TCR-mediated activation promotes GITR upregulation in T cells and resistance to glucocorticoid-induced death
Yifan Zhan1,
David P. Funda1,2,
Alison L. Every1,
Petra Fundova1,2,
Jared F. Purton3,4,
Douglas R. Liddicoat3,
Timothy J. Cole3,
Dale I. Godfrey4,
Jamie L. Brady1,
Stuart I. Mannering1,
Leonard C. Harrison1 and
Andrew M. Lew1
1 Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia
2 Division of Immunology and Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, Vídenská 1083, 142 20, Prague 4, Czech Republic
3 Department of Biochemistry & Molecular Biology and 4 Department of Microbiology & Immunology, University of Melbourne, Royal Parade, Melbourne 3010, Australia
Correspondence to: A. M. Lew; E-mail: lew{at}wehi.edu.au
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Abstract
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T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR/). We also compared the action of GC on GITR+ and GITR T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR/ mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR/ cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (108 to 106 M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.
Keywords: apoptosis, dexamethasone, GITR, glucocorticoid, T cell activation
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Introduction
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GCs are commonly used as an immunosuppressive and anti-inflammatory agent and they have several effects on T lymphocytes (1). One prominent effect of GC on T cells is to induce cell death (2). GC-induced cell death requires glucocorticoid receptor (GR)-mediated gene transactivation (1,3) and can work via the mitochondria-dependent pathway on which many anti-apoptotic molecules such as Bcl-2, Bcl-XL and IAPs (inhibitors of apoptosis) act. GC can inhibit NF-
B, either directly or by I-
B upregulation, and thereby induce cell death. GC can also result in cell death indirectly by regulating prostaglandin synthesis and nitric oxide generation. Conversely, GC can also inhibit T-cell apoptosis, particularly activation-induced apoptosis, partly by inhibiting upregulation of FasL (2,4). Two molecules induced by GC may mediate this anti-apoptotic effect of GC. One is glucocorticoid-induced leucine zipper (GILZ) that blocks activation-induced FasL upregulation and subsequent apoptosis (5). The other is the glucocorticoid-induced TNF receptor (GITR) (6).
GITR was identified as a new member of the TNF receptor superfamily, by comparing gene expression in untreated and DEX-treated murine T-cell lines (6). GITR can also be induced when T cells are activated (6). Although mouse GITR is induced by either GC engagement or T-cell activation, its human homologue (hGITR/AITR) is upregulated only by activation (7). Therefore, the requirements for GR signaling in inducing GITR expression by T cells remain moot. Functionally, overexpression of GITR in T cell hybridomas inhibited activation-induced cell death but not death induced by apoptotic signals derived from Fas triggering, DEX treatment, or UV irradiation. Primary T cells from GITR-deficient mice were also more susceptible to activation-induced apoptosis than GITR wild-type T cells (8). This evidence indicates that GITR was particularly critical for protection from activation-induced cell death. However, cell activation can also antagonize GC-mediated cell death (2). The molecular basis for the interaction between GC- and activation-induced cell death pathway is still not clear and GITR could be the missing link.
GITR has received great attention recently because it is constitutively and exclusively expressed at high levels on CD25+CD4+ regulatory T cells in naive mice (9,10), albeit that T-cell activation will result in GITR upregulation. Signaling through GITR was proposed to break self-tolerance (10). Stimulation of CD25+CD4+ cells through GITR by anti-GITR antibody abrogates the suppressive effect of such cells on the proliferative responses of CD25CD4+ and CD8+ cells in vitro (9). More importantly, removal of GITR-expressing T cells from transferred cells or administration of anti-GITR antibody resulted in autoimmune disease in animal models (10). These findings point to a critical role for GITR in immune regulation. Given that CD25+CD4+ cells induced by recent activation events are different from regulatory T cells that constitutively express CD25, it is plausible that the ontogeny of CD4+ cells that constitutively express GITR may be also different from activation-induced GITR+ T cells. It remains untested whether T cells that constitutively express GITR can develop in the absence of GR signaling, considering that GITR was first identified as a GC-induced gene. Furthermore, it is also unclear whether this unique subset of CD4+ cells is a target of GC action.
A role for GC in T-cell development has been implicated from the work of Ashwell and colleagues (1113). Previously, we used GR/ reconstituted mice to investigate the role of GC in T-cell development and found that T-cell development and selection were unaffected by the absence of GR signaling (14,15), a result that has been confirmed by others using independently generated GR/ mice (16). In the current study, we tested whether the development of GITR-expressing T cells requires GR signaling in GR/ mice. We then tested the regulation of GITR expression on T cells from GR+/+ and / mice by DEX and anti-CD3 stimulated activation in vitro and analyzed the interrelationship between GITR upregulation and cell fate including apoptosis, proliferation and cytokine production.
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Methods
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Mice
GR+/ mice on a GRMB strain background were backcrossed to the C57BL/6 background greater than nine times. Most GR/ mice die neonatally due to respiratory complications. Therefore, in some experiments, Rag1/ mice reconstituted with fetal liver cells from GR/ and GR+/+ mice were used. To produce reconstituted mice, adult C57BL/6 GR+/ mice were crossed to produce fetal GR/ mice. Fetal mice were removed 14 days after the detection of a plug and liver suspensions were prepared. Fetal tail tissue was genotyped by PCR. Six- to eight-week-old B6 Rag2/ mice were irradiated with a dose of 4 Gy, and then injected with either GR+/+ or GR/ fetal liver cells intravenously after a further 2 h delay. Each recipient received at least 2 x 106 fetal liver cells. Blood samples were taken prior to sacrifice to confirm reconstitution. In rare cases, a few GR/ mice survive to adulthood and when available these were used in this study, together with age/sex-matched GR+/+ littermate.
Flow cytometry
Cell suspensions were prepared from the thymus, spleen and lymph nodes of mice in cold PBS containing 2% FCS, enumerated and stained with combinations of the following antibodies for 20 min at 4°C, before analysis on either one laser, three color FACScan (BD Biosciences, Palo Alto, CA) or a two-laser, four-color LSR (BD Biosciences): anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-T-cell receptor (TCR)
ß (clone H57-597), anti-CD62L (clone MEL-14), anti-CD44 (clone IM7), anti-CD25PE (clone PC61) (all antibodies were purchased from BD Biosciences). Anti-GITR mAb was kindly provided by Professor S. Sakaguchi (Kyoto University, Japan) and was biotinylated in our laboratory. Unconjugated rat anti-mouse CD16 (2.4G2 clone) was used during flow cytometry to block non-specific FcR-mediated binding.
Cell culture
Sterile lymphocytes were isolated and in some instances labeled with CFSE (Molecular Probes, Eugene, OR) by incubation at 10 x 106 cells/ml in 5 µM CFSE for 10 min at 37°C, followed by two washes in PBS containing 1% BSA. Cells were cultured at 5 x 106/ml in DMEM (Life Technologies Inc., Rockville, MD), 10% FCS (CSL, Melbourne, Australia), 2 mM GlutaMax (Life Technologies Inc.) and 50 µg/ml penicillin/streptomycin (Life Technologies Inc.) with either 108 to 106 M DEX (Sigma, St Louis, MO), 10 µg/ml immobilized anti-CD3, or both, for 14 days. Cultured cells were stained for CD4, CD8 and GITR. The numbers of cell divisions after days 14 were determined by the intensity of the CFSE fluorescence.
T-cell enrichment
Lymph node cells were prepared from pooled lymph nodes of four GR+/+ and GR/ mice. Lymph node cells were stained with PE-conjugated anti-CD8 antibody. Stained cells after washing were incubated on ice with anti-PE microbeads for 15 min. Washed cells were applied to autoMACS (Miltenyi Biotec, Germany) and CD8+-enriched cells were positively selected. Purity of the enriched population after double passage through the column was generally >98%.
Apoptosis assay
DEX-induced apoptosis of lymphocytes was measured with a well established method (17). Briefly, the cells were harvested from cultures and washed once with 2% FCSPBS. Cells were then resuspended in 1 ml hypotonic propidium iodide (PI) solution (50 µg/ml in 0.1% sodium citrate and 0.1% Triton-100, Sigma). The cells were placed at 4°C in the dark overnight before flow cytometric analysis.
PCR and Southern blotting analysis
Genotyping of mice at the GR gene locus by PCR or Southern blot analysis was performed as previously described (14,15) except that a 32P-labeled riboprobe was generated from the 1.4 kb HindIII genomic fragment using a Riboprobe kit (Promega, Madison, WI).
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Results
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Differential regulation of GITR expression by T cells in response to anti-CD3 and DEX in GR+/+ and GR/ mice
Because GR/ mice do not generally survive postnatally due to respiratory insufficiency, chimeric mice were generated whereby irradiated Rag-deficient mice were repopulated with GR/ or GR+/+ littermate fetal liver cells. Groups of mice were screened at 612 weeks following reconstitution. The GR genotypes of the hematopoietic cells in reconstituted mice were confirmed by PCR of thymocyte DNA (Fig. 1a) and Southern blot (data not shown). As expected, in GR/ fetal liver reconstituted mice, cells of the wild-type genotype were also detected (e.g. GR+/+ stromal cells from host Rag/ mice that were resistant to low-dose irradiation). Nevertheless, the majority of cells from reconstituted mice were GR/. The resistance of T cells from GR/ reconstituted mice to DEX-mediated cell death also confirmed the GR status (see below). A few GR/ mice survive to adulthood. In experiments with adult bona fide GR/ mice, genotypes were also checked by PCR (Fig. 1b). All experiments were repeated three times with reconstituted mice and confirmed with adult GR/ mice.

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Fig. 1. GR status of reconstituted and adult GR/ mice. (a) PCR analysis was performed on genomic DNA prepared from the thymocytes of mice reconstituted with GR+/+ or GR/ fetal liver cells after 8 weeks. The plot shows the PCR products of three reconstituted mice from each group. (b) PCR analysis was performed on genomic DNA prepared from the thymocytes of adult GR/ mice and sex/age-matched GR+/+ littermate controls. The plot shows the PCR products of three reconstituted mice from each group.
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It has been reported that both DEX and anti-CD3 can upregulate GITR expression on T cells. To test whether GITR upregulation requires GR signaling, GR/ and GR+/+ cells were isolated and stimulated in vitro with DEX, anti-CD3, or both. From the spleens of GR/ and +/+ reconstituted mice, GITR-negative cells were purified by FACS sorting and cultured. Most surviving T cells in the unstimulated cultures remained GITR negative (Fig. 2a). Stimulation with anti-CD3, with or without DEX (108 to 106 M), resulted in a dramatic increase in the percentages of both CD4+ and CD8+ GITR+ T cells from both GR/ and +/+ mice. By 40 h, all T cells had upregulated GITR. As expected, most of the GR+/+ lymphocytes cultured with DEX alone (107 M) died and only a small proportion of remaining live cells had high GITR expression (data not shown). To test DEX-induced GITR upregulation further, purified CD8+ T cells (without constitutive high expression of GITR) from GR+/+ and GR/ adult mice were stimulated with a range of doses of DEX (108 to 106 M) or anti-CD3 (10 µg/ml). After 20 h, when a significant proportion of cells was still alive, there was no significant upregulation of GITR on live non-stimulated CD8+ T cells. While anti-CD3 antibody stimulated high levels of GITR expression on nearly all CD8+ T cells (Fig. 2b), GC was not effective at inducing GITR upregulation. Interestingly, the intensity of GITR staining was higher on cells that had proliferated than on undivided cells in the same culture (Fig. 2c).

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Fig. 2. (a) GITR upregulation by sorted GITR negative CD4+ and CD8+ T cells from GR+/+ and / reconstituted mice in vitro. Cells were cultured at 5 x 106 cells/well in 2 ml in 24-well plates in medium alone, DEX (107 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. Cells were cultured for 1 to 3 days. The data from 40 h culture are plotted. The percentage of GITR+ T cells is shown in bold. (b) GITR upregulation by purified CD8+ T cells from adult GR+/+ and / mice in vitro. Cells were cultured at 5 x 106 cells/well in 1 ml in 24-well plates in medium alone, DEX (108 to106 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. Cells were cultured for 20 h and the plots show the data from 106 M DEX culture. The dashed line shows the GITR staining of unstimulated cells. (c) GITR expression on proliferated CD8+ T cells. CSFE-labeled LN cells from GR+/+ and GR/ reconstituted mice were cultured for 3 days as in Fig. 2(A). Gated proliferated CD8+ T cells from anti-CD3 antibody cultures were plotted for GITR expression and CSFE profile. The mean fluorescence intensity of GITR staining of CSFElow (proliferated >2 divisions) and CSFEhigh unproliferated is also shown. The mean fluorescence intensity of GITR staining of unstimulated CD8+ T cells is <30 in both GR+/+ and GR/ mice (data not shown).
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T-cell death in GR/ mice
As GITR has been reported to function primarily as an anti-apoptotic molecule, we tested whether GITR expression would render cells resistant to cell death induced by DEX and anti-CD3. Not surprisingly, DEX treatment resulted in the loss of live T cells (as well as other leukocytes) in GR+/+ mice but not in GR/ mice (Fig. 3a). Upon stimulation with anti-CD3 antibody, T cells from both adult GR/ and +/+ mice behaved similarly. The numbers of viable cells were reduced slightly in anti-CD3 antibody stimulated cultures of both adult GR/ and +/+ mice, compared with those in the unstimulated cultures (Fig. 3a). The reduction was consistent in all the experiments but not statistically significant. In GR+/+ mice, the level of cell death induced by DEX was diminished in the presence of anti-CD3 antibody on day 2 (and day 3) of culture. The presence of more viable cells after culture with both DEX and anti-CD3 antibody could not be attributed solely to anti-CD3-induced proliferation, because the proliferation at 2 days of culture was very limited, as determined by CFSE staining (data not shown).

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Fig. 3. Cell death in culture. (a) Recovery of live cells: splenocytes from three individual adult GR/ and GR+/+ mice were cultured at 5 x 106/well in 2 ml in 24-well plates in the presence of medium alone, DEX (107 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. Cells were cultured for 1 to 3 days and live cells (trypan blue excluding) were counted. Data from day 2 cultures are shown. The numbers of CD4+ and CD8+ T cells were calculated from the total cell numbers and the percentage of subsets in the culture. An asterisk indicates P-value <0.05, compared with untreated cultures. (b) Apoptosis of CD8+ T cells: CD8+ T cells were purified and cultured at 5 x 106 cells/well in 1 ml in 24-well plates in medium alone, DEX (108 to106 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. Apoptosis of CD8+ T cells was assayed. The numbers in the plots show the % of cell death at 20 h after culture. (c) Recovery of GITR+ and GITR CD4+ T cells: splenocytes from adult GR/ and GR+/+ mice were cultured at 5 x 106/well in 1 ml in 24-well plates in the presence of medium alone, DEX (106 M). Cells were cultured for 20 h and then harvested, stained with CD4 and GITR. The gated living CD4+ T cells (PI negative) were plotted for their GITR expression. The mean percentages of GITR+ cells from duplicated cultures are shown.
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To examine T-cell apoptosis directly, purified CD8+ T cells (without constitutive GITR expression) from adult GR+/+ and GR/ mice were treated with DEX and anti-CD3. Indeed, consistent with the live cell recovery in Fig. 3(a), DEX (108 to 106 M) induced greater cell death above background (Fig. 3b). CD8+ T cells from GR/ adult mice were resistant to GC-mediated apoptosis and did not show significant death above background.
Another interesting observation was that the constitutive expression of GITR by a subset of CD4+ cells did not confer significant resistance to DEX-mediated killing. In bulk cultures of splenocytes that contained substantial numbers of CD4 cells with constitutive high GITR levels (1020% of total CD4 cells), stimulation with DEX resulted in a dramatic loss of live cells from adult GR+/+ mice. The proportion of GITR+ CD4+ T cells over GITR CD4+ T cells was not greatly changed by DEX treatment (Fig. 3c). These findings indicated that CD4+ T cells that had constitutively high levels of GITR from GR+/+ mice had no clear survival advantage in the presence of DEX.
Cytokine production by T cells and proliferation of T cells in GR/ mice
We tested here the production of IFN-
and IL-2 by T cells stimulated with anti-CD3 antibody. IFN-
production was substantially suppressed by DEX in GR+/+ reconstituted splenocytes but not in GR/ reconstituted splenocytes (Fig. 4). Considering that the difference in the cell numbers between anti-CD3 antibody treatment and anti-CD3 antibody /DEX treatment was not greatly different (Fig. 3a), especially in day 23 cultures, the differences in cytokine production were unlikely to be due to DEX-mediated cell death, but rather due to suppression of cytokine production by T cells. Despite the fact that DEX markedly suppressed anti-CD3-induced cytokine production by GR+/+ T cells, cell proliferation was not significantly inhibited. In fact, in GR+/+ mice, anti-CD3 and DEX together can be more stimulatory than anti-CD3 alone (Fig. 5). For example, the numbers of divisions were consistently greater in anti-CD3 and DEX-stimulated cultures than in culture with anti-CD3 antibody alone. This was the case for both CD8 T cells and CD4 T cells. However, we cannot discount the possibility that cells that had divided further had become more resistant to DEX-mediated cell death and selectively accumulated in the cultures. As expected, in the absence of GR signaling, DEX did not inhibit T-cell proliferation by anti-CD3 antibody (Fig. 5). T cells from GR+/+ mice proliferated to a similar extent as the cells from GR/ mice in the presence of 107 M DEX and anti-CD3 antibody. Notably, CD8 T cells proliferated faster than CD4 cells in response to anti-CD3 antibody in the same culture and cells did not proliferate without anti-CD3 antibody.

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Fig. 4. Cytokine production. Splenocytes adult GR/ mice and GR+/+ littermate were cultured at 5 x 106/well in 2 ml in 24-well plates in the presence of medium alone, DEX (107 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. Supernatants were harvested at 1 day after culture for IL-2 and IFN- assays. The data represent the mean and SD of three individual mice per group. Asterisks indicate P < 0.01, compared with anti-CD3 antibody stimulated culture without DEX.
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Fig. 5. Proliferation of CD8+ (upper panel) and CD4+ cells (lower panel) in culture. CSFE-labeled splenocytes from adult GR/ mice and GR+/+ controls were cultured at 5 x 106/well in 2 ml in 24-well plates for 3 days in the presence of medium alone, DEX (107 M), anti-CD3 (10 µg/ml, immobilized) or both DEX and anti-CD3 antibody. The numbers in the panels indicate the percent of proliferated cells.
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Normal development of GITR+ T-cell subsets in GR/ mice
In both the GR+/+ and GR/ reconstituted mice, the proportions (% ± SD) of GITR+CD4+ T cells of total CD4 T cells in lymph nodes were comparable (9.5 ± 1.9 vs 10.1 ± 1.4, respectively) (Fig. 6a). Similar proportions of CD25+CD4+ cells were seen in GR+/+ and GR/ reconstituted mice (11.5 ± 1.0 vs 9.3 ± 1.1, respectively) (Fig. 6a). However, when gated CD4+ cells were plotted for expression of GITR and CD25 (Fig. 6a-iii), only two thirds of those CD4+ cells expressing high levels of GITR expressed CD25, regardless of GR genotype. In both GR+/+ and GR/ reconstituted mice, CD25CD4+ cells expressed lower levels of GITR (mean fluorescence intensity = 62 ± 4) compared with CD25+CD4+ cells (mean fluorescence intensity = 79 ± 6). CD8 T cells from both GR+/+ and GR/ reconstituted mice were either negative or very low for GITR expression. There were very few GITR+ cells (<1%) in the TCR
ß population (data not shown).

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Fig. 6. Development of GITR-expressing T cells. (a) Lymph node cells from rag-deficient mice reconstituted with GR+/+ or / fetal liver cells for 8 weeks were stained for TCR ß, GITR, CD25, and CD4 or CD8. (b) Thymocytes from reconstituted mice were stained for CD4, CD8 and GITR.
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The presence of GITR+ cells in the thymus was also compared between the GR+/+ and GR/ reconstituted mice (Fig. 6b). Only CD4+CD8 cells expressed appreciable levels of GITR and the percentage of GITR+ CD4+CD8 cells was comparable between the GR+/+ mice (4.8 ± 0.9) and GR/ reconstituted mice (6.4 ± 0.2). Very few cells of the other thymic subpopulations (CD4CD8, CD4CD8+, CD4+CD8+) expressed GITR. Results from the adult GR/ mice were similar to those from reconstituted mice (data not shown).
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Discussion
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Most of the interest in GITR in T-cell biology has focused on its expression and function by regulatory T cells (particularly CD25+CD4+ cells). Because GITR can be induced by GC signaling and TCR-mediated activation, the signaling requirement for the development of T regulatory cells has not been resolved. By using GR/ mice, we observed that the development of CD4 T cells (in thymic and peripheral compartments) that constitutively expressed GITR is not dependent on GR signaling. It remains to be elucidated how this subpopulation regulated their GITR expression. Enforced expression of Foxp3, a marker reported to be specific for regulatory T cells, has increased expression of GITR but not other activation markers, OX40 and CTLA-4 on CD25CD4+ T cells and these GITR+CD25CD4+ cells can be regulatory (18). This suggests that the GITR expression by this subpopulation may differ from that induced by GC and classical anti-CD3 induced activation. As GITR functions mainly as an anti-apoptotic molecule, it is interesting to determine whether constitutively expressed GITR confers protection from DEX-induced cell death. A recent report shows that CD4+CD25+ T cells (with constitutive expression of GITR) were indeed relatively resistant to cell death (19). However, cells constitutively expressing GITR showed no clear resistance to DEX-mediated cell death in our study at least at 20 h. Our data were more in line with the initial report that GITR overexpression in a CD4+ T cell line protected from TCR-induced apoptosis but not from DEX-induced cell death (6).
A somewhat neglected aspect of GITR research is its regulation on the majority of T cells (including CD8+ and CD4+ T cells that do not have high constitutive expression of GITR) upon stimulation. Despite GITR being firstly identified to be induced by GC and activation (6), we found that a range of doses of DEX induced very limited GITR upregulation on T cells, whereas anti-CD3 induced high levels of GITR expression. Both CD4+ and CD8+ subsets from both GR+/+ and GR/ reconstituted mice upregulated GITR expression in response to anti-CD3 antibody. This indicates that activation-induced GITR expression is also independent of GR signaling. As mentioned above, there is compelling evidence for the role of GITR in protecting against TCR-activation induced cell death. When mice are made genetically deficient in GITR, their primary T cells become more susceptible to activation-induced cell death (8). Moreover, over-expression of GITR in a T cell line conferred the ability to resist cell death induced by TCR-mediated activation (6). GITR transfection however, did not protect against death induced by other stimuli like Fas triggering, UV irradiation or DEX treatment (6). Thus, it seems (from these reports and our own findings in wild-type mice) that GITR upregulation may be more important in protecting T cells against death induced by TCR activation than that from DEX treatment. How is it then that GITR upregulation during T-cell activation might protect against DEX-induced killing? There are at least two explanations. Firstly, pro-survival molecules (e.g. bcl-x) other than GITR are induced by T-cell activation (20) and these may then protect against DEX-induced death. Secondly, TCR-activation induced death pathways and steroid-induced death pathway are mutually antagonistic (2). This second tenet would predict that in GR/ mice, there should be enhanced cell death from TCR activation compared with wild-type T cells. We did not find this in our experimental setting. A tentative explanation is that mature T cells, unlike T cell hybridomas (2) and immature lymphocytes (21), are relatively resistant to anti-CD3 mediated apoptosis and thus GC-mediated protection from TCR-mediated apoptosis is not absolutely required for cell survival.
Besides cell death, cytokine production and cell proliferation were also compared between GR+/+ and GR/ mice under stimulation of both DEX and anti-CD3 antibody. We demonstrated previously that T cells from GR+/+ and GR/ reconstituted mice proliferate equally to stimulation with anti-CD3 antibody (14). Here we showed that addition of DEX at concentration of 107 M did not inhibit the anti-CD3 antibody-induced proliferation of GR+/+ T cells but significantly suppressed cytokine production. At higher doses (106 M) of DEX, proliferation of T cells from GR+/+ but not GR/ reconstituted mice was suppressed (data not shown). Given that the growth factor IL-2 was greatly reduced in the presence of 107 M DEX, it was quite surprising that GR+/+ T cells in the co-presence of anti-CD3 antibody and DEX proliferated equally well, if not better, than cells in the absence of DEX. Not surprisingly, DEX did not affect proliferation of T cells from GR/ reconstituted mice. The relationship between GITR signaling and proliferation is not clear. We observed that uniform upregulation of GITR by anti-CD3-stimulated T cells occurred in the first 24 h and thus was prior to cell proliferation. However, we did observe that T cells that have divided expressed higher levels of GITR on their surface than undivided cells. It has been reported that GITR-deficient T cells proliferated more when given a T-cell stimulus (8). On the other hand, agonistic anti-GITR antibody does lead to the proliferation of T cells, including CD25+CD4+ T cells that normally do not proliferate, in a dose-dependent fashion in the presence of IL-2 (10). Anti-GITR antibody also enhanced anti-CD3-stimulated proliferation of CD28-deficient T cells (10). How GITR signaling influences cell proliferation remains to be elucidated.
An integral part of GITR signaling includes interaction with its natural ligand (GITRL). Human GITRL was identified as a member of the TNF family expressed on endothelial cells. Mouse GITRL was recently identified (22). Purified mouse GITRL can enhance proliferation and thus was proposed as a costimulatory molecule for T cells. Mouse GITRL was expressed on B cells, macrophages and dendritic cells but not on T cells. Mouse GITRL seems not to influence cell death directly. In our study, even though most of the T cells were derived from GR/ fetal livers, GC could still act on host cells that were GR+/+ (e.g. stromal cells, macrophages) and would be expected to express GITRL. In bulk cultures from GR/ reconstituted mice, a small proportion of cells upregulated GITR upon DEX stimulation alone (data not shown). However, there was no upregulation of GITR in GR/ T cells when the starting populations were sorted for GITR T cells (Fig. 2A). This would indicate that in the unsorted bulk cultures, the modest effect of DEX on GR/ T cells was not cell autonomous (i.e. indirectly elicited via contaminating GR+/+ macrophages, dendritic cells and other non-T cells from the original Rag-deficient host). Indeed, data from genuine adult GR/ mice showed that DEX has no effect on GITR expression, cell death, cell proliferation and cytokine production. As GITR is associated with both anti-apoptotic (cell activation and proliferation) and pro-apoptotic (cell death) processes, the potential of cells constitutively expressing GITR to regulate immune responses positively or negatively warrants more investigation. Given that GC are commonly used agents for the treatment of immuno-inflammatory disease, studies of GC-induced molecules such as GITR may reveal molecular mechanisms by which GC exert their anti-inflammatory and immunosuppressive functions. Moreover, the implication of our finding that T-cell activation leads to GITR upregulation and resistance to GC-induced cell death is that GC is less effective in suppressing T-cell mediated immune responses (e.g. transplantation rejection, rheumatoid arthritis) once the T cells are activated.
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Acknowledgements
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We are grateful for funding support by Program grants from the NIH, National Health & Medical Research Council of Australia and Juvenile Diabetes Research Foundation.
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Notes
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Transmitting editor: A. Kelso
Received 22 February 2004,
accepted 23 June 2004.
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References
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- Ashwell, J. D., Lu, F. W. and Vacchio, M. S. 2000. Glucocorticoids in T cell development and function. Annu. Rev. Immunol. 18:309.[CrossRef][ISI][Medline]
- Zacharchuk, C. M., Mercep, M., Chakraborti, P. K., Simons, S. S. Jr and Ashwell, J. D. 1990. Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic. J. Immunol. 145:4037.[Abstract/Free Full Text]
- Riccardi, C., Cifone, M. G. and Migliorati, G. 1999. Glucocorticoid hormone-induced modulation of gene expression and regulation of T-cell death: role of GITR and GILZ, two dexamethasone-induced genes. Cell Death Differ. 6:1182.[CrossRef][ISI][Medline]
- Yang, Y., Mercep, M., Ware, C. F. and Ashwell, J. D. 1995. Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: inhibition of Fas ligand expression by retinoic acid and glucocorticoids. J. Exp. Med. 181:1673.[Abstract]
- D'Adamio, F., Zollo, O., Moraca, R. et al. 1997. A new dexamethasone-induced gene of the leucine zipper family protects T lymphocytes from TCR/CD3-activated cell death. Immunity 7:803.[ISI][Medline]
- Nocentini, G., Giunchi, L., Ronchetti, S. et al. 1997. A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis. Proc. Natl Acad. Sci. USA 94:6216.[Abstract/Free Full Text]
- Kwon, B., Yu, K. Y., Ni, J. et al. 1999. Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand. J. Biol. Chem. 274:6056.[Abstract/Free Full Text]
- Ronchetti, S., Nocentini, G., Riccardi, C. and Pandolfi, P. P. 2002. Role of GITR in activation response of T lymphocytes. Blood 100:350.[Abstract/Free Full Text]
- McHugh, R. S., Whitters, M. J., Piccirillo, C. A., Young, D. A., Shevach, E. M., Collins, M. and Byrne, M. C. 2002. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[ISI][Medline]
- Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. and Sakaguchi, S. 2002. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3:135.[CrossRef][ISI][Medline]
- Vacchio, M. S. and Ashwell, J. D. 2000. Glucocorticoids and thymocyte development. Semin. Immunol. 12:475.[CrossRef][ISI][Medline]
- Vacchio, M. S., Papadopoulos, V. and Ashwell, J. D. 1994. Steroid production in the thymus: implications for thymocyte selection. J. Exp. Med. 179:1835.[Abstract]
- Vacchio, M. S., Lee, J. Y. and Ashwell, J. D. 1999. Thymus-derived glucocorticoids set the thresholds for thymocyte selection by inhibiting TCR-mediated thymocyte activation. J. Immunol. 163:1327.[Abstract/Free Full Text]
- Purton, J. F., Zhan, Y., Liddicoat, D. R., Hardy, C. L., Lew, A. M., Cole, T. J. and Godfrey, D. I. 2002. Glucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice. Eur. J. Immunol. 32:3546.[CrossRef][ISI][Medline]
- Purton, J. F., Boyd, R. L., Cole, T. J. and Godfrey, D. I. 2000. Intrathymic T cell development and selection proceeds normally in the absence of glucocorticoid receptor signaling. Immunity 13:179.[ISI][Medline]
- Brewer, J. A., Khor, B., Vogt, S. K. et al. 2003. T-cell glucocorticoid receptor is required to suppress COX-2-mediated lethal immune activation. Nat. Med. 9:1318.[CrossRef][ISI][Medline]
- Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F. and Riccardi, C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271.[CrossRef][ISI][Medline]
- Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[CrossRef][ISI][Medline]
- Chen, X., Murakami, T., Oppenheim, J. J. and Howard, O. M. 2004. Differential response of murine CD4+CD25+ and CD4+CD25 T cells to dexamethasone-induced cell death. Eur. J. Immunol. 34:859.[CrossRef][ISI][Medline]
- Broome, H. E., Dargan, C. M., Krajewski, S. and Reed, J. C. 1995. Expression of Bcl-2, Bcl-x and Bax after T cell activation and IL-2 withdrawal. J. Immunol. 155:2311.[Abstract]
- Ashwell, J. D., King, L. B. and Vacchio, M. S. 1996. Cross-talk between the T cell antigen receptor and the glucocorticoid receptor regulates thymocyte development. Stem Cells 14:490.[Abstract]
- Tone, M., Tone, Y., Adams, E., Yates, S. F., Frewin, M. R., Cobbold, S. P. and Waldmann, H. 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Natl Acad. Sci. USA 7:7.[CrossRef]