Therapeutic prime-boost vaccination decreased the expression of regulatory T cells. (A) FACS analysis of regulatory T cells in splenocytes of vaccinated mice with MCA304 tumors. Splenocytes collected from Figure 3B on day 33 were stained with anti-CD4, CD25, Foxp3 and rat IgG2a (isotype control) antibodies and analyzed by FACS. Representative expression of CD25 and Foxp3 gated on CD4+ cells is shown. Results are representative of three independent experiments. (B) The MCA304 tumor samples from Figure 5 were stained with antibodies specific for anti-CD4 and anti-Foxp3 and counterstained with 4',6-diamidino-2-phenylindole. The number of positive cells were counted and plotted in C. Tumor sections were quantitated for ≥2 mice per group with a minimum of two sections per tumor for each staining condition, with five fields per section used for counting.