A long-standing question in the field of tumor immunotherapy is how Th2 cytokines block tumor growth. Their antitumor effects are particularly prominent when they are secreted continuously in tumors, suggesting that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. In this study, we show that local production of IL-33 establishes a high number of type 2 innate lymphoid cells (ILC2s) with potent antitumor activity. IL-33 promotes secretion of a massive amount of CXCR2 ligands from ILC2s but creates a tumor microenvironment where tumor cells express CXCR2 through a dysfunctional angiogenesis/hypoxia/reactive oxygen species axis. These two signaling events converge to reinforce tumor cell–specific apoptosis through CXCR2. Our results identify a previously unrecognized antitumor therapeutic pathway wherein ILC2s play a central role.

Cytokines have the ability to recognize and destroy cancer cells directly or indirectly by regulating the immune system and have been developed as cancer treatments (13). Most cytokines approved as drugs for patients with cancer include the Th1 cytokines (IL-2, IFN-γ, TNF-α, and GM-CSF) and other IFN family members (IFN-α and IFN-β) with various proinflammatory cytokines that are in clinical trials (IL-7, IL-12, IL-15, IL-18, and IL-23). Th2 cytokines, such as IL-4 and IL-13, show potent antitumor activities in animal models (4, 5). Although the mechanisms of action underlying these observations have been controversial for 25 y, it is clear that tumorigenicity is lacking in tumor cells engineered to secrete a Th2 cytokine and that this type of tumor rejection can occur in a T cell–independent manner (68), suggesting that modifications of the tumor microenvironment elicited by Th2 cytokines may be toxic directly to tumor cells or unfavorable for tumor growth.

IL-33 is classified as a Th2 cytokine but it plays pleiotropic roles in virtually all types of immune cells and in a variety of nonimmune cells (9, 10). Three studies have provided evidence supporting involvement of IL-33 in antitumor immune responses, that is, transgenic expression, injection as a vaccine adjuvant, or overexpression in tumor cells of IL-33, and increased antitumor immunity by promoting CTL responses to tumor cells (1113). However, it is unknown whether type 2 innate lymphoid cells (ILC2s), which respond vigorously to IL-33, are involved in IL-33–mediated antitumor responses. In this study, we dissected the therapeutic pathways initiated by local production of IL-33 and demonstrate that IL-33 creates a tumor microenvironment wherein ILC2s play an indispensable role in massive tumor cell apoptosis.

Female C57BL/6 and BALB/c mice, 7–9 wk of age, were purchased from Orient Bio–Charles River. Rag2−/−γc−/−, MyD88−/−, IL-13−/−, ΔdblGATA-1 (14), CXCR2−/−, and Thy 1.1 congenic mice were bred and housed at the University of Ulsan. All mice were maintained in pathogen-free conditions. The Animal Care Committee of the University of Ulsan approved these studies.

The EL4–IL-33 cell line was generated as described previously (15), and the B16F10–IL-33 and CT2–IL-33 cell lines were generated in the same way. The cells were maintained in DMEM, with 10% FBS, penicillin/streptomycin (100 U/ml), and Zeocin (50 μg/ml, Invitrogen). C57BL/6 mice were inoculated s.c. with EL4-Vec or EL4–IL-33 cells (5 × 106 cells/mouse) and B16F10-Vec or B16F10–IL-33 cells (2.5 × 106 cells/mouse). BALB/c mice were inoculated with CT26-Vec or CT26–IL-33 cells (3 × 106 cells/mouse). Tumor diameters were measured two to three times every week using engineering calipers (Mitutoyo).

The following FITC-, PE-, PerCP-, Cy5-, or allophycocyanin-conjugated mAbs to mouse proteins were purchased from BD Biosciences or eBioscience: CD3, CD4, CD8, CD11b, CD11c, B220, CD31, CD90, CD117 (c-Kit), CD127 (IL-7Rα), Ly-6A/E (Sca-1), TER119, F4/80, Gr-1, Thy1.1, Thy1.2, IL-13, GATA3, Siglec-F, annexin V, and CXCR2. FITC-conjugated α–smooth muscle actin (α-SMA) was purchased from Santa Cruz Biotechnology. Anti-CD4 (GK1.5), anti-CD8 (2.43), and anti–Gr-1 (RB6-8C5) mAbs were purified from ascites. Control rat IgG was purchased from Sigma-Aldrich. The following cytokines were purchased from PeproTech: recombinant murine Flt3 ligand (Flt3L), stem cell factor (SCF), IL-7, and IL-33. The following neutralization mAbs and blocking reagents were purchased: anti–IL-5 and anti–IL-13 mAbs from R&D Systems; SB225002 from Tocris Bioscience; halofuginone and clodronate-containing liposome (clodrolip) from Sigma-Aldrich; anti–asialo ganglio-N-tetraosylceramide (asialo GM1) from eBioscience; recombinant collagenase IV from Life Technologies; and anti–GM-CSF mAbs from PeproTech.

Tumors were removed surgically, cut into 1- to 2-mm3 pieces, and digested with 5–10 ml DMEM containing collagenase I (1 mg/ml) and DNase I (0.5 mg/ml, Sigma-Aldrich) in a shaking incubator at 37°C for 1 h. The digested tumor tissues were passed through a 40-μm cell strainer to obtain a single-cell suspension. Whole tumor cells were isolated using Ficoll density-gradient centrifugation. Briefly, 5 ml whole tumor suspension in PBS was layered carefully over 3 ml Ficoll in a 15-ml conical centrifuge tube, centrifuged for 30 min (400 × g) at 25°C, and washed. Finally, the cells were suspended in FACS buffer and kept on ice until use. Spleen and bone marrow (BM) cells were prepared, as described (16, 17). Isolated cells were used for FACS analysis and/or sorting ILC2s. Isolated tumor and BM cells were depleted of lineage-positive cells (CD3+, CD4+, CD8+, B220+, CD11b+, CD11c+, Gr-1+, and Ter119+) using a lineage cell depletion kit (Miltenyi Biotec), according to the manufacturer’s instructions to purify ILC2s (1820). Lineage-depleted cells were incubated with fluorochrome-conjugated anti–Sca-1, anti–IL-7Rα, and anti–c-Kit mAbs at 4°C for 20 min. Finally, the cells were washed twice with 1% BSA in PBS buffer and sorted using a FACSAria III (BD Biosciences).

Single-cell suspensions were washed and resuspended in FACS buffer (1% BSA and 0.05% sodium azide in PBS). The cells were preincubated in a 2.4G2 mAb at 4°C for 10 min, followed by staining with appropriate mAbs. The cells were washed twice with FACS buffer and analyzed using the FACSCanto system (BD Biosciences). Data were analyzed using FlowJo software (version 10).

EL4-Vec, EL4–IL-33, CT26-Vec, CT26–IL-33, B16F10-Vec, and B16F10–IL-33 cells were cultured in DMEM in a six-well plate (5 × 105 cells/well) for 3 d. The culture supernatants were collected, and IL-33 levels were determined by ELISA (R&D Systems), according to the manufacturer’s instructions. Purified ILC2s were similarly cultured in StemPro-34 medium (Life Technologies) containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) in a six-well plate (1 ×105 cells/well) for 5–7 d. Levels of IL-5, IL-13, and GM-CSF in culture supernatants were measured using a CBA kit (BD Biosciences) and those of CXCL1 and CXCL2 were measured by ELISA (R&D Systems). Levels of IL-5, IL-13, GM-CSF, CXCL1, and CXCL2 in tumor homogenates were measured similarly. Samples were read at 450 nm using a SpectraMax M2 spectrophotometer (Molecular Devices).

Harvested tumor masses were fixed in 10% formalin and embedded in paraffin. Five-μm--thick serial sections were cut, placed on a charged slide, and stained with H&E or Masson’s trichrome. The paraffin sections were deparaffinized and rehydrated in a graded alcohol series for immunohistochemistry. The sections were washed in distilled water and boiled in a microwave oven in sodium citrate buffer (10 mM sodium citrate containing 0.05% Tween 20 [pH 6]) for 20 min to retrieve the epitopes. The slides were equilibrated in PBS and incubated with blocking solution for 1 h at room temperature, followed by an overnight incubation with FITC–anti-annexin V and PE–anti-CXCR2 in staining buffer (PBS containing 0.2% BSA) at 4°C. All specimens were mounted with Fluoromount-G (SouthernBiotech). Tumors were embedded in OCT compound (Sakura Finetek) and snap frozen in liquid nitrogen to detect tumor vessels. Sections (5 μm thick) were fixed with acetone for 10 min, air dried, and incubated with blocking solution for 1 h at room temperature. The slides were incubated overnight with FITC–anti-α-SMA and PE–anti-CD31 in staining buffer at 4°C. After washing, the slides were mounted with Fluoromount-G. Images were obtained using an Olympus FluoView FV1200 confocal microscope. Cell density was determined by measuring the cell stained pixel density using Adobe Photoshop version 7.0 software (Adobe Systems).

Tumors were harvested, frozen, and stored at −80°C before analysis. Total RNA was extracted using the RNeasy kit (Qiagen). cDNAs were transcribed from 1 μg RNA using Moloney murine leukemia virus reverse transcriptase (Promega). Quantitative real-time PCR was performed using the SYBR Green PCR master mix (Applied Biosystems) and the ABI Prism 7500 version sequence detection system (Applied Biosystems). We used the following primer sequences: IL-1β forward (5′-GCC TCG TGC TGT CGG ACC CA-3′) and reverse (5′-TGA GGC CCA AGG CCA CAG GT-3′); IL-13 forward (5′-GCA AGG CCC CCA CTA CGG TC-3′) and reverse (5′-AAG GGG CCG TGG CGA AAC AG-3′); tissue inhibitor of metalloproteinase-1 forward (5′-GGC CCC CTT TGC ATC TCT GGC-3′) and reverse (5′-TGC GGC ATT TCC CAC AGC CT-3′); lysyl oxidase forward (5′-ACT TGG TGC TGA GGA AGG GCC A-3′) and reverse (5′-TGC CGT GAA ACC TCA CAA TGG GG-3′); collagen I forward (5′-ACC CCC AAA GAC GGG AGG GC-3′) and reverse (5′-GGG GCC AGG CAG GGA AAC TC-3′); collagen VI forward (5′-GAG GCC ACG AGA TAG AAG GAG GAG-3′) and reverse (5′-CAC GGC GCA GGG ATC GAC TGT G-3′); thrombospodin-1 forward (5′-CCG GGT TAC TGA GCC CCG GT-3′) and reverse (5′-TCG GCC CAC ACA GCG TC-3′); matrix metalloproteinase (MMP)-2 forward (5′-TTG GGA CGT GGG ACC CCG TTA-3′) and reverse (5′-AGA ACC CGC AGA GGT CCG GC-3′); MMP-9 forward (5′-TAC AGG CGC CCC CTC ACC TG-3′) and reverse (5′-GGC GGA GTC CAG CGT TGC AG-3′); MMP-12 forward (5′-AGG TAT CTG CCT GTG GGG CTG C-3′) and reverse (5′-TCC GAC CTC TGG GGC ACT GCT CT-3′); hypoxia-inducible factor (HIF)-1α forward (5′-TCA CTG GGA CTG TTA GGC TGG GA-3′) and reverse (5′-GCT GCC GGC GAC ACC ATC AT-3′); vascular endothelial growth factor (VEGF) forward (5′-GGC TGC TGT AAC GAT GAA G-3′) and reverse (5′-CTC TCT ATG TGC TGG CTT TG-3′); platelet-derived growth factor-B forward (5′-CTG GGA CAT CCA GGG AGC AGC-3′) and reverse (5′-CCA CAC TCT TGC CGA CGC CC-3′); Ang-2 forward (5′-CTA CCA ACA ACA ACA GCA TCC-3′) and reverse (5′-CTC CCT TTA GCA AAA CAC CTT C-3′); TGF-β1 forward (5′-GCG TGC TAA TGG TGG ACC GC-3′) and reverse (5′-CAG CAA TGG GGG TTC GGG CA-3′); HIF-2α forward (5′-GTC AAC CTC AAG TCG CGG AC-3′) and reverse (5′-GCT GGA TTG GCT CAC ACA TG-3′); and soluble VEGF receptor 1 forward (5′-GAA CCT GCT CCT CAA GAA CG-3′) and reverse (5′-CCT TTT TGT TGC AGT GCT CA-3′).

Tumors were obtained 10 d after inoculation, homogenized in 1 ml PBS, and digested with 6 N HCl overnight at 110°C. After filtering the hydrolysate through a 45-μm syringe filter (Millipore), 50 μl citrate-acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid [pH 6.0]) and 100 μl chloramine-T solution (282 mg chloramine-T, 2 ml n-propanol, 2 ml H2O, and 16 ml citrate-acetate buffer) were added to samples for 15 min at room temperature. Next, 100 μl Ehrlich’s solution (Sigma-Aldrich) was added and the mixture was incubated at 65°C for 15 min. The samples were read at 550 nm in a SpectraMax M2 spectrophotometer.

An active form of FLAG-tagged mouse IL-33 (amino acids 109–266) fused to the OPG signal peptide (MNKWLCCALLVLLDIIEWTTQ) was amplified using 5′-CTC GAG CGG ATG AAC AAG TGG CTG TGC TGC GCA CTC CTG GTG CTC CTG GAC ATC ATT GAA TGG ACA ACC CAG AGC ATC CAA GGA ACT TCA CTT TTA ACA-3′ (NheI) and 5′-GCT CTA GAC TAC TTA TCG TCG TCA TCC TTG TAA TCG ATT TTC GAG AGC TT-3′ (XbaI) primers and cloned into the pShuttle2 vector (Clontech). After PI-SceI/I-CeuI double digestion of the pShuttle2 vector, the IL-33 expression cassette was cloned into the Adeno-X viral DNA (Clontech). The recombinant adenoviral DNA was digested with the SwaI restriction enzyme and was transformed into Escherichia coli, amplified, and purified. The purified DNA was digested with the PacI restriction enzyme and transfected into Adeno-X 293 cells. The infected cell lysate was harvested 7–10 d after transfection. The amplified virus underwent two additional amplification rounds. The amplified virus was purified with an Adeno-X virus purification kit (Clontech) and the adenovirus titer was determined with an Adeno-X rapid titer kit (Clontech). A control empty vector was generated as described above. A mixture of Adeno–IL-33 (2 × 107 CFU/mouse) in PBS was injected into tumors with an insulin syringe at 2-d intervals.

Flag-tagged mouse CXCL1 and CXCL2 were amplified using 5′-GGA ATT CAT GAT CCC AGC CAC CCG CTC TTC-3′ (EcoRI), 5′-GCT CTA GAC TAC TTA TCG TCG TCA TCC TTG TAA TCC TTG GGG ACA CCT TTT AGC ATC TT-3′ (XbaI), 5′-GGA ATT CAT GGC CCC TCC CAC CTG CCG GC-3′ (EcoRI), and 5′-GCT CTA GAC TAC TTA TCG TCG TCA TCC TTG TAA TCG TTA GCC TTG CCT TTG TTC AG TAT-3′ (Xba I), respectively, and cloned into the pLVX-puro vector (Clontech). Recombinant lentiviral DNAs were transformed into E. coli, amplified, and purified. The purified lentiviral DNAs were transfected into Lenti-X 293T cells using the Lenti-X HTX packing system (Clontech) to produce lentivirus. Lentiviral titers were determined in culture supernatants with the Lenti-X p24 rapid titer kit (Clontech). A control empty vector was generated as described above. Lenti-CXCL1 and Lenti-CXCL2 (5–50 × 105 CFU/mouse) in PBS were injected into tumors with an insulin syringe at 2-d intervals.

Levels of intratumoral reactive oxygen species (ROS) were detected using chloromethyl dichlorodihydrofluorescein diacetate (21), according to the manufacturer’s instructions (Molecular Probes). Cells isolated from tumors were treated with chloromethyl dichlorodihydrofluorescein diacetate (10 μM/sample) at 4°C for 30 min, washed with FACS staining buffer, and analyzed with the FACSCanto system.

EL4 cells were cultured in DMEM in the presence of PBS or 100 μM H2O2 (Sigma-Aldrich) for 24 h. The cells were stained with anti-CXCR2 and annexin V mAbs, and the CXCR2+annexin V+ cells were analyzed by flow cytometry.

Tumor-bearing mice were injected i.p. with pimonidazole hydrochloride (60 mg/kg; Hypoxyprobe), and tumors were harvested 1 h after treatment. Tumor hypoxia was evaluated with the Hypoxyprobe-1 kit (Hypoxyprobe) according to the manufacturer’s instructions.

Tumor-bearing mice were injected i.v. with 100 μl 1% Evans blue (Sigma-Aldrich), as described previously (22), and tumors were harvested 30 min after treatment. Evans blue was extracted from tumors with formamide (1 ml/sample) overnight at 56°C. The samples were read at 630 nm in a SpectraMax M2 spectrophotometer, and the Evans blue concentration was calculated based on a standard curve of known amounts of Evans blue.

Mice were injected i.p. with anti-CD4 (200 μg/mouse), anti-CD8 (200 μg/mouse), or control IgG (200 μg/mouse) at 5-d intervals starting from the day of tumor cell inoculation to deplete CD4+ and CD8+ T cells (16). Tumor-bearing mice were injected i.p. with anti–Gr-1 (200 μg/mouse) and clodrolip (50 μg/mouse) at 2-d intervals beginning 10 d after inoculation to deplete neutrophils and macrophages. Tumor-bearing mice were injected i.p. with anti-asialo GM1 (50 μg/mouse) on days −2, 0, 3, 10, and 17 (day 0: tumor cell inoculation) to deplete NK cells.

GraphPad Prism 5 (GraphPad Software) was used to analyze and present all data. The two-tailed unpaired Student t test was used for comparisons, and p < 0.05 was considered significant. Tumor growth over time was evaluated by linear trend analysis. Data are presented as mean ± SEM.

To test the effect of local IL-33 production on tumor growth, we generated EL4 lymphoma cells engineered to secrete IL-33 (herein referred to as EL4–IL-33) (Fig. 1A). EL4–IL-33 cells failed to form a distinguishable tumor mass (Fig. 1B). Histological analysis of EL4–IL-33 tumors showed a heavy infiltration of granulocytes and macrophages with marked tumor fibrosis (Fig. 1C). Gr-1Siglec-F+ eosinophils were the major cell population in the immune infiltrate (∼50%) (Fig. 1D, 1E). We confirmed severe fibrosis in EL4–IL-33 tumors using Mason’s trichrome stain and by measuring hydroxyproline content (Fig. 1F). Consistent with this result, fibrosis-related genes, including IL-1β, IL-13, tissue inhibitor of metalloproteinase-1, lysyl oxidase, collagen I, collagen VI, thrombospodin-1, MMP-2, MMP-9, MMP-12, and TGF-β were upregulated in EL4–IL-33 tumors (Fig. 1G).

FIGURE 1.

Characterization of EL4–IL-33 tumors. (A) Levels of IL-33 secreted from EL4-Vec and EL4–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d, and concentrations of IL-33 in culture supernatants were measured by ELISA. (B) C57BL/6 mice were injected s.c. with EL4-Vec or EL4–IL-33 cells (5 × 106 cells/mouse). Tumor growth was measured two to three times weekly (n = 5/group). (C) Tumor histological sections. Samples were harvested 15 d after inoculation, and sections were stained with Mason’s trichrome. Original magnification ×100 for the upper panels and ×400 for the lower panels. (D and E) Tumors were removed 28 d after inoculation, and tumor eosinophils were analyzed by FACS. Representative FACS plots (D) and percentages of eosinophils (E) are presented (n = 5/group). (F) Quantities of hydroxyproline are presented as micrograms per 130 mg 15-d tumor (n = 5/group). (G) Real-time PCR analysis of gene expression in tumors harvested 15 d after inoculation (n = 4–12/group). Studies were repeated at least twice. **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 1.

Characterization of EL4–IL-33 tumors. (A) Levels of IL-33 secreted from EL4-Vec and EL4–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d, and concentrations of IL-33 in culture supernatants were measured by ELISA. (B) C57BL/6 mice were injected s.c. with EL4-Vec or EL4–IL-33 cells (5 × 106 cells/mouse). Tumor growth was measured two to three times weekly (n = 5/group). (C) Tumor histological sections. Samples were harvested 15 d after inoculation, and sections were stained with Mason’s trichrome. Original magnification ×100 for the upper panels and ×400 for the lower panels. (D and E) Tumors were removed 28 d after inoculation, and tumor eosinophils were analyzed by FACS. Representative FACS plots (D) and percentages of eosinophils (E) are presented (n = 5/group). (F) Quantities of hydroxyproline are presented as micrograms per 130 mg 15-d tumor (n = 5/group). (G) Real-time PCR analysis of gene expression in tumors harvested 15 d after inoculation (n = 4–12/group). Studies were repeated at least twice. **p < 0.01, ***p < 0.001 between the two groups.

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The Th2 cytokines IL-5 and IL-13 promote differentiation of eosinophils and fibrosis, respectively. Indeed, high levels of IL-5 and IL-13 were detected in EL4–IL-33 tumors (Fig. 2A). These high levels of IL-5 and IL-13 as well as those of GM-CSF in EL4–IL-33 tumor indicate IL-33–induced intratumoral expansion of ILC2s in response to IL-33 (19, 2325). Indeed, a flow-assisted cell sorting analysis showed markedly increased accumulation of LinSca-1+c-Kit+IL-7Rα+ or LinCD90+IL-13+GATA3+ ILC2s (19, 2326) in EL4–IL-33 tumors (Fig. 2B, 2C). To investigate the ability of tumor-contained ILC2s to produce IL-5, IL-13, and GM-CSF, we stimulated EL4–IL-33 tumor-derived ILC2s with IL-33, IL-7, Flt3L, and SCF. Purified tumor ILC2s proliferated vigorously (data not shown), as previously shown (23), and secreted high levels of IL-5, IL-13, and GM-CSF in response to IL-33 (Fig. 2D). BM-derived ILC2s displayed basically the same pattern of cytokine secretion in response to IL-33 (Fig. 2E), indicating that the tumor LinSca-1+c-Kit+IL-7Rα+ cells were genuine ILC2s.

FIGURE 2.

ILC2s are required for IL-33–inhibited tumor growth. (A) Levels of IL-5, IL-13, and GM-CSF in tumors harvested 15 d after inoculation. (B and C) ILC2s were analyzed from 15-d tumors. Representative FACS plots for LinIL-7Rα+c-Kit+Sca-1+ and LinCD90+IL-13+GATA3+ cells, and their respective percentages are presented (n = 6/group). (D and E) ILC2s were sorted from EL4–IL-33 tumors (D) and BM (E), and 1 × 104 cells/well were cultured in StemPro-34 medium containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) for 5–7 d. Levels of IL-5, IL-13, and GM-CSF were measured in culture supernatants. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 2.

ILC2s are required for IL-33–inhibited tumor growth. (A) Levels of IL-5, IL-13, and GM-CSF in tumors harvested 15 d after inoculation. (B and C) ILC2s were analyzed from 15-d tumors. Representative FACS plots for LinIL-7Rα+c-Kit+Sca-1+ and LinCD90+IL-13+GATA3+ cells, and their respective percentages are presented (n = 6/group). (D and E) ILC2s were sorted from EL4–IL-33 tumors (D) and BM (E), and 1 × 104 cells/well were cultured in StemPro-34 medium containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) for 5–7 d. Levels of IL-5, IL-13, and GM-CSF were measured in culture supernatants. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

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To investigate whether ILC2s are required for the antitumor effect of IL-33, we adoptively transferred wild-type (WT) ILC2s to Rag2−/−γc−/− mice, which are deficient in ILC2s (19). EL4–IL-33 tumors grew normally in control Rag2−/−γc−/− mice, whereas their growth was markedly suppressed in mice that received WT ILC2s (Fig. 3A). The transferred ILC2s expanded markedly in EL4–IL-33 tumors (Fig. 3B), accompanied by the accumulation of eosinophils and collagen deposition (Fig. 3C, 3D), indicating that the characteristic phenotypic changes observed in EL4–IL-33 tumors were caused mainly by ILC2s. We next asked whether signaling through ST2, the IL-33 receptor, in ILC2s was sufficient for the antitumor effect of IL-33. MyD88−/− mice, which have defective ST2 signaling (9), were adoptively transferred with WT ILC2s. Growth of EL4–IL-33 tumors was completely restored in MyD88−/− mice, whereas transfer of WT ILC2s induced regression of tumor growth, intratumoral accumulation of ILC2s and eosinophils, and tumor fibrosis in MyD88−/− mice (Fig. 3E–H), indicating that ST2 signaling in cells other than ILC2s is not required for IL-33–mediated inhibition of tumor growth. Tumors formed by CT26 (herein referred to as CT26–IL-33) and B16F10 (herein referred to as B16F10–IL-33) cells, to which the IL-33 gene was introduced, displayed essentially the same characteristics seen in EL4–IL-33 tumors (Fig. 4). Taken together, our results suggest that local production of IL-33 elicits accumulation of ILC2s in tumors, which subsequently inhibits tumor growth, accumulation of eosinophils, and deposition of collagen.

FIGURE 3.

ILC2 MyD88 is required for IL-33–mediated inhibition of tumor growth. (AD) ILC2s (1 × 105 cells/mouse) sorted from WT BM cells were adoptively transferred to Rag2−/−γc−/− mice and inoculated immediately with EL4–IL-33 cells. (A) Tumor growth curves (n = 6/group). (B and C) The percentages of ILC2s and eosinophils were determined in 27-d tumors (n = 6/group). (D) Quantities of hydroxyproline are presented as micrograms per 130 mg 15-d tumor (n = 5/ group). (EH) MyD88−/− mice received WT ILC2s (1 × 105 cells/mouse) immediately after inoculation with EL4–IL-33 cells. (E) Tumor growth curves (n = 5/group). (F and G) The percentages of ILC2s and eosinophils in 27-d tumors (n = 5/group). (H) Hydroxyproline quantities are presented as micrograms per 300 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 3.

ILC2 MyD88 is required for IL-33–mediated inhibition of tumor growth. (AD) ILC2s (1 × 105 cells/mouse) sorted from WT BM cells were adoptively transferred to Rag2−/−γc−/− mice and inoculated immediately with EL4–IL-33 cells. (A) Tumor growth curves (n = 6/group). (B and C) The percentages of ILC2s and eosinophils were determined in 27-d tumors (n = 6/group). (D) Quantities of hydroxyproline are presented as micrograms per 130 mg 15-d tumor (n = 5/ group). (EH) MyD88−/− mice received WT ILC2s (1 × 105 cells/mouse) immediately after inoculation with EL4–IL-33 cells. (E) Tumor growth curves (n = 5/group). (F and G) The percentages of ILC2s and eosinophils in 27-d tumors (n = 5/group). (H) Hydroxyproline quantities are presented as micrograms per 300 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

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FIGURE 4.

CT26–IL-33 and B16F10–IL-33 cells do not form visible tumors. (A) Levels of IL-33 secreted from CT26-Vec and CT26–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d and IL-33 concentrations were measured in culture supernatants. (B) BALB/c mice were injected s.c. with CT26-Vec or CT26–IL-33 cells (3 × 106 cells/mouse). Tumor growth curves are shown (n = 5/group). (C and D) Percentages of ILC2s and eosinophils in 23-d tumors (n = 5/group). (E) Histological analysis of 15-d tumor sections. Mason’s trichrome stain. Original magnification ×100 for the upper panels and ×400 for the lower panels. (F) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). (G) Levels of IL-33 secreted from B16F10-Vec and B16F10–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d and IL-33 concentrations were measured in culture supernatants. (H) C57BL/6 mice were injected s.c. with B16F10-Vec or B16F10–IL-33 cells (2.5 × 106 cells/mouse). Tumor growth curves are shown (n = 5/group). (I and J) Percentages of ILC2s and eosinophils in 29-d tumors (n = 5/group). (K) Histological analysis of 15-d tumor sections. Mason’s trichrome stain. Original magnification ×100 for the upper panels and ×400 for the lower panels. (L) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, *p < 0.05, ***p < 0.001 between the two groups.

FIGURE 4.

CT26–IL-33 and B16F10–IL-33 cells do not form visible tumors. (A) Levels of IL-33 secreted from CT26-Vec and CT26–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d and IL-33 concentrations were measured in culture supernatants. (B) BALB/c mice were injected s.c. with CT26-Vec or CT26–IL-33 cells (3 × 106 cells/mouse). Tumor growth curves are shown (n = 5/group). (C and D) Percentages of ILC2s and eosinophils in 23-d tumors (n = 5/group). (E) Histological analysis of 15-d tumor sections. Mason’s trichrome stain. Original magnification ×100 for the upper panels and ×400 for the lower panels. (F) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). (G) Levels of IL-33 secreted from B16F10-Vec and B16F10–IL-33 cells. Cells (1 × 104/well) were cultured for 3 d and IL-33 concentrations were measured in culture supernatants. (H) C57BL/6 mice were injected s.c. with B16F10-Vec or B16F10–IL-33 cells (2.5 × 106 cells/mouse). Tumor growth curves are shown (n = 5/group). (I and J) Percentages of ILC2s and eosinophils in 29-d tumors (n = 5/group). (K) Histological analysis of 15-d tumor sections. Mason’s trichrome stain. Original magnification ×100 for the upper panels and ×400 for the lower panels. (L) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, *p < 0.05, ***p < 0.001 between the two groups.

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To determine whether local production of IL-33 in established tumors blocks tumor growth, intratumoral infections with an adenovirus containing the IL-33 gene (Adeno–IL-33) were performed. Injections of Adeno–IL-33 significantly suppressed EL4 tumor growth and induced marked accumulation of ILC2s and eosinophils, as well as deposition of collagens (Fig. 5). Similar to EL4–IL-33 tumors, local production of IL-33 in CT26 and B16F10 tumors after the Adeno–IL-33 infection induced the same phenotypic changes experienced by EL4 tumor after Adeno–IL-33 infection (Fig. 6). These results indicate that the expansion of ILC2s induced by IL-33 suppresses growth of established tumors.

FIGURE 5.

Regression of tumor growth by intratumoral injection of Adeno–IL-33. Tumor-bearing mice received intratumoral injections of Adeno-Vec or Adeno–IL-33 (2 × 107 CFU/mouse) at 2-d intervals 10 d after inoculation with EL4 cells. (A) Tumor growth curves (n = 6/group). (B and C) Percentages of ILC2s and eosinophils in 31-d tumors (n = 6/group). (D) Quantities of hydroxyproline are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 5.

Regression of tumor growth by intratumoral injection of Adeno–IL-33. Tumor-bearing mice received intratumoral injections of Adeno-Vec or Adeno–IL-33 (2 × 107 CFU/mouse) at 2-d intervals 10 d after inoculation with EL4 cells. (A) Tumor growth curves (n = 6/group). (B and C) Percentages of ILC2s and eosinophils in 31-d tumors (n = 6/group). (D) Quantities of hydroxyproline are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

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FIGURE 6.

Regression of CT26 and B16F10 tumor growth following intratumoral injection of Adeno–IL-33. Tumor-bearing mice received intratumoral injections of Adeno-Vec or Adeno–IL-33 (2 × 107 CFU/mouse) at 2-d intervals beginning 10 d after inoculation with CT26 cells (AD) or B16F10 cells (FH). (A and E) Tumor growth curves (n = 5/group). (B, C, G, and H) Percentages of ILC2s and eosinophils in 22-d CT26 tumors or 18-d B16F10 tumors (n = 6/group). (D and H) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01 between the two groups.

FIGURE 6.

Regression of CT26 and B16F10 tumor growth following intratumoral injection of Adeno–IL-33. Tumor-bearing mice received intratumoral injections of Adeno-Vec or Adeno–IL-33 (2 × 107 CFU/mouse) at 2-d intervals beginning 10 d after inoculation with CT26 cells (AD) or B16F10 cells (FH). (A and E) Tumor growth curves (n = 5/group). (B, C, G, and H) Percentages of ILC2s and eosinophils in 22-d CT26 tumors or 18-d B16F10 tumors (n = 6/group). (D and H) Hydroxyproline quantities are presented as micrograms per 100 mg 15-d tumor (n = 5/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01 between the two groups.

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We hypothesized that a mediator secreted by ILC2s is responsible for IL-33–mediated inhibition of tumor growth. We first investigated the antitumor activities of IL-5, IL-13, and GM-CSF, which are the major cytokines secreted by ILC2s in IL-33–secreting tumors (Fig. 2A). Although intratumoral neutralization of IL-5 markedly inhibited accumulation of eosinophils in EL4–IL-33 tumors (Supplemental Fig. 1B), it had a minimal effect on tumor growth (Supplemental Fig. 1A). Similarly, no difference in the growth kinetics of CT26–IL-33 tumors in WT versus that in eosinophil-deficient mice was observed (Supplemental Fig. 1C, 1D). These results are consistent with previously reported data showing that differentiation of eosinophils in tumors induced by IL-5 does not suppress tumor growth (27). Intratumoral neutralization of IL-13 or deleting the IL-13 gene in the host also did not induce any evident regrowth of IL-33–secreting tumors (Supplemental Fig. 2A, 2B). Additionally, inhibiting intratumoral collagen synthesis and facilitating intratumoral collagen degradation also failed to restore growth of EL4–IL-33 tumors (Supplemental Fig. 2C, 2D). Thus, these results indicate that production of IL-13 by ILC2s and the subsequent fibrosis are not involved in IL-33–mediated inhibition of tumor growth. Finally, intratumoral neutralization of GM-CSF caused no visible change in the growth pattern of EL4–IL-33 tumors (Supplemental Fig. 3).

ILC2s are implicated in producing CXCR2 ligands during lung injury caused by influenza virus (25). As high levels of CXCL1 and CXCL2 were present in EL4–IL-33 tumors (Fig. 7A), we examined the ability of tumor-derived ILC2s to produce CXCL1 and CXCL2 in response to IL-33. ILC2s isolated from EL4–IL-33 tumors or BM released large quantities of CXCL1 and CXCL2 in response to IL-33 in the presence of IL-7, Flt3L, and SCF (Fig. 7B, 7C). Importantly, a high proportion of cells in EL4–IL-33 tumors expressed CXCR2 and annexin V (Fig. 7D, 7E). An immunohistochemical analysis showed that CXCR2 and annexin V expression was restricted mainly to cells in the inner mass of tumors (Fig. 7F), indicating that CXCR2 is expressed on tumor cells rather than tumor stromal cells and that CXCR2+ tumor cells were undergoing massive apoptosis. To provide undisputed evidence that CXCR2-expressing cells are annexin V+ tumor cells, Thy1.2+ EL4 or Thy1.2+ EL4–IL-33 tumor cells were transplanted into Thy1.1 congenic mice. CXCR2 expression was barely detectable in EL4 tumors (Fig. 7G). However, a high proportion of Thy1.2+ EL4–IL-33 tumor cells and a small proportion of Th1.2 stromal cells in EL4–IL-33 tumors expressed CXCR2 (Fig. 7G). Importantly, most Thy1.2+ EL4–IL-33 tumor cells but not Th1.2 stromal cells were annexin V+ (Fig. 7G), confirming that apoptosis in EL4–IL-33 tumors was CXCR2+ tumor cell specific. Treatment with the CXCR2 antagonist SB225002 largely restored tumorigenicity in EL4–IL-33 cells but did not affect growth of EL4-Vec tumors (Fig. 7H). Overall, our results suggest that ILC2s induce tumor cell-specific apoptosis through CXCR2.

FIGURE 7.

CXCR2 signaling is responsible for IL-33–mediated inhibition of tumor growth. (A) Levels of CXCL1 and CXCL2 in 15-d tumors. (B and C) ILC2s were sorted from EL4–IL-33 tumors (B) and BM (C), and 1 × 104 cells/well were cultured in StemPro-34 medium containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) for 5–7 d. Levels of CXCL1 and CXCL2 were measured in culture supernatants. (DF) Tumors were harvested 15 d after inoculation (n = 5/group). (D) A representative CXCR2-stained FACS histogram and the percentages of CXCR2-expressing cells are presented. Blue line indicates EL4–IL-33; gray line indicates isotype control; red line indicates EL4-Vec. (E) A representative annexin V–stained FACS histogram and percentages of annexin V+ cells. (F) Immunohistochemical staining for CXCR2 (red) and annexin V (green) in 10-d tumor sections. Original magnification ×80. (G) Thy1.1+ congenic mice were inoculated with Thy1.2+ EL4-Vec or EL4–IL-33, and tumors were harvested 10 d after inoculation. Thy1.2+ tumor cells were gated and dot plots for CXCR2+/annexin V+–stained tumor cells and their percentages are presented (n = 5/group). (H) Tumor-bearing mice received PBS or SB225002 (20 μg/mouse) at 2-d intervals beginning 10 d after inoculation. Tumor growth curves are shown (n = 6/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 7.

CXCR2 signaling is responsible for IL-33–mediated inhibition of tumor growth. (A) Levels of CXCL1 and CXCL2 in 15-d tumors. (B and C) ILC2s were sorted from EL4–IL-33 tumors (B) and BM (C), and 1 × 104 cells/well were cultured in StemPro-34 medium containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) for 5–7 d. Levels of CXCL1 and CXCL2 were measured in culture supernatants. (DF) Tumors were harvested 15 d after inoculation (n = 5/group). (D) A representative CXCR2-stained FACS histogram and the percentages of CXCR2-expressing cells are presented. Blue line indicates EL4–IL-33; gray line indicates isotype control; red line indicates EL4-Vec. (E) A representative annexin V–stained FACS histogram and percentages of annexin V+ cells. (F) Immunohistochemical staining for CXCR2 (red) and annexin V (green) in 10-d tumor sections. Original magnification ×80. (G) Thy1.1+ congenic mice were inoculated with Thy1.2+ EL4-Vec or EL4–IL-33, and tumors were harvested 10 d after inoculation. Thy1.2+ tumor cells were gated and dot plots for CXCR2+/annexin V+–stained tumor cells and their percentages are presented (n = 5/group). (H) Tumor-bearing mice received PBS or SB225002 (20 μg/mouse) at 2-d intervals beginning 10 d after inoculation. Tumor growth curves are shown (n = 6/group). Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

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ROS induce CXCR2 expression on tumor cells (28). Interestingly, we found that aberrant angiogenesis was associated with severe hypoxic conditions, ROS production, and CXCR2 expression in EL4–IL-33 tumors. Gross observations revealed a lower density of RBCs and severe defects in permeability in EL4–IL-33 tumors (Fig. 8A–C), indicating that blood vessels in EL4–IL-33 tumors were not fully functional. Consistent with the lower degree of angiogenesis in EL4–IL-33 tumors, these tumors expressed lower levels of angiogenesis-promoting genes (HIF-1α, VEGF, platelet-derived growth factor-B, and TGF-β) but higher levels of angiogenesis-inhibiting genes (HIF-2α and soluble VEGF receptor) (29, 30) (Fig. 8E). Abnormal vessel formation in EL4–IL-33 tumors was also characterized by a paucity of pericytes covering the endothelial cell layer (Fig. 8F–H). As a result of nonfunctional vessel formation, EL4–IL-33 tumors had severe hypoxia (Fig. 8I), which was associated with higher ROS production (Fig. 8J). To examine whether ROS could directly induce CXCR2 expression in EL4 cells, we treated EL4 cells with H2O2 in vitro. As expected, H2O2 significantly upregulated CXCR2 expression in EL4 cells (Fig. 8K, 8L). Importantly, CT26–IL-33 and B16F10–IL-33 tumors also produced higher levels of ROS and contained CXCR2+ cells, with most of them being annexin V+ (Fig. 9). Overall, these results suggest that CXCR2 expression on tumor cells is associated with a hypoxic tumor microenvironment created as a result of IL-33–induced aberrant angiogenesis.

FIGURE 8.

Association between dysfunctional angiogenesis and CXCR2 expression in EL4–IL-33 tumors. (A) Gross observations of tumor vasculature 14 d after inoculation. (B and C) Permeability assay for tumor vessels. Evans blue permeability was observed in 15-d tumors 30 min after i.v. injection of Evans blue (B), and Evans blue was extracted from the tumors (C). Data are presented as nanograms per 100 mg tumor (n = 5/group). (D and E) Real-time PCR analysis for the expression of angiogenesis-related genes in 12-d tumors. (FH) Immunohistochemical analysis for vasculature in 10-d tumors. Frozen tumor sections were double stained for CD31 (red) and α-SMA (green) (F). CD31+ and α-SMA+ vessel areas were calculated on randomly selected fields. Images were generated at ×400 original magnification. (G) Percentages of CD31+ vessel areas (n = 9). (H) Ratios of α-SMA+ to CD31+ vessel area (n = 12). (I) Evaluation of tumor hypoxia. Pimonidazole hydrochloride was injected into mice 10 d after inoculation and tumors were harvested 1 h later. Data are presented as percentages of the area of pimonidazole pixels in tumor sections (n = 5/group). (J) ROS levels in cells isolated from 10-d tumors. Data are presented as a mean fluorescence intensity (MFI) (n = 5/group). (K and L) EL4 cells were cultured in the presence of PBS or H2O2 for 24 h and CXCR2 expression was analyzed. (K) Representative CXCR2-stained FACS histogram. Blue line indicates H2O2; gray line indicates isotype control; red line indicates PBS. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 8.

Association between dysfunctional angiogenesis and CXCR2 expression in EL4–IL-33 tumors. (A) Gross observations of tumor vasculature 14 d after inoculation. (B and C) Permeability assay for tumor vessels. Evans blue permeability was observed in 15-d tumors 30 min after i.v. injection of Evans blue (B), and Evans blue was extracted from the tumors (C). Data are presented as nanograms per 100 mg tumor (n = 5/group). (D and E) Real-time PCR analysis for the expression of angiogenesis-related genes in 12-d tumors. (FH) Immunohistochemical analysis for vasculature in 10-d tumors. Frozen tumor sections were double stained for CD31 (red) and α-SMA (green) (F). CD31+ and α-SMA+ vessel areas were calculated on randomly selected fields. Images were generated at ×400 original magnification. (G) Percentages of CD31+ vessel areas (n = 9). (H) Ratios of α-SMA+ to CD31+ vessel area (n = 12). (I) Evaluation of tumor hypoxia. Pimonidazole hydrochloride was injected into mice 10 d after inoculation and tumors were harvested 1 h later. Data are presented as percentages of the area of pimonidazole pixels in tumor sections (n = 5/group). (J) ROS levels in cells isolated from 10-d tumors. Data are presented as a mean fluorescence intensity (MFI) (n = 5/group). (K and L) EL4 cells were cultured in the presence of PBS or H2O2 for 24 h and CXCR2 expression was analyzed. (K) Representative CXCR2-stained FACS histogram. Blue line indicates H2O2; gray line indicates isotype control; red line indicates PBS. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups.

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FIGURE 9.

Association between ROS, CXCR2 expression, and apoptosis in CT26 and B16B10 tumor cells. CT26 (AD) and B16F10 tumors (EH) were harvested 10 d after inoculation and analyzed. (A and E) ROS levels in cells isolated from 10-d tumors. Data are presented as mean fluorescence intensity (MFI) (n = 5/group). (B and F) Representative CXCR2- and annexin V–stained FACS plots. (C, D, G, and H) Percentages of CXCR2+ cells (C and G) and CXCR2+annexin V+ cells (D and H) are shown. Studies were repeated at least twice. **p < 0.01, ***p < 0.001 between the two groups.

FIGURE 9.

Association between ROS, CXCR2 expression, and apoptosis in CT26 and B16B10 tumor cells. CT26 (AD) and B16F10 tumors (EH) were harvested 10 d after inoculation and analyzed. (A and E) ROS levels in cells isolated from 10-d tumors. Data are presented as mean fluorescence intensity (MFI) (n = 5/group). (B and F) Representative CXCR2- and annexin V–stained FACS plots. (C, D, G, and H) Percentages of CXCR2+ cells (C and G) and CXCR2+annexin V+ cells (D and H) are shown. Studies were repeated at least twice. **p < 0.01, ***p < 0.001 between the two groups.

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We next explored whether CXCR2 ligands secreted by ILC2s are sufficient to suppress tumor growth independently of those derived from other host cells. WT ILC2s were adoptively transferred to Rag2−/−γc−/− mice, and EL4–IL-33 cells were inoculated s.c. Treatment with SB225002 substantially restored tumorigenicity of EL4–IL-33 cells (Fig. 10A). Interestingly, intratumoral expansion of ILC2s was not affected (Fig. 10B), but CXCR2 expression and apoptosis of tumor cells decreased to a basal level by blocking CXCR2 signaling (Fig. 10C), indicating that CXCR2 signaling is critical for maintaining CXCR2 expression on tumor cells. To test this hypothesis, EL4 tumors were injected with lentivirus containing the CXCL1 (Lenti-CXCL1) or CXCL2 (Lenti-CXCL2) gene. Lenti-CXCL1 and Lenti-CXCL2 were equally effective at suppressing tumor growth (Fig. 10D) with increased numbers of CXCR2+ apoptotic tumor cells (Fig. 10E), indicating that CXCR2 signaling contributes to its own expression on tumor cells. Delivery of a mixture of these two virus types had a marginal influence on tumor growth and apoptosis of CXCR2+ tumor cells compared with that of a single virus type (Fig. 10E). We next wanted to confirm the ability of ILC2s to kill EL4 tumor cells in an in vitro coculture system. Indeed, IL-33–activated ILC2s induced stronger apoptosis of EL4 cells in a CXCR2-specific way than did unactivated ILC2s (Fig. 10F, 10G).

FIGURE 10.

CXCR2 ligands secreted by ILC2s are sufficient to regress EL4–IL-33 tumor growth. (AC) ILC2s (1 × 105 cells/mouse) sorted from BM cells were adoptively transferred to Rag2−/−γc−/− mice and inoculated immediately with EL4–IL-33 cells. Tumor-bearing mice received PBS or SB225002 (20 μg/mouse) at 2-d intervals beginning 10 d after inoculation. (A) Tumor growth curves (n = 5/group). (B and C) Tumors were harvested 22 d after inoculation and the percentages of ILC2s (B) and annexin V+ CXCR2+ cells (C) were analyzed (n = 5/group). (D and E) Tumor-bearing mice received intratumoral injections of Lenti-Vec, Lenti-CXCL1, Lenti-CXCL2, or a combination of Lenti-CXCL1 and Lenti-CXCL2 (5–50 × 105 CFU/mouse) at 2-d intervals beginning 10 d after inoculation. (D) Tumor growth curves (n = 5/group). (E) Percentages of annexin V+CXCR2+ cells in 21-d tumors (n = 4/group). (F and G) ILC2s (1 × 104 cells/well) were sorted from BM and cocultured with EL4 cells (1 × 105 cells/well) in DMEM containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) and SB225002 (50 ng/ml). After 24 h, EL4 cells were stained for CXCR2 and annexin V. (F) FACS plots. (G) Percentages of annexin V+ cells. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

FIGURE 10.

CXCR2 ligands secreted by ILC2s are sufficient to regress EL4–IL-33 tumor growth. (AC) ILC2s (1 × 105 cells/mouse) sorted from BM cells were adoptively transferred to Rag2−/−γc−/− mice and inoculated immediately with EL4–IL-33 cells. Tumor-bearing mice received PBS or SB225002 (20 μg/mouse) at 2-d intervals beginning 10 d after inoculation. (A) Tumor growth curves (n = 5/group). (B and C) Tumors were harvested 22 d after inoculation and the percentages of ILC2s (B) and annexin V+ CXCR2+ cells (C) were analyzed (n = 5/group). (D and E) Tumor-bearing mice received intratumoral injections of Lenti-Vec, Lenti-CXCL1, Lenti-CXCL2, or a combination of Lenti-CXCL1 and Lenti-CXCL2 (5–50 × 105 CFU/mouse) at 2-d intervals beginning 10 d after inoculation. (D) Tumor growth curves (n = 5/group). (E) Percentages of annexin V+CXCR2+ cells in 21-d tumors (n = 4/group). (F and G) ILC2s (1 × 104 cells/well) were sorted from BM and cocultured with EL4 cells (1 × 105 cells/well) in DMEM containing Flt3L (20 ng/ml), SCF (20 ng/ml), and IL-7 (10 ng/ml) in the presence or absence of IL-33 (20 ng/ml) and SB225002 (50 ng/ml). After 24 h, EL4 cells were stained for CXCR2 and annexin V. (F) FACS plots. (G) Percentages of annexin V+ cells. Studies were repeated at least twice. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

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The data presented so far indicate that CXCR2 signaling is involved directly in tumor cell apoptosis. To exclude the possibility that CXCR2 signaling in host cells plays a role in tumor cell apoptosis, tumorigenicity of CT26–IL-33 cells was examined in CXCR2−/− mice. No difference in tumor growth kinetics was observed between WT and CXCR2−/− mice (Supplemental Fig. 4A). Additionally, massive apoptosis of CXCR2+ tumor cells occurred in the CT26–IL-33 tumors of CXCR2−/− mice (Supplemental Fig. 4B). These results strongly suggest that CXCR2-mediated tumor cell apoptosis does not involve host cells other than ILC2s. The following evidence supports this hypothesis: systemic depletion of macrophages or neutrophils failed to recover EL4–IL-33 tumor growth (Fig. 11A, 11B), and no regrowth of B16F10–IL-33 tumors was evident after depleting lymphoid cells such CD4+ T cells, CD8+ T cells, or NK cells (Fig. 11C–E). Furthermore, no difference in immune cell composition was observed between the draining lymph nodes and spleen of mice inoculated with EL4–IL-33 and EL4 cells (Fig. 11F, 11G). These results suggest that IL-33–mediated inhibition of tumor growth has no effect on adaptive antitumor immunity. This interpretation was further supported by our observation that EL4–IL-33 cells inhibited growth of EL4 tumors when a mixture of these two cell types was inoculated at the same site but not when EL4 cells were inoculated at another site (Fig. 11H). Taken together, our results show that IL-33–mediated inhibition of tumor growth is a tumor-restricted signaling event. Overall, we have shown that local production of IL-33 in tumors induced marked intratumoral expansion of ILC2s, resulting in various changes in the tumor microenvironment.

FIGURE 11.

Immune cells are not involved in IL-33–mediated inhibition of tumor growth, and local production of IL-33 does not affect systemic antitumor immunity. (A and B) Tumor-bearing mice received intratumoral injections of PBS or clodrolip (50 μg/mouse) and control Ig or anti–Gr-1 mAbs (5 μg/mouse) at 2-d intervals beginning 10 d after inoculation with EL4 or EL4–IL-33 cells to deplete macrophages. Tumor growth curves are shown (n = 5–6/group). (C and D) C57BL/6 mice were inoculated with B16F10-Vec or B16F10–IL-33 cells, and control Ig, anti-CD4, or anti-CD8 mAb (200 μg/mouse) was injected every 5 d starting from the day of inoculation. Tumor growth curves are shown (n = 5/group). (E) C57BL/c mice received control Ig or anti-asialo GM1 (50 μg/mouse) on days −2, 0, 3, 10, and 17. Mice were inoculated with B16B10-Vec or B16F10–IL-33 cells on day 0. Tumor growth curves are shown (n = 5/group). (F and G) Changes in immune cell compositions in the draining lymph node (DLN) and spleen were analyzed 7 d after inoculation with EL4 or EL4–IL-33 cells (n = 8–10/group). (H) One group of mice was inoculated in the left flank with EL4-Vec, EL4–IL-33, or a mixture of an equal number of EL4 and EL4–IL-33 cells. The other group was inoculated with EL4 cells on both flanks or with EL4 cells in the left flank and EL4–IL-33 cells in the right flank. Tumor growth curves are shown (n = 5/group). Studies were repeated at least twice. *p < 0.05 between the two groups.

FIGURE 11.

Immune cells are not involved in IL-33–mediated inhibition of tumor growth, and local production of IL-33 does not affect systemic antitumor immunity. (A and B) Tumor-bearing mice received intratumoral injections of PBS or clodrolip (50 μg/mouse) and control Ig or anti–Gr-1 mAbs (5 μg/mouse) at 2-d intervals beginning 10 d after inoculation with EL4 or EL4–IL-33 cells to deplete macrophages. Tumor growth curves are shown (n = 5–6/group). (C and D) C57BL/6 mice were inoculated with B16F10-Vec or B16F10–IL-33 cells, and control Ig, anti-CD4, or anti-CD8 mAb (200 μg/mouse) was injected every 5 d starting from the day of inoculation. Tumor growth curves are shown (n = 5/group). (E) C57BL/c mice received control Ig or anti-asialo GM1 (50 μg/mouse) on days −2, 0, 3, 10, and 17. Mice were inoculated with B16B10-Vec or B16F10–IL-33 cells on day 0. Tumor growth curves are shown (n = 5/group). (F and G) Changes in immune cell compositions in the draining lymph node (DLN) and spleen were analyzed 7 d after inoculation with EL4 or EL4–IL-33 cells (n = 8–10/group). (H) One group of mice was inoculated in the left flank with EL4-Vec, EL4–IL-33, or a mixture of an equal number of EL4 and EL4–IL-33 cells. The other group was inoculated with EL4 cells on both flanks or with EL4 cells in the left flank and EL4–IL-33 cells in the right flank. Tumor growth curves are shown (n = 5/group). Studies were repeated at least twice. *p < 0.05 between the two groups.

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IL-33 is thought to be a danger signal (also called an alarmin) released early after injury to the epithelium and endothelium. Depending on the context, the IL-33 released can have inflammatory or anti-inflammatory effects. ILC2s are an important target of IL-33 and play a pivotal role in allergy and helminth expulsion (19, 20, 23, 24, 31), obesity (32), and tissue repair (25, 33). Systemic administration of IL-33 results in eosinophilia, mucosal hyperplasia, and organ fibrosis (9, 10, 34), which are likely to be mediated mainly by ILC2-secreted IL-5 and IL-13, respectively. In this study, we showed that local production of IL-33 not only induces these typical phenotypic changes within tumors mediated by ILC2s but also creates a tumor microenvironment unfavorable for tumor growth. In particular, two signaling events, such as aberrant angiogenesis and secretion of CXCR2 ligands, converged at a point leading to active tumor cell apoptosis. First, tumor vessels that form in a milieu of abundant IL-33 are characterized by poor pericyte coverage and impaired tumor perfusion. Our data show that expression of genes critical in pericyte recruitment, differentiation, and vascular stability (35) is downregulated in IL-33–secreting tumors (Fig. 8D). As these and other genes whose expression is regulated by these genes also involve close interactions between endothelial cells and pericytes, the aberrant features of the tumor blood vessels seem to be associated with dysfunctional endothelial cell layers. As a consequence, impaired oxygen delivery causes severe oxidative stress inside tumors (Fig. 8I). This hypoxic condition, in turn, results in intratumoral production of high ROS levels, which seem to be a key trigger for expression of CXCR2 on tumor cells (Fig. 8K, 8L, Supplemental Fig. 3). Second, continuous local production of IL-33 elicits massive intratumoral proliferation and activation of ILC2s. Among various mediators released from activated ILC2s, CXCR2 ligands sustain expression of CXCR2 on tumor cells but also induce apoptosis (Fig. 8A–E). A schematic diagram for the antitumor therapeutic pathway initiated by IL-33 is summarized in Fig. 12.

FIGURE 12.

A schematic diagram for the mechanism underlying the inhibited tumor growth initiated by IL-33. Local production of IL-33 results in dysfunctional angiogenesis, which renders the tumor microenvironment severely hypoxic. Thus, ROS levels increase, inducing expression of CXCR2 on tumor cells. IL-33 elicits massive expansion of ILC2s and production of CXCR2 ligands from these cells. The two signaling events converge to reinforce tumor cell–specific apoptosis via CXCR2.

FIGURE 12.

A schematic diagram for the mechanism underlying the inhibited tumor growth initiated by IL-33. Local production of IL-33 results in dysfunctional angiogenesis, which renders the tumor microenvironment severely hypoxic. Thus, ROS levels increase, inducing expression of CXCR2 on tumor cells. IL-33 elicits massive expansion of ILC2s and production of CXCR2 ligands from these cells. The two signaling events converge to reinforce tumor cell–specific apoptosis via CXCR2.

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Agents causing DNA damage, such as oxidative stress and chemotherapeutic drugs, trigger stress-induced senescence or cell death (36). Studies have demonstrated that CXCR2 signaling causes cellular senescence in a p53-dependent manner, and that expression of its ligands is upregulated during oncogene-induced senescence. These CXCR2 ligands act in a paracrine loop to reinforce senescence and upregulation of CXCR2 expression (37). In addition to the self-amplification loop of senescence via self-signaling, CXCR2 is an important component of the inflammatory network activated by IL-6 during senescence (38). In our experimental system, ROS seemed to be the early trigger for CXCR2 expression on tumor cells, and its expression was maintained by CXCR2 ligands provided by ILC2s. CXCR2 ligands may exert multiple paracrine effects, including reinforcement of senescence (J. Kim, W. Kim, and B. Kwon, unpublished data) and apoptosis, as well as augmentation of the innate immune response. However, coordinated control of signaling events converging on tumor cell apoptosis through CXCR2 is thought to be the main pathway for disabling the tumor growth induced by IL-33. It is unknown how CXCR2 signaling results in tumor cell apoptosis, but one possibility is that inhibiting signaling molecules is involved in tumor cell survival (39).

DNA vaccination with IL-33 adjuvants in conjugation with a human papilloma virus DNA vaccine is highly effective for regressing established tumors, probably by enhancing a tumor Ag-specific T cell response (12). Similar mechanisms, such as the IL-33–induced increase in IFN-γ and perforin effector activities induced by NK cells and CD8+ T cells, act on eradicating IL-33–secreting tumors (13). The reason for the discrepancy between these results and ours is not known, but we suspect that the concentration of IL-33 present within tumors is a critical factor determining the tumor microenvironment. One study (13) reported that markedly increased numbers of NK cells and armed CD8+ T cells but no distinguishable change in myeloid cells is a prominent feature of IL-33–secreting tumors. Thus, higher doses of IL-33 are likely to promote the effector function of CD8+ T cells (12, 13, 40, 41), whereas lower doses of IL-33 seem to induce vigorous proliferation of ILC2s, as shown in the present study. Our results suggest that ILC2-governed responses may override the CD8+ immune response enhanced by IL-33 due to the dominance of a Th2 microenvironment. Nevertheless, it is important to determine whether tumor cell killing by ILC2s results in long-lasting antitumor immunity. More importantly, it is necessary to test the therapeutic efficacy of cells with tumor tropism (i.e., mesenchymal stem cells) that are engineered to express IL-33.

It seemed that the localized antitumor response elicited by IL-33 was not achievable by systemic delivery of cytokines. Indeed, we showed that injecting IL-33–secreting tumor cells or delivering IL-33 using viral vectors into tumor masses regressed the established tumors (data not shown; Fig. 2). Interestingly, local production of effector molecules, such as CXCL1 or CXCL2, resulted in tumor cell apoptosis and subsequent tumor regression (Fig. 10D). Based on these findings in animal models, IL-33– or CXCR2 ligand–based therapy may be useful to test clinically for relatively confined, but surgically inaccessible, tumors, such as certain brain tumors and i.p. malignancies. The potent antitumor activity of IL-33 against a wide variety of tumor cell types is particularly attractive with regard to exploring its efficacy as a human antitumor agent. One of the critical features of this approach is that IL-33 is produced only at the tumor site, thereby producing a strong immune response with no systemic toxicity. This approach may also be useful to treat cancer in combination with traditional chemotherapy or radiotherapy.

We thank our laboratory members for help.

This work was supported by grants from the National Research Foundation of Korea (funded by the Republic of Korea Ministry of Education, Science, and Technology Grants 2009-0094050, NRF-2013R1A1A3013568, and NRF-2014R1A2A2A01005652) and the Korean Health Technology Research and Development Project, Republic of Korea Ministry of Health and Welfare Grant HI13C1325.

The online version of this article contains supplemental material.

Abbreviations used in this article:

Adeno-

adenoviral

asialo GM1

asialo ganglio-N-tetraosylceramide

BM

bone marrow

Flt3L

Flt3 ligand

HIF

hypoxia-inducible factor

ILC2

type 2 innate lymphoid cell

Lenti-

lentiviral

MMP

matrix metalloproteinase

ROS

reactive oxygen species

SCF

stem cell factor

α-SMA

α–smooth muscle actin

VEGF

vascular endothelial growth factor

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data