inhibition of nr4a receptors enhances antitumor immunity ... · receptors, consisting of three...

Tumor Biology and Immunology

Inhibition of Nr4a Receptors Enhances AntitumorImmunity by Breaking Treg-Mediated ImmuneToleranceSana Hibino, Shunsuke Chikuma, Taisuke Kondo, Minako Ito, Hiroko Nakatsukasa,Setsuko Omata-Mise, and Akihiko Yoshimura

Abstract

Enhanced infiltration of regulatory T cells (Treg) into tumortissue is detrimental to patients with cancer and is closely asso-ciated with poor prognosis as they create an immunosuppressivestate that suppresses antitumor immune responses. Therefore,breaking Treg-mediated immune tolerance is important whenconsidering cancer immunotherapy. Here, we show that the Nr4anuclear receptors, key transcription factors maintaining Treggenetic programs, contribute to Treg-mediated suppression ofantitumor immunity in the tumor microenvironment. Mice lack-ingNr4a1 andNr4a2 genes specifically in Tregs showed resistanceto tumor growth in transplantation models without exhibitingany severe systemic autoimmunity. The chemotherapeutic agentcamptothecin and a common cyclooxygenase-2 inhibitor were

found to inhibit transcriptional activity and induction of Nr4afactors, and they synergistically exerted antitumor effects. Geneticinactivation or pharmacologic inhibition of Nr4a factorsunleashed effector activities of CD8þ cytotoxic T cells andevoked potent antitumor immune responses. These findingsdemonstrate that inactivation of Nr4a in Tregs breaks immunetolerance toward cancer, and pharmacologic modulation of Nr4aactivity may be a novel cancer treatment strategy targeting theimmunosuppressive tumor microenvironment.

Significance: This study reveals the role of Nr4a transcriptionfactors in Treg-mediated tolerance to antitumor immunity, withpossible therapeutic implications for developing effective anti-cancer therapies. Cancer Res; 78(11); 3027–40. �2018 AACR.

IntroductionCD4þCD25þ regulatory T cells (Treg), characterized by tran-

scription factor forkhead box P3 (Foxp3) expression, play criticalroles in maintaining immunologic self-tolerance and homeosta-sis (1, 2). Tregs provide dominant regulation over self-reactiveconventional T cells by inhibiting their expansion and activation(3). Tumor cells are derived from self-tissues, but they could berecognized as nonself and eradicated by the immune systembecause of the expression of tumor-specific mutated genes. How-ever, tumor cells with fewer immunogenic mutations are close to"self" and likely evade immune surveillance, resulting in tumorprogression. In this case, mechanisms for maintaining self-toler-ance, such as Tregs, may play undesired roles by establishingimmune tolerance against tumors (4, 5). In addition, tumor tissueitself creates a niche that supports survival and function of Tregs(6). An increased abundance of Tregs and a decreased ratio ofintratumoral CD8þ cytotoxic T cells (CTL) to Tregs have beenshown to predict a poor prognosis in various types of humancancers (7). Tregs behave asmajor obstacles in clinical application

of cancer immunotherapy, such as tumor vaccines or immunecheckpoint blockade (4, 8). Therefore, breaking immunosuppres-sion by Tregs is important for successful cancer therapy.

Depletion of Tregs or specific disruption of Treg functions isactually thought to be a promising strategy to enhance antitumorimmunity (4, 8, 9). Chemical inhibitors targeting signal path-ways or molecules that contribute to the suppression activities ofTregs have been intensively studied (10–13). For example, themaster transcription factor Foxp3 is an attractive target, and P60,a peptide inhibitor of Foxp3, was reported to inhibit Tregfunction and improve tumor vaccine efficiency (14). However,there is not enough evidence to show that Foxp3 alone issufficient to be targeted for disruption of Treg function becausemultiple factors work cooperatively with Foxp3 to maintain Tregphysiology (15, 16).

We recently discovered that the Nr4a family of nuclear orphanreceptors, consisting of three isoforms (Nr4a1, Nr4a2, andNr4a3), redundantly play essential roles in Treg developmentand function via their ability to directly transactivate Foxp3expression (17–19). Thymic Treg development was completelyinhibited in mice lacking all three of the Nr4a factors on T cells(CD4-Cre Nr4a1fl/fl Nr4a2fl/fl Nr4a3�/�), and they died within 3weeks because of systemic multiorgan autoimmunity (18). Inaddition, Nr4a factors have been shown to be highly expressed onmature Foxp3þ Tregs and necessary for maintaining Treg stabilityand suppressive activities (19). Of note, Nr4a factors not onlyregulate Foxp3 expression but also globally regulate the Treg-specific transcriptional program. Therefore, Nr4a-deficient Tregsshowed not only reduced expression of Foxp3, but also globaldysregulation of Treg signature genes, including Foxp3-indepen-dent genes such as Ikzf4 (Eos). Thus, we hypothesize that Nr4a

Department of Microbiology and Immunology, Keio University School of Med-icine, Tokyo, Japan.

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

Corresponding Author: Akihiko Yoshimura, Keio University School of Medicine,35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3483;Fax: 81-3-5360-1508; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-3102

�2018 American Association for Cancer Research.

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factors are a promising target for cancer immunotherapy todisrupt Treg function within tumors.

In this study, using themouse tumor transplantationmodel,wefound that selective deletion ofNr4a1/Nr4a2within Foxp3þTregssignificantly suppressed tumor growth and induced potent anti-tumor immune responses. We then searched for pharmacologicmodulators of Nr4a factors and identified two well-known drugs:the classical chemotherapeutic agent camptothecin (CPT) as theinhibitor of Nr4a transcriptional activity and cyclooxygenase(COX)-2 inhibitors, such as the celecoxib analogue SC-236, asthe inhibitor of Nr4a transactivation. These drugs synergisticallyexerted potent antitumor immune responses against mousetumor models in an Nr4a/Treg-dependent manner. We proposethat Nr4a factors play important roles in Treg-mediated suppres-sion of antitumor immunity, and they are attractive therapeutictargets for cancer immunotherapy.

Materials and MethodsMice

All experiments using mice were approved by the InstitutionalAnimal Care and Use Committee (IACUC; approval number08004) of Keio University and performed according to IACUCguidelines. The Nr4a1- and Nr4a2-floxed mice and Foxp3YFP-Cre

knockin mice were previously described (19). C57BL/6J micewere purchased from Tokyo Laboratory Animals Science. IFNg-venus reporter mice were reported previously (20). All mice weremaintained on aC57BL/6 genetic background and kept in specificpathogen-free conditions at Keio University.

Cell lines and cultureAll cell lines were obtained between 2008 and 2014. Authen-

ticated 3LL (Lewis lung carcinoma) tumor cells were obtainedfrom the Japanese Collection of Research Bioresources Cell Bank.MC38 (colon adenocarcinoma) tumor cells were kindly providedby Dr. James P. Allison (Department of Immunology, MD Ander-son Cancer Center, Houston, TX). Human embryonic kidney293T cells were obtained from the ATCC. 3LL cells were main-tained in RPMI1640 supplemented with 10% FBS and 1% pen-icillin/streptomycin. MC38 and 293T cells were maintained inDMEM supplemented with 10% FBS and 1% penicillin/strepto-mycin. All cell lines were frozen down at early passages (<7) andused in the experiments within five passages after thawing. Thesecells were not further authenticated by our laboratory; however,routine confirmation of in vitro growth properties, morphology,tumor formation in the C57BL/6 syngeneic mouse strain (3LL,MC38), and transfection efficiency (293T) provided evidence ofcorrect cell identity. They were negative for known mouse patho-gens, including Mycoplasma.

Drugs(S)-(þ)-Camptothecin and 7-Ethyl-10-hydroxycamptothecin

(SN-38) were purchased from Tokyo Chemical Industry. Prosta-glandin E2 (PGE2) and thymidine were purchased from NacalaiTesque. Z-VAD-FMK was purchased from Peptide Institute. CPT-11 (irinotecan HCl trihydrate) was purchased from Biochempart-ner. SC-236 was purchased from Sigma-Aldrich. H-89 was pur-chased from Cayman Chemical. Mitomycin-C was purchasedfromKyowaHakkoKirin. The ScreeningCommittee of AnticancerDrugs inhibitor kits (http://gantoku-shien.jfcr.or.jp/) containing363 compounds were kindly provided by a Grant-in-Aid for

Scientific Research in the Priority Area "Cancer" from theMinistryof Education, Culture, Sports, Science and Technology (Tokyo,Japan).

Fluorescence-activated cell sortingThe following fluorescently labeled antibodies were purchased

fromBioLegend, eBioscience, or TONBObiosciences: CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), CD3e (145-2C11), CD11b(M1/70), CD11c (N418), CD45.1 (A20), CD80 (B7-1; 16-10A1),CD86 (B7-2; GL1), Foxp3 (FJK-16s), IFNg (XMG1.2), TNFa(MP6-XT22), IL4 (11B11), CD107a (1D4B), CD152 (CTLA-4;UC10-4B9), Ki-67 (16A8), and Nr4a1 (12.14). Dead cells weregated-out using Fixable Viability Dye eFluor 780 (eBioscience).For intracellular cytokine staining, cells were stimulated with 50ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 500ng/mL ionomycin (Sigma-Aldrich) in the presence of Brefeldin A(eBioscience) for 4 hours. Cells were stained for surface antigens,then fixed/permeabilized with Foxp3 fixation/permeabilizationconcentration and diluent (eBioscience), and stained for nuclearproteins or cytokines. For phospho-CREB (pCREB) staining, cellswere fixed/permeabilized with 4% paraformaldehyde and ice-cold 90% methanol, and stained with pCREB (Ser133) (87G3)rabbit monoclonal antibody (Cell Signaling Technology) for 1hour, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (HþL; Thermo Fisher Scientific). Stained samples weremeasured using a BD Canto II (BD Biosciences), and FlowJosoftware (Tree Star) was used for data analysis.

Mouse tumor modelsBefore transplantation into mice, 3LL and MC38 tumor cells

were grown to 80% confluence, and then counted and suspendedin PBS. Each mouse was subcutaneously injected with 2.5 � 105

3LL or 5.0 � 105 MC38 tumor cells into the right flank on day 0.Tumor growth was monitored every 3 to 4 days throughout theexperiment. Tumor volume was calculated using the followingformula: 0.5 � ab2 (a, major axis; b, minor axis). For inhibitortreatment experiments, mice were injected intraperitoneally withCPT-11 (75mg/kg) and/or theCOX-2 inhibitor SC-236 (3mg/kg)on days 6 and 9. For CD8þ T-cell depletion, mice received an i.p.injection of 400 mg CD8þ T-cell–depleting antibody (TIB211) 3times per week beginning 6 days after tumor implantation.

Isolation of immune cells from tumorAt the specified time points after tumor implantation and drug

treatment, subcutaneous tumors were excised, minced, anddigested with 1mg/mL collagenase D (Roche) and 100 mg/mLDNase I (Roche) at 37�C for 1hourwith gentle shaking.Cellswerepassed through a 70-mm filter, following low speed centrifugation(400 rpm, 5 minutes) to remove cell debris and epithelial cells.Red blood cell lysis was then performed if necessary, and cellswere washed twice with PBS and used for further experiments.

Mouse T-cell isolationSpleen and lymph node cell suspensions were prepared from

the indicated mice, and CD4þ T cells were isolated using amouseCD4þT Cell Isolation Kit and autoMACS Pro (Miltenyi Biotec).CD4þCD62Lþ-na€�ve T cells were prepared as previously described(21). For preparation of CD4þCD25þ Tregs, isolated CD4þ Tcells were stained with allophycocyanin (APC)-conjugated anti-mouse CD25 antibody, followed by positive selection with anti-APC microbeads using autoMACS. Over 95% of the sorted

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CD4þCD25þ cells expressed Foxp3. When Foxp3YFP-Cre knockinmice were used, MACS-isolated CD4þ cells were stained withPerCP-cy5.5 anti-mouseCD4antibody, andCD4þYFPþ cellsweresorted using a SONY SH800 cell sorter (Sony).

Culture of primary T cellsT-cell receptor (TCR) stimulation was performed by incubating

T cells with anti-CD28Ab (57.31; 2 mg/mL) and plate-coated anti-CD3e Ab (145-2C11; 4 mg/mL). Th-skewing conditions in thisstudy were described previously (17). Retrovirus transduction toprimary T cellswasperformed asdescribedpreviously (16, 21). Allcultures were performed in RPMI1640 supplemented with 10%FBS, 1% penicillin/streptomycin, 100 nmol/L nonessential ami-no acids, 2 mmol/L glutamine, and 55 mmol/L 2-mercaptoetha-nol (Invitrogen).

Adoptive transfer of TregsIsolated Tregs were activated using Dynabeads Mouse T-Acti-

vator CD3/CD28 (Thermo Fisher Scientific), according to themanufacturer's instructions. At the specified time points, 2.5 �105 cells were transferred intravenously into the tumor-bearinghost.

In vitro suppression assayResponder T cells (CD45.1þCD4þCD25� cells) were prepared

fromC57BL/6J CD45.1 congenicmice and labeled with 1 mmol/Lcarboxyfluorescein diacetate succinimidyl ester (CFSE; Invitro-gen). CFSE-labeled responder cells (5�104)were coculturedwithunlabeled CD45.1� Tregs at the indicated ratio in the presence ofDynabeads Mouse T-activator CD3/CD28 (Thermo Fisher Scien-tific) in round-bottomed 96-well dishes at a concentration of 1bead per cell. The CFSE dilution of CD45.1þ responder cells wasmeasured using flow cytometry 96 hours later.

mRNA preparation and quantitative RT-PCRTotal RNA was extracted using RNAiso Plus (Takara Bio) or

ReliaPrep RNACellMiniprep System (Promega), and subjected toreverse transcription (RT) using a High Capacity cDNA Synthesiskit (Thermo Fisher Scientific). PCR analysis was performed usingan iCycler iQ multicolor real-time PCR detection system (Bio-Rad) and SsoFast EvaGreen Supermix (Bio-Rad). All primer setsyielded a single product of the correct size. Relative expressionlevels were normalized to 18S rRNA. Sequences for primers usedin this study are available upon request.

Luciferase assay293T cells were seeded on 24-well plates and transfected with

pCMV expression plasmids and pGL4-luciferase plasmids, 200 ngeach, using polyethylenimine. Twenty-four hours after transfec-tion, the indicated chemicals were added to the culture andincubated for an additional 18 hours. Cells were lysed in 100 mLlysis buffer, and luciferase activity wasmeasured using a luciferasesubstrate kit (Promega) and bioluminescence-monitoring appa-ratus (CL96; Churitsu). For each transfection, 200 ng b-galacto-sidase was added as an internal control. Luciferase assay–baseddrug screening was performed as described in SupplementaryMethods.

Statistical analysisFor comparison of two groups, statistical analysis was per-

formed using a Student t test or a Mann–Whitney U test. ANOVA

with Tukey post hoc test was used for multiple comparison experi-ments. P values < 0.05 were considered to indicate statisticalsignificance (�, P < 0.05; ��, P < 0.01; and ��, P < 0.001).

ResultsTreg-specific deletion of Nr4a1/Nr4a2 restricts tumor growth

Human effector Treg fraction (Foxp3hiCD45RA�CD25hi; frac-tion II) has been shown to be highly suppressive and dominant intumor tissues, among the three fractions of human Tregs (9, 22).Interestingly, we confirmed that Nr4a factors are most highlyexpressed in the fraction II population of human Tregs (Supple-mentary Fig. S1A and S1B). This suggests thatNr4a factors canbe atarget for modulating tumor-associated Tregs.

Then, to investigate the role of Nr4a/Tregs in antitumor immu-nity, we prepared Treg-specific Nr4a1/Nr4a2 double-conditionalknockoutmice (Foxp3YFP-Cre Nr4a1fl/flNr4a2fl/flNr4a3þ/þ; hereaftercalled Nr4a-DcKO mice). We used Foxp3YFP-Cre Nr4a1þ/

þNr4a2þ/þNr4a3þ/þ as the WT control. Unlike Nr4a-triple KO(Foxp3YFP-Cre Nr4a1fl/flNr4a2fl/flNr4a3�/�) mice (19), Nr4a-DcKOmice did not show any spontaneous inflammation, suggestingthat systemic Nr4a3 sufficiency suppresses autoimmunity. Over-all, the T-cell population in the thymus and periphery, includingthe Treg fraction in CD4þ T cells, was normal in Nr4a-DcKOmice(Supplementary Fig. S2A and S2B). However, Tregs from Nr4a-DcKO mice had significantly lower expression levels of Treg-signature genes, such as Foxp3, CD25, andCTLA-4, at bothmRNAand protein levels (Supplementary Fig. S2C and S2D). We alsoconfirmed that in vitro suppression activity of Nr4a-DcKO Tregswas attenuated compared with WT (Supplementary Fig. S2E). Inaddition, at steady state, IFNgþ cell fraction was increased withinboth CD4þ and CD8þ conventional T cells in Nr4a-DcKO mice(Supplementary Fig. S2F, top and middle plots). We did not findany IL4-producing CD4þ cells inNr4a-DcKOmice, suggesting theabsence of Th2-type inflammation that is characteristic in TKOmice (Supplementary Fig. S2F, bottom plots).

To examine the effect of the Nr4a1/Nr4a2 deletion in Tregs onantitumor immunity, we performed a mouse subcutaneoustumor transplantation model using the syngeneic 3LL tumor cellline. Although tumor growth rates were indistinguishablebetween WT and Nr4a1- or Nr4a2-single cKO mice, Nr4a-DcKOmice showed a remarkable delay of tumor growth (Fig. 1A). Theestablished tumor was almost completely eradicated in someNr4a-DcKO mice, but not in WT mice (Fig. 1B). The tumor-resistant phenotype ofNr4a-DcKOmicewas also confirmedwhenthe MC38 (colon adenocarcinoma) cell line was transplanted(Fig. 1C–E).

Enhanced antitumor immunity in Nr4a-DcKO miceWe then assessed the contribution of immune cells to the

suppression of tumor growth observed in Nr4a-DcKO mice. Asshown in Fig. 2A, transfer of in vitro–activated WT Tregs into 3LLtumor–bearing Nr4a-DcKO mice almost completely abolishedtumor growth inhibition, suggesting that delayed tumor growthwas due to functional defects of Nr4a-DcKO Tregs. In addition,antibody-mediated depletion of CD8þ CTLs in Nr4a-DcKO micealso accelerated tumor growth, suggesting that CD8þCTLs are themain effector population that restricts tumor growth in Nr4a-DcKO mice (Fig. 2B).

Based on these findings, we further examined immuneresponses that occurred in 3LL tumor–bearing mice. In tumor-draining lymph nodes (TDLN), the fraction of CD8þ T cells was

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significantly increased in Nr4a-DcKO mice compared with WTmice (Fig. 2C), and cells expressing the proliferationmarker Ki-67were also increased among these CD8þ T cells, suggesting theactive expansion of CD8þ CTLs in TDLNs (Fig. 2D). On the otherhand, accumulation of Foxp3þ Tregs in TDLNs was inhibited inNr4a-DcKOmice (Fig. 2E). In addition, Foxp3 protein expressionwas also significantly downregulated in TDLN Tregs of Nr4a-

DcKOmice (Fig. 2F). In tumor tissue, enhanced T-cell infiltrationwas observed in Nr4a-DcKO mice (Fig. 2G, top plots). Thefraction of intratumoral Foxp3þ Tregs was not affected (Fig.2G, bottom plots), but the CD8þ/Treg ratio was significantlyhigher in Nr4a-DcKO mice compared with the WT mice (Fig.2H). In addition, the antitumor effector CD8þCTLs, characterizedby the expression of effector cytokines (IFNg and TNFa) and

Figure 1.

Nr4a-DcKO mice showed resistance to tumor growth. A, WT and Nr4a-DcKO mice (n ¼ 8–12/group) and Nr4a1- and Nr4a2-single cKO mice (n ¼ 5/group) weresubcutaneously injected with 2.5 � 105 3LL cells on day 0, and tumor growth was monitored. B, Tumor growth in individual mice in A. C, WT and Nr4a-DcKOmice (n¼ 8–12/group)were subcutaneously injectedwith 5.0� 105MC38 cells on day0, and tumor growthwasmonitored.D, Tumor growth in individualmice inC.E,Representative images of MC38 tumors collected from WT and Nr4a-DcKO mice described in C and D on day 22. �, P < 0.05; �� , P < 0.01; and ��, P < 0.001compared with WT; data are presented as the mean � SD.

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Figure 2.

Inactivation of Nr4a receptors in Foxp3þ Tregs enhances antitumor immune responses. A, In vitro–activated WT Tregs were transferred into 3LL tumor–bearingNr4a-DcKO mice on days 6 and 11. Tumor weights were measured on day 18 (n ¼ 5–7/group). B, a-CD8 antibodies were i.p. injected into 3LL tumor–bearingNr4a-DcKOmice on days 6, 9, 13, and 16. Tumor weights were measured on day 18 (n¼ 5–7/group). TDLNs (C–F) and tumor tissues (G–I) of 3LL tumor–bearingWTand Nr4a-DcKO mice (n ¼ 5–8/group) were analyzed on day 20. C, Frequency of CD4þ/CD8þ T cells. The plots show gated CD3þ T cells. D, CD8þ T-cellfraction inCwas further analyzed for Ki-67 expression.E,Frequency of CD4þFoxp3þT cells. Theplots showgatedCD4þT cells.F,CD4þFoxp3þTreg fraction inEwasfurther analyzed for Foxp3 protein expression. Numbers in the histograms indicate mean fluorescence intensities. G, Frequency of tumor-infiltrating CD4þ/CD8þ

(top) and CD4þ Foxp3þ Tregs (bottom). The bottom plots show gated CD4þ T cells. H, Ratio of tumor-infiltrating CD8þ T cells to Tregs. I, Frequency oftumor-infiltrating effector T cells (CD8þ-IFNgþ, -TNFaþ, -CD107aþ, and CD4þIFNgþ). The plots show either gated CD8þ or CD4þ T cells. C–G and I, Representativefluorescence-activated cell sorting (FACS) plots or histograms and bar graphs summarizing the FACS data are shown. � , P < 0.05 and �� , P < 0.01 comparedwith WT; n.s., not significant; data are presented as the mean � SD.






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degranulation marker CD107a, as well as IFNgþTh1 CD4þ T cellswere drastically increased inNr4a-DcKOmice (Fig. 2I), suggestingthe induction of potent antitumor immune responses. Similarresults were obtained from the experiments using MC38 tumorcells (Supplementary Fig. S3A–S3C). We also confirmed thatenhanced immune responses in Nr4a-DcKO mice occurred in atumor antigen-specific manner by examining in vitro recallresponses (Supplementary Fig. S3D).

We then examined the mechanism of effector CTL enhance-ment by Nr4a-DcKO Tregs. Previous research demonstrated thatintratumoral Tregs suppress costimulatory signals from antigen-presenting cells by CTLA-4–mediated transendocytosis of B7-molecules CD80/CD86, which in turn suppress CD8þ CTL effec-tor function (3, 23–25). We observed significant upregulation ofCD80 expression on tumor-infiltrating dendritic cells (DC), aswell as downregulation of CTLA-4 on tumor-infiltrating Tregs, inNr4a-DcKO mice (Supplementary Fig. S3E–S3G). Collectively,these results strongly support our hypothesis that inhibition ofNr4a factors in Tregs breaks immune tolerance against tumor cellsand facilitates antitumor activities of tumor-infiltrating effectorCTLs.

Identification of CPT as a functional Nr4a inhibitorCrystal structure analysis revealed that Nr4a factors function as

ligand-independent transcription factors, and their pharmacolog-ic antagonists have not been identified (26). Therefore, we con-ducted reporter assay-based drug screening to identify functionalNr4a inhibitors from a chemical library (Methods summarized inSupplementary Fig. S4A). We looked for compounds that inhib-ited the transcriptional activity of Nr4a2 and identified CPT as apotentNr4a inhibitor (anoverviewand results of the screening aredescribed in detail in Supplementary Fig. S4B and S4C). CPT is aDNA topoisomerase I inhibitor that was shown to be effective in abroad spectrum of tumors (27). CPT inhibited not only Nr4a2,but also Nr4a1 and decreased Nr4a response element (NBRE)reporter activity in a dose-dependent manner (Fig. 3A). CPT alsosuppressed Nr4a2-induced transactivation of Foxp3-promoterreporter activity (Fig. 3B). Although the CPT analogue topotecaninhibited Nr4a activity similarly to CPT, other topoisomeraseinhibitors, such as daunorubicin or etoposide, did not, suggestingthat suppression of Nr4a by CPT is independent of inhibition oftopoisomerase activity (Supplementary Fig. S4D). In addition,CPT did not affect the transcriptional activity of other nuclearreceptors such as estrogen receptor a, thyroid-hormone receptor

b, or peroxisomeproliferator-activated receptor g (SupplementaryFig. S4E), revealing that CPT is not a general transcription inhib-itor, but is specific to Nr4a.

To further confirm the screening result, we next examined theeffect of CPT onmouse primary T cells. CPT treatment suppressedFoxp3 protein expression induced by ectopic expression of Nr4a2in na€�ve CD4þ T cells (Fig. 3C), demonstrating that CPT alsoinhibits Nr4a2 transcriptional activity in T cells. In addition,under in vitro helper T-cell differentiation conditions, CPT potent-ly suppressed induction of Foxp3þ induced Treg (iTreg) cellswhile it promoted induction of IFNgþ Th1 cells in a TGFb-independent manner, at both mRNA and protein levels (Fig.3D; Supplementary Fig. S5A and S5B). Because deletion of Nr4ain T cells robustly suppresses iTreg differentiation while promot-ing Th1 differentiation (17), these results indicate that CPTtreatment mimicked the effects of Nr4a knockdown in T cells.Similar results were obtained in the presence of the apoptosisinhibitor Z-VAD-fmk, suggesting that these effects are not a resultof cytotoxic effects of CPT (Supplementary Fig. S5C).

We then examined the effect of CPT on the stability of Tregs invitro. We treated CD4þCD25þ Tregs (>95% Foxp3þ Treg) withCPT in the presence of thymidine and Z-VAD-fmk to inhibit cellproliferation and apoptosis, respectively. CPT significantlyreduced the expression of Foxp3 and other Treg-related genes ina dose-dependent manner (Fig. 3E and F). CPT-induced destabi-lization of Foxp3 expressionwas not observed in Tregs fromNr4a-DcKO mice, suggesting that CPT reduces Foxp3 through theinhibition of Nr4a factors (Fig. 3G). On the other hand, theexpression of Nr4a factors themselves was not affected by CPTtreatment (Fig. 3E), which is consistent with our proposal thatCPT inhibits transcriptional activity of Nr4a factors, but not theirexpression.

In vivo effects of CPT on Foxp3þ Tregswere also examined usingCPT-11 (irinotecan), a water-soluble, less toxic prodrug of CPT(28). Because CPT-11 exerted its effect after being metabolizedinto SN-38 (7-Ethyl-10-hydroxy-camptothecin) in vivo, we con-firmed that SN-38 inhibited Nr4a activity in vitro similarly to CPT(Supplementary Fig. S5D and S5E). We also confirmed that anysignificant side effects, such as weight loss or immunosuppres-sion, were not observed in na€�ve mice treated with the dose ofCPT-11 used in this study (Supplementary Fig. S5F and S5G).AlthoughCPT-11 treatment did not affect the population of T-cellsubsets including Tregs in the thymus and periphery (Supple-mentary Fig. S5H and S5I), the Foxp3 expression levels in Tregs

Figure 3.Identification of classical chemotherapeutic agent CPT as Nr4a inhibitor. A, Effects of CPT on NBRE-luciferase activity transactivated by Nr4a1/Nr4a2. B, Effects ofCPT on Foxp3 promoter-luciferase activity transactivated by Nr4a2. C, Effect of CPT on Foxp3 induction in WT na€�ve CD4þ T cells by ectopic expressionof Nr4a2. Na€�ve CD4þ T cells were transduced with retroviral vectors encoding for IRES-GFP (empty) and Nr4a2-IRES-GFP (Nr4a2). CPT was added 24 hours aftervirus transduction. The plots show gated CD4þGFPþ cells. D, Effects of CPT on Foxp3 (top) and IFNg expression (bottom) under iTreg and Th1 conditions,respectively. E,Quantitative RT-PCR analysis of mRNA for the indicated genes in CD4þCD25þWTTregs, cultured with or without CPT in the presence of Z-VAD-fmk(50 mmol/L) and thymidine (2 mmol/L) for 72 hours. Gene expression was normalized to 18S rRNA levels. F, Flow cytometry profiles of the indicatedproteins inWT Tregs described in E. Dose-dependent effect of CPTwas tested.G, Effect of CPT on Foxp3 protein expression inWT and Nr4a-DcKO Tregs treated asdescribed inE. One representative result (left) anddata pooled from three independent experiments (right) are shown.H,Effects of CPTonFoxp3expression in Tregsin vivo. The CPT-derivative CPT-11 (75 mg/kg) was i.p. administered to WT mice twice at intervals of 3 days. Thymus and spleens of these mice wereanalyzed 6 days after the second administration (n¼ 5/group).A–G, The indicated concentration of CPTwas added to the culture throughout the experiment. Underculture conditions without CPT, an equivalent volume of DMSO was added only as a control. Data represent four (A–F) or three (G) independent experiments.As for A, B, and E, each experiment was performed in triplicate. C, D, F, and H, Representative fluorescence-activated cell sorting (FACS) plots or histogramsand bar graphs summarizing the FACS data are shown. Numbers in the histograms of F and H indicate mean fluorescence intensities (MFI). For the bar chartin F andG, MFIs of CPT-untreatedWT Tregs are set as one. � , P < 0.05; ��, P < 0.01; ��, P < 0.001 comparedwith the untreated group unless otherwise indicated; n.s.,not significant; data are presented as the mean � SD.






were significantly decreased (Fig. 3H). These data indicate thatCPT destabilizes Foxp3 expression both in vitro and in vivo. Takentogether, in addition to the well-defined chemotherapeuticeffects, our data revealed a previously unidentified inhibitoryactivity of CPT on Nr4a function.

COX-2 inhibitor targets the PGE2–Nr4a axis on Tregs in tumormicroenvironment

COX-2 is often constitutively overexpressed in a variety oftumors, and its enzymatic product PGE2 contributes to tumorprogression either by direct effects on tumor cells or through theformation of a tumor-promoting microenvironment (29). Inter-estingly, in intestinal tumors, PGE2 has been shown to induceNr4a2, thereby supporting survival and proliferation of tumorcells, and COX-2 inhibitors abolished such effects (30, 31).Therefore, we investigated the effect of PGE2 and COX-2 inhibi-tors on Nr4a1/Nr4a2 expression in Tregs. Transient stimulationwith PGE2 alone for 3 hours upregulated both Nr4a1 and Nr4a2expressions in Tregs in a dose-dependent manner (Fig. 4A and B).In addition, continuous PGE2 stimulation for 18 hours alsoenhanced Nr4a1/Nr4a2 expression in Tregs that were maintainedby the TCR stimulation (Fig. 4C). Induction ofNr4a expression byPGE2 was dependent on cAMP/PKA (protein kinase A), becausePKA inhibitor H-89 abolished these effects (Fig. 4B and C). Ofnote, PGE2 also enhanced expression of Foxp3 and Ikzf4 (Eos),direct targets of Nr4a, in WT Tregs, but not in Nr4a-DcKO Tregs(Fig. 4D). These data indicate that PGE2 promotes the expressionof Nr4a factors and regulates Nr4a-dependent gene expression inTregs.

Both 3LL and MC38 tumor cells constitutively express COX-2and produce PGE2 (32, 33). Consistently, treatment of Tregs withMC38 culture supernatant significantly induced Nr4a expressionin vitro; however, this was attenuated by pretreating MC38 cellswith a COX-2 inhibitor (Fig. 4E). These data indicate that tumor-derived PGE2 actually induces Nr4a expression. In an in vivomodel, Tregs from the spleen of 3LL tumor–bearing mice showedhigher Nr4a expression than that of tumor-free mice, which wasabolished by administration of SC-236, a structural analogue ofthe clinically usedCOX-2 inhibitor celecoxib (Fig. 4F). Expressionof the Nr4a target genes Foxp3 and Ikzf4 was also upregulated inTregs in tumor-bearing mice, but was reduced by SC-236 treat-ment (Fig. 4F). In addition, SC-236 treatment downregulatedNr4a1 expression in tumor-infiltrating Tregs and also suppressedCREB (cAMP response element binding protein) activation eval-uated by the phosphorylation levels (Fig. 4G). Taken together,these results suggested that tumor-derived PGE2 inducescAMP/PKA activation, which turns on the transcription of Nr4afactors and their target genes in Tregs, and this regulatory axis canbe targeted by a COX-2 inhibitor in vivo.

CPT and the COX-2 inhibitor synergistically elicit antitumorimmune responses

To examine the effect of the above-identified Nr4a inhibitorson antitumor immunity in vivo, we treated 3LL tumor–bearingmice with CPT-11 and SC-236, according to the experimentaloutline shown in Fig. 5A. We found that each drug alonesignificantly suppressed 3LL tumor growth, and they showedmore potent antitumor effect when used in combination(Fig. 5B). Depletion of CD8þ T cells cancelled the inhibitionof tumor growth by the inhibitors (Fig. 5C), indicating thatthe antitumor effects induced by CPT-11 and SC-236 were

dependent on CD8þ T cells. A significant increase in thefrequency of Ki-67þCD8þ T cells in TDLNs was observedwhen the mice were treated with these compounds (Fig. 5D,top plots, and E). Combination treatment synergisticallyreduced not only the Treg population but also the Foxp3protein levels in Tregs within TDLNs (Fig. 5D, bottom plots).Of note, combination therapy also synergistically enhancedproduction of IFNg from CD8þ effector T cells within the tumor(Fig. 5F, top plots). Increase in the frequency of IFNgþTh1CD4þ T cells was also confirmed in the inhibitor-treated mice(Fig. 5F, bottom plots). Similar results were obtained in experi-ments using MC38 tumor cells (Supplementary Fig. S6A–S6C).These data support our hypothesis that pharmacologic inhibi-tion of Nr4a potently enhances antitumor immune responses.

Therapeutic effects of CPT and the COX-2 inhibitor depend oninhibition of Nr4a-mediated Treg function

To gain mechanistic insights, we characterized gene expressionof Tregs from TDLNs of 3LL tumor–bearing mice treated with orwithout Nr4a inhibitors. Expressions of Nr4a1/Nr4a2 and Treg-signature genes, including Foxp3, Il2ra (Cd25), Ikzf4 (Eos), andCtla4, were significantly downregulated by the treatment (Fig.6A). The suppression activity of Tregs from tumor-bearing micewas higher than that of Tregs from tumor-freemice, but complete-ly attenuated by treatment with the inhibitors (Fig. 6B; Supple-mentary Fig. S7A). These results indicate that Nr4a inhibitorscould reverse Treg activity that was enhanced by the tumor-bearing conditions.

Then to confirm that antitumor effects of the CPT-11/SC-236combination depend on the inhibition of Nr4a in Tregs, weperformed adoptive Treg transfer experiments. The 3LL tumor–bearingmice were treated with theseNr4a inhibitors, and in vitro–activated Tregs were then adoptively transferred. As shown in Fig.6C and D, WT Treg transfer significantly diminished antitumoreffects evoked by the drugs. Conversely, transfer of Nr4a-DcKOTregs did not reverse the therapeutic effects in the same situation(Fig. 6C andD). In addition, as observed in Nr4a-DcKOmice, theCPT-11/SC-236 treatment dramatically increased the expressionlevels of the costimulatory molecule CD80 in tumor-infiltratingDCs, which was also abolished by the transfer of WT Tregs, butnot by Nr4a-DcKO Tregs (Supplementary Fig. S7B). Taken togeth-er, these data strongly support our conclusion that that the com-bination of CPT and a COX-2 inhibitor exerts potent antitumorimmune responses in an Nr4a/Treg-dependent manner in vivo.

DiscussionIn this study, we revealed that Nr4a receptors are involved in

Treg-mediated immune tolerance against tumor cells, and geneticinactivation or pharmacologic inhibition of these factors couldevokeCD8þCTL-dominant potent antitumor immune responses.We propose two mechanisms of CTL augmentation by Nr4ainhibition in Tregs. First is the impairment of Treg-mediatedsuppression of CD8þ T-cell proliferation. Tregs have been shownto suppress proliferation of CD8þ CTLs by depriving IL2 becauseTregs constitutively express CD25, which consists of the high-affinity IL2 receptor (34). Consistently, we observed decreasedexpressionofCD25 inNr4a-DcKOTregs (Supplementary Fig. S2Cand S2D) and reduced suppression activity compared with WTTregs (Supplementary Fig. S2E). Therefore, reduced expression ofCD25 in Nr4a-DcKO Tregs could be a mechanism for enhanced


Hibino et al.




Figure 4.

COX-2 inhibitor targets PGE2–Nr4a axis on Tregs in tumor microenvironment. A–E, RNA was purified from the CD4þCD25þ Tregs cultured under the in vitroconditions described below, and mRNA expression of the indicated genes was measured by quantitative RT-PCR. A,WT Tregs were serum-starved for 1 hour, andthen stimulated with the indicated concentration of PGE2 for 3 hours. Dose-dependent effect of PGE2 was tested. B, Tregs were serum-starved for 1 hour,and then stimulatedwith PGE2 (25mmol/L) for 3 hours following pretreatmentwithDMSOvehicle or PKA inhibitor H-89 (10 mmol/L) for 1 hour.C, Tregswere culturedwith or without 25 mmol/L PGE2 and H-89 (10 mmol/L) under TCR stimulation for 18 hours. D, WT or Nr4a-DcKO Tregs were cultured as described inC. Expression levels of PGE2-untreated Tregswere set as one. E,WTTregswere incubatedwith control media or tumor cell supernatant (TSN) under TCR stimulationfor 18 hours. Tumor cell supernatantwasderived fromMC38 tumor cells cultured in the presenceor absence of theCOX-2 inhibitor SC-236 (10mmol/L).F,QuantitativeRT-PCRanalysis ofmRNA for the indicated genes in CD4þCD25þWTTregs, isolated fromspleens of tumor-free (no tumor), and 3LL tumor–bearingmice treatedwithor without SC-236. Tregs from three mice per group were pooled for one sample. Intraperitoneal administration of SC-236 (3 mg/kg) was performed ondays 6 and 9, and mice were analyzed on day 14. G, Nr4a1 expression and phosphorylation level of CREB on tumor-infiltrating Tregs from 3LL tumor–bearing micetreated with or without SC-236 (n ¼ 4/group) as described in F. Representative histograms and bar graphs summarizing the fluorescence-activatedcell sorting (FACS) data are shown. Numbers in the histograms indicate mean fluorescence intensities (MFI). FMO, fluorescence minus one. A–E, Under culturecondition without PGE2, an equivalent volume of ethanol was added only as a control. A–F, Gene expression was normalized to 18S rRNA levels. Data arerepresentative of three independent experiments, each performed in triplicate. � , P < 0.05; �� , P < 0.01; �� , P < 0.001 compared with the untreated group unlessotherwise indicated; n.s., not significant; data are presented as the mean � SD.






Figure 5.

Combination therapywith CPT-11 and COX-2 inhibitor SC-236 synergistically induced potent antitumor immune responses.A, Schematic of the experimental design.WT mice were subcutaneously injected with 2.5 � 105 3LL tumor cells (day 0). CPT-11 (75 mg/kg) and/or SC-236 (3 mg/kg) were i.p. injected on days 6 and 9.B–F, 3LL tumor–bearingmice were left untreated or treated as described inA and analyzed on day 14 (n¼ 6–8/group). B, Tumor growthwasmonitored throughoutthe experimental period. C, a-CD8 antibodies were i.p. injected on days 5, 7, and 10. Tumor weights were measured. D, Frequency of CD4þ/CD8þ (top) andCD4þFoxp3þ (bottom) T cells in TDLNs. The plots show either gated CD3þ or CD4þ cells, respectively. The rightmost histogram shows gated CD4þFoxp3þ cells,and the numbers indicate mean fluorescence intensities (MFI) of Foxp3. E, The CD3þCD8þ T-cell fraction in D was further analyzed for Ki-67 expression.F, Frequency of tumor-infiltrating effector T cells (CD8þIFNgþ and CD4þIFNgþ). The plots show either gated CD8þ or CD4þ cells.D–F, Representative fluorescence-activated cell sorting (FACS) plots and bar graphs summarizing the FACS data are shown. � , P < 0.05; �� , P < 0.01; �� , P < 0.001 compared with the untreatedgroup unless otherwise indicated; data are presented as the mean � SD.


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Figure 6.

Therapeutic effects of CPT-11 and the COX-2 inhibitor SC-236 are dependent on inhibition of Treg function governed by Nr4a. A and B, 3LL tumor–bearingWTmicewere left untreated or treated with combination of CPT-11/SC-236 as described in Fig. 5A, followed by isolation of TDLN Tregs. Data are representative of threeindependent experiments involving 4 to 6mice per group.A,Quantitative RT-PCR analysis of mRNA for the indicated genes. Gene expressionwas normalized to 18SrRNA levels. Each experiment was performed in triplicate. B, In vitro suppression assay. Suppression of CFSE-labeled CD4þCD25�CD45.1þ cells (responderT cells; Tresp) by CD45.1� Tregs from tumor-free (no tumor) and 3LL tumor–bearing mice treated with or without inhibitors. Cells were stimulated with anti-CD3/28beads for 96 hours. Numbers in histograms represent percentages of undivided cells gated on CD4þCD45.1þ. C and D, 3LL tumor–bearing mice were leftuntreated or treated with combination of CPT-11/SC-236 as described in Fig. 5A. On the day after drug administration (day 10), in vitro–activatedWT or Nr4a-DcKOTregs were transferred. These mice were analyzed on day 15 (n ¼ 5–8/group). C, Tumor weights. D, Frequency of tumor-infiltrating effector T cells (CD8þ-IFNgþ,-TNFaþ, -CD107aþ). The plots show gated CD8þ T cells. Representative fluorescence-activated cell sorting (FACS) plots and bar graphs summarizing theFACS data are shown. � , P <0.05; �� , P <0.01; �� , P <0.001 comparedwith the untreated group unless otherwise indicated; n.s., not significant; data are presented asthe mean � SD.






CD8þCTL expansion at the TDLNs of DcKO mice. The secondmechanism is the failure of downregulation of costimulatorymolecules on DCs through CTLA-4. In a mouse pancreatic cancermodel, Jang and colleagues showed that Treg depletion evokes aCD8þ CTL-dependent antitumor immune response in a CD11cþ

DC-dependent manner (25). We also confirmed reduced expres-sion of CTLA-4 in Nr4a-DcKO Tregs and a drastic upregulation ofCD80 expression on tumor-infiltrating DCs in Nr4a-DcKO mice(Supplementary Fig. S3E–S3G). These observations suggest thatNr4a inhibition abolishes Treg-mediated functional control ofDCs. Taken together, Nr4a factors seem to affect various aspects oftumor immunity regulated by Tregs.

In this study,we identified the classical chemotherapeutic agentCPT as an inhibitor of Nr4a transcriptional activity. It is notablethat CPT treatment replicated the phenotype of Nr4a deletion inTregs both in vitro and in vivo. However, it remains unclear howCPT inhibits Nr4a activity, for example, whether or not CPTphysically interacts with Nr4a. Although we could not find struc-ture–activity relationships among the drugs we screened here,amodiaquine (AQ) and chloroquine (CQ), Nr4a agonists thatdirectly bind to the ligand-binding domain of Nr4a2 (35), con-tain an quinoline skeleton similar to CPT. According to thatreport, AQ and CQ share a 4-amino-7-chloroquinoline entity,and other tested quinoline compounds without that structure donot affect Nr4a activities. Another report suggested that Nr4aagonists and antagonists may bind to the same site, yet thedifference in the form of protein–drug interaction determineswhether they work as an activator or as an inhibitor (36). There-fore, we speculate that the quinoline skeleton is important for thebinding between the drug and Nr4a protein, but the structure ofother skeletons or side chains in the drug may determine itsfunction. Future studies are needed to explain the detailed mech-anism of interaction between Nr4a and CPT.

The COX-2/PGE2 pathway is involved in multiple aspects oftumor pathology; they directly promote tumor growth, regulateangiogenesis, and affect tumor-associated immune cells such asmacrophages, myeloid-derived suppressor cells, and T cells (29).In this study, we demonstrated that tumor-derived PGE2 inducedNr4a factors and their target genes on Tregs in a cAMP/PKA-dependentmanner, and their expression could bemodulated by acommon COX-2 inhibitor. Previous studies have shown thatPGE2 and its downstream cAMP/PKA pathway play importantroles in the maintenance of CD4þ T-cell biology, including Tregsuppression activities (37–40). This is also supported by our datademonstrating that suppressive activities of Treg cells wereenhanced by exposure to tumor-bearing conditions, but wereattenuated by COX-2 inhibitor (Fig. 6B; Supplementary Fig. S7A).In addition, Nr4a is well identified as a target molecule of cAMP/CREB in the signaling pathways involved in various physiologicand pathologic phenomena, such as hepatic glucose metabolismor the stress response in neurons (41, 42). Collectively, wespeculate that Nr4a factors function as a downstream regulatorof PGE2–cAMP–PKA signaling, thereby contributing to the for-mation of an immunosuppressive tumor microenvironment byTregs, and this regulatory axis canbe targetedbyCOX-2 inhibitors.

The recent success of cancer immunotherapy includingimmune checkpoint blockade, such as anti–PD-1 and anti–CTLA-4 antibody therapies, triggered the investigation of drugsthat activate antitumor immunity from already existing drugs(43, 44). Both CPT and COX-2 inhibitors were well defined aseffective antitumor drugs directly acting on tumor cells (27, 45),

but here we revealed that they also enhance antitumor immunityby inhibiting Nr4a/Treg function. A similar finding was recentlyreported for cyclin-dependent kinase 4 and6 (CDK4/6) inhibitors(13). CDK4/6 inhibitors such as abemaciclib have been shown tobe effective against several solid tumors, and their primary mech-anism of action is thought to be inhibition of cell-cycle progres-sion in tumor cells. Goel and colleagues (13) revealed thatabemaciclib also promotes antitumor immunity by selectivelysuppressing the proliferation of Tregs and increasing the immu-nogenicity of tumor cells. Their study and our present study willaccelerate the search for anticancer drugs that promote bothtumor cell death and antitumor immunity.

Nr4a factorsmay play important roles in not only Tregs but alsoCTLs. Mognol and colleagues reported that Nr4a factors areinvolved in the establishment of cancer-induced exhaustion intumor-infiltrating CD8þ CTLs (46). This study suggested a pos-sibility that Nr4a inhibition may also facilitate the reactivation ofexhausted CTLs like anti–PD-1 antibody, in addition to theinhibitory effects on Tregs. Another article showed that Nr4a1 isinvolved in the function and differentiation of a long-lived subsetof tissue-resident memory CD8þ (TRM) T cells (47). Tumor-infiltrating CD8þ T cells were reported to exhibit characteristicsof TRM cells (48). Thus, further study is necessary to elucidate thedetailed roles of Nr4a factors in antitumor CTLs.

In conclusion, we revealed thatNr4a factors play crucial roles invarious parts of Treg function in the tumor microenvironment,and these mechanisms could be applied to tumor therapy usingclinically established compounds. Nr4a factors show great prom-ise as effective therapeutic targets of cancer immunotherapy.

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

Authors' ContributionsConception and design: S. Hibino, A. YoshimuraDevelopment ofmethodology: S.Hibino, S. Chikuma, T. Kondo, A. YoshimuraAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Hibino, T. Kondo, M. Ito, S. Omata-Mise,A. YoshimuraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Hibino, H. Nakatsukasa, S. Omata-Mise,A. YoshimuraWriting, review, and/or revision of the manuscript: S. Hibino, S. Chikuma,T. Kondo, A. YoshimuraAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Chikuma, A. YoshimuraStudy supervision: A. Yoshimura

AcknowledgmentsWe thank N. Shiino, C. Ohkura, Y. Tokifuji, and Y. Hirata for their

technical assistance. This work was supported by JSPS KAKENHI (S)17H06175 (to A. Yoshimura), Advanced Research & Development Programsfor Medical Innovation (AMED-CREST) JP17gm0510019 (to A. Yoshimura),the Takeda Science Foundation (to A. Yoshimura), the Uehara MemorialFoundation (to A. Yoshimura), the SENSHIN Medical Research Foundation(to A. Yoshimura), and Grant-in-Aid for Scientific Research on InnovativeAreas17H05801 (to S. Chikuma).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 10, 2017; revised January 17, 2018; accepted March 15,2018; published first March 20, 2018.


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Hibino et al.




2018;78:3027-3040. Published OnlineFirst March 20, 2018.Cancer Res Sana Hibino, Shunsuke Chikuma, Taisuke Kondo, et al. Breaking Treg-Mediated Immune ToleranceInhibition of Nr4a Receptors Enhances Antitumor Immunity by

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