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Human TH17 Immune Cells Specific for the Tumor Antigen
MAGE-A3 Convert to IFN-g–Secreting Cells as They
Differentiate into Effector T Cells In Vivo
le
ne Senellart2,
Ahmed Hamaï1, Pascale Pignon1, Isabelle Raimbaud1, Karine Duperrier-Amouriaux1, He
2
2
2
1
Sandrine Hiret , Jean-Yves Douillard , Jaafar Bennouna , Maha Ayyoub , and Danila Valmori1,3
Abstract
The role of TH17 cells in cancer is being investigated, but the existence of tumor antigen–specific TH17 cells has
yet to be ascertained. Here, we report the first description of a spontaneous TH17 (IL-17þ) response to the
important tumor antigen MAGE-A3, which occurred concurrently with a TH1 (IFN-g þ) response in a lung cancer
patient. MAGE-A3–specific interleukin (IL)-17þ T cells were mainly CCR7þ central memory T cells, whereas
IFN-g þ cells were enriched for CCR7 effector memory T cells. An assessment of the fine specificity of antigen
recognition by these T cells indicated that the CCR6þCCR4þ and CCR6þCXCR3þ fractions contained the same
TH17/TH1 population at early and late differentiation stages, respectively, whereas the CCR6CXCR3þ fraction
contained a distinct TH1 population. These findings are important because they suggest a differentiation model in
which tumor antigen–specific CD4þ T cells that are primed under TH17 polarizing conditions will progressively
convert into IFN-g–secreting cells in vivo as they differentiate into effector T cells that can effectively attack
tumors. Cancer Res; 72(5); 1059–63. 2012 AACR.
Introduction
TH17 cells have been recently defined as a distinct subset
involved in the pathogenesis of inflammatory autoimmune
diseases, but also in host protection against extracellular
bacteria, fungi, and protozoa (1). Consistent with a physiologic
role of TH17 in protecting mucosal surfaces such as the gut,
lungs, and skin, the subset has been shown to be prevalent at
these locations (2). In humans, TH17 cells are found among
memory populations, suggesting that they differentiate in
response to antigen in vivo, but little direct evidence of their
antigen specificity has been reported (3). In this context, a
recent report has suggested that generation of intestinal TH17
requires microbiota but not microbial derived antigens (4). In
addition, it has been recently proposed that interleukin (IL)17–secreting T cells represent a transient phenotype of populations that tend to convert to IFN-g–producing cells (5).
et de la Recherche
Authors' Affiliations: 1Institut National de la Sante
dicale, Unite
1102; 2Department of Medical Oncology, Institut de
Me
rologie de l'Ouest, Saint Herblain; and 3Faculty of Medicine, UniCance
versity of Nantes, Nantes, France
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
M. Ayyoub and D. Valmori share senior authorship.
Corresponding Authors: Maha Ayyoub, INSERM U1102, Institut de
rologie de l'Ouest, 44800 Saint Herblain, France. Phone: 33(0)24Cance
067-9726; Fax: 33(0)24-067-9763; E-mail: [email protected];
and Danila Valmori, [email protected]
doi: 10.1158/0008-5472.CAN-11-3432
2012 American Association for Cancer Research.
www.aacrjournals.org
The role of TH17 in cancer is being investigated. Several
recent findings indicate a beneficial role for TH17 in antitumor
immunity. Among them are the positive association between
intratumoral TH17 and IFN-g effector cells, CTL and natural
killer cells, reported for some human tumor types (6) along
with evidence that murine transgenic T cells polarized in vitro
to TH17 induce tumor regression following in vivo transfer (7).
Other data, however, depict a more complex picture and
suggest instead a negative effect. These include the reported
proangiogenic and protumoral activity of the TH17 signature
cytokine IL-17, documented by early studies in immune-deficient mice (8), reports of association between the prevalence of
tumor-associated TH17 and bad clinical outcomes in some
tumor types (9), and the close relationship between TH17 and
FOXP3þ Treg, that play opposite immune functions (10–12).
As the role of TH17 in cancer is still to be ascertained, no
evidence for the existence of TH17 specific for human tumor
antigens has been yet provided. In this study, we report the first
description of TH17 specific for the tumor antigen MAGE-A3 in
a patient affected by lung cancer. Together, our results support
a differentiation model in which MAGE-A3–specific CD4þ
T cells primed under TH17 polarizing conditions progressively
convert into IFN-g–secreting as they differentiate into effector
cells in vivo.
Materials and Methods
Patients' samples and cell sorting
Peripheral blood samples were collected from lung cancer
patients seen at the CLCC Rene Gauducheau upon written
informed consent and approval by the Institutional Review
Board (Comite de Protection des Personnes Ouest 2–Angers).
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Hamaï et al.
CD4þ T cells were enriched by positive selection from peripheral blood mononuclear cells by magnetic cell sorting (Miltenyi
Biotec). For ex vivo flow cytometry cell sorting, enriched CD4þ
T cells were stained with fluorochrome-labeled mAb (from BD
Biosciences unless indicated otherwise) specific for CD45RA,
CCR7, CD25 (Beckman Coulter), CD127 (eBioscience), CCR4,
CCR6, and CXCR3 and sorted into the indicated populations to
high purity (>97%; FACSAria; BD Biosciences).
In vitro stimulation and functional assessment of
MAGE-A3–specific CD4þ T cells
Total CD4þ T cells or ex vivo sorted subpopulations were
stimulated in vitro with a pool of 20 to 24 amino acid long
peptides overlapping by 10 amino acids and covering the
MAGE-A3 sequence (Supplementary Table S1), in the presence
of irradiated autologous antigen-presenting cells (APC), and
cultured in the presence of recombinant human IL-2 (Chiron).
Days 10 to 14 cultures were assessed in a 4-hour intracellular
cytokine staining assay using mAb specific for IFN-g (BD
Biosciences) and IL-17 (eBioscience) following stimulation
with the MAGE-A3 peptide pool and analyzed by flow cytometry (FACSAria; BD Biosciences). In some experiments, cells
were stained with cytokine-specific mAb together with mAb
specific for RORg/gt and T-bet (eBioscience; according to the
manufacturer's instructions). To determine the fine specificity
of antigen recognition, aliquots of CD4þ T-cell cultures were
stimulated in the absence or the presence of the peptide pool or
of individual MAGE-A3 peptides and IL-17, and IFN-g were
assessed in 24-hour culture supernatants by ELISA (R&D
Systems and Invitrogen, respectively).
Results and Discussion
The human tumor antigen MAGE-A3, of the cancer/testis
antigen group (13), is frequently expressed in lung cancers (14)
and a MAGE-A3–based anticancer vaccine is currently being
evaluated in lung cancer patients (15). We assessed circulating
lymphocytes from 38 lung cancer patients (Supplementary
Table S2) for spontaneous CD4þ T-cell responses to MAGEA3. To this end, we stimulated isolated circulating CD4þ T
cells from the patients with a pool of long overlapping peptides
spanning the entire MAGE-A3 protein (Supplementary Table
S1) and assessed the cultures 12 days later for IFN-g and IL-17
production in response to the Ag. We detected significant
proportions of specific CD4þ T cells secreting IFN-g in 3
patients (Fig. 1A). In one of them, patient NA171, a significant
IL-17 response was also detected. Simultaneous assessment of
IFN-g and IL-17 secretion revealed 3 distinct subpopulations, 2
major ones secreting IL-17 or IFN-g alone and one cosecreting
IL-17 and IFN-g (Fig. 1B).
CD4þ memory T cells expressing the chemokine receptor
CCR7, called central memory (CM), represent a reservoir at an
early differentiation stage that recirculate in lymphoid organs,
whereas CCR7 populations, called effector memory (EM), are
at a more advanced differentiation stage and can localize in
peripheral tissues (16). To address the in vivo differentiation
stage of MAGE-A3–specific TH17, we assessed them in conventional (CD25) CM and EM CD4þ T cells isolated ex vivo by
flow cytometry cell sorting (Fig. 2A). Because of the reported
relationship between TH17 and Treg (CD25þCD127; ref. 12),
we also assessed them in memory Treg (MTreg). MAGE-A3–
specific cells secreting IL-17 alone were mostly found in CM,
whereas those secreting IFN-g alone or with IL-17 were
enriched in EM (Fig. 2B). We did not detect MAGE-A3–specific
cells in MTreg.
Expression of other chemokine receptors distinguishes
CD4þ T cell subsets with different migratory ability and
effector functions. Whereas expression of CXCR3 characterizes
TH1 and CCR4 TH2, CCR6 has been reported to characterize
TH17 and Treg (17). To further characterize MAGE-A3–specific
TH17, we assessed them in CD4þ T cell populations sorted
ex vivo based on the expression of CCR6, CXCR3, and CCR4
(Fig. 3A). Cells secreting IL-17 in response to MAGE-A3 were
almost exclusively found in the CCR6þCCR4þ fraction, whereas IFN-g–secreting cells were found in the CCR6þCXCR3þ and
CCR6CXCR3þ fractions (Fig. 3B). To support the identification of MAGE-A3–specific TH17 and TH1 cells, we assessed the
expression of the lineage-specific transcription factors RORgt
and T-bet, associated respectively with TH17 and TH1, in
MAGE-A3–specific cells, by combined staining of Ag-stimulated subpopulations with antibodies against cytokines and
transcription factors. As expected, we detected higher expression levels of RORgt in the CCR6þCCR4þ fraction than in the
þ
þ
Figure 1. CD4 T-cell responses to MAGE-A3 in lung cancer patients. Circulating CD4 T cells were stimulated in vitro with a pool of long, overlapping peptides
spanning the entire MAGE-A3 protein. Day 12 cultures were assessed following stimulation in the absence or presence of the peptide pool by intracellular
cytokine staining. A, the proportions of CD4þ T cells producing IFN-g and IL-17 in response to MAGE-A3 are shown for all patients (n ¼ 38, symbols
correspond to individual patients). Responses greater than 0.2 were considered significant. B, dot plots for patient NA171 are shown.
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Cancer Res; 72(5) March 1, 2012
Cancer Research
Spontaneous TH17 Responses to MAGE-A3 in Lung Cancer
þ
þ
þ
Figure 2. MAGE-A3–specific CD4 T cells are detected in conventional CM and EM CD4 T cells but not in MTreg. A, CD4 T cells from patient NA171 were
stained with mAb specific for CD45RA, CCR7, CD25, and CD127, and memory (CD45RA) cells were sorted into MTreg (CD25þCD127low), conventional CM
(CD25CCR7þ), and conventional EM (CD25CCR7) populations. B, sorted populations were stimulated in vitro with the MAGE-A3 peptide pool and
assessed 12 days later for IFN-g and IL-17 production in response to stimulation with the Ag by intracellular cytokine staining.
other populations (Fig. 3C and D). Expression of T-bet was
inversely correlated with that of RORgt and was instead higher
in the CXCR3þ fractions.
To further clarify the relationship between the identified
MAGE-A3–specific populations, we assessed their fine speci-
ficity. We initially assessed total CD4þ T cells with MAGE-A3
peptides and detected reactivity against 3 peptides, 141–160,
241–260, and 271–290 (Fig. 4A). We then assessed the populations isolated according to chemokine receptors expression
with the active peptides. We detected reactivity to peptide
Figure 3. MAGE-A3–specific TH17
and TH1 cells are detected in CD4þ
T-cell populations with distinct
chemokine receptors expression
profiles. A, CD4þ T cells from patient
NA171 were stained with mAb
specific for CD45RA, CCR7, CD25,
CD127, CCR4, CCR6, and CXCR3,
and conventional memory
(Mconv, CD25CD45RA) cells
were sorted into CCR6þCCR4þ,
CCR6þCXCR3þ, CCR6CCR4þ,
and CCR6CXCR3þ populations. B,
sorted populations were stimulated
with the MAGE-A3 peptide pool and
cultures were assessed as in Fig. 2B.
C and D, cultures were assessed,
following stimulation with the MAGEA3 peptide pool, for RORgt and T-bet
expression and for IL-17 and IFN-g
production with specific mAb in an
intracellular staining assay.
Examples of dot plots for
CCR6þCCR4þ and CCR6þCXCR3þ
cultures are shown in C and the mean
fluorescence intensity (MFI) of RORgt
and T-bet staining is summarized in D
for MAGE-A3–specific IL-17þ,
IL-17þ/IFN-g þ, and IFN-g þ cells,
defined as in B, in the indicated
responder cultures.
www.aacrjournals.org
Cancer Res; 72(5) March 1, 2012
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Hamaï et al.
Figure 4. Fine specificity of MAGEþ
þ
A3–specific IL-17 and IFN-g
CD4þ T cells. A, CD4þ T cells from
patient NA171 were stimulated in
vitro with the MAGE-A3 peptide
pool. Aliquots of day 14 cultures
were stimulated in the absence or
presence of the peptide pool or of
individual MAGE-A3 peptides, as
indicated, and IFN-g and IL-17
were measured in 24-hour culture
supernatants by ELISA.
Responses to single peptides were
considered significant when the
cytokine level was more than 3-fold
the background cytokine level
detected in the absence of peptide
(indicated by the dotted line for
each cytokine). B, CD4þ T cells
from patient NA171 were sorted
into the indicated subpopulations
according to chemokine receptors
expression as in Fig. 3A and
stimulated in vitro with the MAGEA3 peptide pool. Aliquots of the
cultures were stimulated in the
absence or in the presence of the
indicated MAGE-A3 peptides and
IFN-g and IL-17 were assessed in
24-hour culture supernatants by
ELISA.
141–160 in the CCR6þCCR4þ fraction and found the same
reactivity in the CCR6þCXCR3þ fraction (Fig. 4B). In contrast,
in the CCR6CXCR3þ fraction the reactivity was distinct and
directed against peptides 241–260 and 271–290. Together,
these results indicated that the CCR6þCCR4þ and
CCR6þCXCR3þ fractions contained the same MAGE-A3–
specific TH17/TH1 population at early (CM) and late (EM)
differentiation stages, respectively, whereas the CCR6CXCR3þ
fraction contained a distinct TH1 population. To further support the conclusion that MAGE-A3 141–160-specific CD4þ T
cells in this patient represented a single population, we isolated
them based on CD154 upregulation following antigen stimulation and expanded them in vitro under clonal conditions. We
obtained several MAGE-A3 141–160-specific clones that secreted IL-17 and/or IFN-g (Supplementary Fig. S1A and S1B). In
addition to recognizing peptide MAGE-A3 141–160, the clones
recognized autologous DC incubated with a recombinant
MAGE-A3 protein but not with a control protein (Supplemen-
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Cancer Res; 72(5) March 1, 2012
tary Fig. S1C). T-cell receptor (TCR) analysis of the clones using
anti-TCR Vb mAb revealed that they all used Vb2 (data not
shown) and molecular analysis of the TCR b chain mRNA from 7
clones with specific primers further confirmed that they used
the TCR beta variable gene (TRBV) 20-1, showed that they all
used a unique TCR beta joining gene (TRBJ) and displayed an
identical CDR3b (Supplementary Fig. S1D).
Together, the findings reported here show for the first
time that TH17 specific for a common tumor antigen can be
found in cancer patients as part of their spontaneous
immune response to the autologous tumor. In addition, they
support a recently proposed differentiation model in which
CD4þ T cells primed in vivo under TH17 conditions progressively convert into IFN-g–secreting as they differentiate into
effector cells (5).
The significance and potential impact of tumor antigen–
specific TH17 responses in lung cancer warrant further investigation, as both positive and negative correlations between the
Cancer Research
Spontaneous TH17 Responses to MAGE-A3 in Lung Cancer
presence of tumor-associated IL-17–secreting cells and survival have been reported (18, 19), a discrepancy that may be
explained by the involvement of cells other than those of
adaptive antitumor immunity (e.g., IL-17–secreting g/d T cells)
as recently suggested (20). In favor of the antitumor potential of
adaptive TH17 immunity, it has been recently shown that, in a
B16 melanoma model, transfer of in vitro polarized antitumor
TH17 lines controlled tumor growth better than TH1 lines, an
effect that was dependent on IFN-g and independent of IL-17
(7). The existence of spontaneously arising tumor antigen–
specific TH17 cells in patients with lung cancer, along with
their penchant to convert into IFN-g–secreting cells as
they differentiate into effectors, therefore encourages the
development of immunotherapeutic approaches aimed at
their amplification.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors are grateful to Dr. Gerd Ritter for providing the recombinant
MAGE-A3 and Melan-A proteins.
Grant Support
The study was supported by the Cancer Research Institute, the Ludwig
Institute for Cancer Research, the Institut National du Cancer (France), and
the Cancerop^
ole ^Ile de France (France).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
Received October 14, 2011; revised December 1, 2011; accepted December 29,
2011; published OnlineFirst January 17, 2012.
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