Figure 2. Synergistic activation of PEC with anti-CD40 and CpG, anti-CD40 and MPL, and CpG and MPL.

A, B: Synergy between anti-CD40 and CpG. PEC were collected from naïve C57BL/6 mice (“naïve”) or mice injected with 0.5 mg anti-CD40 (“anti-CD40”) three days earlier. The adherent PEC were then cocultured with B16 cells in the absence or presence of CpG at the indicated concentrations for 48 hours. The wells were pulsed with [³H]-TdR (1μCi/well) for the last 6 hours of incubation to measure proliferation of B16 cells (A). Results are presented as the mean total number of β-counts over 5 minutes ± SE. Supernatants from the coculture of PEC and B16 cells were tested for nitrite concentration using Griess test (B). Data shown are represented as mean nitrite concentration ± SE. C, D: Synergy between anti-CD40 and MPL. PEC were collected from naïve C57BL/6 mice (“naïve”) or mice injected with 0.5 mg anti-CD40 (“anti-CD40”) three days earlier. The adherent PECs were cocultured with B16 cells in absence or presence of MPL at the indicated concentrations. The tumoristatic (C) and NO-inducing (D) effects of PEC are shown (mean ± SE). E, F: Synergy between CpG and MPL. PEC were collected from naïve C57BL/6 mice. Adherent PEC were cocultured with B16 cells in the absence or presence of CpG, MPL or their combination at the indicated concentrations for 48 hours. The tumoristatic (E) and NO-inducing (F) effects of PEC are shown (mean ± SE). The data were analyzed by two-way ANOVA followed by Bonferroni test. In A, B, C and D: *P <0.00125, **P<0.00025 for the difference between anti-CD40+CpG vs anti-CD40 or CpG alone at the indicated dose of CpG, and between anti-CD40+MPL vs anti-CD40 or MPL alone at the indicated dose of MPL. In E and F: *P<0.0005 for the indicated combination of CpG + MPL vs CpG or MPL alone. The experiment was carried out in triplicate and repeated three times.