Generation of lymphoblastoid cell lines and autologous EBV-specific CTLs from patients with persisting active EBV infection.

(A) The time required for the establishment of LCLs, using the B95-8 laboratory strain of EBV, from the 8 patients affected by CAEBV infection, was comparable to that required by a series of 36 healthy donors. (B) The kinetics of each CTL line generated from the 8 patients affected by CAEBV infection using weekly stimulations with autologous LCLs in the presence of IL-2. (C) The killing activity of the EBV-specific CTLs, as assessed in a standard chromium release assay. HBS-2 target cell lines (■), mismatched LCLs (*), and autologous LCLs (●) were labeled with ⁵¹Cr and incubated for 4 hours with CTL at the E/T ratios indicated. Killing of autologous LCLs was significantly higher compared with mismatched LCL and HSB-2, a LAK-sensitive EBV-negative T-cell lymphoma that provides a measure of lymphokine-activated killer cell–mediated killing. Means of the percent specific chromium release from target cells ± SD of the 8 CTL lines are presented. (D) The perforin (left) and granzyme A (right) expression of 2 representative CTL lines. CTLs were stained for intracellular perforin or granzyme using PE-labeled antiperforin or antigranzyme antibodies (thick line) versus isotype control (thin line) and analyzed by flow cytometry. Upper and lower panels represent the staining of a representative CD8⁺ CTL line and of the CD3⁺ TCRγδ⁺ cells generated from patient 2, respectively.