This application protocol describes a reliable and reproducible protocol for the generation of monocyte-derived dendritic cells (Mo-DCs). Using high-quality reagents that ensure cell viability, the resulting Mo-DCs can be used for downstream phenotypic and functional characterization.
Mo-DCs can be generated using various protocol formats, manually and automatically, and at various scales. Here we describe the procedure for 6-well plates as an example.
Resuspend cells in 1.5 mL of complete medium supplemented with the 2-fold concentration of IL-4 and GM-CSF and put back into the original culture.
Assess the viability, yield, and absolute cell count of mature Mo-DCs (mMo-DCs) by flow cytometry via light scatter signals and PI fluorescence. For the gating strategy, see the figure below.
Dendritic cells have a distinct morphology characterized by many cellular processes. To evaluate whether monocytes assume this morphology during differentiation and maturation to Mo-DCs, we recommend analyzing cells on days 0, 6, and 7 of cell culture, both by microscopy and flow cytometry.
On day 0, cells had a spherical shape, which is normal for monocytes. After 6 days, cells showed cytoplasmic protrusions, which were even more pronounced after maturation to day 7. Moreover, size and granularity of the cells increased during differentiation and maturation, which is reflected in increased forward and side scatter signals in the flow cytometry analysis.
|Cell surface antigen||Clone||Fluorochrome|
Flow cytometry analysis of cell surface markers showed that in vitro generated Mo-DCs assumed the typical DC phenotype. During differentiation, monocytes down-regulated the expression of CD14. In addition, mMo-DCs expressed various DC markers that are involved in the formation of immunological synapses between DC
and naive T cells, including the costimulatory proteins CD80 and CD86, the cell adhesion molecule CD54, and
the antigen-presenting molecules MHC I (HLA-ABC) and MHC II (HLA-DR). Mature Mo-DCs also expressed the DC activation markers CD83, CD25, and CD40, which were up-regulated accordingly. CCR7, which is required for migration of DCs to draining lymph nodes, was also up-regulated. The highest expression levels of the antigen uptake receptors CD209 and CD206 were detected on imMo-DCs, corresponding to their antigen uptake function.
Antigen uptake by antigen-presenting cells can occur specifically via receptor-mediated endocytosis or non-specifically via pinocytosis or phagocytosis. To assess the pinocytosis capacity of in vitro generated Mo-DCs in comparison to monocytes, cells are cultured at 37 °C for up to 1 h in the presence of FITC-dextran. Non-specific binding of FITC-dextran to the cell surface is determined by incubating samples on ice throughout the procedure. The MFI of FITC is then analyzed by flow cytometry after various time points, subtracting the MFI of samples on ice from the MFI of samples incubated at 37 °C.
Immature Mo-DCs had the highest antigen uptake capacity, reflected in a continuous increase in MFI over the entire time course. Antigen uptake was decreased when Mo-DCs were matured for an additional 2 days (day 9). Monocytes showed negligible antigen uptake capacity compared to Mo-DCs. Additionally, flow cytometry analysis showed that imMo-DCs exhibited the highest expression of the antigen uptake receptors CD206 and CD209. These results correlate with the finding that imMo-DCs lose their antigen uptake function upon maturation.
Upon activation of DCs in the periphery, the cells migrate to the draining lymph nodes where they encounter naive T cells. Migration depends on the expression of CCR7 on the DC surface11. The CCR7 ligand CCL19 acts as a chemoattractant for DCs and is expressed in the lymph node areas characterized by high T cell densities.
CCR7 expression was up-regulated during Mo-DC maturation. mMo-DCs migrated towards the CCL19 stimulus in a dose-dependent manner.
After migration to the lymph nodes, DCs can induce the proliferation of naive T cells. The capacity of
monocytes and Mo-DCs to induce T cell proliferation can be tested in an mixed lymphocyte reaction (MLR) assay. To this end, allogeneic naive CD45RA+CD45RO– T cells are isolated to high purities by MACS®
Technology, and their plasma membranes are labeled with CellTrace™ Violet. After coculturing with monocytes or Mo-DCs for 7 d, the number of T cells and the CellTrace Violet staining is analyzed by flow cytometry. As with each division of the T cells the dye gets more diluted in the plasma membrane, an increase in unlabeled cells indicates a high T cell proliferation rate.
Serial dilution of Mo-DCs/monocytes for MLR assay.
|Mo-DC/monocyte density (cells/mL)||Number of Mo-DCs/monocytes per well||Ratio of Mo-DCs/monocytes to T cells |
(number of T cells is constant at 50,000)
In contrast to monocytes, both imMo-DCs and mMo-DCs increased the number of CellTrace Violet–
negative T cells in the culture. This correlated with higher T cell numbers, as determined by cell counting.
T cell proliferation was highest when mMo-DCs were used as antigen-presenting cells, which is in line with
the result from the immunophenotyping showing that receptors involved in T cell priming are up-regulated on Mo-DCs upon maturation.
In general, DCs have the capacity to secrete both pro-inflammatory and immunoregulatory cytokines, depending on the stimulus received. T cell–mediated CD40L stimulation of Mo-DCs induces the production
of TH1-polarizing IL-12, but also the secretion of the immunosuppressive IL-1012-14. However, for studies towards the development of cancer therapies it is desirable to generate Mo-DC populations secreting high amounts of IL-12 and low amounts of IL-10 as these cells generate a more effective antitumor response via induction of TH1 cells.
Mo-DCs can be stimulated under various conditions to determine their capacity to secrete IL-12 and IL-10. They can be incubated with soluble Human CD40-Ligand, which form multimers in vitro, or they can be cocultured with a J558L cell line expressing CD40L.
Stimulation with soluble CD40L led to production of high levels of IL-12 and low levels of IL-10. Coculture with CD40L-expressing J588L cells resulted in an overall increase in secretion of both IL-12 and IL-10. However, the ratio of IL12/IL-10 was higher after stimulation with soluble CD40L than after coculture with the cell line. Thus, recombinant human CD40L multimers represent an attractive alternative to CD40L-transfected cell lines, allowing for CD40 stimulation of Mo-DCs under defined conditions.
|Column||Max. number of labeled cells||Max. number of total cells||Separator|
VarioMACS, SuperMACS II
VarioMACS, SuperMACS II
|autoMACS||2×108||4×109||autoMACS Pro, autoMACS|
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