Figure 6.

Immunological Analysis of the Mice Immunized with the KALA-OVA-LPs

(A) C57BL/6J mice were immunized subcutaneously once with KALA-OVA-LPs at a dose of 25 μg OVA. At 1 days before and 1, 3, and 6 days after the immunization, anti-CD4, anti-CD8, or isotype control antibody was intraperitoneally administered at a dose of 200 μg. Fluorescent-labeled target cells (OVA_257–264 pulsed, CFSE^High) and control cells (no peptide pulsed, CFSE^Low) were injected intravenously at 1 week after immunization. The OVA-specific lysis was calculated from the target cell/control cell ratio measured by flow cytometer 20 hr after injection. Data are mean + SEM (n = 3). Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. **p < 0.01 versus isotype control group. (B and C) C57BL/6J mice were immunized subcutaneously with KALA-OVA-LPs at a dose of 25 μg OVA twice every 7 days. At 7 days after the second immunization, splenocytes were harvested and re-stimulated by (B) OVA_257–264 or (C) OVA_323–339, followed by an additional incubation with the protein transport inhibitor. Populations of cells in the spleen, which were positive in IFNγ (B and C), IL-4, and IL-17 (C) were quantified by flow cytometry after the immunostaining for intracellular cytokines. Each dot represents the percentage of cytokine-positive cells in an individual mouse. Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. *p < 0.05 and **p < 0.01 versus non-treated group.