BALB/c mice (n=3) were primed with HIV-1 Gag DNA vaccine in the presence or absence of agonistic anti-4-1BB Ab. A, Representative FACS plot showing the frequency of Gag-tetramer specific CD8 T cells in blood (top panel) and Gag-specific CD4 and CD8 T cell responses in spleen (lower panel) at 2 weeks following the DNA prime. In the lower panel representative plot, the 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. B and C, Summary of Gag-specific CD4 and CD8 T cells respectively following DNA prime. Bars represent the mean absolute number ± SEM per spleen. D, Kinetics of 4-1BB expression on CD4 and CD8 T cells. Total splenocytes were activated in vitro with soluble anti-CD3 for the indicated time periods. Cells were harvested and 4-1BB expression on CD4 (CD3+ CD4+) and CD8 (CD3+ CD8+) T cells were analyzed by flow cytometry. Open histograms correspond to cells stained with anti-4-1BBAb and shaded histograms correspond to cells stained with anti-rat IgG2a isotype control. Percentage of CD4+ 4-1BB+ or CD8+ 4-1BB+ T cells are indicated on the plot. *, p < 0.05.