Transcriptional regulation of Th2 cell differentiation

Abstract

CD4 T helper 2 (Th2) cells have critical functions in immune responses against extracellular parasites and are involved in asthma and other allergic diseases. The differentiation of naïve CD4 T cells into Th2 cells is initiated from T-cell receptor and cytokine-mediated signaling followed by upregulation of GATA3 and activation of signal transducer and activator of transcription 5 (STAT5), two indispensable events for this differentiation process. In this review, regulation of GATA3 expression and STAT5 activation and functions of these two transcription factors in inducing the expression of Th2 cytokines, cytokine receptors as well as epigenetic modification at Th2 cytokine locus are summarized. Furthermore, I present positive and negative regulatory networks important for Th2 cell commitment, selective growth of committed Th2 cells and suppression of alternative lineage fates. Finally, the difference between in vitro and in vivo Th2 differentiation is discussed.

CD4 T helper (Th) cells have critical functions in regulating adaptive immune responses. Mossman and Coffman¹ first recognized the existence of Th1 and Th2 cells. Although Th1 cells are critical for cell-mediated immunity, Th2 cells are involved in humoral immune responses.^2,3 Now, it is known that naïve CD4 T cells have at least four distinct fates, Th1, Th2, Th17 and induced regulatory T (iTreg) cells, to choose from when they receive signals triggered by antigens and cytokines.⁴

CD4 T helper 2 (Th2) cells are important for immune responses against extracellular parasites and involved in the development of asthma and other allergic diseases. By secreting a variety of signature cytokines, Th2 cells help B cells to make IgE⁵ (through interleukin (IL)-4), induce alternative macrophage activation⁶ (through IL-4/IL-13), recruit eosinophils⁷ (through IL-5), activate mast cells (through IL-9⁸) and act on epithelial cells (through IL-9,⁹ IL-13^10–12 and amphiregulin¹³). Th2 cells also produce IL-10, IL-21 and IL-25, which are also involved in regulating the magnitude of Th2 responses.

Cytokine milieu is an important determinant for Th cell differentiation. Together with T-cell receptor (TCR) ligation, IL-4-mediated signaling promotes Th2 differentiation.^14,15 IL-4 activates the signal transducer and activator of transcription 6 (STAT6) leading to the induction of the transcription factor GATA3.^16,17 IL-2-mediated STAT5 activation is also critical for Th2 cell differentiation.^14,18,19 In this review, I discuss how GATA3 expression and STAT5 activation are regulated and how these two molecules, in collaboration with other transcription factors, induce fully differentiated Th2 cells.

GATA3 and STAT5 are Indispensable for Th2 Differentiation

GATA3 has important functions at multiple stages of CD4 T cell development.²⁰ It is also recognized as the master regulator of Th2 cells.^16,17 The expression of GATA3 is upregulated during Th2 differentiation.^17,21,22 Enforced expression of GATA3 in Th1 cells induces IL-4 and endogenous GATA3 expression.^21,23 On the other hand, a dominant-negative form of GATA3 suppresses Th2 cytokine expression and blocks induction of airway hyperreactivity.²⁴ Gata3 conditional knockout studies show that Th2 differentiation, both in vitro and in vivo, completely depends on GATA3 expression.^25,26 In differentiated Th2 cells, continuous GATA3 expression is essential for the production of IL-5 and IL-13, but not IL-4.²⁵

STAT5 family members include STAT5a and STAT5b, encoded by two related genes head to head in the genome.²⁷ They are critical for cytokine-mediated T-cell proliferation and survival.²⁸ IL-2 is the most potent inducer of STAT5 activation and IL-2-mediated STAT5 signaling is required for Th2 cell differentiation in vitro.^18,19 Although STAT5b is still expressed in STAT5a single knockout cells, such cells have profound defects in Th2 cell differentiation both in vitro and in vivo indicating there is a dose effect of STAT5 activation during Th2 differentiation.^18,19,29,30

Enforced expression of either GATA3 or a constitutively active STAT5a in Th1 cells results in IL-4 production and co-expression of these two molecules maximizes the Th2-inducing effect.¹⁹ On the other hand, the constitutively active STAT5a fails to induce IL-4 in GATA3-deficient cells²⁵ and anti-IL-2 blocks the ability of GATA3 to promote IL-4 expression.¹⁸ Therefore, both GATA3 expression and STAT5 activation are necessary for Th2 cell differentiation in vitro³¹ and possibly in vivo.

Regulation of GATA3 Expression and STAT5 Activation

STAT6 is the major signal transducer in IL-4-mediated Th2 cell differentiation.^32–34 IL-4 can be either provided exogenously or produced by naïve CD4 T cells in response to low-dose peptide stimulation.³⁵ CD4 T cells deficient in STAT6 fail to develop into IL-4-producing cells in vitro and STAT6 activation is necessary and sufficient for inducing high expression levels of GATA3.^36,37 STAT6 may also be involved in chromatin remodeling at the Il4/Il13 locus control region (LCR).³⁸ However, some in vivo Th2 responses can be obtained in the absence of STAT6^39–41 but such Th2 differentiation still requires GATA3 expression,^25,26 suggesting that either GATA3 can be induced by IL-4/STAT6-independent pathway or in vivo Th2 differentiation in some cases only requires basal levels of GATA3 expression found in activated CD4 T cells.

Low-dose peptide stimulation of naïve CD4 T cells induces IL-4/ STAT6-independent early GATA3 expression to a certain level.³⁵ Such GATA3 induction is not observed when cells are stimulated with high-dose peptide, possibly because a strong Erk activation blocks the induction. The detail mechanism through which TCR-mediated signaling induces GATA3 is unknown. NF-κB1 has been shown to have an important function in regulating GATA3 expression.⁴² Bcl-3, as the partner of NF-κB1, directly binds to the promoter of the Gata3.⁴³ The role of NF-κB1 in low-dose peptide-induced GATA3 expression remains to be determined.

Notch signaling is important for Th2 differentiation.^44,45 It has been shown that Jag1/Notch interaction induces Th2 differentiation, whereas Dll1/Notch interaction results in Th1 polarization.⁴⁴ A Notch/CSL-binding site was identified in the alternative distal promoter of Gata3, which is located ∼10kb upstream of the regular Gata3 promoter,⁴⁶ suggesting Notch signaling directly regulates GATA3 expression. A recent report shows that TCF-1/β-catenin may have an important function in regulating IL-4-independent early GATA3 expression in some settings but the dominant transcription starting site of Gata3 is downstream of the proximal promoter.⁴⁷ Most recently, transcription factor Dec2 has been shown to have an important function in Th2 differentiation through forming a positive regulatory feedback loop with GATA3.⁴⁸ GATA3 induces Dec2 expression and in turn Dec2 upregulates GATA3. Dec2 directly binds to the Gata3 promoter. In Dec2-deficient cells, GATA3 induction is impaired; when GATA3 is deleted from Th2 cells, Dec2 expression gradually decreases. The initial signaling responsible for early Dec2 upregulation, just as for early GATA3 induction, has not been determined.

GATA3 induces its own expression.²³ In fact, our unpublished ChIPseq data showed that GATA3 strongly binds to multiple sites at Gata3 locus extending up to 1 Mb 3′ of Gata3, suggesting the regulatory elements for GATA3 expression can be far apart.

TCR-mediated signaling transiently inhibits cytokine signaling including IL-2-mediated STAT5 and IL-4-mediated STAT6 activation.⁴⁹ Indeed, STAT5 activation by IL-2 is evident after 24h of T-cell activation when low strength of TCR signaling is provided, but IL-2 signaling remains suppressed at this time point if T cells receive strong TCR stimulation.³⁵ Therefore, low strength of T-cell activation is critical for the initiation of Th2 cell differentiation through both GATA3 upregualtion and STAT5 activation.

Many cytokines including IL-2, IL-7 and TSLP can induce STAT5 activation. IL-2 is mainly produced by CD4 T cells after activation, whereas IL-7 is constitutively expressed by stromal cells. TSLP can be produced by epithelial cells, mast cells and basophils during the initiation of in vivo Th2 responses.^50–52 These STAT5 activators can be potential initiators for Th2 responses in vivo as GATA3 is induced by T-cell activation and only limited amounts of GATA3 may be required for IL-4 production.¹⁹ Interestingly, both Notch pathway and NF-κB pathway, which are important for inducing GATA3, have also been reported to regulate the expression of IL-2 and CD25,^53,54 and thus IL-2-mediated STAT5 activation.

Other Transcription Factors Involved in Th2 Differentiation

Besides GATA3 and STAT5, many other transcription factors are also involved in regulating IL-4 production and Th2 differentiation. Growth factor independent 1 (Gfi-1) is a STAT6-dependent immediate early gene induced by IL-4.⁵⁵ TCR activation also induces Gfi-1, but IL-4 substantially prolongs its expression. Gfi-1 is important for cytokine-mediated growth of Th2 cells but has a minimal effect on the growth of other Th cells. Thus, Gfi-1 selects GATA3^hi cells to grow. It seems that Gfi-1 regulates molecules both upstream and downstream of STAT5 activation.^55,56

Many transcription factors directly act on Il4 promoter. IL-4 production by Th2 cells requires TCR-mediated Ca²⁺ signaling. Indeed, NFAT1 has been shown to bind to the Il4 promoter.⁵⁷ C-Maf is selectively upregulated in Th2 cells and in its absence, the production of IL-4 but not other Th2 cytokines is diminished.⁵⁸ JunB, expressed at high levels in Th2 cells, collaborates with c-Maf to induce IL-4 production by directly acting on the Il4 promoter.⁵⁹ The expression of JunB may depend on a Th2-specific transcription factor Dec2.⁴⁸ IRF-4 is also required for Th2 cell differentiation.^57,60 It has been shown that IRF-4 functions both upstream and downstream of GATA3 and the latter is through its direct binding to the Il4 promoter.

Notch signaling regulates GATA3 expression as discussed earlier, but Notch/CSL-binding site has also been found in the DNase I hyper-sensitivity site (HS) V of the Il4 gene⁴⁴ suggesting it may regulate IL-4 production directly. Ikaros is important for Th2 cell differentiation as Ikaros-deficient cells fail to produce IL-4.⁶¹

Cross-Regulation between Transcription Factors of Different Lineages

T-bet is the Th1 master regulator.⁶² T-bet expression is induced by IFNγ suggesting IFNγ/T-bet forms a powerful amplification loop for Th1 differentiation.⁶³ IL-12-mediated STAT4 activation is also important for Th1 responses.^64,65 Together with IL-18, IL-12 is able to induce IFNγ production in a TCR-independent manner.^66,67 Runx3 expression is upregulated in Th1 cells and it binds to the Ifng promoter.^68,69 Eomesodermin (Eomes),⁷⁰ another T-box family member, is responsible for IFNγ production in T-bet-deficient CD8 T cells. Therefore, transcription factors T-bet, STAT4, Runx3 and Eomes are involved in IFNγ production.

Gata3 deletion in Th2 cells results in elevated IFNγ production²⁵ and such IFNγ induction is due to the activation of Runx3-Eomes pathway independent of IL-12/STAT4 and IFNγ/T-bet (our unpublished data). In addition, GATA3 deficiency in Th2 cells reverses the suppression of STAT4 expression by IL-4, consistent with an earlier report that GATA3 downregulates STAT4 when it is over-expressed in Th1 cells.⁷¹

On the other hand, Th1 transcription factors suppress Th2 differentiation. T-bet is able to suppress GATA3 expression.²² In addition, T-bet inhibits GATA3-mediated IL-5 production by directly interacting with GATA3 protein.⁷² Runx3 suppresses IL-4 production in Th1 cells through its binding to HSIV site of the Il4 locus.⁶⁸

Strong STAT5 activation is critical for Th2 differentiation as discussed earlier. STAT5a-deficient cells have been shown to be hyperresponsive to IL-12, which results in Th1 differentiation.⁷³ Consistent with this, T-bet expression is suppressed when a constitu-tively active form of STAT5a is introduced into Th1 cells.¹⁹ STAT5 also inhibits the expression of Th17-related molecules, including RORγt and IL-17.⁷⁴

Gfi-1, a positive regulator of Th2 cell expansion, suppresses IFNγ production in Th1 cells⁵⁶ and IL-17 production in Th17 cells.⁷⁵ Blimp-1 is induced in Th2 cells and it inhibits the expression of IFNγ and IL-2.^76–78 Ikaros-deficient ‘Th2’ cells express increased levels of both T-bet and IFNγ suggesting a main function of Ikaros during Th2 differentiation is to suppress Th1-related genes.⁶¹

Amplification of Th2 Responses Through Multiple Positive Feedback Loops

As discussed above, IL-4/IL-4R/STAT6/GATA3 and IL-2/STAT5 pathways are two important pathways for Th2 differentiation. In vivo, IL-4 can be produced by basophils,⁴⁰ NKT cells,⁷⁹ memory Th2 cells and some undefined accessory cells,⁸⁰ whereas, IL-2 is produced by naïve CD4 T cells shortly after T-cell activation. IL-7 and TSLP may substitute IL-2 as STAT5 activators in vivo. STAT5 has been shown to bind to the HSII and HSIII sites in the intron 2 of Il4^18,19 and GATA3 binds to HSVa.⁸¹ Thus, both GATA3 and STAT5 directly act on the Il4 gene to promote its expression. IL-4 produced by CD4 T cells further enhances IL-4Rα expression and upregulates GATA3. Therefore, the induction of IL-4 expression in CD4 T cells through IL-4/IL-4R/STAT6/GATA3 pathway provides a powerful positive feedback loop for Th2 differentiation.

IL-33Rα (also known as T1/ST2 or IL-1R-like 1), an IL-1R super-family member, is preferentially expressed in differentiated Th2 cells.^82,83 Blocking IL-33 signaling reduces eosinophilic airway inflammation⁸⁴ and IL-33Ra-deficient mice show decreased responses to Schistosoma mansoni egg antigen.⁸⁵ IL-33 stimulation of IL-33Rα^high Th2 cells results in IL-13 but not IL-4 production in a TCR-independent manner.⁸⁶ Thus, IL-33 has an important function in amplifying Th2 responses. Interestingly, continuous IL-33Rα expression requires both GATA3 expression and STAT5 activation; GATA3 and STAT5 directly bind to the Il1rl1 gene, which encodes IL-33Rα.⁸⁶

Besides directly acting on the Il4 and Il1rl1 genes, GATA3 and STAT5 pathway cross-regulate each other at multiple levels. Th2 cells express higher levels of CD25 (IL-2Rα) than Th1 cells and such expression may partly depend on c-Maf,⁸² which is a potential target of GATA3. GATA3 also directly binds to intron 1 of Il2ra in Th2 cells suggesting GATA3 may directly regulate CD25 expression (our unpublished data). Thus, GATA3 regulates IL-2/STAT5 signaling by modulating IL-2R expression.

On the other hand, STAT5 regulates IL-4Rα expression during T-cell activation.⁸⁷ In addition, STAT5 has an important function in maintaining GATA3 expression in differentiated Th2 cells.⁸⁶ Furthermore, GATA3 has also been reported to regulate its own expression²³ and STAT5 has a critical function in maintaining CD25 expression.⁸⁸ Therefore, the IL-2/STAT5 pathway and the IL-4/STAT6/GATA3 pathway crosstalk at different levels and the collaboration between STAT5 and GATA3 resulting in full Th2 differentiation (Figure 1).

Figure 1.

Positive feedback loops for Th2 cell differentiation regulated by GATA3 and STAT5. Both IL-4 and weakly stimulated TCR-mediated signaling induce the expression of Th2 master regulator GATA3. In addition, Notch and/or other signaling pathways may regulate GATA3 expression. TCR activation also results in IL-2 production and IL-2Rα upregulation. IL-2 activates STAT5 but its function may be substituted or compensated in vivo by IL-7 and TSLP, the other two STAT5 activators. GATA3 collaborates with activated STAT5 to induce IL-4 production. C-Maf, possibly regulated by GATA3, further enhances the expression of IL-4. In addition, GATA3 and STAT5 regulate the expression of both IL-2R and IL-4R. Elevated IL-4 and IL-2 production together with upregulation of IL-2R and IL-4R provide powerful positive feedback loops for promoting Th2 polarization as well as selective growth of committed Th2 cells.

Epigenetic Modification at the IL4/IL13 Locus in Th2 Cells

Il4 and its congener Il13 genes, flanked by Rad50 and Kif3a, are closely linked on human chromosome 5q31 and the syntenic region on mouse chromosome 11. The LCR for Il4-Il13 lies in a 25 kb region 3′ of Rad50, which is ∼20 kb 5′ of Il13.⁸⁹ Il5 is on the other side of Rad50, however, its expression may not be controlled by the LCR in Rad50. GATA3-binding sites are found in the promoters of the Il5⁹⁰ and Il13^91,92 suggesting GATA3 serves as a transcription activator for IL-5 and IL-13 transcription. Indeed, when GATA3 is removed from Th2 cells, IL-5 and IL-13 expression are completely abolished.²⁵

Transcriptional regulation of signature cytokine expression depends not only on expression or activation of specific transcription factors, but also on chromatin epigenetic modification and accessibility of cytokine genes. During Th2 differentiation, chromatin remodeling and epigenetic modification occur at the Il4/Il13 locus. Across the Il4/Il13 locus, a series of Th2-specific DNase I HS sites have been identified. Among these sites, conserved non-coding sequence 1^93,94 located at Il4-Il13 intergenic region, HSII in the intron 2 of Il4⁹⁵ and HSV and HSVa⁹⁶ (also known as conserved non-coding sequence 2) at 3′ of the Il4 coding region are particularly important. HS IV is accessible in both Th1 and Th2 cells and this element seems to be the target site for silencing IL-4 in Th1 cells.⁹⁷ Runx3 binding to this site offers a potential mechanism for IL-4 repression.⁶⁸ One particular site within the LCR, RHS7, becomes hypersensitive to DNase I and is demethylated at the initiation of Th2 but not Th1 differentiation.^89,98 Deletion of RHS7 diminishes but does not abolish the production of IL-4 and IL-13 in Th2 cells.⁹⁹

GATA3 is responsible for chromatin remodeling at Il4/Il13 locus by inducing DNase I HSs.^23,100,101 STAT5 induces accessibility at HSII site of the Il4 locus.¹⁹ IL-2/STAT5 signaling is also partially responsible for demethylation of RHS7 in LCR during Th2 differentiation.⁹⁸ Our unpublished data suggest GATA3 strongly binds to the HSII and three different sites within LCR in addition to HSVa indicating that GATA3 and STAT5, through their direct binding to two critical regulatory elements HSII and LCR, collaborate in chromatin remodeling of Th2 cytokine locus.

GATA3 may also be involved in regulating DNA CpG methylation. Dnmt-1, a DNA methyltrasferase, has an important function in silencing Il4 locus and Dnmt-1-deficient cells aberrantly express IL-4 without upregulaing GATA3.¹⁰² Likewise, methyl CpG-binding domain protein-2-deficient Th1 cells produce IL-4 even when GATA3 expression level is low.¹⁰³ Indeed, GATA3 is able to block the ability of methyl CpG-binding domain protein-2 to bind to methyl CpG, suggesting GATA3 upregulation may reverse inhibition of IL-4 production caused by CpG methylation at Il4 locus.

Histone modifications are also critical for gene regulation. Trimethylation at histone 3 lysine 4 (H3K4me3) is associated with active gene loci and H3K27me3 with silenced gene loci. At the Il4/Il13 locus, H3K4me3 modification was found in Th2 cells but H3K27me3 in Th1 cells.¹⁰⁴ Histone H3K4 methyltransferase MLL is required for maintaining H3K4 modification at Il4/Il13 locus in Th2 cells¹⁰⁵ whereas H3K27 methyltransferase EZH2 is responsible for suppressive H3K27me3 modification at Il4/Il13 locus in Th1 cells.¹⁰⁶ The functions of GATA3 and STAT5 in regulating histone modifications are not known.

Although Th2 cells are capable of producing both IL-4 and IL-13, at single cell level, the expression of each cytokine from one of two alleles may be different, resulting in heterogenous cytokine expression pattern and monoallelic expression.¹⁰⁷ The actual induction of cytokine expression from a given allele seems to be stochastic and probabilistic,¹⁰⁸ and differential state of chromatin structure and gene accessibility at different alleles may determine the probability of gene expression.¹⁰⁹ Therefore, IL-4 non-producers can still be Th2 cells because they may be expressing IL-5 or IL-13 and/or they may express IL-4 at a different time point.

IL-4/STAT6-Dependent and -Independent Th2 Differentiation In Vivo

IL-4/STAT6 signaling is critical for Th2 differentiation in vitro. Although some in vivo Th2 responses including that to Trichuris muris infection¹¹⁰ and to some allergens¹¹¹ are IL-4/STAT6 dependent, other responses, such as to Nippostrongylus brasiliensis and S. mansoni infection, are IL-4 independent.^{39–41,112,113} However, STAT6 may still be required for the migration of Th2 cells to the lung tissue¹¹² as well as for the generation of Th2 memory cells⁴¹ and IL-4/STAT6 remains critical for inducing IgE production in B cells.

GATA3 is critical for in vivo Th2 responses even in the model in which IL-4 is dispensable,²⁵ suggesting that either there is an IL-4-independent pathway for GATA3 induction or the basal expression of GATA3 is sufficient for in vivo Th2 cell differentiation. Notch⁴⁴ and β-catenin⁴⁷ signaling pathways may be important for in vivo Th2 responses and GATA3 upregualtion, however, whether these pathways are responsible for IL-4-independent in vivo Th2 responses in general has not been determined.

Low-dose peptide stimulation of naïve CD4 T cells in vitro results in IL-4-independent GATA3 upregulation as discussed above.³⁵ Recently, it has been reported that S. mansoni egg product, Omega-1, is the key factor for inducing Th2 responses and this product downmodulates dendritic cell (DC) functions and suppresses IL-12 production by DCs.^114,115 Basophils are also reported to be critical for many in vivo Th2 responses by directly presenting antigens to CD4 T cells.^111,116,117 Interestingly, basophils express lower levels of MHCII than DCs consistent with the idea that low strength of TCR signaling preferentially induces Th2 responses.

In addition to presenting antigens, basophils produce many Th2-prone cytokines, including IL-4 and TSLP. STAT5 can be activated by TSLP suggesting TSLP/STAT5 pathway may serve as an initiation step of Th2 differentiation under certain conditions. However, just as both IL-4-dependent and -independent Th2 responses exist in vivo, TSLP may be required for some but not all in vivo Th2 responses. Whether in vivo Th2 responses require either IL-4 or TSLP needs further investigation.

Concluding Remarks

GATA3 expression and STAT5 activation are two major elements for Th2 differentiation definitely in vitro and possibly in vivo. Many other transcription factors are also involved in regulating or fine-tuning Th2 responses. Full Th2 cell differentiation is achieved through three mechanisms: Th2 lineage commitment, selective growth of differentiating Th2 cells and suppression of alternative lineage fates.³¹ GATA3, STAT5, NFAT1, IRF-4, c-Maf, JunB and Dec2 can directly act on the Il4 and/or Gata3 gene to control their expression (Th2 lineage commitment); GATA3, STAT5, c-Maf and Gfi-1 regulate either the expression of CD25 (IL-2Rα), IL-4Rα and IL-33Rα or responsiveness of Th2 cells to cytokines (selective growth of differentiating Th2 cells); GATA3, STAT5, Ikaros, Blimp-1 and Gfi-1 suppress the expression of molecules of other lineages, including IFNγ, STAT4, T-bet, Eomes, RORγt and IL-17, many of which can block Th2 differentiation (suppression of alternative lineage fates). These three mechanisms constitute powerful positive feedback loops, which promote terminal differentiation of Th2 cells.

Much has been learned from simplified in vitro Th2 differentiation models. However, the transcriptional regulation in developing Th2 cells in response to helminth infections and allergens is far from being understood. In addition, whether many epigenetic modifications observed in Th2 cells cultured in vitro represent modifications that would occur during in vivo Th2 differentiation is unknown. The fact that IL-4 is required for in vitro Th2 differentiation but not for many in vivo Th2 models suggests the complexity of in vivo situations. More factors such as cytokines including TSLP,⁵⁰ IL-25⁸⁰ and IL-33,¹¹⁸ and many cell types including basophils, DCs, mast cells and NKT cells influence in vivo Th2 differentiation. Thus, it is likely that many in vivo Th2 responses differ from each other because of unique activation of certain Th2 components in different models. Generation of indicator mice reporting the expression or activation of key transcription factors and developing sensitive assays handling small cell numbers will greatly help investigate the transcriptional regulation of Th2 differentiation in vivo.