Dataset Information

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

ABSTRACT: Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-?1, and the Notch ligand DLL4 differentiate within 3 days into CD1a⁺Langerin⁺cells containing Birbeck granules. RNA sequencing of these monocyte-derived LCs (moLCs) confirmed gene expression of LC-related molecules, pattern recognition receptors, and enhanced expression of genes involved in the antigen-presenting machinery. On the protein level, moLCs showed low expression of costimulatory molecules but prominent expression of C-type lectin receptors. MoLCs can be matured, secrete IL-12p70 and TNF-?, and stimulate proliferation and cytokine production in allogeneic CD4⁺ and CD8⁺ T cells. In regard to vaccine testing, a recently characterized glycomimetic Langerin ligand conjugated to liposomes demonstrated specific and fast internalization into moLCs. Hence, these short-term in vitro?generated moLCs represent an interesting tool to screen LC-based vaccines in the future.

SUBMITTER: Bellmann L

PROVIDER: S-EPMC7758629 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

Bellmann Lydia L Zelle-Rieser Claudia C Milne Paul P Resteu Anastasia A Tripp Christoph H CH Hermann-Kleiter Natascha N Zaderer Viktoria V Wilflingseder Doris D Hörtnagl Paul P Theochari Maria M Schulze Jessica J Rentzsch Mareike M Del Frari Barbara B Collin Matthew M Rademacher Christoph C Romani Nikolaus N Stoitzner Patrizia P

The Journal of investigative dermatology 20200606 1

Langerhans cells (LCs) in the skin are a first line of defense against pathogens but also play an essential role in skin homeostasis. Their exclusive expression of the C-type lectin receptor Langerin makes them prominent candidates for immunotherapy. For vaccine testing, an easily accessible cell platform would be desirable as an alternative to the time-consuming purification of LCs from human skin. Here, we present such a model and demonstrate that monocytes in the presence of GM-CSF, TGF-β1, a ...[more]

PMID: 32522485

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Project description:The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.

Dataset Information

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

Publications

Notch-Mediated Generation of Monocyte-Derived Langerhans Cells: Phenotype and Function.

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