Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells

Abstract

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

Minor Histocompatibility Antigens on Canine Hemopoietic Progenitor Cells

“…In our animal model the incubation of DCs with CpG oligonucleotides had no effect on the uptake and presentation of KLH to autologous T cells. Finally, maturation of DCs was tried using poly(I:C), which was reported to induce maturation in human DCs (21). We observed decreased uptake and presentation of KLH by poly(I:C)-treated DCs, resulting in decreased stimulation of autologous T cells.…”

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA

“…We found that DCs activated by poly(I:C 12 U) electroporation yielded higher numbers of IFN-g-producing cells com- À melanoma cell line 938-mel were lysed. As previous studies have shown that human monocyte-derived DCs passively pulsed with poly(I:C) induce stimulation of antigen-specific CD8 + T cells, 21 we also investigated the capacity of Melan-A-A2 peptideloaded DCs activated by passive pulsing with poly (I:C 12 U) to activate naïve CD8 + T cells. Expansion of antigen-specific CD8 + T cells in vitro was quantified by tetramer analysis, 7 days after the third stimulation.…”

Efficient In Vitro Expansion of Human Immunodeficiency Virus (HIV)-Specific T-Cell Responses bygagmRNA-Electroporated Dendritic Cells from Treated and Untreated HIV Type 1-Infected Individuals

“…Recently, we (47,48,61) and others (9,15,22,28,40,54,57) have shown that transfection with mRNA is more effective than mRNA lipofection, peptide pulsing, or viral transduction to generate primary (65) and memory (57) responses. Furthermore, we demonstrated that DC from treatment-naïve HIV-1-seropositive subjects can efficiently be transfected with HIV gag and env mRNA, derived either from consensus subtype B or autologous viral or proviral HIV, and that these DC readily trigger autologous CD4 ϩ and CD8 ϩ T cells to release IFN-␥ and IL-2 in a short-term ex vivo enzyme-linked immunospot (ELISPOT) assay (60).…”