Summary Adenosine is a well‐described anti‐inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi‐professional antigen‐presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28‐mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T‐cell priming by professional antigen‐presenting cells (dendritic cells) and semi‐professional antigen‐presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP‐70 as well as Akt and ERK1/2 in naïve αCD3/CD28‐stimulated CD8 cells. Consequently, αCD3/CD28‐induced calcium‐influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane‐proximal T‐cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T‐cell priming and differentiation in peripheral organs.
The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.
phenyl]pyridin-2-yl}sulfanyl)acetamide] is the most potent and selective adenosine A 2B receptor (A 2B AR) agonist known to date. Therefore, it has been widely used for in vitro and in vivo experiments. In the present study, we investigated the binding and functional properties of BAY60-6583 in various native and recombinant cell lines with different A 2B AR expression levels. In cAMP accumulation and calcium mobilization assays, BAY60-6583 was found to be significantly less efficacious than adenosine or the adenosine derivative NECA. When it was tested in human embryonic kidney (HEK)293 cells, its efficacy correlated with the A 2B expression level of the cells. In Jurkat T cells, BAY60-6583 antagonized the agonistic effect of NECA and adenosine as determined in cAMP accumulation assays. On the basis of these results, we conclude that BAY60-6583 acts as a partial agonist at adenosine A 2B receptors. At high levels of the physiologic agonist adenosine, BAY60-6583 may act as an antagonist and block the effects of adenosine at A 2B receptors. This has to be considered when applying the A 2B -selective "agonist" BAY60-6583 in pharmacological studies, and previous research results may have to be reinterpreted.
2-Amino[1,2,4]triazolo[1,5-c]quinazolines were identified as potent adenosine receptor (AR) antagonists. Synthetic strategies were devised to gain access to a broad range of derivatives including novel polyheterocyclic compounds. Potent and selective A AR antagonists were discovered, including 3,5-diphenyl[1,2,4]triazolo[4,3-c]quinazoline (17, K human A AR 1.16 nm) and 5'-phenyl-1,2-dihydro-3'H-spiro[indole-3,2'-[1,2,4]triazolo[1,5-c]quinazolin]-2-one (20, K human A AR 6.94 nm). In addition, multitarget antagonists were obtained, such as the dual A /A antagonist 2,5-diphenyl[1,2,4]triazolo[1,5-c]quinazoline (13 b, K human A AR 51.6 nm, human A AR 11.1 nm), and the balanced pan-AR antagonists 5-(2-thienyl)[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 c, K human A AR 131 nm, A AR 32.7 nm, A AR 150 nm, A AR 47.5 nm) and 9-bromo-5-phenyl[1,2,4]triazolo[1,5-c]quinazolin-2-amine (11 q, K human A AR 67.7 nm, A AR 13.6 nm, A AR 75.0 nm, A AR 703 nm). In many cases, significantly different affinities for human and rat receptors were observed, which emphasizes the need for caution in extrapolating conclusions between different species.
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