In-vitro stimulation of T cells was conducted by co-culturing 5 × 106 splenocytes of immunized mice with 5 × 106 splenocytes of naïve mice, which were loaded with Trp-2, MAGE-A5 and gp100 peptides  and irradiated at 30 Gy, in the presence of IL-2 (30 U/ml; Amersham Pharmacia, Freiburg, Germany) in 24-well plates. After 7 days, T cells were harvested and characterized by FACS analysis as described above.