Enhancing the T-cell Stimulatory Capacity of Human Dendritic Cells by Co-electroporation With CD40L, CD70 and Constitutively Active TLR4 Encoding mRNA

Abstract

The effectiveness of the dendritic cell (DC) vaccination protocols that are currently in use could be improved by providing the DCs with a more potent maturation signal. We therefore investigated whether the T-cell stimulatory capacity of human monocyte-derived DCs could be increased by co-electroporation with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) encoding mRNA. We show that immature DCs electroporated with CD40L and/or caTLR4 mRNA, but not those electroporated with CD70 mRNA, acquire a mature phenotype along with an enhanced secretion of several cytokines/chemokines. Moreover, these DCs are very potent in inducing naive CD4(+) T cells to differentiate into interferon-gamma (IFN-gamma)-secreting type 1 T helper (Th1) cells. Further, we assessed the capacity of the electroporated DCs to activate naive HLA-A2-restricted MelanA-specific CD8(+) T cells without the addition of any exogenous cytokines. When all three molecules were combined, a >500-fold increase in MelanA-specific CD8(+) T cells was observed when compared with immature DCs, and a >200-fold increase when compared with cytokine cocktail-matured DCs. In correlation, we found a marked increase in cytolytic and IFN-gamma/tumor necrosis factor-alpha (TNF-alpha) secreting CD8(+) T cells. Our data indicate that immature DCs genetically modified to express stimulating molecules can induce tumor antigen-specific T cells in vitro and could prove to be a significant improvement over DCs matured with the methods currently in use.

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy

“…This theory is supported by the observations of Chen et al, who also reported a drop in transfection efficiency 2 mRNAs were coelectroporated in DCs compared to electroporation of each of these mRNAs alone [26]. This was also reported by Bonehill and colleagues who reported that upon electroporation of DCs with TriMix the number of mRNA-expressing DCs was always slightly decreased when two or three mRNAs were co-electroporated, in comparison to the electroporation of each mRNA alone [16]. This might indicate that there is a limit to the amount of mRNA that can be transferred into the cells.…”

“…In accordance to our previous reports, mRNA sonoporation as such, without incorporation of additional immune stimulants, readily results in a distinct up-regulation of maturation marker expression at the DC surface. This in contrast to mRNA electroporation, which is considered the golden standard for mRNA transfer into DCs in vitro, where no effects on the DC maturation status were observed [16,21]. Two possible mechanisms might explain this sonoporation-induced maturation.…”

“…The vectors pGEM-Ii80tOVA, pST1-eGFP-bis, pST1-caTLR4, pST1-mouse CD40-L and pST1-mouse CD70 were previously described [16,18,21]. Before in vitro transcription, pGEM and pST1 vectors were linearized with the restriction enzymes Spe I and Sap I, respectively.…”

“…Therefore, we performed sonoporations with antigen mRNA and TriMix mRNA. TriMix is a mixture of 3 mRNAs, encoding CD40-ligand, a constitutively active form of TLR4 and CD70 (a co-stimulatory molecule required for effective CD8 + T cell priming) [16]. Co-delivery of these 3 nucleic acid sequences was already shown to modulate the DCs' functionality, resulting in APCs that display a more mature, T cell activating phenotype [17].…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Nevertheless, studies in the field of cancer immunotherapy have demonstrated that neither sono-nor electroporation results in strong activation of immune cells. In fact, both techniques require additional stimulation with adjuvants to induce therapeutically beneficial immune responses [79,80]. This might indicate that cytosolic PRRs are less immunogenic than endosomal TLRs.…”