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egulatory T cells (Tregs) are a vital component of selftolerance (1, 2); however, the signaling events that govern
Treg development are not well defined. Treg development and
function is dependent upon the forkhead-winged helix transcription factor FoxP3, which is coregulated by signaling molecules downstream of T-cell receptors and cytokine receptors. At
the same time, robust antigen receptor and cytokine receptor
stimulation activates PI3K, which negatively regulates FoxP3
expression (3, 4). This occurs when PI3K activates protein
kinase B (PKB), which in turn phosphorylates and inactivates
forkhead-box O (Foxo) transcription factors that promote
FoxP3 expression (57). Consistent with this model, conditional deletion of Foxo1 and Foxo3 results in reduced numbers
of thymic- and peripheral-derived Tregs (7, 8). Moreover, the
Tregs that eventually populate Foxo1/3-deficient animals are
hyperproliferative, skew toward differentiated effector lineages, and lack suppressive functions. Within the context of
CD4+ T-cell biology, a critical molecule regulated by Foxo1 is
Krppel-like factor 2 (KLF2) (9, 10), a zinc-finger transcription factor that is also inactivated in a PI3K-dependent manner
(11). KLF2 maintains T-cell homeostasis, in part by promoting
expression of sphingosine-1-phosphate receptor 1 (S1P1) (12
14). Surprisingly, S1P1 suppresses expression of FoxP3, as evidenced by increased numbers of thymus-derived Treg (tTregs)
and induced Treg (iTreg) cells after T cell-specific excision in
S1P1 gene-targeted animals (15, 16). On the one hand, studies
using Foxo1/3-deficient mice suggest that KLF2 promotes Treg development and/or function, whereas reports using S1P1-deficient
animals predict an opposing outcome. To clarify this apparent
contradiction and gain further insight into Treg biology, we compared gene-targeted mouse models that excised Klf2 within the
T-cell vs. Treg compartments. We now report that (i) KLF2 is
selectively required for the generation of iTregs but not tTregs,
(ii) KLF2 is necessary during the inductive phase of iTreg
development to promote FoxP3 transcription, and (iii) iTreg production can be augmented by stabilizing KLF2 protein levels with
pharmaceutical drugs.
www.pnas.org/cgi/doi/10.1073/pnas.1323493111
Results
Treg Frequencies Are Maintained in Klf2 Gene-Targeted Mice. KLF2
is a zinc-finger transcription factor that has a documented role in
controlling naive T-cell migration patterns (1214). Its expression
is promoted by Foxo1, a PKB-regulated transcription factor that
has also been reported to control T-cell circulation (9, 10).
Moreover, Foxo1 and Foxo3 promote Treg development and
function; mice lacking these transcription factors have reduced
percentages of FoxP3+ T cells (8), especially at 3 wk of age (7),
and fail to develop functional Tregs. To determine whether Foxo1
and Foxo3 were acting through Klf2 to control Treg biology, we
examined regulatory T cells in Klf2 gene-targeted animals. Using
Lck-cre transgenic mice that specifically excised floxed alleles of
Klf2 (Klf2fl/fl) within the T-cell compartment (Lck-cre; Klf2fl/fl), we
found no obvious Treg defects, as determined by CD4+CD25+
FoxP3+ T-cell frequencies in primary and secondary lymphoid
tissues (Fig. 1A). Likewise, Treg frequencies in the thymus of
neonates seemed normal (Fig. 1B), which would suggest that
Foxo-mediated Treg development was not dependent upon KLF2.
Recent reports have demonstrated that S1P1 suppresses generation of Tregs (15, 16), and because KLF2 promotes S1P1 expression in the CD4+ T-cell lineage (1214), we decided to
examine the relationship between KLF2 and S1P1 relative to Treg
development. Using Vav-cre transgenic mice to thoroughly excise
floxed alleles within the T-cell compartment, including early stages
of thymocyte development, we found that S1P1 gene-targeted mice
(Vav-cre; S1P1fl/fl or Vav-cre; S1P1fl/fl; Klf2fl/fl) expressed increased
frequencies of Tregs, whereas this phenotype was not present in
animals lacking KLF2 alone (Vav-cre; Klf2fl/fl) (Fig. 1C). From
these results we conclude that KLF2 does not contribute to the
overt Treg homeostatic defects observed in Foxo1/3 or S1P1 genetargeted mice.
Significance
Regulatory T cells (Tregs) are crucial for preventing autoimmunity, and thus discovering an efficient means of generating
antigen-specific Tregs is a medical priority. To this end, we
demonstrate that transcription factor Krppel-like factor 2
(KLF2) is necessary for the generation of antigen-induced Tregs
and their in vivo counterpart, peripheral Tregs. Moreover,
pharmaceutical drugs that stabilize KLF2 protein levels during
the transition from CD4+CD25 T cell to CD4+CD25+FoxP3+
Treg augment production of these tolerizing lymphocytes.
Results from this study indicate that KLF2 is a viable target for
altering Treg development, which may significantly impact
patients prescribed statins.
Author contributions: S.K.P., T.M.A., and E.S. designed research; S.K.P., W.R., D.M., D.C.,
P.L.C., K.L.H., and V.V.P. performed research; T.M.A. contributed new reagents/analytic
tools; S.K.P., D.M., P.L.C., and E.S. analyzed data; and E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
IMMUNOLOGY
Spleen
Ms LN
10
10
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0
2
10
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0 10
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11
14
0
0 10
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0 10
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10
103
10
10
10
10
10
10
0
2
10
10
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0 10
10
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10
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10
15
10
0 10
control
0 10
10
15
10
12
10
cKO
0 10
10
10
10
0 10
10
10
10
CD25
control
Klf2 cKO
4.9
5.0
5.1
FoxP3
4.5
CD25
control
10
11.3
dKO
Klf2 cKO
S1P1 cKO
5
10
23.2
10
9.4
10 4
10 4
10 4
10 4
10 3
10 3
10 3
10 3
10 2
10 2
10
24.7
CD3/CD28
TGF (ng/ml)
10
800
800
10 2
10 3
10 4
10 5
10 2
10 3
10 4
10 5
CD25
40
400
0
10
10
10
10
0.6
20
10
10
10
10
10
10
10
10
10
10
10
800
1.3
600
1.2
600
10
0
0
10
10
10
10
CFSE+
cKO
10
10
10
10
10
10
10 2
0
10 2
10 3
10 4
10 5
10 5
10 2
10 3
10 4
10 5
10 5
5.1
0.6
10 4
10
10
CD3
+CD28
+ TGF
10
10
10
10
10 2
10 3
10 4
10 5
10 2
10 3
10 4
10
10
10
10
10 5
10
CFSE+
10 4
10 4
10 3
10
10
20
0
0
10 2
10 3
10 4
10 5
4.7
10 2
10 3
10
10
cKO
10 4
10
10
10
10
10
10
control
0.9
10 5
10 2
10 3
10 4
10 5
10 5
10
10 4
10 4
P<0.0001
50
40
30
20
20
10
CD3/CD28 +
TGF (10ng/ml)
+
+
800
500
CFSE-
CD3/CD28
+ TGF
10 5
60
100
60
40
80
40
10
0.9
60
10 5
CD3/CD28
100
P=0.005
CD25
10
10 3
10 4
CD25
CD3
+CD28
10 3
control
54.6
10 5
CFSE-
10 3
1.0
10 4
10 2
0.3
10 5
0.9
10 2
LN
10
10 5
10 4
10
200
200
10
10 4
400
400
10
FoxP3
Ax
LN
M
en
Sp
le
us
ym
10
800
10 3
Th
10
10
9580 | www.pnas.org/cgi/doi/10.1073/pnas.1323493111
10
0.8
10 4
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10 3
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10 5
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400
Klf2 cKO
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control
S1P1 cKO
800
800
200
30
0
1000
control
200
200
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200
0.4
10 5
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ro
10 4
10
nt
10 3
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co
10 2
18.2
% iTregs (relative
to CD4+ T cells)
400
FoxP3
10 5
12.7
400
% iTregs (relative
to CD4+ T cells)
10 4
2.1
400
IL-17
10 3
600
0.9
600
CD3/CD28
+ TGF
CD3/CD28
10
600
600
10 2
10
FoxP3
600
0.6
400
100
0
2
10
10
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600
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800
0.6
10
10
10
10
800
600
400
control
200
200
10
11.1
300
10.1
400
200
+
+
400
600
FoxP3
10
cK
10
11
10
10
Ax LN
5
cK
10
ro
10
nt
FoxP3
Thymus
5
co
600
12.0
400
iKO
11.6
400
200
200
200
80
10 3
40
10
20
10
6.1
cKO
10 2
0.6
0
0
10 2
10 3
10 4
GATA-3
10 5
10
10
10
10
IL-4
60
10
10
10
10
CD3/CD28
TGF
tamoxifen
10 2
10 3
+
-
10 4
10 5
0
0
10
10
10
FoxP3
+
+
-
10
10 2
10 3
10 4
10 5
+
+
+
Fig. 2. KLF2 is required for the ex vivo generation of iTregs. (A) Ex vivo
generation of iTregs from splenic CD4+CD25 T cells derived from Klf2fl/fl
(control) or Lck-cre; Klf2fl/fl (cKO) mice. Histograms display FoxP3+ T-cell
frequencies. This experiment was repeated four times. (B) iTreg development
using CD4+CD8CD25 thymocytes from Klf2fl/fl (control) vs. Lck-cre; Klf2fl/fl
(cKO) mice. Representative contour plots (Upper) and a chart of accumulated
data (Lower) from four mice per cohort are shown. This experiment was repeated three times. (C) Ex vivo generation of iTregs using cocultured CD4+
CD25 T cells harvested from Klf2fl/fl (CFSE+) and Lck-cre; Klf2fl/fl (CFSE) mice.
Contour plots and pooled results for Treg induction (CD3+CD28+TGF) are
shown. This experiment was repeated three times. (D) CD4+ T cells from Klf2fl/fl
(control) vs. Lck-cre; Klf2fl/fl (cKO) mice were evaluated for their propensity to
skew toward the TH2 lineage and secrete IL-4. (Left) Histogram overlays of
GATA-3 expression after culturing conditions that favor CD4+ T-cell differentiation toward a neutral (solid gray), TH2 (gray line), or iTreg lineage (black
line). (Right) Generation of intracellular cytokines after CD4+ T-cell activation
(Left) vs. iTreg differentiation (Right). This experiment was performed twice.
(E) Tamoxifen-induced excision of Klf2 after ex vivo generation of iTregs using
CD4+CD25 T cells from T2cre vs. T2cre; Klf2fl/fl animals. T cells from T2cre;
Klf2fl/fl mice excised >95% Klf2 as measured by RT-PCR. This experiment was
repeated twice.
Pabbisetty et al.
A
80
60
60
40
40
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80
10
10
10
10
100
Ms LN
10
10
30
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20
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60
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Helios
30
P=0.003
OVA-induced Tregs
20
P=0.01
10
P=0.3
30
P=0.5
0
Ax
Sp
LN
LN
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le
Ax
s
M
OT2; Klf2fl/fl
60
LN
LN
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le
NT control
Non-induced Tregs
Sp
P=0.3
90
B
OT2+ CD4+ FoxP3+ cells (x104)
0.3
20
20
100
Spleen
Helios-low Treg
numbers (x104)
100
Thymus
80
% Helios-low
Regulatory T cells
100
C
100
Thymus
80
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60
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10
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Pabbisetty et al.
67"45"
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40'#"
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30
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Ax
20
10 5
40
10 4
0.5
40
Sp
60
40
10 3
0.2
40
50
Th
60
10 2
40
Helios-low Treg
numbers (x104)
80
% Helios-low
Regulatory T cells
100
IMMUNOLOGY
B
0.04
0.6
0.06
0.03
0.003
0.0025
0.02
0.002
0.0015
0.01
0.001
0.5
0.05
0.4
0.04
0.3
0.03
0.2
0.02
0.1
0.01
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S3
N
C
S1
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Pr
o
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S2
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C
S1
N
.
C
om
Pr
S2
0.004
0.0035
H4Ac immunoprecipitation
WT CD3
cKO CD3
WT CD3 + TGF
Fig. 4. KLF2 directly promotes FoxP3 transcription within the iTreg lineage.
(A) ChIP assays using antibody directed against KLF2 after lysis of wild-type
CD4+CD25 T cells cultured under conditions that promote T-cell activation
(black) vs. iTreg differentiation (white). Isotype control antibody (gray) is
included for iTreg ChIP assays. DNA primers are specific for the promoter or
enhancer elements (CNS1-3) of FoxP3. KLF2 binding to the regulatory
regions of FoxP3 is graphed relative to input amounts of genomic DNA.
These experiments were performed three times. (B) Wild-type (WT) and
KLF2-deficient (cKO) CD4+CD25 T cells were cultured under activating
(black, gray) vs. iTreg-inducing conditions (white, blue) before performing
ChIP assays using antibody directed against acetylated histone H4. This experiment was conducted twice.
9582 | www.pnas.org/cgi/doi/10.1073/pnas.1323493111
that was most evident after T-cell activation (Fig. 5G). All of
these results are consistent with KLF2 stability being a key
rate-determining step during Treg induction, and for that
reason we repeated transfection/induction experiments using
a mutant form of KLF2 that is no longer recognized by the E3
ubiquitin ligase complex, SCFFBW7 (35). SCFFBW7 is a multisubunit RING-finger ligase that targets proteins for 26S proteasomal destruction, and in this context KLF2 has recently been
identified as a substrate in endothelial cells (35). Despite the fact
that several E3 ubiquitin ligases have a documented role in
promoting KLF2 degradation (3436), we found that CD4+
CD25+FoxP3+ frequencies were enhanced when CD4+CD25 T
cells were transfected with the mutant form of KLF2 before ex
vivo Treg induction (Fig. 5H). From this we conclude that KLF2
degradation is a key facet of CD4+ T-cell conversion that can
be modified to enhance the generation of i/pTregs. Of note,
overexpression of KLF2 using pharmaceutical drugs during
the inductive phase of iTreg production did not impair suppressive activity or cell viability (Fig. S6 A and B). Likewise,
elevated levels of KLF2 did not affect ex vivo Treg suppression (Fig. S6C), which suggests that KLF2 and the proteins
that interface with this transcription factor are potential immunotherapeutic targets.
Discussion
Numerous reports have demonstrated that Foxo1 promotes expression of KLF2, which in turn drives expression of S1P1 within
the T-cell compartment. For this reason, it has been difficult to
reconcile the dichotomy in Treg development that occurs in
Foxo1 vs. S1P1 gene-targeted animals; in the former, tTreg and
pTreg populations are decreased, whereas in the latter, both
Treg lineages are increased. Because KLF2 bridges these two
signaling components, we were surprised to discover that KLF2
is not required for the generation of tTregs and is only necessary
for pTreg (and ex vivo iTreg) development. tTregs arise from
self-reactive CD4+CD8+ thymocytes (37), and because these
cells do not yet express KLF2 (KLF2 is initially expressed at the
single-positive thymocyte stage), it is evident that other Foxo1/
3-transcribed factors are necessary to establish CD4+CD25+
FoxP3+ thymocytes. Interestingly, S1P1 thymic expression patterns are similar to those of KLF2, which leads us to speculate
that S1P1 modifies FoxP3 expression after the initial generation
of tTregs. If true, this would suggest that KLF2 and its transcriptional target, S1P1, regulate pTreg levels by initiating FoxP3
transcription and limiting its continued expression, respectively.
This temporal separation in FoxP3 regulation may explain the
counterintuitive Treg phenotypes displayed by Foxo1 and S1P1
gene-targeted animals.
With regard to the molecular mechanisms that underpin iTreg
and pTreg development, current results indicate that KLF2 is
absolutely necessary during the initial stages of Treg induction
but is redundant once FoxP3 is stably expressed. We propose
that KLF2 is incorporated into a signaling complex that provides
transcriptional accessibility to the entire FoxP3 locus, after which
additional nuclear factors stabilize the open conformation in
a KLF2-independent manner. Consistent with a previous report
(38), this larger signaling complex may include histone acetyltransferases that reverse p300/CBP-associated factor-mediated
FoxP3 gene silencing in nave CD4+ T cells. Importantly, we
demonstrate that KLF2 is a rate-limiting factor during this inductive process that can be targeted with drugs to enhance iTreg
production via increased KLF2 stability. Perhaps because KLF2
is a transcription factor, its regulation is typically described in
terms of mRNA levels and Foxo1-mediated transcription; however, current results indicate that KLF2 protein levels are far
more relevant when describing biological events in postactivated
lymphocytes. Case in point, concurrent with TCR stimulation
that promotes KLF2 degradation, KLF2 proteolysis is partially
inhibited by TGF-mediated signaling events that favor iTreg
production over other CD4+ T-cell differentiation outcomes.
Moreover, this block in TCR-mediated KLF2 degradation is
Pabbisetty et al.
10
0.1
10
+
+
10
0.3
10.6
10 4
10
10
10
10
10
10
0
10
10
10
10
10
10
10
0.2
10
10
10
10
10
10
0.2
10 4
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
KLF2
tubulin
10
10
10
10
10
10 3
10
10
FoxP3
0.4
10
10
10
10
10
10
1.6
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10 5
0.3
10 4
0.8
control
10 3
10
10
1.2
10 2
10 2
0
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
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40
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20
#!"
!"
CD3+ CD28
TGF
simvastatin
P<0.0001
P=0.7
'()*'(#&"
+,-."
+
-
/012"
+
+
344"
+
+
-
+
+
+
55
68
45
47
2.0 g
4.0 g
13
14
12
12
4
2
0
Transfected 0 0.5 1
KLF2 vector ( g)
control
CD25
SMURF1-/-
TGF
Vector
KLF2
103
102
0
102
0
010
0.5
10
TGF
+
+
+
+
+
10
104
10
10
10
104
010
104
104
105
10
10
control
SMURF1-/-
25
0.07
ng
10
104
103
104
105
SMURF1-/-
WT+TGF
SMURF1-/+TGF
27.2
103
2
10
0
0102
WT
KLF2-dm 2 g
105
17.2
103
10
0
103
10
KLF2-dm 1.5 g
105
13.5
0102
103
10
0
phospho-Smad2/3
KLF2-dm 1 g
105
103
0.5
0.25
010
CD25
+
12.2
1.0
0.75
0.0005
50
10
0
KLF2-dm 0.5 g
105
FoxP3
10
KLF2
tubulin
103
Vector
TGF
5.6
104
<0.0001
75
0
CD3+ CD28
KLF2 0.5 g
105
8.2
104
103
1.0
Empty
105
0.6
104
FoxP3
1.5
Empty
105
tubulin
KLF2 protein levels
(relative to tubulin)
1.0 g
0.5 g
%CD4+CD25+FoxP3+
T cells
empty vector
14
FoxP3
KLF2
tubulin
18
0.5
CD3+ CD28 - + + + + + +
- - + + + + +
TGF
supplement
CD25
L
LY Y
+P
ra sta D
pa tin
m
yc
in
60
&!"
simvastatin ( M)
CD25
FoxP3
%CD4+CD25+FoxP3+
Thymocytes
80
0
TGF
CD25
P<0.0001
P=0.09
0.5
0.25
CD3+ CD28
10 2
1.0
0.75
cKO
10
10 4
12.7
10 4
10 5
cKO
10 5
4.4
10 4
10
10 4
+
+
2
10 5
2.5
10 4
10 3
10 3
10
+
+
1
10 5
1.3
10 4
10
0
10
0.3
10 4
+
+
0.5
10 5
control
10 4
36.5
10 4
10
+
+
0
CD3+ CD28
TGF
simvastatin ( M)
10 5
10
+
+
+
10
10 4
FoxP3
+
-
10
0
0102
103
104
105
0102
103
104
105
CD25
Fig. 5. Increased KLF2 expression correlates with enhanced generation of iTregs. (A) Ex vivo generation of iTregs using CD4+CD25 T cells from Lck-cre
(control) vs. Lck-cre; Klf2fl/fl (cKO) littermates in the presence of simvastatin. (Upper) Contour plots of iTregs (FoxP3+CD25+) after induction of splenic T cells.
(Lower) Same assay performed in triplicate using CD4+CD8CD25 thymocytes from control (gray) vs. cKO (black) mice. Statistical differences between control
groups (gray numbers) and KLF2-deficient populations (black numbers) are shown. Both sets of experiments were repeated three times. (B and C) A comparison between iTreg induction and KLF2 protein levels. (B) (Left) FACS analysis of KLF2-replete (control) vs. KLF2-deficient (cKO) CD4+CD25 T cells cultured
under suboptimal iTreg conditions with or without increasing concentrations of simvastatin. (Right) Immunoblots of KLF2/tubulin and corresponding densitometry plots using total cell lysate from CD4+CD25 T cells cultured (12 h) as specified. Both sets of experiments were repeated three times. (C) (Left)
Contour plots of iTregs using wild-type CD4+CD25 T cells cultured with the indicated compounds (LY, LY294002; PD, PD98059; statin, simvastatin). Activated,
CD3 + CD28; TGF-induced, CD3 + CD28 + TGF. (Right) Western blots of KLF2/tubulin and corresponding densitometry data from CD4+CD25 T cells
cultured for 12 h under the specified conditions. These experiments were repeated four times. (D) iTreg production after transfection of wild-type CD4+CD25
T cells with KLF2 vector. Left panel: densitometry plot of KLF2 protein levels (relative to tubulin) 24 h after transfection with pcDNA-KLF2. (Right) Contour
plots of T cells after KLF2 transfection (24 h, vector concentrations indicated) and 48 h iTreg induction. This experiment was repeated twice. (E) Quantification
of ex vivo iTreg production using splenic CD4+CD25 T cells harvested from wild-type (gray bars) vs. SMURF1-deficient (black bars) animals. Error bars (SD) and
P values are shown. n = 2 experiments, performed in triplicate. (F) Histogram overlays displaying Smad2/3 phosphorylation after TGF- stimulation of CD4+
CD25 T cells harvested from wild-type vs. SMURF1-deficient mice. This experiment was performed once, in triplicate. (G) KLF2/tubulin immunoblot and
corresponding densitometry plot of 12-h cultured CD4+ T cells harvested from wild-type vs. SMURF1-deficient mice. This experiment was repeated twice. (H)
Contour plots of CD4+ T cells after transfection [24 h with pcDNA3 (empty), pcDNA3-KLF2 (KLF2), or pcDNA3-KLF2-dm (KLF2-dm)] and 48 h iTreg induction.
Percentages of iTregs are included. This experiment was repeated twice.
IMMUNOLOGY
CD3+ CD28
TGF
simvastatin
Flow Cytometry and Cell Sorting. Intracellular and surface staining was performed using standard procedures. For cell quantification, CaliBRITE Beads
(BD Biosciences) were added to individual tubes before acquisition. CD4+
CD25+ T cells were isolated using a Treg isolation kit (Miltenyi Biotec)
according to the manufacturers instructions.
ChIP Assays. FACS-sorted nave CD4+CD25 T cells were activated and cultured for 4 d. ChIP assays were performed using the EZ-ChIP kit (Millipore)
according to the manufacturers protocol. Immunoprecipitations were performed using anti-Klf2 antibody, anti-H4Ac or rabbit IgG control antibody.
The following primer sets were used to amplify the DNA from KLF2 pulldowns: Foxp3 Promoter F 5-CTC TGT GGT GAG GGG AAG AA-3; Foxp3Promoter R 5CGC AGA CCT CGC TCT TCT AA-3; Foxp3 CNS1 F 5- ACG TAT
CTC TCT AGT GGG TCT GGA-3; Foxp3 CNS1 R 5- TGA GGA ACA GTG CAG
GAC AG-3; Foxp3 CNS2 F 5-CTA GCC ACT TCT CGG AAC GA-3; Foxp3 CNS2 R
5-CAG CTT CCT GCA CTG TCT GTT-3; Foxp3 CNS3 F 5- GCC CAC ACC TCT TCT
TCC TT-3; Foxp3 CNS3 R 5- GGG ACC CAT AAA CCA CTT CC-3.
21. Thornton AM, et al. (2010) Expression of Helios, an Ikaros transcription factor family
member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol 184(7):34333441.
22. Gottschalk RA, Corse E, Allison JP (2012) Expression of Helios in peripherally induced
Foxp3+ regulatory T cells. J Immunol 188(3):976980.
23. Zabransky DJ, et al. (2012) Phenotypic and functional properties of Helios+ regulatory
T cells. PLoS ONE 7(3):e34547.
24. Weiss JM, et al. (2012) Neuropilin 1 is expressed on thymus-derived natural regulatory
T cells, but not mucosa-generated induced Foxp3+ T reg cells. J Exp Med 209(10):
17231742, S1.
25. Yadav M, et al. (2012) Neuropilin-1 distinguishes natural and inducible regulatory T
cells among regulatory T cell subsets in vivo. J Exp Med 209(10):17131722, S1S19.
26. Mucida D, et al. (2005) Oral tolerance in the absence of naturally occurring Tregs.
J Clin Invest 115(7):19231933.
27. Sun CM, et al. (2007) Small intestine lamina propria dendritic cells promote de novo
generation of Foxp3 T reg cells via retinoic acid. J Exp Med 204(8):17751785.
28. Josefowicz SZ, et al. (2012) Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature 482(7385):395399.
29. Zheng Y, et al. (2010) Role of conserved non-coding DNA elements in the Foxp3 gene
in regulatory T-cell fate. Nature 463(7282):808812.
30. Wang Z, et al. (2008) Combinatorial patterns of histone acetylations and methylations
in the human genome. Nat Genet 40(7):897903.
31. Sen-Banerjee S, et al. (2005) Kruppel-like factor 2 as a novel mediator of statin effects
in endothelial cells. Circulation 112(5):720726.
32. Parmar KM, et al. (2005) Statins exert endothelial atheroprotective effects via the
KLF2 transcription factor. J Biol Chem 280(29):2671426719.
33. Bu DX, et al. (2010) Statin-induced Krppel-like factor 2 expression in human and
mouse T cells reduces inflammatory and pathogenic responses. J Clin Invest 120(6):
19611970.
34. Xie P, et al. (2011) Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for
ubiquitination and degradation in human lung cancer H1299 cells. Biochem Biophys
Res Commun 407(1):254259.
35. Wang R, et al. (2013) FBW7 regulates endothelial functions by targeting KLF2 for
ubiquitination and degradation. Cell Res 23(6):803819.
36. Zhang X, Srinivasan SV, Lingrel JB (2004) WWP1-dependent ubiquitination and
degradation of the lung Krppel-like factor, KLF2. Biochem Biophys Res Commun
316(1):139148.
37. Lio CW, Hsieh CS (2011) Becoming self-aware: The thymic education of regulatory T
cells. Curr Opin Immunol 23(2):213219.
38. Xiong Y, et al. (2012) Polycomb antagonizes p300/CREB-binding protein-associated
factor to silence FOXP3 in a Kruppel-like factor-dependent manner. J Biol Chem
287(41):3437234385.
39. Khattri S, Zandman-Goddard G (2013) Statins and autoimmunity. Immunol Res
56(2-3):348357.
40. Lee JS, et al. (2006) Klf2 is an essential regulator of vascular hemodynamic forces in
vivo. Dev Cell 11(6):845857.
41. Yamashita M, et al. (2005) Ubiquitin ligase Smurf1 controls osteoblast activity and
bone homeostasis by targeting MEKK2 for degradation. Cell 121(1):101113.
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9584 | www.pnas.org/cgi/doi/10.1073/pnas.1323493111
Pabbisetty et al.
Supporting Information
Pabbisetty et al. 10.1073/pnas.1323493111
unstimulated
CD3/CD28 + TGF
Relative number of
CD4+ CD25+ FoxP3+ cells
72
Overlay
87
Helios
14
86
60
Thymus
2
13
28
Relative number of
CD4+ CD25+ FoxP3+ cells
40
Spleen
15
17
24
15.7
12.0
16.1
15.9
Neuropilin
D
30&!"
<0.0001
0.0002
25%#"
0.01
1.0
0.0002
20%!"
15$#"
10$!"
5 #"
Th
y
Sp mus
le
Ax en
M LN
s
L
Th N
ym
Sp us
le
Ax en
M LN
s
LN
0 !"
Helios-low
Thymus
<0.0001
% CD4+ CD25+ FoxP3+ T cells
Ms LN
Helios
Neuropilin
Ax LN
Spleen
Ms LN
Neuropilin-low
CD44
CD69
CD71
CD152
Fig. S1. Flow cytometric analysis of antibodies used to distinguish thymus-derived regulatory T cell (tTreg) vs. induced/peripheral Treg (i/pTreg) populations.
(A) Intracellular expression of Helios (Upper) vs. surface expression of Neuropilin (Lower) after gating on CD4+CD25+FoxP3+ splenic T cells. Representative
overlay histograms are included (ex vivo generated iTregs = open histogram, splenic Tregs = gray histogram). All cells were derived from C57BL/6 mice. n = 2
experiments. (B) Intracellular staining for Helios expression (Upper) vs. surface expression of Neuropilin (Lower) on CD4+CD25+FoxP3+ T cells under steady-state
conditions. T cells were harvested from the thymus, spleen, mesenteric lymph nodes (Ms LN), and axillary lymph nodes (Ax LN) of C57BL/6 mice. The frequency
of Tregs that expressed low levels of the respective markers is shown. n = 4 experiments. (C) Graph displaying the percentage of Tregs (CD4+CD25+FoxP3+) that
expressed low levels of Helios (black bars) vs. Neuropilin (gray bars). Data from four mice were averaged; error bars (SD) and P values comparing thymic and
secondary lymphoid organs are included. n = 4 experiments. (D) Expression of activation markers on Helioshigh Tregs isolated from various tissues under
homeostatic conditions. Select surface receptors that are typically up-regulated after T-cell activation were examined on CD4+CD25+FoxP3+Helioshigh T cells
freshly harvested from Lck-cre; Klf2fl/fl (black line) and littermate control (solid gray) mice and displayed as a histogram overlay. n = 3 mice per cohort.
1 of 4
30
P=0.07
20
P=0.08
P=0.03
P=0.005
10
0
IL-4
IL-5
IL-13
GATA3
Fig. S2. Frequency of CD4+ T cells expressing TH2-associated cytokines and GATA3 transcription factor. Lymphocytes harvested from the mesenteric lymph
nodes of wild-type (open circles) vs. Lck-cre; Klf2fl/fl (black squares) mice were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin for 6 h,
then examined for intracellular expression of outlined factors. Averaged values (dashes) and P values are displayed. n = 3 mice per cohort.
10
10
10
65
010
CD4
105
10
10
10
10
1
2
10
10
10
10
10
10
10
0
10
100
10 5
cKO
80
60
20
0
0
10 2
10 3
10 4
150
120
120
90
60
10 5
60
30
0
unstim
CD3
PMA
P=0.07
P=0.01
P=0.6
0
control
cKO
control
cKO
uninfected
LCVM d8
CFSE
wildtype
96
10
10
3
2
10
10
10
0.8
99
1
2
10
10
10
10
0
99
010
10
10
10
IFN-
0.6
P=0.01
control nTreg
cKO nTreg
25
20
20000
15
1.2
0.8
0.8
IgG1
0.6
0.4
0.4
0.2
50
40
25
0.3
0.2
6:1
12
12:1
25
25:1
50
50:1
0.4
0.3
0.4
0.2
0.2
0.1
0.1
0
1
0
0.2
0.2
0.15
0.15
14 24
1.0
IgG2a
0
3
0.6
0.5
0.4
0
1
14 24
0.1
0.1
0.05
0.05
0
1
14 24
0.45
IgG2b
0.4
0.8
IgG3
0.4
0.35
0.7
0.6
0.9
0.8
0.3
0.3
0.6
0.25
0.5
0.4
25000
1.2
0.7
0.5
Effector:Target Ratio
30000
IgM
80
3:1
30
0.9
0.8
0.6
0.3
Total Ig
0.7
0.6
75
0.9
0.8
100
Lck cKO
120
010
10 4
40
010
5
10
10
0
10 3
98
010
10
10
CD8
10
0
10 2
OD 405nm
IL-17
10
010
180
40
60
20
180
40
PM
10
0
60
103
Differentiated
CD4+ T cells
D2
10
0
C
control
80
im
100
/C
10
34
st
un
10
10
D3
10
TH17
10
TH1
0.2
0.4
0.2
0.15
0.3
0.2
0.1
0.2
14 24
0.1
0.05
0.1
0
1
14 24
0
1
14 24
15000
10
10000
5000
12
25
50
Tregs
alone
Fig. S3. Lck-cre; Klf2fl/fl mice have a functional T-cell effector compartment. (A) T cells from wild-type vs. Lck-cre; Klf2fl/fl (Lck cKO) mice were stimulated with
PMA plus ionomycin to determine whether these cells had differentiated into pathogenic TH1 (IFN-+) or TH17 (IL-17+) effector cells in vivo. Ex vivo differentiated CD4+ T cells (Upper) were included for staining purposes. n = 3 experiments. (B) Klf2-deficient T-cell proliferation as a function of carboxyfluorescein
succinimidyl ester (CFSE) dilution (Left) and thymidine incorporation (Right). Splenic T cells from Klf2fl/fl (control) and Lck-cre; Klf2fl/fl (cKO) littermates were
stimulated, and proliferation was analyzed by flow cytometry. Histogram overlay: solid gray, untreated; gray line, anti-CD3e (0.5 g/mL); black line, anti-CD3e
(5 g/mL). n = 3 experiments. Alternatively, CD4+ T cells from Klf2fl/fl (black bars) and Lck-cre; Klf2fl/fl (gray bars) littermates were stimulated as indicated, and
[3H]-thymidine incorporation was used to measure proliferation. Error bars indicate SD. P > 0.1 for all events. n = 2 experiments. (C) Quantification of splenic
CD8+ T cells in Klf2fl/fl (control) vs. Lck-cre; Klf2fl/fl (cKO) mice. T-cell numbers were averaged from cohorts of uninfected mice (black bars) or day-8 lymphocytic
choriomeningitis virus (LCMV)-infected mice (gray bars). n = 7 mice per group, error bars are SD. (D) Cytolytic efficiency of CD8+ T cells harvested from Klf2fl/fl
(solid black line) vs. Lck-cre; Klf2fl/fl (dashed gray line) littermates. CD8+ T cells from LCMV-primed mice were cocultured with RMA-S target cells that were
precoated with an LCMV agonist peptide. Target cell lysis (release of intracellular and membrane-associated dyes) was measured by flow cytometry. Percent
viable target cells = (% experimental CFSE+PHK26+ RMA-S cells/% CFSE+PHK26+ RMA-S cells cocultured with nave CD8+ T cells) 100. Error bars show SD; P
values >0.05 except as indicated. n = 7 mice per infected group. This experiment was performed once. (E) T-dependent Ig class-switching. Trinitrophenyl (TNP)specific Ig concentrations in the sera of a Klf2fl/fl mouse (black lines) vs. an Lck-cre; Klf2fl/fl littermate (gray lines) after challenge with TNP-OVA. Mice were
rechallenged with TNP-OVA at day 14. n = 2 experiments. (F) Suppression of Klf2+ T-cell proliferation using CD4+CD25+ Tregs from Klf2fl/fl (solid black line) vs.
Lck-cre; Klf2fl/fl (dashed gray line) mice. Averaged [3H]-thymidine incorporation SD is shown. No significant variation (P > 0.05) between groups was detected.
n = 2 experiments.
2 of 4
FoxP3
+
+
24
Lck-cre; Klf2fl/fl
control
CD3+CD28
TGF
inhibitor
+
+
-
+
+
+
+
rapamycin
LY
% FoxP3+CD25+ cells
50
control
40
Lck-cre; Klf2fl/fl
30
20
10
0
F
G
+T Y
+L
n
ci
F y
G am
+T rap
+
F
G
+T
ed
at
tiv
ac
CD25
Fig. S4. Ex vivo generation of iTregs using CD4+CD25 T cells harvested from KLF2-sufficient (control) or KLF2-deficient (Lck-cre; Klf2fl/fl) mice. (A) Representative contour plots of live-gated, CD4+CD25 T cells cultured under the specified conditions for 72 h. Frequency of FoxP3+CD25+ T cells is indicated. LY,
LY294002. (B) Percent iTregs generated after 72 h. culture with activating media (CD3+CD28) alone or with additional reagents. These results are averaged
from two independent experiments (n = 1 per condition); variance is indicated.
Fig. S5. Simvastatin elevates transcriptional and posttranslational levels of KLF2. (A) Relative Klf2 mRNA expression in naive CD4+CD25 T cells, CD4+CD25+
FoxP3+ Tregs, or CD4+CD25+FoxP3+ Tregs cultured with simvastatin for 24 h. Error bars (SD) and P values are shown. n = 2 experiments. (B) Immunoblot of
KLF2/tubulin and corresponding densitometry plot using total cell lysate from CD4+CD25 T cells, CD4+CD25+FoxP3+ Tregs with or without simvastatin (24 h) or
B cells from a CD19cre; Klf2fl/fl mouse (negative control for KLF2). n = 3 experiments.
3 of 4
Fig. S6. Ectopic expression of KLF2 does not impair Treg function or survival. (A) Enhanced expression of KLF2 during the inductive stage of iTreg production
does not negatively affect suppressor functions. Ex vivo suppressor assays were conducted using iTregs generated in the presence of CD3+CD28+TGF with
or without simvastatin, rapamycin, or LY294002. Each condition was done in duplicate (mean and variance are shown) using three different ratios of effector T
cells: iTregs. This experiment was performed twice. (B) Elevated KLF2 expression during iTreg production does not impact ex vivo cell survival. iTregs generated
with or without simvastatin were cultured in standard media containing IL-2, and cell survival (cell membrane-intact, 7AAD-) was assessed 72 h later by flow
cytometry. Initial input CD4+CD25 T cells were harvested from WT (left columns) or SMURF1/ (right columns) animals. This experiment was performed once
in quadruplicate. Error bars (SD) and P values (Student t test) are shown. (C) Increased KLF2 expression does not impair SMURF1-deficient Treg suppressor
activity. Ex vivo suppressor assays were conducted using splenic Tregs harvested from SMURF1+ vs. SMURF1/ animals. Error bars (SD) and P values (Student t
test) are shown. n = 2 experiments performed in triplicate.
4 of 4