IFNγ inhibits the therapeutic activity of peptide vaccination and decreases the capacity of CD8 T cells to recognize tumor cells. (A) Therapeutic effects induced by Trp2₁₈₀TriVax against 7-day-established subcutaneous B16 tumors in WT and IFNγ^−/− mice. Mice (4/group) were inoculated with tumor and vaccinated intravenously with TriVax (200 μg of Trp2₁₈₀ peptide, 100 μg of anti-CD40 mAb, and 50 μg of poly-IC) on days 7 and 18 (arrows). Nonvaccinated mice (No Vax) were included as controls. Tumor sizes are presented as mean tumor areas in square millimeters. Points, mean for each group; bars, SD. (B) Mice (4/group) received B16 cells intravenously and were vaccinated with Trp2₁₈₀TriVax or Ova₅₅TriVax, as described. On day 28, the numbers of B16 pulmonary nodules were evaluated in individual mice. Horizontal lines, means of each group. (C) Freshly isolated purified CD8 T cells from Trp2₁₈₀TriVax-immunized WT mice were evaluated for cytolytic activity against various targets: Trp2₁₈₀ peptide-pulsed and unpulsed EL4 cells (triangles), nontreated B16, B16 incubated with 100 U/mL IFNγ for 6 or 24 hours (circles), and B16/IFNγ cells (24 hours) pulsed with Trp2₁₈₀ peptide (diamonds). (D) Cytokine-release EliSpot assays using purified CD8 T cells from Trp2₁₈₀TriVax-immunized WT mice were performed using several stimulator cells: Trp2₁₈₀ peptide-pulsed and unpulsed EL4, IFNγ-treated (100 U/mL, 24 hours), and nontreated B16. P values were calculated with 2-way ANOVA (A,C) or unpaired Student t tests (B,D). Experiments were repeated 3 times with similar results.