IMMUNOLOGY

REVIEW ARTICLE

Transcriptional regulation of T helper type 2 differentiation

Gap Ryol Lee Department of Life Science, Sogang University, Seoul, Korea

doi:10.1111/imm.12216 Received 12 September 2013; revised 29 October 2013; accepted 13 November 2013. Correspondence: Gap Ryol Lee, Department of Life Science, Sogang University, 1 Shinsudong, Mapo-ku, Seoul 121-742, Korea. Email: [email protected] Senior author: Gap Ryol Lee

Summary Considerable progress has been made in recent years towards our understanding of the molecular mechanisms of transcriptional regulation of T helper type 2 (Th2) cell differentiation. Additional transcription factors and chromatin-modifying factors were identified and shown to promote Th2 cell differentiation and inhibit differentiation into other subsets. Analyses of mice lacking several cis-regulatory elements have yielded more insight into the regulatory mechanism of Th2 cytokine genes. Gene deletion studies of several chromatin modifiers confirmed their impact on CD4 T-cell differentiation. In addition, recent genome-wide analyses of transcription factor binding and chromatin status revealed unexpected roles of these factors in Th2-cell differentiation. In this review, these recent findings and their implication are summarized. Keywords: cis-regulatory element; differentiation; epigenetics; T helper type 2; transcription factor.

Introduction CD4 T cells orchestrate immune responses against various pathogens. Upon T-cell receptor (TCR) stimulation, naive CD4 T cells differentiate into many different effector cells.1,2 T helper type 1 (Th1) cells produce interferon-c (IFN-c), activate macrophages, and mediate immune responses against intracellular bacteria. Th2 cells produce interleukin-4 (IL-4), IL-5 and IL-13, and mediate immune responses against multicellular parasites. In addition, Th2 cells mediate various allergic responses including asthma and atopic dermatitis. Th17 cells produce IL-17 and mediate immune responses against extracellular bacteria and fungi. Th1 and Th17 cells are also involved in the development of autoimmune diseases. Follicular helper T cells express IL-21 and help the maturation of B cells in the germinal centre. Interleukin-9-producing Th9 cells and IL-22-producing Th22 cells were recently added to the list of effector CD4 T cells. Upon TCR stimulation, naive CD4 T cells can also differentiate into regulatory T (Treg) cells. These are known as induced Treg (iTreg) cells, while thymus-derived Treg cells are referred to as natural Treg (nTreg) cells. Treg cells maintain immune homeostasis by suppressing excessive immune responses against self-antigens, innocuous antigens and commensal bacteria in the gut. During TCR stimulation, the differentiation of CD4 T cells is primarily influenced by cytokines, although other factors, such as antigen dose and TCR affinity, also play a 498

role.1,2 Hence, IL-12 and IFN-c drives Th1, IL-4 drives Th2, IL-6 and transforming growth factor-b drive Th17, and transforming growth factor-b and IL-2 drive iTreg cell differentiation. These cytokines bind to cell surface cytokine receptors and activate Janus kinases, which in turn activate signal transducers and activators of transcription (STATs). The TCR and cytokine signals induce the so-called ‘master’ transcription factors that drive CD4 T-cell differentiation into each subset: T-bet for Th1, GATA-3 for Th2, RORct for Th17, and Foxp3 for iTreg differentiation. STATs and master regulators cooperate in many ways to induce effector cell differentiation. The classic view of ‘one master regulator in one subset’ has been challenged in recent years,2,3 because each subset shows enormous plasticity in its master regulator expression and its functional properties, especially in vivo. Hence, GATA-3 and T-bet are expressed together in in vitro differentiated primary human Th1 cells.4,5 In addition, in vitro polarized Th2 cells can express both GATA3 and T-bet when transferred into mice subsequently infected with lymphocytic choriomeningitis virus.6 Furthermore, the expression of Th1 and Th2 signature genes is highly heterogeneous and is dependent on two polarizing signals.7 Therefore, CD4 T effector cells, including Th2 cells, are flexible in their functional properties, and can adapt to a changing environment. This review will cover recent findings on transcriptional regulation of Th2 cell differentiation, focusing on transcription factors, cis-regulatory elements and epigenetics. ª 2013 John Wiley & Sons Ltd, Immunology, 141, 498–505

Th2 transcription Transcription factors GATA-3 GATA-3 is selectively expressed in Th2 cells and is necessary and sufficient to induce Th2 differentiation, as demonstrated by antisense and transgenic approaches.8 When ectopically introduced, GATA-3 drives Th2 differentiation even in Th1 cells in an IL-4- and STAT6-independent manner.9,10 Expression of a dominant-negative form of GATA-3 suppresses Th2 cytokine expression and inhibits induction of airway hyper-reactivity.11 Conditional deletion of GATA-3 causes a defect in Th2 differentiation in vitro and in vivo.12,13 Continuous expression of GATA-3 is also required for the maintenance of Th2 differentiation.14 GATA-3 exerts its effect via many different mechanisms. These include, but are not limited to, transcriptional activation of many Th2-related genes, interaction with other transcription factors, and epigenetic modification. To begin with, GATA-3 transactivates signature cytokine genes (il4, il5 and il13 genes) of Th2 cells, and other genes crucial for Th2 differentiation. GATA-3 directly binds to the il5 and the il13 promoters and transactivates these genes.15,16 In cooperation with STAT5, GATA-3 binds to hypersensitive site II (HSII)/IE enhancers of the il4 gene and induces its transactivation.17 GATA-3 also regulates Th2-related genes globally.18,19 Recent genome-wide analysis of GATA-3-binding sites showed that GATA-3 binds to a large number of genes in Th2 cells.18,19 GATA-3 binding sites in different lineage cells are lineage-specific, suggesting that the global GATA-3-binding depends on cofactors.18 Indeed, it has been shown that GATA-3 requires cooperation with STAT6 for its binding to target sites in Th2 cells.19 Next, GATA-3 inhibits the expression or function of signalling molecules and transcription factors of other subsets. Given the co-expression of master transcription factors in a cell as described above, this mode of action is particularly important for maintaining the cell identity. Hence, GATA-3 inhibits the expression of STAT4 and IL12 receptor b2, thereby inhibiting IL-12/STAT4 signalling, which is a well-known pathway for Th1 differentiation.20,21 Moreover, GATA-3 physically interacts with Tbet, and they mutually antagonize each other’s function.22 When T-bet is phosphorylated at Y525 by Itk, it is induced to interact with GATA-3 and inhibit its function.22 Recent genome-wide analysis of GATA-3 binding sites in Th2 and Th1 cells showed that GATA-3 binds to different sets of sites when it binds in combination with T-bet in Th1 cells, compared with when it binds alone in Th2 cells.5,23 This explains the role of T-bet in sequestering GATA-3 away from its target sites, to drive Th1 differentiation.5,23 GATA-3 also interacts with Runx3.24,25 Runx3 in cooperation with T-bet binds to the ifng ª 2013 John Wiley & Sons Ltd, Immunology, 141, 498–505

promoter and il4 silencer (HSIV), stimulates IFN-c production, and inhibits IL-4 production.26 Hence, GATA-3 interaction with Runx3 inhibits Th1 differentiation. As GATA-3 is the key transcription factor for Th2 differentiation, it is also a key target for negative regulation during differentiation into other subsets.22,25,27 Finally, GATA-3 induces chromatin remodelling in the Th2 cytokine locus, which contains the il4, il13 and il5 genes. The role of GATA-3 in chromatin remodelling was first suggested by experiments showing that ectopic expression of GATA-3 in Th1 cells induces DNase I hypersensitive sites at the Th2 cytokine locus.9,10 GATA-3 binds to either the co-activator or the co-repressor to activate or repress loci in a locus-dependent manner. GATA-3, in a complex with Chd (a major component of NuRD chromatin remodelling complex) and p300 histone acetyltransferase, binds to the Th2 cytokine locus to induce chromatin remodelling, whereas the GATA-3/Chd complex binds to histone deacetylase to repress the tbx21 (encoding T-bet) locus in Th2 cells.28 It has also been shown that GATA-3, in cooperation with MTA2, a component of NuRD complex, binds to and inhibits the Th2 cytokine locus in Th1 cells, whereas in Th2 cells it binds to and inhibits the ifng locus.29 In Th2 cells, GATA-3 also recruits EZH2 (H3K27me3 methyltransferase) to the ifng locus, causing its inhibition.30 These studies show that GATA-3 mediates both activation and repression of chromatin in any given locus in a cell-context-dependent manner.

STATs STAT6 is activated by IL-4 receptor signalling, and is a major signal transducer in IL-4-mediated Th2 cell differentiation.31–34 STAT6-deficient mice are defective in Th2 cell differentiation.31–34 A constitutively active form of STAT6, and a tamoxifen-induced dimerization of the STAT6-estrogen receptor fusion protein, induce GATA-3 expression in an IL-4-independent manner,35,36 suggesting that STAT6 plays a critical role in the induction of GATA-3 during Th2 cell differentiation. STAT6 induces GATA-3 expression by replacing the Polycomb group (PcG) complex with the Trithorax group (TrxG) complex at the gata3 locus.37 However, STAT6-independent Th2 differentiation was also reported in certain situations, particularly in in vivo infection models, suggesting that alternative pathways may exist.38–40 Recent genome-wide analysis of STAT6 revealed that it binds to a large number of target sites in Th2 cells.41 Furthermore, STAT6 and STAT4 play a major role in shaping the global enhancer landscapes in Th2 and Th1 cells, respectively.41 This ability of STAT6 and STAT4 is superior to the master transcription factors GATA-3 and T-bet, respectively. Moreover, a similarly limited role in shaping the enhancer landscapes has been shown for RORct and Foxp3 in Th17 and Treg cells, respec499

G. R. Lee tively.42,43 Based on these results, it was proposed that there is a regulatory hierarchy or division of work in the complex network of transcription factors. Hence, TCRinduced pioneering factors [such as nuclear factor of activated T cells (NFAT) and activator protein 1 (AP1)] and environment-sensing STAT factors cooperate to shape the global enhancer landscapes, whereas master transcription factors exploit them and focus on controlling chromatin remodelling and gene expression of a small number of signature genes for their respective subsets.43–45 STAT5 is critical for cytokine-mediated T-cell proliferation and survival.46 It is activated by a variety of cytokines, including IL-2, IL-7 and thymic stromal lymphopoietin. Enhanced STAT5 signalling leads to Th2 differentiation without up-regulating GATA-3 expression,47 suggesting that IL-2/STAT5 signalling is an alternative to the IL-4/STAT6 pathway for inducing Th2 differentiation. The IL-2-activated STAT5 binds to the HSII/IE region of the il4 locus and cooperates with GATA-3 to increase the accessibility and expression of the il4 gene.47,48 STAT5 also induces IL-4R (a or b) and amplifies IL-4 signalling,49 and STAT5 ChIP-seq and GATA-3 ChIp-seq show overlapping binding patterns of these proteins at the Th2 cytokine loci.49 Recently, STAT3 was shown to be important in Th2 cell differentiation.50 It is required by STAT6 for interaction with relevant gene loci in the developing Th2 cells.50 In STAT3-deficient Th2 cells, STAT6 cannot interact with its target loci.50 Hence, multiple STAT proteins appear to be involved in Th2 differentiation. The relative contributions of these STAT proteins and their cross-talk await further study.

Other transcription factors This review describes only some of the many transcription factors known to be important in Th2 cell differentiation. Readers are advised to refer to more comprehensive reviews.1,2 In addition, the signalling pathways downstream of the TCR have recently been reviewed by Yamane and Paul.51 In addition to GATA-3 and STAT proteins, many other transcription factors are also involved in Th2 differentiation. For example, c-Maf is selectively up-regulated in Th2 cells and stimulates IL-4 but not IL-13 and IL-5.52,53 JunB is highly expressed in Th2 cells and cooperates with c-Maf to induce il4 expression.54 Dec2 is also highly expressed in Th2 cells and promotes Th2 cytokine expression by inducing the expression of JunB, GATA-3 and CD25.55 IFN regulatory factor-4 is also involved in Th2 differentiation, and cooperates with NFATc2 to activate the il4 promoter.56 In the absence of IFN regulatory factor 4, IL-4 cannot induce Th2 differentiation and GATA3 cannot be up-regulated.56 Notch signalling regulates IL-4 expression by binding to the HSV enhancer of the 500

il4 gene.57 Notch also binds the gata3 promoter and directly induces GATA-3 expression.58,59 Gfi-1, a transcription factor induced by STAT6, promotes Th2 cell expansion by selectively enhancing the proliferation of GATA-3-high-expressing cells.60 TCF-1 is a transcription factor induced by Wnt/b-catenin signalling, together with b-catenin it promotes IL-4-independent Th2 differentiation by inducing GATA-3 expression.61 Furthermore, special AT-rich sequence-binding protein-1 (SATB1) has been shown to recruit b-catenin to the gata3 promoter.62 Collectively, these studies show that Th2 differentiation is a complex process that involves many different transcription factors and signalling pathways. A simplified diagram of the transcription factors involved in Th2 differentiation is shown in Fig. 1.

Cis-regulatory elements The Th2 cytokine locus, harbouring il4, il13 and il5 genes, contains many different types of regulatory elements (Fig. 2). Studies using DNase I hypersensitive sites (DHSs) analysis and DNA sequence comparison to screen distal regulatory regions in the Th2 cytokine locus have identified several DNase I hypersensitive sites: HSI, HSII/IE, HSIII, HSIV, HSV/CNS2 and HSVa in the il4 locus; CNS1/ HSS1-3 in the il4-il13 intergenic region; conserved GATAresponsive element (CGRE)/HS1, HS2 and HS3 in the il13 locus; and the Th2 locus control region (LCR), comprising RHS4, RHS5, RHS6 and RHS7, in the rad50 gene.63–65 The function of many of these sites was analysed at various levels, including the molecular, cellular and organism levels, using transgenic mice and mice in which the regulatory regions were deleted.63–65 These studies revealed that the Th2 cytokine locus contains several different types of regulatory sites that play critical and distinct roles in Th2 differentiation as described below.

Gene-specific enhancers Studies using transgenic and DHS-deleted mice17,66 showed that among the DHSs, HS(V+Va) and HSII/IE have gene-specific enhancer function. Deletion of HSII/IE caused a drastic reduction of IL-4 expression and formation of repressive chromatin mark at the il4 locus.17 GATA-3 and STAT5 bind to this region and increase the accessibility of the il4 locus and transcription of the il4 gene.47,48 IL4P-luc-HS(V+Va) transgenic mice showed enhancement of il4 transcription,67 and deletion of HS (V+Va) caused a reduction of IL-4 but had a marginal effect on IL-5 and IL-13 production.66 HSV binds to GATA-3, NFAT1 (NFATc2) and Notch.57,68 Of note, recent studies using HSV-deleted mice showed that this region mainly influences IL-4 expression in follicular helper T cells rather than Th2 cells.69,70 Collectively, these studies have shown that HSII/IE and HS(V+Va) ª 2013 John Wiley & Sons Ltd, Immunology, 141, 498–505

Th2 transcription IL-4R

TCR

NFAT NFκB

AP1

IL-2R

STAT6

STAT5 Gfi-1

Enhancer landscapes

T-bet GATA3 Runx3

SATB1

YY1

c-Maf Transcriptional activation of il4 and Th2-related genes

JunB Chromatin remodeling Chromosomal interaction

Dec2

Silencer HSIV is a conserved region located at the 3′ untranslated region of the il4 gene, and is formed in naive, Th1 and Th2 cells.63,64 The il4 promoter activity in transgenic mice containing IL4P-luc-HSIV was repressed,67 and HSIVdefective mice showed aberrant expression of IFN-c in Th2 cells,26 suggesting that HSIV functions as a silencer. The transcription factor Runx3 binds at the HSIV region and inhibits IL-4 expression in Th1 cells,72 and GATA-3 interacts with Runx3 to prevents its function.24

rad50

il5

CNS1 IL4P HSII

CGRE IL13P

RHS4 RHS5 RHS6 RHS7

Th2LCR IL5P

predominantly affect IL-4 expression but not IL-13 and IL-5 expression. Likewise, deletion of CGRE (1
6 kb upstream of the il13 gene) caused a reduction of IL-13 but not IL-4 and IL-5 expression.17 GATA-3, in cooperation with c-Myb, binds to CGRE and recruits MLL (H3K4 methyltransferase, catalytic subunit of TrxG) for histone modification of the il13 locus.71 Hence it seems that HSII, HS(V+Va) and CGRE enhancers act locally to influence only nearby genes.

il13

Gene-specific enhancer

GATA3

Coordinate regulator

STAT6

Silencer

YY1

Promoter

STAT5

Gene Chromosomal interaction

c-Maf

HSIV HS(V+Va)

Figure 1. A simplified diagram of T helper type 2 (Th2) differentiation. The roles of T-cell receptor- (TCR), interleukin-4 receptor- (IL-4R) and IL-2R-induced transcriptions factors are shown with special emphasis on GATA3 and signal transducer and activator of transcription 6 (STAT6). The thickness of the arrows indicates the relative importance of the transcription factors.

il4

Coordinate regulators

Figure 2. Cis-acting regulatory elements in the T helper type 2 (Th2) cytokine locus. Regulatory elements (coloured rectangles) are categorized based on their function, and colour-coded. Binding of transcription factors (coloured circles) to the regulatory elements is shown. Long-range chromosomal interactions between Th2 locus control region (LCR) and the promoters of Th2 cytokine genes are shown by blue lines.

Deletion of CNS1, a highly conserved region located in the intergenic region between the il4 and il13 genes, causes a reduction of all three Th2 cytokines (IL-4, IL-5 and IL-13), suggesting that CNS1 coordinately regulates all of them.73,74 CNS1 has been shown to bind to GATA3.75 The Th2 LCR encompassing RHS5, RHS6 and RHS7 is peculiar in that it is scattered among the introns of the 3′ region of the rad50 gene.76,77 Deletion of the entire Th2 LCR caused almost complete reduction of all three Th2 cytokines, suggesting that Th2 LCR is essential for

the coordinate regulation of Th2 cytokine genes.78 The function of individual RHSs was also analysed using deletional approaches.79,80 Deletion of RHS7 caused a modest reduction of IL-4 and IL-13 but not IL-5.79 The transcription factor YY1 binds to RHS7 and recruits GATA-3.81 It also mediates chromatin remodelling and intrachromosomal interactions at the Th2 cytokine locus.81 Deletion of RHS(4 + 5) had no effect on the expression of Th2 cytokine genes.80 Deletion of RHS6 caused the biggest

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G. R. Lee reduction of all three Th2 cytokines among individual RHSs.80 These results suggest that individual RHSs in the LCR have both redundant and distinct properties in the regulation of the Th2 locus. Further studies are needed to determine how the individual RHSs regulate the Th2 locus, and further elucidate the mechanisms of transcription factor binding to these RHSs. Unlike the gene-specific local enhancers described above, CNS1 and Th2 LCR affect the expression of all three cytokine genes. Hence, the molecular mechanisms for these coordinate regulators may differ from those for gene-specific enhancers. A key feature of the coordinate regulation is that it controls the expression of multiple genes from long distance. One clue as to a possible molecular mechanism for the coordinate regulation of all three cytokine genes came from a chromosome conformation capture assay (3C) showing that Th2 LCR is in close spatial proximity to the promoters of the three Th2 cytokine genes by forming loops.82 This strongly supports the idea that distal regulatory elements can regulate multiple genes by direct contact with the promoters. RHS7 is necessary for intrachromosomal interactions, because its deletion disrupts the binding of Th2 LCR to the promoters.79 These long-range chromosomal interactions may be mediated by transcription factors such as GATA-3, STAT6, YY1 and SATB1 binding at the Th2 LCR.81–84 Likewise, in Th1 cells, CCCTC-binding factor (CTCF), cohesion, and T-bet play critical roles in the formation of intrachromosomal interactions at the ifng locus.85,86 Hence, many different transcription factors appear to be involved in chromosomal interactions at different loci. How these interactions are mediated and regulated at the molecular level remains to be determined.

Epigenetic regulation This review covers the recent findings on the role of epigenetic modifiers in Th2 cell differentiation. On the topic of the epigenetic regulation of the Th2 cytokine locus, readers are advised to refer to a comprehensive review by Wilson et al.65

DNA methylation The first indication that epigenetic alteration affects Th1/ Th2 differentiation came from studies using inhibitors of epigenetic mechanisms. Inhibitors of DNA methylation (5-Aza-C) and histone deacetylation (Trichostatin A) enhanced the production of Th1 and Th2 cytokines.87 The role of DNA methylation was further examined using a gene-targeting approach. Deletion of DNMT1 (maintenance DNA methyltransferase 1) caused increased Th2 inflammation in an allergic airway inflammation model, and increased IL-4 production in an in vitro Th2 differentiation culture.88 Two studies on the effects of DNMT3a 502

(de novo DNA methyltransferase) deletion were reported. One study showed that DNMT3a deficiency resulted in a stronger propensity to express both IL-4 and IFN-c due to selective hypomethylation of the Th2 cytokine and the ifng loci,89 whereas the other study showed a similar result but a stronger effect on IL-13 expression.90 In addition, mice deficient in MBD2 (methyl-CpG-binding domain protein 2), which binds to methylated DNA and helps partition methylated genes into silent chromatin, displayed an increase in IFN-c and Th2 cytokine expression in Th2 cells.91 These results support the suggestion that DNA methylation has a critical function in the regulation of Th2 cytokine gene expression.

Histone methylation The role of histone modification was also examined using gene-targeting approaches. H3K4me3 is a histone mark of active chromatin, catalysed by TrxG proteins. Mice heterozygous for MLL (H3K4 methyltransferase, a component of TrxG) have defective maintenance of Th2 cytokines and GATA-3 expression in memory Th2 cells.92 In addition, deletion of menin, another component of TrxG, caused defective GATA-3 maintenance.37 Deletion of mel-18, a Polycomb repressor complex 1 (PRC1) protein that binds to H3K27me3, was found to impair GATA-3 induction and Th2 cell differentiation.93 Deletion of Bmi1, another component of PRC1, caused impaired survival of memory Th2 cells.94 Hence, it seems that both TrxG and PRC have a similar effect on Th2 cell differentiation, although they may exert their function at different stages of the process, namely effector versus memory stage. However, the detailed molecular mechanisms of TrxG and PcG complex involvement in Th2 cell differentiation remain to be elucidated, given the opposite role of these complexes in Drosophila development.95 H3K9 methylation marks constitutive heterochromatin in pericentric and telomeric regions. Th2 cells deficient in SUV39H1 histone methyltransferase, which participates in H3K9me3 methylation, and Th2 cells deficient in HP1a, which binds to H3K9me3, no longer silence the Th1 locus, and express Th1 genes when they are re-cultured under Th1-polarizing conditions.96 In addition, Th2 cells deficient in G9a, an H3K9me2 methyltransferase, showed defective Th2 cytokine expression and increased IL-17a expression.97 Collectively, these results emphasize the important function of histone methylation in Th2 cell differentiation. Recently, the Chip-seq (chromatin immunoprecipitation followed by deep sequencing) assay revolutionized the method of retrieving genome-wide epigenetic information from cells. The Chip-seq assay with several subsets of T helper cells showed that the signature cytokine loci were decorated with active histone marks in each subset, whereas cytokine loci of other subsets were ª 2013 John Wiley & Sons Ltd, Immunology, 141, 498–505

Th2 transcription decorated with repressive histone marks.98 However, master transcription factor loci do not follow this pattern. The transcription factor loci showed both active and repressive marks in the same loci, called bivalent histone marks.98 This puzzling result may provide a mechanistic explanation for the co-expression of these transcription factors in a cell and the plasticity of CD4 T cells.98

ATP-dependent chromatin remodelling factor Studies on the role of ATP-dependent chromatin remodelling complexes in Th2 cells have found that BRG1, a catalytic subunit of SWI/SNF remodelling complexes, is recruited to the Th2 cytokine and the gata3 loci.99,100 Moreover, knockdown of BRG1 caused defective Th2 cytokine expression and chromatin remodelling of the locus.99 Overall, these data strongly support the idea that epigenetic mechanisms play a critical role in Th2 cell differentiation.

Perspective Since the introduction of the concept of the Th1/Th2 dichotomy in mid-1980s, considerable progress has been made in the field. Many different subsets of CD4 effector cells have been found, and their functional properties have been characterized. These studies have extended our understanding of the immune system as well as fundamental properties of cell differentiation. Future work needs to focus on elucidating the complex interactions between transcription factors and chromatin remodelling factors with cis-regulatory sequences to drive CD4 T-cell differentiation. Transcriptional regulation of Th2 differentiation is an excellent model system for investigating these interactions because of the many known players in the system and tractable differentiation methods. The outcome of such studies will not only elucidate a paradigm of gene regulation in cell differentiation, but also facilitate the discovery of novel strategies for the treatment of immune diseases.

Acknowledgements This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (2013-035242) and by a Sogang University Research Grant of 2013.

Disclosures The author has no potential conflicts of interest.

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