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Cancer Research Priority Report 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). 1059 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. 1060 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 1061 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- 1062 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. References 1. Chen Z, O'Shea JJ. Th17 cells: a new fate for differentiating helper T cells. Immunol Res 2008;41:87–102. 2. Kolls JK. Th17 cells in mucosal immunity and tissue inflammation. Semin Immunopathol 2010;32:1–2. 3. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia A, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol 2007;8:639–46. 4. 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