Epigenetic Control of the foxp3 Locus
in Regulatory T Cells
Stefan Floess1[, Jennifer Freyer1[, Christiane Siewert1[, Udo Baron2, Sven Olek2, Julia Polansky1, Kerstin Schlawe1,
Hyun-Dong Chang3, Tobias Bopp4, Edgar Schmitt4, Stefan Klein-Hessling5, Edgar Serfling5, Alf Hamann1,
1 Experimentelle Rheumatologie, Charite ´ Universitaetsmedizin Berlin, Berlin, Germany, 2 Epiontis, Berlin, Germany, 3 Deutsches Rheuma-Forschungszentrum, Berlin,
Germany, 4 Institut fu ¨r Immunologie, Johannes-Gutenberg-Universita ¨t, Mainz, Germany, 5 Abteilung fu ¨r Molekulare Pathologie, Pathologisches Institut, Wuerzburg,
Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development
and function of CD4þregulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself
contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an
evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity.
Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as
histone modifications within the conserved region in ex vivo isolated Foxp3þCD25þCD4þTregs, but not in naı ¨ve
CD25?CD4þT cells. Partial DNA demethylation is already found within developing Foxp3þthymocytes; however, Tregs
induced by TGF-b in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural
Tregs, these TGF-b–induced Foxp3þTregs lose both Foxp3 expression and suppressive activity upon restimulation in
the absence of TGF-b. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow
the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic
applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.
Citation: Floess S, Freyer J, Siewert C, Baron U, Olek S, et al. (2007) Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 5(2): e38. doi:10.1371/journal.pbio.
Regulatory T cells (Tregs), which have been shown to play a
pivotal role in the maintenance of self-tolerance within the
immune system, were described originally as CD4þT cells
constitutively expressing CD25 . More recently, the fork-
head transcription factor Foxp3 has been shown to be
specifically expressed in Tregs and to be a central control
element in Treg development and function . Mutation or
deletion of the gene encoding Foxp3 causes severe auto-
immune disease in mice and humans, due to a failure to
generate CD25þCD4þTregs [3,4], whereas ectopic expression
of Foxp3 in conventional T cells confers suppressive activity
[4,5]. These findings provided compelling evidence that
Foxp3 acts as a master switch controlling the development
and function of Tregs; however, the molecular mechanisms
leading to its induction remain largely unknown. Recently, an
initial characterization of the human FOXP3 promoter
revealed a basal, T cell–specific promoter containing several
NF-AT and AP-1 binding sites, which are positively regulating
FOXP3 expression after triggering of the T cell receptor
Occurrence of autoimmunity in thymectomized mice
provided initial evidence that Foxp3þCD25þCD4þTregs are
generated as an individual lineage within the thymus .
Using mice harboring a GFP-Foxp3 fusion protein-reporter
knockin allele, Fontenot et al. could show that Foxp3
expression becomes prominent in CD4 single-positive (SP)
thymocytes (;83% of Foxp3gfpþthymocytes) [7,8]. In addition
to the thymic generation of Foxp3þTregs, peripheral
conversion of Foxp3?CD25?CD4þT cells into Foxp3þTregs
has recently been demonstrated by tolerogenic antigen
application in vivo [9–11] or upon activation in the presence
of TGF-b in vitro [12–17]. To what extent these induced
populations of Tregs acquire a stable phenotype correspond-
ing to that of natural, thymus-derived Tregs is, however,
An emerging paradigm in understanding the development
of stable cellular lineages emphasizes the role of epigenetic
mechanisms for the permanent, heritable fixation of distinct
gene expression patterns. Molecular mechanisms of epige-
netic imprinting include selective demethylation of CpG
motifs and histone modifications as shown for cytokine genes
[18–20]. Whether Treg differentiation also involves elements
of epigenetic regulation has not been studied thus far. We
therefore investigated whether epigenetic alterations such as
DNA methylation and histone modifications of the foxp3 locus
correlate with Foxp3 expression. The selective association of
chromatin remodeling with a stable Treg phenotype suggests
Academic Editor: Philippa Marrack, National Jewish Medical and Research Center,
United States of America
Received October 20, 2006; Accepted December 6, 2006; Published January 30,
Copyright: ? 2007 Floess 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.
Abbreviations: bp, base pair; ChIP, chromatin immunoprecipitation; FACS,
fluorescence-activated cell sorting; IL, interleukin; kb, kilobase; LN, lymph node;
SP, single positive; Treg, regulatory T cell
* To whom correspondence should be addressed. E-mail: Huehn@drfz.de
[ These authors contributed equally to this work.
PLoS Biology | www.plosbiology.orgFebruary 2007 | Volume 5 | Issue 2 | e380169
P PL Lo oS S BIOLOGY
a role of epigenetic imprinting in the establishment of a
committed regulatory cell type.
CD25þCD4þTregs Display a Stable Foxp3 Expression
It is assumed that Foxp3þCD25þCD4þTregs represent an
individual lineage exhibiting a stable phenotype. To prove
experimentally the stability of Foxp3 expression in natural
Tregs on a cellular level, we adoptively transferred sorted
CD25þCD4þT cells after CFSE (carboxy fluorescein diacetate
succinimide ester) labeling into syngeneic recipients. Four-
teen days after transfer, more than 95% of CFSEþcells,
including those that had divided once or twice according to
loss of CFSE, were still Foxp3þ(Figure 1), supporting data
from a recent publication using a lymphopenic transfer
model . Having shown that Foxp3 is stably expressed in
CD25þCD4þTregs, we next asked whether epigenetic mod-
ifications of the foxp3 locus might account for the main-
tenance of the long-term identity of Foxp3þTregs.
Epigenetic Modifications of the foxp3 Locus in
The lymphoproliferative disorder of scurfy mice is com-
pletely rescued by transgenic complementation with a 30.8-
kilobase (kb) genomic fragment containing the foxp3 gene
from wild-type mice . This indicates that most key
regulatory elements required for proper Foxp3 expression
are located within the transgene including the entire gene, as
well as 12.5 kb and 2.8 kb of 59 and 39 flanking sequences,
respectively. We therefore focused our analysis of epigenetic
modifications on sequences from the 30.8-kb region and
selected specific regions for methylation analysis based on
CpG density (Figure 2A): Overlapping amplicons 1 and 2 map
upstream from exon-1, amplicons 3 and 4 align to the seventh
intron. No CpG-rich regions were observed within the Foxp3
promoter located at the putative 59 end of exon-2b, 6.1 kb
upstream from the first coding exon [6,22].
We sorted CD25þCD4þTregs and conventional CD25?CD4þ
T cells from secondary lymphoid organs of male mice. Male
mice were chosen to avoid potential artifacts due to random
X chromosome inactivation since Foxp3 is encoded on the X
chromosome . As expected, the vast majority of sorted
CD25þCD4þTregs were Foxp3þ, whereas less than 1% of
CD25?CD4þT cells expressed Foxp3 (Figure 2B).
The methylation status of the foxp3 locus was analyzed by
bisulphite sequencing (see Material and Methods). Interest-
ingly, striking differences between CD25þCD4þTregs and
conventional CD25?CD4þT cells could be observed. CpG
motifs within amplicons 1 and 2 displayed a high degree of
methylation (;100%) within conventional CD25?CD4þT
cells, but were almost completely demethylated within
CD25þCD4þTregs (Figure 2C and Table S1). No significant
differences were observed for amplicons 3 and 4, showing
that the demethylation process is not a random event, but is
confined to defined regions as was recently found for the
interleukin 2 (IL-2) promoter . Together, our findings
suggest that demethylation of CpG motifs within selected
elements of the foxp3 locus enable stable Foxp3 expression in
CD25þCD4þTregs. This view is supported by recent findings
with human natural killer (NK) cells, which up-regulated
FOXP3 expression in response to IL-2 only after treatment
with the demethylating agent 5-aza-29deoxycytidine, demon-
strating that this gene was constitutively repressed in non-
Tregs by a mechanism involving DNA methylation .
Differentially Methylated Element of the foxp3 Locus Is
Evolutionarily Conserved and Possesses Transcriptional
The differentially methylated element covered by ampli-
cons 1 and 2 is conserved between mice and humans (77.3%
sequence identity). This is not the case for the region covered
by amplicons 3 and 4, which essentially showed no signs of
differential DNA methylation. In silico analysis of the
differentially methylated, conserved region predicts a num-
ber of binding sites for transcription factors, including ATF/
CREB, C/EBPc, Elk-1, Ets-1, Evi-1, Foxp3, GATA-4, NFATc,
NF-jB, SMAD-4, STAT-1, TCF-4, and TTF1 (Figure S1),
indicating that these factors might be involved in the
induction of Foxp3 expression in CD25þCD4þTregs.
To analyze whether the same region harbors transcrip-
tional activity, we cloned a 1,160–base pair (bp) element
containing the differentially methylated element covered by
amplicons 1 and 2 into the pGL3 luciferase vector in front of
a minimal SV40 promoter. The luciferase vector was trans-
fected into a murine CD4þT cell line, and transfected cells
were either left unstimulated or were stimulated with PMA
for 24 h, followed by measurement of luciferase activity. PMA
treatment mimics part of the signals generated after TCR
triggering, which have been shown to be essential for Foxp3
expression . Interestingly, significant luciferase activity was
only observed in stimulated cells transfected with the vector
containing the conserved element of the foxp3 locus, but not
in cells transfected with the control vector (Figure 3). Similar
results showing a 5-fold to 7-fold induction of luciferase
activity after stimulation were also obtained with ex vivo
isolated CD25þCD4þTregs, albeit much lower transfection
Regulatory T cells play a pivotal role in the maintenance of self-
tolerance within the immune system by preventing autoimmunity
or excessive activation of the T cells that respond to pathogens
(naı ¨ve and effector T cells). They differentiate within the thymus, but
can also be de novo induced in the rest of the body. Mechanisms
determining development of a stable regulatory T cell lineage are
unknown. Our study provides evidence for a critical role of
epigenetic modifications in the locus coding for the forkhead
transcription factor Foxp3, which acts as a master switch controlling
regulatory T cell development and function: An evolutionarily
conserved region within the non-coding part of the gene contains
CpG motifs, which are completely demethylated in regulatory T
cells, but methylated in naı ¨ve and effector T cells, whereas we
observed an inverse occurrence of acetylated histones, another
epigenetic chromatin modification. Regulatory T cells induced in
vitro—which, in contrast to natural regulatory T cells, do not display
a stable regulatory T cell phenotype—display only incomplete DNA
demethylation despite high Foxp3 expression. Our data suggest
that expression of Foxp3 must be stabilized by epigenetic
modification to result in a permanent suppressor cell lineage, a
finding of significant importance for therapeutic applications
involving induction or transfer of regulatory T cells and for the
understanding of long-term cell lineage decisions.
PLoS Biology | www.plosbiology.orgFebruary 2007 | Volume 5 | Issue 2 | e380170
efficiencies were achieved with these primary murine T cells
(unpublished data). Initial experiments targeting the func-
tional activity of selected transcription factors, for which a
binding site in the differentially methylated, conserved
element has been predicted, showed a reduced luciferase
activity if transcription factors of the STAT family were
inhibited by decoy oligonucleotides (unpublished data),
confirming recently published data . Together, our data
assuredly show that the differentially methylated, conserved
element of the foxp3 locus possesses transcriptional activity.
Histone Modifications Indicate Opening of the foxp3
Locus in CD25þCD4þTregs
DNA demethylation is often linked to acetylation or
methylation of histones, other key features of chromatin
remodeling . To investigate whether this also holds true
for the aforementioned region of the foxp3 locus, we
performed chromatin immunoprecipitation (ChIP) experi-
ments using antibodies specific for acetylated histone H3,
acetylated histone H4, and trimethylated lysine 4 of histone
H3 (H3K4). Subsequently, the precipitated DNA was used as a
template for amplifying the differentially methylated region
of the foxp3 locus by quantitative real-time PCR. Indeed, in
CD25þCD4þTregs, the region of interest showed a stronger
association with modified histones when compared with
conventional CD25?CD4þT cells (Figure 4). Major differ-
ences were observed for the acetylated and trimethylated
histone H3, whereas minor differences were found for the
acetylated histone H4. Together, the ChIP data disclose that
within conventional CD25?CD4þT cells, the foxp3 locus is
packed in a more condensed, inaccessible chromatin struc-
ture, whereas it is located within open euchromatin in
Demethylated CpG Motifs in Developing Foxp3þ
Having shown that peripheral CD25þCD4þTregs display a
characteristic methylation status of the foxp3 locus, we next
sought to determine whether this also could be observed in
developing Tregs. Generation of Foxp3þcells in the thymus
occurs preferentially at the CD4 SP stage or during transition
to this stage . We therefore isolated CD25þand CD25?
subsets of CD4 SP thymocytes from male mice. As expected,
approximately 80% of CD25þCD4 SP thymocytes were
Foxp3þ, whereas less than 1% of CD25?CD4 SP thymocytes
expressed Foxp3 (Figure 5A). Only CD25þ, not CD25?CD4 SP
thymocytes, displayed demethylated CpG motifs within the
regions covered by amplicons 1 and 2, whereas CpG motifs in
amplicons 3 and 4 were again fully methylated in both subsets
(Figure 5B and Table S1). As expected, in double-negative
(DN) and double-positive (DP) thymocytes, which show hardly
any Foxp3 expression [7,8], CpG motifs within the regions
covered by amplicons 1 and 2 were completely methylated
(Table S1), supporting our assumption that Foxp3 expression
is developmentally regulated and requires an opening of the
foxp3 locus. When compared to peripheral CD25þCD4þTregs,
in which the CpG motifs within amplicons 1 and 2 were
almost completely demethylated (mean degree of methylation
,3%), CD25þCD4 SP thymocytes showed a clearly reduced
degree of demethylated DNA (mean degree of methylation
;50%) with individual CpG motifs being even completely
methylated (Table S1). These stark differences cannot simply
be explained by the fact that, among CD25þCD4 SP
thymocytes, only 80% are Foxp3þcompared to 95% within
the peripheral counterpart (Figures 2B and 5A). Rather, it
indicates that the locus does not become fully opened until
completion of maturation and exit from the thymus.
Weak CpG Demethylation Correlates with Poor Foxp3
Stability in TGF-b–Induced Tregs
A critical issue for application of Tregs in therapeutic
approaches is the availability of large numbers of cells.
Recent publications have reported that conventional
CD25?CD4þT cells can be converted into Foxp3þTregs by
stimulation in the presence of TGF-b [12–17]. However, the
stability and in vivo efficacy of these cells have not been
thoroughly tested so far. Analysis of the accessibility of the
foxp3 locus might provide an additional clue, aside from the
mere expression of Foxp3, as to the extent to which a
permanent conversion into a Treg lineage did occur. We
therefore analyzed the methylation status of the foxp3 locus
from CD25?CD4þT cells, which had been activated and
cultured for 6 d in the presence of TGF-b. On day 6, more
than 98% of TGF-b–cultured cells were Foxp3þ, whereas
control cells cultured under Th1 conditions showed only
approximately 1% Foxp3 expression (Figure 6A). As ex-
Figure 1. Stable Foxp3 Expression in CD25þCD4þTregs
CD25þCD4þTregs were isolated ex vivo from pooled spleen and LN single-cell suspensions, labeled with CFSE, and then transferred into syngenic 8-wk-
old recipients (2 3 106/mouse). Before transfer, sorted cells were analyzed for intracellular Foxp3 expression by FACS. Fourteen days after transfer,
splenocytes of recipient mice were stained for CD4, CD25, and Foxp3, and analyzed by FACS. About 0.1% of total splenocytes and, accordingly, 3% of
total Foxp3þsplenocytes were donor derived (CFSEþ). Representative dot and histogram plots from four independently analyzed mice were selected.
Numbers display frequency of cells within indicated populations. Comparable results were obtained for cells isolated from peripheral or mesenteric LNs
(unpublished data). The bars in the left and right graphs indicate the marker gate for Foxp3þcells. The box in the middle graph indicates the region that
was used to gate for CD4þCFSEþcells. These gated cells (CFSEþ) are depicted in the right graph.
PLoS Biology | www.plosbiology.org February 2007 | Volume 5 | Issue 2 | e380171
pected, within cultured Th1 cells, all analyzed CpG motifs
were completely methylated (Figure 6B and Table S1). In
contrast, cell culture in the presence of TGF-b led to a clearly
visible demethylation of CpG motifs within the region
covered by amplicons 1 and 2, whereas CpG motifs in
amplicons 3 and 4 again were fully methylated. However, the
degree of demethylation was far less pronounced compared
to naturally occurring peripheral CD25þCD4þTregs (Figure
2C). This prompted us to investigate whether such a weak
degree of CpG demethylation might correlate with persistent
expression of Foxp3 in TGF-b–induced Tregs. Therefore, we
restimulated TGF-b–cultured Foxp3þcells for another 6 d in
the absence of TGF-b followed by the analysis of intracellular
Foxp3 expression. As a control, we cultured ex vivo isolated
Foxp3þCD25þCD4þTregs under comparable conditions.
Whereas ex vivo CD25þCD4þTregs maintained high Foxp3
levels after cell culture for 6 d, TGF-b–induced Tregs have
lost Foxp3 expression during the 6-d restimulation period to
variable degrees, and initial results point toward an almost
complete loss of the partial demethylation of the CpG motifs
within amplicons 1 and 2 in the restimulated TGF-b cultures
(Figure 7, and unpublished data). To rule out selective
outgrowth of Foxp3?cells or enhanced cell death of Foxp3þ
cells during restimulation, we performed TGF-b cultures with
CD25?CD4þT cells from GFP-Foxp3 reporter mice , which
allowed sorting of TGF-b–induced Foxp3þTregs to a purity
greater than 99% before restimulation. In other control
experiments, we either labeled TGF-b–induced Tregs with
CFSE before restimulation or did spiking experiments with
Foxp3?T cells. In none of these control experiments was
outgrowth of Foxp3?cells or massive cell death observed
(unpublished data), confirming our previous assumption that
Foxp3 expression in TGF-b–induced Tregs is lost during
restimulation in the absence of TGF-b. Importantly, loss of
Foxp3 expression was strictly associated with loss of
suppressive activity when tested in in vitro proliferation
assays (Figure S2). Viewed as a whole, our data strongly
suggest that complete demethylation of CpG motifs within
the foxp3 locus is required to stabilize both Foxp3 expression
and suppressive capacity.
Figure 2. Selectively Demethylated CpG Motifs within the foxp3 Locus in CD25þCD4þTregs Isolated from Secondary Lymphoid Organs
(A) Schematic view on the foxp3 locus depicts exon-intron structure and position of selected amplicons (Amp 1–4). Shown is the distribution and
position of individual CpG motifs within the amplicons.
(B) CD25þCD4þand CD25?CD4þT cells were sorted from spleens and LNs pooled from 20 male BALB/c mice. FACS analysis shows the sort purity (upper
panel) and Foxp3 expression in sorted subsets (lower panel). Numbers display frequency of cells within indicated populations. The bars in the lower
graphs indicate the marker gate for Foxp3þcells. The vertical and horizontal lines in the upper graphs indicate the quadrant used to identify the
(C) Methylation pattern of selected amplicons of the foxp3 locus in CD25þCD4þTregs and conventional CD25?CD4þT cells. The amplicons are
subdivided by horizontal lines each representing an individual CpG motif. ESME software  is used for normalization and quantification of
methylation signals from sequencing data by calculating ratios of T and C signals at CpG sites. Data are condensed to methylation information at CpG
positions forming matrices of consecutive CpGs. The methylation status of individual CpG motifs within the four amplicons is color coded according to
the degree of methylation at that site. The color code ranges from yellow (0% methylation) to blue (100% methylation) according to the color scale on
the right. CpG motifs from amplicon 2 overlapping with motifs in amplicon 1 were excluded. Due to sequencing problems, the CpG motif 37 from
amplicon 4 is not listed. One representative experiment out of two individual experiments is shown.
PLoS Biology | www.plosbiology.org February 2007 | Volume 5 | Issue 2 | e380172
The forkhead box transcription factor Foxp3 has been
identified as a specific molecular marker for Tregs, and its
expression is essential for programming Treg development
and function . Although it is widely accepted that Foxp3þ
Tregs represent a stable population mainly generated as a
separate lineage in the thymus, conclusive data on the
molecular mechanisms maintaining stable Foxp3 expression
are not available. We here provide evidence that epigenetic
modifications of the foxp3 locus are required to enable long-
term identity of Foxp3þTregs.
We have identified an element within the 59 untranslated
region of the foxp3 locus, TSDR (Treg-specific demethylated
region), which displays demethylated CpG motifs both in
developing thymic as well as in mature, peripheral murine
Foxp3þCD25þCD4þTregs. Interestingly, the differentially
methylated element is evolutionarily conserved. Preliminary
analyses using cells from human peripheral blood also
showed a differential methylation of CpG motifs within the
conserved element of the foxp3 locus when conventional
CD25?CD4þT cells and CD25highCD4þTregs were compared,
implying that this region and its epigenetic regulation is of
functional importance (unpublished data).
In addition to DNA demethylation, acetylated histones H3
and H4 as well as trimethylated histone H3 were associated
with the conserved region in CD25þCD4þTregs, but not in
conventional CD25?CD4þT cells. Similar histone modifica-
tions have frequently been reported to concur with DNA
demethylation, e.g., as described for the loci encoding the
active cytokines interferon-c (IFN-c) and IL-4 in differ-
entiated Th1 and Th2 cells, respectively [19,20]. These data
suggest that in terminally differentiated Tregs, epigenetic
modifications of the foxp3 locus allow persistent expression of
The human FOXP3 promoter has recently been mapped to
the putative 59 end of exon-2b, 6.1 kb upstream from the first
coding exon . However, other studies have reported
promoter activity upstream from exon-1 close to the differ-
entially methylated region analyzed in the current study
[26,27] corresponding to the Foxp3 mRNA species AY357712
and AY357713. We could show here by performing luciferase
assays that the differentially methylated region itself possesses
transcriptional activity. Together these data suggest that the
evolutionarily conserved element might belong to an alter-
native TATA-less promoter, which contributes to the
regulation of Foxp3 expression.
The differential methylation status of the foxp3 locus in
Tregs appears to be a new example for epigenetic regulation
of cell lineage differentiation. Although almost all cells in an
individual contain the same complement of DNA code,
higher organisms must impose and maintain different
patterns of gene expression in the various types of differ-
entiated cells. Most gene regulation is transitory, depending
on the current state of the cell and changes in external
stimuli. Persistent regulation, on the other hand, is a primary
role of epigenetics: heritable regulatory patterns that do not
alter the basic genetic coding of the DNA. DNA methylation
is the archetypical form of epigenetic regulation; it serves as
the stable memory for cells and performs a crucial role in
maintaining the long-term identity of various cell types. Our
finding that evolutionarily conserved sequences within the
Figure 4. Increased Histone Acetylation and K4 Trimethylation in
ChIP assays were performed with CD25þCD4þTregs (filled bars) and
conventional CD25?CD4þT cells (open bars) sorted from spleens and LNs
pooled from 20 male BALB/c mice. DNA fragments binding to acetylated
or trimethylated histones were immunoprecipitated using antibodies
directed against acetylated histone H3, acetylated histone H4, or
trimethylated histone H3 at position K4. A rabbit isotype immunoglo-
bulin G (IgG) served as control. Precipitated DNA was quantified by real-
time PCR with primers specific for the differentially methylated region of
the foxp3 locus. Sample PCR products were set in relation to input DNA.
One representative experiment out of two individual experiments is
Figure 3. Differentially Methylated, Conserved Element of the foxp3
Locus Possesses Transcriptional Activity
RLM-11–1 cells were transfected with control vector (pGL3 promoter) or
Foxp3-CE vector containing the conserved element (CE). Cells were
stimulated with PMA for 24 h. Control cells were left unstimulated.
Results given are relative luciferase light units (RLUs) normalized for
Renilla luciferase activity (mean 6 standard deviation; n¼3). Results are
representative for two independent experiments.
PLoS Biology | www.plosbiology.org February 2007 | Volume 5 | Issue 2 | e380173
foxp3 locus are completely and selectively demethylated upon
differentiation into persistent Tregs suggests an important
role of epigenetic fixation for this phenotype.
Moreover, this seems to be the first report that a
transcription factor acting as a master switch for a certain
subpopulation is itself subject to epigenetic control. The role
of transcription factors such as T-bet or GATA-3 for the
polarization of Th1 and Th2 cells, respectively, has been
carefully studied, and their interplay with epigenetically
regulated regions in the respective cytokine genes is seen as a
major factor in the acquisition of cytokine memory [18,19].
However, foxp3 appears to be the first known example, at least
in the immune system, in which the transcriptional regulation
of a master transcription factor itself involves epigenetic
A crucial finding of this study is that chromatin remodeling
of the foxp3 locus does not merely correlate with Foxp3
expression. Rather, our current data provide first exper-
imental evidence that the completely demethylated status of
the evolutionarily conserved region is only confined to stable
Treg populations, such as the naturally occurring, thymus-
derived CD25þCD4þcells, and might indeed be a prerequisite
for the permanent commitment to the suppressor cell
lineage. This assumption is based on the analysis of TGF-b–
Figure 6. TGF-b–Induced Tregs Harbor Partially Demethylated CpG Motifs within the foxp3 Locus
(A) CD25?CD4þT cells sorted from spleens and LNs pooled from 20 male BALB/c mice were cultured for 6 d in the presence of TGF-b. Cells cultured
under Th1 conditions were used as control. Within the starting population, less than 1% of the cells were Foxp3þ(unpublished data). On day 6, cultured
cells were sorted by FACS for CD25þcells. FACS analysis shows the purity of sorted subsets (upper panel) and Foxp3 expression in gated CD25þcells
derived from indicated cultures (lower panel). Numbers display frequency of cells within indicated populations. The bars in the lower graphs indicate
the marker gate for Foxp3þcells. The vertical and horizontal lines in the upper graphs indicate the quadrant used to identify the CD4þCD25þ/?subsets.
(B) Methylation pattern of selected amplicons of the foxp3 locus in sorted CD25þcells derived from indicated cultures. The methylation status of
individual CpG motifs within the four amplicons is color coded as described in Figure 2. One representative experiment out of two individual
experiments is shown. n.a., not analyzed.
Figure 5. Demethylated CpG Motifs within the foxp3 Locus in CD25þCD4 SP Thymocytes
(A) CD25þCD4þCD8?and CD25?CD4þCD8?subsets were sorted from thymocytes pooled from 30 male BALB/c mice. FACS analysis shows purity of
sorted subsets (upper panel) and Foxp3 expression in gated CD25?and CD25þsubsets of CD4þSP thymocytes (lower panel). Numbers display
frequency of cells within indicated populations. The bars in the lower graphs indicate the marker gate for Foxp3þcells. The vertical and horizontal lines
in the upper graphs indicate the quadrant used to identify the CD4þCD25þ/?subsets.
(B) Methylation pattern of selected amplicons of the foxp3 locus in CD25þand CD25?CD4 SP thymocytes. The methylation status of individual CpG
motifs within the four amplicons is color coded as described in Figure 2. One representative experiment out of two individual experiments is shown.
PLoS Biology | www.plosbiology.orgFebruary 2007 | Volume 5 | Issue 2 | e38 0174
induced Tregs, which, despite Foxp3 expression and sup-
pressive properties, have not acquired a terminally differ-
entiated phenotype and have lost both Foxp3 expression and
suppressive capacity upon restimulation in the absence of
TGF-b. This indicates that the recently postulated positive
autoregulatory loop involving up-regulation of endogenous
TGF-b expression and subsequent Foxp3-dependent down-
regulation of Smad7, a negative regulator of TGF-b signaling,
is not sufficient to induce stable Foxp3 expression in vitro
. As TGF-b–induced Tregs display only weakly demethy-
lated CpG motifs within the conserved region of the foxp3
locus, a more complete CpG demethylation might be the key
for a stable Foxp3 expression, similar to what has recently
been reported for IL-2 .
The findings of this study have important implications with
respect to clinical applications. First, determination of the
methylation status might allow a better identification and
quality control of Tregs considered for cellular therapy
concepts of autoimmune diseases, graft-versus-host diseases,
or transplant rejections. Temporary expression of FOXP3
can be detected in activated T cells lacking regulatory
function, especially in the human system [28,29]. Analysis of
the methylation status of the foxp3 locus promises to be a
more reliable marker for the successful conversion of
conventional CD4þT cells into a stable population of
Second, detection of demethylated foxp3 sequences might
allow the development of novel diagnostic tools for the
and/or activity and disease status has been observed in a
number of recent studies. Decreased activity and/or number of
Tregs has been noted to be associated with myasthenia gravis,
autoimmune polyglandular syndrome type II, ulcerative colitis,
of Tregs was observed in patients with a variety of malignant
cancers [37–39], and might be involved in tumor progression
[40–42]. Most notably, presence of Tregs, as defined by gene
patients [40,41]. However, the analytical value of flow cytom-
etry, immunohistological, and mRNA expression analysis of
CD25 and Foxp3 as accurate diagnostic tools is blurred by both
ambiguity of the markers and instability of the biological
materials. In contrast, our current data show that DNA
demethylation at the foxp3 locus, both in mice and humans,
strictly coincides with the generation of stable Tregs. There-
fore, measurement of the methylation status of the foxp3 locus
could present a more reliable and objective criterion for the
identification and quantification of Tregs. Moreover, DNA
methylation is intrinsically a more stable parameter than
mRNA expression or protein synthesis, and can be accurately
quantified . Therefore, we believe that establishment of a
measurement system for the methylation status of the human
foxp3 locus may provide a novel diagnostic tool both in tumor
and in autoimmune disease patients.
Materials and Methods
Mice. BALB/c mice were bred at the BfR (Bundesinstitut fuer
Risikobewertung, Berlin, Germany) and used at 6–12 wk of age. All
animal experiments were performed under specific pathogen-free
conditions and in accordance with institutional, state, and federal
Antibodies, staining reagents, and cytokines. The following anti-
bodies were produced in our laboratory: anti-FcR II/III (2.4G2), anti-
CD4 (GK1.5), anti-CD3 (145.2C11), anti-CD28 (37.51), and anti–IL-4
(11B11). The following antibodies and secondary reagents were
purchased from BD PharMingen (San Diego, California, United
States): anti-CD4 (RM4–5), anti-CD19 (1D3), anti-CD25 (7D4), anti-
CD8 (53–6.7), anti-CD25 (PC6.1), anti-CD62L (Mel-14), streptavidin,
and appropriate isotype controls. The PE anti-mouse Foxp3 staining
set was purchased from eBioscience (San Diego, California, United
States). All microbeads were obtained from Miltenyi Biotec (Bergisch
Gladbach, Germany) and all cytokines from R & D systems
(Minneapolis, Minnesota, United States).
Flow cytometry. Cytometric analysis was performed as previously
described  using a FACS Calibur or a LSRII (BD Biosciences, Palo
Alto, California, United States) and the CellQuest software. Dead cells
were excluded by PI (propidium iodide) or DAPI (diamidopheny-
lindole) staining (Sigma, St. Louis, Missouri, United States). Intra-
cellular Foxp3 staining was performed with the PE anti-mouse Foxp3
staining set according to the manufacturer’s instructions.
Ex vivo cell sorting. CD4þT cells were isolated from pooled spleen
and lymph node (LN) single-cell suspensions by using anti–CD4-FITC
plus anti-FITC multisort microbeads and the AutoMACS magnetic
separation system (Miltenyi Biotec). After release of beads according
to the manufacturer’s instructions, CD25þand CD25?cells were
separated using anti–CD25-APC plus anti-APC microbeads. Thymic
single-cell suspensions were sorted for CD8þand CD8?cells using
anti-CD8 microbeads and the AutoMACS magnetic separation
system. MACS-sorted CD8?thymocytes were subsequently stained
using anti–CD4-FITC, anti–CD25-APC, and anti–CD19-PE, and
sorted into CD25þand CD25?subsets of CD4 SP thymocytes as well
as into CD19?DN thymocytes by fluorescence-activated cell sorting
(FACS) (FACSAria; BD Bioscience). Magnetic-activated cell sorting
(MACS)-sorted CD8þthymocytes were stained using anti–CD4-FITC
Figure 7. TGF-b–Induced Foxp3þTregs Loose Foxp3 Expression upon
(A) CD25?CD4þT cells were cultured for 6 d in the presence of TGF-b as
described in Figure 6. On day 6, cultured cells were sorted by FACS for
CD25þcells and analyzed for intracellular Foxp3 expression by FACS
(upper panel). Sorted CD25þcells from the same TGF-b-cultures were
restimulated for another 6 d in the absence of TGF-b. On day 6, cultured
cells were analyzed for intracellular Foxp3 expression by FACS (lower
panel). Numbers display frequency of cells within indicated populations.
(B) Ex vivo isolated CD25þCD4þTregs were analyzed for intracellular
Foxp3 expression before (upper panel) and after 6 d in vitro culture
(lower panel) by FACS. Numbers display frequency of cells within
indicated populations. One representative experiment out of two
individual experiments is shown.
PLoS Biology | www.plosbiology.orgFebruary 2007 | Volume 5 | Issue 2 | e380175
and anti–CD8-PerCP, and sorted into DP thymocytes by FACS. All
subsets were sorted to a purity of greater than 98%.
Adoptive transfer of sorted CD25þCD4þT cells. Sorted CD25þCD4þ
T cells were labeled with CFSE (Molecular Probes, Eugene, Oregon,
United States) as described before . A total of 2 3 106CFSE-
labeled cells were adoptively transferred into syngenic recipients.
Fourteen days after transfer, single-cell suspensions of spleen,
peripheral, and mesenteric LNs were prepared and stained for
CD4, CD25, and Foxp3 as described above.
TGF-b–induced Tregs. CD4þT cells were isolated from pooled
spleen and LN single-cell suspensions by using anti–CD4-FITC plus
anti-FITC multisort microbeads and the AutoMACS magnetic
separation system (Miltenyi Biotec). After release of beads according
to the manufacturer’s instructions, CD25þcells were depleted by using
anti–CD25-APC plus anti-APC microbeads. To avoid the expansion of
precommitted Foxp3þTregs, we excluded the majority of residual
Foxp3þTregs from the CD25?CD4þT cell fraction by sorting for
CD62Lhighcells using anti-CD62L microbeads. MACS-sorted
CD62LhighCD25?CD4þT cells were stimulated for 3 d using plate-
lg/ml anti–IL-4, 20-ng/ml IFN-c and 5-ng/ml IL-12 were added to the
medium. For TGF-b cultures, 5-ng/ml TGF-b and 10-ng/ml IL-2 was
used. After 3 d, cells were removed from the stimulus, transferred to
non-coated plates, and cultured for another 3 d. All cell culture was
done with RPMI 1640 (GIBCO, San Diego, California, United States)
supplemented with 10% FCS (Sigma). On day 6, cultured cells were
stained using anti–CD25-APC and sorted for CD25þcells by FACS
(FACSAria). Foxp3 expression of sorted CD25þcells was analyzed by
intracellular staining. For restimulation experiments, TGF-b–induced
Tregs were sorted for CD25þcells by FACS (FACSAria), and sorted
CD25þcells were stimulated for 3 d using plate-bound anti-CD3 (6 lg/
ml) and anti-CD28 (4 lg/ml) plus IL-2 (10 ng/ml). After 3 d, cells were
removed from the stimulus, transferred to non-coated plates, and
cultured for another 3 d. On day 6, cultured cells were stained for
CD25 and Foxp3 as described above.
Culture of ex vivo isolated CD25þCD4þTregs. CD25þcells were
enriched from pooled spleen and LN single-cell suspensions by using
anti–CD25-FITC, anti-FITC microbeads, and the AutoMACS mag-
netic separation system (Miltenyi Biotec). MACS-enriched CD25þT
cells were subsequently stained using anti–CD4-PerCP and anti–
CD62L-APC, and sorted for CD62LhighCD25þCD4þTregs by FACS
(FACSAria). CD62LhighTregs were used to avoid the expansion of
Foxp3?CD25þeffector T cells. FACS-sorted CD62LhighCD25þCD4þ
Tregs were stimulated for 3 d using plate-bound anti-CD3 (6 lg/ml)
and anti-CD28 (4 lg/ml) plus IL-2 (40 ng/ml), followed by transfer to
non-coated plates and culture for another 3 d. On day 6, cultured
cells were stained for CD25 and Foxp3 as described above.
In vitro suppression of naı ¨ve CD4þT cell proliferation. The assay
was performed as previously described . Proliferation of naı ¨ve
CD62LhighCD25?CD4þresponder cells was evaluated according to
Bisulphite sequencing. Genomic DNA was isolated from purified T
cells using the DNeasy tissue kit (Qiagen, Valencia, California, United
States) following the supplier’s recommendations. Sodium bisulphite
treatment of genomic DNA was performed according to Olek et al.
 with minor modifications, resulting in the deamination of
unmethylated cytosines to uracil, whereas methylated cytosines
remain unchanged. In a subsequent PCR amplification, uracils were
replicated as thymidines. Thus, detection of a ‘‘C’’ in sequencing
reactions reflects methylation of the genomic DNA at that site.
Detection of a ‘‘T’’ at the same site reflects instead the absence of a
methyl modification of the genomic cytosine.
PCRs were performed on MJ Research thermocyclers (Waltham,
Massachusetts, United States) in a final volume of 25 ll containing 13
PCR Buffer, 1-U Taq DNA polymerase (Qiagen), 200 lM dNTPs, 12.5
pmol each of forward and reverse primers, and 7 ng of bisulphite-
treated genomic DNA. The amplification conditions were 95 8C for 15
min and 40 cycles of 95 8C for 1 min, 55 8C for 45 sec, and 72 8C for 1
min, and a final extension step of 10 min at 72 8C. PCR products were
purified using ExoSAP-IT (USB Corp, Staufen, Germany) and
sequenced in both directions applying the PCR primers and the
ABI Big Dye Terminator v1.1 cycle sequencing chemistry (Applied
Biosystems, Foster City, California, United States), followed by
capillary electrophoresis on an ABI 3100 genetic analyzer. Trace files
were interpreted using ESME, which normalizes sequence traces,
corrects for incomplete bisulphite conversion, and allows for
quantification of methylation signals . For each sample, both
PCR amplification and sequencing was repeated once. The following
primers (59 to 39 direction) were used for both PCR amplification of
bisulphite converted genomic DNA and sequence reactions: Amp 1
(fw: AGGAAGAGAAGGGGGTAGATA; rev: AAACTAACATTC-
CAAAACCAAC), Amp 2 (fw: ATTTGAATTGGATATGGTTTGT;
rev: AACCTTAAACCCCTCTAACATC), Amp 3 (fw: AGAGGTT-
GAAGGAGGAGTATTT; rev: ACTATCTATCCAATTCCCCAAC),
and Amp 4 (fw: TGGTTGTTTTGGAGTTTAGTGT; rev: CACTTTTC-
Construction of the luciferase reporter vector. The differentially
methylated, conserved element (CE) of the foxp3 locus was amplified
by PCR using the mouse BAC RPCIB731D08143Q2 (RZPD) as a
template and the following primers: 59-GATCGGTACCTTGTCC-
CAGGAGAGCGGG-39, and 59-GATCCCCGGGCCCATATGGCTG-
The amplified 1,160-bp element was cloned via Asp718 and XmaI
into the pGL3 promoter vector (Promega, Madison, Wisconsin,
United States) in front of the minimal SV40 promoter to generate
Luciferase assay. RLM-11–1 cells , which were kindly provided
by Marc Ehlers (Deutsches Rheuma-Forschungszentrum [DRFZ],
Berlin, Germany), or ex vivo isolated CD25þCD4þTregs were
transfected using 4 lg of pGL3 promoter vector (control) or pGL3-
Foxp3-CE. Synthetic Renilla luciferase reporter vector (pRL-TK, 0.2
lg; Promega) was used as an internal control for transfection
efficiency. Four hours after transfection via nucleofection (Amaxa
Cologne, Germany), RLM-11–1 cells were stimulated with PMA (10 ng/
ml; Sigma) or with PHA (20 ng/ml; Sigma) plus ionomycin (1 lM;
Sigma) in case of ex vivo isolated Tregs. After 18–24-h culture in
IMEM (GIBCO), cells were harvested and luciferase activity was
measured using the dual luciferase assay system (Promega). Data were
normalized to the Renilla luciferase activity.
ChIP. ChIP analysis was carried out according to the manufac-
turer’s protocol (Upstate/Millipore, Billerica, Massachusetts, United
States). Cells (1–5 3 106) were fixed with 1% formaldehyde, and
chromatin was fragmented by ultrasound. For all ChIP samples,
sheared chromatin was precleared by incubation with ProteinA-
agarose/salmon sperm DNA (Upstate/Millipore). Subsequently, chro-
matin was immunoprecipitated by overnight incubation at 4 8C with
4-lg antibodies (rabbit isotype, #2027, Santa Cruz Biotechnology
[Santa Cruz, California, United States]; anti–acetyl-histone H3, #06–
599, Upstate/Millipore; anti–acetyl-histone H4, #06–866, Upstate/
Millipore; and anti–trimethyl-K4-histone H3, #07–473, Upstate/
Millipore) followed by incubation with Protein A-agarose/salmon
sperm DNA for 1 h. Precipitates were defixed and DNA was purified
by using the NucleoSpin Extract II kit (Macherey-Nagel, Du ¨ren,
Germany). The amount of immunoprecipitated DNA was quantified
by real-time PCR with LightCycler (Roche Applied Science, Basel,
Switzerland) using SYBR Green and the following primer pair (59 to
39 direction): Foxp3 (331 bp) fw: GACTCAAGGGGGTCTCA; rev:
Sample PCR products were set in relation to the input DNA using
the following expression: Eð½DNAinput??½DNAIP?Þ.
Bioinformatics. Genomic sequences spanning the foxp3 locus were
analyzed using the alignment software vista (http://pipeline.lbl.gov/
servlet/vgb2), allowing the identification of conserved regions. Tran-
scription factor binding sites were identified using the TRANSFAC
database  and the search tool MATCH .
Figure S1. CpG Motifs and Transcription Factor Binding Sites within
Differentially Methylated, Conserved Element of the foxp3 Locus
Alignment of mouse (upper row) and human (lower row) genomic
sequences corresponding to amplicons 1 and 2 of the foxp3 locus.
Individual CpG motifs are shown in red, and putative transcription
factor binding site core elements, which were identified using the
TRANSFAC database and the search tool MATCH, are illustrated as
Found at doi:10.1371/journal.pbio.0050038.sg001 (25 KB PDF).
Figure S2. Suppressive Capacity of TGF-b–Induced Tregs
CD25?CD4þT cells were cultured essentially as described in Figure 6.
Cultured cells were incubated with CFSE-labeled naı ¨ve
CD25?CD62LhighCD4þresponder T cells at a ratio of 1:1 in the
presence of CD90-depleted APCs and anti-CD3 for 3 d. Proliferation
of naı ¨ve responder cells was evaluated according to CFSE dilution.
Histograms show CFSE staining of responder T cells cultured alone
(positive) or cocultures with ex vivo isolated CD25þCD4þTregs (ex
vivo), with T cells cultured for 6 d under neutral conditions (neutral),
with T cells cultured for 6 d in the presence of TGF-b (TGF-b), with T
PLoS Biology | www.plosbiology.orgFebruary 2007 | Volume 5 | Issue 2 | e380176
cells cultured for 6 d under neutral conditions and restimulated
under neutral conditions (neutral/neutral), and with T cells cultured
for 6 d in the presence of TGF-b followed by restimulation under
neutral conditions (TGF-b/neutral). Representative data from one out
of three independent experiments with similar results are depicted.
Found at doi:10.1371/journal.pbio.0050038.sg002 (72 KB PDF).
Table S1. Methylation Status of Individual CpG Motifs within the
Summary of all DNA methylation analyses performed for indicated
subsets. Shown is the degree of methylation (%) for individual CpG
motifs. CpG motifs from amplicon 2 overlapping with motifs in
amplicon 1 were excluded. n.a., not analyzed.
Found at doi:10.1371/journal.pbio.0050038.st001 (72 KB PDF).
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-
ber for the forkhead transcription factor Foxp3 is AF277994.
We thank Alexander Hellwag, Epiontis GmbH, who performed
Author contributions. S. Floess, A. Hamann, and J. Huehn
conceived and designed the experiments. S. Floess, J. Freyer, C.
Siewert, U. Baron, J. Polansky, K. Schlawe, H. D. Chang, T. Bopp, and
S. Klein-Hessling performed the experiments. S. Floess, U. Baron, S.
Olek, E. Schmitt, E. Serfling, A. Hamann, and J. Huehn analyzed the
data. S. Olek contributed reagents/materials/analysis tools. A. Ha-
mann and J. Huehn wrote the paper.
Funding. This work was supported by the Wilhelm Sander
Foundation, by the German Federal Ministry of Education and
Research (BMBF) (NGFN II, SIPAGE, FKZ 01GS0413), and by the
Deutsche Forschungsgemeinschaft (SFB633 and SFB650).
Competing interests. We herewith state a competing financial
interest for the authors S. Olek and U. Baron, who are a founder and
an employee of the company Epiontis, respectively.
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