Simultaneous ex vivo generation of cytomegalovirus pp65 and Epstein-Barr virus specific cytotoxic T-lymphocyte from human umbilical cord blood

AIM: To investigate the possibility of simultaneously ex vivo generating cytomegalovirus (CMV) pp65 and Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) from human umbilical cord blood (CB). METHODS: Mononuclear cell derived from CB (CBMC) was used to construct EBV-transformed B-lymphoblastoid cell lines (BLCL). Then BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. CBMC from the same CB donor were stimulated with pp65-expressing BLCL (BLCLpp65) weekly for 5~6 weeks. Chromium release assays (CRA) were performed to detect the specific cytotoxicity of the CTL against EBV and CMV. RESULTS: Western blot analysis and immunocytochemistry confirmed that BLCLpp65 could simultaneously express CMVpp65 and EBV antigen. CRA results showed that the generated CTL possessed specific cytotoxic against EBV and CMV, and the cytotoxicity was mediated by CD₈⁺ CTL. CONCLUSION: BLCLpp65 can be used as antigen-presenting cells to stimulate expansion of EBV and CMV specific CTL simultaneously from the predominantly native T cell population in CB.