title: Generation of CMRF-44+ monocyte-derived dendritic cells: insights into phenotype and function.
abstract: The CMRF-44 monoclonal antibody (MoAb) recognizes an intermediate stage of blood dendritic cell (DC) differentiation as well as mature CD83+ blood DC. Here we describe the use of the CMRF-44 MoAb to monitor the in vitro development of DC-like cells from peripheral blood mononuclear cells. Neither granulocyte-macrophage colony-stimulating factor (GM-CSF) nor GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) supported the development of CMRF-44+ cells. However, GM-CSF plus interleukin (IL)-4 generated a substantial number of CMRF-44+ cells among the heterogeneous CD14- myeloid cell population, produced after 7 or 10 days of culture. The addition of TNF-alpha to GM-CSF+IL-4 on the fifth day of culture enhanced the generation of CMRF-44+ cells from days 7 to 14. A concentration of 50 U/mL of IL-4 was sufficient to allow the development of CMRF-44+ cells. The presence of GM-CSF was essential, but a wide range of concentrations (50-800 U/mL) was effective for supporting IL-4-induced generation of CMRF-44+ cells. TNF-alpha at concentrations of 20 or 50 ng/mL induced a maximal increase in the number of CMRF-44+ cells. The CMRF-44+ DCs generated in the presence of GM-CSF+IL-4 were large, irregularly shaped cells with variable CD1a expression and have CD83 transcripts but no CD83 surface expression. Additional TNF-alpha treatment induced prominent dendritic processes and surface expression of CD83 on CMRF-44+ DCs. The CMRF-44+ DCs generated in GM-CSF+IL-4 showed higher allostimulatory activity than CMRF-44 cells but were less efficient at processing and presenting soluble antigen to T-lymphocyte lines. TNF-alpha treatment reduced antigen uptake but increased the allostimulatory activity of CMRF-44+ DCs. CMRF-44+ DC differentiation from blood CD14+ monocytes was not radiosensitive and thus does not involve cell division. We conclude that the MoAb CMRF-44 identifies both intermediate and fully mature stages of monocyte-DC differentiation and may be a useful marker in establishing the optimal timing for antigen loading of in vitro-generated monocyte-derived DCs.
authors.affiliation: Haematology/Immunology Research Group, Christchurch Hospital, New Zealand.
authors.forename: S
authors.initials: S
authors.lastname: Vuckovic
authors.forename: D B
authors.initials: DB
authors.lastname: Fearnley
authors.forename: S I
authors.initials: SI
authors.lastname: Mannering
authors.forename: J
authors.initials: J
authors.lastname: Dekker
authors.forename: L F
authors.initials: LF
authors.lastname: Whyte
authors.forename: D N
authors.initials: DN
authors.lastname: Hart
mesh_terms: D000911:Antibodies, Monoclonal; D017951:Antigen Presentation; D015703:Antigens, CD; D018949:Antigens, CD1 / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000502:physiology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006651:Histocytochemistry; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis; D015847:Interleukin-4 / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis; D009000:Monocytes; D010641:Phenotype; D013601:T-Lymphocytes / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000097802:CD83 Antigen
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018949:Antigens, CD1; D000954:Antigens, Surface; D003470:Culture Media; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007136:Immunoglobulins; D015847:Interleukin-4; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D014409:Tumor Necrosis Factor-alpha; D000097802:CD83 Antigen