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Molecular Endocrinology, doi:10.1210/me.2003-0390
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Molecular Endocrinology 18 (3): 687-695
Copyright © 2004 by The Endocrine Society

Resistance of Single-Positive Thymocytes to Glucocorticoid-Induced Apoptosis Is Mediated by CD28 Signaling

Jens van den Brandt, Dapeng Wang and Holger M. Reichardt

Institute of Virology and Immunobiology, University of Würzburg, 97078 Würzburg, Germany

Address all correspondence and requests for reprints to: Holger M. Reichardt, Institute of Virology and Immunobiology, University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, Germany. E-mail: holger.reichardt{at}mail.uni- wuerzburg.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 CONCLUSIONS
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids administered in pharmacological doses potently induce apoptosis in immature double-positive thymocytes. In contrast, single-positive thymocytes are completely resistant. We now provide evidence that this difference can be attributed to CD28 signaling. When taken into culture, single-positive thymocytes also become sensitive to glucocorticoid-induced apoptosis, which can be prevented by enforced CD28 engagement using a novel type of antibody. This is achieved, at least in part, by transcriptional regulation of apoptosis-related genes such as Bcl-XL via a calcium- and phosphatidylinositol 3 kinase-dependent pathway. Accordingly, deficiency of CD28 in genetically engineered mice leads to an increased sensitivity of single-positive thymocytes toward glucocorticoid-induced cell death in vivo. Taken together, we have identified CD28 signaling in the thymus as a key player in determining the differential sensitivity of double-positive and single-positive cells to glucocorticoid action.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 CONCLUSIONS
 MATERIALS AND METHODS
 REFERENCES
 
APOPTOTIC STIMULI INITIATE signal transduction pathways in cells that eventuate in their self- destruction via a series of biochemical suicide steps (1). This process of programmed cell death is obligatory for sustaining tissue homeostasis as well as normal development and, if disrupted, results in cancer and autoimmune diseases. Two major apoptotic pathways have been identified: the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway. Although initiated by different types of stimuli, both pathways appear to share common downstream components such as caspases, a family of aspartate- specific cysteine proteases. Stimulation of death receptors primarily results in the recruitment of caspase-8 and the formation of the so-called DISC. In contrast, other stimuli, such as DNA damage, activate the mitochondrial pathway by altering the delicate balance of proapoptotic and antiapoptotic proteins. After formation of the apoptosome, a multimeric Apaf-1 and cytochrome c complex, both pathways converge on the activation of caspase-3, the effector caspase that is considered the point of no return in the apoptotic pathway. Although caspases have long been considered to be indispensable for the initiation of apoptosis, evidence for alternative caspase-independent pathways is now accumulating (2).

Although glucocorticoid-induced cell death was one of the first apoptotic processes to be recognized (3), it is still one of the least understood (4). Glucocorticoids (GCs) regulate gene expression by binding to the GC receptor (GR), a member of the nuclear receptor superfamily (5). Evidence that induction of thymocyte apoptosis strictly depends on transcriptional activation comes from the analysis of GR knock-in mice (6). A genetically engineered point mutation renders the GR unable to bind to DNA while retaining the ability to interact with other proteins. Thymocytes derived from these mutant mice are completely resistant to GC-induced apoptosis, indicating that activation of gene expression is the crucial step in GC-mediated cell death. However, although it is clear that transcriptional regulation is necessary for the induction of apoptosis, the genes responsible for initiating the cell death program still await identification. Concerning the execution phase of apoptosis, information is less scarce. It has been shown that GC-induced apoptosis proceeds via the mitochondrial pathway and that Bcl-2 family members are involved. Overexpression of the antiapoptotic Bcl-2 protein protects thymocytes from GC-induced apoptosis, whereas its disruption accelerates it (7, 8). In addition, mice doubly deficient for the proapoptotic proteins Bax and Bak were found to be completely resistant to GC-induced apoptosis (9). With regard to the role of caspases in GC-mediated apoptosis, controversial results were obtained. Although experiments with pharmacological inhibitors have implicated caspases 3, 8, and 9 (10, 11), none of the knockout mouse strains deficient for individual caspase enzymes shows any defect in this process (12, 13, 14). Thus, the contribution of these enzymes to GC-induced apoptosis is unclear. The current model of GC-induced cell death, therefore, is one in which the GR, in a DNA-binding-dependent manner, up- regulates the expression of undetermined proteins. This subsequently disturbs the balance of pro- and antiapoptotic Bcl-2 family members in the mitochondrial membrane, resulting in the release of apoptogenic factors and/or caspase activation. Cleavage of downstream substrates then leads to cell shrinkage, DNA fragmentation, and finally cell death.

Activation of mature T cells requires engagement of both the antigen-specific T cell receptor (TCR) and a costimulatory molecule such as CD28 (15). The finding that CD28 is expressed on thymocytes indicates that it may also play a role during T cell development (16). In rats and humans, CD28 is expressed at different levels on double-positive (DP) and single-positive (SP) thymocytes (16). Furthermore, the natural ligands of CD28, B7-1 (CD80), and B7-2 (CD86) are restricted to thymic epithelial and dendritic cells within the corticomedullary and the medullary region of the thymus (17). Together with the finding that expression of CD28 increases during the course of T cell maturation, it can be assumed that CD28 primarily plays a role in the more advanced stages of T cell development. With regard to thymocyte apoptosis, a cross-talk of GC signaling and the TCR complex has been described. Ashwell and colleagues (18) were the first to provide evidence that locally synthesized GCs could antagonize TCR-induced apoptosis in fetal thymus organ culture. Later, Wagner et al.(19) reported that immature CD3- thymocytes were protected from GC- induced apoptosis by CD28 signaling in the absence of TCR engagement. Although the latter study suggests that CD28 might play a role in thymocyte apoptosis, the physiological relevance of this study is disputable. In particular, immature CD3- thymocytes reside exclusively in the outer cortical regions of the thymus where no CD28 ligands are expressed (17). Furthermore, an analysis of CD28-deficient mice failed to provide any support for a role of CD28 in protecting immature thymocytes from GC-induced cell death (20). Therefore, an important function for CD28 in protecting the majority of DP thymocytes from GC- induced apoptosis appears unlikely. However, the role of CD28 in mature SP thymocytes that are in close contact to B-7-expressing medullary cells has not yet been investigated.

Although the TCR and CD28 are known to cooperate in T cell activation, their shared and individual signaling pathways are unknown (15). Unfortunately, conventional {alpha}CD28 antibodies do not elicit a response in T cells without TCR costimulation, making it difficult to dissect the two signaling pathways. However, a novel so-called "superagonistic" {alpha}CD28 rat monoclonal antibody (JJ316) was recently developed and stimulates T cells through CD28 in the absence of TCR ligation (21). This antibody was shown to activate the protein kinase C {theta}-NF-{kappa}B pathway in a manner indistinguishable from TCR/CD28 costimulation. In contrast, inducible tyrosine phosphorylation of ZAP-70 and TCR{zeta}, both hallmarks of TCR-mediated signaling, is not detectable after treatment with the "superagonistic" {alpha}CD28 antibody (22). JJ316 is therefore a useful tool to study selectively downstream events of CD28 signaling in cell culture. Using this novel antibody we found that CD28 engagement rescues SP thymocytes from GC-induced cell death by regulating expression of apoptosis-related genes. Thus, CD28 signaling provides a convincing explanation for the finding that mature thymocytes residing in the medulla of the thymus are protected from GC-induced cell death, whereas DP cells in the cortex readily undergo apoptosis under the same conditions.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
CONCLUSIONS
 MATERIALS AND METHODS
 REFERENCES
 
Differential Sensitivity of SP Thymocytes to GC-Induced Apoptosis in Vivo and in Vitro
Thymocyte subsets differ in their sensitivity to GC-induced apoptosis, but the underlying mechanism has not yet been identified (23). To address this question, we compared GC-induced cell death in vivo and in vitro. Young Lewis rats were injected ip with different concentrations of the synthetic GC dexamethasone (dex) and were compared with uninjected controls. Twenty hours later the total number of cells per thymus as well as the number of DP and mature SP thymocytes was determined. As expected, dex treatment led to a dose-dependent reduction in the total number of cells (Fig. 1AGo). This effect could be exclusively attributed to a reduction in DP cells, because the number of CD4+ and CD8+ SP thymocytes remained unchanged (Fig. 1BGo). This confirms that SP thymocytes are resistant to GC-induced apoptosis in vivo.



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  		Fig. 1. GC-Induced Thymocyte Apoptosis in Vivo and in Vitro

A and B, Lewis rats were injected ip with the indicated concentrations of dex or PBS as a control, and the total number of thymocytes as well as the number of DP and TCRhigh CD4+ and CD8+ SP thymocytes per thymus was determined by flow cytometry. The average cell number in control animals was set at 100%. Error bars represent the SEM for three independent experiments. C and D, Thymocytes were isolated from Lewis rats and cultured at 1 x 106 cells/ml in 1 ml RPMI+ medium for 20 h. Subsequently, the number of live cells was determined flow cytometrically by staining with annexin V, {alpha}CD4, and {alpha}CD8{alpha} followed by gating. Error bars represent the SEM for five independent experiments.

 
To study GC-mediated cell death in vitro, thymocytes from young Lewis rats were cultured for 20 h in the presence of 10-9 to 10-7 M dex (24). Apoptosis was determined by flow cytometry using annexin V in combination with monoclonal {alpha}CD4 and {alpha}CD8 antibodies. Binding of annexin V to phosphatidylserine, a membrane lipid that becomes externalized during the early events of programmed cell death, can be demonstrated by fluorescence-activated cell sorting (FACS) and is widely considered to be a reliable indicator of apoptosis. Similar to the in vivo experiment, dex treatment of cultured thymocytes led to a strong reduction in the absolute number of live cells in a dose-dependent manner (Fig. 1CGo). However, in contrast to the in vivo studies, we found a dramatic difference in regard to the different thymocyte subsets. In cell culture, apoptosis was induced not only in DP but also in both subsets of SP thymocytes (Fig. 1DGo). Almost 90% of the CD4+ and CD8+ SP cells underwent apoptosis at the highest concentration of dex. Collectively, these experiments demonstrate that SP thymocytes are selectively protected from GC-induced cell death in vivo.

CD28 Signaling Protects SP Thymocytes from GC-Induced Apoptosis
Signaling through the TCR as well as CD28 was previously shown to antagonize GC-mediated apoptosis (18, 19). Therefore, we wondered whether one of these pathways might rescue SP thymocytes from GC- induced apoptosis in cell culture. To separate the effects elicited via the two cell surface molecules, we used a series of monoclonal antibodies. Engagement of the {alpha}ßTCR alone was achieved with the antibody R73, whereas for costimulation the conventional {alpha}CD28 antibody JJ319 was used together with R73. Treatment with the superagonistic {alpha}CD28 antibody JJ316 allowed us to elicit CD28 signaling in the absence of TCR engagement and stimulation with JJ319 alone served as a control, because this antibody is unable to signal via CD28 by itself (21).

Thymocytes were stimulated for 20 h using the aforementioned antibodies, and cell survival was determined by staining with annexin V. TCR engagement as well as costimulation induced massive apoptosis (Fig. 2AGo). In contrast, stimulation with the {alpha}CD28 antibodies JJ316 and JJ319 alone did not result in enhanced cell death (Fig. 2AGo). Next, we cultured thymocytes in the presence of 10-8 M dex. As shown before, this treatment induces apoptosis in the vast majority of thymocytes. However, after costimulation and in the presence of the superagonistic {alpha}CD28 antibody JJ316, a small but statistically significant fraction (P < 0.001 for costimulation and P < 0.05 for JJ316) of the cells was rescued from apoptosis (Fig. 2AGo). This finding was obtained irrespective of the dex concentration used (10-9 to 10-7 M, data not shown). In the case of JJ316 treatment, the number of live cells was almost 3-fold higher than controls. In contrast, neither TCR signaling alone nor the presence of the control {alpha}CD28 antibody JJ319 had any effect on dex-induced thymocyte apoptosis. Taken together, this suggests that whereas signaling via {alpha}ßTCR induces cell death in thymocytes, engagement of CD28 can protect a small subpopulation from GC-induced apoptosis.



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  		Fig. 2. Effect of TCR and CD28 Signaling on Thymocyte Apoptosis

A, Total thymocytes were cultured in the presence of the antibodies R73 (TCR), R73+JJ319 ({alpha}TCR/{alpha}CD28, costim), conventional {alpha}CD28 (JJ319), or superagonistic {alpha}CD28 (JJ316). All stimulations were performed either in the absence or presence of 10-8 M dex. The number of annexin V- live cells was determined flow cytometrically. Relative changes in the number of live cells are indicated. Statistical significance was determined using the Student’s t test. Error bars represent the SEM for four independent experiments. con, Control. B, Total thymocytes and magnetically sorted CD4 and CD8 SP thymocytes were cultured in the presence of JJ316, 10-8 M dex, or a combination of both. In the case of DP thymocytes, cell numbers were determined from total thymocyte cultures by gating. Statistical analysis was performed using the Student’s t test. Error bars represent the SEM for five independent experiments for each thymocyte subtype. n.s., Not significant.

 
Subsequently, we analyzed whether engagement of CD28 protects a specific thymocyte subset from GC-induced apoptosis. Total thymocytes were cultured in the presence of the {alpha}CD28 antibody JJ316, 10-8 M dex or a combination of both. Cell death in DP cells was assessed flow cytometrically by staining with annexin V, {alpha}CD4, and {alpha}CD8 followed by gating. As shown in Fig. 2BGo, almost all DP cells were killed by dex treatment, whereas the presence of JJ316 had no effect. To study apoptosis in SP thymocytes, we used magnetically purified CD4 SP and CD8 SP cells. After 20 h, roughly 80% of these cells had undergone apoptosis in response to 10-8 M dex. Concomitant treatment with JJ316 protected the majority of CD4 SP as well as CD8 SP cells from cell death, whereas the antibody alone had no effect. This suggests that CD28 signaling selectively protects SP thymocytes from GC-induced apoptosis.

CD28 Signaling Prevents the Loss of Bcl-XL in CD4 SP Thymocytes during Culture
In search of the potential mechanism by which CD28 signaling interferes with GC-induced apoptosis, we studied the expression of several anti- and proapoptotic Bcl-2 family members (9, 25, 26). First, we compared the protein levels between freshly isolated DP and CD4 SP thymocytes as well as CD4 SP cells cultured in the presence or absence of the {alpha}CD28 antibody JJ316. Bcl-2 expression is low in DP cells, becomes up-regulated in SP cells, and remains largely unchanged during culture (Fig. 3AGo). In contrast, Bcl-XL is expressed at high levels in DP cells and becomes slightly down-regulated after maturation (Fig. 3AGo). However, after 20 h of cell culture, Bcl-XL expression is completely lost from CD4 SP thymocytes. Importantly, this down-regulation of Bcl-XL is prevented by CD28 signaling as elicited by JJ316 (Fig. 3AGo). With regard to proapoptotic proteins, we found comparable expression of Bax in all samples. In contrast, Bak was expressed at higher levels in CD4 SP as compared with DP cells and was reduced during culture in the presence of JJ316. Taken together, sustained Bcl-XL expression along with a reduction of Bak could explain how CD28 signaling prevents GC-induced apoptosis in mature SP thymocytes.



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  		Fig. 3. Expression of Anti- and Proapoptotic Bcl-2 Family Members

A, Whole-cell extracts from freshly isolated DP and CD4 SP thymocytes as well as cultured CD4 SP cells in the absence or presence of JJ316 were analyzed by Western blot. Samples were normalized to p56-Lck. B, RNA isolated from cells treated as described above was analyzed by RT-PCR. cDNA-probes were normalized to hypoxanthine phosphoribosyltransferase. One representative experiment of three is shown for both the Western blot and the RT-PCR analyses.

 
Because CD28 is known to control gene expression (27), we studied the transcriptional effects of JJ316 treatment. RNA derived from CD4 SP cells cultured in the presence or absence of JJ316 was analyzed by RT-PCR. In agreement with the Western blot results, we found unchanged mRNA expression of bcl-2 and bax after stimulation with JJ316, whereas bcl-XL expression was strongly up-regulated and bak expression was slightly reduced (Fig. 3BGo). This result shows that the observed changes in the protein levels are linked to transcriptional regulation exerted by CD28 signaling and strongly suggests that control of apoptosis-related genes such as Bcl-XL and Bak by CD28 contributes to the resistance of mature SP thymocytes toward GC-induced cell death.

CD28 Regulates the Expression of Bcl-XL in SP But Not in DP Thymocytes
Because it is not possible to detect changes in protein expression in individual cells by Western blot, we also performed flow cytometric analyses of intracellular Bcl-XL expression. This technique allows the relative amount of a specific protein to be detected in individual cells by comparing the fluorescence intensity after staining with a specific and a control antibody (isotype control).

Intracellular Bcl-XL levels were examined in magnetically purified CD4 and CD8 SP cells. As shown in Fig. 4AGo, Bcl-XL protein is clearly detectable in both types of SP thymocytes as indicated by the right-hand shift of the peak for the fluorescein isothiocyanate (FITC)- conjugated Bcl-XL antibody (solid line) as compared with the isotype control (dotted line). We then analyzed SP cells cultured in the absence or presence of the superagonistic {alpha}CD28 antibody JJ316. In agreement with the Western blot studies, Bcl-XL protein decreases in CD4 as well as CD8 SP thymocytes after 20 h of cell culture. In contrast, JJ316-mediated CD28 signaling results in sustained Bcl-XL expression in the majority of SP cells during culture (Fig. 4AGo). The presence of a small shoulder at the position of the isotype control indicates that roughly 10% of the cells did not respond to the antibody treatment and therefore lack Bcl-XL protein. Whether this is due to insufficient expression of CD28 on the surface of a subpopulation of the SP cells or a technical problem is not known.



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  		Fig. 4. Regulation of Bcl-XL in Thymocytes by CD28 Signaling on the Single-Cell Level

A, CD4 and CD8 SP thymocytes were cultured in the absence or presence of JJ316 and analyzed flow cytometrically after intracellular staining for Bcl-XL (solid line). An isotype-matched antibody served as a control (dotted line). B, DP and CD4 SP thymocytes were cultured in the absence or presence of JJ316 and analyzed flow cytometrically for the expression of Bcl-XL and Bcl-2 after intracellular staining (solid line). Isotype-matched antibodies served as a control (dotted line). One representative experiment of three is shown for each analysis.

 
To confirm that the observed effect is specific for the regulation of Bcl-XL in SP cells, we performed two control experiments. First, we found that the level of Bcl-XL expression in DP cells remains largely unchanged during culture, both in the absence and presence of JJ316 (Fig. 4BGo). Second, the amount of Bcl-2 in CD4 SP thymocytes also remains unaltered in cell culture irrespective of whether JJ316 is present (Fig. 4BGo). Taken together, this clearly indicates that Bcl-XL but not Bcl-2 is specifically down-regulated in SP thymocytes during culture, and this down-regulation can be prevented in the majority of cells by enforced CD28 signaling.

Protection of CD4 SP Cells from GC-Induced Apoptosis Depends on Calmodulin and Phosphatidylinositol 3 Kinase (PI3K) Signaling
To get a deeper insight into the molecular mechanism by which CD28 interferes with GC-induced apoptosis, we investigated the role of calcium and PI3K signaling using a pharmacological approach. Signal transduction by PI3K is prevented by the specific inhibitor LY294002, whereas calcium signaling via calmodulin is repressed by Cyclosporin A (CsA). To check whether either of these inhibitors may impair survival by itself, we cultured CD4 SP cells in the presence or absence of 100 ng/ml CsA or 30 µM LY294002 and analyzed their viability by staining with annexin V and propidium iodine. No difference in the percentage of live cells was observed between the three groups (control, 89%, n =4; CsA, 92%, n =3; LY294002, 89%, n =3), indicating that the inhibitors alone have no effect on cell survival.

As depicted in Fig. 5AGo, approximately 80% of the CD4 SP cells cultured in the presence of 10-8 M dex underwent apoptosis, which could be prevented by concomitant treatment with JJ316. However, in the presence of either CsA or LY294002, enforced CD28 signaling by JJ316 was no longer able to protect CD4 SP thymocytes from GC-induced apoptosis (Fig. 5AGo). Western blot analysis confirmed that in the presence of both inhibitors, CD28 signaling was also unable to prevent the loss of Bcl-XL during cell culture (Fig. 5BGo). Taken together, this strongly suggests that CD4 SP thymocytes are rescued from GC-induced apoptosis via a calcium- and PI3K-dependent signaling pathway.



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  		Fig. 5. Pharmacological Interference with Thymocyte Apoptosis

A, Apoptosis in CD4 SP cells was induced with 10-8 M dex in the absence (dex) or presence (d/J) of JJ316. The same experiment was repeated adding CsA at 100 ng/ml or the PI3K inhibitor LY294002 (LY) at 30 µM to the cultures. After 20 h, the number of annexin V- live cells was determined flow cytometrically. Statistical analysis was performed using the Student’s t test. Error bars represent the SEM for five (LY), six (CsA), and nine (control, con) independent experiments. B, Whole-cell extracts from cells treated as for panel A were analyzed by Western blot for the expression of Bcl-XL. ß- Actin was used as a loading control. One representative experiment of three is shown.

 
CD28-Deficient CD4 SP Thymocytes Show Increased Sensitivity to GC-Induced Apoptosis in Vivo
To confirm our findings in an in vivo model, we analyzed Bcl-XL expression as well as the sensitivity of CD4 SP thymocytes toward GC-induced apoptosis in CD28 knockout mice (28). Although we were unable to demonstrate a significant difference in Bcl-XL expression between SP thymocytes derived from homozygous mutants and heterozygous littermates (data not shown), we found a clear difference concerning their resistance to GC action. Mice of both genotypes were injected with 5 mg/kg dex, and 20 h later the absolute number of DP, CD4 SP, and CD8 SP thymocytes was determined by flow cytometry. As expected, dex treatment led to a severe reduction in the total number of DP cells in heterozygous CD28 knockout mice, whereas SP cells were largely resistant (+/-, con vs. dex; Fig. 6Go). However, in homozygous mutants, not only DP but also a significant fraction of the SP thymocytes underwent apoptosis (-/-, con vs. dex; Fig. 6Go). This indicates that, in CD28 knockout mice, SP cells are more susceptible to dex-induced apoptosis than in heterozygous control mice, which supports our model that, in SP thymocytes, signaling via CD28 confers resistance to GC-induced apoptosis.



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  		Fig. 6. GC-Induced Thymocyte Apoptosis in CD28 Knockout Mice

Homozygous CD28 knockout mice and heterozygous littermate controls were injected with dex at a concentration of 5 mg/kg and the number of DP, CD4, and CD8 SP thymocytes was determined 20 h later by flow cytometry after gating. Statistical analysis was performed using the Student’s t test. Error bars represent the SEM for three independent experiments. con, Control.

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 CONCLUSIONS
MATERIALS AND METHODS
 REFERENCES
 
It has long been recognized that DP thymocytes are highly sensitive to GC-induced cell death (3). When these hormones are administered in pharmacological doses or released at high levels under stressful conditions, DP cells readily undergo apoptosis resulting in thymic atrophy. In contrast, mature SP thymocytes are largely resistant to GC-action in vivo, although the reason for this difference had not yet been identified (23). We now provide compelling evidence that CD28 accounts for the differential sensitivity of the thymocyte subpopulations to GC treatment in vivo. First, CD28 signaling occurs exclusively in late stages of thymocyte maturation because expression of its ligands B7-1 and B7-2 is restricted to the corticomedullary and medullary region of the thymus where only SP cells are found (17). The majority of DP cells, on the other hand, reside in the cortex where B7-molecules are not to be found. Second, removal of SP cells from their thymic microenvironment renders them sensitive to GCs, with almost 90% undergoing apoptosis after dex treatment in culture. Thus, signal transduction in the thymus occurring in situ has to account for the protective effect against GC action. Third, enforced CD28 signaling in cell culture using a novel antibody that is able to stimulate CD28 independently of TCR engagement is able to restore resistance of CD4 SP thymocytes to GC-induced cell death. Taken together, this suggests that CD28 signaling in SP thymocytes protects these cells from hormone action in the thymic medulla after maturation. Fourth, SP thymocytes in CD28 knockout mice (28) show an increased sensitivity to GC-induced apoptosis. This is consistent with our hypothesis that it is CD28 signaling that protects SP thymocytes from GC action in vivo. Finally, we provide evidence that sustained expression of Bcl-XL and down-regulation of Bak by transcriptional regulation via a calcium- and PI3K-dependent pathway may play an important, although not exclusive, role in this process.

Whereas our initial model could be fully confirmed in cell culture, it appears to be only partially valid in vivo. First, approximately half of the SP thymocytes still remain resistant to GC treatment in CD28 knockout mice. This could indicate that CD28 signaling is not the only factor contributing to the protection of SP thymocytes or that compensatory mechanisms, e.g. signaling via related costimulatory molecules, are operative in CD28 knockout mice. Second, in contrast to the prediction of our model, we were unable to demonstrate a significantly lower expression of Bcl-XL in CD28-deficient SP thymocytes as compared with SP cells from control mice. Therefore, alterations in other proteins have to contribute to the decreased survival of CD28-deficient SP thymocytes after treatment with dex.

In summary, we propose the following revised model. After maturation, SP thymocytes migrate into the medulla where they come into close contact to B7-expressing epithelial and dendritic cells. Here, CD28 signaling leads to an altered expression of Bcl-XL and other so-far-unidentified antiapoptotic genes, thereby protecting SP cells from GC-induced apoptosis. This suggests that the topology of DP and SP thymocytes is directly linked to their differential sensitivity to GC-mediated cell death. Furthermore, this model also explains how GCs can contribute to shaping the TCR repertoire by inducing apoptosis in DP thymocytes while not affecting already selected mature SP cells residing in the same anatomic structure (29). Thus, by interfering with GC action, CD28 signaling appears to be indirectly involved in TCR repertoire formation in the thymus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 CONCLUSIONS
 MATERIALS AND METHODS
REFERENCES
 
Animal Experimentation
All animals were kept in individually ventilated cages. Lewis rats (Charles River, Sulzfeld, Germany) were propagated in our own animal facility and used for the experiments at 10–12 wk of age. CD28 knockout mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained by interbreeding heterozygous with homozygous mice. Genotyping was performed by PCR. All experiments have been conducted in accordance with accepted standards of humane animal care.

Antibodies and Reagents
The following antibodies and reagents used for flow cytometry, cell purification, and stimulation were obtained from Becton Dickinson (Heidelberg, Germany) unless otherwise indicated: Ox8-FITC (rCD8{alpha}), Ox8-bio (rCD8{alpha}), Ox38-phycoerythrin (PE) (rCD4), Ox35-Cy (rCD4), SA-PE, annexin V-FITC, R73 (rTCRß), JJ316 (rCD28), JJ319 (rCD28), H57-FITC (mTCRß), L3T4-PE (mCD4), LY2-bio (mCD8{alpha}), Bcl-2-FITC and isotype control, Bcl-XL-FITC and isotype control (Biozol, Eching, Germany). Antibodies used for Western blot analysis were obtained from Santa Cruz Biotechnology (Heidelberg, Germany) unless otherwise indicated: Bcl-2, Bcl-XL, ß-actin, p56-lck, Bak, and Bax (Becton Dickinson). Dex was obtained from Sigma (Taufkirchen, Germany), and CsA and LY294002 from Calbiochem (Schwalbach, Germany).

Isolation and Culture of Thymocytes
Thymocytes were isolated by passing the freshly isolated thymus through a nylon mash followed by repeated washings with PBS. After counting, the thymocytes were resuspended in RPMI+ medium (Invitrogen, Karlsruhe, Germany) at a concentration of 106 cells/ml and cultured in 12-well plates in an incubator at 37 C and 5% CO2.

If the cells were to be stimulated by {alpha}CD28 antibodies, the plates were precoated with sheep-antimouse-Ig in carbonate buffer (Roche, Mannheim, Germany) for 1 h at 37 C and JJ316 and JJ319 directly added at 5 µg/ml into the medium. For TCR stimulation, the plates were incubated with the {alpha}TCR antibody R73 at a concentration of 2 µg/ml in balanced salt solution medium for 2 h at 4 C, followed by repeated washings. Dex was diluted from sterile x1000 stocks in PBS.

Purification of CD4 and CD8 SP Thymocytes
Separation of CD4 SP cells was achieved by magnetic cell sorting starting from 6 x108 total thymocytes (Miltenyi, Bergisch Gladbach, Germany). Aliquots of 1.5 x 108 cells resuspended in balanced salt solution/BSA were stained with Ox8-FITC followed by incubation with {alpha}FITC-coupled magnetic beads. CD8+ cells were first crudely depleted by three concomitant incubation steps in a magnet, each time followed by discarding the bound cells. Afterward the presorted cells were combined and further purified over an LD column according to the manufacturer‘s instructions (Miltenyi). The flow-through containing the CD8- cells was stained with Ox38-PE, followed by incubation with {alpha}PE-coupled magnetic beads. Selection of CD4+ cells was achieved by passing the labeled cells over a LS column followed by repeated washings. CD8 SP thymocytes were similarly purified by first depleting CD4+ cells followed by positive selection of CD8-expressing cells. The average yield of 1 x 107 SP cells corresponds to about 2% of the starting material. Purity was assessed by FACS analysis and routinely found to be greater than 99%.

FACS Analysis
Flow cytometry was performed on a FACSCalibur machine (Becton Dickinson). For intracellular staining, cells were first labeled extracellularly, followed by fixation in 4% paraformaldehyde and permeabilization in a saponin-containing buffer. Cells were then stained with an antibody against the intracellular antigen or an appropriate isotype control. All FACS data were analyzed using the CellQuest software provided by the manufacturer (Becton Dickinson).

Western Blot Analysis
Cells were collected by centrifugation and solubilized in radioimmunoprecipitation buffer. Equal amounts of protein were separated on a 15% SDS-PAGE gel, transferred onto a polyvinylidene difluoride membrane, and stained with the indicated antibodies. All secondary antibodies were peroxidase-coupled and the blots were developed using ECL as a substrate (Amersham, Freiburg, Germany). Normalization of the blots was achieved by staining the blots with antibodies against ß-actin or p56-Lck.

RT-PCR Analysis
RNA was isolated by acid phenol extraction and cDNA synthesized by oligo(dT) priming starting from 0.5–1.0 µg of total RNA as previously described (30). PCR was performed using established primer pairs; the sequences are available upon request. Products were resolved on a 1.5% agarose gel and normalization was achieved on the basis of hypoxanthine phosphoribosyltransferase mRNA expression.


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas Hünig for advice concerning the antibody JJ316, Dr. Fred Lühder for help with the genotyping of the CD28 knockout mice, Kirsty McPherson for critical reading of the manuscript, and Melanie Schott and Katrin Voss for excellent technical help.


   FOOTNOTES
 
This work was supported by grants from the VolkswagenStiftung and the Deutsche Forschungsgemeinschaft (Re1631/1).

Abbreviations: CsA, Cyclosporin A; dex, dexamethasone; DP, double-positive; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; GC, glucocorticoid; GR, glucocorticoid receptor; PE, phycoerythrin; PI3K, phosphatidylinositol 3 kinase; TCR, T cell receptor; SP, single-positive.

Received for publication October 9, 2003. Accepted for publication December 19, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 CONCLUSIONS
 MATERIALS AND METHODS
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