Fig. 2.

B16-F10 melanoma cells transfected to express signals 2 and 3 in vitro cause activation of primary T and NK cells. (A–C) B16-F10 cells were transfected with 5-3-49 PBAE/DNA nanoparticles encoding IL-12, 4-1BBL, or both. Secreted IL-12 was measured by ELISA, and surface-bound 4-1BBL was measured by flow cytometry. (D) Transfection with a mixture of the 4-1BBL and IL-12 plasmids results in a synergistic effect greater than the additive effects of each plasmid on its own. (E and F) Across different doses of total plasmid per well, the effect (IFN-γ secretion) of 4-1BBL and IL-12 plasmids in combination is consistently higher than the added effects of 4-1BBL transfection alone and IL-12 transfection alone. For A–D, statistically significant differences were measured by one-way ANOVA with Dunnett posttests comparing to the control (100% Ctrl). All bar graphs show mean ± SE. Four (n = 4) replicates were used per group. For F, a two-way ANOVA was performed, with Dunnett posttests comparing to the control (1:1 Additive Effects). Asterisk colors correspond to the group found to be significantly different from the control at that dose. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.