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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 Inghirami^2,*

^* Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, NY 10016; Department of Anatomic Pathology, University of Torino, Torino, Italy; Divisions of Pediatric Hematology/Oncology and ^§ Hematology/Oncology, Department of Medicine, New York University Medical Center, New York, NY 10016; and ^¶ Department of Microbiology, Miami University, Miami, FL 33101

	Abstract

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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.

	Introduction

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During development, thymocytes undergo a series of selections that shape the T cell repertoire of Ag specificities. The result is the generation of an immune system that is capable of recognizing a large number of Ags and of discriminating between self and non-self Ags (1). The mechanisms regulating thymic negative selection and programmed cell death (PCD),³ however, have been only partially elucidated. In the last few years, compelling data have indicated that several members of the TNF receptor (TNFR) superfamily may be involved in thymic selection, but their exact contribution is still controversial. In fact, the role of TNFR1 in thymic development is largely unclear (2, 3), and contradictory data have emerged from the analysis of Fas (4, 5, 6), because no definitive evidence has yet been provided concerning the expression of Fas ligand in the thymus (7). Also, the precise roles of CD40 ligand (CD40L) (8), CD28 (9), CTLA-4 and CD30 (10) in T cell ontogeny are still not well determined.

CD30, a member of the TNFR family that is expressed after activation by both B and T lymphocytes (11), was first identified in Reed-Sternberg cells of Hodgkin’s disease (12). Relatively few CD30⁺ cells are present in the thymic medulla and in peripheral lymphoid organs; rather, they are primarily localized within the interfollicular areas and less frequently at the rim of germinal centers (13, 14). In the thymus, CD30 mRNA is highly expressed (11), but only low levels of CD30 can be detected in the cytoplasm of CD4⁺/CD8⁺ thymocytes (T. Nguyen, unpublished observation). More recently, Romagnani et al. have demonstrated a small but clearly detectable fraction of CD4⁺/CD8⁺ thymocytes coexpressing CD30, CD45RO, and IL-4R (15). The triggering of CD30 requires a specific ligand (CD30L) that is highly expressed on medullary thymic epithelial cells and on Hassal’s corpuscles (15). The role of CD30 in thymic development has been suggested recently by studies in CD30^-/- mice, which have an impaired negative selection (10). These findings, however, have not been confirmed in vitro using wild-type (WT) thymocytes (3, 16). In contrast, emerging data in mature T cells and cell lines indicate that CD30 engagement in vitro has pleiotropic effects, resulting in enhanced cell proliferation, cell growth arrest, or PCD (17, 18).

The molecular pathways regulating the pleiotropic effects of CD30 and other receptors of the TNFR family involved in immune system regulation, cell proliferation/differentiation, and PCD (19, 20) have been partially identified during the last few years. Indeed, many TNFR family members can mediate apoptosis through their death domain (21). These death domains interact with adaptor molecules like Fas-associated protein with death domain (22), TNFR1-associated death domain, and receptor-interacting protein (23). In turn, Fas-associated protein with death domain interacts with downstream cell death effector molecules, such as caspase 8 (FADD-like ICE (FLICE)) (24, 25, 26), leading to PCD (27) by direct cleavage of caspase 3 or by cytochrome c release and subsequent activation of caspase 9. The mitochondrial release of cytochrome c is blocked by Bcl-2 (28). CD30 lacks a definitive death domain. However, its cytoplasmic domains interact with other adaptor molecules, including TNFR-associated factor 1 (TRAF1), TRAF2, TRAF3 (18, 29), and TRAF5 (30). TRAF2, directly or in association with TRAF1, can lead to NF-B (31) or c-Jun activation (32, 33).

In the present study, we investigate the biological role of CD30 in T cell development through its overexpression in thymocytes. We demonstrate that, after cross-linking via anti-CD30 Abs or via CD30L, thymocytes of CD30 transgenic (Tg) mice are induced to undergo PCD. This process triggers caspases 1 and 3 and is totally prevented by Bcl-2 overexpression. More importantly, we show that, with subliminary doses of staphylococcal enterotoxin B (SEB) superantigen or peptide Ag, CD30 Tg mice have an enhanced deletion of thymocytes. These findings, even if obtained in conditions of forced overexpression, indicate that CD30 plays an important role in thymic negative selection.

	Materials and Methods

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Generation of CD30 Tg mice

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, a generous gift from Dr. D. R. Littman, Skirball Institute, New York University Medical Center, New York, NY) containing the minimal CD4 enhancer (339 bp), the minimal murine CD4 promoter (487 bp), the transcription initiation site, and 70 bp of the untranslated first exon and part of the first intron of the murine CD4 gene (34). The transgene was released with NotI, injected into the pronucleus of fertilized eggs from (C57BL/6 x DBA/2)F₁ hybrid donors, and subsequently transferred to pseudopregnant CD-1 mice. Tg progenies were characterized by Southern blot and/or PCR analyses. Six founders were identified and used to generate six lines (backcrossed to C57BL/6, BALB/c, and DBA/2 strains). The heterozygous offspring from 956 and 986 CD30 Tg mice were used for all of the experiments.

Animals and cell preparation

Mice were housed in the Berg and Skirball Institute Animal Facilities of the New York University School of Medicine. CD30 Tg mice were crossed to Bcl-2-25 Wehi (The Jackson Laboratory, Bar Harbor, ME) and to DO.11.10 ß-TCR Tg mice (35) on a BALB/c background (kindly provided by Dr. J. Lafaille, Skirball Institute, New York University School of Medicine. Double Tg mice were identified by PCR or with mAbs against CD30 (11) and Vß8.1–8.2 TCR (PharMingen, San Diego, CA) by FACS analysis. All experiments were performed using mice at 4–8 wk of age.

Single-cell suspensions were obtained in complete medium (RPMI 1640 medium supplemented with 10% bovine FCS, 50 µg/ml streptomycin, 50 infectious units/ml penicillin, 1 mM L-glutamine, and 5 x 10^-5 M 2-ME). Bone marrow (BM) samples were obtained by flushing the femur and tibia cavities with cold sterile PBS supplemented with heparin (5000 U/ml) and peripheral blood leukocytes from tail vein blood.

Southern blot analysis and PCR

For Southern blot analysis, 10-µg aliquots of genomic DNA were digested, electrophoresed, denatured, and transferred to nitrocellulose, as described previously (36). CD30 gene products were evaluated on BamHI-digested DNA using the ³²P-labeled CD30 cDNA probe.

Total RNA was purified with an RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. cDNA was obtained from total RNA (5 x 10⁶ cells) after reverse transcription using hexanucleotide oligonucleotide primers (Boehringer Mannheim, Indianapolis, IN) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Bethesda, MD), as described previously (36).

The efficiency and quality of each individual cDNA preparation was tested by PCR amplification using specific oligonucleotides recognizing mouse GAPDH. The characterization of genomic DNA and/or mRNA expression of CD30, TRAF1, TRAF2, and FLICE-like inhibitory protein (FLIP) were performed by PCR (CD30-forward primer (FP): 5'-ATGAGCGCCCTACTCACCGCAGC, CD30-backward primer (BP): 5'-GGATCCAAGCTTTCAGTAACACAGGAGAAAGGAGCCGG; TRAF1-FP: 5'-CAGGGTACCATGGCCTCCAGCTCAGCCCCTG, TRAF1-BP: 5'-CAGGGATCCCTAAGCACTAGTGTCCACAATG; TRAF2-FP: 5'-CAGGGATCCCTAGAGTCCTGTTAGGTCC, TRAF2-BP: 5'-CAGGGTACCATGGCTGCAGCCAGTGTGA; FLIP-FP: 5'-TACAAGGGATTACACAGGCA, FLIP-BP: 5'-GTTATGTCATGTGACTTGGG).

Cell culture and in vivo experiments

In vitro CD3 and/or CD30 cross-linking was performed by culturing isolated thymocytes (5 x 10⁵) for 24 h in 96-well microtiter plates precoated overnight at 4°C with specific anti-CD3 (145-2C11, kindly provided by Dr. J. A. Bluestone, Ben May Institute, Chicago, IL) and anti-CD30 (X63 and CD30.1) (11) Abs (20 µg/ml) or with purified polyclonal hamster Ig (20 µg/ml, PharMingen). Alternatively, thymocytes were cocultured with 4% paraformaldehyde-fixed P815 cells or with P815 cells transfected with murine CD30L cloned into the pBMG-His vector (CD30L-P815) (11). For induction of apoptosis, PMA (10 ng/ml), dexamethasone (10^-6M), ionomycin (1 µg/ml), cycloheximide (30 µg/ml), and actinomycin D (4 µg/ml) were all obtained from Sigma (St. Louis, MO); anti-Fas Ab (1 µg/ml, clone Jo-2) was obtained from PharMingen. Inhibitors of PCD were N-tosyl-L-phenylalanine (TPCK) (100 µM, Sigma, added to thymocytes 15 min before the start of the culture), Z-YVAD-cmk (500 µM, Bachem, King of Prussia, PA), and Z-DEVD-fmk (500 µM, Enzyme System Products, Dublin, CA).

For in vivo experiments, SEB was obtained from Sigma; the OVA peptides 323–339 (ISQAVHAAHAEINEAGR) and 324–334 (SQAVHAAHAEI) (35) were synthesized by standard fluorenylmethoxycarbonyl chemistry at Seaver Laboratory (Skirball Institute, New York University Medical Center).

Flow cytometric analysis

For flow cytometric analysis, 0.5–1 x 10⁶ cells were incubated with appropriately diluted biotin-, FITC-, PE-, or tricolor-conjugated Abs. Purified hamster Ig was used as a negative control. Samples were then fixed and analyzed by FACScan (Becton Dickinson, Mountain View, CA).

The Abs used in this study included the following: anti-CD30-PE (clone mCD30.1, PharMingen), anti-Thy1.2-FITC (clone 30-H12, Becton Dickinson), anti-CD4-FITC (clone CT-CD4, Caltag, Burlingame, CA), anti-CD8-TRI (clone CT-CD8a, Caltag), anti-CD44-PE (clone IM7, PharMingen), anti-CD25-biotin (clone 3C7; PharMingen), anti-CD24 (clone M1/69; PharMingen), anti-CD3 (clone 2C11), anti-B220-FITC (clone RA3–6B2, PharMingen), anti-Fas (clone Jo-2, PharMingen), anti-Vß8.1/Vß8.2, Vß8.3, Vß3, Vß11-FITC (PharMingen), and KJ1.26 (a gift of Dr. P. Marrack, Howard Hughes Medical Institute, Denver, CO). PE-conjugated streptavidin (Vector Laboratories, Burlingame, CA) was used to reveal biotin-conjugated Ab staining. In some experiments, cells were washed in cold PBS and subsequently incubated with annexin V-FITC (Boehringer Mannheim, 2 µl/1 x 10⁶ cells) in staining buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, and 5 mM CaCl₂).

For DNA content determination, cells were fixed in 70% alcohol (1 h at 4°C), washed, and incubated with RNase (1 mg/ml, 10 min at room temperature) and propidium iodide (PI) (50 µg/ml, 15 min at room temperature, Calbiochem, San Diego, CA).

Caspase 3 and 9 cleavage assays

Thymocytes were isolated from Tg mice and incubated for 8 h in the presence of medium alone, precoated polyclonal hamster Ig, anti-CD3 and/or anti-CD30 (X63) (all at 10 µg/ml), or soluble anti-Fas Abs (1 µg/ml). Caspase activity was measured using CPP32/caspase-3 and MCH6/caspase-9 fluorometric protease assay kits (Chemicon International, Temecula, CA). The cleavage of DEVD-AFC and LEHD-AFC substrates was measured by a fluorescent plate reader.

Multiprobe RNase protection assay

The detection and quantification of multiple mRNA transcripts after CD30 or Fas cross-linking were investigated using a RiboQuant multiprobe RNase protection assay system (PharMingen) according to the manufacturer’s instructions. Briefly, total RNA (10 µg/reaction) was incubated with radiolabeled [-³²P]UTP probes overnight at 56°C in hybridization buffer. After RNase digestion (45 min at 30°C), samples were treated with proteinase K, extracted, precipitated, and resuspended with the loading buffer. Protected probes were resolved on acrylamide gel and exposed.

Immunoprecipitation and Western blot analysis

Thymocytes (1 x 10⁷) were washed and lysed. After spinning, supernatants were precleared twice with 30 µl of protein G-Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) for 30 min at 4°C and subsequently incubated for 2 h at 4°C with anti-CD30 Abs followed by mouse anti-hamster Ig (2 µg, PharMingen). A total of 30 µl of protein G was added for 2 h at 4°C; after washing (three times, 30 min, 4°C), samples were boiled, loaded on a 12% acrylamide gel, and blotted onto a nitrocellulose membrane. The membrane was first blocked with 1% BSA in PBS with 0.1% Tween 20 and subsequently incubated (for 1 h at room temperature) with anti-CD30 (10 µg/ml, 1 h at room temperature). After five washes, proteins were detected with biotin-conjugated mouse anti-hamster Ig (for 1 h at room temperature), followed by peroxidase-conjugated avidin-biotin complex. Membranes were developed with the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

Electrophoretic mobility shift assay

Cells were spun and washed with ice-cold PBS; low osmolarity buffer 10 mM Tris (pH 7.4), 10 mM NaCl, and 3 mM MgCl₂) was added to the cell pellet. After resuspension, the cells were pelleted and resuspended in cold RSB plus 10% glycerol, 0.25% Nonidet P-40, 1 mM DTT, and 1 mM PMSF. Cells were then lysed by pipetting and vortexing. Nuclei were recovered by centrifugation, and nuclear extraction buffer (20 mM HEPES (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 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 1 µg/ml aprotinin) was added. After incubation for 45 min at 4°C, the samples were centrifuged and supernatants were recovered. A total of 10 µg of the nuclear extracts was incubated (for 30 min at room temperature) in a solution containing 20 mM HEPES, 40 mM KCl, 1 mM EDTA, 1 mM MgCl₂, 0.5 mM DTT, 5% glycerol, 2 µg of poly(dC) (Promega, Madison, WI), 1 mM AMP, 1 µg of sonicated single-stranded herring sperm DNA (Life Technologies), and a ³²P-labeled double-stranded NF-B-specific oligonucleotide (5'-AGCTTGGGGACTTTCCCAGCCG). Cold competitor assays were conducted by adding a 100-fold molar excess of the unlabeled double-stranded oligonucleotide to the reaction mixture. Samples were separated on 6% polyacrylamide gels in 0.2x TRIS-borate-EDTA (TBE) buffer. Gels were dried and exposed.

Kinase assay

For kinase assay, 5 x 10⁶ thymocytes were treated with media alone, anisomycin (1 µg/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 EDTA, 2 mM EGTA, 1 mM Na₃VO₄, 25 mM ß-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 15% v/v glycerol, and 1% v/v Triton X-100), and centrifuged. Supernatants were recovered and incubated with 0.8 µg of rabbit anti-c-Jun N-terminal kinase Ab (Santa Cruz Biotechnology, Santa Cruz, CA) for 90 min at 4°C and subsequently with protein G-Sepharose. Beads were collected, washed twice with lysis buffer and twice with reaction buffer (25 mM HEPES (pH 7.4), 25 mM ß-glycerophosphate, 25 mM MgCl₂, 2 mM DTT, and 0.1 mM Na₃VO₄), and incubated for 30 min at 30°C in 30 µl of reaction buffer with 3 µg of purified GST-c-Jun 1–79(1–79) (a kind gift of Dr. R. J. Schneider, New York School of Medicine), 4 µCi of [-³²P]ATP, and ATP (20 µM). Reactions were stopped with 2x loading buffer, boiled for 5 min, run on SDS-PAGE, and exposed.

	Results

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Tg mice constitutively expressing CD30 Ag in T cells

To generate CD30 Tg mice constitutively expressing CD30 in T lymphocytes, a construct containing the murine CD30 coding region was placed under the murine CD4 minimal promoter in the presence of the CD4 enhancer. This promoter is lacking the CD4 silencer region and is transcriptionally active not only in CD4 cells but also in CD8 single positive (SP) T cells (34). Six founder mice were obtained and used to establish six lines (Fig. 1A). All CD30 Tg mice were fertile and healthy (80 wk). Gross examination on autopsy of the CD30 Tg mice, at 2–12 wk of age, demonstrated normal organ development. The only difference was the larger size (2-fold weight increase) of the spleen and mesenteric lymph nodes (LNs) of CD30 Tg mice. All six lanes behaved similarly. Most of the experiments were done on two lines (956 and 986).

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Characterization of CD30 Tg mice. A, Southern blot analysis. Genomic DNAs of 12 mice obtained from six different founder (+) and normal control littermates (-) 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 (+) and control littermates (-). 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.

Flow cytometry on cells obtained from primary and secondary lymphoid tissues was used to study the expression of CD30 in T lymphocytes. In control mice, CD30⁺ cells represented a small but detectable fraction of the total lymphoid cells compared with the strong expression observed in all T lymphocytes derived from different CD30 Tg lines. More importantly, the CD30 overexpression was not limited to early T cell precursors, but was also found in mature CD4 and CD8 SP T cells (Fig. 1D). These findings are in agreement with those previously reported using different genes driven by the minimal murine CD4 promoter-enhancer construct (34). Normal proportions of thymic CD4⁺/CD8⁺ double positive (DP) and CD4⁺/CD8^- or CD4^-/CD8⁺ SP lymphocytes (Table I) and of CD24⁺, CD44⁺, CD25⁺, ß-TCR⁺, CD3⁺, NK1.1⁺, and CD11b⁺ cells were identified. However, CD30 Tg animals had a decreased ratio of mature T vs B cells in the spleen and LNs, as well as increased CD4/CD8 ratios (Table I; R.C., manuscript in preparation). Finally, no coexpression of CD30 was demonstrated on B220⁺ or CD11b⁺ cells.

The thymi of the CD30 Tg animals have a normal size and number of total cells (Table I). In addition, the percentages of spontaneous cell death of thymocytes of CD30 Tg and WT mice cultured in vitro over time (24, 48, and 72 h) were comparable (data not shown). To evaluate a possible role of CD30 in thymic PCD, we investigated the effect of CD30 cross-linking on control and CD30 Tg thymocytes in vitro, with and without CD3 cross-linking. CD3 cross-linking alone induced a similar increase in PCD over spontaneous cell death in both groups. On the contrary, CD30 cross-linking induced a substantial increment in PCD only in CD30 Tg thymocytes when thymocytes were incubated with specific anti-CD30 Ab or CD30L-transfected P815 (CD30L-P815) cells. Finally, simultaneous cross-linking of CD3 and CD30 had synergistic effects in CD30 Tg thymocytes (Fig. 2A), even with low concentrations of anti-CD3 Ab (1 µg/ml and 10 µg/ml of anti-CD30 Ab) or low concentrations of anti-CD30 Ab (0.1 or 1 µg/ml and 10 µg/ml of anti-CD3 Ab) (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. CD30-induced in vitro apoptosis of thymocytes. A, Thymocytes (5 x 105/well) from CD30 Tg and control littermates were incubated (24 h) with medium alone or in the presence of P815, CD30L-P815 (1 x 105 cells/well), immobilized anti-CD3 (2C11), immobilized anti-CD30 (X63), immobilized anti-CD3+ purified hamster Ig, or immobilized anti-CD3+ anti-CD30 Abs. Cells were then stained with PI and analyzed by FACS. The percentages of PCD (% PCD) are shown as means ± SD of cells with hypodiploid DNA content, as described in Materials and Methods. Data are from five mice for each group (*, p < 0.01; **, p < 0.001). B, CD30 induces apoptosis in DP thymocytes. Thymocytes 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 cytometry. The gate was designed on CD4+/CD8+ thymocytes (A), on CD4+/CD8- thymocytes (B), and on CD4-/CD8+ thymocytes (C). The percentages of total cells are indicated in each quadrant.

CD30-mediated PCD leads to activation of caspases 1 and 3 and does not require NF-B and c-Jun activation

To investigate whether CD30 overexpression could modify the overall sensitivity of thymocytes to PCD, we first used dexamethasone, radiation, PMA, ionomycin, and Fas activation as apoptotic stimuli. No significant differences were documented between control and CD30 Tg thymocytes (data not shown). We subsequently compared the CD30-mediated PCD with those obtained with other proapoptotic agents (dexamethasone, CD3, and Fas cross-linking). This approach was designed to identify any similarity with other well-characterized PCD pathways. With the exception of cycloheximide, all antiapoptotic agents tested, including actinomycin D, TPCK, and iodoacetamide, abrogated the effect mediated by CD30 cross-linking. Comparative analysis showed that the CD30-mediated PCD is most closely related to the PCD mediated by anti-CD3. The anti-Fas- and dexamethasone-induced apoptosis differed in that they were enhanced or blocked by cycloheximide, respectively (Fig. 3).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. CD30-induced PCD activates caspases 1, 3, and 9. A–D, Thymocytes (5 x 105/well) purified from CD30 Tg mice were incubated in the presence of four different apoptotic stimuli: dexamethasone (D) (10-6 M), immobilized anti-CD3 (2C11) or soluble anti-Fas (Jo-2, 1 µg/ml) Abs, or CD30L-P815 cells. Cycloheximide (30 µg/ml), actinomycin D (2.5 µg/ml), TPCK (100 µM), iodoacetamide (1 µg/ml), Z-YVAD-cmk (500 µM), and Z-DEVD-fmk (500 µM) 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 ± SD of cells with hypodiploid DNA content. Data are from four mice for each group. E, Thymocytes from CD30 Tg mice were incubated for 8 h in the presence of medium alone, precoated polyclonal hamster Ig, anti-CD3 and/or anti-CD30 (X63) (all at 10 µg/ml), or soluble anti-Fas Ab (1 µg/ml). Caspase activities were calculated according to the following formula: Relative activation = 100 x ([S - M]/M), where S equals the value obtained from the stimulated sample and M equals the value obtained from the medium alone.

Despite considerable progress in the understanding of PCD, little is known about the mechanisms regulating gene and protein expression of many of the molecules involved in this process. To better characterize whether CD30-mediated PCD requires the transcription modulation of key apoptotic genes, we investigated the mRNA expression of a series of genes involved in apoptosis and signal transduction, using RNase protection and RT-PCR. Thymocytes of CD30 Tg and control mice were cross-linked with anti-Fas or CD30L-P815 cells. As shown in Fig. 4A, no significant changes in the expression of the tested genes could be documented in both WT and CD30 Tg mice after either CD30 cross-linking or Fas activation. Using RT-PCR, we also investigated the mRNA expression of three additional genes, TRAF1, TRAF2, and FLIP. No significant changes were identified for TRAF1 and TRAF2 during the culture (0–24 h). However, a slight increase in FLIP mRNA transcription was seen after Fas cross-linking (data not shown).

View larger version (51K): [in this window] [in a new window]   FIGURE 4. CD30-induced apoptosis does not modify the mRNA levels of several apoptosis-related molecules and does not activate NF-B or c-Jun. A, Thymocytes (5 x 106) from CD30 Tg mice were cultured in presence of P815 or CD30L-P815 (1 x 106) cells or in the presence of anti-Fas Ab (Jo-2, 1 µg/ml). Cells were harvested at different intervals, and an RNase protection assay was performed, as described in Materials and Methods. The intensity of the bands was calculated with ID Image Analysis Software and normalized to the control gene GAPDH. A representative image from one of four experiments is shown. B, CD30 Tg thymocytes (5 x 106) were incubated for the indicated intervals with PMA (100 ng/ml), P815, or CD30L-P815 cells (ratio 5:1). Nuclear extracts (10 µg) were incubated with 32P-radiolabeled oligoprobe containing the NF-B binding site, and gel mobility shift assays were performed as described in Materials and Methods. One of six representative experiments is shown. The OD of individual bands was obtained using ID Image Analysis Software. The mean ratio of the corresponding bands was calculated: CD30L-P815/P815 = 2/1. C, Thymocytes isolated from CD30 Tg mice and control littermates were incubated with anisomycin (1 µg/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 phosphorylation was performed as described in Materials and Methods, with purified GST-c-Jun fusion protein as a substrate.

It has been demonstrated recently that functional or genomic inactivation of TRAF2 does not have any effect on NF-B activation but reduces c-Jun phosphorylation (32, 33), proving that TRAF2 activates c-Jun. To investigate whether CD30-mediated PCD is associated with TRAF2 activation and subsequently with a possible c-Jun activation, CD30 Tg thymocytes were cultured with CD30L-transfected and control P815 cells and subsequently lysed at different intervals (Fig. 4C). Next, c-Jun N-terminal kinase protein was immunoprecipitated and incubated with recombinant c-Jun in the presence of [-³²P]ATP. In contrast to the anisomycin-treated cells, the c-Jun in CD30 Tg thymocytes was not activated after CD30 cross-linking. These findings suggest that c-Jun activation and, most likely, TRAF2 activation do not occur in CD30-mediated PCD.

CD30-induced PCD is inhibited by Bcl-2

The fate of thymocytes is controlled by a complex equilibrium of antagonistic signals. Bcl-2 and related proteins have been demonstrated to play a role in thymic differentiation and selection (37, 38). To investigate whether CD30-mediated PCD could be modulated by Bcl-2 protein, CD30 Tg mice were crossed to Bcl-2-25 Wehi Tg mice, which overexpress Bcl-2 in early and mature T lymphocytes. The effect of multiple proapoptotic agents, including radiation, dexamethasone, PMA, ionomycin, anti-Fas, and anti-CD30, was studied to compare the cell survival of thymocytes obtained from double (Bcl-2/CD30 Tg) and single Bcl-2 or CD30 Tg mice. Cells from Bcl-2/CD30 double Tg mice were totally resistant to the PCD induced by CD30 engagement, as well as to radiation and chemical agents. In contrast, Fas-mediated PCD was only partially abrogated by Bcl-2 overexpression (Fig. 5). These findings suggest that Fas- and CD30-mediated PCD act on at least partially independent pathways.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Inhibition of CD30-induced PCD by Bcl-2 overexpression. Thymocytes (5 x 105/well) from Bcl-2 Tg (Bcl-2+), CD30 Tg (CD30+), and Bcl-2+/CD30+ Tg mice were incubated for 24 h with medium alone or in the presence of dexamethasone (10-6 M), PMA (100 ng/ml), ionomycin (1 µg/ml), P815 or CD30L-P815 cells (ratio of 5:1), or anti-Fas Ab (Jo-2, 1 µg/ml). -irradiated thymocytes (225 rad) were also studied. Cells were harvested, stained with PI, and analyzed by FACS. Percentages of apoptosis (% PCD) are expressed as means ± SD of percentages of cells with hypodiploid DNA content. Four mice were included in each group.

To examine whether CD30 is involved in negative selection, we analyzed thymocyte deletion by SEB, which binds to MHC class II molecules and activates/deletes T cells bearing TCRs containing Vß3, 7, 8.1, 8.2, 8.3, or 17 (39). CD30 Tg and control mice were injected i.p. with 5 µg for 3 alternate days. At 1 day after the last injection, the animals were sacrificed and studied for the deletion of Vß8- and Vß3-bearing CD4⁺CD8^- SP thymocytes. As controls, Vß11⁺ T cells were analyzed. The SEB-induced deletion of Vß8⁺ and Vß3⁺ thymocytes was significantly higher in CD30 Tg animals compared with control animals (Fig. 6A). No significant changes in the percentage of Vß11⁺ T cells were seen in both groups.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Thymic deletion is enhanced in CD30 Tg mice. A, CD30 Tg mice and control littermates (6–8 wk old) were injected i.p. with SEB (5 µg) three times on alternate days and killed on day six. Thymocytes were stained with anti-CD4-PE, CD8-tricolor, and anti-Vß8.1–8.2-FITC, with anti-Vß8.3-FITC, or with anti-Vß11-FITC Abs and analyzed by FACS. The percentages of deletion were evaluated by gating on the CD4+CD8- thymocytes and determining the proportion of Vß8.1–8.2–8.3+ and Vß3+ cells. The percentage of deletion was calculated by gating on CD4+ SP thymocytes and calculating the proportion of Vß8+, 3+, and 11+ according to the following formula: percentage of deletion = 100 x ([p-pi]/p), where p equals the percentage of CD4+ SP Vß8+ or CD4+ SP Vß3+ cells in uninjected mice and pi equals the percentage of CD4+ SP Vß8+ or CD4+ SP Vß3+ 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 µg 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 ± SD of three mice). The number in each quadrant represents the corresponding 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this report, we have described the generation and characterization of a Tg mouse model overexpressing CD30 in thymocytes and peripheral T cells. We chose this approach because, in normal thymocytes, CD30 expression is confined to a distinct but very small subpopulation of CD4⁺/CD8⁺ thymocytes, which limits the possibility of evaluating the effects of CD30. Using our Tg model, we could demonstrate that the engagement of CD30 alone induces apoptosis in thymocytes. More strikingly, CD30 engagement shows a synergistic effect with the concomitant stimulation of the TCR. Our data are in accordance with those obtained in CD8/CD30-transfected T cell hybridomas, in which apoptosis requires the multimerization of CD30 cytoplasmic domains and concomitant signal(s) via TCR (18). The costimulatory functions of CD30 also have been demonstrated in mature T cells (40) and in T cell lines (41). The fact that we saw an enhanced PCD in our CD30 Tg mice after cross-linking of overexpressed CD30 alone could be due to a direct effect of CD30 in thymocytes per se, or alternatively to a potentiation by the overexpressed CD30 of subliminal triggering of the TCR/CD3 complex. Overall, these findings demonstrate that CD30 may act as a costimulatory signal to TCR-MHC interactions during thymic selection.

To support this hypothesis, we used two in vivo models to show the role of CD30 in thymic PCD. The deletion of specific ß-TCR-bearing T cells by SAgs has been widely used to study negative selection. Using this approach, we were able to demonstrate that the injection of SEB is associated with a greater degree of specific deletion of Vß8⁺ and Vß3⁺ CD4⁺/CD8⁺ thymocytes in CD30 Tg than in control littermates. In addition, experiments with the CD30 and DO11.10 ß-TCR double Tg mice clearly showed that a dose of OVA peptide that produces only partial deletion of the single TCR Tg CD4⁺/CD8⁺ thymocytes causes a greater deletion in TCR/CD30 double Tg mice. These results demonstrate that both SAg and peptide Ags become more effective at deleting specific thymic cell populations when CD30 expression is enhanced, thereby supporting the role of CD30 in negative selection. Nevertheless, its physiological role in normal animals still remains to be proven.

A similar conclusion has been proposed recently using CD30-deficient mice (10). It is of interest that in CD30^-/- mice, T cell abnormalities have been documented in both the thymus and periphery for T cells bearing the TCR Tg specific for MHC class I Tla molecules, and in the thymus, but not in the periphery, for CD8⁺ T cells bearing the H-Y Ag-specific TCR. In contrast, CD4⁺ thymocytes or peripheral T cells that normally are deleted 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)-mediated deletion, which is not impaired in CD30^-/- mice, is defective in CD40^-/- 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 engagement 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 importantly, CD30L protein has been demonstrated recently in thymic medullary epithelial cells and in Hassal’s corpuscles (15). Thus, the availability of CD30L on thymic medullary stromal cells may control the destiny of CD30⁺ thymocytes. Furthermore, in normal thymocytes, the modulation of CD30 expression could also be important. In fact, even if there are high levels of mRNA transcripts in the thymus, CD30 is detectable on the surface of a very small portion of thymocytes (15). Therefore, it is possible that the transient and "ad hoc" regulation of the expression of CD30 on stimulated thymocytes could be a key step leading to CD30 triggering by CD30L in a well-defined thymic microenvironment. This limited expression may also account for the lack of effect that anti-CD30 Abs have in vitro on normal thymocytes (3, 16). Alternatively, 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 case, considering that CD30 Tg mice have a normal number of thymocytes, CD30 Tg thymocytes might initially be positively selected and then efficiently deleted. In this way, an increased negative selection would be able to compensate for the increased number of positively selected thymocytes. This model is in accordance with the notion that CD30 can act as a costimulatory molecule, enhancing TCR-related effects in T cell activation and/or differentiation (40).

The molecular mechanisms initiating and controlling negative selection have not yet been clarified. Autoreactive T cell clones are deleted as a consequence of TCR/self Ag/MHC complex interactions (1, 45), but additional signals are necessary; the overall result is due to the net intracellular changes induced by multiple external signals (46). Together with CD30, other molecules seem to be involved in thymic development. However, the precise contribution of other TNFR family members is still unclear. Fas has been studied extensively to identify its role in thymic development. Although the relevance of Fas to the shaping of the T cell repertoire of peripheral T lymphocytes is unquestioned (4, 5, 6, 47), some doubts remain concerning its role in thymic selection (7). Using TCR Tg and deficient lpr mice, several authors have concluded that Fas actually does not function in thymic selection (2, 4, 48), but that it may participate in the PCD of those thymocytes unable to generate productive ß-TCR (47). TNFR1, which is involved in a variety of immunological phenomena, appears to be involved primarily in the regulation of the peripheral T cell repertoire rather than thymic development (2, 49). CD40, which is expressed on thymic epithelial cells, also may have a role in thymic development. In fact, the administration of anti-CD40L to mice interferes with negative selection (8). However, CD40 has been demonstrated to provide costimulatory effects only for the proliferation of CD4⁺ cells, and not for thymic apoptosis (50). Furthermore, coactivation of CD28 via CD80 and/or CD86, which alone is not sufficient to induce PCD in purified DP thymocytes, can provide the necessary in vivo costimulation leading to thymic cell death, possibly by up-regulating CD30, as described in peripheral T cells (9, 40, 51). Finally, other costimulatory molecules or alternative pathways may be operational (16), because CD28 does not appear to be absolutely required for positive and negative selection (52). The data obtained with CTLA-4^-/- mice (53, 54) and more recently using blocking anti-CTLA Ab suggest that the engagement of CTLA-4 produces an additive effect to the TCR-MHC interactions contributing to the regulation of TCR-mediated selection of T cell repertoires (55).

In this study, we have also attempted to identify whether CD30-mediated PCD shares any similarities with other known apoptotic pathways. Our findings indicate that the PCD mediated by either anti-CD3 or CD30 has common features and appears different from dexamethasone- and Fas-mediated pathways. In fact, both CD3 and CD30 pathways are not influenced by cycloheximide, in contrast to those of Fas and dexamethasone, in which cycloheximide has enhancing (56) and inhibitory effects, respectively. Interestingly, the Z-YVAD-cmk and Z-DEVD-fmk peptides were able to inhibit CD30-mediated PCD as well as all other PCDs, and the involvement of caspases 3 and 9 after CD30 engagement was demonstrated by cleavage of the specific substrates DEVD-AFC and LEHD-AFC, respectively.

Compelling data demonstrated the significance of Bcl-2 and Bcl-x_L in different types of thymic PCD (6, 37, 38). In fact, Bcl-2 overexpression completely blocks the PCD caused by radiation and dexamethasone, whereas Fas-mediated PCD is only partially prevented by Bcl-2. Two alternative Fas signaling pathways have been identified recently (57). In the so called type I pathway, a large amount of caspase 8 is recruited to the death-inducing signal complex upon cross-inking of Fas. The activated caspase 8 directly cleaves other downstream caspases, such as caspase 3, and triggers mitochondrial damage through the cleavage of BID (58, 59); this, in turn, activates a proteolytic cascade involving caspase 9. In the type II pathway, only a small amount of caspase 8 is recruited to the death-inducing signal complex; the activation of the apoptotic cascade is slower and primarily involves mitochondrial damage. Thymic Fas-mediated PCD appears to trigger the type I pathway; thus, it is not surprising that Fas-mediated PCD is blocked only partially by the overexpression of Bcl-2, which is capable of preventing mainly the mitocondrial damage. In contrast, the engagement of downstream caspases during CD30-mediated PCD is totally abrogated by the overexpression of Bcl-2, indicating that this pathway may share similarity with Fas type II.

Several members of the TRAF family, including TRAF2 and TRAF5, have been shown to bind to the cytoplasmic domain of CD30 (29), activating NF-B (31). The deletion of this CD30 C-terminal domain (66 aa) results in the abrogation of PCD in hybridomas (18). The availability of TRAF2 can be modulated by the activation of CD30 and TNFR1, resulting in an enhanced degradation of TRAF2 and in a subsequent suppression of TRAF2-dependent antiapoptotic pathways (60, 61). Because two groups have recently demonstrated that NF-B activation, but not c-Jun phosphorylation, can be achieved normally after TNFR1 engagement in TRAF2^-/- and TRAF2 dominant negative Tg mice (32, 33), we argued that, similarly to TNFR1-TRAF2 interactions, CD30 may recruit TRAF2 during PCD in the thymus, resulting in NF-B or c-Jun activation. Indeed, we were able to demonstrate only very moderate NF-B nuclear transposition and no c-Jun phosphorylation when CD30 Tg thymocytes were engaged via CD30. Overall, these findings suggest that TRAF2 is not activated during CD30-mediated PCD. However, we cannot exclude the possibility that NF-B may play some role in CD30-mediated PCD, because it has been demonstrated previously that NF-B activation may be masked or undetectable in the presence of apoptosis (62). Further studies using crosses between CD30 Tg and NF-B^-/-, TRAF2^-/-, or TRAF2 dominant negative mice will help to characterize the role of NF-B and TRAF2 in CD30-mediated PCD. In addition, more studies are necessary to precisely characterize the molecular interactions of CD30 with TCR or other receptors during the induction of thymic PCD.

	Acknowledgments

 
We sincerely thank Elena Mazzone and Zhanqing Yan for their excellent technical assistance. We also thank Drs. R. G. Goodwin, J. Lafaille, and J. Tiesinga for helpful discussions and critical review of the manuscript.

	Footnotes

 
¹ These studies were supported in part by National Cancer Institute Research Grants CA-64033 (to G.I.) and AG-04980 (to G.J.T.). A.P. and G.P. are recipients of National Cancer Institute, National Institutes of Health Training Grant CA-2T32CA09454.

² Address correspondence and reprint requests to Dr. Giorgio Inghirami, Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address:

³ Abbreviations used in this paper: PCD, programmed cell death; TNFR, TNF receptor; CD40L, CD40 ligand; TRAF, TNFR-associated factor; Tg, transgenic; SEB, staphylococcal enterotoxin B; FP, forward primer; BP, backward primer; BM, bone marrow; LN, lymph node; DP, double positive; SP, single positive; PI, propidium iodide; WT, wild type; FLIP, Flice-like inhibitory protein; TPCK, N-tosyl-L-phenylalanine; SAg, superantigen.

Received for publication February 12, 1999. Accepted for publication April 20, 1999.

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