BALB/c mice (n=5) were primed with three different doses of HIV-1 Gag DNA vaccine in the presence or absence of agonistic anti-4-1BB Ab at week 0 and boosted at week 12. A, Gag-tetramer specific CD8 T cells in blood were analyzed at 2 weeks following the DNA prime. The bars represent the means ± SEM. B, Representative FACS plot showing Gag-specific CD4 and CD8 T cell responses at one week following the MVA boost. Left quadrants represent CD4 T cells (CD3+, CD8-) and right quadrants represent CD8 T cells (CD3+, CD8+). Numbers indicate Gag-specific IFN-γ+ CD4 or CD8 T cells expressed as a percent of respective total cells. Gag ID: Gag immunodominant peptide; PP3: Peptide Pool 3; PP4: Peptide Pool 4. C, Summary of Gag-specific CD4 T cells at 1 week following the MVA boost. Data from two independent experiments (Exp.1 and Exp.2) with the 10μg and 100μg DNA priming doses are shown. Bars represent the mean absolute number per spleen (spleens from each group were pooled for analyses except for Exp.2 with 100μg DNA priming dose). D, Summary of Gag-specific CD8 T cells analyzed at 1 week following the MVA boost. Left panel, Gag-tetramer specific CD8 T cells in blood (pooled data). Right panel, Mean absolute number of IFN-γ producing CD8 T cells per spleen is plotted (spleens from each group were pooled for analyses except for Exp.2 with 100μg DNA priming dose). Data from two independent experiments (Exp.1 and Exp.2) with the 10μg and 100μg of DNA priming dose are shown. E, Adjuvant effect of anti-4-1BB agonistic Ab is CD4 dependent. For in vivo depletion of CD4 T cells, mice (n=3) were injected intraperitoneally with 200μg of GK1.5 antibody one day before and after DNA prime along with or without the agonistic anti-4-1BBAb. Gag-tetramer specific CD8 T cells were evaluated in blood for both WT and CD4 depleted mice at the indicated time points following MVA boost. The results are represented as means ± SEM. *, p < 0.05.