Effect of CD4+T cell depletion on the immunogenicity of 4a-CRT/E6E7L2 DNA vaccine. (A) Schematic diagram of the treatment schedule. Briefly, 5-8 weeks old female C57BL/6 mice (5 mice/group) treated with 200 μg/mouse of anti-mouse CD4 depletion 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). (B) Success of CD4+ T cell depletion was verified by peripheral blood CD4 staining followed by flow cytometry analysis. 1 week after depletion initiation, 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 at a 2-week interval. (C) One week after the last vaccination, serum was collected for the analysis of HPV16 E7-specific antibody response by ELISA. (D) HPV16 E7-specific CD8+ T cell responses were analyzed by staining PBMCs with HPV16 E7aa49-57 peptide loaded H2-Db tetramer followed by flow cytometry analysis. (E) Two weeks after last vaccination, the mice were challenged with 5 × 104 TC-1 cells subcutaneously. Tumor growth was monitored twice a week.