This study focuses on the differentiation of dendritic cells (DCs) from human monocytes in vitro. It explores the effects of various treatments on the maturation and tolerogenic properties of DCs.
To elicit an immune response, dendritic cells, DCs, present an antigen displayed with MHC class-II and the co-stimulatory molecules CD80 and CD86. Based on immune response characteristics, they are subdivided into immature, mature, and tolerogenic DCs.
To generate various dendritic cell subsets in vitro, begin with a multi-well plate containing human monocytes — immune precursor cells in a complete medium containing growth factors and the cytokine IL-4 — a signaling molecule.
Incubate the cells for a prolonged duration. The growth factors and IL-4 induce monocyte proliferation and differentiation into immature DCs.
Immature DCs possess pattern recognition receptors, PRRs, and express low MHC class-II and co-stimulatory molecules, exhibiting a diminished capacity to elicit an immune response.
Treat one well containing immature DCs with immunomodulatory agents — vitamin D3 and glucocorticoids. These immunomodulatory agents downregulate MHC class-II and co-stimulatory molecules' expression. This leads to differentiation into tolerogenic DCs — immuno-regulatory cells that prevent exaggerated or undesirable immune responses.
Treat another well containing immature DCs with lipopolysaccharide, LPS — an antigen. LPS interacts with cells' toll-like receptor-4 — a PRR, initiates a signaling cascade, and differentiates them into mature cells. These cells express high MHC class-II, co-stimulatory molecules, and PRRs — efficient for antigen processing and presentation.
Observe the differentially-treated wells for distinct morphological characteristics representing the dendritic cell subtypes.
Seed four sets of CD14+ monocytes in a concentration of 0.3 to 0.5 x 106 per milliliter of cell culture medium of IL-4 in six-well plates. Incubate the cells in a tissue culture incubator at 37 degrees Celsius with 5% CO2. On day 4, remove 850 microliters of medium from the culture, and centrifuge.
Aspirate the supernatant, and resuspend the pellet in 1 milliliter of cell culture medium. Add the cell mixture back to the culture. On day 5, add 1 microliter of vitamin D3 stock and 1 microliter of dexamethasone stock per milliliter of medium to two of the sets to generate tolerogenic moDCs.
On day 6, add 200 nanograms per milliliter of GM-CSF and 200 nanograms per milliliter of IL-4 to all of the sets. Add 1 microgram per milliliter of LPS to one of the sets treated with only GM-CSF and IL-4 to generate mature moDCs.
Add 1 microgram per milliliter of LPS to one set of the tolerogenic modes moDCs to generate LPS-tolerogenic moDCs. Finally, on day 7, harvest the different types of moDCs by flushing the culture dish with PBS-EDTA.
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