A_2B adenosine receptor mediates effects of adenosine on DC differentiation. (A) Selective agonists to A₁, A_2A, and A₃ adenosine receptors do not affect DC differentiation. Immature DCs were generated from human monocytes in the presence of the indicated concentrations of receptor-selective agonists or NECA. Agonist concentrations were 10-fold higher than their K_d for the respective receptor. Results are representative of 3 experiments. (B) Selective antagonists to A_2B receptor but not to A₁, A_2A, or A₃ adenosine receptors reverse NECA-induced alteration of DC differentiation. Immature DCs were generated from human monocytes in the presence of 100 μM NECA and selective antagonists at indicated concentrations. Antagonist concentrations were 10-fold higher than their K_i for the respective receptor. The proportion of CD14^lowCD14⁺ cells was measured by flow cytometry. Average values from 3 different experiments are shown. Error bars denote SE. (C) Effect of NECA on cytokine secretion by DCs from mouse HPCs is not reproduced in cells from A_2B^−/− knockout animals. Mature DCs were generated from wild-type or A_2B^−/− knockout mouse HPCs with or without 100 μM NECA with 20 ng/mL TNF-α added for 24 hours for maturation. Culture supernatants from the maturation step were collected, and secreted cytokines were measured by ELISA. Average values from 3 different experiments are shown. Error bars denote SE.