BALB/c mice (n=10) were primed and boosted with indicated DNAs at week 0 and week 4 respectively. A, Gag-tetramer specific CD8 T cells in spleen. B, Gag-specific IFN-γ producing CD8 T cells in spleen as evaluated by intracellular cytokine staining. C, Representative FACS plots showing Gag-specific CD8 T cells that co-produce IFN-γ and IL-2 (cells were gated on CD3 and CD8). Numbers indicate Gag-specific CD8 T cells either secreting IFN-γ or IFN-γ and IL-2 expressed as a percent of total CD8 T cells. D, Summary of the frequency of IFN-γ+ IL-2+ CD8 T cells in spleen. The bars represent mean frequency ± SEM. The Gag group was immunized with 20μg of Gag plasmid and the m4-1BBL-cis group with 40μg of Gag-m4-1BBL plasmid. The m4-1BBL-trans and SPD-4-1BBL-trans groups were immunized with 20μg of the respective plasmids pre-mixed with 20μg of Gag plasmid. *, p <0.05; and **, p <0.001. NA, Not available.