siRNA4⁺ EBV-CTLs retain their immunophenotype, cytotoxic activity, and EBV-antigen specificity. (A) The immunophenotype of NT and irr-siRNA⁺ EBV-CTLs and of siRNA4⁺ EBV-CTLs cultured in the presence of FK506. Means ± SD are shown for the 6 CTL lines. No significant phenotypic differences were observed for siRNA4⁺ CTLs expanded in the presence of FK506. (B) The results of a standard ⁵¹Cr release assay of NT and irr-siRNA⁺ EBV-CTLs and of irr-siRNA⁺ and siRNA4⁺ EBV-CTLs cultured in the presence of FK506. Targets were K562, autologous LCLs, and allogeneic LCLs. Shown is the CTL/tumor cell ratio of 20:1. Bars represent the mean ± SD of the EBV-CTLs generated from 6 donors. No significant differences in cytotoxic activity were observed for siRNA4⁺ CTLs versus NT or irr-siRNA⁺ CTLs. (C) The frequencies of multimers recognizing latent EBV-associated antigens (EBNA3B-AVF and EBNA3B-IVT) in NT (left plots) and irr-siRNA⁺ (middle plots) EBV-CTLs and in siRNA4⁺ (right plots) EBV-CTLs cultured in the presence of FK506 for 3 weeks in 1 representative donor. (D) The frequency of CTLs responding to the indicated EBV-specific peptides assessed by IFNγ ELIspot assay in another representative donor.