Figure 3. Proliferative impairment of rAAV-induced transgene product–specific CD8⁺ T cells.

(A) Splenocytes of BALB/c mice immunized with 10¹¹ gc AAV2/7gag or AAV2/7GFP and boosted 2 months later with 10¹¹ vp AdC68gag were analyzed by ICS at 10 days after AdC68gag boost. (B) Thy1.2⁺ BALB/c mice were immunized with 10¹¹ gc AAV2/7gag or 10¹⁰ vp AdC68gag; 1 month later splenic CD8⁺ cells were stained with 5 μM CFSE and transferred to Thy1.1⁺ mice that had been immunized i.p. with Vacgag (open histogram) or VacGFP (filled histogram). Three days later, splenocytes were stained with antibodies to CD8 and Thy1.2 and the gag-specific tet and analyzed for levels of CFSE in gag-tet⁺CD8⁺Thy1.2⁺ T cells. (C) Mice were primed with 10¹¹ gc AAV2/7gag, followed 2 months later by 10¹⁰ vp AdC68gag or 200 μg gag DNA vaccine and additionally boosted 2 months later with 10¹⁰ vp AdHu5gag or 10⁶ PFU MVAgag; controls received no rAAV. Ten days after the second boost, splenocytes were analyzed by ICS. (D) Mice were primed with 10¹¹ gc rAAV2/7gag and boosted 2, 4, 7, 24, and 28 weeks later with 10¹⁰ vp AdC68gag; controls received no rAAV. At 10 days after the boost, splenocytes were analyzed by ICS. (E) Mice were immunized with different doses of AAV2/7gag and 1 month later boosted with 10¹⁰ vp AdC68gag; controls received no rAAV. At 10 days after the boost, splenocytes were analyzed by ICS. In A and C–E, the frequency of gag-specific CD8⁺ T cells that produced IFN-γ in response to the gag peptide is shown. Background frequencies (less than 0.1%) were subtracted before plotting. Error bars represent SD for 5 mice.