CpG + anti-OX40 + anti-CTLA4 induces antitumor IFN-γ–producing T cells and decreases the percentage of Treg cells. (A-F) Using the 2-tumor site model, mice bearing A20 tumors were either left untreated, treated with CpG alone, or treated with CpG + anti-OX40 + anti + CTLA4, using the same therapeutic protocol as previously described. Four days after the end of treatment, mice were killed and spleens and tumors were harvested separately and made into a single cell suspension. (A,B) Total splenocytes were cocultured in vitro in the presence of irradiated A20 tumor cells plus anti-CD28 mAb for 24 hours. CD4 and CD8 T cells were assayed for CD44 and intracellular IFN-γ expression. (A) Data from one representative mouse of each group shows IFN-γ expression according to CD44 expression. (B) Average percentage of CD4 and CD8 T cells expressing intracellular IFN-γ of each group (n = 3 mice per group). * indicates a statistically significant difference (P < .05). (C,D) Total cells from the tumor were cocultured in vitro in the presence of irradiated A20 tumor cells plus anti-CD28 mAb for 24 hours. Intracellular expression of IFN-γ for CD4 and CD8 T cells is shown for one representative mouse of each group (C) and as the average of each group (n = 3 mice per group; D). (E,F) Total cells from the tumor were directly analyzed for Treg subpopulation. The proportion of intracellular FoxP3 expressing CD4 T cells is shown for one representative mouse of each group (E) and as the average of each group (n = 3 mice per group; F).