Deoxypodophyllotoxin Induces a Th1 Response and Enhances the Antitumor Efficacy of a Dendritic Cell-based Vaccine

Abstract

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-κB.

Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma.

Results: DPT promoted the activation of CD8(+) T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-γ production and induction of cytotoxic T lymphocyte activity.

Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

Keywords: CTL activity; DC-based vaccination; Dendritic cells; Deoxypodophyllotoxin; Interleukin-12.

Figure 1

DPT is not cytotoxic to DCs. Chemical structures of deoxypodophyllotoxin (DPT) (A) On day 6, the cells were cultured under standard conditions for another 24 h in the presence of 1 nM to 1,000 nM DPT and harvested. The cells were gated on CD11c⁺ cells, and analyzed by 2-color flow cytometry using AnnexinV/PI staining kit [H₂O₂ (200 µM positive control)] (B) This result is representative of 3 experiments that gave similar results.

Figure 2

DPT induces the expression of MHC class I and II, co-stimulatory molecules in a dose-dependent manners and decreased endocytic capacity in DCs. The DCs were cultured under standard conditions for 24 h in the presence of 1 to 100 nM DPT or 200 ng/ml LPS, harvested, and analyzed by 2-color cytometry. The cells were first gated on CD11c⁺. Medium represents the untreated control and LPS represents the positive control. DPT was added to the DCs for 24 h at concentrations of 1 to 100 nM (A) FITC-dextran uptake was analyzed by CD11c⁺-PE/dextran-FITC-positive cells using flow cytometry. DCs (1×10⁵ cells) were treated with DPT (100 nM) or LPS (200 ng/ml) for 24 h (B) Endocytic activity of the control was determined after incubation at 4℃. The numbers represent the percentage of FITC-dextran/CD11c⁺-PE double positive cells. Histogram of Fig. 2B was shown (thick line; endocytic activity control at 4℃, thin line: endocytic activity at 37℃).

Figure 3

DPT induces the production of IL-12 through MAPK and NF-κB in DCs. DCs were generated by stimulating immature DCs with 1 to 100 nM DPT or 200 ng/ml LPS (Positive control) for 24 h. After 24 h, the production of IL-12 and IL-10 was measured by flow cytometry (A) Analysis of IL-12 p70 and IL-10 production by magnetic bead-purified DC (1×10⁶ cells) using ELISA. ^**p<0.01 for a comparison with medium control (B) For MAPK and NF-κB activation analysis, DCs were pretreated with 100 nM DPT for 15, 30, and 45 min. The cell lysates were prepared and blotted with anti-phosho ERK1/2 and anti-phosho-p38 Ab. The nuclear extracts were blotted with anti-p65 Ab. The results are derived from 1 experiment with triplicate samples (C) DCs were pretreated with medium alone, LPS (positive control, 200 ng/ml), and DPT (100 nM) for 6 h. After 6 h, RNA was isolated and evaluated for TLR gene expression by quantitative real-time PCR. ^**p<0.01 for a comparison with medium control (D). The expression level of TLR4 after 24 h by stimulation was measured by flow cytometry (E).

Figure 4

DPT-treated DCs induced the differentiation of T cells to a Th1 response. CD8⁺ T cell proliferation was analyzed by flow cytometry and presented as the percentage of dividing cells. A negative control (CD8⁺ T cells in untreated DC alone), a specific Ag control (1 µM OVA_257-264) and a positive control (CD8⁺ T cells in 200 ng/ml LPS) were created for each experiment (A) To analyze cytokine production, CD4⁺ T cells of DO11.10 Tg mice and CD8⁺ T cells of OT-1 Tg mice were co-cultured in medium, LPS, or DPT-treated DC with or without OVA at 1:10 DC/T cell ratio for 48 h. After 48 h of expansion, IFN-γ (B), IL-4 (C) and IL-2 (D) were measured by ELISA in culture supernatants. Data are the mean±S.D. of 4 independent experiments. ^**p<0.01 for a comparison with medium control.

Figure 5

DPT treatment of DCs increased expression of CCR7 and migration to DLN. DCs were treated with medium or DPT (100 nM) for 12 h. After 12 h, RNA was isolated and evaluated for CCR 7 (A) and CCR 3 (B) gene expression by quantitative real-time PCR. Fold differences compared with medium alone are depicted after normalization with the housekeeping gene. ^**p<0.01 for a comparison with medium control. Fluorescence-activated cell sorter analysis was performed on cells from DLN isolated 24 h after subcutaneous injection of DCs alone, B16-TP-DCs, and DPT treated DCs with 1×10⁶ cells, bead-purified, CFSE-labeled CD11c⁺ DC. The incidence of labeled DC migration to DLN was expressed as a percent of fluorescent cells (C) This result is representative of 3 experiments that gave similar results. ^**p<0.01 for a comparison with DC alone.

Figure 6

DPT-DCs enhance anti-tumor therapeutic efficacy. C57BL/6 mice were injected subcutaneously with 1×10⁵ B16F10 cells on day 0. The tumor-implanted C57BL/6 mice were divided into 4 groups (each group containing 10 mice) and injected in situ with DCs alone (◆), B16-TP-DCs (□), DPT-DCs (○), and HBSS (█), respectively. Injection of 1×10⁶ purified DCs/0.2 ml was performed on day 3, 7, and 14. The length and width of the tumor mass were measured with calipers at 3, 7, 14, and 28 days. Tumor-implanted mice in each group were observed for survival time (A) Inhibition of tumor growth in melanoma-implanted C57BL/6 mice by therapy group (B) Splenic lymphocyte isolation from killed tumor-implanted mice (n=5) 5 days after final injection with HBSS, purified DCs, B16-TP-DCs, and DPT-DCs co-cultured with inactivated B16F10 cells for 7 days in the presence of recombinant murine IL-12 (20 U/ml) and then collected as CTL effector cells and incubated at the indicated E:T ratio. CTL activity was determined by LDH release assay with a CytoTox 96 non-radioactive cytotoxicity assay kit (C). ELISA analysis of IFN-g (D) and IL-2 (E) in the supernatants of 2×10⁶/ml lymphocytes isolated at 5 days after the last injection and restimulated with inactivated B16F10 cells in vitro at a 10:1 ratio. The DCs were cultured under standard conditions for another 24 h in the presence of B16-TP or 100 nM DPT, harvested, and analyzed by 2-color cytometry (F). Data are representative of 3 independent experiments. Mice were monitored for tumor growth and survival. (^*p<0.05, ^**p<0.01, DPT-DCs vs. HBSS).