Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.     

Recombinant Mouse PAP Has pH-Dependent Ectonucleotidase Activity and Acts through A1-Adenosine Receptors to Mediate Antinociception

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A₁-adenosine receptor (A₁R) activation. In this study, we purified the secretory isoform of mouse (m)PAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0). In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP) at an acidic pH (pH 5.6). The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day) antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund''s Adjuvant (CFA) inflammatory pain model. These antinociceptive effects were transiently blocked by the A₁R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX), suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models.     

CD39-adenosinergic axis in renal pathophysiology and therapeutics

Extracellular ATP interacts with purinergic type 2 (P2) receptors and elicits many crucial biological functions. Extracellular ATP is sequentially hydrolyzed to ADP and AMP by the actions of defined nucleotidases, such as CD39, and AMP is converted to adenosine, largely by CD73, an ecto-5′-nucleotidase. Extracellular adenosine interacts with P1 receptors and often opposes the effects of P2 receptor activation. The balance between extracellular ATP and adenosine in the blood and extracellular fluid is regulated chiefly by the activities of CD39 and CD73, which constitute the CD39-adenosinergic axis. In recent years, several studies have shown this axis to play critical roles in transport of water/sodium, tubuloglomerular feedback, renin secretion, ischemia reperfusion injury, renal fibrosis, hypertension, diabetic nephropathy, transplantation, inflammation, and macrophage transformation. Important developments include global and targeted gene knockout and/or transgenic mouse models of CD39 or CD73, biological or small molecule inhibitors, and soluble engineered ectonucleotidases to directly impact the CD39-adenosinergic axis. This review presents a comprehensive picture of the multiple roles of CD39-adenosinergic axis in renal physiology, pathophysiology, and therapeutics. Scientific advances and greater understanding of the role of this axis in the kidney, in both health and illness, will direct development of innovative therapies for renal diseases.     

Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production

Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment.     

Ecto-5'-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line

Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5'-NT/CD73. Quercetin was able to inhibit the ecto-5'-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (alpha,beta-methyleneadenosine-5'-diphosphate), an ecto-5'-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5'-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.     

Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line

Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5′-NT/CD73. Quercetin was able to inhibit the ecto-5′-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (α,β-methyleneadenosine-5′-diphosphate), an ecto-5′-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5′-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.     

Coexpression of ecto-5'-nucleotidase/CD73 with specific NTPDases differentially regulates adenosine formation in the rat liver

Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5'-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high "inflammatory" (500 μM) and low "physiological" (1 μM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

Characterization of Ectonucleotidases in Human Medulloblastoma Cell Lines: ecto-5′NT/CD73 in Metastasis as Potential Prognostic Factor

Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5′NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5′NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5′NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5′NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.     

The role of the CD39–CD73–adenosine pathway in liver disease

Extracellular adenosine triphosphate (ATP) is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. The ectonucleotidases CD39/ectonucleoside triphosphate diphosphohydrolase‐1 and CD73/ecto‐5′‐nucleotidase are cell‐surface enzymes that breakdown extracellular ATP into adenosine. This drives a shift from an ATP‐driven proinflammatory environment to an anti‐inflammatory milieu induced by adenosine. The CD39–CD73–adenosine pathway changes dynamically with the pathophysiological context in which it is embedded. Accumulating evidence suggests that CD39 and CD73 play important roles in liver disease as critical components of the extracellular adenosinergic pathway. Recent studies have shown that the modification of the CD39–CD73–adenosine pathway alters the liver's response to injury. Moreover, adenosine exerts different effects on the pathophysiology of the liver through different receptors. In this review, we aim to describe the role of the CD39–CD73–adenosine pathway and adenosine receptors in liver disease, highlighting potential therapeutic targets in this pathway, which will facilitate the development of therapeutic strategies for the treatment of liver disease.     

A highly potent CD73 biparatopic antibody blocks organization of the enzyme active site through dual mechanisms

The dimeric ectonucleotidase CD73 catalyzes the hydrolysis of AMP at the cell surface to form adenosine, a potent suppressor of the immune response. Blocking CD73 activity in the tumor microenvironment can have a beneficial effect on tumor eradication and is a promising approach for cancer therapy. Biparatopic antibodies binding different regions of CD73 may be a means to antagonize its enzymatic activity. A panel of biparatopic antibodies representing the pairwise combination of 11 parental monoclonal antibodies against CD73 was generated by Fab-arm exchange. Nine variants vastly exceeded the potency of their parental antibodies with ≥90% inhibition of activity and subnanomolar EC₅₀ values. Pairing the Fabs of parents with nonoverlapping epitopes was both sufficient and necessary whereas monovalent antibodies were poor inhibitors. Some parental antibodies yielded potent biparatopics with multiple partners, one of which (TB19) producing the most potent. The structure of the TB19 Fab with CD73 reveals that it blocks alignment of the N- and C-terminal CD73 domains necessary for catalysis. A separate structure of CD73 with a Fab (TB38) which complements TB19 in a particularly potent biparatopic shows its binding to a nonoverlapping site on the CD73 N-terminal domain. Structural modeling demonstrates a TB19/TB38 biparatopic antibody would be unable to bind the CD73 dimer in a bivalent manner, implicating crosslinking of separate CD73 dimers in its mechanism of action. This ability of a biparatopic antibody to both crosslink CD73 dimers and fix them in an inactive conformation thus represents a highly effective mechanism for the inhibition of CD73 activity.     

Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.     

Human Homologue of Seta Binding Protein 1 Interacts with Cathepsin B and Participates in TNF-Induced Apoptosis in Ovarian Cancer Cells

Extracellular adenine nucleotide hydrolysis in the circulation is mediated by the action of an NTPDase (CD39, apyrase) and of a 5′-nucleotidase (CD73), presenting as a final product, adenosine. Among other properties described for adenine nucleotides, an anti-cancer activity is suggested, since ATP is considered a cytotoxic molecule in several tumour cell systems. Conversely, some studies demonstrate that adenosine presents a tumour-promoting activity. In this study, we evaluated the pattern of adenine nucleotide hydrolysis by serum and platelets from rats submitted to the Walker 256 tumour model. Extracellular adenine nucleotide hydrolysis by blood serum and platelets obtained from rats at, 6, 10 and 15 days after the subcutaneous Walker 256 tumour inoculation, was evaluated. Our results demonstrate a significant reduction in ATP, ADP and AMP hydrolysis in blood serum at 6, 10 and 15 days after tumour induction. In platelets, a significant reduction in ATP and AMP hydrolysis was observed at 10 and 15 days after tumour induction, while an inhibition of ADP hydrolysis was observed at all times studied. Based on these results, it is possible to suggest a physiologic protection mechanism against the tumoral process in circulation. The inhibition in nucleotide hydrolysis observed probably maintains ATP levels elevated (cytotoxic compound) and, at the same time, reduces the adenosine production (tumoor-promoting molecule) in the circulation.     

Influence of plasma total antioxidant ability on lipid and protein oxidation products in plasma and erythrocyte ghost obtained from developing and adult rats pretreated with two vitamin K formulations

Degradation of adenine nucleotides in myocardial cells has important physiological implications associated with the regulation of the high-energy phosphate precursor pool and the production of adenosine. Adenosine may be released as from cells or, following adenine nucleotides release, they may be metabolized and rapidly converted to adenosine via the action of an ectoenzyme cascade formed by an ATP diphosphohydrolase and a 5'-nucleotidase. Thyroid hormones are known to have profound effects on the cardiovascular system, as demonstrated by the changes accompanying both hypothyroidism and hyperthyroidism. We previously reported that thyroid hormone significantly increases the ecto-5'-nucleotidase (CD73) activity and expression in C6 glioma cells culture. The object of the present study was to evaluate the extracellular adenosine production from AMP in cardiomyocytes and also the effect of (T3) on activity and expression of the enzyme, CD73. Primary cultures of rat ventricular neonatal cardiac myocytes were submitted to increasing doses of T3 for 24 h. Cell viability and purity were estimated by measuring the release of lactate dehydrogenase (LDH) activity and immunofluorescence cell staining, respectively. CD73 activity was measurement using a malachite green method and RT-PCR was used to analyze enzyme expression. T3 stimulated CD73 activity and expression of the cells, suggesting that this effect could promote an increase in adenosine formation and, therefore, has an important modulatory role in the elicitation of responses that serve to restore the tissue oxygen supply-to-demand ratio back to normal.     

Activity and expression of ecto-5′-nucleotidase/CD73 are increased by thyroid hormones in vascular smooth muscle cells

Extracellular nucleotides ATP, ADP, AMP and adenosine are well known signaling molecules of the cardiovascular system that are involved in several physiological processes: cell proliferation, platelet aggregation, inflammatory processes and vascular tonus. The levels of these molecules are controlled by ecto-NTPDases and ecto-5′-nucleotidase/CD73 (ecto-5′-NT/CD73) actions, which are responsible for the complete ATP degradation to adenosine. The thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), play important roles in the vascular system promoting vasodilatation. Here we investigated the influence of thyroid hormones on the enzyme cascade that catalyzes the interconversion of purine nucleotides in vascular smooth muscle cells (VSMC). Exposure of VSMCs to 50nM T₃ or T₄ did not change ATP and ADP hydrolysis significantly. However, the same treatment caused an increase of 75% in AMP hydrolysis, which was time-dependent but dose-independent. Moreover, T₃ treatment significantly increased ecto-5′-NT/CD73 mRNA expression, which suggests a genomic effect of this hormone upon ecto-5′-NT/CD73. In addition to the importance of the ecto-5′-NT in cell proliferation and differentiation, its overexpression could result in higher extracellular levels of adenosine, an important local vasodilatator molecule.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.