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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
The Anti-Inflammatory Effect of Normothermic Machine Perfused Human Kidneys on Monocyte-Derived Dendritic Cells
Laura W D Knijff1,2, Asel S Arykbaeva2,3, Sandra W van der Kooij1
1Department of Nephrology, Leiden University Medical Center, Leiden, the Netherlands.
Background:
The immunomodulatory activity of normothermic machine perfusion (NMP) on donor organs may be reflected by the release of immune-regulating factors into the perfusate. Dendritic cells (DCs) are potent antigen-presenting cells and are able to sense these factors. We hypothesized that activation of DCs could be used to monitor the release of these mediators into the perfusate.
Methods:
Human monocytes from buffy coats were differentiated into monocyte-derived DCs (moDCs). Perfusate was obtained from human donor kidneys deemed unsuitable for transplantation (n = 15) that underwent 6 h of NMP. MoDCs were incubated for 24 h with perfusate collected at different timepoints. DC supernatant was analyzed with Luminex and ELISA. MoDCs were analyzed with flow cytometry for maturation markers and co-cultured with allogenic T cells.
Results:
Incubation with perfusate resulted in moDCs with a reduced surface expression of CD86, without affecting other maturation markers. Analysis of DC supernatant showed there was a specific increase of IL-10 production upon incubation with perfusate. As a consequence, moDCs incubated with perfusate showed lower T cell stimulatory capacity.
Conclusion:
This study showed that the activation status of DCs can be used to monitor the release of factors into the perfusate during NMP with an anti-inflammatory DC phenotype and function. It demonstrates the feasibility of a cell-based model to determine immune regulatory effects during ex vivo organ perfusion.
