CD30 Overexpression Enhances Negative Selection in the Thymus and Mediates Programmed Cell Death Via a Bcl-2-Sensitive Pathway1

Roberto Chiarle,*† Antonello Podda,*‡ Gabriel Prolla,§ Eckhard R. Podack,¶ G. Jeanette Thorbecke,* and Giorgio Inghirami2*

The biological function of CD30 in the thymus has been only partially elucidated, although recent data indicate that it may be involved in negative selection. Because CD30 is expressed only by a small subpopulation of medullary thymocytes, we generated transgenic (Tg) mice overexpressing CD30 in T lymphocytes to further address its role in T cell development. CD30 Tg mice have normal thymic size with a normal number and subset distribution of thymocytes. In vitro, in the absence of CD30 ligation, thymocytes of CD30 Tg mice have normal survival and responses to apoptotic stimuli such as radiation, dexamethasone, and Fas. However, in contrast to controls, CD30 Tg thymocytes are induced to undergo programmed cell death (PCD) upon cross-linking of CD30, and the simultaneous engagement of TCR and CD30 results in a synergistic increase in thymic PCD. CD30-mediated PCD requires caspase 1 and caspase 3, is not associated with the activation of NF-␬B or c-Jun, but is totally prevented by Bcl-2. Furthermore, CD30 overexpression enhances the deletion of CD4؉/CD8؉ thymocytes induced by staphylococcal enterotoxin B superantigen and specific peptide. These findings suggest that CD30 may act as a costimulatory molecule in thymic negative selection. The Journal of Immunology, 1999, 163: 194–205.

uring development, thymocytes undergo a series of se- cise roles of CD40 ligand (CD40L) (8), CD28 (9), CTLA-4 and lections that shape the T cell repertoire of Ag specific- CD30 (10) in T cell ontogeny are still not well determined. D ities. The result is the generation of an immune system CD30, a member of the TNFR family that is expressed after that is capable of recognizing a large number of Ags and of dis- activation by both B and T lymphocytes (11), was first identified criminating between self and non-self Ags (1). The mechanisms in Reed-Sternberg cells of Hodgkin’s disease (12). Relatively few regulating thymic negative selection and programmed cell death CD30ϩ cells are present in the thymic medulla and in peripheral 3 (PCD), however, have been only partially elucidated. In the last lymphoid organs; rather, they are primarily localized within the few years, compelling data have indicated that several members of interfollicular areas and less frequently at the rim of germinal cen- the TNF receptor (TNFR) superfamily may be involved in thymic ters (13, 14). In the thymus, CD30 mRNA is highly expressed (11), selection, but their exact contribution is still controversial. In fact, but only low levels of CD30 can be detected in the cytoplasm of the role of TNFR1 in thymic development is largely unclear (2, 3), CD4ϩ/CD8ϩ thymocytes (T. Nguyen, unpublished observation). and contradictory data have emerged from the analysis of Fas (4– More recently, Romagnani et al. have demonstrated a small but 6), because no definitive evidence has yet been provided concern- clearly detectable fraction of CD4ϩ/CD8ϩ thymocytes coexpress- ing the expression of in the thymus (7). Also, the pre- ing CD30, CD45RO, and IL-4R (15). The triggering of CD30 re- quires a specific ligand (CD30L) that is highly expressed on med- ullary thymic epithelial cells and on Hassal’s corpuscles (15). The *Department of Pathology and Kaplan Comprehensive Cancer Center, New York role of CD30 in thymic development has been suggested recently † University Medical Center, New York, NY 10016; Department of Anatomic Pathol- by studies in CD30Ϫ/Ϫ mice, which have an impaired negative ogy, University of Torino, Torino, Italy; Divisions of ‡Pediatric Hematology/Oncol- ogy and §Hematology/Oncology, Department of Medicine, New York University selection (10). These findings, however, have not been confirmed Medical Center, New York, NY 10016; and ¶Department of Microbiology, Miami in vitro using wild-type (WT) thymocytes (3, 16). In contrast, University, Miami, FL 33101 emerging data in mature T cells and cell lines indicate that CD30 Received for publication February 12, 1999. Accepted for publication April 20, 1999. engagement in vitro has pleiotropic effects, resulting in enhanced The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cell proliferation, cell growth arrest, or PCD (17, 18). with 18 U.S.C. Section 1734 solely to indicate this fact. The molecular pathways regulating the pleiotropic effects of 1 These studies were supported in part by National Cancer Institute Research Grants CD30 and other receptors of the TNFR family involved in immune CA-64033 (to G.I.) and AG-04980 (to G.J.T.). A.P. and G.P. are recipients of Na- system regulation, cell proliferation/differentiation, and PCD (19, tional Cancer Institute, National Institutes of Health Training Grant CA-2T32CA09454. 20) have been partially identified during the last few years. Indeed, 2 Address correspondence and reprint requests to Dr. Giorgio Inghirami, Department many TNFR family members can mediate through their of Pathology and Kaplan Comprehensive Cancer Center, New York University (21). These death domains interact with adaptor mol- School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address: ecules like Fas-associated with death domain (22), TNFR1- [email protected] associated death domain, and receptor-interacting protein (23). In 3 Abbreviations used in this paper: PCD, programmed cell death; TNFR, TNF recep- tor; CD40L, CD40 ligand; TRAF, TNFR-associated factor; Tg, transgenic; SEB, turn, Fas-associated protein with death domain interacts with staphylococcal enterotoxin B; FP, forward primer; BP, backward primer; BM, bone downstream cell death effector molecules, such as marrow; LN, lymph node; DP, double positive; SP, single positive; PI, propidium (FADD-like ICE (FLICE)) (24–26), leading to PCD (27) by direct iodide; WT, wild type; FLIP, Flice-like inhibitory protein; TPCK, N-tosyl-L-phenyl- alanine; SAg, superantigen. cleavage of caspase 3 or by cytochrome c release and subsequent

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 195

activation of caspase 9. The mitochondrial release of cytochrome CAGGGATCCCTAGAGTCCTGTTAGGTCC,TRAF2-BP:5Ј-CAGGGTAC c is blocked by Bcl-2 (28). CD30 lacks a definitive death domain. CATGGCTGCAGCCAGTGTGA; FLIP-FP: 5Ј-TACAAGGGATTAC Ј However, its cytoplasmic domains interact with other adaptor ACAGGCA, FLIP-BP: 5 -GTTATGTCATGTGACTTGGG). molecules, including TNFR-associated factor 1 (TRAF1), Cell culture and in vivo experiments TRAF2, TRAF3 (18, 29), and TRAF5 (30). TRAF2, directly or ␬ In vitro CD3 and/or CD30 cross-linking was performed by culturing iso- in association with TRAF1, can lead to NF- B (31) or c-Jun lated thymocytes (5 ϫ 105) for 24 h in 96-well microtiter plates precoated activation (32, 33). overnight at 4°C with specific anti-CD3 (145-2C11, kindly provided by Dr. In the present study, we investigate the biological role of CD30 J. A. Bluestone, Ben May Institute, Chicago, IL) and anti-CD30 (X63 and ␮ in T cell development through its overexpression in thymocytes. CD30.1) (11) Abs (20 g/ml) or with purified polyclonal hamster Ig (20 ␮g/ml, PharMingen). Alternatively, thymocytes were cocultured with 4% We demonstrate that, after cross-linking via anti-CD30 Abs or via paraformaldehyde-fixed P815 cells or with P815 cells transfected with mu- CD30L, thymocytes of CD30 transgenic (Tg) mice are induced to rine CD30L cloned into the pBMG-His vector (CD30L-P815) (11). For undergo PCD. This process triggers caspases 1 and 3 and is totally induction of apoptosis, PMA (10 ng/ml), dexamethasone (10Ϫ6M), iono- prevented by Bcl-2 overexpression. More importantly, we show mycin (1 ␮g/ml), cycloheximide (30 ␮g/ml), and actinomycin D (4 ␮g/ml) ␮ that, with subliminary doses of staphylococcal enterotoxin B were all obtained from Sigma (St. Louis, MO); anti-Fas Ab (1 g/ml, clone Jo-2) was obtained from PharMingen. Inhibitors of PCD were N-tosyl-L- (SEB) superantigen or peptide Ag, CD30 Tg mice have an en- phenylalanine (TPCK) (100 ␮M, Sigma, added to thymocytes 15 min be- hanced deletion of thymocytes. These findings, even if obtained in fore the start of the culture), Z-YVAD-cmk (500 ␮M, Bachem, King of conditions of forced overexpression, indicate that CD30 plays an Prussia, PA), and Z-DEVD-fmk (500 ␮M, Enzyme System Products, important role in thymic negative selection. Dublin, CA). For in vivo experiments, SEB was obtained from Sigma; the OVA pep- tides 323–339 (ISQAVHAAHAEINEAGR) and 324–334 (SQAVHAA Materials and Methods HAEI) (35) were synthesized by standard fluorenylmethoxycarbonyl Generation of CD30 Tg mice chemistry at Seaver Laboratory (Skirball Institute, New York University Medical Center). The 1.6-kb fragment encompassing the complete open reading frame of the murine CD30 gene (11) was cloned (SacI-SalI) in a plasmid (CD4-hCD2, Flow cytometric analysis a generous gift from Dr. D. R. Littman, Skirball Institute, New York Uni- ϫ 6 versity Medical Center, New York, NY) containing the minimal CD4 en- For flow cytometric analysis, 0.5–1 10 cells were incubated with ap- hancer (339 bp), the minimal murine CD4 promoter (487 bp), the tran- propriately diluted biotin-, FITC-, PE-, or tricolor-conjugated Abs. Purified scription initiation site, and 70 bp of the untranslated first exon and part of hamster Ig was used as a negative control. Samples were then fixed and the first intron of the murine CD4 gene (34). The transgene was released analyzed by FACScan (Becton Dickinson, Mountain View, CA). with NotI, injected into the pronucleus of fertilized eggs from (C57BL/6 ϫ The Abs used in this study included the following: anti-CD30-PE (clone mCD30.1, PharMingen), anti-Thy1.2-FITC (clone 30-H12, Becton Dick- DBA/2)F1 hybrid donors, and subsequently transferred to pseudopregnant CD-1 mice. Tg progenies were characterized by Southern blot and/or PCR inson), anti-CD4-FITC (clone CT-CD4, Caltag, Burlingame, CA), anti- analyses. Six founders were identified and used to generate six lines (back- CD8-TRI (clone CT-CD8a, Caltag), anti-CD44-PE (clone IM7, PharMin- crossed to C57BL/6, BALB/c, and DBA/2 strains). The heterozygous off- gen), anti-CD25-biotin (clone 3C7; PharMingen), anti-CD24 (clone M1/ spring from 956 and 986 CD30 Tg mice were used for all of the 69; PharMingen), anti-CD3 (clone 2C11), anti-B220-FITC (clone RA3– ␤ ␤ experiments. 6B2, PharMingen), anti-Fas (clone Jo-2, PharMingen), anti-V 8.1/V 8.2, V␤8.3, V␤3, V␤11-FITC (PharMingen), and KJ1.26 (a gift of Dr. P. Mar- Animals and cell preparation rack, Howard Hughes Medical Institute, Denver, CO). PE-conjugated streptavidin (Vector Laboratories, Burlingame, CA) was used to reveal Mice were housed in the Berg and Skirball Institute Animal Facilities of biotin-conjugated Ab staining. In some experiments, cells were washed in the New York University School of Medicine. CD30 Tg mice were crossed cold PBS and subsequently incubated with annexin V-FITC (Boehringer to Bcl-2-25 Wehi (The Jackson Laboratory, Bar Harbor, ME) and to Mannheim, 2 ␮l/1 ϫ 106 cells) in staining buffer (10 mM HEPES (pH 7.4), DO.11.10 ␣␤-TCR Tg mice (35) on a BALB/c background (kindly pro- 140 mM NaCl, and 5 mM CaCl2). vided by Dr. J. Lafaille, Skirball Institute, New York University School of For DNA content determination, cells were fixed in 70% alcohol (1 h at Medicine. Double Tg mice were identified by PCR or with mAbs against 4°C), washed, and incubated with RNase (1 mg/ml, 10 min at room tem- ␤ CD30 (11) and V 8.1–8.2 TCR (PharMingen, San Diego, CA) by FACS perature) and propidium iodide (PI) (50 ␮g/ml, 15 min at room tempera- analysis. All experiments were performed using mice at 4–8 wk of age. ture, Calbiochem, San Diego, CA). Single-cell suspensions were obtained in complete medium (RPMI 1640 medium supplemented with 10% bovine FCS, 50 ␮g/ml streptomy- Caspase 3 and 9 cleavage assays Ϫ5 cin, 50 infectious units/ml penicillin, 1 mM L-glutamine, and 5 ϫ 10 M 2-ME). Bone marrow (BM) samples were obtained by flushing the femur Thymocytes were isolated from Tg mice and incubated for8hinthe and tibia cavities with cold sterile PBS supplemented with heparin (5000 presence of medium alone, precoated polyclonal hamster Ig, anti-CD3 ␮ ␮ U/ml) and peripheral blood leukocytes from tail vein blood. and/or anti-CD30 (X63) (all at 10 g/ml), or soluble anti-Fas Abs (1 g/ ml). Caspase activity was measured using CPP32/caspase-3 and MCH6/ Southern blot analysis and PCR caspase-9 fluorometric protease assay kits (Chemicon International, Te- mecula, CA). The cleavage of DEVD-AFC and LEHD-AFC substrates was For Southern blot analysis, 10-␮g aliquots of genomic DNA were digested, measured by a fluorescent plate reader. electrophoresed, denatured, and transferred to nitrocellulose, as described previously (36). CD30 gene products were evaluated on BamHI-digested Multiprobe RNase protection assay DNA using the 32P-labeled CD30 cDNA probe. Total RNA was purified with an RNA isolation (Qiagen, Valencia, The detection and quantification of multiple mRNA transcripts after CD30 CA) according to the manufacturer’s instructions. cDNA was obtained or Fas cross-linking were investigated using a RiboQuant multiprobe ϫ 6 RNase protection assay system (PharMingen) according to the manufac- from total RNA (5 10 cells) after reverse transcription using hexanucle- ␮ otide oligonucleotide primers (Boehringer Mannheim, Indianapolis, IN) turer’s instructions. Briefly, total RNA (10 g/reaction) was incubated with ␣ 32 and Moloney murine leukemia virus reverse transcriptase (Life Technol- radiolabeled [ - P]UTP probes overnight at 56°C in hybridization buffer. ogies, Bethesda, MD), as described previously (36). After RNase digestion (45 min at 30°C), samples were treated with pro- The efficiency and quality of each individual cDNA preparation was teinase K, extracted, precipitated, and resuspended with the loading buffer. tested by PCR amplification using specific oligonucleotides recognizing Protected probes were resolved on acrylamide gel and exposed. mouse GAPDH. The characterization of genomic DNA and/or mRNA Immunoprecipitation and Western blot analysis expression of CD30, TRAF1, TRAF2, and FLICE-like inhibitory protein (FLIP) were performed by PCR (CD30-forward primer (FP): 5Ј- Thymocytes (1 ϫ 107) were washed and lysed. After spinning, superna- ATGAGCGCCCTACTCACCGCAGC, CD30-backward primer (BP): 5Ј- tants were precleared twice with 30 ␮l of protein G-Sepharose 4B (Phar- GGATCCAAGCTTTCAGTAACACAGGAGAAAGGAGCCGG; TRAF1- macia Biotech, Piscataway, NJ) for 30 min at 4°C and subsequently incu- FP: 5Ј-CAGGGTACCATGGCCTCCAGCTCAGCCCCTG, TRAF1-BP: 5Ј- bated for2hat4°Cwith anti-CD30 Abs followed by mouse anti-hamster CAGGGATCCCTAAGCACTAGTGTCCACAATG; TRAF2-FP: 5Ј- Ig (2 ␮g, PharMingen). A total of 30 ␮l of protein G was added for2hat 196 CD30 TRANSGENIC MICE AND THYMIC NEGATIVE SELECTION

4°C; after washing (three times, 30 min, 4°C), samples were boiled, loaded Flow cytometry on cells obtained from primary and secondary on a 12% acrylamide gel, and blotted onto a nitrocellulose membrane. The lymphoid tissues was used to study the expression of CD30 in T membrane was first blocked with 1% BSA in PBS with 0.1% Tween 20 and lymphocytes. In control mice, CD30ϩ cells represented a small but subsequently incubated (for1hatroom temperature) with anti-CD30 (10 ␮g/ml,1hatroom temperature). After five washes, were detected detectable fraction of the total lymphoid cells compared with the with biotin-conjugated mouse anti-hamster Ig (for1hatroom tempera- strong expression observed in all T lymphocytes derived from dif- ture), followed by peroxidase-conjugated avidin-biotin complex. Mem- ferent CD30 Tg lines. More importantly, the CD30 overexpression branes were developed with the enhanced chemiluminescence system was not limited to early T cell precursors, but was also found in (Amersham, Arlington Heights, IL). mature CD4 and CD8 SP T cells (Fig. 1D). These findings are in Electrophoretic mobility shift assay agreement with those previously reported using different genes driven by the minimal murine CD4 promoter-enhancer construct Cells were spun and washed with ice-cold PBS; low osmolarity buffer 10 ϩ ϩ mM Tris (pH 7.4), 10 mM NaCl, and 3 mM MgCl2) was added to the cell (34). Normal proportions of thymic CD4 /CD8 double positive pellet. After resuspension, the cells were pelleted and resuspended in cold (DP) and CD4ϩ/CD8Ϫ or CD4Ϫ/CD8ϩ SP lymphocytes (Table I) RSB plus 10% glycerol, 0.25% Nonidet P-40, 1 mM DTT, and 1 mM and of CD24ϩ, CD44ϩ, CD25ϩ, ␣␤-TCRϩ, CD3ϩ, NK1.1ϩ, and PMSF. Cells were then lysed by pipetting and vortexing. Nuclei were re- CD11bϩ cells were identified. However, CD30 Tg animals had a covered by centrifugation, and nuclear extraction buffer (20 mM HEPES decreased ratio of mature T vs B cells in the spleen and LNs, as (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 1 mM PMSF, 1 mM sodium vanadate, 1 ␮g/ml leupeptin, 10 well as increased CD4/CD8 ratios (Table I; R.C., manuscript in ␮g/ml soybean trypsin inhibitor, and 1 ␮g/ml aprotinin) was added. After preparation). Finally, no coexpression of CD30 was demonstrated incubation for 45 min at 4°C, the samples were centrifuged and superna- on B220ϩ or CD11bϩ cells. tants were recovered. A total of 10 ␮g of the nuclear extracts was incubated (for 30 min at room temperature) in a solution containing 20 mM HEPES, ␮ Cross-linking of overexpressed CD30 induces PCD in 40 mM KCl, 1 mM EDTA, 1 mM MgCl2, 0.5 mM DTT, 5% glycerol, 2 g of poly(dC) (Promega, Madison, WI), 1 mM AMP, 1 ␮g of sonicated thymocytes 32 single-stranded herring sperm DNA (Life Technologies), and a P-labeled The thymi of the CD30 Tg animals have a normal size and number ␬ Ј double-stranded NF- B-specific oligonucleotide (5 -AGCTTGGGGACTT of total cells (Table I). In addition, the percentages of spontaneous TCCCAGCCG). Cold competitor assays were conducted by adding a 100- fold molar excess of the unlabeled double-stranded oligonucleotide to the cell death of thymocytes of CD30 Tg and WT mice cultured in reaction mixture. Samples were separated on 6% polyacrylamide gels in vitro over time (24, 48, and 72 h) were comparable (data not 0.2ϫ TRIS-borate-EDTA (TBE) buffer. Gels were dried and exposed. shown). To evaluate a possible role of CD30 in thymic PCD, we Kinase assay investigated the effect of CD30 cross-linking on control and CD30 Tg thymocytes in vitro, with and without CD3 cross-linking. CD3 ϫ 6 For kinase assay, 5 10 thymocytes were treated with media alone, cross-linking alone induced a similar increase in PCD over spon- anisomycin (1 ␮g/ml 30 min), P815, or CD30L-P815. Cells were washed, lysed (20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM PMSF, 5 mM taneous cell death in both groups. On the contrary, CD30 cross- ␤ EDTA, 2 mM EGTA, 1 mM Na3VO4,25mM -glycerophosphate, 50 mM linking induced a substantial increment in PCD only in CD30 Tg NaF, 10 mM sodium pyrophosphate, 15% v/v glycerol, and 1% v/v Triton thymocytes when thymocytes were incubated with specific anti- X-100), and centrifuged. Supernatants were recovered and incubated with CD30 Ab or CD30L-transfected P815 (CD30L-P815) cells. Fi- 0.8 ␮g of rabbit anti-c-Jun N-terminal kinase Ab (Santa Cruz Biotechnol- ogy, Santa Cruz, CA) for 90 min at 4°C and subsequently with protein nally, simultaneous cross-linking of CD3 and CD30 had synergis- G-Sepharose. Beads were collected, washed twice with lysis buffer and tic effects in CD30 Tg thymocytes (Fig. 2A), even with low twice with reaction buffer (25 mM HEPES (pH 7.4), 25 mM ␤-glycero- concentrations of anti-CD3 Ab (1 ␮g/ml and 10 ␮g/ml of anti- phosphate, 25 mM MgCl2, 2 mM DTT, and 0.1 mM Na3VO4), and incu- CD30 Ab) or low concentrations of anti-CD30 Ab (0.1 or 1 ␮g/ml ␮ ␮ bated for 30 min at 30°C in 30 l of reaction buffer with 3 g of purified and 10 ␮g/ml of anti-CD3 Ab) (data not shown). GST-c-Jun (1–79) (a kind gift of Dr. R. J. Schneider, New York School of Medicine), 4 ␮Ci of [␥-32P]ATP, and ATP (20 ␮M). Reactions were Immunophenotypic characterization of apoptotic thymocytes stopped with 2ϫ loading buffer, boiled for 5 min, run on SDS-PAGE, and was performed on CD30 Tg thymocytes cocultured with CD30L- exposed. P815-transfected cells. After 24 h of culture, a conspicuous frac- tion of the DP thymocytes showed a dimmer staining for both CD4 Results and CD8 (Fig. 2B). Because it is known that apoptotic DP thymo- Tg mice constitutively expressing CD30 Ag in T cells cytes express CD4low/CD8low Ags, we triple-stained CD30 Tg thy- To generate CD30 Tg mice constitutively expressing CD30 in T mocytes with annexin V, anti-CD4, and anti-CD8 Abs to investi- lymphocytes, a construct containing the murine CD30 coding re- gate the apoptotic population. Virtually all CD4ϩ/CD8ϩ gion was placed under the murine CD4 minimal promoter in the thymocytes were annexin Vϩ (93%). In contrast, a very small presence of the CD4 enhancer. This promoter is lacking the CD4 number of SP CD4ϩ or CD8ϩ cells were found to be annexin Vϩ silencer region and is transcriptionally active not only in CD4 cells (Fig. 2B). Overall, these findings demonstrate that CD4ϩ/CD8ϩ but also in CD8 single positive (SP) T cells (34). Six founder mice cells, but not CD4ϩ/CD8Ϫ or CD4Ϫ/CD8ϩ thymocytes, are sen- were obtained and used to establish six lines (Fig. 1A). All CD30 sitive to CD30-mediated PCD in vitro. Tg mice were fertile and healthy (Յ80 wk). Gross examination on autopsy of the CD30 Tg mice, at 2–12 wk of age, demonstrated CD30-mediated PCD leads to activation of caspases 1 and 3 ␬ normal organ development. The only difference was the larger size and does not require NF- B and c-Jun activation (ϳ2-fold weight increase) of the spleen and mesenteric lymph To investigate whether CD30 overexpression could modify the nodes (LNs) of CD30 Tg mice. All six lanes behaved similarly. overall sensitivity of thymocytes to PCD, we first used dexameth- Most of the experiments were done on two lines (956 and 986). asone, radiation, PMA, ionomycin, and Fas activation as apoptotic To characterize the expression of the CD30 transgene, CD30 stimuli. No significant differences were documented between con- mRNA transcripts and protein expression were studied by RT- trol and CD30 Tg thymocytes (data not shown). We subsequently PCR and immunoprecipitation-Western blot analyses, respec- compared the CD30-mediated PCD with those obtained with other tively. CD30 transcripts could be identified in all lymphoid organs proapoptotic agents (dexamethasone, CD3, and Fas cross-linking). of CD30 Tg mice and were present at a considerably higher level This approach was designed to identify any similarity with other than in normal littermates (Fig. 1B). Tg thymocytes expressed high well-characterized PCD pathways. With the exception of cyclo- levels of CD30 protein (Fig. 1C), running as a band of ϳ110 kDa. heximide, all antiapoptotic agents tested, including actinomycin D, The Journal of Immunology 197

FIGURE 1. Characterization of CD30 Tg mice. A, Southern blot analysis. Genomic DNAs of 12 mice obtained from six different founder (ϩ) and normal control littermates (Ϫ) were digested with BamHI, electrophoresed, transferred, and hybridized with a CD30 cDNA probe. Numbers of CD30 gene copies, calculated after scanning using ID Image Analysis Software (Kodak Digital Science, Eastman Kodak, Rochester, NY), were: lines 986 and 963 (30), line 968 (20), line 956 (24), line 965 (10), and line 967 (6). B, RT-PCR analysis for CD30 mRNA. Total RNA was extracted from the thymus, BM, spleen, LNs, and liver of CD30 Tg mice (ϩ) and control littermates (Ϫ). After reverse transcription, PCR was performed using CD30- and GAPDH- (control gene) specific oligoprimers. C, CD30 immunoprecipitation and Western blot analysis. Cell lysates of thymocytes from CD30 Tg and control littermates (WT) were immunoprecipitated with anti-CD30 Abs (X63 and CD30.1 Abs) or purified hamster Ig, blotted, and developed with anti-CD30 Abs (all lanes). D, Single-cell suspensions from the thymus, spleen, and LN of CD30 Tg mice and control littermates (WT) were stained with anti-Thy 1.2-FITC and anti-CD30-PE Abs. Numbers correspond to percentages of cells in each quadrant. Data are representative of six similar experiments.

TPCK, and iodoacetamide, abrogated the effect mediated by CD30 CD3. The anti-Fas- and dexamethasone-induced apoptosis differed cross-linking. Comparative analysis showed that the CD30-medi- in that they were enhanced or blocked by cycloheximide, respec- ated PCD is most closely related to the PCD mediated by anti- tively (Fig. 3). 198 CD30 TRANSGENIC MICE AND THYMIC NEGATIVE SELECTION

Table I. Lymphocyte subsets in CD30 Tg micea

CD30 Tg Control

Total cells % Total cells %

Thymus (n ϭ 9) (ϫ 106) 117 Ϯ 32 127 Ϯ 36 CD4ϩCD8ϩ 87.1 Ϯ 2.6 83.8 Ϯ 4.5 CD4ϩCD8Ϫ 8.1 Ϯ 2.4 9.9 Ϯ 3.7 CD4ϪCD8ϩ 1.7 Ϯ 0.6 2.8 Ϯ 0.7 CD4ϪCD8Ϫ 2.9 Ϯ 0.9 3.7 Ϯ 1.4 Spleen (n ϭ 9) (ϫ 106) 102 Ϯ 20 70 Ϯ 13 Thy-1.2ϩ 8.6 Ϯ 1.7 22 Ϯ 4.8 B220ϩ 64.1 Ϯ 9.1 63 Ϯ 4.1 CD4ϩCD8Ϫ 5.6 Ϯ 1.1 12 Ϯ 1.7 CD4ϪCD8ϩ 1.8 Ϯ 0.4 6 Ϯ 1.1 LNs (n ϭ 10) Thy-1.2ϩ 37.5 Ϯ 7.1 57.9 Ϯ 7.1 B220ϩ 52.5 Ϯ 14.9 35.3 Ϯ 3.1 CD4ϩCD8Ϫ 28.7 Ϯ 7.1 40.8 Ϯ 8.7 CD4ϪCD8ϩ 8.8 Ϯ 2.1 21.2 Ϯ 4.2

a Cells from thymi, spleens and mesenteric LNs were isolated, counted, stained with Thy-1.2-FITC, B220-FITC, or anti-CD4-FITC and anti-CD8- tricolor, and analyzed in a FACScan. Mice were 5–9 wk old. Data are expressed as means Ϯ SD. Statistical analysis was done by Student’s t test. Percentages of Thy-1.2ϩ cells in the spleen and LNs were significantly lower in CD30 Tg mice than in control littermates ( p Ͻ 0.0001).

When Z-YVAD-cmk and Z-DEVD-fmk peptides, which are in- tein was immunoprecipitated and incubated with recombinant c- hibitors of caspase 1- and caspase 3-like activities respectively, Jun in the presence of [␥-32P]ATP. In contrast to the anisomycin- were used, both abrogated PCD via dexamethasone, anti-CD3, anti- treated cells, the c-Jun in CD30 Tg thymocytes was not activated Fas (Fig. 3, A–C), and CD30-mediated PCD. These findings dem- after CD30 cross-linking. These findings suggest that c-Jun acti- onstrate that caspase 1 and caspase 3 are activated after CD30 vation and, most likely, TRAF2 activation do not occur in triggering and play a role in this apoptotic pathway (Fig. 3D). CD30-mediated PCD. Furthermore, it was observed that caspase 3- and caspase 9-like activation occur during the engagement of CD30 with anti-CD30 Ab alone or in combination with anti-CD3 Ab (Fig. 3E). CD30-induced PCD is inhibited by Bcl-2 Despite considerable progress in the understanding of PCD, lit- The fate of thymocytes is controlled by a complex equilibrium of tle is known about the mechanisms regulating gene and protein antagonistic signals. Bcl-2 and related proteins have been demon- expression of many of the molecules involved in this process. To better characterize whether CD30-mediated PCD requires the tran- strated to play a role in thymic differentiation and selection (37, scription modulation of key apoptotic genes, we investigated the 38). To investigate whether CD30-mediated PCD could be mod- mRNA expression of a series of genes involved in apoptosis and ulated by Bcl-2 protein, CD30 Tg mice were crossed to Bcl-2-25 signal transduction, using RNase protection and RT-PCR. Thymo- Wehi Tg mice, which overexpress Bcl-2 in early and mature T cytes of CD30 Tg and control mice were cross-linked with anti-Fas lymphocytes. The effect of multiple proapoptotic agents, including or CD30L-P815 cells. As shown in Fig. 4A, no significant changes radiation, dexamethasone, PMA, ionomycin, anti-Fas, and anti- in the expression of the tested genes could be documented in both CD30, was studied to compare the cell survival of thymocytes WT and CD30 Tg mice after either CD30 cross-linking or Fas obtained from double (Bcl-2/CD30 Tg) and single Bcl-2 or CD30 activation. Using RT-PCR, we also investigated the mRNA ex- Tg mice. Cells from Bcl-2/CD30 double Tg mice were totally re- pression of three additional genes, TRAF1, TRAF2, and FLIP.No sistant to the PCD induced by CD30 engagement, as well as to significant changes were identified for TRAF1 and TRAF2 during radiation and chemical agents. In contrast, Fas-mediated PCD was the culture (0–24 h). However, a slight increase in FLIP mRNA only partially abrogated by Bcl-2 overexpression (Fig. 5). These transcription was seen after Fas cross-linking (data not shown). findings suggest that Fas- and CD30-mediated PCD act on at least Several studies have demonstrated that CD30 cross-linking partially independent pathways. leads to NF-␬B activation in an appropriate cellular context (31). To study whether the engagement of CD30 results in NF-␬B ac- tivation in thymocytes, NF-␬B fractions were studied by electro- CD30 is involved in thymic negative selection phoretic mobility shift assay after CD30 cross-linking using anti- To examine whether CD30 is involved in negative selection, we CD30 Ab or CD30L-P815-transfected cells. Only a slight increase analyzed thymocyte deletion by SEB, which binds to MHC class in the nuclear translocation of NF-␬B proteins could be demon- II molecules and activates/deletes T cells bearing TCRs containing strated when thymocytes were cocultured with CD30L-P815 cells ␤ (Fig. 4B). V 3, 7, 8.1, 8.2, 8.3, or 17 (39). CD30 Tg and control mice were ␮ It has been demonstrated recently that functional or genomic injected i.p. with 5 g for 3 alternate days. At 1 day after the last inactivation of TRAF2 does not have any effect on NF-␬B activa- injection, the animals were sacrificed and studied for the deletion ␤ ␤ ϩ Ϫ tion but reduces c-Jun phosphorylation (32, 33), proving that of V 8- and V 3-bearing CD4 CD8 SP thymocytes. As con- ϩ TRAF2 activates c-Jun. To investigate whether CD30-mediated trols, V␤11 T cells were analyzed. The SEB-induced deletion of ϩ ϩ PCD is associated with TRAF2 activation and subsequently with a V␤8 and V␤3 thymocytes was significantly higher in CD30 Tg possible c-Jun activation, CD30 Tg thymocytes were cultured with animals compared with control animals (Fig. 6A). No significant ϩ CD30L-transfected and control P815 cells and subsequently lysed changes in the percentage of V␤11 T cells were seen in both at different intervals (Fig. 4C). Next, c-Jun N-terminal kinase pro- groups. The Journal of Immunology 199

FIGURE 2. CD30-induced in vitro apoptosis of thymocytes. A, Thymocytes (5 ϫ 105/well) from CD30 Tg and control littermates were incubated (24 h) with me- dium alone or in the presence of P815, CD30L-P815 (1 ϫ 105 cells/well), immobilized anti-CD3 (2C11), immobilized anti-CD30 (X63), immobilized anti-CD3ϩ purified hamster Ig, or immobi- lized anti-CD3ϩ anti-CD30 Abs. Cells were then stained with PI and analyzed by FACS. The per- centages of PCD (% PCD) are shown as means Ϯ SD of cells with hypodiploid DNA content, as described in Materials and Meth- ods. Data are from five mice for p Ͻ ,ءء ;p Ͻ 0.01 ,ء) each group 0.001). B, CD30 induces apopto- sis in DP thymocytes. Thymo- cytes from CD30 Tg mice were cultured for 24 h in the presence of CD30L-P815-transfected cells (ratio of 5:1), harvested, stained with anti-annexin V-FITC, anti- CD4-PE, and anti-CD8-tricolor Abs, and analyzed by flow cytom- etry. The gate was designed on CD4ϩ/CD8ϩ thymocytes (A), on CD4ϩ/CD8Ϫ thymocytes (B), and on CD4Ϫ/CD8ϩ thymocytes (C). The percentages of total cells are indicated in each quadrant.

To evaluate whether the negative selection induced by peptide increase (Fig. 6B). Overall, these findings demonstrate that CD30 engagement of the TCR is also enhanced by overexpression of overexpression enhances the deletion of specific thymocytes by CD30, DO11.10 Tg mice and CD30/DO11.10 double Tg mice bacterial superantigen (SAg) as well as by peptide Ags. were injected i.p. for 3 days with low amounts (20 ␮g) of the specific OVA and control peptide. The decrease in the total num- ber of thymocytes and in the percentage of CD4ϩ/CD8ϩ cells was Discussion much greater in CD30/DO11.10 double Tg mice compared with In this report, we have described the generation and characteriza- single DO11.10 Tg mice. Alternatively, the same amount of con- tion of a Tg mouse model overexpressing CD30 in thymocytes and trol peptide induced only a slight thymocyte deletion, but similar peripheral T cells. We chose this approach because, in normal deletions were obtained in both animal groups when a higher thymocytes, CD30 expression is confined to a distinct but very amount of specific peptide (120 ␮g) was used. It is also clear from small subpopulation of CD4ϩ/CD8ϩ thymocytes, which limits the the staining profiles for the TCR Tg, with the clonotypic mAb possibility of evaluating the effects of CD30. Using our Tg model, KJ1.26, that the deleted thymocytes from the double Tg mice were we could demonstrate that the engagement of CD30 alone induces primarily those with low to intermediate expression of the Tg apoptosis in thymocytes. More strikingly, CD30 engagement TCR, whereas the moderate-high KJ1.26ϩ cells showed a relative shows a synergistic effect with the concomitant stimulation of the 200 CD30 TRANSGENIC MICE AND THYMIC NEGATIVE SELECTION

FIGURE 3. CD30-induced PCD activates caspases 1, 3, and 9. A–D, Thymocytes (5 ϫ 105/well) purified from CD30 Tg mice were incubated in the presence of four different apoptotic stimuli: dexamethasone (D) (10Ϫ6 M), immobilized anti-CD3 (2C11) or soluble anti-Fas (Jo-2, 1 ␮g/ml) Abs, or CD30L-P815 cells. Cycloheximide (30 ␮g/ml), actinomycin D (2.5 ␮g/ml), TPCK (100 ␮M), iodoacetamide (1 ␮g/ml), Z-YVAD-cmk (500 ␮M), and Z-DEVD-fmk (500 ␮M) were used as inhibitors. The cells were harvested after 24 h of culture, stained with PI, and analyzed by FACS. The percentages of PCD (% PCD) were calculated as means Ϯ SD of cells with hypodiploid DNA content. Data are from four mice for each group. E, Thymocytes from CD30 Tg mice were incubated for8hinthepresence of medium alone, precoated polyclonal hamster Ig, anti-CD3 and/or anti-CD30 (X63) (all at 10 ␮g/ml), or soluble anti-Fas Ab (1 ␮g/ml). Caspase activities were calculated according to the following formula: Relative activation ϭ 100 ϫ ([S Ϫ M]/M), where S equals the value obtained from the stimulated sample and M equals the value obtained from the medium alone.

TCR. Our data are in accordance with those obtained in CD8/ tant signal(s) via TCR (18). The costimulatory functions of CD30 CD30-transfected T cell hybridomas, in which apoptosis requires also have been demonstrated in mature T cells (40) and in T cell the multimerization of CD30 cytoplasmic domains and concomi- lines (41). The fact that we saw an enhanced PCD in our CD30 Tg The Journal of Immunology 201

FIGURE 4. CD30-induced apo- ptosis does not modify the mRNA levels of several apoptosis-related molecules and does not activate NF-␬B or c-Jun. A, Thymocytes (5 ϫ 106) from CD30 Tg mice were cultured in presence of P815 or CD30L-P815 (1 ϫ 106) cells or in the presence of anti-Fas Ab (Jo-2, 1 ␮g/ml). Cells were harvested at dif- ferent intervals, and an RNase pro- tection assay was performed, as de- scribed in Materials and Methods. The intensity of the bands was cal- culated with ID Image Analysis Software and normalized to the con- trol gene GAPDH. A representative image from one of four experiments is shown. B, CD30 Tg thymocytes (5 ϫ 106) were incubated for the in- dicated intervals with PMA (100 ng/ ml), P815, or CD30L-P815 cells (ra- tio 5:1). Nuclear extracts (10 ␮g) were incubated with 32P-radiola- beled oligoprobe containing the NF-␬B binding site, and gel mobility shift assays were performed as de- scribed in Materials and Methods. One of six representative experi- ments is shown. The OD of individ- ual bands was obtained using ID Im- age Analysis Software. The mean ratio of the corresponding bands was calculated: CD30L-P815/P815 ϭ 2/1. C, Thymocytes isolated from CD30 Tg mice and control litter- mates were incubated with anisomy- cin (1 ␮g/ml, 30 min), P815, or CD30L-P815 cells (ratio 5:1) for the indicated intervals. Cell lysates were immunoprecipitated with anti-JNK Ab; the kinase assay for c-Jun phos- phorylation was performed as de- scribed in Materials and Methods, with purified GST-c-Jun fusion pro- tein as a substrate. 202 CD30 TRANSGENIC MICE AND THYMIC NEGATIVE SELECTION

by endogenous retroviral SAg (Mls-2a) were not affected. Such differences may depend upon different TCR affinities or avidities for the Ag-MHC complex, or on the developmental stage of T cells undergoing negative selection (3). Alternatively, it is possible that costimulatory molecules, which modulate the interactions between thymocytes and APCs (thymic stroma and/or B cells), can also regulate this phenomenon. In fact, retroviral SAg (Mls-2a)-medi- ated deletion, which is not impaired in CD30Ϫ/Ϫ mice, is defective in CD40Ϫ/Ϫ animals and in animals treated with anti-CD40L (8). CD30 Tg mice have a normal number of thymocytes, which have normal survival in vitro; therefore, overexpression of CD30 per se does not lead to any detectable increase in PCD within the thymus. On the contrary, the engagement of CD30 in vitro does result in an increase in thymocyte PCD. These findings suggest that CD30 overexpression is not constitutively active, and its en- gagement depends upon the availability of CD30L, which could be the limiting factor in vivo. In fact, CD30L is not only expressed in B cells (42, 43) but is also transcribed in activated BM-derived macrophages and thymic stroma-derived cell lines (44). More im- portantly, CD30L protein has been demonstrated recently in thy- mic medullary epithelial cells and in Hassal’s corpuscles (15). Thus, the availability of CD30L on thymic medullary stromal cells may control the destiny of CD30ϩ thymocytes. Furthermore, in normal thymocytes, the modulation of CD30 expression could also be important. In fact, even if there are high levels of mRNA tran- FIGURE 5. Inhibition of CD30-induced PCD by Bcl-2 overexpression. scripts in the thymus, CD30 is detectable on the surface of a very 5 ϩ ϩ Thymocytes (5 ϫ 10 /well) from Bcl-2 Tg (Bcl-2 ), CD30 Tg (CD30 ), small portion of thymocytes (15). Therefore, it is possible that the and Bcl-2ϩ/CD30ϩ Tg mice were incubated for 24 h with medium alone Ϫ6 transient and “ad hoc” regulation of the expression of CD30 on or in the presence of dexamethasone (10 M), PMA (100 ng/ml), iono- stimulated thymocytes could be a key step leading to CD30 trig- mycin (1 ␮g/ml), P815 or CD30L-P815 cells (ratio of 5:1), or anti-Fas Ab gering by CD30L in a well-defined thymic microenvironment. (Jo-2, 1 ␮g/ml). ␥-irradiated thymocytes (225 rad) were also studied. Cells were harvested, stained with PI, and analyzed by FACS. Percentages of This limited expression may also account for the lack of effect that apoptosis (% PCD) are expressed as means Ϯ SD of percentages of cells anti-CD30 Abs have in vitro on normal thymocytes (3, 16). Al- with hypodiploid DNA content. Four mice were included in each group. ternatively, the lack of numerical abnormalities in our Tg mice may be due to the possibility that the overexpression of CD30 may enhance negative selection as well as positive selection. In this mice after cross-linking of overexpressed CD30 alone could be case, considering that CD30 Tg mice have a normal number of due to a direct effect of CD30 in thymocytes per se, or alternatively thymocytes, CD30 Tg thymocytes might initially be positively se- to a potentiation by the overexpressed CD30 of subliminal trig- lected and then efficiently deleted. In this way, an increased neg- gering of the TCR/CD3 complex. Overall, these findings demon- ative selection would be able to compensate for the increased num- strate that CD30 may act as a costimulatory signal to TCR-MHC ber of positively selected thymocytes. This model is in accordance interactions during thymic selection. with the notion that CD30 can act as a costimulatory molecule, To support this hypothesis, we used two in vivo models to show enhancing TCR-related effects in T cell activation and/or the role of CD30 in thymic PCD. The deletion of specific ␣␤- differentiation (40). TCR-bearing T cells by SAgs has been widely used to study neg- The molecular mechanisms initiating and controlling negative ative selection. Using this approach, we were able to demonstrate selection have not yet been clarified. Autoreactive T cell clones are that the injection of SEB is associated with a greater degree of deleted as a consequence of TCR/self Ag/MHC complex interac- specific deletion of V␤8ϩ and V␤3ϩ CD4ϩ/CD8ϩ thymocytes in tions (1, 45), but additional signals are necessary; the overall result CD30 Tg than in control littermates. In addition, experiments with is due to the net intracellular changes induced by multiple external the CD30 and DO11.10 ␣␤-TCR double Tg mice clearly showed signals (46). Together with CD30, other molecules seem to be that a dose of OVA peptide that produces only partial deletion of involved in thymic development. However, the precise contribu- the single TCR Tg CD4ϩ/CD8ϩ thymocytes causes a greater de- tion of other TNFR family members is still unclear. Fas has been letion in TCR/CD30 double Tg mice. These results demonstrate studied extensively to identify its role in thymic development. Al- that both SAg and peptide Ags become more effective at deleting though the relevance of Fas to the shaping of the T cell repertoire specific thymic cell populations when CD30 expression is en- of peripheral T lymphocytes is unquestioned (4–6, 47), some hanced, thereby supporting the role of CD30 in negative selection. doubts remain concerning its role in thymic selection (7). Using Nevertheless, its physiological role in normal animals still remains TCR Tg and deficient lpr mice, several authors have concluded to be proven. that Fas actually does not function in thymic selection (2, 4, 48), A similar conclusion has been proposed recently using CD30- but that it may participate in the PCD of those thymocytes unable deficient mice (10). It is of interest that in CD30Ϫ/Ϫ mice, T cell to generate productive ␣␤-TCR (47). TNFR1, which is involved in abnormalities have been documented in both the thymus and pe- a variety of immunological phenomena, appears to be involved riphery for ␥␦ T cells bearing the TCR Tg specific for MHC class primarily in the regulation of the peripheral T cell repertoire rather I Tla molecules, and in the thymus, but not in the periphery, for than thymic development (2, 49). CD40, which is expressed on CD8ϩ T cells bearing the H-Y Ag-specific TCR. In contrast, thymic epithelial cells, also may have a role in thymic develop- CD4ϩ thymocytes or peripheral T cells that normally are deleted ment. In fact, the administration of anti-CD40L to mice interferes The Journal of Immunology 203

FIGURE 6. Thymic deletion is enhanced in CD30 Tg mice. A, CD30 Tg mice and control lit- termates (6–8 wk old) were injected i.p. with SEB (5 ␮g) three times on alternate days and killed on day six. Thymocytes were stained with anti-CD4- PE, CD8-tricolor, and anti-V␤8.1–8.2-FITC, with anti-V␤8.3-FITC, or with anti-V␤11-FITC Abs and analyzed by FACS. The percentages of deletion were evaluated by gating on the CD4ϩCD8Ϫ thy- mocytes and determining the proportion of V␤8.1– 8.2–8.3ϩ and V␤3ϩ cells. The percentage of dele- tion was calculated by gating on CD4ϩ SP thymocytes and calculating the proportion of V␤8ϩ, 3ϩ, and 11ϩ according to the following formula: percentage of deletion ϭ 100 ϫ ([p-pi]/p), where p equals the percentage of CD4ϩ SP V␤8ϩ or CD4ϩ SP V␤3ϩ cells in uninjected mice and pi equals the percentage of CD4ϩ SP V␤8ϩ or CD4ϩ SP V␤3ϩ thymocytes in mice after the injection). At least four mice were included in each group. B, DO.11.10 Tg and CD30/DO.11.10 double Tg mice were injected ␮ i.p. with 20 g of specific peptide (OVA323–339)or control peptide (OVA324–334) for 3 consecutive days. On day 4, thymocytes were isolated and stained with CD4-PE and CD8-tricolor or with KJ1.26 Abs using biotin-conjugated anti-mouse and PE-streptavidin as secondary fluorochrome. The number above each panel refers to the total number of thymocytes (mean Ϯ SD of three mice). The number in each quadrant represents the correspond- ing percentages of cells. In the histograms, the dashed lines indicate the boundaries of the TCRlow population. Representative results obtained from one of three mice are shown.

with negative selection (8). However, CD40 has been demon- the necessary in vivo costimulation leading to thymic cell death, strated to provide costimulatory effects only for the proliferation of possibly by up-regulating CD30, as described in peripheral T cells CD4ϩ cells, and not for thymic apoptosis (50). Furthermore, co- (9, 40, 51). Finally, other costimulatory molecules or alternative activation of CD28 via CD80 and/or CD86, which alone is not pathways may be operational (16), because CD28 does not appear sufficient to induce PCD in purified DP thymocytes, can provide to be absolutely required for positive and negative selection (52). 204 CD30 TRANSGENIC MICE AND THYMIC NEGATIVE SELECTION

The data obtained with CTLA-4Ϫ/Ϫ mice (53, 54) and more re- molecular interactions of CD30 with TCR or other receptors dur- cently using blocking anti-CTLA Ab suggest that the engagement ing the induction of thymic PCD. of CTLA-4 produces an additive effect to the TCR-MHC interac- tions contributing to the regulation of TCR-mediated selection of Acknowledgments T cell repertoires (55). We sincerely thank Elena Mazzone and Zhanqing Yan for their excellent In this study, we have also attempted to identify whether CD30- technical assistance. We also thank Drs. R. G. Goodwin, J. Lafaille, and J. mediated PCD shares any similarities with other known apoptotic Tiesinga for helpful discussions and critical review of the manuscript. pathways. Our findings indicate that the PCD mediated by either anti-CD3 or CD30 has common features and appears different References from dexamethasone- and Fas-mediated pathways. In fact, both CD3 and CD30 pathways are not influenced by cycloheximide, in 1. von Boehmer, H., and P. Kisielow. 1993. Lymphocyte lineage commitment: in- struction versus selection. Cell 73:207. contrast to those of Fas and dexamethasone, in which cyclohexi- 2. Sytwu, H. K., R. S. Liblau, and H. O. McDevitt. 1996. The roles of Fas/APO-1 mide has enhancing (56) and inhibitory effects, respectively. In- (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity 5:17. terestingly, the Z-YVAD-cmk and Z-DEVD-fmk peptides were 3. Page, D. M., E. M. Roberts, J. J. Peschon, and S. M. Hedrick. 1998. TNF re- able to inhibit CD30-mediated PCD as well as all other PCDs, and ceptor-deficient mice reveal striking differences between several models of thy- the involvement of caspases 3 and 9 after CD30 engagement was mocyte negative selection. J. Immunol. 160:120. 4. Singer, G. G., and A. K. Abbas. 1994. The Fas antigen is involved in peripheral demonstrated by cleavage of the specific substrates DEVD-AFC but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. and LEHD-AFC, respectively. Immunity 1:365. Compelling data demonstrated the significance of Bcl-2 and 5. Van Parijs, L., A. Ibraghimov, and A. K. Abbas. 1996. The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance. Immunity 4:321. Bcl-xL in different types of thymic PCD (6, 37, 38). In fact, Bcl-2 6. Van Parijs, L., D. A. Peterson, and A. K. Abbas. 1998. The Fas/Fas ligand overexpression completely blocks the PCD caused by radiation pathway and Bcl-2 regulate T cell responses to model self and foreign antigens. and dexamethasone, whereas Fas-mediated PCD is only partially Immunity 8:265. 7. Castro, J. E., J. A. Listman, B. A. Jacobson, Y. Wang, P. A. Lopez, S. Ju, prevented by Bcl-2. Two alternative Fas signaling pathways have P. W. Finn, and D. L. Perkins. 1996. 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