Splenic lymphocytes collected from the 8 immunized chickens were stained with 5 mM CFSE (Invitrogen) in pre-warmed PBS for 10 min, washed three times, and suspended in RPMI 1640 containing 2 mM L-glutamine, 100 IU mL -1 penicillin, 100 IU mL -1 streptomycin, and 10% foetal bovine serum (R10 medium). Cells were plated at 10 6 well -1 in 24-well plates with RPMI 1640 medium, before stimulation for 5d with 100 mg mL -1 of the nine peptides generated from NP and the unrelated peptide N 71-78 . Cells were then washed with PBS and stained using anti-CD8-PE before flow cytometric analysis, which was performed on Cytomics FC500 MCL(Beckman) and analysed with its embedded software CXP. Two repeats were performed simultaneously for each peptide during the flow cytometric analysis.