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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

Pro-inflammatory cytokines are secreted by protamine–RNA-stimulated DCs. The concentrations of pro-inflammatory cytokines in supernatants taken from CD1c+ DCs and pDCs stimulated overnight with medium or IL-3, R848, poly I:C or CpG-C, or 15 µg/ml protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl were measured by ELISA. The concentration ± SEM of IL-12p70 (a) from 9 to 11 CD1c+ DC donors, IFN-α (b) from 6–10 pDC donors, or IL-10 and TNF-α (c) from 5-11 CD1c+ DC and 4–8 pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.01), or ***(p < 0.001)
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Fig3: Pro-inflammatory cytokines are secreted by protamine–RNA-stimulated DCs. The concentrations of pro-inflammatory cytokines in supernatants taken from CD1c+ DCs and pDCs stimulated overnight with medium or IL-3, R848, poly I:C or CpG-C, or 15 µg/ml protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl were measured by ELISA. The concentration ± SEM of IL-12p70 (a) from 9 to 11 CD1c+ DC donors, IFN-α (b) from 6–10 pDC donors, or IL-10 and TNF-α (c) from 5-11 CD1c+ DC and 4–8 pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.01), or ***(p < 0.001)

Mentions: The cytokine profile of activated DCs plays an important role in the skewing of naïve T cells. We therefore compared the release of pro-inflammatory cytokines from purified DCs stimulated overnight with protamine–RNA complexes formed in water or increasing salt concentrations. All three protamine–RNA complex formations induced fivefold to tenfold more IL-12p70 than the positive control poly I:C (Fig. 3a). pDCs secreted comparable levels of IFN-α upon stimulation with the positive control CpG-C and protamine–RNA complexes (Fig. 3b). In accordance with the results in Fig. 2, the smaller protamine–RNA complexes induced the strongest response in pDCs.Fig. 3


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)

Pro-inflammatory cytokines are secreted by protamine–RNA-stimulated DCs. The concentrations of pro-inflammatory cytokines in supernatants taken from CD1c+ DCs and pDCs stimulated overnight with medium or IL-3, R848, poly I:C or CpG-C, or 15 µg/ml protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl were measured by ELISA. The concentration ± SEM of IL-12p70 (a) from 9 to 11 CD1c+ DC donors, IFN-α (b) from 6–10 pDC donors, or IL-10 and TNF-α (c) from 5-11 CD1c+ DC and 4–8 pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.01), or ***(p < 0.001)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Pro-inflammatory cytokines are secreted by protamine–RNA-stimulated DCs. The concentrations of pro-inflammatory cytokines in supernatants taken from CD1c+ DCs and pDCs stimulated overnight with medium or IL-3, R848, poly I:C or CpG-C, or 15 µg/ml protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl were measured by ELISA. The concentration ± SEM of IL-12p70 (a) from 9 to 11 CD1c+ DC donors, IFN-α (b) from 6–10 pDC donors, or IL-10 and TNF-α (c) from 5-11 CD1c+ DC and 4–8 pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.01), or ***(p < 0.001)
Mentions: The cytokine profile of activated DCs plays an important role in the skewing of naïve T cells. We therefore compared the release of pro-inflammatory cytokines from purified DCs stimulated overnight with protamine–RNA complexes formed in water or increasing salt concentrations. All three protamine–RNA complex formations induced fivefold to tenfold more IL-12p70 than the positive control poly I:C (Fig. 3a). pDCs secreted comparable levels of IFN-α upon stimulation with the positive control CpG-C and protamine–RNA complexes (Fig. 3b). In accordance with the results in Fig. 2, the smaller protamine–RNA complexes induced the strongest response in pDCs.Fig. 3

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