Ebv-specific t-cell lines for adoptive transfer to pmrd allograft recipients

We have developed an efficient method for generating Epstein-Barr virus (EBV)-specific T-cell lines using clinical-grade reagents for prophylactic use against donor derived lymphoproliferative disease (LPD) in patients receiving an allograft from a partially matched related donor (PMRD). In a cohort of 249 consecutive patients who received a PMRD-BMT, 13 developed EBV-LPD. The probability of developing EBV-LPD was 14% at two years. Conventional therapies were unsuccessful. Donor PBMCs were transformed with EBV virus and utilized to stimulate donor PBLs in the presence of low dose IL-2. In a period of 5 weeks, we were able to culture 60–70 × 106 cells, which is sufficient for adoptive immunotherapy. The EBV-specific T-cells showed great efficacy & specificity eradicating donor EBV-infected cells in-vitro. We tested our EBV-specific T-cell lines in cytolytic assays and found EBV target cell killing to consistently be in the range of 65–95%, while killing of patient PHA-transformed blasts was negligible. As a control, we utilized K562 cells, and found killing to be in the range of 15–25% at an E:T ratio of 50:1. The immunophenotype was predominately CD3+/8+, CD45RO+ (memory), HLA-DR+, CD62L- (activation), CD49d+ (ability to cross endothelium), and CD57- (terminal differentiation). Intracellular cytokine staining showed a Th1 profile with TNF-α and IFN-γ positivity in the CD8+ population. We will utilize these EBV-specific T-cells prophylactically for adoptive transfer in PMRD recipients.