In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

Plasmacytoid dendritic cells, or pDCs, are immune cells that respond to infection by producing the cytokine type-I interferon.

To generate pDCs in vitro, take a suspension of common lymphoid progenitor cells, or CLPs, isolated from mouse bone marrow. CLPs can differentiate into lymphocytes and dendritic cells.

Add the cell suspension over a layer of feeder cells — adherent stromal cells — irradiated with gamma rays to inhibit proliferation. The feeder cells provide cytokines and cell-cell contact required for the differentiation of CLPs.

Add an adequate concentration of Fms-like tyrosine kinase 3 ligand, or FL — a bivalent cytokine that promotes pDC development — and incubate.

The CLPs migrate between the feeder cells and proliferate to form groups of cells, termed cobblestone areas.

The FL in the medium binds to the Flt3 receptor — a tyrosine kinase receptor — on the CLPs. The binding induces receptor dimerization and the autophosphorylation of the tyrosine residues on the cytoplasmic domain.

Cytoplasmic adaptor proteins bind to these residues and become phosphorylated, transducing the signal to the downstream signaling components. The signaling cascade causes the differentiation of CLPs into pDCs — small round cells that grow in suspension — as well as conventional dendritic cells, or cDCs — larger spindle-shaped adherent cells.

Collect the cells. Add antibodies against pDC- and cDC-specific surface markers. Analyze the cells using flow cytometry.

Cells positive for pDC-specific markers and negative for cDC-specific markers indicate successful pDC development.

To analyze the common lymphoid progenitors, or CLPs, by flow cytometry, next, F_c block the cells with anti-CD16/32 for one to two minutes, followed by simultaneous staining of the cells with the appropriate antibody cocktail.

After 15 minutes on ice, wash the cells in three milliliters of FACS buffer, and resuspend the pellet in 300 microliters of fresh FACS buffer. Then, filter the cell suspension through a 40-micrometer strainer, and immediately sort the cells on a flow cytometer using the appropriate filters and voltages, collecting the cells of interest in a 15-milliliter tube containing 8 milliliters of complete RPMI.

To generate a highly-enriched pDC population, it is crucial to sort out the right population of CLPs from harvested bone marrow cell for the in vitro culture with AC-6 feeder system.

At the end of the sort, spin down the cells, and resuspend the CLP cell pellet with enough complete RPMI to obtain a cell density of about 5 times 10 to the 4th cells per milliliter. Next, seed 500 cells per well into the 12-well plate containing the AC-6 feeder cells, and supplement the co-cultures with 100 nanograms per milliliter FLT3 ligand. Then, incubate the cells at 37 degrees Celsius and 5% carbon dioxide with periodic visual monitoring of the dendritic cell, or DC development, under a microscope.

On day 12, harvest the cells in the co-culture supernatant, and wash each well one time with half a milliliter of complete RPMI medium, pooling the resulting supernatants and washes. Then, add half a milliliter of fresh medium to each well, and use a cell scraper to detach the adherent cells. Combine the detached cells with the floating cells, and centrifuge the resulting cell suspension, resuspending the pellet in 50 microliters of FACS buffer.

Then, count the cells and stain them for CD11c, CD11b, and B220 expression after F_c blocking, as just demonstrated. The co-cultured cells can then be analyzed for the presence of conventional CD11c positive, CD11b positive, B220 negative, and plasmacytoid CD11c positive, CD11b negative, B220 positive DC populations.