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The Journal of Immunology, 2002, 168: 1770-1779.
Copyright © 2002 by The American Association of Immunologists

Inhibitor of Apoptosis Protein from Orgyia pseudotsugata Nuclear Polyhedrosis Virus Provides a Costimulatory Signal Required for Optimal Proliferation of Developing Thymocytes1

María S. Robles2,*, Esther Leonardo*, Luis Miguel Criado*, Manuel Izquierdo{dagger} and Carlos Martínez-A.*

* Department of Immunology and Oncology, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Madrid Campus de Cantoblanco, Madrid, Spain; and {dagger} Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Cientificas-Universidad de Valladolid, Facultad de Medicina, Ramón y Cajal, Valladolid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-{kappa}B and reduced cytoplasmic levels of its inhibitor, I{kappa}B{alpha}. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Programmed cell death, or apoptosis, is a genetically regulated physiological mechanism with an essential role in the development and homeostasis of many tissues in multicellular organisms. The basic steps in the apoptotic pathway are conserved throughout evolution (1). Execution of the apoptotic program requires activation of caspases, a family of proteases whose activity is regulated in the cell by activators and inhibitors. Inhibitors of apoptosis proteins (IAPs)3 are members of a family of endogenous inhibitors of caspases identified in mammals (2) and represent an evolutionarily conserved family of antiapoptotic gene products. IAPs block cell death during development, as well as apoptosis triggered by diverse stimuli. Thus, they are key players in the regulation of apoptotic pathways (3, 4). IAP structure is characterized by a 70-amino-acid motif present in one to three tandem copies (baculoviral IAP repeat (BIR)). The BIR domain is required for IAP antiapoptotic activity, including caspase inhibition, and is also needed for interaction with proapoptotic factors in Drosophila (5, 6). Several IAPs also have a C-terminal ring zinc finger motif that differs from BIR in both structure and function. Depending on the cellular context and the specific IAP, the ring motif appears necessary for antiapoptotic function. Whereas this motif is reported to be essential for baculoviral IAP function, some human IAPs maintain their antiapoptotic function in its absence (6).

Although the mechanism by which IAPs inhibit apoptosis is not well established, several reports provide evidence of biochemical events controlled by these inhibitors (3, 4). Some mammalian and insect IAPs bind to and inhibit certain activated caspases, and IAP overexpression blocks apoptosis by inhibiting proteolytic activation of procaspases (7, 8, 9, 10, 11, 12). Insect IAPs also interact with and inhibit apoptotic inducers, leading indirectly to blockage of caspase activation (6, 13).

Certain IAPs are involved in signal transduction by modulating the activity of the NF-{kappa}B transcription factor, which is linked to the role of IAPs in cancer. Overexpression of human c-IAP1, c-IAP2, and XIAP induce NF-{kappa}B activity. Conversely, NF-{kappa}B can regulate expression of some IAP genes (14, 15), suggesting a regulated positive feedback loop between IAP expression and NF-{kappa}B activation. IAP regulation of NF-{kappa}B activity mediates both an antiapoptotic effect and IAP involvement in cell cycle regulation and tumor progression (4). Up-regulation and mutation of some human IAPs are thus associated to the development of tumors and other disorders, respectively. For example, both human and murine survivin are implicated in the regulation of cell division through interaction with certain essential mitotic cell elements such as microtubule spindles (16), and human survivin is up-regulated in several types of tumors (17).

IAP from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP) was one of the first two IAPs identified, based on its functional capacity to complement a mutant form of the broad-range caspase inhibitor p35 (18). Despite their similar antiapoptotic function, p35 and OpIAP differ in structure and mechanism of action. OpIAP contains two BIR domains and a C-terminal ring finger, which are absent in p35; it functions upstream of p35, preventing caspase activation (18). Although OpIAP is an inhibitor that acts in insect cells, studies have shown that ectopic OpIAP expression can also inhibit apoptosis in mammalian cells (19, 20). This suggests a common mechanism of action for the different IAPs.

Immune system development and homeostasis require a regulated balance of proliferation, differentiation, and cell death. TCR-controlled apoptosis (activation-induced cell death (AICD)) and death by neglect are fundamental in thymic development, and AICD controls homeostasis of the peripheral immune system (1). AICD as well as T lymphocyte activation and proliferation are controlled by TCR triggering, but full activation requires costimulation by other receptors (21). Costimulatory signals provided by ligand engagement to the lymphocyte surface coreceptor CD28 allow complete T cell activation (22). The TCR/CD28 signaling pathway ultimately leads to activation of specific inducible transcription factors (23), one of the most important of which is the Rel/NF-{kappa}B family, which is critical for T cell response regulation (24). NF-{kappa}B activity is controlled by the I{kappa}B family of inhibitors, which retain NF-{kappa}B as latent complexes in the cytoplasm by masking its nuclear localization signal. Although the mechanisms and mediators involved in NF-{kappa}B activation after TCR/CD28 triggering are not fully understood, the CD28 signal is reported to increase NF-{kappa}B activation, cooperating with TCR stimulation to activate the I{kappa}B kinase kinase (IKK) complex (25). Activated IKK phosphorylates I{kappa}B, mainly the I{kappa}B{alpha} form, which is then ubiquitinated and subsequently degraded by the proteosome, leading to NF-{kappa}B activation (26).

The IAPs may have an essential role in immune system development and homeostasis, because they are important endogenous antiapoptotic proteins in mammals and are also implicated in cell division and proliferation. Conservation of both structure and function among different IAP family members prompted us to study the consequences of baculoviral OpIAP expression in thymocyte development and homeostasis. Our results show that OpIAP induces NF-{kappa}B nuclear translocation in developing thymocytes and leads to their complete activation and proliferation in the absence of a CD28 costimulatory signal. In addition, OpIAP blocks TCR-triggered apoptosis in these cells. This study provides the first in vivo data for IAP regulation of NF-{kappa}B activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic (Tg) mice

OpIAP cDNA was subcloned into the BamHI site of the p56lck human growth hormone (hGH) vector. A 6.2-kb NotI fragment containing the lck proximal promoter, OpIAP cDNA, and an hGH sequence was prepared by gel purification and diluted in injection buffer (10 mM Tris (pH 7.5), 0.12 mM EDTA) to a final concentration of 2 mg/ml. Tg mice were generated by pronuclear microinjection of fertilized oocytes from (C57BL/6 x CBA)F1 mice. Microinjected eggs were transferred at the two-cell stage to recipient pseudopregnant OF1 females. Offspring were analyzed by PCR and transgenesis was confirmed by Southern blot analysis.

PCR detection and Southern blot

OpIAP Tg mice were initially typed by PCR using hGH-3 (5'-GCACACGCTGAGCTAGGTTCCC-3') and hGH-4 (5'-CATAGACGTTGCTGTCAGAGGC-3') primers that amplified a 415-bp fragment of the transgene construct. As internal control, we used primers that amplified a 386-bp fragment of thyroid-stimulating hormone {beta} (TSH{beta}), TSH{beta}-5' (5'-TCCTCAAAGATGCTCATTAG-3') and TSH{beta}-3' (5'-GTAACTCACTCATGCA-3'). Transgene integration numbers were evaluated by Southern blot. Genomic DNA from tails of F1 founder offspring was isolated with a kit (Invitrogen, San Diego, CA). After BamHI digestion, genomic DNA (10 µg) was separated in agarose gels and alkaline transferred to {zeta}-Probe membranes (Bio-Rad, Hercules, CA). The membranes were hybridized with a random-priming, 32P-labeled full-length OpIAP cDNA probe.

Western blot analysis

Cytosolic extracts of cell suspensions from several lymphoid organs and sorted thymocytes were prepared by incubation (30 min, 4°C) in extraction buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mM EDTA, 10 mM NaH2PO4, 1% Nonidet P-40, 0.4 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were centrifuged to deplete nuclei. Cytosolic extracts (30 µg/lane) were resolved in 7% (for poly(ADP-ribose) polymerase (PARP) immunoblots) or 10% (for OpIAP, I{kappa}B{alpha} Bcl-xL, and Bcl-2 immunoblots) SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Western blot was performed using rabbit anti-OpIAP, anti-I{kappa}B{alpha} (New England Biolabs, Beverly, MA; 1/500 dilution), anti-Bcl-xL (BD Transduction Labs, Lexington, KY), or anti-Bcl-2 Abs (BD PharMingen, San Diego, CA) and developed with HRP-conjugated anti-rabbit IgG Ab (DAKO, Glostrup, Denmark; 1/1000 dilution). Anti-mouse {beta}-actin Ab (Sigma-Aldrich, St. Louis, MO) was used to confirm protein loading. For PARP detection, blots were probed with an anti-PARP mAb (BD PharMingen; 1/750 dilution) followed by HRP-conjugated anti-mouse IgG (DAKO).

Flow cytometry and cell sorting

Single-cell suspensions were obtained from lymphoid organs of control and Tg animals and minced through a 150-µM nylon mesh, and viable cells were enumerated by trypan blue exclusion. For flow cytometric analysis, cells were stained with saturating Ab concentrations at 4°C. Directly conjugated mAbs were used against CD3-(FITC), CD4-(FITC), CD4-(PE), CD8-(tricolor), CD44-(PE), CD25-(FITC), CD69-(FITC), and CD95-(PE) (BD PharMingen; and Southern Biotechnologies, Birmingham, AL). Biotin-conjugated anti-TCRV{beta}11 Ab (BD PharMingen) was developed using streptavidin-PE (Southern Biotechnologies). Flow cytometry and multiparameter analysis were performed on an Epics XL cytometer (Coulter, Miami, FL). Dead cells were excluded by gating on forward and side light scatter. Cell suspensions from Tg thymus were stained with anti-CD4 (FITC) and CD8 (PE) mAbs, and cells were sorted by FACS (Coulter Epics Altra).

Induction of apoptosis in vivo and in vitro

In vivo apoptosis experiments were performed using 8- to 10-wk-old mice that received a single i.p. injection of PBS (control), antigenic peptide (influenza virus A/NT/60/68 nucleoprotein residues 366-374, ASNENMADM) (19 nmol), or anti-CD3{epsilon} (2C11) Ab (BD PharMingen; 20 µg) in PBS. After 24 h, lymphoid organ cell suspensions were prepared as above. To analyze PARP processing, thymocytes were isolated and cultured (1 h, 37°C). Early apoptosis was studied by injecting the same dose of Ag peptide (19 nmol), and thymocyte cell death was analyzed 90 min later. PARP processing and DNA degradation were analyzed directly in total thymus. For dexamethasone treatment in vitro, thymocytes from Tg mice were cultured for the indicated periods alone or with dexamethasone (200 nM) in the presence or absence of lactacystin (20 µM).

DNA fragmentation assays

DNA fragmentation was analyzed as described (27), followed by incubation with 10 µg of RNase A (30 min, 37°C) and subsequent electrophoresis in a 2% agarose gel.

Caspase assays

Cytosolic extracts (containing 1 mg/ml of cytosolic protein) were diluted 5-fold in assay buffer (25 mM HEPES (pH 7.5), 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (w/v), 10% sucrose, 10 mM DTT, and 0.1 mg/ml OVA) and incubated (37°C, 2 h) with the fluorescent substrate Ac-DEVD-AMC (10 µM) for caspase-3. The reactions were terminated by addition of HPLC buffer (H2O-acetonitrile (75:25), 0.1% trifluoroacetic acid). Cleaved substrate fluorescence was determined by C18 reverse-phase HPLC using fluorescence detection (338 nm excitation, 455 nm emission). Control experiments confirmed that substrate release was linear with time and protein concentration under these conditions.

Cell preparation and proliferation assays

Single-cell thymocyte suspensions were prepared as above in complete medium (RPMI 1640 with 10% FCS, 10 µM mercaptoethanol, 100 mM L-glutamine, 10 mM HEPES, and antibiotics) and then plated (4 x 105 cells/well) in 96-well, flat-bottom microtiter plates precoated overnight with PBS or anti-CD3{epsilon} at several concentrations. Triplicate samples were cultured (48 h and 96 h, 37°C) alone or with anti-CD28 (0.35 µg/ml; BD PharMingen). Tritiated thymidine (1 µCi/well) was added, cells were harvested after 8 h, and radioisotope incorporation into DNA was determined.

ELISA

In vitro IL-2, IFN-{gamma}, IL-4, IL-10, and TNF-{alpha} production were measured in ELISA in culture supernatants from thymocytes, unstimulated or stimulated with anti-CD3 alone or in combination with anti-CD28, on plates precoated with appropriate anti-mouse Abs (Endogen, Boston, MA). Quantification was done by comparison to standards supplied. In vivo cytokine production was measured from mouse serum 2 h after antigenic peptide injection. ELISA was performed as described above.

Nuclear protein extraction and EMSAs

To obtain nuclear extracts, thymocytes were incubated (10 min, 4°C) in buffer A (10 mM HEPES (pH 7.6), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40) before centrifugation to pellet the nuclei. Nuclei were lysed by incubation (30 min, 4°C, with agitation) in buffer B (20 mM HEPES (pH 7.6), 20% glycerin, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA). After centrifugation, supernatants containing nuclear proteins were separated and frozen at -70°C. EMSA was performed using a 32P-labeled, double-stranded oligonucleotide bearing the {kappa}B site of the human IL-2 promoter (5'-GATCGGGATTTCACCT-3'). DNA binding reactions (12.5 µl) containing 5 µg of nuclear extracts, 2 µg of poly(dI-dC), and 2.5 mM EDTA, 2.5 mM DTT, and 2.5% glycerol in 50 mM Tris (pH 8) were incubated for 10 min at 4°C). A total of 100,000 cpm of radiolabeled oligonucleotide probe were then added and incubated (30 min, 4°C). The resulting nucleoprotein complexes were resolved by electrophoresis in native 4% polyacrylamide gels (in 0.5x Tris-buffered EDTA) at 120 V for 2.5 h, then visualized and quantified with a Storm Phosphoimager (Molecular Dynamics, Sunnyvale, CA). For Ab competition experiments, we used anti-p65 Ab (1 µl; Oncogene Research Products, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p56lckpr OpIAP Tg mice

Tg mice were generated expressing IAP from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus. Transgene expression was specifically targeted to T cells using the p56lck proximal promoter, which is more active in immature thymocytes compared with peripheral T cells (28). The Tg construct was generated by inserting an 800-bp cDNA coding for OpIAP downstream of the 3.2-kb fragment of the p56lck promoter and upstream of a 2.1-kb genomic hGH sequence that confers stability to the mRNA (28). The linearized vector was microinjected into mouse oocytes, and 12 founder mice were characterized by PCR analysis of tail genomic DNA. By backcrossing founders with C57BL/6 mice, several independent Tg lines were generated, two of which were used in this study, one derived from founder H72 with low (two to three) and one from founder H51 with high (~10) transgene integrations, as determined by Southern blot (Fig. 1GoA). Transgene expression was analyzed by Western blot using a polyclonal anti-OpIAP antiserum. High OpIAP protein levels were detected in thymus, whereas levels in peripheral lymphoid organs were lower in both Tg lines (Fig. 1GoB), in agreement with promoter activity data. Higher OpIAP protein expression was found in immature (double-positive (DP)) thymocytes compared with mature (single-positive (SP)) thymocytes, according to the relative promoter activities (Fig. 1GoC).



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  		FIGURE 1. Generation of p56lckpr-OpIAP Tg mice. A, Southern blot of genomic DNA from OpIAP Tg mice. BamHI-digested genomic tail DNA was hybridized with a full-length OpIAP cDNA probe. Different amounts of purified OpIAP cDNA were used as control. F1 offspring of several founders (indicated by numbers) were analyzed. *, Founders selected to establish the two Tg lines. B, Western blot analysis of OpIAP protein expression in lymphoid organs from wt and OpIAP Tg mice (line H51). Blots of cytosolic extracts were probed with polyclonal anti-OpIAP antiserum. C, OpIAP protein expression in cytosolic extracts from SP and double DP thymocytes. Western blot analysis was performed with cytosolic extracts from fluorescence-labeled sorted thymocytes. Equivalent protein loading was confirmed by probing with {beta}-actin (bottom).

 
OpIAP expression does not affect total cellularity of thymus (Table IGo) or of peripheral lymphoid organs (data not shown). In flow cytometry, expression of cell surface markers (CD3, CD4, CD8, CD44, CD25, CD69, TCR{alpha}{beta}, B220, CD95, and CD28) showed no major alterations in the composition of thymocyte (Fig. 2Go and Table IGo) or peripheral T cell subpopulations (data not shown) in Tg mice. Nonetheless, more detailed analysis showed that the CD4+/CD8+ ratio in TCRhigh Tg thymocytes was reduced, due to an increase in the CD8+ subpopulation (Table IGo).


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  		Table I. Thymic composition of 8-wk-old wt and OpIAP tg mice

 


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  		FIGURE 2. OpIAP expression does not affect thymic development in Tg mice. The histograms show flow cytometry analysis of thymus from wt and OpIAP Tg mice stained with anti-CD4 and -CD8 mAb. The percentages of the four thymocyte subpopulations (CD4+, CD8+, CD4+CD8+, and CD4-CD8-) are indicated in each quadrant.

 
OpIAP Tg thymocytes show increased nuclear NF-{kappa}B and decreased cytoplasmic I{kappa}B{alpha} levels

The human IAP family members c-IAP2, c-IAP1, and XIAP can trigger NF-{kappa}B activation when overexpressed in cell lines (12, 14, 15). Although the mechanism by which these human IAPs induce NF-{kappa}B activity is unknown, it appears to be mediated by phosphorylation and degradation of I{kappa}B{alpha}, the principal NF-{kappa}B inhibitory protein (14). The conservation of structure and antiapoptotic function between baculoviral OpIAP and cellular IAPs (4) prompted us to analyze whether OpIAP affects signaling pathways leading to NF-{kappa}B modulation. EMSA using nuclear extracts from wild-type (wt) and OpIAP Tg thymocytes allowed direct quantification of nuclear NF-{kappa}B in these cells. Nuclear extracts from OpIAP Tg thymocytes showed increased levels (3- to 4-fold) of NF-{kappa}B p65 complex compared with wt thymocytes. The presence of p65 in this complex was shown by Ab competition assays (Fig. 3GoA). These data indicate that OpIAP expression increases nuclear translocation of the transcriptionally active NF-{kappa}B p65 complex during Tg thymocyte development.



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  		FIGURE 3. Cytosolic I{kappa}B{alpha} levels and NF-{kappa}B nuclear translocation in OpIAP Tg thymocytes. A, EMSA of nuclear NF-{kappa}B proteins. Thymic nuclear extracts from wt and OpIAP Tg mice were analyzed using a radiolabeled {kappa}B oligonucleotide probe. For Ab competition experiments, nuclear extracts were preincubated with NF-{kappa}B, a p65 Ab, or an irrelevant Ab. B, Western blot analysis of I{kappa}B{alpha} expression in thymocytes from two wt and three OpIAP Tg mice. Cytosolic extracts were probed with anti-I{kappa}B{alpha} Ab. Equivalent protein loading was confirmed by probing with {beta}-actin. C, Dex induces OpIAP degradation in a proteasome-dependent manner. Cytosolic extracts from OpIAP Tg thymocytes treated with Dex for different culture periods, alone or in the presence of the proteasome inhibitor lactacystin, were analyzed in Western blot using anti-OpIAP antiserum. Numbers below the figure indicate the remaining OpIAP protein levels as a percentage of levels in untreated thymocytes, obtained by densitometric quantification. Nonspecific bands indicate protein load and are used for normalization.

 
In unstimulated cells, NF-{kappa}B is retained in the cytoplasm, forming complexes with I{kappa}B inhibitory proteins, including I{kappa}B{alpha}. Stimulation induces phosphorylation and subsequent degradation of I{kappa}B{alpha}, allowing NF-{kappa}B translocation to the nucleus (29). As mammalian c-IAP2 is reported to regulate NF-{kappa}B activity by inducing I{kappa}B{alpha} degradation in the TNF signaling pathway (14), we analyzed whether the increase in nuclear NF-{kappa}B in OpIAP Tg thymocytes correlated with alterations in I{kappa}B{alpha} levels. Western blot analysis of cytosolic lysates from wt and OpIAP thymocytes using a polyclonal anti-I{kappa}B{alpha} Ab revealed a 2- to 5-fold reduction in the specific 40-kDa I{kappa}B{alpha} protein band in OpIAP thymocytes compared with wt littermates (Fig. 3GoB). These results indicate that OpIAP expression during thymocyte development leads to a reduction in cytoplasmic I{kappa}B{alpha} levels and an increase in nuclear NF-{kappa}B complex. This provides the first in vivo evidence of OpIAP involvement in control of the NF-{kappa}B signaling pathway during thymocyte development.

Apoptosis induction by Dex and etoposide in thymocytes is reported to lead to loss of endogenous IAPs by autoubiquination and degradation (30). Because ubiquitin protein ligase activity is mediated by the IAP ring domain and OpIAP Tg thymocytes showed no resistance to Dex-induced in vitro apoptosis (M.S.R., unpublished observations), we studied whether OpIAP is also degraded under these conditions. OpIAP thymocytes were cultured with Dex (200 nM) alone or in the presence of the proteasome inhibitor lactacystin (20 µM). As described for endogenous IAPs, Dex induces OpIAP degradation (45% decrease) at 4.5 h after stimulation (Fig. 3GoC). At 9 h, OpIAP levels were reduced by 55%. Lactacystin addition inhibits Dex-induced OpIAP degradation. Baculoviral OpIAP expressed in thymocytes is thus degraded similarly to the endogenous IAPs in response to Dex in a proteasome-dependent manner (30).

Optimal proliferation of mature OpIAP thymocytes after anti-CD3 stimulation

Previous studies showed that NF-{kappa}B translocation is induced in thymocytes after TCR/CD28 costimulation and that this is crucial for thymocyte activation, proliferation, and cytokine production (31, 32). Because OpIAP thymocytes show elevated NF-{kappa}B translocation to the nucleus, we studied whether this increase affected activation and proliferation of these cells. In vitro proliferation assays were performed with thymocytes isolated from OpIAP Tg mice and their wt littermates. Thymocytes were activated for 48 h with different concentrations of plastic-bound monoclonal anti-CD3 Ab (Fig. 4GoA). Compared with wt thymocytes, OpIAP Tg thymocytes showed significantly (p <= 0.01) increased proliferation (more than 2-fold at any anti-CD3 concentration tested). Proliferation of anti-CD3-stimulated OpIAP thymocytes, quantified by [3H]thymidine uptake 96 h after stimulation, was comparable to that of wt thymocytes activated with the anti-CD3 and anti-CD28 combination (Fig. 4GoB). Whereas anti-CD28 costimulation increased wt thymocyte proliferation at any anti-CD3 concentration tested, almost no effect was observed in OpIAP thymocytes (Fig. 4Go, A and B) at optimal anti-CD3 doses (>=1 µg/ml). Similar results were obtained after addition of IL-2 or a combination of IL-2 and anti-CD28 (Fig. 4GoB). The CD28 costimulatory signal is implicated in IL-2 production (33). Addition of IL-2 thus increases the proliferative response of wt cells, rendering it comparable to that of OpIAP thymocytes. These results indicate that OpIAP expression in thymocytes replaces the requirement for CD28 costimulatory signals to achieve a maximum proliferative response, probably due to its capacity to induce NF-{kappa}B activation.



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  		FIGURE 4. Optimal proliferation of OpIAP thymocytes in the absence of CD28 costimulation. A, Thymocytes from wt ({square}) and OpIAP Tg ({circ}) mice were treated for 48 h with different concentrations of plate-bound anti-CD3{epsilon} Ab, alone ({square} and {circ}) or with anti-CD28 Ab ({blacksquare} and •). Values are means ± SD [3H]thymidine incorporation for 8 h, for wt (n = 3) and OpIAP (n = 3) mice. B, Thymocytes were treated for 96 h with immobilized anti-CD3 Ab (1.25 µg/ml) or with anti-CD3 plus anti-CD28, alone or in the presence of IL-2. Bars represent [3H]thymidine incorporation in the last 8 h of culture for wt (n = 3) and OpIAP (n = 3) mice.

 
Increased activation and cytokine production in stimulated OpIAP thymocytes

Proliferative responses are strictly dependent on IL-2 synthesis and the expression of high-affinity receptors for this growth factor. Therefore, we used flow cytometry to analyze the expression of IL-2R {alpha}-chain (CD25) as well as the early activation marker CD69 in stimulated wt and OpIAP thymocytes. Because the anti-CD3-induced proliferation detected in cultured thymocyte suspensions is due to proliferation of mature thymocytes, we focused on the activation of mature CD4+ and CD8+ SP thymocyte subpopulations. Surface expression of CD25 and CD69 was undetectable in unstimulated CD4+ and CD8+ cells from both wt and OpIAP Tg thymus (Fig. 5GoA and data not shown). As predicted, both CD4+ and CD8+ SP thymocytes showed CD25 induction after anti-CD3 stimulation; however, this increase was greater in cells from OpIAP Tg mice compared with wt. The differences in CD25 expression between wt and OpIAP thymocytes were nonetheless abrogated after CD28 costimulation. In addition, after anti-CD3 stimulation, CD69 was expressed at higher levels in both CD4+ and CD8+ OpIAP Tg thymocytes compared with wt cells (Fig. 5GoB). In contrast to CD25, the differences in CD69 expression between OpIAP Tg and wt thymocytes persisted even after CD28 costimulation. In mature Tg thymocytes, maximum expression of both activation markers was thus observed after anti-CD3 stimulation. Only a slight increase in CD69 expression was induced by costimulation, in contrast to wt thymocytes in which the CD28 signal greatly increases expression of both markers.



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  		FIGURE 5. OpIAP thymocytes are fully activated after anti-CD3 stimulation. Thymocytes were stimulated with immobilized anti-CD3{epsilon} (1.25 µg/ml) or anti-CD3 plus anti-CD28 and then analyzed by flow cytometry for surface CD25, CD69, CD8, and CD4 expression. A, CD25 expression in unstimulated and stimulated mature thymocytes (CD8+ or CD4+) from wt and OpIAP Tg mice 24 h after stimulation. B, Surface CD69 expression in CD4+ and CD8+ mature thymocytes from wt and OpIAP Tg mice, 18 h after CD3 or CD3/CD28 stimulation.

 
Increased OpIAP thymocyte proliferation may be due to augmented CD25 expression, to enhanced IL-2 production, or to a combination of both. Secreted IL-2 was thus measured by ELISA in thymocyte culture supernatants. After anti-CD3 stimulation, OpIAP thymocytes secreted 2- to 3-fold more IL-2 than wt cells. Costimulation with anti-CD28 mAb had no significant effect on OpIAP Tg cells, whereas IL-2 secretion increased 2- to 3-fold in wt cells (Fig. 6GoA). OpIAP expression in thymocytes thus leads to simultaneous maximum secretion of IL-2 and expression of its receptor (CD25) in the absence of costimulatory signals. We also measured the production of IFN-{gamma}, a lymphokine whose production is specific to activated T cells. After CD3 stimulation, very high IFN-{gamma} levels were detected in OpIAP Tg thymocyte culture supernatants, whereas no secretion was detected in wt cultures (Fig. 6GoB). T cell costimulation is known to enhance IFN-{gamma} production. We found a slight increase in IFN-{gamma} levels in wt cultures after CD28 costimulation, although this increase did not approximate those seen in OpIAP thymocyte cultures. IL-4, IL-10, and TNF-{alpha} were undetectable in wt or OpIAP Tg culture supernatants, independent of the stimulation (anti-CD3 or CD3/CD28) (data not shown).



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  		FIGURE 6. OpIAP thymocytes produce higher IL-2 and IFN-{gamma} levels after anti-CD3-induced stimulation. Thymocytes from wt and OpIAP Tg mice were stimulated with plastic-bound anti-CD3{epsilon} (1.25 µg/ml) alone or in combination with anti-CD28. A, After 24 h, IL-2 levels in culture supernatants were measured by ELISA. B, IFN-{gamma} supernatant levels in the same cultures measured 48 h after stimulation. Bars represent cytokine concentration (mean ± SD) in wt (n = 3) and OpIAP (n = 3) Tg thymocyte cultures. Cytokine levels were below detection limits (<=15 pg/ml) in unstimulated cultures.

 
OpIAP inhibits both Ag-induced and TCR-triggered apoptosis in vivo

In addition to their role in NF-{kappa}B activation, IAPs were originally described as negative regulators of apoptosis, blocking caspase activation and activity (4). Because OpIAP inhibits apoptosis when expressed ectopically in mammalian cells, we tested whether OpIAP also had an effect in thymic apoptosis, studying positive and negative selection in a TCR Tg mouse model. F5 TCR Tg mice express the V{beta}11, V{alpha}4 TCR from a cytotoxic T cell clone (F5) that recognizes a peptide of the influenza virus nucleoprotein in the context of MHC class I Db, H-2Db (34). The majority of DP and SP CD8+ thymocytes and CD8+ peripheral T lymphocytes express the Tg TCR. OpIAP Tg mice were crossed with F5 TCR mice to generate F5TCR/OpIAP double Tg mice. No differences were found in V{beta}11 expression levels or CD8+ frequencies in F5TCR/OpIAP mice compared with F5 TCR mice, either in thymus or peripheral lymphoid organs (data not shown).

The effect of OpIAP in negative selection was analyzed using the F5TCR model, in which an i.p. injection of antigenic peptide induced 50% thymocyte depletion, mainly of DP cells, after 24 h (Fig. 7GoA), concurring with previous reports (27, 35). In contrast, F5TCR/OpIAP mice were resistant to peptide-induced depletion in thymus, in that no significant difference was observed in total thymocyte numbers compared with peptide- and PBS-injected mice. Comparison of thymocyte subpopulations showed a slight reduction in the absolute number of DP OpIAP thymocytes.



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  		FIGURE 7. TCR-induced apoptosis is prevented in OpIAP Tg thymocytes. A, Graph shows total number of thymocytes and thymic subpopulations in F5 TCR control and F5TCR/OpIAP Tg mice after PBS or F5 peptide injection. B, PARP processing in thymocytes from antigenic peptide-injected mice. Extracts of cultured thymocytes were analyzed in Western blot using an anti-PARP Ab. Arrows indicate unprocessed (115-kDa) and processed (85-kDa) PARP. C, The figure shows caspase-3 activity, expressed in arbitrary fluorescence units (A. U.), measured in thymocyte lysates 14 h after antigenic peptide injection in wt (n = 3) and OpIAP Tg (n = 3) mice. The differences are statistically significant (p < 0.1; Student’s t test). D, Western blot analysis of Bcl-2 (top) and Bcl-xL (middle) protein in total thymus lysates (wt and Tg) from mice at 14 h after F5 peptide or PBS injection. Equivalent protein loading was confirmed by probing with {beta}-actin (bottom).

 
The mechanisms underlying the inhibition of Ag-induced thymic apoptosis were analyzed by studying the proteolysis of a characteristic caspase substrate, PARP. PARP proteolysis was measured in thymocytes cultured at 37°C for 1 h after isolation, a period required for detection of PARP processing; apoptotic thymocytes are rapidly eliminated by macrophages in vivo, and PARP proteolysis cannot be visualized directly (35, 36). In contrast to F5 TCR thymocytes, in which PARP cleavage was detected as the 85-kDa processed form, no PARP processing was observed in thymocytes from F5TCR/OpIAP mice after antigenic peptide injection (Fig. 7GoB). Using a specific substrate, we also analyzed caspase-3 activity in thymocyte lysates from Ag peptide-treated mice. OpIAP expression led to a reduction in caspase-3 activity after antigenic peptide injection (Fig. 7GoC), which correlates with the inhibition of PARP processing described above.

Some antiapoptotic members of the Bcl-2 family are also described to play a role in negative selection. Although the results reported in the literature are controversial, it is suggested that, whereas Bcl-2 can inhibit Ag-mediated negative selection (37), Bcl-xL has no effect on thymocyte negative selection (38, 39). Because OpIAP Tg mice show inhibited thymocyte negative selection, we analyzed whether this might be due to an alteration in Bcl-xL or Bcl-2 protein levels that could improve survival of thymocytes. Western blot analysis showed that OpIAP did not interfere with Bcl-2 expression in thymus, in that no difference in protein levels was detected between wt and OpIAP Tg mice injected with PBS (Fig. 7GoD). At 14 h after Ag peptide injection, no difference was detected in Bcl-2 protein levels between wt and OpIAP Tg thymus. In contrast, Bcl-xL levels were slightly reduced in thymus of PBS-injected mice expressing OpIAP compared with wt (Fig. 7GoD). In addition, whereas the Ag peptide injection induced a decrease in Bcl-xL protein levels in wt thymus at 14 h, no significant reduction was observed in Tg thymus. Loss of Bcl-xL protein is described to precede thymocyte apoptosis after anti-CD3 treatment (32), due to down-regulation of Bcl-xL gene transcription in DP thymocytes. The Ag peptide-induced loss of Bcl-xL observed in wt thymocytes is prevented by OpIAP expression, because protein levels remain similar after treatment (Fig. 7GoD). OpIAP-mediated inhibition of negative selection thus appears not to be related to an increase or up-regulation in the antiapoptotic proteins Bcl-2 or Bcl-xL. These data concur with the lack of difference between wt and Tg mice in spontaneous apoptosis of cultured thymocytes (data not shown).

Our results show that OpIAP inhibits thymic negative selection and that this correlates with reduced proteolytic activity of caspases in Tg thymocytes. It is known that caspases are activated during negative selection (35, 36), but this is the first evidence that an IAP family member inhibits in vivo TCR-mediated apoptosis in thymocytes.

Similar results were obtained in experiments in which anti-CD3 Ab was injected in wt and OpIAP Tg mice. At 24 h after i.p. injection of anti-CD3, a dramatic reduction (>20-fold) in total thymocyte numbers was found in wt mice, whereas less depletion was seen in OpIAP Tg mice. After anti-CD3 injection, OpIAP Tg thymus thus contained nearly 3-fold more thymocytes than those of their wt littermates (Fig. 8GoA). The difference between wt and Tg mice was more marked in the DP population, corresponding to those thymocytes in which anti-CD3 specifically induced apoptosis. These data indicate that OpIAP also inhibits in vivo anti-CD3-induced thymocyte apoptosis.



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  		FIGURE 8. OpIAP inhibits anti-CD3 and early Ag-induced thymocyte apoptosis. A, Number of thymocytes in different subpopulations from wt and OpIAP Tg mice after i.p. anti-CD3 Ab injection. Total thymocytes in control (PBS-injected) animals were 120 x 106 ± 10 x 106 (wt) and 108 x 106 ± 6 x 106 (OpIAP Tg). Results are representative (mean ± SD) of three different experiments. B, Representative DNA fragmentation patterns in total thymus extracts from wt and OpIAP Tg mice at 90 min after PBS or F5 peptide injection. C, Caspase-3 activity detected in thymocyte lysates at 90 min after PBS or F5 peptide treatment in wt (n = 3) and OpIAP Tg (n = 3) mice.

 
We also analyzed the early Ag peptide-induced deletion of immature thymocytes at 90 min, when Ag peptide injection (19 nmol) caused significant induction of thymocyte apoptosis in wt mice. This can be detected by the characteristic oligonucleosomal DNA fragmentation (Fig. 8GoB). OpIAP expression prevents this early thymocyte apoptosis, because no DNA degradation was detected in thymus of Ag peptide-treated OpIAP Tg mice (Fig. 8GoB). Furthermore, analysis of caspase-3 activity using a specific substrate revealed a 30% reduction of this activity in lysates of thymocytes from treated OpIAP Tg mice compared with those from wt mice (Fig. 8GoC). In addition, the absence of cells containing subdiploid DNA in the DP thymocyte population of treated OpIAP Tg mice (data not shown) supports our previous results, indicating that OpIAP prevents early, specific DP thymocyte apoptosis induced by Ag peptide. These data lead us to suggest that the OpIAP-mediated inhibition of thymocyte deletion induced by antigenic peptide is not affected by peripheral T cell activation. Indeed, this early apoptosis is due to specific peptide recognition in thymus, as previously described (40).

OpIAP enhances cytokine production and increases mature thymocyte numbers after in vivo TCR stimulation

The F5 TCR Tg mouse model was used to test whether OpIAP increases in vivo TCR-induced activation of mature thymocytes, as described in vitro. In contrast to immature thymocytes, TCR engagement in mature thymocytes induces activation (27). After antigenic peptide injection, the number of mature (CD4+ and CD8+) thymocytes did not change significantly in F5 TCR mice. In contrast, F5TCR/OpIAP Tg animals show an increase in mature thymocyte numbers, mainly in CD8+ cells (Fig. 7GoA).

Serum cytokine levels were also increased in peptide-injected F5 TCR/OpIAP mice compared with F5 TCR animals (a 6-fold increase in IL-2 and >=15-fold for IFN-{gamma}), without significant change in IL-4 levels (Fig. 9Go). These data correlate with the increase in in vitro proliferation and activation of OpIAP Tg thymocytes after anti-CD3 stimulation.



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  		FIGURE 9. Increased serum cytokine levels in peptide-injected F5TCR/OpIAP Tg mice. The graph shows serum IL-2, IFN-{gamma}, and IL-4 levels measured by ELISA 2 h after F5 peptide injection in wt (n = 3) and OpIAP Tg (n = 3) mice. Cytokine levels were below detection limits (<=15 pg/ml) in PBS-injected mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IAPs inhibit cell death by blocking procaspase activation and caspase activity, but some also modulate the activity of the transcription factor NF-{kappa}B. Most reports that have provided data on the biochemical functions of these inhibitors have focused on tissue cultures and cell lines (3, 4). Because the IAP family members are highly conserved among species, we analyzed the consequences of the expression of one of the first IAPs described (OpIAP) on IAP-regulated processes during thymus development and homeostasis. Our study demonstrates that OpIAP expression in developing thymocytes causes elevated NF-{kappa}B nuclear translocation, concomitant with an increase in I{kappa}B{alpha} degradation. These data concur with earlier reports showing that human c-IAP2 overexpression in T cell lines activates NF-{kappa}B by targeting I{kappa}B{alpha} degradation (14). As for endogenous IAPs, Tg thymocyte-expressed OpIAP is degraded in a proteasome-dependent manner in response to Dex treatment (30). Thus, the regulation and function of OpIAP is similar to that described for endogenous IAPs.

The mechanism by which OpIAP expression leads to decreased I{kappa}B{alpha} levels and increased NF-{kappa}B nuclear translocation during thymocyte development is still unknown. Recent studies established a functional relationship between the IAP ring finger motif and ubiquitin ligase activity (41). Mammalian c-IAP2 is reported to have ubiquitin activity and to promote in vitro ubiquitination of caspase-3 and -7 (42). Moreover, ring-dependent ubiquitin protein ligase activity of c-IAP1 and XIAP is described in thymocytes undergoing apoptosis by Dex (30). Similarly to these endogenous IAPs, OpIAP in Tg thymocytes is also degraded after Dex treatment. Because OpIAP also contains a ring domain, similar ubiquitin ligase activity could be envisaged. This allows us to speculate that OpIAP expression during thymocyte development may promote I{kappa}B{alpha} ubiquitination, a prerequisite for its degradation, leading in turn to NF-{kappa}B nuclear translocation. Further biochemical studies are required to confirm this hypothesis.

CD28 ligation potentiates rapid and persistent I{kappa}B{alpha} degradation by inducing the kinase activity of the IKK complex (25). The active IKK complex phosphorylates I{kappa}B{alpha}, which is in turn ubiquitinated and then degraded by the proteasome. OpIAP may provide a CD28-independent costimulatory signal by inducing I{kappa}B{alpha} degradation, possibly through ubiquitination, as suggested above. Conversely, OpIAP may act upstream of I{kappa}B{alpha} ubiquitination, for instance, by inducing IKK activation. It is reported that IKK activation after TCR/CD28 triggering is promoted by its recruitment to the T cell membrane through protein kinase C {theta} (23). Although this interaction seems to be minimal after TCR triggering, it is significantly augmented after CD28 costimulation. Thus, it can be speculated that, similar to human c-IAP1 and c-IAP2, which are recruited to the TNF-{alpha} signaling complex by interaction with TNFR-associated factor proteins (12, 43), OpIAP may interact with some components of the TCR signaling complex. This would facilitate IKK recruitment and activation, leading to I{kappa}B{alpha} degradation after phosphorylation and subsequent NF-{kappa}B nuclear translocation.

The NF-{kappa}B transcription factor is essential in T cell response regulation. Its activity is induced in thymocytes and mature T lymphocytes after TCR engagement (24). TCR triggering alone is unable to induce complete functional NF-{kappa}B activation, which occurs after TCR/CD28 costimulation, leading to optimal activation and proliferation (23, 25, 26). Because developing thymocytes from OpIAP Tg mice showed increased NF-{kappa}B nuclear translocation, we analyzed their TCR-mediated responses. Our results show that OpIAP expression in thymocytes abrogates the requirement for a costimulatory signal to achieve maximum activation and proliferation. In Tg thymocytes, anti-CD3 stimulation is thus sufficient to induce optimal proliferation and expression of activation markers (CD25 and CD69) as well as cytokine production (IL-2 and IFN-{gamma}). This may be due to the fact that the regulatory regions of CD28-controlled genes such as IL-2, IFN-{gamma}, CD69, and IL-2R harbor NF-{kappa}B-controlled CD28 response element sequences (44). Our data concur with those reported for in vivo inhibition of NF-{kappa}B in thymocytes, describing decreased proliferation and cytokine production (31, 32, 45). The in vivo data also support this hypothesis, in that Ag peptide-injected F5TCR/OpIAP Tg mice showed higher IL-2 and IFN-{gamma} levels in serum compared with F5 TCR littermates, although no differences were found in IL-4 levels. Although the IL-4 gene is also transcriptionally regulated by NF-{kappa}B (45), OpIAP expression does not alter its production, possibly due to differences in the regulatory requirements of these NF-{kappa}B-controlled genes. Inhibition of NF-{kappa}B activity in T cells is reported to lead to a reduction in IFN-{gamma} production and to a preferential impairment of the type 1 (Th1) T cell response compared with the type 2 response (46). Because NF-{kappa}B activity is increased in thymus and higher IFN-{gamma} serum levels are detected after Ag peptide treatment in OpIAP Tg mice, an increased type 1 T cell-mediated response may be found in these mice. Future experiments studying the in vivo immune response in OpIAP Tg mice would offer additional information regarding this point.

NF-{kappa}B also has an important role in immature thymocyte differentiation (47). NF-{kappa}B activity determines DP thymocyte maturation to CD8+ SP cells, reported in Tg mice expressing a nonphosphorylable mutant I{kappa}B{alpha} form that impedes NF-{kappa}B activation (31, 48, 49). Despite increased NF-{kappa}B nuclear translocation in OpIAP thymocytes, no major alterations were found in thymocyte subpopulations. Nonetheless, more detailed analysis of OpIAP thymocytes showed that the CD4+:CD8+ ratio was reduced in the CD3high population, due to an increase in the number of CD8+ vs CD4+ cells. Consistent with our data are those described in Tg mice expressing the mutant I{kappa}B{alpha}, which show a CD4+/CD8+ ratio in the TCRhigh thymocyte population (32, 47).

It is widely accepted that apoptosis helps establish the thymic T cell repertoire by controlling thymocyte ontogeny. Whereas immature DP thymocytes expressing low-affinity TCR fail to be positively selected and die by "neglect," self-reactive DP thymocytes bearing a high-affinity TCR are eliminated by apoptosis during negative selection (50). In addition to its role in NF-{kappa}B activation described here, OpIAP acts as an important apoptosis inhibitor when expressed ectopically in mammalian cells (5). To determine whether OpIAP expression inhibits TCR-induced thymocyte apoptosis, we used in vivo anti-CD3 mAb administration to study polyclonal TCR-mediated thymocyte apoptosis and F5TCR/OpIAP Tg mice to study MHC class I-restricted negative selection. The results are in full agreement in both models: OpIAP expression inhibits TCR-induced apoptosis in Tg thymocytes. The blockage observed concurs with the inhibition of caspase activity and PARP processing in F5TCR/OpIAP thymocytes after peptide injection. In addition to the induction of thymocyte negative selection, antigenic peptide injection also leads to activation of peripheral T cells, although F5 peptide-induced thymocyte deletion is due mainly to peptide-specific recognition in thymus (40). To avoid indirect effects of activated peripheral T cells in thymocyte apoptosis, we studied early apoptosis by analyzing DNA oligonucleosomal fragmentation, one of the first detectable features of apoptosis (27). Our studies revealed a lack of DNA degradation in OpIAP Tg thymocytes after treatment, correlating with a considerable reduction in caspase-3 activity. These results concur with our previous data on late apoptosis in OpIAP Tg mice. Although we cannot eliminate the possible influence of the periphery on this thymocyte apoptosis, activated peripheral T cells are very unlikely to produce an effect in the early thymocyte apoptosis analyzed here. In addition, because OpIAP does not prevent spontaneous cell death of cultured thymocytes from Tg mice, the OpIAP-mediated resistance of thymocyte apoptosis after antigenic peptide or anti-CD3 treatment may be due to a specific blockage of a signal inducing negative selection.

Another endogenous antiapoptotic protein, Bcl-2, is also described to inhibit antigenic peptide-induced thymocyte apoptosis (37). Our study demonstrates that OpIAP-mediated blockage of negative selection is independent of Bcl-2 and Bcl-xL, because the levels of both proteins in thymus remain similar after Ag peptide injection compared with wt and OpIAP Tg mice. It is not known how OpIAP induces the decrease in thymic Bcl-xL. Reduced levels are observed in the thymus of Tg mice compared with wt, although this does not affect OpIAP-mediated blockage of negative selection.

Many studies have shown that caspase activation is associated with TCR-induced apoptosis in thymocytes and that caspase inhibition blocks TCR-induced thymocyte apoptosis (negative selection) (35, 36, 51, 52). Studies in several mouse models showed that caspase inhibitors lacking endogenous counterparts, such as p35 and CrmA, block thymocyte apoptosis and/or negative selection (35, 53), but the data described here represent the first evidence implicating IAPs in in vivo TCR-induced apoptosis. Although it was recently reported that overexpression of human XIAP in murine Tg thymocytes inhibits Dex- and Fas-induced apoptosis in vivo, no data on in vivo TCR-induced thymocyte death were presented (54). Our study reveals a novel role for an IAP family member in the activation and proliferation of developing thymocytes in vivo. A positive effect on NF-{kappa}B pathway regulation is also seen in OpIAP-expressing Tg thymocytes. It has recently been shown that XIAP-deficient mice have no defect in either apoptosis or proliferation in the immune system (55). Compensatory mechanisms involving up-regulation of other IAPs were proposed to explain the lack of obvious phenotype in these mice. Our overexpression approach circumvents these redundancy and compensatory processes and provides data regarding IAP involvement in thymocyte apoptosis and proliferation.

In conclusion, the OpIAP Tg mouse model provides evidence for a broad spectrum of IAP functions, not only as an apoptosis inhibitor by blocking caspase activation or function, but also by inducing I{kappa}B{alpha} degradation and NF-{kappa}B nuclear translocation. Moreover, as for other endogenous IAPs, OpIAP is degraded in a proteasome-dependent manner in Tg thymocytes undergoing Dex-induced apoptosis in vitro. This suggests similar regulation for baculoviral and mammalian IAPs. Interestingly, OpIAP expression during thymocyte development significantly increases thymocyte activation and proliferation after TCR triggering, replacing the CD28 costimulatory signal required for optimal thymocyte responses. Considering the effect of OpIAP on proliferation and activation, it would be of interest to analyze the propensity of the Tg mice for lymphoproliferative and/or autoimmune disease, as well as their tendency toward tumor development.


   Acknowledgments
 
We thank J. A. Garcia-Sanz for critical reading of the manuscript, A. Brás and J. L. Torán for helpful discussions, A. Grandien for the transgenic construct, P. Pallarés and J. M. Fernández Toro for animal work, J. P. Albar for OpIAP antiserum, M. C. Moreno-Ortíz and I. López-Vidriero for flow cytometry analysis, S. Martínez-Martínez and J. M. Redondo for technical help in EMSA, and C. Mark for editorial assistance. We also thank Dr. D. Kioussis (Medical Research Council, London, U.K.) for kindly providing F5 TCR mice.


   Footnotes
 
1 This work was supported by Grants from the Dirección General de Investigación, Ministry of Science and Technology. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research and the Pharmacia Corporation. M.S.R. received fellowships from the Consejo Superior de Investigaciones Científicas/Pharmacia and the Regional Government of Madrid (Comunidad Autónoma de Madrid). Back

2 Address correspondence and reprint requests to Dr. María S. Robles, Department of Immunology and Oncology, Laboratory 411, Centro Nacional de Biotecnología, Campus de Cantoblanco, Universidad Autónoma, E-28049 Madrid, Spain. E-mail address: msrobles{at}cnb.uam.es Back

3 Abbreviations used in this paper: IAP, inhibitor of apoptosis proteins; BIR, baculoviral IAP repeat; OpIAP, IAP generated from the Orgyia pseudotsugata nuclear polyhedrosis virus; AICD, activation-induced cell death; IKK, I{kappa}B kinase kinase; hGH, human growth hormone; Tg, transgenic; TSH{beta}, thyroid-stimulating hormone {beta}; PARP, poly(ADP-ribose) polymerase; SP, single-positive; DP, double-positive; wt, wild type; Dex, dexamethasone. Back

Received for publication July 20, 2001. Accepted for publication November 30, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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