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Abstract
BackgroundDendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC). Recently, Dauer and co-workers presented a modified protocol for differentiation of human monocytes into mature DCs within 48 hours (Fast DC). Here we report a functional comparison of the two strategies for generation of DCs from human monocytes with adaptions for large-scale clinical use.MethodsThe Elutra Cell Selection System was used to isolate monocytes after collection of leukapheresis product. The enriched monocytes were cultured in gas permeable Teflon bags with IL-4 and GM-CSF for 24 hours (Fast DC) or 5 days (Standard DC) to obtain immature DCs. The cells were then transfected with mRNA from the leukemia cell line Jurkat E6 by electroporation and incubated for additional 24 h or 2 days in the presence of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and PGE2) to obtain mature DCs.ResultsMature Fast DC and Standard DC displayed comparable levels of many markers expressed on DC, including HLA-DR, CD83, CD86, CD208 and CCR7. However, compared to Standard DC, mature Fast DC was CD14high CD209low. Fast DC and Standard DC transfected with Jurkat E6-cell mRNA were equally able to elicit T cell specifically recognizing transfected DCs in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets.ConclusionOur results indicate that mature Fast DC are functional antigen presenting cells (APCs) capable of inducing primary T-cell responses, and suggest that these cells may be valuable for generation of anti-tumor vaccines.
Highlights
Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC)
DCs can be differentiated from various cellular sources, including CD34+ progenitor cells from bone marrow (BM) and cord blood (CB), and monocytes obtained from peripheral blood mononuclear cells (PBMCs) [5]
Collection of peripheral blood mononuclear cells PBMCs were collected by leukapheresis from patients with advanced prostate cancer included in clinical trials of DC vaccines [12] using Cobe Spectra (Gambro BCT)
Summary
Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC). When activated by danger signals the DCs start differentiation towards a mature penotype and migration to the T cell dependent areas of secondary lymphoid organs. During this process, they lose the capacity for Ag-capturing and upregulate major histocompatibility complex (MHC)- and costimulatory molecules for stimulation of naive T cells [2,3,4]. By using the Elutra® Cell Separation System (Gambro BCT), up to 20 × 109 PBMCs can be elutriated within one hour, which makes this device convenient for large scale isolation of monocytes for further generation of DCs for clinical purposes. Immature DCs can be generated from monocytes by culturing for 5 days in serum-free medium containing IL-4 and GM-CSF [7,8], and further matured using proinflammatory cytokines as described previously [9]
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