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GATA3-Driven Th2 Responses Inhibit TGF-beta1-Induced FOXP3 Expression and the Formation of Regulatory T Cells
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Author Summary
Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells. The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check. The current study focuses on the role of two key transcription factors-FOXP3 and GATA3-in controlling the commitment of these cells. We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells. We show that IL-4-induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter. We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.
It is shown that Th2 responses prevent the generation of inducible Tregs. This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter. This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.

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Introduction
Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently. Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively. A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations. The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa. Specifically interleukin 4 (IL-4)-induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2]. GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3]. Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5]. A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4-dominated responses can break antigen-specific immune tolerance [7]. Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.
The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens. Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery. These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines. Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions. These induced Treg (iTreg) cells were reported to express FOXP3 [10]. The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14]. TGF-beta has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-betaRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell-dependent multiorgan inflammatory disease [16]. Although the effect of TGF-beta on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.
The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively. We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo. We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.

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FOXP3 Induction in T Cell Subsets
It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).
Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.

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FOXP3 and GATA3 Kinetics in Differentiating Cells
The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-gamma, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.

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IL-4 Inhibits TGF-beta-Mediated iTreg Commitment
IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage. We therefore asked whether IL-4 is able to inhibit TGF-beta induction of FOXP3 during the priming of naive T cells. Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta and/or IL-4 and harvested after 5 d. IL-4 efficiently repressed the TGF-beta-mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B). Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3]. However, at low concentration, IL-4 was able to marginally induce FOXP3 expression. Of note, GATA3 was also induced in the presence of TGF-beta at high IL-4 concentration (Figure 5A and 5B). The IL-4-mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-beta, which demonstrates that IL-4 as well as TGF-beta signaling were functional under these conditions (Figure 5C). Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D). Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo. A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25. We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19]. In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2). Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25- cells, but a real decrease of CD4+CD25+ T cells. In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo. To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity. In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C). Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).

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The Role of TGF-beta in T Cell Differentiation
Although TGF-beta-reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-beta-mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A). It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3- cells and thus decrease the relative percentage of FOXP3+ cells. However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3-. In fact both populations showed similarly enhanced proliferation (Figure 6A). Furthermore the TGF-beta-mediated induction of FOXP3 expression was not caused by overgrowth of a CD25-FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells. CD25 was down-regulated in TGF-beta-treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-beta and IL-4.
The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B). In line with the previous findings, the IL-4-producing cells and the FOXP3 expressing cells are nonoverlapping populations. Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression. The expression kinetic of naive T cells exposed to IL-4 and TGF-beta demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4-8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.
These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-beta signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.

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GATA3 Is a Negative Regulator of FOXP3 Expression
FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3. Besides GATA3's well-known positive effect on gene regulation, GATA3's repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells. To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3. After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-beta. TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel). TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-DeltaDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A). In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3). The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data). These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22]. To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25- cells were isolated, activated with OVA in the presence or absence of TGF-beta, and Foxp3 expression was analyzed after 4 d. The naive CD4+CD25- cells were Foxp3- (unpublished data). As described for the human cells, TGF-beta dramatically up-regulated Foxp3 in the DO11.10 littermate control mice. In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-beta and OVA (Figure 7B). All mice produced similar amounts of TGF-beta; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-beta signaling.
Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.

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GATA3 Represses the FOXP3 Promoter
To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered. The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24]. This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation. The functional relevance of this site was studied using a FOXP3-promoter construct [24]. We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity. The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A). Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B). To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site. This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3-) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C). Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay. HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site. After the pull-down, GATA3 binding was detected by Western blot. Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach. Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E). These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter. To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F). Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.

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Discussion
The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells. After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.
In accordance with other studies, we found that CD4+CD25- cells were able to up-regulate FOXP3 [12,27]. Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile. In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-beta to induce iTreg cells [10,28-34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3. This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation. In this competitive process TGF-beta appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38]. In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-beta showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data). Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42-45]. Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.
The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-beta-mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype. To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4. This has been shown to increase the effect of the cytokine in vivo [19]. Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment. Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells. But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression. IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17-producing T cells (Th17) [46,47]. Similar effects have been recently described for IL-6, which, combined with TGF-beta, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49]. Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.
IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25- [50]. In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression. Importantly, these concentrations were not sufficient to induce GATA3 expression. IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].
Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4-dependent inhibition of FOXP3 could be mediated by GATA3. In fact, GATA3-inducing IL-4 concentrations repressed TGF-beta-mediated FOXP3 expression, whereas IL-4 as well as TGF-beta signaling were intact. This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation. Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3. Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells. This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice). In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-beta. Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%-25% lower frequency compared to wild-type DO11.10 mice. Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin. Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52]. In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55]. Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.
Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region. A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24]. Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3. This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments. Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells. It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57-59]. Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.
The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).
In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4. GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter. We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression. These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.

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failed

overexpression

induce

repressed

expression

binding

low

expression

inhibit

compete

binding

negatively regulated

acts as an inhibitor

expression

repressing

promoter

repressing

expression

induction

up-regulate

induction

up-regulate

TGF-beta

up-regulated

up-regulated

T-bet

promoting

induction

TGF-beta

contribute

drop

enhanced

expression

induce

expression

induce

mediated

inhibition

inducing

mediated

TGF-beta

expression

high

expressing

induction

overexpression

induce

expression

overexpression

regulate

expression


Input:
Mice.
Normal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323-339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8-12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.

expressing


Input:
Isolation of CD4+ T cells.
CD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was >/= 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.

secretion

high

secretion


Input:
Quantitative real-time PCR.
The PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5'-GAA ACA GCA CAT TCC CAG AGT TC-3'; FOXP3 reverse primer 5'-ATG GCC CAG CGG ATG AG-3'; EF-1alpha forward primer and reverse primer as described [61]; GATA3 forward primer 5'-GCG GGC TCT ATC ACA AAA TGA-3' and rwd 5'-GCT CTC CTG GCT GCA GAC AGC-3'. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Quantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5'- AGG TCA AAC AGG AAG ACA GAC GTA-3' and 5'-TCACACCCAAGAACAAGCACA-3'; Smad-7, 5'-GAA ACC GGG GGA ACG AAT TAT-3' and 5'-CGC GAG TCT TCT CCT CCC A-3'; TGF-ss1, 5'-TGA CGT CAC TGG AGT TGT ACG G-3' and 5'-GGT TCA TGT CAT GGA TGG TGC-3'. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 degreesC, followed by 40 cycles of 15 s at 95 degreesC and 60 degreesC for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative DeltaDeltaCT method.


Input:
Inducible murine Treg culture.
Naive CD4+ T cells (CD4+, CD62L+, and CD25-) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 x 105 T cells were co-cultured with 2.5 x 104 bone marrow-derived dendritic cells [62] and 0.01 mug/ml OVA323-339 peptide (Ansynth) in the presence or absence of 20-ng/mul rhTGF-ss1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.

expression


Input:
In vitro T cell differentiation.
CD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 mug/ml, Okt3, IgG1) and anti-CD28 (2 mug/ml) in Th1 conditions: 25 ng/ml IL-12, 5 mug/ml anti-IL-4 (R&D systems); in Th2 conditions: 25 ng/ml IL-4, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12 (R&D systems); or in Treg conditions: 10 ng/ml TGF-beta, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12, 5 mug/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).

depletion


Input:
Cloning of the FOXP3 promoter and construction of mutant constructs.
The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 -511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.


Input:
Transfections and reporter gene assays.
T cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 mug of the FOXP3 promoter luciferase reporter vector and 0.5 mug phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 muL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.


Input:
RNA isolation and cDNA synthesis.
RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.


Input:
Recombinant TAT proteins.
The cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.

lacking

two zinc fingers


Input:
TAT-GATA3 transduction.
CD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-beta (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.

expression


Input:
Intracellular cytokine staining.
T cells were stimulated with 2 x 10-7 M PMA and 1 mug/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4-8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.


Input:
Flow cytometry.
For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4degreesC in staining buffer with the following antibodies: anti-CD152-PE (CTLA-4; BD), anti-PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcgammaRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4'',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.

expression

expression


Input:
Western blotting.
For FOXP3 analysis on the protein level, 1 x 106 CD4+CD25- cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 degreesC. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3-31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),


Input:
Administration of cytokines and antibodies in vivo.
Age- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 mug rmIL-4, 50 mug anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 mug rmIL-4 plus 50 mug anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R&D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.

expression

expression

expression


Input:
ChIP.
ChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3-31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 degreesC for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region -246 to -511 and was performed using the following primers: 5'-gtgccctttacgagt catctg-3' and 5'-gtgccctttacgagtcatctg-3'. The PCR products were visualized using an ethidium bromide gel.


Input:
Pull-down assay.
CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37degreesC. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 mug of poly(dI-dC) (Sigma) and 70 mul of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 degreesC. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 degreesC for 10 min, and the eluants on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).


Input:
Supporting Information
Phenotype of In Vitro Differentiated T Cells
After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).
(1.0 MB AI).
Click here for additional data file.
In Vivo Treatment of Mice with IL-4 Antibody-Cytokine Complexes
B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A-F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A-F) are shown as mean +/- SD. The data are representative of three independent experiments.
(369 KB AI).
Click here for additional data file.
Effect of IL-4 on Already Existing Natural or Inducible Treg Cells
(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean +/- SD of three independent experiments.
(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-beta, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).
(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25- was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 x 104 CD4+CD25+ nTreg cells were added to 5 x 104 CD4+CD25- and 5 x 104 irradiated PBMCs. The results are representative of three independent experiments.
(269 KB AI).
Click here for additional data file.
Schematic Structure of the FOXP3 Gene and Location of the GATA3 Sites
The scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5'untranslated region (UTR). Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow.
(529 KB AI).
Click here for additional data file.

expression

expression

expression

expression

expression

espression

increasing


Input:
Th2 Cells Cannot Induce FOXP3 Expression
(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-beta as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean +/- SD of 4 independent experiments.
(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-beta, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean +/- SD of four independent experiments.

Expression

Induce

Expression


Input:
Th2 Cells Do Not Express FOXP3
(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-beta. FOXP3 expression was measured after 5 d in culture. The dot blots are representative of three independent experiments.
(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed. Data are representative of three independent experiments.

Express

expression

expression


Input:
Th2- or IL-4-Producing Cells Lack FOXP3
(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation. CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25- (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO-CD25- subsets (central panel). A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B). The dotted gray line indicates the IC background level. The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4. Data are representative of three independent experiments.

Producing

expression

expression

expression

expression

secreting

stained

stained

Lack


Input:
FOXP3 Induction During the Differentiation Process
(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta (5 ng/ml) or IL-4 (25 ng/ml). The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression. Bars show the mean +/- SD of three independent experiments.
(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure. Data are representative of three independent experiments.

expression

expression

staining

staining

Induction


Input:
Effect of IL-4 on FOXP3 Induction
(A) A statistical analysis was performed with six donors on day 5 (TGF-beta (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors. Statistical analysis was performed using the Dunnett test. Statistical significance is indicated by asterisks (*p </= 0.05, **p </= 0.01, Dunnett).
(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-beta (5 ng/ml) with an increasing concentration of IL-4, as indicated. Cells were harvested for mRNA quantification after 5 d.
(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-beta (10ng/ml), and IL-4 (100 ng/ml) as indicated. After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6. Total STAT6 and GAPDH served as internal control.
(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.

staining

staining

phosphorylated

phosphorylated

Induction

increasing

Effect

Induction


Input:
IL-4 Inhibits TGF-beta-Mediated iTreg Commitment
CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-beta, and IL-4, as indicated. After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B). Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p </= 0.01, ***p </= 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-beta. Data are representative of three independent experiments.

staining

staining


Input:
GATA3 Acts as a Negative Regulator of FOXP3 Expression
(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells. GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel). Data are representative of four independent experiments.
(B) CD4+CD25- T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-beta for 96 h. Surface CD4 and intracellular FOXP3 were measured by FACS. These data are representative of three independent experiments.
(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-beta expression. Bars show the mean +/- SD of three independent experiments.

presence

expression

expression

expression

TGF-beta


Input:
GATA3 Represses the Human FOXP3 Promoter
(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.
(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean +/- SD of three independent experiments.
(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.
(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides-transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.
(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The "input" represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.

binding

promoter

expression

amounts

Represses

Promoter


Input:
Phenotype of In Vitro Differentiated T Cells
After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A). In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).
(1.0 MB AI).


Input: Click here for additional data file.


Input:
In Vivo Treatment of Mice with IL-4 Antibody-Cytokine Complexes
B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404). Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression. Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A-F). Numbers indicate percentages of CD4+ CD25high CD3+ cells. Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A-F) are shown as mean +/- SD. The data are representative of three independent experiments.
(369 KB AI).

expression

expression

expression

expression

expression


Input: Click here for additional data file.


Input:
Effect of IL-4 on Already Existing Natural or Inducible Treg Cells
(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis. The results shown represent the mean +/- SD of three independent experiments.
(B) iTreg cells were induced in vitro. FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-beta, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).
(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells. CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration. After vigorous washing, their suppressive capacity on responder CD4+CD25- was tested. IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells. 1 x 104 CD4+CD25+ nTreg cells were added to 5 x 104 CD4+CD25- and 5 x 104 irradiated PBMCs. The results are representative of three independent experiments.
(269 KB AI).

espression

increasing


Input: Click here for additional data file.


Input:
Schematic Structure of the FOXP3 Gene and Location of the GATA3 Sites
The scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5'untranslated region (UTR). Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow.
(529 KB AI).


Input: Click here for additional data file.


Input:
Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells
Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis. In this study, we show that the transforming growth factor-beta (TGF-beta) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells. This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter. Inactivation of the gene encoding RUNX cofactor core-binding factor-beta (CBFbeta) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3. The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2-/- mice. Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro. Circulating human CD4+ CD25high CD127- T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-beta mRNA compared with CD4+CD25- cells. Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells. These data demonstrate Runx transcription factors as a molecular link in TGF-beta-induced Foxp3 expression in iT reg cell differentiation and function.

binding

RUNX binding sites

binding

RUNX binding sites

expression

expression

expression

expression

colocalized

T reg cells

colocalized

T reg cells

Inactivation

core-binding factor-beta

suppression

core-binding factor-beta

suppression

reduced

Inactivation

expression

reduced

suppression

expression

siRNA

siRNA

induces

TGF-beta

expression

prerequisite

binding

mediated

suppression

induced

TGF-beta

expression

link

Runx

induced

expressed

expressed

expressed

TGF-beta


Input:
INTRODUCTION
Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006). Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007). A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006).
Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood. It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-kappaB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006). The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming. GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007). It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007). Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003). The crucial role of TGF-beta in their generation has been demonstrated.
The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-alpha [CBFalpha], and polyoma enhancer-binding protein-2alpha [PEBP2alpha]) contains three members, RUNX1 (AML1/CBFA2/PEBP2alphaB), RUNX2 (AML3/CBFA1/ PEBP2alphaA), and RUNX3 (AML2/CBFA3/PEBP2alphaC). They are essential transcriptional regulators of different developmental pathways. RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997). RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002). RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007). RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005).
RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999). The Runt domain-containing protein constitutes the alpha-chain partner of the heterodimeric CBF complex. RUNX proteins heterodimerize with the non-DNA-binding partner, CBFbeta, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993). The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements. The effects of the RUNX-CBFbeta complex regulation are clearly cell lineage and stage specific. They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005).
Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-beta, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells. This study demonstrates that RUNX1 and RUNX3 induced by TGF-beta are involved in the development and suppressive function of Foxp3+ iT reg cells.

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The role of RUNX1 and RUNX3 transcription factors in TGF-beta-mediated iT reg cell generation
To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells. Stimulation with anti-CD2/3/28 mAbs or TGF-beta alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A). In contrast, the combination of both TGF-beta and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells. This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.
To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B). Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-beta-mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA. The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).
The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated. Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed. We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells. On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).
The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry. FOXP3 was only slightly reduced after RUNX1 silencing. Transfection of siRNA for RUNX3 had a stronger effect. The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D). Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2). The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3. In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-gamma in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-beta did not show any significant difference (Fig. S3).
To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127- CD25high T reg cells and compared them with CD4+ CD127+ CD25- T cells. Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25- cells. As expected, IL-10, TGF-beta, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A). There was no difference in RUNX1 mRNA expression between these two cell subsets. We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006). Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).

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RUNX1 and RUNX3 bind to the FOXP3 promoter
Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS). All three binding sites are conserved between human, mouse, and rat (Fig. S4). To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3. After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A). To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay. Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors. The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody. We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B). Although there was a similar and high degree of binding to the -333 and -287 sites, a lower degree of binding was detected when the oligonucleotide containing the -53 site in the Foxp3 promoter was used. This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B). The binding to the two single binding sites at -333 and -287 was comparable to the mixture of all three oligonucleotides. Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFbeta to FOXP3 promoter during the differentiation of naive T cells toward T reg cells. Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-beta as a Foxp3-inducing stimulation. Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFbeta were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C). Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.

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Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3
To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors. An increase in luciferase activity was observed only when the FOXP3 promoter (-511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A). The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1. Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (-511 to +176) luciferase construct were mutated (Fig. 4 A). In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-beta for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.
To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination. No reduction in luciferase activity was observed when the -53 site was mutated and only a slight reduction when either the -287 or -333 site was mutated (Fig. 4 B). However, mutating the -53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter. Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h. This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-beta. Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).

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Role of CBFbeta in the induction of Foxp3
CBFbeta, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences. To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene. Retinoic acid and TGF-beta synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007). To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFbeta, naive CD4+ CD8- T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-beta. After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-gamma expression. TGF-beta induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells. Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-beta and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-beta doses. The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-beta induction of Foxp3 in T reg cells (Fig. 5 A). There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-gamma were neutralized (Fig. S6).
The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined. Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2-/- mice. 6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B). There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C). These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.

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CBFbeta, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells
Even though Cbfb-deficient CD4+ T cells had impaired induction of Foxp3 after stimulation with anti-CD3/28 mAbs and TGF-beta, sufficient numbers of Foxp3+ cells could be generated to permit analysis of their suppressive activity. Purified naive CD4+ T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice (Cd45.2) harboring a Foxp3-ires-GFP allele (Bettelli et al., 2006) were stimulated in vitro with anti-CD3/28 mAbs, IL-2, and TGF-beta. After 3 d, Foxp3+-GFP+ cells were sorted by flow cytometry and mixed with CFSE-labeled naive CD45.1+ CD4+ cells at ratios of 1:4, 1:2, and 1:1. The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days. CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A). CbfbF/+ CD4-cre CD4+ T cells activated in the presence of TGF-beta showed a clear suppression of T cell proliferation that became even more apparent when an increased ratio of FOXP3+/CD25- cells was used (Fig. 6 B). The suppression was significantly reduced when cells from CbfbF/F CD4-cre mice were used, demonstrating that TGF-beta-induced Runx complexes are important for the suppressive activity of Foxp3+ T reg cells (Fig. 6 B). As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-beta were mixed with CFSE-labeled naive CD45.1+ CD4+ cells. No suppression could be observed in all control groups without TGF-beta at all tested ratios (Fig. 6 C). The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation. Foxp3+ and Foxp3- cells generated from both CbfbF/F CD4-cre and control cells all displayed similar proliferation rates and cell death as measured by CFSE dilution and annexin V staining, respectively (Fig. S7, A and B).
In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells. We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA. Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb. Cells in which RUNX1 and RUNX3 were knocked down showed markedly lower suppressive activity compared with control iT reg cells at a T reg/CD4+ T responder cell ratio of 1:20, but not when the T reg/CD4+ T responder cell ratio was increased to 1:5 (Fig. 6 D). These results demonstrate the important role of RUNX1 and RUNX3 not only for the induction of FOXP3, but also for the suppressive capacity of iT reg cells both in humans and in mice. The data suggest both quantity and quality of T reg cells are hampered. Reduced intrinsic suppressive capacity of iT reg cells was demonstrated in mice, because Foxp3-GFP+ cells were FACS sorted and same numbers of iT reg cells are included in control experiments. In the suppression experiment with human cells, reduced suppressive activity was caused by reduced FOXP3 expression in T cells by siRNA inhibition of RUNX1 and RUNX3.

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DISCUSSION
This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-beta. TGF-beta mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells. There are three putative RUNX binding sites in the proximal FOXP3 promoter. One binding site was predicted as a binding site for RUNX2. Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites. This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family. The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development. RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003). Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent. The most striking effect was observed when all three binding sites were mutated. We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.
TGF-beta promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells. It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massague, 2003). It was shown that TGF-beta is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007). RUNX transcription factors are targets of the TGF-beta superfamily and they are involved in the TGF-beta pathway. They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003). TGF-beta can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004). It was shown that RUNX2 regulates the expression of TGF-beta type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved. The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-beta inhibition of leukemic cell growth. RUNX3 plays an important role in TGF-beta-mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-beta and hyperproliferation of the gastric mucosa (Blyth et al., 2005). The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1. This could be dependent on the stage of the cells and organ from which they were isolated.
We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins. For this reason, we decided to use CbfbetaF/F CD4-cre mice. Foxp3 induction by TGF-beta is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice. Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue. It inhibits the IL-6-driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007). We observed an increased number of Foxp3+ cells by retinoic acid and TGF-beta compared with TGF-beta treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice. In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2-/- mice. These data in mice confirm the human data that RUNX proteins play an important role for TGF-beta-dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells. As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-beta and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells. In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.
In a recent study, the role of Runx-CBFbeta was investigated in nT reg cell development in the thymus (Rudra et al., 2009). It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx-CBFbeta complexes. Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study. The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment. Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo. This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007). Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non-T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.
The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcon-Riquelme, 2003). A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002). Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003). RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004). These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004). Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation. Runx3/Cbfbeta are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007). Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002). Runx1 regulates the transitions of developing thymocytes from the CD4- CD8- double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007). Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages. In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated. Furthermore, it was shown that Runx1 activates IL-2 and IFN-gamma gene expression in conventional CD4+ T cells by binding to their respective promoter. RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-gamma production and up-regulation of T reg cell-associated molecules (Ono et al., 2007).
It has been shown that Foxp3 also influences Th17 differentiation. Specifically, Foxp3 physically interacts with RORgammat, and this interaction inhibits RORgammat function (Zhou et al., 2008). This relationship of RORgammat and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006). Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORgammat to activate transcription (Zhang et al., 2008).
The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005). Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007). All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role. We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3. RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-beta pathway is of particular interest.
Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-kappaB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008). NFAT forms a complex with AP-1 and NF-kappaB and regulates the expression of IL-2, IL-4, IFN-gamma, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007). The NFAT-AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006). It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-beta response element was identified in the Foxp3 gene or in the surrounding regions. The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24-48 h after stimulation. The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006). RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008). Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.
In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function. The induction of the transcription factors RUNX1 and RUNX3 by TGF-beta and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.

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