Activation of the iCasp-9 suicide gene abrogates cytokine production and long-term expansion of IL-2 and IL-15 transgenic CTLs. EBV-CTLs transduced with either iC.ΔCD34/IL-2v or iC.ΔCD34/IL-15v vectors were plated at 106 cells/well and incubated with or without the CID AP20187 (50 nM). Panel A illustrates the production of transgenic cytokines IL-2 or IL-15 by iC.ΔCD34/IL-2v and iC.ΔCD34/IL-15v EBV-CTLs before and after exposure to the CID. Neither IL-2 nor IL-15 cytokines could be detected in the supernatants from CTLs treated with the CID. Data represent the mean (± SD) of 4 experiments. Panel B illustrates the long-term expansion of EBV-CTLs transduced with either iC.ΔCD34/IL-2v or iC.ΔCD34/IL-15v vectors and stimulated once a week with autologous LCLs. Viable cells were counted by trypan blue exclusion once a week before EBV-LCL restimulation. Exposure to a single dose of CID (50 nM) ablated CTL expansion, while nonexposed CTLs continued to expand. Data represent mean (± SD) of 4 experiments. Panel C illustrates a representative experiment in which IL-2 and IL-15 transgenic CTLs obtained 24 hours after CID exposure were sorted based on the expression of Annexin-V. These CTLs were then cultured without any further addition of CID and stimulated with EBV-LCLs. Annexin-V/7ADD staining showed that these cells progressed to a late stage of apoptosis/necrosis (Annexin-V+/7ADD+) by days 7 to 9. Panel D shows that CID induced elimination of CD34+ cells for IL-2 or IL-15 transgenic CTLs even in their resting phase, 7 days after the last antigen stimulation.
