Antigen dependence, phenotype, and cytotoxicity of EBV-CTLs are unaffected by transduction with CNb30. (A) Transduced and untransduced EBV-CTLs were grown as normal for 4 stimulations. Proliferation was monitored from the day of stimulation 5. Mean and SEM shown (n = 5). (Ai) Growth of CTLs continuing to receive weekly stimulation with autologous LCLs and exogenous IL-2. (Aii) CTLs that receive no LCL stimulation do not proliferate, even with continuing supplement of 100 U/mL IL-2. (Aiii) CTLs that do not receive LCL stimulation or IL-2 do not proliferate. There is no difference in proliferation between transduced and untransduced CTL lines under any of these conditions. (B) EBV-CTLs were assessed for expression of surface markers 4 days after their fifth stimulation with autologous LCLs. Mean and SEM shown (n = 5). (Bi) Percentage of expression of CD3, CD4, CD8, CD16/56, and CD25 in untransduced and CNb30-transduced lines. (Bii) Distribution of memory subsets in untransduced and CNb30-transduced lines. CM indicates central memory (CD45RO⁺, CD62L⁺); EM, effector memory (CD45RO⁺, CD62L⁻); naive (CD45RO⁻, CD62L⁺); and TD, terminally differentiated (CD45RO⁻, CD62L⁻). (C) A ⁵¹Cr release cytotoxicity assay was performed to assess cytotoxicity against autologous or allogeneic LCL targets of EBV-CTL lines cultured for 2 to 3 weeks in the presence or absence of CN inhibitors (n = 5). Mean and SEM cytotoxicity at an effector-target ratio of 30:1 are shown. (Ci) After culture in the absence of CN inhibitors. (Cii) After 2 to 3 weeks of culture in 10 ng/mL FK506. (Ciii) After 2 to 3 weeks of culture in 200 ng/mL CsA. No effect of CN inhibitors was detected on cytotoxicity of transduced or untransduced EBV-CTLs. CN inhibitors were present during the cytotoxicity assay in panels Cii and Ciii.