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

Sköld AE, van Beek JJ, Sittig SP, Bakdash G, Tel J, Schreibelt G, de Vries IJ - Cancer Immunol. Immunother. (2015)

Bottom Line: We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus

Concentration of NaCl determines size, but not charge, when forming protamine–RNA complexes. protamine–RNA complexes (pR) were formed in water, 25 mM NaCl, or 50 mM NaCl, particle size was evaluated by dynamic light scattering (a, b), and the zeta potential was investigated to determine the charge of the complexes (c)
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Fig1: Concentration of NaCl determines size, but not charge, when forming protamine–RNA complexes. protamine–RNA complexes (pR) were formed in water, 25 mM NaCl, or 50 mM NaCl, particle size was evaluated by dynamic light scattering (a, b), and the zeta potential was investigated to determine the charge of the complexes (c)

Mentions: It has long been known that negatively charged RNA has the ability to bind strongly to substances with positive charge [24]. A protein that has been used to complex nucleic acids is the polybasic protein protamine [17, 21, 25]. The physical characteristics of complexes formed when mixing anionic RNA to cationic protamine is dependent on the ratio between the components and on ionic content [26]. Since CD1c+ DCs and pDCs differ in their ability to take up and respond to particles [27] and express different TLRs [4], we produced protamine–RNA complexes in a ratio of 2:1 in 0, 25, or 50 mM NaCl and investigated their size by dynamic light scattering (Fig. 1a–b). The complexes formed in water or low salt concentration were <200 nm in diameter, while higher salt concentration formed complexes of >500 nm. The particle charge remained relatively constant between the formulations, ranging between 30 and 40 mV (Fig. 1c).Fig. 1


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

Sköld AE, van Beek JJ, Sittig SP, Bakdash G, Tel J, Schreibelt G, de Vries IJ - Cancer Immunol. Immunother. (2015)

Concentration of NaCl determines size, but not charge, when forming protamine–RNA complexes. protamine–RNA complexes (pR) were formed in water, 25 mM NaCl, or 50 mM NaCl, particle size was evaluated by dynamic light scattering (a, b), and the zeta potential was investigated to determine the charge of the complexes (c)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612318&req=5

Fig1: Concentration of NaCl determines size, but not charge, when forming protamine–RNA complexes. protamine–RNA complexes (pR) were formed in water, 25 mM NaCl, or 50 mM NaCl, particle size was evaluated by dynamic light scattering (a, b), and the zeta potential was investigated to determine the charge of the complexes (c)
Mentions: It has long been known that negatively charged RNA has the ability to bind strongly to substances with positive charge [24]. A protein that has been used to complex nucleic acids is the polybasic protein protamine [17, 21, 25]. The physical characteristics of complexes formed when mixing anionic RNA to cationic protamine is dependent on the ratio between the components and on ionic content [26]. Since CD1c+ DCs and pDCs differ in their ability to take up and respond to particles [27] and express different TLRs [4], we produced protamine–RNA complexes in a ratio of 2:1 in 0, 25, or 50 mM NaCl and investigated their size by dynamic light scattering (Fig. 1a–b). The complexes formed in water or low salt concentration were <200 nm in diameter, while higher salt concentration formed complexes of >500 nm. The particle charge remained relatively constant between the formulations, ranging between 30 and 40 mV (Fig. 1c).Fig. 1

Bottom Line: We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus