Cytolytic activity of Gag-specific CD8⁺ T cells. Groups of BALB/c mice were immunized with 5 × 10¹¹ vp of AdC68gag37 and tested 2 months later. (1) Additional mice were primed with 5 × 10¹⁰ vp of AdC68gag37 and boosted with 5 × 10¹⁰ vp of AdHu5gag37 and tested 3 months after the boost (2). Target cells (P815) were coated overnight with 5 μg/mL Gag peptide (■) or an equal concentration of a control peptide (□). Before the assay, frequencies of Gag-specific CD8⁺, T cells were determined by MHC-gag peptide tetramer staining. Lymphocytes were adjusted to equal numbers of the tetramer-positive CD8⁺ cells and cocultured at serial dilutions with ⁵¹Cr-labeled target cells for 6 hours. Supernatants were harvested and tested in a gamma counter. Percent specific lysis was calculated as described.⁹  The origin of the lymphocytes is indicated in the upper part of the graphs. LN, lymph nodes; PL, peritoneal lavage. Error bars show standard deviations for the percent of specific lysis tested in 3 separate samples.