Induction of cytotoxicity and redirected cytolysis correlates with the expression of DAP10. T cells were activated and expanded as described in “Materials and methods.” Cells were pre-enriched using MACS on day 0 and then cultured until 24 to 48 hours prior to use (at various time points in culture days 0, 3, 7, 10, 14, and 21) when they were purified by FACS. Cells were rested for 24 to 48 hours in cRPMI and IL-2 (300 U/mL) and then used for either cytotoxicity assays (A) or redirected cytolysis assays (B), or freshly sorted cells were used to harvest RNA for RT-PCR (C). For cytotoxicity assays (A), targets were the U266 cell line. Either no antibody was used (▪) or isotype control antibody (▨) or anti-NKG2D antibody (□) was added. For redirected cytolysis experiments (B), CD8⁺ T cells at various times in culture were combined with P815 cells alone (▦) or with P815 cells that were incubated with isotype control antibodies (□), anti-NKG2D (▨), anti-CD3 ( ) or anti-NKG2D and anti-CD3 (▪). For panels A and B, antibodies were used at 20 μg/mL, E/T = 10:1, and error bars indicate standard deviation. For PCR experiments (C), DAP10-specific primers were used and the amplified product was separated on a 0.9% agarose gel. (D) For Western blotting experiments, purified CD8⁺ cells were isolated at various times in culture and extracts (50 μg) were separated on a 15% acrylamide gel, then transferred to PVDF membranes, and probed using either a DAP10 or γ-tubulin–specific antibody (loading control). Results are representative of 2 or more individual experiments.