Kinetics of the Gag-specific CD8+ T-cell response. (A) Top panels: Groups of 5 BALB/c mice injected intramuscularly with 1011 vp of AdC68gag37 or 5 × 1011 vp of AdHu5gag37 vector were killed at different times. Lymphocytes isolated from indicated compartments were tested for Gag-specific IFN-γ–producing CD8+ cells by intracellular cytokine staining and analyzed by 2-color flow cytometry.6 Results show frequencies of CD8+ cells producing IFN-γ as a percentage of all CD8+ cells from a representative experiment (top panels). Frequencies of Gag-specific CD8+ T cells in BALB/c mice immunized with 5 × 1011 vp of AdC68gag37 and boosted 2 months later with 106 plaque forming units (pfu) of a recombinant vaccinia virus expressing gag (VVgag). Weeks after immunization refer to the boost. Each experiment was conducted 2 to 4 times and the data shown are representative. (B) The same experiment was conducted with cells from mice immunized with 5 × 1011 vp of Adhu5gag37 vector. C57Bl/6 mice were infected with 2 × 105 pfu of LCMV intraperitoneally or immunized with 1010 vp of Adhu5 expressing LCMV glycoprotein intramuscularly. Db/GP33 tetramer-positive CD8 T cells were enumerated in the peripheral blood mononuclear cells by MHC tetramer staining at the indicated time points. n is 3 for LCMV and n is 2 for Adhu5-GP33. Similar results were observed for Adhu5-GP given intravenously or intraperitoneally (data not shown). (C) Mice were vaccinated with 1011 vp of E1-deleted, E1-deleted, and E3-deleted or E1-deleted, E3-deleted, and E4-deleted AdC8 vector expressing Gag37. T-cell frequencies of spleens of individual mice were analyzed at different times. The graphs show frequencies of Gag-specific CD8+ T cells for individual mice (■) and mean frequencies (X) (± SD).
