CMV-specific CIK cells maintain their Ag specificity and acquire HLA-unrestricted cytotoxicity. (A) Peripheral blood lymphocytes from HLA-A*0201 seropositive donors were expanded for 21 days in the presence of IFN-γ, OKT3, and IL-2, and the percentage of anti–CMV-specific cells was measured at the end of culture by tetramer binding. CMV-specific CIK cells were isolated by immunomagnetic selection and expanded onto irradiated allogeneic feeder layer for further 2 weeks. At the end of the culture, CD56⁺ CMV-specific CIK cells were immunoselected. The dot plots of CD3-CD56 and HLA-A*0201/pp65_495-503 tetramer-CD8 staining, obtained during a representative experiment, are shown. (B) Cytotoxic activity of expanded CMV-specific CIK cells was determined on unpulsed, CMV-pulsed, EBV-pulsed autologous T–PHA-induced blasts and on unpulsed allogeneic T–PHA-induced blasts in calcein-release assays. The data were obtained from 3 independent experiments and were analyzed by Student t test; ***P < .005. (C-D) MHC-unrestricted cytotoxic activity of expanded CMV-specific CIK cells against a panel of malignant cell lines (C) or against freshly isolated myeloid leukemic cells (D) was determined in calcein-release assays. The results show the mean ± SD values of 3 independent experiments.