The Journal of Experimental Medicine
JEM © The Rockefeller University Press $8.00
Vol. 203, No. 4, April 17, 2006 1021–1031 www.jem.org/cgi/doi/10.1084/jem.20052333
Cytokines play essential roles in the control of
immune systems; they not only act as growth
factors but also regulate the diff erentiation,
maintenance, and activation of naive, eff ector,
and memory state of immune cells. Their cyto-
plasmic signal transduction pathways are well
defi ned. Upon binding of cytokines to their re-
ceptors and subsequent receptor dimerization,
receptor-associated JAKs become activated and
phosphorylate tyrosine residues in the cytoplas-
mic domains of receptors, which serve as the
binding sites for Src homology 2 (SH2) domain
of STAT molecules. After phosphorylation of
STATs by JAKs, STATs dimerize and translo-
cate into the nucleus to induce transcription of
cytokine- responsive genes (1, 2).
The cytokine milieu and their intracellular
signaling molecules are also involved in naive
CD4+ Th diff erentiation. It is well established
that IL-12/STAT4 and IL-4/STAT6 are nec-
essary for Th1 and Th2 diff erentiation, respec-
tively. In addition, IFN-γ–STAT1 pathway is
also necessary for Th1 diff erentiation (3, 4).
The molecular mechanism for generating Th3
regulatory cells, which is a unique Th cell sub-
set that primarily secretes TGF-β1, is poorly
understood. TGF-β1 secreted from Th3 cells
provides help for IgA induction and has sup-
pressive properties for both Th1 and Th2 cells
(5, 6). Because TGF-β1 KO mice exhibited se-
vere multiorgan infl ammations (7, 8), TGF-β1
has been thought to be an important immune
regulatory cytokine. TGF-β1 is also suggested
to be involved in the regulatory function of
CD4+ CD25+ regulatory T cells (9, 10), though
the molecular mechanism of TGF-β1 induc-
tion in such regulatory-type T cells remains to
be elucidated. Because production of TGF-β1
Loss of SOCS3 in T helper cells
resulted in reduced immune responses
and hyperproduction of interleukin 10
and transforming growth factor–β1
Ichiko Kinjyo,1 Hiromasa Inoue,2 Shinjiro Hamano,3 Satoru Fukuyama,1,2
Takeru Yoshimura,1 Keiko Koga,1 Hiromi Takaki,1 Kunisuke Himeno,3
Giichi Takaesu,1 Takashi Kobayashi,1 and Akihiko Yoshimura1
1Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, 2Research Institute for Diseases
of the Chest, Graduate School of Medical Sciences, and 3Department of Parasitology, Faculty of Medical Sciences,
Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan
Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal
transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse
studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2)
skewing and lead to hypersensitivity to allergic diseases. To defi ne the physiological roles of
SOCS3 in T cells, we generated T cell–specifi c SOCS3 conditional knockout mice. We found
that the mice lacking SOCS3 in T cells showed reduced immune responses not only to
ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection.
In vitro, SOCS3-defi cient CD4+ T cells produced more transforming growth factor (TGF)-훃1
and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like
differentiation. We found that STAT3 positively regulates TGF-훃1 promoter activity de-
pending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation
assay revealed that more STAT3 was recruited to the TGF-훃1 promoter in SOCS3-defi cient
T cells than in control T cells. The activated STAT3 enhanced TGF-훃1 and IL-10 expression
in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these
fi ndings, we propose that SOCS3 regulates the production of the immunoregulatory cyto-
kines TGF-훃1 and IL-10 through modulating STAT3 activation.
Abbreviations used: ChIP, chro-
cKO, conditional KO; LIF, leu-
kemia inhibitory factor; SH2,
Src homology 2; SBE, STAT3-
binding element; SOCS, sup-
pressor of cytokine signaling.
The online version of this article contains supplemental material.
1022 SOCS3 REGULATES IL-10 AND TGF-β1 PRODUCTION | Kinjyo et al.
is greatly enhanced by IL-4 and IL-10 in Th cells, while sup-
pressed by IFN-γ (11), cytokine signals may play critical roles
in the induction and regulation of TGF-β1 production.
In the physiologic condition as well as in pathological
conditions, functions of cytokines are strictly controlled. Cy-
tokine signaling pathways are negatively regulated by the
family of proteins called suppressors of cytokine signaling
(SOCSs), which are characterized by the presence of an SH2
domain and a COOH terminal conserved domain termed
the SOCS-box. Several reports have indicated that SOCS
proteins are necessary for regulation of normal immune re-
sponses (12). Among them, SOCS3, which associates with
the tyrosine kinase Lck, calcineurin, and CD28, has been
shown to inhibit IL-2 production during T cell activation
(13–16). During Th diff erentiation, SOCS3 is selectively ex-
pressed in Th2 cells, whereas SOCS1 expression is higher in
Th1 than in Th2 cells (17, 18). In the analysis of Lck pro-
moter-driven SOCS3-transgenic mice, the high expression
of SOCS3 in Th cells led to skewing to Th2-type diff erentia-
tion. This is probably because SOCS3 binds to IL-12Rβ2
and inhibits IL-12–mediated STAT4 activation, thereby
blocking Th1 development (18, 19). Importantly, SOCS3
levels were high in T cells from allergic disease patients (18).
These observations implied that SOCS3 might be crucial for
Th cell diff erentiation and activation. However, as most of
these conclusions have been drawn by overexpression studies
or in pathological conditions such as asthma and atopy, analy-
sis of SOCS3-defi cient mice has been necessary to clarify the
physiological function of SOCS3 in T cells more precisely.
Because mice lacking SOCS3 die during embryogenesis as
the result of a placental defect by an enhanced activation of
the leukemia inhibitory factor (LIF) signaling pathway (20,
21), we generated T cell–specifi c SOCS3-defi cient (condi-
tional KO [cKO]) mice by a conditional gene targeting ap-
proach using Cre-loxP system (22). We showed that not only
Th2-type responses in OVA-induced asthma model but also
immune responses against Leishmania major infection were re-
duced in cKO mice. In vitro analysis of T cells demonstrated
that SOCS3-defi cient CD4+ T cells produced more TGF-
β1 and IL-10, but less IL-4 than WT T cells, suggesting a
preferential Th3-like diff erentiation. We found that STAT3
bound to the TGF-β1 promoter and elevated the promoter
activity, and SOCS3 deletion enhanced STAT3 recruitment
to the promoter. It has been shown that STAT3 also binds to
the IL-10 promoter and elevates IL-10 gene expression (23).
In conclusion, we propose that STAT3 and SOCS3 recipro-
cally regulate Th cell function and diff erentiation by control-
ling the induction of the immunosuppressive cytokine,
TGF-β1, and IL-10.
Generation of T cell–specifi c SOCS3-defi cient mice
To delete the SOCS3 gene in a T cell–specifi c manner,
proximal p56Lck promoter-cre transgenic mice were crossed
with SOCS3-fl ox/fl ox mice (22) (Fig. 1 A). Resulting
SOCS3-fl ox/fl ox: Lck-cre Tg mice are designated as cKO
mice. SOCS3 WT alleles, fl oxed alleles, and the Lck-cre trans-
gene were determined by PCR using genomic DNA from
tails and CD4+ T cells. A 380-bp product corresponding to
the nondeleted fl oxed alleles was amplifi ed by primer set of a
and b from tail DNA of SOCS3-cKO and WT-fl ox/fl ox
mice (C57BL/6 showed a 280-bp fragment because of the
lack of fl oxed alleles). An approximately 1.2-kbp fragment
was amplifi ed from DNA isolated from CD4+ T cells of
WT-fl ox/fl ox mice by the primer set of a and c, whereas a
250-bp fragment corresponding to the deleted allele was am-
plifi ed from SOCS3-cKO mice CD4+ T cells (Fig. 1 B).
Deletion of the SOCS3-fl ox gene was specifi c to T cells, and
no deletion was observed in B cells and DCs in cKO mice
(Fig. 1 C). Next, to confi rm the deletion of mRNA, we per-
formed RT-PCR analysis in IL-6–stimulated splenic CD4+
T cells from SOCS3-cKO mice. Although SOCS3 was
Figure 1. Generation of T cell–specifi c SOCS3-defi cient mice.
(A) Schema of SOCS3 fl oxed and deleted loci. Exon 2 was fl anked by two
LoxP sites (arrowheads). (B) PCR genotyping of fl oxed alleles using the
primer set of a and b against tail genome and deleted alleles using the
primer set of a and c against genomic DNA of CD4+ T cells from indicated
mice. (C) PCR detection of undeleted and deleted fl oxed alleles using
primer set a and c against genomic DNA from B cells, DCs, and CD4+
T cells. (D) RT-PCR analysis for mRNA expression of SOCS3, IL-6R, and
glyceraldehydes-3-phoshate dehydrogenase (G3PDH) in IL-6–stimulated
MACS purifi ed splenic CD4+ T cells from cKO and WT mice. (E) Western
blotting analysis for SOCS3 and phosphorylated STAT3 in splenic CD4+ T cells.
JEM VOL. 203, April 17, 2006
induced after IL-6 stimulation in WT CD4+ T cells and the
expression of IL-6 receptor was at an almost equal level,
SOCS3 mRNA was undetectable in CD4+ T cells from
SOCS3-cKO mice (Fig. 1 D). Western blotting analysis us-
ing antibody specifi c for SOCS3 also confi rmed the absence
of SOCS3 protein in CD4+ T cells from SOCS3-cKO mice
(Fig. 1 E). Thus, we concluded that cre-mediated deletion of
SOCS3 occurred effi ciently and specifi cally in T cells in
We examined IL-6–mediated STAT3 activation in
SOCS3-defi cient T cells. As shown in Fig. 1 E, IL-6–induced
STAT3 activation was enhanced and prolonged in SOCS3-
defi cient CD4+ T cells. This confi rmed a negative regulatory
function of SOCS3 for the gp130–STAT3 pathway. As in
macrophages (22), SOCS3 defi ciency in T cells did not much
aff ect IL-10–induced STAT3 activation (unpublished data).
Next, we examined development of T cells in SOCS3
cKO mice. Total mononuclear cell numbers of lymphoid or-
gans such as thymus, spleen, and lymph nodes in SOCS3-cKO
mice were almost the same as those in WT mice. Flow cyto-
metric analysis revealed that the ratio of CD4+ or CD8+ SP
cells was not altered in SOCS3 cKO mice, although the num-
bers of CD4− CD8− DN cells were slightly higher in SOCS3-
cKO mice (unpublished data). T cell numbers, the CD4/CD8
Figure 2. Reduced Th2 responses of SOCS3-defi cient T cells in
OVA/alum immunized mice. (A) Analyses of serum OVA-specifi c IgG1,
IgG2a, and IgE titers in cKO and WT mice. Plasma samples were taken from
mice (n = 5) at indicated days after immunization with OVA/alum on days
0 and 14. Ab titers were measured by ELISA and endpoint analysis. Data
indicate mean ± SD. (B) Mice (n = 9 for each group) immunized with
OVA/alum were aerosol challenged with OVA. Airway responsiveness was
determined by the acetylcholine-dependent change in airway pressure in
saline-treated control and OVA-sensitized/challenged WT and SOCS3-cKO
mice. Provocative concentration 200 (PC200), the concentration
at which airway pressure is 200% of its baseline value. Data indicate
mean ± SD. (C) Cell counts in bronchoalveolar lavage fl uid. *, P < 0.05 by
analysis of variance with Bonferroni correction. Data indicate mean ± SD.
(D) Cytokine profi les of Th1 type (IFN-γ), Th2 type (IL-4 and IL-5), and
TGF-β1 and IL-10. Splenic CD4+ T cells isolated from OVA-immunized mice
were restimulated with or without OVA ex vivo for 48 h. Cytokine levels
were determined by ELISA. Data indicate mean ± SD in one representative
experiment with fi ve mice per group out of three independent experiments.
1024 SOCS3 REGULATES IL-10 AND TGF-β1 PRODUCTION | Kinjyo et al.
ratio, and other T cell markers (TCRβ, CD25, CD69,
CD62L) were not altered in the spleen and lymph node of
cKO mice (unpublished data). Therefore, we concluded that
SOCS3 does not play an essential role in T cell development.
Reduced Th2-type response in SOCS3-cKO mice
Previously, we reported that constitutive expression of SOCS3
in T cells causes preferential Th2 diff erentiation of CD4+ T
cells, resulting in hyper IgE production and enhanced OVA-
induced airway hypersensitiveness (18). Thus, we investigated
the eff ect of SOCS3 deletion in T cells on OVA immuniza-
tion. After mice were immunized with OVA and alum as an
adjuvant on days 1 and 14, we examined the Ig levels and
cytokine production. Total IgG1 and IgG2a levels before
immunization were almost the same between WT and cKO
mice (unpublished data). As shown previously (24), OVA/
alum immunization signifi cantly enhanced Th2-mediated Ig
(IgG1 and IgE) production (Fig. 2 A). Interestingly, SOCS3-
cKO mice produced lower levels of IgG1 and IgE than WT
mice did, although Th1-mediated IgG2a production was sim-
ilarly low between WT and cKO mice (Fig. 2 A). Refl ecting
reduced IgE levels, SOCS3-cKO mice exhibited lower sensi-
tivity to airway responsiveness and reduced eosinophil infi ltra-
tion in BAL fl uids in cKO mice after OVA challenge compared
with WT mice (Fig. 2, B and C). These data confi rmed that
SOCS3 levels in Th cells alter type 2 responses in vivo.
Next, cytokine production by OVA restimulation from
splenic CD4+ T cells was examined. When CD4+ T cells
from OVA/alum-immunized mice were restimulated with
OVA in vitro, the Th2-type signature cytokines such as IL-4
and IL-5 were highly produced, whereas in the Th1-type
signature cytokine, IFN-γ levels were very low (Fig. 2 D).
Levels of these Th2-type cytokines from CD4+ T cells were
not signifi cantly diff erent between WT and SOCS3 cKO
mice (Fig. 2 D). However, in several separate experiments,
we always observed that T cells from cKO mice immunized
with OVA/alum produced a slightly lower amount of IL-4
than those from WT mice (Fig. 2 D). We did not observe
enhanced IFN-γ production in T cells from SOCS3-cKO
mice, suggesting that loss of SOCS3 in Th cells did not en-
force Th1 skewing. In contrast with Th2-type cytokines,
TGF-β1 and IL-10 levels were higher in SOCS3-cKO mice
than in WT mice (Fig. 2 D). We also confi rmed high mRNA
expression levels of TGF-β1 and IL-10 by RT-PCR (un-
published data). During in vitro restimulation, no signifi cant
diff erence in the proliferation was observed between WT
and SOCS3-defi cient T cells (unpublished data). These re-
sults indicate that loss of SOCS3 expression in T cells resulted
in lower Th2-type immune responses, which was accompa-
nied with reduced IL-4 levels; however, production of TGF-
β1 and IL-10, but not IFN-γ, was enhanced.
Altered immune responses to L. major infection
in SOCS3-cKO mice
To further determine the role of SOCS3 in Th cell diff erentia-
tion and function, we compared the immune responses against
L. major, the intracellular protozoan parasite infection, the res-
olution of which is strictly dependent on Th1-type immune
responses (25, 26). Because genetic background of both WT
and cKO mice was 129 × C57BL/6 mixed, these mice are
generally expected to be resistant to L. major compared with
susceptible BALB/c mice. Mice were infected subcutaneously
in the right hind footpad with 107 L. major promastigotes, and
lesion development was monitored for 8 wk. Both WT and
cKO mice were resistant to L. major, which was judged by
footpad swelling (Fig. 3 A). However, the number of parasites
remaining in the lesion at 6 wk after infection was higher in
cKO mice than in WT mice (Fig. 3 B). Moreover, serum an-
tibody levels after L. major infection were lower in cKO mice
than in WT mice after infection (Fig. 3 C), suggesting that im-
mune responses against L. major were reduced in cKO mice.
We examined cytokine production in CD4+ T cells from
right popliteal LN at 4 wk after L. major infection (Fig. 3 D).
After in vitro stimulation with L. major antigen, the IFN-γ
level from SOCS3-defi cient CD4+ T cells was comparable
to that of WT CD4+ T cells, suggesting that eff ective Th1
diff erentiation occurred in cKO mice. IL-4 levels were too
low to compare between WT and cKO mice. Next, we
measured IL-10 and TGF-β1 levels in the same supernatant.
As shown in Fig. 3 D, enhanced production of IL-10 and
TGF-β1 was observed in CD4+ T cells from SOCS3-cKO
mice. Similar higher expression of IL-10 and TGF-β1 in the
CD4+ T cells of SOCS3-cKO mice than in WT T cells was
confi rmed by RT-PCR (Fig. 3 E). These data support our
notion that SOCS3-defi cient T cells possess higher potential
to produce IL-10 and TGF-β1 than WT T cells.
Cytokine production from in vitro–differentiated
SOCS3-defi cient T cells
To elucidate the reason why IL-10 and TGF-β1 were ele-
vated in CD4+ T cells from SOCS3-cKO mice, we analyzed
in vitro Th cell diff erentiation. Purifi ed CD4+ T cells were
stimulated under Th0, Th1, and Th2 skewing conditions for
7 d and restimulated with plate-bound anti-CD3ε and anti-
CD28 antibodies. TCR-mediated tyrosine phosphorylation
of cellular proteins and ERK activation in T cells were not
signifi cantly altered in cKO mice (Fig. S1, available at http://
thermore, there was no signifi cant diff erence in proliferation
between WT and SOCS3-defi cient CD4+ T cells after TCR
stimulation (unpublished data). Neither IFN-γ nor IL-4 was
detected in the culture supernatant of both SOCS3-defi cient
and WT T cells under Th0 conditions. Under Th1 diff eren-
tiating condition, the IFN-γ level was slightly reduced in
SOCS3-defi cient T cells compared with WT T cells (Fig.
4 A). In Th2 diff erentiating condition, IL-4 from SOCS3-
defi cient T cells was also signifi cantly lower than that of WT
T cells (Fig. 4 A). In contrast, IL-10 and TGF-β1 levels were
higher in SOCS3-defi cient CD4+ T cells than in WT CD4+
T cells (Fig. 4 B). In Th0 and Th1 conditions, IL-10 and
TGF-β1 levels were very low in both WT and SOCS3-
defi cient T cells (Fig. 4 B and not depicted).
JEM VOL. 203, April 17, 2006
We next examined Th3 diff erentiation, which has been
induced in vitro by culturing CD4+ T cells in the presence of
IL-4, IL-10, and TGF-β1 (11, 27). As previously described,
TGF-β1 levels were especially enhanced in the Th3 condi-
tion compared with the Th2 condition (Fig. 4 B). Under this
Th3 condition, SOCS3-defi cient CD4+ T cells produced
higher levels of IL-10 and TGF-β1 than WT CD4+ T cells
(Fig. 4 B). Collectively, SOCS3 defi ciency caused enhanced
production of TGF-β1 and IL-10, but reduced production
of IL-4 in CD4+ T cells not only in vivo but also in vitro.
STAT3 elevates TGF-훃1 promoter activity
The inhibitory eff ect of SOCS3 is relatively specifi c to STAT3
among six STATs. Therefore, we next investigated whether
STAT3 could directly regulate the TGF-β1 promoter activ-
ity. The 4.1-kb fragment of the 5′-fl anking region of the
murine TGF-β1 gene was fused to the luciferase expression
vector, and promoter activity was examined in HEK293 cells
by transient transfection. Luciferase gene expression was in-
duced not only by high glucose and TGF-β1 itself as de-
scribed previously (28) but also by LIF, suggesting that this
4.1-kb 5′-fragment of the TGF-β1 gene contained STAT3
responsive elements (Fig. 5 A). Co-expression of exogenous
Figure 3. Reduced Th1 responses of SOCS3-defi cient T cells in
L. major infection. (A) Footpad swelling after L. major infection. BALB/c
(a susceptible strain), WT, and SOCS3-cKO mice were inoculated in the right
hind footpad with L. major promastigotes and the size of the footpad lesion
was monitored. Data shown are mean ± SD and are representative of three
independent experiments. BALB/c mice were killed at 4 wk for ethical
reasons. (B) The number of parasites remaining in the footpads 6 wk after
infection. (C) Serum IgG1, IgG2a, and IgE levels against L. major antigen in
infected mice. Samples were collected from WT (open squares) and cKO
(closed squares) mice 4 wk after infection. Total IgG1, IgG2a, and IgE titers
were determined by ELISA. (D) Cytokine production by CD4+ T cells of the
right popliteal LN from WT and cKO mice 4 wk after L. major infection. CD4+
T cells were cultured with irradiated naive WT splenocytes with (black bar) or
without (gray bar) L. major antigen for 70 h. Concentrations of IFN-γ, IL-4,
IL-10, and TGF-β1 in the culture supernatant were measured by ELISA. Data
indicate mean ± SD of triplicate samples from fi ve mice per group in one
representative experiment out of three independent experiments. (*, P <
0.01). (E) IFN-γ, IL-10, and TGF-β1 mRNA levels determined by RT-PCR
using total RNA from CD4+ right popliteal LN 4 wk after L. major infection.
Figure 4. Cytokine production from in vitro–differentiated CD4+
T cells. IFN-γ and IL-4 production from in vitro–differentiated Th0/Th1/
Th2 cells (A), and IL-10 and TGF-β1 production from Th0/Th2/Th3 cells (B).
Naive CD4+ T cells were cultured under various differentiation conditions
for 7 d as described in Materials and methods. After restimulation with
anti-CD3ε mAb and anti-CD28 mAb for 24 h for IFN-γ, and with IL-4 and
IL-10 for 72 h for TGF-β1, culture supernatants were collected and ana-
lyzed by ELISA. Data indicate mean ± SD of triplicate cultures in one rep-
resentative experiment out of three independent experiments. *, P < 0.01.
1026 SOCS3 REGULATES IL-10 AND TGF-β1 PRODUCTION | Kinjyo et al.
WT STAT3 enhanced LIF-mediated TGF-β1 promoter ac-
tivity in a dose-dependent manner (Fig. 5 A). Furthermore,
constitutive active form of STAT3 (STAT3c) (29) also en-
hanced TGF-β1 promoter activity similar to LIF stimulation
(Fig. 5, A and B), suggesting that STAT3 positively regulates
TGF-β1 promoter. We also confi rmed that STAT3 elevated
TGF-β1 promoter activity in the lymphoid cell line by using
Jurkat cell (unpublished data).
As shown in Fig. 5 B, a reporter assay using a series of
5′-deletion mutants revealed that the LIF-responsive elements
were present upstream of −1755. By searching for potential
STAT3-binding sites with the consensus sequences, TTC/
A(N)3G/TAA (30), two candidates of STAT3-binding sites
were identifi ed in the 4.1-kb TGF-β1 5′-fl anking region.
The two sites were at positions −3155 and −2515 upstream
of the transcription initiation site in the TGF-β1 promoters
designated STAT3-binding element (SBE)-1 and SBE-2, re-
spectively. To determine the signifi cance of these elements,
mutations were introduced into the SBE-1 and/or SBE-2
sites. A mutant promoter lacking both SBE-1 and SBE-2
did not respond to LIF stimulation anymore, whereas con-
structs containing a single SBE site still responded to STAT3
(Fig. 5, B and C). These results indicate that the two SBE
sites of the TGF-β1 promoter are important for STAT3-
To confi rm STAT3 binding to the TGF-β1 promoter in
T cells, chromatin immunoprecipitation (ChIP) assay was
performed (Fig. 5 D). The chromatin–DNA complex was
immunoprecipitated with anti-STAT3 antibody; then,
STAT3 binding to the TGF-β1 promoter was analyzed using
pairs of specifi c primers spanning the STAT3 binding sites.
The SBE site of the c-fos promoter was used as a positive
control of STAT3 recruitment (31). As shown in Fig. 5 D,
STAT3 was actually bound to the TGF-β1 promoter region
containing SBE-1 site in T cells in an IL-6–dependent man-
ner. These data indicate that TGF-β1 is a direct downstream
target of STAT3.
STAT3 positively regulates TGF-훃1 and IL-10 induction
in CD4+ T cells
To address whether STAT3 is critical for IL-10 and TGF-β1
production in CD4+ T cells, we introduced a constitutive
active form of STAT3 (STAT3c) or a dominant negative
STAT3 (dNSTAT3) (32) into CD4+ T cells using a bicis-
tronic retroviral vector pMX carrying IRES-GFP (33) (Fig.
6 A). The retrovirus vectors were infected into nonpolar-
ized CD4+ T cells, which were stimulated with plate-bound
anti-CD3ε mAb and anti-CD28 mAb, and on day 4, GFP-
positive cells were sorted. The expression of myc-STAT3
in the sorted GFP-positive CD4+ T cells was confi rmed by
Western blotting (Fig. 6 B). These cells were cultured under
Th0 or Th3 diff erentiating condition for 7 d and analyzed
for cytokine production upon restimulation. We found
that introduction of STAT3c into CD4+ T cells resulted in
higher TGF-β1 and IL-10 production (Fig. 6 C). In con-
trast, dNSTAT3-GFP introduced to CD4+ T cells showed
less production of TGF-β1 and IL-10 (Fig. 6 C). These data
indicate that STAT3 activation is positively involved in the
production of TGF-β1 and IL-10 in CD4+ T cells.
Next, STAT3 recruitment to the TGF-β1 promoter was
compared between WT and SOCS3-defi cient CD4+ T cells
using the ChIP assay. As shown in Fig. 6 D, STAT3 was
recruited to the TGF-β1 promoter region under the Th3,
but not Th0, diff erentiating conditions. Importantly, more
STAT3 was recruited to the TGF-β1 promoter in SOCS3-
defi cient CD4+ T cells than in WT CD4+ T cells under the
Th3 diff erentiating condition. These data suggest that SOCS3
probably regulates the production of TGF-β1 through ap-
propriate tuning of STAT3 activation in CD4+ T cells.
D I S C U S S I O N
Previously, we reported that forced expression of SOCS3 in
T cells resulted in Th2 skewing. SOCS3 expression levels are
high in T cells from patients with asthma and atopy. There-
fore, we concluded that high SOCS3 levels are related to
pathological conditions, especially Th2-type diseases (18).
However, the role of SOCS3 in physiological conditions
has not been clarifi ed. Here, we generated T cell–specifi c
SOCS3-cKO mice and found that the Th2 immune re-
sponses in SOCS3-cKO mice were actually reduced. How-
ever, this is not the result of higher Th1 responses. Our
SOCS3-defi cient CD4+ T cells showed higher TGF-β1 and
IL-10 production compared with control WT CD4+ T cells.
Thus, we suspect that reduced Th2 responses in SOCS3-
cKO mice may be the result of immunosuppression by these
two immunoregulatory cytokines.
We proposed that SOCS3 inhibits Th1 diff erentiation by
suppressing IL-12–mediated signaling (18). We found that
IL-12–induced STAT4 phosphorylation was actually en-
hanced in SOCS3-defi cient T cells compared with WT
T cells (unpublished data). However, similar or only slightly
reduced IFN-γ production occurred in CD4+ T cells from
SOCS3 cKO mice compared with WT mice (Figs. 3 D and
4 A). Furthermore, delay of parasite clearance and reduced
production of antibodies were observed in cKO mice during
L. major infection (Fig. 3, B and C). This may be the result of
immunosuppressive eff ect of TGF-β1 and IL-10 produced
from T cells during infection. Regulatory roles of SOCS3-
defi cient T cells in other immune reactions should be defi ned
in future studies.
Recently, regulatory functions of Th cells have been ex-
tensively studied. CD4+ CD25+ regulatory T (T reg) cells
are recognized as naturally occurring T reg cells and exhibit
immunosuppressive abilities by a mechanism that is depen-
dent on cell-to-cell contact through the interaction of
CTLA-4 with CD86 (34). Though TGF-β1 is shown to be
one of the mechanisms of the immunosuppressive eff ects of
T reg cells (10) and Foxp3 has been shown to be an essential
transcription factor in the generation and function of T reg
cells (35), we did not fi nd any change in the number of CD4+
CD25+ T reg population or Foxp3 expression between
SOCS3-defi cient and WT T cells (unpublished data).
JEM VOL. 203, April 17, 2006
However, regulatory function of SOCS3-defi cient T reg
cells remains to be investigated.
Previous studies have identifi ed another subset of T reg
cells; Tr1 cells (T reg cell 1), which are induced in vitro by
repeated antigen stimulation of T cells in the presence of IL-
10 (36, 37). Tr1 cells produce high levels of IL-10 rather than
TGF-β1 (38). The additional subset of T reg cells is Th3,
which is induced by orally administered antigens. Th3 cells
Figure 5. STAT3 directly enhances TGF-훃1 promoter activity.
(A) HEK293 cells were transfected with 0.2 μg of pTGF4.1-luc, a reporter
gene containing ?4.1 kb TGF-β1 promoter region, and 0.1 μg of β-
galactosidase expression vector (β-gal) together with 0, 0.2, 0.6 μg WT-
STAT3 or STAT3c expression vector. 1 d after transfection, cells were
stimulated with 10 ng/ml LIF or 10 ng/ml TGF-β1 and luciferase activity
was measured after 8 h. Luciferase activities were normalized by β-gal
activities and expressed as fold induction to control cultures defi ned as
1.0. STAT3 expression levels determined by Western blotting analysis as
shown (bottom). (B) Localization of the STAT3 responsive elements in the
TGF-β1 promoter. HEK293 cells were transiently transfected with 0.2 μg
of plasmid containing various fragments of TGF-β1 promoter region and
0.1 μg of β-gal expression vector. 1 d after transfection, cells were
stimulated with 10 ng/ml LIF or 10 ng/ml TGF-β1 for 8 h and harvested.
(C) Effects of point mutations introduced into the SBE-1 and SBE-2
elements. HEK293 cells were transiently transfected with WT or mutant
pTGF4.1-luc plasmids and β-gal plasmid. Cells were stimulated with
10 ng/ml LIF and luciferase activities were measured. Luciferase activities
were normalized by β-gal activities and expressed as fold induction to
control cultures defi ned as 1.0. (D) ChIP assay was performed using chro-
matin from WT CD4+ T cells treated with IL-6 for 3 h and immunopre-
cipitated with antibody against STAT3. The fi nal DNA extractions were
amplifi ed using pairs of primers that cover the STAT3 binding site (SBE-1)
in the TGF-β1 or c-fos promoter region. G3PDH levels were determined
by PCR using samples before immunoprecipitation as input control.
Luciferase activities normalized by β-gal activity are shown as the
means ± SD of three to fi ve experiments.
1028 SOCS3 REGULATES IL-10 AND TGF-β1 PRODUCTION | Kinjyo et al.
can be distinguished from Th2 cells by cytokine profi les; Th3
cells produce much more TGF-β1 and IL-10 and less IL-4
than Th2 cells (11, 39). Because SOCS3-defi cient CD4+ T
cells produce more TGF-β1 and IL-10, they are more likely
to exhibit a Th3-like phenotype.
In this study, we demonstrated that STAT3 directly binds
to the promoter region of TGF-β1 and elevates TGF-β1
production in T cells. It has already been shown that IL-10 is
up-regulated by STAT3 (23). We showed that constitutive
active form of STAT3 enhanced TGF-β1 and IL-10 produc-
tion in T cells. Furthermore, we showed that a dominant
negative STAT3 suppressed TGF-β1 production (Fig. 6 C).
Therefore, STAT3 could be a positive regulator of Th3-type
diff erentiation. STAT3 being required for Th3 is unlike
STAT4 and STAT6 being required for Th1 and Th2, respec-
tively, because basal transcription of TGF-β1 and IL-10 is
not completely dependent on STAT3. However, STAT3 is
an important regulatory factor for Th3 diff erentiation because
STAT3 is essential for the immunosuppressive function of
IL-10 in macrophages (40) and IL-10 is usually necessary for
induction of Th3 in vitro. Collectively, STAT3 seems to
positively regulate induction and/or diff erentiation of Th3.
A question that remains unsolved is what kind of cyto-
kines are regulated by SOCS3 during Th3-like phenotype
induction. Previously, IL-4 has been shown to induce SOCS3
expression in Th2 cells (17). However, IL-4–induced STAT6
phosphorylation levels were not aff ected in SOCS3-defi cient
T cells (unpublished data). Therefore, it is unlikely that
SOCS3 directly regulates IL-4 signaling. Because STAT3 is
strongly activated by IL-10, we compared IL-10–induced
STAT3 activation between WT and SOCS3-defi cient T cells.
In SOCS3-defi cient T cells, however, IL-10–mediated
STAT3 activation was not much aff ected (unpublished data).
This is probably because SOCS3 does not bind to the IL-10
receptor (22). In contrast, we observed stronger and pro-
longed STAT3 activation in response to IL-6 and IL-27 in
SOCS3-defi cient T cells (Fig. 1 E and not depicted).
Furthermore, STAT3 recruitment to the TGF-β1 promoter
under Th3 diff erentiation condition was enhanced in SOCS3-
defi cient CD4+ T cells. Although we could not conclude
that IL-6 is responsible for the Th3-like phenotype of
SOCS3-defi cient CD4+ T cells, these results suggest that
STAT3 is hyperactivated in SOCS3-defi cient T cells during
T cell diff erentiation, and this is the result of the hypersensi-
tivity to autocrine or paracrine cytokines that activate STAT3.
Identifi cation of these cytokines other than IL-10, which
modulate TGF-β1 and IL-10 production, will be important
for understanding of the regulation of Th3 diff erentiation.
Another possibility for answering the unsolved question
is that SOCS3 aff ects TCR signaling. SOCS3 has been shown
to be able to interact with tyrosine kinase Lck, calcineurin,
and CD28 (13–16). The level of SOCS3 expression is signifi -
cantly high in resting CD4+ T cells and rapidly decreased af-
ter TCR stimulation (unpublished data). Some reports have
shown that the strength of TCR stimulation is an important
factor for Th diff erentiation. Although we could not detect
apparent diff erences in proliferation, tyrosine phosphoryla-
tion of cellular proteins, and ERK activation between
SOCS3-defi cient and WT T cells in response to TCR stimu-
lation (Fig. S1 and not depicted), the absence of SOCS3 in
naive CD4+ T cells may permit some stronger TCR signal-
ings, which might lead to higher IL-10 and TGF-β1 secre-
tion at an early stage of T cell activation, thereby leading to
large diff erences at later stages of T cell diff erentiation.
Although the more detailed molecular basis of the hyper-
production of TGF-β1 and IL-10 in SOCS3-defi cient T
cells has remained elusive, our biochemical analyses suggest
Figure 6. Retroviral transduction of STAT3 mutants modulates
TGF-훃1 and IL-10 production. (A) Schematic structure of the retroviral
pMX vectors containing mutant STAT3, either myc-STAT3c (constitutive
active form) or myc-dNSTAT3 (dominant negative form). (B) GFP-positive
cells were sorted from infected T cells and the expression levels of exoge-
neous myc-STAT3 were examined by Western blotting. (C) IL-10 and
TGFβ1 production from infected CD4+ T cells after Th3 differentiation.
GFP-positive cells were cultured in the presence of IL-4, IL-10, and TGF-
β1 for 7 d and restimulated with anti-CD3ε mAb and anti-CD28 mAb and
cytokines in the culture supernatants were measured by ELISA. Data
shown are mean ± SD of triplicate samples from four independent exper-
iments. (D) ChIP assay to compare STAT3 recruitment to TGF-β1 promoter
(SBE-1 site) between Th0 and Th3 differentiated T cells from WT and cKO
mice. Anti-STAT3 Ab immunoprecipitates were used as templates for PCR
cells. A non-SBE region near the transcription initiation sites was ampli-
fi ed as a negative control. Ratios of the bands intensity of SBE-1 PCR
products and those of control (G3PDH) in two independent experiments
are plotted (right).
JEM VOL. 203, April 17, 2006
that SOCS3 regulates TGF-β1 and IL-10 production by
suppressing STAT3 activity. Thus, we propose that STAT3
and SOCS3 reciprocally regulate Th2/Th3 diff erentiation.
Therefore, suppression of SOCS3 expression in T cells may
possibly be one of the ways to introduce tolerance for auto-
immune diseases or to ameliorate allergic diseases.
MATERIALS AND METHODS
Generation of T cell–specifi c SOCS3-disrupted mice. SOCS-3 fl ox/
fl ox mice (22), and Lck-cre transgenic mice (41) have been described else-
where. Lck-cre transgenic mice (C57BL/6 background) were bred with
SOCS3 fl ox/fl ox mice (129 × C57BL/6 background) to generate mice in
which SOCS3 was deleted in a T cell–specifi c manner. Genotypings were
performed by PCR as described previously (22). Off spring carrying both lck-
cre and fl oxed SOCS3 genes (Lck-Cre:SOCS3 fl ox/fl ox; cKO) and the fl oxed
SOCS3 gene (SOCS3 fl ox/fl ox; WT) were used for intercrossing and further
analyses. Littermate controls were used for all experiments. CD4+ T cells,
splenic B cells, and DCs were isolated by MACS sorting as described previ-
ously (42). Mice were kept in specifi c pathogen-free facilities in the Collab-
orative Station Animal Facility of Kyushu University. All experiments using
these mice were approved by and performed according to the guidelines of
the Animal Ethics Committee of Kyushu University, Fukuoka, Japan.
OVA/alum immunization and assay for airway hyperresponsiveness.
Alhydrogel (alum; Al(OH)3gel) (LSL) was mixed with a predetermined quan-
tity of OVA grade V (Sigma-Aldrich) and incubated at room temperature for
20 min. After centrifugation of the mixture at 14,000 g for 10 min, superna-
tants were used for immunization as described previously (24). Mice (8–12
wk old) were immunized with 0.1 ml of OVA (10 μg) in PBS and absorbed
to alum. Boosting inoculations were performed in the same fashion 2 wk
later. For airway hyperresponsiveness (AHR) and eosinophil infi ltration as-
say, mice received aerosol challenge containing either saline or 1% OVA for
20 min/d on days 26–28 (18, 43). On day 30, 36 h after the last aerosol chal-
lenge, mice were ventilated and AHR to acetylcholine aerosol was measured.
Serum levels of total and OVA-specifi c Ig was analyzed by ELISA with rat
anti–mouse Ig (Serotec Ltd.). Ab titers were determined by endpoint analysis.
For cytokine assays, splenocytes from immunized mice on day 28 were cul-
tured ex vivo in the presence of OVA. Culture supernatants were harvested
after 48 h and analyzed for IL-4, IFN-γ, TGF-β1, and IL-10 by ELISA.
L. major infection and cytokine assay. Infection of L. major was performed
as described previously (44). Mice were infected s.c. in the right footpad
lesion with 107 stationary phase of L. major (MHOM/SU/73-5-ASKH).
Footpad swelling was monitored weekly by a vernier caliper and compared
with the thickness of the uninfected left footpad. 6 wk after infection, the
footpad parasite burdens were quantifi ed by homogenizing tissue in 3 ml of
medium 199 supplemented with 10% FCS containing 2 mM glutamine, 10
mM Hepes, and 100 μl/ml gentamicin. Aliquots were diluted serially across
96-well plates and scored at 1 wk for the presence of motile promastigotes.
4 wk after L. major infection, CD4+ T cells (5 × 105/200 μl/well) from the
right popliteal LN were stimulated with or without L. major antigens (equiv-
alent to 5 × 105 promastigotes) in the presence of irradiated (30 Gy) spleno-
cytes for 70 h. Culture supernatants were collected and analyzed for IL-4,
IFN-γ, TGF-β1, and IL-10 by ELISA. Total RNA was prepared from
MACS-purifi ed CD4+ T cells of popliteal LN 4 wk after L. major infection,
and the expression level of G3PDH was fi rst evaluated as an internal control.
The pair of primers for TGF-β1 was forward, 5′-T G A C G T C A C T G G A G T-
T G T A C G G -3′ and reverse, 5′-G G T T C A T G T C A T G G A T G G T G C -3′.
The expression levels of IFN-γ and IL-10 were assessed using appropriate
pairs of primers described previously (44).
In vitro T cell diff erentiation assay. For in vitro T cell diff erentiation as-
says, CD4+ T cells (106 cells/ml) purifi ed by MACS columns (Miltenyi Bio-
tec) from splenocytes after depletion of red blood cells were cultured in
RPMI 1640 containing 10% FCS and stimulated with 1 μg/ml of plate-
bound anti-CD3ε mAb, 1 μg/ml anti-CD28 mAb, and 1 ng/ml mIL-2
(PeproTech) supplemented with anti–IFN-γ and anti–IL-4 antibodies for
Th0 diff erentiation, 10 ng/ml mIL-12 (PeproTech) for Th1 diff erentiation,
10 ng/ml mIL-4 (PeproTech) for Th2 diff erentiation, or 10 ng/ml mIL-4,
10 ng/ml mIL-10 (PeproTech), and 10 ng/ml hTGF-β1 (PeproTech) for
Th3 diff erentiation (11). Cells were collected after 7 d and washed, and an
equal numbers of viable cells (106 cells/ml) were restimulated with plate-
bound anti-CD3ε mAb and 1 μg/ml anti-CD28 mAb in the absence of any
additional cytokines. Supernatants were collected 24 h later, and the produc-
tion of cytokines was measured in duplicate by ELISA (Genzyme). For mea-
surement of TGF-β1 production, secondary stimulation was done in a
serum-free medium in which Nutridoma SP (Roche Diagnostic) was substi-
tuted for FCS. Supernatant was collected 72 h after secondary stimulation for
TGF-β1 measurement using mTGF-β1 ELISA kit (Promega).
Construction of reporter plasmids. PCR was done to generate the
TGF-β1 promoter plasmid by using mouse genomic DNA as a template.
The nucleotide sequence of the mTGF-β1 promoter has been submitted to
GenBankTM/EBI Data Bank under accession no. L42456.1. A 4.1-kb
XhoI–EcoRI fragment corresponding to nucleotides from −3245 to +845
relative to the determined transcriptional start site of TGF-β1 gene was sub-
cloned into a pGV-basic2 vector (TOYOINKI), pTGF4.1-luc. Reporter
plasmids, including a series of deletion mutants of the TGF-β1 promoter,
were generated by excision at restriction enzyme recognition sites as follows:
−2977 (NcoI), −1755 (HindIII), −1072 (SmaI), −585 (NcoI). To con-
struct SBE-1mt, SBE-2mt, and SBE-1mt/2mt, point mutations were intro-
duced to the two STAT binding elements (SBE-1/SBE-2) by PCR using
the KOD-plus polymerase (TOYOBO) and the sequences are as follows:
SBE-1mt, 5′-G C A G A C G C T G G G A C T G A -3′ and SBE-2mt, 5′-T T C T C-
T G A C C G G G A C C A T T T T -3′ (mutated sites are underlined). The sub-
cloned PCR products were sequenced to confi rm that the products were the
authentic promoter fragments.
Transfection and luciferase assay. HEK293 (105 cells) were seeded on
six-well plates, cultured for 24 h, and transfected with various amounts of an
expression vector of WT-STAT3-pCDNA3 or STAT3c-pRcCMV along
with 0.2 μg of TGF-β1-pGVbasic2 and 0.1 μg of β-galactosidase (β-gal)
plasmid by the calcium phosphate coprecipitation method. Some of them
were stimulated with LIF (10 ng/ml) or TGF-β1 (10 ng/ml) for 8 h. Cells
were harvested in 40 μl lysis buff er. Luciferase assay was performed using a
luciferase substrate kit (Promega) and luciferase activity was read in Packard
luminometer. Luciferase activity was normalized by the internal control
β-gal activity, and shown as the means ± SD of three to fi ve experiments.
ChIP assay. ChIP assay was performed in 107 mouse T lymphocytes. Cells
were fi xed with 1% formaldehyde at 37°C for 10 min after IL-6 stimula-
tion as described previously (31). Cells were washed, suspended in SDS
lysis buff er, and sonicated for 30-s pulses four times using a sonicator (Bi-
oruptor; Cosmo Bio Co.). Samples were incubated with 5 μg anti-STAT3
antibody (C-20; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After
adding salmon sperm DNA and protein A–Agarose Slurry (UBI), the im-
munoprecipitates were sequentially washed with low-salt buff er, high-salt
buff er, LiCl buff er and twice with TE buff er. The DNA–protein complex
was eluted into elution by heating at 65°C for 6 h. Proteins were digested
by proteinase K and RNA was removed by addition of 10 μg of RNase A.
DNA was recovered by extraction with phenol and chloroform and ethanol
precipitation and subjected to PCR analysis. To estimate the DNA content
in the soluble chromatin samples, DNA was similarly extracted from
sonicated samples and used as a template for G3PDH gene amplifi cation.
Promoter-specifi c primers were as follows; mTGF-β1, SBE-1 forward:
5′-T G A C T A A C G G C A C T G A G G A G G C T G C -3′, SBE-1 reverse: 5′-T G G-
A A A C A G G T C T A T C T T C T A C C T A -3′, which amplify 311-bp fragments
fl anking the STAT3 binding element. For negative control, 5′ franking
region close to the transcription initiation site was amplifi ed by forward:
1030 SOCS3 REGULATES IL-10 AND TGF-β1 PRODUCTION | Kinjyo et al.
5′-G T G C C T C C T T G T A T C C G C T A A A G C T C T C -3′ and reverse: 5′-A C-
T A C T A A A G C C G G T G A C C A A C C A A A G -3′. The mouse c-fos primers for
the positive control of STAT3-ChIP are forward: 5′-T C T G C C T T T C C C-
G C C T C C C C -3′ and reverse: 5′-G G C C G T G G A A A C C T G C T G A C -3′.
Retroviral constructs and transduction to primary T cells. The
STAT3c-IRES-GFP-pMX, dNSTAT3-IRES-GFP-pMX, and empty GFP-
pMX plasmids (a gift from T. Kitamura, Tokyo University, Tokyo, Japan)
were transfected into a packaging cell line, Plat-E (33), using FuGENE6
(Roche Diagnostic), and after incubation for 48 h, the culture supernatant
were harvested. CD4+ enriched T cells were stimulated with 1 μg/ml anti-
CD3ε mAb and 1 μg/ml anti-CD28 mAb for 24 h and infected with the vi-
ruses by adding the viral containing supernatants in the presence of 0.6
μg/ml polybrene (Sigma-Aldrich). The infected CD4+ T cells were ex-
panded in the medium supplemented with 100 U/ml rIL-2 for 4 d. GFP-
positive cells were collected by a cell sorter (EPICS ALTRA; Beckman
Coulter) and restimulated with 1 μg/ml anti-CD3ε mAb and 1 μg/ml anti-
CD28 mAb or Th3-inducing condition for 72 h. Culture supernatants were
harvested after 48 h to analyze TGF-β1 and IL-10 production by ELISA.
Online supplemental material. Fig. S1 shows tyrosine phosphorylation
of cellular proteins (anti-pY blot) and ERK activation in CD4+ T cells from
WT and cKO mice after TCR stimulation. Online supplemental material is
available at http://www.jem.org/cgi/content/full/jem.20052333/DC1.
We thank Dr. J. Takeda, Dr. M. Kubo, and Dr. S. Hori for their advice. We also thank
Mr. N. Kinoshita, Ms. M. Othsu, Ms. Y. Yamada, Mr. M. Sasaki, and Ms. E. Fujimoto
(Technical Support Center, Medical Institute of Bioregulation) and Ms. T. Yoshioka
for technical support and Ms. Y. Nishi for manuscript preparation.
This work was supported by special grants-in-aid from the Ministry of
Education, Science, Technology, Sports, and Culture of Japan, Yamanouchi
Foundation for Research on Metabolic Disorders, Takeda Science Foundation and
the Uehara Memorial Foundation. I. K. is supported by Postdoctoral Fellowship from
the Japan Society for the Promotion of Science.
The authors have no confl icting fi nancial interests.
Submitted: 21 November 2005
Accepted: 10 March 2006
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