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Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
Bright expression of CD91 identifies highly activated human dendritic cells that can be expanded by defensins
Monica Cappelletti1, Pietro Presicce, Francesca Calcaterra
1Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy; Laboratory of Clinical and Experimental Immunology, Humanitas Clinical and Research Centre, Rozzano, Italy.
Insights
Defensins, via the CD91 receptor on dendritic cells (DCs), promote immune cell activation. This suggests targeting the CD91/defensin pathway could enhance anti-infective and anti-tumour immunity.
Area of Science:
- Immunology
- Cell Biology
- Dendritic Cell Biology
Background:
- CD91 is a scavenger receptor on immune cells; its ligands, defensins, are involved in immune responses.
- CD91 is expressed on human monocyte-derived dendritic cells (moDCs), and defensins activate these cells in vitro.
- Two moDC subsets, CD91(dim) and CD91(bright), exhibit differential CD91 expression levels.
Purpose of the Study:
- To investigate the role of CD91 expression levels on moDC subsets.
- To determine the effect of defensins and lipopolysaccharide (LPS) on moDC subset frequency and activation.
- To explore the potential of the CD91/defensin axis as a therapeutic strategy.
Main Methods:
- Flow cytometry analysis of CD91 expression on moDC subsets.
- In vitro stimulation of moDCs with recombinant human neutrophil peptide-1 (rHNP-1), recombinant human β defensin-1 (rHBD-1), and lipopolysaccharide (LPS).
- Assessment of activation and maturation markers (CD80, CD40, CD83, HLA-DR) following stimulation.
Main Results:
- CD91(bright) moDCs, a minor subset, displayed higher activation and maturation markers than CD91(dim) moDCs.
- rHNP-1 and rHBD-1 increased CD91(bright) moDC frequency by ~50%, while LPS decreased it by ~35%.
- Defensins upregulated moDC activation markers, with CD91(bright) moDCs maintaining their status, unlike LPS-treated cells.
Conclusions:
- Defensins promote differentiation into activated CD91(bright) dendritic cells.
- The CD91/defensin axis represents a potential therapeutic target for enhancing antimicrobial and anti-tumour immunity.
- Differential responses of moDC subsets to defensins and LPS highlight subset-specific functions.
Abstract:
CD91 is a scavenger receptor expressed by different immune cells and its ligands defensins have been demonstrated to contribute to immune responses against infections and tumours. We previously demonstrated that CD91 is expressed on human monocyte-derived dendritic cells (moDCs) and that human defensins stimulate in vitro the activation of these cells. In this study, we observed that CD91 is expressed at different levels on two distinct moDC subsets: CD91(dim) and CD91(bright) moDCs. Although CD91(bright) moDCs represented a small proportion of total moDCs, this subset showed higher levels of activation and maturation markers compared with CD91(dim) moDCs. The frequency of CD91(bright) moDCs increased by ~ 50% after in vitro stimulation with recombinant human neutrophil peptide-1 (rHNP-1) and recombinant human β defensin-1 (rHBD-1), while lipopolysaccharide (LPS) stimulation decreased it by ~ 35%. Both defensins up-regulated moDC expression of CD80, CD40, CD83 and HLA-DR, although to a lower extent compared with LPS. Notably, upon culture with rHNP-1 and rHBD-1, CD91(bright) moDCs maintained their higher activation/maturation status, whereas this was lost upon culture with LPS. Our findings suggest that defensins promote the differentiation into activated CD91(bright) DCs and may encourage the exploitation of the CD91/defensins axis as a novel therapeutic strategy to potentiate antimicrobial and anti-tumour immune response.
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