Vγ2 T cell clones expressing CD56 are cytotoxic against Daudi cells. γδ T cell clones were generated expansion of a single cell by phosphoantigen in the presence of irradiated feeder cells. Expression of the Vγ2 chain of the TCR, CD56, and NKG2D were determined by flow cytometry. Figure 7A shows CD56 staining of a CD56+ Vγ2+ clone versus a CD56- Vγ2+ clone. Cytotoxicity of each clone was analyzed using a flow cytometry-based assay. Target Daudi cells were labeled with CFSE and cultured with cloned γδ cells at Effector:Target ratios of 10:1, 1:1, and 0.1:1. After 4 hours of incubation, the reactions were stopped with paraformaldehyde and PKH26-labeled Daudi cells were added to each tube. Specific lysis was calculated using the following formula: (CFSE-labeled control ÷ PKH26-labeled control) - (CFSE-labeled target ÷ PKH26-labeled reference) × 100. Assays were performed with triplicate samples and standard deviation was < 15% of the mean. The specific lysis values at E:T of 10:1 are listed for each clone. Cytotoxicity panels for 3 representative clones showing high, medium, and low/no lysis are also shown.