FIGURE 6.
Impairment of CD4, CD40L, or CD28 ablates the IFN-γ-producing T cell response to AAV2/rh32.33.nLacZ and restores β-gal expression in skeletal muscle. C57BL/6, CD40L−/−, and CD28−/− mice were injected with 1011 GC of vector in combination with either the CD4-depleting Ab GK1.5, CD40L blocking Ab MR-1, or the CD40 agonist FGK45. Mice were sacrificed 28 days postinjection with vector, and lymphocytes were isolated from the spleen. Following stimulation with the AAVrh32.33 capsid library or the nLacZ dominant peptide, T cell responses were determined by IFN-γ ELISPOT (primary axis). Lymphocytes isolated from whole blood were stained using the PE-conjugated H-2Kb-ICPMYARV tetramer together with a FITC-conjugated anti-CD8 Ab to determine the percentage of nLacZ-specific CD8+ T cells in the total CD8+ T cell population (secondary axis). X-Gal histochemical staining was performed on sectioned skeletal muscle. Representative sections from four mice per group are shown. Data are mean ± SD for four mice per group, and represent five independent experiments performed using common controls. Statistical analysis compared AAV2/rh32.33 alone in wild-type C57BL/6 mice with AAV2/rh32.33 given in combination with GK1.5, MR-1, FGK45 in CD40L−/− and CD28−/− mice. *, p < 0.001, by ANOVA Student-Newman-Keuls test.