G-specific CD4 T-cell response in Gcf-immune mice.

<p>(A) Mice were immunized twice with Gcf s.l., once with FI-RSV by food-pad injection, or 1×10⁵ PFU of live RSV A2 via i.n. route, and then challenged with RSV three weeks after final immunization. Lung mononuclear cells were prepared from the lungs of the same group of mice (<i>n</i> = 6) four days after challenge and then stimulated with G(183–195) peptide in the presence of Brefeldin A. Cells were stained for CD4, CD44, and IFN-γ, and analyzed by flow cytometry. Cells gated for CD4 expression are shown in each dot plot and the percentages represent the frequency of G-specific IFN-γ-positive cells The numbers in the upper right quadrant indicate the percentage of IFN-γ+ cells among total lung CD4+ cells after subtracting the percentages by isotype control staining of the same samples. (B) Average data represent mean ± SD (<i>n</i> = 6). The results are a representative of two independent experiments. (C) Cytokine levels in the lung supernatant were assessed at day 5 post-challenge by multiplex antibody-based assay (FlowCytomix).