A polysaccharide extracted from Grifola frondosa enhances the anti-tumor activity of bone marrow-derived dendritic cell-based immunotherapy against murine colon cancer

Abstract

We previously isolated the novel heteropolysaccharide maitake Z-fraction (MZF) from the maitake mushroom (Grifola frondosa), and demonstrated that MZF significantly inhibited tumor growth by inducing cell-mediated immunity. In this study, we demonstrated that MZF upregulated the expression of CD80, CD86, CD83, and MHC II on bone marrow-derived dendritic cells (DCs) and significantly increased interleukin-12 (IL-12) and tumor necrosis factor-alpha production by DCs in a dose-dependent manner. MZF-treated DCs significantly stimulated both allogeneic and antigen-specific syngenic T cell responses and enhanced antigen-specific interferon-gamma (IFN-gamma) production by syngenic CD4(+) T cells; however, MZF-treated DCs did not affect IL-4 production. Furthermore, the enhancement of IFN-gamma production in CD4(+) T cells, which was induced by MZF-treated DCs, was completely inhibited by the addition of an anti-IL-12 antibody. These results indicate that MZF induced DC maturation and antigen-specific Th1 response by enhancing DC-produced IL-12. We also demonstrated that DCs pulsed with colon-26 tumor lysate in the presence of MZF induced both therapeutic and preventive effects on colon-26 tumor development in BALB/c mice. These results suggest that MZF could be a potential effective adjuvant to enhance immunotherapy using DC-based vaccination.

Fig. 1

MZF induces IL-12 and TNF-α production and expression of DC maturation markers. a, b DCs (1 × 10⁶ cells/ml) were cultured in RPMI-1640 medium containing 10% FBS and MZF (0–400 μg/ml) or LPS (100 ng/ml) with or without polymyxin B (poly B) for 24 h. TNF-α and IL-12 in the culture supernatants were measured by ELISA. The data shown are representative of three experiments. Values are expressed as mean ± SE (n = 5). *p < 0.05 versus control without poly B. ^§ p < 0.05 versus control with poly B. ^# p < 0.05, poly B-treated group versus non-treated group. c DCs (1 × 10⁶ cells/ml) were stimulated with MZF (0–400 μg/ml) or LPS (100 ng/ml) for 24 h. Expression of CD80, CD86, CD83, and MHC II (I-A^d) on CD11c⁺ cells was determined by flow cytometry and demonstrated by representative histogram profiles. Numbers reflect the percentage of positive cells

Fig. 2

Binding of MZF by DCs requires β-glucan and mannan recognition. a Internalization of Fl-MZF by DCs. DCs were cultured in RPMI-1640 medium containing 10% FBS with Fl-MZF (400 μg/ml) at 37°C. Cells were collected at various time periods and then washed, fixed, and observed by fluorescence microscopy. b Binding of MZF by DCs. DCs (2 × 10⁵ cells/tube) were incubated on ice for 1 h in PBS containing 3% FBS and Fl-MZF at various concentrations. After incubation, the cells were washed and fixed. Fl-MZF⁺ DCs were identified by flow cytometry. c Laminarin, mannan, and anti-Dectin-1 Ab inhibited the DC recognition of Fl-MZF. Ice-cold PBS containing 3% FBS with or without inhibitors (laminarin, mannan and dextran; 1 mg/ml), anti-Dectin-1 Ab (2A11), or isotype Ab (rat IgG2bκ) was added to the DCs (2 × 10⁵ cells/tube) and incubated on ice for 1 h before the addition of Fl-MZF (400 μg/ml). Cells were incubated on ice for 1 h, washed, and fixed. Fl-MZF⁺ DCs were identified by flow cytometry. The data shown have been normalized to the percentages of Fl-MZF⁺ CD11c⁺ cells of uninhibited control. Values are expressed as mean ± SE, n = 3; *p < 0.05

Fig. 3

Allostimulatory activity of MZF-treated DCs stimulates allogeneic T cells. Unstimulated or MZF-stimulated DCs were treated with 50 μg/ml mitomycin C for 1 h and cultured with allogeneic C3H/HeN CD4⁺ T cells at a density of 1 × 10⁵ cells/well in U-bottom 96-well microtiter plates in 5% CO₂ at 37°C for 72 h. a Cell proliferation was measured with WST-8 reagent. b, c IL-12 and IFN-γ levels in supernatants were analyzed by ELISA. Data (n = 5) represent the mean ± SE of two separate experiments. *p < 0.01 versus control

Fig. 4

MZF increases DC Ag-presenting activity. a DCs were stimulated with MZF (400 μg/ml) for 24 h in the presence or absence of KLH (100 μg/ml). DCs were washed and cultured with KLH-primed T cells (1 × 10⁵ cells/well) in U-bottom 96-well microtiter plates for 72 h. Cell proliferation was measured with WST-8 reagent. *p < 0.05 versus all other groups. b DCs were stimulated with MZF at various concentrations for 24 h in the presence or absence of KLH. DCs were washed and cultured with KLH-primed or unprimed T cells (1 × 10⁵ cells/well) for 72 h. ^§ p < 0.05 versus KLH-DCs (MZF 0 μg/ml) co-cultured with KLH-T cells. ^# p < 0.05 versus KLH-DCs (MZF 0 μg/ml) co-cultured with unprimed T cells. c Levels of IL-12, IFN-γ, and IL-4 in the culture supernatants (DCs/T cells ratio, 0.1:1) were then measured by ELISA. d KLH + MZF-DCs were cultured with KLH-T cells (DCs/T cells ratio, 0.1:1) in the presence of anti-IL-12 Ab (C17.8) or isotype Ab (JES3-19F1) for 72 h. IFN-γ levels in the culture supernatants were then measured by ELISA. *p < 0.05 versus KLH-DCs co-cultured with KLH-T cells. Data (n = 4) represent the mean ± SE of two separate experiments. ND not detected

Fig. 5

Preventive and therapeutic effect of MZF in a subcutaneous colon-26 tumor model. a, b Preventive effect of MZF. BALB/c mice were treated with three s.c. injections (a week apart) of 2 × 10⁵ DCs prepared as follows: DC, Lysate-DC, LPS + Lysate-DC, or MZF + Lysate-DC. A week after the final DC injection, colon-26 cells (1 × 10⁵ cells/mouse) were s.c. inoculated. The tumor growth (a) and survival rates (b) were monitored (control, n = 14; other groups, n = 7). c, d Therapeutic effect of MZF. Colon-26 cells (1 × 10⁵ cells/mouse) were s.c. inoculated into BALB/c mice on day 0. On days 3, 10, and 17, mice were treated by s.c. injections of 2 × 10⁵ DCs. The tumor growth (c) and survival rates (d) were monitored (control, n = 14; other groups, n = 7). a, c The Mann–Whitney U test was used to analyze the statistical significance of tumor growth on day 21. *p < 0.05 versus all other groups; ^# p < 0.05 versus control. b, d Survival rates are represented using Kaplan–Meier curves