Dendritic cell subsets generated from CD34+ hematopoietic progenitors can be transfected with mRNA and induce antigen-specific cytotoxic T cell responses.

Human dendritic cells (DCs) generated in culture from either monocytes or CD34+ hematopoietic progenitor cells (CD34-HPCs) have been used in cancer immunotherapy protocols with encouraging results. Yet an optimal strategy for the delivery of antigen(s) to DCs still remains to be established. Recent studies demonstrated the feasibility of mRNA transfection to load monocyte-derived DCs. It is not known, however, whether DCs derived by culturing CD34-HPC with GM-CSF and TNF-alpha for 9 days (CD34-DCs) can be efficiently transduced with mRNA. Here we show that clinical-grade CD34-DCs generated after 8 days of culture can be transfected with mRNA without significant alteration of cell viability. About 90% of cells transfected with GFP-RNA express GFP 24 h post-transfection. Remarkably, transfected CD34-DCs retain high levels of GFP expression for at least 14 days. CD34-DCs transfected with Flu-MP RNA were highly efficient in inducing the proliferation of Flu-MP-specific CD8+ T cells as measured by tetramer staining. Furthermore, the stimulated CD8+ T cells produced IFN-gamma upon antigenic stimulation and were able to kill targets pulsed with Flu-MP peptide. Both DC subsets in CD34-DCs, CD1a+-DC (Langerhans cells) and CD14+-DC (interstitial DC), were equally transfected with GFP-RNA, and yielded Flu-specific cytotoxic T cells upon transfection with Flu-MP RNA. Thus, RNA can be used to deliver antigens to two distinct myeloid DC subsets in CD34-DC cultures.