GENERATION OF CMRF-44(-DERIVED DENDRITIC CELLS - INSIGHTS INTO PHENOTYPE AND FUNCTION() MONOCYTE)

The CMRF-44 monoclonal antibody (MoAb) recognizes an intermediate stag e 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 t he in vitro development of DC-like cells from peripheral blood mononuc lear cells. Neither granulocyte-macrophage colony-stimulating factor ( GM-CSF) nor GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) suppor ted the development of CMRF-44(+) cells. However, GM-CSF plus interleu kin (IL)-4 generated a substantial number of CMRF-44(+) cells among th e heterogeneous CD14(-) myeloid cell population, produced after 7 or 1 0 days of culture. The addition of TNF-alpha to GM-CSF+IL-4 on the fif th day of culture enhanced the generation of CMRF-44(+) cells from day s 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 essent ial, but a wide range of concentrations (50-800 U/mL) was effective fo r 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 nu mber of CMRF-44(+) cells. The CMRF-44(+) DCs generated in the presence of GM-CSF+IL-4 were large, irregularly shaped cells with variable CD1 a 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 gener ated in GM-CSF+IL-4 showed higher allostimulatory activity than CMRF-4 4(-) cells but were less efficient at processing and presenting solubl e antigen to T-lymphocyte lines. TNF-alpha treatment reduced antigen u ptake but increased the allostimulatory activity of CMRF-44+ DCs. CMRF -44+ DC differentiation from blood CD14(+) monocytes was not radiosens itive and thus does not involve cell division. We conclude that the Mo Ab CMRF-44 identifies both intermediate and fully mature stages of mon ocyte-DC differentiation and may be a useful marker in establishing th e optimal timing for antigen loading of in vitro-generated monocyte-de rived DCs.