Heterologous prime-boost amplifies PSA-specific T cell responses. BALB/c males (5/group) were treated every two weeks with: buffer (Control), PROSTVAC-V (VVV), PROSTVAC-F (FFF) or received a PROSTVAC-V prime followed by 2 PROSTVAC-F boosts (VFF). Pooled splenocytes were assayed for PSA-specific responses by IFNγ ELISPOT (A, B) and cytotoxic activity by flow cytometry (% CD107+ IFNγ+ CD8 T cells) (C). Anti-PSA IgG titers were determined by ELISA for each individual mouse (D). For ELISPOT, splenocytes were restimulated with CD4 or CD8 PSA-specific peptides or controls (controls not shown) at indicated concentrations. Responses that were too numerous to count were displayed as 1000 spots/million cells. Statistical significance was determined by Two-way-ANOVA with Tukey post-test at 0.01 μM. ****P < 0.001 compared to control or homologous dosing (A & B). To identify cytotoxic CD8+ T cells, splenocytes were restimulated overnight with a PSA CD8-specific peptide in the presence of anti-CD107 antibody. Graphs show representative data of four independently performed experiments.