Stimulation of PBMC with PHA (2 µg/ml for 72 hr), CD3 (CA17-2A12; [47] ) (0,5 µg/ml for 96 hr) and human IL-2 (200 U/ml for 7 days) was performed at a concentration of 5 × 10 5 cells/ml in RPMI-1640 supplemented with 10% fetal calf serum (FCS "Gold"; PAA Laboratories, Coelbe, Germany), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, non-essential amino acids and 1 mM sodium pyruvate (GIBCO/Invitrogen, Karlsruhe, Germany). T cells were isolated from either stimulated or unstimulated PBMC by positive MACS sorting following the manufacturer's instructions (Miltenyi Biotec, Ber-gisch-Gladbach, Germany). Stimulated and unstimulated PBMC were incubated with mouse anti-canine CD4 (CA13-1) and rat anti-canine CD8 mAbs (Dog 10-8) [48] and subsequently with bead-coupled anti-mouse Fc mAb which also reacts with rat IgG.