Differential Expression of Interleukin-17A
and -17F Is Coupled to T Cell Receptor
Signaling via Inducible T Cell Kinase
Julio Gomez-Rodriguez,1Nisebita Sahu,2,4Robin Handon,1Todd S. Davidson,3Stacie M. Anderson,1
Martha R. Kirby,1Avery August,4and Pamela L. Schwartzberg1,*
1National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
2Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
3National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
4Center for Molecular Immunology & Infectious Disease, Department of Veterinary & Biomedical Sciences, Pennsylvania State University,
University Park, PA 16802, USA
T helper 17 (Th17) cells play major roles in autoimmu-
nity and bacterial infections, yet how T cell receptor
(TCR) signaling affects Th17 cell differentiation is
relatively unknown. We demonstrate that CD4+
T cells lacking Itk, a tyrosine kinase required for full
TCR-induced phospholipase C-g (PLC-g1) activa-
tion, exhibit decreased interleukin-17A (IL-17A)
expression in vitro and in vivo, despite relatively
normal expression of retinoic acid receptor-related
orphan receptor-gT (ROR-gT) and IL-17F. IL-17A
factor of activated T cells (NFAT). Conversely,
decreased TCR stimulation or calcineurin inhibition
preferentially reduced IL-17A expression. We further
found that the promoter of Il17a but not Il17f has
a conserved NFAT binding site that bound NFATc1
in wild-type but not Itk-deficient cells, even though
of IL-17A and IL-17F in vivo. Our results suggest that
sion and the differential regulation of Th17 cell cyto-
kines through NFATc1.
One of the hallmarks of adaptive immune responses is the differ-
entiation of CD4+T helper cells into distinct effector populations
that are required for orchestrating responses to infection. First
recognized and best studied are the T helper 1 (Th1) and Th2
cell subclasses, which produce interferon-g (IFN-g) and inter-
leukin-4 (IL-4), respectively, and have distinct effector functions.
However, it is now appreciated that there are multiple effector
cell populations that can result from activation of naive CD4+
T cells (Zhou et al., 2009; Zhu and Paul, 2008). One of these, the
Th17 cell lineage, has recently been recognized for its major role
in autoimmunity and responses to bacterial infections (Bettelli
et al., 2007; Weaver et al., 2007). Th17 cells were first identified
by their ability to produce IL-17A, a cytokine that helps recruit
neutrophils and is important for driving inflammatory responses.
Inthemouse, Th17cellsdifferentiate inresponseto transforming
growth factor-b1 (TGF-b1) and IL-6 (Bettelli et al., 2006; Mangan
et al., 2006; Veldhoen et al., 2006); additionally, IL-21 helps
promote Th17 cell differentiation (Korn et al., 2007; Nurieva
via a pathway that requires signal transducer and activator of
transcription-3 (STAT-3), turn on expression of the key transcrip-
tion factors retinoic acid receptor-related orphan receptor-gT
(ROR-gT) and RORa, which are critical for expression of Il17a,
as well as the closely linked Il17f gene, and Il21 and Il22 (Ivanov
et al., 2006; Yang et al., 2008b).
regulating lineage-specific transcription factors and the differen-
tiation of distinct effector CD4+T cell populations (Zhu and Paul,
2008). However, in the Th1-Th2 paradigm, it is well established
that signaling from the T cell receptor (TCR) also contributes to
the development and establishment of cell fate. T cells need to
be activated through the TCR in order to produce effector cyto-
strated that varying conditions of TCR ligation induce differential
patterns of cytokines both in vitro and in vivo (Constant and
Bottomly, 1997). Furthermore, distinct components of TCR
and, more distally, transcription factors (Glimcher and Murphy,
2000; Mowen and Glimcher, 2004). How TCR signaling affects
IL-17 production is relatively unknown. Moreover, whether these
signaling pathways contribute to the regulation of the distinct
Th17 cell cytokines is unclear.
The Tec family tyrosine kinase inducible T cell kinase (Itk) is a
critical modulator of TCR signaling, where it functions to regulate
phospholipase C-g (PLC-g) activation, as well as actin polariza-
tion and cell adhesion (Berg et al., 2005). Mutations affecting Itk
reduce TCR-induced PLC-g phosphorylation and downstream
Ca2+mobilization—these defects are worsened by mutations
affecting both Itk and the related resting lymphocyte kinase (Rlk,
also known as Txk) (Berg et al., 2005; Liu et al., 1998; Schaeffer
et al., 1999). Accordingly, cells from Itk?/?and Rlk?/?Itk?/?mice
Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc. 587
show decreased responses to TCR stimulation that are associ-
ated with impaired TCR-induced activation of the Ca2+-sensitive
nuclear factor of activated T cell (NFAT) transcription factors (Fo-
well et al., 1999; Liao and Littman, 1995; Schaeffer et al., 2001).
in T cell function, permitting evaluation of T cell function under
et al., 2007; Schwartzberg et al., 2005).
To evaluate how TCR signaling affects Th17 cell differentia-
tion, we examined cytokine production by TCR-stimulated
sorted naive CD4+T cells isolated from Itk?/?, Rlk?/?Itk?/?,
and wild-type (WT) mice. We find that Itk?/?cells showed
reduced expression of IL-17A, but, surprisingly, expression of
the other Th17 cell cytokines including the closely linked Il17f
gene is relatively intact. Moreover, Itk?/?cells showed normal
expression of the transcription factors RORgT and RORa.
of IL-17A was only poorly rescued by expression of a constitu-
tively activated STAT-3 construct. Instead, IL-17A expression
could be rescued by treatment with a Ca2+ionophore or expres-
sion of an activated mutant of NFATc1. Conversely, specific
decreases in expression of IL-17A (yet relatively normal expres-
sion of IL-17F) could be recapitulated by either low-dose TCR
stimulation or treatment with low-dose inhibition of Calcineurin.
Consistent with these findings, we show that the region
upstream of the promoter of Il17a, but not Il17f, has a NFAT
binding site that is conserved across species and is occupied
by NFATc1 in WT but not Itk?/?Th17 cells, despite both
promoters having open chromatin conformations. Finally, Itk?/?
mice also showed evidence for impaired production of IL-17A
in vivo, despite relatively normal expression of IL-17F. Together,
these data argue that the expression of IL-17A is specifically
coupled to TCR signaling via Itk-mediated regulation of NFAT.
Itk?/?CD4+T Cells Show Defective IL-17A Production
To evaluate how TCR signaling affects Th17 cell differentiation,
we examined intracellular cytokine production from sorted naive
(CD44loCD62Lhi) CD4+T cells isolated from Itk?/?, Rlk?/?Itk?/?,
and WT mice that were stimulated with anti-CD3 plus anti-
CD28 in the presence of WT antigen-presenting cells (APCs)
and polarizing cytokines. Both Itk?/?and Rlk?/?Itk?/?CD4+
T cells were capable of differentiating into IFN-g-producing cells
under Th1 cell differentiation conditions (IL-12 plus anti-IL-4),
although at slightly lower percentages than WT cells (Figure 1A).
In contrast, after exposure to TGF-b1 and IL-6, potent inducers
of Th17 cell differentiation, there were marked reductions in the
percentage of cells that had differentiated into IL-17A-producing
cells from both Itk?/?(16.5% ± 1.9%) and Rlk?/?Itk?/?(11% ±
1.9%) animals as compared to WT cells (50.4% ± 4.0%)
(Figure 1A). Similar results were seen with CD8+T cells, which
can also differentiate to produce IL-17A under these conditions
(data not shown). Similar to a recent report (Veldhoen et al.,
2009), we found that growth in the Iscove’s modified Dulbecco’s
media (IMDM), which contains higher amounts of aryl hydrocar-
bons, improved Th17 cell differentiation (Figure 1A, data points
in red). However, Itk?/?cells were defective in differentiation to
IL-17A-producing cells in either media. To focus our evaluation
on the effects of Itk and TCR signaling during the differentiation
to IL-17A-producing cells, we restimulated cells with PMA and
ionomycin in these studies. However, the defects in IL-17A
expression were even more severe when CD4+T cells were
restimulated with anti-CD3 plus anti-CD28 (Figure 1B), arguing
that Itk?/?and Rlk?/?Itk?/?cells show defects both in priming
to IL-17A-producing cells and in TCR-induced expression of
Itk?/?and Rlk?/?Itk?/?CD4+T cells have defects in TCR-
induced proliferation (Berg et al., 2005; Liao and Littman, 1995;
Schaeffer et al., 1999), which can be severe in the case of
Rlk?/?Itk?/?T cells (cell yields were less than 10% those of WT
cells under Th17 cell culture conditions; see Figure 1A). To eval-
uate the possibility that decreased IL-17A production resulted
from poor proliferation, we stained cells with carboxyfluorescein
succinimidyl ester (CFSE) to follow cell division. Although Itk?/?
cells did exhibit reduced cell division, decreased percentages
of cells producing IL-17A were observed at each division
These results suggested that the defect in IL-17A expression in
flected an actual defect in IL-17A production. However, to mini-
mize the effects of decreased cell proliferation, we focused our
further studies on Itk?/?rather than Rlk?/?Itk?/?cells.
Previous studies have shown that Itk deficiency alters thymic
development and selection so that a large number of innate-
type memory phenotype CD8+T cells develop in Itk?/?mice
(Berg, 2007). To rule out the possibility that altered development
contributes tothe reducedproduction of IL-17A,wesorted naive
WT and Itk?/?CD4+T cells, activated them under null conditions
with blocking cytokine antibodies, and then retrovirally trans-
ducedthemwitharetrovirus expressingmurineItkfor 1day prior
to exposing them to Th17 cell-inducing cytokines. Re-expres-
sion of Itk completely rescued the defect in IL-17A production
in Itk?/?cells during Th17 cell differentiation (Figure 1D). Thus,
efficient production of IL-17A requires Itk at the time of differen-
tiation and does not appear to result from altered development.
Decreased IL-17A Message in Itk-Deficient Cells
To evaluate potential mechanisms for the decreased IL-17A
production, we examined Il17a mRNA expression by quantita-
tive-RT-PCR (q-RT-PCR) after 3.5 days of stimulation in RPMI
media. Il17a message was markedly decreased in Itk?/?cells,
demonstrating that the reduction in IL-17A production occurred
at the level of Il17a mRNA (Figure 2A). Surprisingly, however,
expression of the message of other Th17 cell cytokines,
including Il22 and Il21, appeared normal at this time of analysis.
Indeed, expression of the closely linked Il17f gene was relatively
intact, whereas expression of Il17a was consistently reduced at
all times examined from 24 to 84 hr after stimulation (Figure 2A
and data not shown). The differential effects on IL-17A and IL-
17F were further evaluated by intracellular staining for cytokine
production and ELISA for secreted cytokines (Figures 2B–2D).
Although intracellular staining did reveal statistically significant
reductions in IL-17F production (Figure 2C, p < 0.001), the differ-
ence was much less than seen for IL-17A (Itk?/?82% WT values
for IL-17F, versus 32% for IL-17A) and may reflect other defects
Itk Couples TCR Activation to IL-17A Expression
588 Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc.
in Itk?/?cells that, for example, may affect protein translation.
Similar results were seen with secreted cytokines as evaluated
by ELISA (Figure 2D), where the differences in IL-17A secretion
between WT and Itk?/?were much greater than the differences
in IL-17F secretion. These results suggested that there may be
distinct features of the regulation of the individual Th17 cell cyto-
Itk?/?Mice Show Impaired Production of IL-17A In Vivo
Evaluation of allergic asthma responses have suggested that
IL-17A contributes to pathology whereas IL-17F may be protec-
tive. Mice deficient in IL-17F show exacerbated responses
associated with increased Th2 cell cytokine production in
a model of allergic asthma, whereas mice deficient in IL-17A
show decreased responses (Yang et al., 2008a). Interestingly,
Itk?/?mice also have decreased responses to allergic asthma
that have previously been associated with reduced Th2 cell
eller and August, 2003). To determine whether defects in IL-17A
production were also observed in vivo, we examined responses
in a model of allergic asthma (Figure 3A). Evaluation of IL-17
Figure 1. Reduced IL-17A Production from
Itk?/?and Rlk?/?Itk?/?CD4+T Cells
(A) Naive (CD62LhiCD44lo) CD4+T cells were stim-
ulated with 1 mg/ml anti-CD3 and 3 mg/ml CD28
in the presence of WT T depleted splenocytes
under either Th1 cell- or Th17 cell-inducing condi-
tions for 3.5 days, restimulated with PMA and ion-
omycin for 4 hr and cytokine production analyzed
by intracellular staining. Cell yields at end of
culture are indicated in parentheses below each
flow plot. Data are representative of four experi-
ments for Rlk?/?Itk?/?cells and over ten for Itk?/?
cells. The right panel shows percentages of
IL-17A CD4+T cell producers from individual
experiments after differentiation for 2–3 days
(experiments in IMDM indicated in red). Statistics
were calculated with paired Student’s t test.
(B) Naive (CD62LhiCD44loCD25?) CD4+T cells
were stimulated as above, but restimulated with
anti-CD3 plus anti-CD28 prior to intracellular cyto-
IL-17A CD4+T cell producers from four experi-
(C)NaiveCD4+T cells werelabeled withCFSE and
thendifferentiatedand stained asabove. Forover-
lay, see Figure S1.
(D) Naive CD4+T cells were stimulated for 2 days
under Th0 conditions, then infected with a control
(MIGR) or MIGR-Itk-expressing retroviral vector
for 24 hr prior to exposure to Th17 cell-inducing
cytokines. Two days later, cells were restimulated
with PMA and ionomycin and stained for expres-
sion of IL-17A and IFN-g.
expression confirmed that Il17a mRNA
was more severely reduced than Il17f in
lungs of challenged Itk?/?mice, even
when normalized for decreased numbers
of T cells (Figure 3B). Thus, T cells from
Itk?/?mice showed decreased expres-
sion of Il17a despite relatively normal Il17f expression in vivo,
as well as in vitro.
Altered Responses to Cytokines
To further understand the nature of the defect in IL-17A produc-
tion in Itk?/?cells, we evaluated responses to different cytokine
milieus. Defects inIL-17A production wereobserved in response
to multiple cytokines, including IL-21 plusTGF-b1,IL-1plusIL-6,
TGF-b1 plus IL-1, and TGF-b1 plus IL-6 and IL-23, suggesting
that the decrease in IL-17A production was a universal defect
that occurred in response to many, if not all, Th17 cell-inducing
cytokines conditions (Figure 4A, Figure S2, and data not shown).
We next evaluated the response of Itk?/?cells to individual
cytokines. Exposure of CD4+T cells to TGF-b1 induces their
differentiation to Forkhead box P3 (FoxP3)-expressing induced
T regulatory (iTreg) cells, especially in the context of IL-2 (Chen
et al., 2003; Davidson et al., 2007). Itk?/?CD4+T cells were
able to differentiate normally to FoxP3-expressing cells under
these conditions, suggesting that responses to TGF-b1 were
normal (Figure 4B). In contrast, whereas exposure of WT CD4+
Itk Couples TCR Activation to IL-17A Expression
Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc. 589
cells (presumably because of low amounts of TGF-b1 in the
serum or produced by cells in the culture because this differenti-
ation could be blocked by anti-TGF-b), Itk?/?cells completely
failed to differentiate into IL-17A-producing cells under these
conditions (Figure 4A). In this experiment, Itk?/?CD4+T cells
also produced lower amounts of IFN-g in response to IL-6 in
the presence of anti-TGF-b. These results suggest that Itk-
deficient cells do not respond optimally to IL-6 upon activation.
IL-6activatesSTAT-3, atranscription factor thatisrequiredfor
expression of RORgT, a key transcription factor required for
sion of an activated STAT-3 mutant can drive activated T cells to
express RORgT and produce more IL-17A (Yang et al., 2007). To
evaluate whether loss of Itk affected STAT-3, we examined
STAT-3 phosphorylation, which is required for its activation. In
response to IL-6, either alone or under Th17 cell stimulation
conditions (in combination with TGF-b1), Itk-deficient T cells ex-
(Figure 4C). Nonetheless, we observed normal Rorc and Rora
mRNA in Itk?/?cells, suggesting that this decreased phosphor-
ylation of STAT-3 was not sufficient to affect expression of these
transcription factors (Figure 4D). Although we did observe early
decreases in the expression of IL-21, which is also induced by
IL-6 (Korn et al., 2007; Nurieva et al., 2007a; Wei et al., 2007;
Zhou et al., 2007), these delays were only transient (data not
shown). Moreover, whereas retroviral transduction of cells with
an activated STAT-3 mutant increased expression of IL-17A in
WT cells, it only minimally rescued IL-17A expression in Itk-defi-
cient cells (Figure 4E). Thus, altered STAT-3 activation did not
appear to be the major cause of defective IL-17A production in
Itk?/?cells, suggesting that Itk helps regulate IL-17A expression
by a different or additional mechanism.
Figure 2. Itk Is Required for Efficient Transcription of Il17a, but Not Il17f
(A) Naive CD4+T cells were differentiated under Th17 cell conditions, and mRNA was analyzed by qRT-PCR after 3.5 days (84 hr) of differentiation (mean of 5
experiments ± SEM). Statistics were calculated with the paired Student’s t test. Similar results were seen at 48 hr.
(B) Intracellular staining for IL-17A and IL-17F of cells differentiated for 2 days under Th17 cell conditions.
(C) Scatterplots of total IL-17F CD4+T cell producers (single IL-17F plus double IL-17F plus IL17-A producers) from the subset of experiments, shown in
Figure 1A, where IL-17F was measured. Experiments in IMDM are indicated in red.
(D) IL-17A and IL-17F secreted by WT and Itk?/?CD4+T cells differentiated for 48 hr, as determined by ELISA (IL-17A: WT 3940 ± 374 versus Itk?/?256 ± 73 pg/
ml; IL-17F: WT 99700 ± 5400 versus Itk?/?89700 ± 5390 pg/ml). Statistics were calculated with paired Student’s t test.
Itk Couples TCR Activation to IL-17A Expression
590 Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc.
TCR Signaling Affects IL-17A Production
Itk is required for full TCR-induced activation of PLC-g and down-
signaling affects IL-17A expression, we differentiated WT CD4+
T cells in the presence of decreasing amounts of anti-CD3. We
found that optimal production of IL-17A required high-dose anti-
CD3 stimulation (Figure 5A). Intriguingly, lowering the dose of anti-
CD3 stimulation preferentially affected the expression of IL-17A
over IL-17F, similar to what we observed in Itk?/?cells. To deter-
mine whether altered TCR signaling contributes to the effects we
see in Itk?/?cells, we differentiated cells with anti-CD3 in the pres-
polarized Itk?/?CD4+T cells, whereas expression of IL-17F was
only minimally affected (Figure 5B). In contrast, stimulation with
anti-CD3 plus PMA, which rescues ERK activation in Itk?/?
T cells, did not rescue IL-17A production (data not shown). These
results suggest that defects in TCR-induced Ca2+mobilization
A Role for NFATc1 in Regulation of IL-17A
In T cells, Ca2+signaling regulates activation and expression of
a critical series of transcription factors, the NFATs, which are
dephosphorylated by calcineurin, leading to their nuclear
localization and activation (Gwack et al., 2007; Winslow et al.,
2003). TCR stimulation in conjunction with costimulation further
upregulates expression of NFATc1, which autoregulates its own
expression (Nurieva et al., 2007b). We and others have previ-
ously shown that Itk?/?T cells exhibit defects in NFAT activation
and nuclear localization, as well as the induction of NFATc1
expression observed upon activation with TCR and costimula-
tion (Fowell et al., 1999; Nurieva et al., 2007b; Schaeffer et al.,
2001). To evaluate whether NFAT activation affects IL-17A
expression, we differentiated cells under Th17 cell conditions
in the presence of increasing amounts of the calcineurin inhib-
itors FK506 or Cyclosporin A (CsA), which affect NFAT activa-
tion. Although treatment of cells with high amounts of FK506
affected both IL-17A and IL-17F (data not shown), exposure
of cells to low amounts of FK506 preferentially affected expres-
sion of IL-17A (Figure 5C). Similar results were obtained with
CsA (Figure S3). Thus, the extent of calcineurin inhibition
appears to specifically affect expression of IL-17A compared
To specifically evaluate the role of NFAT in regulating expres-
sion of IL-17, we examined the regions 20 kb upstream of the
Il17a and Il17f transcriptional start sites, as well as the entire
genes for potential NFAT binding sites. Although both promoters
showed multiple potential NFAT consensus binding sites in the
mouse, only the Il17a upstream region contained an NFAT
binding site that was conserved across species (located 3085
bp upstream of the first exon, Figure 6A and Figure S4A). Evalu-
ation of mouse Il17a promoter-luciferase constructs inthe Jurkat
T cell line revealed that expression of a 3.5 kb construct that
included the potential NFAT binding site was specifically
increased by coexpression of a constitutively active NFATc1
mutant (Figure 6B). This mutant is constitutively localized to
the nucleus because of mutations affecting the negative-regula-
tory phosphorylation sites and does not require TCR activation
for its nuclear localization (Neal and Clipstone, 2003). However,
expression of activated NFATc1 did not affect expression of a
similar-length construct derived from the Il17f promoter (Fig-
ure S4B). Furthermore, deletion of the conserved putative NFAT
was a bona fide NFAT binding site.
To evaluate whether this NFAT binding site was used in vivo,
we performed chromatin immunoprecipitation (ChIP) studies
by amplifying this region after crosslinking and immunoprecipi-
tating NFATc1, a major NFAT member expressed in mature
T cells. ChIP analyses of WT cells differentiated under Th17
cell conditions demonstrated a large enrichment of NFATc1
binding to the conserved NFAT binding site in the Il17a promoter
(Figure 6C). However, we saw no enrichment of amplification in
samples from Itk?/?cells. Nonetheless, both WT and Itk?/?cells
did show binding of acetylated histone H3 as well as K4-trime-
thylated histone H3 to the proximal promoter region upstream
of the transcriptional start sites of both Il17a and Il17f genes,
as well as to multiple conserved noncoding regions (CNS) in
this locus (Figure S4C, Figure 7A, and data not shown). These
results suggest that the entire Il17 gene locus had undergone
epigenetic modification consistent with open chromatin in both
WT and Itk?/?cells. Thus, Itk?/?cells show a selective defect
Figure 3. Itk?/?Mice Show Impaired Production of Il17a In Vivo
(A) Lung sections (PAS stained) from naive and OVA-challenged WT and Itk?/?
(B) Expression of Il17a and Il17f mRNA in lungs from immunized and OVA-
challenged WT and Itk?/?mice.
fornormalization of Tcell numbers. Means± SEMfrom 12micefrom four inde-
pendent experiments are shown. Statistics were calculated with the paired
Student’s t test.
Itk Couples TCR Activation to IL-17A Expression
Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc. 591
in the binding of NFATc1 to the Il17a promoter despite having an
open chromatin conformation.
Finally, to determine whether the defect in NFATc1 activation
causes the impaired Il17a transcription in Itk?/?cells, we trans-
duced cells with a retroviral construct expressing activated
NFATc1 construct rescued IL-17A production in Itk?/?CD4+
T cells, supporting the idea that defective NFAT activation is the
cause of the decreased Il17a mRNA in these cells (Figure 7B).
Our results suggest that Itk specifically couples effective TCR
signaling to the expression of IL-17A through its effects on the
activation and expression of NFATc1. This defect in IL-17A
expression is seen in response to multiple cytokines and is not
the result of altered development given that it can be rescued
Figure 4. IL-17A Production in Itk?/?CD4+T
Cells Is Reduced in Response to Multiple
Cytokines, despite Normal Expression of
Rorc and Rora
(A and B) Naive CD4+T cells were differentiated in
the presence of the indicated cytokines (plus anti-
IL-4, anti-IL-12, and anti-IFN-g), and cytokine
production was evaluated by intracellular staining
for IL-17A plus IFN-g (A) or IL17-A plus Foxp3 (B).
Data are representative of over three experiments.
(C) T cells were stimulated with IL-6 plus TGF-b1
for the indicated times, lysed, and immunoblotted
with anti-phospho-STAT3. Lower panels show
total STAT3 levels. Two examples show the range
of STAT-3 phosphorylation across six experi-
cell conditions for 84 hr and examined for Rorc and
Rora mRNA by qRT-PCR (mean of five experiments
± SEM). Similar results were seen at 48 hr.
(E) IL-17A production is poorly rescued in Itk?/?
T cells by expression of constitutively active
catedretroviral vector,and GFP+cells wereexam-
ined for cytokine production after stimulation
under Th17 cell conditions. Results are from the
same experiment as Figure 1D for comparative
purposes but are representative of 2–3 indepen-
dent retroviral transduction experiments.
by re-expression of Itk in preactivated
cells. Our results agree with recent
reports showing that high-dose CsA
blocks IL-17A expression (Liu et al.,
2005; Zhang et al., 2008a) and that inter-
ference with NFAT DNA binding activity
can affect IL-17A production (Hermann-
Kleiter et al., 2008). However, we demon-
strate here that, interestingly, expression
of other Th17 cell cytokines, including
the closely linked Il17f gene, is not
affected to the same extent as Il17a in
Itk?/?T cells. Consistent with these
observations, we find that low-dose
FK506 or CsA preferentially affects IL-17A and that there is
a conserved NFAT binding site upstream of the Il17a promoter,
but not in the region 20 kb upstream of the Il17f gene. These
results suggest that expression of Il17a is particularly sensitive
to the strength of TCR signaling, requiring full activation of
Ca2+-mediated pathways, in addition to signals from cytokines
required for the induction and activation of RORgT and STAT3.
Consistent with this idea, we find that optimal expression of IL-
17A requires high doses of anti-CD3 plus anti-CD28 or high-
dose antigen stimulation (data not shown). Whether there are
other factors that more specifically affect IL-17F production
remains an interesting question. Intriguingly, sequence analyses
demonstrate a conserved NF-kB binding site upstream of the
start of the Il17f gene. Consistent with our data that the absence
of Itk less severely affects IL-17F expression, wehave previously
found that activation of Itk?/?CD4+T cells in the presence of
APCs has only modest effects on NF-kB activation (Schaeffer
Itk Couples TCR Activation to IL-17A Expression
592 Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc.
et al., 2001). We also note that high doses of FK506 or CsA can
IL-17A); however, these effects could result from secondary
effects of NFATs or these inhibitors on expression of other
genes, cell proliferation, or cell viability.
Recent data demonstrate that in addition to RORgT (Ivanov
factor 4 (Brustle et al., 2007), Runt-related transcription factor 1
(Runx1) also participates in the regulation of Il17a via a complex
that can affect RORgT inhibition by FoxP3 (Zhang et al., 2008b).
NFATs have been shown to couple with other transcription
factors to differentially affect T cell functional outcomes (Her-
mann-Kleiter et al., 2008; Hu et al., 2007). Whether NFAT family
members affect transcription of Il17a through secondary effects
on these other transcription factors will be of interest. Our obser-
vation that multiple CNS in the Il17 locus bind acetylated Histone
H3 in Itk?/?T cells suggests that the Il17 locus is in an open
chromatin conformation, which would be consistent with the
proper expression and activation of these other transcription
factors (Kouzarides, 2007). Indeed, we see normal expression
of RORgT, RORa, IRF4, and Runx1 upon differentiation of cells
fromItk?/?mice (this paper and unpublished data). Nonetheless,
we cannot rule out other contributions—although constitutively
activated NFATc1 greatly improves IL-17A production in Itk?/?
CD4 cells, other factors may also participate in pathways
affecting IL-17A expressionin thesecells.For example, although
we have not seen effects on SOCS3 expression (unpublished
data), SOCS3 is known to affect STAT3 phosphorylation and
Th17 cell differentiation (Chen et al., 2006). However, given the
mild decreases in STAT3 phosphorylation, normal expression
of Rorc and Rora, and minimal rescue with activated STAT3
constructs that we observed, it is likely that the major effects
we see on IL-17A production result from effects on NFAT.
Why IL-17A specifically requires maximal TCR signaling for its
expression is not clear but could be related to the functions of
IL-17A, which is a powerful mediator of inflammation, strongly
inducing inflammatory chemokines and neutrophil recruitment
(Dong, 2008; Ouyang et al., 2008). Indeed, recent comparisons
of IL-17A and IL-17F function indicated that IL-17A played
2008a). Intriguingly, the responses of Itk?/?mice in a model of
allergic asthma resemble those of mice deficient in IL-17A, but
vation, we find that Il17a expression is also decreased in lungs
from Itk?/?mice relative to WT mice, even when we normalize to
T cell numbers. Perhaps a secondary requirement for strong
TCR signals provides a safety check to help regulate IL-17A
rial infections, particularly in the gut, can also lead to pathological
effects and more strongly promote autoimmunity than IL-17F.
These data support the idea that there may be subpopulations
be distinctly regulated. Indeed, regulation of IL-22, another Th17
cell cytokine, appears to be particularly sensitive to aryl hydro-
carbons (Veldhoen et al., 2008). It is also of interest that, in Th2
cell clones, differential sensitivity of IL-4 and IL-5 to CsA has
been shown, linking differential regulation of Th2 cell cytokines
to NFAT activation (Bohjanen et al., 1990; Naora et al., 1994).
Our results suggest that cytokine production by Th17 cells, like
the Th1 and Th2 effector cell lineages, is affected by the type
and strength of TCR signals they receive. These findings open
a new window in understanding the factors that control the
expression of cytokines by this important lineage, which may
be important for understanding therapeutic approaches to
inflammatory and autoimmune diseases.
Itk?/?(Liao and Littman, 1995), Rlk?/?Itk?/?(Schaeffer et al., 1999), and WT
mice, backcrossed five generations on C57BL/6 background, were used
between 7 and 9 weeks of age. Patterns of cytokine production were
confirmed and in vivo challenges were performed with animals backcrossed
to C57BL/6 micefor ten to 12generations.Animal husbandry and experiments
were performed in accordance with approved protocols by the National
Human Genome Research Institute’s Animal Use and Care Committee or
the Office of Research Protection’s Institutional Animal Care and Use
Committee at Pennsylvania State University.
Isolation of Naive CD4+T Cells and Cell Culture
T cells were purified by T cell isolation columns (R&D) and then stained with
anti-CD25-PE, anti-CD4-PerCPCy5.5, anti-CD8-APC, anti-CD44-FITC, and
anti-CD62L-Pacific blue (eBioscience) and sorted on a FACsAria to obtain
Figure 5. IL-17A Production Is Affected by TCR and NFAT Activation
(A) WT naive CD4+T cells were differentiated in the presence of varying
amounts of anti-CD3 (plus 3 mg/ml anti-CD28) and stained for IL-17A and
(B) WT and Itk?/?naive CD4+T cells were differentiated with anti-CD3 plus
anti-CD28 or anti-CD3 plus ionomycin and stained for cytokine production.
(C) WT naive CD4+T cells were differentiated with anti-CD3 plus anti-CD28, in
the absence or presence of increasing amounts of FK506.
(A)–(C) represent three or more experiments, with varying concentrations of
antibodies and inhibitors.
Itk Couples TCR Activation to IL-17A Expression
Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc. 593
naive CD4+CD44loCD62Lhior CD4+CD44loCD62LhiCD25?at a purity greater
than 99%. Similar results were obtained with either type of naive population.
Cells wereculturedinRPMI1640orIMDM supplemented with10% Fetal Calf
Serum (Hyclone), 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml strepto-
mycin, and 5 mM 2-B-mercaptoethanol (Invitrogen). Sorted naive CD4+T cells
nocytes as APCs for 1–4 days in 48-well plates containing 1 mg/ml of anti-CD3
anti-IL-4, anti-IFN-g, and anti-IL-12; IL-21 and TGF-b1 used 100 ng/ml of IL-21
plus 5 ng/ml of TGF-b1, anti-IL-4, anti-IFN-g, and anti-IL-12; IL-6 used
20 ng/ml of IL-6 plus anti-IL-4, anti-IFN-g, and anti-IL-12; and IL-1a/b
and IL-6 used 100 ng/ml of either IL-1a or IL-1b plus 20 ng/ml IL-6, anti-IL-4,
anti-IFN-g, and anti-IL-12. IL-23 was used at 20 ng/ml. Cytokines were
purchased from Peprotech, with the exception of TGF-b and IL-23, which were
from R&D. Cytokine antibodies were from BioXcell. The calcineurin inhibitors
CsA (Sigma) and FK506 (Sigma) were added to the naive CD4 T cells and APC
cocultures for 30 min before stimulation with Th17 cell cocktails, and the cells
were cultured for 48 hr. Cells were cultured with 1 mg/ml of anti-CD3 plus 1 mM
ionomycin for 48 hr under Th17 cell conditions.
For intracellular staining, cells were differentiated for 2–3.5 days, then stimu-
lated with 50 ng/ml of PMA (Sigma) and 1 mg/ml of ionomycin (Sigma) or
with 1 mg/ml anti-CD3 and 3 mg/ml anti-CD28 in presence of golgi-plug for
4 hr. Intracellular cytokines were stained with anti-IL-17A, -IL-17F, -IFN-g, or
-FoxP3 (eBioscience). Data acquisition was done on an LSRII (BD Biosci-
ences) and analyzed by FlowJo software (Tree Star). ELISAs were performed
on supernatants from 48 hr cultures with Mouse IL-17A (eBioscience) and Duo
Set IL-17F (R&D) ELISAs.
CD4+CD25?CD44loCD62Lhi(3 3 106cells/ml) were incubated in PBS contain-
ing 2.5 mM of CFSE (Molecular Probes) for 10 min (room temperature), which
was quenched by adding 1 ml of FBS for 1 min. Cells were washed twice
with complete RPMI and stimulated for 3 days.
Reverse-Transcription Quantitative PCR
TotalRNA waspreparedfrom differentiated Tcells atdiffering times after stim-
ulation and from lungs from immunized and OVA-challenged WT and Itk?/?
mice with Trizol reagent (Invitrogen) and RNeasy Mini kit (QIAGEN). cDNA
was synthesized with the Taqman Reverse kit (Applied Biosystems). Quantita-
tive RT-PCR was performed on a 7500 Fast Real-Time PCR instrument
(Applied Biosystems) with either TaqMan Universal PCR Master Mix (Applied
Biosystems) for Il17a, Il17f, Il21, Il22, and Rorc (Applied Biosystems) or Plat-
inum SYBR Green qPCR Supermix (Invitrogen) for Rora (50-TCTCCCTGCGCT
CTCCGCAC-30and 50-TCCACAGATCTTGCATGGA-30). 18srRNA and Thy1
were used for normalization for in vitro-differentiated CD4+T cells and for
lungs, respectively (Thy1 was used to normalize for T cells numbers in the
Figure 6. IL-17A Expression Is Linked to NFAT Activation
(A) Conserved NFAT and ROR binding sites (Yang et al., 2008b) across species in the 20 kb upstream of the Il17a gene, as predicted with the Mulan software at
NCBI DCODE. See Figure S4A for sequence of the conserved NFAT binding site.
DNFAT (potential NFAT binding site ?3085 to ?3077 deleted) in the presence or absence of caNFATc1-pMIGR plasmid. After 24 hr, luciferase activity was quan-
tified and normalized to Renilla luciferase. Results are expressed as fold increase in luciferase activity relative to pGL3-basic plasmid. Means ± SEM of triplicate
samples from four experiments are shown. Statistics were calculated with the paired Student’s t test.
(C) ChIP using NFATc1 antibody and amplifying the region around ?3085 bp from the annotated first exon. Data are representative of duplicate experiments and
were normalized to input value and expressed as fold enrichment relative to normal mouse sera.
Itk Couples TCR Activation to IL-17A Expression
594 Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc.
lungs). The data in differentiated CD4+T cells was expressed relative to naive
CD4+T cells; whereas mRNA expression in lungs from treated animals was
expressed relative to a WT-challenged mouse. The data were expressed as
2?DDCTwith the ABI 7500 SDS 1.3.1 software.
Ovalbumin-Induced Airway Hypersensitivity
Mice were sensitized with ovalbumin (Sigma-Aldrich) complexed to aluminum
hydroxide (10 mg ovalbumin/1 mg alum; Pierce) intraperitoneally in a total
volume of 200 ml on days 0 and 5. Mice were later challenged intranasally
with ovalbumin from days 12 through 15 (at a concentration of 2 mg/ml, for
atotalof 40mg totalexposure).Development of allergic asthma wasconfirmed
by analyzing airway hyperresponsiveness (AHR) on day 16 with a custom-
made mechanical ventilator as previously described. Mice were then
sacrificed, and lungs were used for RNA or sectioned and stained by
periodic acid-Schiff (PAS) as detailed (Ferrara et al., 2006; Muellerand August,
ChIP assays were performed with EZ-Magna ChIP A (Upstate) as recommen-
ded by the manufacturer. In brief, after being fixed in 1% formaldehyde, T cells
were lysed for 10 min at room temperature. Chromatin was sheared by soni-
cation in Sonicator 3000 (Misonix). Lysates equivalent to 2 3 106cells were
used per immunoprecipitation at 4?C overnight with 5 mg of anti-NFATc1,
(sc-7294, Santa Cruz Biotechnology), anti-acetylated Histone H3 (06-599B,
Upstate), anti-trimethyl K4 Histone H3 (39159, Active Motif), or preimmune
mouse or rabbit IgG antibodies. Enrichment of chromatin was analyzed with
Platinum SYBR Green qPCR Supermix (Invitrogen) and the 7500 Fast Real-
Time PCR instrument (Applied Biosystems). Data were normalized to input
values and expressed as fold enrichment relative to normal mouse or rabbit
Figure 7. The IL-17 Locus Has an Open Chromatin
Conformation, and caNFATc1 Rescues the IL-17A
Defect in Itk?/?Cells
(A) ChIP using acetylated histone H3 antibody and ampli-
fying regions justupstream ofthetranscriptionalstartsites
were normalized to input value and expressed as fold
enrichment relative to normal rabbit sera. Data are the
mean ± SEM of four independent experiments.
(B) Production of IL-17A after retroviral transduction with
an activatedNFATc1 construct. Data are from one of three
The primer sequences used for qPCR analysis of the
NFAT binding site in Il17a promoter are 50-AATAGATTC
AACAG-30. Primer sequences for CNS 2, 3, and 7 are
indicated in (Akimzhanov et al., 2007). Conserved
NFAT binding sites were found with Mulan software at
the National Center for Biotechnology Information’s
Retrovirus Production and Infection
from D. Littman), ca-NFATc1-IRES-GFP-pMIGR (gift of
N. Clipstone), and ITK-IRES-GFP-pMIGR (gift of L. Berg)
plasmids (12.5 mg) were used to transfect 293 T cells
with Fugene (Roche). After 48 hr, retroviral supernatants
Sorted naive CD4+T cells were cocultured with T
depleted APCs under Th0 conditions for 48 hr. Retrovirus
supernatants were added to the cells and spun at
2500 rpm for 1.5 hr at room temperature with 8 mg/ml of
polybrene (Sigma). After 24 hr, infected cells were differentiated under Th17
cell conditions for 48 hr and stained for intracellular cytokines.
After stimulation for the indicated times, 1 3 106CD4 T cells were lysed in
Laemmli buffer.Proteinswereseparated in8%SDS-PAGE geland transferred
to nitrocellulose membranes, which were blocked and incubated with either
anti-phospho-STAT3 or anti-STAT3 (9145L, 9132L, Cell Signaling Technology)
as per the manufacturer’s instructions, washed, incubated with horseradish
peroxidase (HPR)-labeled goat anti-rabbit (Amersham Pharmacia), and devel-
oped with the enhanced chemiluminescence detection system (Amersham
Luciferase Reporter Assays
DNA fragments corresponding to ?3500 to +1 from mouse Il17a and Il17f
promoters were subcloned into pGL3-basic (Promega) and designated as
tial NFAT binding site (?3085 to ?3077) from Il17ap plasmid by overlapping
PCR. Jurkat E6 cells (6 3 106) were electroporated with 20 mg luciferase
reporter construct and 1 mg pRL-TK (Renilla luciferase) plasmid with or without
5 mg of caNFATc1-pMIGR plasmid by a BTX ECM 830 electroporator (BTX
Technologies). After 24 hr, cells were lysed according to the manufacturer,
and luciferase activity was quantified in triplicate in reaction mixtures contain-
ing 15 ml lysate and 100 ml reagent from Dual-Luciferase Reporter Assay
System (Promega) with Lumat LB 9507 luminometer (Berthold Technologies).
Each transfection was done in triplicate (or duplicate for Figure S4).
Results were expressed as mean ± standard error of the mean (SEM). Statis-
tical differences between the analyzed groups were calculated with the paired
Itk Couples TCR Activation to IL-17A Expression
Immunity 31, 587–597, October 16, 2009 ª2009 Elsevier Inc. 595
Student’s t test. Values of p < 0.05 are considered significant. Graphs were
done in Excel (Microsoft) and Prism (GraphPad).
We would like to thank the Schwartzberg laboratory, J. Zhu, H. Yamane and J.
Cote-Sierra foradviceand helpfuldiscussions;D.Littman,N.Clipstone,andL.
Berg for retroviral constructs and mice, J. Fekecs and D. Leja for graphics
assistance, and J. O’Shea, J. Zhu, J. Cannons and W. Paul for critical reading
of the manuscript. This work was supported by funding from the intramural
research program of the NHGRI, NIH (to PLS) and NIH grant A1051626 (to AA).
Received: June 1, 2009
Revised: July 11, 2009
Accepted: July 15, 2009
Published online: October 8, 2009
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