Ex vivo IFN-γ ELISPOT analysis of T-cell reactivity in HLA-A2/K^b mice vaccinated with a huCYP1B1-encoding DNA construct. (A) HLA-A2/K^b mice were primed and boosted twice with PLG-microparticle–encapsulated v/huCYP1B1d3 or control vector or were not vaccinated. Twelve days after the second boost, CD8⁺-enriched spleen cells were tested for reactivity against EL4-A2/K^b cells infected with vaccinia encoding huCYP1B1 (vac-huCYP1B1), vaccinia wild-type (vac-wt), or noninfected control. Results are shown as IFN-γ spot-forming cells (SFCs)/10⁶ CD8⁺ T cells. (B) The same spleen cells from vaccinated and control mice were used to measure IFN-γ release in response to EL4-A2/K^b cells untreated or pulsed with the CYP190 peptide. (C) IFN-γ ELISPOT analysis of spleen cells from mice primed and boosted twice with v/huCYP1B1d3 in PLG microparticles. EL4-A2/K^b cells pulsed with the HLA-A*0201–binding human epitope CYP190 or the K^b-binding shared mouse/human epitope CYP77 were used as stimulators. In all assays, PHA was used as a positive control (data not shown), and IFN-γ background secretion was determined without stimulation.