Figure 3. Improved immunization in HLA-A2 transgenic mice prolongs animal survival time and prevents lung metastases.

HLA-A2 transgenic mice were subcutaneously immunized twice with peptide A2-5 (50 μg/mouse), Th epitope peptide, CpGODN (10 μg/mouse) and ISA and then the anti-tumor effects were monitored. A. Splenocytes were harvested and incubated with peptide A2-5 or irrelevant peptide (10 μg/ml). IFN-γ secreting cells were detected by IFN-γ ELISPOT assay. Error bars, SD. ***P < 0.001. B. Splenocytes harvested from peptide immunized mice were stimulated with 10 μg/ml of peptidesfor 6 hr in the presence of PE-conjugated anti-CD107a. After stimulation, FITC-conjugated anti-CD8 antibody was used to detect CD8⁺ T cell. The percentage of CD107a⁺CD8⁺ cells in individual immunized groups was determined by flow cytometry. C. Irradiated EL4-TAL6-A2 or EL4-TAL6 cells (2 × 10⁴) were used to stimulate splenocytes for 2 hr. The percentage of CD107a⁺CD8⁺ cells was determined by flow cytometry as panel B described. D. After peptides immunization, EL4-TAL6-A2 cells (2 × 10⁵ cells) were inoculated through the i.v. routes. Lung tissues from each group were collected for tumor nodule detection 20 days after tumor implantation. E. Mice survival time was monitored and analyzed. *, P < 0.05; **, P < 0.01. F. Melanoma B16 or B16-TAL6-A2 cells (B16 cells with expression of TAL6 and HLA-A2) were intravenously injected in naïve and ISA/A2-5/Th/CpG immunized HLA-A2 transgenic mice. Whole lungs were examined.