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Microenvironment and Immunology IL-17A Produced by gd T Cells Promotes Tumor Growth in Hepatocellular Carcinoma Shoubao Ma 1 , Qiao Cheng 1 , Yifeng Cai 2 , Huanle Gong 1 , Yan Wu 1 , Xiao Yu 1 , Liyun Shi 3 , Depei Wu 2 , Chen Dong 4 , and Haiyan Liu 1,2 Abstract Interleukin (IL)-17A is expressed in the tumor microenvironment where it appears to contribute to tumor development, but its precise role in tumor immunity remains controversial. Here, we report mouse genetic evidence that IL-17A is critical for tumor growth. IL-17Adecient mice exhibited reduced tumor growth, whereas systemic administration of recombinant mouse IL-17A promoted the growth of hepatocellular carcinoma. The tumor-promoting effect of IL-17A was mediated through suppression of antitumor responses, especially CD8 þ T- cell responses. Furthermore, we found that IL-17A was produced mainly by Vg 4 gd T cells, insofar as depleting Vg 4 gd T cells reduced tumor growth, whereas adoptive transfer of Vg 4 gd T cells promoted tumor growth. Mechanistic investigations showed that IL-17A induced CXCL5 production by tumor cells to enhance the inltration of myeloid-derived suppressor cells (MDSC) to tumor sites in a CXCL5/CXCR2dependent manner. IL-17A also promoted the suppressive activity of MDSC to reinforce suppression of tumoral immunity. Moreover, we found that MDSC could induce IL-17Aproducing gd T cells via production of IL-1b and IL-23. Conversely, IL-17A could also enhance production of IL-1b and IL-23 in MDSC as a positive feedback. Together, our results revealed a novel mechanism involving cross-talk among gd T cells, MDSCs, and tumor cells through IL-17A production. These ndings offer new insights into how IL-17A inuences tumor immunity, with potential implications for the development of tumor immunotherapy. Cancer Res; 74(7); 196982. Ó2014 AACR. Introduction The cytokine interleukin (IL)-17A is a proinammatory cytokine that was identied almost two decades ago. More recently, IL-17Aproducing CD4 þ T cells have been shown to be distinct from classical Th1 and Th2 cells, thus designated as Th17 cells (1, 2). In addition to Th17 cells, several other cell types are described as sources for IL-17A, including CD8 þ T cells (3), gd T cells (4), natural killer T (NKT; ref. 5), and lymph tissue inducer cells (LTi cells; ref. 6). IL-17A binds to and signals through IL-17 receptor A (IL-17RA), which is ubiquitously expressed in hematopoietic tissues, various myeloid cells, epithelial cells, broblasts, and endothelial cells. The ligation of IL-17/IL-17R results in the release of proinammatory cytokines, chemokines, and matrix metalloproteinases (MMP) to further stimulate the inammatory cascade. IL-17A and IL-17Aproducing cells have been found in many types of human cancers and murine models. However, the role of IL-17A and IL-17Aproducing cells in tumor development is controversial. A series of reports have suggested that they have potent antitumor functions. Martin-Orozco and colleagues showed that IL-17A / were more susceptible to developing lung melanoma, and adoptively transferred tumor-specic Th17 cells prevented tumor development (7). Moreover, Th17-polarized cells were found to be more effective than Th1 cells in eliminating large established tumors (8). IL-17Apro- ducing CD8 þ T cells also reduced the volume of large estab- lished tumors and could differentiate into long-lasting IFN-g producers (9), suggesting that IL-17A and IL-17Aproducing cells are protective against tumor development. However, other reports have suggested potent protumor functions for IL-17A and IL-17Aproducing cells. IL-17A overexpression in tumor cell lines promotes angiogenesis and tumor growth in immunodecient mice (10), and IL-17A can promote tumor growth by enhancing angiogenesis in immunocompetent hosts (11). IL-17A can induce IL-6 production, which in turn pro- motes tumor growth in a Stat-3dependent pathway (12). Moreover, adoptive transfer of Th17 cells induced by TGF-b and IL-6 promoted tumor growth (13). It has also been reported that the development of tumors was inhibited in IL-17RA / mice (14). Therefore, the role of IL-17A in tumor development Authors' Afliations: 1 Laboratory of Cellular and Molecular Tumor Immu- nology, Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University; 2 Cyrus Tang Hema- tology Center, Jiangsu Institute of Hematology, First Afliated Hospital of Soochow University, Suzhou; 3 Department of Basic Medical Science, Key Lab of Inammation and Immunoregulation, School of Medicine, Hangzhou Normal University, Hangzhou, PR China; and 4 Department of Immunology, MD Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for Y. Cai: Department of Hematology, The Afliated Hospital of Nantong University, Nantong 226001, PR China. Corresponding Author: Haiyan Liu, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu 215123, PR China. Phone: 86-0512-6588-0235; Fax: 86-0512-6588-0235; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-2534 Ó2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1969 on July 15, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 13, 2014; DOI: 10.1158/0008-5472.CAN-13-2534
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Page 1: IL-17A Produced by gd T Cells Promotes Tumor Growth in ... · Microenvironment and Immunology IL-17A Produced by gd T Cells Promotes Tumor Growth in Hepatocellular Carcinoma Shoubao

Microenvironment and Immunology

IL-17A Produced by gd T Cells Promotes Tumor Growth inHepatocellular Carcinoma

Shoubao Ma1, Qiao Cheng1, Yifeng Cai2, Huanle Gong1, Yan Wu1, Xiao Yu1, Liyun Shi3, Depei Wu2,Chen Dong4, and Haiyan Liu1,2

AbstractInterleukin (IL)-17A is expressed in the tumor microenvironment where it appears to contribute to tumor

development, but its precise role in tumor immunity remains controversial. Here, we report mouse geneticevidence that IL-17A is critical for tumor growth. IL-17A–deficientmice exhibited reduced tumor growth,whereassystemic administration of recombinant mouse IL-17A promoted the growth of hepatocellular carcinoma. Thetumor-promoting effect of IL-17A wasmediated through suppression of antitumor responses, especially CD8þ T-cell responses. Furthermore, we found that IL-17Awas producedmainly byVg4 gd Tcells, insofar as depletingVg4gd T cells reduced tumor growth, whereas adoptive transfer of Vg4 gd T cells promoted tumor growth.Mechanistic investigations showed that IL-17A induced CXCL5 production by tumor cells to enhance theinfiltration of myeloid-derived suppressor cells (MDSC) to tumor sites in a CXCL5/CXCR2–dependent manner.IL-17A also promoted the suppressive activity of MDSC to reinforce suppression of tumoral immunity. Moreover,we found that MDSC could induce IL-17A–producing gd T cells via production of IL-1b and IL-23. Conversely,IL-17A could also enhance production of IL-1b and IL-23 in MDSC as a positive feedback. Together, our resultsrevealed a novel mechanism involving cross-talk among gd T cells, MDSCs, and tumor cells through IL-17Aproduction. These findings offer new insights into how IL-17A influences tumor immunity, with potentialimplications for the development of tumor immunotherapy. Cancer Res; 74(7); 1969–82. �2014 AACR.

IntroductionThe cytokine interleukin (IL)-17A is a proinflammatory

cytokine that was identified almost two decades ago. Morerecently, IL-17A–producing CD4þ T cells have been shown tobe distinct from classical Th1 and Th2 cells, thus designated asTh17 cells (1, 2). In addition to Th17 cells, several other celltypes are described as sources for IL-17A, including CD8þ Tcells (3), gd T cells (4), natural killer T (NKT; ref. 5), and lymphtissue inducer cells (LTi cells; ref. 6). IL-17A binds to and signalsthrough IL-17 receptor A (IL-17RA), which is ubiquitouslyexpressed in hematopoietic tissues, various myeloid cells,

epithelial cells, fibroblasts, and endothelial cells. The ligationof IL-17/IL-17R results in the release of proinflammatorycytokines, chemokines, and matrix metalloproteinases (MMP)to further stimulate the inflammatory cascade.

IL-17A and IL-17A–producing cells have been found inmanytypes of human cancers and murine models. However, the roleof IL-17A and IL-17A–producing cells in tumor development iscontroversial. A series of reports have suggested that they havepotent antitumor functions. Martin-Orozco and colleaguesshowed that IL-17A�/� were more susceptible to developinglung melanoma, and adoptively transferred tumor-specificTh17 cells prevented tumor development (7). Moreover,Th17-polarized cells were found to be more effective than Th1cells in eliminating large established tumors (8). IL-17A–pro-ducing CD8þ T cells also reduced the volume of large estab-lished tumors and could differentiate into long-lasting IFN-gproducers (9), suggesting that IL-17A and IL-17A–producingcells are protective against tumor development. However,other reports have suggested potent protumor functions forIL-17A and IL-17A–producing cells. IL-17A overexpression intumor cell lines promotes angiogenesis and tumor growth inimmunodeficient mice (10), and IL-17A can promote tumorgrowth by enhancing angiogenesis in immunocompetent hosts(11). IL-17A can induce IL-6 production, which in turn pro-motes tumor growth in a Stat-3–dependent pathway (12).Moreover, adoptive transfer of Th17 cells induced by TGF-band IL-6 promoted tumor growth (13). It has also been reportedthat the development of tumors was inhibited in IL-17RA�/�

mice (14). Therefore, the role of IL-17A in tumor development

Authors' Affiliations: 1Laboratory of Cellular and Molecular Tumor Immu-nology, Jiangsu Key Laboratory of Infection and Immunity, Institutes ofBiology and Medical Sciences, Soochow University; 2Cyrus Tang Hema-tology Center, Jiangsu Institute of Hematology, First Affiliated Hospital ofSoochow University, Suzhou; 3Department of Basic Medical Science, KeyLabof Inflammation and Immunoregulation, School ofMedicine,HangzhouNormal University, Hangzhou, PR China; and 4Department of Immunology,MD Anderson Cancer Center, Houston, Texas

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for Y. Cai: Department of Hematology, The AffiliatedHospital of Nantong University, Nantong 226001, PR China.

Corresponding Author: Haiyan Liu, Institutes of Biology and MedicalSciences, Soochow University, Suzhou, Jiangsu 215123, PR China.Phone: 86-0512-6588-0235; Fax: 86-0512-6588-0235; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-2534

�2014 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1969

on July 15, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 13, 2014; DOI: 10.1158/0008-5472.CAN-13-2534

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and the endogenous source of IL-17A in tumor microenviron-ment remain to be defined.

Under physiologic conditions, gd T subsets, including Vg1(15), Vg4 (16), Vg6Vd1 (17), and Vg5Vd1 dendritic epidermalT cells (18), have the ability to produce IL-17A. Although IL-17A–producing gd Tcells havebeendescribed to play important rolesin immunopathologic diseases, such as collagen-induced arthri-tis, psoriasis, and microbial infection, the presence and func-tional relevance of IL-17A–producing gd T cells during tumordevelopment arenot fully understood.Myeloid-derived suppres-sor cells (MDSC) are a heterogeneous cell population consistingof immature myeloid cells and myeloid progenitor cells thathave been noted to directly contribute to the negative regulationof immune responses during tumor development (19). MDSCsinhibit proliferation and IFN-g production of tumor-specificCD8þ T cells by a variety of mechanisms. On the basis of theexpressions of Ly6G and Ly6C, MDSCs are subdivided into twosubsets: monocytic (CD11bþLy6G�Ly6Chigh) and granulocytic(CD11bþLy6GþLy6Clow) MDSCs (20, 21). They are morpholog-ically different and function through distinct mechanisms ofsuppression.

In the current study, we first examined the role of IL-17Ain tumor development with three murine hepatocellularcarcinoma models. We next identified Vg4 gd T cells as themajor source of IL-17A in the tumor microenvironment.Further studies revealed that IL-17A facilitated the MDSCs'infiltration through the CXCR2-dependent manner. More-over, MDSCs could further promote IL-17A production by gdT cells through the secretion of IL-1b and IL-23. Our resultsthus revealed a tumor-promoting role of IL-17A through theinterplay among gd T cells, MDSCs, and tumor cells in thetumor microenvironment.

Materials and MethodsMice

C57BL/6 mice were purchased from Shanghai LaboratoryAnimal Center (Shanghai, PR China). C57BL/6 IL-17A�/�micewere provided byDr. ChenDong (MDAndersonCancer Center,Houston, TX). TCRd�/� and OT-I mice were provided by Prof.Zhinan Yin (Nankai University, Tianjin, PR China). IL-1R1�/�

mice were provided by Dr. Liyun Shi (Hangzhou NormalUniversity, Hangzhou, PR China). Mouse care and experimen-tal procedures were performed under specific pathogen—free(SPF) conditions. Age (8–12 week or indicated)–matched malemice were used in the experiments. All animal protocols wereapproved by the Institutional Laboratory Animal Care and UseCommittee at Soochow University (Suzhou, PR China).

Cell lines and reagentsThe Hepa1–6 murine hepatocellular carcinoma cell line was

obtained from the American Type Culture Collection andcultured in Dulbecco's Modified Eagle Medium (DMEM) sup-plemented with 10% FBS, 2 mmol/L L-glutamine, 100 IU/mLpenicillin/streptomycin, 1 mmol/L sodium pyruvate, 1 mmol/Lnonessential amino acids, 2.5 � 10�5 mol/L 2-ME, and 10mmol/L HEPES at 37�C, 5% CO2. Recombinant mouse (rm)IL-17, IL-1b, and IL-23werepurchased fromPeproTech. Recom-binant human IL-2 (rhIL-2) was purchased from Beijing Four

Rings Bio-Pharmaceutical. Antibodies against CD3, CD4, CD8,CD27, TCRgd, NK1.1, CD44, CD62L, CD11c, CD11b, F4/80, Gr-1,CCR6, CXCR2, CXCR4, RORgt, IL-17RA, IL-17, IFN-g , and TNF-awere all purchased from BioLegend. Antibodies against Vg4,Ly6G, and Ly6C were purchased from Sungene Biotech. Mouseregulatory T cell (Treg) staining kit was purchased fromeBioscience. Purified anti-mouse IL-17A (17F3), rat anti-mouseCD8a monoclonal antibody (mAb; 2.43), rat anti-mouse Gr-1mAb (RB6–8C5), hamster anti-mouse T cell receptor (TCR) Vg4mAb (UC3), and their isotype controls were purchased fromSungeneBiotech.Anti-mouse IL-1bmAb(B122) andanti-mouseIL-23 p19 mAb (G23-8) were purchased from eBioscience.Purified anti-mouse CD3e mAb (145-2C11) and anti-mouseCD28mAb (37.51) were purchased fromBDBiosciences. MouseIL-17A ELISA kit was purchased fromeBioscience.Mouse IFN-gand TNF-a ELISA kits were purchased from Dakewe Biotech.CXCR2-specific antagonist SB-265610 and CXCR4 antagonistAMD3100 were purchased from Sigma-Aldrich.

Establishment and assessment of murine hepatocellularcarcinoma models

For subcutaneous tumor model, 7� 106 Hepa1–6 cells wereinjected subcutaneously into 8- to 12-week-old wild-type (WT)or IL-17�/� mice, and tumor growth was monitored every 3days. Mice were sacrificed after 3 to 4 weeks from tumorinoculation. Orthotopic hepatocellular carcinoma model wasperformed as described earlier withminormodification (22); inbrief, anesthetized mice were performed with surgical proce-dures, and then 1 � 106 Hepa1–6 cells in 20 mL PBS wereimplanted intrahepatically. The mice were sacrificed 2 weekslater, and the recognizable tumors were measured with finedigital calipers and tumor volume was calculated by thefollowing formula: tumor volume ¼ 0.5 � width2 � length.For diethylnitrosamine (DEN)-induced hepatocellular carci-noma model, 15-day-old male mice were injected intraperito-neally with a single dose of 25 mg/kg DEN (Sigma-Aldrich).After 8 or 10months on normal chow,mice were sacrificed andtheir livers were removed to analyze tumor size, number oftumor nodules, and histology.

Cell preparationSpleen cells were prepared by gently crushing the tissues to

release the cells. Preparations were filtered to remove debrisand washed twice with PBS before resuspending in RPMI-1640complete medium. Livers were perfused with PBS and pro-cessed into single-cell suspensions, and lymphocytes wereseparated on a 40% Percoll (GE Healthcare) gradient. Erythro-cytes were lysed. Cell counts were performed on a Coulter Z1cell counter (Beckman Coulter).

Flow cytometryFor cell-surface staining, cell samples were stained with

fluorescent dye–conjugated mAb against selected markers for30minutes on ice. For intracellular cytokine staining, cells werestimulated for 5 hours with phorbol 12-myristate 13-acetate(PMA; 50 ng/mL) and ionomycin (500 ng/mL) in the presence ofbrefeldin A (10 mg/mL; BD Biosciences). Cells were harvested,washed, and stained with surface molecule antibodies in the

Ma et al.

Cancer Res; 74(7) April 1, 2014 Cancer Research1970

on July 15, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 13, 2014; DOI: 10.1158/0008-5472.CAN-13-2534

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presence of FcR-block (eBioscience). After the wash, cells werefixed with 4% paraformaldehyde and permeabilized with 1%saponin (Sigma), and then stained with cytokine-specific orcontrol isotype antibodies for 30 minutes on ice. Data wereacquired on a FACSCalibur (BD Biosciences) and analyzedusing FlowJo software (FlowJo). The CD3þCD8þ T cells,gd T (CD3þTCR-gdþ) cells, CD11bþGr-1þMDSCs, CD11bþ

Ly6GþLy6Clow granulocytic MDSCs, and CD11bþLy6G�Ly6Chigh

monocytic MDSCs from spleens or tumor—infiltrating lympho-cytes (TIL) of tumor-bearing mice were sorted using a BDFACSAria III cell sorter (BD Biosciences).

In vivodepletion of CD8T cells, Vg4 gd T cells, andMDSCsTo deplete CD8þ T cells, mice were treated with intravenous

injection of anti-CD8–specificmAb (clone 2.43, 200 mg/mouse)on days �1 and 7 before and after tumor inoculation. Todeplete Vg4 gd T cells, mice were treated with intravenousinjection of anti-Vg4–specific mAb (clone UC3, 200 mg/mouse)on days �5 and �1 before tumor inoculation. Depletion wasconfirmed by fluorescence-activated cell sorting (FACS) anal-ysis of TCR Vg4 expression from peripheral blood cells (Sup-plementary Fig. S4A). To deplete MDSCs, a single dose of 0.25mg Gr-1 mAb (RB6–8C5) was administered intravenously ondays 0, 4, 8, and 12 after tumor implantation. For MDSCdepletion by gemcitabine treatment, gemcitabine (LC Labo-ratories) was injected intraperitoneally at 100mg/kg on days 0,3, 6, 9, and 12 after tumor implantation. Depletion was con-firmed by flow cytometry.

In vivo blocking of chemokine receptor expressionsB6 WT mice were treated with rmIL-17A (1 mg/mouse)

before tumor inoculation, then mice were treated with theCXCR2-specific antagonist SB-265610 (Sigma) at 2 mg/kg/dthrough intraperitoneal injection for 2 weeks [dimethyl sulf-oxide (DMSO) as vehicle], or a CXCR4 antagonist AMD3100(Sigma) at 5 mg/kg/d through intraperitoneal injection for 2weeks (PBS as vehicle).

Vg4 gd T-cell expansion and adoptive transferSplenocytes from B6 WT mice and IL-17A�/� mice were

cultured with plate-coated Vg4-specific mAb (clone UC3, 10mg/mL) and rhIL-2 (100 U/mL) for 8 days as described previ-ously (23). Expanded Vg4 cells were confirmed by FACSanalysis (Supplementary Fig. S4B) and 1� 106 cells per mousewere intravenously transferred into B6 TCRd�/�mice 24 hoursbefore tumor inoculation.

T-cell proliferation and intracellular cytokineproductionsFor proliferation assay, purified splenic CD8þ T cells from

OT-I mice were labeled with a 5 mmol/L CellTrace CFSE CellProliferation Kit (Invitrogen) in PBS with 2% fetal calf serum(FCS) for 10 minutes at 37�C. The labeling reaction wasquenched by addition of cold RPMI-1640 medium with 10%FCS, and cells were washed twice with PBS with 2% FCS toremove excess 5,6-carboxyfluorescein diacetate succinimidylester (CFSE). Then, purified MDSCs from spleens of B16-ovatumor-bearing mice were cocultured with CFSE-labeled

splenic CD8þ T cells at a ratio of 1:3 loaded with SIINFEKL(10 mg/mL) in the presence of a different dose of rmIL-17A.The proliferation of CD8þ T cells was evaluated 3 days laterwith CFSE dilution by flow cytometry. In some experiments,10 mg/mL IL-17A mAb was added. The proliferation index wascalculated using FlowJo software (FlowJo). For CD8þ T cellsproliferation determined by the incorporation of [3H]-thymi-dine in the cell coculture, [3H]-thymidine (1 mCi/well; ShanghaiInstitute of Physics, Chinese Academy of Sciences, Shanghai,PR China) was added to triplicate wells for the final 16 to18 hours before harvesting, and the proliferations of the CD8þ

T cells were determined using a liquid scintillation counter(PerkinElmer Instruments). For intracellular cytokine produc-tions, sorted MDSCs, granulocytic MDSCs, and monocyticMDSCs were cocultured with polyclonal-stimulated (5 mg/mLanti-CD3e and 1mg/mL anti-CD28) CFSE-labeled splenic CD8þ

T cells at a ratio of 1:3 in the presence of a different dose ofrmIL-17A for 3 days. Before harvest, brefeldin A (10mg/mL)wasadded for the last 5 hours of coculture. Thereafter, the expres-sion of TNF-a and IFN-g in CD8þ T cells was analyzed by FACSanalysis. The levels of TNF-a and IFN-g in supernatants weremeasured by ELISA.

Cell migration assaysHepa1–6 cells were treated with different doses of rmIL-17

for 48 hours, to neutralize IL-17A, anti-mouse IL-17A mAb (10mg/mL) was added; thereafter, the culture supernatants wereharvested, centrifuged, and stored at�80�C. For cellmigration, 5� 104 MDSCs were seeded onto the top chamber of Transwellfilters (8mmol/L;Costar). Thefilterswereplaced ina24-well platecontaining rmIL-17A–pretreated tumor supernatants or culturemedium with same dose of rmIL-17. To neutralize CXCR2,MDSCs were preincubated in cell culture medium withCXCR2-specific antagonist SB-265610 (10 mg/mL) for 30minutesat 37�C. Migrated MDSCs were counted (5–7 fields/well, tripli-cate for each experimental groups) 6 to 8 hours after incubation.

IL-17–producing gd T-cell differentiationPurified gd T cells from TILs of tumor-bearing mice were

stimulated with IL-1b (50 ng/mL) and IL-23 (50 ng/mL).MDSCs, granulocytic MDSCs, and monocytic MDSCs wereadded to the culture on day 0 at a ratio of 1:1. Three days aftercoculture, supernatants were collected for measurement of IL-17A by ELISA. The remaining cells were directly stained forintracellular IL-17A in the presence of brefeldin A for 4 hours.In some experiments, 20 mg/mL IL-1b mAbs (eBioscience), 20mg/mL IL-23 p19mAbs (eBioscience), or the combination wereadded. To assess the effect of IL-17 on MDSC-enhanced IL-17–producing gd T-cell polarization, gd T cells were coculturedwith MDSCs in the presence of a different dose of rmIL-17Awithout IL-1b and IL-23 stimulation, IL-17 mAb (10 mg/mL), orisotype immunoglobulin G (IgG; 10 mg/mL) was added to theculture. Cells were harvested and examined for IL-17A expres-sion by intracellular staining 3 days later.

Cytotoxicity assayCTL activity was determined by using a CytoTox 96

Non-Radioactive Cytotoxicity Assay kit (Promega) based on

IL-17A Produced by gd T Cells in Hepatocellular Carcinoma

www.aacrjournals.org Cancer Res; 74(7) April 1, 2014 1971

on July 15, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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release of lactate dehydrogenase from target cells. To obtaintumor-specific CTLs, WT or knockout tumor-free mice werechallenged with Hepa1–6 cells for 14 days. Splenocytes (2� 106

cells/mL) from these tumor-bearing mice were harvested andwere stimulated with Hepa1–6 tumor lysates and rhIL-2 (100U/mL) for 4 to 6 days. Then, tumor-specificCTLswereharvestedand washed for cytotoxicity assay. Briefly, 6� 103 Hepa1–6 cellsin 50 mL were added to 50 mL of various numbers of tumor-stimulated splenocytes that had been plated in U-bottomed 96-microwell plates to obtain target:effector cell ratios of 1:2, 1:4, or1:8. After 4 hours of incubation, supernatant (50 mL) wascollected from each well and added to 50 mL of reconstitutedsubstrate mix for 30 minutes in the dark at room temperature.Enzymatic reaction was stopped by adding stop solution.Absorbance was recorded at 490 nm. Spontaneous release wasdetermined from wells with targets only and total release fromwells with targets plus 1% Triton X-100. Results are expressed aspercentage of cytotoxicity, using the formula: percentage ofcytotoxicity ¼ (experimental � effector spontaneous � targetspontaneous)/(targetmaximum� target spontaneous)� 100%.

Reverse transcriptase PCR and real-time PCRTotal RNA was extracted from tumors or cells by using

TRIzol reagent (Invitrogen). First-strand cDNA was synthe-sized from 1 mgmRNA using reverse transcriptase (Fermentas)and oligo(dT) primers. For TCR-Vg profiling, the resultingcDNA was amplified under the following reaction conditions:denaturation at 94�C, annealing at 55�C, and extension at 72�Cfor 38 cycles. Real-time PCR was performed with an EppendorfRealplex PCR system using SYBR Green PCR Master Mix(Applied Biosystems). The expression was normalized to theexpression of the housekeeping geneGAPDH. The primers usedare described in Supplementary Table S1.

ELISA and cytometric bead arrayBlood was collected from tumor-bearing mice and serum

was separated out by centrifugation at 3,000 rpm for 15minutes at 4�C, and supernatant (serum) was transferred intoa new tube and frozen at �80�C until needed. Supernatantsfrom the coculture system as described previously were col-lected. Serum levels of IL-17A, IFN-g , and TNF-a were mea-sured using ELISA kits according to the manufacturer'sinstructions. Mouse Th1/Th2/Th17 cytokines were measuredby cytometric bead array (CBA; BD Biosciences) according tothe manufacturer's instructions.

Statistical analysisAll data were analyzed by Student t test and were expressed as

mean � SEM. Data were analyzed using GraphPad Prism 5software forWindows (GraphPad Software) and differenceswereconsidered statistically significant when P < 0.05. The signifi-cance levels are marked as: � , P < 0.05; ��, P < 0.01; ���, P < 0.001.

ResultsIL-17A promotes tumor growth in hepatocellularcarcinoma murine models

To dissect the role of IL-17A in hepatocellular carcinomadevelopment, we compared tumor growth inWTand IL-17A�/�

mice in three murine hepatocellular carcinoma models (Fig. 1).The tumor growth was markedly reduced in IL-17A�/� micecompared withWTmice, whereas the administration of rmIL-17A promoted tumor growth in subcutaneous hepatocellularcarcinoma model (Fig. 1A and B). We further used orthotopicmodel to examine the role of IL-17A in the development ofhepatocellular carcinoma (Fig. 1C and D). On day 14 after theimplantation, IL-17A�/� mice exhibited decreased size oftumor nodules when compared with WT mice, and WT micetreated with rmIL-17A exhibited increased tumor growthcompared with PBS control. The DEN-induced tumorigenesiswas also compared among WT mice, rmIL-17A–treated WTmice, and IL-17A�/� mice 8 months after the DEN injection(Fig. 1E andF). Themaximumsize of tumors and thenumber oftumor nodules were significantly decreased in IL-17A�/�micecompared with WT mice. Treatment of WT mice with rmIL-17A enhanced the tumor growth compared with no treatmentcontrol. These results demonstrated that IL-17A promotedtumor growth in murine hepatocellular carcinoma models.

IL-17A impairs antitumor immunityTo understand how IL-17A mediates tumor protection, we

first investigatedwhether IL-17A could directly promote tumorgrowth in vitro (Supplementary Fig. S1). Hepa1–6 cells expresslow levels of IL-17R at both protein and mRNA levels (Supple-mentary Fig. S1A and S1B). However, IL-17A had no directeffect on tumor cell viability (Supplementary Fig. S1C) andapoptosis (Supplementary Fig. S1D). These results suggestedthat IL-17A promoted hepatocellular carcinoma developmentnot by directly affecting tumor cell growth, but throughmodulating the antitumor immune responses.

To study how IL-17A affected tumor immunity and favoredtumor development, we used in situ hepatocellular carcinomamodel for further mechanistic analysis. TILs from tumor-bearing mice were collected 14 days after the tumor implan-tation and subjected to analysis by flow cytometry. ComparedwithWTmice andmice treatedwith rmIL-17, the infiltration ofCD8þ T cells was significantly increased in IL-17A�/� mice(Fig. 2A). The CD8þ T cells with memory phenotypes alsoincreased in IL-17A�/� mice (Fig. 2B). Furthermore, the per-centages of IFN-gþCD8þT cells (Tc1) were significantly higherin IL-17A�/� mice than WT and rmIL-17–treated mice (Fig.2C). On the other hand, the percentages of IFN-gþCD4þ T cellsshowed no significant difference among the three groups,indicating that the IL-17A may only affect CTL responses. Theserum levels of IFN-g , IL-2, and TNF-awere also elevated in IL-17A�/� mice as compared with the WT mice and were furtherreduced by the rmIL-17 treatment (Fig. 2D). Using Hepa1–6cells as targets, splenocytes and sorted CD8þ T cells fromtumor-bearing IL-17A�/�mice showed enhanced killing activ-ity, compared with those from WT mice (Fig. 2E). Tumorgrowth was significantly increased to the similar levels byCD8þ T-cell depletion in bothWT and IL-17A�/�mice, furtherdemonstrating a major role of CTL response in antitumorimmunity (Supplementary Fig. S1E–S1G). We next analyzedthe myeloid populations infiltrated in the tumor. We foundthat the percentages of dendritic cells, macrophages, andneutrophils were not significantly changed in IL-17A�/� mice

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(Fig. 2F). However, the percentages of both monocytic andgranulocytic MDSCs were apparently reduced in IL-17A�/�

mice. Moreover, the administration of rmIL-17 increased thetumor infiltrations of the MDSCs. These findings suggest thatIL-17A impaired antitumor responses, especially CTLresponses, possibly through promoting the recruitment ofMDSCs.

Vg4 gd T cells are the main source of IL-17A duringhepatocellular carcinoma developmentTo examine which cell subsets are the major IL-17A–pro-

ducing cells during hepatocellular carcinoma development, wefirst analyzed the IL-17A production by different cell popula-tions infiltrated in the tumor (Supplementary Fig. S2A). gd Tcells exhibited the highest percentage of IL-17þ cells (24.5%).We then compared the IL-17A production by Th17 cells and gdT cells (Fig. 3A). Of all the IL-17A–producing cells, about 60%were gd T cells, while only 20% were Th17 cells (Fig. 3B). Theabsolute numbers of IL-17A–producing gd T cells were about4-fold higher than the Th17 cells (Fig. 3C). Therefore, the gd T

cells are the major IL-17A–producing cells during hepatocel-lular carcinoma development. Because IL-17A–producing gd Tcells have been reported as CD27�CCR6þ (15), further analysisdid show that the IL-17A–producing gd T cells were RORgtþ,CCR6þ, and CD27� (Fig. 3D).

To further identify themain IL-17A–producing subsets of gdT cells, we then examined the Vg repertoire of the gd T cells intheTILs by reverse transcriptase PCR (RT-PCR; SupplementaryFig. S2B). Data showed that the gd T cells expressed all Vg1,Vg2, Vg4, Vg5, Vg6, and Vg7 genes. FACS analysis showed thatabout 75% of IL-17A–producing gd T cells were Vg4 gd T cells(Fig. 3E). Taken together, our results showed that Vg4 gd T cellswere the main source of IL-17A during hepatocellular carci-noma development.

Vg4 gd T cells promote tumor growth through IL-17Aproduction

To address whether gd T cells could affect tumor growth, wefirst compared the hepatocellular carcinoma growth inTCRd�/� and WT mice (Supplementary Fig. S3A). TCRd�/�

Figure 1. IL-17A promotes tumorgrowth in hepatocellular carcinomamurine models. A–D, Hepa1–6cells (7 � 106) were inoculatedsubcutaneously (A and B) orintrahepatically (C and D) into WTmice treated with or without rmIL-17 and IL-17�/� mice. Data shownare the mean tumor volume � SDand gross morphology of thetumors. E–G, WT and IL-17�/�

mice were given a single injectionof DEN at 15 days of age. After 6months, WT mice were given rmIL-17 every 2 weeks for 2 months.Data shown are the numbers oftumor nodules and maximal tumorsizes, representative tumormorphology, and liver histology in8-month-old DEN-treated mice.The data are representative ofthree independent experiments,each using 4 to 5 mice per group(�, P < 0.05; ��, P < 0.01).

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mice exhibited reduced tumor growth as compared with theWT mice. The serum IL-17 levels were also significantlydecreased in TCRd�/� tumor-bearing mice (SupplementaryFig. S3B). The percentages of memory CD8þ T cells and Tc1cells were also significantly increased in TCRd�/� mice (Sup-plementary Fig. S3C and S3D). These results suggested that gdT cells promoted hepatocellular carcinoma growth throughsuppressing the antitumor immune responses.

To directly define the role of IL-17A–producing Vg4 gd Tcells in hepatocellular carcinoma, we first in vivo depleted Vg4gd T cells using the anti-Vg4 TCR mAb (clone UC3) beforetumor implantation. About 97% of the Vg4 gd T cells weredepleted using this antibody (Supplementary Fig. S4A). Asshown in Fig. 4A, depletion of Vg4 gd T cells resulted insignificant reduction in tumor volumes in comparison with

WT mice treated with the control antibody, suggesting apromoting role of Vg4 gd T cells in tumor growth. Interestingly,Vg4 gd T-cell depletion in IL-17A�/� mice did not furthersuppress the tumor growth, indicating that IL-17A played a keyrole in Vg4 gd T cell–mediated tumor development. FACSanalysis showed that IL-17Aþ gd T cells were significantlyreduced in Vg4 gd T cell–depleted mice (Fig. 4B), while weobserved similar levels of Th17 cells in both Vg4 gd T cell–depleted mice and WT mice (Fig. 4C). Moreover, depletion ofVg4 gd T cells could significantly reduce the serum IL-17Alevels (Fig. 4D). These results confirmed that Vg4 gd T cellswere the main source of IL-17A during hepatocellular carci-noma development. The infiltrations of effector and memoryCD8þT cells in tumorswere significantly increased in Vg4 gd Tcell–depleted mice (Fig. 4E). Moreover, the Tc1 cells were also

Figure 2. IL-17A impairs antitumor CD8þ T-cell responses. TILs and serum from tumor-bearing mice of orthotopic hepatocellular carcinoma modelwere collected 14 days after tumor implantation. A, the percentages of total T cells, CD4þ T cell, CD8þ T cell, gd T cell, NKT cell, and NK cell subsets.B, the percentages of effector and memory CD4þ or CD8þ T cells. C, the percentages of IFN-g–producing lymphocytes, CD4þ, or CD8þ T cells. D,serum levels of IL-2, IFN-g , and TNF-a. E, cytotoxic activity of splenocytes and sorted CD8þ T cells. F, the percentages of myeloid populations fromTILs of tumor-bearing mice. Data represent mean� SEM. Results shown are the representatives of three independent experiments (�, P < 0.05; ��, P < 0.01;���, P < 0.001). DC, dendritic cells.

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significantly increased in Vg4 gd T cell–depleted mice com-paredwith themice treatedwith the control antibody (Fig. 4F),while Vg4 gd T-cell depletion in IL-17A�/� showed no signif-icant difference. Taken together, the data demonstrated thatVg4 gd T-cell depletion resulted in reduced endogenous IL-17Aand tumor growth with enhanced CD8þ T cell–mediatedantitumor response.To further demonstrate the critical role of IL-17A–produc-

ing Vg4 gd T cells in tumor development, we reconstitutedTCRd�/� mice with cultured Vg4 gd T cells either fromWT orIL-17A�/�mice followed by tumor implantation (Fig. 4G). Thepurity of the cultured Vg4 gd T cells was more than 70% (Fig.4SB). Transferring WT Vg4 gd T cells in TCRd�/� miceexhibited similar tumor growth as the WT mice. However,transferring IL-17A�/� Vg4 gd T cells showed significantlyreduced tumor growth, suggesting a promoting role of Vg4 gdT cells in hepatocellular carcinoma through IL-17A produc-tion. Flow cytometry analysis showed increasedmemory CD8þ

T-cell infiltration in IL-17A�/� Vg4 gd T cell–reconstitutedmice (Fig. 4H). Moreover, the percentage of Tc1 cells signifi-cantly increased in IL-17A�/�Vg4 gd Tcell–reconstitutedmiceas compared with the WT mice (Fig. 4I). Interestingly, no IFN-g–producing T cells were detected in WT Vg4 gd T cell–reconstituted mice. These results further demonstrated thatVg4 gd T cells could suppress CD8þ T-cell responses andpromote tumor growth through IL-17A production.

IL-17A promotes the MDSCs infiltration to the tumorsites in a CXCR2-dependent mannerTo further dissect the immunosuppressive mechanism of

IL-17A, we first evaluated the direct effect of IL-17A on CD8þ

T cells and showed no effect on either cytokine productionsor proliferations of CD8þ T cells (Supplementary Fig. S5). Wealso screened the IL-17R expressions on the immune cells(Supplementary Fig. S6). Almost all the MDSCs expressedIL-17R with expressions of IL-17RA and IL-17RC at the tran-scription level. Therefore, IL-17A could function on MDSCsthrough IL-17R.

It has been previously reported that the CXCL5–CXCR2 andCXCL12–CXCR4 interactions serve as intrinsic mechanismsfor the recruitment of MDSCs into tumors (24). Tumor-infil-tratingMDSCs expressed bothCXCR2 andCXCR4 (Fig. 5A).Wealso found that IL-17–treated tumor cells upregulated a groupof chemokine expressions in a dose-dependent manner, espe-cially CXCL5 and CXCL12 (Fig. 5B). Next, we examined theCXCL5 and CXCL12 productions in the tumor microenviron-ment and observed elevated CXCL5 expression in WT tumorscompared with IL-17A�/� tumors (Fig. 5C), while CXCL12exhibited no difference. In vivo administration of CXCR4antagonist, AMD3100, also exhibited no effect on tumorgrowth (Supplementary Fig. S7A). On the basis of these results,we hypothesized that IL-17A may induce CXCL5 productionby the tumor cells to facilitate the MDSCs migration to thetumor site.

To address this hypothesis, we performed in vitro cellmigration assays. Adding different concentrations of IL-17Ain the culture media at the lower chamber did not facilitate themigration of MDSCs, suggesting that IL-17A may not have thedirect effect on MDSCmigrations (Fig. 5D). However, when wetreated the tumor cells with IL-17A and added the tumorsupernatants at the lower chamber, the migration of MDSCssignificantly increased in a dose-dependent manner. The

Figure 3. Vg4 gd T cells are the mainsource of IL-17A within tumorsduring hepatocellular carcinomadevelopment. A, representativeFACS profiles of IL-17Aexpressions by CD4þ and gd Tcells. B, the percentages of Th17and IL-17A–producing gd T cells ofall IL-17A–producing cells. C,absolute numbers of Th17 andIL-17A–producing gd T cells. D,RORgt, CD27, and CCR6expressions of IL-17A–producinggd T cells. E, the percentage of Vg4-positive cells of IL-17A–producinggd T cells. Data are representativeof at least three independentexperiments. ��, P < 0.01.

IL-17A Produced by gd T Cells in Hepatocellular Carcinoma

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increase of MDSC migration was completely blocked whenneutralizing anti-IL-17 antibody was added during the IL-17Atreatment, suggesting that the chemokine secreted by tumorcells induced by IL-17A treatment promoted the migration ofMDSCs. To determine whether the interaction between CXCL5and CXCR2was involved, the antagonist of CXCR2 (SB-265610)was used to treat MDSCs to block its interaction. The antag-onist of CXCR2 completely blocked the increased migration of

MDSCs. These data demonstrated in vitro that IL-17A couldinduce CXCL5 production by the tumor cells, therefore facil-itate the infiltration of the MDSCs to the tumor sites throughCXCL5–CXCR2 interactions.

To further demonstrate the role of CXCL5–CXCR2 inter-action in MDSCs migration and tumor growth promoted byIL-17A, we treated the mice with CXCR2 antagonist togetherwith rmIL-17 and observed a significant reduction in tumor

Figure4. Vg4 gd T-cell depletion reduces tumor growth,whereas adoptive transfer of Vg4 gd T cells promotes tumor growth.WTand IL-17�/�micewere treatedwith intravenous injection of anti-Vg4–specific mAb on days �5 and �1 before tumor implantation. Fourteen days after the implantation, mice weresacrificed and tumor volumewas calculated. A, tumor volumes and the grossmorphology of intrahepatic tumors. B, the percentage of IL-17A–producing gd Tcells. C, the percentage of Th17 cells. D, serum levels of IL-17A. E, the percentage of effector and memory CD4þ or CD8þ T cells. F, the percentage of theIFN-g–producing lymphocytes, CD4þ T cells, and CD8þ T cells. G, TCRd�/� mice were reconstituted with cultured Vg4 gd T cells either from WT or IL-17�/�

mice followed by tumor implantation. Tumor volume and the gross morphology of tumors were shown. H, the percentage of effector and memory CD4þ

or CD8þ T cells. I, the percentage of the IFN-g–producing lymphocytes, CD4þ T cells, andCD8þ T cells (ND, not detected). The data shown are representativeof three independent experiments, each using 4 to 5 mice per group. Data are shown as mean � SEM (ns, P > 0.05; �, P < 0.05; ��, P < 0.01; ���, P < 0.001).Ab, antibody.

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growth compared with rmIL-17 treatment alone (Fig. 5E).Accordingly, CXCR2 antagonist treatment also decreasedthe MDSCs at the tumor sites (Fig. 5F) and increased the

percentages of memory and Tc1 cells (Fig. 5G and H). Takentogether, the results demonstrated that IL-17A promotedMDSCs infiltration to the tumor sites in a CXCR2-dependent

Figure 5. IL-17A promotes themigration of MDSCs to the tumorsites. A, the expression of CXCR4and CXCR2 on tumor-infiltratingMDSCs. B, the chemokineexpression levels of Hepa1–6 cellstreated with indicated dose of rmIL-17 for 48 hours. C, the chemokineexpression levels from tumor tissuesof WT or IL-17�/� mice. D, in vitromigration of MDSCs in response toIL-17 and IL-17–treated tumorsupernatants. E, mice werepretreated with or without rmIL-17and were then treated with a CXCR2-specific antagonist, SB-265610. Thecalculated tumor volumes wereshown. F, the percentage of myeloidpopulations with or without SB-265610 treatment. G, the percentageof effector and memory CD4þ orCD8þ T cells with or without SB-265610 treatment. H, the percentageof IFN-g–producing lymphocytes,CD4þ T cells, and CD8þ T cells withor without SB-265610 treatment. Thedata are representative of threeindependent experiments, eachusing 4 to 5 mice per group. Data areshown as mean �SEM (�, P < 0.05;��, P < 0.01; ���, P < 0.001). ns, notsignificant.

IL-17A Produced by gd T Cells in Hepatocellular Carcinoma

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manner through the upregulation of CXCL5 production bythe tumor cells.

IL-17A promotes the suppressive functions of MDSCsTo examine the direct effect of IL-17A on the suppressive

functions of MDSCs, we sorted Gr-1þCD11bþ MDSCs from thespleens of the tumor-bearing mice, and cocultured with CFSE-labeled OT-I CD8þ T cells stimulated with SIINFEKL with orwithout rmIL-17 (Fig. 6A). [3H]-thymidine incorporation assaywas also performed to measure the proliferation of the OT-ICD8þ T cells (Fig. 6B). We found that IL-17A significantlyenhanced the suppressive function of MDSCs in terms ofinhibiting T-cell proliferation. Furthermore, neutralizing anti-IL-17 antibody partially blocked this increase in MDSCs' sup-pressive activity. IL-17A also promoted the suppressive effectsof MDSCs in terms of inhibiting IFN-g and TNF-a productionsby the CD8þ T cells in a dose-dependent manner (Fig. 6C).

Furthermore, the suppressive function wasmainly mediated bythe monocytic MDSCs and IL-17 treatment could furtherenhance it (Fig. 6D).

To determine whether MDSCs were essential for IL-17A–mediated immune suppression in vivo, we treated mice withrmIL-17 followed by anti-Gr-1 antibody to depleteMDSCs (25).Depletion of MDSCs in the rmIL-17A–treated mice led to thereduced tumor growth in comparison with rmIL-17 and con-trol mAb–treated mice (Fig. 6E). The infiltration of MDSCs atthe tumor sites was significantly reduced (Fig. 6F). Memoryand Tc1 cells were also increased after MDSCs depletion (Fig.6G and H). Selectively depleting MDSCs by gemcitabine treat-ment (26) showed the similar results (Supplementary Fig. S7B–S7E). Taken together, the results demonstrated that IL-17Aimpaired the antitumor immune responses through promot-ing the migrations and suppressive activities of the MDSCs atthe tumor sites.

Figure 6. IL-17A promotes the immunosuppressive functions of MDSCs. A–C,MDSCs from the spleens of B16-ova tumor-bearing mice were cocultured withCFSE-labeled OT-I CD8þ T cells (1:3 ratio) loaded with SIINFEKL (10 mg/mL) with or without rmIL-17(100 ng/mL) or anti-IL-17 antibody (10 mg/mL). Theproliferation of CD8þ T cells was evaluated 3 days later with CFSE dilution by flow cytometry (A). [3H]-thymidine (1 mCi/well) was added to triplicate wellsfor the final 16 to 18 hours before harvest, and the proliferations of the CD8þ T cells were determined using a liquid scintillation counter (PerkinElmerInstruments; B). The expressions of IFN-g and TNF-a in CD8þ T cells were analyzed by flow cytometry (C). D, sorted granulocytic MDSCs andmonocytic MDSCs from the spleens of B16-ova tumor-bearing mice were separately cocultured with OT-I CD8þ T cells (1:3 ratio) loaded with SIINFEKL(10mg/mL)with orwithout rmIL-17 (100ng/mL) for 3days. The levels of TNF-a and IFN-g in supernatantsweremeasuredbyELSIA. E,WTmicewerepretreatedwith or without rmIL-17 (1 mg/mouse) and IL-17–treated mice were then treated with 0.25 mg Gr-1 mAb (RB6–8C5) on days 0, 4, 8, and 12 after tumorimplantation. At day 14 after the tumor implantation, mice were sacrificed and tumor volume was calculated. F, the percentage of MDSCs in TILs wasexamined by flow cytometry. G, the percentage of effector and memory CD4þ or CD8þ T cells. H, the percentage of IFN-g–producing lymphocytes,CD4þ T cells, andCD8þ T cells from TILs. The data are representative of three independent experiments, each using 4 to 5mice per group. Data are shown asmean �SEM (�, P < 0.05; ��, P < 0.01).

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MDSCs induce IL-17A–producing gd T cellsMDSCs have been shown to promote Th17 cell differenti-

ation (26).We have observed a reduction of both Th17 cells andIL-17A–producing gd T cells after MDSC depletion (Fig. 7Aand Supplementary Fig. S7F). Moreover, CXCR2 antagonist,SB-265610, treatment to block MDSCs migration to the tumorsites also reduced the Th17 as well as IL-17A–producing gdT cells (Fig. 7B).To examine the role ofMDSCs in inducing IL-17A–producing

gd T cells, we cultured the sorted gd T cells in the presence orabsence of MDSCs (Fig. 7C). MDSCs significantly promoted theIL-17A production by the gd T cells, although the percentage ofIL-17A–producing gd T cells was slightly lower than that fromthe IL-1b and IL-23 induction. This induction of IL-17A–pro-ducing gd Tcells byMDSCs couldbepartiallyblockedbyanti-IL-1b or anti-IL-23 antibodies or both. The absence of IL-1R on thegd T cells also decreased the percentage of IL-17A–producingcells induced by MDSCs to about 50%. These results demon-strated that MDSCs could promote IL-17A production by gd Tcells through IL-1b and IL-23. The amount of IL-17A detected in

the culture supernatants also demonstrated the same results(Fig. 7D). Further analysis showed that both monocytic andgranulocytic MDSCs could induce IL-17A production by gd Tcells (Fig. 7E and F). However, granulocyticMDSCs could induceslightly higher percentage of IL-17A–producing gd T cells andhigher level of IL-17A production. When we examined the Vg4usage of these in vitro–induced gd T cells by FACS analysis, onlyabout half of the IL-17A–producing gd T cells wereVg4-positive,no matter how they were induced (Supplementary Fig. S8). Thediscrepancy of IL-17A–producing gd T-cell subsets in vivo and invitro still needs further investigation.

Interestingly, combining MDSCs, IL-1b, and IL-23 synergis-tically promoted the IL-17A production by gd T cells (Fig. 7C),suggesting that IL-17A produced by gd T cells may furtherpromote the induction by MDSCs. We then added rmIL-17 inthe coculture of sorted gd T cells and MDSCs (Fig. 7G). Theresults showed that IL-17A could significantly increase thepercentage of IL-17A–producing gd T cells in a dose-depen-dent manner. Adding anti-IL-17 antibody could block thisincrease of induction. Furthermore, the real-time PCR results

Figure 7. MDSCs induce IL-17A–producing gd T cells. A, thepercentage of IL-17A–producingcells after MDSC depletion. B, thepercentage of IL-17A–producingcells after CXCR2 antagonisttreatment. C–F, purified gd T cellsfrom TILs of tumor-bearing micewere stimulated with IL-1b andIL-23. MDSCs (C and D) or sortedmonocytic and granulocyticMDSCs (E and F) were added to theculture on day 0 at a ratio of 1:1.Three days after coculture,percentages of IL-17A–producinggd T cells weremeasured (C and E).In some experiments, anti-IL-1bmAbs, anti-IL-23 p19 mAbs, or thecombination were added. TheIL-17A in the culture supernatantswas measured by ELISA (D and F).G, purified gd T cells from TILs oftumor-bearing mice werecocultured with MDSCs in thepresence of indicated dose of IL-17, anti-IL-17 mAb, or isotype IgG.Cells were harvested andexamined for IL-17A expression.H, purified MDSCs from TILs oftumor-bearing mice were treatedwith indicated dose of rmIL-17 for48 hours; the mRNA expression ofIL-1b and IL-23 was examined byquantitative RT-PCR. The dataare representative of threeindependent experiments. Data areshown as mean�SEM (�, P < 0.05;��, P < 0.01; ���, P < 0.001).

IL-17A Produced by gd T Cells in Hepatocellular Carcinoma

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showed that IL-1b and IL-23 expressions of MDSC cells weresignificantly elevated during rmIL-17 treatment (Fig. 7H), alsoin a dose-dependent manner, suggesting that IL-17A couldincrease IL-1b and IL-23 productions by the MDSCs to furtherpromote the IL-17A production by gd T cells.

DiscussionIn the current study, usingmurine hepatocellular carcinoma

models, we provided evidence for a promoting role of IL-17A intumor development. We also demonstrated that the interac-tions among gd T cells, MDSCs, and tumor cells form a positiveloop through IL-17, IL-1b, and IL-23 as well as CXCL5 in thetumor microenvironment to suppress the antitumor immuneresponses and promote tumor growth (Supplementary Fig. S9).

In support of our findings, one clinical study performed byZhang and colleagues reported that increased intratumoral IL-17A–producing cell density was associated with highmortalityand reduced survival in patients with hepatocellular carcino-ma (27), implying a promoting role of IL-17A in tumor pro-gression. A recent study also found that high expression of IL-17 and IL-17RE associated with poor prognosis of hepatocel-lular carcinoma (28). IL-17A has also been found to promotehepatocellular carcinoma metastasis via MMP2 and 9 expres-sion (29).

Th17 cells have been shown to promote antitumor CTLresponses (7). A recent clinical study also found that increasedcirculating Th17 cells after transarterial chemoembolizationcorrelated with improved survival in stage III hepatocellularcarcinoma (30). Although IL-17 is one of the major cytokinesproduced by Th17 cells, the function of IL-17 and Th17 maydiffer during tumor development. First, Th17 cells producemany proinflammatory cytokines other than IL-17A, includingIL-17F, IL-21, and IL-22. Moreover, in the tumor microenvi-ronment, Th17 cells can also express IL-2, IL-9, GM-CSF(granulocyte macrophage colony—stimulating factor), IFN-g ,and TNF-a. These cytokines may have distinct functions fromIL-17A to mediate biologic activities of Th17 cells. Further-more, it has been shown that Th17 cells are long-lived andretain a stem cell–like molecular signature, and can give rise toTh1-like effector cell progeny in vivo (31). Second, IL-17A can beproduced by different types of cells other than Th17 cells. Untilnow, it has been shown that IL-17A is produced by a variety ofinnate and adaptive immune cell types. Thus, the functions ofIL-17A and Th17 cells may differ under different pathologicconditions.

One of the key findings in this study is that in the tumormicroenvironment, IL-17A was predominantly produced by gdT cells. gd T cells represent a major source of IL-17A duringlung infection by Mycobacterium tuberculosis (32) and liverinfection by Listeria monocytogenes (16). It has been recentlyreported that in both transplantable sarcoma model andmethylcholanthrene-induced primary tumor model, the majorcellular source of IL-17A was also gd T cells (33). These reportsand our findings together suggest that gd T cells could be animportant source of IL-17A during infection and tumor devel-opment. In consistent with our findings, one study reportedthat tumor formation was suppressed in TCRd�/� mice in

subcutaneous hepatocellular carcinoma model (34). We fur-ther identified Vg4 gd T cells as the main subset of gd T cellsthat produced IL-17A within tumors. Vg4 gd T cell–derivedIL-17Ahas been shown to negatively regulateNKTcell functionin Con A–induced fulminant hepatitis (35). To our knowledge,this was the first report demonstrating that Vg4 gd T cellsplayed promoting role in hepatocellular carcinoma throughIL-17A production. Recent studies also demonstrated thatIL-17A–producing gd T cells were required for optimal anti-tumor responses during immunogenic chemotherapy andradiotherapy (36, 37). Cell death triggered by chemotherapyor radiotherapy may elicit a pattern of "danger signals" thatdramatically change the tumor microenvironment. IL-17A–producing gd T cells could be one of the first responders tothese signals to contribute to the antitumor immune response.Therefore, the cellular components and the cytokine milieu inthe tumor microenvironment could be critical for the functionof IL-17A–producing gd T cells.

In the present study, we observed reduced tumor-infiltratingMDSCs in IL-17A�/� mice compared with WT mice. A recentstudy by He and colleagues found that the development oftumors was inhibited in IL-17R�/� mice, and IL-17 increasedthe number of MDSCs in tumors (14). We also demonstratedthat MDSCs were critical for IL-17A–induced immune sup-pression and IL-17A could enhance the immunosuppressiveactivity of MDSCs. We also demonstrated, for the first time,thatMDSCs could induce IL-17A–producing gd T cells throughIL-1b and IL-23 productions. We have also noticed that bothmonocytic and granulocytic MDSCs were recruited to thetumor site by IL-17A, while monocytic MDSCs were moresuppressive of the CTL functions and granulocytic MDSCsseemed to induce more IL-17–producing gd T cells. Thesefindings provided a novel mechanism for IL-17A–regulatingimmune responses through MDSCs and tumor cells.

In summary, the present study demonstrated a novel mech-anism of IL-17A–regulating immune responses in hepatocel-lular carcinoma. Our data suggest that IL-17A may be thecritical cytokine in the tumor microenvironment that could betargeted for tumor therapy. Three agents neutralizing IL-17Aor antagonizing its receptor are in development for autoim-mune diseases (38). Our data demonstrating the role of IL-17Ain tumor development warrant future investigations of IL-17–targeted therapies in tumor treatment.

Disclosure of Potential Conflicts of InterestC. Dong has honoraria from Speakers Bureau of Bristol-Myers Squibb and is a

consultant/advisory boardmember ofGlaxoSmithKline. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Ma, D. Wu, H. LiuDevelopment of methodology: S. Ma, Y. Cai, X. Yu, C. DongAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Ma, Y. Wu, L. ShiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ma, Q. Cheng, H. LiuWriting, review, and/or revision of the manuscript: S. Ma, D. Wu, C. Dong,H. LiuAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Cai, H. GongStudy supervision: H. Liu

Ma et al.

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AcknowledgmentsThe authors thank Dr. Zhinan Yin from Nankai University for providing

TCRd�/�and OT-I mice and for helpful discussions.

Grant SupportThis work has been supported by the grants from National Natural Science

Foundation of China (91029703, 81072436, and 81273268), the project fundingfrom Suzhou city (SWG0904 and SZS201109), Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions, Qing Lan project of

Jiangsu Province, Jiangsu Provincial Innovative Research Team, and Program forChangjiang Scholars and Innovative Research Team in University (IRT1075).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received September 5, 2013; revised January 7, 2014; accepted January 14, 2014;published OnlineFirst February 13, 2014.

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2014;74:1969-1982. Published OnlineFirst February 13, 2014.Cancer Res   Shoubao Ma, Qiao Cheng, Yifeng Cai, et al.   Hepatocellular Carcinoma

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