4 × 106 T-cells, which were isolated from spleens of naïve C57BL/6 mice by panning of B lymphocytes with anti-IgG+M antibodies (Dianova, Hamburg, Germany), were co-cultivated with 2 × 105 DCs and 105 irradiated (100 Gy) tumor cells in 24-well plates for three days. TrAbs were added at 1 μg/ml. Cells were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-Glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, 10 mM HEPES and 50 μM 2-mercaptoethanol. CD69 and CTLA-4 surface expression of CD4+ and CD8+ T cells was determined by flow cytometry at 0, 24, 48 and 72 h using the mAbs listed above. The percentage of positive cells was determined in comparison to corresponding isotype controls.