Vaccine-PGE₂ DCs do not express viral sensors and effector molecules and express and present tumor antigens after mRNA electroporation. (A) DCs were matured for 48 hours with the conventional cytokine cocktail (cDCs), combined vaccines (BCG, Typhim, and Influvac; vaccine DCs), poly(I:C), or with separate preventive vaccines. mRNA levels of PKR, RIG-I, and 2,5-OAS were determined using quantitative PCR 48 hours after maturation. (B) DCs were matured for 48 hours with the conventional cytokine cocktail or combined vaccines (BCG, Typhim, and Influvac; vaccine DCs) with PGE₂. DCs were electroporated with mRNA encoding gp100 or tyrosinase. After 4 hours, gp100 or tyrosinase protein expression was determined by FACS analysis. Filled curves represent staining with specific antibody; and thin-lined curves represent the isotype control. Numbers indicate percentage of cells expressing the antigen. Data are a representative experiment of 3 performed. Average antigen expression (mean ± SEM) of 3 experiments was 73 ± 7 for gp100 and 76 ± 3 for tyrosinase on vaccine PGE₂ DCs, and 65 ± 7 for gp100 and 73 ± 4 for tyrosinase on cDCs. (C) A total of 50 000 gp100:280-specific CD8⁺ T cells were coincubated with 7000 cDCs or vaccine PGE₂ DCs 4 hours after electroporation with gp100 mRNA or tyrosinase mRNA as a control, and T-cell activation was analyzed by measurement of CD69 surface expression (left), IFN-γ production (middle), and IP-10 production (right). Data are mean ± SEM of 1 representative experiment of 3 performed in triplicate (CD69 and IFN-γ) or 1 representative experiment (IP-10). *P < .05.