gm-csf promotes antitumor immunity by inducing …...research article gm-csf promotes antitumor...

Research Article

GM-CSF Promotes Antitumor Immunity byInducing Th9 Cell ResponsesIl-Kyu Kim1,2, Choong-Hyun Koh1, Insu Jeon2, Kwang-Soo Shin1, Tae-Seung Kang2,Eun-Ah Bae2, Hyungseok Seo2, Hyun-Ja Ko3,4, Byung-Seok Kim5,Yeonseok Chung5, and Chang-Yuil Kang1,2

Abstract

GM-CSF as an adjuvant has been shown to promote anti-tumor immunity inmice andhumans; however, theunderlyingmechanismofGM-CSF–induced antitumor immunity remainsincompletely understood. In this study, we demonstrate thatGM-CSF potentiates the efficacy of cancer vaccines throughIL9-producing Th (Th9) cells. GM-CSF selectively enhancedTh9 cell differentiation by regulating the COX2–PGE2 pathway

while inhibiting the differentiation of induced regulatory T(iTreg) cells in vitro and in vivo. GM-CSF–activated monocyte-derived dendritic cells converted tumor-specific na€�ve Th cellsinto Th9 cells, and delayed tumor growth by inducing antitu-mor CTLs in an IL9-dependent manner. Our findings reveal amechanism for the adjuvanticity of GM-CSF and provide arationale for the use of GM-CSF in cancer vaccines.

IntroductionTumor-specificCD4þT cells trigger tumor immune surveillance

by providing costimulation and cytokines required for breakingCD8þ T-cell tolerance (1, 2). Various studies have attempted tofind MHC II–restricted tumor epitopes (3) and have adoptedtumor-specific CD4þ T cells for the treatment of cancer (4, 5).Among Th cell subsets, Th1 cells have been considered the mostefficacious in rejecting tumors directly or indirectly through themechanismof activatingmacrophages andCTLs (3, 6). The role ofTh2 cells in tumor immunity is controversial, although Th2 cellsare known to exert antitumor effects by activating eosinophils, Bcells, andnatural killer cells in severalmodels (3, 7, 8). In contrast,regulatory T (Treg) cells create an immunosuppressive microen-vironment promoting cancer development (9). Th17 cells inhibittumors by acquiring a Th1-like phenotype in vivo (10). A distinctTh subset producing IL9 (Th9 cells) has been found to play a rolein autoimmunity in the lung, central nervous system, andgut (11, 12). Several cytokines and transcription factors areassociated with the development of Th9 cells (11, 12), but a

master transcription factor for Th9 cells has yet to be identified.Th9 cells are more potent than other Th subsets in melanomarejection via mast cells (13) or via activation of dendritic cells(DC) to cross-present tumor antigens (14, 15). A hyperprolifera-tive featuremediated by the PU.1–TRAF6–NF-kBpathway conferspersistence on Th9 cells with cytotoxicity directed by Eomes andgranzymes in the tumor microenvironment (16). Thus, boostingTh9 responsesmay be a useful strategy for the treatment of cancer;this strategy is under investigation (11, 15, 17, 18).

GM-CSF was identified as a hematopoietic growth factor caus-ing granulocyte and macrophage colony formation. It is used forDC generation from bone marrow cells or monocytes in vitro. Atsteady state, however, GM-CSF- or GM-CSFR–deficient miceexhibit no defects in the development of myeloid cells, with theexception of alveolar macrophages and specific DC subsets innonlymphoid tissues (19–21). Instead, GM-CSF plays a role intissue inflammation and autoimmune diseases, including rheu-matoid arthritis, multiple sclerosis, asthma, psoriasis, and type Idiabetes. Blockade of GM-CSF is in clinical trials for the treatmentof rheumatoid arthritis andmultiple sclerosis (21–23).GM-CSF ismainly produced by T cells, B cells, epithelial cells, and fibroblastsupon activation (22, 23). During inflammation, upregulatedGM-CSF acts on macrophages and DCs to generate additionalproinflammatory cytokines, which, in turn, affect Th responses(21, 24). Indeed, GM-CSF controls the development and/orfunction of Th1 and Th17 cells in a mouse model of multiplesclerosis (25–27) and also controls Th2 cell function in mouseasthma models (28, 29) in a context-dependent manner.

In tumor immunity, this immune-stimulating activity ofGM-CSF to self in autoimmune diseases has been exploited byusing GM-CSF as an adjuvant to inhibit tumors. Irradiated B16melanoma cells transduced with the GM-CSF gene were used as acancer vaccine and elicited potent antitumor immune responsesin mice (30). On the basis of this finding, autologous cancervaccines engineered to secrete GM-CSF (GVAX) and tumor pep-tide and DC vaccines with recombinant GM-CSF have beenclinically tested (23, 31). Some trials demonstrated increasedimmunogenicity and phagocyte and lymphocyte infiltration into

1Laboratory of Immunology, Research Institute of Pharmaceutical Sciences,College of Pharmacy, Seoul National University, Seoul, Republic of Korea.2Department of Molecular Medicine and Biopharmaceutical Science, GraduateSchool of Convergence Science and Technology, Seoul National University,Seoul, Republic of Korea. 3Academy of Immunology and Microbiology, Institutefor Basic Science, Pohang, Republic of Korea. 4Department of IntegrativeBiosciences and Biotechnology, Pohang University of Science and Technology,Pohang, Republic of Korea. 5Laboratory of Immune Regulation, ResearchInstitute of Pharmaceutical Sciences, College of Pharmacy, Seoul NationalUniversity, Seoul, Republic of Korea.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Chang-Yuil Kang, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul 08826, Republic of Korea (South). Phone: 822-880-7860;Fax: 822-885-1373; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-18-0518

�2019 American Association for Cancer Research.

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the vaccination sites, whereas other studies showedminimal or noeffects. One potential explanation for the poor outcomes is thatGM-CSF induces the recruitment and expansion of myeloid-derived suppressor cells (MDSC; ref. 32). Alternatively, oncolyticGM-CSF–encoding modified herpes simplex virus (T-VEC) andcombination with checkpoint blockades have shown improvedoutcomes and are under clinical investigation (33–35).

GM-CSF adjuvant augments the recruitment and activation ofDCs, which likely help eradicate cancer through the presentationof tumor antigens to T cells. The function of GM-CSF as anadjuvant was supported by the fact that the enhanced antitumoractivity byGM-CSF disappeared whenCD4þ or CD8þ T cells weredepleted (30). However, the types of Th responses that contributeto this effect remain unclear. In addition, because GM-CSFR isbroadly expressed by myeloid cells (22, 23), different myeloidcell types could be involved in this process. Ly6ChiCCR2þ mono-cytes and monocyte-derived dendritic cells (moDCs) mediateGM-CSF–induced pathogenesis under tissue inflammation(27, 36). Thus, the role of this population in tumor immunityinduced by GM-CSF needs to be defined.

In this study, we dissected Th responses in tumor immunitywhen GM-CSF was administered as an adjuvant and found thatGM-CSF selectively induced Th9development, particularly by actingon moDCs, leading to the promotion of tumor-specific CTL re-sponses that inhibited tumor growth in an IL9-dependent manner.

Materials and MethodsMice

Male and female C57BL/6n, BALB/c mice were purchasedfrom Charles River Laboratories. C57BL/6j, OT-II, DO11.10, andCcr2–/–were from The Jackson Laboratory. CD45.1 congenicmicewere kindly provided by Jae-Ouk Kim (International VaccineInstitute, Seoul, Republic of Korea).Micewere used at 6–10weeksof age and gender-matched in all experiments; mice were bredat Seoul National University (Seoul, Republic of Korea) underspecific pathogen-free conditions. All animal experiments wereapproved by the Institutional Animal Care and Use Committeeat Seoul National University (Seoul, Republic of Korea).

Tumor models and cellular analysesA total of 106 B16F10-OVA cells (kindly provided in 2006

by Kenneth Rock, University of Massachusetts Medical School,Boston,MA), 2�105TC-1 cells (express E6/E7oncoproteins fromHPV16; ATCC in 2006), or 3� 105 CT26-hHER2/neu cells (ATCCin 2002 and transduced hHER2/neu in 2004) negative forMycoplasma contamination (e-Myco Mycoplasma PCR DetectionKit; iNtRON in 2016) were subcutaneously injected into the flankof mice and tumor-bearing mice were randomly allocated tocontrol or treatment groups in all experiments. Tumor cell lineswere validated by their morphology, growth kinetics, and antigenexpression before injection and tumor cells with three to fourpassageswere used for inoculation. Sample sizeswere determinedby Resource Equationmethod. On day 4 after tumor inoculation,some mice were intraperitoneally treated with cognate 50 mg ofOVA (Sigma-Aldrich), total 100 mg of E6/E7 peptide library, orhHER2/neu peptide library with or without 500 ng of carrier-freerecombinant GM-CSF (R&D Systems) incubated for up to 3 hoursin complete Freund's adjuvant (CFA; Sigma-Aldrich) at a 1:1volume ratio. Carrier-free recombinant IL9 was from R&D Sys-tems, and 100 ng of IL9was applied on day 4 and 50 ng of IL9was

applied every other day from day 6 to 16. The sequences of thepeptide library, excluding the kinase domain of HER2/neu andconsisting of 7 or 8 overlapping peptides, were designed using thepeptide library design and calculator tool fromSigma-Aldrich andordered from GenScript. Two injections of 100 ng recombinantGM-CSF were additionally given on days 3 and 5 after tumorimplantation. Tumor volumewas calculated as 0.5236� length�width � height by digital caliper. CD4þ T cells were sortedfrom tumor-draining lymph nodes (TdLN) of B16F10-OVAtumor-bearing mice and stimulated with mitomycin-treatedT-cell–depleted total splenocytes (TdS) plus OVA323-339 peptide(ISQAVHAAHAEINEAGR, 10 mg/mL; Anygen) overnight or for 3days for mRNA or protein expression analysis, respectively. Iso-lation of tumor-infiltrating lymphocytes (TIL) was describedpreviously (15).

For analyzing the cytokine-inducing capacity of moDCs andconventional DCs (cDCs), 100 mg of OVA plus CFA was intra-peritoneally injected into na€�ve or 4-day established B16F10-OVA–bearing mice, and splenic moDCs and cDCs were isolated8 days later and cocultured with na€�ve or total OVA-specific CD4þ

T cells plus OVA323-339 peptide (10 mg/ml) for 48 hours.For DC vaccine experiments, 1.5 � 105 moDCs or cDCs were

isolated from spleens as described above, loaded overnight with10 mg/ml OVA323-339 peptide plus or minus recombinantGM-CSF, and intravenously injected into 8-day establishedB16F10-OVA–bearingmice that had received2�106OVA-specificna€�ve CD4þ T cells one day prior. The equivalent number ofmoDCs loaded with the E6/E7 peptide library (10 mg/ml) wastransferred into 5-day established TC-1–bearing mice. Tumorvolumewasmeasured three times perweek.DonorCD45.2þVa2þ

Th cells were recovered 4 days after DC transfer to analyze Th celldifferentiation in CD45.1þ tumor–bearing hosts.

Reagents and cytokine neutralizationFor analyzing cell proliferation, 5,6-carboxyfluorescein diace-

tate succinimidyl ester (CFSE) or CellTrace Violet (CTV, 5 mmol/Leach; Invitrogen) was used to label cells according to the man-ufacturer's instructions. All microbeads (anti-mCD4, mCD11b,mCD11c, hCD3, and biotin) for positive or negativeselection were purchased from Miltenyi Biotec. Lipopolysaccha-ride (0.5 mg/ml) was from Sigma-Aldrich andG-CSF,macrophagecolony-stimulating factor (M-CSF; 20 ng/mL each) were fromPeproTech. Anti-OX40L (RM134L; BioLegend), anti-TL1A(293327; R&D Systems), and GITR-Fc (Alexis Biochemicals)were used at 10 mg/ml. Synthetic PGD2 (200 nmol/L) and PGE2(50 nmol/L) were from Cayman Chemical.

For neutralization of GM-CSF, mice were intraperitoneallytreated with 200 mg of anti-GM-CSF (MP1-22E9) every 3 daysstarting from day 0. For neutralization of IL9, 200 mg of anti-IL9(MM9C1) was injected intraperitoneally every other day startingfrom a day before treatment with DC or peptide vaccines. Forneutralization of IFNg , 200 mg of anti-IFNg (R4-6A2) was intra-peritoneally administered every two days from day 4 to 16 aftertumor inoculation. Rat IgG for GM-CSF and IFNg blockades ormouse IgG (Sigma-Aldrich) for anti-IL9 was used as a control.

In vitro Th cell differentiationOVA-specific na€�ve CD4þCD44loCD62LhiCD25– T cells were

sorted from enriched CD4þ cells by FACSAria III (BD Biosciences).Purified 5� 104 na€�ve CD4þ T cells were cocultured with 2� 105

TdS or 1.25 � 104 moDCs or cDCs plus OVA323-339 peptide

GM-CSF Drives Th9 and Antitumor Immunity

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(1 mg/ml) for 3 days under specific Th-polarizing conditions.Cells were grown in RPMI1640 culture medium supplementedwith 10% FBS, 2.5% HEPES, 1% sodium pyruvate, 1% nones-sential amino acids, 1% penicillin/streptomycin, and 0.1%2-mercaptoenthanol (all from Gibco by Life Technologies) inthe 96-well round bottom plate. moDCs and cDCs were isolatedfrom CD11b- and CD11c-enriched TdS. The concentration ofGM-CSF was titrated and used at 20 ng/mL. Polarizing condi-tions for each Th subset were as follows: Th1 (4 ng/mL IL12),Th2 (10 ng/mL IL4), Th9 (5 ng/mL TGFb plus 10 ng/mL IL4),Th17 (5 ng/mL TGFb plus 20 ng/mL IL6), and iTreg (5 ng/mLTGFb). All cytokines were purchased from eBioscience exceptTGFb, which was from PeproTech.

Flow cytometryFITC-conjugated antibodies tomouse CD3e (145-2C11), Ly6C

(HK1.4), B220 (RA3-6B2), Foxp3 (FJK-16s; Invitrogen) andhuman CD45RA (HI100), Foxp3 (236A/E7; Invitrogen); PE-con-jugated antibodies tomouse CCR2 (475301; R&D Systems), IL13(eBio13A; Invitrogen), IL17 (TC11-18H10.1), Va2 (B20.1),DO11.10 TCR (KJ1-26; Invitrogen) and human IL9 (MH9A4);PerCP-Cy5.5–conjugated antibodies to mouse CD3e (145-2C11), CD62L (MEL-14), and I-A/I-E (M5/114.15.2); PE/Cy7-conjugated antibodies tomouse CD44 (IM7), Ly6G (1A8), CD19(6D5), CD4 (RM4-5) and human CD4 (RPA-T4), CD45RO(UCHL1); APC-conjugated antibodies to mouse CD4 (RM4-5),CD11c (N418), CD25 (PC61), CD45.2 (104), IFNg (XMG1.2),IL9 (RM9A4) and human CD3 (OKT3), CD25 (BC96); andAPC/Cy7-conjugated antibody to mouse CD8a (53-6.7), F4/80(BM8) and human CD3 (OKT3), Pacific blue–conjugated anti-body to mouse CD4 (RM4-5), CD11b (M1/70), Granzyme B(GB11) and human CD4 (RPA-T4) were used. All the antibodieswere from BioLegend unless otherwise indicated. Cells werewashed and surface molecules stained for 15 minutes at 4�C,followed by intracellular staining. For cytokine staining, cells wererestimulated with phorbol 12-myristate 13-acetate (50 ng/mL;Sigma-Aldrich) and ionomycin (500 ng/mL; Sigma-Aldrich) plusGolgiPlug (BD Biosciences) for 4 hours, followed by fixation andpermeabilization by Cytofix/Cytoperm Kit (BD Biosciences). ForFoxp3 staining, cells were permeabilized with Foxp3 Staining Kit(eBioscience) according to the manufacturer's instructions. Sam-ples were acquired with FACSCalibur or FACSAria III (BD Bios-ciences) and data were analyzed with FlowJo software (Tree Star).

ELISAThe following cytokines in culture supernatants weremeasured

by ELISA kits according to the manufacturer's instructions: IL9(Invitrogen) and IFNg (BD Biosciences).

Quantitative real-time PCR assayTotal RNA was isolated using TRIzol reagent (Invitrogen) and

reverse-transcribed by SuperScript Reverse Transcriptase andoligo(dT) nucleotides (Invitrogen). Resultant cDNA was further quan-tified with a SYBR Green Real-Time PCR Kit (Takara) and Light-Cycler Optical System (Roche). The values of gene expressionwere normalized to the amount ofHprt1 expression. Primers usedin analyses were as follows: mouse Il9 forward, 50-AAC GTG ACCAGC TGC TTG TGT-30; mouse Il9 reverse, 50-CTT GAT TTC TGTGTG GCA TTG G-30; mouse Ifng forward, 50-ACA GCA CTC GAATGT GTC AGG TA-30; mouse Ifng reverse, 50-ATT CGG GTG TAGTCA CAG TTT TCA-30; mouse Il17a forward, 50-CCG CAA TGA

AGA CCC TGA TAG-30; mouse Il17a reverse, 50- TCA TGT GGTGGT CCA GCT TTC-30; mouse Foxp3 forward, 50-GGA TGA GCTGAC TGC AAT TCT G-30; mouse Foxp3 reverse, 50-GTA CCT AGCTGCCCTGCATGAG-30; mouseCox-2 forward, 50-CCCACAGTCAAA GAC ACT CAG GTA-30; mouse Cox-2 reverse, 50-CCA GGCACC AGA CCA AAG AC-30; mouse Ptgds forward, 50-GTT CCGGGA GAA GAA AGC TGT A-30; mouse Ptgds reverse, 50-CTT GGTCTC ACA CTG GTT TTT CC-30; mouse Ptges forward, 50-AAG CCTTTT TTCCTGCGT TTT A-30; mouse Ptges reverse, 50-TCT AAC TCCAGC AAC TT-30; mouse Ptgis forward, 50-GGC TCC TTC TTT TCCTCC TCA A-30; mouse Ptgis reverse, 50-CTG TGG GAG TGT GGTCAT CTG T-30; mouseHprt1 forward, 50-AAG ACT TGC TCG AGATGT CAT GAA-30; mouse Hprt1 reverse, 50-ATC CAG CAG GTCAGC AAA GAA-30.

Human samplesHuman peripheral blood was obtained from healthy volun-

teers who were previously informed and provided consent toexperimental procedures using their samples in accordance withthe Declaration of Helsinki. Peripheral blood mononuclearcells (PBMC) were prepared by BD Vacutainer CPT tubes (BDBiosciences) according to the manufacturer's instructions. Aftercentrifugation, na€�ve CD3þCD4þCD45RAþCD45RO–CD25– Tcells were sorted from enriched CD3þ cells by flow cytometry.The mitomycin-treated allogeneic CD3-depleted cells were usedas the antigen-presenting cell (APC) counterparts. For human Th9cell differentiation, cells were cultured with soluble anti-CD3(5 mg/ml, OKT3; BioLegend) and soluble anti-CD28 (2 mg/ml,CD28.2; BioLegend) plus hTGFb (5 ng/mL; PeproTech), hIL4(20 ng/mL; eBioscience), and hIL2 (20 ng/mL; eBioscience).Sorted na€�veCD4þ T cells were cultured in the presence or absenceof hGM-CSF (40 ng/mL; PeproTech) and analyzed for IL9 expres-sion three days later. The collection of human samples and allhuman experiments were approved by the ethical committee ofSeoul National University (Seoul, Republic of Korea; institu-tional review board no. 1712/001-003).

CTL assayCytotoxicity was measured as described previously (15)

with some modifications. Splenocytes and TdLN cells fromB16F10-OVA tumor-bearing mice were prepared on day 13 andstimulated with an MHC-I–restricted tumor epitope mix ofOVA257-264 (SIINFEKL) and gp10025-33 (EGSRNQDWL, 1 mg/mleach; Anygen) peptides. After a 5-day stimulation, equivalentnumbers of live cells were cocultured for 4 hours with51Cr-labeled B16F10-OVA tumor cells that had been loaded for1 hour with peptide mix for the use as targets. Specific target celllysis was calculated as [(Sample lysis count per minute (cpm) –spontaneous lysis cpm) / (Triton X-100 lysis cpm – spontaneouslysis cpm)] � 100 (%) using Wallac 1470 Wizard Automaticg-Counter (PerkinElmer). For intracellular stainingofCD8þCTLs,effector cells were additionally stimulated with the peptide mixplus GolgiPlug for 4 hours and analyzed by flow cytometry.

Adenoviral construct productionCox-2 DNA was amplified using a house mouse Ptgs2 cDNA

ORF clone (oMu19313, GenScript) and the following primers:mouse Cox-2 forward (KpnI); 50- GAG CTC GGT ACC GCC ACCATG CTC TTC-30; mouse Cox-2 reverse (NotI), 50-GAG GCT GATGCG GCC GCT TAT CAC TTA TCG TC-30. Sequencing primerswere as follows:mouseCox-2first, 50-TGCTGTTCCAATCCATGT

Kim et al.

Cancer Immunol Res; 7(3) March 2019 Cancer Immunology Research500




CA-30; mouseCox-2 second, 50-GGTGAAACTCTGGACAGACA-30; mouse Cox-2 third, 50-GAG TAC CGC AAA CGC TTC TC-30;mouse Cox-2 fourth, 50-TGT CTG TCC AGA GTT TCA CC-30. ThePCR product was cloned into the pShuttle-CMV-EGFP-C vector(13887; Addgene) with restriction enzymes (KpnI, NotI; bothfrom Enzynomics) and T4 ligase (NEB). Then, PmeI (NEB)-digested plasmid DNA was subjected to homologous recombi-nation with the pAdeasy-1 adenovirus vector (Agilent Technolo-gies) by electroporation. Final plasmid products were transfectedinto human embryonic kidney (HEK) 293 cells to generateAdMock-GFP or AdCox-2-GFP. A total of 106 plaque-formingunits of adenoviruswere added to a 96-well round-bottom cultureplate.

Statistical analysisStatistical analyses were performed using Prism software

(GraphPad). Unpaired two-tailed Student t test was used andresults with a P value of <0.05 were considered statisticallysignificant. Data are represented as mean � SEM.

ResultsThe adjuvant effect of GM-CSF in cancer vaccines is IL9-dependent

To investigate the mechanism of GM-CSF adjuvanticityin vivo, we employed a therapeutic cancer vaccine model using

B16F10 melanoma expressing whole ovalbumin (OVA) as amodel tumor antigen. Groups of C57BL/6 mice were subcuta-neously injected with B16F10-OVA, and OVA emulsified inCFA was administered 4 days after tumor inoculation. Treat-ment with OVA plus CFA slightly inhibited tumor growth butfailed to control tumors. However, when GM-CSF was added tothe vaccine, its efficacy was increased and tumor growth wassignificantly delayed (Fig. 1A). We further analyzed tumor-specific CD4þ T cells in the TdLNs and found that IL9 expres-sion was enhanced by GM-CSF treatment (Fig. 1B; Supplemen-tary Fig. S1A). In contrast, addition of GM-CSF significantlyreduced IFNg production and showed little effects on IL17Aand Foxp3 expression by CD4þ T cells in this setting. Asreported previously (13), administration of IL9 also signifi-cantly inhibited tumor growth comparable with GM-CSF treat-ment in B16F10-OVA challenged mice (SupplementaryFig. S1B). This observation led us to hypothesize that IL9 mightbe involved in the enhanced antitumor immunity in mice givenGM-CSF. To test this hypothesis, we additionally treated themice with a neutralizing antibody to IL9 and found that anti-IL9 reversed the antitumor effect of GM-CSF (Fig. 1C). Fur-thermore, we verified IL9-mediated adjuvanticity of GM-CSF intherapeutic models of TC-1 cancer and CT26-HER2/neu carci-noma with a cognate peptide library vaccine (Fig. 1D and E).These results demonstrate a role of IL9 in mediating antitumorimmunity induced by GM-CSF in vivo.

Figure 1.

GM-CSF potentiates therapeutic vaccine efficacy in tumor-bearing mice via IL9. A, Tumor size in mice subcutaneously injected with B16F10-OVA tumors andintraperitoneally treated with or without 50 mg of OVA plus CFA and GM-CSF adjuvant (n¼ 5). B, IL9 and IFNg production by TdLN CD4þ T cells prepared10 days after tumor inoculation. Sorted cells were stimulated with mitomycin-treated TdS plus OVA323-339 peptide (10 mg/mL) for 3 days. C, Tumor size in micesubcutaneously inoculated with B16F10-OVA tumors and intraperitoneally treated with 50 mg OVA plus CFA and then given GM-CSF or anti-IL9 (n¼ 6). Tumorsize in mice subcutaneously injected with TC-1 tumors (D) or CT26-HER2/neu tumors (E) with or without intraperitoneal administration of 100 mg of cognatepeptide library plus CFA and GM-CSF adjuvant (n¼ 5). Data represent two independent experiments. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001 by Student t test.






GM-CSF promotes Th9 cell differentiation in vitroThe observed role of IL9 in GM-CSF–induced tumor inhibition

prompted us to investigate whether GM-CSF plays a role in thedifferentiation of Th9 cells. For this, we sorted na€�ve CD4þ T cellsfromOVA-specific TCR transgenicmice and stimulated themwithT-cell–depleted total splenocytes (TdS) and cognate peptideunder Th9 conditions. Addition of GM-CSF significantlyincreased the frequency of Th9 cells as early as day 3 (Fig. 2AandB). Accordingly, the amount of IL9 in the culture supernatantswas significantly increased by GM-CSF treatment (Fig. 2C). TheGM-CSF receptor b chain is not expressed on T cells, whereas theGM-CSF receptor a chain is known to be expressed on activated Tcells (24). However, addition of GM-CSF did not increase thefrequency of IL9-expressing CD4þ T cells stimulated by anti-CD3plus anti-CD28 in the absence of APCs (Fig. 2D), suggesting thatGM-CSF enhances Th9 cell differentiation by acting on APCs. Tofurther investigate which cell type is primarily responsible for theincrease inGM-CSF–induced Th9differentiation,we isolatedAPCpopulations and measured their ability to induce Th9 cells inresponse to GM-CSF (Supplementary Fig. S2). We found thatamong the APC subpopulations,MHC IIþmoDCs and cDCswereresponsive to GM-CSF in enhancing Th9 differentiation (Fig. 2E).Although moDCs induced substantial numbers of Foxp3þ Tregcells under Th9 conditions, GM-CSF suppressed Treg cell differ-entiation. The increase in Th9 differentiation was possibly due tothe enhanced maturation and T-cell–proliferating capacity ofAPCs caused by GM-CSF. However, T-cell proliferation was notaffected by GM-CSF treatment and the addition of LPS rather

inhibited Th9 cell development (Supplementary Fig. S3A andS3B). Unlike GM-CSF, G-CSF and M-CSF did not promote Th9differentiation, although they enhanced IL13 expression (Sup-plementary Fig. S3B and S3C). In addition, we observed thatGM-CSF potentiated the differentiation of human Th9 cells in thepresence of APCs (Fig. 2F and G).

Next, we determined the effect of GM-CSF on the differentia-tion of other Th subsets. In contrast to Th9, GM-CSF had little orno effect on Th1 and Th17 differentiation when T cells wereactivated by TdS and cognate peptide under Th1 or Th17 condi-tions (Fig. 3A and B). Moreover, GM-CSF did not induce IL9production from Th1 and Th17 cells (Supplementary Fig. S4).Under Th2 and Th9 conditions, the induction of IL9þ, IL13þ, andIL9þIL-13þ cells were all augmented by GM-CSF. We found thatGM-CSF inhibited iTreg cell generation and produced a small butevident population of IL9þFoxp3– cells (Fig. 3A and B). This effecton the differentiation of Th subsets was similar when moDCs orcDCs were used as APC counterparts except that moDCs slightlyincreased, whereas cDCs significantly decreased Th17 differenti-ation in response to GM-CSF (Supplementary Fig. S3D). Collec-tively, these results suggest that GM-CSF favors the differentiationof Th9 cells, but inhibits that of iTreg cells, presumably bymodulating the function of moDCs and cDCs.

GM-CSF triggers IL9-producing Th cells via activation of DCsin vivo

To determine whether GM-CSF affects moDCs and cDCs toinduce Th9 cells in vivo, we immunized C57BL/6 mice with OVA

Figure 2.

GM-CSF enhances mouse and human Th9 cell differentiation in the presence of APCs.A, Intracellular staining (ICS) of IL9 and Foxp3 in OVA-specific na€�ve CD4þ

T cells stimulated with TdS plus OVA323-339 peptide (1 mg/mL) with or without GM-CSF (20 ng/mL) under Th9 conditions. B, The percentages of IL9-producing Tcells gated on CD4þ cells during the culture. C, IL9 in accumulated day 3 supernatants was measured by ELISA. D, The percentages of IL9-producing T cells inna€�ve CD4þ T cells differentiated under anti-CD3, anti-CD28, and Th9 conditions. E, ICS of IL9 and Foxp3 in OVA-specific na€�ve CD4þ T cells stimulated with eachAPC subpopulation and OVA323-339 peptide for 3 days under Th9 conditions. F, The expression of IL9 and FOXP3 in human na€�ve CD4þ T cells stimulated withT-cell–depleted cells plus anti-CD3 (5 mg/mL) and anti-CD28 (2 mg/mL) with or without hGM-CSF (40 ng/mL) under Th9 conditions. Numbers in quadrantindicate each percent cell. G, The percentages of human IL9-producing T cells gated on CD4þ T cells. Each dot represents an individual donor. Data represent atleast two independent experiments (�� , P < 0.01; �� , P < 0.0001 by Student t test; N.S., nonsignificant).

Kim et al.





plus CFA and treated them with control IgG or neutralizing anti-GM-CSF.We isolatedmoDCsor cDCs from themice andanalyzedtheir IL9-inducing capacity.NeutralizationofGM-CSF significant-ly inhibited the capacity of moDCs to induce IL9 from na€�ve andtotal CD4þ T cells, whereas the IFNg-inducing capacity wasincreased or unaffected (Fig. 4A and B). For cDCs, IL9 inductionfrom total CD4þ T cells, but not from na€�ve CD4þ T cells, wasslightly inhibited by anti-GM-CSF.

To examinewhether GM-CSF directly acts onmoDCs and cDCsto promote IL9-inducing capacity, we isolated moDCs and cDCsfrom mice immunized with OVA plus CFA, loaded the cells withOVA323-339 peptide in the presence or absence of exogenousGM-CSF, and adoptively transferred them into recipients thathad received OVA-specific na€�ve CD4þ T cells one day before. Wefound that GM-CSF–activated moDCs and cDCs significantlyincreased IL9 expression in the donor Th cells compared withGM-CSF–untreated controls (Fig. 4C). Accordingly, IL9 proteinproduction by recovered Th cells was potentiated by GM-CSF–activated DCs, but IFNg production was not significantly affected(Fig. 4D). Taken together, these results demonstrate that GM-CSFstimulates moDCs and cDCs that are more potent for inducingTh9 in vivo.

GM-CSF–activatedmoDCs favor antitumor immunity in an IL9-dependent manner

Next, we determined whether GM-CSF also regulates the cyto-kine-inducing activity of moDCs and cDCs in the tumor micro-environment. Consistently, the IL9-inducing capacity of moDCsand cDCs from B16F10-OVA tumor-bearing mice was reduced byanti-GM-CSF, whereas IFNg induction was increased or unaffect-

ed from na€�ve or total CD4þ T cells (Fig. 5A and B). We furtherinvestigated the cellularity ofmyeloid populations includingDCsand macrophages in the setting of cancer vaccination.Ly6ChiCCR2þ monocytes and moDCs accumulated in the spleenin response to OVA plus CFA from around a week after tumorinoculation, whereas cDCs were not changed (Fig. 5C; Supple-mentary Fig. S5A and S5B). Vaccination with OVA plus CFA alsoled to the early accumulation of neutrophils and the late accu-mulation of macrophages during tumor progression, andGM-CSF treatment did not further accelerate accumulation ofthese cells (Supplementary Fig. S5A and S5B). Thus, we nextdetermined whether CCR2þ monocytes contribute to GM-CSF–induced antitumor immunity. We challenged WT and CCR2–deficient mice with B16F10-OVA melanoma and treated themwith OVA plus CFA with or without GM-CSF, and we found thatthe antitumor effect ofGM-CSFdisappeared in theCCR2-deficienthosts (Fig. 5D). In addition, IL9 production by tumor-specific Thcells was not augmented and IFNg production was also notaffected by GM-CSF in CCR2-deficient mice (Fig. 5E). Together,these results suggest that CCR2þ monocytes and moDCs play arole in GM-CSF–induced IL9 production and tumor inhibition.

To show thedirect actionofGM-CSFonDCs inpotentiating IL9production and antitumor activity, we adoptively transferredOVA-specific Th cells and OVA323-339 peptide–loaded moDCs orcDCs, with or without GM-CSF treatment, into B16F10-OVAtumor-bearing mice. GM-CSF–treated moDCs more efficientlyinduced IL9 expression in coinjected tumor-specific na€�ve Th cellsin the tumor microenvironment compared with GM-CSF–untreated moDCs (Fig. 5F). In contrast, GM-CSF–treated cDCsdid not augment IL9 expression; instead, these cells increasedIFNg expression in transferred tumor-specific Th cells comparedwith GM-CSF–untreated cDCs. GM-CSF treatment also manipu-lated moDCs to inhibit the induction of Foxp3–expressing Tregcells, whereas GM-CSF–treated cDCs increased Foxp3 expressionin tumor-specific Th cells (Fig. 5F). IL17A expression in transferredTh cells was not changed by GM-CSF treatment. We furtherinvestigated whether the transfer of GM-CSF–stimulated moDCsleads to tumor growth inhibition in the therapeutic modeland whether its activity is IL9-dependent. As depictedin Fig. 5G, GM-CSF–treated moDCs delayed tumor growth com-pared with GM-CSF–untreated moDCs. Tumor peptide–loadedcDCs also suppressed tumor growth; however, GM-CSF treatmentdidnot further increase its antitumor activity. To examinewhetherthe enhanced antitumor activity of GM-CSF–treated moDCs wasdue to IL9, we adopted a neutralizing anti-IL9 and found thattreatment with anti-IL9 completely reversed the tumor growthinhibition by GM-CSF–treated moDCs (Fig. 5H). We alsoobserved that E6/E7 peptide–loaded, GM-CSF–activated moDCssignificantly inhibited TC-1 tumor growth in an IL9-dependentmanner (Supplementary Fig. S6). Collectively, these results indi-cate that GM-CSF renders moDCs to induce IL9 expression ratherthan Foxp3 in tumor-specific Th cells, which leads to tumorgrowth inhibition in an IL9-dependent fashion.

During tumor development, heterogeneous myeloid popula-tions accumulate in the spleen and tumor tissue. To determinewhether moDCs and cDCs accumulate locally within tumorsand respond to GM-CSF to induce IL9, we analyzed TILs fromB16F10-OVA tumors. Along with tumor progression, moDCs,but not cDCs, significantly accumulated in the tumor (Supple-mentary Fig. S7A). Then, we isolated each tumor-infiltrating DCpopulation and compared their IL9-inducing capacity ex vivo.

Figure 3.

GM-CSF selectively promotes Th9 cell differentiation and inhibits iTreg cellgeneration in vitro.A,OVA-specific na€�ve CD4þ T cells were differentiatedinto each Th subset with or without GM-CSF under appropriate Th-polarizingconditions. Three days later, effector cytokines and the transcription factorFoxp3 were analyzed by flow cytometry. B, The percentages of single ordouble effector cytokine–producing or Foxp3-expressing T cells amongdifferentiated Th cells in A are depicted. Numbers in quadrant indicateeach percent cell. Data represent at least two independent experiments(� , P < 0.05; �� , P < 0.01 by Student t test).






Tumor-infiltratingmoDCs also selectively induced IL9 expressionin tumor-specific Th cells in response to GM-CSF (SupplementaryFig. S7B), suggesting that this phenomenonmay occur in the localtumor microenvironment.

IL9 induced by GM-CSF facilitates antitumor CTL responsesPrevious reports have demonstrated that IL9 or Th9 cells

promote tumor-specificCTLresponses toeradicate tumors(14,15,18). Thus, we analyzed tumor-specific CTL responses in ourtherapeutic B16F10-OVA model treated with tumor-specific Thcells and moDCs or cDCs. In accordance with the tumorgrowth inhibition, lymphoid cells from the recipients of GM-CSF–activated moDCs showed more potent cytotoxicity thanthose from the recipients of GM-CSF–untreated moDCs, whichwas comparable with those from cDC recipients (Fig. 6A). Anti-IL9 treatment reversed the enhancement of tumor-specific CTLresponses in the recipients of GM-CSF–stimulated moDCs. Con-sistently, expression of granzyme B in CD8þ T cells was signifi-cantly increased in the recipients of GM-CSF–stimulated moDCs,and this was almost completely abolished by anti-IL9 (Fig. 6B).These results together suggest that IL9 induced by GM-CSF–activated moDCs triggers antitumor CTL responses in vivo.

GM-CSF promotes Th9 cell differentiation via regulation of theCOX2–PGE2 pathway

Next, we sought to determine the underlying mechanism bywhich GM-CSF promotes Th9 cell differentiation. Studies haveshown that engagement of TNF receptor superfamily members

including OX40, DR3, and GITR enhances Th9 cell differentia-tion (15, 37, 38). To test whether GM-CSF–mediated Th9 celldifferentiation depends on these molecules, we employed block-ing antibodies to each interaction, and found that individualantibodies or the combination of antibodies did not reverse Th9differentiation induced by GM-CSF (Supplementary Fig. S8A andS8B).

GM-CSF regulates glucose and lipid metabolism in immunecells (39) and COX2 and its metabolic products block Th9development (40). Therefore, we next investigated the role ofCOX2-mediated lipidmetabolism in Th9 differentiation inducedby GM-CSF. The addition of GM-CSF decreased the expression ofCOX2 and several downstream prostaglandin (PG) synthasesunder moDC or cDC stimulation (Fig. 7A), and adenoviraltransduction of the COX2 gene to moDCs or cDCs significantlyabolished the increase in Th9 induction by GM-CSF (Fig. 7B andC). Moreover, when the COX2 metabolic product PGE2, but notPGD2, was added to cultures, it significantly reduced the frequen-cy of Th9 cells induced by GM-CSF without affecting cell prolif-eration (Fig. 7D and E). Collectively, these results demonstratethat GM-CSF potentiates Th9 cell differentiation by regulating theCOX2–PGE2 synthesis pathway.

DiscussionAlthough the use of GM-CSF for tumor immunotherapy has

been extensively studied in mice and humans, the role of Thresponses in GM-CSF–induced antitumor immunity is relatively

Figure 4.

GM-CSF triggers IL9 production in vivo by activating moDCs and cDCs. IL9 and IFNg expression in OVA-specific na€�ve CD4þ T cells (A) and total CD4þ T cells (B)stimulated ex vivo by either moDCs or cDCs plus OVA323-339 peptide (10 mg/ml) for 48 hours that had been isolated from OVA plus CFA-immunized syngeneicmice treated with or without anti-GM-CSF antibody. C and D,OVA323-339 peptide–loaded moDCs or cDCs treated with or without GM-CSF (20 ng/mL) overnightwere transferred into mice that had received OVA-specific na€�ve CD4þ T cells one day before. Four days later, transferred Th cells were recovered and analyzedfor IL9, IFNg , IL17A, and Foxp3 expression by qPCR (C) and ELISA (D; n¼ 3; � , P < 0.05; ��, P < 0.01; �� , P < 0.0001 by Student t test). Data represent twoindependent experiments.

Kim et al.





unappreciated. In this study, we demonstrate that GM-CSFtriggers antitumor immunity by enhancing the differentiation ofTh9 cells and by concomitantly inhibiting the generation of iTregcells in therapeutic cancer vaccine and DC vaccine models. Thiseffect ofGM-CSFwasmediated throughmoDCs andwas found toinduce potent tumor-specific CTL responses that suppressedtumor growth in an IL9-dependent manner. The underlyingmechanism was that GM-CSF inhibited COX2-mediated lipidmetabolism and PGE2 production, which consequently increasedTh9 cell differentiation. Hence, we have elucidated the missinglink between GM-CSF and its antitumor activity and identified apathway for Th9 cell development.

Proinflammatory properties of GM-CSF have been implicatedin diverse autoimmune diseases (23). Inflammatory responses

during tumor immune surveillance can be beneficial to the host;however, tumors exploit the host immune system to advancetumor growth. Myeloid inflammation induced by MDSCs andtumor-associated macrophages (TAM) is the representative ofthem, and tumor-derived or exogenous GM-CSF has been shownto trigger the recruitment and differentiation of MDSCs in thetumor microenvironment (32, 41–43), which might accountfor the inconsistent results in previous clinical studies usingGM-CSF (31, 41). In line with this result, we also observed thatneutralization of endogenous GM-CSF led to decreased tumorburden. However, administration of GM-CSF does not alwaysexhibit the same detrimental effect observed with endogenousGM-CSF. Indeed, we and other groups have shown the tumor-inhibiting effects ofGM-CSF, suggesting that (i) physiologic levels

Figure 5.

GM-CSF–activated moDCs inhibit tumor growth by inducing IL9 expression rather than Foxp3 in tumor-specific Th cells. moDCs and cDCs were isolated frommouse spleens 8 days after OVA plus CFA treatment that was 4 days after B16F10-OVA tumor inoculation. Control IgG or anti-GM-CSF antibody wasadministered intraperitoneally every 3 days from day 0. IL9 and IFNg expression were measured by qPCR in OVA-specific na€�ve CD4þ T cells (A) or total CD4þ Tcells (B) stimulated ex vivowith sorted moDCs or cDCs plus OVA323-339 peptide (10 mg/mL) for 48 hours. C, The percentages and absolute numbers ofaccumulated CD11bþLy6ChiLy6G–CCR2þ splenic monocytes in tumor-bearing mice treated with or without OVA plus CFA and GM-CSF adjuvant.D, Tumor size inB16F10-OVA tumor-bearingWT or CCR2–deficient mice treated with 50 mg of OVA plus CFA with or without GM-CSF adjuvant (n¼ 5). E, IL9 and IFNgproduction by TdLN CD4þ T cells isolated from tumor-bearingWT or CCR2–deficient mice. F, IL9, IFNg , IL17A, and Foxp3 expression in transferred OVA-specificna€�ve CD4þ T cells activated by OVA323-339–loadedmoDCs or cDCs treated with or without GM-CSF (20 ng/mL) overnight in B16F10-OVA tumor-bearing mice,and tumor growth (G) in mice that received these cells (n¼ 5).H, Somemice additionally received control IgG or anti-IL9, and tumor sizes were measured(n¼ 6). Data represent at least two independent experiments (� , P < 0.05; �� , P < 0.01; ��, P < 0.0001 by Student t test; N.S., nonsignificant).






of GM-CSF are not sufficient to induce a specific antitumorimmune response or (ii) certain conditions are needed to facilitatethe antitumor activity of GM-CSF.

MDSCs are defined as CD11bþGr-1þ cells with suppressiveactivity and consist of a heterogeneous set of immature mye-loid cells (44). These include CCR2þ monocytes and mono-cyte-derived cells, and indeed, we observed that IFNg produc-tion by Th cells in response to tumor peptide stimulationwas enhanced in CCR2-deficient mice. However, administra-tion of GM-CSF licensed CCR2þMHC IIþ moDCs to driveTh9 cell differentiation, which created favorable conditionsfor tumor inhibition via IL9. Thus, this result suggests thatCCR2þMHC IIþ moDCs are a prerequisite for exerting antitu-mor activity of GM-CSF, which was achieved by vaccinationwith tumor antigen plus CFA in our experimental setting.Moreover, these data explain why the antitumor effect ofGM-CSF–secreting tumor vaccine disappeared in Gr-1–depletedhosts (45). Further studies are needed to determine the relevantadjuvant in humans that selectively induces the recruitment ofCCR2þMHC IIþ moDCs.

The frequency of HLA-DRþ classical monocytes in PBMCs mayserve as a biomarker for responsiveness to anti-PD-1 therapy (46).This population corresponds to moDCs in our mouse study,implying that combination with GM-CSF may exhibit a synergis-tic antitumor effect in responders to anti-PD-1 therapy; indeed,this is under investigation (33–35). Vaccination with GM-CSFadjuvant did not appear to abrogate the suppressive functions oftumor-associated neutrophils (TAN) because CCR2-deficientmice given this therapy accumulated neutrophils and no antitu-mor effect was observed. Thus, combinations with checkpointblockades or agents selectively depleting TANs would be benefi-cial for eliciting the full therapeutic efficacy of GM-CSF in patientswith cancer.

GM-CSF potentiated the differentiation of Th9 cells ratherthan Th1 cells, thought to be the most potent antitumorigenicTh cells, to inhibit tumor growth. However, the appropriateinduction of type I responses at the early phase of tumorprogression is also needed because IFNg is critical for thedifferentiation of monocytes into moDCs (47, 48), which weremediators of GM-CSF–induced Th9 differentiation in vivo;indeed, neutralization of IFNg reversed the antitumor effect ofGM-CSF (Supplementary Fig. S9). Furthermore, GM-CSF inhib-ited the generation of iTreg cells, which are abundant in thetumor microenvironment, and elicited IL9–producing Th cells.This result indicates that the effect of GM-CSF on tumor-specificTh responses has dual advantages of blocking suppressor T cellsand promoting effector T cells to eliminate tumors. In addition,GM-CSF increased IL13 production by Th cells, a factor drivingthe expansion and activation of MDSCs (44), and the additionof neutralizing anti-IL13 may therefore reinforce the antitumoractivity of GM-CSF.

It remains a possibility that GM-CSF enhanced Th9 cell differ-entiation by facilitating differentiation of monocytes intomoDCs. However, GM-CSF is dispensable for moDC differenti-ation in vivo (49). In addition, although GM-CSF can affect thepersistent expression of MHC-II molecules on moDCs andincrease antigen presentation, it did not promote the differenti-ation of Th1 and Th17 effector cells. From this result, we suggestthat GM-CSF may alter moDC functions to selectively increaseTh9 cell differentiation. Meanwhile, consistent with a previousstudy (50), moDCs were defective in inducing the proliferationand expansion of Th cells, whereas cDCs efficiently induced T-cellproliferation upon antigenic stimulation. Thus, the role of cDCsmust not be ignored, with moDCs promoting Th9 cell develop-ment in response to GM-CSF and cDCs potentially playing a rolein Th9 cell proliferation in vivo.

Figure 6.

GM-CSF–activated moDCs induce potent tumor-specific CTL responses in an IL9-dependent manner. Spleen and TdLN cells were isolated from B16F10-OVAtumor–bearing mice that received OVA-specific na€�ve CD4þ T cells and DC vaccine as in Fig. 5G, stimulated ex vivowith OVA257-264 and gp10025-33 peptides(each 1 mg/mL) for 5 days, and then used as effector cells. Control IgG or anti-IL9 was administered intraperitoneally every 2 days starting one day before DCtransfer. A, Target cell lysis was measured using 51Cr-pulsed B16F10-OVA tumors. B, ICS of granzyme B in live CD8þ T effector cells after 5-day culture withpeptide mix. Each dot represents an individual mouse (n¼ 3 or 4). Data represent at least two independent experiments (� , P < 0.05; �� , P < 0.01 by Studentt test).

Kim et al.





We showed that GM-CSF facilitated Th9 cell differentiation viaregulation of the COX2–PGE2 pathway. In line with this result, aprevious study revealed that COX2 and its metabolic productsinhibit Th9 development by decreasing IL17RB expression (40).Further studies are needed to determine how the COX2–PGE2pathway regulates IL17RB expression, whether it affects othertranscription factors or signaling pathways related to Th9 differ-entiation, and how Th9 development might be used to manip-ulate Th cells with antitumor activity.

In this study, we have elucidated a mechanism by whichGM-CSF elicits antitumor immune responses. As an adjuvantfor cancer vaccines, GM-CSF promotes Th9 cell differentiationand inhibits iTreg cell generation, particularly through itseffects on moDCs. This leads to the generation of potent

antitumor CTL responses that suppress tumor growth in anIL9-dependent fashion. On the basis of these results, wesuggest the frequency of moDCs as a predictive biomarker forGM-CSF therapy and IL9 or Th9 as a biomarker for its effec-tiveness. Furthermore, as the adoptive transfer of Th9 cells hasbeen shown to eradicate advanced tumors (16), GM-CSFtherapy can be applied with a combination of other immu-notherapies to treat cancer that is susceptible to Th9 cellresponses. Collectively, our findings identify GM-CSF as a Th9inducer and provide the rationale for the use of GM-CSF incancer vaccines.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Figure 7.

GM-CSF potentiates Th9 cell differentiation by regulating COX2 expression and COX2-mediated PGE2 production. A, Expression of COX2 and prostaglandinsynthases in moDC or cDC cultures with or without GM-CSF (20 ng/mL) was measured by qPCR. B, COX2 and IL9 expression in 24-hour cultures with OVA-specific na€�ve CD4þ T cells and moDCs or cDCs plus OVA323-339 peptide (1 mg/mL) with or without GM-CSF in the presence of adenovirus encoding the Cox-2gene or a mock control. C, IL9 in accumulated supernatants of CD4þ T cells stimulated with moDCs (top) or cDCs (bottom) as in B for 3 days.D, ICS of IL9production by CFSE-labeled OVA-specific na€�ve CD4þ T cells stimulated with moDCs or cDCs plus OVA323-339 peptide for 3 dayswith or without GM-CSF in thepresence of vehicle, PGD2 (200 nmol/L) or PGE2 (50 nmol/L). E, IL9 in accumulated supernatants of CD4þ T cells stimulated with moDCs (top) or cDCs (bottom)as inD for 3 days. Data represent at least two independent experiments (� , P < 0.05; �� , P < 0.01; �� , P < 0.0001 by Student t test).






Authors' ContributionsConception and design: I.-K. Kim, C.-Y. KangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): I.-K. Kim, C.-H. Koh, K.-S. Shin, T.-S. KangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I.-K. Kim, B.-S. KimWriting, review, and/or revision of the manuscript: I.-K. Kim, Y. Chung,C.-Y. KangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.-H. Koh, I. Jeon, K.-S. Shin, E.-A. Bae, H. Seo,H.-J. KoStudy supervision: C.-Y. Kang

AcknowledgmentsThis research was supported by a grant from the Basic Science Research

Program (NRF-2015R1A2A1A10055844), Bio & Medical TechnologyDevelopment Program (NRF-2016M3A9B5941426) through the National

Research Foundation of Korea funded by the Ministry of Science, ICT &Future Planning, and Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by the Ministry of Education(2016R1A6A3A01007625).

We thank J.-O. Kim (International Vaccine Institute) for CD45.1 congenicmice, J.V. Snick (Ludwig Institute for Cancer Research, New York, NY) for theantibody to IL9 (MM9C1), K. Shortman (The Walter and Eliza HallInstitute of Medical Research) for the antibody to GM-CSF (MP1-22E9), andKang's laboratory members for technical supports.

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 indicatethis fact.

Received July 30, 2018; revisedNovember 9, 2018; accepted January 28, 2019;published first February 6, 2019.

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2019;7:498-509. Published OnlineFirst February 6, 2019.Cancer Immunol Res Il-Kyu Kim, Choong-Hyun Koh, Insu Jeon, et al. ResponsesGM-CSF Promotes Antitumor Immunity by Inducing Th9 Cell

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