E7-specific CD8⁺ T cell immune responses and antitumor effect generated by vaccination with E7 DNA mixed with DNA encoding antiapoptotic or proapoptotic proteins. The pcDNA3 (no insert) mixed with pSG5-BCL-xL was used as a negative control. (a) Representative figure of the flow-cytometry data. The data presented in this figure are from one representative experiment of three performed. (b) Bar graph depicting the number of antigen-specific IFN-γ–secreting CD8⁺ T cell precursors per 3 × 10⁵ splenocytes (mean ± SD). (c) In vivo tumor-prevention experiment. Mice were immunized with pcDNA3-E7 mixed with pSG5 encoding BCL-xL, caspase-3, or no insert. The pcDNA3 (no insert) mixed with pSG5-BCL-xL was used as a negative control. One week after the last vaccination, mice were subcutaneously challenged with 5 × 10⁴ TC-1 cells per mouse in the right leg. (d) In vivo Ab-depletion experiments to determine the contribution of various lymphocyte subsets on the tumor protection generated by the coadministration of pcDNA3-E7 and pSG5-BCL-xL DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge. (e) In vivo tumor-treatment experiment. Mice received 10⁴ TC-1 tumor challenge and were immunized 3 days later with pcDNA3-E7 mixed with pSG5 encoding BCL-xL, caspase-3, or no insert. In vivo tumor protection, Ab depletion, and tumor-treatment experiments were performed three times. Casp, caspace.