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Immunity
Article
Nuclear Factor-kB Modulates Regulatory T CellDevelopment by Directly RegulatingExpression of Foxp3 Transcription FactorMeixiao Long,1,2 Sung-Gyoo Park,1,2 Ian Strickland,1,3 Matthew S. Hayden,1 and Sankar Ghosh1,2,*1Department of Immunobiology and Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA2Department of Microbiology & Immunology, Columbia University, College of Physicians & Surgeons, New York, NY 10032, USA3Present address: Medimmune, Cambridge CB21 6GH, UK*Correspondence: sg2715@columbia.edu
DOI 10.1016/j.immuni.2009.09.022
SUMMARY
Naturally derived regulatory T (Treg) cells arecharacterized by stable expression of the transcrip-tion factor Foxp3 and characteristic epigeneticimprinting at the Foxp3 gene locus. Here, we foundthat enhancing nuclear factor (NF)-kB activity viaa constitutive active inhibitor of kB kinase b (IKKb)transgene in T cells led to increased number ofFoxp3+ cells in the thymus and can rescue Foxp3expression in thymocytes deficient in other pleio-tropic signaling molecules. Enhancing the signalstrength of the NF-kB pathway also induced Foxp3expression in otherwise conventionally selectedT cells. NF-kB directly promoted the transcriptionof Foxp3, and upon T cell receptor (TCR) stimulation,c-Rel, a NF-kB family member, bound to Foxp3enhancer region, which is specifically demethylatedin natural Treg cells. Hence, NF-kB signalingpathway is a key regulator of Foxp3 expressionduring natural Treg cell development.
INTRODUCTION
Regulatory T (Treg) cells play a crucial role in maintaining self-
tolerance by suppressing autoreactive T cells (Liston and
Rudensky, 2007; Sakaguchi et al., 2007). Treg cells were first
identified as CD4+CD25+ T cells, because depletion of these
cells resulted in widespread autoimmune and/or inflammatory
diseases (Sakaguchi et al., 1995). Foxp3, a forkhead transcrip-
tion factor family member, has been identified as the master
regulator for the development and function of CD4+CD25+ Treg
cells. Deficiency of Foxp3 results in the deficiency of
CD4+CD25+ Treg cells and leads to severe multiorgan autoim-
mune diseases in mice and humans (Fontenot et al., 2003; Hori
et al., 2003). Ectopic expression of Foxp3 enables both conven-
tional CD4+CD25� T cells and CD8+ T cells to exhibit similar
anergic phenotypes and acquire immunosuppressive functions
(Hori et al., 2003; Yagi et al., 2004; Zheng and Rudensky, 2007).
Naturally derived Treg cells are generated in the thymus and
are considered a unique T cell lineage characterized by their
ability to stably express Foxp3 in vivo (Sakaguchi, 2004).
I
Antigenic stimulation of conventional CD4+ T cells in vitro in
the presence of the cytokine TGF-b can also lead to Foxp3
expression and the acquisition of suppressor function in periph-
eral conventional T cells. However, these induced Treg cells
(iTreg cells) exhibited unstable phenotypes, as indicated by the
fact that most lose Foxp3 expression after restimulation with
antigen in the absence of exogenous TGF-b (Floess et al., 2007).
How Foxp3 expression is induced and regulated during
natural Treg cell development remains poorly understood. So
far, it has been established that T cell receptor (TCR) stimulation
and signals from common gamma cytokine receptors are
required for the induction of Foxp3 (Huehn et al., 2009). A
stepwise model of Treg cell development and Foxp3 expression
suggests that Treg cell precursors first undergo TCR stimulation
with a signaling intensity above a cut-off threshold that primes
these precursors to gain the ability to respond to second-step
signals, such as stimulation by cytokines or costimulatory
molecules (Liston and Rudensky, 2007). TCR activation has
been found to induce binding of transcription factors such as
NFAT, AP1, CREB, and ATF to either the Foxp3 promoter or
the intronic enhancer element in T cells (Kim and Leonard,
2007; Tone et al., 2007). Recently, three highly conserved
noncoding sequences (CNS) in the Foxp3 locus have been
identified and named CNS1 to 3 (Kim and Leonard, 2007; Tone
et al., 2007). CNS1 is in the promoter region and shows extensive
chromatin remodeling in both natural Treg cells and iTreg cells.
CNS2 and CNS3 are located in the intronic region of Foxp3.
CNS2, the TGF-b-specific enhancer, contains binding sites for
NFAT and SMADs and is involved in TGF-b-induced Foxp3
expression in iTreg cells. CNS3 contains a highly conserved
CpG-rich island that is fully demethylated only in natural Treg
cells but not in iTreg cells. The CNS3 region exhibits enhancer
activity in response to TCR stimulation alone, and transcription
factors such as CREB or ATF bind to it only when the CpG island
is demethylated. Artificially induced DNA demethylation of this
region by chemical reagents in conventional T cells or T cell lines
leads to stable FOXP3 expression and a Treg cell phenotype.
Therefore, demethylation of the CpG island in the CNS3 region
is considered a hallmark of natural Treg cells that confers the
ability to stably express Foxp3 in vivo.
The TCR signals required for Treg cell development and Foxp3
expression are different from those required for positive
selection of conventional T cells. Deficiency of several signaling
molecules downstream of TCR stimulation leads to selective
mmunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc. 921
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
impairment in Foxp3+ Treg cells whereas the development of
conventional T cells is seemingly normal or less affected. These
signaling molecules include TAK1, Bcl10, CARMA1, PKCq, and
IKK-b (Medoff et al., 2009; Gupta et al., 2008; Schmidt-Supprian
et al., 2003, 2004; Wan et al., 2006). Additionally, a mutation of
linker for activation of T cells (LAT), which selectively affects
PLC-g-binding activity, also nearly eliminated the Foxp3+
population in both the thymus and the periphery, whereas
conventional T cells still developed normally (Koonpaew et al.,
2006). Interestingly, all of these molecules are pleiotropic
signaling intermediates in TCR-induced NF-kB activation
pathways (Schmidt-Supprian et al., 2004). Therefore, we
wondered whether the NF-kB signaling pathway might have
a role in regulating Foxp3 expression and influencing natural
Treg cell development. In this paper, we report that increased
NF-kB activity led to an increased percentage and absolute
numbers of Foxp3+ cells in the thymus. These Foxp3+ cells
exhibited a demethylation pattern of the CpG island in the
CNS3 enhancer region that was characteristic of natural Treg
cells. In contrast, inhibiting NF-kB activity led to a striking
reduction in the number of thymic Treg cells. Restoring NF-kB
activity rescued Foxp3 expression in thymocytes deficient in
TAK1 and CARMA1. Selectively enhancing NF-kB pathway
signaling strength induced Foxp3 expression in otherwise
conventionally selected T cells. We also found that NF-kB
directly promoted transcription of Foxp3. c-Rel, an NF-kB family
member, bound to the CpG island in the CNS3 enhancer when it
was still methylated, whereas other generic transcription factors,
such as ATF and CREB, could occupy the binding sites within the
CpG island only when it is demethylated. Thus, our results
suggest that NF-kB signaling pathway plays an important role
in Foxp3 expression and natural Treg cell development.
RESULTS
NF-kB Activity in Thymocytes Correlates with ThymicFoxp3 ExpressionTo investigate the role of the NF-kB pathway in regulatory T cell
development and Foxp3 expression, we utilized two transgenic
mouse models in which NF-kB activity can be either enhanced
or reduced (Jimi et al., 2008; Voll et al., 2000). The IKKEE
transgenic (IKKEE-Tg) mice express a constitutively active
mutant of IKK-b kinase under the control of a proximal Lck
promoter. A second transgenic line, IkBa-SR, expresses
a mutated nondegradable IkBa by a similar strategy. We had
observed thymic atrophy and a severe decrease in the CD4
single-positive (SP) thymocytes in IKKEE-Tg mice. The pheno-
type was much more dramatic in the homozygous IKKEE-Tg
mice (which contain two copies of the transgene, as previously
described [Jimi et al., 2008]), in which the CD4 SP thymocyte
number was reduced to less than 20% of wild-type mice and
peripheral mature CD4 cells were nearly completely absent.
The heterozygous IKKEE-Tg mice showed approximately 50%
reduction in the number of both thymic and splenic CD4 SP
cell populations (Jimi et al., 2008).
We examined the thymic development of Treg cells in both
IKKEE and IkBa-SR transgenic mice by intracellular staining of
Foxp3+ cells. In both homozygous and heterozygous IKKEE-Tg
mice, the Foxp3+ population in the CD4 SP thymocytes was
922 Immunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc.
approximately 20% compared to approximately 4% in wild-type
mice (Figures 1A and 1B). Considering the reduction of CD4 SP
thymocyte numbers in heterozygous IKKEE-Tg mice, there was
still a more than 2-fold increase in absolute numbers of Foxp3+
CD4 SP thymocytes (Figure 1B; Jimi et al., 2008). In contrast,
IkBa-SR mice showed an approximately 50% reduction in
both percentage and absolute numbers of Foxp3+ CD4 SP
thymocytes (Figures 1A and 1B). We also found that in IKKEE
transgenic mice, approximately 8% of CD8 SP thymocytes ex-
pressed Foxp3 (Figure 1A). Overnight in vitro stimulation with
IL-7 induced Foxp3 expression in wild-type CD8 SP thymocytes,
but not in IkBa-SR transgenic CD8 SP thymocytes (Figure 1A;
Figure S8 available online). In the spleen of IKKEE-Tg mice, the
percentage of Foxp3+ CD4 cells was increased to approximately
30%–50% (Figures 1C and 1D). Most thymic Foxp3+ cells in
IKKEE-Tg mice did not express CD25, although CD25 expres-
sion in Foxp3+ cells increased in the periphery (Figures 1A and
1C). Thus, although the NF-kB pathway positively regulates
Foxp3 expression, it does not appear to control the development
of other features of natural Treg cells such as CD25 expression.
Enhancing NF-kB Signal Rescues Thymic Foxp3Expression in TAK1- or CARMA1-Deficient MiceGenetic deficiencies of several pleiotropic signaling molecules
were found to severely impair the development of Foxp3+ Treg
cells, whereas the development of conventional T cells was
relatively unaffected or less affected. Among them, PKCq,
CARMA1, Bcl10, and TAK1 are closely related signaling mole-
cules and contribute to antigen receptor-mediated activation
of NF-kB (Rawlings et al., 2006; Schulze-Luehrmann and Ghosh,
2006). A LAT mutation that selectively impairs PLC-g-binding
activity also impairs NF-kB activation via PLC-g1-PKC pathway
(Koonpaew et al., 2006). However, in addition to the NF-kB
pathway, these molecules also contribute to the activation of
other signaling pathways, such as the JNK and NFAT pathway.
To test whether impaired Treg cell development caused by
deficiency in these pleiotropic molecules is indeed mediated
by defective NF-kB activity, we crossed IKKEE-Tg mice to
CARMA1-deicient (Card11�/�) mice or T cell-specific TAK1
conditional-deficient (Lck-Cre-Map3k7fl/fl and Cd4-Cre-
Map3k7fl/fl) mice. As previously reported, we found that the
TAK1 conditional deficiency, generated by expressing Cre
through Lck or Cd4 promoter, led to the complete absence of
Foxp3 expression in thymocytes (Figure 2A). However, constitu-
tive NF-kB activity delivered by the IKKEE transgene completely
rescued Foxp3 expression in TAK1-deficient thymocytes to
an amount similar to that of IKKEE transgenic thymocytes
expressing wild-type TAK1 (Figure 2A). Similar to TAK1 wild-type
IKKEE-Tg mice, there were also a substantial proportion of
Foxp3+ cells in CD8 SP thymocytes from TAK1-deficient
IKKEE-Tg mice (Figure 2A). Restoring the NF-kB pathway,
however, did not rescue the survival and/or proliferation
deficiency of thymocytes (as shown by the fact that peripheral
T cell numbers remained dramatically reduced in IKKEE-Tg
Cd4-Cre-Map3k7fl/fl mice) to an amount similar to that of Cd4-
Cre-Map3k7fl/fl mice without the IKKEE transgene (Figure 2B).
In Card11�/� mice, the IKKEE transgene again rescued Foxp3
expression in single-positive thymocytes (Figure 2C). Foxp3
expression was also restored in the splenocytes from IKKEE-Tg
9.02 1.68
15.67
0.081 6.27e-3
7.529
1.75 0.6
13.79
WT IkB-SR TgIKKEE-Tg (HET)
Foxp3
CD25
CD4 SPthymocyte
WT+IL7
IkB-SR Tg+ IL7
IKKEE-Tg (HET)no stimulation
CD8 SPthymocyte
18.2 2.66
3.45
0.61 1.72
1.42
1.77 3.25
IKKEE-HOMO
IKKEE-HET
WT
IkB-SRTg
Spleen
Foxp3+cellsinCD4SP
thymocytes(%)
IKKEE-HOMO
IKKEE- HET
WTIkB-SR
Tg
Thymus
WT
IKKEE-HOMO
IKKEE-HET
IkB-SRTg
CD4+Foxp3+cells(X106 )
IKKEE-HOMO
IKKEE-HET
WTIkB-SR
Tg
AA BB
CC DD
Foxp3+cellsinCD4SP
thymocytes(%)
CD4+Foxp3+cells(X106 ) Thymus
Spleen
WT IkB-SR TgIKKEE-Tg (HET)
Foxp3
CD25
CD4+splenocyte
15.2 14.4
9.67
7.82 0.16
0.85
1.65 11.1
2.44
0.031 0.037
2.2
6.19 14.2
1.79
0 0
0.26
CD8+splenocyte
0
4
8
12
16
20
24
28
0.0
0.2
0.4
0.6
0.8
1.0
0
20
40
60
0
1
2
3
4
Figure 1. Activity of NF-kB Pathway Is
Correlated with Foxp3 Expression in
Thymus
(A) Foxp3 expression of thymocytes from wild-
type (WT), IKKEE, and IkBa-SR transgenic mice
were analyzed by flow cytometry. The WT and
IkBa-SR CD8 SP thymocytes were stimulated
in vitro with IL-7 overnight before analysis, and
other cells were analyzed directly after isolation
from mice. Numbers in quadrant indicate
percentage.
(B) The average percentage and absolute number
of Foxp3-expressing cells of CD4 SP thymocytes
are shown in the histogram. Graphs show
mean ± SD; n = 3 (IKKEE HET), 4 (WT), or 2 (IKB-SR
and IKKEE HOMO).
(C) Splenocytes from WT, IKKEE, and IkBa-SR
transgenic mice were analyzed by flow cytometry.
Numbers in quadrant indicate percentage.
(D) The average percentage and absolute number
of Foxp3-expressing cells of CD4+ splenocytes
are shown in the histogram. Graphs show
mean ± SD; n = 3 (IKKEE-HET), 4 (WT), or 2
(IKB-SR and IKKEE-HOMO). IKKEE-homo,
homozygous IKKEE-Tg mice; IKKEE-HET, hetero-
zygous IKKEE-Tg mice; IKB-SR, IkBa-SR
transgenic mice.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
Card11�/�mice (Figure 2C). Our data therefore strongly suggest
that NF-kB deficiency is responsible for impaired thymic Foxp3
expression and Treg cell development caused by a deficiency
of pleiotropic signaling molecules such as CARMA1 or TAK1.
Increasing NF-kB Signal Induced Foxp3 Expressionin Otherwise Conventionally Selected ThymocytesIt has been proposed earlier that a unique TCR signaling pattern,
different from that required for positive selection of conventional
T cells, was required for Treg cell development and Foxp3
expression. In TCR (non-self-reactive) transgenic mice deficient
in RAG1 or RAG2, which express the transgenic TCR only
because of defective recombination of endogenous TCR chains,
only conventional T cells were selected and there were no
CD4+CD25+ Treg cells in the thymus and periphery because
there were no high-affinity ligands available in the thymus for
the transgenic TCR to provide the signal needed for selection
of Treg cells (Thorstenson and Khoruts, 2001). We crossed
Immunity 31, 921–931, D
IKKEE-Tg mice to MHC class II-restricted
TCR (OT-II) and MHC class I-restricted
TCR (P14) transgenic mice that were
also deficient in RAG1 to investigate
whether selectively enhancing the NF-kB
pathway is enough to induce Foxp3
expression in otherwise conventionally
selected thymocytes. We found that in
Rag1�/� OT-II TCR transgenic mice,
there were no Foxp3+ cells in the thymo-
cytes (Figure 3A, top). The IKKEE
transgene, however, induced Foxp3
expression in the CD4 SP thymocyte
compartment of Rag1�/� OT-II TCR
transgenic mice (Figure 3A, top). Foxp3-
expressing cells were also detectable in the spleen and lymph
nodes of Rag1�/� OT-II TCR Tg mice (Figure 3A, bottom and
data not shown). We think that these Foxp3+ cells were gener-
ated by peripheral conversion, because it has been reported
that CD4+CD25+ Treg cells can be induced from peripheral
CD25�CD4+ T cells in RAG-deficient TCR transgenic mice
(Thorstenson and Khoruts, 2001). The IKKEE transgene cannot
bypass the requirement for basal TCR signals to induce Foxp3
expression because no Foxp3 expression was detected in the
thymus of Rag1�/� mice bearing the IKKEE transgene but not
OT-II TCR transgene (Figure S6).
In the P14-TCR Tg Rag1�/� mice, we detected no Foxp3-ex-
pressing cells in either thymus or spleen (Figure 3B, right top
and bottom). However, enhancing NF-kB activity with the IKKEE
transgene led to Foxp3 expression in a fraction of CD8 SP
thymocytes and peripheral mature CD8+ T cells (Figure 3B, left
top and bottom). After ex vivo stimulation with IL2 and IL-7,
no CD8 SP thymocytes expressing Foxp3 was detected in
ecember 18, 2009 ª2009 Elsevier Inc. 923
0.21 0
4.87
4.73 0.13
4.3
0.097 0.055
3.53
17.2 1.02
1.968
14.4 2.48
3.48
0.23 0.054
3.55
6.2 0.75
7.14
0 0
5.65
CD25
Foxp3
Map3k7fl/fl
CD4-Cre
Map3k7fl/fl
Lck-Cre
IKKEE-Tg WTCD8 SP thymocytesCD4 SP thymocytes
IKKEE-Tg WT
0.27 0
0.95
0.51 0.055
1.89
4.02 0.15
1.85
11.8 0.73
2.22
0 0
0.14
0.88 0.16
0.46
1.72 0.015
0.21
12.2 2.8
6.73
CD25
Foxp3
CD4+splenocytes
CD8+splenocytes
CD8 SPthymocytes
C4 SPthymocytes
IKKEE-Tg WT
CD25CD8
Foxp3
CD4
A
B
C Card11-/-
Map3k7fl/fl / CD4-Cre
Total splenocytes CD4+ splenocytes
IKKEE-Tg
WT
28.9 11.4
11.2
1.07 0.11
0.84
0.94 3.23
9.47
2.32 0.082
1.71
Figure 2. Restoring NF-kB Activity Rescues Foxp3 Expression in Thymocytes Deficient of TAK1 and CARMA1
(A and B) Thymocytes (A) or splenocytes (B) from Cd4-Cre-Map3k7fl/fl or Lck-Cre-Map3k7fl/fl mice with or without IKKEE transgene were analyzed for Foxp3
expression by flow cytometry. Numbers in quadrant indicate percentage. Data shown are representative of three independent experiments.
(C) Thymocytes or splenocytes from CARMA1-deficient (Card11�/�) mice with or without IKKEE transgene were analyzed by flow cytometry for Foxp3
expression. Numbers in quadrant indicate percentage. Data shown are representative of two independent experiments.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
P14-TCR Tg Rag1�/� mice. In contrast, in the presence of the
IKKEE transgene, approximately 10% of P14-TCR Tg Rag1�/�
CD8 SP thymocytes were induced to express Foxp3
(Figure 3B, middle). These results suggested that elevated
NF-kB activity could be the crucial difference in TCR
signaling events that determine conventional versus Treg cell
development.
Foxp3+ T Cells from IKKEE Transgenic Mice Are NotGenerated by Peripheral ConversionAn alternative explanation for the increased Foxp3+ cell
percentage in the thymus of IKKEE transgenic mice could be
that these Foxp3+ cells were actually generated from peripheral
expansion or conversion of conventional T cells, which later
migrated back to the thymus.
To test this possibility, we crossed IKKEE-Tg mice to
Foxp3-RFP knockin mice in which Foxp3-expressing cells
were marked with RFP expression. We sorted the peripheral
CD4+Foxp3+ versus CD4+Foxp3� cells by the criteria of RFP
expression and adoptively transferred each group of cells into
Rag1�/� recipient mice. The spleens of recipient mice were
harvested 1 month later. We found that IKKEE-Tg CD4+Foxp3�
T cells did not spontaneously convert to Foxp3+ cells in the
recipient (Figure 4A), nor did they selectively accumulate in the
thymus and convert to Foxp3+ cells (Figure S7). Additionally,
the CD4+Foxp3� T cells bearing an IKKEE transgene expanded
924 Immunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc.
to a much lesser extent in the Rag1�/� recipient compared to
wild-type counterparts (Figure 4A; Figure S3). We also did not
observe excessive proliferation of IKKEE-Tg CD4+Foxp3+
T cells. Instead, they were almost undetectable 1 month after
transfer, although they maintained Foxp3 expression to a similar-
extent as did wild-type counterparts (Figure 4A). Transferred
peripheral IKKEE-Tg Foxp3� did not migrate back to thymus,
either (Figure S7). Therefore, increased Foxp3+ cell percentage
in IKKEE-Tg thymocytes was most probably due to increased
thymic output, rather than peripheral conversion or expansion.
Furthermore, to exclude the possibility that the NF-kB
pathway selectively promotes the expansion or survival of
Foxp3+ thymocytes, we performed a BrdU incorporation assay
and annexin V staining on Foxp3+ and Foxp3� thymocyte
populations from IKKEE-Tg mice and wild-type mice. We did
not observe excessive proliferation or reduced apoptosis of
IKKEE Tg CD4+Foxp3+ cell thymocytes or of CD4+CD25+Foxp3�
cells (which contains the putative immediate thymic Treg cell
precursors) in the IKKEE-Tg mice (Figures S4 and S5). Thus,
the increase of thymic Foxp3+ cells observed in IKKEE-Tg
mice was not caused by increased proliferation or survival.
IKKEE Transgene Induces Characteristic EpigeneticImprinting Pattern of Natural Treg Cells in Foxp3 LocusNatural Treg cells are considered to be a separate T cell lineage
and maintain Foxp3 expression throughout their lifespan in vivo
0 0.088
2.37
2.38 0.15
12.6
0 0
42.7
11.9 0.78
9.87
4.21 0.033
24.6
0 0.12
9.39
Foxp3
Rag1-/-
P14 TgCD8+ thymocytes
Rag1-/ -
P14 TgCD8+ thymocytes+IL7, IL2
CD25
B
Rag1-/-
P14 TgCD8+ splenocytes
IKKEE-Tg
CD25
WT
Rag1-/ -
OTII TgCD4+ thymocytes
Foxp3
A
Rag1-/ -
OTII TgCD4+ splenocytes
3.12 3.69
5.65
8.46 4.26
14.3
23.3 0.33
5.76
0.16 0.078
21.2
IKKEE-Tg WT
Figure 3. Enhancing NF-kB Activity Leads to Foxp3 Expression in
Otherwise Positively Selected Conventional T Cells
(A) Thymocytes or splenocytes from Rag1�/� OTII-TCR transgenic mice with
or without IKKEE transgene were analyzed by flow cytometry for Foxp3
expression. Numbers in quadrant indicate percentage. Data shown are
representative of four independent experiments.
(B) Thymocytes or splenocytes from Rag1�/�P14-TCR transgenic mice with or
without IKKEE transgene were analyzed by flow cytometry for Foxp3 expres-
sion. In the middle panel, thymocytes were treated with IL-7 and IL-2 overnight
before analysis. Numbers in quadrant indicate percentage. Data shown are
representative of two independent experiments.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
in an IL-2- and TGF-b-dependent (low-level) manner. A highly
conserved CpG-rich region has been found in the CNS3 intronic
enhancer region of Foxp3 locus. In natural Treg cells, this CpG
island was found to be fully demethylated, whereas in conven-
tional T cells and even TGF-b-induced Treg cells, it remained
fully methylated (Kim and Leonard, 2007). Several lines of
evidence suggested that demethylation of this CpG island in
the CNS3 enhancer region corresponds with the stability of
Foxp3 expression in natural Treg cells. To test the methylation
status of the CNS3 region in Treg cells induced in IKKEE mice,
we again sorted Foxp3+ cells from both IKKEE-Tg mice and
wild-type mice and performed bisulfite sequencing analysis as
described previously (Kim and Leonard, 2007). We found that
I
in IKKEE-Tg Foxp3+ CD4 cells, the CNS3 enhancer CpG island
was fully demethylated, similar to wild-type Treg cells, whereas
the CNS3 enhancer CpG island in Foxp3� CD4+ T cells from
IKKEE-Tg mice remained fully methylated (Figure 4B). Therefore,
increased NF-kB activity in IKKEE-Tg mice increased Foxp3
expression in thymocytes by promoting the characteristic
demethylation of the CpG-rich region within the CNS3 enhancer
that is a hallmark of natural Treg cells. These IKK-Tg Foxp3+
cells, however, were not as suppressive when measured by
in vitro suppression assay (Figure S2), which indicated that
excessive NF-kB activity might be detrimental to the suppressive
function of Treg cells.
Increased NF-kB Activity Did Not Enhance Common-g
Cytokine SensitivityCommon gamma cytokines, primarily IL-2 and IL-7, also play
an indispensable role in natural Treg cell development as evi-
denced by the fact that common cytokine receptor g chain (gc)
deficiency leads to the complete disappearance of Treg cells
in mice. JAK and STAT5 signaling pathways, which are acti-
vated by common gamma cytokines, have also been found to
be essential for Foxp3 expression and Treg cell development.
The NF-kB pathway has been found to be involved in the
induction of components of common gamma receptors in
lymphocytes (Bellavia et al., 2000; Vallabhapurapu et al.,
2008). Therefore, increased NF-kB pathway signals may
promote Foxp3 expression in developing thymocytes via
increasing the gc receptor expression and sensitivity. Both IL-2
and IL-7 have been reported to induce Foxp3 expression in
thymic CD4+CD25hiFoxp3� Treg cell precursors (Burchill et al.,
2008). Therefore, we checked the expression of IL-2Ra,
IL-2Rb, and IL-7Ra by flow cytometry in thymocytes. In
IKKEE-Tg mice, these receptors were not increased in DN, DP,
CD4 SP, or CD8 SP thymocyte compartments (Figure 5A). We
further fractionated the CD4+ and CD8+ SP thymocytes from
IKKEE-Tg mice into Foxp3+ and Foxp3� subpopulations but
did not note increased expression of gc receptors in either of
these groups. In fact, there was a substantial reduction in the
CD25 (IL-2Ra)+ CD122 (IL-2Rb)+ double-positive population in
IKKEE-Tg mice.
Recently, it has been reported that Treg cells not only express
high amount of IL-2 receptors, but also have a lower threshold for
IL-2 signaling (Yu et al., 2009). It has also been found that TCR
signaling can modulate STAT5 phosphorylation and common
gamma cytokine signaling (Welte et al., 1999). Therefore,
although enhanced NF-kB signaling did not increase the expres-
sion of a gc receptor, it could directly lower the threshold for
common cytokine signaling. To test this possibility, we examined
the STAT5 phosphorylation status in thymocytes in response
to IL-2 or IL-7 stimulation. We found that thymocytes from
IKKEE-Tg mice did not show spontaneous STAT5 phosphoryla-
tion in vivo (Figure 5B). Within the CD4 SP thymocyte compart-
ment, the CD25+CD122+, CD25�CD122+, and CD25�CD122�
subpopulations also showed a similar response to IL-2 in
IKKEE-Tg mice compared to wild-type mice (Figure 5C).
Therefore, our data suggest that enhanced NF-kB signaling
intensity does not promote Foxp3 expression through increasing
common gamma chain cytokine signaling.
mmunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc. 925
33.9 6.14
5.65
0.43 0.13
4.93
24.8 12.8
1.71
0.66 0.96
10.6
Foxp3
0.013 0.74 0.19 2.14
TCRb
IKKEE Foxp3+ IKKEE Foxp3-A
B
IKKEE-Tg WT
Foxp3+
Foxp3-
WT Foxp3+ WT Foxp3-
CD4
CD25
Figure 4. IKKEE-Tg Foxp3+ Cells Are Not Generated from
Peripheral Conversion and Mimic Natural Treg Cell
Epigenetically
(A) IKKEE-Tg versus wild-type (WT) mice were crossed to Foxp3-RFP
knockin mice. CD4+Foxp3+ RFP+ versus CD4+Foxp3� T cells were
sorted from IKKEE-Tg or WT mice and were then adoptively transferred
into Rag1�/� mice. Splenocytes were harvested 4 weeks later from
recipient mice. Representative flow cytometry plots showed frequency
of TCRb+CD4+ donor population of total splenocytes and percentage
of Foxp3 expression of TCRb+CD4+ donor cells. Numbers in quadrant
indicate percentage. Data shown are representative of two indepen-
dent experiments (totally at least four recipient mice in each group).
(B) CD4+Foxp3+ RFP+ versus CD4+Foxp3� T cells were sorted from
IKKEE-Tg or WT mice and then were subjected to bisulfite mutation
analysis. Methylation status of nine CpG sites (indicated by the
number at the top of the plot, as described in Kim and Leonard
[2007]) on the CpG island in Foxp3 CNS3 enhancer region were
analyzed. Open circle, demethylated CpG site; filled circle, methyl-
ated CpG site. Data shown are from two independent experiments.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
The NF-kB Pathway Directly Regulates Foxp3TranscriptionWe next investigated whether the NF-kB signaling pathway
could directly regulate Foxp3 gene transcription. Sequence
analysis has revealed three CNSs in the Foxp3 locus
(Figure 6A). CNS1 corresponds to the promoter region, CNS2
is the intronic enhancer that contains the TGF-b-responsive
element, and CNS3 is the TCR-responsive enhancer that
contains a highly conserved CpG-rich region and binding sites
for multiple transcription factors, such as CREB and ATF (Kim
and Leonard, 2007; Tone et al., 2007). In T cells, a reporter
construct with the promoter alone showed only minimal activity
upon TCR stimulation. A reporter construct with the promoter
region plus CNS3 responded strongly to TCR stimulation
in a demethylation-dependent manner, whereas a reporter
construct containing the promoter region plus CNS2
responded to TCR stimulation only in the presence of TGF-b.
To investigate the effect of the NF-kB pathway on Foxp3
expression, we utilized the reporter construct containing the
CNS3 and promoter region, which responds to TCR stimula-
tion. We cotransfected Jurkat T cells with the reporter
construct and either an IkBa-SR expression construct to
suppress the NF-kB pathway or an IKKEE expression construct
to enhance the NF-kB signal. The transfected Jurkat cells were
then stimulated with PMA and ionomycin for 17 hr. Cotransfec-
tion with IkBa-SR markedly reduced reporter activity after PMA
and ionomycin stimulation whereas cotransfection with IKKEE
constructs markedly induced the reporter activity even without
PMA and ionomycin stimulation (Figure 6B). We also found that
PMA and ionomycin and cotransfection of IKKEE can activate
the reporter construct by acting on the Foxp3 promoter
alone (Figure S1), indicating that the NF-kB-responsive element
for acute transcription of the Foxp3 gene is located in the
promoter region.
926 Immunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc.
f
l
,
l
,
l
,
r
Next we examined whether NF-kB family transcription
factors can directly bind to the regulatory regions of the
Foxp3 gene by chromatin immunoprecipitation (ChIP).
Analysis of the sequence revealed several potential
NF-kB sites in the Foxp3 locus. Three of these sites
were in the CNS3 enhancer region, located within approximately
500 bp, and were named kb1, kb2, and kb3 (Figure 6A). Addi-
tionally, two adjacent NF-kB binding sites were also identified
in the promoter region. We stimulated the Jurkat T cells with
PMA and ionomycin for 3 hr and used an antibody against p65
or c-Rel for chromatin IP. We found that c-Rel binding to the
kb1 site within the CNS3 enhancer region was dramatically
induced after PMA and ionomycin stimulation; the binding o
promoter sites by c-Rel was also induced to approximately
7-fold after stimulation (Figure 6C). We also found that c-Re
can bind to the Foxp3 promoter and kb1 site in the CNS3
enhancer of primary mouse CD4+ T cells (Figure 6D). This
binding pattern was similar to what we observed in Jurkat cells
albeit less dramatic. However, we did not detect a substantia
increase in p65 binding in any of these sites, which indicates
that c-Rel might function as a c-Rel-p50, but not c-Rel-p65
dimer. Additionally, we assayed c-Rel binding of the Foxp3
enhancer in 293 cells, a non-T cell line, and DPK cells, a DP
thymocyte cell line, and found that c-Rel did not bind to the
kb1 site or Foxp3 promoter in these cells (Figure 6E and data
not shown). Thus, the ability of c-Rel to bind to the methylated
CpG island in the CNS3 enhancer in T cells developed beyond
DP stage makes it a candidate for being the pioneer transcrip-
tion factor that initiates chromatin remodeling that is character-
istic of natural Treg cells.
DISCUSSION
Here, we have provided evidence that the NF-kB signaling
pathway plays a crucial role in regulating Foxp3 expression
during thymic Treg cell development. Deficiency of severa
pleiotropic signaling molecules, including TAK1, Bcl10
CARMA1, PKCq, and LAT, has been found to cause severely
impaired Treg cell development, whereas it does not affect, o
CD4SP CD8 SP DN DP
IL7
IL2
Beforetreatment
Blue line: WTRed line: IKKEE-Tg
B
IKKEE-Tg
WT
CD122(IL-2Rb)
CD25(IL2Ra)
Red line:IKKEE-Tg thymocytesBlue line:Wild-type thymocytes
A
0.39
0.72
92.8
1.35
2.41
pSTAT5CD25
CD122
IKKEE-Tg
WT
Red line:CD122+CD25+Blue line:CD122+CD25-Green line:CD122-CD25-
C
79.9 0.34
0.23
82.3 10.6
1.42
12.9 0.41
0.56
21.9 46.8
4.71
0.68 0.021
0.28
9.45 0.94
34.2
11.6 15.8
2.77
2.64 0.21
0.51
44.7 0.4
0.4
35.6 0.22
0.29
0.1 7.39e-3
0.34
6.3 0.55
41.6
DN DP CD4+Foxp3+ CD4+Foxp3- CD8+Foxp3+ CD8+Foxp3-
CD4+CD25+Foxp3-
CD127 (IL-7Ra)
pSTAT5
Figure 5. NF-kB Pathway Does Not Induce Foxp3 Expression by Increasing Sensitivity to Common Gamma Cytokines
(A) Flow cytometry analysis of CD127 (IL-7Ra), CD122 (IL-2Rb), and CD25 (IL-2Ra) expression in different thymocyte populations from IKKEE-Tg and wild-type B6
mice. All mice were crossed to Foxp3-GFP knockin mice and Foxp3 expression was identified by GFP expression. Numbers in quadrant indicate percentage.
Data shown are representative of three independent experiments.
(B) STAT5 phosphorylation in different thymocyte populations from IKKEE-Tg versus wild-type B6 mice. Thymoyctes were analyzed either directly after isolation
or after 30 min of IL2 (5 ng/ml) or IL7 (2 ng/ml) treatment. Data shown are representative of two independent experiments.
(C) IL-2-stimulated STAT5 phosphorylation in CD4 SP thymocyte subpopulations expressing both of CD122 and CD25, CD122 only, or neither CD122 and CD25.
Numbers in the left panel indicate percentage. Data shown are representative of two independent experiments.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
affects to a lesser extent, conventional T cell development
(Medoff et al., 2009; Gupta et al., 2008; Koonpaew et al., 2006;
Schmidt-Supprian et al., 2004; Wan et al., 2006). Signaling
events mediated by these molecules, therefore, could be specif-
ically required for natural Treg cell development. Interestingly,
most of these signaling molecules are linked to TCR-induced
NF-kB activation. We found that restoring NF-kB activity with
the IKKEE transgene can rescue Foxp3 expression in TAK1- or
CARMA1-deficient thymocytes that otherwise showed the
most dramatic impairment in Treg cell development. It is
possible that deficiency of NF-kB signaling may be the common
mechanism underlying impaired Foxp3 expression caused by
I
deficiency of all of the aforementioned pleiotropic signaling
molecules. We previously reported that CD4 SP thymocytes,
most of which would mature into conventional T cells, exhibited
weaker NF-kB activity compared to other thymic subpopula-
tions. Therefore, a stronger NF-kB pathway signal could be the
unique signaling event that is specifically required for Foxp3
expression and thymic Treg cell development. To further test
this hypothesis, we studied Rag1�/� TCR transgenic mice. In
these mice, thymocytes express only one type of TCR and are
uniformly selected into conventional T cells, which indicates
that Foxp3 expression and Treg cell development require TCR
signaling that is different from positive selection of conventional
mmunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc. 927
A
B C D E
Figure 6. NF-kB Can Directly Regulate Foxp3 Gene Expression
(A) Schematic representation of Foxp3 promoter region (CNS1) and enhancer region (CNS3). Bottom: the CpG island within CNS3 region and three potential
NF-kB binding sites around that region.
(B) Luciferase assay with reporter plasmids containing Foxp3 promoter fragment (CNS1) and intronic enhancer fragment (CNS3) in Jurkat cells. Expression
plasmids of IKKEE or IkBa-SR were cotransfected as indicated. Cells were either unstimulated or stimulated with PMA and ionomycin (PI) for 17 hr before
analysis. Graphs show mean ± SD. n = 3, data are representative of two independent experiments.
(C) Chromatin IP analysis of c-Rel binding in the Foxp3 promoter and enhancer region in unstimulated or PMA and ionomycin-stimulated Jurkat cells. One poten-
tial NF-kB binding site in the promoter region and three potential binding sites in CNS3 enhancer region (kb1–3) were analyzed. Graphs show mean ± SD. n = 3,
data are representative of two independent experiments.
(D) Chromatin IP analysis of c-Rel binding in the Foxp3 promoter and enhancer region in unstimulated or PMA and ionomycin-stimulated primary mouse CD4+
T cells. Graphs show mean ± SD. n = 3, one independent experiment.
(E) Chromatin IP analysis of c-Rel binding in the Foxp3 promoter and enhancer region in unstimulated or PMA and ionomycin-stimulated HEK293 cells. The kb1
site specific primers were used for analysis. Graphs show mean ± SD. n = 3, data are representative of two independent experiments.
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
T cells. We found that Foxp3 expression could be induced in
these ‘‘one-conventional TCR only’’ thymocytes by enhancing
NF-kB activity through introducing an IKKEE transgene. Again,
our data support the notion that a stronger NF-kB signal is the
difference in TCR signaling that is uniquely required for Foxp3
expression during thymic T cell development.
928 Immunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc.
How TCR-triggered signaling events affect the fate decision of
developing thymocytes is under intense investigation. In all of
the suggested models, TCR signaling strength plays a key role:
low-affinity TCR engagement leads to positive selection of
conventional T cells, whereas high-affinity engagement triggers
negative selection. Treg cell development, however, may require
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
an intermediate TCR signaling strength. Different TCR
downstream signaling pathways have also been found to play
distinct roles in positive and negative selection. Impaired
calcineurin and ERK activity specifically affect positive selection
(Fischer et al., 2005; Neilson et al., 2004), whereas the JNK and
p38 MAPK pathways are involved in negative selection (Gong
et al., 2001). Therefore, the preferential enhancement of certain
TCR signaling pathways may also play an important role in
thymocyte fate decision. Our findings suggest that the signal
strength of the NF-kB pathway (i.e., selectively enhanced by
the heterozygous IKKEE transgene) sets a threshold for thymo-
cyte fate decision. This signal, of proper intensity, in addition to
TCR signaling typical of positive selection, is required for
Foxp3 expression induction and natural Treg cell development.
Studies in IKKb- and c-Rel-p50-deficient mice all pointed to
the particular importance of the NF-kB pathway in Treg cell
development. However, the NF-kB pathway is also crucial for
IL-2 production (Lai et al., 1995; Schmidt-Supprian et al.,
2003). Therefore, the observed deficiency in Treg cell develop-
ment could be attributed to the impaired IL-2 production by
conventional T cells rather than a cell-intrinsic effect in differen-
tiating Treg precursor cells. We think that the former explanation
is unlikely. First, there is redundancy in the effect of common
gamma cytokines in thymic Treg cell development. IL-2
deficiency alone reduced, but did not completely ablate, thymic
Foxp3+ cells (Fontenot et al., 2005). Additionally, the complete
disappearance of thymic Treg cells in CARMA1 and TAK1
deficiencies, which could be rescued by restoring NF-kB
activity, cannot be explained by IL-2 deficiency. Second, the
IKKEE transgene rescued Foxp3 expression in TAK1 conditional
deficiency, but it did not rescue the impaired maturation of SP
thymocytes into peripheral T cells, which are a major source of
IL-2. Furthermore, Rag1�/� TCR transgenic mice have no thymic
Treg cells but the production of IL-2 by conventional T cells is
normal. Selectively increasing the NF-kB signal restored Foxp3
expression of thymocytes in these mice, which suggests that
the NF-kB pathway plays a crucial cell-intrinsic role in the induc-
tion of Foxp3 expression during thymic Treg cell development.
The Foxp3+ cells in our IKKEE-Tg mice did not express CD25
and exhibited poor peripheral homeostatic proliferation and
survival when compared to wild-type Treg cells. However, they
did gain the ability to stably express Foxp3 and showed the
unique DNA demethylation of the CpG island within the CNS3
enhancer, which is a hallmark of natural Treg cells (Kim and
Leonard, 2007). Therefore, our data suggest that although the
NF-kB pathway is crucial for induction of Foxp3 expression, it
does not account for other aspects of Treg cell development.
Other signals, such as stimulation from common gamma
cytokines, might be responsible for these Foxp3-independent
features of natural Treg cells, and limiting the availability of these
signals might explain why the majority of Foxp3+ thymocytes in
IKKEE-Tg mice did not express CD25. We also found that
IKKEE-Tg Foxp3+ cells were not as suppressive when measured
by in vitro suppression assay (Figure S2). It has been reported that
in vitro engagement of CD28, together with TCR stimulation,
abrogates the suppressor function of natural Treg cells in the clas-
sical in vitro suppression assay system that we used (Sakaguchi,
2004;Takahashi et al., 1998). Work from our laboratory and others
have established that CD28 is an important cofactor for
I
enhancing NF-kB signaling during TCR ligation. Therefore, we
speculate that increased NF-kB activity may have abrogated
in vitro suppression capacity in our IKK-Tg Foxp3 Treg cells.
We found that c-Rel bound to the Foxp3 promoter and CNS3
enhancer region after TCR stimulation. Interestingly, the c-Rel
binding site in the CNS3 enhancer was located within a CpG-rich
region that remains methylated in the Jurkat T cells that we
studied. Other generic transcription factors that have binding
sites in the same region, such as CREB and ATF, only bind after
the CpG island is demethylated (Kim and Leonard, 2007). c-Rel
has been found to play an essential role in initiating chromatin
remodeling in IL-2 promoter in primary CD4+ T cells (McKarns
and Schwartz, 2008; Rao et al., 2003). Interestingly, it has been
reported that rapid demethylation of the CpG sites in the
promoter region occurred after T cell activation both in vitro
and in vivo, which can, in turn, affect the transcriptional expres-
sion of IL-2 (Bruniquel and Schwartz, 2003; Thomas et al., 2005).
It is thus likely that in both the IL-2 promoter and the CNS3
enhancer of Foxp3, c-Rel plays a role in recruiting chromatin-
modifying complexes to the regulatory sequences and
stabilizing an open chromatin conformation, further facilitating
the recruitment of enzymes responsible for CpG demethylation.
Additionally, in the thymus, c-Rel expression was found to be
very low in the preselected CD4+CD8+ DP thymocytes but was
strongly induced after positive selection (Moore et al., 1995).
Consistent with this finding, we observed that c-Rel cannot
bind to the CNS3 enhancer region in DPK cells, a DP thymocyte
cell line. Therefore, we speculate that c-Rel could act as
a pioneer transcription factor that initiates chromatin remodeling,
thus facilitating DNA methylation of the Foxp3 locus. This
process likely takes place during the CD4+ SP thymocyte stage,
shortly before the appearance of Foxp3+ thymocytes in the CD4+
SP compartment.
In summary, our results suggest that the NF-kB pathway is
a key regulator of Foxp3 induction during thymic Treg cell
development, and preferential increase in NF-kB activity
promotes Foxp3 expression and Treg cell lineage differentiation.
Such preferential increase in NF-kB activity could be achieved
by complementing TCR stimulation by a combination of signals
from certain costimulatory molecules and cytokines, such as
CD28, CD27, and TNF-a (Watts, 2005). Thus, the NF-kB pathway
may also be a sensor of peripheral inflammatory events. An
excessive inflammatory response may enhance NF-kB
activation in thymocytes via production of cytokines such as
TNF-a. This, together with an increased production of IL-2,
which is an indicator of T cell response, could favor Treg cell
development over conventional T cell development as well as
promote negative selection of conventional T cells. Therefore,
the NF-kB pathway may play a crucial role in shifting thymic
output from conventional T cells to regulatory T cells during an
excessive inflammatory response.
EXPERIMENTAL PROCEDURES
Mice
The IKKEE-Tg, IkBaSR-Tg, Map3k7fl/fl, Card11�/�, and the Foxp3-RFP
reporter (FIR) mice have been described previously (Egawa et al., 2003; Jimi
et al., 2008; Voll et al., 2000; Wan et al., 2006; Wan and Flavell, 2005; Xie
et al., 2006). These mice were backcrossed to B6 strain for more than six
generations. Mice were housed at the Yale Animal Resource Center, and
mmunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc. 929
Immunity
NF-kB Regulates Foxp3 Expression in Treg Cells
experiments were performed in accordance with the guidelines of the National
Institutes of Health (NIH) and protocols approved by the Institutional Animal
Care and Use Committee.
Flow Cytometry and Cell Sorting
FACS analysis was performed as previously described (Jimi et al., 2008).
Intracellular Foxp3 staining was done with the kit from eBioscience according
to the manufacturer’s protocols. Phospho-STAT5 staining was done as
previously described (Long and Adler, 2006). Stained cells were analyzed on
a FACSCalibur or an LSRII station (Becton Dickinson). The sortings were
done in a MoFlow cell sorter (Dako Cytomation) or a VantageSE station
(Becton Dickinson, customized and equipped with a 568 nm laser).
Adoptive Transfer
CD4+RFP+Foxp3+ or CD4+RFP�Foxp3� T cells were purified by flow cytome-
try from Foxp3-RFP reporter mice with or without the IKKEE transgene. 2 3105
cells were transferred into T cell-deficient recipient mice by i.v. injection. The
spleens of recipient mice were harvested 4 weeks later, and single-cell
suspensions were analyzed for frequency of TCRb+CD4+ cells and Foxp3
expression.
DNA Methylation Analysis
Cell populations were isolated from male mice and methylation analysis was
carried out via bisulphite sequencing protocol as described before (Clark
et al., 2006). The CpG-rich region of CNS3 intronic enhancer region of
FOXP3 gene was amplified by PCR with primers described before: DS-inner
FW, 50-TTTTGGGTTTTTTTGGTATTTAAGA-30; and DS-inner RV, 50- TTAACCA
AATTTTTCTACCATTAAC-30 (Kim and Leonard, 2007). The PCR product was
cloned with the ZeroBlunt PCR Cloning kit (Invitrogen) and was sequenced.
Chromatin Immunoprecipitation
Jurkat cells were stimulated with or without PMA (20 ng/ml) and ionomycin
(500 ng/ml) for 3 hr and were then fixed with 1% formaldehyde for 15 min at
room temperature, washed twice with ice-cold PBS, and resuspended in
ChIP lysis buffer. The lysates were subsequently sonicated to shear the
genomic DNA into around 200 bp fragments via a Bioruptor sonicator. To
ensure uniformly small fragment sizes, we sonicated for a total of 7.5 min (15
times for 30 s with a 30 s rest between each pulse) for all experiments. ChIP
analysis was performed on the supernatants with cRel and p65 antibodies
via methods as described before (Long et al., 2006). The primer sequences
for ChIP analysis were as follows: human promoter c-Rel site primer, FW: AT
TAGAAGAGAGAGGTCTGCGGCT, RV: GGCTTGTGGGAAACTGTCACGTAT;
human CNS kb1 site primer, FW: TCAGATGACTCGTAAAGGGCAAAG, RV:
TCTCTCTCTCTGTGTTTCTCCT; human CNS kb2 site primer, FW: CATA
ATCTGTGTCCCAGAAACATCCC, RV: GGCTTCCTGCACTGTCTGTT; human
CNS kb3 site primer, FW: AGCTGCCTGACTTTCAGATGGTTC, RV:
GTCCCAAAGTCTCAGTATGTGTAGGC.
Reporter Assay
The Foxp3 reporter constructs were kindly provided by W.J. Leonard at NIH
(Kim and Leonard, 2007) and were subcloned into PGL4 basic reporter
construct. We transfected 1.5 3 106 cells with 1.5 mg of luciferase reporter
plasmid and 0.4 mg of phRL-TK (Promega) as an internal control via the poly-
ethylenimine (PEI) method. 1.5 mg of IKKEE, IkBaSR, or control empty expres-
sion plasmid was cotransfected as indicated. The transfected cells were then
cultured in 24-well plates and were stimulated with PMA (10 ng/ml) and
ionomycin (500 ng/ml) for 17 hr before analysis as indicated.
SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures and eight
figures and can be found with this article online at http://www.cell.com/
immunity/supplemental/S1074-7613(09)00500-7.
ACKNOWLEDGMENTS
The authors would like to thank R. Flavell and A. Rudensky for providing the
Foxp3 RFP and GFP reporter mice. The TAK1-floxed mice were obtained
930 Immunity 31, 921–931, December 18, 2009 ª2009 Elsevier Inc.
from the laboratory of M. Schneider. The CARMA1-deficient mice were ob-
tained from the laboratory of D. Littman. The Foxp3 reporter constructs were
kindly provided by W.J. Leonard. The work reported in this manuscript was
supported by NIH R37 AI33443 and RO1 AI068977. M.L. was supported by
NIH T32AI007019-33. M.S.H. was also supported by NIH/NIGMS MSTP grant
GM07205.
Received: April 21, 2009
Revised: July 22, 2009
Accepted: September 22, 2009
Published online: December 17, 2009
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