Interleukin-27 Exerts Its Antitumor Effects by Promoting ... · Corresponding Author: Takayuki Yoshimoto, Department of Immunoregula- tion, Institute of Medical Science, Tokyo Medical

Tumor Biology and Immunology

Interleukin-27 Exerts Its Antitumor Effects byPromoting Differentiation of HematopoieticStem Cells to M1 MacrophagesYukino Chiba, Izuru Mizoguchi, Junichi Furusawa, Hideaki Hasegawa,Mio Ohashi, Mingli Xu, Toshiyuki Owaki, and Takayuki Yoshimoto

Abstract

The interleukin IL27 promotes expansion and differentiation ofhematopoietic stem cells into myeloid progenitor cells. Manytumor-infiltrating myeloid cells exert immunosuppressive effects,but we hypothesized that themyeloid cells induced by IL27wouldhave antitumor activity. In this study, we corroborated this hypoth-esis as investigated in two distinct mouse transplantable tumormodels. Malignant mouse cells engineered to express IL27 exhib-ited reduced tumor growth in vivo. Correlated with this effect was asignificant increase in the number of tumor-infiltrating CD11bþ

myeloid cells exhibiting a reduced immunosuppressive activity.Notably, these CD11bþ cells were characterized by an activatedM1macrophage phenotype, on the basis of increased expression ofinducible nitric oxide synthase and other M1 biomarkers. In vivodepletion of these cells by administering anti–Gr-1 eradicated the

antitumor effects of IL27. When admixed with parental tumors,CD11bþ cells inhibited tumor growth and directly killed the tumorin a nitric oxide-dependentmanner. Mechanistically, IL27 expand-ed Lineage�Sca-1þc-Kitþ cells in bone marrow. Transplant experi-ments in Ly5.1/5.2 congenic mice revealed that IL27 directly actedon these cells and promoted their differentiation into M1 macro-phages, which mobilized into tumors. Overall, our results illus-trated how IL27 exerts antitumor activity by enhancing the gener-ation of myeloid progenitor cells that can differentiate into anti-tumorigenic M1 macrophages.

Significance: These findings show how the interleukin IL27exerts potent antitumor activity by enhancing the generation ofmyeloid progenitor cells that can differentiate into antitumori-genic M1 macrophages. Cancer Res; 78(1); 182–94. �2017 AACR.

IntroductionAlthough immunotherapeutic approaches against cancer are

promising new treatments, their effectiveness can be furtherimproved by overcoming the immunosuppression induced bytumors. These immunosuppressive mechanisms includeimmune checkpoint molecules, regulatory T cells, and immu-nosuppressive myeloid cells. Generally, tumor-bearing hostsand cancer patients have increased infiltration of immunosup-pressive myeloid cells, including tumor-associated macro-phages, M2 macrophages, and myeloid-derived suppressorcells (MDSC; refs. 1, 2). Macrophages are characterized byhigh plasticity and diversity with two major phenotypes,namely M1 (classically activated) and M2 (alternatively acti-vated) macrophages (3). The M1 macrophage phenotype ischaracterized by the production of pro-inflammatory cyto-kines, reactive oxygen species, and nitric oxide (NO) and thepromotion of helper T (Th) 1 immune responses, resulting instrong microbicidal and tumoricidal activity. In contrast, the

M2 macrophage phenotype is characterized by immunoregu-latory functions leading to the promotion of tissue remodelingand tumor progression.

IL27 is an IL12 family cytokine that consists of two distinctsubunits, Epstein-Barr virus-induced gene 3 and p28, and itsreceptor (R) is composed of IL27Ra (WSX-1/TCCR) and gly-coprotein 130 (4, 5). It is one of the pleiotropic cytokines withboth pro- and anti-inflammatory properties acting on variouscell types. IL27 promotes the early induction of Th1 differen-tiation and generation of cytotoxic lymphocytes, but it inhibitsthe differentiation to Th2 and Th17 cells and suppresses theproduction of pro-inflammatory cytokines (4, 5). Because wefirst demonstrated the antitumor efficacy of IL27 in 2004 usinga transplanted mouse tumor genetically engineered to secreteIL27 as a preclinical tumor model (6), accumulating evidencesuggests that IL27 has potent antitumor activity through mul-tiple mechanisms, depending on the characteristics of individ-ual tumors (7, 8). Moreover, we recently demonstrated thatIL27 can promote the expansion and differentiation of hemato-poietic stem cells (HSC) into myeloid progenitor cells and thatit plays a critical role in the control of parasite infection duringemergency myelopoiesis (9, 10).

IL27 was also demonstrated to augment the productionof immunosuppressive cytokine IL10 and enhance expressionof the immune checkpoint molecules such as programmeddeath-1 ligand 1 (PD-L1) and T-cell immunoglobulin andmucin domain-3 (TIM-3) in lymphocytes and macrophages(11–15). In addition, IL27 was recently shown to upregulateanother immunosuppressive molecule, CD39, together withPD-L1 and IL10 in macrophages, resulting in acquisition of

Department of Immunoregulation, Institute of Medical Science, Tokyo MedicalUniversity, Shinjuku-ku, Tokyo, Japan.

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

Corresponding Author: Takayuki Yoshimoto, Department of Immunoregula-tion, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku,Shinjuku-ku, Tokyo 160-8402, Japan. Phone: 81-3-3351-6141; Fax: 81-3-3351-6250; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0960

�2017 American Association for Cancer Research.

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immunoregulatory properties by tumor-associated macro-phages in various cancer patients (16, 17). These results indi-cate that macrophages may play an inhibitory role in theexertion of antitumor activity by IL27. However, myeloabla-tion by treosulfan, a chemotherapy drug used to induce a severeor complete depletion of bone marrow (BM) cells includingcells other than macrophages, was recently shown to mostlyabolish the antitumor effects of IL27 in mice bearing non-small cell lung cancer (18). Moreover, IL27 inhibited thedevelopment of M2 macrophages from the human monocyticcell line U937 differentiated by phorbol 12–myristate 13–acetate and subsequently polarized by IL4 (19). Therefore, ourunderstanding of the role of macrophages in the exertion ofantitumor activity by IL27 remains incomplete.

Although the mouse transplantable tumor model geneticallyengineered to secrete IL27 significantly decreased tumor growth(20, 21), we initially noticed that the number of tumor-infil-trating cells, including CD11bþ myeloid cells greatly increased.Therefore, we wondered whether these myeloid cells would beprotumorigenic or antitumorigenic. In the present study, toclarify this, we used two distinct mouse transplantable tumormodels, B16F10 melanoma and MC38 colon adenocarcinoma.Our results clearly revealed a novel mechanism whereby IL27exerts antitumor activity by promoting expansion and differ-entiation of HSCs into antitumorigenic M1 macrophages intumor-bearing mice.

Materials and MethodsCell lines and mice

Mouse melanoma cell line B16F10 and colon carcinoma cellline MC38 were kindly provided by Dr. Nariuchi (University ofTokyo, Tokyo, Japan) in 2007 and by Dr. Kawakami (KeioUniversity, Tokyo, Japan) in 2014, respectively. These cell lineswere expanded in our laboratory, frozen immediately, andstored in liquid nitrogen to ensure that cells used for experi-ments were passaged for fewer than 6 months. Early passagecells were used for all experiments. Cell line authentication wasnot routinely performed. But cell lines were tested routinely foridentity by appearance, staining with Hoechst 33258 (Dojindo,Kumamoto, Japan), or growth curve analysis in 3H-thymidineincorporation assay, and validated to be mycoplasma free.These cell lines were cultured at 37�C under 5% CO2/95% airin RPMI-1640 (Sigma) containing 10% FCS and 100 mg/mLkanamycin (Meiji Seika, Tokyo, Japan). BM cells and Line-age�Sca-1þc-Kitþ (LSK) cells were cultured in the same RPMI-1640 medium additionally supplemented with 50 mmol/L2 mercaptoethanol (Gibco). OT-I mice, C57BL/6 (CD45.2) mice,and C57BL/6 (CD45.1) mice were maintained in specific patho-gen-free conditions. All experiments were approved by the AnimalCare and Use Committee of Tokyo Medical University and wereperformed in accordance with our institutional guidelines.

Antibodies and reagentsMonoclonal antibodies (mAb) for mouse c-Kit (2B8), Sca-1

(D7), CD3e (145-2C11), CD8a (53-6.7), CD19 (6D5), CD49b(DX5), Gr-1 (RB6-8C5), TER119/erythroid cell (TER-119),CD11c (N418), CD11b (M1/70), F4/80 (BM8), NK1.1(PK136), B220 (RA3-6B2), FceRIa (MAR-1), macrophage col-ony-stimulating factor receptor (M-CSFR; AFS98), Flt3(A2F10), CD16/32 (2.4G2), CD150 (TC15-12F12.2), MHC

class II I-A/I-E (M5/114.15.2), Ly6G (1A8), Ly6C (HK1.4),CD45.1 (A20), and CD45.2 (104) were purchased from Bio-Legend. mAbs against mouse CD4 (GK1.5), CD34 (RAM34),and CD86 (GL1) were purchased from eBioscience. APC-Cy7–conjugated streptavidin, PerCP/Cy5.5-conjugated streptavidin,and Brilliant Violet 510-conjugated streptavidin were pur-chased from BioLegend and used to reveal staining with bio-tinylated Abs. Mouse recombinant IL27 was prepared as atagged single-chain fusion protein by flexibly linking EBI3 top28 using HEK293-F cells (Life Technologies), as describedpreviously (22). Mouse recombinant stem cell factor (SCF)and IL3 were purchased from PeproTech. Depletion mAb toGr-1 (RB6-8C5) was obtained from BioXCell. NG-monomethylL-arginine (L-NMMA) was purchased from Sigma-Aldrich.

Preparation of transfectantsB16F10 and MC38 tumor cells were transfected with the

expression vector of a FLAG-tagged single-chain fusion proteinof IL27–expression vector in p3xFLAG-CMV-14 vector (Sigma-Aldrich; ref. 6) or empty vector as control using Fugene 6 (Roche)and selected with Geneticin (G418). Resultant respective trans-fectants were designated as B16F10-IL27 or MC38-IL27 andB16F10-Vector or MC38-Vector, respectively.

Transplantable mouse tumor modelsTumor cells (1 � 106 cells/mouse) of B16F10 or MC38 were

injected subcutaneously into the right flank of mice (n ¼ 3–5).Tumor growth was monitored twice a week with an electroniccaliper and expressed as volume (mm3) by calculating using thefollowing volume equation: 0.5(ab2), where a is the long diameterand b is the short diameter.

Flow cytometryOn days 13 to 22 after tumor implantation, tumor was har-

vested frommice (n¼ 3–5),minced into small pieces, and treatedwith collagenase. Subsequently, tumor-infiltrating mononuclearcells were purified from it by Percoll (GE Healthcare) densitygradient centrifugation. Then, flow cytometry was performed on aFACS Canto II (BD Biosciences), and data were analyzed usingFlowJo Software (Tree Star). Cell number was counted by usingflow cytometry. The following cell surface markers were used toidentify M1 and M2 macrophages (Supplementary Fig. S1): M1macrophages, Class IIhighCD86þCD45.2þCD11bþF4/80þ; M2macrophages, Class IIlowCD86þCD45.2þCD11bþF4/80þ (1, 23).

Quantitative real-time RT-PCRTotal RNA was prepared from the tumor-infiltrating mononu-

clear cells ofmice (n¼ 4) using an RNeasyMini Kit (Qiagen), andcDNAwas prepared using oligo(dT) primer and SuperScript III RT(Thermo Fischer Scientific). Real-time quantitative PCR was per-formed using SYBR Premix Ex Taq II and a Thermal Cycler Dicereal-time system according to the manufacturer's instructions(Takara). Glyceraldehyde-3-phosphate (GAPDH) was used as ahousekeeping gene to normalize mRNA. Relative expression ofreal-time PCR products was determined by using the DDCt meth-od to compare target gene andGAPDHmRNAexpression. Primersused in this study are listed in Supplemental Table S1.

Measurement of immunosuppressive activityOn days 13 to 20 after tumor implantation, tumor was har-

vested from mice (n ¼ 3 or 4), minced into small pieces, and

Differentiation to Antitumorigenic M1 Macrophages by IL27

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treated with collagenase; tumor-infiltrating mononuclear cellswere purified from it by using Percoll density gradient centrifu-gation. Tumor-infiltrating myeloid cells were further isolated byAutoMACS Pro after staining with anti-CD11b magnetic beads(Miltenyi Biotec). Immunosuppressive activity was then assessedby the ability to inhibit ovalbumin (OVA)-specific CD8þ T-cellproliferation. Spleen cells (5 � 105 cells/200 mL) obtained fromOT-I T-cell receptor transgenic mice were stimulated with OVA(0.3mg/mL) in the presence of increasing cell numbers (1, 3, and10� 104 cells) of CD11bþmyeloid cells for 48 h and pulsed with3H-thymidine for last 8 hours, and the incorporation of 3H-thymidine was measured in triplicate.

Depletion experimentAfter tumor (2 � 105 cells/mouse) implantation, 0.3 mg of

anti–Gr-1 mAb (RB6-8C5) or control rat IgG was intraperitone-ally administered to mice (n ¼ 5) three times a week to depletemyeloid cells. Tumor growth was monitored twice a week. Thedepletion level was confirmed by analyzing blood samplesobtained from the tail vein.

Admixture experimentCD11bþmyeloid cells (1� 106 cells) were purified from tumor

of mice (n¼ 4–6) on days 13 to 22 after tumor implantation. Thecells were admixed with parental tumor (2� 105 cells) in 200 mLPBS and then immediately injected into the right flank of mice.Tumor growth was monitored twice a week.

Killing assayCD11bþmyeloid cells were purified from tumor of mice (n¼

4 or 5) on days 13 to 22 after tumor implantation. Parentaltumor cells (1 � 106 cells/50 mL of 50% fetal calf serum/RPMI-1640) were labeled by incubating in 100 mCi 51Cr for 45 minat 37�C and then washed three times. Labeled target cells(1 � 104 cells/200 mL) were incubated with purified CD11bþcells (1 � 105 cells) in a 96-well U-bottomed plate at 37�C for4 hours. Supernatants then were analyzed in a scintillationcounter. The percentage of lysis was determined in triplicateas (experimental count� spontaneous count)� 100/(maximalcount � spontaneous count). Results were expressed as thepercentage of specific lysis. The effect of inducible nitric oxidesynthase (iNOS) inhibitor was analyzed in the presence orabsence of L-NMMA (100 mmol/L).

Measurement of NO productionNO production by CD11bþ myeloid cells (1 � 105 cells/200

mL) purified from tumor ofmice (n¼ 4or 5) on days 17 to 21 aftertumor implantation was determined by measuring nitrite(NO2

�), a stable and non-volatile by-product of NO, producedin culture supernatants for 48 hours using a Griess reagent kit(Thermo Fischer Scientific). The Griess reagent and nitrite stan-dards or supernatants were mixed, incubated, and then read at520 and 590 nm as a reference tomeasure nitrite levels. The effectof iNOS inhibitor was analyzed in the presence or absence ofL-NMMA (100 mmol/L).

Preparation of LSK cellsSpleen and BM Lineage� cells were enriched by negative selec-

tion using an AutoMACS Pro with a combination of magneticbeads conjugated with mAbs against CD3e, CD4, CD8a, Gr-1,TER119, CD11b, CD11c, NK1.1, B220, and FceRIa. Cells were

then stained with mAbs against c-Kit, and Sca-1 was used for LSK.Sorting was performed on a MoFlo XDP (Beckman Coulter).

In vitro differentiation assayFor analysis of multipotent myeloid potential, LSK cells were

purified from BM cells in tumor-bearing mice (n ¼ 3) on days17 to 20 after tumor implantation or untreated mice. The sortedLSK cells (2.5 � 102/200 mL) were cultured with 10 ng/mLSCF and 10 ng/mL IL3 in 96-well plates for 7 days. Half ofthe medium was replaced every 3 days, with cytokines added.The following cell surface markers were used to identify respec-tive cells (Supplementary Fig. S1): neutrophil, Ly6GþCD11bþ

or Gr-1þCD11bþ; macrophage, F4/80þCD11bþ; mast cell,c-KitþFceR1aþCD11b�; basophil, CD49bþFceR1aþCD11b�.

Adoptive cell transferBM LSK cells (5 � 103) purified from C57BL/6 (CD45.1)

congenic mice were intravenously transferred into sublethallyirradiated (4 Gy) C57BL/6 (CD45.2) recipient mice (n ¼ 4). Onthe next day, tumor cells (1 � 106 cells) of B16F10-IL27 orB16F10-Vector were injected subcutaneously into the right flankof resulting mice. Tumor growth was monitored twice a week.

ELISASerumprotein level of TGFb1was determined by ELISA accord-

ing to the manufacturers' instructions (R&D Systems).

Statistical analysisData are presented as mean � SEM. Statistical analyses were

performed by using the two-tailed Student t test for comparisonsof two groups and one-way ANOVA and Tukey–Kramer multiplecomparison test for comparing more than three groups usingGraphPad Prism 7 (GraphPad Software Inc.). P < 0.05 wasconsidered to indicate a statistically significant difference.

ResultsIL27 augments the infiltration of CD11bþ myeloid cells intotumor

As reported previously (20), melanoma B16F10 tumor cellstransfected with IL27–expression vector (B16F10-IL27) showedgreat inhibition of tumor growth compared with those trans-fected with control empty vector (B16F10-Vector; Fig. 1A andB). During analysis of antitumor activity of IL27, we initiallynoticed that the percentage and total number of mononuclearcells infiltrating into tumors were significantly increased byIL27 (Fig. 1C and D). Implantation of tumors also increased thespleen weight regardless of B16F10-IL27 or B16F10-Vector (Fig.1B), implying augmented myelopoiesis by tumor growth.Among the various types of cells, FACS analyses revealed thatIL27 greatly increased the percentage and number of not onlyCD8þ T cells but also CD11bþ myeloid cells in tumor pertumor volume, as well as per tumor weight (Fig. 1E and F). Asimilar increase of tumor-infiltrating CD11bþ myeloid cellswas observed with another tumor model, MC38 colon carci-noma (Supplementary Fig. S2).

Given that tumor-infiltratingmyeloid cells are generally immu-nosuppressive cells, such as M2 macrophages and MDSCs, it isimportant to investigate the properties of IL27–medaited infil-trating myeloid cells, their role in induction of antitumor effects,

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Figure 1.

IL27 augments the infiltration of CD11bþ myeloid cells into tumor. A, B16F10-IL27 or B16F10-Vector tumor was implanted and tumor growth was monitored.B–D, On day 13, tumor and spleen were harvested, and their weights were measured (B), single-cell suspension was then prepared from the tumorand subjected to flow cytometer analysis (C), and total number of tumor-infiltrating mononuclear cells was counted (D). E, Representative dot plotsof individual cell populations are shown. F, Their cell numbers were counted, and calculated per tumor volume and also per tumor weight. Data are shownas mean � SEM (n ¼ 3 or 4) and are representative of six independent experiments. � , P < 0.05; �� , P < 0.001.






and the molecular mechanism whereby IL27 induces such aphenomenon.

IL27 promotes the differentiation into M1 macrophages intumor-bearing mice

M1 and M2 macrophages have antitumorigenic activity andprotumorigenic activity, respectively (3). Therefore, we nextexamined the phenotype of these IL27–mediated tumor-infil-trating myeloid cells. On day 13 after tumor implantation,mononuclear cells were purified from tumor and subjected tocell surface analysis by using FACS. IL27 greatly increased thepercentage and number of monocytic (Mo�)MDSCs (1, 2) andmacrophages, but decreased those of granulocytic (Gr�)MDSCs(1, 2), in tumor per tumor volume, as well as per tumorweight (Fig. 2A and B). Among these tumor-infiltrating macro-phages, IL27 greatly increased the percentage and number of M1macrophages (Class IIhighCD86þCD45.2þCD11bþF4/80þ) ascompared with those of M2 macrophages (ClassIIlowCD86þCD45.2þCD11bþF4/80þ; Fig. 2C–E). The ratio ofM1/M2 macrophages was significantly increased, indicatingthe enhanced bias toward M1 macrophages by IL27 (Fig. 2Dand E). Consistent with this, mRNA expression analysesrevealed that CD11bþ myeloid cells purified from tumor ofB16F10-IL27 tumor-bearing mice express more M1 macro-phage markers, such as iNOS, interferon regulatory factor 8(IRF8), and IL12p40, but less M2 macrophage markers, such asariginase-1 (Arg-1), chitinase 3-like-3 (Ym1), and found ininflammatory zone 1 (Fizz1), compared with those of B16F10-Vecter tumor-bearing mice (Fig. 2F). Similar results wereobtained with the MC38 tumor model (Supplementary Fig.S3). These results suggest that IL27 promotes differentiationinto M1 macrophages in tumor-bearing mice.

Because IL27–transduced tumor grows at a much slower ratethan control tumor, not only the tumor size but also the tumormicroenvironment might be different between them and there-fore affect the number and phenotype of tumor-infiltratingcells. To examine this possibility, we tried to adjust the tumorsize to reach the same size by changing the conditions such asthe cell number of tumors to inject, and then analyzed thetumor-infiltrating cells by flow cytometry. Under these condi-tions, we could still observe that IL27 much less but signifi-cantly enhances the percentage and number of tumor-infiltrat-ing cells including macrophages, especially with M1 phenotype(Supplementary Fig. S4).

IL27 inhibits differentiation into immunosuppressivemacrophages in tumor-bearing mice

Generally, tumor-infiltrating myeloid cells are immunosup-pressive (1). Therefore, we next examined immunosuppressiveactivity of tumor-infiltrating myeloid cells. On day 16 aftertumor implantation, mononuclear cells were purified fromtumor, and CD11bþ myeloid cells were further purified fromthem and subjected to immunosuppressive activity, which wasassessed by the ability to inhibit OVA-specific CD8þ T-cellproliferation. Myeloid cells purified from tumor of B16F10-Vector tumor-bearing mice showed marked inhibitory activityagainst the antigen-specific proliferation (Fig. 3). In contrast,myeloid cells purified from tumor of B16F10-IL27 tumor-bearing mice showed significantly reduced inhibitory activitycompared with those of control tumor-bearing mice (Fig. 3).Similar results were obtained with the MC38 tumor model

(Supplementary Fig. S5). Thus, IL27 inhibits the generationof immunosuppressive myeloid cells such as M2 macrophagesin tumor-bearing mice, but it promotes the generation of M1macrophages.

IL27-induced tumor-infiltrating myeloid cells have potentantitumor effects

To further explore the role of myeloid cells in B16F10-IL27tumor-bearing mice in the IL27-mediated antitumor activity,depletion experiments of myeloid cells by treatment with anti-Gr-1 mAb were performed. Treatment of mice with anti–Gr-1mAb almost completely depleted Gr-1þCD11bþ myeloidcells in blood (Fig. 4A). Under these conditions, depletion ofmyeloid cells in B16F10-Vector tumor-bearing mice greatlyinhibited the tumor growth, as generally expected (Fig. 4A).In contrast, depletion of myeloid cells in B16F10-IL27 tumor-bearing mice significantly promoted the tumor growth(Fig. 4B). These results suggest that IL27 exerts antitumoractivity via myeloid cells.

IL27-induced tumor-infiltrating myeloid cells exert directantitumor effects by killing tumor through NO

To further examine the effect of IL27 onmyeloid cells in tumor-bearingmice, admixture experiments ofmyeloid cells with paren-tal B16F10 tumor were performed. Consistent with the results ofdepletion experiments, admixture of CD11bþmyeloid cells puri-fied from tumor of B16F10-Vector tumor-bearing mice withparental B16F10 tumor greatly promoted the tumor growth (Fig.5A). In contrast, admixture of CD11bþ myeloid cells purifiedfrom tumor of B16F10-IL27 tumor-bearing mice with parentalB16F10 tumor markedly inhibited the tumor growth (Fig. 5A).Again, tumor-infiltratingmyeloid cells are quite different betweenB16F10-IL27 and B16F10-Vector tumor-bearingmice, being anti-tumorigenic versus protumorigenic cells.

To further evaluate the therapeutic potential of the CD11bþ

myeloid cells purified from tumors of B16F10-IL27 tumor-bearing mice, the antitumor efficacy of these myeloid cells onpre-existing tumor model was examined. When mice wereimplanted with parental B16F10 tumor and their tumor sizereached 5 mm in diameter on day 5, these CD11bþ myeloidcells were both intravenously and intratumorally injected intothese mice with the pre-existing tumor. By using these injec-tions, we could observe a tendency to inhibit the tumor growth(Supplementary Fig. S6).

Next, to investigate the molecular mechanism whereby IL27augments antitumor activity via myeloid cells, the possibilityof direct killing of tumors by macrophages was examined byusing a 51Cr-releasing assay. CD11bþ myeloid cells purifiedfrom tumor of B16F10-IL27 tumor-bearing mice showed great-ly augmented killing activity compared with those of B16F10-Vector tumor-bearing mice (Fig. 5B). Correlating with theaugmented killing activity, myeloid cells purified from tumorof B16F10-IL27 tumor-bearing mice produced more NO thanthose of B16F10-Vector tumor-bearing mice (Fig. 5C). Treat-ment with NOS inhibitor, L-NMMA, almost completelyblocked the killing activity of myeloid cells purified fromtumor of B16F10-IL27 tumor-bearing mice (Fig. 5D). Similarresults were obtained with the MC38 tumor model (Supple-mentary Fig. S7).

Thus, IL27 promotes the generation of antitumorigenic mye-loid cells rather than protumorigenic myeloid cells in tumor-

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Figure 2.

IL27 promotes the differentiation into M1 macrophages in tumor-bearing mice. A and B, Single-cell suspensions prepared from tumor on day 13 aftertumor implantation were analyzed by flow cytometry. Representative dot plots of Gr-MDSCs (CD45.2þCD11bþLy6GþLy6Cint), Mo-MDSCs(CD45.2þCD11bþLy6G�Ly6Chi), and macrophages (CD45.2þCD11bþF4/80þ) are shown, and their cell numbers were counted and calculated per tumor volumeand also per tumor weight. C, Representative dot plots of M1 macrophages (Class IIhighCD86þCD45.2þCD11bþF4/80þ) and M2 macrophages (ClassIIlowCD86þCD45.2þCD11bþF4/80þ) are shown. D and E, Their cell numbers were counted, and calculated per tumor volume (D) and also per tumorweight (E), together with respective ratio of M1/M2 macrophages. Data are shown as mean � SEM (n ¼ 3 or 4) and are representative of more than sixindependent experiments. F, mRNA expression of markers for M1 and M2 macrophages in purified CD11bþ myeloid cells from tumor-bearing mice on day 17was analyzed by real-time RT-PCR. Data are shown as mean � SEM (n ¼ 4) and are representative of more than six independent experiments. � , P < 0.05;�� , P < 0.01; �� , P < 0.001.






bearing mice, and the antitumorigenic myeloid cells exert directkilling activity against tumor through NO.

IL27 induces the expansionof LSK cellswith enhanced ability todifferentiate into M1 macrophages

Generally, tumor-bearing mice have increased production oftumor-derived cytokines such as G-CSF and GM-CSF, resulting in

the promotion of myelopoiesis in the BM and spleen (1). Con-sistent with this phenomenon, tumor-bearing mice have spleno-megaly (Fig. 1B). Therefore, we investigated the effects of IL27 onthe myelopoiesis in tumor-bearing mice. Similar to the resultsobtained by using the malaria infection model (10), IL27increased the percentage and number of LSK cells in both BMand spleen (Fig. 6A).Moreover, IL27 also increased the percentageand number of the most primitive HSC population, long-termHSC (LT-HSC; ref. 24), but not multipotent progenitor (MPP)in both BM and spleen (Fig. 6B). Thus, IL27 directly affectsLSK cells and LT-HSCs in the BM to promote the myelopoiesisin transplantable tumor models.

Next, the multipotency of LSK cells in the BM of tumor-bearing mice was examined under in vitro myeloid celldifferentiation conditions. LSK cells purified from the BM ofB16F10-Vector tumor-bearing mice had less ability to differ-entiate into myeloid cells such as mast cells, basophils, neu-trophils, and macrophages compared with those purified fromuntreated mice (Fig. 6C and D). However, LSK cells purifiedfrom the BM of B16F10-IL27 tumor-bearing mice had agreater ability to differentiate into these myeloid cells com-pared with those of B16F10-Vector tumor-bearing mice (Fig. 6Cand D). Interestingly, BM LSK cells from B16F10-IL27 tumor-bearing mice had an enhanced ability to differentiate intoM1 macrophages compared with those from B16F10-Vectortumor-bearing mice (Fig. 6E). These results suggest that IL27promotes the expansion and generation of LSK cells, whichhave an enhanced ability to differentiate into myeloid cells,especially with the M1 macrophage phenotype.

IL27 directly acts on BM LSK cells and promotes the expansionand differentiation into M1 macrophages in vivo

To further examine the direct effect of IL27 on BM HSCsto differentiate into M1 macrophages in vivo, adoptive cell

Figure 3.

IL27 inhibits the differentiation into immunosuppressivemacrophages in tumor-bearing mice. On day 16 after tumor implantation, mononuclear cellswere purified from tumor, and CD11bþ myeloid cells were further purifiedand subjected to immunosuppressive activity, which was assessed by theability to inhibit OVA-specific CD8þ T-cell proliferation. Data are shown asmean � SEM (n ¼ 3 or 4) and are representative of more than sixindependent experiments. � , P < 0.05; ��, P < 0.01; ��, P < 0.001.

Figure 4.

IL27–induced tumor-infiltrating myeloid cells havepotent antitumor effects.A,Mice were treatedwith anti–Gr-1 mAb or control IgG, and deletion level of neutrophilswas analyzed in blood by flow cytometry on day 7 aftertumor implantation. B, Tumor growth was monitoredwith time course. Data are shown asmean� SEM (n¼ 5)and are representative of three independentexperiments. � , P < 0.05.

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transfer of BM LSK cells purified from CD45.1 congenic miceinto sublethally irradiated CD45.2 recipient mice was per-formed. Tumor cells of B16F10-IL27 or B16F10-Vector werethen implanted, and tumor growth was monitored. As shownbefore (Fig. 1A), IL27 significantly inhibited tumor growth (Fig.7A). On day 20 after tumor implantation, tumor-infiltratingcells were analyzed. IL27 greatly increased the percentage andnumber of CD45.1þ cells and macrophages infiltrating intotumors (Fig. 7B and C). Consistent with the in vivo results (Fig.2) and in vitro differentiation results (Fig. 6C–E), IL27 markedlyincreased the percentage and number of M1 macrophages intotumor (Fig. 7D). Thus, IL27 directly acts on the BM LSK cellsand promotes the expansion and differentiation into antitu-morigenic M1 macrophages in tumor-bearing mice.

DiscussionAccumulating evidence suggests that IL27 possesses potent

antitumor activity through multiple mechanisms depending onthe characteristics of individual tumors (7, 8). The present resultsindicate that IL27 has another novel mechanism to exert potentantitumor activity. IL27 promotes the expansion and differenti-ationofHSCs intomyeloidprogenitor cells, whichhave enhancedpotential to differentiate into antitumorigenic M1 macrophageswith increased expression of iNOS, IRF8, and IL12p40 butreduced expression of Arg-1, Ym1, and Fizz1 in tumor-bearingmice. Blocking experiments using the iNOS inhibitor, L-NMMA,further revealed that the CD11bþ myeloid cells purified fromIL27-tumor-bearing mice directly kill tumors in a highly NO-dependentmanner,whereaswhether other potentialmechanismsmay also exist remains to be elucidated. This is consistent with ourprevious report showing that IL27 augments the production ofNO in thioglycollate-elicited peritoneal macrophages in synergywith lipopolysaccharide through STAT1, NF-kB, andMAP kinases

(25). We also reported that IL27 greatly induced IRF1 and IRF8expression in mouse melanoma B16F10-expressing WSX-1, lead-ing to inhibition of its tumor growth (21). Importantly, IRF8 is acritical transcription factor for the development of monocytes,dendritic cells, eosinophils, and basophils in synergy with PU.1(26), whereas IRF8 inhibits CCAAT-enhancer-binding protein aactivity to suppress the generationof neutrophils (26). IRF8 is alsoa critical transcription factor for induction of iNOS expression incollaboratingwith IRF1 (27) orNF-kB (28), resulting in enhancedNOproduction. In addition, IRF8 is an integral negative regulator(29) against the development of MDSCs by promoting sponta-neous apoptosis and turnover in myeloid cells (30) and byrepressing GM-CSF expression in T cells (31). Thus, IL27-medi-ated promotion of HSCs to expand and differentiate into anti-tumorigenic M1 macrophages with enhanced iNOS expressionpresumably is highly dependent on IRF8, but this possibilityremains to be further clarified.

Of note, the present results indicate that properties of tumor-infiltrating myeloid cells in the presence or absence of IL27 arequite different. Myeloid cells in IL27 tumor-bearing mice areantitumorigenic with the M1 macrophage phenotype, whereasthose in control tumor-bearing mice are protumorigenic with theM2 macrophage phenotype (Figs. 2–5A). Myeloid cells in IL27tumor-bearing mice were demonstrated to kill parental tumorsdirectly in a NO-dependent manner (Fig. 5B–D). Moreover, IL27increased the percentage and number of LSK cells in both BM andspleen of tumor-bearing mice (Fig. 6A and B). We previouslydemonstrated that IL27 directly acts on LSK cells, especiallyLT-HSCs, and promotes their expansion and differentiation intophenotypically similar LSK cells but distinct myeloid progenitors(10). LSK cells in the BM of control tumor-bearing mice have areduced ability to ex vivodifferentiate intomyeloid cells comparedwith LSK cells in the BM of untreated mice, possibly due to theimmunosuppressive effects of myeloid cells on LSK cells in the

Figure 5.

IL27-induced tumor-infiltrating myeloid cells exertdirect antitumor effects by killing tumor throughNO. A, CD11bþ myeloid cells purified from tumor ofB16F10-IL27 or B16F10-Vector tumor-bearing micewere admixed with parental B16F10 tumor, and tumorgrowth was monitored with time course. Data areshown as mean � SEM (n ¼ 6) and are representativeof more than three independent experiments. B and C,On day 18 after tumor implantation, killing activity ofCD11bþ myeloid cells against parental B16F10 tumorwas measured by 51Cr release assay (B), and NOproduction in the culture supernatant was measuredin the presence or absence of NOS inhibitor, L-NMMA(C). D, The effect of inhibition of NOS activity byL-NMMA on the killing activity was examined. Dataare shown as mean � SEM (n ¼ 4 or 5) and arerepresentative of more than five independentexperiments. � , P < 0.05; �� , P < 0.001.






tumor-bearing mice (Fig. 6C and D). In contrast, LSK cells in theBM of IL27-tumor-bearingmice have a greatly increased ability toex vivo differentiate intomyeloid cells including M1macrophagescompared with those of control tumor-bearing mice (Fig. 6C–E).Finally, adaptive cell transfer experiments revealed that IL27 actsdirectly on the LSK cells in the BM in vivo and promotes theexpansion and differentiation into M1 macrophages, mobilizingthem in tumor (Fig. 7).

Although the infiltration of CD8þ T cells into IL27–treatedtumors is dominant in B16F10 melanoma model (Fig. 1F),the infiltration of CD4þ T cells is dominant in MC38 coloncarcinoma model (Supplementary Fig. S2C). Although themolecular mechanism whereby IL27 induces such distinctdominancy remains elusive, IL27 also greatly induces theinfiltration of CD11bþ myeloid cells into tumor in both mod-els irrespectively of CD8þ or CD4þ T cell infiltration. One of the

Figure 6.

IL27 induces the expansion of LSK cellswith enhanced ability to differentiate intoM1 macrophages. A and B, On day 13 aftertumor implantation, cell populations of LSK(A), LT-HSC, and MPP (B) were analyzedby flow cytometry. Data are shown asmean � SEM (n ¼ 3 or 4) and arerepresentative of more than sixindependent experiments. C and D, Themultipotency of LSK cells in the BM ofB16F10-IL27 or B16F10-Vector tumor-bearing mice on day 19 after tumorimplantation together with untreated micewas examined under in vitro myeloid celldifferentiation conditions into mast cells,basophils, neutrophils, and macrophages.E, Cell number of M1 and M2 macrophageswas counted and the ratio of M1/M2macrophages was calculated. Data areshown as mean � SEM (n ¼ 3) and arerepresentative of more than threeindependent experiments. � , P < 0.05;�� , P < 0.01; �� , P < 0.001.

Chiba et al.





possible reasons for the different effects by IL27 on the tumor-infiltrating cells may be ascribed to the different microenvi-ronment situations such as distinct places and stages whereIL27 acts on these cells. In the case of myeloid cells, IL27directly acts on the BM cells and promotes the differentiationinto M1 macrophages following the infiltration into tumor(Fig. 6 and 7). On the other hand, IL27 likely acts on naiveT cells in the spleen or lymph node to promote the differen-

tiation and proliferation of them (32). Moreover, IL27 seem-ingly induced high M1/M2 ratio and NO production of thetumor-infiltrating CD11bþ myeloid cells in the MC38 modelcompared with those in the B16F10 model (Fig. 1C and 5C;Supplementary Figs. S3C and S7C). Correlating to these results,the killing activity of the CD11bþ myeloid cells augmentedby IL27 in relation to that by control vector in the MC38model was higher than that in the B16F10 model (41.8 �

Figure 7.

IL27 directly acts on BM LSK cells and promotesthe expansion and differentiation into M1macrophages in vivo. A, BM LSK cells purifiedfrom CD45.1 congenic untreated mice wereadoptively transferred into sublethally irradiatedCD45.2 recipient mice, and then tumor cells ofB16F10-IL27 or B16F10-Vector were implanted,and tumor growth was monitored with timecourse. B and C, On day 20 after tumorimplantation, percentage and number ofCD45.1þ or CD45.2þ cell population (B) andmacrophages (C) were measured. D,Representative dot plots of M1 and M2macrophages are shown, their cell numberswere counted, and ratio of M1/M2 macrophageswas calculated. Data are shown as mean � SEM(n ¼ 4) and are representative of more thanthree independent experiments. � , P < 0.05.






18.9-folds vs. 7.1 � 4.2-folds; Fig. 5B; Supplementary Fig. S7B).Further studies are necessary to clarify the different effects byIL27 between these two tumor models.

Because IL27 induces upregulation of immunosuppressivemolecules such as PD-L1, CD39, TIM-3, and IL10 (11–17), thepossibility that IL27 may induce protumorigenic effects hasbeen discussed (8, 33). However, by using histopathologicalanalyses, Airoldi and colleagues (18) observed that IL27 treat-ment increases the infiltration of granulocytes and macro-phages into tumor using xenograft models of human lungadenocarcinoma and squamous cell carcinoma cell linesCalu-6 and SK-MES in immunodeficient mice. To assess wheth-er these granulocytes and macrophages may account for theantitumor effects of IL27, mice were pretreated with myeloa-blative doses of treosulfan. The myeloablation significantlyabolished the antitumor effect of IL27, although this treatmentinduced a severe or complete depletion of BM cells, whichaffects the cellularity of peripheral cells other than macro-phages (18). Yao and colleagues (19) also recently reportedthat IL27 inhibits the development of M2 macrophage fromthe human monocytic cell line U937 differentiated by phorbol12–myristate 13–acetate and subsequently polarized by IL4in vitro. Consistent with the present results, these two reportsfurther support the possibility that macrophages play a criticalrole in exerting antitumorigenic effects rather than protumori-genic effects by IL27 in tumor-bearing mice.

Given that IL27 is a multifunctional cytokine with a double-edged sword, both antitumor and protumor effects of IL27 areconceivably expected. Indeed, we observed enhanced mRNAexpression of immune checkpoint molecules such as PD-L1 andTIM-3 by in tumor-infiltrating CD11bþ myeloid cells (Supple-mentary Fig. S8A and S8B) as reported (14, 15, 17). Similartendency of the up-regulation of PD-1 mRNA expression by IL27was also observed, although its expression level even in steadystate of myeloid cells seemed to be low (Supplementary Fig. S8Aand S8B; ref. 34). Similar upregulation of TGFb1 mRNA expres-sion by IL27 was observed in tumor-infiltrating CD11bþmyeloidcells, but its serum level was rather decreased by IL27 (Supple-mentary Fig. S8C). Because IL27 was recently reported to inhibitthe functions of TGFb1, including induction of fibrosis andepithelial–mesenchymal transition (35, 36), which contributeto antitumor effects, further study is necessary to clarify the roleof TGFb1 in the exertion of antitumor effect by IL27. BecauseB16F10 tumor does not express endogenous one of IL27R sub-units, WSX-1, IL27 does not directly act on B16F10 tumor cells toinhibit the immunosuppressive functions of the tumors (21).Rather, IL27 induces antitumor activity via acting on other cellsincluding T cells, NK cells, macrophages, and endothelial cells inthe tumor microenvironment (7, 8). Thus, the overall antitumorpotency of IL27 is highly likely to result from the sum of itspositive and negative effects on tumors within the tumor micro-environment. Therefore, if the IL27 therapy could be combinedwith the immune checkpoint blockade byusing antibody (37),wewould expect a synergistic antitumor effect between them. Thispossibility is currently under investigation.

IL27 belongs to the IL12 cytokine family including IL12,IL23, and so on. IL12 plays a critical role in the induction ofTh1-type immune responses through STAT4 activation, and itwas demonstrated to induce strong antitumor effects viamainly IFN-g production in various preclinical models (38).However, the clinical trials of IL12 have been limited by

systemic toxicities such as hepatomegaly, leukopenia, andgastrointestinal toxicity (39–42). IL23 plays a key role inautoimmune and inflammatory diseases through STAT3/STAT4 activation via IL23/IL17 immune axis (43). IL23 wasdemonstrated to promote tumorigenesis by inducing inflam-mation, which inhibits adaptive immune surveillance (44).Nevertheless, multiple studies have also shown the antitumoreffects by IL23 in various mouse tumor models (45–47).Thus, the role of IL23 in tumor procession is still controversial.On the other hand, IL27 activate STAT1 and STAT3 throughits respective receptor subunits, WSX-1 and gp130, respectively(48, 49). IL27 was previously reported to induce direct anti-proliferative effects on melanoma cells via STAT1 signaling asdoes IFN-g (21). IL27 has very low toxicity, probably becauseof low induction of IFN-g (6, 47). IL27 treatment does notinduce severe liver injury with increased levels of aspartate andalanine aminotransferases (6, 47), and transgenic mice con-stitutively overexpressing IL27 can survive for almost thewhole life span (50). Such a low toxic property of IL27 is ofbenefit to its clinical application.

Taken together, our present findings clearly indicate thattumor-infiltrating myeloid cells play a critical role in exertionof antitumor effects by IL27, although these myeloid cells showhigh expression of immune checkpoint molecules. To ensurepatient safety, therefore, combination therapy of IL27 withmonoclonal antibodies against these immune checkpointmolecules could be a more promising and effective therapeuticstrategy against cancer.

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

Authors' ContributionsConception and design: Y. Chiba, T. YoshimotoDevelopment of methodology: Y. Chiba, I. Mizoguchi, T. YoshimotoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. OhashiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. OhashiWriting, review, and/or revision of themanuscript: Y. Chiba, M. Xu, T. Owaki,T. YoshimotoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Furusawa, T. OwakiStudy supervision: T. Yoshimoto

AcknowledgmentsThe authors thank Dr. H. Nariuchi (University of Tokyo) and

Dr. Y. Kawakami (Keio University) for the B16F10 mouse melanomacell line and MC38 colon carcinoma cell line, respectively, and also thankR. Tsunoda (Tokyo Medical University) for his encouragement. This studywas supported in part by a grant-in-aid (to T. Yoshimoto) and the PrivateUniversity Strategic Research Based Support Project (to T. Yoshimoto)from the Ministry of Education, Culture, Sports, Science, and Technology,Japan, by Research Grant from Princess Takamatsu Cancer Research Fund(to T. Yoshimoto), and by Research Fellowship for Young Scientists (DC2)from Japan Society for the Promotion of Science (to Y. Chiba).

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 April 4, 2017; revised August 25, 2017; accepted October 27, 2017;published OnlineFirst November 1, 2017.

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Chiba et al.




2018;78:182-194. Published OnlineFirst November 1, 2017.Cancer Res Yukino Chiba, Izuru Mizoguchi, Junichi Furusawa, et al. Differentiation of Hematopoietic Stem Cells to M1 MacrophagesInterleukin-27 Exerts Its Antitumor Effects by Promoting

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Interleukin-27 Exerts Its Antitumor Effects by Promoting ... · Corresponding Author: Takayuki Yoshimoto, Department of Immunoregula- tion, Institute of Medical Science, Tokyo Medical