Lymphocyte preparation, culture and T cell adoptive transfer

The lymphocytes were isolated from the heparinized blood from SPF pigs vaccinated with the avirulent strain, 05HAS68. Briefly, lymphocytes (>85% purity) were prepared by sequential centrifugation on Ficoll and Percoll gradients. After isolation, cells (1 × 10⁶ cells/ml) were incubated with heat-inactive 05HAS68 cells (1 × 10⁶ cells/ml) for 3 days at 37 °C in a 5% CO2 atmosphere. The specific T lymphoblasts were prepared by sequential centrifugation on Ficoll gradients. After washing, the cells were resuspended in RPMI1640 and 1.8 × 10⁹ cells or mixed with 5 ml plasma from the piglets vaccinated with avirulent 05HAS68 and were then transferred to naïve piglets (n = 6) via injection into the ear vein.