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The discovery of the specification of CD4+ helper T cells to discrete effector ‘lineages’ represented a watershed event in conceptualizing mechanisms of host defense and immunoregulation. However, our appreciation for the actual complexity of helper T-cell subsets continues unabated. Just as the Sami language of Scandinavia has 1000 different words for reindeer, immunologists recognize the range of fates available for a CD4+ T cell is numerous and may be underestimated. Added to the crowded scene for helper T-cell subsets is the continuously growing family of innate lymphoid cells (ILCs), endowed with common effector responses and the previously defined ‘master regulators’ for CD4+ helper T-cell subsets are also shared by ILC subsets. Within the context of this extraordinary complexity are concomitant advances in the understanding of transcriptomes and epigenomes. So what do terms like ‘lineage commitment’ and helper T-cell ‘specification’ mean in the early 21st century? How do we put all of this together in a coherent conceptual framework? It would be arrogant to assume that we have a sophisticated enough understanding to seriously answer these questions. Instead, we review the current status of the flexibility of helper T-cell responses in relation to their genetic regulatory networks and epigenetic landscapes. Recent data have provided major surprises as to what master regulators can or cannot do, how they interact with other transcription factors and impact global genome-wide changes, and how all these factors come together to influence helper cell function.
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Han-Yu Shih
Giuseppe Scium!
e
Amanda C. Poholek
Golnaz Vahedi
Kiyoshi Hirahara
Alejandro V. Villarino
Michael Bonelli
Remy Bosselut
Yuka Kanno
Stefan A. Muljo
John J. O’Shea
Transcriptional and epigenetic
networks of helper T and innate
lymphoid cells
Authors’ addresses
Han-Yu Shih
1
, Giuseppe Scium!e
1
, Amanda C. Poholek
1
, Golnaz
Vahedi
1
, Kiyoshi Hirahara
1,2
, Alejandro V. Villarino
1
, Michael Bonelli
1
,
Remy Bosselut
3
, Yuka Kanno
1
, Stefan A. Muljo
4
, John J. O’Shea
1
1
Molecular Immunology and Inflammation Branch,
National Institute of Arthritis, and Musculoskeletal and Skin
Diseases, National Institutes of Health, Bethesda, MD, USA.
2
Department of Advanced Allergology of the Airway, Chiba
University, Chiba, Japan.
3
Laboratory of Immune Cell Biology, Center for Cancer
Research, National Cancer Institute, Bethesda, MD, USA.
4
Laboratory of Immunology, National Institute of Allergy
and Infectious Diseases, National Institutes of Health,
Bethesda, MD, USA.
Correspondence to:
John O’Shea
Molecular Immunology and Inflammation Branch National
Institute of Arthritis, and Musculoskeletal and Skin Diseases
National Institutes of Health
Building 10, Bethesda, MD 20892, USA
Tel.: +1 301 496 6026
Fax: +1 301 402 0012
e-mail: osheajo@mail.nih.gov
Acknowledgements
This study was supported by the NIH Intramural Research
Programs of NIAMS, NIAID, and NCI. The authors have
no conflicts of interest to declare.
This article is part of a series of reviews
covering Transcriptional and Epigenetic
Networks Orchestrating Immune Cell
Development and Function appearing in
Volume 261 of Immunological Reviews.
Summary: The discovery of the specification of CD4
+
helper T cells to
discrete effector ‘lineages’ represented a watershed event in conceptualiz-
ing mechanisms of host defense and immunoregulation. However, our
appreciation for the actual complexity of helper T-cell subsets continues
unabated. Just as the Sami language of Scandinavia has 1000 different
words for reindeer, immunologists recognize the range of fates available
for a CD4
+
Tcellisnumerousandmaybeunderestimated.Addedtothe
crowded scene for helper T-cell subsets is the continuously growing fam-
ily of innate lymphoid cells (ILCs), endowed with common effector
responses and the previously defined ‘master regulators’ for CD4
+
helper
T-cell subsets are also shared by ILC subsets. Within the context of this
extraordinary complexity are concomitant advances in the understanding
of transcriptomes and epigenomes. So what do terms like ‘lineage com-
mitment’ and helper T-cell ‘specification’ mean in the early 21st century?
How do we put all of this together in a coherent conceptual framework?
It would be arrogant to assume thatwehaveasophisticatedenough
understanding to seriously answer these questions. Instead, we review the
current status of the flexibility of helper T-cell responses in relation to
their genetic regulatory networks andepigeneticlandscapes.Recentdata
have provided major surprises as to what master regulators can or cannot
do, how they interact with other transcription factors and impact global
genome-wide changes, and how all these factors come together to influ-
ence helper cell function.
Keywords: transcription factors, gene regulation, epigenetics, T cells, ILCs, cell identity
Introduction: functional specification of CD4
+
helper
T cells
The existence of T cells was first recognized in the 1960s
(1, 2), and their division into helper (CD4
+
) and cytotoxic
(CD8
+
) T cells was appreciated in 1970s (15). It was not
until the late 1980s that the dualism between type 1 and 2
responses of CD4
+
helper T-cell subsets was first proposed
(6, 7). Type 1 helper T (Th1) cells produce the signature
cytokine interferon c(IFN-c) and play a pivotal role in
mounting immunity against intracellular pathogens (8, 9).
Type 2 helper T (Th2) cells produce interleukin-4 (IL-4),
IL-5, and IL-13 and are important against helminth
Immunological Reviews 2014
Vol. 261: 23–49
Printed in Singapore. All rights reserved
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public domain in the USA.
Immunological Reviews
0105-2896
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 23
infections and for helping B cells to produce immunoglobu-
lin E (IgE) antibodies (10).
Just as T and B cells or CD4
+
and CD8
+
T cells were
viewed as distinct lineages, the notion that these subsets of
cytokine-secreting CD4
+
T cells were distinct lineages was
driven by the recognition that with repeated rounds of stim-
ulation the distinctive cytokine production was stabilized
concomitant with extinction of alternate cytokine programs.
This view was strengthened in the late 1990s and early
2000s by the findings that each subset expressed a master
regulator transcription factor (TF) that was necessary and
sufficient for fate determination (1115). First came the
identification of GATA-3 in Th2 cells followed by T-bet in
Th1 cells. Thus, a helper T-cell lineage paradigm evolved to
be viewed as having at least two key attributes: expression
of a signature cytokine and a master regulator TF. Depend-
ing upon your perspective though, it was either edifying or
perplexing that the expression of the master regulators was
controlled by the signature cytokines: the process is clearly
self-reinforcing (16). In addition, it was appreciated that the
gene expression programs for Th1 and Th2 cells extended
beyond just cytokines, as differentiating Th1 and Th2 cells
downregulated TFs and receptors for cytokines that pro-
moted alternative fates (IL-4R in Th1 cells and IL-12R in
Th2 cells) (17, 18).
As recognized by the noted American philosopher, Yogi
Berra, ‘You can observe a lot just by watching’. And so it
was with CD4
+
T-cell subsets; immunologists began to
observe a number of new options available for CD4
+
T cells.
This recognition, which continues at a dizzying pace, began
with the designation of T-helper 17 (Th17) cells (15, 19
21). As implied by the name, these cells produce IL-17A
and IL-17F but also IL-21 and IL-22. They may also express
the immunoregulatory cytokine IL-9, which can also be
expressed by Th2 and Th9 cells; however, its functional sig-
nificance for Th17 cells is uncertain (2226). Th17 cells can
also express the immunoregulatory cytokine IL-10 perhaps
as a self-imposed negative feedback loop that can be seen in
Th1 cells as well (27, 28). Identification of a subset of T
cells that produce IL-17 was notable for a number of
reasons. As one of the evolutionarily oldest cytokines, IL-17
is important for host defense against extracellular bacteria
and fungi; this is vividly illustrated in the disease Job’s
syndrome (2931). IL-17 is also important for activation of
complement and increase in IgA production from B cells
(32, 33). Moreover, Th17 cells provided an important
‘missing link’ in pathogenesis of autoimmunity (3436).
Surprisingly, in a mouse model of arthritis, IL-17A is crucial
for autoantibody formation (37). Interestingly, within the
Th17 lineage, there is heterogeneity manifested as different
degrees of pathogenicity (38, 39). With the recognition of
Th17 cells, it was edifying that they expressed a master reg-
ulator, retinoid orphan receptor ct (RORct) (encoded by
Rorc); although another related factor RORacan also contrib-
ute, with a minor role, to differentiation of IL-17-producing
cells (40). However, IL-22 is produced by Th17 cells,
another T-cell subset, termed Th22 cells, selectively pro-
duces this cytokine (4143). Th22 cells differ from ‘con-
ventional’ Th17 cells, as they express low levels of RORct,
high levels of T-bet, and mediate protection against Citrobact-
er rodentium (44). In addition, a new subset closely related to
Th2 cells, termed Th9 cells, has been identified, which par-
ticipates in regulation of allergic inflammation, tumor
immunity and, recently, immunopathology (45, 46). As
indicated by the name, these cells produce IL-9, expression
of which is dependent upon TGF-band IL-4. They express a
different ‘master regulator’, PU.1, along with IRF4 and
GATA-3 (4749).
The preceding lineages of helper T cells were all defined
by their production of an eponymous cytokine; however,
one effector subset is not defined in this manner. Such cells
are called follicular helper T cells (Tfh cells); unlike other
subsets, Tfh cells are defined by their location. They are
found in B-cell follicles and germinal centers and provide
help for an efficient antibody production. When dysregulat-
ed, Tfh cells can contribute to autoantibody formation as
exemplified in the sanroque mutant mouse (50). Their signa-
ture cytokine is IL-21, which is also produced by other cells
and thus Tfh cells cannot be uniquely defined by their pro-
duction. Likewise, they express a master regulator TF, Bcl6,
but the expression of this factor is by no means absolutely
limited to Tfh cells (5158).
Any student of immunology will appreciate that in addi-
tion to the array of immune cells with effector functions,
there are also many types of ‘suppressor’ cells. Although
suppressor cells have a checkered history (59), it is now
understandable in retrospect given the multitude of cells and
mechanisms that mediate immunosuppressive functions.
This is certainly true of CD4
+
T cells, with multiple subsets
of CD4
+
T cells being endowed with repressive functionality
(60). The phenomenology of regulatory function was sim-
plified by the recognition of Forkhead box P3 (FoxP3) as
the master TF that is necessary for the development of these
critical regulatory T cells (Treg cells) (61). These cells can
arise in the thymus (thymic Treg or tTreg cells), periphery
(pTreg cells), or can be induced in vitro (62). However,
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24 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
some aspects of Treg cell biology defied the emerging
master regulator/signature cytokine view of specification:
they are functionally critical, but the molecular basis of their
regulatory activity remains incompletely understood. Fur-
thermore, there is evidence that a network of TFs is
required for the Treg cell gene expression program (63,
64). Moreover, FoxP3-expressing Treg cells are not the only
regulatory T cells; multiple types of Foxp3-negative regulatory
T cells have been identified and termed Th3, Tr1, or Tr35 cells
(6568), although the identity of these cells remains some-
what imprecise. These cells produce critical anti-inflammatory
cytokines like TGFb, IL-10, or IL-35, but these are by no
means signature cytokines. On the contrary, many cells,
including effector T cells, broadly produce these cytokines
(69). Even among CD4
+
CD25
+
Foxp3
+
Treg cells, there is
heterogeneity. For example, there are fat- and muscle-resident
Treg cells (70, 71).
Added to this complexity is the recognition that mole-
cules, like perforin, which are expressed by effector cells
have regulatory functions, serving to mediate and limit
effector function (72, 73). Thus, what defines the identity
of regulatory cells and precisely how they exert their immu-
nosuppressive effect encompasses a variety of factors acting
in diverse cells that employ different mechanisms to exert
regulatory function.
CD4
+
T cells have issues with boundaries
Despite views of different T-cell subsets as stable, self-rein-
forcing, terminally differentiated lineages, there was also
evidence early on of a more fluid view of immunoregula-
tion (7476). Much has been written on this topic and
there are many examples of flexibility, so only a few strik-
ing cases will be pointed out. Even though ‘Th17 cells’ were
quickly anointed as a separate lineage, it is well known that
they can make IFN-c, a Th1 cytokine (7782). Indeed, the
current view is that Th17 cells represent a heterogeneous
collection of cells, some of which are pathogenic and
express T-bet, GM-CSF, and other factors, and others that
express IL-10 and are not pathogenic (38, 39). Th2 cells
exhibit plasticity too, and can be reprogrammed into GATA-
3
+
T-bet
+
cells, that produce both IL-4 and IFN-cfollowing
viral infection (83). By their nature, iTreg cells are prone to
Foxp3 instability and can produce effector cytokines (84).
The extent to which tTreg cells are plastic is still the subject
of some debate, although as will be discussed, epigenetic
mechanisms have been identified that help explain their
stability (8587).
Tfh cells are among the hardest cells to characterize as a
simple, distinct ‘lineage’. They do not have a unique pattern
of signature cytokine secretion and have the ability to pro-
duce cytokines of other lineages. Tfh-like cells generated in
vitro can be reprogrammed to make IFN-c(88), and Tfh-like
features are present early in Th1 differentiation (55). This
flexibility is not limited to in vitro differentiation. During hel-
minthic infections, IL-4-producing cells in the lymph nodes
are located in germinal centers, blurring the boundary of
Tfh and Th2 cells (57, 89, 90). Conversely, during a Th1-
type bacterial infection, Tfh cells express IFN-c(57). While
this complicates a simple view of helper T-cell differentia-
tion, it also makes some sense. After all, a major role of
CD4
+
helper T cells is to provide help in particular for B
cells to mount humoral responses. They need not help B
cells in just one way, using a limited palette of cytokines.
Though the emerging consensus is that many differenti-
ated CD4
+
T cells retain at least some degree of plasticity, it
has been assumed that the boundary between CD4
+
and
CD8
+
T cells constitute a more formidable boundary and
these two subsets are true lineages. However, even these ‘ter-
minally differentiated’ cells show more flexibility than previ-
ously assumed. CD4
+
T-cell commitment per se appears not to
be fixed and helper cells can acquire cytolytic functions;
more on this shortly (91, 92). Suffice it to say, that it is
increasingly difficult to argue that differentiated CD4
+
T cells
necessarily produce a selective, fixed transcriptomic program.
CD4
+
T cells: you are not alone!
An additional development in the field that needs to be con-
sidered in discussions of helper T-cell lineage commitment is
that they are no longer the only lymphoid cell subset that
exhibits selective cytokine production. Along with CD4
+
T cells, multiple innate lymphoid cell (ILC) subsets have
been recently identified and divided into three main groups
corresponding to Th1-, Th2- and Th17-associated cytokine
production (93, 94) (Fig. 1). Long recognized as professional
IFN-cproducers are conventional natural killer (NK) cells,
which represent the first Type 1 ILC (ILC1) described. Ini-
tially identified for their spontaneous cytotoxic activity (95
99), NK cells represent a major innate source of IFN-cpro-
duced rapidly before the onset of an adaptive immune
response. In vivo studies have demonstrated that NK cell-pro-
duced IFN-cis important against infections by intracellular
bacteria, parasites, and viruses (100102). In addition to
conventional NK cells, other tissues contain IFN-c-producing
lymphoid cells endowed with lower or no killing activity
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Immunological Reviews 261/2014 25
Shih et al !Toward the elucidation of the helper T-cell regulome
(103105); these cells are also termed ILC1 (94). Thus, in
addition to IFN-c-producing Th1 cells, the IFN-c-producing
lymphocytes include a/bCD8
+
T cells, NKT cells, and c/dT
cells (106, 107).
The innate source of Th2 cytokines has been of interest
for a number of years. Basophils and mast cells can produce
IL-4 (108111), as can NKT cells (112). More recently,
ILCs that produce IL-13 and IL-5 have been identified by
three independent groups and termed nuocytes, natural
helper cells, and innate type 2 helper cells (ILC2 cells),
although the cells identified do not necessarily correspond
precisely to the same subsets (113116). ILC2 functions can
be elicited by IL-25 and IL-33 and can amplify type 2
responses (117). Similarly, whereas mast cells are known to
produce IL-9 (118120), ILC2 are now recognized as the
major producers of IL-9 in the lung (121).
Production of IL-17 and IL-22 in ILC was characterized in
2009, and it is now recognized as ILC3 represent an impor-
tant source of these two cytokines in the earlier phases of
infection (122124). Lymphoid tissue inducer (LTi) cells
and cells expressing NKp46 (currently named NCR
+
ILC3)
belong to these groups and altogether participate in the
development of lymphoid tissues, regulation of epithelium
barrier function, host defense against Citrobacter rodentium, and
shape T-cell responses (125131). Beyond ILC, and other
lymphocytes, such as cd-T cells and NKT cells, expression
of ‘type 3’ cytokines has been described also in neutrophils
(132140).
Production of IL-10 is not limited to T cells but includes
many other cells such as myeloid, B, and NK cells (69,
141144). Bone marrow-derived stromal cells also produce
IL-10 and have suppressor functions. Parenthetically, IL-2,
GATA-3
T-bet
Bcl-6
NK
Liver
ILC1
Gut
ILC1
Thymic
ILC1
ILC2
NCR+
ILC3
CCR6-
ILC3
CD4-
LTi
CD4+
LTi
Rorγt
FoxP3
Eomes
Th1
Th2
Th22
Th17
Treg
Tfh
IL-4
IL-5
IL-13
IFN-γ
IL-17
IL-10
TGF-β
IL-21
IL-22
IL-5
IL-13
IFN-γ
IL-17
IL-22
A
No expression/Not reported
Expression can be acquired after development
Required for development or functionally critical
“Master regulator”
T-bet Eomes FoxP3RorγtGATA-3 PlzfNfil3 Blimp1Bach2Bcl6
Treg
Th17/22
Th1
Th2
NK
ILC3
ILC2
ILC1
CTL
Tfh
Myeloid
B
NKT
γδT
B
Fig. 1. Shared expression of key transcription factors (TFs) in helper T cells and innate lymphoid cells. (A). CD4
+
T cells specify to diverse
subsets that orchestrate immune responses through their selective ability to produce certain cytokines. In the last few years however, it has
become increasingly clear that innate lymphoid cells also exhibit this functional specification. Accordingly, helper T cells and innate lymphoid
cells (ILCs) share a number of key TF that drive selective transcriptomic programs. For instance, T-bet, a key TF for Th1 cells, is also expressed in
IFN-c-producing ILC1 but is also required for development of NCR
+
ILC3. The requirement for Eomes distinguishes NK cells from the other
ILC1. Gata3 not only controls Th2 polarization but also terminal differentiation of ILC2, whereas Rorct is required for development of Th17/22
and ILC3. (B). The usage of different TFs among various immune cells is depicted using a heat map based on current literature.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
26 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
the prototypic T-cell growth factor, is produced by non-T
cells including dendritic cells (145) and by a specific ILC1
subset (146). So the bottom line is that selective cytokine
production is hardly the sole domain of CD4
+
T cells: it
appears that no cytokine is produced exclusively by T cells
and furthermore CD4
+
T-cell lineages are not the only
immune cells that have the capacity to selectively produce
restricted cytokine programs. This appreciation has profound
implications for the concept of cell identity and specifica-
tion, and the role of TFs as we consider exactly what it is
required for helper T-cell differentiation.
Transcription factors acting across immune cell fates
It is famously stated, accurately or otherwise, that Eskimos
have more than 100 words for snow and ice. Similarly,
Sami speakers of Lapland are said to have hundreds of
words to describe reindeer as well as snow. This may be a
reasonable metaphor for immunologists in the early 21st
century. We have become very good at paying attention to
the enormous range of subtle and not so subtle differences
among populations of immune cells. The challenge, of
course, is to move beyond simple descriptions and to pro-
vide solid molecular and mechanistic explanations that
explain and predict the actions of lymphoid cells in terms of
the patterns of gene expression and regulatory networks.
For this reason, it is useful in thinking about the specifi-
cation of CD4
+
T cells to keep firmly in mind that innate
and adaptive lymphoid cells share common bone marrow
progenitors and share many functionalities. This is certainly
true with respect to selective production of cytokines. The
fact that so many immune cells have the capacity to discrim-
inately express virtually all of the cytokines produced by
helper T cells implies that this capacity and the attendant
machinery is in place prior to the specification of ILCs and
T cells (including c/d, NKT, CD4
+
, and CD8
+
cells). In
other words, the capacity to effect specialized gene expres-
sion as it relates to cytokines genes must arise earlier in
ontogeny than diversification of lymphocytes from other
products of hematopoietic stem cells (HSC). The functional-
ities of T cells and ILCs are likely to be superimposed upon
pre-existing programs.
While defining the precise relationships between the dif-
ferent cells is still a work in progress, we do know the iden-
tities of a number of TFs that are fundamentally important
for HSC development and fitness. Those factors set the stage
for generation of differentiated immune cells. Factors
include: Ikaros, E2A, Pu.1, Bcl11a, as well as Hox, Runx,
and Gata family members, and all are important contributors
to early events in hematopoiesis (147151) and lymphocyte
specification as well.
Deciphering lineage specification
The extraordinary variety of immune cells is coordinated by
the regulatory network of TFs, which shapes cell features
and identity. In this network, some TFs can define and/or
preserve boundaries among lineages. However, the same TFs
can be ‘recycled’ during differentiation by switching their
expression on and off, serving distinct functions at different
times. They can be also shared among the different lineages,
making the boundaries of lineage-defining TFs blur and dif-
ficult to distinguish (Fig. 2).
A major determinant of T-cell development is Notch1;
T-cell differentiation is completely blocked in the absence of
this factor, with resultant expansion of B cells in the thymus
(152, 153). A constitutively active form of Notch promotes
expansion of T cells in the bone marrow at the expenses of B
cells (154). Notch signaling though is not just important for
T cells; ILCs, DCs, and splenic marginal zone B cells are also
affected by the absence of Notch. T-cell factor 1 (TCF-1)
(encoded by Tcf7) is induced by Notch and it is also
required for generation of T cells and specific ILC subsets
(155158).
If Notch, at least to some extent, is the switch for B/T-cell
fate, E2A, a basic helixloophelix (bHLH) TF controls T/ILC
bifurcation. Multiple steps of T-cell development in the thy-
mus require the activity of this TF or the related protein HEB
(159163). Bcl11b is yet another factor that is important for
double-negative thymocytes, repressing genes expressed in
stem cells and preventing the expression of NK-cell lineage
genes (164). Fate choice between a helper versus cytotoxic T
cells is controlled by the mutually antagonistic actions of Th-
inducing POZ-Kruppel factor (ThPOK), encoded by the zinc
finger and BTB domain containing 7b (Zbtb7b) gene, and the
related protein LRF (165) and Runx (166169); (170).
By inhibiting the transcriptional activity of E proteins, the
inhibitor of DNA binding (Id)-2 (a bHLH protein) pro-
motes generation of all ILC (114, 171173). Deletion of
E2A in Id2
"/"
mice is sufficient to restore generation of NK
cells (174), while overexpression of Id3 promotes NK cell
development at the expense of T/B lymphocytes in an in vi-
tro system (175). Unlike ILCs, Id2 deletion is not sufficient
to abrogate development of thymic invariant NKT (iNKT)
cells, due to the redundant role of Id3 in promoting iNKT
lineage specification (176).
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 27
Shih et al !Toward the elucidation of the helper T-cell regulome
The basic leucine zipper TF encoded by Nfil3 (also called
E4bp4) was initially proposed as the first TF specifically
required for NK cell development (177, 178). Surprisingly,
during viral infection, activating receptors and pro-inflam-
matory cytokines can drive generation of fully competent
NK cells in absence of Nfil3 (179). However, requirement of
Nfil3 is not restricted to NK cells and broadly contributes to
the differentiation of other ILC1 subsets, and CD8a
+
DC,
IgE class switching in B cells and regulation of cytokine pro-
duction in CD4
+
T cells (180186). Moreover, an expanded
role has been attributed to the signature TF for iNKT
development, PLZF (promyelocytic leukemia zinc finger pro-
tein) (encoded by Zbtb16, a member of the POK family). It
is expressed by a precursor that generates all helper ILCs,
with the exception of NK and LTi cells (187). Finally, thy-
mocyte selection-associated high mobility group box (Tox)
is a factor that is important for both CD4
+
T cells and LTi
cells (188, 189). Tox
"/"
mice show decreased LNs and Pe-
yer’s patches, and absence of NK cells; whether other ILC
are affected has not been investigated.
STATs and lymphoid development and differentiation
Cytokine signaling is a critical determinant of the lymphoid
differentiation programs. Signal transducer and activator of
transcription (STAT) family includes seven members (STAT1-
4, STAT5A, STAT5B, and STAT6) able to transmit signals
from most cytokines and to regulate unique spectra of gene
sets. The advent of ChIP-seq technology, which has rapidly
advanced over the last few years, has quickly expanded the
knowledge of the molecular functions of STATs on T cell.
Here, we review some of the main concepts concerning the
role of STATs in lymphocyte differentiation.
Among the different STATs, STAT3, and STAT5 cover a
wide spectrum of functions, even beyond the hematopoietic
system. There are two Stat5 genes, Stat5a and Stat5b, which
play a non-redundant role in mammary gland development
and growth hormone signaling, respectively (190193).
Deletion of both alleles typically results in growth retardation
and perinatal lethality due to anemia (194). STAT5 is a
critical factor for the hematopoietic system and the entire
lymphoid compartment, controlling HSC fitness, lymphoid
Th1
NK/ ILC1
ILC2
Th2
Th17/22
ILC3
Treg
Tfh
T-bet
Gata3 Rorgt FoxP3 Bcl6
Tox
Gata3 STAT5 Notch
Eomes
E2A/Id2
STATs
Fig. 2. Transcription factor (TF) network: the Aspen Grove. Subsets of CD4
+
T cells and innate lymphoid cells (ILCs) exhibit the same or
similar functionalities. Presumably, this is because they employ many of the same TFs working in concert to effect similar programs. As a
metaphor for this problem, we considered that though aspen trees appear to separate trees, they are, in fact, connected by a shared root system.
We posit that the fundamental network of TFs might function analogously such that the same ‘root system’ (i.e. TF network) is used as cells
differentiate from hematopoietic stem cell to the multiplicity of lymphoid cells, some of which express a T-cell receptor and some of which do
not. Many TFs act deeply below the surface (Notch, GATA-3, STAT5) being fundamental for generation and maintenance; others are important
for acquisition of more specific functions later on. Regardless though, even if it appears that one is looking at different ‘trees’, one does not have
to dig very deep to find the common elements. The Pando grove is the largest known aspen grove located in Utah (USA), and comprises more
than 100 acres and 43 000 trees. It is estimated to be 80 000 years old.
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28 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
cell development/homeostasis and, later on, Th polarization
(195197). Its relevance relates to the importance of c-kit
(stem cell factor) and IL-7 signaling for HSC and lymphoid
development, and IL-15 signaling for generation of conven-
tional NK cells and homeostasis of memory T cells (198,
199).
STAT5 Chip-seq data revealed that regulation of homeo-
stasis during Th polarization occurs through direct binding
of STAT5 to genes important for proliferation and anti-
apoptotic activity (including cyclin genes and Bcl2). STAT5
controls Treg homeostasis and generation, directly by regu-
lating the Il2ra and Foxp3 genes generating a positive loop in
which stable expression of Foxp3 is influenced by expres-
sion of IL-2 receptor (200). STAT5 is essential for both Th1
and Th2 cell differentiation by transmitting IL-2 signals
(201). On the other hand, IL-2, through STAT5, suppresses
formation of Tfh and Th17 cells (202206). STAT5 can
directly inhibit Il17a and promote FoxP3 expression by
competing with STAT3 (207). In summary, STAT5 is a criti-
cal TF for lymphocytes at all stages of their differentiation.
Elucidation of the stage-specific versus unique functions of
STAT5 is still being resolved.
Many of the paradigms concerning the role of STATs have
been developed by the plethora of evidence concerning
helper T-cell polarization (208). In the initial rigid mono-
lithic view of Th polarization, each STAT (except STAT2)
was argued to be associated with a given T-cell fate. While
some STATs are more easily linked to particular T-cell subset
(e.g. STAT4 and STAT6 with Th1 and Th2, respectively), it
is now recognized that each subset can be influenced by
multiple STATs. A good example is provided by Tfh cell
development, which is promoted by the complementary
actions of STAT1, STAT3, and STAT4 (55, 209214).
Among the ways STATs promote specific helper features
is through direct interaction and activation of ‘master regu-
lator’ TF genes. Like STAT5 and Foxp3, STATs directly regu-
late Tbx21,Gata3, and Rorc. STATs regulate hundreds of other
genes, including many other ‘lineage-specific’ loci including
cytokines, cytokine receptors, chemokines, and chemokine
receptors (215217), microRNAs (218), and lincRNAs
(219, 220) (see section below).
Although the role of STATs has been relatively poorly
characterized in ILC, especially in terms of defining targets
by Chip-seq, it is likely that they will regulate many of the
key loci that contribute to ILC function, especially those
that are shared with T cells. It will be of great interest to
dissect shared and unique actions. NK cells express high
basal levels of STAT4, and their effector functions are
highly affected in STAT4-deficient mice (221). At the same
time, STAT3 deficiency in ILC3 impairs their ability to pro-
duce IL-22 and IL-17 (222). Whether STAT6 can partici-
pate in regulation of effector functions in ILC2 has not
been characterized yet. However, the two main cytokines
involved in ILC2 activation, IL-25 and IL-33, do not use
STAT6 for their signaling.
Function of helper cell master regulators beyond Th
differentiation
The classical helper T-cell master regulators, T-bet, GATA-3,
and Rorct, have functions beyond this restricted role. Even
though T-bet (encoded by Tbx21 gene), initially described as a
Th1 specific TF (11) and an important factor for acquisition
of type 1 features in Th cells, it is also expressed in CD8
+
T
cells, NKT cells, conventional NK cells/ILC1, specific ILC3
subsets, myeloid cells, and B cells (223). Th1 responses and
development of tissue-specific ILC1, along with effector func-
tions of CD8
+
T cells, conventional NK cells, and NKT cells
are all T-bet dependent (224229). Global profiling of T-bet
binding and its impact on transcription and epigenetics has
now been accomplished (230). T-bet binds the Ifng,Il12rb2,
and Cxcr3 loci, and promotes expression of these genes. The
integration of T-bet binding and transcriptional profiling in
T-bet-deficient cells suggests that only 6% of genes bound by
T-bet are transcriptionally regulated by this factor, but overall
the number of genes positively or negatively regulated by
T-bet are comparable.
T-bet also seems to be important for IL-22 production in
Th22 cells (44), and it is relevant for generation of NCR
+
ILC3 (224, 231). ILC3 expressing T-bet can acquire the
ability to produce IFN-cand can convert to ‘pure’ type 1
ILC, but the requirement for development implies a function
beyond regulation of IFN-c(107).
CD8
+
T cells and conventional NK cells illustrate the
importance of another, non-redundant T-box TFs, Eomeso-
dermin (Eomes). In CD8
+
T cells, the fine-tuned regulation
of T-bet and Eomes expression can direct fate to the mem-
ory versus effector cells (232). High expression of Eomes is
a hallmark of conventional NK cells among the other ILC1,
expressing T-bet only and differing for cytokine production
(146, 229, 233).
GATA-3 plays a broad role in lymphoid development.
During T-cells development in the thymus, expression of
Gata3 is finely regulated. Notch, Tcf1, and T-cell receptor
(TCR) signaling are important for its induction, while E2A
proteins restrain GATA-3 expression (234). Beyond its role
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Immunological Reviews 261/2014 29
Shih et al !Toward the elucidation of the helper T-cell regulome
in T-cell lineage commitment, GATA-3 is important to drive
generation of CD4
+
T cells at the expense of CD8
+
T cells,
both by inducing ThPOK expression (168) and by repress-
ing Runx3 (235). GATA-3’s role in Th2 cells is well appre-
ciated, being induced by IL-2 and IL-4 in a STAT5 and
STAT6-dependent manner, respectively. GATA-3 is also
important for ILC2 cell differentiation and is also required
for maintenance and maturation of a lineage-specific ILC2
precursor in the bone marrow (236238). Global gene
expression analysis reveals similar function of GATA-3 in
ILC2 and Th2 cells regulating the same pattern of cytokines
and receptors (239).
An unbiased analysis of GATA-3 in Th2 cells suggests that
60% of the genes that require GATA-3 for transcription also
exhibit GATA-3 binding, arguing for a direct mode of
action in a relatively large proportion of genes (240).
Rorct, encoded by Rorc, is essential for generation of
Th17 cells, but like T-bet and GATA-3, it too has broad
functions in ILCs and other cells (131). Rorct is important
for survival of DP thymocytes and expression of Bcl-xL
(241, 242). It is also important for the lymphoid organo-
genesis and generation of ILC3 (104, 241243), NKT
(244), and cd-T cells (15). It is also expressed in non-lym-
phoid cells, including neutrophils, another source of IL-17
(133). The genome-wide characterization of Rorct binding
argues that this protein has a relatively focused mode of
action serving as modulator rather than a master TF in the
conventional sense. In fact, Rorct binding is associated with
modest changes in gene expression in Th17 cells relative to
Th0 cells (217). The atypical nuclear factor I kappa B family
member, IkBf(encoded by Nfkbiz) acts in concert with
Rorct to promote Th17 differentiation (245). The role of
Nfkbiz in ILCs has not been explored, but it would not be
surprising if it is relevant for these cells.
The importance of FoxP3, other Forkhead Box proteins,
and their actions have been intensively reviewed and are not
being discussed here. Interested readers are referred to many
other outstanding reviews of this important topic (246
248).
Repressors abound
Also of interest in terms of helper T-cell function are three
key repressors Blimp-1, Bach2, and Bcl6 (249, 250). Identi-
fied first in B cells, these TFs are in fact expressed in many
cell types. Perhaps more interestingly, they create a tran-
scriptional network that can regulate one another (251). In
B, T, and NK cells, Blimp-1 is associated with terminally
differentiated cells (252258). In B cells, it is the master
regulator of plasma cell formation, suggesting that Blimp-1
controls gene programs that drive a highly differentiated
state (259, 260). Both Bcl6 and Bach2 can repress Blimp-1,
suggesting early and inappropriate activation of Blimp-1 is
detrimental to the cellular differentiation process (52, 261,
262). In the absence of Bach2, plasma cells form too early,
and both germinal center responses and class switch recom-
bination are impaired (263, 264).
A critical role for Bach2 in T cells was described recently,
where Bach2 acts to restrain effector T-cell differentiation by
suppressing Blimp-1 and other targets (265, 266). This is
especially critical in Treg cells, where increased Bach2 levels
control effector T-cell genes and prevent the development of
a lethal autoimmunity (265, 266).
In contrast to Blimp-1, Bcl6 is considered to be the mas-
ter regulator of germinal center reactions (267, 268). In
addition to controlling the DNA damage response and cell
cycle checkpoints in GC B cells, a major role of Bcl6 is to
suppress Blimp-1 and plasma cell development until somatic
hypermutation and class switch recombination are com-
pleted (249, 269, 270). In T cells, Bcl6 is proposed as the
master TF required for Tfh cell formation (5254). Blimp-1
can also repress Bcl6, and overexpression of Blimp-1 results
in severely impaired Tfh responses (52). Mutations in all
three of these TFs are associated with lymphomagenesis,
further emphasizing the critical role these factors play in
controlling cellular differentiation (249).
Although these factors are members of different families,
they work in a similar fashion. All have N-terminal protein
protein interaction domains, with C-terminal DNA-binding
domains. Blimp-1 recruits co-repressors such as G9a and
HDAC1/2 and induces repressive marks like H3K9 methyla-
tion (271273). Bach2 and Bcl6 both have BTB protein
protein binding domains that mediate proteinprotein
interaction, and function as homodimers, or interact with
each other. In addition, they bind other TFs and recruit
co-repressor complexes (274, 275). Bach2 was identified in
B cells in a pull-down with MafK, and has a bZIP DNA-
binding domain that can bind DNA elements that are well
known to also bind AP-1 family members (275, 276).
While mainly described as a repressor, examples of Bach2
acting as an activator have been described (277). Bcl6 has a
zinc finger DNA-binding domain, and recruits the co-repres-
sor complexes SMRT, NCOR, and BCOR (278, 279). New
models suggest Bcl6 can repress transcription by two distinct
but simultaneous mechanisms (280). Bcl6 can repress pro-
moter regions by depletion of activating marks, and addition
of repressive marks via a ternary complex with BCOR and
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30 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
SMRT/NCOR. A second mechanism acts on a different set of
genes to switch enhancers from an active to a poised config-
uration by recruiting the deacetylating SMRTHDAC3 com-
plexes and opposing the action of the histone
acetyltransferase (HAT) p300 (280). Although the basics of
how these factors repress have been established, the target
genes they each act on in specific cell types and conditions
still remain unclear. Far more work is needed to fully
understand the role these factors play by modulating the
epigenetics of chromatin to control gene expression and
cellular differentiation.
More players in the TF network
TCR signals are essential for initiation of CD4
+
T-cell differ-
entiation and signal strength biases T-cell programming
toward divergent differentiating directions. In this setting,
nuclear factor of activated T cells, adapter-related protein
complex 1 (AP-1) (encoded by Fos and Jun), and nuclear fac-
tor-jB (NF-jB) among other TFs are important regulators
of gene expression (281). While ILCs do not express anti-
gen receptors, a variety of receptors including Ly49, NKG2,
and integrin family members can provide signals that acti-
vate that the aforementioned TFs, which presumably activate
many of the same target genes (282, 283).
Other TFs including basic leucine zipper transcription fac-
tor (BATF), which can form AP-1 complexes, and a ternary
complex with interferon regulatory factor 4 (IRF4), are also
essential for Th differentiation (284289). In Th17 cells,
BATF and IRF4 are globally co-localized in the genome and
both required for remodeling chromatin landscape for depo-
sition of other TFs (217, 290). Along with STAT3, BATF,
and IRF4, influence genome-wide histone acetyltransferase
p300 occupancy in Th17, whereas RORct has minimal
effects (217). Increasing evidence suggests BATF and IRF4
are ‘pioneer factors’ for permission of lineage specification.
However, how these pioneer TFs from TCR signaling inter-
act with polarizing TFs regulated by cytokines to tune the
gene expression remains unclear.
Maf was originally identified as a Th2-associated TF, but
is induced by IL-6, IL-27, and STAT3 and so is expressed in
Th17, Tfh, and Tr1 cells (292, 293). Maf has been reported
to be a positive regulator of IL-10 (293). It is also induced
by TGFband directly inhibits Il22 (294).
Due to the exposure to the mucosal barrier, generation of
Th17 cells and ILC3 subsets share many other common fea-
tures, such as dependency on bacteria, environmental fac-
tors, and dietary components. Aryl hydrocarbon receptor
can affect expression of IL-17 and IL-22 in T cells, and it is
also required for the generation of ILC3 cells (295299).
Dietary stress, such as vitamin A deprivation, highly impacts
ILC3 generation (300), while Th17 generation is favored
(301). Finally, dietary salt can enhance IL-23-mediated
Th17 differentiation by regulating serum glucocorticoid
kinase 1 (SGK1). One action of SGK1 is to deactivate the TF
Foxo1 (302, 303). SGK1 can also promote Th2 and repress
Th1 cell differentiation (304).
At the risk of overwhelming readers, it should be clear from
the above that numerous TFs work in concert to drive gene
expression. While it may seem like an impossibility to sort
out their discrete, cell- and stage-specific functions, Chip-seq
technology does provide a high-throughput means to experi-
mentally identify potential direct targets of TFs. Using gen-
ome-editing technology it should be feasible to introduce
specific binding-site mutations and prove causality of some of
these DNA-binding events. We are in our infancy of such
studies, and the data and work ahead will be overwhelming;
nonetheless, it should be possible to identify precise functions
amidst this apparent cacophony. But wait, it is not just about
TFs acting on protein-coding genes.
Gene expression and epigenetic controls
While key TFs working in a combinatorial fashion are essen-
tial elements for cell specification, their ‘substrate’, DNA, is
anything but a passive participant with respect to control of
gene expression. DNA is packaged into nucleosomes and
chromatin, and variety of DNA and chromatin modifications
contribute to the accessibility of DNA. The regulatory mech-
anisms that promote or restrict DNA accessibility include:
DNA methylation, histone modifications, nucleosome posi-
tioning or remodeling, chromatin insulators, and long-dis-
tance chromatin interactions. All of these factors weave a
complicated network now referred to as the epigenome that
contributes each unique cell identity and fate determination.
By analogy, the RNA within a cell is neither linear nor
naked; therefore, RNA-binding proteins and the epitran-
scriptome will need to be considered.
A major challenge in the field though is to understand
how epigenetic modifications allow or prevent TF access to
key sites in the genome. Alternatively, TFs can also modify
the epigenetic landscapes (so-called pioneer factors) (305,
306) (Fig. 3). In addition, it is now well appreciated that
the control of lineage-specific programming extends far
beyond the small portion of the genome that encodes
conventional genes that give rise to proteins. Only a tiny
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Immunological Reviews 261/2014 31
Shih et al !Toward the elucidation of the helper T-cell regulome
portion of the genome encodes such genes (<2%); a consid-
erably greater portion of the genome is transcribed and
these diverse RNAs generate many products large and small,
including microRNAs (miRNAs), enhancer RNAs (eRNAs),
and long non-coding RNAs (lncRNAs). Emerging data indi-
cate that these products themselves are important in control-
ling gene expression. In addition, chromatin accessibility
and architecture also function as ‘switches’ that regulate dis-
tal gene activities by facilitating or excluding TF binding to
cis-regulatory elements including promoters and enhancers.
Promoters are DNA sequences located upstream of transcrip-
tion start sites (TSS) and are essential for transcription by
recruiting the transcriptional apparatus. Enhancers regulate
gene expression also by recruiting TFs and are ‘distal’ in
terms of linear distance from genes; because of looping of
DNA and higher order chromatin conformations, enhancers
can bring TFs to promoters. An important goal is to
integrate the action of TF networks with modifications of
epigenetic landscapes, signaling pathways and cellular
metabolism. This is an active area of research that has
already yielded a number of surprises. Traditionally, defin-
ing how gene transcription was influenced by the epigenetic
landscape was a significant challenge; however, thanks to
the development of deep-sequencing technology and bioin-
formatic methods, nowadays it is reasonably straightforward
to measure genome-wide gene expression for both coding
and non-coding RNAs, TF binding, and epigenetic dynam-
ics. We will briefly summarize the current views of
H3K4me1
LDTF
Pioneer TF
P300
H3K4me1
P300
H3K27AcH3K27Ac
Promoter
Active Enhanceosome
EZH2
or
H3K27me3 H3K27me3
EZH2
Repressive Chromatin
Fig. 3. Transcription factors (TFs) and the multistep process of remodeling heterochromatin to create active enhancer landscapes. Pioneer
TFs first scan the genome and then bind their cognate recognition DNA sequences even though these regions are still in a closed configuration.
The binding of pioneer TFs facilitates the recruitment of histone modifiers and chromatin remodelers to label these regions with active markers
like H3K4 mono-methylation, as well as decompaction of chromatin structure by nucleosome depletion or replacement. Up to this step, the
enhancers are accessible but remain poised prior to binding of lineage-determining TFs (LDTFs) binding. Once the LDRFs are induced or activated
following appropriate stimuli, they occupy previously marked poised enhancer regions and recruit the histone acetyltransferase p300, which
catalyzes H3K27 acetylation to finalize the activation of enhanceosome. These structures further ‘enhance’ activation of target genes through
physical interactions and chromatin looping. On the other hand, repressors can also bind these open sites and promote inaccessible chromatin
configuration.
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32 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
epigenetic regulation and their roles in programming cellu-
lar differentiation using examples pertinent to lymphocyte
biology and provide instances in which we have begun to
understand how TFs modify the epigenome.
DNA methylation
DNA methylation modifies cell development and differentia-
tion by attracting specific proteins or making DNA less
accessible to TF binding (307). DNA methylation at the fifth
carbon of cytosine (5mC) occurs mainly at CpG dinucleo-
tides that are abundant across the genome (approximately
70% of promoters contain high frequency of CpG sites,
termed CpG islands). DNA methylation can repress gene
activity through recruiting repressor complexes that contain
methyl CpG-binding domain. It can also simply prevent
interaction with some DNA-binding proteins that can either
activate or repress transcription. Methylated CpG islands also
influence nucleosome positioning (308, 309). Methylation
of cytosine is catalyzed de novo by DNA methyltransferases
(DNMT) 3A and DNMT3B, and then maintained by DNMT1
during mitosis. Absence of DNMT1 in naive CD4
+
T cells
results in abnormal cytokine expression (310).
Methylated DNA has been argued to be among the most
stable epigenetic marks; however, there are multiple exam-
ples in lymphocytes of rapid or active demethylation. For
instance, the Il2 locus is quickly demethylated upon T-cell
activation (311), and the Ifng,Il4, and Il17 loci are demethy-
lated during differentiation of Th1, Th2, and Th17 cells,
respectively (312316). These loci remain methylated in
cells that are differentiated to opposing fates (e.g. the Ifng
locus is methylated in Th2 cells). In addition, demethylation
of Foxp3 and other Treg signature genes is important for sta-
bilization of Treg fate (317319). At present, there are no
comprehensive, genome-wide comparisons of DNA methyl-
ation among the different helper T-cell subsets and the con-
sequences on transcription are not well known.
The role of DNA methylation has also been studied in
ILCs, but no comprehensive maps have been provided. DNA
methylation is important for the regulation of Ly49 genes in
NK cells, a collection of loci clustered on chromosome 6
(320, 321). These genes are variably expressed by different
mouse strains (322) and are subject to allelic exclusion
(323). However, precisely how other characteristic features
of ILCs are or are not controlled by methylation has not
been determined.
The biochemical basis of DNA demethylation has been
elusive, and while it has been proposed that the loss DNA
methyl-groups could occur simply by dilution during cell
division, this does not explain the rapid demethylation of
the Il2 locus that occurs independent of cell proliferation
(311, 324). Recently, new insights into the processing of
methylated DNA have emerged. Instead of a simple erasure
of the methyl group, 5mC is sequentially converted into
5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC),
and 5-carboxylcytosine (5caC) through oxidation by ten-ele-
ven translocation (TET) proteins-catalyzed oxidations (325).
In mouse embryonic stem cells (ESCs), Tet1 and Tet2 are
highly expressed, whereas in differentiated cells, Tet2 and
Tet3 are the major TET enzymes. The 5fC and 5caC are fur-
ther removed by thymine DNA glycosylase and the base
excision repair pathway.
The dynamic 5mC oxidation forms can regulate gene
expression by further modulating protein binding land-
scapes. For instance, MeCP2 recognizes both 5mC and
5hmC, whereas repressive complexes recruiter, methyl CpG-
binding domain protein 1 (MBD1) and MBD2 only bind
5mC but not 5hmC. MBD2 has been linked to regulate
demethylation of immune-related genes (326328). MBD2
mediates demethylation and TET2 binding of a CpG-rich
region upstream FoxP3, which is critical for FoxP3 expres-
sion in thymic Treg (tTreg) cells (327, 328). Hence,
dynamic 5mC oxidation forms and the proteins each of
them recruits can be important for lineage specification.
Despite our very incomplete understanding of the gen-
ome-wide state of DNA methylation in lymphocytes in
health and diseases, a number of new techniques are now
or becoming available that will help fill the gaps in our
knowledge. Currently, genome-wide DNA modification of
each 5mC oxidation form can be identified at single base
resolution by combining bisulfite-based chemical reactions
with deep sequencing, including bisulfite-sequencing (BS-
seq), oxidative bisulfite-sequencing (oxBS-seq), Tet-assisted
bisulfite sequencing (Tab-seq), and chemical modification-
assisted bisulfite sequencing (fCAB-seq) (reviewed in 325).
Interestingly, the demethylation intermediates are enriched
at regulatory elements (329331). For instance, 5hmC is
enriched at promoters with ‘bivalent’ histone modifications
as well as active enhancers (332336). Comprehensive mea-
surement of genome-wide methylation remains technically
challenging and is costly. As a result, a comprehensive DNA
methylation map of relevant cytokine-producing subsets is
lacking; however, it will surely be the case that many
immune response-related genes are tightly regulated by
DNA methylation/demethylation.
Alterations in DNA methylation are not just relevant to
our basic understanding of helper T-cell differentiation but
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Immunological Reviews 261/2014 33
Shih et al !Toward the elucidation of the helper T-cell regulome
also may be relevant to the pathogenesis of immunologic
diseases (337), especially systemic lupus erythematosus
(338341). Drugs that affect DNA methylation can cause
lupus in humans and also in mouse models (342, 343).
Nucleosome positioning and histone modifications
Nucleosomes are the basic units of chromatin that contains
a histone octamer wrapped by 147 base pairs of DNA. The
dynamic nucleosome positioning and histone modifications
play key roles in determining chromatin accessibility to TF
binding. At cis-regulatory elements, such as promoters and
enhancers, nucleosomes are usually depleted or replaced by
more dynamic histone variants like H2A.Z and H3.3. There-
fore, cis-regulatory elements are usually more accessible and
sensitive to DNA nucleases and can be predicted by DNaseI
or Micrococcal nuclease hypersensitivity.
The mechanisms for nucleosome positioning are compli-
cated and not fully understood. In addition to DNA
sequence preference, both ATP-dependent chromatin remo-
delers and transcription machinery are also involved in the
localization of nucleosome positioning (reviewed in 344).
SWI/SNF complex, one of the ATP-dependent chromatin
remodelers, can loosen nucleosomes using the energy from
ATP hydrolysis and cause nucleosome depletion or sliding.
SWI/SNF complexes are essential for remodeling chromatin
at multiple stages of T-cell development in thymus while
receiving external signals from pre-TCR and TCR (345) and
for the bifurcation of CD4/CD8 SP T cells by silencing CD4
expression (346). This mechanism is also relevant to helper
T-cell differentiation. In Th1 differentiation, Brahma-related
gene 1 (BRG1), one of SWI/SNF complex subunits, targets
Ifng locus for nucleosome remodeling in a STAT4-dependent
manner (347). In cooperation with STAT4, BRG1 also regu-
lates Il12rb2 gene expression in Th1 cells (348). BRG1 is
also required for full activation of Treg ability to suppress
autoimmunity (349).
Post-translational modifications of histone proteins create
epigenetic codes that mark distinct chromatin status and
function (350352). Histone H3 lysine 4 trimethylation
(H3K4me3) and H3K36me3 mark active transcription;
H3K4me3 modification is highly enriched at TSS regions,
while H3K36me3 modification preferentially spreads across
the transcribing gene body. Conversely, histone methylation
of H3K9, H4K20, and H3K27 are linked to gene repression.
High H3K4 mono-methylation (H3K4me1) and low
H3K4me3 modifications are recognized as general features
of enhancers, in which ‘active’ enhancers can be distin-
guished from ‘poised’ enhancers by H3K27 acetylation and
acetyltransferase, p300 and/or CBP binding. The roles of
other histone acetyltransferases such as PCAF and GCN5 are
not yet known in T cells. Using these marks, studies have
revealed global enhancer landscape that will be discussed
later. In addition, histones can be modified by many other
post-translational modifications including phosphorylation,
ubiquitination, and sumoylation; we are just beginning to
decipher all of the elements of the ‘histone code’.
Although particular histone marks has been associated with
either promoting or repressing transcription and even
splicing, genetic evidence in mammals is for the most part
lacking.
H3K27 trimethylation is catalyzed by histone methyltrans-
ferase Ezh1 or Ezh2, a subunit of polycomb repressive com-
plex 2 (PRC2). Ezh2 has been linked to various types of
cancers including prostate cancer, breast cancer, and leuke-
mia (353355). In CD4
+
T cells, Ezh2 is important for
modulating Tbx21 and Gata3 expression in Th1 and Th2
cells, respectively (356, 357). It suppresses Eomes expres-
sion (357) and stabilizes T-bet levels through both tran-
scriptional and post-translational regulation in Th1 cells
(358). In Th9 cells, TGF-b-activated Smad proteins displace
Ezh2 from the Il9 locus promoting expression of the
encoded cytokine (359). In Treg cells, Ezh2 is induced and
recruited to FoxP3-bound regions of the genome following
inflammatory stimuli. This results in increased H3K27
trimethylation and repression of nearby genes (360). In
Th17 cells, a DNA-binding protein called Jarid2 is required
for recruitment of PRC2 to its chromatin targets, which
include Il22,Il10,Il9, and Atf3 (361). PRC2 is generally
thought to lead to PRC1 recruitment; however, the role of
PRC1 in T cells is not known. In addition to nuclear func-
tions, however, Ezh2 also controls TCR-dependent actin
polymerization (362).
H3K9me3 is another important repressive mark that
recruits heterochromatin protein (HP) for gene silencing.
During T-cell differentiation, Th2 cell commitment requires
H3K9me3 involved repression of Th1 loci (363). In addi-
tion, H3K9me3 also controls CD8
+
T-cell memory progres-
sion by Blimp-1-dependent recruitment of G9a histone
methyltransferase to the Il2ra and Cd27 loci (271).
The genome-wide enumeration of permissive and repres-
sive histone marks in helper T cells has been obtained and
helps explain several features of distinctive gene expression
in helper T cells (364). As expected, characteristic genes
associated with lineage commitment have the predicted
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34 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
accessible marks in their respective lineage and repressive
marks in opposing lineages. However, genes that encode
key regulatory TF s including Tbx21,Gata3,Rorc,Prdm1, etc.
have more complex features. The combination of H3K4me3
and H3K27me3 modifications, so-called ‘bivalent’ domains,
is indicative of genes that are poised for expression (364).
In principle, this could provide an explanation for the plas-
ticity of TF expression. Other TFs like Bcl6 reveal a different
pattern; the epigenetic marks surrounding this locus show
that the Bcl6 gene accessible in all subsets (55). This helps
explain the fact that multiple T-cell subsets can acquire fea-
tures of Tfh cells. Thus, the epigenetic landscape of genes
encoding master regulators may allow flexibility in expres-
sion and thereby permit the blurring lineages, allow fine
tuning or provide sub-specialization.
As discussed, a critical issue is defining the factors respon-
sible for creation and modification of epigenetic landscapes.
STAT proteins are one important class of TFs that regulate
lineage-specific expression profiles by shaping histone modi-
fication patterns. In Th1 cells, STAT4 is essential for promot-
ing genome-wide H3K4me3 modification for activated
genes, whereas in Th2 cells, a major aspect of STAT6’s
action is to influence the removal of repressive H3K27me3
modification on poised loci (216). In addition, analysis of
epigenetic marks and transcription activities of STAT4 target
genes reveals that STAT4 can regulate histone modifications
or transcription independently. That is, for STAT4-bound
genes, only a very small proportion (4%) is STAT4-depen-
dent in terms of both histone modifications and transcrip-
tion. In contrast, 11% shows STAT4-dependence with
respect to transcription only and another 20% shows
STAT4-dependence for epigenetic modifications only. These
observations suggest STAT4 impacts cell phenotype in vari-
ous ways. This point is particularly important because STATs
were first identified as gene activators; however, identifica-
tion of STAT4-dependent repressive markers with genome-
wide analysis suggests a role for STAT4 as a transcriptional
repressor as well as its more widely recognized role as a
transcriptional activator.
Epigenetic modifications communicate with transcrip-
tional machinery through certain ‘histone code readers’.
For example, bromodomain and extraterminal (BET) pro-
teins can recognize acetylated histones. BET proteins,
including BRD2, BRD3, BRD4, and BRDT, provide a bridge
on chromatin to connect histone modifiers, chromatin rem-
odelers, and Mediator complex for gene regulation (365).
BRD4, for instance, can recruit the positive transcription
elongation factor b (P-TEFb) complex to promote phos-
phorylation of paused RNA polymerase II for mRNA elon-
gation. BET proteins have been proposed to be target for
cancer therapy because they regulate oncogenesis-related
growth factors such as c-myc in cancer cells (366). Of
note, BET proteins also play a role in the regulation of
pro-inflammatory cytokines and chemokines as well as T-
cell differentiation. Targeting BET proteins with small mole-
cule inhibitors suppresses the production of IL-1b, IL-6, IL-
12a, CXCL9, and CCL12 from bacterial endotoxin-stimu-
lated macrophages (367). In addition, BRD2 and BRD4
control Th17 differentiation through direct binding to Il17
locus (368). Treatment with BET inhibitors suppresses both
Th1- and Th17-induced autoimmune pathology in mice
(368, 369).
Enhancer landscapes
Enhancers are DNA elements essential for gene regulation by
controlling promoter activity from a distance as far as a
megabase away. It is believed that enhancers are brought
into proximity of promoters by looping of DNA and in this
way contribute to the precise spatial and temporal regulation
of gene expression profiles during development and differ-
entiation. Therefore, identifying functional enhancers and
understanding the mechanisms for their dynamic activities is
likely to be key in deciphering basis of cellular specification
and the acquisition of specialized functions. The other side
of the coin is to characterize transcriptional repressor or
silencer elements in DNA.
For many years, the identification of enhancer elements
was an arduous task. One strategy to identify candidate enh-
ancers was through computational approaches, seeking con-
served non-coding sequences. An alternative approach was
DNase hypersensitivity assays based on the property of enh-
ancers as being nucleosome-depleted to allow for TF bind-
ing. This was only done on small portions of the genome
and was validated by cloning candidate sequences into
reporter constructs that may not reflect the endogenous
chromatin context. In a limited number of circumstances,
their in vivo function was established genetically by deleting
the sequences in engineered mouse models.
In lymphocytes, cytokine loci are regulated by compli-
cated enhancer structures that fine-tune gene expression
under various stimulations or defines lineage specificity
(370). For instance, enhancer activity from CNS2 on Il4
locus is critical for IL-4 expression specifically in Tfh cells
but not in Th2 cells (371). Similarly, CNS1 in the FoxP3
locus is required for differentiation of pTreg cells but not
for tTreg cells (317).
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Immunological Reviews 261/2014 35
Shih et al !Toward the elucidation of the helper T-cell regulome
The identification of chromatin signatures at enhancers
using high-throughput sequencing has profoundly affected
the field of chromatin biology. As described previously,
enhancers are highly associated with high H3K4me1 and
low H3K4me3 modifications (372), and the activity of these
enhancers are reflected by H3K27Ac modification and depo-
sition of the acetyltransferase p300 (372375). These
enhancer characteristics have been used to identify numer-
ous putative enhancers and to track the dynamics of enhan-
cer activity during cell development and reprogramming
(376, 377). For instance, comparing H3K4me1 and p300
binding patterns in macrophages with or without lipopoly-
saccharide (LPS) treatment suggests that LPS-induced enh-
ancers marked by p300 are labeled with H3K4mel prior to
LPS stimulation (377). Genome-wide analysis of H3K27
acetylation has been used to track dynamic enhancer activity
in heart, brain, and liver tissues during mouse development
(376). Recently, H3K27 acetylation has been used to iden-
tify a cluster of lineage-specific enhancers (378), which will
be discussed later.
With the ability to enumerate one class of distal enhanc-
ers, questions arise as to what factors are responsible for the
appearance of these sites and what factors employ these sites
to exert their effect. At present, the answers to these ques-
tions for lymphocytes are limited. Nonetheless, some sur-
prises have already emerged. Master regulatory factors, or
lineage-determining TFs (LDTFs), have been argued to be
important for determining the lineage-specific enhancer
landscape (379). These LDTFs recognize essential cis-regula-
tory elements and mark them through histone modification
and/or nucleosome positioning that alters the accessibility
for other factors. For instance, PU.1, a key LDTF essential
for development of hematopoietic cells, can coordinate with
other regulatory factors to ‘prime’ enhancer candidates for
complete composition of active enhanceosome (380). In
addition, it has been characterized that PU.1 can maintain
enhancer structure through maintaining H3K4me1 modifica-
tion (377). As appealing as this model is, the situation for
CD4
+
T cells is more complicated.
During CD4
+
T-cell differentiation, the expectation might
be that LDTFs like T-bet, GATA-3, Rorct, and FoxP3 might
be the major drivers of the selective enhancer landscapes. In
fact though, the lack of these factors had minimal impact on
the global profiles of enhancer landscape in Th1, Th17, and
Treg cells, respectively (217, 299, 381, 382). This calls into
question whether these factors are indeed master regulators
as they are subservient to STATs and Foxo1, for example.
Based on current data, it appears that the LDTFs for T-helper
cell subsets exert their effect on a preset chromatin land-
scape. Indeed some master regulators, like T-bet, have lim-
ited action on distal enhancers and preferentially exert their
affect more proximally directly on genes. Similarly, FoxP3
binds to regions that are already accessible in naive CD4
+
T
cells, the stage prior to Treg differentiation and FoxP3
expression. However, FoxP3 leads the road for Ezh2 to mark
FoxP3-bound regions with H3K27me3 once Ezh2 is upregu-
lated upon inflammatory stimuli. Therefore, FoxP3 is not
the pioneer factor to permit chromatin accessibility, but
rather it is one of the ‘directing’ factors for selective gene
expression and cell fate. Given the limited ability of LDTFs
to shape the enhancer landscape for T-helpers (381, 382), a
useful strategy was to identify computationally factors that
generated the accessibility of LDTFs. A recently developed
assay of transposase accessible chromatin, ATAC-seq, which
allows evaluation of chromatin accessibility as well as TF
footprints on small amount of cells, provides a new avenue
to assess the identity and hierarchy of gene regulators
(383).
If master regulators are not the major factors that drive
creation of the distinctive ‘switches’ in T cells, then who are
the drivers and what are the master regulators doing? Inter-
estingly, STATs were found to have a much more profound
effect on lineage-specific chromatin landscape than T-cell
master regulators. More specifically, STAT1/STAT4 and
STAT6 binding motifs are enriched in Th1- and Th2-specific
active enhancers, respectively, in both mouse and human
(381, 384). Within more than 9000 murine Th1-specific
active enhancers, only 17% are T-bet dependent, while 60%
are STAT1- and/or STAT4 dependent (381). Importantly,
exogenous expression of T-bet or GATA-3 fails to fully res-
cue the defective chromatin landscapes caused by STAT defi-
ciency. Similarly, during Th17 differentiation, the presence
of STAT3 as well as BATF and IRF4 is more critical for the
establishment of lineage-specific enhancer landscapes than
the presence of RORct (217).
With advanced bioinformatic assistance, a new family of
enhancers called ‘super’ or ‘stretch’ enhancers (SEs) have
been recently identified (378, 385, 386). SEs represent
sequences across several kilobases that contain multiple dis-
continuous enhancer domains bound by key TFs, Mediator
complex, and intense deposition of p300 or H3K27 acetyla-
tion. Mutation of the Mediator complex, inhibiting Brd4 or
any key TFs results in reduced expression of SE-related
genes. Comparison of SEs patterns in various cell types
revealed that SEs play a significant role in defining cell iden-
tity (378, 385, 386). For instance, in ESCs, SEs are enriched
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36 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
at genes essential for ESCs. Therefore, it is intriguing to
utilize SE patterns to distinguish diverse hematopoietic lin-
eages, especially for CD4
+
T and ILC subsets. With the abil-
ity to identify enhancers genome-wide, an obvious next
question is what they regulatehold that thought for now.
We return to this issue later.
Non-coding RNAs
Although only 2% of genome encodes messages for pro-
teins, recent whole transcriptome RNA sequencing data
suggest that over 80% of genome may be actively tran-
scribed. While there is considerable debate surrounding this
topic, it clearly begs the question why there are so many
RNAs generated that do not produce proteins. This question
has been partially answered by discovery of new RNA roles
within various important biological processes (387). Arrays
of small RNAs (<30nt), including microRNA (miRNA) and
piwi-associated RNA, function as gene repressors by binding
to complementary RNA sequences and recruiting silencing
complexes that either act at the posttranscriptional or trans-
lational level, respectively (388). Recently, a new focus of
the RNA field is deciphering the function of eRNAs and
lncRNAs that are largely unknown.
lncRNA
lncRNAs are transcripts longer than 200 nucleotides that lack
a functional open reading frame. Most lncRNAs are believed
to be produced in the similar way as mRNAs in the sense
that both of them are transcribed by RNA polymerase II,
modified by 5’ capping and 3’ polyadenylation and undergo
splicing and sometimes exported to the cytoplasm. Recently,
the maturation of high-throughput RNA-seq methods
enhanced the progress of lncRNA identification and brings
us to a new level of viewing fundamental biology in the
cell. More than 10 000 lncRNAs have been identified in
mammals, but only a few have been functionally character-
ized (389, 390).
Despite this paucity of knowledge, the criticality of lncR-
NAs has been established. Perhaps the most striking example
is the role of Xist, a lncRNA essential for X chromosome
inactivation (391). In addition, lncRNAs have roles in
imprinting, chromatin remodeling, and constructing chro-
matin architecture. Recently, several lncRNAs were identified
to function as scaffold for recruiting histone modifiers. For
instance, HOTTIP, a approximately 4 kb lincRNA transcribed
upstream of HoxA gene clusters, can regulate its target genes
through direct interactions by chromatin loop formation
and through introduction of histone methyltransferase MLL
complex by direct interactions with WDR5, a subunit of
MLL complex. These actions drive H3K4 trimethylation and
facilitate transcription of HOTTIP target genes (392).
lncRNAs can also antagonize protein or miRNA function
through physical interactions. lncRNA GAS5, for instance,
binds to the DNA-binding domain of the glucocorticoid
receptor (GR) to inhibit GR-induced gene activation
{Kino:2010dt}. ecCEBPA RNA can physically target DNA
methyltransferase DNMT1 to prevent local DNA methylation
(393). Recently, a new class of abundant circularized
lncRNA molecules ‘sponge up’ miRNAs in the cells to neu-
tralize their activity (394, 395). Disruption of novel lncR-
NAs by knockdown in vitro or knockout in vivo results in cell
abnormality or death, arguing that lncRNAs are functionally
essential rather than just byproducts from transcription
machinery (389, 396).
Emerging data are beginning to show just how important
lncRNAs are essential for immune cells. During lymphocyte
development, expression of lncRNAs on antigen receptor
loci (also called germline transcription or sterile transcrip-
tion) is essential for recombinase accessibility to target
recombination signal sequences to reassemble V(D)J gene
segments (397). In germinal center B cells, sterile transcrip-
tion of switch regions is predictive of immunoglobulin iso-
type class switch recombination (398). NeST (also known
as TMEVPG1 or LincR-Ifng-3’AS), a 45 kb lincRNA located
adjacent downstream Ifng locus, controls susceptibility to
Theiler’s virus and Salmonella infection in mice through epi-
genetic regulation of the IFN-clocus (399). NeST is
expressed specifically in Th1 and CD8
+
T but not NK cells,
and the expression is dependent on Th1 factors STAT4 and
T-bet (400). Like HOTTIP, NeST regulates gene expression
through the recruitment of WDR5 and its associated H3K4
methylation (399). In Th2 cells, an antisense lncRNA, lincR-
Ccr2-5’AS, is important for regulating gene expression
across this chemokine locus, which contains the Ccr1,Ccr2,
Ccr3, and Ccr5 genes. These chemokines are required for
Th2 migration to lung and are downregulated after knock-
ing down LincR-Ccr2-5’AS (220). Another lncRNA that is
involved in immune responses is lincRNA-Cox2, which pos-
itively and negatively regulates distinct clusters of immune
genes. lncRNA-Cox2 can repress genes through its interac-
tion with heterogeneous nuclear ribonucleoprotein A/B and
A2/B1 (401).
The array of lncRNAs produced by subsets of T cells has
recently been cataloged by deep sequencing of both poly-A
+
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Immunological Reviews 261/2014 37
Shih et al !Toward the elucidation of the helper T-cell regulome
and total transcriptomes within differentiating T cells at vari-
ous stages, and 1524 lncRNAs were identified in total
(220). Among these lncRNAs, 464 were expressed by dou-
ble-negative thymocytes, 515 in double- and single-positive
thymocytes, and 646 in naive and/or differentiated CD4
+
helper T-cell subsets. The expression of these lncRNAs was
highly dynamic during thymocyte development and helper
T-cell differentiation as compared to mRNA expression, and
therefore provides a new way of thinking about functional
cell identity. A number of these newly identified lncRNAs
are STAT-dependent in their expression.
eRNAs
Another exciting discovery in RNA field is the identification
of transcripts originated from enhancers, termed eRNAs.
eRNAs are non-coding RNAs transcribed bidirectionally
from enhancers and are generally 5’-capped, non-spliced,
and non-polyadenylated (402405). eRNAs are essential for
transcriptional regulations as well as loop formation for
enhancerpromoter interactions. The expression of eRNAs
can be induced by external stimuli and their expression cor-
relates well with neighbor gene expression (406409). The
evolving view is that eRNAs are active participants in estab-
lished accessibility of protein-coding genes. Using cap analy-
sis of gene expression, the FANTOM project has mapped
genome-wide TSS across hundreds of cell types (410). Inter-
estingly, enhancers that identified by the combination of
H3K27ac, H3K4me1, and p300 correlate well with the pro-
duction of bidirectional eRNAs, while TSSs for protein-cod-
ing genes are more biased toward one direction (411).
Therefore, the expression of eRNAs can be another indicator
for the prediction of active enhancers.
miRNAs
Numerous miRNAs are recognized as critical regulators to
fine-tune gene expression. They are encoded in the genome
and transcribed by RNA polymerase II to generate primary
miRNA (pri-miRNA) transcripts, which are then processed
sequentially by two members of RNase III type endonucleas-
es, Drosha and Dicer. The mature ~21mer miRNAs are
bound by Argonaute proteins to form miRNA-induced
silencing complexes (miRISCs) to target complementary
mRNAs in a sequence-specific fashion. miRNAs modulate
target mRNA levels through various mechanisms including
blocking translation, mRNA deadenylation followed by 5’
decapping, and enhancing mRNA degradation (388).
Importantly, each of these ‘tiny pieces’ can target more than
one gene; vice versa, each gene can be regulated by more than
one miRNA, therefore creating a complicated regulatory net-
work. Thus, the regulatory logic of miRNAs is analogous to
TFs, except that as far as we know, miRNAs repress gene
expression in general.
miRNAs have been shown to dramatically influence the
homeostasis of immune systems. T-cell specific deletion of
Drosha or Dicer causes abnormal T-cell differentiation and
autoimmunity (412414). Interestingly, in the absence of
Dicer, Th2 differentiation cultures contain T cells that aber-
rantly express IFN-c, suggesting that one or more miRNAs
restrict Th2 cell plasticity (412). Individual miRNAs also
have been shown to influence effector cell differentiation
and stability. miR-155, for instance, is involved in the
development of Th17 and Treg cells under the regulations
of key regulators like STAT3 (218) and FoxP3 (415). miR-
155 regulates IL-2 production for Treg cell maintenance by
suppressing cytokine signaling 1 (Socs1), a negative regulator
of IL-2 signaling (416). miR-155 also controls TGF-bsig-
naling molecules SMAD2 (417) and SMAD5 (418), Ets1, a
negative regulator of Th17 differentiation (419), c-Maf, and
Jarid2 (361, 420). miR-155-deficient mice are protected
from EAE and CIA (416, 421423), but develop enteric and
lung inflammation (420). miR-146a and miR-29 are essen-
tial for suppression of Th1 differentiation; miR-29 does this
by directly targeting IFNc, T-bet, and Eomes (424, 425).
miR-146a inhibits Th1 responses through regulating Treg
cell activity. More specifically, miR-146a keeps STAT1
expression in check, which would otherwise unleash IFNc
expression. Deficiency of miR-146a in T cells leads to over-
expression of IFNc- and Th1-mediated pathology
(426). miR-146a also targets IL-1 receptor-associated kinase
1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6),
two molecules involved in NFjB activation. De-repression
of IRAK1 and TRAF6 leads to NFjB-mediated TCR hyper-
responsiveness, followed by upregulation of IFN-cin effec-
tor T cells (427).
Other miRNAs, mir-10a, miR-181, miR-210, and miR-
17~92 cluster, are also involved in various immune regula-
tions. miR-10a can restrain conversion of iTreg into Tfh by
targeting Bcl-6 and is also involved in suppression of Th17
differentiation (428). miR-181 modulates T-cell responses
mainly by targeting several phosphatases critical for TCR
signaling (429431). miR-210 regulates Th17 differentia-
tion in hypoxia by targeting HIF-1a, a key TF for Th17
polarization (432). Finally, miR-17~92 cluster regulates IL-
10 production in Treg cells and Tfh differentiation (433,
434).
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
38 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
Higher order chromatin conformation
Beyond the previously mentioned epigenetic mechanisms,
another aspect of chromosome biology is also critical for
gene expression and cell identity, namely the three-dimen-
sional chromatin conformation. It has been appreciated that
enhancers regulate gene activity through physical interac-
tions with promoters. These interactions require chromatin
folding that excludes intervening genes and specifies enhan-
cer targets. As the enhancers can function in a location-inde-
pendent manner, analyzing enhancerpromoter interactions
has become critical for identifying putative targets of an
enhancer without getting into laborious genetic modifica-
tions. More importantly, the three billion base pair, 2-m
long genome is complexly packaged in nuclei that are only
a couple micrometers in diameter (reviewed in 435). How
this compact architecture permits the tightly regulated gene
expression is intriguing in terms of understanding what
switches regulate which circuits. Mapping these connections
is key to deciphering the logic of lymphocyte function.
Currently, chromosome conformation capture (3C) and
its derivative methods are prevalently used for determining
chromatin spacial organization. In the past decade, the
development of 3C-based methods, including 4C, 5C, Hi-C,
and ChIA-PET, has broadened our access to chromatin archi-
tecture from local loops to global interactions (436). 4C is
chromosome conformation capture-on-chip or circular
chromosome conformation capture, using inverse PCR to
genome-widely identify regions interacting with interest bait
(one-to-all); 5C is chromosome conformation capture car-
bon copy, using multiplex primers during ligation-mediated
amplification (many-to-many). Hi-C is amplifying ligation
junction by introducing biotin and pulling down (all-to-
all), while ChIA-PET is chromatin interaction analysis with
paired-end tag sequencing, combining chromatin immuno-
precipitation and Hi-C. The basis of 3C involves formalde-
hyde-crosslinking and ligation of DNA fragments that are
nearby in three-dimensional space in the nucleus. The
advantages of 3C technology include that it can detect DNA
folding at molecular level [high resolution as compared to
imaging three-dimensional fluorescence in situ hybridization
(3D-FISH)], and it can be incorporated with modern
sequencing techniques to study genome-wide chromosome
topology (436). Furthermore, 3D-FISH is low throughput
and can only look at a few genes at a time.
Global mapping of DNA proximity reveals a hierarchic
chromatin organization that aggregates active and inactive
genes in euchromatin and heterochromatin compartments,
respectively (437). Within these compartments are meg-
abase-scale globules termed topologically associated domains
(TADs) that have stable boundaries that are invariant within
different cell types and are conserved between species (437
439). Within each TAD are numerous submegabase-scale
long-distance interactions that are dynamic and cell type
specific (440). TADs that contain repressive genes are often
associated with nuclear peripheral lamina regions as well as
H3K9 and H3K27 methylation (439, 441, 442). Hence,
identification of cell type-specific interactome is informative
for understanding the regulation of gene expression and cell
specification.
With regard to the mechanisms, both TFs and global
chromatin organizers are essential for the formation of cell
type-specific chromatin architecture. It has been shown that
the long-distance structure domains consist of colocalizing
of CTCF and cohesin, whereas dynamic enhancerpromoter
interactions are regulated by Mediator and cohesin (440).
Master TFs and Polycomb proteins are also reportedly
involved in the formation of cell type-specific chromatin
architecture. In mouse pluripotent stem cells, lineage-spe-
cific master TFs, Nanog, Sox2, and Oct4, orchestrate chro-
matin conformations with the help of Polycomb proteins.
Depletion of one master regulator or Polycomb subunit dis-
rupts local DNA contacts but not the large-scale chromo-
some topology (443, 444).
Several studies have demonstrated cell type-specific and
stimulus-inducible chromatin architectures on cytokine loci
(445449). For instance, the Th2 cytokine (Il4,Il5, and
Il13) locus forms a cell type-specific interacting center that
recruits the promoters of these genes in CD4
+
T and NK
cells but not in B cells or fibroblasts (447). Interestingly,
upon Th2 activation, this locus further develops from basal
status with limited contacts into a more complicated ‘cage-
like’ chromatin architecture in a special AT-rich sequence
binding protein 1 (SATB1)-dependent manner (448). Simi-
larly, the Ifng locus possesses lineage-specific DNA contacts
across 100 kb specifically in Th1 cells that facilitate IFN-c
expression (445, 446). The Th1-specific interacting hub on
Ifng locus is framed by two CTCF/cohesin-binding sites
anchor to another CTCF/cohesin site within the first intron
of Ifng gene. Knockdown of CFCF or cohesin results in
reduction in long-distance interactions and IFNcproduction
(445, 446). T-cell lineage-specific TFs, T-bet and GATA-3,
respectively, are also essential for the looping on Th1 and
Th2 cytokine loci (445, 447). Based on the chromatin sig-
nature, Ifng gene is surrounded by multiple enhancers (a
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Immunological Reviews 261/2014 39
Shih et al !Toward the elucidation of the helper T-cell regulome
good example of a super-enhancer) and most of which are
within the loop created by CTCF/cohesin, suggesting this
factor can help define the boundaries of super-enhancer
architecture.
The Ifng and Il4/Il3/Il5 loci contrast with genes rapidly
activated by TNF in which the enhancerpromoter interac-
tions are present prior to stimulation, suggesting that the
chromatin conformation sets the stage for rapid responses of
extrinsic stimuli (449). Furthermore, the genome-wide
mapping of promoterenhancer interactomes reveals that
global gene expression is fine-tuned by tissue-specific enh-
ancers, even for those genes that are not cell type specific.
For instance, within near 5000 promoter interactions shared
by B cells and ES cells, up to 90% use at least one cell type-
specific enhancer (450). These enhancers, however, are
associated with lineage-determining factors.
Evidence also reveals that expression of co-regulated genes
can be coordinated through inter-chromosomal interactions
(451). During mouse T-cell differentiation, the dynamic
inter-chromosomal interactions between cytokine loci pro-
vide a new mechanism for genomic regulation. For exam-
ple, Ifng locus on chromosome 10 interacts with Th2
cytokine on chromosome 11 in naive CD4
+
T cells, in
which both genes are inactive. This interaction further dis-
sociates once the cell differentiated into Th1 or Th2 cells,
suggesting a co-regulation or ‘poised’ nuclear organization
for lineage-specific genes (452). Similarly, the Th2 locus is
also shown to interact with Il17 locus to restrain Th17 dif-
ferentiation (453).
Conclusions
More than three decades ago, the term master regulator was
introduced to describe ‘a gene that occupies the very top of
a regulatory hierarchy’ (454). This concept was introduced
roughly at the same time when ‘lineages’ of CD4
+
helper T
cells were first recognized. Master regulator tacitly implies
that these factors dominantly specify cell lineage. The classi-
cal example is the myogenic TF MyoD, which is essential
for muscle cell differentiation and can turn on myogenic
genes when introduced into heterologous cells. Initially, it
seemed appropriate to view helper T-cell lineages and cog-
nate master regulators in the same way. However, much has
changed over the last 30 years. There are many more fates
for CD4
+
T cells and likewise the array of cytokines pro-
duced by ILCs has also expanded. These discoveries high-
light the limitations of a one lineage-one master regulator
model for explaining the diversity of functions of lymphoid
cells. More accurate is the appreciation that the establish-
ment of each immune cell type requires multiple key TFs
that coordinately regulate aspects of their specialized func-
tions. In this way, more than one master regulator can be
expressed in more than one cell type. Moreover, multiple
cells can exhibit the same functionality (e.g. production of
IFN-cor IL-17), and not surprisingly, these cells express
many of the same factors. However, master regulators like
T-bet appear to be functionally critical in different ways in
different cells. GATA-3 and Rorct are important at multiple
steps in lymphocyte differentiation; their function is not
limited to cytokine production alone. Therefore, the notion
of master regulators, at least based on the traditional defini-
tion, needs to be revised with respect to diverse immune
cell populations that have distinct functions and gene
expression. Superimposed upon selective cytokine produc-
tion are other functionalities of immune cells and their abil-
ity to localize in diverse tissues. Consequently, lymphoid
populations express more than one master regulator, and
diverse types of cells can express the same master regulator.
This observation limits the notion that a single TF defines a
specific cell population. A more accurate view is to think
about the superimposition of functionalities that can coexist.
Thus, the combinatorial action of TFs is probably a more
appropriate way of visualizing how these factors specify
gene expression programs.
In addition to thinking about how TFs act on genes, one
also needs to consider how chromatin states affect the action
of TFs. Accumulating evidence indicates that cell identity is
established by converging signals provided by epigenetic
traits accumulated from the action of pioneer TFs, not mas-
ter regulators, and the consequence of past environmental
stimuli that alter the epigenetic landscape to imprint ‘mem-
ory’ and in this way alter TF deposition. For instance, the
process of differentiation of HSCs into effector immune cells
requires multiple steps of chromatin remodeling and epige-
netic reprogramming. However, the connections between
these events are only partially understood. An important
challenge will be to track the dynamic appearance of epige-
netic marks along cell differentiation and activation, to
understand the interpretation of each epigenetic mark, to
identify the hierarchy and/or the combination of TFs for
cell identity.
Also, genes represent only a tiny portion of the genome;
most of the genome represents different kinds of switches,
many of which are themselves transcribed into RNA, but
not into proteins. Understanding what factors are responsi-
ble for the creation of these switches and what controls
their activation state is an important challenge. Clarifying
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
40 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
the role of key TFs in creating the switches and how the
switches influence TFs access to the genome are important
questions to resolve. The advent of deep-sequencing tech-
nologies now allows comprehensive, genome-wide views of
chromatin states in lymphocytes, along with assessment of
TF binding and measurements of the transcriptome that go
far beyond the small portion of the genome that encodes
conventional protein-coding genes. With improved ability
to edit the genome with efficient technologies like TALENs
or Crispr/Cas9, along with rich resources like ENCODE
(http://www.encode-roadmap.org), enumeration and func-
tional dissection of the switches is now within reach. Defin-
ing TF networks and how they affect or employ enhancer
landscapes will undoubtedly provide a more sophisticated
understanding of diverse lymphoid populations in health
and disease.
References
1. Miller JF, Mitchell GF. The thymus and the
precursors of antigen reactive cells. Nature
1967;216:659663.
2. Mitchell GF, Miller JF. Immunological activity of
thymus and thoracic-duct lymphocytes. Proc Natl
Acad Sci USA 1968;59:296303.
3. Cantor H, Boyse EA. Functional subclasses of
T-lymphocytes bearing different Ly antigens. I.
The generation of functionally distinct T-cell
subclasses is a differentiative process
independent of antigen. J Exp Med
1975;141:13761389.
4. Kisielow P, et al. Ly antigens as markers for
functionally distinct subpopulations of
thymus-derived lymphocytes of the mouse.
Nature 1975;253:219220.
5. Shiku H, et al. Expression of T-cell differentiation
antigens on effector cells in cell-mediated
cytotoxicity in vitro. Evidence for functional
heterogeneity related to the surface phenotype of
T cells. J Exp Med 1975;141:227241.
6. Mosmann TR, Cherwinski H, Bond MW, Giedlin
MA, Coffman RL. Two types of murine helper T
cell clone. I. Definition according to profiles of
lymphokine activities and secreted proteins. J
Immunol 1986;136:23482357.
7. Mosmann TR, Coffman RL. TH1 and TH2 cells:
different patterns of lymphokine secretion lead to
different functional properties. Annu Rev
Immunol 1989;7:145173.
8. North RJ, Jung Y-J. Immunity to tuberculosis.
Annu Rev Immunol 2004;22:599623.
9. Reiner SL, Locksley RM. The regulation of
immunity to Leishmania major. Annu Rev
Immunol 1995;13:151177.
10. Pulendran B, Artis D. New paradigms in type 2
immunity. Science 2012;337:431435.
11. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman
CG, Glimcher LH. A novel transcription factor,
T-bet, directs Th1 lineage commitment. Cell
2000;100:655669.
12. Zheng W, Flavell RA. The transcription factor
GATA-3 is necessary and sufficient for Th2
cytokine gene expression in CD4 T cells. Cell
1997;89:587596.
13. Hori S. Control of regulatory T cell development
by the transcription factor Foxp3. Science
2003;299:10571061.
14. Fontenot JD, Gavin MA, Rudensky AY. Foxp3
programs the development and function of
CD4+CD25+regulatory T cells. Nat Immunol
2003;4:330336.
15. Ivanov II, et al. The orphan nuclear receptor
RORct directs the differentiation program of
proinflammatory IL-17+T helper cells. Cell
2006;126:11211133.
16. Lighvani AA, et al. T-bet is rapidly induced by
interferon-gamma in lymphoid and myeloid
cells. Proc Natl Acad Sci USA 2001;98:15137
15142.
17. Szabo SJ, Dighe AS, Gubler U, Murphy KM.
Regulation of the interleukin (IL)-12R beta 2
subunit expression in developing T helper 1
(Th1) and Th2 cells. J Exp Med 1997;185:817
824.
18. Rogge L, et al. Selective expression of an
interleukin-12 receptor component by human T
helper 1 cells. J Exp Med 1997;185:825831.
19. Bettelli E, et al. Reciprocal developmental
pathways for the generation of pathogenic
effector TH17 and regulatory T cells. Nature
2006;441:235238.
20. Veldhoen M, Hocking RJ, Atkins CJ, Locksley
RM, Stockinger B. TGFbeta in the context of an
inflammatory cytokine milieu supports de novo
differentiation of IL-17-producing T cells.
Immunity 2006;24:179189.
21. Miossec P, Korn T, Kuchroo VK. Interleukin-17
and type 17 helper T cells. N Engl J Med
2009;361:888898.
22. Veldhoen M, et al. Transforming growth
factor-beta “reprograms” the differentiation of T
helper 2 cells and promotes an interleukin
9-producing subset. Nat Immunol 2008;9:1341
1346.
23. Schmitt E, et al. IL-9 production of naive CD4+
T cells depends on IL-2, is synergistically
enhanced by a combination of TGF-beta and
IL-4, and is inhibited by IFN-gamma. J Immunol
1994;153:39893996.
24. Nowak EC, et al. IL-9 as a mediator of
Th17-driven inflammatory disease. J Exp Med
2009;206:16531660.
25. Gessner A, Blum H, Rollinghoff M. Differential
regulation of IL-9-expression after infection
with Leishmania major in susceptible and
resistant mice. Immunobiology 1993;189:419
435.
26. Elyaman W, et al. IL-9 induces differentiation of
TH17 cells and enhances function of FoxP3+
natural regulatory T cells. Proc Natl Acad Sci
2009;106:1288512890.
27. Jankovic D, et al. Conventional T-bet(+)Foxp3(-)
Th1 cells are the major source of host-protective
regulatory IL-10 during intracellular protozoan
infection. J Exp Med 2007;204:273283.
28. Esplugues E, et al. Control of TH17 cells occurs in
the small intestine. Nature 2011;475:514518.
29. Minegishi Y, et al. Molecular explanation for the
contradiction between systemic Th17 defect and
localized bacterial infection in hyper-IgE
syndrome. J Exp Med 2009;206:1291
1301.
30. Ma CS, et al. Deficiency of Th17 cells in hyper
IgE syndrome due to mutations in STAT3. J Exp
Med 2008;205:15511557.
31. Milner JD, et al. Impaired T(H)17 cell
differentiation in subjects with autosomal
dominant hyper-IgE syndrome. Nature
2008;452:773776.
32. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17
and Th17 cells. Annu Rev Immunol
2009;27:485517.
33. Hirota K, et al. Plasticity of Th17 cells in Peyer’s
patches is responsible for the induction of T
cell-dependent IgA responses. Nat Immunol
2013;14:372379.
34. Hueber W, et al. Effects of AIN457, a fully
human antibody to interleukin-17A, on psoriasis,
rheumatoid arthritis, and uveitis. Sci Transl Med
2010;2:52ra72.
35. Genovese MC, et al. LY2439821, a humanized
anti-interleukin-17 monoclonal antibody, in the
treatment of patients with rheumatoid arthritis: a
phase I randomized, double-blind,
placebo-controlled, proof-of-concept study.
Arthritis Rheum 2010;62:929939.
36. Leonardi C, et al. Anti-interleukin-17 monoclonal
antibody ixekizumab in chronic plaque psoriasis.
N Engl J Med 2012;366:11901199.
37. Wu H-J, et al. Gut-residing segmented
filamentous bacteria drive autoimmune arthritis
via T helper 17 cells. Immunity 2010;32:815
827.
38. Lee Y, et al. Induction and molecular signature
of pathogenic TH17 cells. Nat Immunol
2012;13:991999.
39. Ghoreschi K, et al. Generation of pathogenic T
(H)17 cells in the absence of TGF-bsignalling.
Nature 2010;467:967971.
40. Yang XO, et al. T helper 17 lineage
differentiation is programmed by orphan nuclear
receptors RORaand RORc. Immunity
2008;28:2939.
41. Duhen T, Geiger R, Jarrossay D, Lanzavecchia A,
Sallusto F. Production of interleukin 22 but not
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 41
Shih et al !Toward the elucidation of the helper T-cell regulome
interleukin 17 by a subset of human
skin-homing memory T cells. Nat Immunol
2009;10:857863.
42. Eyerich S, et al. Th22 cells represent a distinct
human T cell subset involved in epidermal
immunity and remodeling. J Clin Invest
2009;119:35733585.
43. Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits
H. Identification of a human helper T cell
population that has abundant production of
interleukin 22 and is distinct from T(H)-17, T
(H)1 and T(H)2 cells. Nat Immunol
2009;10:864871.
44. Basu R, et al. Th22 cells are an important source
of IL-22 for host protectionagainst
enteropathogenic bacteria. Immunity
2012;37:10611075.
45. Schmitt E, Bopp T. Amazing IL-9: revealing a
new function for an “old” cytokine. J Clin Invest
2012;122:38573859.
46. Tan C, Gery I. The unique features of Th9 cells
and their products. Crit Rev Immunol
2012;32:110.
47. Chang H-C, et al. The transcription factor PU.1 is
required for the development of IL-9-producing
T cells and allergic inflammation. Nat Immunol
2010;11:527534.
48. Dardalhon V, et al. IL-4 inhibits TGF-b-induced
Foxp3+T cells and, together with TGF-b,
generates IL-9+IL-10+Foxp3"effector T cells.
Nat Immunol 2008;9:13471355.
49. Jabeen R, et al. Th9 cell development requires a
BATF-regulated transcriptional network. J Clin
Invest 2013;123:46414653.
50. Vinuesa CG, et al. A RING-type ubiquitin ligase
family member required to repress follicular
helper T cells and autoimmunity. Nature
2005;435:452458.
51. Crotty S. Follicular helper CD4 T cells (TFH).
Annu Rev Immunol 2011;29:621663.
52. Johnston RJ, et al. Bcl6 and Blimp-1 are
reciprocal and antagonistic regulators of T
follicular helper cell differentiation. Science
2009;325:10061010.
53. Yu D, et al. The transcriptional repressor Bcl-6
directs T follicular helper cell lineage
commitment. Immunity 2009;31:457468.
54. Nurieva RI, et al. Bcl6 mediates the development
of T follicular helper cells. Science
2009;325:10011005.
55. Nakayamada S, et al. Early Th1 cell
differentiation is marked by a Tfh cell-like
transition. Immunity 2011;35:919931.
56. Wei L, Laurence A, Elias KM, O’Shea JJ. IL-21 is
produced by Th17 cells and drives IL-17
production in a STAT3-dependent manner. J Biol
Chem 2007;282:3460534610.
57. Reinhardt RL, Liang H-E, Locksley RM.
Cytokine-secreting follicular T cells shape the
antibody repertoire. Nat Immunol 2009;10:385
393.
58. Hsu H-C, et al. Interleukin 17-producing T
helper cells and interleukin 17 orchestrate
autoreactive germinal center development in
autoimmune BXD2 mice. Nat Immunol
2008;9:166175.
59. Germain RN. Special regulatory T-cell review: a
rose by any other name: from suppressor T cells
to Tregs, approbation to unbridled enthusiasm.
Immunology 2008;123:2027.
60. Sakaguchi S, Yamaguchi T, Nomura T, Ono M.
Regulatory T cells and immune tolerance. Cell
2008;133:775787.
61. Zheng Y, Rudensky AY. Foxp3 in control of the
regulatory T cell lineage. Nat Immunol
2007;8:457462.
62. Abbas AK, et al. Regulatory T cells:
recommendations to simplify the nomenclature.
Nat Immunol 2013;14:307308.
63. Hill JA, et al. Foxp3
transcription-factor-dependent and -independent
regulation of the regulatory T cell transcriptional
signature. Immunity 2007;27:786800.
64. Fu W, et al. A multiply redundant genetic switch
“locks in” the transcriptional signature of
regulatory T cells. Nat Immunol 2012;13:972
980.
65. Collison LW, et al. The inhibitory cytokine IL-35
contributes to regulatory T-cell function. Nature
2007;450:566569.
66. Gagliani N, et al. Coexpression of CD49b and
LAG-3 identifies human and mouse T regulatory
type 1 cells. Nat Med 2013;19:739746.
67. Battaglia M, Gregori S, Bacchetta R, Roncarolo
MG. Tr1 cells: from discovery to their
clinical application. Semin Immunol
2006;18:120127.
68. Awasthi A, et al. A dominant function for
interleukin 27 in generating interleukin
10-producing anti-inflammatory T cells. Nat
Immunol 2007;8:13801389.
69. Saraiva M, O’Garra A. The regulation of IL-10
production by immune cells. Nat Rev Immunol
2010;10:170181.
70. Burzyn D, et al. A special population of
regulatory T cells potentiates muscle repair. Cell
2013;155:12821295.
71. Feuerer M, et al. Lean, but not obese, fat is
enriched for a unique population of regulatory T
cells that affect metabolic parameters. Nat Med
2009;15:930939.
72. Crome SQ, Lang PA, Lang KS, Ohashi PS. Natural
killer cells regulate diverse T cell responses.
Trends Immunol 2013;34:342349.
73. Magnani CF, et al. Killing of myeloid APCs via
HLA class I, CD2 and CD226 defines a novel
mechanism of suppression by human Tr1 cells.
Eur J Immunol 2011;41:16521662.
74. Messi M, Giacchetto I, Nagata K, Lanzavecchia A,
Natoli G, Sallusto F. Memory and flexibility of
cytokine gene expression as separable properties
of human T(H)1 and T(H)2 lymphocytes. Nat
Immunol 2003;4:7886.
75. Zhou L, Chong MMW, Littman DR. Plasticity of
CD4+T cell lineage differentiation. Immunity
2009;30:646655.
76. O’Shea JJ, Paul WE. Mechanisms underlying
lineage commitment and plasticity of helper
CD4+T cells. Science 2010;327:10981102.
77. Zielinski CE, et al. Pathogen-induced human
TH17 cells produce IFN-cor IL-10 and are
regulated by IL-1b. Nature 2012;484:514518.
78. Mukasa R, et al. Epigenetic instability of cytokine
and transcription factor gene loci underlies
plasticity of the T helper 17 cell lineage.
Immunity 2010;32:616627.
79. Boniface K, et al. Human Th17 cells comprise
heterogeneous subsets including
IFN-gamma-producing cells with distinct
properties from the Th1 lineage. J Immunol
2010;185:679687.
80. Annunziato F, et al. Phenotypic and functional
features of human Th17 cells. J Exp Med
2007;204:18491861.
81. McGeachy MJ, et al. TGF-beta and IL-6 drive the
production of IL-17 and IL-10 by T cells and
restrain T(H)-17 cell-mediated pathology. Nat
Immunol 2007;8:13901397.
82. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia
A, Sallusto F. Interleukins 1beta and 6 but not
transforming growth factor-beta are essential for
the differentiation of interleukin 17-producing
human T helper cells. Nat Immunol 2007;8:942
949.
83. Hegazy AN, et al. Interferons direct Th2 cell
reprogramming to generate a stable GATA-3(+)
T-bet(+) cell subset with combined Th2 and Th1
cell functions. Immunity 2010;32:116128.
84. Yang XO, et al. Molecular antagonism and
plasticity of regulatory and inflammatory T cell
programs. Immunity 2008;29:4456.
85. Hori S. Regulatory T cell plasticity: beyond the
controversies. Trends Immunol 2011;32:295
300.
86. Hori S. Lineage stability and phenotypic plasticity
of Foxp3regulatory T cells. Immunol Rev
2014;259:159172.
87. Bailey-Bucktrout SL, Bluestone JA. Regulatory T
cells: stability revisited. Trends Immunol
2011;32:301306.
88. Lu KT, et al. Functional and epigenetic studies
reveal multistep differentiation and plasticity of
in vitro-generated and in vivo-derived
follicular T helper cells. Immunity
2011;35:622632.
89. Glatman Zaretsky A, Taylor JJ, King IL, Marshall
FA, Mohrs M, Pearce EJ. T follicular helper cells
differentiate from Th2 cells in response to
helminth antigens. J Exp Med 2009;206:991
999.
90. King IL, Mohrs M. IL-4-producing CD4+T cells
in reactive lymph nodes during helminth
infection are T follicular helper cells. J Exp Med
2009;206:10011007.
91. Cheroutre H, Husain MM. CD4 CTL: living up to
the challenge. Semin Immunol 2013;25:273
281.
92. Mucida D, et al. Transcriptional reprogramming
of mature CD4helper T cells generates distinct
MHC class II-restricted cytotoxic T lymphocytes.
Nat Immunol 2013;14:281289.
93. Spits H, Di Santo JP. The expanding family of
innate lymphoid cells: regulators and effectors of
immunity and tissue remodeling. Nat Immunol
2011;12:2127.
94. Spits H, et al. Innate lymphoid cells a proposal
for uniform nomenclature. Nat Rev Immunol
2013;13:145149.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
42 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
95. Rosenberg EB, et al. Destruction of human
lymphoid tissue-culture cell lines by human
peripheral lymphocytes in 51Cr-release cellular
cytotoxicity assays. J Natl Cancer Inst
1974;52:345352.
96. Kiessling R, Klein E, Pross H, Wigzell H.
“Natural” killer cells in the mouse. II. Cytotoxic
cells with specificity for mouse Moloney
leukemia cells. Characteristics of the killer cell.
Eur J Immunol 1975;5:117121.
97. Kiessling R, Klein E, Wigzell H. “Natural” killer
cells in the mouse. I. Cytotoxic cells with
specificity for mouse Moloney leukemia cells.
Specificity and distribution according to
genotype. Eur J Immunol 1975;5:112117.
98. Herberman RB, Nunn ME, Holden HT, Lavrin
DH. Natural cytotoxic reactivity of mouse
lymphoid cells against syngeneic and allogeneic
tumors. II. Characterization of effector cells. Int J
Cancer 1975;16:230239.
99. Herberman RB, Nunn ME, Lavrin DH. Natural
cytotoxic reactivity of mouse lymphoid cells
against syngeneic acid allogeneic tumors. I.
Distribution of reactivity and specificity. Int J
Cancer 1975;16:216229.
100. Orange JS, Wang B, Terhorst C, Biron CA.
Requirement for natural killer cell-produced
interferon gamma in defense against murine
cytomegalovirus infection and enhancement of
this defense pathway by interleukin 12
administration. J Exp Med 1995;182:
10451056.
101. Sher A, Oswald IP, Hieny S, Gazzinelli RT.
Toxoplasma gondii induces a T-independent
IFN-gamma response in natural killer cells that
requires both adherent accessory cells and tumor
necrosis factor-alpha. J Immunol
1993;150:39823989.
102. Scharton TM, Scott P. Natural killer cells are a
source of interferon gamma that drives
differentiation of CD4+T cell subsets and
induces early resistance to Leishmania major in
mice. J Exp Med 1993;178:567577.
103. Vosshenrich CAJ, et al. A thymic pathway of
mouse natural killer cell development
characterized by expression of GATA-3 and
CD127. Nat Immunol 2006;7:12171224.
104. Satoh-Takayama N, et al. Microbial flora drives
interleukin 22 production in intestinal NKp46+
cells that provide innate mucosal immune
defense. Immunity 2008;29:958970.
105. Takeda K, et al. TRAIL identifies immature
natural killer cells in newborn mice and adult
mouse liver. Blood 2005;105:20822089.
106. Schoenborn JR, Wilson CB. Regulation of
interferon-gamma during innate and adaptive
immune responses. Adv Immunol 2007;96:41
101.
107. Klose CSN, et al. A T-bet gradient controls the
fate and function of CCR6-RORct+innate
lymphoid cells. Nature 2013;494:261265.
108. Sokol CL, Barton GM, Farr AG, Medzhitov R. A
mechanism for the initiation of allergen-induced
T helper type 2 responses. Nat Immunol
2008;9:310318.
109. Min B, et al. Basophils produce IL-4 and
accumulate in tissues after infection with a
Th2-inducing parasite. J Exp Med
2004;200:507517.
110. Motomura Y, et al. Basophil-derived
interleukin-4 controls the function of natural
helper cells, a member of ILC2s, in lung
inflammation. Immunity 2014;40:758771.
111. Voehringer D, Shinkai K, Locksley RM. Type 2
immunity reflects orchestrated recruitment of
cells committed to IL-4 production. Immunity
2004;20:267277.
112. Lantz O, Bendelac A. An invariant T cell receptor
alpha chain is used by a unique subset of major
histocompatibility complex class I-specific CD4+
and CD4-8- T cells in mice and humans. J Exp
Med 1994;180:10971106.
113. Neill DR, et al. Nuocytes represent a new innate
effector leukocyte that mediates type-2
immunity. Nature 2010;464:13671370.
114. Moro K, et al. Innate production of T(H)2
cytokines by adipose tissue-associated c-Kit(+)
Sca-1(+) lymphoid cells. Nature 2010;463:540
544.
115. Price AE, et al. Systemically dispersed innate
IL-13-expressing cells in type 2 immunity. Proc
Natl Acad Sci 2010;107:1148911494.
116. Saenz SA, et al. IL25 elicits a multipotent
progenitor cell population that promotes T(H)2
cytokine responses. Nature 2010;464:1362
1366.
117. Halim TYF, et al. Group 2 innate lymphoid cells
are critical for the initiation of adaptive T helper
2 cell-mediated allergic lung inflammation.
Immunity 2014;40:425435.
118. Hultner L, et al. In activated mast cells, IL-1
up-regulates the production of several
Th2-related cytokines including IL-9. J Immunol
2000;164:55565563.
119. Stassen M, et al. Murine bone marrow-derived
mast cells as potent producers of IL-9:
costimulatory function of IL-10 and kit ligand in
the presence of IL-1. J Immunol
2000;164:55495555.
120. Stassen M, et al. IL-9 and IL-13 production by
activated mast cells is strongly enhanced in the
presence of lipopolysaccharide: NF-kappa B is
decisively involved in the expression of IL-9. J
Immunol 2001;166:43914398.
121. Wilhelm C, et al. An IL-9 fate reporter
demonstrates the induction of an innate IL-9
response in lung inflammation. Nat Immunol
2011;12:10711077.
122. Cupedo T, et al. Human fetal lymphoid
tissue-inducer cells are interleukin 17-producing
precursors to RORC+CD127+natural killer-like
cells. Nat Immunol 2009;10:6674.
123. Takatori H, et al. Lymphoid tissue inducer-like
cells are an innate source of IL-17 and IL-22. J
Exp Med 2009;206:3541.
124. Sonnenberg GF, Monticelli LA, Elloso MM,
Fouser LA, Artis D. CD4(+) lymphoid
tissue-inducer cells promote innate immunity in
the gut. Immunity 2011;34:122134.
125. Hepworth MR, et al. Innate lymphoid cells
regulate CD4+T-cell responses to intestinal
commensal bacteria. Nature 2013;498:113117.
126. Qiu J, et al. Group 3 innate lymphoid cells
inhibit T-cell-mediated intestinal inflammation
through aryl hydrocarbon receptor signaling and
regulation of microflora. Immunity
2013;39:386399.
127. Vonarbourg C, et al. Regulated expression of
nuclear receptor RORct confers distinct
functional fates to NK cell receptor-expressing
RORct(+) innate lymphocytes. Immunity
2010;33:736751.
128. Sawa S, et al. Lineage relationship analysis of
RORgammat+innate lymphoid cells. Science
2010;330:665669.
129. Luci C, et al. Influence of the transcription factor
RORgammat on the development of NKp46+cell
populations in gut and skin. Nat Immunol
2009;10:7582.
130. Sanos SL, et al. RORgammat and commensal
microflora are required for the differentiation of
mucosal interleukin 22-producing NKp46+cells.
Nat Immunol 2009;10:8391.
131. Eberl G. Development and evolution of RORct+
cells in a microbe’s world. Immunol Rev
2012;245:177188.
132. Zindl CL, et al. IL-22-producing neutrophils
contribute to antimicrobial defense and
restitution of colonic epithelial integrity during
colitis. Proc Natl Acad Sci 2013;110:12768
12773.
133. Taylor PR, et al. Activation of neutrophils by
autocrine IL-17A-IL-17RC interactions during
fungal infection is regulated by IL-6, IL-23,
RORct and dectin-2. Nat Immunol
2014;15:143151.
134. Hoshino A, et al. MPO-ANCA induces IL-17
production by activated neutrophils in vitro via
classical complement pathway-dependent
manner. J Autoimmun 2008;31:79
89.
135. Li L, et al. IL-17 produced by neutrophils
regulates IFN-gamma-mediated neutrophil
migration in mouse kidney ischemia-reperfusion
injury. J Clin Invest 2010;120:331342.
136. Michel M-L, et al. Identification of an
IL-17-producing NK1.1(neg) iNKT cell
population involved in airway neutrophilia. J Exp
Med 2007;204:9951001.
137. Cui Y, et al. Major role of gamma delta T cells in
the generation of IL-17+uveitogenic T cells. J
Immunol 2009;183:560567.
138. Ito Y, et al. Gamma/delta T cells are the
predominant source of interleukin-17 in affected
joints in collagen-induced arthritis, but not in
rheumatoid arthritis. Arthritis Rheum
2009;60:22942303.
139. Roark CL, French JD, Taylor MA, Bendele AM,
Born WK, O’Brien RL. Exacerbation of
collagen-induced arthritis by oligoclonal,
IL-17-producing gamma delta T cells. J Immunol
2007;179:55765583.
140. Cua DJ, Tato CM. Innate IL-17-producing cells:
the sentinels of the immune system. Nat Rev
Immunol 2010;10:479489.
141. O’Garra A, Chang R, Go N, Hastings R,
Haughton G, Howard M. Ly-1 B (B-1) cells are
the main source of B cell-derived interleukin 10.
Eur J Immunol 1992;22:711717.
142. Zhang X, Majlessi L, Deriaud E, Leclerc C,
Lo-Man R. Coactivation of Syk kinase and MyD88
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 43
Shih et al !Toward the elucidation of the helper T-cell regulome
adaptor protein pathways by bacteria promotes
regulatory properties of neutrophils. Immunity
2009;31:761771.
143. Siewe L, Bollati-Fogolin M, Wickenhauser C,
Krieg T, Muller W, Roers A. Interleukin-10
derived from macrophages and/or neutrophils
regulates the inflammatory response to LPS but
not the response to CpG DNA. Eur J Immunol
2006;36:32483255.
144. McGuirk P, McCann C, Mills KHG.
Pathogen-specific T regulatory 1 cells induced in
the respiratory tract by a bacterial molecule that
stimulates interleukin 10 production by dendritic
cells: a novel strategy for evasion of protective T
helper type 1 responses by Bordetella pertussis. J
Exp Med 2002;195:221231.
145. Granucci F, et al. Inducible IL-2 production by
dendritic cells revealed by global gene expression
analysis. Nat Immunol 2001;2:882888.
146. Daussy C, et al. T-bet and Eomes instruct the
development of two distinct natural killer cell
lineages in the liver and in the bone marrow. J
Exp Med 2014;211:563577.
147. Aggarwal R, Lu J, Pompili VJ, Das H.
Hematopoietic stem cells: transcriptional
regulation, ex vivo expansion and clinical
application. Curr Mol Med 2012;12:3449.
148. Kumano K, et al. Notch1 but not Notch2 is essential
forgenerating hematopoietic stem cells from
endothelial cells. Immunity 2003;18:699711.
149. Burns CE, Traver D, Mayhall E, Shepard JL, Zon
LI. Hematopoietic stem cell fate is established by
the Notch-Runx pathway. Genes Dev
2005;19:23312342.
150. Ikawa T. Genetic and Epigenetic Control of Early
Lymphocyte Development. Immunol: Curr. Top.
Microbiol, 2014.
151. Novershtern N, et al. Densely interconnected
transcriptional circuits control cell states in
human hematopoiesis. Cell 2011;144:296309.
152. Han H, et al.Induciblegene knockout of
transcription factor recombination signal binding
protein-J reveals its essential role in T versus B lineage
decision. Int Immunol 2002;14:637645.
153. Radtke F, et al. Deficient T cell fate specification
in mice with an induced inactivation of Notch1.
Immunity 1999;10:547558.
154. Pui JC, et al. Notch1 expression in early
lymphopoiesis influences B versus T lineage
determination. Immunity 1999;11:299308.
155. Germar K, et al. T-cell factor 1 is a gatekeeper
for T-cell specification in response to Notch
signaling. Proc Natl Acad Sci 2011;108:20060
20065.
156. Keerthivasan S, et al. b-Catenin promotes colitis
and colon cancer through imprinting of
proinflammatory properties in T cells. Sci Transl
Med 2014;6:225ra28.
157. Mielke LA, et al. TCF-1 controls ILC2 and
NKp46+RORct+innate lymphocyte
differentiation and protection in intestinal
inflammation. J Immunol 2013;191:43834391.
158. Yang Q, et al. T cell factor 1 is required for
group 2 innate lymphoid cell generation.
Immunity 2013;38:694704.
159. Jones ME, Zhuang Y. Acquisition of a functional
T cell receptor during T lymphocyte
development is enforced by HEB and E2A
transcription factors. Immunity 2007;27:860
870.
160. Agata Y, et al. Regulation of T cell receptor beta
gene rearrangements and allelic exclusion by the
helix-loop-helix protein, E47. Immunity
2007;27:871884.
161. Bain G, et al. E2A deficiency leads to
abnormalities in alphabeta T-cell development
and to rapid development of T-cell lymphomas.
Mol Cell Biol 1997;17:47824791.
162. Engel I, Johns C, Bain G, Rivera RR, Murre C.
Early thymocyte development is regulated by
modulation of E2A protein activity. J Exp Med
2001;194:733745.
163. Dias S, M#ansson R, Gurbuxani S, Sigvardsson M,
Kee BL. E2A proteins promote development of
lymphoid-primed multipotent progenitors.
Immunity 2008;29:217227.
164. Wakabayashi Y, et al. Bcl11b is required for
differentiation and survival of alphabeta T
lymphocytes. Nat Immunol 2003;4:533
539.
165. Carpenter AC, et al. The transcription factors
Thpok and LRF are necessary and partly
redundant for T helper cell differentiation.
Immunity 2012;37:622633.
166. Keefe R, Dave V, Allman D, Wiest D, Kappes
DJ. Regulation of lineage commitment distinct
from positive selection. Science
1999;286:11491153.
167. He X, et al. The zinc finger transcription factor
Th-POK regulates CD4 versus CD8 T-cell lineage
commitment. Nature 2005;433:826833.
168. Wang L, et al. The zinc finger transcription
factor Zbtb7b represses CD8-lineage gene
expression in peripheral CD4+T cells. Immunity
2008;29:876887.
169. Setoguchi R, et al. Repression of the
transcription factor Th-POK by Runx complexes
in cytotoxic T cell development. Science
2008;319:822825.
170. Collins A, Littman DR, Taniuchi I. RUNX
proteins in transcription factor networks that
regulate T-cell lineage choice. Nat Rev Immunol
2009;9:106115.
171. Eberl G, Marmon S, Sunshine M-J, Rennert PD,
Choi Y, Littman DR. An essential function for the
nuclear receptor RORgamma(t) in the generation
of fetal lymphoid tissue inducer cells. Nat
Immunol 2004;5:6473.
172. Satoh-Takayama N, et al. IL-7 and IL-15
independently program the differentiation of
intestinal CD3-NKp46+cell subsets from
Id2-dependent precursors. J Exp Med
2010;207:273280.
173. Yokota Y, et al. Development of peripheral
lymphoid organs and natural killer cells depends
on the helix-loop-helix inhibitor Id2. Nature
1999;397:702706.
174. Boos MD, Yokota Y, Eberl G, Kee BL. Mature
natural killer cell and lymphoid tissue-inducing
cell development requires Id2-mediated
suppression of E protein activity. J Exp Med
2007;204:11191130.
175. Heemskerk MH, et al. Inhibition of T cell and
promotion of natural killer cell development by
the dominant negative helix loop helix factor
Id3. J Exp Med 1997;186:15971602.
176. Verykokakis M, Krishnamoorthy V, Iavarone A,
Lasorella A, Sigvardsson M, Kee BL. Essential
functions for ID proteins at multiple checkpoints
in invariant NKT cell development. J Immunol
2013;191:59735983.
177. Kamizono S, et al. Nfil3/E4 bp4 is required for
the development and maturation of NK cells in
vivo. J Exp Med 2009;206:29772986.
178. Gascoyne DM, et al. The basic leucine zipper
transcription factor E4BP4 is essential for natural
killer cell development. Nat Immunol
2009;10:11181124.
179. Firth MA, et al. Nfil3-independent lineage
maintenance and antiviral response of natural
killer cells. J Exp Med 2013;210:29812990.
180. Crotta S, et al. The transcription factor E4BP4 is
not required for extramedullary pathways of NK
cell development. J Immunol 2014;192:2677
2688.
181. Male V, et al. The transcription factor E4 bp4/
Nfil3 controls commitment to the NK lineage
and directly regulates Eomes and Id2 expression.
J Exp Med 2014;211:635642.
182. Seillet C, et al. Differential requirement for Nfil3
during NK cell development. J Immunol
2014;192:26672676.
183. Kashiwada M, Pham N-LL, Pewe LL, Harty JT,
Rothman PB. NFIL3/E4BP4 is a key transcription
factor for CD8adendritic cell development.
Blood 2011;117:61936197.
184. Motomura Y, et al. The transcription factor
E4BP4 regulates the production of IL-10 and
IL-13 in CD4+T cells. Nat Immunol
2011;12:450459.
185. Yu X, et al. TH17 cell differentiation is regulated
by the circadian clock. Science 2013;342:727
730.
186. Kashiwada M, et al. IL-4-induced transcription
factor NFIL3/E4BP4 controls IgE class switching.
Proc Natl Acad Sci 2010;107:821826.
187. Constantinides MG, McDonald BD, Verhoef PA,
Bendelac A. A committed precursor to innate
lymphoid cells. Nature 2014;508:397401.
188. Aliahmad P, de la Torre B, Kaye J. Shared
dependence on the DNA-binding factor TOX for
the development of lymphoid tissue-inducer cell
and NK cell lineages. Nat Immunol
2010;11:945952.
189. Aliahmad P, Kaye J. Development of all CD4 T
lineages requires nuclear factor TOX. J Exp Med
2008;205:245256.
190. Socolovsky M, Fallon AE, Wang S, Brugnara C,
Lodish HF. Fetal anemia and apoptosis of red cell
progenitors in Stat5a-/-5b-/- mice: a direct role
for Stat5 in Bcl-X(L) induction. Cell
1999;98:181191.
191. Teglund S, et al. Stat5a and Stat5b proteins have
essential and nonessential, or redundant, roles in
cytokine responses. Cell 1998;93:841850.
192. Udy GB, et al. Requirement of STAT5b for sexual
dimorphism of body growth rates and liver gene
expression. Proc Natl Acad Sci USA
1997;94:72397244.
193. Liu X, Robinson GW, Wagner KU, Garrett L,
Wynshaw-Boris A, Hennighausen L. Stat5a is
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
44 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
mandatory for adult mammary gland
development and lactogenesis. Genes Dev
1997;11:179186.
194. Cui Y, et al. Inactivation of Stat5 in mouse
mammary epithelium during pregnancy reveals
distinct functions in cell proliferation, survival,
and differentiation. Mol Cell Biol 2004;24:8037
8047.
195. Yao Z, et al. Stat5a/b are essential for normal
lymphoid development and differentiation. Proc
Natl Acad Sci USA 2006;103:10001005.
196. Wei L, Laurence A, O’Shea JJ. New insights into
the roles of Stat5a/b and Stat3 in T cell
development and differentiation. Semin Cell Dev
Biol 2008;19:394400.
197. Wang Z, Bunting KD. STAT5 in hematopoietic
stem cell biology and transplantation. JAKSTAT
2013;2:e27159.
198. Huntington ND. The unconventional expression
of IL-15 and its role in NK cell homeostasis.
Immunol Cell Biol 2014;92:210213.
199. Boyman O, Krieg C, Homann D, Sprent J.
Homeostatic maintenance of T cells and natural
killer cells. Cell Mol Life Sci 2012;69:1597
1608.
200. Miyao T, et al. Plasticity of Foxp3(+) T cells
reflects promiscuous Foxp3 expression in
conventional T cells but not reprogramming
of regulatory T cells. Immunity 2012;36:262
275.
201. Liao W, Lin J-X, Wang L, Li P, Leonard WJ.
Modulation of cytokine receptors by IL-2 broadly
regulates differentiation into helper T cell
lineages. Nat Immunol 2011;12:551559.
202. Laurence A, et al. Interleukin-2 signaling via
STAT5 constrains T helper 17 cell generation.
Immunity 2007;26:371381.
203. Ballesteros-Tato A, et al. Interleukin-2 inhibits
germinal center formation by limiting T follicular
helper cell differentiation. Immunity
2012;36:847856.
204. Oestreich KJ, Mohn SE, Weinmann AS. Molecular
mechanisms that control the expression and
activity of Bcl-6 in TH1 cells to regulate
flexibility with a TFH-like gene profile. Nat
Immunol 2012;13:405411.
205. Nurieva RI, et al. STAT5 protein negatively
regulates T follicular helper (Tfh) cell generation
and function. J Biol Chem 2012;287:11234
11239.
206. Johnston RJ, Choi YS, Diamond JA, Yang JA,
Crotty S. STAT5 is a potent negative regulator of
TFH cell differentiation. J Exp Med
2012;209:243250.
207. Yang X-P, et al. Opposing regulation of the locus
encoding IL-17 through direct, reciprocal actions
of STAT3 and STAT5. Nat Immunol
2011;12:247254.
208. Hirahara K, et al. Mechanisms underlying helper
T-cell plasticity: implications for
immune-mediated disease. J Allergy Clin
Immunol 2013;131:12761287.
209. Schmitt N, et al. IL-12 receptor b1 deficiency
alters in vivo T follicular helper cell response in
humans. Blood 2013;121:33753385.
210. Ma CS, et al. Functional STAT3 deficiency
compromises the generation of human T
follicular helper cells. Blood 2012;119:3997
4008.
211. Nurieva RI, et al. Generation of T follicular
helper cells is mediated by interleukin-21 but
independent of T helper 1, 2, or 17 cell lineages.
Immunity 2008;29:138149.
212. Nakayamada S, et al. Type I IFN induces binding
of STAT1 to Bcl6: divergent roles of STAT family
transcription factors in the T follicular helper cell
genetic program. J Immunol 2014;192:2156
2166.
213. Choi YS, Eto D, Yang JA, Lao C, Crotty S. Cutting
edge: STAT1 is required for IL-6-mediated Bcl6
induction for early follicular helper cell
differentiation. J Immunol 2013;190:30493053.
214. Ray JP, Marshall HD, Laidlaw BJ, Staron MM,
Kaech SM, Craft J. Transcription factor STAT3
and type I interferons are corepressive insulators
for differentiation of follicular helper and T
helper 1 cells. Immunity 2014;40:367377.
215. Durant L, et al. Diverse targets of the
transcription factor STAT3 contribute to T cell
pathogenicity and homeostasis. Immunity
2010;32:605615.
216. Wei L, et al. Discrete roles of STAT4 and STAT6
transcription factors in tuning epigenetic
modifications and transcription during T helper
cell differentiation. Immunity 2010;32:840851.
217. Ciofani M, et al. A validated regulatory network
for Th17 cell specification. Cell 2012;151:289
303.
218. Escobar T, Yu C-R, Muljo SA, Egwuagu CE.
STAT3 activates miR-155 in Th17 cells and acts
in concert to promote experimental autoimmune
uveitis. Invest Ophthalmol Vis Sci
2013;54:40174025.
219. Witte S, Muljo SA. Integrating non-coding RNAs
in JAK-STAT regulatory networks. JAKSTAT
2014;3:e28055.
220. Hu G, et al. Expression and regulation of
intergenic long noncoding RNAs during T cell
development and differentiation. Nat Immunol
2013;14:11901198.
221. Miyagi T, Gil MP, Wang X, Louten J, Chu W-M,
Biron CA. High basal STAT4 balanced by STAT1
induction to control type 1 interferon effects in
natural killer cells. J Exp Med 2007;204:2383
2396.
222. Guo X, et al. Induction of innate lymphoid
cell-derived interleukin-22 by the transcription
factor STAT3 mediates protection against
intestinal infection. Immunity 2014;40:2539.
223. Lazarevic V, Glimcher LH, Lord GM. T-bet: a
bridge between innate and adaptive immunity.
Nat Rev Immunol 2013;13:777789.
224. Scium$e G, et al. Distinct requirements for T-bet
in gut innate lymphoid cells. J Exp Med
2012;209:23312338.
225. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR,
Sleckman BP, Glimcher LH. Distinct effects of
T-bet in TH1 lineage commitment and
IFN-gamma production in CD4 and CD8 T cells.
Science 2002;295:338342.
226. Townsend MJ, et al. T-bet regulates the terminal
maturation and homeostasis of NK and
Valpha14i NKT cells. Immunity 2004;20:477
494.
227. Way SS, Wilson CB. Cutting edge: immunity and
IFN-gamma production during Listeria
monocytogenes infection in the absence of T-bet.
J Immunol 2004;173:59185922.
228. Sullivan BM, Juedes A, Szabo SJ, von Herrath M,
Glimcher LH. Antigen-driven effector CD8 T cell
function regulated by T-bet. Proc Natl Acad Sci
USA 2003;100:1581815823.
229. Gordon SM, et al. The transcription factors T-bet
and Eomes control key checkpoints of natural
killer cell maturation. Immunity 2012;36:5567.
230. Zhu J, et al. The transcription factor T-bet is
induced by multiple pathways and prevents an
endogenous Th2 cell program during Th1 cell
responses. Immunity 2012;37:660673.
231. Rankin LC, et al. The transcription factor T-bet is
essential for the development of NKp46+innate
lymphocytes via the Notch pathway. Nat
Immunol 2013;14:389395.
232. Kaech SM, Cui W. Transcriptional control of
effector and memory CD8(+) T cell
differentiation. Nat Rev Immunol 2012;12:749
761.
233. Klose CSN, et al. Differentiation of type 1 ILCs
from a common progenitor to all helper-like
innate lymphoid cell lineages. Cell
2014;157:340356.
234. Miyazaki K, Miyazaki M, Murre C. The
establishment of B versusT cell identity. Trends
Immunol 2014;35:205210.
235. Xiong Y, et al. Thpok-independent repression of
Runx3 by Gata3 during CD4+T-cell
differentiation in the thymus. Eur J Immunol
2013;43:918928.
236. Mj
osberg J, et al. The transcription factor GATA3
is essential for the function of human type 2
innate lymphoid cells. Immunity 2012;37:649
659.
237. Klein Wolterink RGJ, et al. Essential,
dose-dependent role for the transcription factor
Gata3 in the development of IL-5+and IL-13+
type 2 innate lymphoid cells. Proc Natl Acad Sci
2013;110:1024010245.
238. Hoyler T, et al. The transcription factor GATA-3
controls cell fate and maintenance of type 2
innate lymphoid cells. Immunity 2012;37:634
648.
239. Yagi R, et al. The transcription factor GATA3 is
critical for the development of all
IL-7Ra-expressing innate lymphoid cells.
Immunity 2014;40:378388.
240. Wei G, et al. Genome-wide analyses of
transcription factor GATA3-mediated gene
regulation in distinct T cell types. Immunity
2011;35:299311.
241. Jetten AM, Kurebayashi S, Ueda E. The ROR
nuclear orphan receptor subfamily: critical
regulators of multiple biological processes. Prog
Nucleic Acid Res Mol Biol 2001;69:205247.
242. Sun Z, et al. Requirement for RORgamma in
thymocyte survival and lymphoid organ
development. Science 2000;288:23692373.
243. Kurebayashi S, et al. Retinoid-related orphan
receptor gamma (RORgamma) is essential for
lymphoid organogenesis and controls apoptosis
during thymopoiesis. Proc Natl Acad Sci USA
2000;97:1013210137.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 45
Shih et al !Toward the elucidation of the helper T-cell regulome
244. Michel M-L, et al. Critical role of ROR-ct in a
new thymic pathway leading to IL-17-producing
invariant NKT cell differentiation. Proc Natl Acad
Sci 2008;105:1984519850.
245. Okamoto K, et al. IkappaBzeta regulates T(H)17
development by cooperating with ROR nuclear
receptors. Nature 2010;464:13811385.
246. Luo CT, Li MO. Transcriptional control of
regulatory T cell development and function.
Trends Immunol 2013;34:531539.
247. Ramsdell F, Ziegler SF. FOXP3 and scurfy: how it
all began. Nat Rev Immunol 2014;14:343349.
248. Ohkura N, Kitagawa Y, Sakaguchi S.
Development and maintenance of regulatory
T cells. Immunity 2013;38:414423.
249. Hatzi K, Melnick A. Breaking bad in the germinal
center: how deregulation of BCL6 contributes to
lymphomagenesis. Trends Mol Med
2014;20:343352.
250. Swaminathan S, Duy C, Muschen M.
BACH2-BCL6 balance regulates selection at the
pre-B cell receptor checkpoint. Trends Immunol
2014;35:131137.
251. Nutt SL, Taubenheim N, Hasbold J, Corcoran
LM, Hodgkin PD. The genetic network
controlling plasma cell differentiation. Semin
Immunol 2011;23:341349.
252. Kallies A, et al. A role for Blimp1 in the
transcriptional network controlling natural killer
cell maturation. Blood 2011;117:18691879.
253. Rutishauser RL, et al. Transcriptional repressor
Blimp-1 promotes CD8(+) T cell terminal
differentiation and represses the acquisition of
central memory T cell properties. Immunity
2009;31:296308.
254. Kallies A, Xin A, Belz GT, Nutt SL. Blimp-1
transcription factor is required for the
differentiation of effector CD8(+) T cells and
memory responses. Immunity 2009;31:283295.
255. Shin H, et al. A role for the transcriptional
repressor Blimp-1 in CD8(+) T cell exhaustion
during chronic viral infection. Immunity
2009;31:309320.
256. Martins GA, et al. Transcriptional repressor
Blimp-1 regulates T cell homeostasis and
function. Nat Immunol 2006;7:457465.
257. Kallies A, et al. Transcriptional repressor Blimp-1
is essential for T cell homeostasis and
self-tolerance. Nat Immunol 2006;7:466474.
258. Nutt SL, Fairfax KA, Kallies A. BLIMP1 guides the
fate of effector B and T cells. Nat Rev Immunol
2007;7:923927.
259. Lin Y, Wong K, Calame K. Repression of c-myc
transcription by Blimp-1, an inducer of terminal B
cell differentiation. Science 1997;276:596599.
260. Turner CA, Mack DH, Davis MM. Blimp-1, a
novel zinc finger-containing protein that can
drive the maturation of B lymphocytes into
immunoglobulin-secreting cells. Cell
1994;77:297306.
261. Ochiai K, et al. Plasmacytic transcription factor
Blimp-1 is repressed by Bach2 in B cells. J Biol
Chem 2006;281:3822638234.
262. Shaffer AL, Yu X, He Y, Boldrick J, Chan EP,
Staudt LM. BCL-6 represses genes that function in
lymphocyte differentiation, inflammation, and
cell cycle control. Immunity 2000;13:199212.
263. Muto A, et al. Bach2 represses plasma cell gene
regulatory network in B cells to promote
antibody class switch. EMBO J 2010;29:4048
4061.
264. Muto A, et al. The transcriptional programme of
antibody class switching involves the repressor
Bach2. Nature 2004;429:566571.
265. Tsukumo S-I, et al. Bach2 maintains T cells in a
naive state by suppressing effector
memory-related genes. Proc Natl Acad Sci
2013;110:1073510740.
266. Roychoudhuri R, et al. BACH2 represses effector
programs to stabilize T(reg)-mediated immune
homeostasis. Nature 2013;498:506510.
267. Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM.
Control of inflammation, cytokine expression,
and germinal center formation by BCL-6. Science
1997;276:589592.
268. Ye BH, et al. The BCL-6 proto-oncogene controls
germinal-centre formation and Th2-type
inflammation. Nat Genet 1997;16:161170.
269. Ranuncolo SM, et al. Bcl-6 mediates the germinal
center B cell phenotype and lymphomagenesis
through transcriptional repression of the
DNA-damage sensor ATR. Nat Immunol
2007;8:705714.
270. Phan RT, Dalla-Favera R. The BCL6
proto-oncogene suppresses p53 expression in
germinal-centre B cells. Nature 2004;432:635
639.
271. Shin HM, Kapoor VN, Guan T, Kaech SM, Welsh
RM, Berg LJ. Epigenetic modifications induced by
Blimp-1 Regulate CD8T cell memory
progression during acute virus infection.
Immunity 2013;39:661675.
272. Morgan MAJ, Mould AW, Li L, Robertson EJ,
Bikoff EK. Alternative splicing regulates Prdm1/
Blimp-1 DNA binding activities and corepressor
interactions. Mol Cell Biol 2012;32:34033413.
273. Gyory I, Wu J, Fej$er G, Seto E, Wright KL.
PRDI-BF1 recruits the histone H3
methyltransferase G9a in transcriptional silencing.
Nat Immunol 2004;5:299308.
274. Ahmad KF, et al. Mechanism of SMRT
corepressor recruitment by the BCL6 BTB
domain. Mol Cell 2003;12:15511564.
275. Muto A, et al. Activation of Maf/AP-1 repressor
Bach2 by oxidative stress promotes apoptosis and
its interaction with promyelocytic leukemia
nuclear bodies. J Biol Chem 2002;277:20724
20733.
276. Oyake T, et al. Bach proteins belong to a novel
family of BTB-basic leucine zipper transcription
factors that interact with MafK and regulate
transcription through the NF-E2 site. Mol Cell
Biol 1996;16:60836095.
277. Kobayashi A, et al. A combinatorial code for
gene expression generated by transcription factor
Bach2 and MAZR (MAZ-related factor) through
the BTB/POZ domain. Mol Cell Biol
2000;20:17331746.
278. Ghetu AF, Corcoran CM, Cerchietti L, Bardwell
VJ, Melnick A, Priv$e GG. Structure of a BCOR
corepressor peptide in complex with the BCL6
BTB domain dimer. Mol Cell 2008;29:384391.
279. Huynh KD, Bardwell VJ. The BCL-6 POZ domain
and other POZ domains interact with the
co-repressors N-CoR and SMRT. Oncogene
1998;17:24732484.
280. Hatzi K, et al. A hybrid mechanism of action for
BCL6 in B cells defined by formation of
functionally distinct complexes at enhancers and
promoters. Cell Rep 2013;4:578588.
281. Yamane H, Paul WE. Early signaling events that
underlie fate decisions of naive CD4(+) T cells
toward distinct T-helper cell subsets. Immunol
Rev 2013;252:1223.
282. Vosshenrich CAJ, Di Santo JP. Developmental
programming of natural killer and innate
lymphoid cells. Curr Opin Immunol
2013;25:130138.
283. Lanier LL. NK cell recognition. Annu Rev
Immunol 2005;23:225274.
284. Staudt V, et al. Interferon-regulatory factor 4 is
essentialfor the developmental program of T
helper 9 cells. Immunity 2010;33:192202.
285. Ahyi ANN, Chang HC, Dent AL, Nutt SL, Kaplan
MH. IFN regulatory factor 4 regulates the
expression of a subset of Th2 cytokines. J
Immunol 2009;183:15981606.
286. Schraml BU, et al. The AP-1 transcription factor
Batf controls T. Nature 2010;460:405409.
287. Brustle A, et al. The development of
inflammatory TH-17 cells requires
interferon-regulatory factor 4. Nat Immunol
2007;8:958966.
288. Kwon H, et al. Analysis of interleukin-21-induced
Prdm1 gene regulation reveals functional
cooperation of STAT3 and IRF4 transcription
factors. Immunity 2009;31:941952.
289. Zheng Y, et al. Regulatory T-cell suppressor
program co-opts transcription factor IRF4 to
control T. Nature 2009;458:351356.
290. Li P, et al. BATFJUN is critical for
IRF4-mediated transcription in T cells. Nature
2013;490:543546.
291. Kim JI, Ho IC, Grusby MJ, Glimcher LH. The
transcription factor c-Maf controls the production
of interleukin-4 but not other Th2 cytokines.
Immunity 1999;10:745751.
292. Apetoh L, et al. The aryl hydrocarbon receptor
interacts with c-Maf to promote the differentiation
of type 1 regulatory T cells induced by IL-27. Nat
Immunol 2010;11:854861.
293. Xu J, et al. c-Maf regulates IL-10 expression
during Th17 polarization. J Immunol
2009;182:62266236.
294. Rutz S, et al. Transcription factor c-Maf mediates
the TGF-b-dependent suppression of IL-22
production in TH17 cells. Nat Immunol
2011;12:12381245.
295. Qiu J, et al. The aryl hydrocarbon receptor
regulates gut immunity through modulation of
innate lymphoid cells. Immunity 2012;36:92
104.
296. Lee JS, et al. AHR drives the development of gut
ILC22 cells and postnatal lymphoid tissues via
pathways dependent on and independent of
Notch. Nat Immunol 2012;13:144151.
297. Veldhoen M, Hirota K, Christensen J, O’Garra A,
Stockinger B. Natural agonists for aryl
hydrocarbon receptor in culture medium are
essential for optimal differentiation of Th17 T
cells. J Exp Med 2009;206:4349.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
46 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
298. Veldhoen M, et al. The aryl hydrocarbon
receptor links TH17-cell-mediated autoimmunity
to environmental toxins. Nature 2008;453:106
109.
299. Yosef N, et al. Dynamic regulatory network
controlling TH17 cell differentiation. Nature
2013;496:461468.
300. Spencer SP, et al. Adaptation of innate lymphoid
cells to a micronutrient deficiency promotes type
2 barrier immunity. Science 2014;343:432437.
301. Mucida D, et al. Reciprocal TH17 and regulatory
T cell differentiation mediated by retinoic acid.
Science 2007;317:256260.
302. Wu C, et al. Induction of pathogenic TH17 cells
by inducible salt-sensing kinase SGK1. Nature
2013;496:513517.
303. Kleinewietfeld M, et al. Sodium chloride drives
autoimmune disease by the induction of
pathogenic TH17 cells. Nature 2013;496:518
522.
304. Heikamp EB, et al. The AGC kinase SGK1
regulates TH1 and TH2 differentiation
downstream of the mTORC2 complex. Nat
Immunol 2014;15:457464.
305. Smale ST, Tarakhovsky A, Natoli G. Chromatin
contributions to the regulation of innate
immunity. Annu Rev Immunol 2014;32:489
511.
306. Rothenberg EV. The chromatin landscape and
transcription factors in T cell programming.
Trends Immunol 2014;35:195204.
307. Suzuki MM, Bird A. DNA methylation landscapes:
provocative insights from epigenomics. Nat Rev
Genet 2008;9:465476.
308. Collings CK, Waddell PJ, Anderson JN. Effects of
DNA methylation on nucleosome stability.
Nucleic Acids Res 2013;41:29182931.
309. Fenouil R, et al. CpG islands and GC content
dictate nucleosome depletion in a
transcription-independent manner at mammalian
promoters. Genome Res 2012;22:23992408.
310. Lee PP, et al. A critical role for Dnmt1 and DNA
methylation in T cell development, function, and
survival. Immunity 2001;15:763774.
311. Bruniquel D, Schwartz RH. Selective, stable
demethylation of the interleukin-2 gene enhances
transcription by an active process. Nat Immunol
2003;4:235240.
312. Lee DU, Agarwal S, Rao A. Th2 lineage
commitment and efficient IL-4 production
involves extended demethylation of the IL-4
gene. Immunity 2002;16:649660.
313. Santangelo S, Cousins DJ, Triantaphyllopoulos K,
Staynov DZ. Chromatin structure and DNA
methylation of the IL-4 gene in human TH2
cells. Chromosome Res 2009;17:485496.
314. Schoenborn JR, et al. Comprehensive epigenetic
profiling identifies multiple distal regulatory
elements directing transcription of the gene
encoding interferon-c. Nat Immunol
2007;8:732742.
315. Kim ST, Fields PE, Flavell RA. Demethylation of a
specific hypersensitive site in the Th2 locus
control region. Proc Natl Acad Sci USA
2007;104:1705217057.
316. Thomas RM, Sai H, Wells AD. Conserved
intergenic elements and DNA methylation
cooperate to regulate transcription at the il17
locus. J Biol Chem 2012;287:2504925059.
317. Zheng Y, Josefowicz S, Chaudhry A, Peng XP,
Forbush K, Rudensky AY. Role of conserved
non-coding DNA elements in the Foxp3 gene in
regulatory T-cell fate. Nature 2010;463:808
812.
318. Ohkura N, et al. T cell receptor
stimulation-induced epigenetic changes and
Foxp3 expression are independent and
complementary events required for treg cell
development. Immunity 2012;37:785799.
319. Toker A, et al. Active demethylation of the
Foxp3 locus leads to the generation of stable
regulatory T cells within the thymus. J Immunol
2013;190:31803188.
320. Rouhi A, Gagnier L, Takei F, Mager DL. Evidence
for epigenetic maintenance of Ly49a monoallelic
gene expression. J Immunol 2006;176:2991
2999.
321. Yokoyama WM, Kehn PJ, Cohen DI, Shevach EM.
Chromosomal location of the Ly-49 (A1, YE1/
48) multigene family. Genetic association with
the NK 1.1 antigen. J Immunol 1990;145:2353
2358.
322. Belanger S, Tai L-H, Anderson SK, Makrigiannis
AP. Ly49 cluster sequence analysis in a mouse
model of diabetes: an expanded repertoire of
activating receptors in the NOD genome.
Genetics 2008;9:509521.
323. Held W, Roland J, Raulet DH. Allelic exclusion
of Ly49-family genes encoding class I
MHC-specific receptors on NK cells. Nature
1995;376:355358.
324. Kersh EN, et al. Rapid demethylation of the
IFN-gene occurs in memory but not naive CD8 T
cells. J Immunol 2006;176:40834093.
325. Pastor WA, Aravind L, Rao A. TETonic shift:
biological roles ofTET proteins in DNA
demethylationand transcription. Nat Rev Mol Cell
Biol 2013;14:341356.
326. Aoki K, Sato N, Yamaguchi A, Kaminuma O,
Hosozawa T, Miyatake S. Regulation of DNA
demethylation during maturation of CD4+naive
T cells by the conserved noncoding sequence 1. J
Immunol 2009;182:76987707.
327. Wang L, et al. Mbd2 promotes Foxp3
demethylation and T-regulatory-cell function.
Mol Cell Biol 2013;33:41064115.
328. Lal G, et al. Epigenetic regulation of Foxp3
expression in regulatory T cells by DNA
methylation. J Immunol 2009;182:259273.
329. Song C-X, et al. Genome-wide profiling of
5-formylcytosine reveals its roles in epigenetic
priming. Cell 2013;153:678691.
330. Shen L, et al. Genome-wide analysis reveals TET-
and TDG-dependent 5-methylcytosine oxidation
dynamics. Cell 2013;153:692706.
331. Ziller MJ, et al. Charting a dynamic DNA
methylation landscape of the human genome.
Nature 2013;500:477481.
332. Pastor WA, et al. Genome-wide mapping of
5-hydroxymethylcytosine in embryonic stem
cells. Nature 2012;473:394397.
333. Williams K, et al. TET1 and
hydroxymethylcytosine in transcription and DNA
methylation fidelity. Nature 2011;473:343348.
334. Ficz G, et al. Dynamic regulation of
5-hydroxymethylcytosine in mouse ES cells and
during differentiation. Nature 2012;473:398402.
335. Gu T-P, et al. The role of Tet3 DNA dioxygenase
in epigenetic reprogramming by oocytes. Nature
2012;477:606610.
336. Wu H, et al. Dual functions of Tet1 in
transcriptional regulation in mouse embryonic
stem cells. Nature 2011;473:389393.
337. Bergman Y, Cedar H. DNA methylation dynamics
in health and disease. Nat Struct Mol Biol
2013;20:274281.
338. Altorok N, Sawalha AH. Epigenetics in the
pathogenesis of systemic lupus erythematosus.
Curr Opin Rheumatol 2013;25:569576.
339. Absher DM, et al. Genome-wide DNA
methylation analysis of systemic lupus
erythematosus reveals persistent hypomethylation
of interferon genes and compositional changes to
CD4+T-cell populations. PLoS Genet 2013;9:
e1003678.
340. Corvetta A, Della Bitta R, Luchetti MM,
Pomponio G. 5-methylcytosine content of DNA
in blood, synovial mononuclear cells and
synovial tissue from patients affected by
autoimmune rheumatic diseases. J Chromatogr
1991;566:481491.
341. Richardson B, Scheinbart L, Strahler J, Gross L,
Hanash S, Johnson M. Evidence for impaired T
cell DNA methylation in systemic lupus
erythematosus and rheumatoid arthritis. Arthritis
Rheum 1990;33:16651673.
342. Quddus J, et al. Treating activated CD4+T cells
with either of two distinct DNA
methyltransferase inhibitors, 5-azacytidine or
procainamide, is sufficient to cause a lupus-like
disease in syngeneic mice. J Clin Invest
1993;92:3853.
343. Cornacchia E, Golbus J, Maybaum J, Strahler J,
Hanash S, Richardson B. Hydralazine and
procainamide inhibit T cell DNA methylation and
induce autoreactivity. J Immunol
1988;140:21972200.
344. Struhl K, Segal E. Determinants of nucleosome
positioning. Nat Struct Mol Biol 2013;20:267
273.
345. Chi TH, et al. Sequential roles of Brg, the ATPase
subunit of BAF chromatin remodeling
complexes, in thymocyte development. Immunity
2003;19:169182.
346. Chi TH, et al. Reciprocal regulation of CD4/CD8
expression by SWI/SNF-like BAF complexes.
Nature 2002;418:195199.
347. Zhang F, Boothby M. T helper type 1-specific
Brg1 recruitment and remodeling of
nucleosomes positioned at the IFN-gamma
promoter are Stat4 dependent. J Exp Med
2006;203:14931505.
348. Letimier FA, Passini N, Gasparian S, Bianchi E,
Rogge L. Chromatin remodeling by the SWI/
SNF-like BAF complex and STAT4 activation
synergistically induce IL-12Rbeta2 expression
during human Th1 cell differentiation. EMBO J
2007;26:12921302.
349. Chaiyachati BH, Jani A, Wan Y, Huang H, Flavell
R, Chi T. BRG1-mediated immune tolerance:
facilitation of Treg activation and partial
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 47
Shih et al !Toward the elucidation of the helper T-cell regulome
independence of chromatin remodelling. EMBO J
2013;32:395408.
350. Kanno Y, Vahedi G, Hirahara K, Singleton K,
O’Shea JJ. Transcriptional and epigenetic control of
T helper cell specification: molecular mechanisms
underlying commitment and plasticity*. Annu Rev
Immunol 2012;30:707731.
351. Rivera CM, Ren B. Mapping human epigenomes.
Cell 2013;155:3955.
352. Ruthenburg AJ, Li H, Patel DJ, Allis CD.
Multivalent engagement of chromatin
modifications by linked binding modules. Nat
Rev Mol Cell Biol 2007;8:983994.
353. Ntziachristos P, et al. Genetic inactivation of the
polycomb repressive complex 2 in T cell acute
lymphoblastic leukemia. Nat Med 2012;18:298
301.
354. Kleer CG, et al. EZH2 is a marker of aggressive
breast cancer and promotes neoplastic
transformation of breast epithelial cells. Proc Natl
Acad Sci USA 2003;100:1160611611.
355. Varambally S, et al. The polycomb group protein
EZH2 is involved in progression of prostate
cancer. Nature 2002;419:624629.
356. Koyanagi M. EZH2 and histone 3 trimethyl lysine
27 associated with Il4 and Il13 gene silencing in
TH1 cells. J Biol Chem 2005;280:3147031477.
357. Tumes DJ, et al. The polycomb protein Ezh2
regulates differentiation and plasticity of CD4.
Immunity 2013;39:819832.
358. Tong Q, et al. Ezh2 regulates transcriptional and
post-translational expression of T-bet and
promotes Th1 cell responses mediating aplastic
anemia in mice. J Immunol 2014;192:5012
5022.
359. Wang A, Pan D, Lee Y-H, Martinez GJ, Feng
X-H, Dong C. Cutting edge: Smad2 and Smad4
regulate TGF-b-mediated Il9 gene expression via
EZH2 displacement. J Immunol 2013;191:4908
4912.
360. Arvey A, van der Veeken J, Samstein RM, Feng
Y, Stamatoyannopoulos JA, Rudensky AY.
Inflammation-induced repression of chromatin
bound by the transcription factor Foxp3 in
regulatory T cells. Nat Immunol 2014;15:580
587.
361. Escobar TM, et al. miR-155 activates cytokine
gene expression in Th17 cells by regulating the
DNA-binding protein Jarid2 to relieve
polycomb-mediated repression. Immunity
2014;40:865879.
362. Su I-H, et al. Polycomb group protein ezh2
controls actin polymerization and cell signaling.
Cell 2005;121:425436.
363. Allan RS, et al. An epigenetic silencing pathway
controlling T helper 2 cell lineage commitment.
Nature 2012;487:249253.
364. Wei G, et al. Global mapping of H3K4me3 and
H3K27me3 reveals specificity and plasticity in
lineage fate determination of differentiating
CD4+T cells. Immunity 2009;30:155167.
365. Belkina AC, Denis GV. BET domain co-regulators
in obesity, inflammation and cancer. Nat Rev
Cancer 2012;12:465477.
366. Delmore JE, et al. BET bromodomain inhibition
as a therapeutic strategy to target c-Myc. Cell
2011;146:904917.
367. Nicodeme E, et al. Suppression of inflammation
by a synthetic histone mimic. Nature
2010;468:11191123.
368. Mele DA, Salmeron A, Ghosh S, Huang H-R,
Bryant BM, Lora JM. BET bromodomain
inhibition suppresses TH17-mediated pathology.
J Exp Med 2013;210:21812190.
369. Bandukwala HS, et al. Selective inhibition of
CD4+T-cell cytokine production and
autoimmunity by BET protein and c-Myc
inhibitors. Proc Natl Acad Sci 2012;109:14532
14537.
370. Wilson CB, Rowell E, Sekimata M. Epigenetic
control of T-helper-cell differentiation. Nat Rev
Immunol 2009;9:91105.
371. Harada Y, et al. The 3’ enhancer CNS2 is a
critical regulator of interleukin-4-mediated
humoral immunity in follicular helper T cells.
Immunity 2012;36:188200.
372. Heintzman ND, et al. Histone modifications at
human enhancers reflect global cell-type-specific
gene expression. Nature 2009;459:108112.
373. Rada-Iglesias A, Bajpai R, Swigut T, Brugmann
SA, Flynn RA, Wysocka J. A unique chromatin
signature uncovers early developmental enhancers
in humans. Nature 2011;470:279283.
374. Creyghton MP, et al. Histone H3K27ac separates
active from poised enhancers and predicts
developmental state. Proc Natl Acad Sci
2010;107:2193121936.
375. Visel A, et al. ChIP-seq accurately predicts
tissue-specific activity of enhancers. Nature
2009;457:854858.
376. Nord AS, et al. Rapid and pervasive changes in
genome-wide enhancer usage during mammalian
development. Cell 2013;155:15211531.
377. Ghisletti S, et al. Identification and
characterization of enhancers controlling the
inflammatory gene expression program in
macrophages. Immunity 2010;32:317328.
378. Hnisz D, et al. Super-enhancers in the control of
cell identity and disease. Cell 2013;155:934
947.
379. Natoli G, Ghisletti S, Barozzi I. The genomic
landscapes of inflammation. Genes Dev
2011;25:101106.
380. Heinz S, et al. Simple combinations of
lineage-determining transcription factors prime
cis-regulatory elements required for macrophage
and B cell identities. Mol Cell 2010;38:576
589.
381. Vahedi G, et al. STATs shape the active enhancer
landscape of T cell populations. Cell
2012;151:981993.
382. Samstein RM, et al. Foxp3 exploits a pre-existent
enhancer landscape for regulatory T cell lineage
specification. Cell 2012;151:153166.
383. Buenrostro JD, Giresi PG, Zaba LC, Chang HY,
Greenleaf WJ. Transposition of native chromatin
for fast and sensitive epigenomic profiling of
open chromatin, DNA-binding proteins and
nucleosome position. Nat Methods
2013;10:12131218.
384. Hawkins RD, et al. Global chromatin state analysis
reveals lineage-specific enhancers during the
initiation of human T helper 1 and T helper 2 cell
polarization. Immunity 2013;38:12711284.
385. Whyte WA, et al. Master transcription factors and
mediator establish super-enhancers at key cell
identity genes. Cell 2013;153:307319.
386. Parker SCJ, et al. Chromatin stretch enhancer
states drive cell-specific gene regulation and
harbor human disease risk variants. Proc Natl
Acad Sci USA 2013;110:1792117926.
387. Cech TR, Steitz JA. The noncoding RNA
revolution-trashing old rules to forge new ones.
Cell 2014;157:7794.
388. Baumjohann D, Ansel KM. MicroRNA-mediated
regulationof T helper cell differentiation
andplasticity. Nat Rev Immunol 2013;13:666
678.
389. Sauvageau M, et al. Multiple knockout mouse
models reveal lincRNAs are required for life and
brain development. Elife 2013;2:e01749.
390. Rinn JL, Chang HY. Genome regulation by long
noncoding RNAs. Annu Rev Biochem
2012;81:145166.
391. Penny GD, Kay GF, Sheardown SA, Rastan S,
Brockdorff N. Requirement for Xist in X
chromosome inactivation. Nature 1996;379:131
137.
392. Wang KC, et al. A long noncoding RNA
maintains active chromatin to coordinate
homeotic gene expression. Nature
2012;472:120124.
393. Di Ruscio A, et al. DNMT1-interacting RNAs
block gene-specific DNA methylation. Nature
2013;503:371376.
394. Memczak S, et al. Circular RNAs are a large class
of animal RNAs with regulatory potency. Nature
2013;495:333338.
395. Hansen TB, Kjems J, Damgaard CK. Circular RNA
and miR-7 in cancer. Cancer Res 2013;73:5609
5612.
396. Guttman M, et al. lincRNAs act in the circuitry
controlling pluripotency and differentiation.
Nature 2011;477:295300.
397. Abarrategui I, Krangel MS. Germline
transcription: a key regulator of accessibility and
recombination. Adv Exp Med Biol 2009;650:93
102.
398. Manis JP, Tian M, Alt FW. Mechanism and
control of class-switch recombination. Trends
Immunol 2002;23:3139.
399. Gomez JA, et al. The NeST long ncRNA controls
microbial susceptibility and epigenetic activation
of the interferon-cLocus. Cell 2013;152:743
754.
400. Collier SP, Collins PL, Williams CL, Boothby MR,
Aune TM. Cutting edge: influence of Tmevpg1, a
long intergenic noncoding RNA, on the
expression of Ifng by Th1 cells. J Immunol
2012;189:20842088.
401. Carpe nter S, et al. A long noncoding RNA mediates
both activation and repression of immune response
genes. Science 2013;341:789792.
402. Kim T-K, et al. Widespread transcription at
neuronal activity-regulated enhancers. Nature
2010;465:182187.
403. De Santa F, et al. A large fraction of extragenic
RNA pol II transcription sites overlap enhancers.
PLoS Biol 2010;8:e1000384.
404. Djebali S, et al. Landscape of transcription in
human cells. Nature 2012;489:101108.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
48 Immunological Reviews 261/2014
Shih et al !Toward the elucidation of the helper T-cell regulome
405. Koch L. Non-coding RNA: small RNA determines
silkworm sex. Nat Rev Genet 2014;15:441.
406. Mousavi K, et al. eRNAs promote transcription
by establishing chromatin accessibility at
defined genomic loci. Mol Cell 2013;51:606
617.
407. Li W, et al. Functional roles of enhancer RNAs
for oestrogen-dependent transcriptional
activation. Nature 2013;498:516520.
408. Lam MTY, et al. Rev-Erbs repress macrophage
gene expression by inhibiting enhancer-directed
transcription. Nature 2013;498:511515.
409. Melo CA, et al. eRNAs are required for
p53-dependent enhancer activity and gene
transcription. Mol Cell 2013;49:524535.
410. Kanamori-Katayama M, et al. Unamplified cap
analysis of gene expression on a
single-molecule sequencer. Genome Res
2011;21:11501159.
411. Andersson R, et al. An atlas of active enhancers
across human cell types and tissues. Nature
2014;507:455461.
412. Muljo SA, Ansel KM, Kanellopoulou C,
Livingston DM, Rao A, Rajewsky K. Aberrant T
cell differentiation in the absence of Dicer. J Exp
Med 2005;202:261269.
413. Chong MMW, Rasmussen JP, Rudensky AY,
Rundensky AY, Littman DR. The RNAseIII
enzyme Drosha is critical in T cells for
preventing lethal inflammatory disease. J Exp
Med 2008;205:20052017.
414. Cobb BS, et al. A role for Dicer in immune
regulation. J Exp Med 2006;203:25192527.
415. Kohlhaas S, Garden OA, Scudamore C, Turner M,
Okkenhaug K, Vigorito E. Cutting edge: the
Foxp3 target miR-155 contributes to the
development of regulatory T cells. J Immunol
2009;182:25782582.
416. Lu L-F, et al. Foxp3-dependent microRNA155
confers competitive fitness to regulatory T cells
by targeting SOCS1 protein. Immunity
2009;30:8091.
417. Louafi F, Martinez-Nunez RT, Sanchez-Elsner T.
MicroRNA-155 targets SMAD2 and modulates the
response of macrophages to transforming growth
factor-{beta}. J Biol Chem 2010;285:41328
41336.
418. Rai D, Kim S-W, McKeller MR, Dahia PLM,
Aguiar RCT. Targeting of SMAD5 links
microRNA-155 to the TGF-beta pathway and
lymphomagenesis. Proc Natl Acad Sci
2010;107:31113116.
419. Hu R, et al. MicroRNA-155 confers
encephalogenic potential to Th17 cells by
promoting effector gene expression. J Immunol
2013;190:59725980.
420. Rodriguez A, et al. Requirement of bic/
microRNA-155 for normal immune function.
Science 2007;316:608611.
421. O’Connell RM, et al. MicroRNA-155 promotes
autoimmune inflammation by enhancing
inflammatory T cell development. Immunity
2010;33:607619.
422. Bluml S, et al. Essential role of microRNA-155 in
the pathogenesis of autoimmune arthritis in
mice. Arthritis Rheum 2011;63:12811288.
423. Murugaiyan G, Beynon V, Mittal A, Joller N,
Weiner HL. Silencing microRNA-155 ameliorates
experimental autoimmune encephalomyelitis. J
Immunol 2011;187:22132221.
424. Ma F, et al. The microRNA miR-29 controls
innate and adaptive immune responses to
intracellular bacterial infection by targeting
interferon-c. Nat Immunol 2011;12:861869.
425. Steiner DF, et al. MicroRNA-29 regulates T-box
transcription factors and interferon-cproduction
in helper T cells. Immunity 2011;35:169181.
426. Lu L-F, et al. Function of miR-146a in
controlling Treg cell-mediated regulation of Th1
responses. Cell 2010;142:914929.
427. Yang L, et al. miR-146a controls the resolution
of T cell responses in mice. J Exp Med
2012;209:16551670.
428. Takahashi H, et al. TGF-band retinoic acid
induce the microRNA miR-10a, which targets
Bcl-6 and constrains the plasticity of helper T
cells. Nat Immunol 2012;13:587595.
429. Li G, et al. Decline in miR-181a expression with
age impairs T cell receptor sensitivity by
increasing DUSP6 activity. Nat Med
2012;18:15181524.
430. Palin AC, Ramachandran V, Acharya S, Lewis DB.
Human neonatal naive CD4+T cells have
enhanced activation-dependent signaling
regulated by the microRNA miR-181a. J
Immunol 2013;190:26822691.
431. Li Q-J, et al. miR-181a is an intrinsic modulator
of T cell sensitivity and selection. Cell
2007;129:147161.
432. Wang H, Flach H, Onizawa M, Wei L, McManus
MT, Weiss A. Negative regulation of Hif1a
expression and TH17 differentiation by the
hypoxia-regulated microRNA miR-210. Nat
Immunol 2014;15:393401.
433. Kang SG, et al. MicroRNAs of the miR-17 #92
family are critical regulators of T(FH)
differentiation. Nat Immunol 2013;14:849857.
434. de Kouchkovsky D, Esensten JH, Rosenthal WL,
Morar MM, Bluestone JA, Jeker LT.
microRNA-17-92 regulates IL-10 production by
regulatory T cells and control of experimental
autoimmune encephalomyelitis. J Immunol
2013;191:15941605.
435. Gibcus JH, Dekker J. The hierarchy of the 3D
genome. Mol Cell 2013;49:773782.
436. de Wit E, de Laat W. A decade of 3C
technologies: insights into nuclear organization.
Genes Dev 2012;26:1124.
437. Lieberman-Aiden E, et al. Comprehensive
mapping of long-range interactions reveals
folding principles of the human genome. Science
2009;326:289293.
438. Dixon JR, et al. Topologicaldomainsinmammalian
genomesidentified by analysis of chromatin
interactions. Nature 2012;485:376380.
439. Nora EP, et al. Spatial partitioning of the
regulatory landscape of the X-inactivation centre.
Nature 2012;485:381385.
440. Phillips-Cremins JE, et al. Architectural protein
subclasses shape 3D organization of genomes
during lineage commitment. Cell
2013;153:12811295.
441. Zullo JM, et al. DNA sequence-dependent
compartmentalization and silencing of chromatin
at the nuclear lamina. Cell 2012;149:14741487.
442. Towbin BD, et al. Step-wise methylation of
histone H3K9 positions heterochromatin at the
nuclear periphery. Cell 2012;150:934947.
443. de Laat W, Duboule D. Topology of mammalian
developmental enhancers and their regulatory
landscapes. Nature 2013;502:499506.
444. Denholtz M, et al. Long-range chromatin contacts
in embryonic stem cells reveal a role for
pluripotency factors and polycomb proteins in
genome organization. Cell Stem Cell
2013;13:602616.
445. Sekimata M, et al. CCCTC-binding factor and the
transcription factor T-bet orchestrate T helper 1
cell-specific structure and function at the
interferon-gamma locus. Immunity
2009;31:551564.
446. Hadjur S, et al. Cohesins form chromosomal
cis-interactions at the developmentally regulated
IFNG locus. Nature 2009;460:410413.
447. Spilianakis CG, Flavell RA. Long-range
intrachromosomal interactions in the T helper
type 2 cytokine locus. Nat Immunol
2004;5:10171027.
448. Cai S, Lee CC, Kohwi-Shigematsu T. SATB1
packages densely looped, transcriptionally active
chromatin for coordinated expression of cytokine
genes. Nat Genet 2006;38:12781288.
449. Jin F, et al. A high-resolution map of
three-dimensional chromatin interactome in
human cells. Nature 2013;503:290294.
450. Kieffer-Kwon K-R, et al. Interactome maps of
mouse gene regulatory domains reveal basic
principles of transcriptional regulation. Cell
2013;155:15071520.
451. Fanucchi S, Shibayama Y, Burd S, Weinberg MS,
Mhlanga MM. Chromosomal contact permits
transcription between coregulated genes. Cell
2013;155:606620.
452. Spilianakis CG, Lalioti MD, Town T, Lee GR,
Flavell RA. Interchromosomal associations
between alternatively expressed loci. Nature
2005;435:637645.
453. Kim LK, et al. Oct-1 regulates IL-17 expressionby
directing interchromosomal associations in
conjunction with CTCF in T cells. Mol Cell
2014;54:5666.
454. Ohno S. Major regulatory genes for mammalian
sexual development. Cell 1976;7:315321.
Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
Immunological Reviews 261/2014 49
Shih et al !Toward the elucidation of the helper T-cell regulome
... Innate lymphoid cells (ILCs) play critical roles in tissue homeostasis, barrier integrity and primary host defense and mirror the functionalities of their effector counterparts in the adaptive immune compartment, CD4 + helper T (Th) and CD8 + cytotoxic T lymphocytes (CTL) (7)(8)(9)(10). The similarities between innate and adaptive lymphocyte programming have dramatically accelerated our understanding of ILC regulation using the knowledge accumulated from studies of T cells (11)(12)(13)(14). ...
... Finally, type 3 immunity is governed by RAR-related orphan receptor gamma, RORgt (encoded by the Rorc gene), which controls ILC3 and Th17 lineage specification and cytokine secretion (37, 38). These LDTFs epigenetically activate and stabilize function-related gene expression and, at the same time, inhibit transcription of genes that contribute to alternative cell fates (8,39). ...
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The precise control of cytokine production by innate lymphoid cells (ILCs) and their T cell adaptive system counterparts is critical to mounting a proper host defense immune response without inducing collateral damage and autoimmunity. Unlike T cells that differentiate into functionally divergent subsets upon antigen recognition, ILCs are developmentally programmed to rapidly respond to environmental signals in a polarized manner, without the need of T cell receptor (TCR) signaling. The specification of cytokine production relies on dynamic regulation of cis-regulatory elements that involve multi-dimensional epigenetic mechanisms, including DNA methylation, transcription factor binding, histone modification and DNA-DNA interactions that form chromatin loops. How these different layers of gene regulation coordinate with each other to fine tune cytokine production, and whether ILCs and their T cell analogs utilize the same regulatory strategy, remain largely unknown. Herein, we review the molecular mechanisms that underlie cell identity and functionality of helper T cells and ILCs, focusing on networks of transcription factors and cis-regulatory elements. We discuss how higher-order chromatin architecture orchestrates these components to construct lineage- and state-specific regulomes that support ordered immunoregulation.
... Indeed, the ILC1, ILC2 and ILC3 subsets produce T helper (Th) 1-, Th2-and Th17/22-cytokines, respectively. Furthermore, given their phenotypic, developmental and functional similarities, Natural Killer (NK) cells, the innate counterpart of cytotoxic T lymphocytes, are now grouped together with ILC1s, whereas lymphoid tissue inducer (LTi) cells, belong now to group 3 ILCs (5,7,8). ...
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Innate lymphoid cells (ILCs) were firstly described by different independent laboratories in 2008 as tissue-resident innate lymphocytes mirroring the phenotype and function of T helper cells. ILCs have been subdivided into three distinct subgroups, ILC1, ILC2 and ILC3, according to their cytokine and transcriptional profiles. Subsequently, also Natural Killer (NK) cells, that are considered the innate counterpart of cytotoxic CD8 T cells, were attributed to ILC1 subfamily, while lymphoid tissue inducer (LTi) cells were attributed to ILC3 subgroup. Starting from their discovery, significant advances have been made in our understanding of ILC impact in the maintenance of tissue homeostasis, in the protection against pathogens and in tumor immune-surveillance. However, there is still much to learn about ILC ontogenesis especially in humans. In this regard, NK cell developmental intermediates which have been well studied and characterized prior to the discovery of helper ILCs, have been used to shape a model of ILC ontogenesis. Herein, we will provide an overview of the current knowledge about NK cells and helper ILC ontogenesis in humans. We will also focus on the newly disclosed circulating ILC subsets with killing properties, namely unconventional CD56dim NK cells and cytotoxic helper ILCs, by discussing their possible role in ILC ontogenesis and their contribution in both physiological and pathological conditions.
... The human CD4+ T cell is a heterogeneous population, divided into sub-populations depending on the functions performed. The activities of individual subpopulations result from the type of transcription factors and receptors contained in cells, as well as secreted cytokines, specific for each subpopulation (22). ...
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Lymphocytes play a leading role in regulation of the immune system in lung cancer patients. The recognition of T cells profile may help in prediction of effectiveness of anticancer immunotherapy. The aim of the study was to determine the dominant subpopulation of CD4+ and CD8+ lymphocytes in metastatic and non-metastatic lymph nodes (LNs) of lung cancer patients. LNs aspirates were obtained during EBUS/TBNA procedure and cells were analyzed by flow cytometry. We showed a higher percentage of CD4+ and CD8+ effector memory T cells in the metastatic than in the non-metastatic LNs (28.6 vs. 15.3% and 28.6 vs. 14.0%, p< 0.05). The proportion of CD45RO+ T regulatory cells (CD45RO+ Tregs) was higher in the metastatic LNs than in the non-metastatic ones (65.6 vs. 31%, p< 0.05). We reported the significant differences in T cell subsets depending on the lung cancer metastatic process. We observed that the effector memory T cells were predominant subpopulations in metastatic LNs. Lymphocyte profile in LNs is easy to evaluate by flow cytometry of EBUS/TBNA samples and may reflect the immune status in lung cancer.
... The TF EOMES, T-BET, GATA3, and RORγt, mentioned above, are also referred to as lineage =-defining TFs (LDTFs), since these molecules dictate ILC fates and are required for determining the effector functions of mature ILC subsets [26,27]. LDTFs represent the first layer of ILC regulation, although the establishment of specific developmental programs and effector functions is now seen as the result of complex TF networks rather than the effect of one single "master" regulator [28]. ...
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Innate lymphoid cells (ILCs) are innate lymphocytes playing essential functions in protection against microbial infections and participate in both homeostatic and pathological contexts, including tissue remodeling, cancer, and inflammatory disorders. A number of lineage-defining transcription factors concur to establish transcriptional networks which determine the identity and the activity of the distinct ILC subsets. However, the contribution of other regulatory molecules in controlling ILC development and function is also recently emerging. In this regard, noncoding RNA (ncRNAs) represent key elements of the complex regulatory network of ILC biology and host protection. ncRNAs mostly lack protein-coding potential, but they are endowed with a relevant regulatory activity in immune and nonimmune cells because of their ability to control chromatin structure, RNA stability, and/or protein synthesis. Herein, we summarize recent studies describing how distinct types of ncRNAs, mainly microRNAs, long ncRNAs, and circular RNAs, act in the context of ILC biology. In particular, we comment on how ncRNAs can exert key effects in ILCs by controlling gene expression in a cell- or state-specific manner and how this tunes distinct functional outputs in ILCs.
... Most of the initial findings leading to the identification of novel ILC subsets were built upon the observation that "helper" responses could be found in Rag-deficient mice and, in general, were based on the evidence that non-T lymphocytes could produce large amounts of type 2 and type 3 cytokines (Cupedo et al., 2009;Fallon et al., 2006;Fort et al., 2001;Hurst et al., 2002;Moro et al., 2010;Neill et al., 2010;Price et al., 2010;Takatori et al., 2009). The identification of such innate cells able to perform effector functions similar to those observed in T cells, but in a shorter time frame, led scientists to rethink many aspects of the immune response, ranging from protection against pathogens to cancer immunosurveillance and immunostimulation (Cherrier et al., 2018;Mattiola and Diefenbach, 2019;Shih et al., 2014;. In, roughly, the last 10 years, the ILC field has brought to light a variety of subsets and complexity which parallel those of T cells Vivier et al., 2018). ...
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Innate lymphoid cells (ILCs) and tissue-resident natural killer (NK) cells ensure immunity at environmental interfaces and help maintain barrier integrity of the intestinal tract. This wide range of innate lymphocytes is able to provide fast and potent inflammatory responses that, when deregulated, have been associated with pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). While the presence of tumor-infiltrating NK cells is generally associated with a favorable outcome in CRC patients, emerging evidence reveals distinct roles for ILCs in regulating CRC pathogenesis and progression. Advances in next generation sequencing technology, and in particular of single-cell RNA-seq approaches, along with multidimensional flow cytometry analysis, have helped to deconvolute the complexity and heterogeneity of the ILC system both in homeostatic and pathological contexts. In this review, we discuss the protective and detrimental roles of NK cells and ILCs in the pathogenesis of CRC, focusing on the phenotypic and transcriptional modifications these cells undergo during CRC development and progression.