Mature Dendritic Cells Derived from Human Monocytes Within 48 Hours: A Novel Strategy for Dendritic Cell Differentiation from Blood Precursors

Abstract

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5–7 days of differentiation with GM-CSF and IL-4, followed by 2–3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1α. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-γ. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-γ production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.

Epstein‐Barr virus (EBV) latent membrane protein‐1‐specific cytotoxic T lymphocytes targeting EBV‐carrying natural killer cell malignancies

“…DC were prepared as previously described with slight modification [51]. Briefly, CD8 + T cells were isolated from PBMC using CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and stored at -135 C until use.…”

“…After incubation for 4 or 16 h at 37 C, the radioactivity in the supernatants was counted in a ccounter. The percentage specific 51 Cr release was calculated as 100 Â (experimental release -spontaneous release) / (maximum release -minimum release).…”

The N-N non-covalent domain of the nucleocapsid protein of type 2 porcine reproductive and respiratory syndrome virus enhances induction of IL-10 expression

“…Porcine MoDCs were generated using a previously described protocol for the introduction of human 'fast' DCs (Dauer et al, 2003(Dauer et al, , 2005Guan et al, 2004) and used to determine IL-10 levels following PRRSV infection. Briefly, PBMCs were isolated from the blood of piglets (free of PRRSV, porcine circovirus type 2, classical swine fever virus, pseudorabies virus, swine influenza virus and Mycoplasma hyopneumoniae) and washed three times with complete medium.…”

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between Vγ9Vδ2 T Cells, αβ CD8+ T Cells, Regulatory T Cells, and Dendritic Cells

“…+ cells within only 48 h of in vitro culture (f-DC) (24,42). This strategy has been adapted and validated for large-scale production under GMP conditions in patients with advanced prostate cancer (43), but the ability of f-DC to induce the proliferation of gd T cells and concurrently activate MHC-restricted CD8 + ab T cells is unknown.…”