Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T- cell clones can restore protective immunity after stem cell transplantation . Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblast s as stimulator cells confines this approach to HCMV-seropositive donors an d requires the presence of infectious virus during the stimulation procedur e. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex: vivo for adoptive immunotherapy. Generation of HC MV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using p eptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy i n 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. T he CTLs recognized HCMV-infected autologous fibroblasts. No lysis was obser ved with either non-infected autologous or HLA-mismatched infected fibrobla sts, Staining with tetrameric HLA/peptide complexes revealed significant en richment for peptide-specific T cells of up to 28% and >90% of CD8(+) T cel ls after three and five specific stimulations respectively. In addition, th e expansion rates indicated that ex: vivo generation of >1 x 10(9) HCMV-spe cific T cells was possible after 6-7 weeks when cultures were initiated wit h 1-5 x 10(6) responder cells. Thus, the approach with peptide-pulsed DCs t o generate HCMV-specific CTLs is feasible for clinical application after al logeneic stem cell transplantation.