Doi 10.1146 - Annurev-Immunol-051116-052350

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Review in Advance first posted online

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18 November 2016
D V A
Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

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Th2 Cells in Health and

Disease
Toshinori Nakayama,1,2 Kiyoshi Hirahara,1
Atsushi Onodera,1,3 Yusuke Endo,1
Hiroyuki Hosokawa,1 Kenta Shinoda,1
Damon J. Tumes1,4 and Yoshitaka Okamoto5
1
Department of Immunology, Graduate School of Medicine, Chiba University,

Chiba 260-8670, Japan; email: tnakayama@faculty.chiba-u.jp, hiraharak@chiba-u.jp,
a-onodera@faculty.chiba-u.jp, san3tamariayuyu@chiba-u.jp, hiroyuki@caltech.edu,
kenta.shinoda@nih.gov, Damon.Tumes@sahmri.com, yokamoto@faculty.chiba-u.jp
AMED-CREST, AMED, Chiba 260-8670, Japan
Institute for Global Prominent Research, Chiba University, Chiba 260-8670, Japan
South Australian Health and Medical Research Institute, North Terrace, Adelaide SA 5000,
Australia
Department of Otorhinolaryngology, Graduate School of Medicine, Chiba University,

Chiba 260-8670, Japan
Annu. Rev. Immunol. 2017. 35:5384
Keywords
The Annual Review of Immunology is online at

immunol.annualreviews.org
Th2, allergy, epigenetics, Polycomb, Trithorax, Gata3, pathogenic Th2

cell
This articles doi:

10.1146/annurev-immunol-051116-052350
c 2017 by Annual Reviews.
Copyright
All rights reserved
Abstract
Helper T (Th) cell subsets direct immune responses by producing signature
cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important
in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular
analysis of Th2 cell differentiation and maintenance of function has led to
recent discoveries that have refined our understanding of Th2 cell biology.
Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell
identity. In the context of allergic diseases, memory-type pathogenic Th2
cells have been identified in both mice and humans. To better understand
these disease-driving cell populations, we have developed a model called the
pathogenic Th population disease induction model. The concept of defined
subsets of pathogenic Th cells may spur new, effective strategies for treating
intractable chronic inflammatory disorders.
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INTRODUCTION

The immune system has been described as a supersystem in which multiple processes, including
cell activation, division, differentiation, apoptotic cell death, and anergy, are all engendered from a
single initial stimulus, such as peptide recognition by a T cell (1). Upon antigen peptide recognition
via the T cell receptor (TCR), naive CD4 T cells undergo clonal expansion and differentiation
into functionally distinct effector helper T (Th) cell subsets, including Th1, Th2, and Th17 cells.
These Th cell subsets direct immune responses to promote the control of pathogens via production
of signature cytokines. Th1 cells express the transcription factor T-bet (T-box 21; encoded by the
Tbx21 gene) and produce IFN-, whereas Th2 cells express GATA3 (GATA-binding protein 3;
encoded by the Gata3 gene) and secrete IL-4, IL-5, and IL-13. Th17 cells express ROR-t (RARrelated orphan receptor , encoded by the Rorc gene) and produce IL-17. These Th cell subsets
are involved in inflammatory disorders; Th1 and Th17 cells are important in autoinflammation,
whereas Th2 cells can drive allergic inflammation. Over the last several decades, a large number
of elegant molecular, cellular, and in vivo studies in both mouse and human systems have been
performed in this area, as described in a series of excellent published reviews (212). In this review,
we focus on recent advances in our understanding of Th2 cell biology and the pathogenesis of
Th2 cellmediated allergic inflammatory diseases. In the first section, we describe the molecular
program for Th2 cell differentiation, focusing on its control by cytokines and TCR signaling,
and also the recently well studied mechanistic target of rapamycin (mTOR) signaling pathway. In
the second section, we focus on recent advances in understanding epigenetic regulation of both
the establishment and the maintenance of Th2 cell identity. In the third section, we summarize the
recently defined composition and functional characteristics of GATA3 transcriptional complexes
that coordinate the Th2 genetic program. In the fourth section, we describe and discuss memorytype pathogenic Th2 (Tpath2) cells, with a focus on their induction by IL-33 and their maintenance
in the inflamed lung. Finally, in the fifth section we summarize recent studies analyzing Th2
cell function in allergic inflammatory diseases, including asthma, chronic rhinosinusitis, atopic
dermatitis, and eosinophilic gastrointestinal inflammatory disease. In addition, we present our
recently proposed pathogenic Th population disease induction model. We hope this review will
be useful for immunologists, both in basic sciences and in clinical settings.
MOLECULAR PROGRAM FOR TH2 CELL DIFFERENTIATION

Upon peptide recognition by the TCR, peripheral naive CD4 T cells undergo functional differentiation into distinct Th cell subsets within lymphoid tissue. TCR-mediated signaling, as well as
costimulatory receptor and cytokine receptormediated signaling, are crucial for naive CD4 T
cells to differentiate into distinct Th cell subsets (7, 13, 14). Differentiated effector Th cells migrate
to inflammatory sites in peripheral tissues and produce large amounts of effector cytokines upon
reencounter with their cognate antigens (15). Therefore, the recognition of cognate antigens by
TCRs is an essential initial event that induces a series of cellular and molecular events that are
required for efficient antigen-specific immune responses.
Cytokine-Induced Program
A pioneering series of elegant studies established that IL-4 is a key cytokine that induces Th2 cell
differentiation (28). Among the seven members of signal transducers and activators of transcription (STATs), STAT6 responds to IL-4 stimulation (16). Phosphorylation of STAT6 results in
induction of a set of genes including Gata3, encoding a key transcription factor for Th2 cell differentiation (8, 17, 18). IL-4-dependent STAT6 activationand other signaling pathways, including
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the TCR-mediated extracellular signalregulated kinase (ERK)/mitogen-activated protein kinase

(MAPK) pathwayare required to induce high-level expression of GATA3 protein (13). Moreover, STAT6 activates Th2 cellspecific enhancers and suppresses those promoting alternative
Th cell fates (19). In addition to IL-4, other cytokines, including thymic stromal lymphopoietin
(TSLP), IL-25, and IL-33, are reported to play crucial roles in the induction of Th2 responses
under various in vitro and in vivo conditions (2022). STAT5 activation induced by IL-2 is also
required for Th2 cell differentiation (23). Recently, we found that IL-33 induces IL-5 production
through the p38 MAPK pathway in memory Th2 cells (24).
GATA3 expression is detected in early T cell precursors and plays important roles throughout
T cell development (25). GATA3 also functions in several organs and cell types, and loss-offunction mutations of Gata3 are reported to be associated with the autosomal dominant syndrome
HDR (hypoparathyroidism, deafness, renal disease) (26). GATA3 is therefore thought to have
crucial dose-dependent and cell contextspecific roles in different developmental stages (2527).
The introduction of GATA3 into differentiating Th1 cells induces expression of Th2 cytokines
and repression of IFN- production, accompanied by open chromatin at the Th2 cytokine loci
and closed chromatin at the Ifng locus (8, 28). Conversely, deletion of the Gata3 gene in CD4
T cells prevents their differentiation into the Th2 lineage, causing cells to differentiate toward a
Th1 phenotype in the absence of polarizing cytokines (29, 30). In addition, Gata3-deficient Th2
cells divide less and incorporate less BrdU, indicating GATA3 involvement in the regulation of
Th2 cell proliferation (29). Moreover, GATA3 binding sites have been identified in the Il5 gene
promoter region, and GATA3 directly transactivates the Il5 gene (31). Thus, GATA3 acts as a
master transcription factor for Th2 cell differentiation by inducing chromatin remodeling of the
Th2 cytokine loci, inhibition of Th1 cell differentiation, facilitation of Th2 cell division, and
transactivation of the Il5 gene.
TCR-Mediated Signaling
Antigen dosage and TCR-mediated signal strength have been implicated in the control of Th1 and
Th2 cell fate during differentiation. Bottomly and coworkers demonstrated that low-dose antigen
induces early IL-4 production from naive CD4 T cells and thus promotes Th2 cell generation
(32). In contrast, OGarra and colleagues reported that Th2 cell development was observed at
both very high and very low doses of antigenic peptides, whereas midrange peptide doses directed
the development of Th1 cells (33). Some reports support the former hypothesis that low-level
antigenic stimulation induces Th2 rather than Th1 cell differentiation (34, 35), whereas others
support high-level TCR stimulation favoring Th2 cell differentiation and being required for Th2
cell function (3638). These two sets of conclusions appear contradictory but could be due to
experimental differences in the range of TCR signal strength. In 2010 we proposed a biphasic
Th1/Th2 cell differentiation model in which both low and high amounts of antigen induce Th2
cells, whereas intermediate amounts induce Th1 cells (13), an idea similar to that more recently
proposed by Tubo & Jenkins (39). These investigators used a Listeria monocytogenes infection model
to provide new insight into the relationship between antigen dose and follicular helper T (Tfh)
cell differentiation. They demonstrated that high-dose antigen inhibits Th1 cell differentiation
and facilitates germinal center Tfh (GC-Tfh) cell development (40). They also proposed a model
based on the assumption that TCR signals are not only weak or strong, but can also vary in
qualitative ways (39). Moreover, TCR affinity and dwell time have been reported to affect Tfh cell
differentiation (41).
Preferential roles of the TCR-mediated signal transduction pathways in Th cell differentiation
have also been clarified. Using chemical inhibition or dominant-negative mutants, we and others
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reported that strong activation of the ERK/MAPK cascade is critical for Th2 cell differentiation
and the induction of Th2-driven inflammation in a murine model of asthma (36, 42, 43). The
ERK/MAPK cascade induces Growth factor independent 1 (Gfi-1) and regulates the stability of
GATA3 protein through the ubiquitin/proteasome-dependent pathway (44, 45). Interestingly,
Th1 cell differentiation and Th1 cytokine production are reported to be highly dependent on
the c-Jun N-terminal kinase ( JNK)/MAPK and p38/MAPK cascades, respectively (46, 47). The
Activator protein 1 (AP-1) transcription factor complex driven by the MAPK cascade is also known
to control Th1/Th2 cell differentiation. Jun:Fos heterodimers induce Th1 cell differentiation,
whereas Jun:Jun homodimers induce Th2 cell differentiation (35). Using similar approaches, we
found that Th2 cell differentiation is more dependent on the calcium/calcineurin pathway than
Th1 cell generation (48). Increased IL-4 production is observed in mice deficient in Nuclear
factor of activated T-cells 1 (NFAT1; encoded by the Nfatc2 gene), whereas lymphocytes that
lack NFAT2 (encoded by the Nfatc1 gene) produce less IL-4, indicating that NFAT1 and NFAT2
induce preferential differentiation of Th1 and Th2 cells, respectively (49). The protein kinase C
(PKC)/nuclear factorB (NF-B) pathway is also required for Th2 cell differentiation. In Prkcq
(encoding PKC)-deficient mice, Th2 cellmediated immune responses are markedly impaired
and the Th1 immune response is unaffected (50). However, NF-B has also been shown to control
increased expression of Ifng (51), and PKC has also been reported to have a role in Th17 cell
dependent immune responses (52). The PKC/NF-B pathway therefore appears to be critical not
only in Th2 cell differentiation but also in aspects of Th1 and Th17 cell function. The cross talk
between TCR and cytokine signaling pathways is also involved in determination of naive CD4
T cell fate. Recent reports on Nedd4 family ubiquitin ligases and their activators, Ndfip1 and
Ndfip2, provide an additional layer of regulation of CD4 T cell differentiation (53, 54).

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mTOR Signaling Pathways

In addition to TCR-mediated or cytokine-induced signaling, a recently well analyzed signaling
pathway is the evolutionarily conserved kinase named mTOR, which has a pivotal role in the regulation of cell proliferation, growth, and cellular metabolism (55). mTOR is a critical regulator
that integrates multiple inputs from environmental signals such as nutrients, growth factors, and
cytokines (56, 57). In mammalian systems mTOR exists as two different complexes. The first is
mTOR complex 1 (mTORC1), which contains a scaffolding subunit called regulatory associated
protein of mTOR (RAPTOR), and the second is mTORC2, which has a different scaffolding
protein called rapamycin-insensitive companion of mTOR (RICTOR) (57). The importance of
mTOR in the differentiation of Th cell subsets has recently become clear (56). For example,
mTOR-deficient CD4 T cells, in which both mTORC1 and mTORC2 are disrupted, fail to differentiate into Th1, Th2, or Th17 cells and instead preferentially differentiate into regulatory T
cells (Tregs) expressing the transcription factor Foxp3 (Forkhead box P3; encoded by the Foxp3
gene). Selective inhibition of mTORC1 by deleting the gene for the small GTPase Ras homolog
enriched in brain (Rheb) indicated that the mTORC1 pathway is required for differentiation of
Th1 and Th17 cells but not Th2 cells. However, T cellspecific genetic deletion of Raptor, the
scaffolding subunit of mTORC1, abrogates both T cell priming and Th2 cell differentiation (58).
This indicates potential functional redundancy of GTPase activity or the presence of alternative
pathways for mTORC1 activation in activated T cells. Mechanistically, RAPTOR-mTORC1
has been shown to orchestrate metabolic reprogramming during Th2 cell differentiation (58),
suggesting that the molecular processes required during Th cell differentiation are metabolically
demanding. In contrast to the loss of mTORC1 signaling, deficiency of mTORC2 signaling
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induced by genetic deletion of Rictor suppresses Th2 cell differentiation in vitro and in vivo but
does not affect Th17 cell differentiation (56). mTORC2 contributes to Th2 cell differentiation by
inhibiting the negative feedback inhibitor suppressor of cytokine signaling 5 (SOCS5), which functions as a suppressor of STAT6 (56). Another group suggested that decreased PKC activity and
NF-B-mediated Il4 transcription are responsible for decreased Th2 cell differentiation in Rictordeficient cells because complementation with active PKC restored Th2 differentiation (59). In
addition, another downstream target of mTORC2 called serum and glucocorticoid-regulated kinase 1 (SGK1) was found to be involved in both enhancing commitment to the Th2 cell lineage
and inhibiting Th1 differentiation (60). Thus, both mTORC1-induced metabolic reprogramming
and mTORC2-induced SGK1 pathways function as essential regulators of Th2 cell differentiation. Further study is required to clarify the role of mTORC-induced metabolic reprogramming
during Th2 cell differentiation. In addition, it will be important to determine whether SOCS5,
PKC, NF-B, and SGK1 act independently or cooperatively during Th2 cell differentiation.
ESTABLISHMENT OF TH2 CELL IDENTITY

Acquisition and Maintenance of Cell Identity
To determine how the Th2 cell phenotype is established and maintained, the mechanisms underlying cellular differentiation must first be understood. The control of differentiation from precursor
cells to a particular cell type requires timely regulation of gene expression, which is triggered
by lineage-specifying transcription factors (61). These processes involve epigenetic regulation,
a system of posttranslational histone modifications and DNA methylation. The methylation of
histones H3K27 and H3K9 and DNA (5-methylcytosine) is considered important for gene repression, whereas methylation of H3K4, K36, and K79 or acetylation of H3K9, K14, and K27 is
associated with gene activation (62).
Epigenetic Regulators: Polycomb and Trithorax Proteins

Numerous genes involved in epigenetic regulation have been identified. Many of them encode
members of histone-modifying enzymatic complexes. In particular, members of the Polycomb
group (PcG) and Trithorax group (TrxG) complexes, which possess histone methyltransferase
activity, have been identified as key epigenetic regulators (6366). PcG and TrxG proteins were
originally identified in Drosophila; however, they also play major roles in controlling mammalian
gene expression in various tissues. It has long been thought that PcG and TrxG proteins antagonize
each other to switch off or switch on target gene expression. PcG proteins mediate gene silencing
via the repressive histone mark H3K27me3, whereas TrxG proteins mediate gene activation via
the permissive histone mark H3K4me3; both complexes are often found to regulate the same genes
at different stages of development (67). In addition, emerging evidence indicates that PcG and
TxrG proteins participate in more complex regulatory mechanisms in mammalian tissues (67, 68).
PcG complexes are classified into two canonical types, Polycomb repressive complex 1 (PRC1)
and PRC2, both of which are involved in transcriptional repression (Figure 1a). Enhancer of
zeste (EZH), the enzymatically active subunit of PRC2, methylates H3K27. The PRC1 complex
recognizes trimethylated H3K27, resulting in its colocalization with PRC2. In addition, the Ring
finger protein 1 (RING1) subunit of PRC1 has a ubiquitin ligase activity for histone H2AK119
(69). In contrast, mixed lineage leukemia (MLL) family proteins, major subunits of the TrxG
complex (Figure 1b), have H3K4 methyltransferase activity that induces a chromatin structure
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Polycomb (PcG) proteins
PRC1
PCGF
KMT2A/B
PHC
ASH2
HCF1
H2A-K119 ubiquitination
RBBP4/7
KMT2C/D
KMT2F/G
CBX
RING1A/B
PRC2
Trithorax (TrxG) proteins
MLL1/2
Menin
SUZ12
HCF1?
ASH2
UTX
DPY30
MLL3/4
PA1
RBBP5
DPY30
RBBP5
WDR5
PTIP
NCOA6
EED
EZH1/2
H3-K4 methylation
H3-K27 methylation
CXXC1
ASH2 DPY30
HCF1
SET1A/B
WDR5
WDR82
H3-K4 methylation
RBBP5
WDR5
H3-K4 methylation

PcG
Embryonic
stem cell
TrxG
Gene
PcG
Gene
TrxG
TSS
Poised transcription
PcG
Gene A
Gene
Gene C
Gene B
PcG
TrxG
Gene A
T cell
Gene C
Gene B
TrxG
TSS
TSS
TSS
Gene A
Transcription
Medium
High
Low
Distal
promoter
d
Naive
CD4 T cell
PcG
PcG
Proximal
promoter
PcG
Gene C
PcG
Gata3 gene locus
TrxG
Gata3 mRNA
+
TrxG
IL-4 /TCR
(STAT6 dependent, Menin/TrxG independent)
Effector
Th2 cell
Activation
Gata3 mRNA
+++
H3-K9 acetylation
HAT
HAT
PcG
TrxG
TrxG
TrxG
STAT6
TrxG
STAT6
(STAT6 independent, Menin/TrxG dependent)
Differentiated
(memory) Th2 cell
PcG
H3-K9 acetylation
Maintenance
Gata3 mRNA
+++
H3-K4 methylation
M TrxG M TrxG M TrxG M TrxG M TrxG M TrxG M TrxG M TrxG M TrxG M TrxG
M Menin
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HAT
Histone acetyltransferase
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permissive for transcription. In mammals, six H3K4 methylases have been identified (70). The
H3K4 methylase complex containing MLL1 or MLL2 includes a unique subunit named Menin
(encoded by the Men1 gene in mice). The MLL3- and MLL4-containing complex associates with
the H3K27 demethylase UTX (encoded by the Kdm6a gene in mice). These MLL-associated
complexes appear to mediate H3K4 trimethylation in a gene-specific manner. The SET1A- or
SET1B-containing complex includes the unique WD repeat-containing 82 (WDR82). TrxG proteins function to turn on target genes and/or keep them active, indicating that these proteins are
not always associated with simple gene activation (70).

Spatial Interplay Between PcG and TrxG Proteins

Bivalency, the presence of both H3K27me3 and H3K4me3 at the same gene at the same time, is
the subject of intense research in the field of embryonic stem cells (71). It is postulated that bivalent
genes are poised for expression; however, the functional relevance of PcG and TrxG cooccupancy
remains unclear. Spatial interplay between PcG and TrxG proteins may have an important part
in regulating gene expression at cooccupied genes (Figure 1c) (72). Our genome-wide analysis
using embryonic stem cells and CD4 T cells revealed that binding of Ezh2 and binding of Menin
(central members of the PcG and TrxG complexes, respectively) are reciprocally correlated (72).
In addition, in CD4 T cells, genes with lower expression are frequently associated with PcG and
TrxG binding downstream and upstream of transcription start sites (TSSs), respectively. This
contrasts with highly expressed genes, where PcG and TrxG binding occurs in the reciprocal
pattern (Figure 1c). Interestingly, among the highly expressed genes are many that are important
for T cell development and function (e.g., Gata3, Nfatc1, Fli1, and Gfi1) (72). Thus, spatial patterns
of PcG and TrxG binding may represent a novel mechanism regulating genes important for the
specification and identity of cells.
Figure 1
Polycomb (PcG) and Trithorax (TrxG) complexes in mammals. (a) Polycomb repressive complex 1 (PRC1) and PRC2 are two
canonical types of PcG complex. PRC1 consists of four core subunits: RING1A/B, CBX, PCGF, and PHC (218). RING1A/B
ubiquitinate H2AK119. PCGF2 and PCGF4 are also known as Mel-18 and Bmi1, respectively. PRC2 consists of four core subunits:
EZH1/2, EED, SUZ12, and RBBP4/7 (66, 218). PRC2 methylates histone H3K27 through the SET domain of EZH1/2.
(b) Mammalian cells bear six H3K4 methylases: MLL1 (KMT2A), MLL2 (KMT2B), MLL3 (KMT2C), MLL4 (KMT2D), SET1A
(KMT2F), and SET1B (KMT2G) (66). All of the mammalian complexes share ASH2L, RBBP5, DPY30, HCF1, and WDR5 (green)
(66, 70). MLL1/2 complexes uniquely associate with Menin, MLL3/4 complexes associate with PTIP, PA1, UTX, and NCOA6, and
SET1A/B complexes associate with WDR82 and CXXC1 (blue). (c) Spatial interplay between PcG and TrxG proteins. In embryonic
stem cells, PcG and TrxG proteins are frequently bound in a similar position relative to the transcription start site (TSS). Most of these
genes are poised for transcription. In CD4 T cells, PcG and TrxG proteins bind at discrete positions on either side of the TSS of some
genes. PcG binding downstream and TrxG binding upstream of the TSS are frequently found at genes with lower expression, and PcG
binding upstream and TrxG binding downstream is found at genes with higher transcription levels. ChIP-seq profiles based on datasets
c American Society for Microbiology; https://doi.org/
in GSE73825 are modified from Reference 72 with permission (Copyright
10.1128/MCB.00677-15). (d) Two distinct mechanisms underlying the acquisition and maintenance of Th2 cell identity. In naive
CD4 T cells, the PcG complex binds to the upstream region of the proximal promoter of the Gata3 gene, and the expression of Gata3
mRNA is moderate. After stimulation through T cell receptor in the presence of IL-4, activated STAT6 molecules associate with the
histone acetyltransferase (HAT) complex and bind to the Gata3 gene locus. HAT-dependent histone acetylation spreads to the
upstream region, resulting in dissociation of the PcG complex. A high-level Gata3 mRNA expression is achieved in an
IL-4/STAT6-dependent but Menin/TrxG-independent manner. Once Th2 cells are differentiated, the Menin/TrxG complex binds to
the whole Gata3 gene region, including the upstream region of the proximal promoter. A broad range of H3K9ac and H3K4me3 is
observed. The Menin/TrxG complex bound to the Gata3 locus maintains high-level expression of Gata3 in the absence of IL-4.
Transcription of Gata3 is regulated in an IL-4/STAT6-independent but Menin/TrxG-dependent manner. Modified from Reference 73
c Onodera et al., 2010; originally published in J. Exp. Med.; https://doi.org/10.1084/jem.20100760).
with permission (Copyright
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Epigenetic Regulation of Th2 Cell Identity

Recent transcriptional and epigenetic analyses highlight the importance of understanding similarities and differences between the acquisition and maintenance of Th2 cell identity.

Acquisition of Th2 cell identity. Epigenetic changes at the Gata3 gene locus are essential for
the acquisition and maintenance of Th2 cell identity (8, 73). During Th2 cell differentiation,
binding patterns of PcG and TrxG proteins are dynamically changed at the Gata3 gene locus,
and these epigenetic changes result in GATA3 protein upregulation, which consequently induces
chromatin remodeling at the Th2 cytokine gene loci, including Il4, Il5, and Il13 (8, 73). The Gata3
gene contains distal and proximal promoters. Both basal transcription in naive CD4 T cells and
induced transcription in differentiated Th2 cells are dependent on the proximal promoter (73, 74).
In naive CD4 T cells, PcG proteins bind upstream and TrxG proteins bind downstream of
the Gata3 proximal promoter (Figure 1d) (73). During Th2 cell differentiation, PcG proteins
dissociate from the region upstream of the Gata3 proximal promoter and the binding of TrxG
proteins expands into this region in wild-type but not STAT6-deficient cells. Rapid alterations
in the binding patterns of PcG and TrxG proteins are observed in the region between the Gata3
distal and proximal promoters. We identified two functional STAT6 binding sites within the Gata3
gene locus and found that displacement of PcG by TrxG was induced in a STAT6-dependent
manner (73). Interestingly, these two STAT6 binding sites are located at intronic regions and were
confirmed to be functional by chromatin immunoprecipitation followed by massively parallel
sequencing (ChIP-seq) analysis (75). These results indicate that STAT6 directly binds to the
Gata3 gene locus and induces PcG/TrxG displacement, although the precise mechanism remains
unknown. Our ChIP-seq analysis also identified a GATA3 binding peak close to one of the STAT6
binding sites at the Gata3 gene locus (73, 76). This GATA3 binding site could be a cis-regulatory
element responsible for GATA3-dependent autoactivation of the Gata3 gene (77). T cellspecific
deletion of Menin, a component of the TrxG complex, does not affect Th2 cell differentiation,
suggesting that induction of high-level expression of Gata3 (i.e., acquisition of Th2 cell identity)
is dependent on STAT6 and independent of the Menin/TrxG complex (Figure 1d) (73). A study
of human Th2 cells indicated that STAT6 binding is not easily detected at the GATA3 gene,
although STAT6 knockdown is effective for reducing GATA3 expression (78).
In contrast to TrxG proteins, PcG proteins are proposed to act as a braking system to maintain
appropriate levels of Gata3 expression in CD4 T cells. T cellspecific deletion of Ezh2 results
in hypersensitivity to IL-4-induced Gata3 upregulation and hyperproduction of Th2 cytokines
(79). ChIP-seq analysis of Ezh2 revealed that Ezh2 binding levels are high at the Gata3 gene
locus, whereas they are very low at the Th2 cytokine gene loci, indicating that Ezh2 controls Th2
cytokine expression via direct binding to the Gata3 gene locus. Direct regulation of H3K27me3 by
Ezh2 at the Il4 and Il13 genes has also been proposed to transcriptionally silence these genes in Th1
cells (80); conversely, SUV39H1-dependent H3K9me3 has been found to maintain the silencing
of Th1 cellrelated genes in Th2 cells (81). In contrast, PRC1 deficiency results in decreased
Th2 cytokine production, conflicting with studies on PRC2 components. Deletion of Bmi1, Pcgf2
(encoding Mel-18), or Rnf2 (encoding Ring1B) resulted in decreased IL-4 production (8284).
This can be explained partly by the proliferation limit caused by early senescence, enhanced
apoptotic cell death, and metabolic abnormality in the absence of PRC1 components (85, 86).
Impaired 3-D chromatin structure may also be associated with these phenotypes (87).
Maintenance of Th2 cell identity. How does the switch from PcG to TrxG occupancy at the
regulatory region of Gata3 regulate Th2 cell function? Our analysis of Menin-deficient mice
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that lacked TrxG function revealed that maintenance of Gata3 expression is dependent on the
Menin/TrxG complex and independent of IL-4 and STAT6 in Th2 cells (73). A similar molecular
mechanism was found to operate in vivo using Kmt2a+/ (referred to as MLL1+/ elsewhere in this
review) mice (88). MLL1 binding occurs throughout the Gata3 gene locus in memory Th2 cells,
and Gata3 expression is reduced in MLL1+/ cells. MLL1 maintains high expression of Gata3
in vivo and is responsible for type 2 immune responses. In human Th2 memory cells, MLL and
Menin are reported to form a core transcriptional complex that regulates GATA3 expression (89).
TrxG proteins therefore represent an essential mechanism of transcriptional maintenance in the
memory T cell response (90). In contrast, PRC1 proteins contribute to maintenance of Th2 cell
identity and survival by suppressing apoptosis in vivo (91). Thus, the acquisition and maintenance
of Th2 cell identity are regulated by two distinct mechanisms (Figure 1d) (73). First, during Th2
differentiation, Gata3 upregulation accompanied by PcG/TrxG displacement at the Gata3 gene
locus is induced in an IL-4- and STAT6-dependent manner. Second, in differentiated and memory
Th2 cells, maintenance of high-level Gata3 expression is dependent on the Menin/TrxG complex.
GATA3-Dependent and -Independent Regulation of Th2 Cell Identity

As described above, induction and maintenance of high-level Gata3 expression are tightly regulated by epigenetic machinery, and high-level GATA3 expression is required for maintenance of
Th2 cytokine-producing ability (30, 92). Our recent studies have identified 32 Th2 cellspecific
genes using DNA microarray-based gene expression analysis and ChIP-seq-based epigenetic analysis (76, 93). These include 17 GATA3-targeted and 15 non-GATA3-targeted genes. Most of
the GATA3-targeted genes are downregulated by Gata3 knockdown in Th2 cells, including the
Th2 cytokine genes Il4, Il5, and Il13. Expression of Ccr8, which is reported to be associated
with Th2-mediated diseases in the skin (94), is also highly dependent on GATA3. Therefore,
GATA3 and its target genes are important in the acquisition and maintenance of Th2 cell identity. In contrast, Gata3 knockdown has variable effects on the expression of Th2 cellspecific
but GATA3-nontargeted genes. For example, expression of Tube1 increases more than twofold
compared to control following Gata3 knockdown, whereas the Ptgir and Rnf128 genes show
more than threefold reduction in expression upon Gata3 knockdown. Indeed, Rnf128 encodes the
protein named Gene related to anergy in lymphocytes (GRAIL), which controls expression of
IL-4, indicating that the GATA3-GRAIL axis is associated with Th2 cell identity (95, 96). On
the other hand, the expression of some Th2 cellspecific but GATA3-nontargeted genes (Dusp4,
Plcd1, and S100a1) are upregulated in a GATA3-independent manner in Th2 cells, although their
functions remain unknown. Moreover, the GATA3-targeted gene Tnfrsf8, which plays an important role in the induction of Th2 cellmediated allergic asthma, shows very low dependency on
the expression of GATA3 (97), indicating that GATA3 does not regulate all aspects of Th2 cell
programming.
STRUCTURAL BASIS FOR THE FUNCTION OF GATA3

AND GATA3 COMPLEXES
Structure of GATA3 Protein
Members of the GATA family of transcription factors (GATA16) possess two highly conserved
type IV zinc fingers, each of which is followed by a conserved basic region. The C-terminal zinc
finger (C-finger) is essential for DNA binding, whereas the N-terminal zinc finger (N-finger)
stabilizes binding and physically interacts with other transcription factors. The N-finger and
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DNA binding
Activation of Th2 cytokines
Interaction with other factors
Structure of GATA3 protein
DNA binding
Repression of Th1 development
288
263
342
317
N-finger
443 AA
C-finger
N-finger
Me
P
PP
C-finger
354
SSTEG RECVNCGATS TPLWRRDGTG HYLCNACGLY HKMNGQNRPL IKPKRR LSAA RRAGTSCANC QT T T T TLWRR NANGDPVCNA CGLYYKLH NI N RPL
256
Transactivation
of the Il5 gene
Survival
Homing

Repression of
Th1 development
DNA binding
GATA3
Posttranslational modification
Phosphorylation at
S308/T315/S316
NuRD
Chd4
Methylation of R261
p300
GATA3
Repression
Chd4
Yy1
Ruvbl2
GATA3
GATA3
GATA3
Activation
Activation
PcG
Repression
Hsp60
JunB
(AP-1)
Ets1 GATA3
Activation
Tbx21 (T-bet)
Th2 cytokine genes
Cdkn2c (p18, Ink4c)
Il5
Inhibition of Th1
cell differentiation
Chromatin remodeling
intrachromosomal interactions
Proliferation
Transactivation
Figure 2
Functionally important motifs of GATA3 and GATA3 complexes. (a) GATA3 possesses two zinc fingers: C-terminal zinc finger
(C-finger) and N-terminal zinc finger (N-finger). Both of them are type IV zinc fingers. While both zinc fingers are involved in the
DNA binding activity of GATA3 and activation of Th2 cytokines, the N-finger is not required to repress Th1 cell differentiation. The
amino acids KRR (304306; magenta) have been shown to be involved in T cell survival and homing. The conserved YxKxHxxxRP
(344353; red) motif is indispensable for GATA3 DNA binding and function. Phosphorylation of S308, T315, and S316 (blue) is
important for the organization of the GATA3/Chd4-NuRD complex and repression of IFN- production by Th2 cells. Methylation of
R261 (green) regulates transactivation of the Il5 gene. (b) GATA3 establishes Th2 cell identity via organization of several distinct
complexes. A GATA3/Chd4-NuRD repression complex at the Tbx21 locus represses T-bet-dependent IFN- production, and
phosphorylation of GATA3 at S308, T315, and S316 controls the dissociation of Hdac2 from this complex. A GATA3/Chd4/p300
transcriptional activation complex induces chromatin remodeling at the Th2 cytokine loci and expression of Th2 cytokines. Yy1 has
important roles in the GATA3-mediated intrachromosomal interactions of Th2 cytokine enhancers and promoters. GATA3/Ruvbl2
complex recruits PcG complex at the CDK inhibitor, Cdkn2c locus, and silences its expression to support Th2 cell proliferation.
GATA3 organizes transactivation complex with AP-1 and Ets1 at the proximal promoter of the Il5 gene. An R261 mutated form of the
Gata3 gene that mimics methylated GATA3 has a strong association with Hsp60, and transcriptional elongation of the Il5 gene by
RNA polymerase II is subsequently impaired.
C-finger thus play different roles in the regulation of Th2 cell differentiation (18, 98). Although
both the N-finger and the C-finger are required for the induction of Th2 cytokines, the Nfinger is not involved in Tbx21 and Ifng repression (Figure 2a) (98, 99). The conserved basic
regions, positioned after the zinc fingers, also have important roles in the regulation of Th2 cell
differentiation. A KRR-GATA3 mutant that has a substitution of three amino acids, 304306
(KKR) following the N-finger, with alanines was shown to act as a dominant-negative for Th2
cytokine production, and this was accompanied by reduced T cell survival and altered lymphocyte
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homing (100). We also identified a conserved YxKxHxxxRP (344353; x represents any amino
acid residue) motif adjacent to the C-finger of all GATA family proteins in mice and humans that
is indispensable for GATA3 DNA binding and function, including its transcriptional activity and
the ability to induce open chromatin at the Th2 cytokine loci (Figure 2a) (101).

Organization and Function of GATA3 Complexes

As described above, GATA3 simultaneously exerts several distinct functions during Th2 cell differentiation. Thus, it is logical to assume that GATA3 organizes functionally distinct complexes in
differentiating Th2 cells that positively and negatively regulate gene expression to establish Th2
cell identity. GATA3 organizes a transactivation complex with AP-1 and E26 avian leukemia oncogene 1 5 domain (Ets1) at the Il5 promoter (102). Transcription factor Yin yang 1 (Yy1) associates
with GATA3 and regulates GATA3-mediated interchromosomal interaction of Th2 cytokine promoters and enhancers (Figure 2b) (103). However, the majority of previously reported GATA3
interacting molecules such as Eomesodermin (Eomes), Friend of GATA-1 (FOG1), Lymphoid
enhancer binding factor 1 (LEF1), Repressor of GATA (ROG), Spleen focus forming virus proviral
integration oncogene (PU.1), Sex determining region Y-box 4 (Sox4), and T-bet appear to negatively regulate GATA3 function through inhibition of its DNA binding activity (104109). Recent
reports identified several GATA3 binding partners that support the distinct functions of GATA3.
A GATA3 transcriptional activation complex with Chromodomain helicase DNA-binding protein
4 (Chd4)/p300, and a GATA3 transcriptional repression complex with Chd4-Nucleosome remodeling histone deacetylase (NuRD) cooperatively regulate Th2 differentiation. In addition, a transcriptional repression complex including GATA3/Ruv-B like protein 2 (Ruvbl2)/PcG regulates
Th2 cell proliferation (110). Furthermore, we recently found that posttranslational modifications
of GATA3 control aspects of GATA3 complex formation (99, 111) (Figure 2a,b).
GATA3/Chd4/p300 and GATA3/Chd4-NuRD Complexes

GATA3 promotes the expression of a large number of Th2 cellspecific genes, and represses an
alternate gene subset including Tbx21, a key regulator of Th1 cell fate determination (76, 112).
Chd4 was identified as a central component of two functionally distinct GATA3 complexes (113).
Chd4 is an ATP-dependent chromatin remodeler and a major subunit of the repressive NuRD
complex. Together with GATA3, Chd4 participates in both upregulation of Th2 cytokines
and repression of IFN- in differentiating Th2 cells (113). The GATA3/Chd4 complex binds
to the Th2 cytokine loci and regulates chromatin remodeling through the recruitment of
histone acetyltransferase p300 and subsequent induction of histone H3K9 acetylation at the
Th2 cytokine gene loci. On the other hand, GATA3 recruits the repressive NuRD complex,
including Chd4, Histone deacetylase 2 (Hdac2), and Methyl-CpG binding domain protein 3
(Mbd3) to the Tbx21 locus, leading to diminished T-bet-dependent IFN- expression in Th2
cells. Therefore, GATA3 organizes a GATA3/Chd4/p300 transcription activating complex
at the Th2 cytokine loci and a GATA3/Chd4-NuRD repressive complex at the Tbx21 locus
in Th2 cells, simultaneously regulating Th2 cytokine gene activation and Tbx21 repression
(113). More recently, we identified posttranslational modifications of GATA3 that control the
organization of the repressive GATA3/Chd4-NuRD complex at the Tbx21 locus (99). We
reported simultaneous phosphorylation of GATA3 at S308, T315, and S316 nearby the C-finger,
which induces dissociation of Hdac2 from the GATA3/Chd4 repressive complex. Thymoma viral
proto-oncogene 1 (Akt1) was identified as one of the kinases responsible for this phosphorylation,
and activation of Akt1 causes derepression of Tbx21 and Ifng expression in Th2 cells. In both
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human and murine systems, T-bet-dependent IFN- production from memory Th2 cells appears
to be regulated by activation of Akt and the phosphorylation status of GATA3 (99). Memory
Th2 cells that express IFN- may play an important role in inflammation that includes both
eosinophils and neutrophils (114). Thus, it is possible that Akt-mediated phosphorylation of
GATA3 and subsequent derepression of IFN- production from memory Th2 cells are involved
in Th2-mediated chronic inflammatory disorders in which IFN- is also involved, such as atopic
dermatitis and chronic airway inflammatory disorders.
GATA3/Ruvbl2/PcG Complex

Activated CD4 T cells proliferate more vigorously under conditions that support Th2 differentiation compared to conditions that support Th1 differentiation. Gata3-deficient Th2 cells exhibit
substantially reduced cell division and BrdU incorporation, indicating that GATA3 is involved
in the regulation of Th2 cell proliferation (29). Ruvbl2 was identified in the GATA3 complex
by a unique proteomics approach using affinity purification from the material obtained from
formaldehyde cross-linked GATA3 complexes, in which associated molecules with low affinity
can be identified (110). Ruvbl2 was initially found as a protein interacting with the TATA-box
binding protein (Tbp) in Drosophila, yeast, and humans, belonging to the AAA+ (ATPase associated
with multiple activities) ATPase family, and having an ATPase domain with Walker A and Walker
B motifs. Previous reports identified Ruvbl2 in various chromatin remodeling complexes, such as
Brahma-associated factors (BAF), Ino80, PcG, and Tip60 (encoded by Kat5) complexes (115). In
Th2 cells, Ruvbl2 regulates the binding of GATA3 to the Cdkn2c locus. The GATA3/Ruvbl2 complex is required to recruit PcG gene products and induces a closed chromatin status at the Cdkn2c
locus in Th2 cells. Thus, repression of Cdkn2c expression mediated by the GATA3/Ruvbl2/PcG
complex is one of the key mechanisms promoting proliferation of differentiating Th2 cells
(110).
Methylation of R261 in the N-Finger of GATA3

The GATA3/AP-1/Ets1 transactivation complex directly activates transcription of the Il5 gene
(102). Recently, we identified a novel arginine methylation site nearby the N-finger of GATA3
at R261 and demonstrated its role in the function of GATA3 (111). Although a methylationmimicking GATA3 mutant retains the ability to induce IL-4 and repress IFN- expression, IL-5
production is selectively impaired. This mutant is capable of binding to the Il5 promoter and
inducing chromatin remodeling of the Il5 promoter to the same extent as wild-type GATA3.
However, Heat shock protein 60 (Hsp60) strongly associates with the methylation-mimicking
GATA3 mutant and represses the transcriptional elongation of Il5 by RNA polymerase II. The
methylation-mimicking GATA3 mutant exhibits a dominant-negative effect on GATA3-mediated
transactivation of the Il5 gene, without altering Il4 and Il13 expression (111).
GATA3 therefore organizes functionally distinct complexes with defined functions in differentiating Th2 cells and both positively and negatively regulates the expression of genes to establish
Th2 cell identity (Figure 2b). Lineage commitment involves the activation of specific gene programs and the concomitant suppression of multipotential and alternate lineage gene profiles.
These events are regulated in large part by a limited set of lineage-specific master transcription
factors that functionally cross-antagonize one another. GATA3 is a well-defined example of a
master transcription factor. However, several areas still require clarification, particularly the relationship between extracellular signaling, posttranslational modifications, and the organization of
GATA3 complexes, and how the complexes recognize the specific target loci.
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MEMORY-TYPE Tpath2 CELLS

Functional Heterogeneity of Memory Th2 Cell Subsets

Memory Th cells, such as memory Th2 cells, are considered a key cell population in the pathogenesis of allergic inflammatory disorders (116). However, the specific subpopulations of memory
Th cells and the factors that induce and sustain chronic inflammation remain unclear owing to the
high heterogeneity of memory Th cells and the limited numbers of antigen-specific memory Th
cells recoverable from inflammatory tissues (116, 117). Recently, several investigators identified
phenotypically and functionally distinct memory Th2 cell subsets that produce large amounts of
IL-5, IL-17, or IFN- in addition to IL-4 and IL-13 (94, 105, 118, 119). These noncanonical
memory Th2 cells are critical to the pathogenesis of chronic allergic inflammatory diseases and
the prevention of lymphocytic choriomeningitis virus (LCMV) persistence.
In 2011, we reported that high-level IL-5-producing memory Th2 cells are important drivers
of allergic asthma (105). In our experimental system, depletion of the IL-5-producing memory
Th2 cells leads to reduced eosinophilic lung inflammation and reduced methacholine-induced
airway hyperresponsiveness (AHR). In addition, another group demonstrated that multiple rounds
of cultivation of human cells efficiently generated IL-5-producing Th2 cells (120). The IL-5producing subpopulation can be phenotypically identified by expression of cell surface molecules
and epigenetic status and may play a role in Th2 celldriven pathology (105, 120). Thus, these
unique high-level IL-5-producing memory Th2 cells may represent a novel pathogenic population
that induces eosinophilic inflammation in allergic diseases such as asthma and chronic dermatitis.
Chronic airway inflammation, especially steroid-resistant airway inflammation, is induced experimentally not only by Th2 cells but also by Th17 cells (121, 122). Recent studies have described
a novel subset of Th2 memory/effector cells that coexpress the transcription factor GATA3 and
ROR-t and simultaneously produce both IL-4 and IL-17 (118, 122, 123). IL-17-producing Th2
cells were induced in the inflamed lung and persisted as the dominant IL-17+ T cell population
during the chronic stage of asthma (118). Ovalbumin (OVA)-specific, IL-17-producing Th2 cells
caused enhanced recruitment of eosinophils, neutrophils, macrophages, and lymphocytes into
the airway as compared to classical Th2 or Th17 cells (118, 122). In addition to the findings in
preclinical models, a population of human memory CD4 T cells expressing CD161 and CCR6
also coproduced IL-4 and IL-17, and this population increased significantly in peripheral blood
mononuclear cells (PBMCs) of patients with asthma (123). Therefore, the IL-17-producing Th2
cell subset appears to be another type of Tpath2 cell population in both mice and humans.
Identification of Memory-Type Tpath2 Cells

Among the functionally distinct memory Th2 cell subsets described above, our study based on
the functional clarification of memory Th2 cell subpopulations identified a pathogenic IL-5producing subpopulation that appears to be responsible for the pathology of various Th2-type
chronic inflammatory disorders, including asthma (105, 116). Memory Th cells are divided into
two major subsets: effector memory T (TEM) cells and central memory T (TCM) cells (117).
TEM cells show a CD62Llo CCR7lo phenotype and preferentially reside in the peripheral organs;
in contrast, TCM cells show CD62Lhi CCR7hi phenotype and mainly localize in the lymphoid tissues (117, 124). We found that a substantial proportion of memory Th2 cells express Chemokine
(C-X-C motif) receptor 3 (CXCR3), a well-known chemokine receptor for Th1 cells. Memory
Th2 cells can be subdivided into at least four distinct subpopulations according to their expression
of CXCR3 and CD62L (105). Interestingly, although all four subpopulations of memory Th2
cells produce large amounts of IL-4 and IL-13 in response to antigenic restimulation, only the
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CXCR3lo CD62Llo subpopulation produces IL-5. The production of IL-5 by memory Th2 cells
is tightly regulated by both the status of chromatin at the Il5 gene and the expression of Eomes, a
T-box transcription factor. Eomes is highly expressed in activated and memory CD8 T cells (125).
Eomes suppresses the transcriptional activity of GATA3 via inhibition of GATA3 DNA binding
to the Il5 promoter region in memory Th2 cells (105). Pathogenic IL-5-producing memory Th2
cells exhibit high levels of permissive chromatin modifications, such as H3K9ac and H3K4me3 at
the Il5 gene locus, and express very low levels of Eomes, resulting in a large amount of IL-5 production upon antigenic stimulation. The phenotype of mouse memory-type IL-5-producing Tpath2
cells is ST2hi CXCR3lo CD62Llo CCR4+ CD44+ Sca-1+ ICOS+ IL7R+ (105). Both pathogenic and
nonpathogenic (not producing IL-5) memory-type Th2 cells express IL17RB, TSLP receptor
(TSLPR), Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2),
and CCR8 at similar levels.
Type 2 innate lymphoid cells (ILC2s) are another recently identified cell type that also produce
large amounts of IL-5 and IL-13 in response to IL-33 (126, 127). Importantly, recent studies have
shown that ILC2s can drive eosinophilic airway inflammation in lymphopenic hosts, and that
ILC2s contribute to type 2 immune responses in the lung (128130). Tpath2 cells and ILC2s
share several features, including (a) cell surface molecule expression, (b) effector function in terms
of cytokine production, (c) expression of transcription factors relevant to lineage development (24,
116), and (d) active enhancer landscapes upon worm infection (131). While some key distinctions
have been identified, such as specific production of IL-4 by Tpath2 cells, it will be important
to further establish the molecular and functional differences between these two cell types and to
define how these cell types interact in disease contexts.

IY35CH03-Nakayama
Induction of Memory-Type Tpath2 Cells

We recently demonstrated that the IL-33-suppression of tumorigenicity 2 (ST2)-p38 MAPK axis
is crucial for the induction and enhancement of pathogenicity of memory Th2 cells in allergic
airway inflammation in both mice and humans (24). IL-33 is a newly recognized member of the
IL-1 superfamily and was originally identified as a ligand for the ST2 receptor (also known as
IL1RL1) (22). IL-33 is known to induce strong Th2-type immune responses and eosinophilic
inflammation in the lung and intestine by amplifying and maintaining Th2 cytokine production
(132, 133). IL-33 also connects type 2 immune responses with basal metabolism in vivo by regulating thermogenesis (134). In asthma patients, epithelial cells, endothelial cells, and airway smooth
muscle cells are three major sources of IL-33, especially in the event of tissue damage (132).
Furthermore, recent large-scale genome-wide association studies (GWAS) have shown that the
IL33 and IL1RL1 genes are associated with the onset of asthma (135). Although some evidence
suggests a strong connection between IL-33 and the pathogenesis of allergic inflammation, the
types of cells that IL-33 affects in inflammatory settings are still being identified. We found that
IL-5-producing Tpath2 cells show selective and high ST2 expression and that exposure of memory Th2 cells to IL-33 induces a significant increase in IL-5 production (24). In contrast, IL-33
does not induce IL-4 production by these cells. Interestingly, IL-33-induced IL-5 upregulation
and increased expression of ST2 were not observed in effector Th2 cells or effector and memory
Th1 cells. IL-33-ST2 signaling upregulates IL-5 production and ST2 expression in memory Th2
cells through the induction of chromatin remodeling at the Il5 and Il1rl1 gene loci (24). This
finding is notable because the cytokine IL-33 can induce chromatin remodeling of cytokine and
cytokine receptor genes in T cells without TCR stimulation. Besides IL-33, IL-25 and TSLP
have also been shown to play critical roles in several Th2 cellassociated disease models (129,
136, 137). Like IL-33, a combination of IL-2 and IL-25 can induce IL-5 production in ILC2s
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(138). Consistent with the effect on ILC2s, a combination of IL-2 and IL-25, similar to IL-33,
induces the ability to produce IL-5 in memory Th2 cells (24). Regarding the signal cascades activated by IL-33, the activation of MAPKs and NF-B in mast cells has been reported (22, 133). In
memory Th2 cells, IL-33 selectively activates p38 MAPK, increases the expression of ST2, and
augments the ability to produce IL-5. The administration of neutralizing anti-IL-33 antibodies or
anti-ST2 antibodies to allergic mice inhibits eosinophilic recruitment, Th2 cytokine production,
serum IgE, and mucus hyperproduction (139, 140). Consistent with these findings, Il33/ mice
or Il1rl1/ mice show decreased eosinophil infiltration into the bronchoalveolar lavage (BAL)
fluid and decreased pulmonary inflammation during airway inflammation (141, 142), although
some variations in the results have been reported (143). We used memory Th2 celldependent
airway inflammation models and demonstrated a pathological role for the IL-33-ST2 pathway
(24). In this model, ST2-expressing memory Th2 cells were increased in the lung after antigenic
challenge. Genetic deletion of Il1rl1 in Th2 cells resulted in a significant decrease in mononuclear
cell infiltration into the peribronchiolar regions of the lungs and in the number of inflammatory
eosinophils in the BAL fluid. In addition, deletion of Il33 in recipient mice abrogated the inflammatory response. Since the effector Th2 cells possess little reactivity to IL-33, the IL-33-ST2 axis
appears to operate predominately in the induction of eosinophilic inflammation by memory-type
Tpath2 cells. Therefore, IL-33 is a newly identified factor in the induction and exacerbation of
chronic airway inflammation through memory Th2 cells (Figure 3a).
Maintenance of Memory-Type Tpath2 Cells in the Lung

The maintenance of memory CD4 T cells is incompletely understood, and it is likely that multiple mechanisms differentially contribute to the proliferative renewal and homeostatic turnover of
various memory populations. In secondary lymphoid organs, the maintenance of memory CD4
T cells is dependent on homeostatic TCR signaling and multiple cytokines, including IL-7 and
IL-15 (144146). Studies using mice transgenically altered to allow inducible TCR signaling
blockade have demonstrated that homeostatic (nonspecific) TCR signaling is dispensable for primary effector CD4 T cells to transition to memory cells, but indispensable for memory CD4 T
cell homeostatic turnover and longevity in the secondary lymphoid organs (147). It has also been
reported that a combination of TCR signaling and IL-7 and IL-2 cytokine signaling suppresses
proapoptotic pathways during the formation of human memory CD4 T cells (148). IL-7 is known
to be required during the transition from effector to memory CD4 T cell in the secondary lymphoid organs, and again for long-term maintenance of memory CD4 T cells (145, 149). Memory
CD4 T cells require IL-15 for long-term maintenance, similar to memory CD8 T cells, and the
relative strengths of IL-7 and IL-15 signaling may strikingly alter the rate of homeostatic turnover
of memory CD4 T cells and influence the expansion of secondary responses (146). The sources of
IL-7 are incompletely characterized but have been investigated by multiple groups using several
techniques (150152). The cumulative evidence indicates that stromal tissue in multiple lymphoid
organs, including lymph nodes, spleen, and bone marrow, produces IL-7. Although secondary
lymphoid organs appear to be the primary reservoir for circulating memory cells, a substantial
fraction of memory CD4 T cells traffic to the bone marrow, where they rest and are maintained
indefinitely (151). While CD69 and CD49b expression on these memory cells was reported to
facilitate homing to niches containing IL-7-producing bone marrow stromal cells (153, 154),
memory maintenance in bone marrow is also facilitated by CD11c+ dendritic cells, which may
also provide antigen presentation for secondary responses (155). Given that the memory CD4 T
cells maintained in bone marrow appear to be in a resting state in terms of their gene expression and proliferation (151), they are considered less pathogenic than memory cells maintained in
secondary lymphoid organs.
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Induction
A IR W A Y
Maintenance
Allergen
Memory
Th2 cell
IL-33
Eosinophil
T cell
B cell
TCR

p38
T1/ST2
DC
IL-5
Tpath2 cell
FDC
Memory
Th2 cell
T1/ST2 ++
TCR
IL-5 +++
IL-4
IL-13
Tpath2 cell
IL-7+IL-33+
LEC
iBALT
Inflammatory niche
L U N G T I SSUE
Figure 3
Induction and maintenance of memory-type pathogenic Th2 (Tpath2) cells in the airway. (a) Allergen exposure induces IL-33 secretion
from IL-33+ cells, including epithelial cells and endothelial cells in the airway. IL-33 induces chromatin remodeling at the Il5 and
Il1rl1 loci in an ST2/p38 MAPK-dependent manner in memory Th2 cells. Antigen restimulation induces secretion of greater amounts
of IL-5 from IL-33-activated memory-type Tpath2 cells and thereby exacerbates chronic eosinophilic inflammation. (b) Maintenance
of memory-type Tpath2 cells in chronic airway inflammation. Ectopic lymphoid structures called inducible bronchus-associated
lymphoid tissue (iBALT) are formed in the lung during chronic inflammation and consist of various types of immune cells, including T
cells, B cells, dendritic cells (DCs), and follicular dendritic cells (FDCs). The selective localization and survival of memory-type Tpath2
cells within iBALT are supported by IL-7+ lymphatic endothelial cells (LECs). These IL-7+ LECs are also IL-33+ ; thus, these
IL-7+ IL-33+ LECs may confer the memory Th2 cells with greater pathogenicity. IL-7+ IL-33+ LECs are considered to be the niche
for survival Tpath2 cells and maintenance of their function at sites of chronic inflammation (inflammatory niche).
Recent investigations have demonstrated that subsets of memory CD4 T cells are retained
at specific peripheral sites as tissue-resident memory CD4 T cells and may confer an effective
in situ first line of defense against tissue-specific infections (156, 157). This result suggests that
lung-resident memory CD4 T cells may occupy a distinct compartment in the lung, in contrast
to spleen-resident memory CD4 T cells, which could circulate to multiple tissue sites. Moreover,
our group and others have recently found that lung-resident, antigen-specific memory Th2 cells
are key drivers of lung allergic responses (158, 159). Lung-resident, antigen-specific memory Th2
cells are sufficient to induce asthmatic symptoms, and IL-2 signaling directs a program of tissue
homing migrational cues and instructs the residency of these cells in the lung (158). These findings
are supported by clinical studies in which lung transplants from mildly asthmatic donor patients
transferred airway disease into nonasthmatic recipients, whereas transplantation of a nonasthmatic
lung into an asthmatic patient ameliorated disease (160).
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The mechanisms regulating maintenance of lung-resident memory CD4 T cells are not well
understood. One possibility is that memory CD4 T cells resident in peripheral tissues actively
traffic to secondary lymphoid organs for targeted interactions with IL-7-producing stromal cells
(150). However, parabiosis experiments in which pairs of mice were surgically conjoined and
shared a common circulation provided direct evidence for the retention of memory CD4 T cells
within lung tissues (157). We recently found that the maintenance of lung-resident, antigenspecific memory Th2 cells is dependent on IL-7-producing lymphatic endothelial cells (LECs),
localized within structures called inducible bronchus-associated lymphoid tissue (iBALT) (159).
iBALT is a lymphoid structure induced in the lung in response to inflammation caused by various infectious organisms (161). It consists of separate B cell areas, containing follicular dendritic
cells, dendritic cells, high endothelial venules, and lymphatics. Antigen-specific memory Th2
cells preferentially localized in iBALT and were maintained by an IL-7-dependent but antigenindependent mechanism (159). Notably, IL-7-producing LECs increase in number after lung
inflammation concomitant with the formation of iBALT, suggesting that modification of the lung
microenvironment promotes survival of antigen-specific memory Th2 cells within iBALT. Moreover, analysis of IL-7-producing LECs revealed that this cell population also produces IL-33. It
has recently been shown that loss of tissue-derived cytokines, including IL-33, abrogates the terminal differentiation of Th2 cells in the lung (162), and as outlined above, IL-33 directly instructs
memory-type Tpath2 cells to enhance IL-5 production, leading to eosinophilic inflammation (24,
163). Thus, it appears that IL-7- and IL-33-producing LECs provide a niche for Tpath2 cells that
maintains both their survival and their pathogenicity in the lung (Figure 3b). The frequency of
IL-5-producing Tpath2 cells as a percentage of all memory Th2 cells in our mouse model is 1%
[1.1% 0.4% (mean standard deviation)] in the spleen and 8% (7.7% 2.5%) in the lung
(24, 105). Strikingly, the proportion of IL-5-producing Tpath2 cells in mice with iBALT formation was 10% (10.4% 1.6%) in the spleen and 39% (38.7% 6.2%) in the lung (159).
In humans, substantial numbers (1%) of CD161hi IL-5-producing Tpath2 cells are detected
in PBMCs of patients with eosinophilic gastrointestinal disease (164). Although IL-33-reactive
IL-5-producing Tpath2 cells were few within PBMCs, they were easily detected in polyps of patients with eosinophilic chronic rhinosinusitis (CRS) (24). It should be noted that in the absence of
antigen, the number of lung-resident Tpath2 cells gradually decreases. It is possible that Tpath2
cells can circulate and traffic into other inflamed mucosal sites or lymphoid organs that provide
survival cues necessary for their prolonged lifespan.
TH2 CELLMEDIATED INFLAMMATORY DISEASES

Asthma
Asthma is a common chronic inflammatory disease of the lower airways characterized by airway
inflammation, variable airway obstruction, mucus hyperproduction, and airway hyperresponsiveness (165). The pathophysiology of asthma includes infiltration of various types of inflammatory
cells into the lungs, followed by airway remodeling such as thickened bronchial walls and hyperplasia of the bronchial glands (165). The pathophysiology of allergic airway inflammation is
thought to be shaped mainly by Th2 cells. The impact of the Th2 cytokines, IL-4, IL-5, and IL-13
has been revealed in human asthma, as well as in murine models of allergic airway inflammation
(10). The contribution of Th2 cytokines to the various pathological changes, such as infiltration
of eosinophils, increased mucus production, and fibrosis, is well recognized (10). The pathogenic
role of Th2 cytokines has been further established by the successful use of therapeutic monoclonal antibodies directed against IL-5 (mepolizumab) and IL-13 (lebrikizumab) (166, 167). In
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2015 humanized anti-IL-5 antibodies (mepolizumab and reslizumab) were approved for treatment
of asthma patients with severe eosinophilic disease by the United States Food and Drug Administration (168). As discussed above, IL-5 is the main pathogenic effector cytokine of the Tpath2 cell
subpopulation, capable of inducing eosinophilic inflammation. Therefore, further detailed analysis
of the molecular basis for the production of IL-5 may lead to the discovery of selective therapeutic targets for Tpath2 cellmediated inflammatory disorders. Emerging results from analyses of
numerous molecules involved in Th2 cell differentiation and function have highlighted their roles
in the pathogenesis of allergic asthma. In this section, we focus on (a) the impact of epigenetic
regulation of pathogenicity in allergic airway inflammation, and (b) the pathophysiological roles
of transcription factors in allergic airway inflammation.

Epigenetic regulation involved in the pathology of asthma. Gene expression can be regulated
by enzyme-mediated methylation, acetylation, phosphorylation, and ubiquitination of N-terminal
histones (169). As described above, PcG and TrxG proteins possess methyltransferase activity,
and these complexes shape the pathophysiology of allergic airway inflammation. Loss of Bmi1 or
Ring1B, components of PRC1 complex in CD4 T cells, resulted in decreased Th2 cell generation and impaired allergic airway responses induced by OVA (84, 91). Interestingly, T cellspecific
deletion of Ezh2, a component of the PRC2 complex, results in increased Th2 celldriven lung inflammation in a model of allergic asthma and also spontaneous development of memory phenotype
Th2 cells and increased levels of circulating IgE (79). Thus, Ezh2 may restrain the pathogenicity
of CD4 T cells in allergic airway inflammatory diseases such as asthma. In contrast, the TrxG
proteins positively regulate Th2 cellmediated inflammation, as memory Th2 celldependent allergic airway inflammation is ameliorated in mice with MLL1+/ memory Th2 cells (88). Loss
of the TrxG protein Menin does not cause an obvious defect in Th2 cell differentiation; it does
appear to be required for long-term programming of Th2 cell phenotype. In addition, Menin deficiency impairs differentiation into Th17 cells and ameliorates neutrophilic airway inflammation
(170). Thus, TxrG and PcG proteins are positive and negative epigenetic regulators in the control
of Th cell differentiation, and may be involved in the pathogenesis of Th cellmediated allergic
airway inflammation. Memory Th2 (CD4+ CD45RO+ CCR4+ ) cells from human PBMCs show
unique enhancer landscapes compared to memory Th1 (CD4+ CD45RO+ CCR4 ) cells or naive
CD4 T cells (171). Analysis of enhancers that gain H3K4me2 marks during Th2 cell differentiation revealed significant enrichment near asthma-associated single nucleotide polymorphisms
(SNPs). Moreover, analysis of memory Th2 cells from patients with asthma identified more than
100 asthma-associated enhancers (171). Investigation of the function of these enhancers will be
important to clarify the molecular pathways governing the pathogenesis of asthma.
Epigenetic regulation via DNA methylation also plays a part in establishing the immune phenotype associated with asthma. DNA methylation on cytosines of CpG dinucleotides at the gene
promoters is another system encoding epigenetic information that can repress transcription (172).
CD4 T cells from mice with airway inflammation induced by OVA stimulation have enhanced
methylation at the Ifng promoter, and administration of an inhibitor of DNA methyltransferase
(DNMT) can ameliorate airway inflammation (173). Analysis of PBMCs from asthmatic patients
shows hypomethylation at specific gene loci such as IL13 (174).
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by directly binding to the 3 untranslated region (UTR) of target gene
mRNAs. Recently, important roles of miRNA in regulating various biological functions (175),
including the pathophysiology of allergic airway inflammation, have been revealed (176). Various
miRNAs, including miRNA-24 and miRNA-27, are involved in shaping the pathogenicity of
airway inflammation through modifying CD4 T cell differentiation (176).
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As the involvement of epigenetic regulators in various chronic inflammatory diseases

becomes apparent, epigenetic modulators will become candidates for the next generation of
anti-inflammatory drugs. For example, a small-molecule inhibitor selective for the H3K27me3specific jumonji ( JMJ) subfamily reduces lipopolysaccharide-induced TNF- production by
human macrophages (177). However, further evidence of curative efficacy and safety in clinical
trials is required before drugs are used to treat patients with chronic inflammatory diseases such
as severe asthma.
Transcription factors that regulate the pathology of asthma. Key transcription factors
involved in the differentiation and function of Th2 cells, such as STAT6, GATA3, NFB, interferon regulatory factor 4 (IRF4), and BTB and CNC homology basic leucine zipper transcription factor 2 (Bach2), are reported to influence the pathology of chronic airway
inflammation.
STAT6, a crucial signal transducer downstream of IL-4 and IL-13, shapes the pathogenicity
of allergic airway inflammation. STAT6-deficient mice show ameliorated eosinophil infiltration
into the airway, decreased mucus production, and reduced airway hyperresponsiveness in a model
of airway inflammation using OVA (178). In addition, mice deficient in the negative regulators of
STAT6, SOCS2, or Cytokine inducible SH2-containing protein (Cis) exhibit exacerbated airway
inflammation (179, 180). Interestingly, it has been reported that asthma-associated SNPs are
overrepresented in Th2 cellspecific enhancers containing STAT6 binding motifs (181). Thus,
STAT6 appears to be involved in the pathology of allergic airway inflammation.
GATA3 is a master transcription factor for Th2 cell differentiation (8, 17), and patients with
severe asthma have been reported to have increased numbers of GATA3+ cells in the BAL fluid
compared to patients without obvious clinical symptoms (182). Mice expressing a dominant negative form of GATA3 exhibit a reduction in Th2 cytokine production and ameliorated airway inflammation in a model of asthma (100). ROG, a Poxvirus and zinc finger (POZ) domain-containing
Kruppel-type zinc finger family repressor, inhibits Th2 cell differentiation by preventing GATA3
from binding to DNA in vitro (109, 183). ROG-deficient mice demonstrate exacerbated experimental airway inflammation and enhanced Th2 cytokine production from CD4 T cells (184).
Sox4, a transcription factor induced by Transforming growth factor- (TGF-) stimulation, constrains Th2 cell differentiation by inhibiting GATA3 binding to its target gene loci and inhibits
Th2 cellmediated inflammation, including OVA-induced airway inflammation (108). Recently, a
double-blind, multicenter clinical trial was conducted to evaluate the safety and efficacy of SB010,
a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 mRNA, in patients with allergic asthma (185). Treatment with SB010 attenuates both early- and late-phase
responses of asthma after exposure of allergic asthma patients to allergen (185). New treatment
strategies to develop drugs that target key transcription factors or enzymes within transcription
factor complexes may soon be possible.
NF-B is a constitutively expressed transcription factor in T cells that regulates various
genes encoding cytokines, chemokines, inducible nitric oxide synthase (iNOS), cyclooxygenase-2
(COX-2), and different adhesion molecules involved in asthma pathogenesis. P50 (NF-B1), p52
(NF-B2), p65 (RelA), c-Rel, and RelB are members of NF-B. Among these, c-Rel (encoded by
the Rel gene) is required for the development of airway inflammation and airway hyperresponsiveness induced by OVA in mice (186). Moreover, Rel-deficient mice showed no increase in infiltrating
eosinophils in the BAL fluid and no elevation of serum IgE in a model of asthma (186). Further
evidence for the involvement of NF-B in the development of asthma comes from mice with
CD4+ T cellspecific deficiency of Schnurri-2 (Shn-2), an MHC enhancer binding protein. Shn-2
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deficiency results in constitutive activation of NF-B and increased expression of GATA3 together
with enhanced differentiation of Th2 cells (187) and exacerbation of airway inflammation (188).
NLRP3 (NLR family, pyrin domain containing 3) forms the NLRP3 inflammasome that activates caspase-1 and mediates the release of IL-1 and IL-18. It positively regulates Th2 cell
differentiation in cooperation with IRF4 (189). Nlrp3-deficient mice show ameliorated airway
inflammation in an OVA-induced asthma model (189). The transcription factor Bach2 broadly
regulates CD4 T cell differentiation, and deletion of Bach2 resulted in enhanced differentiation
of Th1, Th2, and Th17 cells (190). Bach2-deficient mice exhibited enhanced infiltration of inflammatory cells together with the increased production of Th2 cytokines from CD4 T cells
(190). Importantly, genetic polymorphisms within the gene locus encoding BACH2 have also
been associated with allergic diseases, including asthma, in humans (191).

Chronic Rhinosinusitis
CRS is an upper respiratory inflammatory disease, often refractory to treatment, that involves
chronic inflammation of the nasal cavities and sinuses with a characteristic pattern of cytokine
production and remodeling of inflamed tissue (192). CRS is one of the most common complications of adult asthma. Upper and lower chronic airway inflammatory diseases are closely
associated with each other because CRS and asthma share similar processes of disease progression
and pathogenicity (193). CRS is divided into three subgroups based on clinical classification:
CRS without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP), and allergic fungal
rhinosinusitis (192). Among these subgroups of CRS, the majority of cases of CRSwNP show
type 2 inflammation with chronic eosinophilic inflammation. In recent years, the incidence of
eosinophilic CRSwNP has been increasing, and it is now frequently referred to as eosinophilic
chronic rhinosinusitis (ECRS) (194, 195). There is also a type of CRSwNP, primarily found in
East Asia, that is not characterized by eosinophilic inflammation but instead shows Th1 cell
skewed inflammation in nasal polyps (196198). ECRS differs from noneosinophilic CRSwNP in
asthma comorbidity rates and postoperative recurrence (194196, 198). Polyps from patients with
ECRS exhibit massive infiltration with CD45RO+ memory CD4 T cells that express ST2 and
IL-17 receptor B (IL17RB) and produce large amounts of IL-5 in response to IL-33 and IL-25
(24, 159, 199). Transcriptomic analysis of memory Th2 (CD4+ CD45RO+ CCR4+ ) cells from
PBMCs of patients with rhinitis also revealed increased expression of IL17RB compared to those
of healthy controls (200). Moreover, Thy1+ IL-7-producing LECs, crucial for local maintenance
of Tpath2 cells, are increased in ectopic lymphoid structures in nasal polyps from patients with
ECRS (159). These recent results indicate that IL-33-induced memory-type Tpath2 cells are
involved in shaping the pathology of ECRS. Further dedicated mechanistic studies of chronic
inflammatory diseases of the upper respiratory airway like ECRS may also help us to understand
the pathology of human chronic inflammatory diseases in the lower airway, such as asthma.
Atopic Dermatitis
Atopic dermatitis is one of the most common chronic inflammatory diseases of the skin, characterized by eczematous skin lesions and intense pruritus (201). Excessive Th2 cell responses to
environmental stimuli are observed in the skin of patients with atopic dermatitis (201). Nishikinezumi Cinnamon/Nagoya (NC/Nga) mice are an experimental model for human atopic dermatitis. These mice develop atopic dermatitis-like skin lesions accompanied by excessive Th2
cell differentiation as a result of hyperactivation of NF-B and AP-1 (202). Dupilumab, a human
monoclonal antibody that blocks IL-4 and IL-13, shows efficacy in patients with severe atopic
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dermatitis (203). These results indicate that Th2 cells are predominantly involved in the pathogenesis of atopic dermatitis. Interestingly, it has been reported that CCR8+ memory Th2 cells
produce large amounts of IL-5 and play a pathogenic role in a mouse model of chronic skin inflammation (94). In patients with atopic dermatitis, IL-5-producing hematopoietic prostaglandin
D synthase (hPGDS+ )CRTH2+ Th2 cells appeared to be involved in the pathogenesis of chronic
allergic inflammation (164). Moreover, like the iBALT in chronic lung inflammation, inducible
skin-associated lymphoid tissue (iSALT) has been reported to shape inflammation of the skin (204).

Eosinophilic Gastrointestinal Disorders

Primary eosinophilic gastrointestinal disorders are diseases that specifically affect the gastrointestinal tract with marked eosinophilic inflammation without obvious causes for eosinophilia (e.g.,
parasitic infections, malignancy, and drug reaction) (205). For example, significantly increased
esophageal expression of IL4 and IL5 mRNA in patients with active eosinophilic esophagitis reflects the crucial role of Th2 cells in regulating the pathology of the allergic gastrointestinal
disorder (206). Moreover, levels of hPGDS+ CRTH2+ Th2 cells, which are prominent IL-5 producers and appear to be a human counterpart of mouse Tpath2 cells, are increased in patients
with eosinophilic gastrointestinal diseases (164). Several groups reported that mRNA expression
of IL33, a key cytokine to enhance type 2 immune responses (22, 132, 133), is increased in the gut
mucosa of patients with ulcerative colitis (UC) (207, 208), indicating the pathogenic roles of IL-33
and Th2 cells in UC. Exogenous IL-33 administration drives eosinophilic intestinal inflammation, accompanied by enhanced production of Th2 cytokines (209). Moreover, GPR15-expressing
Tpath2 cells accumulated in the colon of patients with UC (210).
As discussed above, various research groups have recently highlighted that IL-5-producing
Tpath2 cells play a key pathogenic role in Th2-mediated inflammatory diseases in mouse and
human systems, particularly those with eosinophilic inflammation (24, 94, 105, 164, 210). Thus,
Tpath2 cells maintained within local inflammatory sites are a potential therapeutic target for
intractable allergic diseases.
PATHOGENIC TH POPULATION DISEASE INDUCTION MODEL

It has long been recognized that unbalanced generation of Th1 and Th2 cells is associated with
the pathogenesis of autoimmune diseases and allergic diseases, respectively (211, 212). Based on
the recent discoveries of the role of Tpath2 cells in the pathogenesis of allergic diseases, particularly chronic inflammatory diseases, in various tissues, we proposed the pathogenic Th population
disease induction model (Figure 4) (116). In this model, a pathogenic subpopulation of Th cells
that possess one or more distinguishing effector functionsfor example, atypical expression of
cytokines and chemokines or unique expression of cell surface moleculesis generated in vivo,
particularly in the peripheral nonlymphoid tissues; these cells play crucial roles in the pathogenesis
(induction and persistence) of Th1, Th2, and Th17 cell diseases regardless of the overall population balance of these Th cell subsets. Several defined populations of Tpath2 cells have been
characterized in both mice and humans. CD44hi CD62Llo CXCR3lo CCR4hi CCR8hi IL7Rhi ST2hi
memory-type Tpath2 cells are a key subpopulation for the pathogenesis of chronic airway inflammation (24, 105, 159, 199). CCR8hi Tpath2 cells and CRTH2hi CD161hi hPGDShi Tpath2 cells are
pathogenic subpopulations in chronic atopic dermatitis and eosinophilic gastrointestinal diseases,
respectively (94, 164), and CD45ROhi GPR15hi Tpath2 cells are involved in the pathogenesis of
UC (210). CXCR3 and IL23R are emerging as signature molecules for Tpath1 cells and Tpath17
cells, respectively (213, 214). Importantly, allergen-specific immunotherapy induces preferential
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SLO, NLT
Effector
Memory
Tpath1
Phenotype
Involved
diseases
Ref.
Human
CXCR3hi
Type 1 diabetes
213
Murine
CD44hiCD62LloCXCR3lo
Chronic airway
inflammation
24,
105,
159
24,
159,
199
Organism
CCR4hiCCR8hiIL7RhiST2hi
Th1
Human
CD45ROhiCD69hi
ST2hiIL17RBhi
Eosinophilic
chronic
rhinosinusitis
Human
CD45ROhiCRTH2hi
CCR4hiCXCR3loCCR6lo
CD27lo
Alder pollen
allergy
CCR8hi
Atopic
dermatitis
94
CRTH2hiCD161hi
hPGDShi
Atopic
dermatitis
164
CRTH2hiCD161hi
hPGDShi
Eosinophilic
gastrointestinal
disease
164
Human
CD45ROhi
GPR15hi
Ulcerative
colitis
210
Human
CRTH2hi
CCR6hi
Asthma
118
Human
CD161hi
CCR6hi
Asthma
123
Murine
IL23Rhi
Experimental
encephalitis
214
Tpath2

Murine
Naive
CD4+
T cells
Th2
Human
Human
Tpath2+17
Tpath17
Th17
215
Figure 4
Pathogenic Th population disease induction model. In the secondary lymphoid organs (SLOs), each subset of memory Th cells
(Th1, Th2, and Th17 cells) is generated from the corresponding effector Th cell subset. Emerging results indicate that Tpath cells
are generated in nonlymphoid tissues (NLT), and Tpath cells appear to reside preferentially in the tissues. However, they also
circulate and are detected in the SLOs, such as spleen and lymph nodes. Tpath1, Tpath2, and Tpath17 cells play pathogenic roles
in various inflammatory diseases in an organ-specific manner in both mice and humans. In the case of Tpath2 cells,
CD44hi CD62Llo CXCR3lo CCR4hi CCR8hi IL7Rhi ST2hi memory-type Tpath2 cells and CD45ROhi CD69hi ST2hi IL17RBhi
memory-type Tpath2 cells are key subpopulations for the pathogenesis of chronic airway inflammation in mice and humans,
respectively (24, 105, 159, 199). CCR8hi Tpath2 cells and CRTH2hi CD161hi hPGDShi Tpath2 cells are pathogenic subpopulations in
chronic atopic dermatitis and in eosinophilic gastrointestinal disease (94, 164). CD45ROhi CRTH2hi CCR4hi CXCR3lo CCR6lo CD27lo
Tpath2 cells are a pathogenic population in alder pollen allergy (215). CD45ROhi GPR15hi Tpath2 cells are involved in the
pathogenesis of ulcerative colitis (210). CXCR3 and IL23R appear to be important molecules for Tpath1 cells and Tpath17 cells,
respectively (213, 214).
deletion of CD45ROhi CRTH2hi CCR4hi CXCR3lo CCR6lo CD27lo Tpath2 cells in patients with
alder pollen allergy (215).
SUMMARY AND FUTURE DIRECTIONS

This review focuses on recent advances in our understanding of Th2 cell biology and Th2
cellmediated pathology. Identification of proinflammatory Tpath2 cells may dramatically change
our understanding of the pathogenesis of chronic allergic inflammatory diseases. The concept
of pathogenic Th cell populations can also be expanded to other inflammatory disorders caused
by Th1 and Th17 cells, such as tissue-specific autoimmune diseases. Thus, we believe that the
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pathogenic Th population disease induction model will be helpful for understanding the pathogenesis of various inflammatory disorders, including chronic intractable inflammatory diseases.
In the last decade, several other Th subsets, including Th17, Th9, and Tfh cells, were identified, and both their developmental pathways and their functions within the immune system were
intensely studied. More recently, subsets of ILCs have been identified and actively analyzed in
mouse and human systems. Among the ILC subsets, ILC2 cells share several similarities with Th2
cells, particularly Tpath2 cells, while also maintaining discreet functionalities within the immune
system (24, 116). Thus, one direction for future research is clarification of the molecular mechanisms underlying the induction and functional maintenance of Tpath2 cells, and their roles in the
pathogenesis of various human allergic inflammatory diseases, particularly chronic intractable inflammatory disorders. Regarding the induction and activation of Tpath2 cells within inflammatory
lesions, environmental factors including allergens and infectious microorganisms are reasonable
candidates. Filamentous fungi frequently colonize the sinuses of patients with Th2 cellassociated
airway diseases, including asthma (216). Indeed, repeated exposure of Aspergillus fumigatus induces
severe airway inflammation with massive infiltration of Th2 and Th9 cells in mice (217), indicating
that environmental fungi could be a candidate for the induction and maintenance of Tpath2 cells
in the airway. Moreover, as listed in Figure 4, various types of pathogenic Th cell populations in
different inflammatory diseases have been identified; thus, developing antibodies specific for these
pathogenic Th cell populations will help to establish curative treatments for currently intractable
chronic inflammatory diseases.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENT
We are grateful for Drs. Omid Akbari, Mark Bix, Steven Ziegler, and Magdalene Papadopoulos,
for their critical reading and providing valuable suggestions on the manuscript. We apologize to
colleagues whose excellent work we were unable to include because of space limitations.
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Th2 Cells in Health and

Department of Immunology, Graduate School of Medicine, Chiba University,

AMED-CREST, AMED, Chiba 260-8670, Japan

Department of Otorhinolaryngology, Graduate School of Medicine, Chiba University,

Annu. Rev. Immunol. 2017. 35:5384

The Annual Review of Immunology is online at

Th2, allergy, epigenetics, Polycomb, Trithorax, Gata3, pathogenic Th2

This articles doi:

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MOLECULAR PROGRAM FOR TH2 CELL DIFFERENTIATION

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the TCR-mediated extracellular signalregulated kinase (ERK)/mitogen-activated protein kinase

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

mTOR Signaling Pathways

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ESTABLISHMENT OF TH2 CELL IDENTITY

Epigenetic Regulators: Polycomb and Trithorax Proteins

www.annualreviews.org Th2 Cells in Health and Disease

Polycomb (PcG) proteins

Trithorax (TrxG) proteins

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Gata3 gene locus

(STAT6 dependent, Menin/TrxG independent)

(STAT6 independent, Menin/TrxG dependent)

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Spatial Interplay Between PcG and TrxG Proteins

Epigenetic Regulation of Th2 Cell Identity

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

GATA3-Dependent and -Independent Regulation of Th2 Cell Identity

STRUCTURAL BASIS FOR THE FUNCTION OF GATA3

Structure of GATA3 protein

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Th2 cytokine genes

Cdkn2c (p18, Ink4c)

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Organization and Function of GATA3 Complexes

GATA3/Chd4/p300 and GATA3/Chd4-NuRD Complexes

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Methylation of R261 in the N-Finger of GATA3

MEMORY-TYPE Tpath2 CELLS

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Functional Heterogeneity of Memory Th2 Cell Subsets

Identification of Memory-Type Tpath2 Cells

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Induction of Memory-Type Tpath2 Cells

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Maintenance of Memory-Type Tpath2 Cells in the Lung

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Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

TH2 CELLMEDIATED INFLAMMATORY DISEASES

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

As the involvement of epigenetic regulators in various chronic inflammatory diseases

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

Eosinophilic Gastrointestinal Disorders

PATHOGENIC TH POPULATION DISEASE INDUCTION MODEL

Annu. Rev. Immunol. 2017.35. Downloaded from www.annualreviews.org

SUMMARY AND FUTURE DIRECTIONS