Phenotype and functional activity of human adenosine-differentiated DCs and time course of their differentiation. (A) Immature DCs were generated from human monocytes in the presence of 30 μM NECA to produce about equal proportions of CD1a⁺ and CD1a^lowCD14⁺ cells; these cells were separated by flow sorting into CD1a⁺CD14⁻ and CD1a⁻CD14⁺ cell populations (indicated on a dot plot) and expression of myeloid and DC cell surface markers was assessed by flow cytometry (histograms). Results are representative of 3 experiments. (B,C) Adenosine-differentiated DCs have impaired ability to stimulate proliferation and IFN-γ production by allogeneic T cells. Immature (iDC) and mature (DC) DCs were generated from human monocytes with or without 100 μM NECA and used as stimulators in MLR with allogeneic donor's T cells. MLR was performed as described in the “Methods.” IFN-γ concentrations were measured in MLR supernatants by ELISA. Results are representative of 3 experiments. (C) Average values from 3 different experiments are shown. Error bars denote SE. (D) Time course of generation of CD1a⁺ and CD1a^lowCD14⁺ DCs from human monocytes in the presence of 100 μM NECA. NECA was added to monocytes at day 0, and the proportions of CD1a⁺ and CD1a^lowCD14⁺ DCs were assessed daily by flow cytometry. Average values from 3 different experiments are shown. Error bars denote SE.