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J. Exp. Med. Vol. 206 No. 7 1457-1464
BRIEF DEFINITIVE REPORT
A major advance in understanding the role of
various T cell subsets in disease has been the
recent identification of the Th17 subset, char-
acterized by the production of IL-17A (IL-17),
as well as the related IL-17F cytokine (Weaver
et al., 2006). Just as with the Th1 and Th2
subsets, whose development and propagation
are mediated by specific cytokines, Th17 de-
velopment is selectively induced by a combi-
nation of IL-6 and TGF-, whereas the IL-12
family member IL-23 supports Th17 propaga-
tion (Chen and O’Shea, 2008). The ability of
IL-6 to divert TGF-–stimulated T cells away
from regulatory T cell differentiation and to-
ward Th17 cell differentiation, as well as the
distinct roles of IL-12 and IL-23 in supporting
Th1 and Th17 propagation, respectively, em-
phasizes the high degree of regulation involved
in T cell development and function under
various physiological states.
Th17 responses appear to be physiologi-
cally important in pulmonary bacterial immu-
nity as well as immunity to certain intestinal
pathogens (Khader et al., 2007). The specific
role of IL-17 and other cytokines produced
by Th17 cells in these responses remains to be
completely elucidated, though direct effects
on epithelial cells as well as recruitment of
neutrophils seem to be important factors, de-
pending on the site and nature of the infec-
tion (Khader et al., 2007). Pathologically,
Th17 responses are involved in certain in-
flammatory and autoimmune diseases, includ-
ing inflammatory bowel disease, rheumatoid
arthritis, autoimmune iritis, and central ner-
vous system autoimmune syndromes (Bettelli
et al., 2007). CD8 T cells and non–T cells
have been reported to produce Th17 cyto-
kines (Weaver et al., 2007), including IL-17,
but the role of non–T cell–derived IL-17 re-
mains to be further defined.
The immune system can act as an extrinsic
suppressor of tumors, and the importance of
Th1, characterized by IFN- and type 1 IFNs,
in inhibiting tumor incidence and growth has
been established (Kaplan et al., 1998; Dunn
et al., 2006). Recent studies, however, have
also demonstrated a critical role of certain im-
mune cells, via production of specific cytokines
Abbreviations used: MEF, mouse
embryonic fibroblast; phospho-
Stat3, phosphorylated Stat3.
L. Wang and T. Yi contributed equally to this paper.
IL-17 can promote tumor growth
through an IL-6–Stat3 signaling pathway
Lin Wang,1,4 Tangsheng Yi,2,3,4 Marcin Kortylewski,1 Drew M. Pardoll,5
Defu Zeng,2,3 and Hua Yu1
1Department of Cancer Immunotherapeutics and Tumor Immunology, 2Department of Hematology and Hematopoietic Cell
Transplantation, 3Department of Diabetes and Metabolic Diseases Research, and 4Graduate School of Biological Science,
Beckman Research Institute at City of Hope Medical Center, Duarte, CA 91010
5Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 21231
Although the Th17 subset and its signature cytokine, interleukin (IL)-17A (IL-17), are impli-
cated in certain autoimmune diseases, their role in cancer remains to be further explored.
IL-17 has been shown to be elevated in several types of cancer, but how it might contribute
to tumor growth is still unclear. We show that growth of B16 melanoma and MB49 bladder
carcinoma is reduced in IL-17/ mice but drastically accelerated in IFN-/ mice, contrib-
uted to by elevated intratumoral IL-17, indicating a role of IL-17 in promoting tumor
growth. Adoptive transfer studies and analysis of the tumor microenvironment suggest that
CD4+ T cells are the predominant source of IL-17. Enhancement of tumor growth by IL-17
involves direct effects on tumor cells and tumor-associated stromal cells, which bear IL-17
receptors. IL-17 induces IL-6 production, which in turn activates oncogenic signal transducer
and activator of transcription (Stat) 3, up-regulating prosurvival and proangiogenic genes.
The Th17 response can thus promote tumor growth, in part via an IL-6–Stat3 pathway.
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TH17 RESPONSES PROMOTE TUMOR PROGRESSION VIA A STAT3 PATHWAY | Wang et al.
and tumor angiogenesis (Bromberg et al., 1999; Yu et al.,
2007). Persistent activation of Stat3 in tumor cells and in tu-
mor-associated immune cells also promotes accumulation of
tumor myeloid-derived suppressor cells and tumor regulatory
T cells, leading to tumor immune suppression (Kortylewski
et al., 2005). Although IL-17–producing T cells have been
found in increased numbers within certain tumors (Miyahara
et al., 2008; Sfanos et al., 2008; Zhang et al., 2008), it remains
controversial whether IL-17 promotes or inhibits cancer pro-
gression (Numasaki et al., 2003; Numasaki et al., 2005;
Muranski et al., 2008; Nam et al., 2008; Kryczek et al., 2009;
Xiao et al., 2009; Zhang et al., 2009). Importantly, the un-
derlying mechanisms of IL-17 in modulating tumor growth
is still poorly understood.
or growth factors, in promoting carcinogenesis and tumor
growth (Colombo and Mantovani, 2005). This opposing role
of the immune system in tumor immune surveillance and
cancer promotion is exemplified by a pair of related cyto-
kines: although IL-12 clearly possesses antitumor activity via
both NK activation and Th1/IFN- induction, IL-23 has
been found to promote carcinogenesis (Langowski et al.,
2006). Specific STAT pathways regulate the IL-12/IL-23
balance, with Stat3 coordinately activating IL-23/p19 gene
transcription while inhibiting expression of the IL-12/p35
gene (Kortylewski et al., 2009). Similar to IL-23, IL-17 ex-
pression is also regulated by Stat3 (Chen et al., 2006), which
is an oncogene persistently activated in tumor cells and tumor
stromal cells, promoting tumor cell survival, proliferation,
Figure 1. IL-17 promotes tumor growth, which is mainly mediated by T cells. (A) 105 B16 tumor cells were injected subcutaneously into WT,
IL-17/, IFN-/, and IFN-/IL-17/ B6 mice. Data represent means ± SEM (n = 12 mice per group from three independent experiments). (B) MB49
tumor growth in WT, IL-17/, IFN-/, and IFN-/IL-17/ B6 mice (n = 8 mice from two experiments). (C and D) Intratumoral IL-17 promotes
cancer metastasis. (C) MB49 tumor cells were injected subcutaneously into the four groups of mice as indicated, and lung colonies were enumerated
3 wk later (n = 4; P = 0.01). (D) Representative photos of the lung tissues from each group with arrows indicating lung metastasis are shown. Bars:
(top) 1,000 µm; (bottom) 500 µm. (E) IL-17–mediated tumor-promoting effects are mainly mediated by T cells. Rapid B16 tumor growth in Rag-2/
mice receiving adoptive transfer of IFN-/ T cells was observed compared with significantly reduced growth rates in mice receiving adoptive-trans-
ferred IFN-/IL-17/ T cells. Data represent means ± SEM (n = 11 mice from two experiments).
JEM VOL. 206, July 6, 2009
BRIEF DEFINITIVE REPORT
RESULTS AND DISCUSSION
Th17 cells can promote tumor growth
We assessed the role of IL-17 in modulating tumor growth in
mice with genetically ablated IL-17 alleles (IL-17 KO). Be-
cause IFN-–producing Th1 T cells generally provide anti-
tumor immunity (Kaplan et al., 1998; Dunn et al., 2006) and
are known to restrain Th17 cell development (Harrington
et al., 2005), we also evaluated tumor growth in IFN- KO
mice and in IL-17IFN- double KO mice. Sex- and age-
matched WT, IL-17/, IFN-/, and IFN-/IL-17/
B6 mice were challenged with B16 melanoma. Results from
these experiments showed that tumor growth rate was re-
duced in the B6 IL-17/ mice when compared with WT
B6 controls (Fig. 1 A). As expected from the antitumor role
of IFN- (Kaplan et al., 1998; Dunn et al., 2006), in B6 mice
lacking IFN- alleles, B16 tumors appeared earlier and the
tumor growth rate was drastically increased relative to the
WT (Fig. 1 A). Importantly, in IFN-/IL-17/ mice, tumor
growth was significantly reduced compared with IFN-/
mice (Fig. 1 A), supporting a tumor-promoting role of Th17
cells in the B16 subcutaneous tumor model.
We also evaluated growth of the MB49 bladder carci-
noma in WT, IL-17/, IFN-/, and IFN-/IL-17/
mice. Similar to B16, we observed growth inhibition of MB49
tumors in IL-17/ mice relative to WT controls (Fig. 1 B).
Furthermore, MB49 tumor growth was significantly acceler-
ated in IFN-/ relative to WT mice. Ablating IL-17 di-
minished accelerated tumor growth caused by a lack of IFN-
(Fig. 1 B). However, the tumor growth rate in the IFN-
/IL-17/ B6 mice was higher than in IL-17/ mice.
Thus, the general pattern of tumor growth in IL-17/, IFN-
/, and IFN-/IL-17/ mice was similar for B16 and
MB49 tumors despite their different tissue origins. Subcuta-
neous MB49 tumors also naturally metastasize to lungs; we
therefore assessed whether levels of lung metastasis correlate
with tumor size in the four groups of mice. Ablating IL-17 in
both WT and IFN-/ mice was associated with a decreased
number of lung metastases (Fig. 1, C and D).
To assess whether T cells were a primary source of IL-17
for promoting tumor growth, we reconstituted Rag2/
mice by adoptive T cell transfer with purified T cells from
IFN-/ mice that display high levels of IL-17 production
(Fig. 2), or from IFN-/IL-17/ mice. Tumor growth in
mice receiving IFN-/IL-17/ T cells was significantly
reduced compared with mice given IFN-/ T cells (Fig. 1 E),
suggesting that the increased tumor growth, caused by ele-
vated IL-17 production, was mainly contributed by T cells.
Reciprocal relationship between IL-17 and IFN-
production in tumors
We performed intracellular cytokine staining and in vitro
cytokine production assays to compare the cytokine pro-
file of CD4+ and CD8+ T cells in tumors harvested from
WT, IL-17/, IFN-/, and IFN-/IL-17/ mice.
IL-17 production in B16 tumors was markedly augmented
in the absence of IFN-, with an increase in the number of
Figure 2. Reciprocal relationship between IFN- and IL-17 pro-
duction by tumor-infiltrating T lymphocytes. Single-cell suspen-
sions were prepared from B16 or MB49 tumors harvested 20 d after
tumor implantation. (A) IL-17 expression in CD4+ T cells from B16 (top)
or MB49 (bottom) tumors; flow cytometric patterns shown (one tumor
per group) are representative of three independent experiments. Per-
centages are shown. (B) IL-17 secretion by intratumoral CD4+ T cells
sorted from B16 tumors grown in WT, IL-17/, IFN-/, or IFN-
/IL-17/ mice was assessed by ELISA after a 24-h culture, combin-
ing results from three independent experiments of three to four pooled
tumor samples (P = 0.001). Data represent means ± SEM. (C) Percent-
ages of CD4+ and CD8+ T cells within the total intratumoral leukocyte
population harvested on days 20 (top row) and 14 (second from top
row). (second from bottom row) IFN- expression in CD8+ T cells
derived from tumors of WT or IL-17/ mice. (bottom row) IFN-
expression by tumor-infiltrating CD4+ T cells (representative of three
independent experiments). Percentages are shown. (D) Increased secre-
tion of IFN- by sorted tumor-infiltrating CD8+ (top) and CD4+ (bottom)
T cells, combining results from three independent experiments of
pooled tumor samples (P = 0.002). Data represent means ± SEM.
TIL, tumor-infiltrating T lymphocytes.
TH17 RESPONSES PROMOTE TUMOR PROGRESSION VIA A STAT3 PATHWAY | Wang et al.
cence staining of phosphorylated Stat3 (phospho-Stat3) in
situ. We observed reduced phospho-Stat3 levels in B16 tu-
mors grown in IL-17/ mice compared with WT controls.
In addition, Stat3 activity was drastically increased in tumors
grown in IFN-/ mice, suggesting an inhibitory role of IFN-
on tumor Stat3 activation. Stat3 phosphorylation was dimin-
ished in tumors harvested from IFN-/IL-17/ compared
with IFN-/ mice (Fig. 3 A). Immunofluorescence stain-
ing results were confirmed by Western blotting using an anti-
body for phospho-Stat3 (Fig. 3 B). Collectively, these results
indicated that the levels of IL-17 positively correlated with
Stat3 activity in growing tumors.
We next tested whether increased IL-17 expression af-
fected downstream Stat3 target genes. We found that Bcl
family members, including Bcl-2 and Bcl-xL, were up-regu-
lated in tumors from IFN-/ mice (Fig. 3 C). In addition,
vascular endothelial growth factor and MMP9 were up-reg-
ulated in tumors from IFN-/ mice, and their expression
was reduced in tumors from IFN-/IL-17/ mice (Fig. 3 D).
These results suggested that IL-17 promoted Stat3 activity in
tumors, leading to up-regulation of antiapoptotic and angio-
To investigate whether IL-17 signals directly through
Stat3, we cultured B16 tumor cells, normal fibroblasts, en-
dothelial cells, splenic DCs, and non-DC myeloid cells in
the presence of recombinant mouse IL-17 or control. Re-
sults from these experiments showed that IL-17 indeed in-
duced Stat3 activity (Fig. 4 A). Although IL-17 induced
Stat3 activity in all tested target cells, it did so with signifi-
cantly different kinetics (Fig. 4 A). Normal fibroblasts and
endothelial cells were included to test IL-17 response, as
they are major components of the tumor microenviron-
ment. Similarly, we found that human recombinant IL-17
significantly stimulated Stat3 phosphorylation in several
tumor-infiltrating Th17 cells (Fig. 2 A, top). IL-17 expression
was also elevated in MB49 tumors grown in IFN-/ mice
(Fig. 2 A, bottom). Further, ELISA analyses indicated a >10-
fold increase in secreted IL-17 by B16 tumor–infiltrating
CD4+ T cells in IFN-/ mice, which was again abrogated
in IFN-/IL-17/ double KO mice (Fig. 2 B). Notably,
we observed only minimal levels of IL-17 production by
CD8+ T cells in both B16 and MB49 tumors (Fig. S1).
We observed an increased infiltration of CD4+ and CD8+
T cells in tumors grown in IL-17/ mice (Fig. 2 C, top two
rows). Furthermore, tumor-infiltrating CD8+ and CD4+ T
cells from IL-17/ mice produced more IFN- relative to
WT control mice, as assessed by intracellular cytokine stain-
ing (Fig. 2 C). The increase in IFN- production by both tu-
mor-infiltrating CD4+ and CD8+ T cells from IL-17/ mice
was confirmed by ELISA (Fig. 2 D). IL-17 ablation only
modestly increased the percentage of tumor-infiltrating regu-
latory T cells (Fig. S2 A). It has been reported that IL-17 abla-
tion does not affect the function of regulatory T cells (Yi
et al., 2008). No correlation was observed with Th2-type im-
mune responses as measured by CD4+ T cell–mediated IL-4
production caused by IL-17 and IFN- ablation (Fig. S2 B).
IL-17 activates Stat3 in both tumor and stromal cells
in the tumor microenvironment
The underlying mechanisms by which Th17 promoted tu-
mor growth was still unknown. Because Stat3 activation in
tumor cells and tumor-associated inflammatory cells plays a
critical role in tumor progression by augmenting tumor sur-
vival and tumor angiogenesis, and suppressing antitumor im-
munity (Yu et al., 2007), we explored the possibility that
IL-17 mediates tumor growth via activation of Stat3. Stat3
activity in B16 tumors from WT, IL-17/, IFN-/, and
IFN-/IL-17/ mice was examined by immunofluores-
Figure 3. Tumorigenic effects of IL-17 are associated with Stat3 activation and up-regulation of oncogenic genes. B16 tumors were har-
vested from WT, IL-17/, IFN-/, and IFN-/IL-17/ mice 20 d after tumor challenge. (A and B) Stat3 activation in B16 tumors in vivo is notably
diminished by IL-17 ablation. (A) Frozen tumor sections were stained with antibodies specific to phospho-Stat3 (pStat3; green). Results are representa-
tive of three independent experiments. Bar, 100 µm. (B) Western blot analysis of phospho-Stat3, total Stat3, and -actin in protein extracts prepared
from freshly harvested B16 tumors (representative of three experiments). (C and D) IL-17 deletion inhibits expression of Stat3-downstream oncogenic
genes. Western blot analysis of tumor protein extracts to detect Bcl-xL, Bcl-2, vascular endothelial growth factor, MMP9, and -actin protein levels
(representative of two independent experiments).
JEM VOL. 206, July 6, 2009
BRIEF DEFINITIVE REPORT
tumor cells with recombinant IL-17 only modestly increased
their proliferation (Fig. S5 A), which is consistent with an-
other study (Numasaki et al., 2005), similar treatment of endo-
thelial cells led to increased expression of several Stat3 downstream
genes that are involved in cell migration (Fig. S5 B). In addi-
tion, IL-17–mediated increased secretion of angiogenic fac-
tors by endothelial cells induced endothelial cell migration
in a Stat3-dependent manner (Fig. S5 C). Importantly, neu-
tralizing IL-6 but not IL-10 (another Stat3 activator) with a
blocking mAb reduced Stat3 activation in (mouse embry-
onic fibroblasts) MEFs, DCs, and MB49 tumor cells after
IL-17 stimulation (Fig. 4 D). Similarly, anti–IL-6 antibodies
abrogated IL-17–induced Stat3 activation in human cancer
cells (Fig. S3 B). These results indicated that IL-17 activated
Stat3 through IL-6 in diverse types of cancer and nontrans-
formed cells constituting the tumor stroma.
In vivo IL-6 blockade partially reverses tumor progression
in the setting of high intratumoral IL-17
We first assessed whether IL-17 affected IL-6 production by
tumors in vivo. Freshly harvested B16 tumor cells from WT
human cancer cell lines (Fig. S3 A). We further confirmed
the expression of IL-17 receptor in tumor cells and fibro-
blasts (Fig. S4). These results suggested that induction of
Stat3 activation in tumor cells and elements of the tumor
microenvironment by IL-17 occurred through induction
of an intermediary factor rather than directly.
IL-17 activates Stat3 via IL-6 induction
IL-6 is a Stat3 activator and is elevated in diverse cancers
(Hirano et al., 2000). IL-17 has recently been reported to
stimulate production of IL-6 and Stat3 activation through
a positive feedback loop, in inflammatory cells as well as
fibroblasts, in an autoimmune disease setting (Ogura et al.,
2008). We therefore determined whether IL-6 mediated IL-
17–driven Stat3 activation in a tumor setting. We found that
IL-17 stimulated IL-6 production by B16 and MB49 tumor
cells in vitro, in addition to other cell types that constitute
the tumor stroma such as fibroblasts, endothelial cells, and
DCs (Fig. 4, B and C). The levels of IL-6 per cell produced
by the tested tumor cells were lower than those produced
by fibroblasts and endothelial cells. Although treating the
Figure 4. IL-17 activates Stat3 in tumor and tumor stromal cells through an IL-6–dependent mechanism. (A) The kinetics of Stat3 activation in
B16 melanoma cells, MEFs, endothelial cells (ECP), splenic CD11c+ DCs, or splenic CD11b+ myeloid cells after stimulation with recombinant IL-17. Western
blots of phospho-Stat3, total Stat3, and -actin are shown and are representative of two independent experiments. (B and C) IL-17 induces IL-6 expres-
sion in tumor and various tumor stromal cells. (B) IL-6 levels in B16 cells treated for 24 h with recombinant IL-17 were assessed by quantitative real-time
PCR (left) and ELISA (second from left). IL-6 induction in MB49 tumor cells (middle), MEFs (second from right), and endothelial cells (ECP; right) by recom-
binant IL-17 treatment is shown (from three independent experiments; P < 0.0001). Data represent means ± SEM. (C) Production of IL-6 by CD11c+ DCs
upon IL-17 stimulation, as determined by ELISA (from three independent experiments; P = 0.0002). Data represent means ± SEM. (D) IL-17–induced Stat3
activation is IL-6 dependent. (top) MB49 tumor cells were incubated with IL-17 with or without anti–IL-6 or –IL-10 antibodies, and Western blots were
performed for phospho-Stat3, total Stat3, and -actin protein levels. (bottom) MEFs and splenic CD11c+ DCs were stimulated with IL-17 in the presence
of IL-6–neutralizing antibodies or control IgG (representative of three independent experiments).
TH17 RESPONSES PROMOTE TUMOR PROGRESSION VIA A STAT3 PATHWAY | Wang et al.
level of IL-6 when compared with the IFN-/IL-17/
mice (Fig. 5 A). More detailed analysis of growing tumors
indicated that tumor-associated immune cells (CD45+) were
the main source of IL-6 when compared with the tumor cells
(Fig. 5 B). To directly test whether IL-17 could promote
tumor growth through IL-6–mediated Stat3 activation,
IFN-/ mice were challenged with B16 melanoma, fol-
lowed by administration of IL-6–neutralizing mAbs or con-
trol rat IgG. IFN-/IL-17/ mice challenged with B16
melanoma but without anti–IL-6 antibody treatment were
included as an additional control. Because IL-6 was shown to
promote Th17 cell differentiation, we did not start anti–IL-6
treatment until 6 d after tumor inoculation, a time at which
tumor-infiltrating Th17 cells were detectable (unpublished
data). We found that administration of monoclonal anti–IL-6
but not rat IgG antibodies significantly inhibited tumor
growth in IFN-/ mice (Fig. 5 C), suggesting that IL-17–
induced tumor progression is at least in part mediated by IL-6.
Consistent with a role for IL-6 in mediating tumor Stat3 ac-
tivation and tumor growth promotion, neutralizing IL-6 in
vivo reduced Stat3 activity and the expression of its down-
stream antiapoptotic gene, Bcl-xL, in tumors (Fig. 5 D).
In summary, we have shown that IL-17 can promote
tumor growth. Our results suggested that the IL-17–medi-
ated tumor-promoting role involves a direct effect on tumor
through IL-6 induction, which in turn activates Stat3 in
both tumor as well as nontransformed cells constituting the
tumor microenvironment. IL-6 activation of Stat3 in tumor
cells results in increases in antiapoptotic, proproliferation,
and proangiogenic genes, as well as suppression of certain
proinflammatory genes (Yu et al., 2007). IL-6–induced Stat3
signaling in T cells is also critical in promoting Th17 cell
differentiation and expression (Chen et al., 2006; Harris
et al., 2007). Given that TGF-, the other cytokine involved
in Th17 cell differentiation, is also expressed at high levels in
tumors, IL-17–induced IL-6 production can generate an au-
toamplification loop for Th17 cells in the tumor microenvi-
ronment. Consistent with our findings, several publications
suggested a role of IL-17 in promoting tumor growth
(Numasaki et al., 2003; Numasaki et al., 2005; Nam et al.,
2008; Xiao et al., 2009), although the mechanism was not
well addressed in these studies. However, a recent paper
showed that, in a different tumor system (MC38 sarcoma),
tumors growth was increased in IL-17/ mice (Kryczek
et al., 2009). These findings suggest that the role of IL-17 in
cancer is context and system dependent, like many other cy-
tokines such as TNF-. Additional studies are required to
reveal why IL-17 can have opposite roles in modulating
growth in different tumor systems.
The opposing functions of IFN- and IL-17 as anticarci-
nogenic and protumor growth effector cytokines extend an
emerging concept that the qualitative nature of the immune
response determines whether it will predominantly inhibit
tumor growth or promote cancer development. Recently,
analogous opposing effects of the two IL-12 family mem-
bers, IL-12 and IL-23, in tumor inhibition versus promotion
mice produced relatively high levels of IL-6, which were re-
duced after IL-17 ablation (Fig. 5 A). In addition, tumors
grown in IFN-/ mice produced a markedly increased
Figure 5. Tumor-derived IL-6 mediates tumorigenic effects of IL-17.
(A and B) IL-6 secretion in the tumor microenvironment correlates with IL-
17 levels. (A) Single-cell suspensions prepared from B16 tumors harvested
from the indicated mice were cultured in vitro overnight; supernatants were
then collected and assayed for IL-6 levels, combining four mice from two
independent experiments (WT vs. IL-17/, P = 0.0026; IFN-/ vs. IFN-
/IL-17/, P = 0.005). Data are means ± SEM. (B) Single-cell suspensions
prepared from MB49 tumors were enriched for CD45. Both CD45+ (immune
cells) and CD45 (tumor cells) were cultured overnight before supernatants
were collected for ELISA (n = 4 mice representing three independent experi-
ments; IFN-/ vs. IFN-/IL-17/, P = 0.03). Data are means ± SEM.
(C) 105 B16 tumor cells were injected subcutaneously into IFN-/ and IFN-
/IL-17/ mice. 6 d after tumor implantation, tumor-bearing IFN-/
mice were treated with IL-6–neutralizing antibodies or control rat IgG every
other day. Data are means ± SEM (n = 7 mice from two independent experi-
ments). (D) Western blot analyses of phospho-Stat3, total Stat3, Bcl-xL, and
-actin levels from protein extracts prepared from B16 tumors harvested
from the indicated mice (representative of two independent experiments).
JEM VOL. 206, July 6, 2009
BRIEF DEFINITIVE REPORT
ELISA assays. 105 sorted tumor-infiltrating CD4+, CD8+ T cells were
cultured for 24 h in a U-bottom 96-well plate with plate-bound CD3 and
2 µg/ml of soluble CD28. 106 enriched splenic CD11c+ or cultured cells/ml
were stimulated for 24 h with 10 ng/ml of recombinant IL-17. IFN-, IL-17,
IL-6, and IL-4 concentrations in culture supernatants were measured using
ELISA kits from R&D Systems.
Immunofluorescence staining. 5-µm sections of flash-frozen tumor
specimens were fixed in acetone, permeabilized with methanol, stained with
antibodies specific to tyrosine–phospho-Stat3 (Santa Cruz Biotechnology,
Inc.) and detected with secondary antibodies conjugated Alexa Fluor 488
(Invitrogen), as previously described (Kortylewski et al., 2005). After stain-
ing with Hoechst 33342 (Invitrogen) to visualize cell nuclei, slides were
mounted and analyzed by fluorescence microscopy.
Statistical analysis. The unpaired t test was used to calculate the two-
tailed p-value. The two-way analysis of variance test was used to calculate
the p-value for tumor growth. Data were analyzed using Prism software
(GraphPad Software, Inc.).
Online supplemental material. Fig. S1 shows the production of IL-17 in
tumor-infiltrating CD8+ T cells, determined by intracellular staining and
ELISA. Fig. S2 indicates that ablating IFN- or IL-17 did not significantly
affect the T reg cell percentage and IL-4 production. Fig. S3 shows that IL-17
activates Stat3 in human tumor cell lines in an IL-6–dependent manner.
Fig. S4 confirms IL-17 receptor expression on B16 tumor cells and MEFs.
Fig. S5 depicts the effect of IL-17 on tumor cell proliferation and endothelial
cell migration. Online supplemental material is available at http://www.jem
We thank Dr. Y. Iwakura for generously providing us with IL-17/ mice; Dr. Y. Liu
for superb assistance; the members of the Flow Cytometry Core, the Pathology
Core, and the Animal Facility at the City of Hope Medical Center for their
contributions; and Dr. S. da Costa for editing the manuscript.
This study was supported by the National Institutes of Health (grants
R01CA122976, R01CA115815, and R01 AI066008).
The authors have no conflicting financial interests.
Submitted: 27 January 2009
Accepted: 8 June 2009
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microenvironment determined the balance between these
cytokines by transcriptionally activating IL-23p19 and inhib-
iting IL-12p35 (Kortylewski et al., 2009). The current work
is among the first examples by which a qualitatively distinct
(Th17) endogenous T cell immune response can be cancer
promoting. Further, it adds a new element to the link be-
tween immune responses that either depend on or induce
Stat3 signaling and Stat3-mediated protumor growth.
MATERIALS AND METHODS
Mice. WT and IFN-/ C57BL/6 mice were purchased from the Na-
tional Cancer Institute and the Jackson Laboratory, respectively. The gener-
ation of C57BL/6 IL-17/ mice has been previously reported (Nakae
et al., 2002), and the mice were provided by Y. Iwakura (University of Tokyo,
Tokyo, Japan). Mouse care and experimental procedures were performed
under pathogen-free conditions in accordance with established institutional
guidance and approved protocols from the Research Animal Care Commit-
tee of the City of Hope Medical Center. For tumor challenge, 105 B16 tumor
cells were injected subcutaneously into 8–12-wk-old WT or transgenic mice,
and tumor growth was monitored every other day. For IL-6 neutralization
experiments in vivo, mice were treated with 125 µg anti–IL-6 antibodies
(eBioscience) or control rat IgG (Jackson ImmunoResearch Laboratories)
injected i.v. on days 6, 8, 10, 12, and 14 after tumor inoculation. Mice were
sacrificed after 2–3 wk from tumor inoculation or when tumor volume
exceeded 1.5 cm in diameter.
Cell lines. B16 and MB49 cell lines were originally obtained from Ameri-
can Type Culture Collection and were maintained in our laboratory. Endo-
thelial cell lines derived from mouse prostate were provided by J. Fidler
(M.D. Anderson Cancer Center, Houston, TX).
T cell adoptive transfer. T cells were enriched from IFN-/ or IFN-
/IL-17/ mice. Specifically, non–T cells were depleted with biotin-
labeled mAbs, including anti-B220, Gr-1, Mac-1, CD11c, DX5, and Ter119,
using a magnetic purification system from Miltenyi Biotec. TCR+ T cell
purity was >90%, as determined by flow cytometry.
Preparation of tumor-infiltrating immune cells. The procedure for iso-
lating immune cells from tumors has been described previously (Kortylewski
et al., 2005). In brief, freshly excised tumor tissues were gently minced and
digested for 30 min at 37°C by collagenase D/DNase I (Roche). Cell
suspensions were filtered through a 70-µm cell strainer, and dead cells
were removed by centrifugation at 600 g over a Histopaque gradient
Flow cytometry. The FITC-, PE-, allophycocyanin (APC)-, or Cy7-
APC–conjugated antibodies specific to CD4, CD8, IL-17, and IFN- were
purchased from BD; PE-labeled antibody against mouse Foxp3 was pur-
chased from eBioscience, and PE-labeled antibody for IL-17R was pur-
chased from R&D Systems. Intracellular staining for IL-17 and IFN- was
performed according to the manufacturer’s instructions (BD; Yi et al.,
2008). In brief, cells were stimulated for 5 h with PMA and ionomycin
(Sigma-Aldrich) in the presence of Golgi-Stop reagent (BD). Cells were
harvested, washed, and stained with anti-CD4 or -CD8 in the presence
of FcR-Block (BD). After the wash, cells were fixed, permeabilized, and
stained with cytokine-specific or control isotype antibodies for 30 min on
ice. Multiple-color FACS analysis was performed using a three-laser CyAn
Immunocytometry System (Dako). Dead cells were excluded by the Fixable
Aqua Dead Cell Stain Kit (Invitrogen).
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