Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3⁺ regulatory T cells

Abstract

Recent studies have highlighted the importance of peripheral induction of Foxp3-expressing regulatory T cells (Tregs) in the dominant control of immunological tolerance. However, Foxp3⁺ Treg differentiation from naïve CD4⁺ T cells occurs only under selective conditions, whereas the classical T helper (Th) 1 and 2 effector development often dominate T cell immune responses to antigen stimulation in the periphery. The reason for such disparity remains poorly understood. Here we report that Th1/Th2-polarizing cytokines can potently inhibit Foxp3⁺ Treg differentiation from naïve CD4⁺ precursors induced by TGF-β. Furthermore, antigen receptor-primed CD4⁺ T cells are resistant to Treg induction because of autocrine production of IFNγ and/or IL-4, whereas neutralizing IFNγ and IL-4 not only can potentiate TGF-β-mediated Foxp3 induction in vitro but can also enhance antigen-specific Foxp3⁺ Treg differentiation in vivo. Mechanistically, inhibition of Foxp3⁺ Treg development by Th1/Th2-polarizing cytokines involves the activation of Th1/Th2 lineage transcription factors T-bet and GATA-3 through the canonical Stat1-, Stat4-, and Stat6-dependent pathways. Using IFNγ and IL-4 knockouts and retrovirus-mediated transduction of T-bet and GATA-3, we further demonstrate that enforced expression of the Th1/Th2 lineage-specific transcription factors is sufficient to block Foxp3 induction and Treg differentiation independent of the polarizing/effector cytokines. Thus, our study has unraveled a previously unrecognized mechanism of negative cross-regulation of Foxp3⁺ Treg fate choice by Th1/Th2 lineage activities. In addition, these findings also provide an attainable explanation for the general paucity of antigen-triggered de novo generation of Foxp3⁺ Tregs in the periphery.

The formation of distinct lineages of effector and regulatory T cells (Tregs) from naïve CD4⁺ precursors in response to antigen stimulation is a hallmark of the adaptive immune system (1–3). T helper (Th) 1 and Th2 are the two most predominant types of CD4⁺ effector cells formed during protective immune responses to pathogens as well as pathogenic responses to self or innocuous environmental antigens (1, 2). Besides Th1/Th2, naïve CD4⁺ cells can also differentiate into Tregs after antigen exposure (4–6). Tregs expressing the forkhead winged-helix transcription factor Foxp3 represent a pivotal subset of Tregs for the dominant control of adaptive immune responses and self-tolerance in the periphery (7–9). The indispensable role of Foxp3⁺ Tregs is underscored by rapid onset of fatal aggressive autoimmunity in mice and humans deficient of Foxp3 (9). Furthermore, recent molecular and genetic studies have firmly established that Foxp3 is uniquely expressed in Treg cells and acts as a master transcription regulator for their development and function (10–14). Although Foxp3⁺ Tregs were initially found to be generated during T cell development in the thymus (8), it now becomes clear that these regulatory cells can also be generated from naïve CD4⁺ T cells in peripheral lymphoid tissues (4–6). Induction of Foxp3⁺ Tregs from naïve CD4⁺ T cells in the periphery appears to be largely driven by TGF-β (6). Naïve CD4⁺ T cells from mice and humans have been shown to up-regulate Foxp3 expression and to differentiate into Tregs with strong suppressive activity when they are activated in vitro in the presence of exogenous TGF-β (15, 16). In vivo, de novo generation of Foxp3⁺ Tregs can be induced by different forms of antigenic stimulation. von Boehmer and colleagues reported that chronic stimulation by agonist peptide ligand could instruct naïve T cell receptor (TCR)-transgenic T cells to up-regulate Foxp3 expression and differentiate into suppressive Tregs (17). In the subsequent studies, they further showed that Foxp3⁺ Treg differentiation could also occur in response to a suboptimal dose of agonist ligand targeted specifically to resting DEC205⁺ dendritic cells (18). Similarly, Knoechel et al. (19) demonstrated the formation of Foxp3⁺ Tregs from adoptively transferred TCR-transgenic T cells that were continually exposed to a cognate antigen produced systemically in the host. In addition, Foxp3⁺ Treg induction has also been found in the immune response to transplant antigens (20, 21). However, peripheral induction of Foxp3⁺ Tregs appears to take place only under certain restrictive conditions that are commonly defined as tolerogenic. De novo generation of Foxp3⁺ Treg has not been observed in T cell responses to microbial infections (13, 22) or in an autoimmune/inflammatory environment where Th1/Th2 responses prevail (14, 18). It remains unresolved why Treg induction is overshadowed by Th1/Th2 effector development. Clearly, a better understanding of factors and conditions that favor or oppose de novo generation of Tregs would aid more effective manipulation of T cell immune responses for disease treatment and prevention. In this report we demonstrate that, although the majority of naïve CD4⁺ T cells are capable of activating Foxp3 expression and differentiating into Tregs, the choice to Foxp3⁺ Treg fate is profoundly inhibited by Th1/Th2 activities. The mechanism of Foxp3⁺ Treg fate inhibition involves Th1/Th2-polarizing/effector cytokines IL-12, IFNγ, or IL-4 to activate the central Th1/Th2 differentiation programs leading to the up-regulation of lineage-specific transcription factors T-bet or GATA-3 through Stat1/Stat4- and Stat6-dependent pathways.

Results

Inhibition of Foxp3⁺ Treg Differentiation from Naïve CD4⁺ T Cells by Th1/Th2 Polarization Cytokines.

To investigate regulation of peripheral induction of Foxp3⁺ Tregs, we used in vitro culture systems in which Foxp3 protein expression is directly measured by intracellular FACS staining. Naïve CD4⁺ T cells could undergo robust Foxp3⁺ Treg differentiation when they were activated by polyclonal or antigen-specific stimulation in the presence of exogenous TGF-β. The induction of Foxp3 expression occurred in a TGF-β dose-dependent manner, and with the optimal dose of TGF-β1 >90% of CD4⁺ T cells could differentiate into Foxp3⁺ Tregs with potent suppressor activity [supporting information (SI) Fig. 5]. Thus, it appears that the Foxp3⁺ Treg differentiation potential is fully retained in naïve CD4⁺ T cells after their maturation in the thymus. However, this finding has raised a critical question as to why Th1 and Th2 differentiation often dominate the outcome of naïve CD4⁺ T cell response to antigen stimulation, whereas the development of Tregs is restricted to certain conditions. To address this issue, we carried out a set of experiments by treating naïve CD4⁺ cells with TGF-β1 and Th1- or Th2-polarizing cytokines simultaneously during their activation in response to anti-CD3 stimulation. We found that the addition of Th1 promoting cytokine IL-12 or IFNγ to the cultures resulted in a substantial reduction of the percentage of Foxp3 expression induced by TGF-β1 (Fig. 1A). More strikingly, induction of Foxp3⁺ Tregs was completely inhibited by IL-4, a Th2 differentiation cytokine (Fig. 1A). Recent studies showed that IL-6 can potently antagonize TGF-β-mediated induction of Foxp3⁺ Tregs whereas it promotes Th17 differentiation (23–25). In direct comparison, our experiments showed that IFNγ and IL-12 have a similar level of inhibition as IL-6, whereas IL-4 is much stronger even at lower doses (SI Figs. 6 and 7). To substantiate this finding, we performed further experiments using antigen-presenting cell (APC)-dependent T cell activation systems in which WT CD4⁺ T cells were stimulated with soluble anti-CD3 in the presence of spleen APCs, OT-II transgenic T cells by OVA_323–339 peptide plus spleen APCs, or bone marrow-derived dendritic cells (Fig. 1 B–D). Similar inhibition was observed with all of these conditions, suggesting that the antagonistic effect of Th1/Th2-polarizing cytokines on TGF-β-mediated induction of Foxp3⁺ Tregs is a general phenomenon for peripheral CD4⁺ T cells. It is interesting to note that IL-12 and IFNγ exerted stronger inhibitions on Foxp3 induction in the APC-dependent T cell activation systems, in particular when T cells were stimulated by the cognate peptide ligand (Fig. 1 C and D). To determine whether the inhibition occurred at the transcriptional level, we carried out real-time PCR to measure Foxp3 mRNA expression. Consistently, IL-12, IFNγ, or IL-4 treatment potently inhibited Foxp3 mRNA induction, which correlated well with the decrease of percentages of Foxp3⁺ cells in the cultures (Fig. 1E). To confirm that the diminution of Foxp3 expression reflects a functional block of Treg development, we performed T cell suppression assays. Indeed, the IL-12-, IFNγ-, or IL-4-treated cultures exhibited much lower levels of suppressive activity compared with the control and were directly proportional to the reduction of the frequency of Foxp3-expressing cells (Fig. 1F). Thus, these results have revealed a regulatory role of Th1- or Th2-polarizing cytokines in negative control of peripheral induction of Foxp3⁺ Tregs.

Fig. 1.

Inhibition of Foxp3⁺ Treg differentiation by IL-12, IFNγ, and IL-4. Naïve CD4⁺ T cells from WT mice were stimulated by plate-bound anti-CD3 and soluble anti-CD28 (A) or by soluble anti-CD3 and spleen APCs (B) in the presence of 1 ng/ml TGF-β1. Naïve CD4⁺ T cells from OT-II TCR-transgenic mice were stimulated by OVA_323–339 peptide and spleen APCs (C) or by OVA_323–339 peptide and bone marrow-derived dendritic cells (D) in the presence of 1 ng/ml TGF-β1. Recombinant IL-12 (10 ng/ml), IFNγ (100 ng/ml), or IL-4 (10 ng/ml) was added to the cultures as indicated. Representative FACS plots of anti-Foxp3 and anti-CD25 double staining (Left) and histograms of anti-Foxp3 single stain (Right) are shown. (E) The cells from A were analyzed by real-time PCR analysis for Foxp3 mRNA expression, and relative expression levels are plotted. (F) Suppressive activities of induced Tregs from A were measured by CFSE-based suppression assay with percentage numbers showing target cells divided more than once (average of two independent experiments). (G) Naïve CD4⁺ cells from WT mice were stimulated by anti-CD3/soluble anti-CD28 in the presence of TGF-β1 (≈0.1–10 ng/ml) with or without neutralizing mAbs (10 μg/ml) to IFNγ and IL-4. Histograms of Foxp3 expression are shown.

IFNγ/IL-4 Blockade Promotes both in Vitro and in Vivo Induction of Foxp3⁺ Treg.

In complementary experiments, we observed that the addition of IFNγ- and IL-4-neutralizing Abs to the cultures could result in a substantial enhancement of Foxp3 induction when T cells were stimulated with low amounts of TGF-β1 (0.1–1 ng/ml) (Fig. 1G). Furthermore, IFNγ/IL-4 blocking also led to Foxp3 induction in the absence of exogenous TGF-β (data not shown). To understand the mechanism of this regulation, we performed intracellular cytokine staining for IFNγ and IL-4. The results showed that naïve CD4⁺ T cells could produce low levels of IFNγ and IL-4 in response to TCR stimulation (SI Fig. 8). Consistent with earlier reports (26, 27), IFNγ and IL-4 production was inhibited by high levels of TGF-β1 (SI Fig. 8). However, with lower levels of TGF-β1 the inhibition was incomplete, and a residual amount of IFNγ and/or IL-4 was produced by the stimulated cells (SI Fig. 8). Thus, both exocrine and autocrine forms of IFNγ and IL-4 can effectively counter Foxp3 induction, which are mostly potent when TGF-β levels are low (Fig. 1G). To investigate whether this mechanism operates in antigen-specific T cell response in vivo, we used the Rag^−/− DO11.10 TCR-transgenic system originally described by Thorstenson and Khoruts (28). After adoptive transfer of Rag2^−/− DO11.10 donor T cells, which do not contain any preexisting Foxp3⁺ Tregs, the recipient mice were immunized by i.v. injection of the cognate antigen peptide. To examine the effect of IFNγ/IL-4 on Treg induction, one group of the mice were treated with IFNγ- and IL-4-neutralizing Abs, whereas other groups were treated with control Abs or left untreated. Consistent with the previous report (28), this immunization scheme led to de novo induction of a small population of antigen-specific CD25⁺ Tregs from the donor cells. Using Foxp3 intracellular staining, we confirmed that indeed the majority of these induced CD25⁺ cells coexpressed Foxp3, although some of the responding donor cells were positive for only Foxp3 or CD25 (Fig. 2 A and D). Importantly, administration of IFNγ/IL-4-neutralizing Abs resulted in a 2- to 3-fold increase of the frequency of Foxp3 expression in both spleen and lymph node compartments compared with the controls (Fig. 2 B and E), and a similar level of increase was also shown by enumerating the absolute number of Foxp3⁺ donor cells (Fig. 2 C and F). Therefore, we concluded that IFNγ/IL-4 blockade can also promote antigen-triggered de novo Foxp3⁺ Treg induction in vivo.

Fig. 2.

Neutralizing IFNγ/IL-4 enhances de novo induction of Foxp3⁺ Tregs in vivo. Rag2^−/− DO11.10 CD4⁺ T cells were adoptively transferred to BALB/c mice, and the recipients were immunized with four conditions: no peptide; 25 μg of OVA_323–339 peptide; 25 μg of OVA_323–339 peptide plus control Abs; and 25 μg of OVA_323–339 peptide plus anti-IFNγ/IL-4-neutralizing Abs. Nine days after immunization the spleen (A–C) and peripheral lymph node (D–F) samples were analyzed for Foxp3 and CD25 expression in the KJ1-26⁺CD4⁺ donor T cells. (A and D) Representative FACS plots and histograms showing coexpression of Foxp3 and CD25 and the percentage of Foxp3-expressing KJ1-26⁺CD4⁺ donor cells. (B and E) Statistical representation of Foxp3 induction in the donor cells. (C and F) Enumeration of the absolute number of Foxp3⁺ KJ1-26⁺CD4⁺ cells. Each bar represents the mean value from three individual mice per group. *, P > 0.1; **, P < 0.05.

IFNγ or IL-4 Inhibits Foxp3⁺ Treg Induction by Activating the Central Signaling Pathway for Th1/Th2 Lineage Differentiation.

The finding of antagonistic effect of Th1 and Th2 cytokines on Foxp3⁺ Treg induction prompted us to investigate whether the canonical signaling pathways for Th1/Th2 differentiation are involved. We first examined whether the requirement of Stat1 or T-bet mediates the inhibitory activity of IFNγ on Foxp3⁺ Treg differentiation. We found that, in the absence of Stat1 or T-bet, the inhibition of Foxp3 expression by IFNγ was abrogated in both APC-dependent and -independent T cell stimulation systems (Fig. 3 A and B). Interestingly, the inhibitory effect of IL-12 appears to be mediated by Stat4 and requires IFNγ receptor signaling (SI Fig. 9). Thus, IFNγ is likely to function as a central component of Th1 program in negative regulation of Foxp3 expression. To investigate the Th2 signaling pathway, we used T cells isolated from Stat6 knockout mice. We found that Stat6 deficiency resulted in the loss of Foxp3 inhibition imposed by IL-4 (Fig. 3C). Thus, together, these genetic experiments demonstrated that the activation of a Th1 or Th2 differentiation program is required for IFNγ or IL-4, respectively, to inhibit Foxp3 induction and Treg development from naïve CD4⁺ T cells.

Fig. 3.

IFNγ/IL-4 inhibits Foxp3 induction via the canonical signaling pathways required for Th1/Th2 lineage differentiation. Naïve CD4⁺ T cells from WT mice, Stat1^−/−, and T-bet^−/− were stimulated by soluble anti-CD3/spleen APC (A) or plate-bound anti-CD3/soluble anti-CD28 (B) in the presence of 1 ng/ml TGF-β1. IFNγ (100 ng/ml) was added to the cultures as indicated. (C) Naïve T cells from WT and Stat6^−/− mice were stimulated by plate-bound anti-CD3/soluble anti-CD28 in the presence of 1 ng/ml TGF-β1. IL-4 (10 ng/ml) was added to the cultures as indicated. Representative FACS plots of Foxp3 staining are shown with the percentage numbers averaged from two independent experiments.

Involvement of Th1/Th2 Effector Cytokine-Dependent and -Independent Mechanisms in Preventing Foxp3⁺ Treg Differentiation.

To further investigate the mechanism involved in the negative regulation of Foxp3⁺ Treg fate, we examined whether Th1/Th2 primed cells could be reprogrammed to undergo Foxp3⁺ Treg differentiation in response to TGF-β1 treatment. To test this, we activated naïve CD4⁺ T cells with plate-bound anti-CD3 and soluble anti-CD28 under different polarization conditions: (i) without Th1/Th2 cytokines or blocking Abs as the nonpolarizing condition; (ii) with IL-12 as Th1-polarizing; (iii) with IL-4 as Th2-polarizing; and (iv) with IFNγ- and IL-4-blocking Abs as the neutral condition. After priming, the cells were harvested and restimulated with plate-bound anti-CD3 plus TGF-β1 to induce Foxp3⁺ Treg differentiation. Whereas the cells primed under the neutral condition were able to differentiate into Foxp3⁺ Tregs like naïve cells (Fig. 4 A, 4), Th1 or Th2 primed cells were refractory to Foxp3 induction (Fig. 4A, 2 and 3). Interestingly, the cells primed under the nonpolarizing condition also failed to undergo Foxp3⁺ Treg differentiation (Fig. 4A, 1). Consistent with the results presented in SI Fig. 10, the lack of Foxp3⁺ Treg induction from cells primed by Th1, Th2, and nonpolarizing conditions appeared to correlate with their ability to produce IFNγ and/or IL-4 as detected by intracellular cytokine staining analysis (SI Fig. 10B). To determine whether the impairment of Foxp3⁺ Treg differentiation was primarily caused by the autocrine effect of IFNγ and/or IL-4 in the primed T cells, we conducted another set of experiments by adding IFNγ- and/or IL-4-blocking Abs to the cultures. To our surprise, we found that IFNγ and IL-4 neutralization failed to rescue Foxp3 induction in Th1 or Th2 polarized cells (Fig. 4A, 2 and 3), although the same treatment resulted in a marked restoration of Foxp3 expression in the cells primed by the nonpolarizing condition (Fig. 4A, 1). Thus, these results demonstrated that, even though autocrine IFNγ and IL-4 play an important role in inhibiting Foxp3 induction, an effector cytokine-independent mechanism must operate intrinsically in polarized Th1/Th2 cells to prevent their divergence to the Foxp3⁺ Treg lineage.

Fig. 4.

Negative cross-regulation of Foxp3⁺ Treg development by Th1/Th2 effector cytokines and the lineage-specific transcription factors. (A) Naïve CD4⁺ cells from WT mice were stimulated in primary cultures (1° stimulation) by anti-CD3/anti-CD28 under different priming conditions: 1, nonpolarizing, no neutralizing mAbs or Th1/Th2-polarizing cytokines; 2, Th1-polarizing plus 10 ng/ml IL-12; 3, Th2-polarizing plus 10 ng/ml IL-4; 4, neutral plus neutralizing mAbs (10 μg/ml) to IFNγ and IL-4. The cells were harvested on day 7 and restimulated by plate-bound anti-CD3 and TGF-β1 in secondary cultures (2° stimulation) with or without neutralizing mAbs (10 μg/ml) to IFNγ and/or IL-4 to induce Foxp3⁺ Treg. A rat IgG isotype control was included (control Ab). Representative FACS plots of Foxp3 staining are shown (average of two independent experiments). (B) The cells from A were restimulated in the presence of anti-IFNγ/IL-4 to induce Foxp3⁺ Treg differentiation. Suppressive activities of the induced Tregs were determined by CFSE-based suppression assay (average of two independent experiments). Naïve CD4⁺ cells from WT (C), IFNγ^−/− (D), or IL-4^−/− (E) mice were activated under the neutral condition and transduced with an EGFP bicistronic retroviral vector encoding T-bet or GATA-3. Empty vector was included as control (Vector ctrl). The transduced cells (GFP⁺) were isolated by cell sorting for subsequent experiments. (Top and Middle) Intracellular cytokine staining for IFNγ and IL-4 production. (Bottom) Foxp3 expression induced by anti-CD3 plus TGF-β1 stimulation in secondary culture (average of two independent experiments). (F) The cells from C–E were subjected to real-time PCR analysis for Foxp3 mRNA expression, and relative expression levels are plotted. (G) WT CD4⁺ T cells were transduced with the mutant variants of T-bet and GATA-3, and Foxp3 induction was as in C. Histograms of Foxp3 expression are shown. T-bet ΔDBD, deletion of the T-box DNA binding domain; T-bet ΔAD, deletion of the C-terminal transcription activation domain; GATA-3 ΔNF, deletion of the N-terminal zinc finger DNA binding domain; GATA-3 ΔND, deletion of the N-terminal activation domain.

Inhibition of Foxp3⁺ Treg Differentiation by Th1/Th2 Lineage Transcription Factors T-bet and GATA-3.

T-bet and GATA-3 are well characterized master transcription factors for Th1 and Th2 lineage specification, respectively. These transcription factors not only play a positive role in promoting the permissive lineage fate but also actively repress the opposite fate choice (1, 2). Because polarized Th1/Th2 cells appear to use a cell-intrinsic mechanism to inhibit Foxp3⁺ Treg development, we postulated that T-bet and GATA-3 might constitute a central negative regulatory circuit for such inhibition. To test this hypothesis, we used retroviral vector to ectopically express T-bet or GATA-3 in naïve CD4⁺ activated under the neutral condition (29). After retrovirus transduction, the productively transduced cells were sorted based on their expression of a bicistronic GFP marker carried by the vector. Consistent with previous reports (30), our intracellular cytokine staining showed that an enforced expression of T-bet led to a selective activation of IFNγ production, whereas GATA-3 led to IL-4 production (Fig. 4C Top). To examine the Foxp3⁺ Treg differentiation potential, the sorted cells were restimulated with anti-CD3 in the presence of TGF-β1 as in Fig. 4A. Interestingly, although the empty vector transduced cells could undergo normal differentiation to Foxp3⁺ Tregs, this potential was completely lost in T-bet or GATA-3 transduced cells. Because enforced expression of T-bet or GATA-3 in WT T cells rendered active production of IFNγ or IL-4, we conducted further experiments to rule out that the impairment of Foxp3⁺ Treg differentiation was simply enacted by the effector cytokines. To this end, cytokine-deficient T cells were used. First, IFNγ^−/− cells were transduced with T-bet, GATA-3, and control viruses. The control analyses showed that indeed no IFNγ was detected in the knockout cells transduced with T-bet, whereas the normal level of IL-4 production was induced by GATA-3 transduction (Fig. 4D Top). Consistent with the notion of a cell-intrinsic mode of inhibition, T-bet expression was fully capable of inhibiting Foxp3 expression in the total absence of IFNγ (Fig. 4D Bottom). In complementary experiments with IL-4^−/− cells, we found that ectopic expression of GATA-3 was sufficient to inhibit Foxp3⁺ Treg differentiation in IL-4^−/− cells (Fig. 4E). Thus, these results revealed that Th1/Th2 lineage transcription factors T-bet and GATA-3 could operate independent of their downstream effector cytokines to inhibit Foxp3⁺ Treg fate. In agreement with the inhibition of Foxp3 protein expression, real-time PCR analysis showed that Foxp3 mRNA levels were diminished by the enforced expression of T-bet or GATA-3 (Fig. 4F). To further confirm the specificity of this inhibitory effect, we examined mutations in T-bet and GATA-3 that affect their DNA binding or transcription activation (31, 32). We found that these mutant forms of T-bet and GATA-3 totally failed to inhibit Foxp3 expression (Fig. 4G). Thus, these results provided strong support to the specific requirement of DNA binding and transcription activity of T-bet and GATA-3 in negative cross-regulation of Foxp3⁺ Treg differentiation from naïve CD4⁺ T cells.

Discussion

How peripheral differentiation of Foxp3⁺ Tregs is induced and regulated is of critical importance to understanding the fundamental question of dominant control of immunological tolerance to self as well as to foreign antigens in physiological and pathophysiological settings. In this work we have revealed that induction of Foxp3⁺ Tregs in the periphery is actively opposed by factors that drive Th1/Th2 lineage differentiation. We showed that Th1/Th2 polarization cytokines could effectively inhibit Treg differentiation from naïve cells induced by TGF-β1, whereas blocking of IFNγ/IL-4 could promote Foxp3⁺ Treg differentiation both in vitro and in vivo (Figs. 1 and 2). Using Stat1, Stat6, and T-bet knockout mice, we confirmed that the inhibition of Foxp3⁺ Treg induction required full activation of the central intracellular signaling pathways for Th1/Th2 lineage differentiation (Fig. 3). More importantly, we demonstrated that up-regulation of T-bet or GATA-3 expression, which is associated with and required for Th1/Th2 lineage commitment, was sufficient to block Foxp3 induction in a cell-autonomous manner (Fig. 4). Taken together, our findings have provided a new insight into Th1/Th2 antagonism to Foxp3⁺ Treg induction and mechanisms of cross-regulation of regulatory and effector fate choice (SI Fig. 11).

In addition to intrathymic development, Tregs expressing Foxp3 can also be generated in the periphery from naïve CD4⁺ precursor cells in response to a diverse array of antigens, including both self and non-self antigens (4–6). It has become clear that induction of Foxp3 expression in naïve CD4⁺ precursor cells and commitment to Treg differentiation are largely instructed by TGF-β. However, little is known about other signals that can positively or negatively regulate Treg generation during immune responses. In general, peripheral induction of Foxp3⁺ Tregs appears to be a rare event and is restricted to certain conditions. Unlike Th1 and Th2 effector differentiation, which are the predominant outcomes of most T cell immune responses to microbial infections, innocuous environmental antigens in allergy, or self-antigens in autoimmune inflammation, de novo generation of Foxp3⁺ Tregs has not been observed under these circumstances (13, 14). Three possibilities can be put forward to explain this phenomenon. First is that only some naïve CD4⁺ T cells in the periphery have the potential to activate Foxp3 expression and differentiate into Tregs. Second, TGF-β, the driving factor for peripheral Foxp3⁺ Treg differentiation, is limiting during normal immune responses. Third, Foxp3⁺ Treg differentiation is outcompeted by other T cell lineage fates and/or actively inhibited by negative signals. Using several well defined in vitro CD4⁺ T cell stimulation systems, we now show that the majority of CD62L⁺CD25⁻ naïve CD4⁺ T cells are capable of differentiating into Foxp3-expressing Tregs when they are stimulated by different modes of TCR triggering in the presence of TGF-β1. However, this differentiation potential is strongly antagonized by either Th1 or Th2 lineage differentiation factors. We envisage that, during microbial infection or autoimmunity, early IL-12, INFγ, or IL-4 produced by activated innate cells, such as dendritic cells, natural killer cells, or natural killer T cells, would drive Th1 or Th2 differentiation (1–3). Concomitantly, the activation of Stat1/Stat6 signaling pathways and subsequent up-regulation of Th1/Th2 lineage-specific transcription factors by these cytokines will forcefully inhibit Foxp3 expression in antigen-responding T cells. Moreover, once the initial cohorts of Th1/Th2 effector cells are formed and accumulated at the infection/inflammatory site, they will further reinforce and amplify the inhibition of Foxp3⁺ Treg development through the production of Th1/Th2 effector cytokines. However, it remains possible that Foxp3⁺ Tregs can be induced toward the late stage of inflammatory immune responses when innate activation and Th1/Th2 effector activities have waned (5, 19). In keeping with this, our findings lend further support to the view that the peripheral induction of Foxp3⁺ Tregs might not normally function as a negative feedback mechanism for down-regulating immune responses or limiting immunopathology during ongoing infection or autoimmune/inflammatory flare (13, 14). Thus, to induce Treg differentiation and enforce Foxp3⁺ Treg-mediated dominant tolerance in such circumstances, it is necessary to first block and neutralize Th1/Th2-polarizing and effector cytokines. Indeed, our in vivo experiments showed that IFNγ/IL-4 blockade could substantially enhance naïve T cells to undergo Foxp3⁺ Treg differentiation in response to antigen stimulation (Fig. 2).

The finding of Th1/Th2 antagonism to Foxp3⁺ Treg development has further highlighted recent studies on cross-regulations of CD4⁺ T cell fate decisions in peripheral immune responses (1–3). In addition to Th1 and Th2, T cells producing members of the IL-17 family of cytokines have recently been recognized as a new Th cell lineage, namely Th17 (3). Interestingly, Th17 differentiation has been shown to be instructed by the dual actions of TGF-β and IL-6 (23, 25, 33). Furthermore, Foxp3⁺ Treg development also appears to be antagonized by this Th lineage program. The study by Bettelli et al. (23) showed that while IL-6 promotes the development of Th17 cells, it simultaneously blocks Foxp3⁺ Treg generation induced by TGF-β1. Similarly, we also observed the inhibitory effect of IL-6 on Foxp3 induction in our experiments, albeit less potent than IL-4 (SI Fig. 6). Thus, in light of all these findings, it appears that Foxp3⁺ Treg development is controlled by multipolar cross-regulations rather than a simple dichotomous decision of regulatory versus effector fate choice. More significantly, our study has further revealed that the so-called master lineage transcription factors are at the center of such multipolar cross-regulations. We showed that ectopic expression of T-bet or GATA-3 is sufficient to block Foxp3 induction and Treg differentiation, which requires both the DNA binding and transcription activation activities of these two transcription factors (Fig. 4G). How IL-6 and the Th17 lineage program antagonize Foxp3⁺ Treg differentiation is not fully understood. A more recent study by Ivanov et al. (24) showed that the orphan nuclear receptor family of transcription factor RORγt is synergistically induced by TGF-β and IL-6 signaling and is necessary and sufficient to activate IL-17 expression. Thus, RORγt appears to assume the same status as T-bet and GATA-3 in functioning as the master transcriptional regulator for Th17 effector lineage development. It will be interesting to investigate in future experiments whether RORγt also plays a key role in inhibiting Foxp3⁺ Treg fate during a Th17-dominated response.

In summary, we report here that postthymic development of Foxp3⁺ Tregs in response to antigen stimulation is profoundly antagonized by Th1/Th2 lineage differentiation activities involving both cell-intrinsic and -extrinsic mechanisms. These findings are likely to facilitate further investigation to elucidate the molecular control of Treg fate choice and to manipulate peripheral induction of Foxp3⁺ Tregs for disease treatment.

Materials and Methods

Mice.

C57BL/6 and BALB/c mice were purchased from the National Cancer Institute (Bethesda, MD), OT-II TCR and Rag2^−/− DO11.10 TCR-transgenic mice and Stat1^−/− and T-bet^−/− mice were from Taconic (Germantown, NY), and IL-4^−/−, IFNγ^−/−, IFNγR^−/−, Stat4^−/−, and Stat6^−/− mice were from The Jackson Laboratory (Bar Harbor, ME). All mouse colonies were maintained in a specific pathogen-free barrier facility at the University of Texas M. D. Anderson Cancer Center approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, and procedures conformed to the Institutional Animal Care and Use Committee protocols.

Adoptive Transfer and Immunization.

Rag2^−/− DO11.10 CD4⁺ T cells were adoptively transferred (3–4 × 10⁶ cells per mouse) by i.v. injection into BALB/c mice. Two days later, the recipient mice were immunized by a single i.v. injection of 25 μg of OVA_323–339 peptide in PBS saline; some mice were treated with 0.4 mg of anti-IFNγ- and anti-IL-4-neutralizing mAbs or control Abs by i.p. injection every other day for four times after the cell transfer.

Cell Preparation and Tissue Culture Reagents.

See SI Materials and Methods.

Retroviral Transduction.

MigR1 retroviral vector (carrying a bicistronic EGFP marker)-based constructs encoding the WT murine T-bet and GATA-3 were described in ref. 30, GATA-3 mutants ΔNF and ΔND were in ref. 32, and T-bet mutants ΔDBD and ΔAD were in ref. 31. Retroviral constructs were cotransfected with pCL-Eco packaging plasmid in 293T cells as described (29). The virus supernatants were collected 48 h after transfection. Naive cells were activated by anti-CD3 under the neutral priming condition. Forty-eight hours after activation, the cells were infected with the virus supernatants with 6 μg/ml polybrene (Sigma–Aldrich) under centrifugation (1,328 × g) for 90 min and further cultured for 4–5 days with rIL-2 (50–200 units/ml). The productively transduced GFP⁺ cells were isolated by FACS sorting (>98% purity).

Flow Cytometry.

PerCP-conjugated anti-CD4 (L3T4) and allophycocyanin-conjugated anti-CD25 (PC61) mAbs were used for surface staining. Intracellular staining for Foxp3 was done with phycoerythrin-conjugated anti-mouse Foxp3 (FJK-16s; eBioscience) according to the manufacturer's instruction. DO11.10 transgenic T cells were identified by phycoerythrin-conjugated KJ1-26 clonotypic mAb (BD Pharmingen). Phycoerythrin-conjugated anti-mouse IFNγ (XMG1.2) and anti-mouse IL-4 (11B11) were used for intracellular cytokine staining with FIX/PERM buffers (BD Pharmingen). Flow cytometry acquisition was done with a FACSCalibur (Becton Dickinson), and data analysis was done with FlowJo software (TreeStar).

Suppression Assays.

Standard [³H]thymidine incorporation and FACS analysis for CFSE dilution were used. See SI Materials and Methods for details.

Real-Time PCR.

Real-time PCR was performed by SYBR Green Gene Expression Assays using the Universal PCR Master Mix and ABI-PRISM 7500 Sequence Detector System (Applied Biosystems/PerkinElmer). See SI Materials and Methods for primer sequences.

Western Blot Analysis.

Western blot analysis was performed with anti-Foxp3 (clone 7979; eBioscience) and anti-β-actin (Sigma–Aldrich) mAbs followed by HRP-conjugated goat anti-mouse IgG and ECL detection (Pierce).

Statistical Analysis.

A two-tailed, paired Student t test was used for the analysis.

Abbreviations

Treg

regulatory T cell

Th

T helper

APC

antigen-presenting cell

TCR

T cell receptor.