Fig 5. Analysis of antitumor and immune responses after anti-neu-CpG vaccination plus depletion of T-regs.

(A) BALB-neuT mice were inoculated s.c. on day zero with 10⁶ TUBO cells. On days 9, 16 and 23 animals were injected with anti-CD25, anti-OX40 or anti-GITR (300 μg/injection). Animals were treated with anti-CD25, anti-OX40 or anti-GITR alone or in combination with anti-neu-CpG. On day 10, animals started intratumoral injections of anti-neu-CpG two times a week for three weeks (50 μg/injection). Tumor growth and survival (only survival of control, anti-neu-CpG, anti-neu-CpG+anti-OX40 and anti-neu-CpG+anti-GITR is shown) of all groups was evaluated. (B) Animals that rejected the tumor were challenged with the 10⁶ TUBO cells 70 days after the primary tumor was implanted. A significant P< 0.001 difference was found between anti-neu-CpG, anti-neu-CpG+anti-OX40 groups of treated mice and BALB-neuT mice treated with anti-neu-CpG plus anti-GITR. Six to eight animals were included per group. Data are representative of two experiments. (C) To evaluate the induction of cytotoxic responses, BALB-neuT mice were implanted with 10⁶ TUBO cells on day zero. Tumor was allowed to grow for two weeks. Animals were not-treated or treated with anti-neu-CpG or with anti-neu-CpG plus anti-GITR. For treatment anials were i.t. injected with anti-neu-CpG (50 μg/injection) two times a week for one week. Group of animals that were treated with anti-GITR mAb received a single injection (300 μg/injection). Two weeks after the first injection with anti-neu-CpG animals were sacrificed and splenocytes were stimulated irradiated TUBO cells plus feeder cells for five days in complete media. Cytotoxic activity of restimulated cultures was measured against TUBO, 66.3, A2L2 and RENCA (haplotype irrelevant control cells) in standard 6-hour ⁵¹Cr release assay at 30:1 Effector:Target ratio. Four animals were included per group ±SD.