IL-2, -7, and -15, but Not Thymic Stromal Lymphopoeitin,
Redundantly Govern CD4?Foxp3?Regulatory
T Cell Development1
Kieng B. Vang, Jianying Yang, Shawn A. Mahmud, Matthew A. Burchill,2Amanda L. Vegoe,
and Michael A. Farrar3
Common ? chain (?c)-receptor dependent cytokines are required for regulatory T cell (Treg) development as ?c?/?mice lack
Tregs. However, it is unclear which ?c-dependent cytokines are involved in this process. Furthermore, thymic stromal lympho-
poietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing
CD4?Foxp3?Tregs in the thymus express the IL-2R?, IL-4R?, IL-7R?, IL-15R?, and IL-21R? chains, but not the IL9R? or
TSLPR? chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in
CD4?Foxp3?Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were
capable of inducing the conversion of CD4?CD25?Foxp3?thymic Treg progenitors into CD4?Foxp3?mature Tregs in vitro. To
examine this issue in more detail, we generated IL-2R??/?? IL-7R??/?and IL-2R??/?? IL-4R??/?mice. We found that
IL-2R??/?? IL-7R??/?mice were devoid of Tregs thereby recapitulating the phenotype observed in ?c?/?mice; in contrast, the
phenotype observed in IL-2R??/?? IL-4R??/?mice was comparable to that seen in IL-2R??/?mice. Finally, we observed that
Tregs from both IL-2?/?and IL-2R??/?mice show elevated expression of IL-7R? and IL-15R? chains. Addition of IL-2 to Tregs
from IL-2?/?mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the
predominant role in Treg development, but that in its absence the IL-7R? and IL-15R? chains are up-regulated and allow for IL-7
and IL-15 to partially compensate for loss of IL-2. The Journal of Immunology, 2008, 181: 3285–3290.
development of Tregs. These include signals emanating from the
TCR and the costimulatory molecule CD28 (2–5). In addition, sev-
eral studies have suggested that cytokines play a key role in this
process (6–11). However, which cytokines are involved has re-
mained controversial. For example, it has been known for many
years that mice lacking IL-2, or the IL-2R? or IL-2R? chains,
develop lethal autoimmune disease (12–14). This was initially at-
tributed to defective Treg development as these mice lacked
CD4?CD25?T cells. More recent studies using the transcription
factor Foxp3 as an identifier of Tregs found that young IL-2?/?
mice have relatively normal numbers of
D4?Foxp3?regulatory T cells (Tregs)4that develop in
the thymus play a key role in preventing autoimmune
disease (1). Multiple signals are required to induce the
CD4?Foxp3?Tregs (8, 15, 16). In contrast, both IL-2R??/?and
IL-2?/?? IL-15?/?mice exhibited significant decreases in Treg
numbers, suggesting that IL-2 and IL-15 play a redundant role in
Treg development (8, 9).
Importantly, although Treg differentiation is inhibited in IL-
2R??/?mice, Tregs are not completely absent (8, 9). This raises
the possibility that other cytokines can also drive Treg differenti-
ation. Along these lines, Watanabe et al. (17) have suggested a role
for the cytokine thymic stromal lymphopoietin (TSLP). Specifi-
cally, they suggested that TSLP production by Hassall’s corpuscles
plays an important role in human Treg development (17). Like-
wise, the common ?-chain (?c), which is closely related to the
TSLPR, has also been shown to be involved in Treg development.
For example, we and others have demonstrated that mice lacking
?c are devoid of Tregs (8, 15). The ?c forms a component of
multiple cytokine receptors including those for IL-2, IL-4, IL-7,
IL-9, IL-15, and IL-21 (18). Thus, two key questions in Treg de-
velopment are 1) which ?c-dependent cytokines can induce Treg
development and 2) whether TSLP signals are also involved in this
process. In this study, we demonstrate that developing Tregs ex-
press IL-2R?, IL-7R? and IL-15R? and respond to IL-2, IL-7, and
to a much lesser degree IL-15, by inducing STAT5 activation in
CD4?Foxp3?thymocytes. Similarly, IL-2-induced conversion of
CD4?CD25?Foxp3?thymic Treg progenitors into CD4?Foxp3?
mature Tregs. IL-7 and IL-15 also induced conversion of thymic
Treg progenitors into mature CD4?Foxp3?Tregs, albeit much
less effectively; in contrast, TSLP showed no activity in this con-
version assay. IL-4 signaling also does not appear to play a role in
Treg development as IL-4R??/?? IL-2R??/?mice show com-
parable numbers of Tregs as that seen in IL-2R??/?mice. In con-
trast, IL-2R??/?? IL-7R??/?mice exhibit a developmental
block which mimics that seen in ?c?/?mice. Finally, the
Center for Immunology and Department of Laboratory Medicine and Pathology, Uni-
versity of Minnesota, Minneapolis, MN 55455
Received for publication March 19, 2008. Accepted for publication June 29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a Pew Scholar Award, a Cancer Research
Institute Investigator Award, a Leukemia and Lymphoma Society Scholar Award, and
by National Institutes of Health Grant AI061165 to M.A.F. K.B.V. was supported by
a Supplement to Promote Diversity in Health-Related Research.
2Current address: Integrated Department of Immunology, National Jewish Medical
and Research Center, 1400 Jackson Street, K512C, Denver, CO 80206.
3Address correspondence and reprint requests to Dr. Michael A. Farrar, Center for
Immunology, University of Minnesota, 312 Church Street SE, 6-116 Nils Hasselmo
Hall, Minneapolis, MN 55455. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: Treg, regulatory T cell; ?c, common gamma
chain; p-STAT5, phospho-STAT5; LMC, littermate control; TSLP, thymic stro-
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
expression of IL-7R? and IL-15R? chains is suppressed in mature
Tregs; this suppression does not occur in IL-2?/?or IL-2R??/?
mice, demonstrating that IL-7R? and IL-15R? down-regulation
occurs via an IL-2/IL-2R?-dependent signaling pathway. Our find-
ings demonstrate that IL-2, IL-7, and IL-15, are the critical ?c-
dependent cytokines that are responsible for promoting Treg de-
velopment. In contrast, developing Tregs do not express the
TSLPR?-chain, nor respond to TSLP, by inducing phospho-
STAT5 (p-STAT5). Thus, at least in the mouse, TSLP does not
appear to play a direct role in Treg development.
Materials and Methods
IL-2?/?, IL-2R??/?, IL-7R??/?, and IL-4R??/?mice were obtained from
The Jackson Laboratories. IL-2R??/?, IL-7R??/?, and IL-4R??/?mice
were crossed in our laboratory to obtain IL-2R??/?? IL-7R??/?and
IL-4R??/?? IL-2R??/?mice. Mice used were on a C57BL/6 background
with the exception of the IL-4R??/?and IL-4R??/?? IL-2R??/?mice,
which were on a mixed BALB/c ? C57BL/6 background, and Foxp3-GFP
reporter mice, which were on a mixed C57BL/6 ? 129 background.
Foxp3-GFP reporter mice were provided by Dr. Sasha Rudensky (Univer-
sity of Washington School of Medicine, Department of Immunology, Se-
Flow cytometry and FACS analysis
Mice were sacrificed and lymph node, spleen, and thymus were isolated.
Five million cells were used per staining condition. Cells were first pre-
treated with an Ab that blocks Fc receptor binding (Clone 24G2). Cells
were subsequently stained with the following Abs from eBioscience: CD4-
Alexa 700, CD8-allophycocyanin-Alexa Fluor 750 or CD8-FITC, CD3?-
FITC, CD25-PE-Cy7 (PE-Cy 7), or CD25-allophycocyanin. In addition,
biotinylated Abs for CD122 (IL2R?), CD124 (IL4R?), CD127 (IL7R?),
and IL21R?, were obtained from eBioscience. Abs for IL-9R?, TSLPR?,
and IL-15R? were obtained from R&D Systems. Isotype control Abs for
TSLPR? and IL-15R? were obtained from R&D Systems, while isotype
controls for IL-9R? were obtained from eBioscience. Both the IL-9R? and
TSLPR? were biotinylated according to the manufacturer’s instructions
(Sigma-Aldrich, Cat. no. BTAG-1KT), while the IL-15R? Ab was pur-
chased in a biotinylated form. Streptavidin allophycocyanin from eBio-
science was used as a secondary reagent to reveal staining with biotinylated
Abs. Intracellular Foxp3 staining was done after fixation, permeabilization,
and overnight incubation at 4°C as described previously (8).
To examine whether IL-2 alters IL-7R? and IL-15R? expression on
Tregs in IL-2?/?mice, we purified CD4?splenocytes from IL-2?/?mice
by MACS beads enrichment (Miltenyi Biotec). Purified cells were then
stimulated with IL-2 (100 U/ml) for 4, 8, 12, and 24 h. Cells were then
harvested and stained for IL-7R?, IL-15R?, and Foxp3 expression and
analyzed on an LSR II flow cytometer (BD Biosciences).
Flow cytometry for p-STAT5
Single-cell suspensions were generated from isolated spleens and thymii
from Foxp3-GFP reporter mice. These cell suspensions were pretreated
with an Ab that blocks Fc receptor binding. Cells were then stained for the
surface markers CD4, CD8, and CD25. Five million cells were then serum
starved in 500 ?l of 1? DMEM for 30 min at 37°C, then stimulated with
either 100 U/ml of IL-2 (PeproTech), or 50 ng/ml IL-4 (PeproTech), IL-7
(R&D Systems), IL-9 (R&D Systems), TSLP (R&D Systems), IL-15
(R&D Systems), or IL-21 (PeproTech) for 20 min. After stimulation, the
cells were washed with 1? DMEM to remove all traces of the supernatant;
they were then resuspended in 100 ?l of fixation medium from the Caltag
Fix and Perm kit and incubated at 37°C for 15 min. Afterward, 1 ml of 4°C
100% methanol was added and the cells were incubated overnight at 4°C
in the dark. Intracellular p-STAT5 staining was done using the Caltag Fix
and Perm kit and PE-conjugated anti-p-STAT5 (BD Biosciences). Non-
stimulated cells were used as negative controls.
Treg conversion assay
The conversion assay of Treg progenitors into CD4?Foxp3?Tregs was
conducted as previously described (10). In brief, CD4?CD25?Foxp3?
Treg progenitors were sorted from Foxp3-GFP reporter mice (19) and
placed in culture in the presence of the indicated amounts of cytokine.
Twenty-four hours later cells were stained for CD4 and CD25 and analyzed
for expression of these markers plus Foxp3-GFP using an LSR II flow
To examine the role that different cytokines play in Treg devel-
opment in the thymus, we first analyzed the expression of IL-2R?,
IL-4R?, IL-7R?, IL-9R?, IL-15R?, IL-21R?, and TSLPR? on
CD4?Foxp3?thymocytes and CD4?Foxp3?splenocytes. We
found four basic patterns of cytokine receptor expression. First, the
IL-2R?-chain was selectively expressed on CD4?Foxp3?thymo-
cytes (Fig. 1). This pattern of expression was maintained in splenic
Foxp3?vs Foxp3?T cells. Second, IL-9R? and TSLPR? were
not observed on either Foxp3?or Foxp3?thymocytes. To confirm
that our staining for these receptors was working, we also stained
peritoneal B1 B cells and CD19?pre-B cells, which have previ-
ously been reported to express the IL-9R? and TSLPR? chains,
single positive thymocytes and CD4?Foxp3?splenocytes. Thymus and
spleen cells from 5- to 9-wk-old C57BL/6 mice were harvested and stained
with Abs to CD4, CD8, and Foxp3, to identify distinct thymic and splenic
T cell subsets, as well as with Abs to IL-2R?, IL-4R?, IL-7R?, IL-9R?,
IL-15R?, IL-21R?, and TSLPR?. Shown are flow cytometry histograms of
CD4 single positive (SP) thymocytes (left column) and CD4?splenocytes
(right column). Gray filled in histograms represent staining of the corre-
sponding CD4?Foxp3?population with isotype control Ab. Solid lines
and broken lines represent staining of CD4?Foxp3?and CD4?Foxp3?
cells, respectively. A representative example of four independent experi-
ments is depicted (n ? 17).
?c cytokine-family receptor expression on CD4?Foxp3?
3286CYTOKINE-DEPENDENT Treg DEVELOPMENT
respectively (17, 18). We detected IL-9R? expression on perito-
neal B1 B cells; as expected, we also observed TSLPR? expres-
sion on pre-B cells in the bone marrow, thereby indicating that our
Abs to IL9R? and TSLPR? are capable of detecting expression of
these receptors (data not shown). Third, IL-4R? and IL-21R? were
expressed equally on Foxp3?vs Foxp3?thymocytes and splenic
T cells (Fig. 1). Last, we observed a dynamic expression pattern
for the IL-7R? and IL-15R? chains. Expression of both of these
receptors was observed in CD4?Foxp3?thymocytes, but was sig-
nificantly reduced in splenic Tregs (Fig. 1). Thus multiple ?c-
dependent cytokine receptors, but not the TSLPR?-chain, are ex-
pressed on developing Tregs in the thymus.
To assess whether receptor expression on Tregs correlated with
function, we stimulated cells with IL-2, IL-4, IL-7, IL-9, IL-15,
IL-21, and TSLP and examined STAT5 activation by intracellular
staining for p-STAT5. We focused on STAT5 as previous reports
have indicated that STAT5 plays a critical role in Treg develop-
ment (8, 10, 20). For these studies, we identified Tregs using
Foxp3-GFP reporter mice; similar studies were also conducted us-
ing CD25 as a marker of Tregs. We identified three distinct re-
sponse patterns. First, IL-2 induced robust STAT5 activation in
CD4?Foxp3?thymocytes; splenic Tregs remained highly respon-
sive to IL2 stimulation (Fig. 2). Second, IL-7 and IL-15 induced
modest STAT5 activation in CD4?Foxp3?thymocytes. However,
these responses were almost completely eliminated in CD4?
Foxp3?splenocytes (Fig. 2). Third, IL-4, IL-9, IL-21, and TSLP
did not induce detectable STAT5 activation on CD4?Foxp3?thy-
mocytes (Fig. 2). Similar results were obtained when gating on
CD4?CD25?T cells, with the exception that under those staining
conditions IL-4 led to very weak STAT5 phosphorylation in
CD4?Foxp3?thymocytes and splenocytes (data not shown). A
potential caveat with the IL-15 studies is that IL-15 could be pre-
sented by the IL-15R?-chain via trans presentation in vivo (21). It
is possible, therefore, that our ex vivo stimulation studies may not
haveallowed for optimaltranspresentation
CD4?Foxp3?Tregs. Thus, developing thymic Tregs respond to
IL-2 and IL-7, and to a lesser degree IL-15.
Previous reports have suggested that TSLP plays a role in Treg
development (22). However, TSLPR?/?mice have no reported
defects in Treg develoment or function (23). This latter observa-
tion is consistent with our failure to observe TSLPR expression on
developing Tregs. A potential caveat with our studies is that
TSLPR expression levels may be below the limits of detection by
flow cytometery. Likewise, the amount of STAT5 phosphorylation
induced by TSLP might be below the level that we can detect by
flow cytometry. To explore this in more detail, we examined
CD4?Foxp3-GFP?thymcoytes. These studies indicated that
TSLPR mRNA could be detected at some level in developing
Tregs (data not shown). A key question then is whether this results
in expression of a receptor capable of inducing biological re-
sponses. To address this question, we made use of the recent iden-
tification of CD4?CD25?Foxp3?thymocytes as penultimate Treg
progenitors that can be converted into CD4?Foxp3?mature Tregs
following stimulation with IL-2 (10, 11). For these studies, we
isolated CD4?CD25?Foxp3-GFP?Treg progenitors and stimu-
lated them overnight with either IL-2, IL-7, IL-15, or TSLP. The
cultured cells were then examined for Foxp3-GFP expression. As
shown in Fig. 3, IL2 induced clear conversion of Treg progenitors
into CD4?Foxp3-GFP?Tregs. IL-7 and IL-15 were also capable
of inducing the conversion of Treg progenitors into Foxp3?Tregs,
although they were much less effective than IL-2. In contrast,
CD4?Foxp3?thymocytes and splenocytes. Single-cell suspensions of thy-
mocytes or splenocytes from Foxp3-GFP mice were serum starved for 30
min and then stimulated with IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or TSLP
for 20 min. Cells were then stained with Abs to CD4, CD8, CD25, and
phospho-STAT5 as described in the methods section. Shown are histo-
grams of phospho-STAT5 expression in CD4SP thymocytes (left column)
and CD4?splenocytes (right column). Gray filled in histograms represent
staining of unstimulated Foxp3-GFP?cells. Solid lines and broken lines
represent staining of stimulated Foxp3-GFP?and Foxp3-GFP?cells, re-
spectively. A representative example of two independent experiments is
depicted (n ? 3 mice). Similar results were obtained when using CD25 to
identify Tregs in C57BL/6 mice (n ? 13 mice, data not shown).
Cytokine stimulation and phospho-STAT5 expression on
of CD4?CD25?Foxp3?thymic Treg progenitors into CD4?Foxp3?
Tregs. Sorted CD4?CD25?Foxp3?thymic Treg progenitors from Foxp3-
GFP reporter mice were stimulated with 10 U/ml IL-2, 5 ng/ml IL-7, 100
ng/ml IL-15, and 50 ng/ml TSLP in culture overnight. After 24 h of stim-
ulation, cells were stained with Abs to CD4 and CD25. Shown are contour
plots of Foxp3 vs CD25 for CD4-gated cells (95% of cells were CD4?)
after stimulation with medium alone (top left), TSLP (top middle), IL-2
(top right), IL-7 (bottom left), or IL-15 (bottom right). A representative
example of three independent experiments is depicted.
IL-2, IL-7, and IL-15, but not TSLP, induce the conversion
3287The Journal of Immunology
TSLP stimulation failed to induce conversion of any Treg progen-
itors into Foxp3?Tregs. Thus, even if the TSLPR is expressed at
very low levels on developing Tregs, it is incapable of inducing
Treg differentiation following stimulation with TSLP.
Our observation that IL-2R? and IL-7R? were the predominant
receptors expressed on developing thymocytes suggested that IL-2,
IL-7, and IL-15 were most likely the key ?c-dependent cytokines
that drive Treg development. However, given the expression of the
IL-4R?-chain on developing Tregs, we examined whether IL4-
dependent signals also played a role in this process. IL-4R??/?
mice show no decrease in the percentage of Tregs in the thymus
relative to littermate control (LMC) mice (Fig. 4). Furthermore,
splenic Tregs were also not reduced in IL-4R??/?mice (Fig. 4).
To examine whether IL-2R? and IL-4R?-dependent signals
played a redundant role in Treg development, we generated IL-
2R??/?? IL-4R??/?mice. As previously reported, IL-2R??/?
mice exhibited reduced numbers of Tregs in both the thymus and
spleen; a further reduction was not observed in IL-2R??/?? IL-
4R??/?mice (Fig. 4). These findings strongly suggest that IL-
4R?-dependent signals are not required for Treg development. It is
important to note here that unlike our previous studies, which used
IL-2R??/?mice on the C57BL/6 background, the IL-4R??/?and
IL-2R??/?? IL-4R??/?mice in these experiments are on a
mixed C57BL/6 ? BALB/c background. We have consistently
noticed that the IL-2R??/?mice on the C57BL/6 ? BALB/c back-
ground mice have a more severe phenotype (i.e., fewer Tregs at an
earlier age) than IL-2R??/?on the C57BL/6 background. This
results in IL-2R??/?mice on the mixed background having a re-
duced percentage of Tregs relative to that seen in IL-2R??/?mice
on the C57BL/6 background.
Given the expression of both functional IL-7R? and IL-2R? on
developing Tregs, we predicted that these two cytokine receptors
might both be capable of driving Treg development. Consistent
with our previous report, we found that although total numbers of
T cells are greatly reduced in IL-7R??/?mice, the percentage of
Tregs relative to other T cell subsets was not affected (Fig. 5A) (8).
Thus, IL7R? signaling is not required for Treg development. How-
ever, it remains possible that IL-2R? and IL-7R? can act redun-
dantly to drive Treg development. To test this possibility, we
compared Treg differentiation in IL-7R??/?vs IL-2R??/?? IL-
7R??/?mice. We found that IL-2R??/?? IL-7R??/?mice
showed a significant decrease in Treg numbers when compared
with IL-7R??/?mice (p ? 0.009, Student’s t test) (Fig. 5, A and
B). Importantly, the numbers of Tregs found in IL-2R??/?? IL-
7R??/?mice (thymus ? 195 ? 75; spleen ? 686 ? 136) were
comparable to that which we observed in age-matched ?c?/?mice
(thymus ? 80 ? 22; spleen ? 1500 ? 651) in our previous studies
(Fig. 5B) (8). These experiments demonstrate that IL-2R? and IL-
7R?-dependent cytokines are the only ?c-dependent cytokines re-
quired for Treg development.
IL-7R? and IL-15R? are expressed at quite low levels on ma-
ture splenic Tregs. Thus, it is rather surprising that splenic Tregs
are maintained in young IL-2?/?mice. To examine this further, we
stained CD4?Foxp3?Tregs from LMC and IL-2?/?mice for the
expression of IL-7R? and IL-15R?. We found that CD4?Foxp3?
4R??/?mice. Thymus and spleen were harvested from 4- to 5-wk-old
LMC, IL-4R??/?, IL-2R??/?, and IL-2R??/?? IL-4R??/?mice. Cells
were stained with Abs to CD4, CD8, and Foxp3 to identify Tregs. Shown
are flow cytometry plots of thymus (top panel) or spleen cells (bottom
panel) gated on CD4?T cells. A representative example of six independent
experiments is depicted.
Treg development in IL-4R??/?and IL-2R??/?? IL-
7R??/?mice. A, Thymus and spleen were harvested from 4- to 5-wk-old
LMC, IL-7R??/?, and IL-2R??/?? IL-7R??/?mice. Cells were stained
with Abs to CD4, CD8, and Foxp3 to identify Tregs. Shown are flow
cytometry plots of thymus (top panel) or spleen cells (bottom panel) gated
on CD4?T cells. A representative example of six independent experiments
is depicted. B, Shown are bar graphs representing total numbers of
CD4?Foxp3?Tregs in the thymus (left panel) and spleen (right panel).
Error bars represent SEM; n ? 6 IL-7R??/?and 6 IL-2R??/?? IL-
7R??/?mice; p values were calculated using two-tailed Student’s t test.
Treg development in IL-7R??/?and IL-2R??/?? IL-
CD4?Foxp3?Tregs from IL-2?/?and IL-2R??/?mice compared with
wild type LMC. Splenocytes were isolated from 4- to 5-wk-old LMC,
IL-2?/?and IL-2R??/?mice and stained with Abs for CD4, CD8, and
Foxp3 to identify splenic Tregs. Shown are CD4?Foxp3?gated cells
stained for IL-2R? (left panels), IL-7R? (middle panels), and IL-15R?
(right panels). Gray histograms represent staining of CD4?Foxp3?cells
with isotype control Ab. Solid lines represent histograms of CD4?Foxp3?
T cells from IL-2?/?(top panel) or IL-2R??/?(bottom panel) mice; bro-
ken lines represent staining of CD4?Foxp3?Tregs from wild-type LMC
mice. A representative example of three independent experiments is de-
picted (n ? 6 IL-2?/?and 3 IL-2R??/?mice).
Expression ofIL-2R?, IL-7R?,andIL-15R?
3288CYTOKINE-DEPENDENT Treg DEVELOPMENT
Tregs in IL-2?/?mice expressed significantly higher levels of both
the IL-7R? and IL-15R? chains (Fig. 6). We considered two ex-
planations for these findings. First, it is possible that, in the ab-
sence of IL-2, any splenic Tregs that express higher levels of IL-
7R? or IL-15R? have a competitive advantage and are selectively
expanded. Alternatively, it is possible that IL-2/IL-2R?-dependent
signals actively down-regulate IL-7R? or IL-15R? expression. To
distinguish between these two possibilities, we stained the few
CD4?Foxp3?Tregs in IL-2R??/?mice for IL-7R? and IL-15R?
expression. Once again, we observed increased expression of both
IL-7R? and IL-15R? on Tregs from IL-2R??/?vs LMC mice
(Fig. 6). In IL-2R??/?mice, IL-15R? expression provides no
competitive advantage. Thus, this latter finding strongly suggests
that IL-15R? down-regulation, and likely IL-7R? as well, is due to
IL-2R?-dependent signals. To investigate this further, we took
CD4?splenocytes from IL-2?/?mice and stimulated those cells
with IL-2. We then examined expression of IL-7R? and IL-15R?
chains on CD4?Foxp3?cells (Fig. 7). We observed down-regu-
lation of the IL-7R?-chain as early as 8 h after IL-2 stimulation;
both the IL-7R? and IL-15R? chains were clearly down regulated
after 24 h of IL-2 stimulation. Taken together, these studies indi-
cate that IL-2 dependent signals can negatively regulate IL-7R?
and IL-15R? expression on CD4?Foxp3?Tregs.
These studies identify IL-2, IL-7, and IL-15 as the sole ?c-depen-
dent cytokines required for Treg development in the thymus. Four
pieces of data support this conclusion. First, receptors for these
three cytokines are expressed on developing Tregs in the thymus.
Second, these cells can respond to IL-2 and IL-7 by inducing ro-
bust STAT5 activation. The only other ?c-dependent cytokine re-
ceptors expressed on thymic Tregs are IL-4R? and IL-21R?. How-
ever, IL-4 and IL-21 did not induce STAT5 activation in
CD4?Foxp3?thymocytes and only induced minimal STAT5 ac-
tivation in mature CD4?Foxp3?splenocytes. The role of IL-15 is
somewhat more complicated. We observed only weak STAT5 in-
duction following ex vivo stimulation of thymic CD4?Foxp3?
Tregs with IL-15. This may reflect the absence of accessory cells
in our ex vivo stimulation cultures that would allow for effective
trans presentation of IL-15 to developing Tregs. Third, IL-2 and to
a lesser degree IL-7 and IL-15 were capable of inducing the con-
version of CD4?CD25?Foxp3?Treg progenitors into mature
Foxp3?Tregs. In contrast, TSLP was completely ineffective in
this assay. Finally, we found that IL-2R??/?? IL-7R??/?mice
recapitulated the phenotype reported in ?c?/?mice which are es-
sentially devoid of Tregs. In contrast, the reduction in thymic
Tregs in IL-2R??/?? IL-4R??/?mice was no more severe than
that seen in IL-2R??/?mice. Taken together with our previous
observation that IL-2?/?? IL-15?/?mice have significantly
fewer Tregs that IL-2?/?mice, our findings strongly support the
conclusion that IL-2, IL-7, and IL-15, but not other ?c-dependent
cytokines, can contribute to Treg differentiation in the thymus.
We also examined the role of TSLP on Treg differentiation as
previous studies have suggested that TSLP plays an important role
in human and murine Treg development (17, 22). Our studies rule
out a direct role for TSLP in murine Treg differentiation. First,
consistent with our earlier observation, IL-7R??/?mice show no
reduction in Tregs relative to non-Tregs. Second, we could not
detect expression of TSLPR? on thymic Tregs nor induce STAT5
activation following stimulation of these cells with TSLP. Third
TSLP was incapable of inducing the conversion of thymic Treg
progenitors into CD4?Foxp3?Tregs. These findings demonstrate
that TSLP cannot play a direct role in Treg development. It re-
mains possible that TSLP plays an indirect role by acting on other
cell types that may be involved in promoting Treg differentiation.
However, this function is either not unique to TSLP, or not critical,
as Tregs clearly develop in mice lacking the IL-7R?-chain, which
is a critical component of the TSLPR.
Although our studies demonstrate that IL-2, IL-7, and IL-15 can
redundantly contribute to Treg development and homeostasis, it
seems likely that IL-2 is the relevant cytokine in wild-type mice.
Specifically, we found that expression of IL-7R? and IL-15R?
were significantly increased on Tregs in both IL-2?/?and IL-
2R??/?mice. Ex vivo stimulation of CD4?Foxp3?Tregs from
IL-2?/?mice with IL-2 led to rapid down-regulation of both of
these receptor subunits. Thus, IL-2 plays an important role in ren-
dering CD4?Foxp3?Tregs uniquely responsive to IL-2-dependent
signals in wild-type mice. This most likely serves to link Treg
homeostasis directly to effector T cell activation and IL2 secretion.
Effector T cell IL2 production appears to be critical for Tregs to
expand in step with activated effector T cells and thereby mediate
effective suppression. Supporting this conclusion, IL-2?/?mice,
but not IL-7?/?, or IL-15?/?mice, show signs of T cell activation
and ultimately succumb to lethal multiorgan autoimmune disease.
Thus, although IL-7 and IL-15 are capable of sustaining Treg pop-
ulations in young mice, they are not effective at expanding these
cells sufficiently during ongoing immune responses. Finally, these
findings also have implications for the use of low-level IL-7R?
expression to identify Tregs (24, 25) as this receptor may be up-
regulated under conditions of limited IL-2 availability.
We thank Rachel Agneberg and Jared Liebelt for assistance with animal
husbandry, Paul Champoux for assistance with flow cytometry, and Dr.
Alexander Khoruts for review of the manuscript.
The authors have no financial conflict of interest.
in IL-2?/?mice after ex vivo IL2 stimulation. CD4?T cells were isolated
as described in the Materials and Methods section and cells were stimu-
lated with 100 U/ml IL-2 for 8 or 24 h. Cells were then stained for Foxp3,
IL-7R?, and IL-15R? and analyzed by flow cytometry. Dark unshaded
histogram represents IL-7R? and IL-15R? expression after IL-2 stimula-
tion for the times indicated. Shaded histograms represent nonstimulated
controls. Shown is a representative example of six independent experi-
ments for IL7R? expression and two independent experiments for IL15R?
Expression of IL-7R? and IL-15R? on CD4?Foxp3?Tregs
3289 The Journal of Immunology