Protamine-stabilized RNA as an ex vivo stimulant of primary human dendritic cell subsets.
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.
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.|Department of Oncology-Pathology, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden.
Annette E
http://orcid.org/0000-0002-5309-8860
AE
Sköld
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Jasper J P
JJ
van Beek
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Simone P
SP
Sittig
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Ghaith
G
Bakdash
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Jurjen
J
Tel
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
Gerty
G
Schreibelt
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands. Jolanda.deVries@radboudumc.nl.|Department of Medical Oncology, Radboud University Medical Centre, Geert Grooteplein 26, Route 278, 6525 GA, Nijmegen, The Netherlands. Jolanda.deVries@radboudumc.nl.
I Jolanda M
IJ
de Vries
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000145:classification / Q000276:immunology*; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D008213:Lymphocyte Activation; D011479:Protamines / Q000494:pharmacology*; D012313:RNA / Q000494:pharmacology*; D020871:RNA Stability; D012965:Sodium Chloride / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000502:physiology
D000276:Adjuvants, Immunologic; D016207:Cytokines; D011479:Protamines; D051193:Toll-Like Receptors; D012965:Sodium Chloride; D012313:RNA; D007371:Interferon-gamma
Adjuvants; GMP; Immunotherapy; TLR; mDC; pDC