Selection of EBV-CTLs with a silenced FKBP12 occurs in the presence of FK506. EBV-CTLs were transduced with either the pSUPER.eGFP vector encoding the irrelevant siRNA (irr-siRNA) or pSUPER.eGFP encoding siRNA4. (A) GFP expression (as measure of transduction) of irr-siRNA⁺ (top plots) and siRNA4⁺ (bottom plots) EBV-CTLs stimulated with irradiated EBV-LCLs in the presence of IL-2 (20 U/mL) and FK506 (5 ng/mL) in a representative donor. The plots show a progressive increase of GFP⁺ cells for siRNA4⁺ CTLs, whereas the percentage of GFP⁺ cells is stable for irr-siRNA⁺ CTLs. (B) The data for the percentage of GFP⁺ cells for 6 CTL lines. Bars represent mean ± SD. Shown is the percentage of GFP⁺ cells after the 4th, 7th, and 11th stimulations, which significantly increased over time only for siRNA4⁺ EBV-CTLs, whereas it remained stable for irr-siRNA⁺ EBV-CTLs. (C) The MFI of GFP⁺ cells significantly increased in siRNA4⁺ EBV-CTLs compared with irr-siRNA⁺ cells when cells were maintained in culture in the presence of FK506. Bars represent mean ± SD for 6 CTL lines. (D) The expression of FKBP12 in NT and irr-siRNA⁺ EBV-CTLs and in siRNA4⁺ EBV-CTLs after 5 weeks in culture in the absence or in the presence of FK506, as assessed by WB. FKBP12 is completely undetectable by WB in siRNA4⁺ EBV-CTLs cultured in the presence of FK506.