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

Abstract

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 interleukin (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 receptor-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: Cancer immunology; Dendritic cells; Interleukin-33; Tc9.

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

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

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

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

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

Fig. 6

IL-33 increases BMDC-induced antitumor efficacy in vivo. OT-I mice were injected subcutaneously with 1 × 10⁵ B16-OVA cells. On day 3 after tumor challenge, the mice were randomly divided into groups with five mice per group. One group of mice was treated with IL-33 (250 ng/mouse) via intraperitoneal (i.p.) injection every 3 days. Some mice were administered two weekly subcutaneous immunizations with 1 × 10⁶ treated mDCs with or without i.p. injection of IL-33 (250 ng/mouse) every 3 days. Mice that received PBS served as controls. The experiments were performed twice with a total of 10 mice per group (n = 10). Shown are the tumor growth curves. Data are presented as the mean ± SD of the combined experiments. **P < 0.01