Activation of the iCasp-9 suicide gene significantly eliminates IL-2 and IL-15 transgenic CTLs. EBV-CTLs transduced with either ΔCD34v or iC.ΔCD34/IL-2v or iC.ΔCD34/IL-15v vectors were plated at 10⁶ cells/well and incubated with or without CID AP20187 at 50 nM. Twenty-four hours later cells were collected, stained with CD34-PE antibody, and the percentage of residual transgenic CTLs was evaluated by FACS analysis. Significant reduction of CD34⁺ cells after incubation with CID was obtained only for EBV-CTLs transduced with either iC.ΔCD34/IL-2v or iC.ΔCD34/IL-15 vectors incorporating the iCasp-9 gene. Panel A illustrates ΔCD34 expression in a representative experiment. Panel B summarizes the effects of the CID on 4 different EBV-CTL lines transduced with either iC.ΔCD34/IL-2v, iC.ΔCD34/IL-15v, or ΔCD34v vectors. The y axis represents the mean (± SD) for CD34⁺ CTLs in the CTL lines before or after incubation with CID. The percentage of CD34⁺ cells remained unchanged in control CTLs transduced with the ΔCD34v vector lacking the suicide gene. In contrast, a significant reduction in the percentage of CD34⁺ CTLs was observed for CTLs transduced with either iC.ΔCD34/IL-2v or iC.ΔCD34/IL-15v vectors. Panel C shows that the elimination of transgenic CTLs was mediated by induction of apoptosis as assessed by Annexin-V/7-AAD staining. Panel D shows that the induction of apoptosis/necrosis by CID was dose dependent. CTLs transgenic for IL-2 or IL-15 and selected using CD34 magnetic beads were incubated with different doses of CID, and then 24 hours later the induction of apoptosis/necrosis was evaluated by staining with Annexin-V/7-ADD and FACS analysis.