T helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13 production

Abstract

Interleukin-4 (IL-4), IL-5 and IL-13, the signature cytokines that are produced during type 2 immune responses, are critical for protective immunity against infections of extracellular parasites and are responsible for asthma and many other allergic inflammatory diseases. Although many immune cell types within the myeloid lineage compartment including basophils, eosinophils and mast cells are capable of producing at least one of these cytokines, the production of these “type 2 immune response-related” cytokines by lymphoid lineages, CD4 T helper 2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) in particular, are the central events during type 2 immune responses. In this review, I will focus on the signaling pathways and key molecules that determine the differentiation of naïve CD4 T cells into Th2 cells, and how the expression of Th2 cytokines, especially IL-4 and IL-13, is regulated in Th2 cells. The similarities and differences in the differentiation of Th2 cells, IL-4-producing T follicular helper (Tfh) cells and ILC2s as well as their relationships will also be discussed.

1. Introduction

CD4 T helper (Th) cells are the key players during adaptive immune responses^{1, 2}. Almost thirty years ago, it was first recognized by Mossman and Coffman that there are different subsets of Th cells, namely type 1 T helper (Th1) and type 2 T helper (Th2) cells, which are critically involved in distinct types of immune responses^3–5. With the discovery of a third major CD4 T effector cell lineage, Th17 cells that produce interleukin-17 (IL-17), and a regulatory T cell subset (induced regulatory T (iTreg) or peripherally induced regulatory T (pTreg)) that can be differentiated from naive CD4 T cells outside of the thymus, either in vitro or in vivo, the members of CD4 T cell family have expanded^{2, 6–9}.

Th2 cells are the crucial lymphocytes during adaptive immune responses to the infections of extracellular parasites such as helminths; Th2 cells are also responsible for development of some asthmatic and allergic inflammatory diseases. By secreting IL-4, Th2 cells instruct B cells to produce IgG1 and IgE¹⁰; by producing IL-4 and IL-13, Th2 cells induce alternatively activated macrophage¹¹. Th2 cells also recruit eosinophils via IL-5 production¹² and directly act on epithelial cells and smooth muscle cells through IL-13 production^13–15. Therefore, IL-4, IL-5 and IL-13 are the major effector cytokines produced by Th2 cells during type 2 immune responses. All three cytokine genes, Il4, Il5 and Il13, are located within a genomic segment, which is subjected to the regulation of locus control region (LCR) in the Rad50 gene^{16, 17}.

During CD4 T cell activation through T cell receptor (TCR)-mediated signaling and co-stimulation, cytokine signals received by the activated T cells are deterministic in T cell fate commitment. For example, together with TCR ligation, IL-4-mediated activation of the signal transducer and activator of transcription 6 (STAT6) plays an important role during Th2 cell differentiation ^18–22, although IL-4-independent Th2 cell differentiation may occur in the absence of IL-4 signaling under certain conditions in vivo ^23–27. Both IL-4-dependent and IL-4-independent Th2 differentiation requires the key transcription factor GATA-3 (Figure 1), which is responsible for epigenetic changes in many Th2-specific gene loci and for direct transcription activation^28–31. In addition, IL-2-mediated activation of STAT5 is indispensable for the production of Th2 cytokines possibly through chromatin remodeling of the Th2 cytokine locus as well as maintaining GATA3 expression in already differentiated Th2 cells ^{18, 32–34}. Therefore, GATA3 up-regulation and STAT5 activation are the two key events for Th2 cell differentiation. Th2 cell differentiation and the induction of Th2 cytokines are also regulated by many other transcription factors including NFAT, NFκb and AP-1 family members. Mechanisms for the reinforcement of Th2 cell differentiation include positive feedbacks, inhibition of other alternative lineage choices and selective growth of differentiated Th2 cells.

Figure 1. Transcriptional network and positive feedback regulation during Th2 cell differentiation.

TCR activation and cytokine-mediated signaling are critical during Th2 cell differentiation. TCR stimulation activates NFAT, NFκb and AP-1 family members, resulting in up-regulation of IRF4 expression, which has a general function in T cell activation. Low dose of antigen stimulation accompanied by the up-regulation of Th2 master regulator GATA3 favors Th2 cell differentiation. IL-4-mediated Stat6 activation and other signaling pathways such as Notch-mediated signaling are also capable of inducing GATA3 expression. GATA3 directly mediates epigenetic modifications at the Th2 cytokine locus and cytokine transcription. GATA3 also indirectly regulates Th2 cytokine expression by inducing other transcription factor some of which may further up-regulate GATA3 expression. GATA3 also regulates its own expression. IL-2-mediated Stat5 activation is another key event for Th2 cytokine production. Activated T cells are able to produce both IL-2 and IL-4, and to up-regulate IL-2 and IL-4 receptors, forming a powerful positive feedback loop.

Besides Th2 cells, other lymphoid cells including subsets of γδ T cells, NKT cells, T follicular helper (Tfh) cells and type 2 innate lymphoid cells (ILC2s) are also capable of producing IL-4 and/or IL-13. In fact, in the steady state, ILC2s are the major IL-5-producing cells ^35,36. ILC2s exert similar functions as Th2 cells during type 2 immune responses ^37,38. In fact, the production of IL-13 by T cells is dispensable for type 2 immunity suggesting that there is another importance source of IL-13, most likely from ILC2s ³⁹. While this review will primarily focus on Th2 cell differentiation and the regulation of IL-4/IL-13 production in Th2 cells, the relationships among conventional Th2 cells, IL-4-producing Tfh cells and ILC2s, as well as the regulation of cytokine production in these cell subsets will be also discussed.

2. Signaling pathways involved in Th2 cell differentiation

2.1. IL-4-mediated signaling pathway

IL-4 promotes Th2 cell differentiation mainly by activating STAT6 through tyrosine phosphorylation^20–22. Naïve STAT6-deficient CD4 T cells fail to up-regulate GATA3 expression and thus are not able to develop into Th2 cells in vitro even when IL-4 is exogenously provided. On the other hand, constitutively active STAT6 mutants are capable of replacing IL-4 in driving Th2 cell differentiation^{40, 41}. In addition to up-regulating GATA3 expression, STAT6 is also responsible for chromatin remodeling at the Il4/Il13 LCR region¹⁶. The source of IL-4 during initial immune responses that lead to the differentiation of Th2 cells could be NKT cells⁴², basophils^{43, 44} and CD4 T cells themselves^{45, 46}.

However, under certain circumstances, Th2 cell differentiation can occur in the absence of the IL-4/STAT6 pathway in vivo^23–26, even though STAT6 is still important for Th2 cells migrating to the lung tissue²⁶, for optimal expression of GATA3⁴⁷, and for the maintenance of Th2 memory cells²⁴. IL-4-independent Th2 cell differentiation could be induced by low strength of TCR stimulation with co-stimulation, Notch signaling and/or IL-2-mediated STAT5 activation which will be discussed below.

2.2. TCR-mediated signaling pathway

Strength of TCR signaling is an important factor for determining the fate of T helper cell differentiation⁴⁸. Although cytokine environment can often be the deterministic factor for T helper cell differentiation during infections, TCR signaling strength has a dominant effect over the Th1- or Th2-inducing adjuvants^49–51. Naive CD4 T cells cultured in vitro with low dose peptide stimulation undergo IL-4/STAT6-independent induction of GATA3 within 24 hours, which is essential for the early IL-4 production by these cells⁴⁵. GATA3 is not up-regulated when naïve CD4 T cells receive high dose peptide stimulation. TCR signaling also induces the activation of p38 kinase, which phosphorylates and regulates GATA3 function⁵². The inhibition of GATA3 up-regulation is due to a strong Erk activation by high levels of TCR activation. This is consistent with the finding that resting dendritic cells (DCs) preferentially induce differentiation of Th2 cells⁵³. In fact, omega-1, an important glycoprotein in the Schistosoma mansoni egg, inhibits the activation of DCs thus limits the strength of T cell activation; this may be an important mechanism for how Schistosoma mansoni egg antigen induces Th2 responses in vivo^{54, 55}. Similarly, some in vivo Th2 cell differentiation may depend on basophils, which serves as Th2-promoting antigen presenting cells possibly because of low expression levels of MHC class II on these cells^{44, 56, 57}.

2.3. Co-stimulation of T cell activation

Although TCR co-stimulation mediated by CD28 is required for optimal T cell activation and Th2 cell differentiation, the CD28 effect is mainly through the induction of IL-2 production⁵⁸. IL-2-STAT5 pathway, which will be further discussed below, is important for Th2 cell differentiation at least in vitro^{32, 33}. Whether diminished Th2 responses in CD28-deficient mice are due to reduced IL-2 production is not clear^{59, 60}. On the other hand, CTLA-4 is a negative regulator of Th2 cell differentiation^{61, 62}. ICOS was originally thought to co-stimulate Th2 cell differentiation^{63, 64}, however, ICOSL/ICOS interaction is also known to be important for other T helper cell differentiation, especially for Tfh cells⁹. In addition, OX40 co-stimulation promotes Th2 cell differentiation^{65, 66}. Interestingly, TSLP induces the expression of OX40 ligand on DCs thus to promote Th2 response⁶⁷.

T cell activation and co-stimulation also activate PI3K pathway, which leads to mTOR (mammalian target of rapamycin) activation; mTOR regulates Th cell fate determination^{68, 69}. While mTORC1 signaling is important for Th1 and Th17 differentiation, mTORC2 signaling promotes Th2 cell differentiation partly through activating SGK1 to stabilize JunB protein (an important AP-1 transcription factor for Th2 cell differentiation)^70–73. However, the dispensable role of mTORC1 during Th2 cell differentiation has been called into question. The conclusion that mTORC1 is dispensable for Th2 cell differentiation is based on the results obtained with Rheb-deficient cells (Rheb is considered as an important upstream activator of mTORC1)⁷², however, a study using Raptor-deficient cells (Raptor is the component of mTORC1) indicates that mTORC1 is also important for Th2 cell differentiation⁷⁴. Rheb-deficient cells cultured under Th2 conditions divide slower than wild type cells and there is a Rheb-independent mTORC1 activation; these results are consistent with the notion that mTORC1 is required for metabolic reprogramming and cell proliferation during T cell activation. In addition, PI3K/mTOR pathway is involved in translational regulation of GATA3 protein⁷⁵. Whether the dose of antigen affects preferential activation of either mTORC1 or mTORC2 is not known.

2.4. Notch-mediated signaling pathway

Notch signaling is involved in Th2 cell differentiation^76–78. It seems that different notch ligand may have different function since Dll1/Notch interaction induces Th1 but Jag1/Notch interaction induces Th2 cell differentiation⁷⁶. After activation, intracellular portion of Notch is translocated to the nucleus and forms a complex with CSL protein to induce gene expression. A Notch/CSL binding site is identified in the 3’ of the Il4 gene suggesting that Notch signaling may directly regulate IL-4 expression⁷⁶. In addition, Notch/CSL complex binds to the distal promoter of Gata3 gene⁷⁸. Therefore, Notch signaling may promote Th2 cell differentiation by inducing the expression of both GATA3 and IL-4. Notch signaling may also modulate co-stimulation by affecting PI3K pathway to allow naïve CD4 T cells to respond to low dose of antigen⁷⁹. Furthermore, Notch-mediated signaling pathway regulates the expression of IL-2 and IL-2 receptor α chain^{79, 80}; IL-2-mediated signaling pathway, discussed in detail below, is one of the key signaling pathways for Th2 cell differentiation.

2.5. STAT5-activating signaling pathways

STAT5 family includes STAT5A and STAT5B, which are encoded by two genetically linked genes⁸¹. While low amount of STAT5 activation is necessary and sufficient for the proliferation and survival of all the CD4 T cell subsets^{32, 82}, high levels of STAT5 activation is essential for Th2 cell differentiation both in vitro and in vivo^{32, 33, 83, 84}. Enforced expression of a constitutively active form of STAT5A in Th1 cells results in IL-4 production in these cells without up-regulating GATA3 expression³³.

Several cytokines including IL-2, IL-7 and TSLP can activate STAT5 in T cells. TSLP, produced by epithelial cells, dendritic cells (DCs), and/or basophils, may mediate the initiation of Th2 responses in vivo^{43, 67, 85, 86}. Although the major role of TSLP is to regulate the functions of dendritic cells^{67, 87}, TSLP may also directly act on T cells to promote Th2 cell differentiation^{88, 89}. Other potent STAT5 activators are IL-2 and IL-7. While IL-7 is capable of inducing and/or promoting Th2 differentiation in vitro, its function in vivo has not been carefully studied. IL-2 is mainly a product of activated T cells and thus may serve as an important autocrine cytokine during Th2 differentiation. However, due to a broader function of IL-2, including its effect on regulatory T cells, it is difficult to assess the precise role of IL-2 during Th2 cell differentiation in vivo.

It is known that TCR-mediated signaling transiently suppresses cytokine signaling during initial T cell activation⁹⁰. Both IL-2/STAT5 and IL-4/STAT6 signaling pathways are inhibited 4–6 hours of T cell activation. Interestingly, IL-2-mediated STAT5 activation is detected at the early stages of T cell activation with low dose but not high dose of TCR stimulation⁴⁵. As mentioned above, Notch signaling induces the expression of IL-2 and IL-2 receptor α chain during T cell activation. In fact, all five Th2-promoting signaling pathways triggered by antigen, co-stimulation, Notch, IL-4 and IL-2, respectively, may crosstalk with each other and collaborate to induce Th2 cell differentiation.

2.6. Th2 cell differentiation via a default program

IFNγ produced by Th1 cells is an important cytokine that promotes Th1 cell differentiation^91–93. IFNγ is also produced by NK cells and/or ILC1s, which are the first responders at the innate phase of type 1 immune responses^93–96. In addition, IL-12 produced by antigen presenting cells including DCs plays an important role in driving Th1 cell differentiation both in vitro and in vivo through activating STAT4 in CD4 T cells^97–99. On the other hand, an equivalent DC-derived cytokine that induces Th2 cell differentiation has not been found. Instead, IL-2 and IL-4, two known cytokines driving Th2 cell differentiation, are mainly produced by T cells themselves. Thus, the determination of Th1 and Th2 cell fate appears to be asymmetrical. Even more interestingly, because GATA3 is critical for the development of CD4 T cells at multiple stages¹⁰⁰, it is already expressed by naïve CD4 T cells^{28, 101}. Furthermore, STAT5 is usually activated either by IL-7 at naïve stage or by IL-2 upon T cell activation.

Given that a Th2-biased differentiation program (basal GATA3 expression and STAT5 activation) has pre-existed in naïve CD4 T cells, IL-4/STAT6 signaling, which possibly serve as a reinforcement and/or amplification of Th2 responses, may not be necessary for Th2 cell differentiation, especially at early stages, in many situations in vivo. Indeed, Nippostrongylus brasiliensis and Schistosoma mansoni infection exclusively elicit IL-4-independent Th2 responses^23–27. The requirement of IL-4/STAT6 during some in vivo Th2 responses such as in response to Trichuris muris infection¹⁰² may simply because such infection generates a mixture of Th1 and Th2 responses and IL-4 is required to suppress Th1 responses. Consistent with the notion that Th2 cell differentiation can occur through a default pathway when the major Th1-inducing signals are absent, mice deficient in either Th1 master regulator T-bet¹⁰³ or STAT4/IFNγR doubly deficient mice developed Th2 responses in response to Toxoplasma gondii infection¹⁰⁴, which usually induces robust Th1 responses. However, in the absence of IL-12 alone, T cells fail to default to a Th2 program¹⁰⁵, suggesting that both IL-12 and IFNγ are involved in suppressing the endogenous Th2 program in T cells. Indeed, either IL-12 or IFNγ is able to induce T-bet expression¹⁰⁴.

3. Key transcription factors required for Th2 cell differentiation

3.1. GATA3

GATA3 has been recognized as the master regulator of Th2 cells^28–31. GATA3 is expressed by naïve CD4 T cells but its expression levels are induced during Th2 differentiation^{29, 106, 107}. GATA3 is sufficient to induce the Th2 phenotype since retrovirus-mediated enforced expression of GATA3 in Th1 cultures induces IL-4 production and endogenous GATA3 expression^{106, 108}. On the other hand, knocking down or deletion of GATA3 results in diminished Th2 cell differentiation, whether it is IL-4-dependent or IL-4-independent, both in vitro and in vivo^{28, 30, 31, 109}.

GATA3 promotes Th2 cell differentiation through multiple mechanisms¹¹⁰. The key role of GATA3 is to directly act on the Il4/Il13 gene locus at various sites. Chromatin immune-precipitation followed by high throughput sequencing (ChIP-Seq) analysis with anti-GATA3 reveals genome wide pattern of GATA3 binding¹⁰¹. In Th2 cells, GATA3 binds to the promoters of the Il5 and the Il13 gene including the CGRE site in the Il13 distal promoter^{111, 112}; GATA3 also binds to multiple sites at the LCR region in the Rad50^{113, 114} and several enhancer regulatory elements for the Il4 genes, such as DNase I hypersensitivity II (HSII)^{112, 115} and HSV sites¹¹⁶. GATA3 regulates epigenetic modifications in the Th2 cytokine locus during Th2 cell differentiation. However, in already differentiated Th2 cells, GATA3 is dispensable for IL-4 production although it is still essential for IL-5 and IL-13 production possibly because GATA3 is critical for activating the promoters of these two genes³⁰. Besides its direct action on the Th2 cytokine locus, GATA3 also directly regulate many Th2 specific genes such as Il1rl1 encoding T1/ST2, the IL-33 receptor, and many chemokine receptor genes such as Ccr8¹⁰¹.

3.2. STAT5

As discussed above, a strong STAT5 activation is another key element for Th2 cell differentiation. Co-expression of GATA-3 and a constitutively active form of STAT5A in non-Th2 cells results in a larger proportion of IL-4-producing cells compared to that induced by either GATA3 or active STAT5A alone³³. On the other hand, GATA3 has very limited IL-4-inducing capacity when STAT5 activation is inhibited by anti-IL-2 treatment³² and the active STAT5A is unable to induce IL-4 in Gata3-deficient CD4 T cells³⁰. Therefore, both GATA3 induction and STAT5 activation are essential for Th2 cell differentiation; high STAT5 activation levels may reduce the requirement of GATA3 expression levels for inducing IL-4 expression¹¹⁷.

Mechanistically, STAT5 binds to the HSII and HSIII sites of the Il4 gene, through which it regulates the accessibility of these sites^{32, 33}. ChIP-Seq results confirming STAT5 binding at the HSII site has also revealed another STAT5 binding at the LCR region, which may contribute to inducing IL-4¹¹⁸. Besides directly regulating the Il4/Il13 locus, IL-2/STAT5 induces the expression of IL-4Rα especially at the initial stage of Th2 cell differentiation so that activated T cells may respond to IL-4 to complete the differentiation process¹¹⁸.

3.3. Other transcription factors

In addition to GATA3 and STAT5, there are several other transcription factors that regulate IL-4 production and/or Th2 cell differentiation (Figure 1). IRF-4 is highly up-regulated during T cell activation and it is important for the differentiation and functions of virtually any CD4 T helper subsets including Th2 cells^119–128. In Th2 cells, one important function of IRF4 is to bind to the Il4 promoter to regulate its expression^{121, 122}.

It has been recently reported that c-Maf is highly expressed by Th17 cells^{126, 129, 130} and Tfh cells¹³¹; c-Maf regulates the expression of IL-10, IL-21 and IL-22. However, c-Maf was initially considered as a Th2 specific transcription factor by comparing its expression in Th1 and Th2 cells. Indeed, it controls the production of IL-4 but not other Th2 cytokines¹³². C-Maf directly binds to the promoter of the Il4 gene. C-Maf expression is negatively regulated by a LincRNA, linc-MAF-4, which is highly expressed in human Th1 cells¹³³.

Klf13 collaborates with c-Maf in regulating IL-4 production in T cells^{134, 135}. The action of c-Maf at the Il4 promoter also relies on JunB, which is critical for Th2 cell differentiation⁷⁰. The induction of JunB expression partially depends on Dec2, another Th2-specific transcription factor¹³⁶. Nfil3 (E4BP4), a transcription factor that is critical for the development of NK cells and ILCs^137–142, is highly expressed by Th2 cells and responsible for regulating IL-10 production¹⁴³. In addition, YY1 physically interacts with GATA3 and thus collaborates with GATA3 in chromatin remodeling and cytokine production¹⁴⁴.

IL-4 production and Th2 cell differentiation can also be negatively regulated by many transcription factors. One typical example is Runx3, which binds to the HSIV site of the Il4 gene and suppresses IL-4 production^145–147. Both Eomes, which is expressed by a subset of memory Th2 cells, and Sox4 that is induced by TGFβ suppress GATA3 activity and Th2 cytokine expression^{148, 149}. PU.1, a critical transcription factor regulating myeloid and lymphoid cell development at early stages, is also expressed by subsets of T cells. In Th2 cells, PU.1 is preferentially expressed by IL-4 non-producers suggesting that PU.1 negatively regulates IL-4 production¹⁵⁰.

GATA3 is expressed by some activated regulatory T cells (Tregs) at an intermediate level^151–154, however, such cells do not produce IL-4 suggesting that Foxp3 may suppress the GATA3 function in inducing Th2 cytokines. Interestingly, reduced Foxp3 expression in Tregs results in Th2 cytokine production by these cells¹⁵⁵. Such Foxp3 effect may be particularly important in humans since activated human effector T cells can transiently express Foxp3^{156, 157}.

Therefore, Th2 cell differentiation and cytokine production depend on a network of many transcription factors, which consists of GATA3, STAT5, IRF4, c-Maf, JunB, Dec2, Klf13, E4BP4, YY1, Runx3 and PU.1 etc. Changes in the balance and/or the ratio of these transcription factors may lead to an alteration in Th2 cell fate determination.

4. Mechanisms of reinforcing Th2 cell differentiation

4.1. Positive feedback

A basic principle of T helper cell differentiation is positive feedback mechanism². During Th2 cell differentiation, IL-4 produced by developing Th2 cells may instruct IL-4 non-producers to produce IL-4, and/or enhance the IL-4-producing capacity of the IL-4-producing cells in a paracrine and/or autocrine manner (Figure 1). Not only can IL-4 induce GATA3 expression and thus IL-4 production, it also enhances the sensitivity of developing Th2 cells to IL-4 stimulation by up-regulates IL-4Rα expression. Therefore, IL-4/GATA3/IL-4 and IL-4/IL-4R constitute two powerful positive feedback loops during Th2 differentiation.

It has been reported that GATA3 auto-regulates its expression¹⁰⁸. Our ChIP-Seq data suggest that GATA3 binds to multiple regions at the Gata3 locus across 1 Mb length of DNA¹⁰¹. Therefore, GATA3 may regulate its own expression directly. However, in the absence of functional GATA3 protein, the truncated GATA3 mRNA expression is not reduced when T cells are activated in the presence of IL-4¹⁰¹. This result suggests that GATA3 auto-regulation is minimal when IL-4/STAT6 signaling is present. Nevertheless, GATA3 auto-regulation may be critical to sustain Th2 phenotype when IL-4/STAT6 signaling ceases. Among the GATA3 binding sites at the Gata3 gene, one co-localizes with a T cell specific enhancer for GATA3 expression¹⁵⁸. Even more interesting, this enhancer region overlaps with the dual promoter that mediates the transcription of two long intergenic non-coding RNAs (LincRNAs) into opposite directions¹⁵⁹. Whether the expression of these two LincRNAs is related to the “enhancer” activity needs to be further studied.

GATA3 may indirectly regulate itself by inducing other transcription factors such as Dec2 and IRF4; the expression of Dec2 and IRF4 is reduced when GATA3 is acutely deleted from Th2 cells^{101, 136}. On the other hand, both Dec2 and IRF4 are able to enhance GATA3 expression levels in Th2 cells^{121, 136}.

Positive feedback regulations are also found between the two critical pathways, IL-2/STAT5 and IL-4/STAT6/GATA3, during Th2 cell differentiation. Besides inducing CD25 (IL-2 receptor α chain) expression¹⁶⁰, IL-2-mediated STAT5 activation up-regulates the expression of IL-4Rα during the initial T cell activation¹¹⁸. Furthermore, STAT5 activation is required to maintain GATA3 expression in differentiated Th2 cells³⁴. On the other hand, GATA3 regulates optimal expression levels of CD25 by T effector cells as well as Tregs^{101, 151}.

4.2. Suppression of other lineage fates

CD4 T cells can differentiate into different effector lineages including Th1, Th2 and Th17 cells. As mentioned above, IL-12/STAT4 and IFNγ/STAT1 pathways, both of which induce the master regulator T-bet, are important for Th1 cell differentiation^{92, 97, 98, 103, 161}. Therefore, STAT1/STAT4 activation and T-bet induction are the key events during Th1 cell differentiation. Similarly, Th17 cell differentiation requires STAT3 activation and the induction of RORγt expression^{1, 8}.

Th2 cell differentiation is accompanied by the suppression of other lineage fates. GATA3 deficiency leads to de-repression of STAT4 expression and enforced expression of GATA-3 down-regulates the expression of STAT4 in Th1 cells^{146, 162}. Interestingly, Gata3 deletion during Th2 cell differentiation results in an IL-12/STAT4 and IFNγ/T-bet independent IFNγ production, which is mediated by Runx3/Eomesodermin (Eomes) pathway^{30, 145–147, 163}. It has been shown that T-bet inhibits GATA-3 transcription¹⁰⁷ and suppresses its function through protein-protein interaction¹⁶⁴. T-bet and GATA3 co-expressing cells can be generated in response to parasite infection¹⁶⁵. The physical interaction between GATA3 and T-bet may allow them to suppress each other’s function. Indeed, T-bet and GATA3 binding sites co-localize at many critical Th1- or Th2-specific genes¹⁶⁶. Furthermore, GATA3 is able to silence Tbx21 (encoding T-bet) locus in Th2 cells by its direct binding to a site in the Tbx21 locus with suppressive epigenetic modifications; removing GATA3 results in a substantial reduction of such suppressive modifications at the binding site ¹⁰¹. Thus, GATA3 suppresses Th1 cell fate through multiple mechanisms¹¹⁰.

Strong STAT5 activation is necessary for Th2 differentiation as discussed above. Interestingly, constitutively active STAT5a suppresses T-bet expression when introduced into developing Th1 cells ³³. STAT5 activation also suppresses Th17 differentiation and Tfh cell differentiation ^167–171. Besides GATA3 and STAT5, another transcription factor, growth factor independent 1 (Gfi-1), which is preferentially expressed in Th2 cells, inhibits IFNγ and IL-17 production in Th1 and Th17 cells, respectively ^172–174.

4.3. Selective growth of Th2 cells

IL-12 receptor β2 chain is selectively up-regulated during Th1 cell differentiation ¹⁷⁵, thus, IL-12 plays an important role in selecting committed Th1 cells to grow ¹⁷⁶. However, although IL-4 receptor α chain can be up-regulated by IL-4 ¹⁷⁷, naïve CD4 T cells as well as non-Th2 cells are also able to respond to IL-4 because of constitutive expression of IL-4 receptor on T cells ⁹⁰. Therefore, IL-4 receptor up-regulation is not the major mechanism for selective outgrowth of Th2 cells in response to IL-4 stimulation. Nevertheless, IL-4 up-regulates Gfi-1 expression through STAT6 activation; Gfi-1 preferentially induces the expansion of GATA-3^hi Th2 cells through its actions upstream and downstream of STAT5 activation^{172, 174}.

In fully differentiated Th2 cells, IL-33 receptor α chain (T1/ST2 or IL-1R–like 1) is highly expressed^{178, 179}. Since IL-33 is released during Th2 responses, IL-33 may play a role in promoting the survival and/or expansion of fully differentiated Th2 cells. Indeed, blocking IL-33 signaling results in attenuated eosinophilic airway inflammation and decreased responses to Schistosoma mansoni^{180, 181}. Strikingly, continuous IL-33Rα expression in differentiated Th2 cells depends on both GATA3 expression and STAT5 activation³⁴.

5. Chromatin remodeling and epigenetic modifications at the Il4/Il13 locus

The Il4 and Il13 genes, located at chromosome 5 in human and chromosome 11 in mouse, respectively, are flanked by the Rad50 and Kif3a genes. The locus control region (LCR) of the Il4-Il13 locus is within the 3’ of Rad50 gene¹¹³; the Il5 gene is on the other side of the Rad50. There are GATA3-mediated intrachromosomal interactions between the LCR and the Th2 cytokine gene promoters¹⁸². A potential regulatory element may reside within the Kif3a gene based on the specific-lineage epigenetic markers in Th2 cells in this region¹⁰¹. Transcriptional regulation of Il4/Il13 expression, like transcriptional regulation of any other lineage specific genes, highly depends on chromatin remodeling and epigenetic modifications^{17, 183, 184}.

During Th2 differentiation, several Th2-specific DNase I hypersensitivity (HS) sites are induced at the Il4-Il13 locus¹⁸⁵. These include the HSII site in the intron 2 of the Il4 gene^{112, 186}, HSV and HSVa¹¹⁶ at 3’ of the Il4 coding region, conserved non-coding sequence 1 (CNS1)^{187, 188} which is located at the intergenic region of Il4 and Il13, and several HS sites within the LCR in the Rad50 gene^{113, 189}. Interestingly, the HS IV site is accessible in both Th1 and Th2 cells¹⁸⁵; Runx3 binds to this region in Th1 cells to suppress IL-4 production^{145, 190}. Deletion of the RHS7 (HS7 site within the LCR in the Rad50) site results in reduced IL-4 and IL-13 production but not IL-5 in Th2 cells¹⁹¹. However, deletion of the RHS6 site or the whole LCR affects the expression of all three cytokines^{192, 193}. The requirement of these elements to induce cytokine production seems to vary when different immunization protocols are followed¹⁹² suggesting that the LCR contains both IL-4-depedent and IL-4-independent regulatory elements, consist with the findings that STAT6 binds to both the RHS6 and RHS7 sites¹⁶, but GATA3 only binds to RHS5 and RHS6 not RHS7 of the LCR¹⁰¹. Activated STAT5 binds to the HSII site and induces the accessibility at this region³³. ChIP-Seq analysis also indicates that GATA3 binds the HSII site¹⁰¹. Our unpublished data suggest GATA3 is responsible for the induction and maintenance of HSII accessibility.

Different histone modifications are associated with either gene activation or repression¹⁹⁴. Tri-methylation and di-methylation at the lysine position 4 of histone 3 (H3K4me2 and 3) are usually indicative of active gene loci, whereas, H3K27me3 is a marker for repressed gene loci. The Il4/Il13 locus displays H3K4me3 modification in Th2 cells and H3K27me3 in Th1 cells¹⁹⁵. MLL, a histone H3K4 methyltransferase, is important for maintaining H3K4 methylation at the Il4/Il13 locus in differentiated Th2 cells¹⁹⁶. On the other hand, Ezh2, an H3K27 methyltransferase, is responsible for mediating H3K27me3 modification at the Il4/Il13 and Gata3 loci in Th1 cells^197–199. Deletion of Ezh2 in T cells results in reduced H3K27me3 at the Tbx21 and Gata3 loci, and thus enhanced Th1 and Th2 differentiation. Ezh2 deletion also causes reduced stability of differentiated cells and enhances their plasticity. Besides histone methylation modifications, histone acetylation at the lineage-specific cytokine gene loci has also been described²⁰⁰.

Although GATA3 may regulate the expression of IL-5 and IL-13 through its direct binding to the promoters of the Il5 and Il13 genes^{30, 111, 201, 202}, a main function of GATA3 in Th2 cell fate determination seems to mediate histone modifications at Th1 and Th2 specific gene loci including the Ifng and Il4/Il13 locus^{108, 203, 204}. In the absence of GATA3, H3K27me3 marks around the GATA3 binding site at the Ifng locus is greatly reduced. On the other hand, H3K4me2 marks at the Il4/Il13 locus, especially at the LCR region where GATA3 binds, are diminished when GATA3 is absent. At a genome level, GATA3 binds to ∼2000 genes in Th2 cells¹⁰¹. When GATA3 is deleted, ∼100 genes among these 2000 genes have altered their expression level, however, alteration of histone modifications occurs at the loci of ∼900 genes¹⁰¹. This strongly indicates that GATA3-dependent alterations in epigenetic modifications is not a result of gene expression change.

DNA CpG methylation also affects gene expression²⁰⁵. Deletion of Dnmt-1, a DNA methyltrasferase, or of methyl CpG-binding domain protein-2 (MBD2), results in aberrant expression of IL-4 without affecting GATA3 expression^{206, 207}. Interestingly, GATA3 inhibits the binding of MBD2 to methyl CpG. STAT5 activation is partially involved in de-methylation of the RHS7 site in the LCR during Th2 differentiation¹⁸⁹.

Collectively, both GATA3 and STAT5 strongly bind to key regulatory elements of the Il4/Il13 locus including the HSII site¹¹² and HS sites within the LCR¹⁹¹; both GATA-3 and STAT5 are involved in many aspects of chromatin remodeling at the Il4/Il13 locus including chromatin accessibility, histone modifications and DNA methylation.

6. Induction of IL-4 and IL-13 expression by Th2 cells

TCR-mediated signaling plays an important role in T helper cell differentiation as discussed above. Another critical function of TCR-mediated signaling is to induce cytokine production in already differentiated T helper cells. The most efficient way to stimulate Th2 cells to produce cytokines is through their TCRs or by chemicals such as PMA and ionomycin to mimic TCR-mediated signaling. TCR stimulation activates NFAT family members in a Ca²⁺ dependent manner²⁰⁸. Upon TCR re-stimulation of differentiated Th2 cells, NFAT translocates to the nucleus and binds to the Il4 promoter as well as HSVa region to regulate IL-4 expression^{122, 209}. Besides inducing cytokine production, NFAT proteins are also involved in Th2 cell differentiation²¹⁰.

T helper cells can also produce cytokines in response to cytokine stimulation. IL-18 together with IL-12 induces IFNγ production independent of TCR stimulation^{211, 212}. Similarly, IL-33 in combination of STAT5 activators, such as IL-2, IL-7 and TSLP, induces IL-13 but not IL-4 expression in differentiated Th2 cells^{178, 179}. In addition, Th2 cells express cysteinyl leukotriene receptor 1 and are able to respond to leukotrienes to produce IL-13 possibly through calcium-dependent pathway^{213, 214}.

Whether Th2 cells are stimulated by TCR to produce IL-4 and IL-13, or by cytokines to produce IL-13, not all Th2 cells actually produce IL-4 and/or IL-13 upon stimulation. Although heterogeneity of Th2 cells can be a result of differential epigenetic status of each cytokine allele^{215, 216}, the actual cytokine production in a given cell upon a single round of stimulation may be a stochastic event²¹⁷. Heterogeneous activation of transcription factors such as NFAT or differential assembly of functional transcription factor complex needed for inducing cytokine production may be one of the main explanations^{217, 218}. Thus, IL-4- and/or IL-13-non-producing cells identified after stimulation at one time may express these cytokines in response to another round of stimulation. Therefore, both cytokine-producing and non-producing cells may represent fully differentiated cells. Indeed, IL-4-producers and non-producers from a same Th2 culture express similar levels of GATA3 and have identical chromatin accessibility at the Il4/Il13 locus²¹⁷. Interestingly, this may not be entirely true for IFNγ production in Th1 cells since low- and high-IFNγ-producing cells express different levels of T-bet and seem to have a quantitative memory to produce IFNγ²¹⁹.

7. Relationship between Th2 cells and IL-4-producing Tfh cells

Tfh cells, many found in the germinal centers in the B cell follicle, are critical helper T cells in helping B cells produce antibodies and undergo immunoglobulin (Ig) class switching⁹. IL-4 is essential for the Ig class switching to IgE¹⁰, suggesting that some Tfh cells need to produce IL-4^220–222. Indeed, IL-4-producing CD4 T cells are mainly Tfh cells²²². Interestingly, Tfh cells do not produce IL-13⁴⁷.

GATA3-deficient mice fail to generate type 2 response including IgE class switching³⁰ suggesting that GATA3 is also required for Tfh cells to produce IL-4. However, Tfh cells containing IL-4-producers express low levels of GATA3 suggesting that small amount of GATA3 is sufficient for IL-4 production in Tfh cells^{47, 223}. On the other hand, IL-13-producing Th2 cells are GATA3 high expressors⁴⁷. GATA3 at a low level is sufficient to induce IL-4 but not IL-13 production when there is a strong STAT5 activation³³. However, Tfh cells express low levels of IL-2Rα, and STAT5 activation is paradoxically suppressive for Tfh cell differentiation^{168, 224}.

Since c-Maf is highly expressed by Tfh cells¹³¹ and it is important for IL-4 production in Th2 cells¹³², it is possible that high levels of c-Maf expression by Tfh cells may compensate low GATA3 expression and/or low STAT5 activity for inducing IL-4. Interestingly, deletion of a DNA segment containing both HSV and HSVA results in impaired IL-4 production in both Th2 cells and mast cells¹¹⁶, however, only deleting the CNS2 region at the HSV site dramatically affects IL-4 production in Tfh cells and in naïve CD4 T cells but has a small effect on IL-4 production in conventional effector Th2 cells in tissue^{225, 226}. Consequently, IgE induction is completely abolished in CNS2 deficient mice. Furthermore, CNS2 region is much more active in Tfh cells²²⁵. Since Notch/CSL complex directly binds the CNS2 region⁷⁶, Notch signaling may be constitutively active and critical for IL-4 production in Tfh cells.

The relationship between classical Th2 cells and IL-4-producing Tfh cells is still not certain. An in vitro study suggests that T cells can sequentially gain cytokine producing capacity and follicular helper phenotype²²⁷. That is, conventional Th2 cells can be converted to IL-4-producing Tfh cells. Similarly, Tfh cells can be further differentiated to produce IL-4. In a transfer model, it has been shown that activated T cells that have gained IL-4-producing capacity can later on display a Tfh cell phenotype in a B cell dependent manner²²¹. However, this does not rule out the possibility that some Tfh cells gain IL-4-producing capacity after they have entered germinal center. Alternatively, activated T cells may gain IL-4-producing capacity and features of Tfh cells simultaneously, and their fate to become either classical effector Th2 cells or IL-4-producing Tfh cells may be determined during early T cell differentiation. Indeed, it has been shown that there is an early lineage commitment to either T effector cells or Tfh cells based on their Bcl6 and IL-2Rα expression two days after immunization¹⁶⁸. Bcl6 is transiently induced during Th1 cell differentiation supporting the model of early fate determination²²⁸, however, whether Bcl6-expressed/expressing cells will become Tfh cells later is unknown.

IL-4-producing cells are mainly found in the B cell follicle whereas IL-13-producing cells are found in the tissue⁴⁷; this suggests that Th2 effector cells and IL-4-producing Tfh cells may adopt distinct differentiation programs which involves regulation of GATA3 levels and other transcription factors collaborating with GATA3. Interestingly, it has also been shown that memory T helper cells may originate from Tfh cells²²⁹. A careful genome-wide assessment of the epigenetic status in conventional Th2 cells, IL-4-producing Tfh cells and Th2 memory cells may help understand the relationship between these cells.

8. Relationship between Th2 cells and ILC2

ILC2s are important players during type 2 immune responses particularly at early stages^230–233 and their development requires transcription factors RORα^{234, 235} and GATA3^{38, 236–240}. Gfi-1, also highly expressed by ILC2s, is involved in the development and/or maintenance of ILC2s²⁴¹; without Gfi-1, ILC2s aberrantly express IL-17 similar to the finding in Gfi-1-deficient T cells¹⁷³. ILC2s not only contribute to the expulsion of helminths, they are also involved in allergic lung and skin inflammation^242–246. ILC2s are also involved in adipocyte differentiation and energy metabolism^{35, 247, 248}; whether Th2 cells have similar functions is an important question of interest.

When pharmacologically stimulated by cytokines such as IL-25 or IL-33, ILC2s are able to expel worms in the absence of adaptive immune system. During activation, ILC2s transiently produce IL-9, which in turn maintains ILC2 survival in an autocrine manner^{249, 250}. IL-9 receptor is highly expressed on ILC2s and such expression depends on GATA3^{38, 250}. GATA3 is also critical for the expression of many ILC2-specific genes^{38, 47}, including IL-5 and IL-13 that are also shared by Th2 cells, and GATA3 is indispensable for the maintenance of ILC2 cell numbers in vivo^{38, 237}.

ILC2s and Th2 cells have identical functions and similar requirement of transcriptional machinery in cytokine production (Figure 2) ^{37, 251}. However, due to the lack of an antigen receptor and toll-like receptor, ILC2s mainly respond to cytokines such as IL-33 to produce type 2 cytokines, IL-5 and IL-13, but not IL-4.

Figure 2. Similarity between Th2 cells and ILC2s.

Both Th2 cells and ILC2s are capable of producing a set of cytokines such as IL-5, IL-13 and Amphiregulin (Areg), although ILC2s produce less IL-4 and no parathyroid hormone (Pth). GATA3 is critical for the development, maintenance and functions of both Th2 cells and ILC2s. While ILC2s lack T cell receptor (TCR), they express cytokine receptors found on Th2 cells including IL-33 receptor (T1/ST2), IL-2 receptor and IL-7 receptor. Both Th2 cells and ILC2s produce IL-5 and IL-13 when stimulated through IL-33 and a Stat5 activator such as IL-2 and IL-7. Th2 cells and ILC2s also share expression of specific chemokine receptors including CRTh2, CCR8, CCR1 etc. Similar gene expression between Th2 cells and ILC2s and their dependence on GATA3 is consistent with their similar functions during type 2 immune responses.

ILC2s also respond to cysteinyl leukotrienes to produce cytokines²⁵². Unlike IL-33, cysteinyl leukotriene D4 induces the expression of IL-4 in addition to IL-5 and IL-13. PGD2, a product of mast cells after IgE-mediated degranulation, can induce ILC2 migration and cytokine production through its interaction with CRTH2 expressed by ILC2s²⁵³. Interestingly, basophil-derived IL-4 is able to induce ILC2 proliferation²⁴⁵ and IL-13 production by ILC2s²⁵⁴; this direct effect of IL-4 on ILC2s to produce IL-13 requires further investigation and confirmation by other groups. On the other hand, lipoxin A4 and E-cadherin (a ligand for KLRG1) suppress cytokine production by ILC2s^{255, 256}.

ILC2s and Th2 cells collaborate during type 2 immune responses^257–260. In a lung inflammation model, papain-induced IL-33 activates ILC2s to produce IL-13, which induces the migration and activation of lung dendritic cells (DCs) to the draining lymph nodes²⁶⁰; these migratory DCs then induce Th2 cell differentiation in an IL-4-independent manner. Some ILC2s also express MHC class II through which activate T cells to produce IL-2, and IL-2 acts back on ILC2s to produce type 2 cytokines^{257, 258}. IL-2 has been reported to induce ILC2 proliferation and cytokine release^{246, 249, 261}. Co-culture ILC2s with naïve CD4 T cells results in ILC2 proliferation and cytokine production, which is mediated by IL-2 produced by T cells²⁵⁸. While it is clear that ILC2s are capable of inducing Th2 cell differentiation in vitro possibly through IL-4 production²⁵⁹, whether ILC2 and T cell direct interaction is critical for initiating and promoting Th2 cell differentiation in vivo, or this interaction mainly reflects collaborative relationship of ILC2s and Th2 cells at the effector stage requires further investigation.

9. Summary and conclusions

During Th2 cell differentiation, multiple signaling pathways result in two major critical events: up-regulation of GATA3 expression and activation of STAT5 proteins. Both GATA3 expression and STAT5 activation are essential for Th2 cell differentiation. GATA3 and STAT5 directly act on the Il4/Il13 locus through chromatin remodeling and epigenetic modifications. Several other transcription factors are also involved in Th2 cell differentiation. Th2 cell lineage commitment, determined by a network of transcription factors, is accompanied by blockage of alternative lineage fates and selective growth of already differentiated Th2 cells. Once Th2 cells are fully differentiated, the production of Th2 cytokines including IL-4, IL-5 and IL-13 requires re-stimulation by antigens through TCR, by cytokines such as IL-33, or possibly by other inflammatory molecules such as cysteinyl leukotrienes. Some transcription factors that are important for Th2 lineage commitment may also be involved in regulating Th2 cytokine production upon restimulation.

We have gained much knowledge of Th2 cell differentiation mainly from in vitro studies. However, our understanding of Th2 cell differentiation in vivo is far from complete. A particular example is that while IL-4/STAT6 pathway is absolutely necessary for in vitro Th2 cell differentiation, this pathway is only minimally involved in many in vivo Th2 responses. The complexity of Th2 responses in vivo is partially due to the involvement of other cytokines such as TSLP, IL-25 and IL-33^{85, 262, 263}, and the participation of many immune cells including DCs, ILC2s, NKT cells, basophils, eosinophils and mast cells²⁶⁴. In the future, it is critical to further understand the relationship between conventional Th2 cells and IL-4-producing Tfh cells, and the functional similarity and difference between Th2 cells and ILC2s, particularly during type 2 immune responses in vivo. Two particular intriguing questions are: how GATA3 with various amounts differentially regulates IL-4 and IL-13 in conventional Th2 cells and Tfh cells, respectively; why Th2 cells express both IL-4 and IL-13 but ILC2s mainly produce IL-13 but not IL-4. By simultaneously studying Th2 cells and ILC2s, common and unique pathways/molecules that are involved in regulating type 2 immune responses will be identified; these pathways and molecules may be considered as drug targets for treating Th2- and/or ILC2-related diseases including allergy and asthma.