Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

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.