In vitro differentiation of CD34+ hematopoietic progenitor cells toward distinct dendritic cell subsets of the birbeck granule and MIIC- positive Langerhans cell and the interdigitating dendritic cell type

Abstract

We have demonstrated recently that Birbeck granule-positive Langerhans cells (LC) can be derived from CD34+ peripheral blood progenitor cells in the presence of a seven-cytokine cocktail (CC7–7). Here, we show that the sequential use of early-acting hematopoietic growth factors, stem cell factor, interleukin (IL)-3, and IL-6, followed on day 8 by differentiation in the two-factor combination IL-4 plus granulocytemacrophage colony-stimulating factor (GM-CSF) (CC4GM) is more efficient and allows the cells to be arrested in the LC stage for more than 1 week while continuous maturation occurs in CC7–7. Maturation of LC to interdigitating dendritic cells (DC) could specifically be induced within 60 hours by addition of tumor necrosis factor-alpha (20 ng/mL) or lipopolysaccharide (100 ng/mL). Using LC that had been enriched to greater than 90% CD1a+ cells by an immunoaffinity column, we were able to define clear-cut differences between LC and DC that corroborate data of the respective cells derived from epithelial borders (LC) or from lymph nodes (LN) and spleen (DC). Thus, molecules and functions involved in antigen (AG) uptake and processing were highly expressed in LC, while those involved in AG presentation were at maximum in DC. LC were CD1a+2 DR+2, CD23+, CD36+, CD80-, CD86-, and CD25-, while DC were CD1a+/- DR+3, CD23-, CD36-, CD80+, CD86+2, and CD25+, CD40 and CD32 were moderately expressed and nearly unchanged on maturation, in contrast to monocyte-derived DC. Macropinocytosis of fluorescein isothiocyanate-dextran was dominant in LC, as were multilamellar major histocompatibility complex (MHC) class II compartments (MIICs), which were detected by electron microscopy. The functional dichotomy of these cell types was finally supported by testing the AG-presenting cell function for tetanus toxoid to primed autologous T-cell lines, which was optimal when cells were loaded with AG as LC and subsequently induced to become DC.

Cytokine and chemokine production by CD34+ haemopoietic progenitor cells: detection in single cells

“…Since cells in culture are composed of various amounts of mature and immature cells of different haemopoietic lineages, secretion of secondary cytokines by these cells has to be expected. This has been shown by different reports (Herbst et al, 1996;Bot et al, 1988;Watari et al, 1994). The actual cytokine mixture has unpredictable effects on cell growth, differentiation and function and thus precludes controlled manipulation of the cells in culture.…”

Functional Characterization of CD8+ Antigen-Specific Cytotoxic T Lymphocytes after Enrichment Based on Cytokine Secretion: Comparison with the MHC-Tetramer Technology

“…90% CD8 1 T cells and was used as responder cell population. DC were generated from monocyte-enriched cell fractions as described previously [13]. Briefly, monocytes were isolated from PBMC by plastic adherence.…”

The evolving function of Langerhans cells in adaptive skin immunity

“…6 Studies using LC-like cells derived in vitro from bone marrow or hematopoietic stem cells showed similar results. [7][8][9] The most commonly used assay of LC function in vivo is contact hypersensitivity (CHS) to haptens that are applied directly onto the skin. Mice are sensitized by application of the hapten to their abdomens.…”