Figure 5. Induction of pulmonary IFN-γ and CTL activity in peptide immunized transgenic mice challenged with RSV.

At day 4 post RSV infection, lung homogenates were prepared and the following were measured. (A) IFN-γ expression by ELISA, and (B) the number of CD8⁺ T cells in the lungs by flow cytometry using PE-cy5-labeled anti-CD8 antibody. (C). Enumeration of CTL activity in the lungs of mice immunized with CD8 peptide epitopes and challenged with RSV. Effector lymphocytes isolated from the lungs of mice immunized with peptide 3 (▪), peptide 13 (▴), peptide 14 (▾), or peptide 23 (•) were cultured and supplemented with murine IL-2 in the presence of 2 µg of the same peptide for 5 days. In parallel, cultured pulmonary lymphocytes from vehicle-immunized mice were stimulated with 2 µg of peptide 3 (□), peptide 13 (Δ), peptide 14 (▽), or peptide 23 (ο), respectively, for 4 days. DCs isolated from the tibia of HLA-B6 mice were pulsed with 20 µg per mL of the individual peptides for 2 hours at 37°C and labeled with CFSE and used as targets in the in vitro CTL assay. Un-pulsed DCs served as negative control. The viable effector cells were co-cultured with 10⁴ peptide-loaded target DCs cells at effector∶target ratios of 50∶1, 10∶1, and 0∶1 for five hours. The cell mixtures were labeled with 7-AAD and analyzed by flow cytometry. Results are expressed as mean percentage of 7-AAD/CFSE positive cells normalized with un-pulsed DCs. Six mice were taken in each group. The result is a representative of two independent experiments.