tmpA8D TMP

KLF2 is a rate-limiting transcription factor that can be
targeted to enhance regulatory T-cell production

Sudheer K. Pabbisettya, Whitney Rabacala, Damian Masedaa, Delphine Cendrona, Patrick L. Collinsb, Kristen L. Hoeka,
Vrajesh V. Parekha, Thomas M. Aunea,c, and Eric Sebzdaa,1
Departments of aPathology, Microbiology, and Immunology, and cMedicine, Vanderbilt University School of Medicine, Nashville, TN 37232; and bDepartment
of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, Ontario Cancer Institute, University
Health Network, Toronto, Canada, and approved May 22, 2014 (received for review December 17, 2013)
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
To whom correspondence should be addressed. E-mail: eric.sebzda@vanderbilt.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1323493111/-/DCSupplemental.
PNAS | July 1, 2014 | vol. 111 | no. 26 | 95799584
IMMUNOLOGY
Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells

that maintain self-tolerance by functionally suppressing autoreactive
lymphocytes. The Treg compartment is composed of thymus-derived
Tregs (tTregs) and peripheral Tregs (pTregs) that are generated in
secondary lymphoid organs after exposure to antigen and specific
cytokines, such as TGF-. With regard to this latter lineage, pTregs
[and their ex vivo generated counterparts, induced Tregs (iTregs)]
offer particular therapeutic potential because these cells can be
raised against specific antigens to limit autoimmunity. We now
report that transcription factor Krppel-like factor 2 (KLF2) is
necessary for the generation of iTregs but not tTregs. Moreover,
drugs that limit KLF2 proteolysis during T-cell activation enhance iTreg development. To the authors knowledge, this study
identifies the first transcription factor to distinguish between
i/pTreg and tTreg ontogeny and demonstrates that KLF2 is a
therapeutic target for the production of regulatory T cells.
Spleen
Ms LN
10
10
10
10
10
10
10
0
2
10
10
10
0 10
10
11
14
0
0 10
10
12
10
10
10
0 10
10
10
10
10
10
10
10
10
10
10
10
103
10
10
10
10
10
10
0
2
10
10
10
0 10
10
10
10
10
10
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
In Vivo Development of iTregs Is KLF2-Dependent. We next addressed

the physiological relevance of KLF2 by examining peripheral Treg
(pTreg) populations under homeostatic conditions. Reports indicate that Helios (an Ikaros transcription factor family member)
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
Relative cell number
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
KLF2 Is Required for the Ex Vivo Generation of iTregs. We have

previously demonstrated that KLF2 differentially impacts lymphocyte homeostasis in a lineage-specific manner (17). Therefore, we decided to examine iTreg and tTreg development
independent of one another. Strikingly, we found that KLF2 was
necessary for the ex vivo generation of iTregs. Antigen stimulation in the presence of TGF- induced regulatory T-cell production; however, CD4+ T cells from Lck-cre; Klf2fl/fl mice did
not differentiate into FoxP3-expressing iTregs under these conditions (Fig. 2A). Similarly, KLF2-deficient CD4+CD25 (singlepositive) thymocytes were unable to differentiate into FoxP3+
CD25+ cells, demonstrating that defective iTreg development
was not secondary to aberrant T-cell migration (Fig. 2B). A published report indicated that KLF2-deficient CD4+ T cells secrete
elevated levels of IL-4 after T-cell receptor (TCR) stimulation
200
10 3
Fig. 1. Treg frequencies in Klf2 gene-targeted mice. (A) Percent FoxP3+CD25+

Tregs relative to CD4+ T cells in tissues harvested from Klf2fl/fl (control) vs. Lckcre; Klf2fl/fl (cKO) littermates (6 wk of age). This experiment was repeated four
times. Ms LN, mesenteric lymph nodes; Ax LN, axillary lymph nodes. (B) Percent
Tregs relative to CD4+ thymocytes in 3-wk-old Klf2fl/fl (control) or Lck-cre; Klf2fl/fl
(cKO) littermates. This experiment was performed three times. (C) Proportion of
Tregs present in Klf2fl/fl (control), Vav-cre; S1P1fl/fl (S1P1 cKO), Vav-cre; Klf2fl/fl
(Klf2 cKO), and Vav-cre; S1P1fl/fl; Klf2fl/fl (dKO) littermates (n = 2 mice per group).
FACS plots: flow cytometric analysis of CD4+ T cells in the spleen. % FoxP3+
CD25+ Tregs are indicated. Bar graph: average frequency of Tregs relative to
CD4+ T cells in lymphoid organs. Mice were 7 wk old. This experiment was repeated twice (excluding S1P1 cKO littermates, which were analyzed once).
10
400
10 5
dKO
400
Klf2 cKO
10
10
600
600
control
S1P1 cKO
800
800
200
30
0
1000
control
200
200
200
200
0.4
10 5
10
ro
10 4
10
nt
10 3
10
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
% FoxP3+ CD25+ cells

(relative to CD4+ T cells)
600
0.6
400
100
0
2
10
10
10
600
10
10
10
10
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
(18), raising the possibility that IL-4 was suppressing TGF-

mediated iTreg generation (19). To address this issue, KLF2sufficient and KLF2-deficient CD4+ T cells were cocultured
under iTreg differentiating conditions. As shown in Fig. 2C, only
KLF2-sufficient T cells were capable of producing iTregs, demonstrating the cell-intrinsic nature of this defect. Moreover,
we did not detect increased expression of the TH2-associated
transcription factor, GATA3, or elevated production of IL-4
by KLF2-deficient splenic CD4+ T cells after antigen stimulation (Fig. 2D), indicating that this cytokine was not responsible
for the iTreg defect in the present study. To determine when
KLF2 was necessary during iTreg generation, we used a tamoxifen-inducible cre system (T2-cre; Klf2fl/fl) to excise Klf2 after
TGF-induced FoxP3 expression. As shown in Fig. 2E, Klf2
excision did not affect the stability of FoxP3 expression, indicating that KLF2 was required solely at the induction stage of
iTreg generation.
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.
KLF2 Directly Regulates FoxP3 Expression Within the iTreg Lineage.
Foxp3 expression is orchestrated by transcription factors that

selectively bind to key genetic elements, including the promoter
region and three conserved noncoding sequences (CNS13)
downstream of the transcriptional start site (29). These enhancer
elements are differentially used by iTregs and tTregs; for instance, CNS1 is necessary for the generation of iTregs, whereas it
is dispensable for tTreg production. To determine whether KLF2
bound to these regulatory regions, ChIP assays were performed
using DNA from CD4+ T cells that were TCR-stimulated in the
presence (iTregs) or absence (mock control) of TGF-. As shown
in Fig. 4A, KLF2 bound to the promoter and all three enhancer
A
80
60
60
40
40
0
0
80
10
10
10
10
100
Ms LN
10
10
30
30
20
20
10
10
'()*+,"
-./001"
2,"34"
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ym
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0
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Ax
20
40
20
10
0.06 <0.0001 <0.0001 <0.0001
40
Sp
40
40
Th
60
10
Ax LN
80
60
Helios
OT2- CD4+ FoxP3+ cells (x104)
30
P=0.003
OVA-induced Tregs
20
P=0.01
10
P=0.3
OT2; Lckcre; Klf2fl/fl

P=0.07
30
P=0.5
0
Ax
Sp
LN
LN
en
le
Ax
s
M
OT2; Klf2fl/fl
60
LN
LN
en
le
NT control
Non-induced Tregs
Sp
P=0.3
90
B
OT2+ CD4+ FoxP3+ cells (x104)
0.008 0.002 0.0001
0.3
20
20
100
Spleen
Helios-low Treg
numbers (x104)
100
Thymus
80
% Helios-low
Regulatory T cells

(CD4+ FoxP3+)
100
C
100
Thymus
80
60
60
40
40
20
0
0
80
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100
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10
10
10
10
0
100
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Pabbisetty et al.
67"45"
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le
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0.4
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10 2
MS
Ms LN
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spleen
LN
10 5
thymus
thymus
en
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00
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10
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10 2
10
0
0
0
20
20
ym
30
30
20
0.02
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20
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30
0.2
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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)

(CD4+ FoxP3+)
80
% Helios-low
Regulatory T cells
100
Fig. 3. KLF2 is required to establish a pTreg compartment

in vivo. (A) Expression of Helios within the Treg compartment (CD4+FoxP3+CD25+) of Klf2fl/fl (gray histograms) vs.
Lck-cre; Klf2fl/fl (open histograms) mice (n = 4 mice per
group). Representative histogram overlays are displayed.
Percentage and absolute number of Helioslow regulatory T
cells from Klf2fl/fl (gray bars) vs. Lck-cre; Klf2fl/fl (black bars)
mice. Error bars (SD) and P values are displayed. This experiment was repeated four times. (B) De novo generation
of pTregs in non-TCR transgenic control (white bars), OT2;
Klf2fl/fl (black bars), and OT2; Lck-cre; Klf2fl/fl (gray) mice
placed on ovalbumin-infused drinking water. (Left) Absolute
number of OT2+ CD4+FoxP3+ Tregs isolated from tissues
associated with pTreg generation [spleen, mesenteric lymph
nodes (Ms LN)] as well as control tissue [axillary lymph nodes
(Ax LN)]. (Right) Absolute number of non-TCR transgenic
OT2 CD4+FoxP3+ Tregs isolated from same animals. This
experiment was conducted twice, n = 3 mice per cohort.
Error bars (SD) and P values (Student t test) are indicated. (C)
Comparison of Helios expression within the Treg compartment of Klf2fl/fl (gray histograms, gray bars) vs. FoxP3-cre;
Klf2fl/fl (open black histograms, black bars) mice (n = 3 animals per group). Bar graphs display percentage and absolute
number of Helioslow Tregs, including error bars (SD) and
P values. This experiment was repeated four times.
PNAS | July 1, 2014 | vol. 111 | no. 26 | 9581
IMMUNOLOGY
not significantly impact nontransgenic Treg numbers. Moreover,

low frequencies of CD4+ T cells expressing a TH2-like profile
were identified in the GALT of Lck-cre; Klf2fl/fl mice (Fig. S2),
a phenotype that has previously been associated with a lack of
pTregs in vivo (28). To confirm that KLF2 was necessary for induction but not maintenance of FoxP3 expression within the pTreg
compartment, we next examined Helios expression in Klf2 fl/fl
mice bred onto a FoxP3-cre transgenic background (FoxP3-cre;
Klf2fl/fl). Consistent with our ex vivo results, we found similar frequencies and numbers of Helioslow cells in SLO harvested from
FoxP3-cre; Klf2fl/fl mice and littermate controls (Fig. 3C). These
data indicate that under normal physiological conditions, KLF2 is
necessary during the inductive stage of pTreg development. Despite the lack of pTregs, Lck-cre; Klf2fl/fl mice were healthy and did
not exhibit signs of autoimmunity, including spontaneous T-cell
activation or pathogenic TH1/TH17 differentiation (Fig. S3A).
Moreover, KLF2-deficient T cells maintained their effector functions (Fig. S3 BE), suggesting that autoimmunity was actively repressed by a functional Treg compartment. Direct analysis of Tregs
from Lck-cre; Klf2fl/fl mice confirmed that KLF2-deficient Tregs
retained their suppressive properties (Fig. S3F). Therefore, we
conclude that tTregs are sufficient to prevent autoimmunity under
homeostatic conditions and posit that pTregs are necessary to limit
tissue damage associated with an inflammatory environment.
and Neuropilin 1 (Nrp1, a membrane-bound surface receptor) are

preferentially but not exclusively expressed by tTregs relative to
pTregs (2025). To determine whether either of these molecules
were appropriate surrogates for pTreg identification, we initially
characterized expression patterns within the entire regulatory Tcell compartment. Most FoxP3+CD4+ thymocytes belong to the
tTreg lineage, whereas peripheral FoxP3+CD4+ T cells are composed of both tTregs and pTregs, with a partial preference for
pTregs in mucosal-associated lymph nodes. Consistent with this
phenotype, few HelioslowFoxP3+CD25+ T cells were detected in
the thymus, whereas increased frequencies were found in secondary lymphoid organs (SLO) and after ex vivo Treg induction
(Fig. S1). Although Nrp1low stains also identified iTregs in culture,
expression did not strictly correlate with known pTreg tissue distribution patterns found under steady-state conditions. For this
reason we focused on Helios expression as a proxy to distinguish
between tTreg (Helioshigh) and pTreg (Helioslow) populations. As
shown in Fig. 3A, most FoxP3+CD4+ thymocytes expressed high
levels of Helios, whereas FoxP3+CD4+ T cells in SLO of wild-type
animals contained a spectrum of Helios expression. In contrast,
the majority of FoxP3+CD4+ T cells in Lck-cre; Klf2fl/fl mice were
Helioshigh, both in primary and secondary lymphoid tissues. These
Helioshigh FoxP3+CD25+ T cells were phenotypically quiescent
(Fig. S1D), thus excluding the possibility that KLF2-deficient
Tregs were transiently expressing Helios owing to aberrant
activation (22). Significantly fewer Helioslow Tregs were
present in the SLO of Lck-cre; Klf2fl/fl mice relative to littermate controls, consistent with an in vivo defect in pTreg
generation. To directly assess de novo generation of pTregs,
KLF2 gene-targeted mice were crossed onto a TCR transgenic background (OT2) that recognizes ovalbumin peptide (p323339, OVA) in the context of H2-IAb. OVA-specific (V2+/V5+)
CD4+FoxP3+ Tregs are absent in OT2 animals; however, OT2
mice placed on OVA-infused drinking water develop transgenic
pTregs in gut-associated lymphoid tissues (GALT; e.g., mesenteric
lymph nodes, spleen) (26, 27). In agreement with surrogate Helios
staining results, OT2; Lck-cre; Klf2fl/fl mice were unable to generate
OVA-specific FoxP3+ Tregs (Fig. 3B), although this defect did
elements of TGF-stimulated T cells. Acetylated histone H4

(H4Ac) epigenetically marks transcriptionally permissive areas of
chromatin (30), as demonstrated by H4Ac association with FoxP3
regulatory sites after iTreg induction of wild-type CD4+ T cells
(Fig. 4B). In contrast, minimal H4Ac was recovered from ChIP
assays using KLF2-deficient nuclear extracts. From these assays
we contend that KLF2 is necessary to open compacted chromatin
associated with the FoxP3 locus, thus permitting iTreg development under conditions that favor KLF2 expression.
Drugs That Stabilize KLF2 Expression Enhance iTreg Production.
KLF2 is necessary for the generation of iTregs, raising the

possibility that this transcription factor can be manipulated to
enhance i/pTreg development for clinical purposes. The 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin has
previously been shown to enhance Klf2 expression in endothelium (31, 32) and activated T cells (33), which led us to test
this US Food and Drug Administration-approved drug on T
cells undergoing iTreg induction. Consistent with our previous
experiments, simvastatin increased production of iTregs using
cultured CD4+CD25 T cells or thymocytes (Fig. 5A) in
a manner that paralleled KLF2 expression (Fig. 5B). Furthermore, this direct correlation extended to other inhibitory
compounds that suppressed TCR-mediated down-regulation of
KLF2 (Fig. 5C and Fig. S4). Analysis of simvastatin-treated
Tregs indicated that this drug enhanced/stabilized KLF2 at the
mRNA and protein level (Fig. S5), raising the possibility that
one of these factors was responsible for augmented iTreg
development. Overexpression of KLF2 using an established
mammalian vector did not amplify FoxP3+ T-cell frequencies
(Fig. 5D), which suggests that KLF2 stability rather than transcription was the primary means of regulating KLF2-mediated
events after T-cell activation. To investigate the potential relationship between KLF2 stability and regulatory T-cell development, iTreg production was analyzed using CD4+CD25
T cells from Sma and Mad homologue (SMAD) specific E3
ubiquitin protein ligase 1 (SMURF1) gene-targeted animals.
In keeping with a previous report that demonstrated that
SMURF1 targets KLF2 for ubiquitin-mediated degradation
(34), we found that ex vivo iTreg production was enhanced in
the absence of SMURF1 (Fig. 5E). Importantly, SMURF1
deficiency did not uncouple iTregs requirement for TGF-
(Fig. 5E) or affect the activation status of SMAD molecules
that act directly downstream of TGF-R1 (Fig. 5F). Instead,
SMURF1-deficient cells exhibited a defect in KLF2 stability
B
0.04
0.6
0.06
Fraction of Input (%)
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
0.0005
Control IP: CD3 + TGF
S3
N
C
S1
N
C
Pr
o
S3
S2
Klf2 IP: CD3 + TGF
N
C
S1
N
.
C
om
Pr
Klf2 IP: CD3
S2
Fraction of Input (%)
0.004
0.0035
H4Ac immunoprecipitation
WT CD3
cKO CD3
WT CD3 + TGF
cKO 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.
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
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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.
a bottleneck in iTreg production that can be amplified using

a diverse array of chemical inhibitors. Future work will determine whether drugs that stabilize KLF2 protein levels expand
pTreg populations in vivo, especially during inflammatory outbreaks encountered in autoimmune diseases. In this regard, it is
worth noting that statins, which are currently prescribed to approximately one-quarter of all Americans over the age of 45 y
(www.cdc.gov/nchs/hus.htm), have a documented role in alleviating autoimmune symptoms that are independent of their lipidreducing properties (39). At the same time, there are numerous
situations in which Tregs hinder desired immune responses, such
as those directed against tumors; under these circumstances
pharmaceuticals that stabilize KLF2 protein levels would be
predicted to harm patient outcome. Instead, it may prove advantageous to treat the T-cell compartment with drugs that
promote KLF2 degradation via E3 ubiquitin ligases such as
WWP1 (36), SMURF1 (34), or FBW7 (35).
Pabbisetty et al.
Materials and Methods

Mice. Klf2fl/fl mice were generated as previously described (40). SMURF1
gene-targeted mice (41) were obtained from the laboratory of Ying E.
Zhang (Center for Cancer Research, National Cancer Institute, Bethesda,
MD). Lck-cre mice were purchased from Taconic, and all other mice
were purchased from Jackson Laboratories. All KLF2 gene-targeted
animals were back-crossed a minimum of 10 generations onto a C57BL/6
background. Mice were housed in pathogen-free conditions in accordance with the Institutional Animal Care and Use Committee at Vanderbilt University.
Antibodies. Anti-Thy1.2 (53-2.1), -CD3e (145-2C11), -CD4 (GK1.5), -CD8 (536.7), -CD25 (PC61.5), -CD28 (37.51), -CD44 (IM7), -CD69 (H1.2F3), -CD71
(C2), -IFN- (XMG1.2), -IL4 (11b11), -IL17 (TC11-18H10), -GATA3 (L50-823),
and phospho Smad2/3 (072-670) were purchased from BD Biosciences.
Anti-FoxP3 (FJK-16s) and -CD152 (UC10) were obtained from eBioscience
USA. Anti-Helios (22F6) was purchased from Biolegend. Anti-Neuropilin
was purchased from R&D Systems. Anti-KLF2 and -AcetylHistone H4
PNAS | July 1, 2014 | vol. 111 | no. 26 | 9583
IMMUNOLOGY
CD3+ CD28
TGF
simvastatin
KLF2 protein levels

(H4Ac) were purchased from Millipore. Anti--tubulin was obtained from

Cell Signaling Technologies.
an additional 72 h. Klf2 excision was assessed by RT-PCR, and cells were

analyzed by flow cytometry.
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.
Ex Vivo Induction of Tregs. FACS- or MACS-sorted nave CD4+CD25 cells (2

106 cell/mL) were preincubated with or without LY294002 (10 M), PD98059
(10 M), simvastatin (2 M), or rapamycin (100 nM), activated with platebound anti-CD3e (2 g/mL) and soluble -CD28 (2 g/mL) antibody, then cultured with TGF- (110 ng/mL) in complete medium containing IL-2 (5 ng/mL).
After 24 d of culture cells were collected and stained for FoxP3.
In Vivo Induction of Tregs. Klf2fl/fl, OT2; Klf2fl/fl, and OT2; Lck-cre; Klf2fl/fl mice
were placed on chicken ovalbumin (p323-339)-infused drinking water
(20 mg/mL) for 7 d, after which animals were killed and analyzed for pTreg
development. Antibodies directed against V2 and V5 (BD Biosciences)
were used to identify OVA-specific T cells.
Nucleofection of Quiescent CD4+CD25 T Cells. Transient nucleofection was

performed using the Lonza/Amaxa Mouse T-cell Nucleofector Kit (VPA-1006)
according to the manufacturers instructions. Nucleofection of purified
splenic CD4+CD25 T cells (1 106) from C57BL/6J mice was performed with
the indicated amounts of pcDNA3 (empty vector), pcDNA3-KLF2 (40), or
pcDNA3-dmKLF2 plasmid (35). Cells were subsequently cultured under iTreginducing conditions before FACS analysis.
Ex Vivo Excision of KLF2. A tamoxifen-inducible cre transgenic mouse model

(T2-cre) (42) was used to excise KLF2 after ex vivo generation of Tregs. After
4 d of iTreg culture, cells were treated with 100 nM 4-hydroxy-tamoxifen for
Statistical Analysis. Data were analyzed using a two-tailed Student t test.

P values 0.05 were considered statistically significant.
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41. Yamashita M, et al. (2005) Ubiquitin ligase Smurf1 controls osteoblast activity and
bone homeostasis by targeting MEKK2 for degradation. Cell 121(1):101113.
42. Ruzankina Y, et al. (2007) Deletion of the developmentally essential gene ATR in
adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1(1):
113126.
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
Relative cell number of

CD4+CD25+FoxP3+Helios+ cells
<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.
Pabbisetty et al. www.pnas.org/cgi/content/short/1323493111
1 of 4
% positive CD4+ T cells
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
[3H] Thymidine incorporation

(cpm) x 103
10 4
40
010
5
10
10
0
10 3
98
010
10
10
CD8
10
0
10 2
OD 405nm
IL-17
10
CD8+ T cell number (x106)
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
% viable target cells
10
[3H] Thymidine incorporation

(x103 cpm)
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
Number of Tregs (x103)
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.
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KLF2 is a rate-limiting transcription factor that can be

targeted to enhance regulatory T-cell production

To whom correspondence should be addressed. E-mail: eric.sebzda@vanderbilt.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

PNAS | July 1, 2014 | vol. 111 | no. 26 | 95799584

Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells

In Vivo Development of iTregs Is KLF2-Dependent. We next addressed

Relative cell number

KLF2 Is Required for the Ex Vivo Generation of iTregs. We have

Fig. 1. Treg frequencies in Klf2 gene-targeted mice. (A) Percent FoxP3+CD25+

Relative cell number

% FoxP3+ CD25+ cells

Relative cell number

(18), raising the possibility that IL-4 was suppressing TGF-

KLF2 Directly Regulates FoxP3 Expression Within the iTreg Lineage.

Foxp3 expression is orchestrated by transcription factors that

0.06 <0.0001 <0.0001 <0.0001

OT2- CD4+ FoxP3+ cells (x104)

OT2; Lckcre; Klf2fl/fl

0.008 0.002 0.0001

Relative cell number

Relative cell number

Fig. 3. KLF2 is required to establish a pTreg compartment

PNAS | July 1, 2014 | vol. 111 | no. 26 | 9581

not significantly impact nontransgenic Treg numbers. Moreover,

and Neuropilin 1 (Nrp1, a membrane-bound surface receptor) are

elements of TGF-stimulated T cells. Acetylated histone H4

KLF2 is necessary for the generation of iTregs, raising the

Fraction of Input (%)

Control IP: CD3 + TGF

Klf2 IP: CD3 + TGF

Klf2 IP: CD3

Fraction of Input (%)

cKO CD3 + TGF

TGF -induced TGF -induced TGF -induced TGF -induced

Activated TGF -induced

CD3+ CD28 TGF

KLF2 protein levels

KLF2 protein levels

a bottleneck in iTreg production that can be amplified using

Materials and Methods

PNAS | July 1, 2014 | vol. 111 | no. 26 | 9583

KLF2 protein levels

(H4Ac) were purchased from Millipore. Anti--tubulin was obtained from

an additional 72 h. Klf2 excision was assessed by RT-PCR, and cells were

Ex Vivo Induction of Tregs. FACS- or MACS-sorted nave CD4+CD25 cells (2

Nucleofection of Quiescent CD4+CD25 T Cells. Transient nucleofection was

Ex Vivo Excision of KLF2. A tamoxifen-inducible cre transgenic mouse model

Statistical Analysis. Data were analyzed using a two-tailed Student t test.

1. Sakaguchi S, Miyara M, Costantino CM, Hafler DA (2010) FOXP3+ regulatory T cells in

Relative cell number of

Pabbisetty et al. www.pnas.org/cgi/content/short/1323493111

% positive CD4+ T cells

[3H] Thymidine incorporation

CD8+ T cell number (x106)

Relative cell number

% viable target cells

[3H] Thymidine incorporation

Number of Tregs (x103)

Pabbisetty et al. www.pnas.org/cgi/content/short/1323493111

Pabbisetty et al. www.pnas.org/cgi/content/short/1323493111

Pabbisetty et al. www.pnas.org/cgi/content/short/1323493111