Immunology and Cell Biology (2014) 92, 148–155 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

ORIGINAL ARTICLE

Glucocorticoid-mediated repression of T-cell receptor signalling is impaired in glucocorticoid receptor exon 2-disrupted mice This paper has been corrected since online publication and a corrigendum also appears in this issue.

Douglas R Liddicoat1,2,3, Jared F Purton1,4, Timothy J Cole3,5 and Dale I Godfrey1,5 Studies using glucocorticoid receptor exon 2-disrupted knockout (GR2KO) mice provided strong evidence against an obligatory role for glucocorticoid receptor (GR) signalling in T-cell selection. These mice express a truncated form of the GR that is incapable of transmitting a range of glucocorticoid (GC)-induced signals, including GC-induced thymocyte death. However, one study that suggested that truncated GR function is preserved in the context of GR-mediated repression of T-cell activationinduced genes, challenged earlier conclusions derived from the use of these mice. Because GR versus T-cell receptor (TCR) signalling cross-talk is the means by which GCs are hypothesized to have a role in T-cell selection, we reassessed the utility of GR2KO mice to study the role of the GR in this process. Here, we show that GR-mediated repression of TCR signalling is impaired in GR2KO T cells in terms of TCR-induced activation, proliferation and cytokine production. GC-induced apoptosis was largely abolished in peripheral T cells, and induction of the GC-responsive molecule, interleukin-7 receptor, was also severely reduced in GR2KO thymocytes. Together, these data strongly re-affirm conclusions derived from earlier studies of these mice that the GR is not obligatory for normal T-cell selection. Immunology and Cell Biology (2014) 92, 148–155; doi:10.1038/icb.2013.76; published online 12 November 2013 Keywords: glucocorticoids; glucocorticoid receptor; T-cell receptor; T-cell selection The majority of actions exerted by glucocorticoids (GCs) occur by their binding to the glucocorticoid receptor (GR), a cytosolic, ligandactivated transcription factor, expressed in nearly all nucleated cells. Upon ligand binding, the GR modulates gene expression by two general mechanisms: (1) binding of GC response elements found in the regulatory regions of GC responsive genes (transactivation) and (2) interference with other signalling molecules and transcription factors, mostly by direct protein–protein interaction (transrepression).1 In the immune system, high concentrations of GCs induced by the stress response downregulate T-cell proliferation and cytokine production and induce thymocyte apoptosis.2 Interestingly, T-cell receptor (TCR) stimulation has been shown to rescue thymocytes and peripheral T cells from GC-induced cell death.3–5 The cornerstone of these effects is a complex ‘cross-talk’ between multiple effector molecules key to GC and TCR signalling pathways.1,2 This cross-talk phenomenon prompted a series of investigations exploring the possibility of a functional role for GCs in T-cell selection (reviewed in2,6,7). In the 1990s, a significant role for

GCs in T-cell selection was suggested by experiments using chemical inhibitors of GC synthesis and action, and in particular, the generation of a T-cell-specific, GR anti-sense transgenic mouse line driven by the lck-promoter.8 Despite only an B50% reduction in GR expression in these mice, they exhibited greatly decreased thymic cellularity and a profound blockade of thymocyte development at the CD4 CD8  to CD4 þ CD8 þ transition stage, which suggested that thymocyte development is exquisitely sensitive to suboptimal GR signalling. These studies formed the basis of the ‘mutual antagonism’ hypothesis, whereby GR and TCR signalling cross-talk in thymocytes sets the thresholds for positive and negative selection, thus regulating normal T-cell development.2 This hypothesis was challenged by two studies using an independently developed lck-promoter-driven GR anti-sense mouse line, as well as a neurofilament-promoterdriven GR anti-sense mouse line, where in both cases, an increase (rather than a decrease) in thymic cellularity was reported.9,10 Subsequently, the role of GCs in T-cell selection has been investigated using GR exon 2 knockout (GR2KO) mice.11,12 The

1Department of Microbiology and Immunology, University of Melbourne, Victoria, Australia; 2Department of Immunology, Monash University, Victoria, Australia; 3Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia; 4Department of Immunology, Scripps Research Institute, La Jolla, CA USA 5Co-chief investigators Correspondence: Associate Professor TJ Cole, Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia. E-mail: [email protected] or Professor DI Godfrey, Department of Microbiology and Immunology, University of Melbourne, Grattan St, Parkville, Victoria 3010, Australia. E-mail: [email protected] Received 2 September 2013; accepted 15 October 2013; published online 12 November 2013

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gene-targeting strategy involved disruption of exon 2, the first protein-coding exon of the GR gene, by insertion of a neomycin resistance cassette. GR2KO mice were found to be profoundly resistant to GC signalling, and exhibited severely impaired hypothalamus–pituitary–adrenal axis feedback, as well as perinatal lethality owing to retarded lung maturation.11 Studies using GR2KO mice failed to support a role for GCs in T-cell development. Analysis of the GR2KO fetal thymus, combined with fetal thymus organ culture experiments, revealed seemingly normal thymocyte development and normal positive and negative T-cell selection.12 These results were extended using rare surviving GR2KO adult mice, as well as wild-type (WT) irradiated mice reconstituted with GR2KO fetal liver precursors.13 This study showed no difference between GR2KO and WT mice in terms of peripheral T-cell homoeostasis, including T-cell subset numbers and proportions, proliferative ability, as well as activation and memory status. In strong support of these results, normal T-cell subset numbers and proportions have also been found in three independently generated GR knockout mouse lines, one carrying a global deletion of GR gene exons 1C and 2,14 and two carrying a T-cell-specific deletion of exons 1C and 215 or 316 using the loxP/Cre system. Normal thymocyte development was also observed in GR dimerization mutant mice that were incapable of transmitting GC signals associated with transactivation.17 However, in direct contrast with the abovementioned GR knockout lines, a new report of an independently generated T-cell-specific GR exon 3 knockout mouse line exhibiting defective T-cell development, has re-ignited this field. These mice exhibited a moderate reduction (B50%) in thymic cellularity, an altered T-cell repertoire as indicated by TCR Vb CDR3 analysis and reduced T-cell proliferative responses.18 Further studies will be required to shed light on this intriguing departure from the results of the four previously mentioned GR knockout models. Previously, we reported that while GR2KO mice carry a 39 kDa truncated C-terminal fragment of the GR, they are nonetheless refractory to the GR signalling-dependent functions tested, including GC-induced thymocyte apoptosis and induction of GC-response genes.11,12,19–21 However, one study used cDNA microarray to address whether the truncated GR in this line can still confer GC responsiveness by investigating the effect of GCs on phorbol 12-myristate 13-acetate (PMA)/ionomycin-mediated activationinduced genes in WT versus GR2KO thymocytes.22 From this analysis it was suggested that the GR-mediated transrepression was largely intact in GR2KO thymocytes, suggesting that truncated GR in GR2KO thymocytes retains signalling activity via the C-terminal domain of the GR. The authors therefore called into question the utility of GR2KO mice to study the role of GCs in T-cell selection. Given GR2KO mice represent an important source of evidence in the debate surrounding the role of GR signalling in T-cell selection, and ongoing studies rely on the use of GR2KO mice, we decided to directly assess GC-mediated inhibition of TCR signalling in T cells Alternative first exons

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from these mice. In this study, we measured the effect of GCs on TCR-induced activation, proliferation and cytokine production in WT and GR2KO T cells. We show that GC-mediated repression is clearly impaired in GR2KO T cells, with intermediate effects in GR2 heterozygous T cells, indicating that the truncated GR possesses little biological activity in terms of inhibition of TCR signalling. These findings support the use of GR2KO mice in earlier studies to investigate the hypothesized role of GC signalling in intra-thymic T-cell selection.12,13 Taken together with independent studies using a range of GR mutant mice,14–17 we maintain that mutual antagonism involving GR-mediated inhibition of TCR signalling does not have a key role in intra-thymic T-cell selection.

RESULTS GC-mediated repression of T-cell activation and proliferation is severely impaired in GR2KO mice The GR gene is composed of multiple alternative non-protein-coding first exons, followed by eight protein-coding exons (2–9). The genetargeting strategy in GR2KO mice involved disruption of exon 2 by insertion of a neomycin resistance cassette (Figure 1). Previously, we reported that although GR2KO mice are profoundly GC resistant, western blot analysis using antibodies to the GR C-terminus showed that they carry a 39 kDa truncated C-terminal GR fragment of sufficient molecular weight to contain the ligand-binding domain (LBD), but not a complete or functional DNA-binding domain.19 Although the truncated fragment allows binding of dexamethasone (DEX) at 30–60% the level of WT tissues, GR2KO mice were shown to be refractory to GC signalling, as demonstrated by their inability to induce multiple GC-response genes and their resistance to GCinduced thymocyte apoptosis.11,12,19–21 Prompted by our report of the truncated GR transcript, Mittelstadt and Ashwell22 analysed GR2KO fetal thymocytes by cDNA microarray to explore if the truncated GR can still confer GC responsiveness. From this analysis it was concluded that some types of GR-mediated modulation, in particular transrepression of PMA/ionomycin-mediated activationinduced genes, was preserved in GR2KO thymocytes. To further investigate this, we established experimental conditions that show clear DEX-mediated inhibition of T-cell activation and proliferation in WT mouse splenocytes, and investigated whether this repressive effect persisted in GR2KO splenocytes. Carboxyfluorescein succinimidyl ester (CFSE)-labelled GR2KO splenocytes were stimulated with immobilized anti-CD3 antibody in the presence of a range of concentrations of DEX for 40 h. These cells were then analysed for activation by cell surface staining for interleukin-2 (IL-2) receptor subunit-a (CD25), as well as proliferation (CFSE dilution). Anti-CD3 stimulation upregulated CD25 mean fluorescence intensities (MFIs) on T cells by B2 decades compared to nonstimulated controls (data not shown). As expected, high concentrations of DEX inhibited activation and proliferation in WT

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Figure 1 Structure of the GR gene in GR2KO gene-targeted mice. Schematic diagram of the GR2KO allele with exons shown as black squares (numbered above), and the inserted neomycin resistance cassette shown in grey. The left arrow indicates the position of the normal translation start site (Met1), whereas the right arrow indicates the translation start (Met487) in the gene-targeted allele. Immunology and Cell Biology

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Figure 2 GC-mediated repression of activation and division of splenic T cells is severely impaired in GR2KO mice. DEX dose–response of CFSE-labelled splenic cell cultures from adult GRMB mice treated with plate-bound anti-CD3 for 40 h. (a) Following culture, the cells were stained with anti-CD25, CD8 and CD4 antibodies, and analysed by flow cytometry. At bottom-left of each dot plot, figures representing percentage of cells in each quadrant are shown. Data is representative of results that are summarized in b and c. (b) Percentage of maximally activated (CD25 þ ) cells calculated as described in METHODS. (c) Percentage of maximally proliferated cells as determined by CFSE staining, calculated as described in METHODS. (b,c) Results depict mean±s.e. for splenocyte samples collected from separate mice over three independent experiments. GR þ / þ : n ¼ 6, GR þ / : n ¼ 7 and GR / : n ¼ 7. ** ¼ different to GR þ / þ (Po0.01) by the two-tailed Mann–Whitney test. Data from GR þ /  mice was not statistically compared with the other groups.

splenocytes with 40% inhibition of CD25 expression and B60% inhibition of proliferation at the highest dose of DEX tested (100 nM). However, in GR2KO mice, this repressive effect was almost completely lost, whereas heterozygous mice exhibited intermediate levels of repression (Figure 2).

supernatant by ELISA. As expected, DEX strongly inhibited TCRmediated IL-2 production by WT splenocytes. However, this repressive effect was dramatically inhibited in GR2KO splenocytes, whereas heterozygous splenocytes exhibited an intermediate level of repression (Figure 3d).

GC-mediated repression of cytokine production is severely impaired in GR2KO T cells To further test for GR-mediated transrepression of TCR signalling in GR2KO T cells, we examined the well-documented repressive effect of GCs on interferon-g (IFN-g) production.23–26 Splenocytes were stimulated with immobilized anti-CD3 antibody in the presence of a range of concentrations of DEX for 20 h and IFN-g production was assessed using intracellular cytokine staining. As expected, IFN-g production by WT splenocytes was strongly inhibited by DEX in a dose-dependent manner, with the number of IFN-g producing cells reduced by B80% at the highest dose tested (100 nM). In addition, within the IFN-g þ cell compartment, Dex also strongly inhibited the intensity of IFN-g staining (MFI) by B70%. These repressive effects were largely abrogated in GR2KO splenocytes, whereas heterozygous splenocytes exhibited intermediate levels of GC-mediated repression of TCR-mediated IFN-g production (Figures 3a–c). Like IFN-g, activated GR also inhibits IL-2 production by activated T cells.24–27 We tested whether this repressive effect of GCs on IL-2 persists in GR2KO splenocytes by measuring IL-2 levels in culture

Peripheral T cells from GR2KO mice are refractory to GC-induced apoptosis We have previously reported that GR2KO mouse thymocytes are completely resistant to GC-induced apoptosis.12,13 However, the severity of the knockout phenotypes may differ between different developmental stages, and thus it is important to examine whether this hallmark of GC sensitivity is also absent in peripheral T-cell subsets. To address this question, adult GR2KO and WT splenocytes were cultured with and without 100 nM Dex and the viabilities of splenic T cells were determined using a live lymphocyte gate (Figure 4a). As expected, Dex potently induced apoptosis in WT T cells, reducing cell viabilities to B20% of Veh levels. In contrast, this effect was largely, albeit not completely, abolished in GR2KO mice. Next, to examine activated peripheral T cells, we repeated the above experiment in the presence of anti-CD3 antibody stimulation (Figure 4b). At the early 20 h timepoint, activated WT splenocytes were beginning to show a lower viability than their GR2KO counterparts, and by 40 h Dex decreased WT CD4 þ and CD8 þ splenocyte viabilities to 35% and 60% of Veh levels, respectively. In contrast,

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Figure 3 GC-mediated repression of IFN-g and IL-2 production in splenic T cells is severely impaired in GR2KO mice. DEX dose–response of adult GRMB mouse splenic cell cultures treated with plate-bound anti-CD3 for 20 h. (a) Following culture, cells were stained with anti-IFN-g, CD8 and CD4 antibodies, and analysed by flow cytometry. Percentage of cells in the IFN-g positive gate are shown at top-left of each dot plot. Data is representative of results summarized in b and c. (b) Percentage maximal IFN-g positive cells, as calculated in METHODS. (c) Percentage maximal IFN-g MFI (gating on IFN-g positive cells) as calculated in METHODS. (d) Percentage maximal IL-2 production, determined by ELISA on culture supernatants, calculated as in METHODS. (b–d) Results depict mean±s.e. for splenocyte samples collected from separate mice over three independent experiments. GR þ / þ : n ¼ 6, GR þ / : n ¼ 7 and GR / : n ¼ 6. ** ¼ different to GR þ / þ (Po0.01) by the two-tailed Mann–Whitney test. Data from GR þ /  mice was not statistically compared with the other groups.

Figure 4 Splenic T cells from GR2KO mice are resistant to GC-induced apoptosis. GRMB splenic cells from adult mice were cultured with 100 nM Dex with or without plate-bound anti-CD3 antibody for 20 or 40 h. Following culture cells were stained with anti-CD4 and CD8 antibodies and analysed by flow cytometery using a forward versus side-scatter gate. (a) Resting (un-stimulated) peripheral T cells at 20 h. (b), Anti-CD3-stimulated T cells. At 20 h, all standard errors are below 2%. (a,b) GR þ / þ n ¼ 6, GR /  n ¼ 7. Results depict mean±s.e. for splenocyte samples collected from separate mice over three independent experiments.

activated GR2KO T cells were completely resistant to the apoptotic effect of Dex. Induction of IL-7R by DEX is severely reduced in GR2KO mice To test for residual transactivation in GR2KO mice, Mittelstadt and Ashwell22 cultured GR2KO E18 thymocytes with DEX and compared gene inductions to WT by microarray. Although overall it was found that GC-response genes in GR2KO mice were on average induced to a minor fraction of that seen in WT mice, it was reported that genes

induced to the same degree as WT were nonetheless detectable.22 One such gene encoding the interleukin-7 receptor (IL-7R) a-subunit, was followed-up by qPCR and reported to be induced in GR2KO cells at the same level as WT cells (a subtle 1.5–1.7-fold induction in both cell types), thus raising the possibility that some genes may be normally responsive to GCs in GR2KO mice. In light of prior studies showing induction of six other well-characterized GC-response genes was completely abolished or, at minimum, severely reduced in GR2KO mice, this was a surprising result and thus we further investigated this Immunology and Cell Biology

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treatment for these subsets (Po0.001, Po0.001, Po0.05, respectively, by two-tailed Wilcoxon test). Thus, our findings first support the concept that GC signalling drives a subtle increase in IL-7R expression in WT thymocytes, which is consistent with the earlier study.22 However, in our hands this was largely, although not completely, abrogated in GR2KO mice.

possibility by culturing E18 thymocytes from GR2KO mice with 10 mM DEX for 6 h, followed by analysis of IL-7R expression by flow cytometry. In WT mice, DEX subtly, but significantly, induced IL-7R expression B1.3-fold over vehicle controls (Po0.05, two-tailed Wilcoxon test) and this was the highest induction observed within 8 h (from preliminary dose and time course experiments, not shown). In direct contrast to the previously published results, the induction of IL-7R seen in WT mice was abolished in GR2KO mice (Figure 5a). To further explore this discrepancy, we examined this data in the context of CD4- and CD8-defined thymocyte subsets. In all subsets, DEXmediated induction of IL-7R was significantly reduced in GR2KO compared with WT (Figure 5b and c). In the major population of CD4 þ CD8 þ thymocytes, IL-7R induction was completely abrogated in GR2KO. However, some residual IL-7R induction remained in CD4 CD8 , CD4 CD8 þ and CD4 þ CD8  thymocytes, and indeed this residual induction was found to be statistically significant when comparing the IL-7R geometric MFIs for DEX versus vehicle

DISCUSSION Studies using GR2KO mice have strongly challenged the concept that GR signalling is required for normal T-cell selection.12,13 Previously, we reported that GR2KO mice carry a 39 kDa truncated GR C-terminal fragment containing the LBD.19 Despite this, GR2KO mice are refractory to many GR signalling-dependent functions tested, including GC-induced thymocyte apoptosis and induction of a variety of GC response genes,11,12,19 suggesting that these mice are a valuable means for investigating the role for GCs in T-cell selection. Later, our report of the presence of truncated GR in GR2KO mice

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Figure 5 GC induction of IL-7R expression in GR2KO mouse thymocytes is severely impaired compared with WT. E18 thymocytes from C57BL/6 GR2KO mice, as well as heterozygous and WT littermates were cultured for 6 h with and without 10 mM DEX and analysed for induction of IL-7Ra expression by flow cytometry. (a) The average fold induction of IL-7R geometric MFI by DEX relative to vehicle controls in whole thymocytes for each mouse genotype. (b) Representative profiles of IL-7R expression in whole thymus as well as CD4 and CD8-defined thymocyte subsets following culture in VEH or DEX. SSC, side scatter; FSC, forward scatter. (c) The average fold induction of IL-7R geometric MFI by DEX relative to vehicle controls in CD4 and CD8-defined thymocyte subsets for each mouse genotype. (a and c) Results depict mean±s.e. for thymocyte samples collected from separate mice over five independent experiments. GR þ / þ : n ¼ 6, GR þ / : n ¼ 21 and GR / : n ¼ 14. *Different to WT (Po0.05), **different to WT (Po0.01) by the two-tailed Mann–Whitney test. Data from GR þ /  mice was not statistically compared with the other groups. Immunology and Cell Biology

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prompted a study where GR2KO thymocytes were tested for residual GC responsiveness using a microarray approach.22 To test for transactivation activity, E18 fetal GR2KO thymocytes were cultured with or without DEX for 5 h. In addition, to detect transrepressive activity, this culture protocol was repeated in the presence of PMA/ ionomycin which upregulates many inflammatory signalling pathways and response genes that are targets of some of the transrepressive actions of the GR. Following culture, RNA was isolated and used to probe microarrays containing B10 000 murine cDNAs. The key conclusion drawn from this data was that DEX induced transrepression of PMA/ionomycin-induced gene expression in GR2KO cells to a similar degree as WT cells. In addition, it was reported that although overall gene expression induced by DEX alone (transactivation) in GR2KO mice was reduced, genes that still responded to DEX to a similar extent to those in WT thymocytes could be found. The possibility that GR2KO mice retain normal transrepression in T cells warranted further investigation, because it calls into question the utility of GR2KO mice as a model for the investigation of the potential function that GCs are hypothesized to have in T-cell selection in the thymus. Thus, we carried out fully quantitative tests for transrepression at the level of functional T-cell biology. Initially, the particular functional manifestation of GR-mediated transrepression of TCR signalling which we intended to investigate was its ability to inhibit apoptosis that results from TCR signalling alone. This was a key observation that lead to the theoretical ability of GCs to rescue thymocytes from activation-induced cell death, as proposed by the mutual antagonism hypothesis.2 However, rescue from activationinduced cell death by GCs has only been demonstrated in T-cell hybridomas.28 Furthermore, two previous studies as well as our own investigations involving culture under a broad range of DEX or corticosterone to TCR stimulation ratios (data not shown), show that this does not occur in freshly isolated primary thymocytes in vitro.3,5 In addition to challenging a tenet of the mutual antagonism hypothesis, this also means that GR-mediated rescue of thymocytes from activation-induced cell death could not be tested using GR2KO thymocytes. Instead, we undertook reassessment of GR2KO mice by testing for the well-established transrepressive effects of the GR that are observable in primary T cells.23–26 GR-mediated repression of TCR signalling occurs by many different mechanisms including direct protein–protein interactions between the GR and intracellular signalling molecules and transcription factors, as well as competition with transcription factors for overlapping DNA target sites.1 Multiple important mechanisms of GRmediated repression of TCR signalling involve inhibition of proximal TCR signalling steps such as activation, recruitment and membrane compartmentalization of key signalling molecules TCRx, Lck, Fyn, ZAP-70 and LAT.29–34 In addition, the GR also has a well-documented ability to repress distal TCR signalling molecules, such as transcription factors AP-1 and NF-kB.1,35 In stark contrast to the conclusions from the GR2KO microarray study,22 we observed that transrepression was largely abrogated in GR2KO cells in terms of TCR-induced T-cell activation, proliferation and cytokine production. In addition, GR þ /  mice showed intermediate levels of transrepression, demonstrating a clear relationship between repression and presence of the WT GR allele. We suggest this quantitative analysis of well-characterized GRmediated transrepression effects on TCR signalling stands as the most meaningful assessment of residual activity of truncated GR in GR2KO T cells, and thus an affirmation of the utility of GR2KO mice for study of GR signalling in T-cell selection. Also, consistent with our

result in GR2KO thymocytes, we also show that GC-induced apoptosis is largely abrogated in splenic GR2KO T cells thus providing further evidence of the GC-resistant nature of this mouse strain.11 We are unable to unequivocally explain why our results are so distinct from the GR2KO microarray study22 but one obvious point of differentiation lies in the ways in which T-cell activation is induced in our respective studies. With the aim of examining overall levels of transrepression in GR2KO cells, we have used anti-CD3 antibodymediated stimulation which activates all known points where GR inhibits TCR signalling, including proximal TCR signalling steps,29–34 and provides a physiologically relevant representation of the TCR signalling pathway. In contrast, by the use of PMA and ionomycin, the GR2KO microarray study22 bypassed proximal TCR signalling steps and strongly engaged distal signalling events that are not limited to the TCR signalling pathway. Specifically, PMA targets protein kinase C, whereas ionomycin induces a strong calcium influx. These factors are not limited to the TCR signalling pathway, they can activate cells in a TCR-independent manner and indeed, PMA and ionomycin can act on non-T cells.36,37 Thus, although the data in the GR2KO microarray study suggests the ability of the truncated GR to transrepress, the TCR-independent nature of PMA and ionomycin stimulation makes it difficult to interpret these findings in the context of T-cell selection. Indeed, it is possible that some effects in that study were related to the action of PMA/ionomycin and GC on non-T cells such as thymic stromal cells that would also probably be present in the embryonic thymus suspension cultures. Thus, juxtaposition of these two studies suggests that the ability of the truncated GR to inhibit proximal TCR signalling is impaired, while residual activity in relation to distal signalling events that are common to TCR and other signalling pathways, possibly including in non-T cells, may still exist. Suppression of proximal TCR signalling events would have flow-on effects on downstream signalling events, and subsequent functional readouts. Future studies involving assays of the activity and membrane localization of molecules known to be repressed by WT GR such as TCR-zeta, Fyn, Lck, ZAP-70 and LAT, as well as second messenger pathways such as calcium, PKC and MAP kinase signalling, may shed more light on this possibility. Although this data revealed a severe loss of trans-repression in GR2KO T cells compared with WT T cells for each parameter tested, we consistently observed a low but detectable level of GC-mediated repression of TCR signalling in these cells. Likewise, a low level of residual GC-induced apoptosis was observed in resting peripheral T cells GR2KO mice, whereas it was completely abolished in activated peripheral T cells, and thymocytes.11 Thus, the truncated GR may have minor residual activity, perhaps reflecting that reported in the GR2KO microarray study22 although our data suggest that this is well below normal levels. Towards explaining the residual repression seen in GR2KO mice, given that our western blot analysis of the truncated C-terminal fragment showed it is of sufficient size to contain an intact LBD,19 any transrepression mechanism that is dependent on the LBD alone might reasonably be expected to be intact. In line with this, it was shown that the GR LBD was sufficient for sequestration and subsequent inactivation of c-Jun amino-terminal kinase (JNK).38 Indeed, as JNK is known to specifically stabilize IL-2 mRNA via interaction with elements in its 50 UTR,39 this may be a mechanism by which residual low-level transrepression of IL-2 production is conferred in GR2KO mice. In contrast to the findings of Mittelstadt and Ashwell,22 in this study we found that GC-induction of the IL-7Ra-subunit is largely abolished in GR2KO mice. In the previous study,22 scatter plot Immunology and Cell Biology

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analysis showed that average gene-induction in response to GCs in GR2KO mice was a minor fraction of that seen in WT mice. However, a small portion of genes were apparently induced at similar levels between WT and GR2KO thymocytes.22 This was validated by qPCR for IL-7Ra in E18 thymus and glutamine synthase in E18 lung, each of which were induced subtly (sub-2-fold), but nonetheless similarly, in WT and GR2KO cells. Given that IL-7R transmits essential signals for T-cell development in the thymus,40 we investigated GC-induction of IL-7Ra on the surface of E18 GR2KO thymocytes to a high level of biological replication using flow cytometry, a technique commonly used to detect subtle shifts in expression. Our finding that induction of IL-7Ra in GR2KO thymocytes was largely abolished is consistent with the eight other GC-response genes quantitatively investigated in GR2KO mice to date, all of which show either complete loss of expression, or severe reduction in expression relative to WT.11,19–22 Although the basis for these contrasting results is unclear, the low level of IL-7R induction (o1.7 fold in both studies) leaves little room for measuring reduced responses, and we favour the use of more robustly induced GC response genes to avoid this caveat in future studies. It is also important to note that although the above studies overall reveal a severe loss of transactivation of GC-response genes in GR2KO mice, the small residual amounts of induction seen for some genes such as IL-7R in these mice suggest a minor, but nonetheless noteworthy degree of transactivation is retained. Although the mechanism behind this is uncertain, we envisage two possibilities. First, any hypothetical mechanism where the truncated GR LBD in isolation can confer a transactivation effect may still be active in GR2KO mice, although we are not aware of any such reports in the literature. Second, minor transactivation may occur via the MR (mineralocorticoid receptor), which binds the same response element motif in GC response-gene promoters.41 Previously, MR mRNA has been detected in E18 thymus42 and we have verified this by qPCR in our own laboratory.42 Although subsequent tests for MR-dependent modulation of IL-7R and Glucocorticoid-Induced Leucine Zipper genes in the E18 thymus in our laboratory have been negative, we cannot exclude the possibility of MR-dependent modulation of other target genes. In summary, based on the reassessment of residual GR-mediated repression of TCR signalling in T cells of GR2KO mice in this study, we maintain this line was, and is, valid for investigation of a role for the GR in T-cell selection. With this in mind, previous studies using GR2KO mice,12,13 later confirmed by reports of normal T-cell development in three other independently generated GR-deficient mice,14–16 stand as compelling evidence to suggest that GC signalling is not essential for normal T-cell selection in the thymus. Nonetheless, the recent report of defective T-cell selection in a newly developed T-cell-specific GR exon 3 knockout mouse line18 has re-ignited this debate, and further studies are required to properly understand the role, if any, that GR signalling has in T-cell selection. METHODS Ethics statement These studies were reviewed and approved by, and conducted in accord with the standards of the Monash University Biomedical Sciences Animal Ethics Committee and the University of Melbourne Animal Welfare Committee.

Mice Adult GR2KO mice were generated by gene targeting as described previously,11 by mating GR2KO heterozygous ( þ /–) mice of a combined C57BL/6/129sv genetic background (GRMB). As has been reported previously,19 10–20% of GR /  mice born from crosses of GR þ /  mice on the GRMB survive past Immunology and Cell Biology

birth. These mice were humanely killed between 8–24 weeks of age, together with aged-matched GR þ / þ and GR þ /  littermate controls. Offspring were genotyped for the GR gene locus at three weeks of age by PCR as previously described.12

Primary cell culture and reagents Thymic and splenic cell preparations were cultured in RPMI-1640 medium (Gibco-BRL, Auckland, NZ, USA), 5% FCS (CSL, Melbourne, Australia), 2 mM GlutaMax (Gibco) and 50 mg ml 1 penicillin/streptomycin (Gibco). Cultures were also performed in various concentrations of DEX (Sigma, Castle Hill, Australia) diluted from a 10 2 M solution stored in ethanol or with a (1/1000) dilution of ethanol alone as a vehicle control.

Flow cytometry Cell suspensions were prepared in cold phosphate-buffered saline (PBS) containing 2% FCS, and counted and stained with combinations of the following antibodies for 20 min at 4 1C, before analysis on a two-laser, fourcolour FACScalibur (Becton Dickinson, San Jose, CA, USA): anti-CD8-FITC (clone 53-6.7), anti-CD4-allophycocyanin (APC) (clone RM4–5), anti-CD8PerCP (clone 53-6.7), anti-CD25-PE (clone PC61) and anti-IL-7Ra-PE (clone B12-1) (all antibodies were purchased from BD-PharMingen, San Diego, CA, USA). Unconjugated rat anti-mouse CD16 (2.4G2 clone) was used in all flow cytometry experiments to block non-specific Fc-receptor-mediated binding.

Stimulation of T cells in culture Anti-CD3 stimulation was carried out according to a previously described method.43 Briefly, anti-CD3 (clone 2C11) was diluted to 10 mg ml 1 in sterile PBS and 50 ml per well was used to coat 96-well plates for overnight incubation at 4 1C. Wells were washed with sterile PBS three times and 5  105 splenocytes were added in 200 ml of culture media for 20 or 40 h before being harvested, stained and analysed on a FACScalibur. For cell division experiments, sterile splenic lymphocytes were isolated, washed twice in 0.1% BSA PBS, and then incubated at a concentration of 2  107 cells ml 1 in 0.8 mM CFSE for 10 min at 37 1C. Next, cells were washed in cold 20% FCS RPMI, followed by cold 5% FCS RPMI before culturing in wells that had been coated overnight at 4 1C with 10 mg ml 1 anti-CD3 (2C11) in PBS. Cultured cells were stained for CD4, CD8 and CD25 expression and screened after 40 h for cell division based on the intensity of the CFSE fluorescence. The percent of maximally proliferated cells ¼ [% proliferated cells/% proliferated cells in VEH]  100. The percent of maximally activated cells ¼ [% activated cells/% activated cells in VEH]  100.

Intracellular cytokine staining Following culture, splenic lymphocytes were labelled with cell surface antibodies and washed once in PBS before fixing in 0.5% paraformaldehyde (BDH Chemicals, Poole, UK) in light-protected conditions for 30 min at RT. Cells were washed twice in PBS before incubation in light-protected conditions for 1 h at room temperature with PE-conjugated anti-IFN-g (clone XMG1.2) in PBS containing 0.05% saponin (Sigma-Aldrich, St Louis, MO, USA). Rat IgG1 was used as an isotype control (clone R3–34). Lymphocytes analysed for intracellular cytokine staining were cultured for 2 h in 5 mg ml 1 brefeldin A (Sigma-Aldrich), before cell surface labelling. The percent of maximal IFN-g producing cells ¼ [% IFN-g producing cells/% IFN-g producing cells in VEH]  100. % maximal IFN-g MFI ¼ [IFN-g MFI/IFN-g MFI in VEH]  100, gating on IFN-g þ cells-only.

ELISA for IL-2 IL-2 was detected by sandwich ELISA using the OptEIA set mouse IL-2 kit (BD Biosciences, Mebbourne, VIC, Australia). Briefly, the captured antibody was bound to round-bottomed micro-titer plates that were then blocked with 10% FCS in PBS at 21 1C for 1 h. Sample supernatants and cytokine standards were added and incubated for 2 h at 21 1C. The plates were then washed, followed by addition of the detection layer, biotinylated anti-mouse IL-2. Bound detection antibody was detected with avidin-horseradish peroxidase conjugate. The substrate used was tetramethylbenzidine dihydrochoride (TMB) (Sigma

Glucocorticoid inhibition of T-cell receptor signalling DR Liddicoat et al 155 Chemical Co., St Louis, MO, USA) and results were determined by optical density scanning on a Labsystems Multiscan Multisoft (Helsinki, Finland). The percent of maximal IL-2 production ¼ [IL-2 production / IL-2 production in VEH]  100.

Statistical analysis Statistical analysis was performed using the Mann–Whitney rank sum U-test or the Wilcoxon matched-pairs signed rank test using Prism software version 3.0c (Graphpad, San Diego, CA, USA). In each experiment, only the WT versus GR /  samples were compared statistically.

ACKNOWLEDGEMENTS The authors thank Daniel Pellicci and Konstantinos Kyparissoudis for technical help, Max Walker and David Taylor and staff for animal husbandry assistance, and Morag Young for critical comments on the manuscript. We also thank the Picchi Brothers Foundation for generously supporting our flow cytometry facility. This research was supported by a project grant (#350302) from the National Health and Medical Research Council (NHMRC) of Australia. DIG is supported by an NHMRC Senior Principal Research Fellowship (#1020770). This work is dedicated to the memory of Jared F. Purton.

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