The percentages of IL-4-producing <i>CD4</i>+ T cells.

<p>Fourteen days after the last vaccination three mice per group were euthanized, and splenocytes were collected and characterized for CD4+ T cells using intracellular IL-4 staining followed by flowcytometry analysis. Data represent mean ± SEM (n = 3). ** denote significant differences from controls and all other groups, respectively.</p

Evaluation of the amount of IFN-γ produced in vaccinated mice.

<p>Nine BALB/c female mice per group were vaccinated three times subcutaneously with 100 μg/mouse of P₅₊₄₃₅ long peptide, P₅₊₄₃₅ in combination with CpG, or in combination with both 50 μg/mouse of PADRE and 25 μg/mouse of CpG. Two weeks after the last vaccination, splenocytes from three mice from each group were collected and activated with the long peptide. Immune responses were then determined using an IFN-γ ELISpot assay. The data indicate the mean ± SD. (n = 3). * denotes significant difference from all other groups (P < 0.001).</p

In vivo antitumor effects experiments.

<p>Six mice/group were immunized three times with P₅₊₄₃₅ long peptide alone, long peptide + CpG, or long peptide + PADRE + CpG. Control mice were immunized with PADRE, PADRE + CpG, or PBS. After 14 days the mice were challenged subcutaneously with 5× 10⁵ live TUBO cells. (A) Tumors were measured weekly and sizes were recorded. The values are means of tumor size and error bars indicate SD. (B) The survival times of the mice were analyzed by log-rank (P = 0.117) and Fleming-Harrington tests (P = 0.058) for 80 days. * denotes significant difference from PBS (P < 0.05).</p

The percentages of IFN-γ producing CD4+ and CD8+ T cells.

<p>Flow cytometry data were also analyzed according to the percentage of cytokine-producing cells and dot plots were drawn for each vaccinated group. Quadrants showing dot plot of CD8 and CD4 cells producing IFN-γ percentage. Spleen cells were analyzed using a gating strategy to exclude debris and identify CD4+ and CD8+ T cells. The subsequent analysis was on CD8+ or CD4+ gates to describe IFN-γ-producing T cells.</p