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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

T cell proliferation and IFN-γ production is induced by protamine–RNA-stimulated DCs. DCs were cultured with medium alone or IL-3, R848, or protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. CFSE-labeled allogenic PBLs or T cells were added after overnight culture. a The dilution of CFSE-labeled PBLs was measured on CD3+CD11c−BDCA2− cells by flow cytometry on day 5. Cultured, unstimulated PBLs were used as reference. The mean percentage proliferating cells ± SEM of 6 CD1c+ DC and pDC donors is depicted. b The expression of IFN-γ and IL-10 in day 2 DC:PBL co-cultures was measured with ELISA. Mean concentration ± SEM from 5 to 6 CD1c+ DC and pDC donors is depicted. c, d Intracellular IFN-γ was measured in CD3+CD11c−BDCA2− T cells cultured for 5 days either alone or with DCs stimulated with medium or IL-3, R848, or pR complexes. Results are depicted as the mean percentage IFN-γ+ cells ± SEM from 6 CD1c+ DC and pDC donors (c) or as a representative figure (d). Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.05), or NS (non-significant)
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Fig5: T cell proliferation and IFN-γ production is induced by protamine–RNA-stimulated DCs. DCs were cultured with medium alone or IL-3, R848, or protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. CFSE-labeled allogenic PBLs or T cells were added after overnight culture. a The dilution of CFSE-labeled PBLs was measured on CD3+CD11c−BDCA2− cells by flow cytometry on day 5. Cultured, unstimulated PBLs were used as reference. The mean percentage proliferating cells ± SEM of 6 CD1c+ DC and pDC donors is depicted. b The expression of IFN-γ and IL-10 in day 2 DC:PBL co-cultures was measured with ELISA. Mean concentration ± SEM from 5 to 6 CD1c+ DC and pDC donors is depicted. c, d Intracellular IFN-γ was measured in CD3+CD11c−BDCA2− T cells cultured for 5 days either alone or with DCs stimulated with medium or IL-3, R848, or pR complexes. Results are depicted as the mean percentage IFN-γ+ cells ± SEM from 6 CD1c+ DC and pDC donors (c) or as a representative figure (d). Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.05), or NS (non-significant)

Mentions: After characterizing the effects of protamine–RNA on primary DCs, the subsequent T cell response induced by activated DCs was investigated. Proliferation of CFSE-labeled PBLs was measured in an allogeneic setting. The percentage of dividing CD3+ cells was determined 5 days after addition of protamine–RNA-activated DCs. As a positive control for proliferation, SEB was added (data not shown). R848- and protamine–RNA-treated CD1c+ DCs induced comparable proliferation of T cells, which was significantly higher than untreated DCs (Fig. 5a). Stimulated pDCs also induced a proliferative response in T cells comparable to IL-3-treated cells (Fig. 5a).Fig. 5


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)

T cell proliferation and IFN-γ production is induced by protamine–RNA-stimulated DCs. DCs were cultured with medium alone or IL-3, R848, or protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. CFSE-labeled allogenic PBLs or T cells were added after overnight culture. a The dilution of CFSE-labeled PBLs was measured on CD3+CD11c−BDCA2− cells by flow cytometry on day 5. Cultured, unstimulated PBLs were used as reference. The mean percentage proliferating cells ± SEM of 6 CD1c+ DC and pDC donors is depicted. b The expression of IFN-γ and IL-10 in day 2 DC:PBL co-cultures was measured with ELISA. Mean concentration ± SEM from 5 to 6 CD1c+ DC and pDC donors is depicted. c, d Intracellular IFN-γ was measured in CD3+CD11c−BDCA2− T cells cultured for 5 days either alone or with DCs stimulated with medium or IL-3, R848, or pR complexes. Results are depicted as the mean percentage IFN-γ+ cells ± SEM from 6 CD1c+ DC and pDC donors (c) or as a representative figure (d). Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.05), or NS (non-significant)
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Fig5: T cell proliferation and IFN-γ production is induced by protamine–RNA-stimulated DCs. DCs were cultured with medium alone or IL-3, R848, or protamine–RNA complexes (pR) formed in 0, 25, or 50 mM NaCl. CFSE-labeled allogenic PBLs or T cells were added after overnight culture. a The dilution of CFSE-labeled PBLs was measured on CD3+CD11c−BDCA2− cells by flow cytometry on day 5. Cultured, unstimulated PBLs were used as reference. The mean percentage proliferating cells ± SEM of 6 CD1c+ DC and pDC donors is depicted. b The expression of IFN-γ and IL-10 in day 2 DC:PBL co-cultures was measured with ELISA. Mean concentration ± SEM from 5 to 6 CD1c+ DC and pDC donors is depicted. c, d Intracellular IFN-γ was measured in CD3+CD11c−BDCA2− T cells cultured for 5 days either alone or with DCs stimulated with medium or IL-3, R848, or pR complexes. Results are depicted as the mean percentage IFN-γ+ cells ± SEM from 6 CD1c+ DC and pDC donors (c) or as a representative figure (d). Wilcoxon matched-pair signed-rank tests were performed between indicated groups and are indicated by *(p < 0.05), **(p < 0.05), or NS (non-significant)
Mentions: After characterizing the effects of protamine–RNA on primary DCs, the subsequent T cell response induced by activated DCs was investigated. Proliferation of CFSE-labeled PBLs was measured in an allogeneic setting. The percentage of dividing CD3+ cells was determined 5 days after addition of protamine–RNA-activated DCs. As a positive control for proliferation, SEB was added (data not shown). R848- and protamine–RNA-treated CD1c+ DCs induced comparable proliferation of T cells, which was significantly higher than untreated DCs (Fig. 5a). Stimulated pDCs also induced a proliferative response in T cells comparable to IL-3-treated cells (Fig. 5a).Fig. 5

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