In vitro generation of Epstein-Barr virus-specific cytotoxic T cells in patients receiving haplo-identical allogeneic stem cell transplantation

Use of a partially mismatched related donor (PMRD) is an option for patient s who require allogeneic transplantation but do not have a matched sibling or unrelated donor. Epstein-Barr virus (EBV)-induced lymphoma is a major ca use of mortality after PMRD transplantation. In this study, we present a cl inical grade Culture system for donor-derived EBV-specific cytotoxic T cell s (CTLs) that do not recognize haplo-identical recipient cells. The EBV-spe cific CTLs were tested for cytolytic specificity and other functional prope rties, including ability to transgress into tissues, propensity for apoptos is, degree of clonality, stability of dominant T-cell clones, and Tc and Th phenotypes. The EBV-specific CTLs were routinely expanded to greater than 80 x 10(6) over a period of 5 weeks, which is sufficient for clinical appli cation. A CD8(+) phenotype predominated, and the CTLs were highly specific for donor lymphoblastoid cell lines (LCLs) without killing of recipient tar gets or K562. V beta spectratyping showed an oligoclonal population that wa s stable on prolonged culture. The EBV-specific CTLs were activated (D-rela ted human leukocyte antigen [HLA-DR+], L-selectin(+/-)) and of memory pheno type (CD45RO(+)). Expression of the integrin VLA-4 suggested that these CTL s could adhere to endothelium and migrate into tissues. The Bcl-2 message w as upregulated, which may protect the CTLs from the apoptosis. The first de monstration of overexpression of bcl-2 in human memory CTLs. In addition, w e show that lymphoblastoid cell lines used to generate CTLs are readily gen etically modified with recombinant lentivirus, indicating that genetically engineered antigen presentation is feasible.