Effect of CD4+T cell depletion on the therapeutic antitumor immunity generated by 4a-CRT/E6E7L2 DNA vaccine. (A) Schematic illustration of the experiment. Briefly, 5-8 week old female C57BL/6 mice (5 mice/group) were treated with 100 μg/mouse of anti-mouse CD4 deletion antibody (clone GK1.5) or PBS via intraperitoneal injection for 3 days and the depletion was maintained by weekly injection of the depletion antibody (100 μg/mouse). 5 days after the initiation of the depletion, mice were injected with 1 × 105 TC-1 cells intravenously. 3 days after TC-1 cell injection, mice were vaccinated with 20 μg/mouse of 4a-CRT/E6E7L2 DNA vaccine via intramuscular injection followed by electroporation. The mice were boosted with the same regimen twice with 1-week interval. (B) One week after last vaccination, PBMCs were collected and HPV16 E7-specific CD8+ T cell responses were analyzed by HPV16 E7aa49-57 peptide loaded H2-Db tetramer staining followed by flow cytometry analysis. (C) 13 days after last vaccination, splenocytes were harvested for the detection of HPV-16 E6 and E7-specific CD8+ T cell responses analyzed by IFN-³ intracellular staining. Diagram of the number of TC-1 cell metastasis nodules (D) and the weight of lungs (E).