation to a greater extent be to cleave c-FLIP to its p43 form, which more efficiently re- than does FLIP (28). Both c-FLIP and c-FLIP have been re- L L L S cruits RIP1 and TNFR-associated factor 2, leading to activation of ported to inhibit TRAIL receptor-mediated apoptosis (50–54). c- NF-␬B (58). Consistent with this hypothesis, we observed that FLIP protein is up-regulated within the first day of activation of S RIP1 was in a complex with and proportional to levels of active primary human T cells (45). When cyclohexamide is used during caspases and overall p43FLIP levels (Figs. 3 and 6). Several stud- this time frame, FLIP is down-regulated and cells are sensitive to S ies have revealed a role of Fas signaling in neurite outgrowth (1), Fas-mediated mitochondrial membrane potential loss, despite nor-

fibroblast proliferation (4), maturation of dendritic cells (5), and by guest on October 1, 2021 mal expression of Bcl-2 family members (45). Levels of c-FLIP S hepatocyte regeneration (59). Conceivably, Fas may engage signal diminish over time and by day 6 are nearly undetectable (45). pathways involved with cell growth, such as Erk activation (1). How- Other studies show that restimulation of day 6 effectors with anti- ever, we currently have no evidence that Fas is required for the ini- CD3/CD-28 also leads to up-regulation of c-FLIP protein and S tiation of caspase activity in effector T cells. In fact, preliminary stud- partial protection of mitochondrial integrity, compared with re- lpr stimulation with only anti-CD3 (44). In both studies, increased ies show similar caspase activity is generated in Fas-deficient effector T cells (J. Q. Russell, unpublished observations). c-FLIPS protein expression correlated with decreased caspase-8 processing at the Fas-mediated death-inducing signaling complex The direct connection between TCR ligation and activation of caspases has not yet been identified. A logical consideration is that (44, 45). Other studies indicate a role for elevated c-FLIPS in the survival of various tumor cells, including human gastric cancers expression of a death receptor ligand is up-regulated, which stim- (55, 56) and cells from patients with myelodysplastic syndrome ulates its cognate receptor to activate caspases. This is consistent (57). We propose an alternative mechanism of generating p27FLIP with previous findings showing that Fas ligation can costimulate T by further cleavage of p43FLIP. In addition to the known caspase cell proliferation (6, 21, 60). However, as mentioned above, Fas- cleavage site at Asp376 (LEVD), there is a second potential caspase deficient lpr T cells proliferate normally and activate caspases cleavage site at Asp258 (LIID) that would lead to p27FLIP. We upon TCR stimulation. An alternative view is that TCR activation found that p27FLIP is also a cleavage product in T cell lysates may promote approximation of caspase-8 with c-FLIPL indepen- dently of a death receptor, possibly via lipid raft association as from c-FLIPL transgenic mice as well as from FasL treatment of T cell blasts. The fact that Th2 cells possess more caspase activity suggested for Fas signaling in activated T cells (61). In this regard, ϩ and greater p27FLIP than do Th1 cells is also consistent with pro- c-FLIPL transgenic mouse CD4 T cells manifest increased teolytic generation of this fragment. In addition, quantitative PCR caspase activity (R. C. Budd, unpublished observations) and hy- revealed that c-FLIP message is expressed at very similar levels in perproliferate in response to TCR engagement (62). Th1 and Th2 cells (data not shown). If the p27FLIP that we ob- Caspases (especially caspase-8) and c-FLIPL clearly have an served were to be generated via an alternative splicing mechanism, important role in the growth and development of potentially many then we would expect to see an increase in c-FLIP message in Th2 cell types. This is underscored by the cardiac developmental block cells. p27FLIP may thus arise by two mechanisms in activated T in mice deficient for either c-FLIPL (63) or caspase-8 (64). Defin- cells. ing the substrates of caspases that are critical for cell growth will Our results expand on other recent studies showing a require- represent an important link between the mechanisms of cell growth ment for caspases in T cell function. In addition, human T cells and cell death and may suggest an important central process for bearing a mutation in the caspase-8 gene exhibited decreased IL-2 preventing aberrant growth by linking cell cycling to the same production and CD25 induction upon stimulation (7). Restoration pathways used at a higher intensity for cell death. 4008 EFFECTOR CD4 T CELLS GENERATE MODERATE CASPASE LEVELS

Acknowledgments 24. Wei, Y., T. Fox, S. P. Chambers, J. Sintchak, J. T. Coll, J. M. Golec, L. Swenson, K. P. Wilson, and P. S. Charifson. 2000. The structures of caspases-1, -3, -7 and We thank Colette Charland for technical assistance with flow cytometry, -8 reveal the basis for substrate and inhibitor selectivity. Chem. Biol. 7:423. members of the University of Vermont DNA facility for assistance with 25. Shi, Y. 2002. Mechanisms of caspase activation and inhibition during apoptosis. real-time PCR, and University of Vermont Immunobiology Program mem- Mol. Cell 9:459. bers for helpful discussions and suggestions. 26. Irmler, M., M. Thome, M. Hahne, P. Schneider, Hofmann, K., V. Steiner, J.-L. Bodmer, M. Schroter, K. Burns, C. Mattmann, et al. 1997. Inhibition of death receptor signals by cellular FLIP. Nature 388:190. Disclosures 27. Chang, D. W., Z. Xing, Y. Pan, A. Algeciras-Schimnich, B. C. Barnhart, S. Yaish-Ohad, M. E. Peter, and X. 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