[Invitrogen], 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 50 M -mercaptoethanol, and 10% FCS) before they were used in subsequent in vitro assays. Peptide concentration was 20 mg/ml and 0.20 μl was used per well. ELISpot assays with murine splenocytes were performed using α-mouse IFN-γ mAb (AN18) and biotinylated α-mouse IFN-γ mAb (R4-6A2) as the cytokine-specific capture antibodies. Antibodies were monoclonal antibodies. 5 × 10 5 splenocytes were plated per well.