T helper 17 cells promote cytotoxic T cell activation in tumor immunity.

Abstract

Although T helper 17 (Th17) cells have been found in tumor tissues, their function in cancer immunity is unclear. We found that interleukin-17A (IL-17A)-deficient mice were more susceptible to developing lung melanoma. Conversely, adoptive T cell therapy with tumor-specific Th17 cells prevented tumor development. Importantly, the Th17 cells retained their cytokine signature and exhibited stronger therapeutic efficacy than Th1 cells. Unexpectedly, therapy using Th17 cells elicited a remarkable activation of tumor-specific CD8(+) T cells, which were necessary for the antitumor effect. Th17 cells promoted dendritic cell recruitment into the tumor tissues and in draining lymph nodes increased CD8 alpha(+) dendritic cells containing tumor material. Moreover, Th17 cells promoted CCL20 chemokine production by tumor tissues, and tumor-bearing CCR6-deficient mice did not respond to Th17 cell therapy. Thus, Th17 cells elicited a protective inflammation that promotes the activation of tumor-specific CD8(+) T cells. These findings have important implications in antitumor immunotherapies.

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Immunity
Article
T Helper 17 Cells Promote Cytotoxic T Cell
Activation in Tumor Immunity
Natalia Martin-Orozco,1Pawel Muranski,4Yeonseok Chung,1Xuexian O. Yang,1Tomohide Yamazaki,1Sijie Lu,2
Patrick Hwu,3Nicholas P. Restifo,4Willem W. Overwijk,3and Chen Dong1,*
1Department of Immunology
2Department of Stem Cell Transplantation and Cell Therapy
3Department of Melanoma Medical Oncology
MD Anderson Cancer Center, Houston, TX 77030, USA
4National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
*Correspondence: cdong@mdanderson.org
DOI 10.1016/j.immuni.2009.09.014
SUMMARY
Although T helper 17 (Th17) cells have been found in
tumor tissues, their function in cancer immunity is
unclear. We found that interleukin-17A (IL-17A)-defi-
cient mice were more susceptible to developing lung
melanoma. Conversely, adoptive T cell therapy with
tumor-specific Th17 cells prevented tumor develop-
ment. Importantly, the Th17 cells retained their cyto-
kine signature and exhibited stronger therapeutic
efficacy than Th1 cells. Unexpectedly, therapy using
Th17 cells elicited a remarkable activation of tumor-
specific CD8+T cells, which were necessary for the
antitumor effect. Th17 cells promoted dendritic cell
recruitment into the tumor tissues and in draining
lymph nodes increased CD8a+dendritic cells con-
taining tumor material. Moreover, Th17 cells pro-
moted CCL20 chemokine production by tumor
tissues, and tumor-bearing CCR6-deficient mice
didnotrespondtoTh17celltherapy. Thus,Th17cells
elicited a protective inflammation that promotes the
activation of tumor-specific CD8+T cells. These find-
ings have important implications in antitumor immu-
notherapies.
INTRODUCTION
CD4+Tcells,uponactivationbyantigen-presenting cells(APCs),
differentiate into cytokine-expressing effector helper T (Th) cells,
which are classified as Th1, Th2, Th17, and T follicular helper
(Tfh) cell subsets on the basis of their cytokine secretion and
immune regulatory function. Th17 cells produce the proinflam-
matory cytokines IL-17A, IL-17F, and IL-22 (Dong, 2008). As
the signature cytokine of Th17 cells, IL-17A induces the expres-
sion of several chemokines (CCL2, CCL7, CXCL1, and CCL20)
and matrix metalloproteinases (MMP3 and MMP13); transgenic
overexpression of IL-17A in the lung provokes the induction of
proinflammatorygeneexpressionandtissueinfiltrationbyleuko-
cytes (Park et al., 2005). Conversely, inhibition of IL-17A
signaling leads to impaired host defense against bacterial infec-
tion (Ye et al., 2001) and resistance to autoimmune diseases
(Langrish et al., 2005; Nakae et al., 2003; Park et al., 2005;
Yang et al., 2008).
Th17 cells and IL-17A expression have been found in various
human tumors (Kryczek et al., 2007; Langowski et al., 2006;
Miyahara et al., 2008; Sfanos et al., 2008; Zhang et al., 2008);
however, their function in cancer immunity is unclear. IL-17A
overexpression in tumor cell lines promotes angiogenesis and
tumorgrowthwhenthetumorsareimplantedinimmunodeficient
mice, therefore suggesting a protumor activity (Numasaki et al.,
2003).Incontrast,theexpressionofIL-17Ainahematopoietically
derived tumor was reported to promote tumor protection in im-
muno-competent hosts (Benchetrit et al., 2002). The basis for
this discrepancy has not been understood, and the presence
or absence of the adaptive immune system has been suggested
to account for it (Martin-Orozco and Dong, 2009). Th17 cells
highly express IL-23R; IL-23 is required for the late stage of
Th17 cell development and also functions to expand Th17 cells
and promote their function (Langrish et al., 2005; McGeachy
et al., 2009). Il-23a (p19) mRNA expression has been found in
several human carcinomas (Langowski et al., 2006). Moreover,
IL-23-deficient mice (Il23a?/?and Il12b?/?) have been reported
to be resistant to chemically induced tumors (Langowski et al.,
2006). Paradoxically, the expression of IL-23 at the tumor site
or therapy with dendritic cells expressing IL-23 can induce
potent tumor-specific immunity against melanoma and glioma
(Hu et al., 2006; Overwijk et al., 2006). More recently, it was
shown that Th17 cells could protect against skin melanoma in
a lymphopenic environment (Muranski et al., 2008); however,
because the protection was dependent on IFN-g, presumably
because of conversion of Th17 to Th1 cells, the exact function
of Th17 cells remains unclear.
In the current study, we first analyzed tumor development in
IL-17-deficient mice by using a poorly immunogenic B16-F10
melanoma that colonizes the lung. Additionally, we used adop-
tive transfer of Th17 cells in several tumor prevention and treat-
mentmodels.OurresultsindicatethatIL-17AandTh17cellsplay
a protective role against tumors. Unexpectedly, tumor-specific
Th17 cells triggered a strong CD8+T cell response against the
tumor. Th17 cell therapy promoted dendritic cell (DC) infiltration
into tumor tissues and presentation of tumor antigens in the
tumor-draining lymph nodes. Compared to Th1 cells, Th17 cells
strongly induced CCL20 expression in the tumor tissues and
CCR6 deficiency abrogated the antitumor effects of Th17 cells.
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Our results thus reveal a protective function of Th17 cells in
tumor immunity by eliciting cytotoxic T cell activation.
RESULTS
Enhanced Tumor Growth in the Absence of IL-17
To investigate the role of IL-17 in tumor development in vivo, we
challenged IL-17A-deficient mice (Yang et al., 2008) and wild-
type (WT) age-matched controls on 129xB6 mixed background
with B16-F10 melanoma injected intravenously. On days 14
and 16 after the challenge, Il17a?/?mice exhibited increased
numbers of tumor foci and larger tumors in size when compared
to WT mice (Figure 1A and Figure S1A available online). Consis-
tently, Il17a?/?mice that had been backcrossed to the C57BL/6
background also exhibit increased tumor burdens when
compared to WT C57BL/6 mice (Figure S1B).
Because IL-17 is involved in regulation of tissue inflammation,
lungs from all mice were analyzed by flow cytometry 16 days
after tumor implantation. Compared with WT mice, Il17a?/?
mice had substantially reduced numbers of total CD45+leuko-
cytes (Figure 1B). All subsets of leukocytes including CD4+and
CD8+
Tcells,granulocytes,
CD11c+CD8a+DC were all substantially reduced (Figures 1B
and 1D). In contrast, CD11b+macrophage numbers were not
significantly (p = 0.5) different between the two groups. We
also examined the activation status of T cells from the leukocyte
fractions and found that CD4+T cells from lungs of Il17a?/?mice
showed reduced CD44 expression (54.13% ± 2.29% of WT
versus 40.30% ± 1.9% of KO, p = 0.0036) (Figure 1C). Therefore,
IL-17-deficient mice had fewer leukocytes to the tumor tissue,
and CD4+T cells in the lung were also less activated.
To study how IL-17 deficiency affected leukocyte infiltration
and favored tumor development, we analyzed the mRNA ex-
pression of IL-17-regulated chemokines and their receptors by
lung cells (free of leukocytes) collected from WT and Il17a?/?
and CD11c+CD11b+
and
mice that had B16-F10 tumors for 16 days. Lung cells from
Il17a?/?mice had significantly reduced mRNA expression for
chemokines Ccl20, Ccl2, and Ccl7 (p = 3.01 3 10?6, p = 9.3 3
10?4, and p = 0.049 respectively) when compared with WT
mice (Figure 1E). Interestingly, mRNA expression for Ccr2 (the
receptor for CCL2) and Ccr6 (the receptor for CCL20) were
also found to be significantly reduced in lung cells of Il17a?/?
mice (p = 0.0139 and p = 6.4 3 10?5, respectively) (Figure 1E).
CCR6 protein expression on lung DCs from Il17a?/?mice was
present, but CD8a+DC from lung lymph nodes (LLNs) of WT
mice expressed greater amounts of CCR6 on their surface
compared to those from Il17a?/?mice (MFI 1360 ± 86 for WT
and 980 ± 105, p < 0.05). Our results indicate that IL-17A partic-
ipates in tumor protection, possibly by regulating chemokine-
mediated leukocyte migration into tissues.
Antitumor Th17 Cells Reduce Tumor Growth
in Prevention Models
To further understand the function of Th17 cells in tumor immu-
nity, we used a B16-F10 line that expresses chicken ovalbumin
(B16-OVA) and performed adopted transfer experiments with
OVA-specific Th17 cells. CD4+T cells purified from CD45.1
OT-II transgenic mice were differentiated into Th17 effector
cells in vitro (Chung et al., 2009; Nurieva et al., 2009). In each
experiment, the differentiated Th17 cells typically contained
>35% IL-17A- and/or IL-17F-expressing T cells, with <2%
IFN-g-producing cells as evaluated by intracellular cytokine
staining (Figure 2A). First, we transferred Th17 cells on the
same day as B16-OVA tumor challenge. Mice treated with
Th17 cells contained significantly (p = 0.014) reduced numbers
of tumor colonies in the lung on day 16 compared to control
mice that had not received any T cells (Figure 2B and Fig-
ure S2A). The donor cells were detected in substantial percent-
ages in the lung (average 15% ± 3% SD) and secondary
lymphoid organs at the end point of the experiment, suggesting
Figure 1. IL-17-Deficient Mice Are More Susceptible
to B16-F10 Melanoma Development in the Lung
Il17a?/?(KO) and wild-type (WT) mice were challenged i.v.
with 1 3 105B16-F10 melanoma cells and lungs were
analyzed on days 14 and day 16.
(A) Graphs show the total number of tumor colonies present in
the lung lobes (n = 4, average ± SD).
(B–E) At day 16, lung leukocyte and lung cell fractions were
isolated and processed for FACS analysis or RNA extraction.
(B) Total leukocyte cell numbers from lungs. The numbers
were calculated from the percentages of total live cells that
were gated on CD45+cells (n = 4, average ± SD).
(C) Expression of CD44 on CD45+CD4+T cells from the leuko-
cyte fraction.
(D) Myeloid populations (n = 4, average ± SD).
(E)Chemokineandchemokinereceptorgeneexpressionanal-
yses from lung fractions, which were free of leukocytes, was
performed by RT-PCR. Data shown were normalized to the
reference gene Actb. The lower expression of each gene
was refered as 1. Shown are the averages of four mice after
duplicate analysis per sample (n = 4, average ± SD). Results
shown are from a representative experiment of three, each
using 4–5 mice per group. (* = p < 0.05, ** = p < 0.01).
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that Th17 cells did not suffer deletion and were circulating in
the mice harboring tumors (Figure S2C). To substantiate the
above finding, we used the BWTRP-1 TCR transgenic mouse,
in which CD4+T cells recognize the tyrosinase-related protein 1
(TRP-1), a melanocyte differentiation antigen present in normal
melanocytes and in melanomas (Muranski et al., 2008). Trans-
fer of TRP-1 Th17 cells at the time of the B16 tumor implanta-
tion completely inhibited the tumor growth in the lung
(Figure 2C and Figure S2B). From these results, we conclude
that tumor-specific Th17 cells, regardless of their antigenic
specificities, can protect mice from developing B16-F10 lung
melanoma.
To understand how Th17 cells mediate tumor protection, we
analyzed the cellular composition of the lungs from the mice
described above. OVA-specific Th17 cell-treated tumor-bearing
mice had higher numbers of CD45+leukocytes as well as CD4+
and CD8+T cells in the lungs compared to the control mice
(Figure 2D and Figure S2C) and mice receiving TRP-1-specific
Th17 cells had five times more CD8+T cells over the control
animals (Figures S3A and S3B). Interestingly, CD4+and CD8+
T cells from the mice receiving Th17 cells showed higher CD44
expression than control mice (Figure 2E and Figures S2E and
S3C). When we analyzed the antigen-presenting cells from
leukocyte fractions, we found that Th17 cell-recipient mice
also had increased numbers of CD11c+CD11b+and CD11c+
CD8a+DCs and granulocytes, whereas the numbers of macro-
phages were not altered (Figure 2F and Figure S3D). Therefore,
transferred Th17 cells induced the recruitment of DC as well as
activated CD4+and CD8+T cells to the lung.
To understand the inflammatory regulation by Th17 cells, we
further analyzed the expression of several chemokines and che-
mokine receptor genes by lung cells using real-time PCR. We
analyzed nonfractionated total lung cells (total lung), leukocytes,
and leukocyte-depleted lung cells. We found that the expression
of Ccl20 and Ccl2 was greatly increased in lungs from Th17 cell-
treated mice (Figure 2G), whereas Ccl7 and Cxcl1 (Gro-a)
expression was not increased. Further analysis after cellular
fractionation revealed that the leukocyte fraction from both
Th17 cell-treated and control mice expressed similar levels of
chemokines and chemokine receptors (Figure 2G); however,
the leukocyte-free lung cells of Th17 cell-treated mice showed
greatly increased expression of Ccl20 and Ccl2 compared to
those from control animals (Figure 2G). These results suggest
that Th17 cells in the lung might activate lung and/or tumor cells
to produce chemokines CCL20 and CCL2 that promoted the
recruitment of DCs and activated T cells.
To determine whether tumor-specific Th17 cells protect
against tumors in different tissues other than lung and also to
test a different tumor model, we applied a subcutaneous mela-
noma model and a subcutaneous fibrosarcoma model. Equal
numbers of either Th1 or Th17 OT-II cells were transferred into
C57BL/6 mice on the same day when B16-OVA or MCA205-
OVA cells were implanted subcutaneously. Th17 cells greatly
reduced growth of both B16 and MCA 205 in the skin, but Th1
Figure 2. Antitumor Th17 Cells Prevent
B16-F10 Melanoma Development
(A) Cytokine profiles of CD45.1+OT-II cells polar-
ized to Th17 cells that were used for the transfer.
(B, D, E, and F) C57BL/6 mice were injected i.v.
with 1 3 105B16-OVA cells and 5 million OT-II
Th17 cells.
(B) The graph shows the average of tumor colony
numbers in the lungs from untreated control
mice (Ctrl) and mice treated with Th17 cells
(Th17) (n = 5, average ± SD).
(C) C57BL/6 mice were injected i.v. with 1 3 105
B16/F10 cells and 5 million of TRP-1 Th17 cells.
Number of colonies in the lung (n = 5, average ±
SD).
(D) Either total CD45+or CD4+or CD8+T cells
(n = 5, average ± SD) from the leukocyte fraction.
(E) Expression of CD44 on gated CD45.2+CD4+
(upper panel) or CD45.2+CD8+T cells (lower
panel). Numbers represent the percentages of
CD44hicells.
(F) Numbers of myeloid cell populations from
leukocyte lung fraction were calculated from the
percentages of live cells gated on CD45.2+
CD11c+CD11b+(n = 5, average ± SD).
(G) mRNA gene expression analysis of total-lung-
derived cells with leukocytes, lung cells withtumor
cells and no leukocytes, and leukocyte fractions
was assessed by RT-PCR. Data shown were
normalized to the reference gene Actb. The lower
expressionofeachgenewasreferredas1.Graphs
represent the average values of four mice after
duplicate analysis per sample (n = 4, average ±
SD) (*p < 0.05, **p < 0.01). Results shown are
from one representative experiment of three using
4–5 mice per group.
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cells had very minimal effects when compared with mice
receiving no T cells (Figures S4A and S4C). In the MCA model,
there was no survival advantage of Th17 over Th1 cells because
similar numbers of CD45.1 donor cells were recovered from
draining lymph nodes on day 24 (Figures S4D–S4F). Taken
together, these results indicate that Th17 cells can induce effec-
tive antitumor responses for several tumors types and organ
locations in preventive settings. Th17 cells induce lung cells to
produce CCL2 and CCL20, resulting in DC and activated T cell
recruitment.
Therapeutic Effects of Th17 Cells in Mice
with Established Tumors
We then tested whether the transfer of Th17 cells could also help
eliminate established tumors. We compared Th17 and Th1 cells
Figure 3. Antitumor Th17 Cells Control Es-
tablished B16-F10 Melanoma
(A) Mice were inoculated i.v. with B16-OVA and on
day 5, these mice received 3 3 106Th1 or Th17
cells i.v. Mice were euthanized on day 14 after
tumor challenge for analysis. Shown are the tumor
colonies present in the lung lobes of each group of
mice (n = 4, average ± SD).
(B) Cell numbers from leukocyte lung fractions of
each group of mice, Control (Ctr) cells, Th1 cells,
and Th17 cells were calculated from the per-
centages of live cells gated on CD45.2 (n = 4,
average ± SD).
(C) Expression of CD44 on T cells obtained from
the lung leukocyte fraction (Lung) or from lung
lymph nodes (Lung LN). Cells were gated on
CD45.2 and CD4 (middle panel) or CD8 (lower
panel). Numbers represent the percentages of
CD44hicells.
(D) Total numbers of myeloid cell populations from
leukocyte lung fraction. Numbers of cells were
calculated from the percentages of live cells gated
on CD45.2 (n = 4, average ± SD).
(E) Gene expression analysis of total-lung-derived
cells withleukocytes andleukocytefractions. Data
shown were normalized to the reference gene
Actb. The lower expression of each gene was
referredas1.Graphsrepresenttheaveragevalues
of four mice after duplicate analysis per sample
(n = 4, average ± SD). Results shown are from
one representative experiment of three using 4–5
mice per group (*p < 0.05, **p < 0.01).
in their protection against 5 day estab-
lished pulmonary melanomas. Both Th17
and Th1 cell cytokine production were
confirmed by ICS (Figure S5) before
transfer, and equal numbers of cells
were injected in C57BL/6 mice harboring
B16-OVA.Compared
control mice, those treated with Th1 cells
had 40% fewer tumor foci, whereas Th17
cell-treated ones showed 75% reduction
(Figure 3A). Therefore, both Th1and Th17
celltherapieshelped
growth, with Th17 cells showing greater
withuntreated
controltumor
potency. When we analyzed cytokine production by ICS after
activation with OVA323-339peptide, we found that both Th1 and
Th17 cells maintained their cytokine profiles throughout the
experiment (data not shown).
To compare the inflammatory responses caused by Th1 and
Th17 cell therapy, we analyzed the lung infiltrates of all experi-
mental mice. There was increased recruitment of CD4+T cells
inthelungsofmicereceivingTh1orTh17cells(Figure3B).These
mice also had increased numbers of CD4+T cells in the lung
lymph nodes (LLNs). Both Th1 and Th17 cell-treated mice had
activatedCD4+andCD8+TcellsinlungandLLNswithincreased
expression of CD44 (Figure 3C). However, only Th17 cell-treated
mice had significantly (p = 0.0217 and p = 0.0023) more CD45+
leukocytes and CD8+T cells (p = 0.029 and p = 0.0034) in the
lungs and LLNs (Figure 3B). Further analysis of the myeloid
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populationsshowedthatTh17celltreatmentelicitedthegreatest
infiltration of granulocytes, macrophages, and DC, whereas Th1
cell treatment showed slightly increased DC but reduced macro-
phage numbers when compared to the control mice (Figure 3D).
In addition, mice receiving Th17 cells had increased numbers of
leukocytes in LLNs, particularly CD8a+DCs (data not shown and
Figure S6B).
Our data suggested that Th17 and Th1 cells induced different
antitumor inflammatory responses in the lung. We thus analyzed
the chemokine and chemokine receptor expression of the lungs
andleukocytesfromlungsofmicetreated withTh17or Th1cells.
Elevated expression of Ccl20 and Ccl2 mRNA by the lungs was
present only in Th17 cell-treated mice but not in Th1 cell- or
control-treated mice (Figure 3E). Ccl20 and Ccl2 expression
was predominant in lung cells depleted of leukocytes, which
indicated a direct effect of Th17 cells on lung cell production of
chemokines. Th1 cell-treated lungs showed a reduced expres-
sion of Ccl7. Ccl20 was highly expressed in the leukocyte
fractions from both Th17 and Th1 cell-treated mice, but it was
dominant in Th17 cell-treated mice (Figure 3E). Ccr6 expression
was slightly higher in leukocytes from Th1 cell-treated mice
compared with control mice but was highly increased in Th17
cell-treated mice. Taken together, these results indicate that
Th17 cells can induce an effective antitumor responses for es-
tablishedtumors.AlsotheantitumorTh17cell-inducedresponse
is different and more effective than the one induced by Th1 cells
in the lung.
Th17 Cells Maintain Their Phenotypes In Vivo
We recently demonstrated that adoptive cell therapy with Th17
cells together with total body irradiation in mice was protective
against subcutaneous melanoma; this effect of Th17 cells was
dependent on IFN-g but independent of IL-17 and IL-23 for their
protective effects (Muranski et al., 2008). This study suggests
a possible conversion of Th17 to Th1 cells upon transfer into
the irradiated hosts. Our above data on Th17 versus Th1 cell
function in nonirradiated tumor-bearing mice suggested that
Th17 cells might not require conversion to Th1 cells to exert their
antitumorfunctioninanormal,nonlymphopenicenvironment.To
determine whether Th17 cells were the direct cause of the tumor
protection, we further purified Th17 cells by using our IL-17F-
RFP reporter mice (Yang et al., 2008) and tested their capacity
to protect mice with established B16 lung melanoma. CD4+
T cells from OT-II+IL-17F-RFP mice were cultured in Th17 cell-
polarizing conditions and on day 4, live CD4+RFP+T cells were
sorted (Figure 4A) and transferred into mice bearing lung tumors.
We found that both total and sorted RFP+Th17 cells significantly
(p = 0.00309 and p = 0.018) reduced the number of tumor foci
to similar extents at day 16 (Figure 4B).
To directly evaluate the phenotypic characteristics of trans-
ferred Th17 cells, we labeled Th17 cells generated from OT-II
mice on Rag2?/?background with carboxy-fluorescein diac-
eate-succinimidyl ester (CFDA-SE) and transferred them into
mice bearing lung tumors. On day 4 after the transfer, cell divi-
sion and cytokine production were evaluated in LLNs. We found
that Th17 cells had proliferated (more than 90% of the cells were
CFDA-SE low) and continued to produce IL-17 and IL-17F but
not IFN-g (Figure 4C). We also analyzed cytokine production
from LLN cells recovered on day 14 from mice treated with
TRP-1 Th17 cells by ELISPOT. We found that in response to
TRP-1 peptide restimulation, the predominant response was
IL-17 secretion with very low amounts of IFN-g production (Fig-
ure S3E). Therefore, we conclude that in nonirradiated tumor-
bearing mice, transferred Th17 cells maintained their Th17 cell
Figure 4. Th17 Cells Maintain Their Cyto-
kine Expression in Tumor-Bearing Mice
Purified CD4+T cells from OT-II.IL-17F-RFP
reporter mice were cultured in Th17 cell condi-
tions. On day 4, CD4+RFP+cells were sorted and
injected into C57BL/6 mice bearing 5 day estab-
lished pulmonary B16-OVA tumors.
(A) Sorting strategy for RFP+, IL-17F producing
cells.
(B) Tumor foci from lungs of C57BL/6 mice that
had B16-OVA melanoma and received no treat-
ment (Ctrl), unsorted Th17 (total Th17) cells, or
RFP+sorted (RFP+) cells (n = 4, average ± SD).
(C) Purified CD4+T cells from Rag1?/?OT-II mice
were cultured in Th17 cell conditions and on day
4,cellswerelabeledwithCFDA-SEandtransferred
into C57BL/6 mice bearing 5 day established
pulmonary B16-OVA tumors. LLNs from these
mice were analyzed for proliferation and IL-17, IL-
17F, and IFN-g production on day 4 after transfer.
(D) Mice were inoculated with B16-OVA tumor and
received on the same day Th17 (Th17) cells i.v. or
no T cells. A set of mice from each group was
treated with IFN-g blocking antibodies (a-IFN-g)
on day ?1 and every other day until day 14 after
tumor challenge. Shown are the tumor colonies
present in the lung lobes of each group of mice
(n = 4, average ± SD) (** p < 0.01).
(E) Total cell numbers from leukocyte lung fractions. Number were calculated from the percentages of live cells gated on CD45.2 (n = 4, average ± SD).
(F) Number of myeloid cell populations from leukocyte lung fraction was calculated from the percentage of live cells gated on CD45.2 (n = 4, average ± SD).
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cytokine production after encounter of tumor antigens and did
not convert to a Th1 cell phenotype.
Moreover, we injected IFN-g neutralizing antibodies 1 day
before the transfer of Th17 cells and every other subsequent
day until the endpoint. The same IFN-g antibody regime has
been tested and reported by us to inhibit Th1 cell-mediated
type I diabetes (Martin-Orozco et al., 2009). We found that
blockade of IFN-g did not alter the tumor-protective effect of
Th17 cells (Figure 4D). Moreover, the anti-IFN-g treatment did
not reduce the numbers of total CD45+leukocytes or CD4+or
CD8+T cells in the lung (Figure 4E). There was also no change
in the DC populations in the lungs of Th17 cell-treated mice as
a result of the IFN-g blocking antibody (Figure 4F). Therefore, it
seems IFN-g is not involved in the recruitment of leukocytes to
the lung nor does it play a role in the tumor-protective effect
elicited by Th17 cells. Our results indicate that the Th17 cell
tumor-protection effect was not dependent on these cells con-
verting to the Th1 cell phenotype.
Th17 Cells Enhance the Activation of Tumor-Specific
CD8+T Cells
Although Th17 cells have potent protective effect against B16
melanoma in vivo, we did not find any proapoptotic effect of
IL-17 on B16 cells cultured in vitro (data not shown), suggesting
thatTh17cellsdonotdirectlykillmelanomacells.InTh17butnot
Th1 cell-treated tumor-bearing mice, we observed increased
numbers of CD8+T cells in the lung, suggesting that Th17 cells
may promote the activation or recruitment of tumor antigen-
specific CD8+Tcells.IntheB16-OVAtumormodel,wesearched
for endogenous CD8+T cells reactive against the SIINFEKL
peptide derived from OVA protein by using a Kbtetramer
(OVA-tet). In the lungs of mice that received OT-II-Th17 cells,
there was a distinct population of CD8+OVA-tet+T cells,
?10% and 5% of total CD8+T cells in the prevention or thera-
peutic tumor model, respectively. Such OVA-tet+T cells were
absentinuntreatedcontrolmice(Figures5Aand5B).Incontrast,
Th1cellselicitedasmallpopulationofOVA-tet+Tcellsinthelung
(1.17% ± SD 0.07%) (Figure 5B). We also detected an increased
number of OVA-tet+T cells in mice with flank B16-OVA tumors
that were also treated with Th17 cells (Figure S4B). Altogether,
these results suggest that Th17 cells ‘‘help’’ the activation of
endogenous antitumor CD8+cells and also promote their subse-
quent localization to the tumor sites.
ToanalyzewhetherCD8+Tcellsmediatedtheobservedtumor
protection by Th17 cells, we depleted CD8+T cells in tumor-
bearing mice before they were treated with Th17 cells. Anti-
CD8 was injected on day 4 after B16-OVA tumor challenge and
every 4 days until the end point. The CD8+T cell depletion effi-
ciency was confirmed in blood- and lung-derived cells and
reached 95% and 86%, respectively. Depletion of CD8+T cells
reduced tumor protection mediated by Th17 (from 75% to
40% reduction of tumor foci), resulting in a degree of protection
similar to that of Th1 cells without CD8 depletion (Figure 5C).
Therefore, CD8+T cells in large part mediate the tumor protec-
tion conferred by Th17 cells.
To further assess CD8+T cell regulation by Th17 cells, we
labeled CD8+OT-I T cells with CFDA-SE and transferred them
alone or together with Th17 OT-II cells into mice bearing lung
tumors for 5 days. OT-I T cells, when cotransferred with Th17
cells, proliferated more extensively in LLNs with at least three
more cell divisions than those transferred alone (Figure 6A).
However, OT-I cells proliferated similarly with or without Th17
cell partners in lung or spleen, suggesting that Th17 cell transfer
enhances CD8+T cell priming only in LLNs. In addition, in the
LLNs from mice receiving Th17 cells, there were increased
numbers of OT-I T cells producing IFN-g accompanying their
cell division, with ?30% cells in the fifth division producing
IFN-g. OT-I T cells transferred alone lost their cytokine expres-
sion during their minimal divisions with only 10% IFN-g+T cells
inthethirddivision(Figure6B).Incontrast,cotransferofTh1cells
did not influence OT-I cell division or cytokine production of OT-I
cellsinLLN(FigureS7A).ThesedatademonstratethatTh17cells
promote CD8+T cell proliferation while sustaining their cytokine
expression capacities.
Figure 5. Th17 Cells Elicit Tumor-Specific CD8+T Cell Responses
(A and B) Dot plots show the frequencies of OVA tetramer-staining (Kb-SIINFEKL) cells in CD45.2+CD8+gated T cells obtained from the leukocyte fraction in
mice that received either preventive or therapeutic treatment with Th17 cells. Lower graphs represent the total number of cells from the average of 4 mice
per group (n = 4, average ± SD).
(C) Micewere inoculated i.v. withB16-OVAand on day5mice receivedeither PBS or33 106ofTh1 orTh17 OT-II cells i.v. Depleting antibodiesagainst CD8 were
administered i.p. on day 4 after the tumor inoculation and every 4 days until the endpoint. Shown are the average numbers of tumor colonies present in the lung
lobes of each group of mice (n = 4, average ± SD) (**p < 0.01, * p < 0.05).
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To confirm that IL-17 can influence CD8+T cell priming, we
transferred OT-I cells labeled with CFDA-SE into WT or IL-17?/?
mice that had been inoculated with B16-OVA tumors. We found
that on day 4 after transfer, Il17a?/?mice had reduced numbers
of CFSE-labeled OT-I cells in LLNs (Figure 6C), with lower
production of IFN-g when compared to WT mice (Figure 6D).
This result confirms that IL-17 influences the priming of CD8+
T cells to generate IFN-g-producing effector cells. Taken
together, these results indicate that Th17 cells induce a strong
antitumor CD8 response that participates in the elimination of
the tumor by favoring priming of tumor antigens in tumor-drain-
ing lymph nodes.
Th17 Cells Regulate DC Function in a CCR6-Dependent
Manner
We further investigated the basis of Th17 cell ‘‘help’’ to CD8+
T cells. Th17 cell cytokines could possibly act on CD8+T cells
directly during priming. However, we did not observe an effect
on CD8+T cell proliferation and cytokine production when they
were activated in vitro in the presence of IL-17A, IL-17F, or
Th17cellsupernatants(datanotshown).Alternatively,Th17cells
could indirectly influence CD8+T cell priming by improving
presentation of tumor antigens by DCs. Previous studies have
reported that therapy with DCs loaded with class I peptides or
dead tumor cells protected mice against established B16 solid
tumors (Goldszmid et al., 2003; Lou et al., 2004). Consistent
with this idea, we have found that CD8a+DC doubled in percent-
ages in the lung as early as 3 days after Th17 cell transfer, and
such a result did not occur with Th1 cells (Figure S7). Therefore,
wedecidetoassessthishypothesisbyusingB16melanomathat
coexpresses OVA and GFP and identify DC uptake of tumor
materials and migration to LLNs. Mice were injected with
B16-OVA-GFP and Th17 or Th1 cells, and 72 hr later, LLN
were harvested for flow cytometry analysis of DC uptake of
GFP. High levels of GFP were found in CD8a+DCs from mice
that received Th17 cells, and this signal was higher than Th1
cell-treated mice or controls (Figure 7A). GFP in CD11b+DCs
from mice treated with either Th1 or Th17 cells was also higher
than in control mice (Figure 7A). Moreover, given that the total
numbers of DCs present in LLNs were four times greater in
mice treated with Th17 cells compared to those treated with
Th1 cells or in control mice, the total number of CD8a+DCs
Figure 6. Th17 T Cell Treatment Promotes CD8+Effector T Cell Differentiation
(A and B) Purified CD8+T cells from OT-I mice were labeled with CFDA-SE and transferred into C57BL/6 mice bearing 5 day established pulmonary B16 nodules
(OT-I). On theday of the transfer, one group of mice also receivedOT-II Th17 (OT-I+Th17). On day 3aftertransfer, LLNs,lungs, and spleens from thesemicewere
analyzed for T cell proliferation and IFN-g and IL-2 production.
(A) Histograms show cell division. Numbers in the lower panel indicate numbers of division.
(B) Cytokine production. Numbers inside the plots represent the percentages of dividing OT-I cells that also produced the cytokine. Bar graphs indicate the
percentages of cells that produced cytokines per cell division.
(C and D) CD8+T cells from OT-I mice were labeled with CFDA-SE and transferred into C57BL/6 mice or Il17a?/?(KO) bearing 5 day established pulmonary
B16-OVA. LLNs and inguinal lymph node (ILN) cells from these mice were analyzed for proliferation and cytokine production on day 4 after transfer.
(C) Total numbers of CD8+CFSE+T cells recovered from ILNs and LLNs.
(D)CytokineprofilesofCD8+CFSE+TcellsfromLLNs.ThegraphshowsthetotalnumberofCD8+CFSEloIFN-g+TcellsrecoveredfromLLN(n=3,average±SD).
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containing GFP was ten times more in mice treated with Th17
cells than in control or Th1 cell-treated mice (Figure 7B). There-
fore,Th17cellsprovokedinflammationinthelungthatresultedin
increased numbers of DCs in the lung and DCs containing tumor
materials in LLNs, resulting in improved antitumor CD8+T cell
priming.
Interestingly, we also found that both CD11b+and CD8a+DCs
expressed CCR6 on their surface; however, there was only
increased CCR6 expression on CD8a+but not CD11b+DCs
from mice treated with Th17 cells compared to those from the
control mice (Figure 7C). Given that Ccl20 expression in tumor
tissues was enhanced by Th17 cells, we implanted B16-OVA
tumor in CCR6-deficient (Ccr6?/?) mice and treated them with
OT-II Th17 cells. Tumor colonies in the lung of Ccr6?/?mice
grew similarly to those in WT mice, whereas Ccr6?/?mice did
not respond to treatment with Th17 cells and developed similar
numbers of tumor colonies as the untreated mice (Figure 7D).
Furthermore, CD8a+DC numbers were only increased in the
LLNs of WT mice treated with Th17 cells but not in CCR6-defi-
cient mice (Figure 7E). The total CD4+and CD8+T cells and
CD8a+DCs in the lung of Ccr6?/?mice were not increased by
Th17 cell treatment (Figures S8A and 8SB). However, there
was an increased number of GR1+, CD11b+DCs and macro-
phages in Ccr6?/?mice treated with Th17 cells. Therefore, we
conclude that CCR6 is required for the response to Th17 cell
therapy; the signaling through CCL20 may allow CD8a+DCs
loaded with tumor antigens to prime antitumor CD8+T cells in
LLNs.
DISCUSSION
Although Th17 cells have been found in human tumors, their
physiological function in tumor development has been poorly
defined. By using IL-17-deficient mice in a model of lung mela-
noma, we have provided direct evidence for a protective role
of IL-17 in antitumor responses. Moreover, we show that the
adoptive transfer of tumor-specific Th17 cells protect against
various tumors and, in the lung melanoma model, promote
tumor-specific cytotoxic T cell responses.
The function of IL-17 in tumor immunity has been a controver-
sial subject. The effects of IL-17 on tumor development are
directly influenced by the existence of an adaptive immune
system—in the presence of lymphocytes, IL-17 promotes tumor
rejection, whereas in the absence of them, IL-17 favors tumor
Figure 7. Characterization of DCs from LNs of mice treated with Th17 Cells
(A) C57BL/6 mice were injected i.v. with B16/F10 cells expressing OVA-GFP together with Th1 or Th17 OT-II cells. LLNs were harvested on day 3 for DC analysis
by flow cytometry. Histograms show gated CD11bhiCD11c+(CD11b+) and CD11bintCD11c+CD8a+(CD8a+) DCs.
(B) Lung lymph node cells from (A) were counted, and the total number of cells per DC population was calculated from live gate, CD11c, and CD11b (Total) or live
gate, CD11c, CD11b, and GFP (GFP) (average ± SD).
(C)C57BL/6micewereinjectedi.v.withB16-OVAandTh17OT-IIcells.LLNswereharvestedonday16forDCanalysis.Cellswerelabeledwithantibodiesagainst
CCR6 or isotype rat IgG. Shown are overlay histograms of either CD8a+DC or CD11b+cells. Rat IgG (gray filled histograms), CCR6 from control mice (thin line),
and mice that were treated with Th17 cells (bold line) are shown.
(D) C57BL/6 mice or CCR6-deficient mice (Ccr6?/?) were injected i.v. with B16-OVA and Th17 OT-II cells. Graph shows the average number of tumor colonies
from lung lobes from WT (n = 5) and KO (n = 3) mice (average ± SD).
(E) LLN cells from (D) were analyzed for DC populations. Graphs show the total number of cells from WT (n = 5) and KO (n = 3) mice (average ± SD).
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growth and angiogenesis (Martin-Orozco and Dong, 2009). This
notion is consistent with our current data on Il17a?/?mice and
adoptive transfer of Th17 cells. Our current data also indicate
an active role of IL-17 in tumor immunosurveillance; IL-17 defi-
ciency resulted in reduced leukocyte infiltration into the target
tissue, and these mice were more susceptible to the tumor
development. Also, antitumor CD8+effector T cell differentiation
was compromised in IL-17-deficient mice. In contrast to our
results, IL-23, important in Th17 cell regulation, has been shown
to promote tumor growth and prevent immunosurveillance (Lan-
gowski et al., 2006). There are two possibilities to account for
these discrepancies. First, other cells or factors regulated by
IL-23 may have a distinct function in cancer immunity. It has
been recently described that IL-23 produced by macrophages
activates STAT-3 in the macrophages and Treg cells and
promotes tumor growth (Kortylewski et al., 2009). Second, it is
alsopossiblethattheIL-23-IL-17axisofinflammatoryresponses
may have differential functions in different settings; its regulation
of chronic inflammation may provide a supportive role for certain
types of tumors. Short acute inflammation associated with anti-
tumor response might help the fight against the tumor (Overwijk
et al., 2006). Further studies on different tumor models may
reveal the complex relationships between IL-17-IL-23, inflam-
mation, and cancer.
In our current study, we found that Th17 cells provide better
protection to tumors than Th1 cells, and this difference was
largely due to their unique ability to promote CD8+T cell priming.
Because anti-IFN-g did not influence the protective immunity
mediated by Th17 cells against tumors, CD8+T cells may kill
tumors independent of this cytokine, possibly by utilizing the
cytolytic enzymes. It was also recently shown in subcutaneous
B16 melanoma that transfer of antitumor CD4+Th17 cells
together with whole-body irradiation, vaccination, and IL-2 treat-
ment could control tumor growth. However, the effect of Th17
cell vaccination was dependent on IFN-g and independent of
IL-17 and IL-23 (Muranski et al., 2008). We believe that in a lym-
phopenic environment, conversion of Th17 to Th1 cells occurs;
however, Th17 cells maintain their phenotypes in normal hosts
in the presence or absence of inflammation (Martin-Orozco
et al., 2009; Nurieva et al., 2009). Although large numbers of
Th1 cells did provide some degree of tumor protection in our
model as well, our Th17 donor cells were more effective in
several tumor models tested while maintaining their Th17 cell
cytokine expression profiles. Additionally, when Th17 cells
were purified with the RFP reporter, they efficiently provided
tumor protection comparable to the total Th17 cells. Thus, our
experimental settings including a normal, nonlymphopenic envi-
ronment allow us to more directly address the physiological
function of Th17 cells in cancer.
The protective function of Th17 cells against tumors is prob-
ably due to their ability to enhance inflammatory responses,
andsuchenhancement resultsinincreasedantigen presentation
by DCs. Leukocyte homing to tumors, usually inhibited by the
tumor cells (Gajewski, 2007), was reduced in Il17a?/?mice and
promoted by transfer of tumor-specific Th17 cells. Increased
numbers of DCs infiltrating the lung after Th17 cell transfer
may lead to increased capture of tumor antigens, which are
then presented to tumor-reactive CD8+T cells in the draining
lymph nodes. It has been reported that the presence of
increased numbers of mature DCs within solid tumor masses
induces more effective antitumor immune responses in animal
models (Furumoto et al., 2004) and is associated with improved
prognosis in clinical patients (Lotze, 1997). Moreover, patients
with advanced melanoma that have been treated with GM-CSF
showedageneralincreaseof matureDCs,whichhasbeenasso-
ciated with disease remission and delayed tumor recurrence
(Daud et al., 2008). At this point, our report is the first to show
that Th17 cells influence the generation of CD8+effector
T cells in vivo. Given that CD4+T cell help is essential for the
generation of CD8+memory T cells during the priming phase
of an acute viral infection (Shedlock and Shen, 2003), it is
possible that Th17 cells may participate in this process via the
generation of CD8+antitumor effector-memory T cells. Th17
cells, on one hand, promote proliferation of tumor-specific
CD8+T cells. On the other, they also allow for sustained IFN-g
expression by dividing CD8+T cells, thus preventing them from
becoming ‘‘exhausted.’’
We did not found a direct effect of Th17 cells on CD8+T cells
effectordifferentiation;however,
numbers of DCs carrying tumor-derived material in the lymph
nodes of Th17 cell-treated mice. The predominant DCs recruited
were CD8a+, which are indispensable for cross-presentation of
self-antigens in several tissues and in the lung (den Haan and
Bevan, 2002; Dudziak et al., 2007; Schnorrer et al., 2006). In
comparison to Th1 cell-mediated effects, we found that Th17
cells selectively induced the expression of CCL20 and that
CCR6 deficiency selectively impairs the recruitment of CD8a+
DC, and such results may account for the difference of Th1
and Th17 cells in activation of tumor-specific CD8+T cells and
in their potency of tumor immunity. Previously, Furumoto et al.
reported that overexpression of CCL20 by B16 cells, although
it increased intratumor DC numbers, did not promote tumor
regression but helped the regression of CT26 colon adenocarci-
nomas (Furumoto et al., 2004), suggesting that CCL20-CCR6
might be necessary but not sufficient in the induction of tumor
immunity.
Ourdataaltogethersuggestthefollowingmodel:Th17cellsgo
to the tumor site and, by secreting IL-17, activates residential
cellstoproduceCCL2andCCL20,whichprovokes themobiliza-
tion of DCs and other leukocytes to the tumor site. DCs uptake
tumor antigens in thelung and migrate tothe lymph nodes where
they activate CD8+T cells against the tumor. The new wave of
effector CD8+T cells migrates back to the lung and kills estab-
lished tumors (Figure S9).
Our data suggest that tumor immunosurveillance in the lung in
the steady state is dependent, in part, on IL-17 but more impor-
tantly that tumor-specific Th17 cells may be used in adoptive
T cell therapy. To date, adoptive cell therapy for cancer with
in vitro-expanded, tumor-infiltrating CD8+lymphocytes has
achieved some degree of success in cancer therapy (Dudley
and Rosenberg, 2007; Rosenberg and Dudley, 2004). Together
with the current literature on the crucial function of Th17 cells
in autoimmunity, our results suggest that Th17 cells directed
against tumor antigens may be employed in cancer patients. In
addition, Th17 and CD8+T cells derived from the tumors may
be expanded ex vivo and employed together to enhance cancer
immunity. Therefore, our data demonstrating that Th17 cells
and IL17 participate in antitumor immunity by facilitating T cell
weobservedincreased
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recruitment to the tumor site and CD8+T cell priming and
effector differentiation suggests a new avenue for developing
Th17 cell-based therapy for tumors or chronic viral infections
and as an adjuvant for vaccinations.
EXPERIMENTAL PROCEDURES
Mice
C57BL/6 mice were purchased from the NCI and OT-II (C57BL/6-Tg
(TcraTcrb)425Cbn/J),OT-I(C57BL/6-Tg(TcraTcrb)1100Mjb/J),
(B6.SJL-Ptprca Pepcb/BoyJ), and CCR6 KO (B6.129P2-Ccr6tm1Dgen/J) mice
were purchased from the Jackson Laboratory (Yamazaki et al., 2008).
OT-II.2 RAG2 (C57BL/6-RAG2tm1Alt-TgNOT-II.2) mice were from Taconic
Farms. IL-17A-deficient mice and IL-17F-RFP mice were generated in our
lab (Yang et al., 2008). Homozygous KO and WT animals on the same 129 3
C57BL/6 F1 mixed background were bred and used in some experiments.
For some experiments, IL-17AKO mice that had been backcrossed to
C57BL/6 mice for six generations were used. IL-17F-RFP mice and CD45.1
were crossed to OT-II mice for OT-II.IL-17F-RFP and OT-II- CD45.1 mice
generation, respectively. Mice were maintained in the MD Anderson Animal
Facility and the MD Anderson Cancer Center Institutional Animal Care and
Use Committee approved all animal studies. Rag1?/?BWTRP-1 TCR trans-
genic mice were bred at the National Institutes of Health (NIH).
CD45.1
Induction and Assessment of B16-F10 Lung Melanoma
Six-week-old mice were injected intravenously (i.v.) with 1 3 105wild-type
B16-F10 cells or with B16/F10 transfected with OVA (B16-OVA). For adoptive
transfer experiments, the mice were injected i.v. on the same day with 1 to 3 3
106in vitro differentiated Th17 OT-II cells in the prevention model or 5 days
after the tumor injection in the therapeutic model. At day 14 or 16 after tumor
introduction, mice were sacrificed for enumeration of metastatic lung foci. All
lung lobes were evaluated under a tissue microscope (Leica MZFLIII). In the
experiments in which IFN-g blocking antibody (XMG1.2 clone, BioXcell) or
anti-CD8 (53.6.72 clone, BioXcell) were used, 100 mg/mouse was injected in
100 ml of PBS i.p every 4 days.
Subcutaneous Tumor Induction
Mice were injected subcutaneously (s.c.) in the front of the abdomen with 2 3
105B16-OVA or 5 3105MCA205-OVA cells, and on the same day, some mice
received 3 to 5 3 106Th1 or Th17 OT-II cells. The tumor area was measured
with digital calipers. Mice were sacrificed once tumors reached 200 m2.
In Vitro Th17 and Th1 Cell Polarization
CD4+T cells from spleens and lymph nodes of OT-II mice were differentiated
into Th1 or Th17 cells as described previously (Chung et al., 2006; Nurieva
et al., 2007). When Rag1?/?BWTRP-1 TCR-transgenic cells were used, sple-
nocytes and lymph node cells were cultured with 0.01 mg/ml of TRP-1(106-130)
peptide and irradiated splenocytes and with the same polarizing conditions.
Cells were harvested at day 4 for cytokine analysis and for transfer. Intracel-
lular cytokine analysis was performed on cells that were stimulated with
specific peptide (5 mg /ml OVA or 1 mg/ml or TRP-1) for 5 hr and GolgiStop
and further stained for IL-17, IL-17F, and IFN-g with the BD Cytoperm/wash
kit according to the manufacturer’s instructions (BD Biosciences). Cytokines
were purchased from Peprotec and blocking antibodies were from Bioxcell.
Lung Fractionation and Cell Analysis
Lungs were digested with 1 mg/ml collagenase D for 30 min at 37?C and 5 min
with 0.01 EDTA for prevention of DC-T cell aggregates (Vremec et al., 2000).
The cells were further suspended in LSM lymphocyte separation medium
(MP Biomedicals, LLC) and then centrifuged in accordance with the manufac-
turer’s instructions. The middle section of the gradient was enriched for leuco-
cytes,andthebottomfractionwasenrichedfortumorandlungcells.Leucocyte
fractionswerecountedandusedforflowcytometryanalysisorRNAextraction.
Flow Cytometry
Antibodies against CD45.2, CD4, CD8, CD44, CD62L, CD11c, CD11b, Gr1,
IL-17, IFN-g, and IL-2 were all purchased from BD (BD Biosciences). Poly-
clonal anti-IL-17F (Yang et al., 2008) was labeled with Alexa647 (Invitrogen).
Anti-CD45.1 was purchased from Biolegend. Kbtetramer carrying the
OVA257-264 peptide (SIINFEKL) and Db tetramer carrying human gp10025–
33 (KVPRNQDWL) were both labeled with streptavidin-PE and were made
in-house. Immunofluorescence staining was performed at 4?C except for the
tetramer staining, which wasperformedat roomtemperature for 1hr. Samples
were analyzed in a FACS Canto II, LSR II, or FACScalibur equipped with DIVA
software or CellQuest software, respectively. RFP+cells were sorted in a Mo-
Flow sorter (Cytomation). Files were analyzed with TreeStar Flowjo.
Real-Time PCR
The expression of Ccl20, Ccl2, Ccl7, Ccr6, and Gro-a was performed with
specific primers reported previously (Chang and Dong, 2007; Chang et al.,
2006) and the BioRad SYBRgreen system. Expression was normalized to
the expression of the housekeeping gene Actin (Actb).
CFDA-SE Labeling and Transfer Experiments
Th17cells generated from naive cellsinRag.OT-IImicewerelabeled with5mM
CFDA-SE (Invitrogen). A total of 5 3 106cells were injected i.v. into mice
bearing 5 day established B16-OVA lung tumors. After 4 days, recipient
mice were sacrificed, lung, LLNs, and spleen were harvested, and cells were
restimulated with 5 mg/ml OVA323-339peptide in the presence of Golgi-stop
for 6 hr. ICS for IL-17, IL-17F, and IFN-g was performed with the BD Cytofix/
cytoperm kit.
CD8+TcellsfromOT-Imicewerepurifiedwithanti-CD8beads(Miltenyi)and
the AutoMacs system. After purification, the cells were labeled with CFDA-SE
and 5 3 106cells were injected i.v. into mice bearing 5 day established B16-
OVA lung tumors. On the same day, the mice received i.v. injections of 3 to
5 million Th17 cells or PBS. Lung, LLNs, and spleen were harvested on day
3after transferand restimulated with5mg/ml ofOVA257-264peptide inthepres-
ence of Golgi-plug for 6 hr. ICS staining for IFN-g and IL-2 was performed with
the BD Cytofix/cytoperm kit.
Similar experiments were performed with transfer of purified OT-I cells
labeled with CFDA-SE into IL17 KO or WT mice in the C57BL/6 strain.
Dendritic Cell Analysis
Micewereinjectedi.v.with13105B16-F10expressing OVA-GFPand13106
of either Th1 or Th17 polarized OT-II cells. LLN were harvested at 72 hr and
were digested with 1 mg/ml of collagenase and with 0.01 EDTA for preventing
DC-T cell aggregates. Cells were counted and were incubated with anti-CD16
plus anti-CD32 (FcBlock) for 20 min, additionally labeled with mAb against
CD11b, CD11c, GR1, CD8a, or Isotype Rat IgG, and analyzed by flow cytom-
etry.
Leukocytes from lung fractionation were analyzed similarly for determining
DC, macrophage, and granulocyte populations.
ELISPOT
On day 16 after tumor injection, inguinal lymph node cells from mice that were
treated with TRP-1 Th17 cells were counted and plated in HA plates (Millipore)
previously coated with anti-IFN-g or IL-17 (BD Biosciences). Cells were pulsed
with TRP-1(106-130)peptide and cultured for 18 hr. Plates were blotted with bio-
tinylated anti-IFN-g or IL-17 and developed with avidin-alkaline phosphatase
and 5-bromo-4 chloro-3indolyl phosphate and nitro blue tetrazolium chloride
(BCIP/NBT). ELISPOT dots were counted on a C.T.L. ImmunoSpot with Image
Acquisition 4.4 software for image capture and Immunospot 3 for analysis.
Statistical Methods
All statistical comparisons were analyzed with the Student’s t test.
SUPPLEMENTAL DATA
Supplemental Data include nine figures and can be found with this article on-
line at http://www.cell.com/immunity/supplemental/S1074-7613(09)00451-8.
ACKNOWLEDGMENTS
We thank the entire Dong lab for their help and discussion. This work is sup-
ported in part by grants from the National Institute of Health (to W.W.O. and
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C.D.) and a grant from the Center for Targeted Therapy of MD Anderson
CancerCenter(toC.D.andN.M.O.).C.D.receivedaLeukemia andLymphoma
Society Scholar award and a Trust Fellowship of the MD Anderson Cancer
Center.
Received: October 1, 2008
Revised: June 11, 2009
Accepted: September 4, 2009
Published online: October 29, 2009
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