Efficacy of dendritic cells matured early with OK-432 (Picibanil), prostaglandin E2, and interferon-alpha as a vaccine for a hormone refractory prostate cancer cell line

Abstract

Dendritic cells (DCs) are potent antigen-presenting cells. OK432 (Picibanil) was introduced as a potent stimulator of DC maturation in combination with prostaglandin-E(2) and interferon-alpha. We compared the efficacy of a DC-prostate cancer vaccine using early-mature DCs stimulated with OK432, PGE2 and INF-alpha (OPA) with that of vaccines using other methods. On days 3 or 7 of DC culture, TNF-alpha (T), TNF-alpha and LPS (TL) or OPA were employed as maturation stimulators. DU145 cells subjected to heat stress were hybridized with mature DCs using polyethyleneglycol. T cells were sensitized by the hybrids, and their proliferative and cytokine secretion activities and cytotoxicity were measured. The yields of early-mature DCs were higher, compared to yields at the conventional maturation time (P<0.05). In the early maturation setting, the mean fusion ratios, calculated from the fraction of dual-positive cells, were 13.3%, 18.6%, and 39.9%, respectively (P=0.051) in the T only, TL, and OPA-treated groups. The function of cytotoxic T cells, which were sensitized with the hybrids containing DCs matured early with OPA, was superior to that using other methods. The antitumor effects of DC-DU145 hybrids generated with DCs subjected to early maturation with the OPA may be superior to that of the hybrids using conventional maturation methods.

Keywords: Cancer Vaccines; Dendritic Cells; Immunotherapy; Prostatic Neoplasms.

Fig. 1

Study design of six maturation method combinations, specifically, early maturation with TNF-α only (20 mg/mL), TNF-α (20 mg/mL) plus lipopolysaccharides (LPS) (1 µg/mL), and a cocktail of OK432 (0.1 KE/mL), PGE2 (50 ng/mL), and interferon-α (500 IU/mL) on day 3, compared with conventional maturation with the above agents on day 7 of culture.

Fig. 2

Phenotyping of immature and mature dendritic cells (DC), and DU145 cell lines analyzed by flow cytometry using various surface molecules.

Fig. 3

Dendritic cells (DC)-DU145 hybrids and flow cytometry analysis: (A) Mature DCs displaying positivity for the CD86-FITC area only; (B) DU145 cells were positive for the PKH 26-PI area only; (C) DC-DU145 hybrids within the dual-positive PKH26-PI and CD86-FITC area were counted (arrows). The fusion ratio in the presenting figure was 60.8% with the early maturation OPA set, which was the highest yield.

Fig. 4

Fusion ratios of DU145 and mature DCs obtained with the six maturation methods (n=3). Data on DCs obtained from three different donors (EM, early maturation; CM, conventional maturation; T only, 20 mg/mL TNF-α; TL, 20 mg/mL TNF-α+1 µg/mL LPS; OPA: 0.1 KE/mL OK432+50 ng/mL PGE2+50 ng/mL INF-α).

Fig. 5

Analysis of the functions of DC-DU145 hybrids containing DCs matured with the three sets of stimulating agents and non-hybrid controls: IL-12 secretion measured with ELISA (n=4).

Fig. 6

T-cell stimulatory functions of DC-DU145 hybrids containing mature DCs treated with the three sets of agents and non-hybrid controls (n=4): T cell proliferation assay using a mixed lymphocyte reaction (innate T cells: DC-DU145 hybrids=10:1).

Fig. 7

Efficacy of CTLs sensitized with DC-DU145 hybrids containing mature DCs treated with the three sets of DC stimulation agents and non-hybrid controls: Measurement of INF-γ, a cytotoxic cytokine secreted from sensitized T cells, using ELISA (n=4).

Fig. 8

Cytotoxicity of CTLs sensitized with DC-DU145 hybrids containing early mature DCs treated with three sets of stimulating agents: LDH release assay (n=2).