Cytotoxic T Cell-Mediated Killing Assay

Target cells were labeled with CellTrace Violet (Invitrogen) before use. T cells were co-cultured with 2 × 10³ target cells at varying effector/target ratios for 4–6 h in 96-well round-bottomed plates, followed by active Caspase-3 staining (BD Biosciences)^{6 (link),29} . The blocking of mouse MHC-II and FasL was performed as described previously^{9 (link)}. In human cell killing assays, target cells (CLL cells) were pre-incubated with HLA-II (HLA-DR, -DP, -DQ) blocking antibody (TÜ39) or isotype control mouse IgG2a (both at 2 μg/ml; BD Biosciences) for 20 min at 37°C. In all killing assays, effector/target mixtures in 96-well plates were spun down at 8 g for 2 min prior to the incubation at 37°C; cultures were stained for CD4 or CD8 (to exclude effector cells) and analyzed for active Caspase-3 levels in CellTrace-labeled target cells. Active Caspase-3⁺CellTrace⁺ cells represent apoptotic target cells. % specific killing = % apoptotic target cells in cultures with both effectors and targets - % apoptotic target cells in cultures with targets alone.