c-Rel Controls Multiple Discrete Steps in the Thymic
Development of Foxp3+CD4 Regulatory T Cells
George Grigoriadis1,2., Ajithkumar Vasanthakumar1., Ashish Banerjee1., Raelene Grumont1, Sarah
Overall3, Paul Gleeson3, Frances Shannon4, Steve Gerondakis1,2,5*
1Centre for Immunology, Burnet Institute, Melbourne, Australia, 2Australian Centre for Blood Diseases and Department of Clinical Hematology, Monash University, Alfred
Medical Research and Education Precinct, Melbourne, Australia, 3Bio21, University of Melbourne, Parkville, Australia, 4The John Curtin School of Medical Research,
Australian National University, Canberra City, Australia, 5Department of Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne,
The development of natural Foxp3+CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen
dependent generation of CD25+GITRhiFoxp32CD4+nTreg precursors followed by the cytokine induction of Foxp3. Using
mutant mouse models that lack c-Rel, the critical NF-kB transcription factor required for nTreg differentiation, we establish
that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-
insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an
inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the
subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with
the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a
permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2
dependent induction of Foxp3 without a need for c-Rel reactivation.
Citation: Grigoriadis G, Vasanthakumar A, Banerjee A, Grumont R, Overall S, et al. (2011) c-Rel Controls Multiple Discrete Steps in the Thymic Development of
Foxp3+CD4 Regulatory T Cells. PLoS ONE 6(10): e26851. doi:10.1371/journal.pone.0026851
Editor: Charalampos Babis Spilianakis, University of Crete, Greece
Received August 17, 2011; Accepted October 5, 2011; Published October 31, 2011
Copyright: ? 2011 Grigoriadis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from National Health and Medical Research Council (Program 257502 and Project 543141, Australia, and the
Leukemia and Lymphoma society (SCOR 7015). George Grigoriadis is supported by a scholarship from the Hematology Society of Australia and New Zealand and
The Alfred Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Regulatory T cells (Tregs), CD25+CD4+T lymphocytes that
express the Foxp3 transcription factor, restrict the extent and
duration of T cell mediated immune responses , as well as
maintain peripheral self-tolerance by suppressing auto-reactive T
cells that escape negative selection in the thymus [2,3,4]. The
importance of Tregs in the inhibition of self-reactive T cells is best
illustrated by the severe autoimmune disease that afflict humans
and mice with developmental or functional defects in this T cell
lineage . The majority of Foxp3+CD4 T cells develop in the
thymus soon after birth and are referred to as natural Treg or
nTreg cells . Peripheral Foxp32CD252CD4 T cells can also be
converted by TGFb into Foxp3+CD25+CD4+T cells [7,8], with
these TGFb inducible Tregs (iTregs) possessing immune suppres-
sive properties akin to those of nTregs .
The development of nTregs occurs via a two-step process reliant
on multiple intracellular pathways activated by a combination of T
cell receptor (TCR), CD28 and cytokine receptor mediated signals
[10,11]. The primary developmental step involves antigen selected
Foxp32CD4+cells, a population highly enriched for nTreg
precursors . This initial step in nTreg development is
dependent on signals generated by TCR bound self-peptide/
MHC class II complexes  and B7 ligand/CD28 interactions
. nTregs emerge from a pool of antigen selected thymocytes
that express TCRs with a relatively high affinity for self antigens
. This developmental requirement for nTregs differs from the
fate of conventional CD4 T cells expressing higher affinity TCRs,
which are eliminated by negative selection . The subsequent
conversion of nTreg precursors into functional nTregs involves the
IL-2 and/or IL-15 induction of Foxp3 expression , a step
dependent on the regulation of foxp3 transcription by a number of
different transcription factors.
Foxp3 serves an essential role maintaining a pattern of gene
expression responsible for the immune suppressive properties of
Tregs , whereas the differentiation of nTregs is dictated by other
transcription factors . c-Rel, an NF-kB family member, is a
transcription factor that was recently shown to control nTreg
development [17,18,19]. While mice lacking c-Rel have ,15% of
normal thymic nTreg numbers , the remaining c-rel2/2
Foxp3+nTregs possess relatively normal immune suppressive
properties . c-Rel is thought to control nTreg development in
several ways [18,19,20]. A recent study that reported the frequency
of CD25+GITRhiFoxp32CD4+thymocytes is reduced in c-rel2/2
mice , indicates that c-Rel is required for the generation of
nTreg precursors. Defects in TCR  and CD28  signaling
also reduce nTreg precursor numbers  , a phenotype shared
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with c-rel2/2mice that is consistent with c-Rel being activated in
CD4 T cells through both of these receptors . It remains unclear
whether c-Rel is induced by TCR and/or CD28 signals during this
step in nTreg development. c-Rel has also been implicated in the
control of Foxp3 expression, with the mechanism by which it might
regulate foxp3 transcription a topic of considerable debate. In one
model, c-Rel has been proposed to promote foxp3 transcription by
binding to a CD28 response element in CNS3, a conserved region
withinintron1 ofthe foxp3 genethat isrequired for the generationof
region of the foxp3 59 un-translated region that encompasses a
conserved CpG island , the demethylation of which is necessary
for the heritable maintenance of foxp3 transcription in nTregs .
This finding has led to suggestions that c-Rel might regulate foxp3
transcription by promoting the demethylation of the CpG island.
Finally, c-Rel was shown to activate foxp3 promoter reporter
constructs by binding to unique Rel-NFAT sites that appear to be
required for the formation of an nTreg specific ‘‘enhanceosome’’
. Our finding that Foxp3 expression is normal in the remaining
c-rel2/2thymic nTregs  indicates that c-Rel is probably
dispensable for constitutive Foxp3 expression. Instead, the collective
results linking c-Rel to Foxp3 expression are compatible with a
stable Foxp3 expression in nTregs [24,25]. This putativerole has led
to c-Rel being dubbed a ‘pioneer’ transcription factor .
To better understand how c-Rel promotes nTreg development,
we generated c-rel2/2mice that express a functional GFP-Foxp3
fusion protein encoded by a foxp3 gene that is subject to normal
transcriptional control . These c-rel2/2foxp3gfpmice permitted
a careful analysis of the role(s) c-Rel serves during the two key steps
in nTreg development, including a functional assessment of
whether c-Rel controls Foxp3 expression. Our study confirms that
c-Rel is important for generating normal numbers of nTreg
precursors and reveals that this developmental step is regulated in
a c-rel copy dependent fashion. We also demonstrate that c-Rel is
required for efficient IL-2 and IL-15 dependent conversion of
Foxp32nTreg precursors into Foxp3+nTregs. While c-Rel is
highly expressed in CD25+GITRhiFoxp32CD4+cells, its localisa-
tion to the cytoplasm of these cells is indicative of c-Rel being in an
inactive state and suggests that c-Rel is necessary for the
generation but not the maintenance of nTreg precursors. The
inability of c-rel2/2nTreg precursors to efficiently up-regulate
Foxp3 coincides with a signalling defect downstream of IL-2R that
results in impaired STAT5 phosphorylation following IL-2
stimulation of c-rel2/2nTreg precursors. Despite IL-2 plus IL-7
co-stimulation restoring STAT5 phosphorylation in c-rel2/2
nTreg precursors, a failure of this cytokine combination to rescue
the Foxp3 expression defect in these cells indicates that c-Rel must
control the function of multiple components in the IL-2 signal
transduction pathway required for the optimal induction of Foxp3.
Finally, the finding that IL-2 induction of Foxp3 in nTreg
precursors occurs in the absence of detectable nuclear c-Rel
expression leads us to propose that c-Rel establishes a permissive
state for foxp3 transcription prior to the formation of nTreg
precursors that influences the subsequent cytokine dependent
induction of Foxp3, without a need to re-activate c-Rel.
c-Rel promotes the development of thymic nTreg
precursors via a haplo-insufficient mechanism that is
independent of cell survival
Although c-Rel is expressed at high levels in Foxp3+nTregs, its
cytoplasmic sequestration in these cells, a hallmark of transcriptional
inactivity indicates that c-Rel is dispensable for the maintenance of
Foxp3+cells and instead is necessary for nTreg differentiation .
While the generation of nTregs comprises a two-step process that
initially involves TCR and CD28 signalling, followed by an IL-2
dependent step, it remained unclear exactly when c-Rel is required
during this developmental process. We addressed this question by
and its sub-cellular localization during nTreg development, plus the
consequences an absence of c-Rel has on the different steps of nTreg
differentiation.InitiallynTreg development was compared infoxp3gfp
and c-rel2/2foxp3gfpmice.Consistentwiththe propertiesdisplayed by
the parental c-rel2/2 and foxp3gfpmice , the only phenotypic
defectobserved inthe different mature thymocyte populations of the
c-rel2/2foxp3gfpmutant was a reduction in Foxp3+CD4 regulatory T
cells (Fig. 1A and 1B), which were reduced from 2.1% to 0.4% of
CD4 SP thymocytes. A comparison of thymic CD25+GITRhi-
Foxp32(GFP2)CD4+cells in 4 to 6-week old foxp3gfpand c-rel2/2
foxp3gfpmice, a population highly enriched in wild-type (wt) mice for
nTreg precursors , revealed that the percentage(2.5% and 0.4%
respectively in wt and c-rel2/2mice; Fig. 1A and 1B) and absolute
number (Table S1) of these cells was markedly reduced in c-rel2/2
foxp3gfpmice. This finding, which agrees with a recent report ,
establishes that both the Foxp32nTreg precursor and Foxp3+nTreg
populations are much smaller in the absence of c-Rel.
The thymic CD25+GITRhiFoxp32CD4+and CD25+GITRhi-
Foxp3+CD4+populations in c-rel+/2foxp3gfpmice were found to be
of an intermediate size when compared with age and sex matched
foxp3gfpand c-rel2/2foxp3gfpanimals (Fig. 1A and 1B). Consistent
with published studies showing c-Rel is not essential for the
development of other thymocytes [27,28,29], the size of
populations that include conventional CD4 and CD8 SP
thymocytes, NK and NKT cells was normal in c-rel+/2and c-
rel2/2mice (results not shown). This finding that the copy number
of c-rel influences the sizes of the nTreg precursor and nTreg
populations, establishes that c-Rel regulates nTreg development in
a haplo-insufficient manner.
Despite the failure of enforced Bcl-2 expression to rescue c-rel2/2
thymic nTreg numbers , this study did not address the
possibility that impaired regulation of the cell intrinsic survival
pathway could contribute to the diminished nTreg precursor
population in c-rel2/2mice. The c-Rel dependent induction of
A1 pro-survival gene expression in response to TCR signaling
, coupled with the finding that TGFb limits Bim-dependent
apoptosis of thymic Treg precursors during negative selection
, prompted us to reassess whether c-Rel influenced the
survival of nTreg precursors. With enforced Bcl-2 expression
shown to neutralize Bim-dependent apoptosis during thymocyte
selection , we examined the impact a bcl-2 transgene (bcl-
2Tg) with a pan hemopoietic expression pattern  had on
Although enforced bcl-2Tgexpression protected wt, c-rel+/2and
c-rel2/2thymocytes from Bim-dependent growth factor with-
drawal induced cell death in culture (results not shown), it did
not correct the deficit of CD25+GITRhiFoxp32CD4+cells in c-
rel+/2or c-rel2/2mice (Fig. 2A and 2B). This demonstrates that
the reduced number of nTreg precursors that result from a loss
of c-Rel function is not due to a defect in the cell intrinsic pro-
survival pathway that operates during T cell selection.
c-Rel regulates the IL-2 and IL-15 dependent induction of
Foxp3 in nTreg precursors
While c-Rel is necessary for the generation of nTreg precursors,
emergingevidencethatitmay alsobeimportant inregulating Foxp3
expression during nTreg differentiation [18,19,20] prompted us to
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determine if c-Rel influences the cytokine dependent generation of
Foxp3+cells from Foxp32precursors. This was assessed by
monitoring the IL-2 and IL-15 dependent differentiation of c-rel2/2
nTreg precursors into Foxp3+cells in culture. Initially, equivalent
numbers of CD25+GITRhiFoxp32CD4+thymocytes isolated from
foxp3gfpand c-rel2/2foxp3gfpmice were cultured for 24 hrs in 100 U/
ml of IL-2 as previously described [11,34]. The frequency of un-
stimulated (media alone) nTreg precursors of both genotypes that
express Foxp3 was ,0.5% (Fig. 3A, 3C), confirming a need for IL-2
signaling in the induction of Foxp3. While IL-2 induced Foxp3
expression in ,14% of cultured wt nTreg precursors, a frequency
consistent with published reports , by contrast only 2 to 3%
(61.5 SEM) of c-rel2/2CD25+GITRhiFoxp32CD4+nTreg pre-
cursors up-regulated Foxp3 in response to IL-2 stimulation.
Furthermore, although c-Rel controls the generation of nTreg
precursor numbers in a gene copy dependent manner, c-rel+/2
nTreg precursors unlike their c-rel2/2counterparts differentiate into
Foxp3+cells as efficiently as wt precursors following IL-2 stimulation
(Fig. 3A and 3C). This difference in how c-rel copy number
influences IL-2 dependent and independent nTreg differentiation
indicates that c-Rel controls these two developmental steps via
distinct mechanisms. The reduction in the frequency of c-rel2/2
nTreg precursors able to up-regulate Foxp3 in response to IL-2
stimulation was not due to the increased death of these cells (Figure
S1), nor was this defect IL-2 concentration dependent, given a 100-
fold range in IL-2 levels (50 to 5,000 U/ml) still failed to increase
Foxp3+cell numbers (Figure S2). An inability to correct thiscytokine
dependent step by extending the period of IL-2 stimulation up to
48 hrs(resultsnotshown)alsoestablishedthat the defectwasnotdue
to a delay in initiating Foxp3 expression. Finally, IL-15, another
common c chain (cc) cytokine that can promote the induction of
differentiation of c-rel2/2nTreg precursors (Fig. 3B and 3C).
Collectively, these data show that c-rel2/2nTreg precursors have a
cell intrinsic defect that impacts upon the IL-2 and IL-15 dependent
induction of Foxp3.
Figure 1. nTreg precursor populations in c-Rel deficient mice. Single cell suspensions from the thymi of foxp3gfp, c-rel+/2foxp3gfpand c-rel2/2
foxp3gfpmice stained with antibodies for CD4, CD8, CD25 and GITR were examined by flow cytometry. Representative dot plots from one of six
independent experiments are shown with the percentage of cells in the relevant quadrants indicated. (A) Profiles of CD25 versus Foxp3 expression
(gated on CD4 SP cells) and CD25 versus GITR expression (gated on Foxp32CD4SP cells) for thymocyte populations in wt, c-rel+/2and c-rel2/2mice.
(B) Percentages of thymic nTregs (CD25+Foxp3+CD4SP) and nTreg precursors (CD25+Foxp32CD4SP) in wt, c-rel+/2and c-rel2/2mice. The data
represents the mean values (6SEM with ANOVA p value ,0.0001) compiled from six independent experiments described in panel A.
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The expression of the high affinity trimeric IL-2 receptor
complex on c-rel2/2nTreg precursors is normal
Those cells within the CD25+GITRhiFoxp32CD4+thymocyte
population that express high levels of the IL-2Rb chain (CD122)
account for the majority of nTreg precursors that possess a capacity
to induce foxp3 transcription in response to IL-2 stimulation .
With the NF-kB pathway implicated in controlling CD25 levels on
conventional T cells , we compared the expression of CD25,
CD122 and CD132 (IL-2R common c-chain) on wt and c-rel2/2
expression were equivalent on the wt and c-rel2/2nTreg precursor
populations, while CD132 levels were slightly higher on c-rel2/2
CD25+GITRhiFoxp32CD4+cells. Similar findings were made for
the wt and c-rel2/2Foxp3+CD4+thymocyte populations (results not
shown). While fewer c-rel2/2CD25+GITRhiFoxp32CD4+thymo-
cytes up-regulate CD25 inresponse to IL-2stimulation(Fig. 3A), the
reduction in CD25hicells was mainly restricted to the Foxp3+
population (Fig. 3A). A similar outcome was seen for c-rel2/2
CD25+GITRhiFoxp32CD4+cells stimulated with IL-15 (Fig. 3B).
Collectively our findings indicate that c-Rel does not appear to
promote IL-2 dependent nTreg differentiation by controlling the
expression of the IL-2 receptor complex. Instead, our data points to
c-Rel regulating the IL-2 induction of Foxp3 in CD25+GITRhi-
Foxp32CD4SP thymocytes via a mechanism that operates
downstream of the IL-2 receptor.
IL-2 dependent STAT5 phosphorylation is differentially
regulated in nTreg precursors and nTregs by c-Rel
A number of transcription factors including NFAT, Ets-1 and
STAT5 are important for the induction and maintenance of foxp3
expression [36,37,38]. In the case of STAT5, its IL-2 dependent
phosphorylation by JAK is an essential requirement for activating
this transcription factor . Importantly in the context of this
study, the NF-kB pathway has been shown to regulate STAT5
Figure 2. Bcl-2 transgene expression fails to rescue the deficit of nTreg precursors in c-Rel deficient mice. Thymocyte suspensions from
c-rel+/+bcl-2Tg, c-rel+/2bcl-2Tgand c-rel2/2bcl-2Tgmice were fixed, permeabilized and stained with antibodies for CD4, CD8, CD25, GITR and Foxp3.
Representative dot plots from one of three independent experiments are displayed with the percentage of cells in the relevant quadrants indicated.
(A) Profiles of CD25 versus Foxp3 (gated on CD4 SP cells) and CD25 versus GITR (gated on Foxp32CD4SP cells). Two mice of each genotype were
analyzed in each experiment. (B) Percentages of thymic nTregs (CD25+Foxp3+CD4SP) and nTreg precursors (CD25+Foxp32CD4SP) in c-rel+/+bcl-2Tg, c-
rel+/2bcl-2Tgand c-rel2/2bcl-2Tgmice. The data represents the mean values (6SEM with ANOVA p value ,0.0001) compiled from three independent
experiments described in panel A.
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phosphorylation in conventional CD4 T cells  and CD4 Tregs
 stimulated with common c-chain cytokines. To determine if
the reduced capacity of IL-2 to convert c-rel2/2nTreg precursors
into Foxp3+cells was associated with impaired STAT5 activation,
IL-2 induced STAT5 phosphorylation was examined in wt and c-
rel2/2CD25+GITRhiFoxp32CD4+thymocytes. Whereas IL-2
induced STAT5 phosphorylation in wt nTreg precursors, STAT5
was not phosphorylated in c-rel2/2CD25+GITRhiFoxp32CD4+
cells (Fig. 5A). In contrast to nTreg precursors, levels of IL-2
thymic Foxp3+nTregs (Fig. 5B). These findings indicate that IL-
2 induced STAT5 phosphorylation is differentially regulated in
nTreg precursors and Foxp3+nTregs by c-Rel. IL-7, another
common chain cytokine that efficiently phosphorylated STAT5
in wt and c-rel2/2nTreg precursors (Fig. 5A) but alone cannot
induce Foxp3 expression. But when combined with IL-2
induced normal levels of STAT5 phosphorylation in c-rel2/2
nTreg precursors (Fig. 5A) still failed to correct the cytokine
dependent defect in Foxp3 expression (Fig. 5C). These findings
indicate that impaired IL-2 induced STAT5 phosphorylation is
not the sole reason for the reduced IL-2 dependent conversion
of c-rel2/2nTreg precursors into Foxp3+cells.
c-Rel is restricted to the cytoplasm of nTreg precursors
and is not mobilized to the nucleus by IL-2 signaling
The finding that c-Rel is required for the development of nTreg
precursors and the IL-2 dependent differentiation of these cells into
Foxp3+nTregs indicated that c-Rel must be expressed in nTreg
precursors. This was confirmed by performing Western blotting on
whole cell extracts isolated from CD25+GITRhiFoxp32CD4+
thymocytes, which like Foxp3+nTregs  express high levels of
c-Rel (Fig. 6A). However, an analysis of c-Rel levels in the
cytoplasmic and nuclear fractions from nTreg precursors demon-
strated that c-Rel was restricted to the cytosol of nTreg precursors
(Fig. 6B). This indicates that c-Rel activity is not essential for the
maintenance of these cells and instead is required at a develop-
mental stage preceding the formation of nTreg precursors.
While a need for c-Rel function during the cytokine induction of
Foxp3 in nTreg precursors infers that IL-2 signaling promotes c-
Rel activation in these cells, Western blot analysis of nuclear
Figure 3. The IL-2 dependent differentiation of c-rel2/2nTreg precursors is impaired. Purified CD25+GITR+Foxp32CD4+nTreg precursors
isolated from foxp3gfp, c-rel+/2foxp3gfpand c-rel2/2foxp3gfpmice cultured for 24 hrs in the absence or presence of IL-2 or IL-15 were analysed for
Foxp3 expression (GFP+) using flow cytometry. In each case representative dot plots from one of six independent experiments are shown with the
percentage of CD25hiFoxp3+cells indicated. (A) IL-2 stimulation (B) IL-15 stimulation (C) Percentages of CD25hiFoxp3+cells developing in cultures of
cytokine treated wt, c-rel+/2and c-rel2/2thymic nTreg precursors (CD25+Foxp32CD4SP cells). The data represents the mean values (6SEM with
ANOVA p value ,0.0001) compiled from six experiments described in panels A and B.
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fractions from IL-2 activated nTreg precursors clearly demon-
strates that IL-2 does not induce nuclear c-Rel expression in these
cells (Fig. 6B). The inability of IL-2 to mobilize c-Rel to the
nucleus of nTreg precursors is not because c-Rel is refractory to
activation in these cells, as phorbol ester plus ionomycin co-
stimulation readily induced the nuclear expression of c-Rel in
nTreg precursors (Fig. 6C). Instead these data indicate that the
influence c-Rel exerts on the IL-2 dependent induction of Foxp3
expression is determined earlier in development at a stage that
precedes the formation of nTreg precursors.
The differentiation of nTreg cells in the thymus proceeds via a
two step process that first involves the TCR and CD28 dependent
generation of CD25+GITRhiFoxp32CD4+
followed by the IL-2 or IL-15 induced maturation of these cells
into Foxp3+nTregs . Here we establish that the c-Rel
transcription factor is necessary for both the generation of nTreg
precursors and the subsequent IL-2 induction of Foxp3. c-Rel
appears to exert different levels of control over these two
developmental steps, with the formation of nTreg precursors but
not the cytokine induced expression of Foxp3 controlled by a c-Rel
dose dependent mechanism. Despite a requirement for c-Rel in
the IL-2 induction of Foxp3, this step proceeds independently of
nuclear c-Rel expression and is consistent with a differentiation
model in which c-Rel generates a state of transcriptional
permissiveness during the development of nTreg precursors that
promotes the ensuing IL-2 induced maturation of these cells into
Previous studies had shown that c-Rel is required for the
generation of most Foxp3+nTregs in the thymus [17,18,19,20].
Here we show that the frequency of CD25+GITRhiFoxp32CD4+
thymocytes, a population highly enriched for nTreg precursors is
also reduced 5 to 6-fold in c-rel2/2mice. This finding confirms a
recent report  indicating that c-Rel is required for the
generation of nTreg precursors. Consistent with its regulation of
nTreg precursor development, we demonstrate that c-Rel is
expressed at high levels in CD25+GITRhiFoxp32CD4+cells. With
c-Rel expressed at low levels in CD4+CD8+thymocytes  but
up-regulated in nTreg precursors, the induction of c-Rel during
nTreg differentiation is most likely linked to T cell selection. This
proposition is supported by the finding that c-Rel is induced in
CD69hiTCRbhiCD4+CD8+thymocytes , cells that have
recently undergone positive selection. Interestingly, the generation
of nTreg precursors and consequently Foxp3+nTregs is controlled
in a c-rel copy dependent fashion. c-Rel and other NF-kB regulated
cellular processes are not normally subjected to haplo-insufficient
control , which indicates that c-Rel levels are limiting during
the development of nTreg precursors.
It remains to be determined exactly which signals regulate c-Rel
expression and its activation during the development of nTreg
precursors. Although c-Rel is activated in conventional CD4 T
cells by TCR  and CD28  signaling, a report that TCR
induced T cell proliferation, like nTreg precursor formation is
controlled by c-Rel via a haplo-insufficient mechanism 
indicates that TCR rather than CD28 signaling regulates the c-Rel
dependent generation of nTreg precursors. The localization of c-
Rel to the cytoplasm of nTreg precursors is indicative of
transcriptional inactivity. While we cannot rule out that low
amounts of c-Rel are present in the nucleus of nTreg precursors at
levels sufficient to regulate transcription, this seems unlikely given
the numerous signals that activate c-Rel in different cell types
induce readily detectable levels in the nucleus. Instead we favor a
model in which c-Rel is required for the differentiation of nTreg
precursors rather than maintaining a function in these cells.
Nevertheless, the continued expression of c-Rel in nTreg
precursors points to these cells needing to retain a capacity to
activate c-Rel. This could reflect some remaining developmental
flexibility in CD25+GITRhiFoxp32CD4SP thymocytes or the
need to prepare nTregs for immune responsiveness once the
process of thymic differentiation is complete.
Here we show that c-Rel is also required for the second major
step in nTreg differentiation, the cytokine induction of Foxp3
expression in nTreg precursors. In the absence of c-Rel, only 2–3%
Figure 4. IL-2 receptor expression on nTreg precursors. Total
thymocytes isolated from foxp3gfpand c-rel2/2foxp3gfpmice were pre-
enriched for CD4 SP cells using a CD8 depletion strategy described in
Methods, then stained with antibodies for CD4, GITR, CD25, CD122 and
CD132. Histograms show expression levels (mean fluorescence intensity
or mfi) of the CD25, CD122 and CD132 chains of the IL-2 receptor on wt
(solid line) and c-rel2/2(broken line) nTreg precursors (GITRhi-
Foxp32CD4SP cells). The data shown is representative of three
independent experiments in which 3 mice of each genotype were
used in each experiment.
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of cultured CD25+GITRhiFoxp32CD4+thymocytes up-regulate
Foxp3 in response to IL-2 or IL-15 stimulation. In those c-rel2/2
nTreg precursors that induce Foxp3, its levels in these cells are
normal, indicating that c-Rel controls the capacity of an nTreg
precursor to induce Foxp3 transcription rather than determine
absolute levels of Foxp3 expression. Unlike the generation of nTreg
precursors which c-Rel regulates in a haplo-insufficient fashion, the
c-Rel dependent control of IL-2 induced Foxp3 expression is not
influenced by c-rel copy number. Thisindicates that the events c-Rel
controls during these two distinct phases of nTreg differentiation
exhibit a differential dependence on the cellular levels of this
transcription factor. It also establishes that reduced numbers of
nTregs in c-rel+/2mice reflect the role of c-Rel in generating nTreg
precursors rather than a diminution of the frequency with which
precursors convert into Foxp3+nTregs.
Exactly how c-Rel controls the induction of Foxp3 expression in
nTreg precursors remains unclear. Although multiple sequences
within the foxp3 locus bind c-Rel [18,19,20], of these, only CNS3
has been shown to control the frequency of Foxp3+cells that
develop in the thymus . Like c-rel2/2mice , the
foxp3DCNS3/DCNS3mutant displays a 5-fold reduction in thymic
Foxp3+nTregs , with those remaining nTregs expressing
normal levels of Foxp3. While this data and the findings presented
here provide compelling evidence that c-Rel regulates the
induction of foxp3 transcription through CNS3, it remains to be
established whether c-Rel controls foxp3 expression by directly
binding to CNS3, or instead regulates another transcription factor
that binds this element or other sites within the foxp3 locus. The
finding that c-Rel activates STAT5 by controlling its IL-2 induced
phosphorylation, certainly lends support to the notion that c-Rel
promotes the induction of foxp3 expression by regulating other
transcription factors. However, the inability of IL-7 to correct IL-2
induced Foxp3 expression in c-rel2/2nTreg precursors despite
rescuing the STAT5 phosphorylation defect in these cells,
indicates that c-Rel controls multiple events required for the
induction of Foxp3.
Although the IL-2 induction of Foxp3 expression and STAT5
phosphorylation in nTreg precursors is c-Rel dependent, IL-2 does
not promote the nuclear translocation of c-Rel in these cells. While
it remains possible that c-Rel is constitutively expressed in the
nucleus of nTreg precursors at levels below detection, this scenario
seems unlikely. Instead the following models are offered as
explanations for how c-Rel can regulate the IL-2 induced
maturation of nTregs without needing to re-activate c-Rel in
nTreg precursors. We propose that c-Rel remodels the chromatin
structure of loci such as foxp3 at a stage in thymocyte
differentiation preceding the development of nTreg precursors,
which then influences the subsequent activation of these genes by
other IL-2 regulated transcription factors. Alternatively, c-Rel
could generate a transcriptional memory in nTreg precursors
Figure 5. The IL-2 induced phosphorylation of STAT5 is impaired in c-rel2/2nTreg precursors. Purified nTreg precursors and nTregs
isolated from foxp3gfpand c-rel2/2foxp3gfpmice stimulated with IL-2, IL-7 or IL-2 plus IL-7 for 30 minutes, were fixed, permeabilized and stained with
antibodies to phospho-STAT5. (A) Phospho-STAT5 levels in cytokine stimulated wt and c-rel2/2nTreg precursors (CD25+Foxp32CD4SP thymocytes).
Histograms show expression levels (mfi) of phospho-STAT5 in wt (solid line) and c-rel2/2(broken line) nTreg precursors without (black lines) or in
response to cytokine stimulation (red lines). (B) Phospho-STAT5 levels in IL-2 stimulated wt and c-rel2/2nTregs (CD25+Foxp3+CD4SP thymocytes). (C)
Percentages of CD25hiFoxp3+cells developing in cultures of wt and c-rel2/2nTreg precursors (CD25+Foxp32CD4SP cells) co-stimulated with IL-2 plus
IL-7. Experiments were performed as described in Fig. 3. The data shown in A and B are representative of three independent experiments, using 3
mice of either genotype in each experiment, while the data shown in panel C is based on three independent experiments.
c-Rel and Regulatory T Cell Development
PLoS ONE | www.plosone.org7 October 2011 | Volume 6 | Issue 10 | e26851
through the partial assembly of an RNA polymerase II complex on
the promoters of key target genes that are later activated by IL-2
signaling. This mechanism accounts for how RelA can promote
the delayed induction of inos transcription in L.monocytogenes
infected macrophages . Future studies aimed at identifying
those genes regulated by c-Rel in nTreg precursors and Foxp3+
nTregs will help to shed additional light on the mechanisms by
which c-Rel controls the sequential differentiation of nTregs in
response to TCR and cytokine signaling.
Materials and Methods
All experimental mice are on a C57BL/6 background and were
5 to 9 weeks of age. foxp3gfp, c-rel2/2, and vav-bcl2
transgenic (bcl-2Tg)  mice were maintained as inbred strains. c-
rel+/2foxp3gfpand c-rel2/2foxp3gfpmice were generated by inter-
crossing c-rel2/2and foxp3gfpmice, while c-rel+/2bcl-2Tgand c-rel2/2
bcl-2Tgmice were generated by intercrossing c-rel2/2and bcl-2Tg
mice. Animals were housed in a specific pathogen-free animal
facility. Experiments using mice were approved by the Alfred
Medical Research and Education Precinct Animal Ethics Commit-
tee in accordance with guidelines of the National Health and
Medical Research Council, Australia (Approval number E/0847/
Reagents and antibodies
Recombinant murine IL-2 and IL-15 were from Peprotech
(Princeton, NJ) and murine IL-7 from R&D Systems (Minneapolis,
MN). Phorbol-12-myristate-13-acetate (PMA) and Ionomycin
were from Sigma. The following antibodies were used: anti-mouse
c-Rel , PE or PerCP-Cy5.5-conjugated anti-CD4 (RM4-5; BD
Pharmigen), APC-Cy7-conjugated anti-CD25 (PC61), APC-con-
jugated anti-GITR (DTA-1), Pacific Blue (PB)-conjugated anti-
CD8 (53-6.7), PE-conjugated anti–mouse Foxp3 (FJK-16S),
biotin-conjugated CD122 and biotin conjugated CD132. Strepta-
vidin-conjugated PE-Cy7 was purchased from eBioscience. Anti-
rat IgG conjugated microbeads were obtained from Polysciences.
An analysis of CD25, CD122 and CD132 expression on nTreg
precursors was done using thymocytes from foxp3gfpand c-rel2/
2foxp3gfpmice stained with anti–CD4-PerCP-Cy5.5, anti-CD8-
Pacific Blue, anti-GITR-APC, anti–CD25-PE, anti-CD122-biotin
and anti-CD-132-biotin labelled antibodies. Biotin labelled
antibodies were detected using Streptavidin-PE-Cy7. Intracellular
Foxp3 stains were performed on thymocyte suspensions prepared
from adult bcl2T, c-rel+/2bcl2Tand c-rel2/2bcl2Tmice. Thymocytes
were first stained with anti–CD4-PerCP-Cy5.5, anti-CD8-Pacific
Blue and anti–CD25-APC-Cy7 antibodies, then intracellular
Figure 6. c-Rel expression in nTreg precursors. c-Rel expression in nTreg precursors and nTregs. (A) Whole cell lysates from equivalent numbers
of purified nTreg precursors and nTregs were analysed by Western blotting for c-Rel, Foxp3 and ERK (loading control) expression. The data is
representative of three independent experiments. Purified nTreg precursors cultured in the absence or presence of IL-2 (B) or PMA plus ionomycin (C)
for 2 hrs were subjected to sub-cellular fractionation and Western blots performed on nuclear and cytosolic fractions for c-Rel, ERK (cytosolic loading
control) and histone H3 (nuclear loading control) expression. The data shown in panels B and C is representative of three independent experiments.
c-Rel and Regulatory T Cell Development
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e26851
Foxp3 stains were performed using PE-conjugated anti-Foxp3
antibodies (eBioscience) as previously described . Stained cells
were analysed using a FACSCalibur (Becton Dickinson) or LSR
(Becton Dickinson) and the data analyzed using CellQuest Pro
software (Becton Dickinson). CD25+GITR+Foxp32CD4+nTreg
precursors and CD25+Foxp3+CD4+nTregs were purified from
foxp3gfpand c-rel2/2foxp3gfpmice by first employing an enrichment
step that involved depleting CD8+and CD4+CD8+thymocytes.
Anti-CD8 (Rat anti-mouse CD8a clone YTS-169) labelled
thymocytes were incubated with anti-Rat IgG coupled microbeads.
Followingmagnetic depletion, theunboundcell fraction was stained
with anti–CD4-PerCP-Cy5.5, anti–CD8-Pacific blue, anti–CD25-
APC-Cy7 plus anti-GITR-APC antibodies and CD25+GITRhi-
Foxp32CD4+and CD25+GITRhiFoxp3+CD4+thymocytes isolated
by flow cytometry using a FACSAria (Becton Dickinson). The
purity of both sorted populations (CD25+GITRhiFoxp32CD4+
precursors and CD25+GITRhiFoxp3+CD4+nTregs) was typically
Unless otherwise stated, wt and c-rel2/2CD25+GITRhiFoxp32
CD4+nTreg precursors (56103cells) isolated from foxp3gfpand c-
96-well round bottom plates in 0.1 ml of Dulbecco’s Modified Eagle
Medium and 10% FBS, with or without cytokines (50 U/ml human
IL-2, 50 ng/ml IL-7, IL-2 plus IL-7 or 100 ng/ml of IL-15) as
previously described . Foxp3 expression (GFP+cells) was
analyzed after 24 hours by flow cytometry using an LSR (Becton
Intracellular phospho-STAT5 stains
Phospho-STAT5 was detected as described . Following the
depletion of CD8+thymocytes from foxp3gfpand c-rel2/2foxp3gfp
mice, the remaining cells were incubated in serum-free DMEM and
stimulated with human IL-2 (2500 U/ml) and/or murine IL-7
(50 ng/ml) for 30 mins. Cells were fixed at room temperature using
1.5% paraformaldehyde, washed and then resuspended in PBS/3%
FCS. Cells were made permeable with methanol (20 min at 4uC),
washed with PBS/3% FCS and incubated with Alexa Fluor-647-
conjugated anti-phospho-STAT5 antibody for 30 min at room
temperature. Cells were washed 3 times with PBS/3% FCS,
incubated with10% FCSfor 10 min as a blocking step, stained with
directly conjugated fluorescent antibodies for CD4, CD8, CD25
and GITR and analysed using an LSR (Becton Dickinson).
Equal amounts of total cellular protein isolated from flow purified
populations of CD25+GITRhiFoxp32CD4+and CD25+GITRhi-
and then subjected to electrophoresis and Western blotting as
previously described  using c-Rel-specific and anti-Foxp3
antibodies. Blots were stripped and reprobed with ERK antibodies.
Westernblots onnuclear and
mocytes prepared as previously described  were probed with
antibodies to c-Rel, after which blots were stripped and reprobed with
anti-ERK (Santa Cruz Biotechnology, Inc.) or histone H3–specific
antibodies (Cell Signalling Technology).
Data were subjected to one way ANOVA analysis followed by
Tukey test using the GraphPad Prism software.
culture is normal. The viability of nTreg precursors isolated
from foxp3gfpand c-rel2/2foxp3gfpmice cultured in the presence of
IL-2 for 24 hrs was measured as described . Data is
representative of 3 independent experiments.
The survival of c-rel2/2nTreg precursors in
the Foxp3 expression defect in c-rel2/2nTreg precur-
sors. Purified CD25+GITR+Foxp32CD4+nTreg precursors
isolated from foxp3gfpand c-rel2/2foxp3gfpmice cultured for 24 hrs
in the absence or presence of 50, 500 or 5,000 U/ml of IL-2 were
analysed for Foxp3 expression (GFP+) using flow cytometry.
Shown is the percentage of CD25hiFoxp3+cells (% of input cell
numbers), with the data representing the mean values (6SEM)
compiled from three independent experiments.
Different concentrations of IL-2 fail to rescue
cursors in the thymus.
Absolute numbers of nTreg and nTreg pre-
The authors thank J. Adams (WEHI) and A. Rudensky (Sloan Kettering)
for mice, S. Guzzardi for assistance with animal husbandry and D. Vremec
Conceived and designed the experiments: GG AV AB SG. Performed the
experiments: GG AV AB RG. Analyzed the data: GG AV AB SG.
Contributed reagents/materials/analysis tools: SO PG FS. Wrote the
paper: GG AV AB SG.
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