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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

Protamine–RNA complexes are well tolerated by DCs and induce upregulation of maturation markers and MHC complexes. Purified CD1c+ DCs and pDCs were cultured 18–24 h with 15, 7.5, or 1.5 µg/ml of protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. Untreated CD1c+ DCs or IL-3 treated pDCs were used as negative controls, while R848 was used as a control for TLR7/8 stimulation and poly I:C and CpG-C were used as positive controls for CD1c+ DCs or pDCs, respectively. a The cell viability was determined by flow cytometry. The mean percentage ± SEM of cells negative for live–dead marker from 7–8 CD1c+ DC donors and 6–8 pDC donors is depicted. b, c The relative expression of MHC class I and HLA-DR (b) and CD86 (c) on viable cells was calculated by normalizing the MFI values for each donor against the negative control. The fold increase ± SEM of 6–10 CD1c+ DC and pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed on raw data, comparing against negative control, and are indicated by *(p < 0.05), **(p < 0.01), ***(p < 0.001), or NS (non-significant)
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Fig2: Protamine–RNA complexes are well tolerated by DCs and induce upregulation of maturation markers and MHC complexes. Purified CD1c+ DCs and pDCs were cultured 18–24 h with 15, 7.5, or 1.5 µg/ml of protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. Untreated CD1c+ DCs or IL-3 treated pDCs were used as negative controls, while R848 was used as a control for TLR7/8 stimulation and poly I:C and CpG-C were used as positive controls for CD1c+ DCs or pDCs, respectively. a The cell viability was determined by flow cytometry. The mean percentage ± SEM of cells negative for live–dead marker from 7–8 CD1c+ DC donors and 6–8 pDC donors is depicted. b, c The relative expression of MHC class I and HLA-DR (b) and CD86 (c) on viable cells was calculated by normalizing the MFI values for each donor against the negative control. The fold increase ± SEM of 6–10 CD1c+ DC and pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed on raw data, comparing against negative control, and are indicated by *(p < 0.05), **(p < 0.01), ***(p < 0.001), or NS (non-significant)

Mentions: To evaluate the ability of RNA complexed to protamine to activate DCs, we formulated protamine–RNA complexes with different salt conditions (Fig. 2). Purified DCs were cultured overnight with concentrations ranging from 1.5 to 15 µg/ml of protamine–RNA complexes formed in either 0, 25, or 50 mM NaCl. As a control for cell stimulation, the TLR7/8 ligand R848 was used. Viability and expression of maturation markers were investigated. Unstimulated pDCs do not survive ex vivo; therefore, IL-3-treated cells were used as a negative control [28].Fig. 2


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)

Protamine–RNA complexes are well tolerated by DCs and induce upregulation of maturation markers and MHC complexes. Purified CD1c+ DCs and pDCs were cultured 18–24 h with 15, 7.5, or 1.5 µg/ml of protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. Untreated CD1c+ DCs or IL-3 treated pDCs were used as negative controls, while R848 was used as a control for TLR7/8 stimulation and poly I:C and CpG-C were used as positive controls for CD1c+ DCs or pDCs, respectively. a The cell viability was determined by flow cytometry. The mean percentage ± SEM of cells negative for live–dead marker from 7–8 CD1c+ DC donors and 6–8 pDC donors is depicted. b, c The relative expression of MHC class I and HLA-DR (b) and CD86 (c) on viable cells was calculated by normalizing the MFI values for each donor against the negative control. The fold increase ± SEM of 6–10 CD1c+ DC and pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed on raw data, comparing against negative control, and are indicated by *(p < 0.05), **(p < 0.01), ***(p < 0.001), or NS (non-significant)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Protamine–RNA complexes are well tolerated by DCs and induce upregulation of maturation markers and MHC complexes. Purified CD1c+ DCs and pDCs were cultured 18–24 h with 15, 7.5, or 1.5 µg/ml of protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. Untreated CD1c+ DCs or IL-3 treated pDCs were used as negative controls, while R848 was used as a control for TLR7/8 stimulation and poly I:C and CpG-C were used as positive controls for CD1c+ DCs or pDCs, respectively. a The cell viability was determined by flow cytometry. The mean percentage ± SEM of cells negative for live–dead marker from 7–8 CD1c+ DC donors and 6–8 pDC donors is depicted. b, c The relative expression of MHC class I and HLA-DR (b) and CD86 (c) on viable cells was calculated by normalizing the MFI values for each donor against the negative control. The fold increase ± SEM of 6–10 CD1c+ DC and pDC donors is depicted. Wilcoxon matched-pair signed-rank tests were performed on raw data, comparing against negative control, and are indicated by *(p < 0.05), **(p < 0.01), ***(p < 0.001), or NS (non-significant)
Mentions: To evaluate the ability of RNA complexed to protamine to activate DCs, we formulated protamine–RNA complexes with different salt conditions (Fig. 2). Purified DCs were cultured overnight with concentrations ranging from 1.5 to 15 µg/ml of protamine–RNA complexes formed in either 0, 25, or 50 mM NaCl. As a control for cell stimulation, the TLR7/8 ligand R848 was used. Viability and expression of maturation markers were investigated. Unstimulated pDCs do not survive ex vivo; therefore, IL-3-treated cells were used as a negative control [28].Fig. 2

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