Rapid generation of combined CMV-specific CD4+and CD8+T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4+and CD8+CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-γ (IFN-γ) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 × 108combined CD4+and CD8+CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-γ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4+and CD8+T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4+and CD8+T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4+and CD8+CMV-specific T cells under conditions mimicking good manufacturing practice. (Blood. 2004; 103:3565-3572)