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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 fibroblasts as stimulator cells confines this approach to HCMV‐seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV‐specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV‐specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide‐pulsed dendritic cells as antigen‐presenting cells. HCMV‐specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV‐seropositive and 2 out of 11 HCMV‐seronegative donors. The CTLs recognized HCMV‐infected autologous fibroblasts. No lysis was observed with either non‐infected autologous or HLA‐mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide‐specific T cells of up to 28% and > 90% of CD8+ T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 × 109 HCMV‐specific T cells was possible after 6–7 weeks when cultures were initiated with 1–5 × 106 responder cells. Thus, the approach with peptide‐pulsed DCs to generate HCMV‐specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.