www.nature.com/cmi Cellular & Molecular Immunology

ARTICLE IL-33 drives the antitumor effects of dendritic cells via the induction of Tc9 cells

Ning Liu1, Yuxue Jiang2, Jintong Chen2, He Nan3, Yinghua Zhao2, Xiao Chu2, Alison Wang2, Dongjiao Wang1, Tianxue Qin4, Sujun Gao4, Qing Yi2,5, Ying Yue1 and Siqing Wang2

Dendritic cell (DC) tumor vaccines exert their antitumor effects through the induction of effector T cells. We recently identified Tc9 cells as a new potent antitumor effector T cell subset. However, approaches to direct DCs to preferably prime antitumor Tc9 cells should be further exploited. Here, we demonstrate that the addition of (IL)-33 potently promotes the induction of Tc9 cells by DCs in vitro and in vivo. IL-33 treatment also drives the cytotoxic activities of DC-induced Tc9 cells. Notably, IL-33 treatment enhances cell survival and proliferation of DC-primed CD8+ T cells. More importantly, the addition of IL-33 during in vitro priming of tumor-specific Tc9 cells by DCs increases the antitumor capability of Tc9 cells. Mechanistic studies demonstrated that IL-33 treatment inhibits exhaustive CD8+ T cell differentiation by inhibiting PD-1 and 2B4 expression and increasing IL-2 and CD127 (IL-7 -α, IL-7Rα) expression in CD8+ T cells. Finally, the addition of IL-33 further promotes the therapeutic efficacy of DC-based tumor vaccines in the OT-I mouse model. Our study demonstrates the important role of IL-33 in DC-induced Tc9 cell differentiation and antitumor immunity and may have important clinical implications.

Keywords: Interleukin-33; Dendritic cells; Tc9; Cancer immunology

Cellular & Molecular Immunology (2019) 16:644–651; https://doi.org/10.1038/s41423-018-0166-0

INTRODUCTION activities in vivo.11 These observations suggest that DC tumor Dendritic cells (DCs) are professional antigen-presenting cells vaccines that preferably induce Tc9 cells may potently increase (APCs) and play a crucial role in the induction of antitumor their antitumor effects. immunity.1,2 DCs exert their antitumor effects through the IL-33 is a member of the IL-1 superfamily of and a induction of effector T cells.3 CD8+ T cells (cytotoxic T ligand for suppression of tumorigenicity 2 (ST2) receptor.14,15 lymphocytes or Tc cells) are superior antitumor effector T cells Previous studies reported that IL-33 preferably stimulates antiviral with immediate cytolytic activity against tumor cells.4,5 CD8+ and antitumor CD8+ T cell responses.16,17 IL-33 overexpression in T cells can be differentiated into different Tc cell subsets with tumors inhibits melanoma tumor growth by activating NK and distinct phenotypes, such as Tc1, Tc2, and Tc17 cells.6 In tumor CD8+ T cell responses18,19 and ILC2-mediated tumor cell immunology, Tc1 cells produce -γ (IFN-γ) and kill tumor apoptosis.20 IL-33 also promotes anti-tumor immunity in mice by cells by IFN-γ-mediated or perforin-mediated mechanisms.4 inhibiting the differentiation and immunosuppressive activity of Tc1 cells are also the major antitumor Tc cells primed by regular granulocytic myeloid-derived suppressor cells and activating DCs DCs in tumor therapy.7 Tc2 cells secrete type II cytokines, such as and .21–23 We recently found that dectin-1-activated IL-4, IL-5, and IL-10.4 Tc17 cells secrete IL-17, which promotes both DCs drive potent Th9 cell responses.24 T cell differentiation tumor growth and tumor regression.8–10 depends mainly on the microenvironment.25 We further We recently described a new Tc cell subset Tc9 characterized by found that dectin-1 activation stimulates DCs to overexpress 42 the secretion of IL-9.11 Tc9 cells can be generated in vitro cytokines and costimulatory molecules, including IL-33, which may by culturing naive CD8+ T cells under Tc9-polarizing conditions contribute to the induction of Th9 cells by dectin-1-activated (i.e., anti-CD3/CD28 plus transforming β (TGF-β)/ DCs.24 Given that Th9 and Tc9 cell differentiation may require IL-4).11,12 Tumor-specific Tc9 cells are superior antitumor effector the same or similar polarizing cytokines, we hypothesized that cells.11 Tc9 cells are less cytolytic in vitro than Tc1 cells; however, cytokines overexpressed by dectin-1-activated DCs may also drive Tc9 cells elicit greater antitumor responses than Tc1 cells in vivo.11 the induction of Tc9 cells by DCs. Tc9 cells have a "younger" phenotype and persist much longer In this study, we found that the addition of IL-33 promotes the than Tc1 cells in vivo.13 Furthermore, Tc9 cells can be induction of Tc9 cells by DCs. IL-33 treatment increases Tc9 cell differentiated into Tc1-like effector cells with increased cytolytic survival and proliferation and drives their cytotoxic activities

1Department of Gynecological Oncology, The First Hospital of Jilin University, Changchun 130061, China; 2Department of Cancer Immunology, The First Hospital of Jilin University, Changchun 130061, China; 3Department of Rheumatology and Immunology, China-Japan Union Hospital of Jilin University, Changchun 130033, China; 4Department of Hematology, The First Hospital of Jilin University, Changchun 130061, China and 5Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA Correspondence: Ying Yue ([email protected]) or Siqing Wang ([email protected]) Received: 27 January 2018 Accepted: 15 August 2018 Published online: 1 October 2018

© CSI and USTC 2018 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 645 against tumor cells. In addition, IL-33 treatment during in vitro Quantitative polymerase chain reaction (qPCR) priming of tumor-specific Tc9 cells by DCs increases the antitumor Total RNA was extracted from cells using an EasyPure RNA Kit capability of Tc9 cells. Furthermore, the combined use of DC (TransGen Biotech), and cDNA was synthesized with an All-in-One vaccine and IL-33 further promotes the therapeutic efficacy of DC- First-Strand cDNA Synthesis SuperMix (TransGen). Cd244, Ifng, Il2, based tumor vaccines in the OT-I mouse model. Our study Il9, Irf4, Gzmb, Lag3, Pdcd1, Spi1, St2, and Tbx21 mRNA levels in Tc demonstrates the important role of IL-33 in DC-induced Tc9 cell cells were analyzed. Expression was normalized to the expression differentiation and antitumor immunity and may have important of the housekeeping Gapdh. Primer sets for Ifng, Il9, Irf4, Spi1, clinical implications. and Tbx21 were previously reported.24 Primer sets for Cd244, Il2, Gzmb, Lag3, Pdcd1 and St2 are provided in Supplementary Table 1.

MATERIALS AND METHODS Enzyme-linked immunosorbent assay (ELISA) Mice and cell lines IL-9 and IFN-γ concentrations in culture supernatants were C57BL/6 (H-2b) and OT-I (C57BL/6-Tg(TcraTcrb)1100Mjbn/J) mice detected by ELISA as previously described.24 Capture/detection were purchased from the Jackson Laboratory. All mice were antibodies for IL-9 and IFN-γ were purchased from BD Biosciences. housed and bred under specific pathogen-free conditions at Recombinant mouse IL-9 and IFN-γ, which were used as ELISA Animal Center of The First Hospital of Jilin University. Mice were standards, were purchased from R&D Systems and BD Biosciences, used for experiments at age 6–8 weeks. All animal studies were respectively. Avidin-HRP was purchased from Biolegend. conducted according to the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act of 1986, EU Directive 2010/63/EU for In vivo functional tests of IL-33/ST2 in DC-induced T cell animal experiments, and the ethical guidelines of the Animal differentiation Ethical Committee of First Hospital of Jilin University. BMDCs were pulsed with OT-I OVA peptides (5 μg/mL). Mice were B16 and B16-OVA melanoma cell lines (ATCC) were cultured in administered 2 weekly subcutaneous immunizations with 1 × 106 RPMI 1640 medium (CORNING) supplemented with 10% heat- treated DCs. Mice injected with PBS served as controls. In some inactivated fetal bovine serum (FBS, HyClone), 100 U/mL penicillin experiments, mice were administered IL-33 (250 ng/mouse) every (HyClone) and 100 mg/mL streptomycin (HyClone). Cells were 3 days starting 1 day after the first DC immunization. On day 3 grown in standard (37 °C, 5% CO2) culture incubators. after the second DC immunization, total leukocytes were collected from spleens and lymph nodes. Cells were restimulated with OT-I

1234567890();,: Reagents and Abs OVA peptides (5 μg/mL) for 2 days. Cells from PBS control mice Recombinant mouse granulocyte- colony-stimulat- were untreated. Culture cells and supernatants were collected and ing factor (GM-CSF), TNF-α, IL-1β, IL-4, and IL-2 were purchased analyzed by flow cytometry and ELISA. CD8+ T cells were isolated from Peprotech. Recombinant mouse IL-33 and human TGF-β by magnetic cell sorting (MACS) and analyzed by qPCR. In some were purchased from R&D Systems. The OVA (257–264, SII NFE KL) experiments, total leukocytes collected from spleens and lymph peptide used in the OT-I mouse model was purchased from GL nodes were restimulated with OVA peptide-pulsed BMDCs in the Biochem (Shanghai) Ltd. Functional anti-mouse CD3e and CD28 presence or absence of IL-33 (50 ng/mL) for 2 days. Culture cells antibodies (mAbs) were purchased from eBioscience. and CD8+ T cells isolated by MACS were analyzed by flow cytometry and/or qPCR. Generation of DCs DCs were generated as described previously.24 In brief, bone Cytotoxicity assay marrow (BM) cells were cultured in RPMI 1640 complete medium The cytotoxicity assay was performed as previously described.11 supplemented with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL). At To examine the cytotoxicity of in vitro differentiated Tc9 cells, day 4, the culture medium was replaced with fresh GM-CSF (10 ng/ naive CD8+ T cells from OT-I mice were cocultured with BMDCs mL) and IL-4 (10 ng/mL)-containing medium. At day 7, semi- under Tc9-polarizing conditions in the presence or absence of IL- adherent cells were collected as immature DCs (iDCs) and matured 33 (50 ng/mL) for 2 days. CD8+ T cells were isolated by MACS and by TNF-α (10 ng/mL) and IL-1β (10 ng/mL) (BMDCs) for 48 h. At day used as effector cells. B16-OVA cells labeled with 2.5 μM CFSE 9, the semi-adherent cells were collected as mature DCs (mDCs) were used as target cells, whereas B16 nontarget cells labeled with for further experiments. 0.25 μM CFSE were used as controls. Target cells or nontarget cells were cocultured with effector cells at different ratios for 8 h. Cells Flow cytometry analysis from target and nontarget cell cultures were mixed and analyzed Immunofluorescence surface staining was performed as previously by FACS. Percent specific lysis was calculated as (1-target/ described.24 The fluorescence-labeled mAbs against CD3, CD8, control) × 100%. CD25, CD62L, PD-1, 2B4, LAG3, and Annexin V were purchased from To examine the cytotoxicity of BMDC-primed Tc cells in vivo, BD Biosciences. APC-labeled ST2 Ab was purchased from Biolegend. OT-I mice (n = 4–5/group) were immunized twice (1 week apart) Intracellular staining was performed as previously described.24 with 1 × 106 OT-I OVA peptide-pulsed BMDCs. Mice treated with PE-conjugated or Pacific Blue-conjugated mAbs against IL-9, IFN-γ, PBS served as controls. In some experimental groups, mice were granzyme B (GzmB), and Ki67 were purchased from Biolegend. administered IL-33 (250 ng/mouse) every 3 days starting 1 day After staining, cell samples were analyzed using a BD LSRFortes- after the first DC immunization. On day 2 after the second saTM cytometer. immunization, spleen cells from the mice were restimulated with OT-I OVA peptides for 48 h. CD8+ T cells were isolated by MACS In vitro Tc9 cell differentiation and used as effector cells. B16-OVA cells labeled with 2.5 μM CFSE Naive CD8+ T cells (CD8+CD25-CD62Lhi) were purified by were used as target cells, and B16 nontarget cells labeled with fluorescence activated cell sorting (FACS) from mouse spleens 0.25 μM CFSE were used as controls. and cocultured at 1 × 105 per well with BMDCs (1 × 105/well) in the presence of plate-bound anti-CD3 (2 μg/mL), soluble anti-CD28 (2 Adoptive tumor immunotherapy μg/mL), Tc9-polarizing cytokines IL-4 (10 ng/mL) and TGF-β (1 ng/ Briefly, 5 × 105 B16-OVA cells were injected subcutaneously into mL), and IL-2 (50 ng/mL). Cells from cultures without the addition C57BL/6 mice. To generate Tc9 cells, naive CD8+ T cells from OT-I of TGF-β and IL-4 were used as Tc0 cells. IL-33 (50 ng/mL) was mice were cocultured with BMDCs under Tc9-polarizing condi- added to some cell cultures. After 2 days of culture, cells were tions in the presence or absence of IL-33 for 2 days. Cells from harvested and analyzed by flow cytometry and qPCR. cultures without the addition of Tc9-polarizing cytokines TGF-β

Cellular & Molecular Immunology (2019) 16:644 – 651 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 646 A BMDC BMDC+IL33 B BMDC BMDC+IL33 5.6 7.1 18.9 14.7 60 40 Tc0 ** Tc0 30 ** 40 T cells (%) T + T cells (%) T 20 +

15.9 34.6 9

- 11.2 20.7 20

IL NS GzmB + Tc9 Tc9 + 10 CD8 0 CD8 0

IL-9 Tc0 Tc9 Tc0 Tc9 GzmB CD8 CD8 C D E 15 30 Il9 5 Ifng 6 GzmB 3 Spi1 4 Irf4 4 Tbx21 ** * 4 NS * * NS * 3 3 NS 10 20 4 2 NS 3 2 2

9 (ng/mL) NS 2 NS

IL- 5 10 2 1 NS 1 1 1 Relative expression Relative expression 0 0 0 0 0 0 0 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 F G 4 St2 100 * 3 75 * * BMDC BMDC BMDC+IL33 2 BMDC+IL33 50 NS

1 % Specific lysis 25 Relative expression 0 0 Tc0 Tc9 1:1 5:1 20:1 E:T Ratio Fig. 1 IL-33 drives Tc9 cell differentiation in vitro. Naive CD8+ T cells were cocultured with BMDCs under Tc9-polarizing conditions with or without the addition of IL-33 for 2 days. Cell cultures without (Tc0) the addition of Tc9-polarizing cytokines TGF-β and IL-4 were used as controls. a, b Flow cytometry analysis of IL-9-expressing CD8+ T (Tc9) cells (a) and GzmB +CD8+ T cells (b). Numbers in the dot plots represent the percentages of CD8+IL-9+ T cells and GzmB +CD8+ T cells. Right, summarized results of three independent experiments obtained as reported on the left. c ELISA assessed IL-9 secretion in the cocultures. d–f qPCR analysis of the indicated cytokines (d), transcription factors (e) and St2 (f) in T cells. Expression was normalized to Gapdh and set at 1 in BMDC-induced Tc9 cells. g Naive CD8+ T cells from OT-I mice were cocultured with BMDCs under Tc9-polarizing conditions in the presence or absence of IL-33 for 2 days. B16-OVA-specific cytotoxicity of the cultured CD8+ T cells was examined. The results presented are the mean ± SD of 3–5 independent experiments. NS nonsignificant; *P < 0.05; **P < 0.01

and IL-4 were used as Tc0 cells. At day 3 after tumor injection, Statistical analysis mice were randomly divided into groups and treated with The Student t test (2 groups) and one-way ANOVA (≥3 groups) adoptive transfer of 1 × 106 Tc0 or Tc9 cells. Mice treated with were used to compare various experimental groups. A P-value less PBS served as controls. Tumor development was monitored over than 0.05 was considered significant. time. The mice were sacrificed when the tumor diameter was between 1.5 and 2 cm. Tumor volume was calculated by the following formula: 3.14 × (mean diameter)3/6. RESULTS IL-33 drives DC-induced Tc9 cell differentiation in vitro DC-based tumor therapy We first compared the effects of DCs plus IL-33 and IL-33 alone on BMDCs were pulsed with OT-I OVA peptides (5 μg/mL) for 2–4h Tc9 cell differentiation in vitro. As shown in Supplementary and then harvested for mouse immunization. Briefly, 5 × 105 B16- Fig. 1A&B, IL-33 treatment slightly enhanced the development of OVA were injected subcutaneously into OT-I mice. On day 3 after IL-9-producing Tc9 cells and increased Il9 mRNA expression in Tc9 tumor injection, the mice were randomly divided into groups with cells compared with untreated controls. However, the combina- five mice per group. One group of mice was treated with IL-33 tion of BMDCs and IL-33 further increased Tc9 cell development (250 ng/mouse) via intraperitoneal (i.p.) injection every 3 days. (Supplementary Fig. 1A) and Il9 mRNA expression (Supplementary Some mice were administered two weekly subcutaneous immu- Fig. 1B) compared with cells treated with IL-33 alone. Given that nizations with 1 × 106 treated mDCs with or without i.p. injection we are exploiting new strategies to improve the therapeutic of IL-33 (250 ng/mouse) every 3 days. Mice injected with PBS efficacy of DC-based tumor immunotherapy, we therefore focused served as controls. Tumor development was monitored over time. on the role of BMDCs plus IL-33 in Tc9 cell induction and The mice were sacrificed when the tumor diameter was between antitumor immunity in this study. 1.5 and 2 cm. To further exploit the role of IL-33 in DC-induced Tc9 cell differentiation, naive CD8+ T cells were cocultured with BMDCs

Cellular & Molecular Immunology (2019) 16:644 – 651 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 647 A B BMDC BMDC+IL33 BMDC BMDC+IL33 49.1 59.1 48.5 45.5 120 80 Tc0 * Tc0 + NS 60 80 NS T cells T (%)

+ 40 86.7 97.1 14.3 10.2 Annexin V

40 + * Ki67 T cells (%) T + 20 Tc9

Tc9 CD8 CD8 0 0 Tc0 Tc9 Tc0 Tc9 Ki67 Annexin V CD8 CD8 C F BMDC BMDC+IL33 6 B16-OVA (C57 ) PBS

64.6 62.0 ) 100 3 Tc0

Tc0 mm

NS 3 Tc0+IL33

75 10 4 * ** Tc9 ×

T cells (%) **

+ 50 Tc9+IL33 55.8 40.3 2 ** PD-1 25 Tc9 + Tumor size ( CD8 -1 0 Tc0 Tc9 0 PD 0102030 CD8 Days after tumor challenge D E Pdcd1 Cd127 Cd244 Il2 5 4 2 8 * * 4 3 NS 6 3 ** * * BMDC 2 NS 1 4 2 BMDC+IL33 * 1 1 2 Relative expression 0 0 0 Relative expression 0 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Tc0 Tc9 Fig. 2 IL-33 increases the survival and proliferation of Tc9 cells in vitro. Naive CD8+ T cells were cultured as shown in Fig. 1. a–c Flow cytometry of Ki67+CD8+ (a), Annexin V+CD8+ (b) and PD-1+CD8+ (c) T cells. Numbers in the dot plots represent the percentages of double- positive T cells. Right, summarized results of three independent experiments obtained as reported on the left. d, e qPCR analysis of Pdcd1, Cd127, and Cd244 (d) and Il2 (e) expression in CD8+ T cells. f Naive CD8+ T cells from OT-I mice were cocultured with BMDCs under Tc9- polarizing or Tc0-polarizing conditions with or without the addition of IL-33 for 2 days. Cells (1 × 106 per mouse) were adoptively transferred into B16-OVA-bearing C57BL/6 mice. Mice treated with PBS served as controls. Shown are the tumor growth curves. The results presented are the mean ± SD of 3–5 independent experiments. NS nonsignificant; *P < 0.05; **P < 0.01 under Tc9-polarizing conditions with or without the addition of IL- IL-33 increases Tc9 cell survival and proliferation in vitro 33. Similarly, the addition of IL-33 enhanced BMDC-induced Tc9 We next examined the effects of IL-33 treatment on the survival cell development (Fig. 1a) and increased IL-9 and mRNA and proliferation of Tc9 cells primed by DCs in vitro. We first expression in BMDC-primed Tc9 cells (Fig. 1c, d). In addition, the examined the effects of IL-33 on Tc9 cell proliferation by flow addition of IL-33 enhanced Tc9 cell expression of Tc9-related cytometry analysis of Ki67 (a marker of cell proliferation) transcription factors Spi1 and Irf4 (Fig. 1e) but not Tc1-related expression in cells. As shown in Fig. 2a, IL-33 treatment increased transcription factor Tbx21 (Fig. 1e). Furthermore, IL-33-treated Tc9 Ki67 expression in Tc9 cells. Furthermore, IL-33 treatment reduced cells expressed increased GzmB mRNA and protein compared with apoptosis in Tc9 cells compared with untreated control cells regular Tc9 cells (Fig. 1b, d). Interestingly, the addition of IL-33 (Fig. 2b). enhanced the expression of the IL-33 receptor St2 by Tc9 cells A younger CD8+ T cell phenotype may favor cell survival and (Fig. 1f). Finally, IL-33 treatment increased the cytotoxic activity of proliferation.26 PD-1 is an immune-checkpoint protein and a BMDC-induced Tc9 cells (Fig. 1g). marker of exhaustive T cell differentiation.27 We next examined Next, we analyzed the effects of IL-33 on other Tc1 cell PD-1 expression in Tc9 cells with or without IL-33 treatment. differentiation processes. Naive CD8+ T cells were cocultured with Although Tc9 cells expressed lower levels of PD-1 compared with BMDCs under Tc1-polarizing conditions in the presence or Tc0 cells (Fig. 2c, d), IL-33 treatment decreased PD-1 expression in absence of IL-33. IL-33 slightly promotes BMDC-induced Tc1 cell both Tc0 and Tc9 cells (Fig. 2c, d). In addition, IL-33 treatment differentiation, leading to increased expression of Ifng, GzmB, and downregulated 2B4 (CD244) mRNA levels in Tc9 cells (Fig. 2d and Tbx21 (Supplementary Fig. 2A&B). To compare the role of IL-33 in Supplementary Fig. 3), which is another marker of exhaustive T cell the tumor-specific cytotoxicity of BMDC-primed Tc9 and Tc1 cells, differentiation. Furthermore, IL-33 treatment increased the expres- OT-I Tc9, and Tc1 cells were generated in vitro. The addition of IL- sion of the IL-7 receptor-α gene Cd127 in Tc9 cells (Fig. 2d) and Il2 33 increased the cytotoxic activity of BMDC-induced Tc9 cells but in both Tc0 and Tc9 cells (Fig. 2e), which may contribute to the not Tc1 cells (Supplementary Fig. 2C). Together, these results survival and proliferation of CD8+ T lymphocytes. These data demonstrated that IL-33 drives BMDC-induced Tc9 cell differentia- demonstrated that IL-33 enhances BMDC-primed Tc9 cell survival tion in vitro. and proliferation in vitro.

Cellular & Molecular Immunology (2019) 16:644 – 651 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 648 A B C 10 ** BMDC BMDC + IL33 60 15 * PBS * 5.1 5.8 27.3 ** * 8

T cells **

+ 10 40 ** * 6 * * 4

NS 9 (ng/mL) 20 * - 5 NS IFN-γ (ng/mL) IFN-γ IL IL-9 % of CD8 2 5.6 12.5 24.0 0 0 0 IL9+ IFNγ+ GzmB+

D Il9 Ifng Gzmb IFN-γ 30 * 5 6 ** * PBS 5.5 11.0 21.3 * * 4 * BMDC 4 20 3 BMDC+IL33 2 10 2 1 GzmB

CD8 Relative expression 0 0 0 E F 100 6 * PBS PBS 75 * BMDC BMDC * * 4 BMDC+IL33 * BMDC+IL33 ** * 50 *

2 25 % Specific lysis

Relative expression 0 0 Spi1 Irf4 Tbx21 1:1 5:1 20:1 E:T Ratio Fig. 3 IL-33 promotes the development of BMDC-induced Tc9/1 cells in vivo. OT-I mice were administered two weekly subcutaneous immunizations with 1 × 106 OVA-peptide-pulsed BMDCs. Some mice were administered an intraperitoneal injection of IL-33 every 3 days beginning on the day of the first immunization. PBS served as control. On day 2 after the 2nd immunization, mouse spleen cells were restimulated with OT-I OVA peptides for 2 days. Cells from control mice were untreated. a Flow cytometry of IFN-γ-, IL-9-producing or GzmB- producing CD8+ T cells. Numbers in the dot plots represent the percentages of double-positive Tc cells. b Summarized results of three independent experiments obtained in (a). c ELISA assays of IL-9 and IFN-γ in the cultures. d, e CD8+ T cells were isolated by magnetic cell sorting (MACS). qPCR analyses of Il9, Ifng, and Gzmb (d) and Spi1, Irf4, and Tbx21 (e) in CD8+ T cells. f CD8+ T cells were isolated by MACS. B16- OVA-specific cytotoxicity of CD8+ T cells was examined. Data are representative of three (a) independent experiments or presented as the mean ± SD of three (b–f) independent experiments. *P < 0.05; **P < 0.01

Tumor-specific Tc9 cells are potent antitumor effector cells.11 addition, CD8+ T cells from mice immunized with BMDCs plus IL- We found that IL-33 enhances DC induction of Tc9 cells and 33 expressed higher levels of Spi1 and Irf4 compared with mice promotes their survival and proliferation. Based on these treated with BMDCs or PBS control (Fig. 3e). These results observations, we hypothesized that Tc9 cells primed by DCs plus indicated that IL-33 enhances the induction of Tc9 cells primed IL-33 display more potent antitumor activities compared with by BMDCs in vivo. Tc9 cells primed by DCs alone. To address this issue, OT-I Tc9 We also examined the expression of the antitumor effector cells were primed by BMDCs in the presence or absence of IL-33 molecules IFN-γ and GzmB by CD8+ T cells in mice. Mice in vitro, and cells were used to treat B16-OVA-bearing C57BL/6 immunized with BMDCs plus IL-33 exhibited increased percen- mice. Although Tc9 cells mediated increased inhibition of tages of IFN-γ-producing and GzmB-producing CD8+ T cells melanoma tumor growth compared with Tc0 cells (Fig. 2f), the (Fig. 3a, b) and increased expression levels of IFN-γ and Gzmb in addition of IL-33 during Tc9 cell priming further improved their CD8+ T cells compared with mice treated with BMDCs or PBS antitumor efficacy (Fig. 2f), demonstrating the role of IL-33 in control (Fig. 3c, d). Furthermore, CD8+ T cells from mice the improvement of the antitumor capability of BMDC-primed immunized with BMDCs plus IL-33 expressed higher levels of Tc9 cells. Tbx21 compared with mice treated with BMDCs or PBS control (Fig. 3e), indicating that IL-33 promoted BMDC induction of IL-33 promotes the development of BMDC-induced Tc9/1 cells Tc1 cells in vivo. Notably, the administration of IL-33 increased in vivo the tumor-specific cytotoxicities of CD8+ T cells primed by We next explored the role of IL-33 in DC-induced Tc9 cell BMDCs in vivo (Fig. 3f). Together, these results demonstrated that differentiation in vivo. OT-I mice were immunized with OVA IL-33 stimulates BMDCs to induce Tc9 and Tc1 cells in vivo. peptide-pulsed BMDCs with or without addition of IL-33. Although mice immunized with BMDCs exhibited low levels IL-33 increases the proliferation of Tc cells primed by BMDCs of IL-9-expressing CD8+ T cells (Tc9) that were comparable to in vivo those of PBS control mice (Fig. 3a, b), mice immunized with We next examined the role of IL-33 in ST2 expression by CD8+ BMDCs plus IL-33 exhibited increased levels of Tc9 cells T cells primed by BMDCs in vivo. OT-I mice were immunized by compared with mice immunized with BMDCs or PBS control OVA-peptide-pulsed BMDCs with or without the addition of IL-33. (Fig. 3a, b). ELISA and qPCR further confirmed the increased Mice immunized by BMDCs plus IL-33 exhibited increased expression of IL-9 by CD8+ T cells in mice immunized with BMDCs percentages of ST2-expressing CD8+ (ST2+CD8+) T cells in spleen plus IL-33 compared with BMDCs or PBS control (Fig. 3c, d). In cells (Fig. 4a, b) and increased St2 mRNA expression in CD8+

Cellular & Molecular Immunology (2019) 16:644 – 651 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 649 A B C E St2 100 PBS BMDC BMDC + IL33 40 6 * * ** ** PBS 5.8 8.7 19.2 * 75 30 * 4 BMDC NS

T cells (%) T 50 BMDC+IL33 T cells (%) T

+ 20

NS + 2

ST2 25

+ 10 CD8 ST2

CD8 Relative expression 0 0 CD8 0 D F G PBS BMDC BMDC + IL33 PBS BMDC BMDC + IL33 100 ** 25.931.7 72.4 ** 40.5 25.2 76.8 75 T cells T (%)

+ 50 NS

Ki67 25 + Ki67 Isotype CD8 CD8

CD8 0 Fig. 4 IL-33 increases the proliferation capability of Tc cells in vivo. a–c OT-I mice were immunized, and spleen cells were restimulated as presented in Fig. 3. a Flow cytometry of ST2-expressing CD8+ T (ST2+CD8+) cells in mouse spleen cells. b Summarized results of three independent experiments obtained in (a). c CD8+ T cells were isolated by MACS. qPCR analyses of St2 in CD8+ T cells. (d–g) OT-I mice were immunized as presented in Fig. 3. On day 2 after the 2nd immunization, mouse spleen cells were restimulated with BMDCs or BMDCs plus IL-33 for 2 days. Cells from PBS control mice were untreated. d Flow cytometry of CD8+ T cells in mouse spleen cells. e Summarized results of three independent experiments obtained in (d). f Flow cytometry analysis of Ki67+CD8+ T cells. g Summarized results of three independent experiments obtained in f. Data are representative of three (a, d, f) independent experiments or presented as the mean ± SD of three (b, c, e, g) independent experiments. NS nonsignificant; *P < 0.05; **P< 0.01

A B + + PBS BMDC BMDC + IL33 PD-1+ LAG3 2B4 40 30 30 8.5 28.9 10.6 ** * ** * PBS

T cells 30 NS BMDC + 20 20 * BMDC+IL33 20 10 10 10 PD1 % of CD8 7.6 17.7 7.9 0 0 0 C D ** 2 2 2 10 * NS NS NS LAG3 8 ** 15.0 13.4 7.9 ** ** 6 1 1 1 4 2 Relative expression Relative expression

2B4 0 0 0 0 CD8 Pdcd1 Lag3 Cd244 Il2 Fig. 5 IL-33 inhibits the exhaustive differentiation of BMDC-activated CD8+ T cell in vivo. OT-I mice were immunized as shown in Fig. 3, and lymph node cells were collected. a Flow cytometry analysis of PD-1, LAG3, and 2B4 expression in CD8+ T cells. b Summarized results of three independent experiments obtained in (a). c, d qPCR examined the expression of Pdcd1, Lag3, Cd244 (c) and Il2 (d) in CD8+ T cells. Data are representative of three (a) independent experiments or presented as the mean ± SD of three (b–d) independent experiments. NS nonsignificant; *P < 0.05; **P< 0.01

T cells (Fig. 4c) compared with mice treated with BMDCs or PBS expression in Tc cells from mice treated with BMDCs plus IL-33 controls. However, there was no difference in ST2 expression by compared with BMDCs alone or PBS controls (Fig. 4f, g). These CD8+ T cells from mice immunized with BMDCs compared with results indicated that IL-33 promotes the proliferation of Tc cells PBS controls (Fig. 4a–c). These results demonstrated that IL-33 primed by DCs in vivo. stimulates ST2 expression by CD8+ T cells in vivo. We next examined the phenotype of Tc cells from mice We next examined the effects of IL-33 on the proliferation immunized by BMDCs plus IL-33. BMDC immunization increased of CD8+ T cells primed by BMDCs in vivo. Mice immunized CD8+ T cell expression of PD-1 and LAG3 compared with PBS with BMDCs exhibited comparable percentages of CD8+ T cells controls (Fig. 5a–c). However, immunization with BMDC plus IL-33 compared with PBS control mice (Fig. 4d, e). Interestingly, potently inhibited PD-1, LAG3, and 2B4 expression by CD8+ T cells mice immunized by BMDCs plus IL-33 exhibited significantly compared with BMDC treatment (Fig. 5a–c). Furthermore, mice increased percentages of total CD8+ T cells in mouse spleen immunized with BMDCs plus IL-33 exhibited increased Il2 cells compared with mice receiving BMDCs or PBS control expression in CD8+ T cells compared with mice treated with (Fig. 4d, e). To determine whether IL-33 promoted BMDC-primed BMDCs alone or PBS controls (Fig. 5d). These results indicated that CD8+ T cell proliferation, spleen T cells were analyzed using IL-33 inhibits exhaustive differentiation of CD8+ cells primed by intracellular Ki67 staining. Similarly, IL-33 increased Ki67 BMDCs in vivo.

Cellular & Molecular Immunology (2019) 16:644 – 651 IL-33 drives the antitumor effects of dendritic cells via the induction. . . N. Liu et al. 650 6 PBS IFN-γ- and granzyme-B (GrzB)-producing cytolytic Tc1-like effector B16-OVA (OT-I ) 11 )

3 cells in vivo. These observations suggest that immunization BMDC with DC vaccines plus IL-33 may have both direct and indirect mm

3 BMDC+IL33 effects on the induction of Tc9 and Tc1 cells, which mediate 4 10 IL33 potent antitumor responses.

× In this study, we found that IL-33 treatment promotes cell ** proliferation and inhibits cell apoptosis of BMDC-primed Tc9 cells. 2 2nd Tc9 cells exhibit a “younger” phenotype as demonstrated by the 1st reduced expression of KLRG-1 and higher expression of IL-2 and ** IL-7Rα in Tc9 cells compared with Tc1 cells, which may contribute Tumor size ( Tumor ** 11,31–33 0 to the long-term survival of Tc9 cells in vivo. In this study, α 0102030 we found that IL-33 treatment further increases IL-7R and IL-2 Days after tumor challenge expression and inhibits PD-1 and 2B4 expression in BMDC-primed Tc9 cells. PD-1 is an immune-checkpoint protein that has been Fig. 6 IL-33 increases BMDC-induced antitumor efficacy in vivo. OT-I implicated in tumor immune escape by promoting the apoptosis mice were injected subcutaneously with 1 × 105 B16-OVA cells. On of tumor-specific T cells.27 In addition, 2B4 is another marker for day 3 after tumor challenge, the mice were randomly divided into exhaustive T cell differentiation.34 Thus, our data demonstrated fi groups with ve mice per group. One group of mice was treated the role of IL-33 in the survival and proliferation of DC-induced with IL-33 (250 ng/mouse) via intraperitoneal (i.p.) injection every Tc9 cells. 3 days. Some mice were administered two weekly subcutaneous 6 Previous studies demonstrated that IL-33 promotes the devel- immunizations with 1 × 10 treated mDCs with or without i.p. 35,36 injection of IL-33 (250 ng/mouse) every 3 days. Mice that received opment and immunosuppressive function of Treg cells. We PBS served as controls. The experiments were performed twice with also found that IL-33 treatment fails to improve the antitumor a total of 10 mice per group (n = 10). Shown are the tumor growth efficacy of BMDC vaccines in a B16-OVA OT-II mouse model curves. Data are presented as the mean ± SD of the combined (unpublished data). However, in this study, we found that IL-33 experiments. **P < 0.01 promotes BMDC-induced Tc9 cell responses and antitumor efficacy in an OT-I mouse model. These observations indicated IL-33 increases BMDC-induced antitumor efficacy that IL-33 has different effects on the induction of CD8+ and CD4+ To examine the effects of IL-33 on the antitumor efficacy T cell-mediated antitumor immunity primed by DCs. Further induced by DC vaccines, B16-OVA-bearing OT-I mice were treated studies are necessary to investigate new strategies of blocking with OVA peptide-pulsed BMDCs in the presence or absence the immunosuppressive function of IL-33 on CD4+ T cells but of IL-33. As shown in Fig. 6, BMDCs plus IL-33 induced more increasing its immunostimulatory activity on CD8+ T cell-mediated potent inhibition of melanoma tumor growth than BMDCs or IL-33 antitumor immunity. alone. Nevertheless, IL-33 alone could not inhibit melanoma In conclusion, our study demonstrates that IL-33 drives the growth compared with PBS controls (Fig. 6). These results induction of Tc9 cells by BMDCs. IL-33 treatment increases the demonstrated that IL-33 promoted BMDC-induced antitumor survival and proliferation of BMDC-induced Tc9 cells and immunity in vivo. promotes their antitumor activities. The addition of IL-33 further promotes BMDC-induced antitumor efficacy in an OT-I mouse model. Our study demonstrates the important role of IL-33 in DC- DISCUSSION induced Tc9 differentiation and antitumor immunity and may DC-based immunotherapy is a promising approach for tumor have important clinical implications. therapy, and tumor-specific Tc9 cells are potent antitumor effector T cells.11 Therefore, strategies to drive the induction of tumor-specific Tc9 cells by DCs may have high clinical significance. In this study, we found that the addition of IL-33 ACKNOWLEDGEMENTS potently promotes the generation of Tc9 cells primed by BMDCs This work was supported by funds from National Natural Science Foundation of China (81372536 to S.W., 81502452 to X.C. and 81602485 to Y.Z.). in vitro and in vivo. More importantly, IL-33 treatment further increases the antitumor efficacy induced by BMDC tumor vaccines in OT-I mouse models. Consistently, a recent study AUTHOR CONTRIBUTIONS demonstrated that IL-33 favors the production of antileukemic 28 S.W. and Y.Y. initiated the study. S.W. designed the experiments and wrote the paper. IL-9-producing T cells. Mechanistically, Tc9 cells primed by S.W., N.L., Y.J., J.C., H.N, Y.Z., X.C., A.W., D.W. and T.Q. performed the experiments and DCs plus IL-33 in vitro expressed high levels of IL-9 and statistical analyses. A.W. read and edited the manuscript. Q.Y. and S.G. provided GzmB antitumor effector molecules, and such Tc9 cells exhibit critical suggestions to this study. increased tumor-specific cytotoxicity and antitumor capabilities. In addition, immunization of BMDCs plus IL-33 stimulates potent Tc9 and Tc1 cell responses in vivo. Tumor-specific Tc9 and ADDITIONAL INFORMATION Tc1 cells are both potent antitumor effectors.11 Tc9 cells exhibit The online version of this article (https://doi.org/10.1038/s41423-018-0166-0) longer persistence potential and elicit greater antitumor responses contains supplementary material. compared with Tc1 cells in vivo.11 Thus, our data demonstrate the important role of IL-33 in BMDC-induced Tc9 cell differentiation Competing interests: The authors declare no competing interests. and antitumor efficacy. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims In this study, we found that IL-33 and DCs exert synergistic in published maps and institutional affiliations. effects on the induction of Tc9 and Tc1 cells in vivo. The underlying mechanisms must be further defined. However, IL-33 stimulates DCs to overexpress OX40L,29 which is an inducer of REFERENCES 24,30 Th9 cells. IL-33 itself may be a driver of Tc9 cell differentia- 1. Timmerman, J. M. & Levy, R. vaccines for cancer immunotherapy. 28 tion. Regarding Tc1 cells, previous studies demonstrated that Annu. Rev. Med. 50, 507–529 (1999). + IL-33 enhances the production of IFN-γ-expressing CD8 T cells 2. Randolph, G. J., Ochando, J. & Partida-Sanchez, S. Migration of dendritic cell – in vivo.16 18 More importantly, Tc9 cells can also differentiate into subsets and their precursors. Annu. Rev. Immunol. 26, 293–316 (2008).

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