2. given to the molecular control that dictates the

2. Factors controlling fate of Treg
cells

The
function of regulatory T cells to mediate immune responses, in health and illness
via diverse mechanisms has been extensively documented. However, little
attention has been given to the molecular control that dictates the development
and fate of Treg cells, in vivo. This review, thus aims to discuss
and summarize those regulatory elements that establish the fate regulatory T
cells from a molecular perspective. Based on current scientific knowledge
available in this regard, this paper attempts to briefly outline the multifactorial
influences – signaling pathways, transcription factors, cytokines, nuclear
factors, and epigenetic modifications to shape Treg cells’ destiny.

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2.1
Transcription Factors

A) Foxp3

Pioneering
research work conducted by Hori et al., in 2003 provides critical insights into
the biology of nTreg cells and cellular mechanisms induced by them
to regulate immune homeostasis. Their group was successful in identifying a
particular protein – Foxp3, which functions as the principle regulator for
directing the developmental and functional pathways of Treg cells. Foxp3 (forkhead box
P3) gene is located on the X-chromosome which encodes a unique transcription
factor for CD4+ nTreg cells. The unequivocal role of
mutated Foxp3 gene in life-threatening X-linked autoimmune disorders such as
IPEX/XLAAD was revealed through previous efforts. Taking this lead, they
further investigated the possible contribution made by Foxp3 towards the
development of Treg cells and their role in preventing autoimmune
disorders. Their findings conclude complete blockage of Treg cell
development due to the loss-of-function mutations in the Foxp3 gene. This also
simultaneously comments on the inhibitory functions of nTreg cells
imposed by high levels of  Foxp3
expression, identified in both human and mice CD4+ T cells.  Additional aspect of the study depicts the
potential of naive T cells to transform into Treg cells with a
suppressor phenotype similar to CD4+ nTreg cells upon
retroviral transfer of the Foxp3 gene. Conventional T cells can also be induced
to express Foxp3 by factors such as TGF-?, retinoic acids, rapamycin, and
FTY720, a sphingosine 1-phosphate receptor agonist. Moreover, certain signaling
pathways are critical for inducing Foxp3 expression which involve activation of
NFAT, Smad3 and the Nr4a protein family (Ohkura, Naganari et al., 2013).

In
2003, Jason D. Fontenot and his research team put forth noteworthy real-time
quantitative polymerase chain reaction (qPCR) results with regard to the
expression of Foxp3 in Treg cells. They observed significant
up-regulation of Foxp3 mRNA in freshly isolated CD4+ CD25+ T
cells as compared to CD4+ CD25– T cells, thus stating its
uniqueness as a marker to identify CD4+ CD25+ Treg
cells. They also demonstrated the cardinal requirement of Foxp3 for the thymic
development of CD4+ CD25+ Treg cells with the
help of a model. The model proposes induction of Foxp3 in immature T cells upon
interaction of TCR with MHC class II molecules on self cells, with an affinity
range that lies between positive and negative selection. The strength of TCR
signals during thymic selection, perhaps modified by co-stimulation or the
nature of the antigen-presenting cell, may determine Foxp3 expression and,
thereby, the regulatory phenotype of the T cell. Their results denote loss of
Foxp3 expression in the absence of CD25+ CD4+ Treg
cells and re-enforces the necessity of Foxp3 expression for CD25+
CD4+ Treg cell development. Gain of suppressor function
upon ectopic transfer of Foxp3 in peripheral Treg cells opens new
avenues for treating autoimmunity and preventing transplant rejection in
Foxp3-transduced T cell lines. Further insights into the regulation of Foxp3
expression and function will have profound implications for the understanding
of immune function in health and disease.

To
verify the notion that Foxp3 expression is the driving force for the
differentiation of Treg cells, many labs conducted experiments
resulting in identical outcomes. Upon assessment of Foxp3 expression in mice T
cells, they observed higher expression of Foxp3 in CD25+ CD4+
Treg cells as compared to naive and activated CD4+ CD25?
T cells (24–26). Further evidence to indicate the contribution of Foxp3 in the
differentiation of Treg cells, CD25+ CD4+ Treg
cells in the thymus and peripheral lymphoid organs of mixed bone marrow
chimeras were analysed by the transfer of Foxp3- and WT bone marrow
into T cell deficient mice. The recipient mice were free of
lympho-proliferative disease and immune mediated tissue lesions. CD25+
Treg cells were generated only from Foxp3 sufficient and not
deficient precursor cells, demonstrating the absolute necessity of Foxp3 in Treg
cell differentiation in the thymus (Steven Z. Josefowicz, 1,2,? Li-Fan Lu, 1,3,? and Alexander Y.
Rudensky).

In
addition, retroviral transfer of the Foxp3 gene into activated peripheral CD4+
CD25?  T cells confers
suppressor function and Treg cell surface phenotype (24, 26). In
mice, expression of a Foxp3 transgene presented suppressor activity to CD8 T
cells (25). Thus, on the basis of results obtained from a plethora of
experiments, it can be concluded that Foxp3 acts in a ‘cell extrinsic’ dominant
fashion to negatively regulate the activities of the immune system.

2) GATA-3

The
role of GATA-3 as a “master regulator” to induce the differentiation
of Th0 cells to Th2 cells while simultaneously prohibiting Th1 differentiation
is well documented. However, GATA-3 expression is not exclusive to CD4+
T cells and is reported to influence the biological functions of NK cells (Pai
et al., 2003; Samson et al., 2003). Expression of GATA3 by Foxp3+ Treg
cells can be induced in a cytokine-rich milieu, implying the functional
involvement of GATA3 in Treg cells. Thus, the raptness of GATA3 to
modulate diverse immune responses through different immune cell lineages is
appreciated.

Yunqi
Wang and his team found elevated GATA-3 expression in Treg cells in
comparison to conventional T cells. This expression was suppressed in Treg
cells under Th1 cell polarizing condition, where Treg cell function
is often found tempered (Caretto et al., 2010; Oldenhove et al., 2009).
Conjectures were thus made about the importance of GATA-3 expression in Treg
cells to direct the development and function of Treg cells.
They validated the role of GATA3 expression in Treg cells through GATA3
gene deletion following isolation of Foxp3+ Treg mice
cells. The GATA3 deficient mice developed inflammatory disorders thereby
demonstrating the essential role of GATA3 to stimulate immune suppression. They
also report defective homeostasis and poor immune regulation due GATA-3 deficient
Treg cells, in vivo and in vitro. Their results further prove
increase in the activity of Foxp3 gene due to the binding of GATA3 to the
regulatory region of the Foxp3 locus. These observations were in coherence with
the decreased expression of Foxp3 and Treg cell “signature genes” in
GATA-3 deficient Treg cells. Apart from controlling the fate of
Tregs, combined function of GATA-3 and Foxp3 was vital for Foxp3 expression,
because virtually no Foxp3-expressing cells were detected when both GATA-3 and
Foxp3 were defective. This study collectively reveals an essential function of
GATA-3 in controlling Treg cell function and Foxp3 expression and provides
further insight into immune regulatory mechanisms.

Another
research article published by Elizabeth Wohlfert and her group, explored the
activity of GATA3 in Treg physiology during tissue inflammation in
great detail through an array of experiments. They showed GATA3 levels to be
significantly expressed in both murine and human Treg cells when
subjected to TCR and IL-2 stimulation. When Foxp3+ CD4+ T?cells?were?co-cultured?with?Dendritic
Cells isolated from the spleen?(SpDCs) and?anti-CD3?antibody, approximately 65%?of?the
Treg cells?expressed?high?levels?of?GATA3.?Although addition?of?recombinant?IL-2
to the co-culture did not up-regulate GATA3 expression,?it proved to sustain
prolonged?GATA3?expression. The intrinsic expression of GATA3 by Treg
cells is considered to be a pre-requisite for their accumulation at inflamed
sites. Further, their findings indicate the limitations posed by GATA3 to
polarize Treg cells to effector T cells. The study thus reveals new
dimensions to the complex role GATA3 plays in T cells during Treg
cell development and to control their expression during the episode of inflammation.

3)
Batf3

Batf3 (Basic leucine zipper transcription
factor ATF-like 3) has recently been identified
as a T cell fate-decider, for generating conventional T cells by suppressing Treg
differentiation. Batf3 belongs to AP-1 transcription factor family that
binds to DNA along with c-Jun and nuclear factor of activated T cells (NFAT). Through
an array of experiments, Wonyong Lee et al., justifies the significant role of
Batf3 to negatively regulate Treg differentiation.  Microarray analysis show down-regulation of Batf3 gene in Treg
cells as compared to other CD4+ T cell subsets. Next, to explore the
role of Batf3 in CD4+ T cell differentiation, Batf3 was induced in
various differentiated T cell subsets using a retroviral vector to measure
the expression of common regulators in each subset. Ectopic expression of Batf3
did not alter the transcription of T-bet, Gata3 or ROR?t. However, a significant
reduction in Foxp3 mRNA levels was observed in iTreg cells. Similar
results were obtained upon examination of Batf3 function in vivo.

The most important section of the
study explores the molecular mechanisms underlying Batf3-mediated regulation of
Treg cell differentiation. Batf3
selectively binds to the CNS1 region of the Foxp3 locus. Multiple
AP-1-binding sites have been identified on the promoter CNS1 and
CNS2 of the Foxp3 locus which validates the activity of Batf3 to
negatively control Treg differentiation. Batf3-induced changes were
measured in the activity of CNS1 using a transient reporter assay. The Foxp3
promoter and CNS1 greatly increased luciferase activity upon combined
stimulation with PMA, ionomycin, and TGF-? however, concomitant expression of
Batf3 significantly reduced the activity of CNS1. This effect was abolished
when the AP-1 site was deleted from CNS1. These finding chiefly indicate the
novel function of Batf3 as a transcriptional repressor of
the Foxp3 gene in Treg cells.

2)
Cytokines

The dependence on cytokine signals for tTreg
cells development has been reviewed at great length (12, 13). Most of the data suggests that IL-2 provides the
essential signals to CD25+FOXP3? single positive tTreg precursors to
differentiate into FOXP3+ cells (14). In addition, a recent report established the
requirement of IL-15 signaling for CD25?FOXP3+ precursors to develop into tTreg
cells in vitro and in vivo (15). A key question that however remains unanswered
is which cells secrete IL-2 in the thymus and under what conditions? As
dendritic cells are known to be present in close proximity to developing tTreg
cells and also release IL-2 (18) and are (19) they could be hypothesized as potential
candidates.

The activity of TGF-? in tTreg
development is deemed critical. The present understanding that TGF-? drives
Foxp3 expression in thymocytes for tTreg differentiation is supported
by molecular evidence. TGF-? induces Foxp3 expression via Smad binding to a
conserved Smad–NFAT response element (CNS1). The increase in the level of TGF-?  in neonatal mice is found to be directly proportional
to the increase in thymocyte apoptosis (due to negative selection) resulting in
the production of tTreg cells. Although the relative importance of
IL-2 versus TGF-? in tTreg differentiation versus survival is a matter
of argument, both are necessary for designating cell specific lineage of tTreg
cells.

Development of pTreg cells also demands appropriate
cytokine milieu. In mice TGF-? and IL-2 together drive the transformation of
CD4+CD25?FOXP3? naïve T cells into CD4+CD25+FOXP3+ pTregs (11, 23–25). TGF-? signaling is augmented by
anti-inflammatory cytokines which potentiates pTreg development whereas,
pro-inflammatory cytokines (IL-1?, IL-6, IL-21, IL-23, and/or TNF-?) instead
drive Th17 cell development by enhancing ROR?t expression.(27). Interestingly, activated human Tregs express
high levels of latent TGF-? coupled to latency-associated peptide and bound to
the cell surface protein GARP (33–35). Therefore, Treg cells themselves can
drive the generation of new pTreg cells by providing a source of
TGF-? (36, 37), offering a molecular explanation for a process
termed “infectious tolerance” that has been observed for many years in animal
models of transplantation (38–40). Mucosal DCs are also a rich source of TGF-?
because they express integrin ?v?8, which converts extracellular latent TGF-? to its active
form (41, 42). These cells may therefore be particularly
important for the differentiation of intestinal pTregs that are required for
intestinal homeostasis.

Effective secretion of ATRA by mice mucosal DCs enhances
TGF-?-mediated pTreg generation (44–46) Taking this lead, many studies sought to
determine whether the addition of other cytokines or compounds with TGF-? enhances
the robustness of Treg cell differentiation (43). In humans, suppressive iTreg cells
can be generated with ATRA and TGF-?. (47–49). Similarly, addition of rapamycin enhances
TGF-?-induced FOXP3 expression (49), and although the stability of these cells is
unknown, there is an ongoing clinical trial to test their potential as a
cellular therapy in hematopoietic stem cell transplantation (NCT01634217).

The significance of IL-2 signaling in tTreg
cell development has been reported by numerous studies. This is due to the
development of autoimmunity and reduced Treg numbers in IL-2
deficient mice which demonstrates the role of IL-2 signaling in Treg cell
differentiation, survival and homeostasis. It was suggested that IL-2 is
redundant for Treg differentiation in the thymus, 60,61 but the upregulation of
CD25 on tTreg precursors and subsequent IL-2 dependence suggests
otherwise, Treg cells constitutively express the high-affinity IL-2
receptor alpha (IL-2R?or CD25), and, once in the periphery, are highly
dependent on exogenous IL-2. However, the models for tTreg
differentiation cannot fully explain the functional relationship between Foxp3
and IL-2 expression, which remains controversial. With
the emerging information about the impact of cytokines on regulatory T cell
biology, a variety of cellular immunotherapies are being discovered (Chan and Carter, 2010). However, better understanding is required to dissect the
specific cytokine requirements for tTreg and pTreg cell generation
and homeostasis to fully exploit their clinical applications.

3)
Co-stimulation

TCR signaling of appropriate strength is fundamental
to the development and function of Treg cells in the thymus which
can be controlled by the process of co-stimulation.  A range of co-stimulatory signals have been
described in Treg cells. However, CD28 is the most well
characterized co-stimulatory molecule and was the first molecule discovered to
play a role in Treg-mediated control of autoimmunity (39,40). CD28
is known to promote cellular proliferation, induce IL-2 production and also stabilize
the antiapoptotic molecule Bcl-xL required for T cell survival (37,38). Later,
it was discovered that co-stimulation through CD28 is  impetus to CD25, CD122 expression and remodeling
of the chromatin in the Foxp3 locus (41–43) CD28 can signal through multiple
pathways, but for Treg development, signaling through Lck (lymphocyte-specific
protein kinase) is essential (44). The role of CD28 to promote Treg
cell proliferation, survival, and subsequent suppression of effector T cells
was determined through its targeted deletion in mice tTreg cells. These
CD28 deficient Treg mice developed autoimmune lymphoproliferative
diseases .45,46 Interestingly, these mice had decreased numbers, but not a
complete absence of tTreg cells. 45 This suggests that the
expression of CD28 in differentiating tTreg cells is indeed most important
before precursors express Foxp3.

CD27 is a member of the tumor necrosis
factor receptor superfamily and is commonly expressed in thymocytes from the
pro-T cell stage. 48,49 Studies report, deletion of CD27 in mice resulted in
reduced numbers of Treg cells without affecting the conventional T
cell population. CD27 is proposed to be involved in the development of Treg
cell development by inhibiting mitochondrial apoptosis. It is hypothesised that
CD28 binds to CD70 expressed by thymic dendritic cells and epithelial cells to
rescue developing Tregs from apoptosis and can allow their differentiation.

CTLA4 is a ligand for CD80 and CD86
expressed on APC and is highly expressed on Treg cells in a Foxp3-dependent
manner (51,52) CTLA4-deficient mice suffer from lethal lymphoproliferation which
proves that CTLA4 acts as a negative regulator of T cell-mediated immune
responses. 56 CTLA4 can be induced independently of Foxp3 but Foxp3 amplifies
and stabilizes its expression, which is important for the suppressive function
of Treg cells. 3,57

4) Nuclear Factors

Nuclear transcription factors such as
nuclear factor ?B (NF-?B), Akt, and mechanistic target of rapamycin (mTOR) and E-proteins
are implied to contribute to tTreg cell development and
differentation.

a) mTOR

 mTOR,
a Ser/Thr protein kinase is required in T cells for activation and maintaining
the integrity of homeostasis. The exact mechanism of mTOR signaling in the differentiation
of Treg cells remains unclear however, Treg cells express
high mTORC1 activity as compared to conventional T cells. 87 Several studies
have shown a negative role for mTORC1 and mTORC2. mTORC2 is known to activate
Akt which in turn inhibits Treg differentiation and proliferation
through Fox1 signaling and mTORC1 synergism. 75,76,89–91 Deletion of mTORC1 led
to increased numbers of Treg cells with reduced suppressive function,
confirming the negative role of mTORC1 in Treg differentiation. In
summary, mTOR does play a role in Treg differentiation but appears to negatively
direct the fate of Treg cells.

b) E-Proteins

E-proteins (E12, E47, HEB, and E2-2) are a
family of transcription factors that exhibit a role in thymocyte development,
before lineage commitment and also additionally inhibit the transition from DN
to DP and DP to SP in later thymocyte differentiation. 92–94. This function of
E-proteins led to the hypothesis that they could play a role in tTreg
differentiation, by fine-tuning the TCR signal strength. Deletion of E-proteins
led to increased tTreg differentiation deficiency in E-protein
inhibitors Id2 and Id3 led to reduced tTreg differentiation. This is due to the
binding of E-proteins to the CD25 enhancer locus which suppresses CD25
transcription. In the absence of E-proteins, this suppression is removed leading
to increased CD25 expression and Treg differentiation. 17,97

c) NF-?B

NF-?B family transcription factors are
actively induced by TCR signaling and are required for tTreg development.
Reports suggest loss of mutations in members of the NF-?B signaling pathway
(such as PKC?, CARMA1, Bcl10, and I?B kinase (IKK) 2) causes notable defects in
the generation of tTreg cells. 98–101 c-Rel, a member of the NF-?B family,
directly binds to CNS2, CNS3 of the Foxp3 locus leading to the chromatin
remodeling of these regions for enhancing the transcription of Treg
specific genes. In addition, c-Rel enables the formation of the c-Rel
enhanceosome at the Foxp3 promoter region, driving Foxp3 expression. 43 In its
turn, Foxp3 can interact with c-Rel to downregulate c-Rel-induced activation of
NF-?B, which is required for to maintain a suppressive phenotype in mature Treg
cells. 103 Constitutive expression of the IKK-? complex in WT mice enhanced
NF-?B activity to increase the fraction of tTreg cells. 74

3)
Epigenetic Modifications – DNA Hypomethylation

Epigenetics
is defined as the study of complex interactions between the genome and environment
resulting in genomic changes that affects the activity and expression
of the genes without disrupting the DNA sequence. It acts as an additional
level of control apart from the genomic DNA to regulate the activation/deactivation
of genes by altering the accessibility of transcription factors to the target
genes.

The
most widely studied epigenetic changes include histone modifications that
alters the contact between the DNA sequence and histone proteins, DNA
methylation that adds a methyl group to cytosine bases and nucleosome
positioning that determines the exposure of transcription factors to the DNA
sequence (Pina, B. et al., 1990, Boyes, J. and Bird, A.,1991).
Overall, these modifications have an impact on the chromatin architecture and
thereby control the expression of a myriad of genes. The provision to generate
and maintain functionally distinct cell lineages arises from the study of these
modifications. They are of prime importance as they allow stable expression and
maintenance of lineage-specific heritable genes transferred through generations  (Kim et al., 2009). It
is believed that,
DNA methylation, a reversible epigenetic modification, is inherited through
multiple cell divisions, greatly contributes to cell-lineage determination.

 

Multipotent Hematopoeitic Stem Cells
(HSCs) in the bone marrow differentiates into a variety of blood cells. Under
the influence of epigenetic control, different subtypes of T lymphocytes are
produced from lymphoid progenitor cells by acquiring specific lineage identity
in a lineage restriction form that prevents differentiation into other cell
types 35, 36). Studies
performed on nTreg cells and iTreg cells reveal their
possibility to possess a specific epigenetic features. One such feature
involves the presence of DNA hypomethylated patterns at Treg signature
gene loci, particularly at Foxp3 CNS2 (conserved non-coding region 2), an
enhancer region that drives the transcription of Foxp3 for Treg-cell
lineage Ohkura et al., 20124, 19, 20. The presence
of Treg specific regions in the mice genome have been observed to be
hypomethylated which stimulates the activation of genes independent of Foxp3
expression, during thymic Treg-cell development 4. These findings were
used to speculate the occurrence of epigenetic changes during Treg cell
development in the thymus.

A number of research groups offered
significant clues for suggesting the contribution of epigenetics in Treg
cell development and function. Aras Toker et al., provided insights into the
role of CNS2, an evolutionarily conserved element within the Foxp3 locus,
for the development of Treg cells in the thymus. Their data
demonstrates that Foxp3 Treg
cell lineage commitment begins in the thymus through induction of stable Foxp3 expression
and is completed by CNS2 demethylation in mature
thymic Foxp3+ Treg cells. This property grants a
vast population of Treg cells to retain complete Foxp3
functionality and regulatory capacity under homeostatic and inflammatory
conditions. Demethylation of CNS2-CpGs to 5 hydroxymethylcytosine in developing
Treg cells occurs through an active DNA demethylation pathway
catalyzed by enzymes belonging to the ten-eleven-translocation (Tet) family.
They were also able to show that general TCR stimulation and IL-2 influence was
sufficient to induce DNA demethylation and Foxp3 expression in thymic Treg
cells. This ability was however found missing in conventional CD4SP (CD4 Single
Positive, CD4+CD8-) cells 45.
These facts point towards the presence of epigenetic modulation in the
chromatin architecture of Treg  precursor cells, prior to Foxp3 induction for
ensuring their differentiation to Treg cells. Genome-wide study of
Foxp3-binding sites and DNA methylation status in resting and activated Treg
cells revealed that DNA hypomethylated sites are associated with gene
activation in resting Treg cells, whereas Foxp3 is involved in gene
repression in activated Treg cells 21.
 

 

Another interesting revelation made
by Steven Z. Josefowicz and his team illustrated the ability of TCR stimulation
alone to induce Foxp3 expression even in the absence of the enzyme DNA Methyltransferase.
Morever, they also showed deletion of DNA methylation maintenance enzyme, Dnmt1
induces Foxp3 expression even in CD8+ T cells
upon TCR stimulation, suggesting a role for DNA de-methylation in Foxp3
induction 46.
Collectively, these theories suggests thymic Treg-cell development
involves preconditioning of the chromatin architecture in precursor cells,
followed by the induction of Foxp3 and DNA demethylation.

 

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