Production of IL-12p70 and activation of CD4⁺ T cells in coculture of fucoidan-matured PBDCs and CD4⁺ T cells. (A) Fucoidan-, LPS-, or TNF-α–matured PBDCs (F-PBDC, L-PBDC, or T-PBDC) were harvested and washed twice with media. These DCs were then pretreated with mitomycin C and mixed with CD4⁺ T cells (10⁵) in a 1:25 ratio. CD4⁺ T cells alone (T cells) and mixtures of the cells were then cultured in the presence or absence of neutralizing Abs against CD40 (5 μg) for an additional 1 or 5 days to determine the concentrations of IL-12p70 and IFN-γ, respectively. The concentrations of cytokines in the supernatants were determined by ELISA. (B) CD4⁺ T cells were labeled with CFSE and cocultured with fucoidan-, LPS-, or TNF-α–matured PBDCs in the presence or absence of neutralizing Abs against CD40. After 5 days, proliferation of CD4⁺ T cells was analyzed for CFSE dilution using flow cytometry. (C) PBDCs were pretreated with or without neutralizing Abs against TNF-α and then stimulated with fucoidan, LPS, or TNF-α for 24 hours. After coculture of the DCs and T cells in a 1:25 ratio for 5 days, the concentrations of IFN-γ in the supernatants were measured as in panel A. *P < .05 compared with PBDC alone. **P < .05 compared with that without anti-CD40 neutralizing Abs. ***P < .01.