Immunogenicity of BA46-derived peptides in HHD mice. Mice were immunized intraperitoneally three times at 7-day intervals, with 2 × 10⁶ irradiated (50 Gy) peptide-loaded TAP2-deficient RMA-S-HHD-B7.1 cells. (a) The cells were loaded separately with individual peptides, washed, and pooled before immunization. (b) The cells were loaded with single peptides and injected individually. Spleens were removed on day 10 and splenocytes were restimulated in vitro by 100 μM BA46-derived peptides in opti-MEM for 2 hours at 37°C, 5% CO₂, followed by restimulation of lymphocytes for 4 more days in RPMI-HEPES as described. CTL assays were performed on day 5 with individual BA46-derived peptides loaded on RMA-S-HHD as targets. Unloaded RMA-S-HHD or tyrosinase-loaded targets were used as negative controls. An effector-to-target (E/T) ratio of 50:1 is shown. Black bars represent the targets loaded with BA46 peptides and white bars represent targets loaded with the tyrosinase-specific peptides. Specific lysis of all three peptides, BA46-6, BA46-7, and BA46-9, is statistically significant (P < 0.001) compared with lysis of the tyrosinase peptide. Results represent the average of three similar experiments. T/E ratio, tumor/effector ratio.