In Vitro Cytotoxic T-Cell Assay

The in vitro CTL assay was performed as previous described (21 (link),24 (link)). The splenocytes from immunized mice were re-stimulated with 2μg/ml of AFP₂₁₂ or AFP₄₉₉ peptide for indicated times in the RPMI media containing 20 units/ml of IL-2. The EL4 and EL4-AFP tumor cells were labeled with 1μM and 0.03μM of 5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen), respectively, for 10min at 37°C. After washing, equal numbers (5×10⁴) of CFSE^lo-labeled target EL4-AFP cells and CFSE^hi-labeled EL4 control cells were co-cultured in triplicate with the in vitro peptide-stimulated splenocytes at the indicated E/T ratios for 6 hours. The killing of target EL4-AFP cells was analyzed by flow cytometry. The specific killing activity was calculated using the formula [1-(ratio of CFSE^hi/CFSE^lo in the absence of CTL) / (ratio of CFSE^hi/CFSE^lo in the presence of CTL)] ×100 as previously described (21 (link)).