Administration of CID eliminates iCasp9_M-expressing T cells. (A) On the day of antigen stimulation, F-Casp9_M.I.GFP-transduced CTLs were either untreated or treated with 10 nM CID. Seven days later, the response to CID was measured by flow cytometry for GFP. The percentage of transduced T cells was adjusted to 50% to allow for an accurate measurement of residual GFP⁺ cells after CID treatment. The responses to CID in unselected (top row) and GFP_high-selected CTLs (bottom row) was compared. The percentage of residual GFP⁺ cells is indicated. (B) After overnight incubation with 10 nM CID, F-Casp9_M.I.GFP_high-transduced T cells had apoptotic characteristics such as cell shrinkage and fragmentation by microscopic evaluation. (C) Staining with markers of apoptosis showed that 64% of T cells had an apoptotic phenotype (annexin V⁺, 7-AAD^-, lower right quadrant) and 32% a necrotic phenotype (annexin V⁺, 7-AAD⁺, upper right quadrant). A representative example of 3 separate experiments is shown. (D) A dose-response curve using the indicated amounts of CID (AP20187) shows the sensitivity of F-Casp9_M.I.GFP_high to CID. Survival of GFP⁺ cells is measured on day 7 after administration of the indicated amount of CID. Shown are mean and standard deviation. Similar results were obtained using AP1903, which has proven safe in a clinical trial in healthy volunteers.¹¹