Interleukin-27 Exerts Its Antitumor Effects By

Published OnlineFirst November 1, 2017; DOI: 10.1158/0008-5472.CAN-17-0960

Cancer Tumor Biology and Immunology Research

Interleukin-27 Exerts Its Antitumor Effects by Promoting Differentiation of Hematopoietic Stem Cells to M1 Macrophages Yukino Chiba, Izuru Mizoguchi, Junichi Furusawa, Hideaki Hasegawa, Mio Ohashi, Mingli Xu, Toshiyuki Owaki, and Takayuki Yoshimoto

Abstract

The interleukin IL27 promotes expansion and differentiation of antitumor effects of IL27. When admixed with parental tumors, þ hematopoietic stem cells into myeloid progenitor cells. Many CD11b cells inhibited tumor growth and directly killed the tumor tumor-infiltrating myeloid cells exert immunosuppressive effects, in a nitric oxide-dependent manner. Mechanistically, IL27 expand- þ þ but we hypothesized that the myeloid cells induced by IL27 would ed Lineage Sca-1 c-Kit cells in bone marrow. Transplant experi- have antitumor activity. In this study, we corroborated this hypoth- ments in Ly5.1/5.2 congenic mice revealed that IL27 directly acted esis as investigated in two distinct mouse transplantable tumor on these cells and promoted their differentiation into M1 macro- models. Malignant mouse cells engineered to express IL27 exhib- phages, which mobilized into tumors. Overall, our results illus- ited reduced tumor growth in vivo. Correlated with this effect was a trated how IL27 exerts antitumor activity by enhancing the gener- þ significant increase in the number of tumor-infiltrating CD11b ation of myeloid progenitor cells that can differentiate into anti- myeloid cells exhibiting a reduced immunosuppressive activity. tumorigenic M1 macrophages. þ Notably, these CD11b cells were characterized by an activated M1 Significance: These findings show how the interleukin IL27 macrophage phenotype, on the basis of increased expression of exerts potent antitumor activity by enhancing the generation of inducible nitric oxide synthase and other M1 biomarkers. In vivo myeloid progenitor cells that can differentiate into antitumori- depletion of these cells by administering anti–Gr-1 eradicated the genic M1 macrophages. Cancer Res; 78(1); 182–94. 2017 AACR.

Introduction M2 macrophage phenotype is characterized by immunoregulatory functions leading to the promotion of tissue remodeling Although immunotherapeutic approaches against cancer are and tumor progression. promising new treatments, their effectiveness can be further IL27 is an IL12 family cytokine that consists of two distinct improved by overcoming the immunosuppression induced by subunits, Epstein-Barr virus-induced gene 3 and p28, and its tumors. These immunosuppressive mechanisms include receptor (R) is composed of IL27Ra (WSX-1/TCCR) and gly- immune checkpoint molecules, regulatory T cells, and immu- coprotein 130 (4, 5). It is one of the pleiotropic cytokines with nosuppressive myeloid cells. Generally, tumor-bearing hosts both pro- and anti-inflammatory properties acting on various and cancer patients have increased infiltration of immunosup- cell types. IL27 promotes the early induction of Th1 differen- pressive myeloid cells, including tumor-associated macro- tiation and generation of cytotoxic lymphocytes, but it inhibits phages, M2 macrophages, and myeloid-derived suppressor the differentiation to Th2 and Th17 cells and suppresses the cells (MDSC; refs. 1, 2). Macrophages are characterized by production of pro-inflammatory cytokines (4, 5). Because we high plasticity and diversity with two major phenotypes, first demonstrated the antitumor efficacy of IL27 in 2004 using namely M1 (classically activated) and M2 (alternatively acti- a transplanted mouse tumor genetically engineered to secrete vated) macrophages (3). The M1 macrophage phenotype is IL27 as a preclinical tumor model (6), accumulating evidence characterized by the production of pro-inflammatory cyto- suggests that IL27 has potent antitumor activity through mul- kines, reactive oxygen species, and nitric oxide (NO) and the tiple mechanisms, depending on the characteristics of individ- promotion of helper T (Th) 1 immune responses, resulting in ual tumors (7, 8). Moreover, we recently demonstrated that strong microbicidal and tumoricidal activity. In contrast, the IL27 can promote the expansion and differentiation of hematopoietic stem cells (HSC) into myeloid progenitor cells and that Department of Immunoregulation, Institute of Medical Science, Tokyo Medical it plays a critical role in the control of parasite infection during University, Shinjuku-ku, Tokyo, Japan. emergency myelopoiesis (9, 10). Note: Supplementary data for this article are available at Cancer Research IL27wasalsodemonstratedtoaugmenttheproduction Online (http://cancerres.aacrjournals.org/). of immunosuppressive cytokine IL10 and enhance expression Corresponding Author: Takayuki Yoshimoto, Department of Immunoregula- of the immune checkpoint molecules such as programmed tion, Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjuku, death-1 ligand 1 (PD-L1) and T-cell immunoglobulin and Shinjuku-ku, Tokyo 160-8402, Japan. Phone: 81-3-3351-6141; Fax: 81-3-3351- mucin domain-3 (TIM-3) in lymphocytes and macrophages 6250; E-mail: [email protected] (11–15). In addition, IL27 was recently shown to upregulate doi: 10.1158/0008-5472.CAN-17-0960 another immunosuppressive molecule, CD39, together with 2017 American Association for Cancer Research. PD-L1 and IL10 in macrophages, resulting in acquisition of

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immunoregulatory properties by tumor-associated macro- class II I-A/I-E (M5/114.15.2), Ly6G (1A8), Ly6C (HK1.4), phages in various cancer patients (16, 17). These results indi- CD45.1 (A20), and CD45.2 (104) were purchased from Bio- cate that macrophages may play an inhibitory role in the Legend. mAbs against mouse CD4 (GK1.5), CD34 (RAM34), exertion of antitumor activity by IL27. However, myeloabla- and CD86 (GL1) were purchased from eBioscience. APC-Cy7– tion by treosulfan, a chemotherapy drug used to induce a severe conjugated streptavidin, PerCP/Cy5.5-conjugated streptavidin, or complete depletion of bone marrow (BM) cells including and Brilliant Violet 510-conjugated streptavidin were pur- cells other than macrophages, was recently shown to mostly chased from BioLegend and used to reveal staining with bio- abolish the antitumor effects of IL27 in mice bearing non- tinylated Abs. Mouse recombinant IL27 was prepared as a small cell lung cancer (18). Moreover, IL27 inhibited the tagged single-chain fusion protein by flexibly linking EBI3 to development of M2 macrophages from the human monocytic p28 using HEK293-F cells (Life Technologies), as described cell line U937 differentiated by phorbol 12–myristate 13– previously (22). Mouse recombinant stem cell factor (SCF) acetate and subsequently polarized by IL4 (19). Therefore, our and IL3 were purchased from PeproTech. Depletion mAb to understanding of the role of macrophages in the exertion of Gr-1 (RB6-8C5) was obtained from BioXCell. NG-monomethyl antitumor activity by IL27 remains incomplete. L-arginine (L-NMMA) was purchased from Sigma-Aldrich. Although the mouse transplantable tumor model genetically engineered to secrete IL27 significantly decreased tumor growth Preparation of transfectants (20, 21), we initially noticed that the number of tumor-infil- B16F10 and MC38 tumor cells were transfected with the þ trating cells, including CD11b myeloid cells greatly increased. expression vector of a FLAG-tagged single-chain fusion protein Therefore, we wondered whether these myeloid cells would be of IL27–expression vector in p3xFLAG-CMV-14 vector (Sigma- protumorigenic or antitumorigenic. In the present study, to Aldrich; ref. 6) or empty vector as control using Fugene 6 (Roche) clarify this, we used two distinct mouse transplantable tumor and selected with Geneticin (G418). Resultant respective trans- models, B16F10 melanoma and MC38 colon adenocarcinoma. fectants were designated as B16F10-IL27 or MC38-IL27 and Our results clearly revealed a novel mechanism whereby IL27 B16F10-Vector or MC38-Vector, respectively. exerts antitumor activity by promoting expansion and differentiation of HSCs into antitumorigenic M1 macrophages in Transplantable mouse tumor models tumor-bearing mice. Tumor 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 electronic Materials and Methods caliper and expressed as volume (mm3) by calculating using the Cell lines and mice following volume equation: 0.5(ab2), where a is the long diameter Mouse melanoma cell line B16F10 and colon carcinoma cell and b is the short diameter. line MC38 were kindly provided by Dr. Nariuchi (University of Tokyo, Tokyo, Japan) in 2007 and by Dr. Kawakami (Keio Flow cytometry University, Tokyo, Japan) in 2014, respectively. These cell lines On days 13 to 22 after tumor implantation, tumor was har- were expanded in our laboratory, frozen immediately, and vested from mice (n ¼ 3–5), minced into small pieces, and treated stored in liquid nitrogen to ensure that cells used for experi- with collagenase. Subsequently, tumor-infiltrating mononuclear ments were passaged for fewer than 6 months. Early passage cells were purified from it by Percoll (GE Healthcare) density cells were used for all experiments. Cell line authentication was gradient centrifugation. Then, flow cytometry was performed on a not routinely performed. But cell lines were tested routinely for FACS Canto II (BD Biosciences), and data were analyzed using identity by appearance, staining with Hoechst 33258 (Dojindo, FlowJo Software (Tree Star). Cell number was counted by using Kumamoto, Japan), or growth curve analysis in 3H-thymidine flow cytometry. The following cell surface markers were used to incorporation assay, and validated to be mycoplasma free. identify M1 and M2 macrophages (Supplementary Fig. S1): M1 high þ þ þ þ These cell lines were cultured at 37 Cunder5%CO2/95% air macrophages, Class II CD86 CD45.2 CD11b F4/80 ;M2 þ þ þ þ in RPMI-1640 (Sigma) containing 10% FCS and 100 mg/mL macrophages, Class IIlowCD86 CD45.2 CD11b F4/80 (1, 23). kanamycin (Meiji Seika, Tokyo, Japan). BM cells and Line- þ þ age Sca-1 c-Kit (LSK) cells were cultured in the same RPMI- Quantitative real-time RT-PCR 1640 medium additionally supplemented with 50 mmol/L Total RNA was prepared from the tumor-infiltrating mononu- 2 mercaptoethanol (Gibco). OT-I mice, C57BL/6 (CD45.2) mice, clear cells of mice (n ¼ 4) using an RNeasy Mini Kit (Qiagen), and and C57BL/6 (CD45.1) mice were maintained in specificpatho- cDNA was prepared using oligo(dT) primer and SuperScript III RT gen-free conditions. All experiments were approved by the Animal (Thermo Fischer Scientific). Real-time quantitative PCR was per- Care and Use Committee of Tokyo Medical University and were formed using SYBR Premix Ex Taq II and a Thermal Cycler Dice performed in accordance with our institutional guidelines. real-time system according to the manufacturer's instructions (Takara). Glyceraldehyde-3-phosphate (GAPDH) was used as a Antibodies and reagents housekeeping gene to normalize mRNA. Relative expression of Monoclonal antibodies (mAb) for mouse c-Kit (2B8), Sca-1 real-time PCR products was determined by using the DDCt meth- (D7), CD3e (145-2C11), CD8a (53-6.7), CD19 (6D5), CD49b od to compare target gene and GAPDH mRNA expression. Primers (DX5), Gr-1 (RB6-8C5), TER119/erythroid cell (TER-119), used in this study are listed in Supplemental Table S1. CD11c (N418), CD11b (M1/70), F4/80 (BM8), NK1.1 (PK136), B220 (RA3-6B2), FceRIa (MAR-1), macrophage col- Measurement of immunosuppressive activity ony-stimulating factor receptor (M-CSFR; AFS98), Flt3 On days 13 to 20 after tumor implantation, tumor was har- (A2F10), CD16/32 (2.4G2), CD150 (TC15-12F12.2), MHC vested from mice (n ¼ 3 or 4), minced into small pieces, and

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treated with collagenase; tumor-infiltrating mononuclear cells then stained with mAbs against c-Kit, and Sca-1 was used for LSK. were purified from it by using Percoll density gradient centrifu- Sorting was performed on a MoFlo XDP (Beckman Coulter). gation. Tumor-infiltrating myeloid cells were further isolated by AutoMACS Pro after staining with anti-CD11b magnetic beads In vitro differentiation assay (Miltenyi Biotec). Immunosuppressive activity was then assessed For analysis of multipotent myeloid potential, LSK cells were fi þ by the ability to inhibit ovalbumin (OVA)-speci c CD8 T-cell purified from BM cells in tumor-bearing mice (n ¼ 3) on days 5 proliferation. Spleen cells (5 10 cells/200 mL) obtained from 17 to 20 after tumor implantation or untreated mice. The sorted OT-I T-cell receptor transgenic mice were stimulated with OVA LSK cells (2.5 102/200 mL) were cultured with 10 ng/mL (0.3 mg/mL) in the presence of increasing cell numbers (1, 3, and þ SCF and 10 ng/mL IL3 in 96-well plates for 7 days. Half of 10 104 cells) of CD11b myeloid cells for 48 h and pulsed with 3 3 the medium was replaced every 3 days, with cytokines added. H-thymidine for last 8 hours, and the incorporation of H- The following cell surface markers were used to identify respec- þ þ thymidine was measured in triplicate. tivecells(SupplementaryFig.S1):neutrophil,Ly6G CD11b þ þ þ þ or Gr-1 CD11b ; macrophage, F4/80 CD11b ; mast cell, Depletion experiment þ þ þ þ c-Kit FceR1a CD11b ; basophil, CD49b FceR1a CD11b . After 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 deplete Adoptive cell transfer 3 fi myeloid cells. Tumor growth was monitored twice a week. The BM LSK cells (5 10 ) puri ed from C57BL/6 (CD45.1) depletion level was confirmed by analyzing blood samples congenic mice were intravenously transferred into sublethally n ¼ obtained from the tail vein. irradiated (4 Gy) C57BL/6 (CD45.2) recipient mice ( 4). On the next day, tumor cells (1 106 cells) of B16F10-IL27 or Admixture experiment B16F10-Vector were injected subcutaneously into the right flank þ CD11b myeloid cells (1 106 cells) were purified from tumor of resulting mice. Tumor growth was monitored twice a week. of mice (n ¼ 4–6) on days 13 to 22 after tumor implantation. The cells were admixed with parental tumor (2 105 cells) in 200 mL ELISA PBS and then immediately injected into the right flank of mice. Serum protein level of TGFb1 was determined by ELISA accord- Tumor growth was monitored twice a week. ing to the manufacturers' instructions (R&D Systems).

Killing assay Statistical analysis þ fi n ¼ CD11b myeloid cells were puri ed from tumor of mice ( Data are presented as mean SEM. Statistical analyses were 4 or 5) on days 13 to 22 after tumor implantation. Parental performed by using the two-tailed Student t test for comparisons 6 tumor cells (1 10 cells/50 mL of 50% fetal calf serum/RPMI- of two groups and one-way ANOVA and Tukey–Kramer multiple 1640) were labeled by incubating in 100 mCi 51Cr for 45 min comparison test for comparing more than three groups using at 37 C and then washed three times. Labeled target cells GraphPad Prism 7 (GraphPad Software Inc.). P < 0.05 was 4 fi þ (1 10 cells/200 mL) were incubated with puri ed CD11b considered to indicate a statistically significant difference. cells (1 105 cells) in a 96-well U-bottomed plate at 37Cfor 4 hours. Supernatants then were analyzed in a scintillation counter. The percentage of lysis was determined in triplicate Results þ as (experimental count spontaneous count) 100/(maximal IL27 augments the infiltration of CD11b myeloid cells into count spontaneous count). Results were expressed as the tumor percentage of specific lysis. The effect of inducible nitric oxide As reported previously (20), melanoma B16F10 tumor cells synthase (iNOS) inhibitor was analyzed in the presence or transfected with IL27–expression vector (B16F10-IL27) showed absence of L-NMMA (100 mmol/L). great inhibition of tumor growth compared with those transfected with control empty vector (B16F10-Vector; Fig. 1A and Measurement of NO production B). During analysis of antitumor activity of IL27, we initially þ NO production by CD11b myeloid cells (1 105 cells/200 noticed that the percentage and total number of mononuclear mL) purified from tumor of mice (n ¼ 4 or 5) on days 17 to 21 after cells infiltrating into tumors were significantly increased by tumor implantation was determined by measuring nitrite IL27 (Fig. 1C and D). Implantation of tumors also increased the (NO2 ), a stable and non-volatile by-product of NO, produced spleen weight regardless of B16F10-IL27 or B16F10-Vector (Fig. in culture supernatants for 48 hours using a Griess reagent kit 1B), implying augmented myelopoiesis by tumor growth. (Thermo Fischer Scientific). The Griess reagent and nitrite stan- Among the various types of cells, FACS analyses revealed that dards or supernatants were mixed, incubated, and then read at IL27 greatly increased the percentage and number of not only þ þ 520 and 590 nm as a reference to measure nitrite levels. The effect CD8 T cells but also CD11b myeloid cells in tumor per of iNOS inhibitor was analyzed in the presence or absence of tumor volume, as well as per tumor weight (Fig. 1E and F). A þ L-NMMA (100 mmol/L). similar increase of tumor-infiltrating CD11b myeloid cells was observed with another tumor model, MC38 colon carci- Preparation of LSK cells noma (Supplementary Fig. S2). Spleen and BM Lineage cells were enriched by negative selec- Given that tumor-infiltrating myeloid cells are generally immu- tion using an AutoMACS Pro with a combination of magnetic nosuppressive cells, such as M2 macrophages and MDSCs, it is beads conjugated with mAbs against CD3e, CD4, CD8a, Gr-1, important to investigate the properties of IL27–medaited infil- TER119, CD11b, CD11c, NK1.1, B220, and FceRIa. Cells were 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 tumor and subjected to flow cytometer analysis (C), and total number of tumor-infiltrating mononuclear cells was counted (D). E, Representative dot plots of individual cell populations are shown. F, Their cell numbers were counted, and calculated per tumor volume and also per tumor weight. Data are shown as mean SEM (n ¼ 3 or 4) and are representative of six independent experiments. , P < 0.05; , P < 0.001.

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and the molecular mechanism whereby IL27 induces such a (Supplementary Fig. S5). Thus, IL27 inhibits the generation phenomenon. of immunosuppressive myeloid cells such as M2 macrophages in tumor-bearing mice, but it promotes the generation of M1 IL27 promotes the differentiation into M1 macrophages in macrophages. tumor-bearing mice M1 and M2 macrophages have antitumorigenic activity and IL27-induced tumor-infiltrating myeloid cells have potent protumorigenic activity, respectively (3). Therefore, we next antitumor effects examined the phenotype of these IL27–mediated tumor-infil- To further explore the role of myeloid cells in B16F10-IL27 trating myeloid cells. On day 13 after tumor implantation, tumor-bearing mice in the IL27-mediated antitumor activity, mononuclear cells were purified from tumor and subjected to depletion experiments of myeloid cells by treatment with anti- cell surface analysis by using FACS. IL27 greatly increased the Gr-1 mAb were performed. Treatment of mice with anti–Gr-1 þ þ percentage and number of monocytic (Mo )MDSCs (1, 2) and mAb almost completely depleted Gr-1 CD11b myeloid macrophages, but decreased those of granulocytic (Gr )MDSCs cells in blood (Fig. 4A). Under these conditions, depletion of (1, 2), in tumor per tumor volume, as well as per tumor myeloid cells in B16F10-Vector tumor-bearing mice greatly weight (Fig. 2A and B). Among these tumor-infiltrating macro- inhibited the tumor growth, as generally expected (Fig. 4A). phages, IL27 greatly increased the percentage and number of M1 In contrast, depletion of myeloid cells in B16F10-IL27 tumor- þ þ þ þ macrophages (Class IIhighCD86 CD45.2 CD11b F4/80 )as bearing mice significantly promoted the tumor growth compared with those of M2 macrophages (Class (Fig. 4B). These results suggest that IL27 exerts antitumor þ þ þ þ IIlowCD86 CD45.2 CD11b F4/80 ; Fig. 2C–E). The ratio of activity via myeloid cells. M1/M2 macrophages was significantly increased, indicating the enhanced bias toward M1 macrophages by IL27 (Fig. 2D IL27-induced tumor-infiltrating myeloid cells exert direct and E). Consistent with this, mRNA expression analyses antitumor effects by killing tumor through NO þ revealed that CD11b myeloid cells purified from tumor of To further examine the effect of IL27 on myeloid cells in tumor- B16F10-IL27 tumor-bearing mice express more M1 macro- bearing mice, admixture experiments of myeloid cells with paren- phage markers, such as iNOS, interferon regulatory factor 8 tal B16F10 tumor were performed. Consistent with the results of þ (IRF8), and IL12p40, but less M2 macrophage markers, such as depletion experiments, admixture of CD11b myeloid cells puri- ariginase-1 (Arg-1), chitinase 3-like-3 (Ym1), and found in fied from tumor of B16F10-Vector tumor-bearing mice with inflammatory zone 1 (Fizz1), compared with those of B16F10- parental B16F10 tumor greatly promoted the tumor growth (Fig. þ Vecter tumor-bearing mice (Fig. 2F). Similar results were 5A). In contrast, admixture of CD11b myeloid cells purified obtained with the MC38 tumor model (Supplementary Fig. from tumor of B16F10-IL27 tumor-bearing mice with parental S3). These results suggest that IL27 promotes differentiation B16F10 tumor markedly inhibited the tumor growth (Fig. 5A). into M1 macrophages in tumor-bearing mice. Again, tumor-infiltrating myeloid cells are quite different between Because IL27–transduced tumor grows at a much slower rate B16F10-IL27 and B16F10-Vector tumor-bearing mice, being anti- than control tumor, not only the tumor size but also the tumor tumorigenic versus protumorigenic cells. þ microenvironment might be different between them and there- To further evaluate the therapeutic potential of the CD11b fore affect the number and phenotype of tumor-infiltrating myeloid cells purified from tumors of B16F10-IL27 tumor- cells. To examine this possibility, we tried to adjust the tumor bearing mice, the antitumor efficacy of these myeloid cells on size to reach the same size by changing the conditions such as pre-existing tumor model was examined. When mice were the cell number of tumors to inject, and then analyzed the implanted with parental B16F10 tumor and their tumor size þ tumor-infiltrating cells by flow cytometry. Under these condi- reached 5 mm in diameter on day 5, these CD11b myeloid tions, we could still observe that IL27 much less but signifi- cells were both intravenously and intratumorally injected into cantly enhances the percentage and number of tumor-infiltrat- thesemicewiththepre-existing tumor. By using these injec- ing cells including macrophages, especially with M1 phenotype tions, we could observe a tendency to inhibit the tumor growth (Supplementary Fig. S4). (Supplementary Fig. S6). Next, to investigate the molecular mechanism whereby IL27 IL27 inhibits differentiation into immunosuppressive augments antitumor activity via myeloid cells, the possibility macrophages in tumor-bearing mice of direct killing of tumors by macrophages was examined by þ Generally, tumor-infiltrating myeloid cells are immunosup- using a 51Cr-releasing assay. CD11b myeloid cells purified pressive (1). Therefore, we next examined immunosuppressive from tumor of B16F10-IL27 tumor-bearing mice showed great- activity of tumor-infiltrating myeloid cells. On day 16 after ly augmented killing activity compared with those of B16F10- tumor implantation, mononuclear cells were purified from Vector tumor-bearing mice (Fig. 5B). Correlating with the þ tumor, and CD11b myeloid cells were further purified from augmented killing activity, myeloid cells purified from tumor them and subjected to immunosuppressive activity, which was of B16F10-IL27 tumor-bearing mice produced more NO than þ assessed by the ability to inhibit OVA-specificCD8 T-cell those of B16F10-Vector tumor-bearing mice (Fig. 5C). Treat- proliferation. Myeloid cells purified from tumor of B16F10- ment with NOS inhibitor, L-NMMA, almost completely Vector tumor-bearing mice showed marked inhibitory activity blocked the killing activity of myeloid cells purified from against the antigen-specific proliferation (Fig. 3). In contrast, tumor of B16F10-IL27 tumor-bearing mice (Fig. 5D). Similar myeloid cells purified from tumor of B16F10-IL27 tumor- results were obtained with the MC38 tumor model (Supple- bearing mice showed significantly reduced inhibitory activity mentary Fig. S7). compared with those of control tumor-bearing mice (Fig. 3). Thus, IL27 promotes the generation of antitumorigenic mye- Similar results were obtained with the MC38 tumor model 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 after tumor implantation were analyzed by ﬂow cytometry. Representative dot plots of Gr-MDSCs (CD45.2þCD11bþLy6GþLy6Cint), Mo-MDSCs (CD45.2þCD11bþLy6GLy6Chi), and macrophages (CD45.2þCD11bþF4/80þ)areshown,andtheircellnumberswerecountedandcalculatedpertumorvolume and also per tumor weight. C, Representative dot plots of M1 macrophages (Class IIhighCD86þCD45.2þCD11bþF4/80þ) and M2 macrophages (Class IIlowCD86þCD45.2þCD11bþF4/80þ)areshown.D and E, Their cell numbers were counted, and calculated per tumor volume (D)andalsopertumor weight (E), together with respective ratio of M1/M2 macrophages. Data are shown as mean SEM (n ¼ 3or4)andarerepresentativeofmorethansix independent experiments. F, mRNA expression of markers for M1 and M2 macrophages in puriﬁed CD11bþ myeloid cells from tumor-bearing mice on day 17 was 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.

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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 on the myelopoiesis in tumor-bearing mice. Similar to the results obtained by using the malaria infection model (10), IL27 increased the percentage and number of LSK cells in both BM and spleen (Fig. 6A). Moreover, IL27 also increased the percentage and number of the most primitive HSC population, long-term HSC (LT-HSC; ref. 24), but not multipotent progenitor (MPP) in both BM and spleen (Fig. 6B). Thus, IL27 directly affects LSK cells and LT-HSCs in the BM to promote the myelopoiesis in transplantable tumor models. Next, the multipotency of LSK cells in the BM of tumor- bearing mice was examined under in vitro myeloid cell differentiation conditions. LSK cells purified from the BM of B16F10-Vector tumor-bearing mice had less ability to differentiate into myeloid cells such as mast cells, basophils, neutrophils, and macrophages compared with those purified from fi Figure 3. untreated mice (Fig. 6C and D). However, LSK cells puri ed IL27 inhibits the differentiation into immunosuppressive macrophages in tumor- from the BM of B16F10-IL27 tumor-bearing mice had a bearing mice. On day 16 after tumor implantation, mononuclear cells greater ability to differentiate into these myeloid cells com- were purified from tumor, and CD11bþ myeloid cells were further purified pared with those of B16F10-Vector tumor-bearing mice (Fig. 6C and subjected to immunosuppressive activity, which was assessed by the and D). Interestingly, BM LSK cells from B16F10-IL27 tumor- fi þ ability to inhibit OVA-speci c CD8 T-cell proliferation. Data are shown as bearing mice had an enhanced ability to differentiate into mean SEM (n ¼ 3 or 4) and are representative of more than six independent experiments. , P < 0.05; , P < 0.01; , P < 0.001. M1 macrophages compared with those from B16F10-Vector tumor-bearing mice (Fig. 6E). These results suggest that IL27 promotes the expansion and generation of LSK cells, which bearing mice, and the antitumorigenic myeloid cells exert direct have an enhanced ability to differentiate into myeloid cells, killing activity against tumor through NO. especially with the M1 macrophage phenotype.

IL27 induces the expansion of LSK cells with enhanced ability to IL27 directly acts on BM LSK cells and promotes the expansion differentiate into M1 macrophages and differentiation into M1 macrophages in vivo Generally, tumor-bearing mice have increased production of To further examine the direct effect of IL27 on BM HSCs tumor-derived cytokines such as G-CSF and GM-CSF, resulting in todifferentiateintoM1macrophagesin vivo,adoptivecell

Figure 4. IL27–induced tumor-inﬁltrating myeloid cells have potent antitumor effects. A, Mice were treated with anti– Gr-1 mAb or control IgG, and deletion level of neutrophils was analyzed in blood by ﬂow cytometry on day 7 after tumor implantation. B, Tumor growth was monitored with time course. Data are shown as mean SEM (n ¼ 5) and are representative of three independent experiments. , P < 0.05.

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Differentiation to Antitumorigenic M1 Macrophages by IL27

Figure 5. IL27-induced tumor-infiltrating myeloid cells exert direct antitumor effects by killing tumor through NO. A, CD11bþ myeloid cells purified from tumor of B16F10-IL27 or B16F10-Vector tumor-bearing mice were admixed with parental B16F10 tumor, and tumor growth was monitored with time course. Data are shown as mean SEM (n ¼ 6) and are representative of more than three independent experiments. B and C, On day 18 after tumor implantation, killing activity of CD11bþ myeloid cells against parental B16F10 tumor was measured by 51Cr release assay (B), and NO production in the culture supernatant was measured in the presence or absence of NOS inhibitor, L-NMMA (C). D, The effect of inhibition of NOS activity by L-NMMA on the killing activity was examined. Data are shown as mean SEM (n ¼ 4or5)andare representative of more than five independent experiments. , P < 0.05; , P < 0.001.

transfer of BM LSK cells purified from CD45.1 congenic mice (25). We also reported that IL27 greatly induced IRF1 and IRF8 into sublethally irradiated CD45.2 recipient mice was per- expression in mouse melanoma B16F10-expressing WSX-1, lead- formed. Tumor cells of B16F10-IL27 or B16F10-Vector were ing to inhibition of its tumor growth (21). Importantly, IRF8 is a then implanted, and tumor growth was monitored. As shown critical transcription factor for the development of monocytes, before (Fig. 1A), IL27 significantly inhibited tumor growth (Fig. dendritic cells, eosinophils, and basophils in synergy with PU.1 7A). On day 20 after tumor implantation, tumor-infiltrating (26), whereas IRF8 inhibits CCAAT-enhancer-binding protein a cells were analyzed. IL27 greatly increased the percentage and activity to suppress the generation of neutrophils (26). IRF8 is also þ number of CD45.1 cells and macrophages infiltrating into a critical transcription factor for induction of iNOS expression in tumors(Fig.7BandC).Consistentwiththein vivo results (Fig. collaborating with IRF1 (27) or NF-kB (28), resulting in enhanced 2) and in vitro differentiation results (Fig. 6C–E), IL27 markedly NO production. In addition, IRF8 is an integral negative regulator increased the percentage and number of M1 macrophages into (29) against the development of MDSCs by promoting sponta- tumor (Fig. 7D). Thus, IL27 directly acts on the BM LSK cells neous apoptosis and turnover in myeloid cells (30) and by and promotes the expansion and differentiation into antitu- repressing GM-CSF expression in T cells (31). Thus, IL27-medi- morigenic M1 macrophages in tumor-bearing mice. ated promotion of HSCs to expand and differentiate into antitumorigenic M1 macrophages with enhanced iNOS expression presumably is highly dependent on IRF8, but this possibility Discussion remains to be further clarified. Accumulating evidence suggests that IL27 possesses potent Of note, the present results indicate that properties of tumor- antitumor activity through multiple mechanisms depending on infiltrating myeloid cells in the presence or absence of IL27 are the characteristics of individual tumors (7, 8). The present results quite different. Myeloid cells in IL27 tumor-bearing mice are indicate that IL27 has another novel mechanism to exert potent antitumorigenic with the M1 macrophage phenotype, whereas antitumor activity. IL27 promotes the expansion and differenti- those in control tumor-bearing mice are protumorigenic with the ation of HSCs into myeloid progenitor cells, which have enhanced M2 macrophage phenotype (Figs. 2–5A). Myeloid cells in IL27 potential to differentiate into antitumorigenic M1 macrophages tumor-bearing mice were demonstrated to kill parental tumors with increased expression of iNOS, IRF8, and IL12p40 but directly in a NO-dependent manner (Fig. 5B–D). Moreover, IL27 reduced expression of Arg-1, Ym1, and Fizz1 in tumor-bearing increased the percentage and number of LSK cells in both BM and mice. Blocking experiments using the iNOS inhibitor, L-NMMA, spleen of tumor-bearing mice (Fig. 6A and B). We previously þ further revealed that the CD11b myeloid cells purified from demonstrated that IL27 directly acts on LSK cells, especially IL27-tumor-bearing mice directly kill tumors in a highly NO- LT-HSCs, and promotes their expansion and differentiation into dependent manner, whereas whether other potential mechanisms phenotypically similar LSK cells but distinct myeloid progenitors may also exist remains to be elucidated. This is consistent with our (10). LSK cells in the BM of control tumor-bearing mice have a previous report showing that IL27 augments the production of reduced ability to ex vivo differentiate into myeloid cells compared NO in thioglycollate-elicited peritoneal macrophages in synergy with LSK cells in the BM of untreated mice, possibly due to the with lipopolysaccharide through STAT1, NF-kB, and MAP kinases immunosuppressive effects of myeloid cells on LSK cells in the

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Figure 6. IL27 induces the expansion of LSK cells with enhanced ability to differentiate into M1 macrophages. A and B, On day 13 after tumor implantation, cell populations of LSK (A), LT-HSC, and MPP (B) were analyzed by ﬂow cytometry. Data are shown as mean SEM (n ¼ 3 or 4) and are representative of more than six independent experiments. C and D, The multipotency of LSK cells in the BM of B16F10-IL27 or B16F10-Vector tumor- bearing mice on day 19 after tumor implantation together with untreated mice was examined under in vitro myeloid cell differentiation conditions into mast cells, basophils, neutrophils, and macrophages. E, Cell number of M1 and M2 macrophages was counted and the ratio of M1/M2 macrophages was calculated. Data are shown as mean SEM (n ¼ 3) and are representative of more than three independent experiments. , P < 0.05; , P < 0.01; , P < 0.001.

þ tumor-bearing mice (Fig. 6C and D). In contrast, LSK cells in the Although the infiltration of CD8 T cells into IL27–treated BM of IL27-tumor-bearing mice have a greatly increased ability to tumors is dominant in B16F10 melanoma model (Fig. 1F), þ ex vivo differentiate into myeloid cells including M1 macrophages the infiltration of CD4 T cells is dominant in MC38 colon compared with those of control tumor-bearing mice (Fig. 6C–E). carcinoma model (Supplementary Fig. S2C). Although the Finally, adaptive cell transfer experiments revealed that IL27 acts molecular mechanism whereby IL27 induces such distinct directly on the LSK cells in the BM in vivo and promotes the dominancy remains elusive, IL27 also greatly induces the þ expansion and differentiation into M1 macrophages, mobilizing infiltration of CD11b myeloid cells into tumor in both mod- þ þ them in tumor (Fig. 7). els irrespectively of CD8 or CD4 Tcellinfiltration. One of the

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possible reasons for the different effects by IL27 on the tumor- tiation and proliferation of them (32). Moreover, IL27 seem- infiltrating cells may be ascribed to the different microenvi- ingly induced high M1/M2 ratio and NO production of the þ ronment situations such as distinct places and stages where tumor-infiltrating CD11b myeloid cells in the MC38 model IL27 acts on these cells. In the case of myeloid cells, IL27 compared with those in the B16F10 model (Fig. 1C and 5C; directly acts on the BM cells and promotes the differentiation Supplementary Figs. S3C and S7C). Correlating to these results, þ into M1 macrophages following the infiltration into tumor the killing activity of the CD11b myeloid cells augmented (Fig. 6 and 7). On the other hand, IL27 likely acts on naive by IL27 in relation to that by control vector in the MC38 T cells in the spleen or lymph node to promote the differen- model was higher than that in the B16F10 model (41.8

Figure 7. IL27 directly acts on BM LSK cells and promotes the expansion and differentiation into M1 macrophages in vivo. A, BM LSK cells puriﬁed from CD45.1 congenic untreated mice were adoptively transferred into sublethally irradiated CD45.2 recipient mice, and then tumor cells of B16F10-IL27 or B16F10-Vector were implanted, and tumor growth was monitored with time course. B and C, On day 20 after tumor implantation, percentage and number of CD45.1þ or CD45.2þ cell population (B)and macrophages (C) were measured. D, Representative dot plots of M1 and M2 macrophages are shown, their cell numbers were counted, and ratio of M1/M2 macrophages was calculated. Data are shown as mean SEM (n ¼ 4) and are representative of more than three independent experiments. , P < 0.05.

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18.9-folds vs. 7.1 4.2-folds; Fig. 5B; Supplementary Fig. S7B). systemic toxicities such as hepatomegaly, leukopenia, and Further studies are necessary to clarify the different effects by gastrointestinal toxicity (39–42). IL23 plays a key role in IL27 between these two tumor models. autoimmune and inflammatory diseases through STAT3/ Because IL27 induces upregulation of immunosuppressive STAT4 activation via IL23/IL17 immune axis (43). IL23 was molecules such as PD-L1, CD39, TIM-3, and IL10 (11–17), the demonstrated to promote tumorigenesis by inducing inflam- possibility that IL27 may induce protumorigenic effects has mation, which inhibits adaptive immune surveillance (44). been discussed (8, 33). However, by using histopathological Nevertheless, multiple studies have also shown the antitumor analyses, Airoldi and colleagues (18) observed that IL27 treat- effects by IL23 in various mouse tumor models (45–47). ment increases the infiltration of granulocytes and macro- Thus, the role of IL23 in tumor procession is still controversial. phages into tumor using xenograft models of human lung On the other hand, IL27 activate STAT1 and STAT3 through adenocarcinoma and squamous cell carcinoma cell lines its respective receptor subunits, WSX-1 and gp130, respectively Calu-6 and SK-MES in immunodeficient mice. To assess wheth- (48, 49). IL27 was previously reported to induce direct anti- er these granulocytes and macrophages may account for the proliferative effects on melanoma cells via STAT1 signaling as antitumor effects of IL27, mice were pretreated with myeloa- does IFN-g (21). IL27 has very low toxicity, probably because blative doses of treosulfan. The myeloablation significantly of low induction of IFN-g (6, 47). IL27 treatment does not abolished the antitumor effect of IL27, although this treatment induce severe liver injury with increased levels of aspartate and induced a severe or complete depletion of BM cells, which alanine aminotransferases (6, 47), and transgenic mice con- affects the cellularity of peripheral cells other than macro- stitutively overexpressing IL27 can survive for almost the phages (18). Yao and colleagues (19) also recently reported whole life span (50). Such a low toxic property of IL27 is of that IL27 inhibits the development of M2 macrophage from benefit to its clinical application. the human monocytic cell line U937 differentiated by phorbol Taken together, our present findings clearly indicate that 12–myristate 13–acetate and subsequently polarized by IL4 tumor-infiltrating myeloid cells play a critical role in exertion in vitro. Consistent with the present results, these two reports of antitumor effects by IL27, although these myeloid cells show further support the possibility that macrophages play a critical high expression of immune checkpoint molecules. To ensure role in exerting antitumorigenic effects rather than protumori- patient safety, therefore, combination therapy of IL27 with genic effects by IL27 in tumor-bearing mice. monoclonal antibodies against these immune checkpoint Given that IL27 is a multifunctional cytokine with a double- molecules could be a more promising and effective therapeutic edged sword, both antitumor and protumor effects of IL27 are strategy against cancer. conceivably expected. Indeed, we observed enhanced mRNA expression of immune checkpoint molecules such as PD-L1 and Disclosure of Potential Conflicts of Interest fi þ TIM-3 by in tumor-in ltrating CD11b myeloid cells (Supple- No potential conflicts of interest were disclosed. mentary Fig. S8A and S8B) as reported (14, 15, 17). Similar tendency of the up-regulation of PD-1 mRNA expression by IL27 Authors' Contributions was also observed, although its expression level even in steady Conception and design: Y. Chiba, T. Yoshimoto state of myeloid cells seemed to be low (Supplementary Fig. S8A Development of methodology: Y. Chiba, I. Mizoguchi, T. Yoshimoto and S8B; ref. 34). Similar upregulation of TGFb1 mRNA expres- Acquisition of data (provided animals, acquired and managed patients, þ sion by IL27 was observed in tumor-infiltrating CD11b myeloid provided facilities, etc.): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. Ohashi cells, but its serum level was rather decreased by IL27 (Supple- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mentary Fig. S8C). Because IL27 was recently reported to inhibit computational analysis): Y. Chiba, I. Mizoguchi, H. Hasegawa, M. Ohashi the functions of TGFb1, including induction of fibrosis and Writing, review, and/or revision of the manuscript: Y. Chiba, M. Xu, T. Owaki, – T. Yoshimoto epithelial mesenchymal transition (35, 36), which contribute Administrative, technical, or material support (i.e., reporting or organizing to antitumor effects, further study is necessary to clarify the role data, constructing databases): J. Furusawa, T. Owaki of TGFb1 in the exertion of antitumor effect by IL27. Because Study supervision: T. Yoshimoto B16F10 tumor does not express endogenous one of IL27R subunits, WSX-1, IL27 does not directly act on B16F10 tumor cells to Acknowledgments inhibit the immunosuppressive functions of the tumors (21). The authors thank Dr. H. Nariuchi (University of Tokyo) and Rather, IL27 induces antitumor activity via acting on other cells Dr. Y. Kawakami (Keio University) for the B16F10 mouse melanoma including T cells, NK cells, macrophages, and endothelial cells in cell line and MC38 colon carcinoma cell line, respectively, and also thank the tumor microenvironment (7, 8). Thus, the overall antitumor R. Tsunoda (Tokyo Medical University) for his encouragement. This study potency of IL27 is highly likely to result from the sum of its was supported in part by a grant-in-aid (to T. Yoshimoto) and the Private University Strategic Research Based Support Project (to T. Yoshimoto) positive and negative effects on tumors within the tumor micro- from the Ministry of Education, Culture, Sports, Science, and Technology, environment. Therefore, if the IL27 therapy could be combined Japan, by Research Grant from Princess Takamatsu Cancer Research Fund with the immune checkpoint blockade by using antibody (37), we (to T. Yoshimoto), and by Research Fellowship for Young Scientists (DC2) would expect a synergistic antitumor effect between them. This from Japan Society for the Promotion of Science (to Y. Chiba). possibility is currently under investigation. IL27 belongs to the IL12 cytokine family including IL12, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked IL23, and so on. IL12 plays a critical role in the induction of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Th1-type immune responses through STAT4 activation, and it this fact. was demonstrated to induce strong antitumor effects via mainly IFN-g production in various preclinical models (38). Received April 4, 2017; revised August 25, 2017; accepted October 27, 2017; However, the clinical trials of IL12 have been limited by published OnlineFirst November 1, 2017.

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194 Cancer Res; 78(1) January 1, 2018 Cancer Research

Interleukin-27 Exerts Its Antitumor Effects by Promoting Differentiation of Hematopoietic Stem Cells to M1 Macrophages

Yukino Chiba, Izuru Mizoguchi, Junichi Furusawa, et al.

Cancer Res 2018;78:182-194. Published OnlineFirst November 1, 2017.

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