Functional CD4+ and CD8+ T-Cell Responses Induced by Autologous Mitomycin C Treated Epstein–Barr Virus Transformed Lymphoblastoid Cell Lines

Epstein–Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4+ and CD8+ T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4+ and (granzyme B positive) CD8+ T-cells rosetting with McLCL. Granzymes A and B, IFN-γ, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4+ and CD8+ T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.