Cytomegalovirus (CMV) disease remains a severe complication in patients who have undergone transplantation. Viremia can be prevented and treated by the adoptive transfer of donor-derived CMV-directed T cells. To ensure long-term protection against CMV disease, it is important to transfer CMV antigen-specifi T cells that represent both the CD8 + and the CD4 + subsets. In the present study, we used as stimulators dendritic cells (DCs) that were electroporated with in vitro-transcribed 5 -capped

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... lyadenylated messenger RNA (mRNA) that encoded the CMV pp65 protein (i.e., pp65 mRNA). These DCs could efficientl activate CMVdirected CD8 + T cells, as assayed by tetramer staining, interferon-g production, and cytolytic activity. We also used DCs that were pulsed with a recombinant pp65 protein to activate CMV-directed CD4 + T cells. When DCs were comodifie with pp65 mRNA and pp65 protein, large numbers of CMV-directed CD8 + and CD4 + T cells were generated simultaneously. The approach outlined in the present study can be adapted for a clinical protocol that circumvents potential virus-related biohazards and is available to all patients independently of their human leukocyte antigen haplotype.