TGF-? regulates in vivo expansion of
Foxp3-expressing CD4?CD25?regulatory T cells
responsible for protection against diabetes
Yufeng Peng*†, Yasmina Laouar*†, Ming O. Li*, E. Allison Green‡, and Richard A. Flavell*§¶
*Section of Immunobiology and§Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520; and‡Juvenile Diabetes
Research Foundation?Wellcome Trust Diabetes and Inflammatory Laboratory, Cambridge Institute for Medical Research, Cambridge University,
Cambridge CB2 2XY, United Kingdom
Contributed by Richard A. Flavell, February 4, 2004
CD4?CD25?regulatory T cells are essential in the protection from
organ-specific autoimmune diseases. In the pancreas, they inhibit
actions of autoreactive T cells and thereby prevent diabetes progres-
sion. The signals that control the generation, the maintenance, or the
Here we show that a transient pulse of transforming growth factor
? (TGF-?) in the islets during the priming phase of diabetes is
sufficient to inhibit disease onset by promoting the expansion of
intraislet CD4?CD25?T cell pool. Approximately 40–50% of intraislet
CD4?T cells expressed the CD25 marker and exhibited characteristics
of regulatory T cells including small size, high level of intracellular
CTLA-4, expression of Foxp3, and transfer of protection against
diabetes. Results from in vivo incorporation of BrdUrd revealed that
the generation of a high frequency of regulatory T cells in the islets
is due to in situ expansion upon TGF-? expression. Thus, these
findings demonstrate a previously uncharacterized mechanism by
which TGF-? inhibits autoimmune diseases via regulation of the size
of the CD4?CD25?regulatory T cell pool in vivo.
are highly complex and not foolproof. Models of passive tolerance,
such as thymic deletion of autoreactive T cells or nonreponsiveness
the presence of autoreactive T cells in healthy individuals despite
the absence of the development of organ-specific autoimmune
diseases. T cells endowed with suppressive function to control
thought originally to be a specialized T cell population the effect of
which would be mediated by secreted antigen-specific factors (2).
are delineated into two cell subsets of natural regulatory
(CD4?CD25?) cells that emerge from the thymus (3, 4) and
adaptive regulatory (CD4?CD25?) cells induced in the periphery
to develop suppressive activity (5–7). However, this concept of
dichotomous thymic CD25?versus adaptive CD25?regulatory T
the peripheral generation of CD25?regulatory T cells in vivo and
in vitro (8–12). The finding that suppressive functions are instruc-
tively programmed by the expression of Foxp3 finally provided the
basis for integrating a unified model of regulatory T cell diversity
(13–15). Forced expression of Foxp3 in CD4?CD25?nonregula-
tory T cells, either by retroviral expression or in transgenic mice,
showed acquisition of suppressive activity in vitro and inhibition of
disease in vivo, inducing in a substantial proportion of Foxp3-
bearing cells the expression of CD25 and GITR markers indicating
that expression of Foxp3 within peripheral T cells may convert
nonregulatory CD25?cells into CD25?regulatory cells. The fact
that the expression of the Foxp3 gene during thymic development
alone was not sufficient to protect otherwise Foxp3-null animal
from disease (16) works in favor of this hypothesis. The point of
interest here is that continuous Foxp3 expression in peripheral
ype I diabetes is an autoimmune disease that results from the
failure of tolerance to beta-cell antigens (1). The mechanisms
tissues seems to be necessary either for the maintenance or the
function of regulatory T cells extrathymically. The signals that
control regulatory T cell pool in the periphery, however, remain to
We believe that potential candidates lie within the inflamma-
tory milieu of autoimmune diseases. Several studies have dem-
onstrated that CD4?CD25?regulatory T cells produce elevated
levels of transforming growth factor ? (TGF-?) in both mice and
humans. A key to understanding the cell contact-dependent
immunosuppression by these cells was the recognition that
CD4?CD25?T cells express surface membrane-bound TGF-?
(17). Not only do CD4?CD25?regulatory T cells express latent
TGF-? (18), but they also bear TGF-? in its active configuration
on the cell surface (19). The fact that enhanced TGF-? signaling
receptors reside on the membrane of CD4?CD25?regulatory T
cells underscores the potential for autocrine and?or paracrine
receptor-ligand interactions in these cells. In this study, we
provide direct evidence that TGF-? is a positive regulator of
CD4?CD25?regulatory T cell expansion in vivo. Expression of
TGF-? in the pancreatic cells by using a tetracycline on?off
system shows that a short pulse of TGF-? is sufficient to inhibit
the development of type I diabetes by promoting the expansion
of CD4?CD25?T cells. Detailed analysis with an adoptive
transfer system identified the profile of regulatory T cells,
including their small size, high level of intracellular CTLA-4,
expression of Foxp3, and transfer of protection against diabetes.
This finding provides a significant step forward in our under-
maintained in peripheral tissues.
Materials and Methods
Mice. Plasmid pTet-TTA that encodes the tetracycline-responsive
repressor was described previously (20). pTet-TTA was cleaved at
the SacI–HindIII sites and hCMV promoter was replaced with the
rat insulin promoter type II, creating pRIP-TTA (RIP, rat insulin
promoter), which carries the RIP-TTA element (Fig. 1A). Plasmid
p-TRE-TGF-? (TRE, tetracycline-responsive element) that en-
codes an active form of TGF-?1 under the control of the TRE was
generated by substituting the RIP component of a RIP-TGF-?
plasmid (21) with the TRE element described in detail previously
were purified by using Elutip purification columns (Schleicher &
Schuell) and coinjected to make double transgenic mice in the
nonobese diabetic (NOD) background. Transgenic founders were
screened by using PCR and further selected based on TGF-?
Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; NOD, nonobese dia-
activated cell sorter.
†Y.P. and Y.L. contributed equally to this work.
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2004 by The National Academy of Sciences of the USA
March 30, 2004 ?
vol. 101 ?
expression in pancreas. The primers used for screening transgenic
mice and RT-PCR were as follows: TGF-?, 5?-GACCTCCATA-
GAAGACACC-3? and 5?-AACCCGTTGATGTCCACTTGC-3?;
supplemented food (20 mg?kg) was purchased from Bioserv
(Frenchtown, NJ), and TGF-? expression was repressed by replac-
ing normal chow with doxycycline-supplemented chow and initi-
repressed from birth, the breeding mothers were fed with supple-
mented food since pregnancy. Urine Glucose level was monitored
by using Diastyx sticks (Bayer) on weekly basis. Animals that had
values of ?250 mg?dl on two consecutive occasions were counted
as diabetic and confirmed by One-Touch strips (Lifescan, Milpitas,
CA). All mice were maintained under specific pathogen-free
RT-PCR and Real-Time PCR. Total pancreatic RNA was prepared by
guanidine thiocyanate extraction (23); for all other tissues, RNA
was prepared by using TRIzol reagent (GIBCO?BRL). cDNA was
synthesized with random primers (GIBCO?BRL). TGF-? and
TTA transcripts were amplified with the primers described above.
The expression of Foxp3 was measured by the real-time RT-PCR
with the primers 5?-GGCCCTTCTCCAGGACAGA-3? and 5?-
GCTGATCATGGCTGGGTTGT-3? at a final concentration of
500 nM, and the internal TaqMan probe 5?-FAM-ACTTCATG-
CATCAGCTCTCCACTGTGGAT-BHQ-1–3? at a final concen-
tration of 200 nM. Hypoxanthine phosphoribosyltransferase
(HPRT) was used as an internal reference and measured by using
the primers 5?-CTGGTGAAAAGGACCTCTCG-3? and 5?-
TGAAGTACTCATTATAGTCAAGGGCA-3? at a final concen-
tration of 200 nM, and the TaqMan probe 5?-FAM-TGTTG-
GATACAGGCCAGACTTTGTTGGAT-BHQ-1–3? at a final
concentration of 200 nM. Relative expression of Foxp3 normalized
to HPRT levels was presented.
Cell and Tissue Preparation. Pancreatic tissues were collected and
digested with Liberase RI (Roche Applied Science) at 37°C for
15–20 min. Dissociated islets were hand-picked under dissecting
microscope. To release intraislet lymphocytes, islets were digested
with Trypsin-EDTA for 10 min, followed by treatment with cell
dissociation buffer (GIBCO?BRL) for 15 min at 37°C. Digested
islets were incubated at 37°C for 6–7 h to recover the expression of
cell-surface receptors in RPMI medium 1640 with 10% FCS.
Subsequently, lymphocytes were purified with lympholyte (Ceder-
Fluorescence-Activated Cell Sorting (FACS) and Histology. Expression
of surface markers were analyzed by using anti-CD4 (H129.19),
anti-CD8 (53.67), anti-CD25 (PC61), and V?4 (KT4) (BD Pharm-
ingen). Intracellular staining of CTLA-4 was performed by using
intraislet lymphocytes were first stimulated with phorbol 12-
myristate 13-acetate (50 ng?ml) and ionomycin (1 ?M) for 6 h in
the presence of Golgi Stop (BD Pharmingen). Subsequently, cells
were surface-labeled, fixed, and permeabilized, then stained with
anti-IFN-? (XMG1.2) and anti-IL-4 (11B11) antibodies (BD
For tissue expression of TGF-?, pancreas were fixed in 1%
paraformaldehyde and passed through sucrose gradients before
freezing in Tissue-Tek OCT (Bayer). Five- to 7-?m frozen sections
was detected by using biotinylated anti-TGF-? (A75-7.1, BD
Pharmingen), followed by phosphatase streptavidin and 5-bromo-
4-chloro-3-indolyl phosphate (BCIP)?nitroblue tetrazolium (NBT)
substrate (Zymed). For histology, tissues were prepared by fixation
in 15% formaldehyde, and 5- to 7-?m sections of paraffin-
embedded tissue were stained with hematoxylin and eosin.
Adoptive Transfers. Spleen cells from 6- to 7-month-old NOD
transgenic mice were pooled from each group, and 10 ? 106cells
were transferred to NOD–severe combined immunodeficient
(SCID) recipient mice. Development of diabetes was monitored
every 3 days after transfer for 12 weeks.
When indicated, islets were isolated from transgenic mice and
lymphocytes were prepared as previously described. Transgenic
intraislet lymphocytes (105) were cotransferred with 20 ? 106
diabetogenic splenocytes isolated from NOD mice into NOD-
SCID mice together. Development of diabetes was monitored for
Splenic naive BDC2.5 T cells (CD62LhighCD25low) were selected
with carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells
(2 ? 106) were transferred into transgenic or control mice. Pan-
system. Under the control of a rat insulin promoter (RIP), tetracycline-controlled
transactivator (TTA) is expressed specifically in insulin-producing cells. In the
presence of tetracycline or its analogue doxycycline (Dox), TTA (?Dox) is unable
to bind the TRE and TGF-? transcription is inactive, whereas in the absence of
doxycycline (?Dox) TTA binds TRE and induces TGF-? expression via the cyto-
megalovirus minimal promoter (P min CMV). DNA fragments RIP-TTA and TRE-
TGF-? were coinjected to generate double transgenic mice in NOD background.
doxycycline-supplemented food. Total RNA was isolated from pancreatic tissue.
Lanes 1 and 3, constitutively on for 5 weeks since birth; lanes 2 and 4, constitu-
tively on for 5 weeks followed by 1-week turn-off; lane 5, transgene negative
control; lane 6, constitutively off for 2 months since birth; lanes 7, 8, and 10,
constitutively off for 2 month since birth followed by a turn-on for 4 days (lanes
7 and 10) or 1 week (lane 8); lane 9, spleen RNA controls. Each lane represents a
(red, counterstained by hematoxylin?eosin). (a) Constitutively off for 8 weeks
since birth. (b) Constitutively on for 5 weeks since birth. (c) Constitutively on for
Peng et al.
March 30, 2004 ?
vol. 101 ?
no. 13 ?
by FACS to monitor the intensity of CFSE.
BrdUrd Detection. Eight week-old control and transgenic mice were
treated (i.p.) with 1 mg of BrdUrd (Sigma) for 4 consecutive days.
Subsequently, mice were killed and islets and several lymphoid
organs were removed. Cell suspensions were surface stained, fixed,
and permeabilized in PBS containing 1% paraformaldehyde plus
0.01% Tween 20 for 48 h at 4°C, then treated with 250 units?ml
DNase I (Sigma) for 30 min at 37°C. BrdUrd incorporation was
revealed with anti-BrdUrd mAb (Becton Dickinson).
Transient Expression of TGF-? in the Islets Is Sufficient to Suppress
Diabetes. The development of diabetes in NOD mice occurs as a
result of the early development of lymphoid cell infiltration around
the islets at 3 weeks of age and a late event wherein beta-cell
immune response is countered by regulatory mechanisms (24).
Protection conferred by constitutive expression of TGF-? was
previously reported (21, 25), but constitutive expression of TGF-?
in the islets led to fibrosis that compromised investigations of the
mechanisms underlying immune suppression. To overcome this
limitation, we generated mice in which TGF-? expression can be
induced temporally by the tetracycline regulatory system (Fig. 1A)
(22). We developed two transgenic lines expressing high or low
levels of TGF-? in the pancreas, referred to as line 4 and line 45,
respectively. Data from RT-PCR and histochemistry studies
showed that TGF-? gene and protein expression in both lines can
be turned on and off in the islets efficiently within 1 week after
changing of the diet to a doxycycline-containing food source (Fig.
1 B and C).
Using this system, we were able to control and target the
expression of the transgene at specific stages of diabetes develop-
ment (Fig. 2A). This includes stages from birth to 3 weeks (prein-
3), from 4 to 8 weeks (priming phase of disease; group 4), and from
8 to 60 weeks (beta-cell destruction phase; group 5). Transgenic
mice from groups 1–5 were monitored for diabetes development in
comparison to transgene-negative control littermates during 60
weeks. We found that TGF-? expression significantly inhibited the
development of diabetes in NOD transgenic mice (groups 2–5),
indicating that a short pulse of TGF-? in the islets during either the
priming or effector phase of the disease was sufficient to provide
protection (Fig. 2 B and C). Histological analysis did not reveal any
significant difference in the progression of insulitis between differ-
ent groups of transgenic mice suggesting that TGF-? was unlikely
not shown). To determine effects of TGF-? on the development of
antiislet T cell repertoire, splenic T cells from 6- to 7-month-old
nondiabetic transgenic mice were transferred to NOD-SCID re-
cipient mice, and diabetes development was monitored during 12
weeks posttransfer (Table 1). Mice that had received T cells from
control transgenic mice in which TGF-? expression had been
repressed for entire time period (group 1) developed diabetes
within 3–6 weeks after transfer. In contrast, we found that spleen
that TGF-? inhibits the development of an antiislet repertoire.
Because TGF-?-induced fibrosis was significant in line 4 but
minimal in line 45 (Fig. 1C), and because constitutive transgene
repression was more efficient in line 45 than in line 4 (Fig. 2 B and
C), further investigation of mechanisms of protection by TGF-?
were performed by using line 45.
Neither Antigen Presentation nor Immune Deviation Is Affected by
the immunoregulatory action of TGF-? in protecting against au-
Table 1. Transfer of diabetes
Time posttransfer, weeks
123456789 1011 12
Splenic T cells (10 ? 106) from 6- to 7-month-old nondiabetic transgenic mice (line 4) from different groups (1–5) were transferred
that develop diabetes over the total number of mice. Frequency and onset of diabetes in NOD-SCID recipient mice were similar when
spleen cells from line 45 were transferred.
circle, TGF-? turned on and off (groups 2–5); filled triangle, TGF-? constitutively
turned off (group 1).
Transient expression of TGF-? inhibits diabetes. (A) Summary of the
www.pnas.org?cgi?doi?10.1073?pnas.0400810101 Peng et al.
toreactive T cells in NOD mice. The first event of the autoagressive
immune response is initiated by immature dendritic cells that take
up beta-cell antigens and migrate into pancreatic lymph nodes for
antigenic presentation to naı ¨ve T cells (26). To determine whether
expression of TGF-? influences antigen presentation by antigen-
presenting cells, CFSE-labeled naı ¨ve BDC2.5 T cells were adop-
tively transferred into transgenic mice. BDC2.5 T cells are reactive
to self beta-cell antigen(s); on encountering their specific antigens
in the pancreatic lymph nodes, these cells are primed and activated
(27). Their proliferation therefore reflects the antigen presentation
pancreatic lymph nodes were isolated and the cell division profile
not reduced and, if anything, was more efficient in transgenic mice
compared to control group 1 mice (Fig. 3 A and B), indicating that
antigen presentation is not affected by the expression of TGF-?.
Next we considered suppression of T helper (Th) 1 function
through immune deviation toward a Th2 profile as previously
reported from mouse models of constitutive expression of TGF-?
in NOD mice (25). In these mice, TGF-? modified the profile of
antigens presented by antigen-presenting cells and caused beta-
cell-specific splenic T cells to shift from IFN-?-producing Th1 cells
to IL-4-secreting Th2 cells. Accordingly, we isolated T cells from
islets and analyzed their effector phenotype, Th1 versus Th2, after
a 6-h stimulation with phorbol 12-myristate 13-acetate and iono-
mycin (Fig. 3 C–F). We found no significant changes in the
distribution of IFN?- versus IL-4-expressing T cells in mice with or
without expression of TGF-?. Approximately 10% of the CD4?T
cells expressed IFN-?, whereas IL-4-expressing T cells were barely
detectable, indicating no effect of TGF-? on Th1?Th2 differenti-
ation of intraislet effector T cells.
TGF-? Promotes Expansion of the Foxp3-Expressing CD4?CD25?Reg-
ulatory T Cell Pool. CD4?CD25?regulatory T cells account for
5–10% of the CD4?T cells infiltrating NOD islets, the effect of
which might be responsible for the slow development of diabetes.
To investigate whether TGF-? regulates the homeostasis of the
regulatory T cell pool, the frequency of intraislet CD4?CD25?T
cells was determined (Fig. 4 A–D). We found that exposure to
TGF-? for the first 8 weeks (group 3, Fig. 4C) dramatically
enhanced the frequency of CD4?CD25?T cells among total
intraislet CD4?T cells by 4-fold when compared to control
transgenic mice (group 1, Fig. 4A). Accumulation of intraislet
CD4?CD25?T cells was observed at 8 weeks of age and further
persisted up to 16 weeks of age (Fig. 4D). However, this phenom-
enon was not observed if the expression of TGF-? was initiated
after the priming phase that is between 3 and 8 weeks of age (Fig.
4B). Thus, the expression of TGF-? in the islets during the priming
phase of diabetes is associated with the generation of CD4?CD25?
T cells in the islets and the protection against diabetes.
Regulatory T cells (CD4?CD25?) are most commonly charac-
OX40, GITR, CD62L, and CTLA-4. We found that intraislet
regulatory T cells compared to control regulatory T cells isolated
from pancreatic lymph nodes, as indicated by small size and high
levels of CTLA-4 expression (Fig. 4 E and F).
Recent studies have demonstrated that Foxp3 is selectively
development and function of these cells (13, 14). To investigate the
of Foxp3 mRNA expression were determined by using real-time
quantitative PCR analysis (Fig. 4G). Sorted splenic CD4?CD25?
and CD4?CD25?T cells were used as controls in this experiment.
As previous reported, levels of Foxp3 mRNA showed 30-fold
higher expression in CD4?CD25?T cells compared to the
CD4?CD25?T cells purified from the spleen of NOD mice (Fig.
4G). Consistent with the increased frequency of CD4?CD25?
regulatory T cells in the transgenic islets (Fig. 4C), Foxp3 mRNA
expression showed a 4-fold increase among transgenic intraislet
CD4?T cells compared to the control islet T cells (Fig. 4G). These
observations provide direct evidence that expression of TGF-?
results in the accumulation of Foxp3-expressing CD4?CD25?
regulatory T cells in the islet.
To determine whether these cells with a regulatory T cell
phenotype are endowed with suppressive activity in vivo, an adop-
tive transfer model was used that is commonly applied toward this
end (28). Intraislet T cells were isolated from transgenic or control
mice and then cotransferred with splenic cells from diabetic NOD
diabetogenic splenic cells alone induced severe onset of diabetes in
100% of recipient mice by 5 weeks posttransfer. In contrast,
cotransfer of transgenic intraislets T cells with diabetogenic splenic
cells significantly reduced the development of diabetes and con-
ferred protection on ?50% of recipient mice up to 9 weeks
or T cell differentiation profile. CFSE-labeled purified naı ¨ve BDC2.5 T cells were
injected into 4-week-old transgenic mice (line 45) in which TGF-? was either
turned off (A) or on (B) from birth to 4 weeks of age. Four days posttransfer,
pancreatic lymph node cells were collected and lymphocytes were analyzed by
FACS. Dot plots represent CFSE intensity on gated CD4?T cells. The results are a
representative of eight experiments. Similar results were obtained from line 4.
Intraislet T cells were pooled from five transgenic mice (line 45) and stimulated
with phorbol 12-myristate 13-acetate plus ionomycin for 6 h. Contour plots
TGF-? expression does not affect intraislet antigen-presenting function
Peng et al.
March 30, 2004 ?
vol. 101 ?
no. 13 ?
that TGF-? leads to the generation of a T cell population endowed
with suppressive activity.
et al. (29), indicating that in the presence of sufficient IL-2 to
counteract its suppressive effects on T cell proliferation, TGF-?
showed positive effects on the proliferation and survival of human
T cells that develop potent suppressive activity (29). Accordingly,
the potential of different intraislet T cell subsets to proliferate in
vivo was investigated by using an in vivo BrdUrd incorporation
approach. After 4 days of continuous administration of BrdUrd,
lymphocytes were isolated from islets, spleen, mesenteric lymph
CD25 antibodies (Fig. 5). Among the thymic, splenic, and lymph
node CD4?T cell populations, the distribution of BrdUrd-positive
cells between CD25?and CD25?cells showed no difference in
transgenic compared to control mice (Fig. 5). In contrast, a higher
frequency of proliferating cells was found among transgenic in-
traislet CD4?CD25?T cells in comparison to T cells from trans-
suppressive effects of CD4?CD25?T cells, intraislet CD4?CD25?
T cells showed a complete lack of proliferation in transgenic mice,
whereas intermediate levels of proliferation were observed in cells
from nontransgenic mice (Fig. 5). It is possible, however, that lack
of proliferation of intraislet CD4?CD25?T cells in TGF-? trans-
genic mice is a combined effect of direct exposure to islet TGF-?
expression and suppressor activity of CD4?CD25?regulatory T
cells present at high frequency. Nevertheless, these observations
demonstrate a previously uncharacterized function for TGF-? in
High levels of membrane-bound TGF-? on the surface of
regulatory T cells had linked TGF-? with regulatory T cell
suppressive function. The relationship between both compo-
nents has been proposed to function in a paracrine pathway in
which regulatory T cells produce TGF-? that inhibits T cell
activation. Here we focused on the potential effects of TGF-? on
the regulatory T cell compartment and provided direct evidence
for the expression of CD4 and CD25 markers. Contour plots represents results
TGF-? on for 8 weeks since birth were compared for intracellular expression of
were purified from transgenic line 45 with TGF-? on for 8 weeks since birth (Tg
pos) or from transgenic negative mice (Tg neg), and levels of mRNA of Foxp3
expression were determined by real-time quantitative PCR (filled bars). Splenic
8 weeks since birth, and 105cells were transferred to NOD-SCID recipient mice
together with 20 ? 106splenic cells from diabetic NOD mice (open bars). As
control, 20 ? 106splenic cells from diabetic NOD mice were transferred alone to
recipient mice (filled bars). Diabetes development was monitored for 10 weeks
mice (B) and transgenic line 45 with TGF-? on for 8 weeks since birth (A) were
injected for 4 consecutive days with 1 mg of BrdUrd. Lymphocytes were isolated
from islets, mesenteric lymph nodes, spleen, and thymus, and stained with
anti-CD4 and anti-CD25 and anti-BrdUrd antibodies. Contour plots show the
distribution of BrdUrd versus CD25 expression on gated CD4?T cells. The results
are representative of four pooled mice.
www.pnas.org?cgi?doi?10.1073?pnas.0400810101 Peng et al.
for the expansion of Foxp3-expressing regulatory T cells in- Download full-text
volved in the protection against diabetes.
One consideration is that the timing and duration of TGF-?
expression in the islets influences the accumulation of intraislet
CD4?CD25?T cells. If expression of TGF-? is initiated after 8
weeks of age, CD4?CD25?regulatory T cells no longer accumu-
late. Nonetheless, TGF-? still confers protection against diabetes.
Studies on the dynamics of pathogenic and suppressor T cells in
NOD mice reported that progression of diabetes occurs along with
progressive modulation of T cell activity reflected in reduced
suppressor activity of CD4?CD25?T cells and increased pathoge-
nicity of CD4?CD25?T cells (30). Indeed, CD4?CD25?regula-
delayed the onset of diabetes in adoptive transfer models and
showed decreased production of immunoregulatory factors such as
IL-10. In contrast, the profile of T cell differentiation of
mice (30). Consistent with this, our analysis of BrdUrd incorpora-
tion in CD4?CD25?versus CD4?CD25?intraislets T cells indi-
promoting expansion of the regulatory T cells and the suppression
of pathogenic T cells. Indeed, expression of TGF-? in the islets led
to a diminished repertoire of autoaggressive T cells in the spleens
of transgenic mice. As a result, adoptive transfer of these splenic
cells did not yield rapid diabetes in recipient mice that was found
by using spleen cells from control nontransgenic mice. However,
the frequency of CD4?CD25?T cells was not increased in the
for the induction of type I diabetes because of the inhibitory effects
of regulatory T cells in situ in the islets on the clonal expansion and
activation of autoaggressive T cells. In the normal nontransgenic
animal, such autoaggressive cells expand in the islets and migrate
systemically, and can be recovered in spleen. Nevertheless, we
cannot exclude differential effects of TGF-? on autoaggressive T
will be required to elucidate the fate of autoaggressive cells under
these circumstances. The point of interest here is that TGF-?, a
factor known to be a negative regulator of CD4?CD25?T cells, is
conversely a positive regulator of CD4?CD25?regulatory T cell
pool in vivo.
of regulatory T cells in the periphery (8–12). It is unlikely, at least
in humans, that the thymus can replenish the peripheral pool of
CD4?CD25?regulatory T cells throughout the human lifespan. As
a result of thymic involution, the number of CD4?CD25?regula-
tory T cells should decrease over time, and yet evidence suggests
T cells must be efficiently maintained in the periphery. Our data
revealed a critical role of TGF-? in the expansion of the Foxp3-
expressing CD4?CD25?regulatory T cell pool. Although regula-
tory T cells are most commonly known to be anergic and prone to
apoptosis (31–33), several lines of evidence indicate that they can
vigorously proliferate. Anergic CD4?CD25?T cells proliferate
efficiently when transferred to lymphopenic hosts, and regulatory
T cells generated ex vivo in the presence of IL-2 and TGF-? can
proliferate when transferred to nonlymphopenic syngeneic mice
islets remains to be determined, but multiple factors are known to
impact on TGF-? effects. With sufficient IL-2 to counteract its
suppressive effects, TGF-? enhances the proliferation and survival
of human T cells that develop potent suppressive activities (29).
Moreover, engagement of GITR on CD4?CD25?regulatory T
cells seems able to lead to an intermediate step of reversal anergy
where IL-2-induced proliferation may occur (35, 36). Engagement
of both GITR and TGF-? engagement may therefore allow pro-
liferation of regulatory T cells while preserving their suppressive
After this manuscript was prepared, a paper was published
showing that TGF-? could convert peripheral CD4?CD25?T cells
into Foxp3-expressing CD4?CD25?regulatory T cells in vitro (37).
Accordingly, we cannot exclude the possibility of conversion of a
similar subset of CD4?CD25?T cells to the regulatory intraislet
CD4?CD25?T cells by expression of TGF-? in the pancreas.
Nonetheless, our study provided direct evidence for a previously
uncharacterized function of TGF-? in the expansion of regulatory
T cells in vivo that prevents the development of autoimmune
We thank Drs. Li Wen and F. Suzan Wong for helpful discussions,
Debby Butkus and Cindy Hughes for generating transgenic mice,
Tony Ferrandino and Joanne Daugherty for helpful assistance, and
Fran Manzo for help with manuscript preparation. This work was
supported in part by Diabetes Endocrinology Research Center Award
NIH DK45735. FACS analysis was supported by National Institutes of
Health Program Project AI36529 (to R.A.F.). Y.P. is supported by the
Juvenile Diabetes Foundation. Y.L. is supported by the American
Diabetes Association (research grant to R.A.F.) and was previously
supported by an American Diabetes Association Mentor-Based Post-
doctoral Fellowship (to R.A.F.). M.O.L. is a postdoctoral fellow of the
American Cancer Society. R.A.F. is an Investigator of the Howard
Hughes Medical Institute.
1. Bach, J. F. & Chatenoud, L. (2001) Annu. Rev. Immunol. 19, 131–161.
2. Gershon, R. K. (1975) Transplant Rev. 26, 170–185.
4. Shevach, E. M. (2001) J. Exp. Med. 193, F41–F46.
5. Groux, H., O’Garra, A., Bigler, M., Rouleau, M., Antonenko, S., de Vries, J. E. & Roncarolo,
M. G. (1997) Nature 389, 737–742.
6. Levings, M. K. & Roncarolo, M. G. (2000) J. Allergy Clin. Immunol. 106, S109–S112.
7. Weiner, H. L. (2001) Microbes Infect. 3, 947–954.
8. Thorstenson, K. M. & Khoruts, A. (2001) J. Immunol. 167, 188–195.
9. Asano, M., Toda, M., Sakaguchi, N. & Sakaguchi, S. (1996) J. Exp. Med. 184, 387–396.
10. Zhang, X., Izikson, L., Liu, L. & Weiner, H. L. (2001) J. Immunol. 167, 4245–4253.
11. Jonuleit, H., Schmitt, E., Schuler, G., Knop, J. & Enk, A. H. (2000) J. Exp. Med. 192, 1213–1222.
12. Gregori, S., Casorati, M., Amuchastegui, S., Smiroldo, S., Davalli, A. M. & Adorini, L. (2001)
J. Immunol. 167, 1945–1953.
13. Hori, S., Nomura, T. & Sakaguchi, S. (2003) Science 299, 1057–1061.
14. Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. (2003) Nat. Immunol. 4, 330–336.
15. Khattri, R., Cox, T., Yasayko, S. A. & Ramsdell, F. (2003) Nat. Immunol. 4, 337–342.
16. Khattri, R., Kasprowicz, D., Cox, T., Mortrud, M., Appleby, M. W., Brunkow, M. E., Ziegler,
S. F. & Ramsdell, F. (2001) J. Immunol. 167, 6312–6320.
Sci. USA 100, 10878–10883.
18. Nakamura, K., Kitani, A. & Strober, W. (2001) J. Exp. Med. 194, 629–644.
19. Chen, W. & Wahl, S. M. (2003) Cytokine Growth Factor Rev. 14, 85–89.
R. A. (2002) J. Autoimmun. 19, 9–22.
22. Green, E. A. & Flavell, R. A. (2000) Immunity 12, 459–469.
23. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156–159.
24. Delovitch, T. L. & Singh, B. (1997) Immunity 7, 727–738.
25. King, C., Davies, J., Mueller, R., Lee, M. S., Krahl, T., Yeung, B., O’Connor, E. & Sarvetnick,
N. (1998) Immunity 8, 601–613.
26. Spatz, M., Eibl, N., Hink, S., Wolf, H. M., Fischer, G. F., Mayr, W. R., Schernthaner, G. & Eibl,
M. M. (2003) Cell Immunol. 221, 15–26.
28. Mukherjee, R., Chaturvedi, P., Qin, H. Y. & Singh, B. (2003) J. Autoimmun. 21, 221–237.
29. Yamagiwa, S., Gray, J. D., Hashimoto, S. & Horwitz, D. A. (2001) J. Immunol. 166, 7282–7289.
30. Gregori, S., Giarratana, N., Smiroldo, S. & Adorini, L. (2003) J. Immunol. 171, 4040–4047.
31. Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. &
Sakaguchi, S. (1998) Int. Immunol. 10, 1969–1980.
32. Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. & Sakaguchi, S.
(1999) J. Immunol. 162, 5317–5326.
34. Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A. & Rudensky, A. (2002) Nat. Immunol. 3,
35. McHugh, R. S., Whitters, M. J., Piccirillo, C. A., Young, D. A., Shevach, E. M., Collins, M. &
Byrne, M. C. (2002) Immunity 16, 311–323.
36. Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y. & Sakaguchi, S. (2002) Nat. Immunol. 3,
37. Chen, W., Jin, W., Hardegen, N., Lei, K. J., Li, L., Marinos, N., McGrady, G. & Wahl, S. M.
(2003) J. Exp. Med. 198, 1875–1886.
Peng et al.
March 30, 2004 ?
vol. 101 ?
no. 13 ?