FIG. 2.

Induction of POL-specific and ENV-specific CTLs. (A) Three CB6 F₁ mice were immunized with pcDNA3-POL/ENV followed by vPOL/ENV, both of which express the HBV POL plus ENV proteins. Ten days after the last immunization, the frequency of CD8⁺ T cells that responded to POL and ENV protein ex vivo was measured by intracellular IFN-γ staining. Spleen cells from immunized mice were incubated with the indicated transfectants for 5 h and stained for intracellular IFN-γ. Data are presented as the percentage of CD8⁺ T cells that produce IFN-γ upon stimulation with transfectants that express POL and/or ENV proteins. Representative responses of spleen cells from one mouse are shown. The mean number of antigen-specific CD8⁺ T cells for the three mice is shown in each panel. (B) The frequency of POL-specific and/or ENV-specific CD8⁺ T cells after 5 days of in vitro stimulation with P815POL/ENV was measured by intracellular IFN-γ staining. Cultured cells were incubated with indicated transfectants for 5 h and stained for intracellular IFN-γ. Results are shown as described above. (C) ENV28- to 39-specific CTL clone 6C2 can recognize both P815POL/ENV and P815ENV. 6C2 cells (5 × 10⁵) were incubated with same number of indicated transfectants for 5 h and stained for intracellular IFN-γ. Results are representative of at least three independent experiments. (D) The POL-specific CTL clone POL2 responded to P815POL/ENV but not to P815ENV. POL2 (5 × 10⁵) cells were incubated with same number of indicated transfectants for 5 h and stained for intracellular IFN-γ. Results are representative of at least three independent experiments.