Enhancement of antitumor immunity in mice immunized with OX40L mRNA–cotransfected DCs. (A) Murine DCs (generated from bone marrow precursors in the presence of IL-4 and GM-CSF) were transfected with OX40L or actin (control) mRNA, and OX40L cell-surface expression was determined by flow cytometry. (A, top panel) Isotypic control; (A, bottom panel) staining with anti-OX40L antibody. (B) Mice were inoculated subcutaneously with 2.5 × 104 F10.9 tumor cells. Three days after tumor implantation, mice (n = 10 in each group) were immunized intraperitoneally with 3 × 105 mRNA-transfected DCs twice at 7-day intervals. Tumor growth was evaluated every other day starting on day 10. Mice were killed once the tumor size reached 10 mm in diameter. (C) Splenocytes were isolated from mice vaccinated with TRP-2 mRNA–transfected DCs (TRP-2 – OX40L) or TRP-2/OX40L mRNA–cotransfected DCs (TRP-2 + OX40L). Cells were restimulated for 8 hours using DCs transfected with either TRP-2 mRNA (DC–TRP-2), actin mRNA (DC-actin), or OX40L mRNA (DC-OX40L), with 10 μg/mL Brefeldin A added during the last 4 hours of stimulation. Subsequently, cells were stained with anti-CD4 and anti-CD69 antibodies and analyzed by FACS analysis. Cells were permeabilized and stained with anti–IL-2, anti–IL-4, and anti–IFN-γ antibody. After gating on CD4⁺CD69⁺ cells, intracellular levels of cytokines were determined by flow cytometry. (D) Cytolytic assays. Splenocytes were isolated from naive mice, mice vaccinated with TRP-2 mRNA–transfected DCs (DC–TRP-2), and mice vaccinated with TRP-2/OX40L mRNA–transfected DCs (DC–OX40L + TRP-2). Cells were restimulated once with actin mRNA–transfected DCs (DC-actin), TRP-2 mRNA–transfected DCs (DC–TRP-2), and OX40L mRNA–transfected DCs (DC-OX40L). After 5 days, restimulated cells were analyzed in standard chromium release assays for specific killing of TRP-2 mRNA–transfected DCs (▵), actin mRNA–transfected DCs (▪), or OX40L mRNA–transfected DC (○) at the indicated effector-to-target (E/T) ratios.