of June 13, 2013.
This information is current as
Responses through Both T-bet
STAT1-Activating Cytokines Limit Th17
Alejandro V. Villarino, Eugenio Gallo and Abul K. Abbas
2010; 185:6461-6471; Prepublished online 25
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by guest on June 13, 2013
The Journal of Immunology
STAT1-Activating Cytokines Limit Th17 Responses through
Both T-bet–Dependent and –Independent Mechanisms
Alejandro V. Villarino, Eugenio Gallo, and Abul K. Abbas
Given the association with autoimmune disease, there is great interest in defining cellular factors that limit overactive or misdir-
ected Th17-type inflammation. Using invivo and invitro models, we investigated the molecular mechanisms for cytokine-mediated
inhibition of Th17 responses, focusing on the role of STAT1 and T-bet in this process. These studies demonstrate that, during
systemic inflammation, STAT1- and T-bet–deficient T cells each exhibit a hyper-Th17 phenotype relative to wild-type controls.
However, IL-17 production was greater in the absence of T-bet, and when both STAT1 and T-bet were deleted, there was no
further increase, with the double-deficient cells instead behaving more like STAT1-deficient counterparts. Similar trends were
observed during in vitro priming, with production of Th17-type cytokines greater in T-bet2/2T cells than in either STAT12/2or
STAT12/2T-bet2/2counterparts. The ability of IFN-g and IL-27 to suppress Th17 responses was reduced in T-bet–deficient cells,
and most importantly, ectopic T-bet could suppress signature Th17 gene products, including IL-17A, IL-17F, IL-22, and retinoic
acid-related orphan receptor gT, even in STAT1-deficient T cells. Taken together, these studies formally establish that, down-
stream of IFN-g, IL-27, and likely all STAT1-activating cytokines, there are both STAT1 and T-bet–dependent pathways capable
of suppressing Th17 responses.The Journal of Immunology, 2010, 185: 6461–6471.
output. Among these, the recently identified Th17 subset has been
increasingly linked to autoimmunity with its signature products,
IL-17A and IL-17F, implicated in the pathogenesis of numerous
inflammatory disorders including arthritis, multiple sclerosis, graft-
versus-host disease, and psoriasis (1). Although multiple factors
are known to influence Th17 responses, cytokines have emerged
as the key negative regulators, with IFN-g, the signature product
of Th1 effector cells, considered one of the most potent inhibitors.
Deletion of IFN-g or its receptor leads to increased IL-17 pro-
duction and severe clinical manifestations in many of the exper-
imental models where Th17 cells are considered pathogenic,
including arthritis, experimental autoimmune encephalitis (EAE),
and experimental autoimmune uveitis (2–6). IFN-g deficiency also
results in a hyper-Th17 phenotype during in vitro T cell differ-
entiation, and consistent with a T cell-intrinsic mode of action, the
addition of exogenous IFN-g yields a converse hypo-Th17 phe-
notype (7–9). These findings imply an inverse relation between the
Th1 and Th17 subsets, but it should also be noted that cells pro-
ducing both IL-17A and IFN-g have been reported in numerous
inflammatory settings, and that conversion of IL-17–producing
Th17 cells into IFN-g–producing Th1 cells has been shown to
uring an adaptive immune response, CD4+Th cells
differentiate into multiple effector subsets, each char-
acterized by their transcriptional profile and cytokine
occur both in vivo and in vitro, which points toward develop-
mental plasticity and, perhaps, a linear relation (2, 10–14).
IFN-g exerts its functions through a high-affinity cell surface
receptor that is composed of two chains (IFN-gR1/IFN-gR2) and
is expressed on range of immune and nonimmune cells. In CD4+
T cells, IFN-g propagates a Jak/STAT signaling cascade leading to
robust activation of STAT1 and, to a lesser extent, of STAT3 (15).
Among its principal STAT1-dependent activities in CD4+T cells
is the induction of T-bet, a transcription factor (TF) that is both
necessary and sufficient for Th1 differentiation. T-bet drives ex-
pression of several hallmark Th1 genes, including IL-12Rb2, and
through its ability to drive IFN-g production, it establishes a pos-
itive feedback loop where IFN-g induces T-bet, which, in turn,
triggers more IFN-g (16–20). T-bet deficiency confers resistance
in several models of T cell-dependent autoimmunity, like diabetes,
EAE, and colitis (21–24); but in some inflammatory settings, like
allograft rejection and myocarditis, it leads to increased pathology,
typically characterized by a profound a Th1 defect and a corre-
sponding increase in Th17-type cytokines (13, 25–27). Thus, al-
though generally considered proinflammatory, T-bet–driven Th1
responses can also have anti-inflammatory consequences, best
exemplified by their ability to limit Th17-mediated disease.
Like IFN-g, other STAT1-activating cytokines can affect Th17
responses. One pertinent example is IL-27, which mirrors IFN-g
in three key ways: 1) it is a potent STAT1 activator; 2) it induces
expression of T-bet; and 3) it inhibits key Th17 gene products, like
IL-17A, IL-17F, and retinoic acid-related orphan receptor gT
(RORgT) (28). Consistent with this analogy, IL-27R- and IL-27R-
deficient mice, similar to IFN-g2/2and IFN-gR2/2mice, exhibit
severe pathology and increased Th17 responses in models of
T cell-dependent autoimmunity, including toxoplasmic encepha-
litis and EAE (9, 29, 30). In vitro studies have shown that IFN-g
and IL-27 cannot suppress IL-17 production in the absence of
STAT1, which suggests a common mechanism, but it remains
unclear whether this is due to direct effects (i.e., STAT1 binding to
Th17-associated loci) and or indirect effects (i.e., STAT1 regu-
lating other inductive/inhibitory factors) (9, 29–33). The role of
T-bet in this process is also poorly understood. It is known that
Received for publication April 23, 2010. Accepted for publication September 19,
This work was supported by National Institutes of Health Grant RO1 AI64677 with
Minority Postdoctoral Supplement PA-05-015 (to A.V.V.).
Address correspondence and reprint requests to Prof. Abul. K. Abbas, Department of
Pathology, University of California San Francisco, 513 Parnassus Avenue, HSW 513;
Box 0511, San Francisco, CA 94143. E-mail address: abul.abbas@UCSF.edu
The online version of this article contains supplemental material.
Abbreviations used in this paper: EAE, experimental autoimmune encephalitis; LNs,
lymph nodes; RORgT, retinoic acid-related orphan receptor gT; sOVA, soluble OVA;
TF, transcription factor; WT, wild-type.
by guest on June 13, 2013
IFN-g and IL-27 can each induce expression of T-bet, and that
ectopic T-bet expression can suppress IL-17 production, but
whether this is due to a cell-intrinsic mechanism or its ability to
drive IFN-g–mediated, STAT1-dependent inhibition has not been
resolved (34, 35). Furthermore, although numerous studies have
shown that IL-27 can limit Th17 responses in the absence of T-bet,
thus demonstrating that it is not required for STAT1-mediated
inhibition, the possibility remains that T-bet–dependent mecha-
nisms are still working in parallel to or in concert with STAT1 (9,
30, 31). The data presented in this study address these latter issues,
formally establishing that, downstream of STAT1-activating cy-
tokines, there are two distinct anti-Th17 pathways: first, the pre-
viously described STAT1-dependent, T-bet–independent pathway;
and second, a T-bet–dependent, STAT1-independent pathway.
Materials and Methods
Gene-deficient donor mice were generated by crossing DO11.10 TCR
transgenics (Jackson Laboratories, Bar Harbor, ME) with the following
BALB/c strains: IFN-g2/2(Jackson Laboratories), T-bet2/2(from L.
Glimcher, Harvard University, Cambridge, MA) (36), IL-17A2/2(from Y.
Iwakura, University of Tokyo, Tokyo, Japan) (37), and IL-4Ra2/2(Taconic,
Germantown, NY) (38). STAT12/2mice from Taconic were used in com-
pliance with their Research Cross-breeding Agreement. These were back-
crossed (more than eight generations) onto the BALB/c background and
then bred with either wild-type (WT) or T-bet2/2DO11.10 mice. Soluble
OVA (sOVA)-transgenic mice were generated as described previously (13)
and bred onto WTor IFN-g2/2Rag2-deficient backgrounds (sOVA Rag22/2
or IFN-g2/2sOVA Rag22/2). Genotyping was done by PCR. All animals
were maintained in specific pathogen-free housing at the University of Cal-
ifornia, San Francisco, and experiments were carried out according to
guidelines set by the Institutional Animal Care and Use Committee.
Lymph nodes (LNs) were dissected from 4- to 6-wk-old donor mice and
stained directly ex vivo with fluorochrome-conjugated anti-CD4, anti-
DO11.10, anti-CD44, and anti-CD25 Abs (eBioscience, San Diego, CA).
Naive CD4+DO11.10 TCR+, CD44low, CD252cells were then purified by
high-speed cell sorting (.99% purity) and intravenously injected into
age-/sex-matched recipients (5 3 105cells in 400 ml PBS per host). For
some experiments, WT or gene-deficient donor mice were crossed onto
a Rag2-deficient background (DO11.10 Rag22/2) and CD4+DO11.10+
cells purified by positive selection using magnetic beads (.96% purity;
Dynal Beads; Invitrogen, Carlsbad, CA). No difference was observed be-
tween DO11.10 and DO11.10 Rag22/2donor T cells in terms of pro-
liferation or cytokine production (data not shown).
Ex vivo T cell monitoring
Lymphocytes from recipient mice were restimulated overnight with bone
marrow-derived dendritic cells that were preactivated with LPS (1 mg/ml;
Sigma, St. Louis, MO) and preloaded with OVA peptide (1 mg/ml;
5:1 lymphocyte/dendritic cell ratio). Cultures were then treated with bre-
feldin A (10 mg/ml) for 2 h, fixed (4% paraformaldehyde), permeabilized
(0.25% saponin), and stained with anti-CD4 and anti-DO11.10 in combi-
nation with anti–IFN-g, anti–IL-17A, anti–IL-17F, anti–TNF-a, anti–IL-2,
anti–IL-4, and/or anti–IL-13 Abs (eBioscience). Four-color flow cytometry
was performed on a FACSCalibur instrument and analyzed using Cell-
Quest Pro Software (Becton Dickinson, Franklin Lakes, NJ). Logarithmic
scales were used for all dot plots.
In vitro T cell differentiation
LNs and spleens were dissected from WT or gene-deficient mice, pooled,
and CD4+cells purified by positive selection using magnetic beads. These
were then stimulated with plate-bound anti-CD3ε (1 mg/ml, clone: 17A2)
and soluble anti-CD28 Abs (1 mg/ml, clone: 37.51) at a density of 1 3 106
cells/ml in flat-bottomed 24- or 96-well plates (Sigma/Costar). After 72 h,
they were restimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml;
4 h total with brefeldin A for the final 2 h), and stained for intracellular
cytokine staining as described earlier. For Th17-polarizing conditions,
cultures were supplemented with recombinant human TGF-b (2.5 ng/ml;
R&D Systems, Minneapolis, MN), recombinant mouse IL-6 (10 ng/ml;
eBioscience), and neutralizing anti–IL-4 mAb (10 mg/ml; clone: 11B11;
National Institutes of Health/National Cancer Institute Biological
Resources Branch Preclinical Repository). Where indicated, recombinant
mouse IFN-g or IL-27 was also added (25 ng/ml; eBioscience). All studies
were performed in supplemented tissue culture media (RPMI 1640 with
10% FCS, 1% sodium pyruvate, 1% nonessential amino acids, 0.1%
b-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin; Life
Technologies/Invitrogen, Carlsbad, CA).
For ex vivo studies, CD4+DO11+CD44highcells from recipient mice,
together with naive controls (CD4+DO11+CD252CD44lowcells from
DO11.10 mice), were purified by high-speed cell sorting (2–10 3 104per
group). For in vitro studies, T cells were cultured for 48 h and then col-
lected (no restimulation). For retroviral experiments, T cells expressing
more than one transgene (GFP+Thy1.1+) were purified by high-speed cell
sorting (1–2 3 104per group; Supplemental Fig. 4). For all PCR studies,
mRNA was extracted from purified T cells and converted to cDNA using
oligo-dT priming and SuperScript III reverse transcriptase (100–250 ng
RNA per reaction; Invitrogen). PCR amplification was performed with
SYBR green master mix (10–50 ng cDNA per reaction; Applied Bio-
systems, Foster City, CA) using an iQ5 real-time PCR thermal cycler (Bio-
Rad, Hercules, CA). Primer sequences and relevant information are pro-
vided in Supplemental Table I. Reactions were performed in duplicate, Ct
values normalized to b-actin levels, and fold induction (n . 1) or re-
duction (n , 1) of each gene calculated (DDCt) with respect to the in-
dicated controls (n = 1).
Retroviral Gene Transduction
RORgT cDNA was PCR-amplified from Th17-polarized BALB/c CD4+
T cells using a high-fidelity polymerase (Easy A; Stratagene, Cedar Creek,
TX). PCR products were then digested, ligated into a modified MIG-R1
vector (directly upstream of the internal ribosome entry site and Thy1.1
marker), and recombinant clones with ∼100% sequence homology to the
National Center for Biotechnology Information GenBank mRNA entry
(accession No. AJ132394) were selected for further amplification (re-
striction enzymes and T4 ligase from NEB, Ipswich, MA; PCR sequencing
by Sequetech, Mountain View, CA; Mini- and Maxi-prep kits from Qiagen,
Germantown, MD). MIG-R1 expressing T-bet followed by an IRES and
GFP was generated as described and kindly provided by S. Reiner (Uni-
versity of Pennsylvania, Philadelphia, PA) (19). RORgT and T-bet vectors,
together with corresponding “empty” controls, were transfected into
Phoenix packaging cells (together with pCL-Eco helper plasmid), and the
resulting culture supernatants were used to infect T-bet2/2or STAT12/2
T cells. These were cultured under nonpolarizing conditions (6 anti–IFN-
g) for 36 h, exposed to viral supernatant for 1 h (at 2200 rpm, 19˚C), and
then cultured for an additional 36 h.
Paired Student t test (one-tailed) was used to measure statistical deviation
between experimental groups. In all figures, an asterisk represents signifi-
cant differences (p , 0.05–0.08); when this symbol is not used, p values are
Kinetics of Th1 and Th17 responses during
lymphopenia-induced autoimmune disease
To investigate mechanisms of T cell-mediated autoimmunity, we
have developed a mouse model where naive, TCR-transgenic
T cells (DO11.10) are adoptively transferred into lymphopenic
hosts that express their cognate Ag, chicken OVA, as a soluble
protein in the bloodstream (sOVA Rag22/2). This highly immu-
nogenic environment results in an aggressive T cell response
characterized by distinct waves of effector subsets, with a rapid
expansion of Th17 cells followed by a more prolonged Th1-type
response. Previous work has shown that, in this setting, IFN-g and
T-bet–deficient T cells exhibit a hyper-Th17 phenotype, thereby
illustrating the anti-Th17 capacity of the IFN-g/T-bet axis and
validating this as a robust in vivo model for studying the regula-
tion of Th17 responses (13).
For our basic experimental setup, naive CD4+T cells were
purified from DO11.10 Rag22/2mice and adoptively transferred
6462 STAT1 AND T-bet LIMIT Th17 RESPONSES
by guest on June 13, 2013
into sOVA Rag22/2hosts or, ascontrols, into lymphopenic Rag22/2
hosts that lack the sOVA transgene. From 3 to 10 d later, LNs were
dissected from recipient animals, restimulated overnight, and cy-
tokine production measured by intracellular flow cytometry (IFC).
We found that, soon after transfer (days 3–4), there was a massive
accumulation of Th17 cells in sOVA Rag22/2hosts, with about
half of the donor cells producing IL-17A and a quarter producing
high levels of IL-17F (Fig. 1A). At later time points (days 7–9), this
Th17 response was depressed, as evidenced by the . 2-fold
reductions in IL-17A and IL-17F, whereas a Th1-type response
dominated, as evidenced by sustained production of IFN-g. In
control Rag22/2hosts, therewere few IL-17A– or IL-17F–positive
donor T cells at the early time point, but, surprisingly, these were
readily detected at the later time point, which suggests that, even in
the absence of a high-affinity Ag, lymphopenia itself is permissive
for Th17 differentiation. However, it should also be noted that, in
contrast with sOVA Rag22/2hosts, this “homeostatic” Th17 re-
sponse was slower, lesser in magnitude (in terms of total cytokine-
producing cells), and ultimately, did not elicit autoimmune disease,
which suggest that Ag is still key for determining the kinetics and
pathogenicity of donor T cell responses. In addition, few IFN-g+
donor cells could be detected in Rag22/2hosts (Fig. 1A). Given
that T cells are known to produce IFN-g under such lymphopenic
conditions (39, 40), this finding reflects both the importance of Ag
and the fact that, although day 7 is a late time point for moribund
sOVA Rag22/2hosts, it is a relatively early time point compared
with previous studies on “Ag-independent” homeostatic Th1 re-
To further investigate the inflammatory response in sOVA
Rag22/2mice, we purified donor T cells and used real-time time
PCR to measure Th17-associated cytokines, receptors, and TFs.
Consistent with our IFC measurements, we found the early time
point was associated with prominent induction of IL-17A, IL-17F,
IL-22, and RORgT, whereas the later time point was associated
with a .2-fold decline in all of these transcripts (Fig. 1B). Two
Th17-associted receptors, IL-1R1 and IL-23R, were also strongly
induced, but in those cases, mRNA levels continued to escalate
throughout the course of study. Th1-type transcripts IFN-g, IFN-
gR2, T-bet, and IL-12Rb2 were highly expressed at all time points,
as was IL-21, which is associated with both Th1- and Th17-type
inflammation (Fig. 1B). Together with our IFC studies, these data
suggest a complex relation between Th17 and Th1 responses; there
appears to be some chronology, with the former preceding the latter,
but there is also evidence for concurrent expression of both Th1-
and Th17-type factors within the donor population, if not within
individual donor cells.
To explore the relation between Th17 and Th1 cells in sOVA
Rag22/2hosts, we used IFC to measure coexpression of IL-17A
and IFN-g. Consistent with previous work (13), we found that a
large fraction of donor T cells produced both cytokines, and that,
sOVA Rag22/2hosts. Three to 9 d later, lymphocytes from recipient mice were restimulated ex vivo and cytokine production measured by IFC. Shown is
the percentage of cytokine-positive donor cells (CD4+DO11.10+) at the indicated time points. Day 0 represents cytokine production in naive controls. Data
are compiled from 5 experiments (5–10 mice/group) and shown is the fold change for sOVA Rag22/2hosts comparing the early and late time points. B,
Adoptive transfers were performed as in A. CD4+DO11.10+CD44highdonor cells were purified by high-speed cell sorting and mRNA levels quantified by
real-time PCR. Data are representative of four experiments and are presented as the fold increase (x . 1) or decrease (x , 1) relative to naive controls (x =
1). Shown is the fold change for the indicated mRNAs comparing the early and late time points. C, Adoptive transfers and restimulations are performed as
in A. Dot plots indicate the percentage of donor T cells producing IL-17A and or IFN-g/IL-17F in sOVA Rag22/2hosts. Bottom panels, Histograms denote
the percentage of donor T cells expressing detectable levels of IL-17F (see Supplemental Fig. 1 for additional IL-17F analysis). D, IL-17A/IFN-g and IL-
17A/IL-17F coexpression data are compiled from three to four individual experiments.
Kinetics of Th1 and Th17 responses during systemic autoimmune disease. A, Naive DO11.10 CD4+T cells were transferred into Rag22/2or
The Journal of Immunology6463
by guest on June 13, 2013
over time, these double-positive cells declined with similar ki-
netics to IL-17A single-positive counterparts (Fig. 1C, 1D). We
also found that, in both sOVA Rag22/2and Rag22/2hosts, most
IL-17A+donor cells coexpressed IL-17F. However, there was
a notable difference in the flow cytometry for IL-17A and IL-
17F, with the former exhibiting a bimodal distribution and the
latter exhibiting a more unimodal distribution and a whole-sale
population shift rather than a clear demarcation between positive
and negative events (Fig. 1C, bottom panels). Whether this reflects
the actual protein output or a technical issue (i.e., Ab affinity)
remains unknown, but given this caveat, we performed a detailed
analysis to better gauge IL-17F production. This examination
revealed that, whereas cells expressing low levels IL-17F+cells
were better visualized using histograms than dot plots, all of the
observed trends, including time-dependent inhibition and coex-
pression with IL-17A, were similar between these two types of
analysis (Supplemental Fig. 1). In addition, both show that, al-
though most IL-17A+cells (.60%) expressed detectable levels of
IL-17F, there were some that did not, which is consistent with
published reports on the heterogeneity of Th17 cells (10, 41, 42).
Taken together, these studies demonstrate that, when primed under
lymphopenic conditions, helper T cells can differentiate into “clas-
sic” Th1 and Th17 effectors, as well as a hybrid subset that shares
the defining characteristics of both. They also suggest a hierarchy,
with IFN-g–producing Th1 cells eventually dominating over IL-17–
producing “Th17-like” subsets.
T-bet and STAT1 limit Th17 responses during systemic
To investigate the role of IFN-g in limiting Th17 responses, we
crossed both our donor and recipient mouse strains onto an IFN-
g–deficient background and performed adoptive transfer experi-
ments with each of the four possible donor/host combinations. We
found that host-derived IFN-g had the greatest impact, with pro-
duction of IL-17A and IL-17F greater in IFN-g–deficient hosts
than in WT sOVA Rag22/2counterparts (Fig. 2A, 2B, and Sup-
plemental Fig. 1). A similar trend was observed when donor
T cells were transferred into IFN-g–deficient Rag22/2mice only;
in this study, a combination host and donor-derived IFN-g was
required to limit “homeostatic” Th17 responses (Fig. 2C and Sup-
g2/2DO11.10 donors were adoptively transferred into either WT or IFN-g2/2sOVA Rag22/2hosts. Shown is the percentage of cytokine-positive donor
cells after 7 d. B, Data are compiled from three experiments (see Supplemental Fig. 1 for additional IL-17F analysis). C, Naive CD4+T cells from WT or
IFN-g2/2Rag22/2DO11.10 donors were adoptively transferred into either WTor IFN-g2/2Rag22/2hosts (no Ag). Shown is the percentage of cytokine-
positive donor cells compiled from 3 experiments (n = 3–6 mice/group). B and C, Single asterisk denotes significant differences between the indicated
group and the WT into WT group. Double asterisk denotes significant differences between the indicated group and the WT into IFN-g2/2group. p , 0.05.
T cell and non-T cell-derived IFN-g can limit Th17 responses during systemic autoimmune disease. A, Naive CD4+T cells from WTor IFN-
6464STAT1 AND T-bet LIMIT Th17 RESPONSES
by guest on June 13, 2013
plemental Fig. 1). Consistent with this result, and the idea that
non-T cell-derived IFN-g can play a major role, we noted that
IFN-g+cells were readily detectable in the non-T cell fraction of
our restimulation cultures, and that when WT T cells were trans-
ferred into IFN-g2/2sOVA Rag22/2recipients, the percentage of
IFN-g+donor cells was dramatically reduced (Fig. 2A, 2B, and
Supplemental Fig. 2). In contrast, genetic ablation of IFN-g had
little effect on T cell TNF-a or IL-2 production whether in sOVA
Rag22/2or Rag22/2host (Fig. 2B and data not shown). Together
with previous studies (13), these data indicate that both T cell- and
non-T cell-derived IFN-g can limit Th17-type inflammation in
To better define the mechanism for IFN-g–mediated suppres-
sion, we compared the ability of STAT1- and T-bet–deficient
T cells to generate Th17 responses in sOVA Rag22/2hosts. We
found that, although each exhibited a hyper-Th17 phenotype rel-
ative to WT controls, IL-17 production was greater in T-bet2/2
donors than in STAT12/2counterparts (Fig. 3 and Supplemental
Fig. 1). To ask whether they operate in the same inhibitory
pathway, we generated donor mice lacking both T-bet and
STAT1 (T-bet2/23 STAT12/2DO11), and compared Th17 re-
sponses with those of T-bet– or STAT1-deficient donors. Surpris-
ingly, when both T-bet and STAT1 were deleted, there was no
additive effect, with the double-deficient cells instead behaving
more like STAT12/2cells (Fig. 3A, 3B). The loss of T-bet and or
STAT1 led to similarly profound defects in IFN-g production and
had little impact on TNF-a or IL-2 (Fig. 3 and data not shown).
Taken together, these data imply that hyper-Th17 phenotype of
T-bet–deficient donors cannot be fully explained by a lack of
STAT1-dependent regulation. Furthermore, though STAT1 is
known to be a potent inducer of T-bet, these data also argue that
the milder hyper-Th17 phenotype of STAT1-deficient donors
cannot be fully explained by a lack of T-bet.
Aside from IL-17 production, there were other notable differ-
ences in the behavior of STAT1- and T-bet–deficient donors in
sOVA Ra22/2hosts. Whether T-bet2/2or T-bet+/+, STAT1-defi-
cient T cells did not proliferate to the same extent as either WT or
T-bet2/2counterparts (Fig. 4B). Unlike the hyper-Th17 pheno-
type, this hypoproliferative phenotype cannot be explained by the
loss of IFN-g since, paradoxically, donor T cells were hyper-
proliferative in the absence of IFN-g (Fig. 4A). Compared to T-
bet2/2counterparts, STAT12/2donors also produced more IL-4
and IL-13, two known Th17 inhibitors (Fig. 4C) (7, 8, 43). To
determine the impact of this increased Th2-type response, we
compared IL-17 production from donors lacking STAT1 with that
of donors lacking STAT1 and IL-4Ra, a shared receptor compo-
nent for IL-4 and IL-13. Relative to STAT1-deficient counterparts,
the compound loss STAT1 and IL-4Ra had little effect on IL-17
but did lead to significant reductions in IL-4 and IL-13 (data not
Direct evidence for both T-bet– and STAT1-dependent
regulation of Th17 responses
To complement our in vivo studies, we used an in vitro model
where Th17 differentiation could be monitored in the presence or
absence ofSTAT1-activatingcytokines.Asexpected, we foundthat
WT T cells produced IFN-g and almost no IL-17 in nonpolar-
izing cultures. The inverse was true in Th17-polarizing cultures,
where they produced little IFN-g and significantly more IL-17
(Fig. 5A). Recombinant IFN-g had little impact on WT T cells,
whereas IL-27 had dramatic effects, inducing IFN-g production
and suppressing Th17-type cytokines (Fig. 5A, 5B). IFN-g–de-
ficient T cells produced almost twice as much IL-17 as WT coun-
terparts under Th17 conditions, and in this case, both IFN-g and
IL-27 could suppress the Th17 response (Fig. 5A, 5B). Given that
IFN-g could suppress IL-17 production only in an IFN-g–deficient
exhibit a hyper-Th17 phenotype during systemic
autoimmune disease. A, Naive T cells were puri-
STAT12/2DO11.10 mice and adoptively trans-
percentage of cytokine-positivedonorcells (CD4+
DO11.10+) on day 7 posttransfer. B, Data are
compiled from three to five experiments. Single
asterisk denotes statistically significant differences
between the indicated group and WT donors.
Double asterisk denotes significant differences be-
0.05. See Supplemental Fig. 1 for additional IL-
T-bet– and STAT1-deficient T cells
The Journal of Immunology6465
by guest on June 13, 2013
setting, these data imply that IFN-g responsiveness is rapidly
saturated by autocrine IFN-g production in WT cells. In contrast,
because IL-27 could suppress in either WT or IFN-g2/2cultures,
these data also suggest that its anti-Th17 capacity is independent
of its pro-Th1 capacity and, perhaps, hint at a level of cooperation,
with IL-27 retaining the ability to suppress IL-17 production when
cells become insensitive to IFN-g.
As with the in vivo model, T-bet– and STAT1-deficient T cells
exhibited a hypo-Th1, hyper-Th17 phenotype during in vitro dif-
ferentiation. Under Th17 polarizing conditions, they each produced
T-bet– and STAT1-deficient donor T cells in
sOVA Rag22/2mice. A, Naive T cells were
purified from either WT or IFN-g2/2DO11.10
mice and then adoptively transferred into either
WT or IFN-g2/2sOVA Rag22/2hosts. At 7
d posttransfer, lymphocytes were stained di-
rectly ex vivo for flow cytometry. Shown are the
percentages of total and activated donor T cells
(CD4+DO11.10+; CD25+or CD44high). B, Na-
ive T cells were purified from WT, T-bet2/2,
STAT12/2, or T-bet2/23 STAT12/2DO11
mice and transferred into sOVA Rag22/2hosts.
Shown are the percentages of total and activated
donor T cells after 7 d. A and B, Data are
compiled from three individual experiments. An
asterisk denotes statistically significant differ-
ences between the indicated group and WT
controls. p , 0.05. C, Adoptive transfers were
performed as in B. At 7 d posttransfer, lym-
phocytes were restimulated and stained for IFC.
Shown are the percentages of IL-4+and IL-13+
donor T cells (gated on CD4+DO11.10 TCR+).
Data are representative of three individual
Phenotypic differences between
T-bet2/2, and STAT12/2mice. These were cultured under nonpolarizing or Th17 conditions for 72 h and shown is the percentage of cytokine-positive cells
(CD4+). B, Data are compiled from three to five experiments (Th17 conditions) and are presented as the log2fold change in cytokine-positive cells on
exposure to IFN-g (black bars) or IL-27 (white bars). Untreated groups have a relative value of 0, whereas cytokine-treated groups have values . 0 or , 0,
depending on whether they have an enhancing (x . 0) or inhibitory effect (x , 0). Gray area denotes log2values that are .–1 but ,1. Error bars represent
the SD between all fold changes for each group. Asterisk denotes .2-fold, statistically significant differences between the indicated group and corre-
sponding Th17 controls. p , 0.05.
STAT1-activating cytokines fail to suppress Th17 responses in the absence of STAT1. A, CD4+T cells were purified from WT, IFN-g2/2,
6466STAT1 AND T-bet LIMIT Th17 RESPONSES
by guest on June 13, 2013
more IL-17 than WT counterparts, but again, T-bet deficiency led
to a greater increase, and when both TFs were deleted, the double-
deficient cells behaved like STAT12/2counterparts (Fig. 5A, 5B).
IFN-g had a muted effect in T-bet–deficient cultures, prompting
only small reductions in IL-17A and IL-17F. IL-27 was more
potent, leading to .2-fold reductions in both, though it should be
noted that the level of inhibition was less than what was observed
in WT or IFN-g2/2cultures. Neither IFN-g nor IL-27 impacted
IL-17 production in STAT1-deficient cultures, whether T-bet de-
ficient or sufficient, thereby illustrating the central role of STAT1
in this process (Fig. 5A, 5B).
Consistent with our protein measurements, exogenous IFN-g
had little effect on IL-17A and IL-17F mRNA levels in WT cells
but suppressed both transcripts in IFN-g2/2T cells (Fig. 6). A
similar trend was observed for IL-22, another Th17-associated
cytokine, and for RORgT and RORa, two key Th17-associated
TFs (Fig. 6). As before, IL-27 displayed potent activity in either
WT or IFN-g2/2cells, suppressing IL-17A, IL-17F, and other
Th17-associated transcripts, whereas at the same time promoting
Th1-associated transcripts. IFN-g had little effect on Th17-
associated mRNAs in T-bet–deficient cells, and although IL-27
could still prompt .2-fold reductions, its inhibitory capacity was
less in T-bet2/2cells than in WT or IFN-g2/2counterparts (Fig.
6). As noted earlier, neither IFN-g nor IL-27 affected Th17-
associated transcripts in STAT1-deficient cells (Fig. 4). Also note-
worthy, and consistent with a recent report (44), IL-27 was a
powerful STAT1 and T-bet–independent inducer of IL-21 mRNA
(Fig. 6). Together with our flow cytometry studies, these data
confirm that STAT1-dependent pathways are critical for limiting
Th17 responses. In addition, given the reduced capacity of STAT1-
activating cytokines to suppress in T-bet–deficient cells, they also
suggest a T-bet–dependent, STAT1-independent pathway.
To directly test whether T-bet can suppress Th17 responses
independently of STAT1, we used retroviral gene transduction to
overexpress T-bet and/or RORgT in a STAT1-deficient setting
(Supplemental Fig. 2). As expected, we found that ectopic RORgT
was a potent Th17 stimulus, prompting a dramatic increase in the
percentage of IL-17+cells whether in T-bet–, STAT1-, or double-
deficient T cells (Fig. 7A–C). However, when T-bet was also
introduced, RORgT-driven IL-17 production was significantly de-
creased, and when these “double-infected” cells were cultured
without neutralizing anti–IFN-g mAb, there was a further re-
duction in T-bet–deficient but not STAT1-deficient cells. These
latter findings establish that T-bet can suppress Th17 responses
independently of STAT1, and that, in WT cells, STAT1- and T-
bet–mediated pathways may cooperate in achieving this shared
Consistent with our protein measurements, we also found that
ectopic T-bet had significant impact on RORgT-driven Th17 re-
sponses at the mRNA level, prompting reduced expression of IL-
nonpolarizing or (B) Th17 conditions, and PCR was used to measure the indicated transcripts. Data are pooled from three experiments and presented as the
log2fold change on exposure to IFN-g (black bars) or IL-27 (white bars). Error bars represent the SD between all fold changes, and an asterisk denotes
.1.9-fold, statistically significant differences between the indicated group and Th17 controls. p # 0.08. See Supplemental Fig. 3 for additional PCR
Transcription of Th17-associated genes is influenced by both T-bet– and STAT1-dependent pathways. Naive T cells were cultured under (A)
The Journal of Immunology 6467
by guest on June 13, 2013
17A, IL-17F, and IL-22 in both T-bet2/2and STAT12/2cells.
Removing the anti–IFN-g led to further reductions only in T-bet2/2
cells, which, again, hints at a degree of cooperation between T-bet
and STAT1 in this process (Fig. 8A, 8B). We also noted that the
ability of T-bet to suppress RORgTand RORa was modest by com-
parison, and that it had little influence, positive or negative, on ex-
pression of IL-23R. Thus, although T-bet clearly influences the
output of Th17-type cytokines, it does not appear to do so by
“locking down” transcription of ROR family TFs.
In the preceding studies, we used in vivo and in vitro models of
T cell differentiation to establish that, downstream of IFN-g, IL-27
and likely all STAT1-activating cytokines, there are both STAT1-
and T-bet–dependent mechanisms capable of suppressing Th17
responses. As evidence for STAT1-mediated inhibition, and con-
sistent with published reports (8, 9, 29–31), we demonstrate that
STAT1-deficient T cells exhibit a hyper-Th17 phenotype and are
refractory to the anti-Th17 effects of IFN-g and IL-27. As evi-
dence for T-bet–mediated inhibition, we demonstrate that T-bet–
deficient T cells also exhibit a hyper-Th17 phenotype, that T-bet
deficiency hinders the ability of STAT1-activating cytokines to
suppress Th17 responses, and most importantly, that ectopic T-bet
expression can suppress Th17 responses in the complete absence
of STAT1. Previous studies have also suggested that STAT1 and
T-bet might play independent roles in this process, but because T-
bet is both upstream (an inducer) and downstream (induced by)
of STAT1, they could not definitively exclude the possibility that
cultured under nonpolarizing conditions and transduced with retroviral vectors expressing T-bet (GFP), RORgT (Thy1.1), and/or “empty” controls
(Supplemental Fig. 4). Shown is the percentage of cytokine-positive cells only for those infected with two vectors (CD4+GFP+Thy1.1+). A single asterisk
denotes significant differences between the indicated group and the RORgT-only group; double asterisk denotes significant differences in the absence of
anti–IFN-g. p , 0.05. (D) Coexpression of IL-17A and IFN-g is analyzed for cells expressing T-bet and or RORgT. Data are compiled from three
Direct evidence for T-bet–dependent regulation of Th17 responses. T-bet (A), STAT1 (B), and T-bet–/STAT1-deficient T cells (C) were
“Double-infected” cells were purified by high-speed cell sorting, and PCR was used to measure the indicated mRNAs. Data are representative of three
individual experiments and are presented as the log2fold change relative to the control group (Control-GFP/Control-Thy1.1). Area of each box plot denotes
SD within replicate measurements.
T-bet limits transcription of key Th17-associated genes. T-bet (A) or STAT1-deficient (B) T cells were cultured and transduced as in Fig. 7.
6468STAT1 AND T-bet LIMIT Th17 RESPONSES
by guest on June 13, 2013
T-bet may be arbitrating STAT1-dependent inhibition through its
well-known ability to drive IFN-g production (26, 34). We over-
came the “chicken-and-egg” problem by using retroviral vectors
to restore T-bet expression in either T-bet or STAT1-deficient
T cells, finding that, indeed, there are two pathways at work, with
STAT1 not required for T-bet–mediated inhibition and T-bet not
required for STAT1-mediated inhibition.
Aside from those operating through STAT1, other cytokines
IL-2 and IL-4, which are known to act primarily through STAT5
and STAT6, respectively. Similar to STAT1 deficiency, genetic
ablation of these STATs is associated with increased Th17 re-
sponses, but whether this shared outcome is achieved through a
common mechanism is yet to be resolved (8, 45). The most direct
way that STATs could limit Th17 responses is by binding to
promoter/enhancer regions of Th17-assoctaed genes and thereby
obstructing the transcriptional machinery. There is some evidence
for this, with STAT5 having been shown to bind the promoter of
IL-17A, but whether this interaction is what determines the ability
of STAT5 to suppress IL-17 production was not determined (45).
Likewise, STAT1 has been shown to bind upstream of the RORa
and RORc loci in human HELA cells, but the nature of this in-
teraction, be it stimulatory or inhibitory, and whether it happens in
primary T cells, are questions that remain unanswered (46). An-
other, more indirect way that STATs could impact Th17 responses
is by inducing or promoting the function of auxiliary anti-Th17
factors. There is strong evidence for this because cytokines with
anti-Th17 activity are already known to induce “Th17 inhibitors,”
like T-bet, Ets1, and Gfi-1, and this is not likely to be an ex-
haustive list of indirect targets (47–49). STATs could also in-
fluence Th17 responses by interfering with pro-Th17 TFs or
signaling pathways, as is the case with the ability of IL-27 to
induce expression of SOCS3, which is known to curb STAT3-
dependent Th17 responses (50), the ability of IFN-g and IL-27
to suppress S1P, a receptor known to promote IL-6–driven Th17
responses (51), and the ability of several anti-Th17 cytokines to
suppress RORgT, which is both necessary and sufficient for Th17
differentiation (29–31). Thus, although other regulatory pathways
will likely emerge, it is already clear that cytokines limit Th17
responses through both direct and indirect STAT-driven pathways
that, together, disable multiple steps in the Th17 differentiation
As with the STATs, T-bet could suppress Th17 responses in
a variety of ways. A recent genome-wide mapping of T-bet binding
sites did not reveal significant enrichment near the IL-17A, IL-17F,
IL-22, or ROR loci, making a direct interaction between T-bet and
relevant Th17-associated promoters seem unlikely (52). However,
a direct protein–protein interaction between T-bet and pro-Th17
TFs remains a possibility, especially because T-bet is known to
interact with and thereby limit the function of other TFs, including
GATA-3 and RelA (53, 54). Consistent with this latter point, our
studies demonstrate that T-bet can suppress IL-17 production even
in the face of ectopic RORgT, which is driven by a retroviral
promoter and, thus, is impervious to transcriptional effects. These
data suggest a physical interaction between T-bet and elements of
the Th17 differentiation machinery, if not RORgT itself, though it
should also be noted that T-bet might influence Th17 responses
through more indirect means. Adding further complexity, T-bet
has a functional homolog, Eomes, which is expressed in T cells
and is known to exhibit anti-Th17 activity. Recent studies have
shown that ectopic expression of Eomes can suppress IL-17
production, but whether this is due to a cell-intrinsic effect or its
ability to drive IFN-g–mediated suppression was not resolved
(35). It is also known that, unlike T-bet and STAT1, genetic
ablation of both T-bet and Eomes results in a compound hyper-
Th17 phenotype, but again, the increase in IL-17 production was
mirrored by a corresponding reduction in Th1 responses, making
it unclear whether the phenotype was due to direct effects or a lack
of IFN-g/STAT1-dependent inhibition (26). Based on the data
presented in this paper, we propose that both are true: that T-bet
and Eomes can each limit Th17 responses through at least two
shared mechanisms, one involving STAT1, with IFN-g as an in-
termediary, and the other completely STAT1 independent.
Although our findings establish that STAT1 and T-bet influence
Th17-type cytokines through genetically distinct pathways, we
noted that T cells lacking both TFs did not exhibit an additive, or
compound, phenotype. Instead, the hyper-Th17 phenotype of T-
bet–deficient cells was always more severe than that of STAT1- or
double-deficient counterparts, which, despite the well-known
ability of STAT1 to drive T-bet expression, is also inconsistent
with the notion that they operate within the same pathway. Taken
together, these contradictory observations suggest epistasis, mean-
ing that the loss of STAT1 affects cellular processes that, although
not directly related to Th17 differentiation, impact the overall
quality of T cell responses, thereby hindering production of Th17-
type cytokines. We found that, beyond Th17 responses, T-bet and
STAT1-deficient T cells behaved differently in vivo, with the latter
exhibiting reduced proliferation and increased Th2-type cytokine
production. We also found evidence for epistasis during in vitro
differentiation. Those studies confirmed that STAT1 is required for
IFN-g and IL-27 to suppress IL-17 production, but also showed
that, compared with WT counterparts, expression of many Th17-
type mRNAs was not grossly increased in STAT1-deficient cells,
which, perhaps, indicates posttranscriptional effects (Supplemen-
tal Fig. 3). Thus, although we can still conclude that STAT1 and
T-bet influence Th17 responses through both divergent and con-
vergent mechanisms, it must be noted that genetic dissection of
these pathways was confounded by the wide-ranging effects of
The ability of signature Th1-type factors, like IFN-g, STAT1,
and T-bet, to inhibit signature Th17-type factors, like IL-17, IL-
22, and RORgT, has led to the idea that there is an inverse relation
between the Th1 and Th17 subsets. However, despite this antag-
onism, T cells producing IFN-g and IL-17 are known to occur in
multiple inflammatory settings, which suggests a more nuanced
relation (2, 10–14). This work illustrates both sides of this para-
dox. On one hand, we have shown that T cells produce either IFN-
g or IL-17 when primed in vitro; on the other hand, they can
produce both when primed in vivo (in highly immunogenic sOVA
Rag22/2mice). We also report that, when T-bet and RORgT were
both highly expressed in the same cells, the result is dichotomous,
with some cells expressing one cytokine or the other, but rarely
both (Fig. 1D, 7D). Based on this last finding, and the fact that
T-bet is known to promote long-term Th1 lineage commitment, we
propose that “double-positive” T cells represent a transitional
phase in a linear progression from IL-17–producing Th17 cell to
IFN-g–producing Th1 cell. Inherent to this hypothesis is the idea
that Th17 cells can convert to other subsets, which has strong
experimental support (10), and that such conversion is a part of
normal immune responses, which is now supported by recent studi-
es demonstrating that Th17-type cytokines are required for the
development of Th1 responses during infection (55).
Although Th17 responses can have important, host-protective
functions, it is also widely accepted that, when dysregulated, they
can promote autoimmune disease. Given the current and prospective
use of STAT1-activating cytokines as therapeutics for Th17-
associated pathologies, best exemplified by the use of IFN-b to
treat multiple sclerosis, it is critical to understand exactly how they
The Journal of Immunology 6469
by guest on June 13, 2013
suppress Th17-type inflammation. The studies presented in this
paper provide an important piece of mechanistic information, de-
monstrating that the ability of STAT1 to limit Th17-type responses
is intimately linked to T-bet, a TF that is at once a potent anti-
Th17 effector and, through its ability to drive IFN-g production,
an essential STAT1 stimulus. Although we have focused on CD4+
T cells, which we interrogated in a select few model systems, this
relation between STAT1 and T-bet is likely to impact other IL-17–
producing lineages, such as CD8+T cells or NKT cells, and is
likely to influence Th17 responses in a variety of immune and
autoimmune settings, making these pathways acutely relevant in
the context of inflammatory disease cause and cytokine-based
We thank members of the Abbas, Anderson, Bluestone, and Tang labora-
tories for helpful discussions, and Drs. M. Ansel and S. Katzman for critical
reading of this manuscript. We also thank S. Jiang for cell sorting and
C. Benitez for animal husbandry.
A.V.V. has a patent pending on the anti-inflammatory properties of IL-27.
The other authors have no financial conflicts of interest.
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