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Figure 9: WE-CN promotes CD4+Th1 differentiation and increased HER-2/neu-specific functional CD8+ T cells in spleens from vaccinated mice. (a) CD4+ T cells were purified from the different groups and stimulated with recombinant HER-2/neu protein (10 μg/mL). After 3 days, the expression levels of IFN-γ or IL-4 mRNA were determined by quantitative real-time RT-PCR. The data were normalized to HPRT expression in each sample, and error bars indicate the mean ± SD of six mice per group assessed from three independent experiments. (b) The spleen cells from the different vaccinated groups were stimulated with a peptide pool composed of 10 μg/mL each of peptide 362–370 (EFAGKKI) (BioBasic, Canada) and peptide 404–412 (EEITGYLYI) of the HER-2/neu sequence. After stimulation for 18 hr, the percentage of IFN-gamma-producing CD8+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± SD of five mice from two independent experiments. The symbol * indicates a statistically significant difference compared to control vector-treated mice ( ). The symbol ** indicates a statistically significant difference compared to the HER-2/neu DNA vaccine alone group ( ).