IL-4-mediated inhibition of IFN-{gamma} production by CD4+ T cells proceeds by several developmentally regulated mechanisms

Olivier Wurtz1, Marc Bajénoff1 and Sylvie Guerder1

1 Centre d’Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale/Centre National de la Recherche Scientifique/Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 09, France

Correspondence to: S. Guerder; E-mail: guerder{at}ciml.univ-mrs.fr
Transmitting editor: A. Cooke


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mechanisms by which Th1 and Th2 cells inter-regulate in vivo are still poorly understood. In this study we examined the plasticity of Th1 cell differentiation and how Th2 cells may down-regulate these responses. We show here that IL-4 affects Th1 cell responses by two developmentally regulated mechanisms. During the commitment phase of naive CD4+ T cells, IL-4 inhibits Th1 cell differentiation and induces a reversion of developing Th1 cells to the Th2 lineage. In contrast, for effector Th1 cells IL-4 does not affect the developmental process, but only the transcription of the IFN-{gamma} gene. We further show that the difference in IL-4 responsiveness correlates with a loss, in effector Th1 cells, of IL-4-dependent up-regulation of GATA-3 expression despite normal activation of STAT6. Transient inhibition of IFN-{gamma} production by differentiated effector cells may explain why Th1 and Th2 responses can co-exist in vivo although Th2 effector cells dominate functionally, as observed in some infectious or autoimmune mice models.

Keywords: cellular differentiation, gene regulation, Th1/Th2, transcription factor


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Following stimulation by antigen-presenting cells (APC), CD4+ T cells differentiate into either Th1 or Th2 effector cells. Lineage commitment is critically regulated by the cytokines present during the early phase of T cell activation. Thus, IFN-{gamma} and IL-12 instruct Th1 cell development and inhibit Th2 cell differentiation. The binding of IFN-{gamma} to its receptor and subsequent STAT1 activation will induce T-bet gene expression, the master regulator of Th1 cell differentiation (14). The transcription factor T-bet induces IL12Rß2 chain expression, thus allowing for IL-12-dependent STAT4 activation and subsequent Th1 cell differentiation (2,58). In addition, T-bet inhibits, by yet unknown mechanisms, Th2 cell differentiation (1,7). Conversely, IL-4 instructs Th2 cell differentiation and inhibits Th1 cell differentiation through activation of STAT6 and subsequent induction of the expression of GATA-3, the master regulator of Th2 cell differentiation (915). The transcription factor GATA-3 triggers chromatin remodeling at the IL-4/IL-13 locus, and consequently expression of the Th2 cytokines IL-4, -5 and -13 (13,1517). GATA-3 also inhibits Th1 cell differentiation, likely by direct or indirect inhibition of IFN-{gamma} production and IL12Rß2 gene expression (13,1517).

Due to these different regulatory circuits Th1 and Th2 responses are mutually exclusive in vitro. In vivo, however, Th1 and Th2 responses may co-exist, although one response dominates functionally, suggesting a more complex regulation. This is clearly observed in the mouse model of leishmaniasis. Control of Leishmania major infection correlates with development of a Th1 response, while susceptibility is associated with a dominant Th2 response. In the early phase of the infection, however, both resistant B10.D2 and susceptible BALB/c mice develop a similar Th1 and Th2 response against the parasite L. major (18). While the Th2 response extinguishes within 2 weeks of infection in B10.D2 mice, both responses persist in BALB/c mice (18). More importantly, in BALB/c mice the persistent Th2 response appears to dominate functionally in vivo, thus preventing parasite clearance by antigen-specific Th1 cells. Functional dominance of Th2 responses is also observed in some models of autoimmune diabetes. Indeed, in transgenic mice models of autoimmune diabetes as well as in the NOD mouse strain, anti-islet Th2 response correlates with reduced disease incidence despite the persistence of an islet-specific Th1 response (1921). These observations raised the possibility that Th2 cells may regulate Th1 responses by several different mechanisms, altering the differentiation process as well as the effector phase.

In this study we address this possibility and show that IL-4 affects Th1 cell responses by two developmentally regulated mechanisms. During the commitment phase of naive CD4+ T cells, IL-4 inhibits Th1 cell differentiation and induces a reversion of developing Th1 cells to the Th2 lineage. In contrast, for effector Th1 cells IL-4 does not affect the developmental process, but only the transcription of the IFN-{gamma} gene. We further show that the difference in IL-4 responsiveness correlates with a loss in effector Th1 cells of IL-4-dependent up-regulation of GATA-3 expression despite normal activation of STAT6.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
B10.BR mice were purchased from Harlan France SARL (Gannat, France). Stat6-deficient mice were maintained on a B6 background (11). Six- to 10-week-old mice are used as experimental animals.

Cell purification and in vitro T cell priming
Lymph nodes CD4+ T cells were purified by negative selection using a cocktail of antibodies containing RA36B2 (anti-B220), M1/70.15.11.5HL (anti-CD11b), 24G2 (anti-FcRII/III), H59.101.2 (anti-CD8) and M5/114.15.2 (pan-anti-MHC class II) followed by incubation with sheep anti-rat IgG and sheep anti-mouse IgG magnetic beads (Dynal, Biotech, Oslo, Norway). After magnetic depletion, the selected population was >90% enriched in CD4+ T cells. When indicated naive CD44CD62L+ T cells were purified by FACS sorting using anti-CD44–phycoerythrin and anti-CD62L–FITC (BD PharMingen, La Jolla, CA). For primary stimulation, 5 x 105 CD4+ T cells were incubated with 106 irradiated (24 Gy) T cell-depleted splenocytes as APC and 1 µg/ml of soluble anti-CD3 (145.2C11) and anti-CD28 (37.51) antibodies. At the indicated time point, cells were washed 3 times, and 5 x 105 CD4+ T cells were re-stimulated with plate-bound anti-CD3 (10 µg/ml) and 1 µg/ml of soluble anti-CD28 in the presence of 50 U/ml recombinant murine IL-2 (Peprotech, Princeton, NJ). To induce Th1 differentiation, 3.5 ng/ml recombinant murine IL-12 (Peprotech) and 10 µg/ml anti-IL-4 (11B11) were added to the culture. To induce Th2 differentiation, 10 ng/ml recombinant murine IL-4 (Peprotech) and 10 µg/ml anti-IFN-{gamma} (XMG1-2) were added to the culture. For Th0 conditions, 10 µg/ml of 11B11 and XMG1-2 antibody were added to the cultures. The anti-CD3, anti-CD28, anti-IL-4 and anti-IFN-{gamma} antibodies were produced and purified at the Centre d’Immunologie de Marseille-Luminy.

Antibodies and intracellular FACS staining
The anti-CD4–PerCP, anti-IL4–allophycocyanin and anti-IFN-{gamma}–FITC antibodies were all purchased from BD PharMingen. For intracellular FACS staining, cells were re-stimulated overnight with plate-bound anti-CD3 (10 µg/ml) and 1 µg/ml soluble anti-CD28 in the presence of 50 U/ml of recombinant murine IL-2; 5 µM Monensin (Sigma-Aldrich, St Louis, MO) was added for the last 4 h of stimulation. Quadruple staining were performed as previously described (22).

Protein extraction and electrophoretic mobility shift assays (EMSA)
CD4+ T cells were lysed in buffer A (10 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM DTT and 0.625% NP-40) containing protease inhibitors (0.5 mM PMSF, 2 µg/ml aprotenin, leupeptin, pepstatin A, chymostatin and antipain, 1 mM sodium orthovanadate, 10 mM sodium molybdate, and 1 mM DTT). Cytoplasmic extracts were collected after centrifugation and nuclei were lysed in Nuclear lysis buffer (20 mM HEPES, pH 7.6, 0.4 M NaCl, 1 mM EDTA and 1 mM EGTA) containing protease inhibitors, as described above. For EMSA, 2 µg nuclear proteins were incubated with 5 x 104 32P-end-labeled double-stranded STAT6 oligonucleotides (3'-GTATTTCCC AGAAAAGGAAC-5'; Santa Cruz Biotechnology, Santa Cruz, CA) in 30 µl binding buffer [50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 5% glycerol and 2 µg of poly(dI–dC)]. For supershift experiments, extracts were pre-incubated for 30 min at room temperature with 2 µg STAT6-specific antibody (Santa Cruz Biotechnology) before addition of the labeled probe.

RNA preparation and quantitative RT-PCR
Total RNA was extracted using the High Pure RNA Isolation kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer’s instructions, treated with DNase I (Roche Diagnostic), and reverse transcribed using random primers and Superscript II RT (Life Technology, Grand Island, NY). Real-time PCR was performed on cDNA samples using the SYBR Green system (PE Biosystems, Warrington, UK). Primers used were HPRT sense 5'-AGC CCT CTG TGT GCT AAG G-3', HPRT antisense 5'-CTG ATA AAA TCT ACA GTC ATA GGA ATG GA-3'; T-bet sense 5'-CAA CAA CCC CTT TGC CAA AG-3', T-bet antisense 5'-TCC CCC AAG CAG TTG ACA GT-3'; GATA-3 sense 5'-GAG GTG GAC GTA CTT TTT AAC ATC G-3', GATA-3 antisense 5'-GGC ATA CCT GGC TCC CGT-3'. Cycling conditions were 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, and 40 cycles each corresponding to 15 s at 95°C and 1 min at 60°C. Analysis used the sequence detection software supplied with the instrument. The relative quantitation value is expressed as 2{Delta}cT, where {Delta}CT is the difference between the mean CT value of duplicates of the sample and of the endogenous HPRT control.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IL-4 inhibits Th1 responses by two developmentally regulated mechanisms
To examine the plasticity of Th1 cell differentiation and determine how IL-4 may down-regulate Th1 cell responses, we analyzed the effect of IL-4 on either the differentiation process or the expression by differentiated Th1 cells of effector functions. Several studies have shown that stimulation of naive CD4+ T cells under Th1-polarizing conditions for a period of 48–72 h suffices in inducing the differentiation of effector Th1 cells (23,24). Indeed, 24 h stimulation under Th1-polarizing conditions (IL-12 and anti-IL-4 antibody) of CD4+ T cells isolated from B10.BR mice did not lead to significant differentiation of IFN-{gamma}-producing Th1 cells (Fig. 1A). By 96 h of primary stimulation >50% of the CD4+ T cells were fully differentiated and produced IFN-{gamma} in the absence of IL-4 (Fig. 1A). Differentiation of CD4+ T cells into IL-4-producing Th2 cells follows a similar kinetic (Fig. 1A). Based on these results, we differentiated CD4+ T cells under Th1-polarizing conditions for 24 or 96 h and re-stimulated them for 48 h in the presence of IL-4 while blocking IFN-{gamma} signaling (Th2-polarizing condition). As control, CD4+ T cells were stimulated twice, with the same kinetics, under either Th1- or Th2-polarizing conditions, or Th1-polarized cultures were re-stimulated under neutral conditions (Th0, blocking of IL-4 and IFN-{gamma}). CD4+ T cells that were activated under Th1-polarizing conditions for 24 h, then re-stimulated under Th2-polarizing conditions differentiated exclusively into Th2 effector cells (Fig. 1A). Indeed, we detected no IFN-{gamma}-producing cells and maximal frequency of IL-4-producing cells in such cultures. Likewise, re-stimulation of 96 h-stimulated Th1 cells under Th2-polarizing conditions greatly reduced IFN-{gamma} production by these activated T cells (Fig. 1A). In this case, however, the frequency of IL-4-producing Th2 cells was only minimally increased (Fig. 1A). The cytokine production pattern of 24 or 96 h-stimulated Th1 cells re-stimulated under Th0 conditions was comparable to that of cultures re-stimulated under Th1 conditions, further suggesting that the cytokine profile of Th2 re-stimulated cultures clearly results from IL-4 signaling (Fig. 1A). Finally proliferation of activated CD4 T cells was comparable for the different culture conditions. Thus, the total number of cytokine-producing cells generated in the different re-stimulation conditions was similar (Fig. 1B). As a consequence, changes in the percentage of cytokine-producing cells were mirrored by similar changes in the number of cytokine-producing cells, further suggesting that IL-4 did not induce the selective death of Th1 cells, but rather modulated their cytokine production capacity (Fig. 1B).



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Fig. 1. IL-4 inhibits Th1 cell differentiation by two developmentally regulated mechanisms. (A–C) Total CD4+ T cells (A and B) or FACS-sorted CD62L+CD44 naive CD4+ T cells (C) were activated with soluble anti-CD3 and anti-CD28 antibody under Th1- or Th2-polarizing conditions as indicated (primary stimulation). Twenty-four or 96 h after primary stimulation some of the cells were re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibody under Th1- or Th2-polarizing conditions (secondary stimulation). At the end of the primary (none) and 48 h secondary stimulation IL-4 and IFN-{gamma} production were analyzed by intracellular FACS staining (A and C). (B) The number of cytokine-producing cells was then calculated by multiplying the percentage of cytokine-producing cells by the number of cells recovered in each culture. The number of IFN-{gamma} (gray symbols)- or IL-4 (black symbols)-producing cells obtained following activation of 106 naive T cells is presented. (D) CD4+ T cells isolated from B10.BR mice were stimulated under Th1 conditions for 96 h and re-stimulated under Th1 or Th2 conditions as in (A). At the end of the secondary stimulation period part of the culture was washed and rested in IL-2-containing medium for 48 h. At the end of the 96 h primary stimulation (a), 48 h secondary stimulation (b and c) and 48 h resting period (d and e) IL-4 and IFN-{gamma} production was analyzed by intracellular FACS staining. The percentage of positive cells in each quadrant is indicated (A, C and D). One representative experiment out of three is shown.

 
To ensure that memory cells may not contribute to the observed phenomena we performed similar experiments with FACS-purified naive CD44CD62L+CD4+ T cells. In this case, too, re-stimulation under Th2 conditions of 24 h-stimulated Th1 cells induced a complete reversion to the Th2 phenotype, while a similar re-stimulation of 96 h-stimulated Th1 cells only inhibits IFN-{gamma} production with no significant increase in IL-4-producing Th2 cells (Fig. 1C). Since FACS-sorted naive T cells and total CD4 T cells showed the same response pattern, we used total CD4 T cells in the subsequent experiment.

Collectively, these results suggest that IL-4-mediated regulation of Th1 responses may proceed by different mechanisms that are developmentally controlled. Thus, during the commitment phase IL-4 blocks Th1 cell differentiation and redirects the activated T cells towards the Th2 lineage. In contrast, for differentiated effector Th1 cells IL-4 does not appear to affect the differentiation process, but only the transcription of the IFN-{gamma} gene. The inhibitory effect of IL-4 on IFN-{gamma} production should thus be, in the later case, transient and reversible. To test this possibility we activated CD4+ T cells for 96 h under Th1-polarizing conditions, re-stimulated them for 48 h under Th2 conditions, then washed out the IL-4 and rested the cells for 48 h in the absence of IL-4 or IL-12. We found that the percentage of IFN-{gamma}-producing cells detected in such treated cultures was comparable to that detected in 96 h Th1 cells (Fig. 1D, cf. b and e). Thus, as anticipated, the inhibitory effect of IL-4 on IFN-{gamma} production was transient.

STAT6 activation is necessary for IL-4-mediated inhibition of Th1 responses
The contrasted effects of IL-4 on 24 or 96 h Th1 cells suggested that the signaling pathways activated by IL-4 receptor engagement may be regulated during Th1 cell differentiation. In agreement, Huang et al. showed that highly polarized Th1 cells demonstrate a major impairment in IL-4 signaling (25). Binding of IL-4 to the IL-4 receptor activates pathways involved in regulating proliferation as well as a rapid phosphorylation of the transcription factor STAT6, known to be critical for Th2 cell differentiation (911). We, therefore, determined whether the IL-4 receptor expressed by CD4+ T cells differentiated under Th1 conditions for 24 or 96 h was capable of activating STAT6. For this experiment we performed EMSA on nuclear extracts to directly analyze STAT6 phosphorylation, subsequent nuclear localization and binding of the dimers to STAT6 sites. In CD4+ T cells differentiated under Th2 conditions for 24 or 96 h, IL-4 induces the generation of complexes that bind to the STAT6 probe (Fig. 2, lanes 5 and 10). Binding of these complexes was abolished by pre-incubation of the extracts with a STAT6-specific antibody, indicating that these complexes are STAT6 homodimers (Fig. 2, lanes 6 and 11). IL-4, but not IL-12, induces similar complexes in Th1 cells differentiated for 24 or 96 h under Th1-polarizing conditions (Fig. 2, lanes 2, 3, 7 and 8). Supershift experiments with STAT6-specific antibody confirmed that these complexes are indeed STAT6 dimers (Fig. 2, lanes 4 and 9).



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Fig. 2. The IL-4 receptor is functional in polarized Th1 cells. CD4+ T cells isolated from B10.BR mice were activated for 24 or 96 h with soluble anti-CD3 and anti-CD28 antibody under Th1- or Th2-polarizing conditions as indicated (primary stimulation). At the end of the primary stimulation, the cells were re-stimulated for 30 min with plate-bound anti-CD3 and soluble anti-CD28 antibody under Th1- or Th2-polarizing conditions (secondary stimulation), and nuclear extracts were prepared. Protein extracts (2 µg) were analyzed by EMSA with a STAT6-specific probe in the presence (+) or absence (–) of STAT6-specific antibody. The STAT6-specific complexes are indicated with an arrow.

 
Activation of STAT6 following IL-4 binding to the IL-4 receptor therefore proceeds normally in Th1 cells differentiated for 24 or 96 h under Th1-polarizing conditions. We further determined using STAT6–/– mice whether STAT6 was required for IL-4-mediated inhibition of Th1 responses in 24 or 96 h-stimulated Th1 cells. As expected, the differentiation of naive CD4+ T cells isolated from STAT6-sufficient or STAT6-deficient cells into effector Th1 cells was comparable (Fig. 3). As shown above, re-stimulation, under Th2 conditions, of 24 or 96 h-stimulated STAT6-sufficient Th1 cells induces a 92 and 64% reduction of IFN-{gamma} production, respectively (Fig. 3). In contrast, the addition of IL-4 during re-stimulation of 24 or 96 h-activated STAT6-deficient Th1 cells had only a partial incidence on IFN-{gamma} production (Fig. 3). Indeed, the frequency of IFN-{gamma}-producing cells was reduced by 36 and 29.8% when 24 and 96 h Th1 cells from STAT6-deficient CD4+ T cells were re-stimulated under Th2 conditions respectively (Fig. 3). Partial inhibition of IFN-{gamma} production by IL-4 in STAT6-deficient mice may suggest that IL-4 can repress IFN-{gamma} by both STAT6-dependent and STAT6-independent pathways. Alternatively, partial inhibition may reflect a proliferative or survival defect of STAT6-deficient Th1 cells when deprived of IL-12 (26). Nonetheless, these results indicate that STAT6 plays an essential role in IL-4-mediated regulation of IFN-{gamma} production by both 24 and 96 h Th1 cells. Furthermore, in agreement with the role of STAT6 in Th2 cell differentiation, no IL-4-producing Th2 cells were detected when 24 h-stimulated STAT6-deficient Th1 cells were re-stimulated under Th2 conditions (Fig. 3).



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Fig. 3. STAT6 is necessary for the IL-4-mediated inhibition of Th1 responses. CD4+ T cells isolated from either B10.BR or STAT6–/– mice were activated with soluble anti-CD3 and anti-CD28 antibody under Th1- or Th2-polarizing conditions as indicated (primary stimulation). Twenty-four or 96 h after primary stimulation the cells were re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibody under Th1- or Th2-polarizing conditions (secondary stimulation). At the end of the primary (none) or 48 h secondary stimulation IL-4 and IFN-{gamma} production were analyzed by intracellular FACS staining. The percentage of cytokine-producing cells of one experiment out of three performed is shown.

 
Altogether, these results show that IL-4 receptor engagement leads to STAT6 activation in both 24 and 96 h-stimulated Th1 cells, an event that is necessary in both situations for IL-4-mediated inhibition of IFN-{gamma} production. Nonetheless, despite a normal activation of STAT6, IL-4 was ineffective at reverting 96 h-stimulated Th1 cells, suggesting that downstream events of STAT6 activation may be impaired in these differentiated Th1 cells.

Absence of IL-4-dependent GATA-3 up-regulation in 96 h-stimulated Th1 cells
Activation of STAT6 induces expression of the Th2-specific transcription factor GATA-3 which conditions Th2 cell development (12). We, therefore, examined GATA-3 gene expression by quantitative RT-PCR in the different cultures described above. As previously described, unstimulated T cells express substantial levels of GATA-3 mRNA (14) (Fig. 4a–c). Expression of GATA-3 is maintained for 24 h, then up-regulated in cells stimulated under Th2 conditions and rapidly extinguished in developing Th1 cells (Fig. 4a–c). By 96 h of Th1 cell differentiation, GATA-3 mRNA expression had increased slightly, reaching a level ~30% of that found in naive T cells (Fig. 4b). Addition of IL-4 during the re-stimulation of 24 h-stimulated Th1 cells, a condition that induces reversion of differentiating Th1 cells to the Th2 phenotype, induces a 5.5-fold increase in GATA-3 mRNA expression (Fig. 4a). This up-regulation likely results from IL-4 signaling since it is not observed in 24 h Th1 cells re-stimulated under Th0 conditions (Fig. 4a). In contrast, addition of IL-4 during the re-stimulation of 96 h Th1 cells, a culture condition that does not permit reversion to the Th2 lineage, does not significantly modulate GATA-3 expression by this population (Fig. 4b). In 96 h Th1 cells, therefore, despite a normal activation, STAT6 is unable to further induce GATA-3. The level of GATA-3 was, however, comparable in 24 and 96 h Th1 cells re-stimulated under Th2 conditions, suggesting that the outcome of IL-4 stimulation of 24 and 96 h-stimulated Th1 cells is not solely determined by the absolute level of GATA-3 expression (Fig. 4a and b). We, therefore, analyzed expression of the Th1-specific transcription factor T-bet. Stimulation of naive CD4+ T cells under Th1-polarizing, but not Th2-polarizing, conditions up-regulated T-bet mRNA expression (Fig. 4d–f). A slight down-modulation of T-bet mRNA was consistently observed in 96 h-stimulated Th1 cells (Fig. 4e). This down-modulation was transient and the T-bet mRNA level increased thereafter (Fig. 4f). Likewise, a high level of T-bet mRNA expression is observed following re-stimulation of 24 or 96 h Th1 cells under Th1-polarizing conditions (Fig. 4d and e). T-bet expression is differently regulated in 24 and 96 h-stimulated Th1 cells that were re-stimulated under Th2 conditions (Fig. 4d and e). Thus, a significant increase in T-bet mRNA expression levels was only observed upon re-stimulation of 96 h Th1 cells (Fig. 4d and e).



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Fig. 4. Absence of GATA-3 up-regulation in 96 h-stimulated Th1 cells. Naive CD4+ T cells isolated from B10.BR mice were activated for 24 h (a and d), 96 h (b and e) or 6 days (c and f) with soluble anti-CD3 and anti-CD28 antibody under Th1- or Th2-polarizing conditions. At the indicated time point of primary stimulation the cells were re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibody under Th1-, Th2- or Th0-polarizing conditions. RNA was isolated from naive T cells (NA) or stimulated cells at the end of the primary culture (Th1 and Th2) or 6 h after secondary stimulation (Th1-Th1, Th1-Th2, Th1-Th0 and Th2-Th2). The expression level of GATA-3 (a–c) and T-bet (d–f) was determined by real-time quantitative RT-PCR. The means and SD of relative values normalized for HPRT transcripts of two groups of mice analyzed in one experiment were calculated as indicated in Methods. One representative experiment out of four performed is shown.

 
Collectively, these results show that the difference in IL-4 responsiveness of 24 and 96 h Th1 cells correlates with differences in GATA-3 and T-bet regulation. Thus, reversion of 24 h-activated Th1 cells to the Th2 lineage following re-stimulation under Th2-polarizing conditions correlates with an increased level of GATA-3 expression and a reduced level of T-bet expression. In contrast, in 96 h Th1 cells re-stimulated under Th2 conditions, GATA-3 remains low, while T-bet gene expression is up-regulated.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In several infectious mice models or models of autoimmune diabetes, both Th1 and Th2 responses co-exist, although Th2 responses dominate functionally (1821). These observations raised the possibility that effector Th2 cells may not only alter Th1 differentiation processes, but also the effector phase of the response. We tested this possibility and show here that IL-4 inhibits Th1 responses by two developmentally regulated mechanisms. When present during the commitment phase, IL-4 inhibits Th1 cell differentiation and re-directs the responding T cells towards the Th2 lineage. At the later time point, when the responding cells are differentiated, IL-4 has a transient inhibitory effect on IFN-{gamma} gene expression, but is unable to revert effector cells to the Th2 lineage. Thus, 96 h Th1 cells are irreversible effector Th1 cells, but retain IL-4 responsiveness. Indeed, IL-4 is unable to revert these effector cells to the Th2 lineage, but can modulate cytokine production by these polarized Th1 cells. Transient inhibition of IFN-{gamma} gene expression without altering the differentiation of effector Th1 cells may explain why Th2 responses dominate functionally in vivo.

A critical issue raised by this and a previous study (27) is whether IL-4 directly modulates cytokine production by committed or differentiated Th1 cells. Since most cultures are likely heterogeneous, as suggested by the limited percentage of cytokine-producing cells, it would be possible that addition of IL-4 to Th1-stimulated T cells would lead to selective death of Th1 cells and induce the differentiation of yet uncommitted T cells present in the culture. It is unlikely that this may be the case for 96 h-stimulated Th1 cells since (i) proliferation was comparable in the different re-stimulation conditions, and (ii) the effect of IL-4 on IFN-{gamma} production was, for that population, transient and completely reversible after 48 h culture in the presence of IL-2. Conversely, we cannot conclude from our study that 24 h committed Th1 cells are reversible. Indeed, in addition to committed Th1 cells, 24 h Th1 populations may contain uncommitted cells that could give raise to most Th2 cells when re-stimulated in the presence of IL-4. A definitive answer to the critical issue of whether Th1 cells are truly reversible awaits the development of reliable markers that may identify committed Th1 cells. Nonetheless, the 24 h-stimulated Th1 population shows plasticity, indicating that most T cells from that culture can be directed to the Th2 lineage when exposed to IL-4. Recent in vivo studies indicate that the initial interaction between antigen-specific T cells and antigen-presenting dendritic cells lasts for <24 h (2831). Plasticity of the 24 h-stimulated Th1 population would, therefore, suggest that T cell effector function is not set by the initial T cell stimulation, but may be modulated by the cytokine environment the responding T cells meet within the lymph node.

Further biochemical studies show that differentiation into effector cells and the resulting irreversibility of Th1 cells correlate with alteration of IL-4-dependent regulation of GATA-3 and T-bet. Indeed, despite normal activation, STAT6 is unable to up-regulate GATA-3 and down-regulate T-bet expression in 96 h-stimulated effector Th1 cells. The absence of IL-4-dependent GATA-3 up-regulation in effector Th1 cells is likely not due to increased T-bet expression. Indeed, over-expression of T-bet in developing Th2 cells does not hinder the IL-4-dependent up-regulation of GATA-3 (7). These results suggest that likely STAT6-dependent GATA-3 expression may be repressed in effector Th1 cells. In agreement, Kurata et al. showed that STAT6-dependent up-regulation of GATA-3 is lost in repeatedly stimulated effector Th1 cells (12). How STAT6 regulates GATA-3 expression is still unclear. STAT6 may directly transactivate GATA-3 or allow for the expression of other transcription factors involved in GATA-3 gene transcription. Selective expression, in differentiated effector cells, of inhibitors of these transcription factors may then regulate GATA-3 transcription. Alternatively, epigenetic modification of the GATA-3 locus as a consequence of Th1 cell differentiation may render the GATA-3 gene inaccessible to transcription factors. Whatever the mechanisms are, the inability of STAT6 to up-regulate GATA-3 likely determines irreversibility of effector Th1 cells. Indeed, GATA-3 induces chromatin remodeling of the IL-4/IL-13 locus, a necessary step for the expression of the genes present in that locus (17,3234). In addition, over-expression of GATA-3 inhibits Th1 cell differentiation (14,17). Selective inhibition of STAT6-dependent GATA-3 up-regulation may, therefore, ensure irreversible commitment to the Th1 lineage. We find, however, that IL-4 is fairly efficient at inhibiting IFN-{gamma} production by 96 h-stimulated Th1 cells. This suggests that STAT6 itself or other STAT6-induced factors may negatively regulate transcription of the IFN-{gamma} gene. Further dissection of the STAT6-activated pathway will certainly help clarify these different possibilities.


    Acknowledgements
 
We thank A.-M. Schmitt-Verhulst and L. Leserman for helpful discussions and critical reading of the manuscript, J.-P. Gorvel for the STAT6–/– mice, and J. Imbert and B. Kahn-Perlès for experimental advice. This work was supported by institutional grants to S. G. from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique, and by grants from Association National de la Recherche sur le SIDA. O. W. is supported by a fellowship from La Ligue Nationale contre le Cancer.


    Abbreviations
 
APC—antigen-presenting cell

EMSA—electrophoretic mobility shift assay


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Szabo, S. J., Kim, S. T., Costa, G. L., Zhang, X., Fathman, C. G. and Glimcher, L. H. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655.[ISI][Medline]
  2. Mullen, A. C., High, F. A., Hutchins, A. S., Lee, H. W., Villarino, A. V., Livingston, D. M., Kung, A. L., Cereb, N., Yao, T. P., Yang, S. Y. and Reiner, S. L. 2001. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science 292:1907.[Abstract/Free Full Text]
  3. Lighvani, A. A., Frucht, D. M., Jankovic, D., Yamane, H., Aliberti, J., Hissong, B. D., Nguyen, B. V., Gadina, M., Sher, A., Paul, W. E. and O’Shea, J. J. 2001. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells. Proc. Natl Acad. Sci. USA 98:15137.[Abstract/Free Full Text]
  4. Szabo, S. J., Sullivan, B. M., Stemmann, C., Satoskar, A. R., Sleckman, B. P. and Glimcher, L. H. 2002. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295:338.[Abstract/Free Full Text]
  5. Kaplan, M. H., Sun, Y.-L., Hoey, T. and Grusby, M. J. 1996. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174.[CrossRef][ISI][Medline]
  6. Thierfelder, W. E., van Deursen, J. M., Yamamoto, K., Tripp, R. A., Sarawar, S. R., Carson, R. T., Sangster, M. Y., Vignali, D. A. A., Doherty, P. C., Grosveld, G. C. and Ihle, J. N. 1996. Requirement for Stat4 in Interleukin-12-mediated responses of natural killer and T cells. Nature 382:171.[CrossRef][ISI][Medline]
  7. Afkarian, M., Sedy, J. R., Yang, J., Jacobson, N. G., Cereb, N., Yang, S. Y., Murphy, T. L. and Murphy, K. M. 2002. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat. Immunol. 3:549.[CrossRef][ISI][Medline]
  8. Mullen, A. C., Hutchins, A. S., High, F. A., Lee, H. W., Sykes, K. J., Chodosh, L. A. and Reiner, S. L. 2002. Hlx is induced by and genetically interacts with T-bet to promote heritable TH1 gene induction. Nat. Immunol. 3:652.[ISI][Medline]
  9. Kaplan, M. H., Schindler, U., Smiley, S. T. and Grusby, M. J. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313.[ISI][Medline]
  10. Shimoda, K., van Deursen, J., Sangster, M. Y., Sarawar, S. R., Carson, R. T., Tripp, R. A., Chu, C., Quelle, F. W., Nosaka, T., Vignali, D. A. A., Doherty, P. C., Grosveld, G., Paul, W. E. and Ihle, J. N. 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630.[CrossRef][ISI][Medline]
  11. Takeda, K., Tanaka, T., Shi, W., Matsumoto, M., Minami, M., Kashiwamura, S.-I., Nakanishi, K., Yoshida, N., Kishimoto, T. and Akira, S. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380:627.[CrossRef][ISI][Medline]
  12. Kurata, H., Lee, H. J., O’Garra, A. and Arai, N. 1999. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity 11:677.[ISI][Medline]
  13. Ferber, I. A., Lee, H. J., Zonin, F., Heath, V., Mui, A., Arai, N. and O’Garra, A. 1999. GATA-3 significantly downregulates IFN-gamma production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin. Immunol. 91:134.[CrossRef][ISI][Medline]
  14. Ouyang, W., Ranganath, S. H., Weindel, K., Bhattacharya, D., Murphy, T. L., Sha, W. C. and Murphy, K. M. 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9:745.[ISI][Medline]
  15. Zheng, W. and Flavell, R. A. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587.[ISI][Medline]
  16. Ouyang, W., Lohning, M., Gao, Z., Assenmacher, M., Ranganath, S., Radbruch, A. and Murphy, K. M. 2000. Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment. Immunity 12:27.[ISI][Medline]
  17. Lee, H. J., Takemoto, N., Kurata, H., Kamogawa, Y., Miyatake, S., O’Garra, A. and Arai, N. 2000. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells. J. Exp. Med. 192:105.[Abstract/Free Full Text]
  18. Sommer, F., Meixner, M., Mannherz, M., Ogilvie, A. L., Rollinghoff, M. and Lohoff, M. 1998. Analysis of cytokine patterns produced by individual CD4+ lymph node cells during experimental murine leishmaniasis in resistant and susceptible mice. Int. Immunol. 10:1853.[Abstract]
  19. Scott, B., Liblau, R., Degermann, S., Marconi, L. A., Ogata, L., Caton, A. J., McDevitt, H. O. and Lo, D. 1994. A role for non-MHC genetic polymorphism in susceptibility to spontaneous autoimmunity. Immunity 1:73.[ISI][Medline]
  20. von Herrath, M. G., Guerder, S., Lewicki, H., Flavell, R. A. and Oldstone, M. B. A. 1995. Coexpression of B7.1 and viral (‘self’) transgenes in pancreatic ß-cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes. Immunity 3:727.[ISI][Medline]
  21. Lenshow, D., Herold, K., Rhee, L., Patel, B., Koons, A., Qin, H., Fuchs, E., Singh, B., Thompson, C. and Bluestone, J. 1996. CD28/B7 regulation of Th1 and Th2 subsets in the development of autoimmune diabetes. Immunity 5:285.[ISI][Medline]
  22. Bajenoff, M., Wurtz, O. and Guerder, S. 2002. Repeated antigen exposure is necessary for the differentiation, but not the initial proliferation, of naive CD4+ T cells. J. Immunol. 168:1723.[Abstract/Free Full Text]
  23. Bird, J. J., Brown, D. R., Mullen, A. C., Moskowitz, N. H., Mahowald, M. A., Sider, J. R., Gajewski, T. F., Wang, C. R. and Reiner, S. L. 1998. Helper T cell differentiation is controlled by the cell cycle. Immunity 9:229.[ISI][Medline]
  24. Iezzi, G., Scotet, E., Scheidegger, D. and Lanzavecchia, A. 1999. The interplay between the duration of TCR and cytokine signaling determines T cell polarization. Eur. J. Immunol. 29:4092.[CrossRef][ISI][Medline]
  25. Huang, H. and Paul, W. E. 1998. Impaired interleukin 4 signaling in T helper type 1 cells. J. Exp. Med. 187:1305.[Abstract/Free Full Text]
  26. Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J. and Paul, W. E. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17:701.[CrossRef][ISI][Medline]
  27. Murphy, E., Shibuya, K., Hosken, N., Openshaw, P., Maino, V., Davis, K., Murphy, K. and O’Garra, A. 1996. Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Exp. Med. 183:901.[Abstract]
  28. Miller, M. J., Wei, S. H., Parker, I. and Cahalan, M. D. 2002. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869.[Abstract/Free Full Text]
  29. Stoll, S., Delon, J., Brotz, T. M. and Germain, R. N. 2002. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296:1873.[Abstract/Free Full Text]
  30. Bajenoff, M., Granjeaud, S. and Guerder, S. 2003. The strategy of T cell antigen-presenting cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell activation. J. Exp. Med. 198:715.[Abstract/Free Full Text]
  31. Bousso, P. and Robey, E. 2003. Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nat. Immunol. 4:579.[CrossRef][ISI][Medline]
  32. Takemoto, N., Kamogawa, Y., Jun Lee, H., Kurata, H., Arai, K. I., O’Garra, A., Arai, N. and Miyatake, S. 2000. Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J. Immunol. 165:6687.[Abstract/Free Full Text]
  33. Agarwal, S. and Rao, A. 1998. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9:765.[ISI][Medline]
  34. Avni, O., Lee, D., Macian, F., Szabo, S. J., Glimcher, L. H. and Rao, A. 2002. TH cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat. Immunol. 3:643.[ISI][Medline]