Fig 4. Evaluation of antitumor immune responses induced by anti-neu-CpG.

(A) To evaluate the generation of memory responses, Balb/c and BALB-neuT tumor bearing mice were treated with i.t. injections of ant-neu-CpG (as in Fig 3). Mice that rejected the tumor were challenged with the 10⁶ TUBO cells 70 days after the primary tumor was implanted. Animals were monitored for the development of tumors and survival. (B) To evaluate whether BALB-neuT mice have a similar capacity to develop memory responses to nominal antigens as Balb/c mice, Balb/c and BALB-neuT were implanted with 10⁵ BM-185-EGFP cells. A control group of animals were injected with BM-185-w.t. cells. Mice that rejected the BM-185-EGFP cells were challenged with 10⁵ BM-185-w.t. cells 60 days after injection of the primary tumor. Six animals were included per group. Data are representative of two experiments. (C) To analyze whether T-regs influence the immune responses in BALB-neuT tumor bearing mice, the accumulation of CD4⁺CD25⁺Foxp3⁺ T-regs within the tumor over time was evaluated. BALB-neuT mice were implanted with 10⁶ TUBO cells on day zero. Animals were sacrificed on days 10, 20 and 30. Tumors were extracted at the determined times and stained against CD4, CD25 and Foxp3. (D) The levels of accumulation of CD4⁺Foxp3⁺ T-regs in spleen, LN and within the tumor was evaluated in non-treated and treated anti-neu-CpG BALB-neuT mice. BALB-neuT mice were implanted with 10⁶ TUBO cells on day zero. Tumor was allowed to grow for two weeks. Tumors were then treated with i.t. injections of anti-neu-CpG (50 μg/injection) two times a week for one week. Next day following the last injection, animals were sacrificed. The prevalence of CD4⁺Foxp3⁺ T-regs in LN, spleen and within the tumor was determined. Four animals were included per group ±SD. Data are representative of two experiments.