c-Rel: A pioneer in directing regulatory T-cell lineagecommitment?

Shohei Hori

Research Unit for Immune Homeostasis, RIKEN Research Center for Allergy and Immunology,

Tsurumi, Yokohama, Japan

The transcription factor Foxp3 controls the differentiation and function of Treg, but the

molecular mechanisms that regulate Foxp3 transcription remain elusive. In particular,

signals and factors that open and remodel the Foxp3 locus and imprint developing Treg

with a stable Foxp3 phenotype are largely unknown. Two reports in this issue of the

European Journal of Immunology, together with recent reports published elsewhere,

demonstrate that a member of the NF-jB family transcription factors, c-Rel, is required for

thymic differentiation of Foxp31 Treg. Moreover, c-Rel is shown to regulate Foxp3 tran-

scription directly by binding to cis-regulatory elements at the Foxp3 locus upon TCR/CD28

stimulation, including the promoter and the newly identified conserved non-coding DNA

sequence harboring a ‘‘permissive’’ chromatin status in Treg precursors. These findings

collectively suggest that c-Rel may act as a pioneer transcription factor in initiating Foxp3

transcription in Treg precursors in the thymus.

Key words: Cell differentiation . Gene regulation . NF-kB pathway . Regulatory T cells .

Transcription factors

See accompanying articles by Visekruna et al. and Deenick et al.

A small subpopulation of T lymphocytes known as Treg, which

were originally described as CD41CD251 T cells [1], play a

central role in preventing pathological immune responses

including autoimmunity, inflammation and allergy, and thus

ensure dominant tolerance to self and innocuous environmental

antigens. The transcription factor Foxp3 is predominantly

expressed in these cells and is indispensable for their differentia-

tion and function [2–4]. In support of this notion, a deficiency in

functional Treg was shown to be the primary cause of the

catastrophic autoimmune pathology that develops in Foxp3-

deficient mice [2].

Given the importance of Foxp3 in Treg differentiation and

function, it is essential to understand the mechanisms by which

Foxp3 expression is regulated. Thymic Foxp31 Treg development

occurs mainly at the CD41CD8� single-positive (SP) stage.

Compelling evidence indicates that recognition by developing

thymocytes of self-antigens presented by thymic stromal cells is

required for Foxp3 induction and Treg selection [5]. In addition,

it has also become evident that Foxp31 Treg can also be gener-

ated from peripheral naive CD41 T cells upon ‘‘tolerogenic’’

antigen presentation in vivo or activation in the presence of

TGF-b in vitro (so-called induced iTreg) [6]. Although thymus-

derived Treg and peripherally generated Treg share some func-

tional and phenotypic characteristics, it is becoming clear that

they are different in many aspects, particularly in terms of gene

expression, phenotypic stability and mechanistic requirements for

differentiation [5–8]. Although TCR signals are central for both

thymic and peripheral Treg induction, ‘‘quality’’ of the TCR

signals appears different. While thymic Treg differentiation

requires high-affinity/avidity TCR interactions together with co-

stimulatory signals through CD28, peripheral Treg induction

depends on suboptimal TCR stimulation in the absence of CD28Correspondence: Dr. Shohei Horie-mail: shohei@rcai.riken.jp

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201040372 Eur. J. Immunol. 2010. 40: 664–667Shohei Hori664

Co

mm

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tary

co-stimulation and on TGF-b [5, 6]. Moreover, TGF-b-induced

iTreg exhibit unstable Foxp3 expression, while the majority

(although not all) of natural Foxp31 T cells exhibit stable Foxp3

expression [8, 9]. Importantly, the stable Foxp3 expression in

natural Treg is associated with chromatin remodeling of the

Foxp3 locus, particularly complete demethylation of one of the

evolutionary conserved non-coding sequence (CNS) elements

termed the Treg-specific demethylation region (TSDR) [8]. These

findings collectively suggest that there are multiple, presumably

redundant, pathways leading to Foxp3 induction and that Foxp3

expression itself is not sufficient to commit Treg precursors to the

Treg lineage and to imprint them with permanent Foxp3

expression and Treg function. Thus, Treg lineage is likely deter-

mined by a higher-order regulatory process that operates

upstream of Foxp3, which ensures stable and high-level Foxp3

expression (see [7, 10] for more detailed discussion). Elucidation

of the molecular nature of such a regulatory system, more

specifically the one that opens and remodels the Foxp3 locus,

remains a major challenge in the field [8].

Given the importance of TCR signaling in Treg differentiation,

it is logical to surmise that some signaling pathways and tran-

scription factors downstream of the TCR may be essential in

regulating Treg development. While NFAT, AP-1 and CREB have

been implicated in TCR-mediated Foxp3 induction [5], previous

studies have also suggested an important role of the TCR/CD28-

dependent NF-kB pathway, as mice deficient in the genes

encoding its critical components, PKC-y, Bcl-10, CARMA1 and

IkB kinase 2, have greatly reduced numbers of Foxp31CD4SP

cells in the thymus and in the periphery [11–16]. Importantly,

Barnes et al. [14] have shown that CARMA1-deficient mice

exhibit severely impaired thymic Treg differentiation, yet rela-

tively normal TGF-b-mediated iTreg generation in vitro. This

finding suggests that distinct TCR-signaling pathways are

responsible for Foxp3 induction in thymocytes and in peripheral

T cells, and that the thymic Treg differentiation depends critically

on the NF-kB family transcription factors. It has remained

unclear, however, which NF-kB family members are required,

whether NF-kB proteins directly and/or indirectly regulate the

Foxp3 gene, and how they do so. Two reports in this issue of the

European Journal of Immunology by Deenick et al. [17] and

Visekruna et al. [18], together with four recent reports published

elsewhere [19–22], have now addressed these issues and suggest

that it is c-Rel that pioneers Foxp3 induction and hence Treg

differentiation in the thymus.

A previous study has shown that mice deficient in both c-Rel

and NF-kB1/p50 have reduced numbers of splenic CD41CD251

T cells [23], suggesting that either c-Rel or NF-kB1 or both, are

required for Treg development. Deenick et al. [17], Visekruna

et al. [18] and others have now analyzed mice lacking each

member of the NF-kB proteins known to mediate TCR/CD28

signals and observed that c-Rel-deficient mice have reduced

numbers of Foxp31CD4SP cells in the thymus and in the

periphery [17–19, 21]. In contrast, NF-kB1-deficient mice had

relatively normal numbers of these cells [17, 19, 21], while

radiation chimeras reconstituted with RelA/p65-deficient bone

marrow cells displayed modest reduction in CD251Foxp31

CD4SP thymocyte numbers [19]. The reduced Treg numbers in

c-Rel-deficient mice results from a Treg-intrinsic defect rather

than from the defective IL-2 production by conventional T cells,

because the presence of wild-type cells failed to rescue the

impaired Treg development from c-Rel-deficient bone marrow

cells in mixed bone marrow chimeras [17, 19, 21, 22]. Moreover,

Long et al. [20] showed that NF-kB proteins are the mediator that

translates TCR signals into Foxp3 transcription in thymocytes,

because enhanced NF-kB activity (by transgenic expression of a

constitutive active inhibitor of IkB kinase 2) was sufficient for

Foxp3 induction in CD4SP thymocytes (and surprisingly in

CD8SP thymocytes as well), expressing a monoclonal transgenic

TCR that otherwise does not support Foxp31 Treg differentiation.

These results collectively demonstrate that c-Rel, but not NF-kB1,

is the major NF-kB protein that links TCR signals to Treg devel-

opment in the thymus, although RelA may also contribute to this

process to some extent.

Although all these reports agree on the cell-intrinsic require-

ment of c-Rel for thymic Treg differentiation, the role of c-Rel

in peripheral Treg generation, however, appears controversial.

Isomura et al. [19] found that c-Rel-deficient naı̈ve CD41 T cells

normally upregulated Foxp3 when stimulated in the presence of

TGF-b. Although Visekruna et al. [18] found that TGF-b-mediated

Foxp3 induction in c-Rel-deficient peripheral CD41 T cells is

severely hampered, this defect was not cell autonomous but

rather due to their defective IL-2 production, because addition

of exogenous IL-2 did rescue the impaired Foxp3 induction. In

contrast, Ruan et al. [21] show that iTreg differentiation from c-Rel-

deficient CD41CD25� T cells was impaired to some degree even in

the presence of exogenous IL-2. The reasons for this apparent

discrepancy as well as a role of c-Rel in peripheral Treg generation

in vivo are unclear, but it seems fair to conclude that TGF-b-medi-

ated iTreg generation is not absolutely dependent on c-Rel.

Differentiation of iTreg and pro-inflammatory Th17 cells is

proposed to be interrelated, although counter regulated, because

of a common requirement for TGF-b [24]. In their report,

Visekruna et al. [18] additionally addressed whether c-Rel is

required for Th17 generation. They also found that c-Rel-defi-

cient naı̈ve CD41 T cells are fully capable of differentiating into

Th17 cells when stimulated in vitro in the presence of TGF-b and

IL-6, irrespectively of exogenous IL-2, although it remains to be

addressed whether this is also the case in vivo. To determine a

contribution of c-Rel to iTreg and Th17 generation in vivo,

it would be important to examine the development of Foxp31

T cells and Th17 cells in the intestine of c-Rel-deficient mice, as

the intestine is considered to be a site in which both iTreg and

Th17 cells are generated efficiently to maintain immune home-

ostasis [25].

There are several potential mechanisms by which c-Rel could

promote thymic Treg differentiation. First, c-Rel may promote

Treg survival rather than Foxp3 induction, presumably by indu-

cing genes encoding Bcl-2 pro-survival proteins. Secondly, c-Rel

may induce Foxp3 expression indirectly by affecting other genes

important for Foxp3 expression such as components of common

Eur. J. Immunol. 2010. 40: 664–667 HIGHLIGHTS 665

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

g-receptors; however, these indirect mechanisms are unlikely

because introduction of a bcl-2 transgene failed to correct the

selective reduction in c-Rel-deficient Treg [19] and enhanced

NF-kB activity affected neither the expression of common g-chain

receptors nor the sensitivity to the cytokines [20]. Instead, c-Rel

was found to induce Foxp3 transcription directly by binding to

cis-regulatory elements in the Foxp3 gene, although exactly how

c-Rel does so remains controversial [20–22]. Long et al. [20]

show that c-Rel binds to one of the kB sites in the methylated

TSDR as well as the promoter in Jurkat T cells and in primary

CD41 T cells in a TCR stimulation-dependent manner. They also

suggest that c-Rel may recruit chromatin-modifying complexes to

the methylated TSDR, thereby facilitating its demethylation;

however, Ruan et al. [21] found no evidence for c-Rel binding to

the methylated TSDR in TCR/CD28-stimulated primary CD41

CD25� T cells. The latter, however, propose that c-Rel, together

with RelA, activates the Foxp3 promoter through unique Rel-

NFAT sites by recruiting other transcription factors including

NFAT, Smad and CREB that bind to the distal enhancer as well as

to the promoter, thereby promoting the formation of a Treg-

specific ‘‘enhanceosome’’. In contrast, Zheng et al. [22] analyzed

‘‘permissive’’ and ‘‘non-permissive’’ modifications of histone H3 at

Lys 4 at the Foxp3 locus. They also found that, while natural Treg

exhibit fully permissive features at all the CNS elements and the

promoter, Treg precursors, namely CD41CD81 double-positive

and CD4SP thymocytes, but not B cells, display some permissive

chromatin features only at a newly identified CNS region located

just downstream of the exon 1, but not at other known CNS

regions including the TSDR, suggesting that this element acts

earlier than other CNS regions during Foxp3 induction. A close

examination of this element identified a motif similar to the CD28

response element in the Il2 locus, which was indeed bound by

c-Rel but neither by NF-kB1 nor by RelA upon TCR/CD28

stimulation. Furthermore, deletion of this CNS element (includ-

ing the c-Rel binding site) resulted in impaired generation of

thymic Treg, suggesting that binding of c-Rel to this element

initiates Foxp3 induction in developing Treg.

These findings collectively suggest that c-Rel acts as a ‘‘pioneer’’

transcription factor that initiates Foxp3 transcription in thymic

Treg precursors. It remains to be established exactly how c-Rel

does so and whether it indeed remodels the Foxp3 locus to

promote stable Foxp3 expression in the thymus-derived Treg

lineage. Foxp31 T cells generated in transgenic mice with

enhanced NF-kB signaling showed demethylation of the TSDR

[20], suggesting that c-Rel may be at least sufficient for the

chromatin remodeling of the Foxp3 locus; however, these trans-

genic Foxp31 T cells were predominantly CD25� and lacked full

suppressor function [20]. In addition, c-Rel-deficient Treg, despite

the reduction in numbers, are fully suppressive [17, 19] and show

a gene expression profile similar to wild-type Treg [19]. Thus,

c-Rel may be neither absolutely necessary nor sufficient to specify

many features of the Treg phenotype. Although further studies are

necessary to resolve these issues, the current studies have certainly

provided us with an important clue to further delve into the

molecular mechanisms of Treg lineage commitment.

Acknowledgements: The author is supported by grants-in-aid for

Scientific Research in Priority Areas (19059014) and for Young

Scientists (A) (20689012), the Takeda Science Foundation, the

Naito Foundation and by the Kanae Foundation for the

Promotion of Medical Science.

Conflict of interest: The author declares no financial or

commercial conflict of interest.

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Abbreviations: CNS: conserved non-coding sequence � SP: single

positive � TSDR: Treg-specific demethylation region

Full correspondence: Dr. Shohei Hori, Research Unit for Immune

Homeostasis, RIKEN Research Center for Allergy and Immunology,

1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan

Fax: 181-45-503-7068

e-mail: shohei@rcai.riken.jp

See accompanying articles:

http://dx.doi.org/10.1002/eji.200940260

http://dx.doi.org/10.1002/eji.201040298

Received: 4/2/2010

Accepted: 8/2/2010

Eur. J. Immunol. 2010. 40: 664–667 HIGHLIGHTS 667

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu