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. Author manuscript; available in PMC: 2009 Jun 1.
Published in final edited form as: Immunity. 2008 Jun;28(6):763–773. doi: 10.1016/j.immuni.2008.04.016

Jak3-dependent signals induce chromatin remodeling at the Ifng locus during Th1 differentiation

Min Shi 1, Tsung H Lin 2, Kenneth C Appell 3, Leslie J Berg 4
PMCID: PMC2587400  NIHMSID: NIHMS55827  PMID: 18549798

Summary

Differentiation of naïve CD4+ T cells into Th1 effector cells requires both TCR signaling and cytokines such as IL-12 and IFN-γ. Here we report that a third cytokine signal, mediated by the Jak3/STAT5 pathway, is also required for Th1 differentiation. In the absence of Jak3-dependent signals, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired upregulation of T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

Introduction

After antigen stimulation, naïve CD4+ T cells differentiate into one of several functional classes of effector cells; most frequent among these effector subsets are T helper (Th) type 1 (Th1) and Th type 2 (Th2) cells (Murphy and Reiner, 2002). The distinct patterns of cytokine production by activated Th1 and Th2 effector cells are the consequence of differential gene transcription, a process that is regulated at multiple levels (Ansel et al., 2003; Lee et al., 2006). The first level involves changes in chromatin accessibility. Subsequent to this, specific transcription factors access the open loci, where they bind to regulatory regions and activate gene transcription. Finally, binding of some transcription factors to their cis-acting regulatory elements directs chromatin-remodeling activity, leading to heritable changes in inducible transcriptional activity.

The signaling pathways that contribute to Th differentiation have been well characterized (Ho and Glimcher, 2002; Lee et al., 2006). When naïve CD4+ T cells are initially stimulated, latent transcription factors non-selectively stimulate low levels of both IFN-γ and IL-4 production (Avni et al., 2002). In the presence of IL-12, Th1 differentiation is efficiently induced when IFN-γ stimulates Jak1/Jak2-dependent IFN-γR signaling, leading to the activation of signal transducer and activator of transcription 1 (STAT1), and thereby inducing expression of the Th1-specific transcription factor, T-bet (Afkarian et al., 2002). T-bet up-regulates expression of IFN-γ, as well as the IL-12 receptor β2-subunit (IL-12Rβ2) (Afkarian et al., 2002; Mullen et al., 2001). IL-12, acting via Jak2/Tyk2 to activate STAT4, is not required for initial Th1 differentiation; instead, IL-12 amplifies the Th1 response by augmenting the production of IFN-γ (Yoshimoto et al., 1998). IL-12 signals together with T-bet also act to down-regulate the lineage commitment and cytokine expression of the alternative Th2 subset (Mullen et al., 2001; Ouyang et al., 1998).

Epigenetic regulation of cytokine loci also play an essential role in Th differentiation (Lee et al., 2006). The first definite evidence of chromatin changes occurring during Th differentiation was reported by A. Rao and colleagues, who identified T cell lineage-specific DNAase I hypersensitive sites near the Ifng gene promoter and enhancer (Agarwal and Rao, 1998). These hypersensitive sites denote regions where chromatin structure has been altered, and they reflect the occupancy of these sequences with specific DNA binding proteins. Others have confirmed the importance of epigenetic changes in the regulation of Th1 and Th2 cytokine gene transcription (Bird et al., 1998; Valapour et al., 2002). The chromatin modification most often studied during Th differentiation is histone acetylation (Lee et al., 2006).

Changes in histone acetylation at the cytokine loci have been well documented during Th differentiation (Avni et al., 2002; Chang and Aune, 2005; Fields et al., 2002; Schoenborn et al., 2007). Interestingly, timecourse analysis indicates that the initial increase in histone acetylation at the regulatory regions of both the Il4 and Ifng genes is elicited in response to TCR signaling and is independent of the cytokine milieu (Avni et al., 2002). However, in Th1- or Th2-polarizing conditions, cytokine locus-specific histone acetylation patterns are observed by day 5 post-activation, and are maintained in the differentiated effector cells (Avni et al., 2002; Fields et al., 2002; Morinobu et al., 2004).

STAT4, the transcription factor activated by IL-12 signaling, has been implicated in maintaining histone acetylation at the Ifng locus in Th1 cells (Chang and Aune, 2005). The Th1-specific transcription factor, T-bet, also promotes histone acetylation, as enforced expression of T-bet induces DNase I hypersensitivity and histone hyperacetylation at the Ifng locus in stimulated STAT4-deficient T cells (Fields et al., 2002; Mullen et al., 2001). However, the early events that contribute to Ifng locus epigenetic modification in Th1 cells are still not completely elucidated.

The Jak1/STAT1 and Jak2/STAT4 signaling pathways are known to be important for Th differentiation. In contrast, a third Jak kinase, Jak3, has not previously been implicated in Th1 differentiation. Jak3 is required for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, all of which share a common receptor subunit, γc. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation, maturation, homeostasis and survival (Imada and Leonard, 2000). While it is not surprising that Jak3 is essential for Th2 differentiation, due to its essential role in IL-4 signaling, little is known about the role of Jak3-dependent cytokine signals in Th1 differentiation. In this study we examined this issue by assessing Th1 differentiation and IFN-γ production by naïve CD4+ T cells from Jak3-deficient and STAT5-deficient mice. We complemented these studies with analysis of wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3. Together, these experiments demonstrated that Jak3/STAT5-dependent cytokine signals regulate Th1 differentiation by controlling chromatin remodeling at the Ifng locus.

Results

IFN-γ production by Th1 cells is greatly reduced in the absence of Jak3

To investigate the potential role of Jak3-dependent cytokine signals in Th1 differentiation, we established an in vitro assay using homogenous populations of naïve Jak3−/− CD4+ T cells. To accomplish this, we first crossed Jak3−/− mice to the transgenic line expressing the OT-II TCR (Barnden et al., 1998). Since γc cytokines are required for naïve CD4+ T cell survival in vivo (Lantz et al., 2000), we also introduced a transgene expressing Bcl-2 (Strasser et al., 1994). The resulting Jak3−/− OTII-transgenic Bcl2-transgenic (hereafter referred to as Jak3−/− OT-II Bcl2) mice have peripheral CD4+ T cells that predominantly exhibit a naïve (CD44lo) phenotype (Fig. S1). However, to ensure a starting population of naïve cells, we chose to use CD4+CD8− single-positive (CD4 SP) thymocytes from these mice for our experiments.

CD4 SP thymocytes were isolated from Jak3+/− or Jak3−/− OT-II Bcl2 mice, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with anti-CD3/CD28 antibodies and cultured under non-skewing or Th1-skewing conditions without addition of exogenous IL-2. After four days, cells were restimulated, and IFN-γ was examined by intracellular cytokine staining. As shown in Figure 1a, in the absence of Jak3, the percentage of IFN-γ-producing cells was reduced from an average of 12.5±2.7% to 0.5±0.4% (P < 0.001) and 27.1±4.1% to 7.0±6.8% (P < 0.01) under non-skewing and Th1-skewing conditions, respectively. In addition, the percentage of lymphotoxin α-producing cells was also decreased in the absence of Jak3 (data not shown). Cell division, as assessed by CFSE dilution, was similar between Jak3+/− and Jak3−/− OT-II Bcl2 cells (Fig. 1a), indicating that the reduced Th1 cytokine production in the Jak3-deficient T cells was not due to impaired proliferation.

Figure 1. Differentiation of CD4+ T cells leads to impaired IFN-γ production in the absence of Jak3.

Figure 1

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CFSE-labeled CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated with anti-CD3 plus anti-CD28 antibodies and cultured under non-, Th1- or Th2-skewing conditions for 4 days.

(A) Cells were restimulated with PMA plus ionomycin for 5 hours. Dot-plots show intracellular staining for IFN-γ versus CFSE fluorescence. The graphs show percentages of IFN-γ producing cells from five different experiments, with the means indicated by horizontal bars. Differences between Jak3+/− and Jak3−/− responses are statistically significant, with the p values indicated.

(B, C) Following 4-day culture in Th1- or Th2-polarizing conditions, cells were restimulated with PMA and ionomycin for 24h. B) Supernatants were analyzed for production of IFN-γ, IL-4, IL-5 and IL-10 by ELISA. C) Ifng mRNA levels were analyzed by real-time quantitative PCR. Data are normalized to levels of 18srRNA in each sample.

To confirm these findings, we examined cytokine secretion by Jak3−/− and Jak3+/− OT-II Bcl2 T cells following stimulation in Th1- and Th2-polarizing conditions. As shown in Figure 1b, under Th1-skewing conditions, Jak3−/− CD4 SP cells did not secrete Th2 cytokines (IL-4, IL-5 and IL-10) and produced much less IFN-γ than Jak3+/− cells. As expected based on the role for IL-4 receptor signaling during Th2 differentiation (Murphy and Reiner, 2002), Jak3−/− CD4 SP cells stimulated under Th2-polarizing conditions did not produce detectable levels of IL-4, IL-5 or IL-10 (Fig. 1b).

To determine whether the impaired production of IFN-γ by Jak3−/− CD4 SP cells resulted from reduced levels of Ifng mRNA, we performed real-time quantitative RT-PCR. Consistent with the protein data, Ifng transcript levels are reduced in Jak3−/− CD4 SP cells compared to Jak3+/− cells, following culture under both non-skewing and Th1-skewing conditions (Fig. 1c). Together, these data indicate that Jak3-dependent signals are required for optimal production of IFN-γ in differentiated CD4+ T cells, and suggest that these signals promote maximal transcription of the Ifng gene.

Transcription factors important for Th1 differentiation are independent of Jak3

Our previous studies indicated that T cells from Jak3-deficient mice have intact TCR signaling (Thomis et al., 1997). To examine whether IFN-γ/STAT1 and IL-12/STAT4 pathways are intact in the absence of Jak3, freshly-isolated CD4 SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated with IFN-γ, and assessed for STAT1 tyrosine phosphorylation (Fig. 2a); further, differentiated Jak3+/− and Jak3−/− Th1 cells were assessed for STAT4 phosphorylation in response to IL-12 stimulation (Fig. 2b). As shown, both of these responses are intact in the absence of Jak3. We also examined up-regulation of T-bet, a master transcription factor for Th1 differentiation which is induced by IFN-γ signaling in T cells (Mullen et al., 2001; Szabo et al., 2000). While no T-bet is detected in naïve T cells, Jak3+/− CD4 SP cells up-regulate T-bet mRNA and protein after stimulation in both non-skewing and Th1-polarizing conditions (Fig. 2c, d, and data not shown). Jak3−/− cells fail to induce T-bet under non-skewing conditions, perhaps as a result of their failure to produce any IFN-γ following activation. In contrast, Jak3−/− cells cultured under Th1-skewing conditions express comparable levels of T-bet mRNA and protein compared to the Jak3+/− control cells (Figure 2c, d). Together with our previous studies, these data indicate that three important pathways for Th1 differentiation, TCR signaling, IFN-γ/STAT1, and IL-12/STAT4, are intact in the absence of Jak3-dependent cytokine signals.

Figure 2. STAT1 phosphorylation, STAT4 phosphorylation and T-bet expression are not impaired in Jak3-deficient Th1 cells.

Figure 2

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(A) Freshly-isolated purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated with or without IFN-γ (100ng/ml) for 30 min. Total cell lysates were prepared and blotted for STAT1p and total STAT1. CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were used as a control.

(B) Purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were cultured in Th1-skewing conditions for 4 days. Cells were rested overnight, and then stimulated with or without IL-12 (15ng/ml) for 25 min. Total cell lysates were prepared and blotted for STAT4p and total STAT4.

(C) Purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated under non-, Th1- or Th2-skewing conditions for 4 days. T-bet mRNA levels were analyzed by real-time quantitative PCR. Data are normalized to the levels of 18srRNA in each sample (left panel). Total cell lysates from freshly isolated CD4SP thymocytes (naïve) or Th1-polarized cells (Th1) were prepared and immunoblotted for T-bet and β-actin (right panel).

Pharmacological inhibition of Jak3 leads to impaired IFN-γ production during Th1 differentiation

To rule out the possibility that Jak3−/− T cells are developmentally abnormal, leading to their impaired Th1 differentiation, we examined cytokine production by Jak3+/+ CD4+ T cells treated with a pharmacological Jak3 inhibitor. Compound PS078507 was developed by Pharmacopeia, Inc. (Princeton, NJ), and has an IC50 of 2.1nM for inhibition of Jak3 enzyme activity (Fig. S2a). Inhibitory activity (IC50) on the other Jak kinases was measured at 20nM, 6.3nM, and 12nM for Jak2, Jak1, and Tyk2, respectively, and was >300nM on a panel of 30 additional kinases tested (data not shown). In a cellular assay examining inhibition of IL-2-induced proliferation of human peripheral blood T cells, the IC50 is 73nM (Fig. S2b). To determine the optimal concentration of PS078507 for Jak3 inhibition in murine peripheral CD4+ T cells, we assessed IL-2-induced STAT5 phosphorylation after treatment with varying concentrations of PS078507 (Fig. S2c). PS078507 completely inhibits IL-2-induced STAT5 phosphorylation in Jak3+/+ CD4+ T cells at 625nM. We also found that PS078507 had no effect on T cell proliferation induced by TCR stimulation over the course of a 4-day assay (Fig. 3a). Furthermore, PS078507 had no inhibitory effect on TCR signaling, as assessed by CD69 up-regulation following TCR stimulation of naïve Jak3+/+ CD4+ T cells (Fig. S2d).

Figure 3. Pharmacological inhibition of Jak3 during Th1 differentiation leads to impaired IFN-γ production.

Figure 3

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(A) Purified CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were labeled with CFSE and stimulated with anti-CD3 plus anti-CD28 antibodies for 4 days in the presence of vehicle alone (WT vehicle) or PS078507 at 625nM (Jak3 inhibitor). Histograms show CFSE fluorescence.

(B, C) CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were cultured under non-and Th1-skewing conditions for 4 days, in vehicle alone or PS078507 (625nM). Cells were restimulated with PMA plus ionomycin for 6 hours. B) Dot-plots show IFN-γ intracellular staining versus forward scatter. The graphs below show percentages of IFN-γ producing cells from four different experiments, with the means indicated by horizontal bars. Differences between cells cultured in vehicle alone or PS078507 (Jak3 inhibitor) are statistically significant, with the p values indicated. C) Total cell lysates were prepared and immunoblotted for T-bet and β-actin.

We then tested PS078507 for its effect on Th1 differentiation leading to IFN-γ production. As shown in Figure 3b, inhibition of Jak3 enzymatic activity in Jak3+/+ OT-II transgenic CD4+ T cells greatly diminished the percentage of IFN-γ secreting cells from an average of 19.7±7.3% to 1.9±1.3% (P < 0.01) under non-skewing conditions, and 42.7±8.9% to 19.5±7.0% (P < 0.01) under Th1-skewing conditions (Fig. 3c). As seen with Jak3−/− T cells, T-bet expression was greatly reduced in CD4+ T cells cultured under non-skewing conditions in the presence of PS078507; however Jak3+/+ T cells stimulated under Th1-polarizing conditions showed normal up-regulation of T-bet, even in the presence of the Jak3 inhibitor (Fig. 3d). These findings confirm the results seen with Jak3−/− CD4 SP cells. Together, these data indicate that the impaired production of IFN-γ in Th1 cells differentiating in the absence of Jak3-dependent signals cannot be explained by defects in the known transcription factors required for Ifng gene expression.

In vivo binding of T-bet to the Ifng promoter is dramatically impaired in the absence of Jak3-signaling

The Ifng gene is a direct target of the transcription factor, T-bet (Lovett-Racke et al., 2004). Since T-bet protein levels were not reduced in the absence of Jak3-dependent signals, we considered the possibility that Jak3-dependent signaling might regulate the ability of T-bet to interact with its responsive element in the Ifng promoter. To address this, chromatin immunoprecipitation (ChIP) assays were performed to assess the in vivo binding of T-bet to the Ifng proximal promoter in Jak3+/− versus Jak3−/− OT-II Bcl2 cells (Fig. S4; primer set #1). CD4 SP thymocytes were activated and cultured under Th1-skewing conditions for 4 days. As shown in Figure 4a, naive CD4 SP thymocytes from Jak3+/− or Jak3−/− OT-II Bcl2 mice had no detectable T-bet bound to the Ifng promoter (lane 3 and lane 6). Following Th1 differentiation, T-bet bound to the Ifng promoter was easily detectable in Jak3+/− T cells, but substantially diminished in Jak3−/− T cells (lane 9 and lane 12). We confirmed these findings with Jak3+/+ CD4+ T cells stimulated in Th1-skewing conditions in the presence of the Jak3 inhibitor PS078507 (Fig. 4b). Overall these data correlate with the pattern of IFN-γ production by Jak3−/− T cells, or following Jak3 inhibition, suggesting that reduced production of IFN-γ under these conditions may result from an impaired ability of T-bet to bind to the Ifng promoter, and thereby to activate Ifng gene transcription.

Figure 4. In vivo binding of T-bet to the Ifng promoter is impaired in Jak3-deficient cells.

Figure 4

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(A) Purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were cultured under Th1-skewing conditions for 4 days (lanes 7–12) and compared with freshly isolated cells (naïve, lanes 1–6). ChIP analysis was done with an anti-T-bet antibody (lanes 3, 6, 9, 12) or a rabbit IgG control antibody (lanes 2, 5, 8, 11).

(B) CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were cultured with vehicle alone or PS078507 (625nM) under Th1-skewing conditions for 4 days. ChIP analysis was done with a rabbit IgG antibody control (top) or an anti-T-bet antibody (middle). The location of PCR primers is indicated in Supplemental Figure 4 and primer sequences are shown in Supplemental Table I.

The apparent inability of T-bet to promote transcription of the Ifng gene is not due to a global defect in T-bet activity or function. Jak3+/− and Jak3−/− OT-II Bcl2 CD4 SP thymocytes were activated and stimulated under Th1-polarizing conditions for 4 days, and mRNA levels encoding the IL-12Rβ2 chain, Hlx, and Ets-1 were examined (Fig. S3). Both populations showed comparable expression of transcripts encoding the IL-12Rβ2 chain and Ets-1, whereas Jak3−/− cells had reduced levels of Hlx mRNA. As IL-12Rβ2 and Hlx are both transcriptional targets of T-bet (Mullen et al., 2001; Mullen et al., 2002), these findings indicate that T-bet is transcriptionally competent to promote expression of IL-12Rβ2, but selectively loses the ability to mediate transcription of other genes, such as Ifng and Hlx.

Th1 cells differentiating in the absence of Jak3-dependent signals show reduced Ifng promoter accessibility

The results described above suggested that T-bet function in Th1 cells may be regulated by chromatin structure. To examine this issue further, we examined the accessibility of the Ifng proximal promoter using restriction endonuclease digestion followed by ligation-mediated polymerase chain reaction (LM-PCR; Fig. S4, primer set #2). As shown in Figure 5a, the Ifng promoter in naïve Jak3+/− or Jak3−/− CD4 SP thymocytes is not accessible to endonuclease digestion. Following four days of stimulation under Th1-polarizing conditions, the Ifng promoter shows dramatically increased accessibility in Jak3+/− SP cells. In contrast, we observe a >3-fold reduction in signal from Jak3−/− SP cells, indicating that the Ifng promoter is markedly less accessible in Jak3-deficient T cells cultured under Th1-skewing conditions.

Figure 5. Chromatin accessibility of the Ifng promoter is reduced in Th1 cells differentiated in the absence of Jak3-dependent signals.

Figure 5

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(A, B) Purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated under Th1-skewing conditions for 4 days (lanes 7–12). Freshly-isolated cells (naïve, lanes 1–6) were used for comparison. A) Nuclei were isolated, digested with HinfI, and three-fold dilutions of genomic DNA were subjected to LM-PCR with primers for the Ifng proximal promoter (LM-PCR primer set #2). B) ChIP analysis was performed with an antibody to acetylated histone H3 (lanes 3, 6, 9, 12) or a rabbit IgG control antibody (lanes 2, 5, 8, 11) (H3 primer set #3).

(C) CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were cultured in vehicle alone or with the Jak3 inhibitor (625nM) in Th1-skewing conditions for 4 days. ChIP analysis was performed with a rabbit IgG control antibody (top) or antibody to acetylated histone H3 (middle) (H3 primer set #3).

(D) Purified CD4SP thymocytes from Jak3+/− and Jak3−/− OT-II Bcl2 mice were stimulated under Th1-skewing conditions. Parallel cultures were treated with or without sodium butyrate (SB; 200µM), added at 24h post-stimulation. At day 4, cells were restimulated with PMA and Ionomycin. The graph shows the percentage of cells producing IFN-γ as assessed by intracellular staining. Data are pooled from three independent experiments. Differences between sodium butyrate treated and untreated samples for each genotype of cells are statistically significant, with p values indicated. Differences between sodium butyrate treated Jak3+/− and Jak3−/− cells are not significant.

Using the ChIP assay, we next examined the acetylation status of histone H3 at the Ifng promoter in Th1 cells stimulated in the presence or absence of functional Jak3 (Fig. S4, primer set 3). As shown in Figure 5b, naïve Jak3+/− and Jak3−/− OT-II Bcl2 CD4 SP thymocytes have a low level of histone H3 acetylation at the Ifng promoter. Following four days of culture in Th1-skewing conditions Jak3+/− cells displayed strong hyperacetylation of histone H3 at the Ifng promoter. In contrast, histone H3 acetylation of the Ifng promoter was reduced in Jak3−/− Th1 cells, consistent with the diminished accessibility of this region to restriction endonuclease digestion. These findings were also confirmed with Jak3+/+ T cells stimulated under Th1-polarizing conditions in the presence of the Jak3 inhibitor, PS078507 (Fig. 5c). Furthermore, all findings were confirmed with a second set of PCR primers (Fig. S4, primer set #4; data not shown). Together, these data strongly suggest that Jak3-dependent cytokine signals are required for chromatin remodeling of the Ifng locus during Th1 differentiation.

To further assess the role of histone acetylation in Jak3-dependent IFN-γ production, we pharmacologically-induced histone acetylation of the Ifng promoter using a histone deacetylase (HDAC) inhibitor, sodium butyrate. Addition of sodium butyrate during Th1 differentiation of both Jak3+/− and Jak3−/− OT-II Bcl2 CD4 SP thymocytes resulted in increased production of IFN-γ compared to untreated cells (Fig. 5d). This finding was confirmed with Jak3+/+ CD4+ T cells stimulated in the presence of the Jak3 inhibitor, PS078507 (data not shown). Overall, the data presented here indicate that Jak3-dependent cytokine signals induce IFN-γ production via chromatin remodeling of the Ifng locus.

Jak3-dependent signals act 24–72 hours following activation to promote histone acetylation at the Ifng promoter

Our data indicate that Jak3-dependent signals promote IFN-γ production by inducing histone H3 acetylation. Examining the timecourse of histone H3 acetylation at the Ifng promoter during Th1 differentiation by ChIP assay demonstrated only a low basal level of histone H3 acetylation in the first 24h post-stimulation. Histone H3 acetylation increased at 48h and then increased further at 72h (Fig. 6a). These data indicate that during Th1 differentiation, selective Ifng chromatin remodeling occurs rapidly between 24–72h following activation.

Figure 6. The Jak3-dependent signals are required at 24–72h of stimulation to induce histone acetylation at the Ifng locus.

Figure 6

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(A) CD4+ splenocytes were isolated from Jak3+/+ OTII-transgenic mice and were stimulated under Th1-skewing conditions for 0h, 12h, 24h, 48h and 72h. At each timepoint, ChIP analysis was performed with a rabbit IgG control antibody (top) or an antibody to acetylated histone H3 (H3 primer set #3).

(B) CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were stimulated under Th1-skewing conditions, with vehicle alone or the Jak3 inhbitor (PS078507 at 625nM) added at the indicated timepoints following the initiation of the cultures. All cultures were harvested at day 4, at which time the cells were restimulated and analyzed for IFN-γ production by intracellular cytokine staining. Cell initially cultured in vehicle alone were restimulated in the presence of PS078507 at 625nM (Restim).

(C) CD4+ splenocytes from Jak3+/+ OTII-transgenic mice were stimulated under Th1-skewing conditions in vehicle alone or with PS078507 (625nM) added at the indicated times following the initiation of the cultures. At day 4, ChIP analysis was performed with a rabbit IgG control antibody (top) or an antibody to acetylated histone H3 (middle) (H3 primer set #3). Freshly isolated Jak3+/+ OTII-transgenic cells were used for comparison (naïve).

Use of a small molecule Jak3 inhibitor allowed us to assess the kinetic parameters of the Jak3-dependent signal, and to correlate these data to the changes in histone acetylation. For these experiments, Jak3+/+ CD4+ T cells were stimulated under Th1-polarizing conditions in the absence of inhibitor PS078507, or with inhibitor added at varying times following the initial activation of the T cells. At day 4, cells were restimulated and IFN-γ production was examined by intracellular cytokine staining. Consistent with the histone H3 acetylation pattern, PS078507 inhibited IFN-γ production when cells were treated with the inhibitor starting at 0h, 1h, 3h, 6h, 12h and 24h post-stimulation. However, the inhibitory effect of PS078507 was substantially diminished when inhibitor was applied at 48h, and was ineffective when added at 72h post-stimulation (Fig. 6b). Similarly, acetylation of histone H3 at the Ifng promoter was completely inhibited by PS078507 when added 24h after initial T cell activation, but was ineffective when cells were stimulated under Th1-polarizing conditions for 72h prior to addition of the Jak3 inhibitor (Fig. 6c). These findings indicate that the predominant action of Jak3-dependent signals in promoting optimal IFN-γ production occurs between 24–72h after stimulation, and that these signals regulate Ifng chromatin remodeling during Th1 differentiation.

STAT5 and IL-2 promote optimal IFN-γ production during Th1 differentiation

Following Jak kinase activation, STAT5 translocates to the nucleus where it activates the transcription of target genes (Lin and Leonard, 2000). In human NK cells, a STAT5 binding site has been identified in a distal region of the IFNG gene, 3.6kb upstream of the transcriptional start site. This STAT5 binding site serves as a target for epigenetic modification of the IFNG locus, or as an IL-2-induced transcriptional enhancer (Bream et al., 2004; Gonsky et al., 2004). STAT5 also binds to this region of the IFNG gene in human T cells, where it enhances IFNG gene expression following CD2 stimulation (Gonsky et al., 2004). In murine T cells, STAT5 binding to the Ifng promoter has not previously been examined. However, several Ifng regulatory elements have been identified in these cells through DNase I hypersensitive site (HS) mapping and conserved noncoding sequence (CNS) searching. These elements are IfngCNS-34 (Hatton et al., 2006), IfngCNS-22 (Hatton et al., 2006), IfngCNS-5.5 (Lee et al., 2004; Shnyreva et al., 2004), HS-0.3 (Agarwal and Rao, 1998) and IfngCNS+18 (Shnyreva et al., 2004), which are −34kb, −22kb, −5.5kb, −0.3kb and +18kb from the IFN-γ transcriptional initiation site, respectively. To investigate whether STAT5 binds to any of these regulatory sites, we utilized the ChIP assay (Fig. S4, primer sets #5–10). As shown in Figure 7a, no STAT5 binding is detected to IfngCNS-34 or IfngCNS-22 (Fig. 7a, lanes 1 and 2). Furthermore, although STAT5 binds to the −3.6kb region of the human IFNG gene, we cannot detect STAT5 binding to the corresponding region of the murine Ifng locus (Fig. 7a, lane 4). Interestingly, CD4+ T cells cultured in Th1-polarizing conditions for 48hr exhibit easily detectable STAT5 binding to IfngCNS-5.5 and HS-0.3 following IL-2 stimulation (Fig. 7a, lanes 3 and 5). In addition, we find extremely weak binding of STAT5 to IfngCNS+18 (Fig. 7a, lane 6). These data suggested that the Jak3-STAT5 pathway might directly regulate Ifng gene expression.

Figure 7. STAT5 and IL-2 are required for optimal IFN-γ production during Th1 differentiation.

Figure 7

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(A) Purified CD4+ T cells from Jak3+/+ OTII-transgenic mice were stimulated and cultured in Th1-skewing conditions for 2 days. Cells were rested overnight, and then restimulated with IL-2 (50ng/ml) for 1h. ChIP analysis was performed using an anti-STAT5a antibody. Rabbit IgG antibody was used as a negative control. Primers for each of the indicated Ifng regulatory regions were designed to detect STAT5 binding (STAT5 primer sets #5–10).

(B) Purified CD4SP thymocytes from CD2-Cre transgenic STAT5fl/+ and STAT5fl/fl mice were stimulated under Th1-skewing conditions. Sodium butyrate (200µM) was added at 24h post-stimulation. At day 4, cells were restimulated with PMA and Ionomycin. Bar graphs indicate the percentages of IFN-γ producing cells. Data are representative of two independent experiments with similar results.

(C) CD4+ splenocytes from Jak3+/+ mice were cultured under Th1-skewing conditions for 4 days, in the indicated conditions. Jak3 inhibitor PS078507 was used at 625nM, IL-2 blocking included anti-IL-2 (10µg/ml), anti-CD25 (10µg/ml) and anti-CD122 (10µg/ml) antibodies, recombinant IL-7 (rIL-7, 10ng/ml) was added together with IL-2 blocking antibodies. Cells were restimulated with PMA plus ionomycin for 6 hours. Histograms show IFN-γ intracellular staining.

(D) Purified CD4+ splenocytes from Jak3+/+ mice were stimulated with anti-CD3 plus anti-CD28 antibodies for 24h, rested for 4 hours, then stimulated with medium (first lane), IL-2 or IL-7, at 50ng/ml or 100ng/ml for 15 minutes. Cell lysates were prepared and immunoblotted for STAT5p and total STAT5.

To determine whether STAT5 is required during Th1 differentiation for optimal IFN-γ production, we utilized T cells from conditional STAT5a and STAT5b double-deficient mice (Cui et al., 2004). To accomplish this, mice carrying a floxed STAT5 allele were crossed to CD2-Cre transgenic mice (de Boer et al., 2003). CD4 SP thymocytes were isolated and stimulated under Th1-polarizing conditions. After four days, cells were restimulated and IFN-γ production was assessed by intracellular cytokine staining. Consistent with the data from CD4 SP cells lacking Jak3, STAT5-deficient cells also show impaired differentiation of IFN-γ-producing Th1 effector cells (Fig. 7b). Furthermore, addition of sodium butyrate during the differentiation process promotes greatly enhanced production of IFN-γ in T cells lacking STAT5 (Fig. 7b).

To determine if IL-2 is the cytokine responsible for activating Jak3 and STAT5 during Th1 differentiation and thereby promoting IFN-γ production, we stimulated wild-type (Jak3+/+ STAT5ab+/+) T cells under Th1-polarizing conditions in the presence of anti-IL-2 and anti-IL2 receptor blocking antibodies. As shown in Figure 7c, inhibition of IL-2 signaling is nearly as effective at preventing the differentiation of IFN-γ-producing Th1 cells as is the Jak3 inhibitor, PS078507. We also tested whether addition of exogenous IL-7 could replace IL-2 and promote optimal IFN-γ production. As shown in Figure 7c, IL-7 is only modestly effective at promoting IFN-γ production. The relative activities of IL-2 and IL-7 at inducing IFN-γ production during Th1 differentiation correlate well with their ability to induce STAT5 tyrosine phosphorylation in differentiating Th1 cells (Fig. 7d). These data provide further evidence consistent with a role for STAT5 in promoting IFN-γ production during Th1 differentiation.

Discussion

Th1 versus Th2 cell fate determination and maintenance are regulated by exogenous signals through the TCR and cytokine receptors, which together activate distinct transcription factor networks and promote epigenetic modifications of lineage-specific cytokine loci (Ansel et al., 2003). Here we show that CD4+ T cells require Jak3-dependent signals to produce optimal levels of IFN-γ during Th1 differentiation, independently of effects on cell division. Jak3 activity is not required for TCR signaling, nor for the IFN-γ/STAT1/T-bet and IL-12/STAT4 pathways. Instead, Jak3-dependent signals regulate chromatin remodeling at the Ifng locus, promote histone H3 acetylation, and control the accessibility of T-bet to the Ifng promoter to induce optimal IFN-γ production by Th1 cells.

Helper T cell differentiation is coupled to cell cycle progression. As seen first by Bird and colleagues, IFN-γ is expressed by an increasing frequency of cells with each cell division (Bird et al., 1998), a finding also illustrated in our data (Fig. 1a). Although γc-dependent cytokines have been shown to promote lymphoid proliferation (Sugamura et al., 1995), we find that cells lacking Jak3 (i.e., Jak3−/−), as well as Jak3+/+ cells treated with a small molecule Jak3 inhibitor, are able to proliferate comparably to wild type cells in the context of a 4-day in vitro assay. Thus we conclude that Jak3-dependent signals are directly influencing effector T cell differentiation, rather than proliferation.

Our data indicate that a third cytokine signal, mediated by Jak3 and STAT5, is required during the early stages of Th1 differentiation (prior to 72h). We complemented our studies on Jak3-deficient and STAT5-deficient T cells with experiments on wild type CD4+ T cells treated with a small molecule inhibitor of Jak3 enzymatic activity, PS078507. In all cases, the inhibitor data are completely concordant with the results obtained using Jak3−/− cells, demonstrating that the effects seen are not due to abnormal development of, or compensatory changes in Jak3−/− or STAT5ab−/− cells. Further, the experiments utilizing the Jak3 inhibitor indicate that Jak3 kinase activity is required for its role in promoting Ifng chromatin remodeling.

One potential caveat of the inhibitor experiments is the fact that PS078507 has significant inhibitory activity on Jak1, as well as Jak3. Since Jak1 is a key component of IFN-γ receptor signaling (Muller et al., 1993), and IFN-γ receptor signaling has been implicated in the induction of T-bet expression and thus in Th1 differentiation (Afkarian et al., 2002), inhibition of Jak1 activity by PS078507 could contribute to the impaired Th1 differentiation seen in the presence of the inhibitor. However, several lines of evidence indicate that this scenario is unlikely. First, we detect normal levels of T-bet in wild-type CD4+ T cells cultured under Th1-polarizing conditions in the presence of PS078507. Second, we observe that IFN-γ receptor is totally absent from CD4+ T cells by six hours after initial activation, and further, that IFN-γ-induced STAT1 phosphorylation is also undetectable by three hours after T cell activation (Bach et al., 1995; Van De Wiele et al., 2004) (and data not shown). Since we find that PS078507 still inhibits optimal IFN-γ production when added to Th1-polarizing cultures after 24 hours of activation, it is extremely unlikely that its ability to block this response is due to inhibition of IFN-γ receptor signaling. Thus, the concordance between our findings based on analysis of Jak3−/− T cells with the data using PS078507 provide strong support for a role of Jak3 signaling in Th1 differentiation.

T-bet is considered the master regulator of Th1 lineage commitment (Szabo et al., 2000). T-bet controls Th1 programs by promoting Ifng gene remodeling (Mullen et al., 2002); in addition, T-bet acts to directly induce transcription of the Ifng (Lovett-Racke et al., 2004), IL-12Rβ2 (Mullen et al., 2001), and Hlx (Mullen et al., 2002) genes. With regard to Ifng expression, both of these functions require T-bet binding to the Ifng locus. Interestingly, our data indicate that T-bet protein expression alone is not sufficient for maximal T-bet binding to the Ifng promoter. These findings could be accounted for by two possible mechanisms. First, it is formally possible that a potential post-translational modification of T-bet (Hwang et al., 2005) is impaired in the absence of Jak3-dependent signals. Arguing against this possibility, the expression of the IL-12Rβ2 chain, one of the direct transcriptional targets of T-bet (Mullen et al., 2001), is not impaired in Th1 cells lacking Jak3, indicating that the T-bet protein present in these cells is transcriptionally active.

Instead, we favor the notion that impaired T-bet binding in vivo to the Ifng gene is due to reduced accessibility of the Ifng locus in the absence of Jak3-dependent signals. In previous studies, the importance of T-bet and STAT4 in Ifng chromatin remodeling was demonstrated using T-bet- or STAT4-deficient T cells, or by a retroviral over-expression system (Chang and Aune, 2005; Fields et al., 2002; Mullen et al., 2001); therefore it was not possible to determine precisely when these factors carry out their functions. By using a pharmacological Jak3 inhibitor we find that Jak3-dependent signals function at 24–72h following initial T cell activation. Since TCR signals act within 24h to regulate Ifng epigenetic modification (Avni et al., 2002), these data suggest that Jak3-dependent signals function after TCR signaling to promote chromatin remodeling. The impaired ability of T-bet to bind to the Ifng promoter in Jak3−/− cells further suggests that Jak3-dependent signals act prior to, or synergistically with T-bet, to regulate Ifng gene expression. After 72h, this Jak3-dependent signal is dispensable, and T-bet plus STAT4 are sufficient to reinforce and maintain the open status of the Ifng gene.

Chromatin remodeling at the Ifng locus may occur by STAT5 recruitment of histone acetyltransferases or chromatin remodeling factors, as has been described in other systems (Bertolino et al., 2005; Rascle and Lees, 2003; Shuai, 2000; Xu et al., 2007; Ye et al., 2001). While we have been unable to detect constitutive STAT5 binding to the Ifng locus in differentiating Th1 cells, IL-2 stimulation of T cells 48h after initial activation induces robust binding of STAT5 to Ifng regulatory regions, including IfngCNS-5.5 and HS-0.3. Interestingly, T-bet is also able to bind to these regions (Lee et al., 2004; Shnyreva et al., 2004). As our data indicate that T-bet binding to the Ifng proximal promoter is greatly reduced in the absence of STAT5 activation, we propose that STAT5 functions either before, or synergistically with T-bet, to regulate the chromatin remodeling process.

Early studies provided evidence that IL-2 ‘primes’ T cells for production of both IFN-γ and IL-4 (Seder et al., 1994). More recently, IL-2 receptor signaling via STAT5 has been implicated in the regulation of IL-4 expression and Th2 differentiation, independently of the IL-4/STAT6 pathway (Cote-Sierra et al., 2004; Zhu et al., 2003). These data, together with our findings, suggest the intriguing possibility that IL-2, or another γc-dependent cytokine, may be required in the initial stages of both Th1 and Th2 differentiation for chromatin remodeling at the Ifng and Il4 locus, respectively. This requirement would thereby couple effector CD4+ T cell differentiation with appropriate survival and growth-promoting signals. In our in vitro system, IL-2 is the cytokine activating Jak3 and promoting IFN-γ production during Th1 differentiation. It remains to be determined whether STAT5 is inducing chromatin remodeling at the Il4 locus during Th2 differentiation. Nonetheless, these data demonstrate that optimal effector CD4+ T cell differentiation depends on an additional cytokine signal that is not lineage-specific, but is required for epigenetic regulation of lineage-specific cytokine loci.

Experimental Procedures

Mice and reagents

OTII-transgenic (Barnden et al., 1998) and Bcl2-transgenic (Strasser et al., 1994) mice were purchased from Jackson laboratory (Bar Harbor, ME). Jak3−/− mice (Thomis et al., 1995) were backcrossed to C57BL/6 for ten generations. STAT5a/b double-deficient mice (Cui et al., 2004) and CD2-cre transgenic mice (de Boer et al., 2003) were generously provided by Dr. Joonsoo Kang with the permission of Dr. Lothar Hennighausen and Dr. Dmitris Kioussis, respectively.

Cell isolation, culture, and in vitro T cell differentiation

CD4 SP thymocytes were sorted on a Mo-Flo sorter (Cytomation, Fort Collins, CO) to a purity of >98%. CD4+ splenocytes from were purified (Miltenyi, Auburn, CA) to a purity of >94%. Cells were stimulated with plate-bound anti-CD3 (1µg/ml) plus anti-CD28 (4µg/ml) antibodies (eBioscience, San Diego, CA). Th1 and Th2 differentiation conditions included rIL-12 (5ng/ml) (R&D, Minneapolis, MN) and anti-IL-4 antibody (5µg/ml) (BD Pharmingen), or rIL-4 (20ng/ml) (R&D) and anti-IFN-γ antibody (5µg/ml) (BD Pharmingen), respectively. For non-skewing conditions, no exogenous cytokines or antibodies were added. For all culture conditions, no exogenous IL-2 was added. IL-2 blocking was performed by addition of anti-IL-2 plus anti-CD25 plus anti-CD122 antibodies (BD Pharmingen), each at 10µg/ml. Recombinant IL-7 (rIL-7, eBioscience) was used at 10ng/ml for cell culture and at 50 and 100ng/ml for STAT5 phosphorylation assays.

Intracellular cytokine staining

1×106 T cells cultured under non-skewing or Th1-skewing conditions were restimulated with PMA (5ng/ml) plus ionomycin (375ng/ml) for 5h in a 96-well plate. Golgi Plug was added for the last 4h, and intracellular staining was performed according to the Cytofix/Cytoperm kit protocol (BD Pharmingen).

Cytokine ELISA

3×105 T cells cultured under Th1- or Th2-polarizing conditions were restimulated with PMA (5ng/ml) plus ionomycin (375ng/ml) for 24h. Supernatants were harvested, serially diluted and assayed for IFN-γ, IL-4, IL-5 and IL-10 using cytokine detection kits (BD Pharmingen).

Real-time quantitative PCR

CD4 SP thymocytes were stimulated for 4 days with anti-CD3 and anti-CD28 antibodies under non-, Th1- or Th2-skewing conditions. Total RNA was extracted, cDNA was generated and mRNA levels were analyzed by real-time quantitative PCR as previously described (Miller and Berg, 2002). 18srRNA were used as internal control. Primer sequences are shown in Supplemental Table I.

Western blot

Cells lysates were immunoblotted with antibodies to T-bet (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (BD Pharmingen), phospho-Stat1 (Tyr701) (Cell Signaling Technology, Danvers, MA), total Stat1 (Cell Signaling Technology), phospho-Stat4 (Y693) (Invitrogen, Carlsbad,CA), total Stat4 (Santa Cruz Biotechnology) and phospho-Stat5 (Cell Signaling Technology) antibodies.

Chromatin immunoprecipitation

ChIP assays were performed on 1.5×106 cells using the ChIP Assay Kit (Upstate Cell signaling Solutions, Charlottesville, VA). The antibodies used were anti-T-bet (H-210, Santa Cruz), anti-acetylated histone H3 (Upstate), and anti-Stat5a (R&D). PCR was performed with 5µl from a total of 50µl of the immunoprecipitated DNA with various primers. As a control, the PCR was done directly on input DNA purified from chromatin before immunoprecipitation. Positions of primers are indicated on a map of the murine Ifng locus (Supplemental Fig. 4). Primer sequences are shown in Supplemental Table I. T-bet primer sequences were derived from Lovett-Racke, et al. (Lovett-Racke et al., 2004).

Restriction enzyme accessibility assay

Restriction enzyme accessibility experiments using LM-PCR were performed as previously described (de la Serna et al., 2005). Nuclei were digested with HinfI. Input DNA was monitored by amplifying the Ifng promoter in a region that cannot be digested by HinfI. Primer sequences are shown in Supplemental Table I.

Statistical Analysis

Statistical analysis was performed using the two-tailed paired student’s t test.

Supplementary Material

01

Acknowledgments

We thank Wei Wang and Maria Webb for technical advice, Cara West and Regina Whitehead for assistance, and Joonsoo Kang, Amanda Prince and Wenfang Wu for helpful discussions. This work was supported by grants from the NIH (AI37584 and AI46564). Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used.

Footnotes

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Contributor Information

Min Shi, Dept. Pathology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

Tsung H. Lin, Pharmacopeia Inc., PO Box 5350, Princeton, NJ 08540, USA

Kenneth C. Appell, Pharmacopeia Inc., PO Box 5350, Princeton, NJ 08540, USA

Leslie J. Berg, Dept. Pathology, University of Massachusetts Medical School, Worcester, MA 01655, USA

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