To prepare splenic lymphocytes, all eight pCAGGS-NP immunized chickens were killed by cardiac puncture blood collection. Sterile spleens were collected and meshed through a sieve screen using a syringe plunger to obtain a single-cell suspension in tissue culture medium (RPMI 1640, Gibco BRL NY, USA). Cell suspensions were overlaid onto Histopaque 1077 density gradient medium and centrifuged at 1800 rpm for 20 min at 18uC. Lymphocytes were collected from the interface and washed three times in RPMI, before cells were counted using a trypan blue dye exclusion assay.