Phenotypic and genetic evaluation of human monocyte-derived dendritic cells generated from whole blood for immunotherapy

Yousri M Hussein,Doaa M Hendawy,Abdalrahman N Alghamdy,Nermin Raafat

doi:10.1186/s43042-021-00168-7

Yousri M Hussein, Doaa M Hendawy + Show 2 more

Open Access

https://doi.org/10.1186/s43042-021-00168-7

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Abstract

BackgroundDendritic cells (DCs) recognize different pathogens and cancer cells and activate the adaptive immune response. The generation of effective DC-based cancer vaccines depends on the appropriate differentiation of monocytes in vitro. This study aimed to standardize a protocol for the in vitro differentiation of human peripheral blood monocytes into immature DCs upon treatment with growth factors and generate monocyte-derived DCs (MoDCs). Peripheral blood mononuclear cells were separated from peripheral blood. After monocyte enrichment by plastic adhesion, monocytes were cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate immature DCs. The cells were examined by microscopy. Using flow cytometry, DCs were evaluated for the expression of the CD83 and HLA-DR surface antigens, for the uptake of fluorescein isothiocyanate conjugated dextran, and also for the expression of CD80 and CD86 mRNA.ResultsCD80 and CD86 genes expression was upregulated at day six and exhibited a significant difference (P < 0.05). DCs showed positive expression of the CD83 and HLA-DR surface antigens by flow cytometry and FITC-conjugated dextran uptake.ConclusionThis study represents a preliminary trial to generate immature MoDCs in vitro from blood monocytes collected by the flask adherence method. It offers a panel of surface markers for DCs characterization and provides Immature DCs for experimental procedures after 6 incubation days.

Highlights

Dendritic cells (DCs) recognize different pathogens and cancer cells and activate the adaptive immune response
Human monocytes isolated from Peripheral blood mononuclear cells (PBMCs) differentiate to produce Immature dendritic cells (iDCs) so that DCs are usually generated from blood monocytes in vitro. iDC maturation to mature DCs occurs through an antigen-loading step [15]
Our results demonstrated DC-positive expression of CD83 and HLA-DR by flow cytometry, DCs showed high positive uptake of dextran and significant upregulation of CD80 and CD86 genes expression in DCs compared to monocytes

Summary

Introduction

Dendritic cells (DCs) recognize different pathogens and cancer cells and activate the adaptive immune response. This study aimed to standardize a protocol for the in vitro differentiation of human peripheral blood monocytes into immature DCs upon treatment with growth factors and generate monocyte-derived DCs (MoDCs). After monocyte enrichment by plastic adhesion, monocytes were cultured for 6 days in the presence of granulocytemacrophage colony-stimulating factor and interleukin-4 to generate immature DCs. The cells were examined by microscopy. Dendritic cells (DCs) recognize and uptake various antigens, including tumors and viruses in peripheral tissue. Once activated, they migrate to lymph nodes, where they initiate an adaptive immune response. DCs process the engulfed antigens into peptides, which are presented on major histocompatibility complex (MHC) molecules. Once foreign antigens are presented to the pattern recognition receptors (e.g., toll-like receptors, C-type lectins, or complement receptors) expressed on the cell membrane, iDCs undergo changes and maturation.

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