Characterization of the T regulatory cells and myeloid-derived suppressor cells (MDSC) in the tumor microenvironment following vaccination with CRT/E7 DNA with or without imiquimod in tumor-bearing mice. C57BL/6 mice were divided into four groups (5/group: untreated, imiquimod alone, CRT/E7 alone, and combined group). All mice were inoculated with TC-1 cells (5 × 104/mouse) s.c. over right leg at d0. For the untreated group, mice were regularly followed after TC-1 implantation without specific treatment. For the imiquimod group, each mouse received topical imiquimod (50 mg/mouse) every two days (began at d6 for a total of 6 treatments). For CRT/E7 alone group, each mouse was vaccinated with 2 μg of pcDNA3-CRT/E7 DNA via gene gun at d9, d13, and d17. For the combined treatment group, each mouse received the same treatment schedule as for each monotherapy alone group. Tumors were harvested at d24 for flow cytometric analysis. Tumor cells were either stained with PE-conjugated anti-CD4 (L3T4) and FITC-conjugated anti-CD25 (PC61) mAbs (A & B) or with PE-conjugated anti-CD11b (M1/70) and FITC-conjugated anti-Gr-1 (RB6-8C5) mAbs (C & D). Plots were gated on lymphocyte population. A) Representative flow cytometry data demonstrating the percentage of CD4+CD25+ cells. B) Bar graph representing the percentage of Tregs among the tumor-infiltrating lymphocytes. C) Representative flow cytometry data demonstrating the percentage of CD11b+Gr-1+ cells. D) Bar graph representing the percentage of Tregs among the tumor-infiltrating lymphocytes. C denotes untreated group, and I denotes imiquimod treated group. Representative data from one of three independent experiments are shown. *, P < 0.05; **, P < 0.01.