In Vitro Cytotoxicity Assay

For the in vitro cytotoxicity assay, naïve CD8⁺ T cells were differentiated into CTLs as described above. Fc receptor–positive P815-GFP⁺ target cells (ATCC, TIB-64) were co-cultured with effector CD8⁺ T cells at 0.5:1, 1:1, 1:3 and 1:10 ratios in the presence of soluble anti-CD3 antibody at a total density of 2 x 10⁵ cells/200 μL/well in 96-well U-bottom shaped tissue culture plates. Cells were incubated for 4h at 37°C. After the incubation period, cells were harvested and cell suspension was stained for Annexin V and CD8α, and apoptosis of target cells (GFP⁺) was quantified by flow cytometry. As a negative control, effector and target cells were co-cultured without anti-CD3 antibody.