Heterologous boost vaccination strongly increases p572-specific CD8 T-cell responses in lentivector-primed mice. HHD mice (n = 4-6/group) were immunized with lv-hTERT vector alone or with pY572/IFA boost done 3 weeks later. (A) Enumeration of pY572/pentamer⁺ CD8 T cells by pentamer staining. Representative FACS dot plots for each group is shown (left) and the mean percentage of pY572/pentamer⁺ CD8 T cells after subtraction of irrelevant pentamer (right). *Statistically significant value (P < .05). (B) IFN-γ–producing spleen lymphocytes were detected by an ex vivo IFN-γ ELISpot in response to p572 peptide. Responses from individual mice are shown. (C) The ex vivo cytotoxicity of CD8 T cells was tested against RMAS/HHD cells pulsed with pY572 (left) or against B16/A2 tumor cells (right). Results represent the specific lysis (percentage) plus or minus SD in each immunized group of mice (n = 3 mice/group), after subtraction of nonspecific lysis measured in the presence of nonpulsed RMAS/HHD or wt B16 cells. The CD8 T cells from mice immunized with lv-GFP were used as negative control.