GATA3: A master of many trades in immune regulation

GATA3 in innate and adaptive immunity

The immune system is constantly challenged by pathogens such as bacteria and viruses. Complex strategies have developed to respond to those insults through innate and adaptive immunity, while also maintaining immune homeostasis and self-tolerance. Elucidating the mechanisms that guide immune function under normal and pathological conditions could aid the design of effective therapies to treat a variety of afflictions, including infectious diseases, autoimmunity, inflammatory disorders, and some types of cancers. A central step towards understanding these mechanisms is to define the role of transcription factors that drive immune cell function.

The transcription factor GATA3 has emerged as a critical regulator of both innate and adaptive immunity. GATA3 belongs to the GATA (GATA-binding protein) family comprised of six members - GATA1 to 6 - in mammals. GATA transcription factors recognize the consensus DNA sequence (W)GATA(R) (W = A or T and R = A or G) [1, 2]. Different GATA family members are expressed in a tissue and cell-type specific fashion. Compared to other GATA family members, GATA3 is highly expressed in the hematopoietic compartment, and gene-targeting studies have shown an essential role for GATA3 in the development and function of T cells, B cells, CD1-restricted natural killer T (NKT) cells, natural killer (NK) cells, innate like lymphoid cells (ILCs) ([3–8] and commented in [9]). It should be noted that GATA3 also functions in non-hematopoietic cells in both embryonic and adult tissues including the adrenal glands, kidneys, central nervous system, inner ear, hair follicles, skin and breast tissue. For the purpose of this article, I will focus on the roles of GATA3 in innate and adaptive immunity.

Upon encounter with cognate antigen presented by antigen presenting cells (APCs) and depending on the inflammatory context, CD4+ T cells differentiate into distinct effector subsets including T helper 1 (Th1), Th2, Th17 and Th9 cells to direct discrete immune responses (reviewed in [10–13]) (Figure 1), often in combination with cytolytic CD8+ T cell effectors (reviewed in [14]). GATA3 function in T cells has been studied more extensively than in other cell types of adaptive immunity including B and NKT cells. Although GATA3 was originally identified as a master regulator for Th2 differentiation of CD4+ T cells, increasing evidence suggests that it is critical for the development, differentiation and function of other CD4+ T cell subsets, as well as CD8+ cells. While this review will discuss how GATA3 functions in B and NKT cells, it will focus on GATA3 controlled T cell function. In addition, recent studies point to roles for GATA3 in innate lymphocyte, which will also be discussed in this review. Furthermore, this article will place the findings in the context of earlier studies so as to delineate similarities and differences that provide insight into the multiple mechanisms of action of this versatile transcription factor in different immune cell types.

GATA3 integrates diverse upstream signals to control target gene expression

High levels of GATA3 expression in Th2 cells were first observed by Zheng and Flavell by a cDNA representational difference analysis that compared gene expression in Th1 and Th2 cells [15]. This study also showed that interleukin 4 (IL4), a Th2 cell polarizing cytokine, promoted GATA3 expression. Later findings revealed the importance of other upstream signals in the regulation of GATA3 expression, expanding on studies showed that GATA3 activation can occur independent of the IL4-STAT6 signal [16–18] (Figure 2A). T cell receptor (TCR) stimulation was shown to trigger GATA3 up-regulation in CD4+CD8+ DP thymocytes, as well as in mature CD4+ and CD8+ T cells [19]. IL4 and TCR promote GATA3 expression through different mechanisms: Signaling downstream of IL4, largely mediated by STAT6, enhances the transcription of GATA3 [19], while signaling through the TCR promotes the translation of GATA3 via PI3K-mTOR dependent pathways [20]. IL2 stimulation also up-regulates GATA3 expression in activated CD4+ T cells in a STAT5 dependent manner [21]. IL2 promotes GATA3 expression in both activated CD4+ and activated CD8+ T cells [22]. It remains to be addressed whether IL2 controls GATA3 expression through transcriptional or post-transcriptional mechanisms.

Wnt and Notch signals also regulate GATA3 expression. Activation of Wnt-Frizzle signal induces the accumulation of β-catenin in Th2 cells and promotes GATA3 transcription by recruiting Satb1, β-catenin and the histone acetyltransferase p300 to the GATA3 promoter [23]. Abrogation of Wnt-Frizzled/β-catenin signaling by the Wnt antagonist Dkk1 or small interfering RNA (siRNA) targeting of β-catenin leads to reduced GATA3 expression in Th2 cells [23]. Wnt-driven GATA3 expression has also been shown through the induction of TCF1 and β-catenin binding to the TCF1-binding site upstream of GATA3 exon-1b during the initiation of Th2 cell differentiation [18]. Notch signaling in activated CD4+ T cells promotes GATA3 transcription and Th2 differentiation independent of IL4-STAT6 signal [24–26].

In addition, GATA3 expression has been shown to diminish in Th2 cells when Notch signal is inhibited by the expression of a dominant negative form of Mastermind-like (MAML), a factor necessary for the assembly of the complex that contains Recombination signal binding protein for immunoglobulin kappa J region (Rbpj) and the cleaved intracellular domain of Notch (ICN), which triggers the transcription of target genes [26]. In addition to regulatory mechanisms at the transcriptional and translational levels, GATA3 function is also regulated through post-translational modifications. p38 phosphorylates GATA3 [27] to promote its nuclear translocation [28]. Pharmacological inhibition of p38 activity impairs the nuclear translocation [28] and transactivation activity [27] of GATA3 with reduced Th2 cytokine production in T cells [27]. In addition, pharmacological inhibition of p38 activity blocked the nuclear translocation of GATA3 in cycling long-term hematopoietic stem cells (LTHSCs) and the self-renewal of these cells [29]. Moreover, in type 2 innate lymphoid cells (ILC2), cytokine stimulation induces p38-mediated GATA3 phosphorylation and inhibition of p38 activity by pharmacological drugs blocked such phosphorylation and the production of IL6 by ILC2 [30]. GATA3 is acetylated on lysine 305 (K305) [31], but how this acetylation affects GATA3 function remains controversial: In transgenic models, expression of a hypoacetylated form of GATA3 (KRR to AAA) resulted in defects in T cell survival and homing [31], decreased Th2 cytokine production, and impaired allergic response [32]. Nonetheless, when a GATA3 with the KRR mutation is retrovirally transduced into activated CD4+ T cells, it is able to bind to DNA within the Il4 locus, induce DNase hypersensitive sites, and drive the production of IL4, albeit at moderately reduced levels compared to wild type GATA3 [33]. It is possible this discrepancy is due to the difference in the timing of GATA3 (KRR) expression: It is expressed earlier in transgenic T cells (before T cell activation) than in retrovirally transduced T cells (after T cell activation). These findings nonetheless demonstrate that GATA3 function is modulated by post-translational modifications. How these and other yet-to-be-identified post-translational modifications integrate information from upstream signaling pathways in different cellular context, and the molecular mechanisms by which they impact GATA3 function are open questions.

It should be noted that while the aforementioned signaling pathways impact GATA3 function in immune cells under specific conditions, their functions extend beyond GATA3 regulation. In fact, all of the above mentioned pathways exert pleiotropic effect on T cells. Notch, for example, sensitizes CD4+ T cell to differentiate into multiple Th cell types including Th1, Th2 and Th17 [34]. Notch inhibition by a gamma secretase inhibitor (GSI) or by the expression of dominant negative MAML leads to impaired Th1, Th2 and Th17 differentiation with reduced expression of Ifnγ, Tbx21, Il4, Gata3, Il17α, and Rorc. Notch1 directly binds to Il4, Gata3, Tbx21 loci under Th1, Th2 and Th17 polarizing conditions. In addition, GSI inhibits Th cell differentiation more potently in sub-optimal than in optimal Th polarization conditions [34]. These findings suggest that Notch integrates and amplifies cytokine-derived signals to sensitize the differentiation of not only Th2 but also Th1 and Th17 cells [25, 26, 34–36].

Besides being regulated by exogenous stimuli, GATA3 can initiate an auto-activation feedback loop independent of cytokine stimulation. Retrovirus mediated ectopic expression of GATA3 induced strong expression of endogenous GATA3 in both IL4/STAT6 deficient and Th1 CD4+ T cells [16, 37–39]. Structural and mutational analysis revealed that GATA3 may promote its own expression by acting on a T-cell-specific cis elements within the Gata3 locus [39]. Therefore, GATA3, once highly expressed, is able to ‘lock-in’ a GATA3-promoted program to stabilize Th2 function.

Transcriptional regulation by GATA3

GATA3 controls cellular function predominantly through regulating target gene expression (Figure 2B). For example, to promote Th2 differentiation, GATA3 activates the expression of Th2 cytokines by binding directly to the Il5 and Il13 promoters, the intragenic regions of Il4, and the CGRE region within Il13 locus [40]. To inhibit Th1 differentiation, GATA3 suppresses the expression of IL12Rβ2 [41] and STAT4 [42], both of which are critical for Th1 differentiation [43, 44]. In addition, GATA3 inhibits Eomes expression and IFN-γ production by physically interacting with Runx3 [45], a transcriptional regulator that promotes Th1 differentiation [46]. To reveal targets of GATA3 in T cells, genome-wide analysis has been performed using a combination of ChIP-Seq and RNA-Seq approaches, which enable the identification of DNA sequences bound by GATA3-containing protein complexes as well as the profiling of associated RNA expression [47]. Many GATA3 binding sites, e.g. the ones within Ctla4 and Icos loci, are shared by different T cell subsets including thymocytes, CD4+ T, CD8+ T, Treg cells, Th1, Th2, Th17 and NKT cells. Nonetheless, GATA3 regulates different transcriptional programs depending on the cellular context [47]. For example, GATA3 directly regulates the expression of Th-POK, Notch1, and TCR subunits specifically in the thymocytes and naïve T cell, and it controls the distinct expression of 91, 90, 7, and 43 genes in Th1, Th2, Th17, and iTreg cells respectively. GATA3 thus deploys shared and unique mechanisms to control the function of different T cell subsets. To achieve diverse function in different cell types, GATA3 associates with various co-factors including ETS, RUNX, AP1, TCF11 and FLI1 in a cell type specific manner to either directly regulate target gene expression or modify epigenetic markers, such as the methylation of the histon [47].

GATA3 in the development of T, B and NKT cells

GATA3 controls the function of both adaptive and innate immune cells (Figure 3). The involvement of GATA3 in adaptive immunity, esp. T cell function, has been studied extensively. Early studies in human cells revealed that GATA3 is expressed by early T cell progenitors, and that it binds the human TCR-α enhancer [48], suggesting a role in T cell development. GATA3 was deleted in mice using gene targeting approaches, but these mice die during early embryonic development (day 12) [49], precluding an assessment of the role of GATA3 in lymphocyte development in these mice. To study GATA3 function in T cells, chimeric mice were created by supplementing GATA3-deficient embryonic stem cells into immuno-deficient Rag2 null blastocysts. GATA3 deficient T cells failed to develop in the chimeric mice, whereas B cells developed normally, indicating a requirement for GATA3 in T cell development [49]. In this study, it was difficult to rule out an effect of GATA3^−/− non-lymphoid tissues on T cell lineage commitment because GATA3 null embryonic stems cells contributed to the formation of nonlymphoid tissues including those in the thymus. In subsequent studies, T and B cell development was evaluated in bone marrow chimeric mice reconstituted with GATA3 null fetal liver cells and in a conditional gene targeted model that enabled the deletion of GATA3 specifically in hematopoietic cells [3, 50]. GATA3 deficiency resulted in failed development of T but not B cells in these models [3, 50]. Furthermore, studies in a mouse model engineered to have green fluorescent protein (GFP) under the control of the endogenous GATA3 promoter revealed that GATA3 is expressed in Lin^loc-Kit^hiCD25⁻ early T cell progenitors (ETPs). The generation of ETPs is impaired when GATA3 expression is abated by targeted deletion or an engineered hypomorphism [50]. Therefore, GATA3 is required for T cell lineage commitment, but not for B cell lineage commitment. In fact, GATA3 actively suppresses B cell development. EBF1, a factor critical for B cell development (reviewed in [51]), suppresses GATA3 expression. Blockage of EBF1-mediated suppression of GATA3 leads to elevated GATA3 expression and defective B cell development, which can be rescued by GATA3 deletion [52]. In addition, inducible GATA3 deletion in DN2 thymocytes results in B cell development [53]. Therefore, GATA3 promotes T cell development but suppresses B cell development. These reciprocal functions of GATA3 in T and B cell development appear to be evolutionarily conserved: orthologues of GATA3 are preferentially expressed in T- but not B-cell-like cells in jawless vertebrates, such as lampreys [54].

While the functions of GATA-3 in T cell lineage commitment are not fully understood, GATA3 is suggested to mediate Notch signaling that is important for both promoting T cell development and inhibiting B cell development (reviewed in [55]). Fetal liver precursors cultured with stromal cells expressing Delta-like-1, a Notch ligand, develop into T cells with enhanced GATA3 mRNA expression [56, 57], and this up-regulation was reversed upon Notch ligand removal [57]. TCF1 was reported to control GATA3 expression downstream of Notch signaling during early T cell development. Notch activation induced TCF1 expression, which in turn increased transcription of GATA3 and T cell development independent of Notch signal [58]. While TCF1 promotes GATA3 expression by binding to the regulatory sites upstream of GATA3 exon 1b in Th2 cells [18], whether GATA3 is a direct transcriptional target of the TCF1 during early T cell development remains to be addressed. Activation of Notch signaling by introducing ICN into fetal liver precursors is not sufficient to promote T cell development when GATA3 is deficient. Nonetheless, ICN expression in fetal liver precursors effectively inhibited B cell development when GATA3 was absent [59]. Likewise, while GATA3-deficient DN2 thymocytes could develop into B cells in the absence of Notch signaling, Notch activation prevented B cell development of GATA3-deficient DN2 thymocytes [53]. Taken together, these findings suggest that GATA3 is specifically required for Notch-driven T cell development but dispensable for Notch-mediated inhibition of B cell development.

The function of GATA3 in development extends beyond T cell lineage commitment in the thymus. In a mouse model engineered to delete GATA3 in double negative (DN) thymocytes (GATA3^fl/fl-LckCre mice), development of both CD4+ and CD8+ T cells is abrogated, and this defect was shown to relate to a requirement for GATA3 in TCR β chain-selection during the DN3-DN4 transition [60]. In contrast, when GATA3 is deleted at the double positive (DP) stage of thymocyte development (GATA3^fl/fl-CD4Cre mice), only CD4+ T cell development is abrogated in [22, 60–62], indicating a specific requirement for GATA3 in co-receptor expression during the DP to SP transition in developing T cells.

One of the mechanisms by which GATA3 controls co-receptor expression is by promoting the expression of ThPOK [62], a transcription factor that mediates the up-regulation of CD4 expression and the down-regulation of CD8 expression during DP to SP transition (reviewed in [63]). GATA3 binds to the ThPOK promoter suggesting that ThPOK acts downstream of GATA3 to control CD4SP thymocyte development. Transgenic expression of ThPOK in DP thymocytes, however, failed to restore the development of CD4+ T cells in the absence of GATA3 [62]. Two mutually non-exclusive possibilities may account for these findings: In a first scenario, GATA3 and ThPOK would function inter-dependently in a feedforward loop during CD4SP thymocyte development. Alternatively, GATA3 may also control processes relevant to expression of CD4 and CD8 in a ThPOK independent manner. Evidence exists to support the latter: Enhanced GATA3 expression increased the numbers of TCR^hiCD24^lo thymocytes with a CD4SP-like phenotype in a GATA3 transgenic model [64]. GATA3 was also found to repress the expression of Runx3, a factor that suppresses CD4 expression and promotes CD8 expression (reviewed in [65]), independent of ThPOK [64].

Like conventional CD4+ and CD8+ T cells, regulatory T (Treg) cells, a CD4+ T cell subset that is critical to suppress immune response and maintain self-tolerance (reviewed in [66, 67]), also develop in the thymus. Treg cells are characterized by expression of the X-linked transcription factor Foxp3 (reviewed in [68]). In a mouse model where GFP is knocked into endogenous Foxp3 locus to mark Foxp3-expressing cells, Treg cells are detected in DN, DP and SP (mostly CD4SP) thymocytes [69, 70]. Like conventional CD4+ T cells, Foxp3+ Treg cells also express GATA3 [71]. To investigate the role of GATA3 in thymic development of Treg cells, several research groups deleted GATA3 specifically in Treg cells by crossing GATA3^fl/fl mice with mice bearing a Cre-knocked-in Foxp3 allele [72, 73] or with mice carrying a Foxp3-Cre transgene in a bacterial artificial chromosome (BAC) [74]. The thymic generation of Treg cells in these mice is normal [72–74], indicating that unlike conventional CD4+ T cells, thymic development of Treg cells does not require GATA3.

Invariant NKT (iNKT) cells consist of subset of T cells expressing a semi-invariant TCR a chain (Vα14-Jα18 in the mouse and Vα24-Jα18 in humans). iNKT cells recognize and react to lipid antigens presented in the context of CD1d [75]. iNKT cells develop from DP thymocytes [76, 77], but undergo thymic selection restricted by CD1d instead of MHC [78, 79]. GATA3 is expressed in iNKT cells, and GATA3 deficiency leads to failed development of iNKT cells in a mouse model carrying a GATA3 conditional allele and Cre under the control of the CD4 promoter (GATA3^fl/fl-CD4Cre) [6]. The mechanisms by which GATA3 impacts the development of iNKT cells are not clear. The transcription factors Myc and Myb may be involved, as Myc deletion and Myb deletion in CD4 cells (a Myc^fl/fl-CD4Cre mouse model [80] and a Myb^f/f-CD4Cre mouse model [81], respectively) results in defects in iNKT cell development similar to those observed in GATA3^fl/fl-CD4Cre mice. The finding that GATA3 controls Myc expression in T cells [22] implies that the GATA3-Myc signaling axis is critical in controlling iNKT cell development, a notion that warrants further investigation.

GATA3 functions in the maintenance of T cells in the periphery

In addition to controlling T cell development, GATA3 contributes to the maintenance of mature T cells in the periphery. GATA3 is expressed by mature CD8+ T cells in the periphery under steady state conditions, and recent studies point to a requirement for GATA3 in peripheral maintenance of these cells. CD8+ T cells isolated from GATA3^fl/fl-CD4Cre mice failed to be maintained in syngeneic hosts after adoptive transfer. In addition, CD8+ T cells isolated from GATA3^fl/fl-ERCre mice failed to be maintained in syngeneic recipients after GATA3 deletion in vivo; in this mouse model GATA3 is flanked by flox sites and Cre is expressed under the estrogen promoter (ER), which is pharmacologically induced by tamoxifen treatment. These GATA3-deficient CD8+ T cells were shown to express lower levels of surface IL7Rα, and have associated defects in IL7-driven T cell survival [22]. GATA3 has been shown to bind to the promoter/enhancer region of Il7r gene in CD8+ T cells [22, 47], and because IL7 signal is essential for the maintenance of T cells in the periphery [82], the aforementioned findings suggest that one of the mechanisms by which GATA3 regulates CD8+ T cell maintenance is by regulating IL7 signaling.

GATA3 may also control the peripheral maintenance of naïve CD4+ T cells under steady state conditions because GATA3 expression is readily detectable in mature CD4+ T cells in the periphery. Failed CD4 T cell development in GATA3^fl/fl-CD4Cre mice however precludes the study of GATA3 function in mature CD4+ T cells. Nonetheless, this hypothesis may be addressed using the GATA3^fl/fl-ERCre model, which would allow deletion of GATA3 in mature CD4+ T cells in an inducible fashion [22].

The peripheral maintenance of Treg cells has different requirements from that of other CD4+ T cell subsets, in that Treg cells rely more on IL2 than on IL7 signaling [83–85]. GATA3 is required for the peripheral maintenance of these cells under homeostasis as well as inflammatory conditions [72–74]. IL2Rα (CD25) expression on Treg cells is reduced in the absence of GATA3 [74]. Because GATA3 deletion leads to reduced Foxp3 expression in Treg cells [72–74], and Foxp3 is critical for IL2Rα expression in Treg cells [86–88], a mechanism underlying the requirement of GATA3 for maintenance of Treg cells in the periphery may involve GATA3 regulation of IL2Rα expression via Foxp3 up-regulation. GATA3 controls Foxp3 expression by binding to and promoting the activity of the conserved non-coding DNA sequence (CNS) 2 [72–74], a cis-acting element in Foxp3 locus which is associated with sustained high levels of expression of Foxp3 [89]. GATA3 does not bind to the Foxp3 promoter, CNS1 - a site required for TGF-β-promoted Foxp3 expression, or CNS3 - a site required for Foxp3 induction during thymic development within Foxp3 locus [89], although putative GATA3 binding sites are found in these regions. The DNA binding pattern on Foxp3 locus agrees with the finding that GATA3 is dispensable for the generation of Treg cells in the thymus but is required for optimal Foxp3 expression and the peripheral maintenance of Treg cells. It should be noted that the critical function of GATA3 in Treg cells may not be mediated through Foxp3 exclusively; GATA3 also regulates T cell function by controlling TCR- and cytokine-signaling that are important for Treg cell function.

Distinct aspects of T cell activation and proliferation require GATA3

As discussed above, GATA3 expression is exquisitely regulated by TCR and cytokine stimulation [19–22, 72, 90], which suggests that GATA3 regulates T cell function in response to these stimuli. Indeed, GATA3 deficient CD8+ T cells fail to grow in size (biomass increase) or to proliferate after TCR stimulation or stimulation with cytokines IL2, IL4 and IL15 [22, 91]. A similar requirement for GATA3 in CD4+ T cell proliferation is suggested by a study in which antisense DNA-mediated silencing of GATA3 led to reduced proliferation of a CD4+ T cell line [15]. In addition, GATA3-deficient Th2 cells proliferate poorly compared to wild-type cells [92]. The mechanisms by which GATA3 functions in T cell activation and proliferation are not fully understood; signaling immediately downstream of TCR ligation, such as the activation of NFκB, JNK, p38 and NFAT, is independent of GATA3 [22, 91]. Whereas the mechanisms by which GATA3 impacts cell size are not clear, the up-regulation of Myc, a critical regulator for the metabolism, cell size increase and proliferation of activated T cells [74, 93], was abolished in the absence of GATA3 [22]. It is plausible that the GATA3-Myc signaling axis is central for T cell metabolism and cell size increase, a notion that needs further investigation. This axis may also mediate GATA3’s effects on T cell proliferation because GATA3 deletion led to failed up-regulation of Myc in proliferating T cells upon TCR activation; GATA3 was shown to bind directly to the Myc locus, and ectopic expression of Myc restored the proliferation of GATA3-deficient T cells to a similar extent as wild-type T cells [22]. Myc expression did not, however, fully restore the proliferation of GATA3-deficient T cells, which implies that additional GATA3-dependent pathways are involved. Indeed, the down-regulation of KLF2 [94] and TOB1 [95], transcription factors that suppress T cell proliferation following TCR activation, was impaired in GATA3-deficient T cells [22]. In addition, GATA3 regulates the proliferation of Th2 cells by associating with Ruvbl2 [92] to suppress the expression of a cell cycle inhibitor Cdkn2c (p18) [92, 96]. Whether and how KLF2, TOB1 and other factors interact with GATA3 to control T cell proliferation are questions yet to be addressed.

Cell-type specific roles for GATA3 in T cell effector function

While GATA3 appears to be essential for the proliferation of both CD4+ and CD8+ T cells, its role in T cell effector function is cell-type specific. GATA3 deletion has minimal effects on IFN-γ production by CD8+ T cells in vitro when stimulated through TCR and with cytokines IL2, IL4 and IL15 [22]. In addition, GATA3-deficient CD8+ T cells expresses normal levels of FasL, Granzyme B and perforin when activated in vitro via TCR stimulation and with IL2. Accordingly, GATA3 deletion has minimal effects on the ability of CD8+ T cells to kill MHC-mismatched target cells in vitro [91]. Thus, GATA3 appears dispensable for effector function of CD8+ T cells. Whether GATA3 controls the effector function of CD8+ T cells in vivo is difficult to address since few GATA3-deficient CD8+ T cells could be recovered in vivo during LCMV infection, acute GvHD response [22] or anti-tumor response [91]. Due to this same reason, it is difficult to study the role of GATA3 in the generation and function of CD8+ memory T cells. Nonetheless, in GATA3^fl/fl-CD4Cre mice and in bone marrow chimeric mice containing GATA3^fl/fl-CD4Cre T cells, the populations of atypical central memory (CD62L^hiCD44^hi) and effector memory (CD62L^loCD44^hi) CD8+ T cells appeared decreased and increased respectively upon GATA3 deletion during homeostasis [22], which suggests a role for GATA3 in memory CD8+ T cells.

CD4+ T cells acquire distinct effector functions in the periphery by differentiating into different Th subsets that are defined by their cytokine production (Figure 1). These subsets include Th1, Th2, Th17 and Th9 cells (reviewed in [97]). GATA3 function in Th1 and Th2 cell differentiation has been studied extensively, as discussed above, and is relatively well understood. The involvement of GATA3 in the differentiation and function of other Th cell subsets is less clear. The role of GATA3 in Th17 cell differentiation has not been specifically addressed experimentally. GATA3-deficient Treg cells, however, produce elevated levels of IL17 under inflammatory conditions in vivo [72–74]. In addition, compared to wild-type Treg cells, GATA3-deficient Treg cells are prone to produce IL17 when activated in the presence of IL6 in vitro [74]. Therefore, GATA3 appears to suppress a Th17 differentiation program in Treg cells. It is unclear whether GATA3 does so directly through suppressing a bona fide Th17 differentiation program or indirectly through enhancing Foxp3 expression in Treg cells; indeed, both mechanisms could be at work. The observation that GATA3 binds to Il17 and Rorc loci suggests that GATA3 may directly regulate the Th17 differentiation program independent of Foxp3 expression [47, 72].

Differentiation of the recently identified Th9 cells is promoted by IL4 and TGF-β [98, 99]. In line with IL4 being essential for the generation of Th9 cells, STAT6 was found to be critical for Th9 differentiation [98, 99]. IL4-STAT6 signaling induces the transcription factor IRF4 [100], which in turn promotes Th9 differentiation [101]. Compared to Th2 cells, Th9 cells express much less GATA3 mRNA but similar amounts of GATA3 protein [99, 100]. Both mRNA and protein expression of GATA3 in Th9 cells depends on STAT6 [100]. These findings suggest a potential role for GATA3 in controlling Th9 differentiation. Indeed, GATA3 deletion results in impaired Th9 differentiation in vitro [98]. It should be noted that GATA3 expression level during Th9 differentiation is exquisitely regulated; moderate overexpression of GATA3 in developing Th9 cells leads to Th2 differentiation [100].

GATA3 in innate lymphocytes

While much effort has been focused on the investigation of GATA3 function in cells of adaptive immunity, emerging evidence has revealed critical role for GATA3 in controlling the function of innate cells. Innate lymphoid cells (ILCs) are effectors of innate immunity that control lymphoid tissue remodeling. ILCs share common features including a lymphoid morphology, lack of recombination activating gene (RAG)-dependent rearranged antigen receptors, and lack of phenotypical markers for myeloid and dendritic cells. ILCs can be classified into three groups based on cytokine production. Group 1 ILCs include NK cells, which produce IFN-γ; Group 2 ILCs produce Th2-cytokines including IL5 and IL14; Group 3 ILCs produce Th17-cytokines IL17 and IL22 (Reviewed in [102]). GATA3 is expressed by certain subsets of ILCs and recent studies have shown an impact of GATA3 in the differentiation and function of these cells.

NK cells are a prototypical Group 1 ILC population that mediates early responses against viruses and cancer cells. While expressed in NK cell precursors, GATA3 was initially shown to be largely dispensable for peripheral NK cell development in the SCID mice received GATA3-deficient embryonic HSCs [5]. The same research group later found that GATA3 is highly expressed by IL7Rα+ thymic NK cells whose development requires IL7 signaling. By analyzing SCID mice received GATA3-deficient embryonic HSCs, they found that GATA3 is required for the development of IL7Rα+ thymic NK cells [103]. Thus, the GATA3-IL7Rα signaling axis seems operative in both IL7Rα+ thymic NK cells and, as discussed above, CD8+ T cells [22]. In terms of NK cell function, GATA3-deficient NK cells display an immature phenotype based on cell-surface marker expression, fail to traffic to the liver and produce less IFN-γ, failing to mount an effective response against Listeria monocytogenes infection [5]. The molecular mechanisms underlying GATA3 control of NK cell function are undefined.

ILC2 cells reside at mucosal surfaces, and have been shown to play an important role in intestinal immunity against Helminth infection (e.g., Nippostrongylus brasiliensis) [104] and to regulate tissue repair after acute influenza virus infection [105]. Aberrant function of ILC2 may contribute to inflammation and autoimmunity (e.g., allergic airway inflammation) (reviewed in [106]). ILC2 express cell surface markers including IL7Rα, IL2Rα, IL33R, IL25R, Thy1, and Kit. Importantly, GATA3 is highly expressed in ILC2 [7, 8, 107], and what upstream signals promote GATA3 expression in ILC2 is a question of interest. Notch is expressed at high levels in ILC2, and is important for ILC2 development [108–110]. TCF1 has been shown to be critical for Notch-driven ILC2 generation, and to promote GATA3 expression in ILC2 cells [110, 111], suggesting a Notch-TCF1-GATA3 axis in ILC2. As mentioned above, TCF1 regulates GATA3 expression during early thymocyte development [58] as well as Th2 differentiation [18]. Thus, TCF1 control of GATA3 expression is a shared feature of T cell development, Th2 differentiation and ILC2 generation. Current evidence suggests that Notch-TCF1 signaling controls GATA3 during T cell development and ILC2 generation, whereas Wnt-TCF1 signaling controls GATA3 during Th2 differentiation. Nonetheless, a Wnt-independent, Notch-TCF1-dependent pathway may also contribute to GATA3 expression during Th2 differentiation since the inhibition of Wnt signaling results in reduced but not abolished GATA3 expression during Th2 differentiation [23]. Further investigation is needed to address whether the Notch-TCF1-GATA3 signaling axis controls all three processes of T cell development, CD4 differentiation and ILC2 function.

Expression of GATA3 is vital for ILC2 generation. Ectopic expression of GATA3 promoted surface marker expression and function of human ILC2 [7]. A partial silencing of GATA3 by shRNA in human ILC2 impaired their function [7]. In addition, deletion of GATA3 in developing ILC2 prevented ILC2 generation in mice [8]. It is, however, unclear how GATA3 regulates the generation of ILC2. GATA3 controls ILC2 function likely through multiple mechanisms. A study of GATA3-binding sites in early thymocyte progenitors identified ROR-α as a GATA3 target [112]. Another study demonstrated that GATA3 is essential for the induction of ROR-α in fetal liver cells in the presence of Notch signal [113]. Because ROR-α is critical for ILC2 homeostasis [109, 114], one way by which GATA3 controls early ILC2 development may be through regulating ROR-α expression. Another mechanism through which GATA3 regulates ILC2 function may be through IL7Rα. IL7Rα is highly expressed by ILC2 and is important for ILC2 generation [115]. GATA3 was found to control IL7Rα expression by binding to Il7r locus [22, 47]. How ROR-α, IL7Rα and other factors mediate GATA3 controlled ILC2 function remains to be addressed. In addition to IL7Rα+ NK and ILC2 cells, hematopoietic cell–derived intestinal ILC3 cells require GATA3 because GATA3 deletion leads to defective generation and function of these cells [116]. Therefore, GATA3 regulates the function of ILC1, ILC2 and ILC3 cells, which suggests that GATA3 controls a common progenitor for these ILCs, a question to be addressed. With the recent increased interest in understanding regulation of ILC function, more mechanistic insights into the regulation and the function of GATA3 in ILCs are expected to be revealed in the near future.

Concluding Remarks

The immune function of GATA3 is multifaceted and extends beyond controlling Th2 differentiation. GATA3 is important for T cell development, homeostasis, activation, proliferation and effector functions. In addition, GATA3 controls ILC function. With the development of more sophisticated experimental tools, e.g. lineage-specific knockout mouse models, it is expected that additional functions of GATA3 in immune regulation will be unveiled in the future. More importantly, to fully appreciate the versatile functions of GATA3, we need to understand, on a molecular level, how GATA3 integrates diverse upstream signals into specific genetic programs depending on cellular- and micro-environmental contexts.