Protamine-stabilized RNA as an ex vivo stimulant of primary human dendritic cell subsets

Abstract

Dendritic cells (DCs) are key in connecting innate and adaptive immunity. Their potential in inducing specific immune responses has made them interesting targets for immunotherapeutic approaches. Our research group was the first to exploit the naturally occurring myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in therapeutic vaccination trials against melanoma. To develop primary DC subsets as an optimal vaccine, the identification of a clinically applicable adjuvant activating both subsets is required. Although the expression of pathogen recognition receptors differs distinctly between the DC subsets, both pDCs and mDCs can respond to single-stranded RNA (ssRNA) via Toll-like receptors 7 and 8, respectively. Since ssRNA is easily degraded by RNases, we stabilized anionic RNA by complexing it with the positively charged protein protamine. This leads to the formation of protamine–RNA complexes with varying features depending on ionic content. We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets. Both mDCs and pDCs upregulated maturation markers and produced pro-inflammatory cytokines in a dose-dependent way to the protamine–RNA complexes. This was dependent on endosomal acidification and correlated partly with the uptake of protamine–RNA complexes. Furthermore, both DC subsets induced T cell proliferation and IFN gamma secretion in a beneficial ratio to IL-10. These results indicate that protamine–RNA complexes can be used to stimulate human mDC and pDC ex vivo for use in immunotherapeutic settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1746-9) contains supplementary material, which is available to authorized users.

Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy

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Human blood myeloid and plasmacytoid dendritic cells cross activate each other and synergize in inducing NK cell cytotoxicity

“…Alternatively, both DC subsets can be stimulated with a single TLR agonist. Our group recently established a clinical-grade stimulus that can activate both CD1c + DCs and pDCs in the form of stabilized RNA complexes 43 . The use of such a stimulus would be optimal to exploit the advantages of both DC subsets in a clinical setting.…”

Human CD1c+ DCs are critical cellular mediators of immune responses induced by immunogenic cell death

“…The most effective response was observed for CD1c + DCs. Notably, CD1c + DCs were the only DC subset that, in response to interaction with platinum-treated tumor cells, secreted TNF-α and IL-10, cytokines typically induced upon exposure to different maturation stimuli 49,50 . This myeloid DC population was capable of engulfing platinum-treated tumor cells, responding to activatory signals and inducing T cell proliferation.…”