Intracellular cytokine staining was performed as previously described with minor modifications [28] . Briefly, primed T cells were incubated with peptideloaded T2 cells in the presence of Brefeldin A (eBioscience, San Diego, CA, USA) for 8 h. The cells were stained with a PE-conjugated anti-human CD8 antibody (eBioscience) at 4°C for 30 min. After washing with 1· PBS, the cells were fixed and permeabilized with a permeabilization buffer (eBioscience) according to the manufacturer's instructions. The cells were finally stained with an FITC-conjugated anti-human IFN-c antibody at 4°C for 30 min in the dark, and the fluorescent signals were detected by flow cytometrical analysis (BD FASC Aria II, BD Bioscience). T2 cells loaded with previously identified non-responsive peptide (N160 LQLPQGTTL), were used as a negative experimental control [2] .