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Glioblastoma (GB) is the most common primary brain tumor in adults and carries a dismal prognosis, despite the best available treatment. The 2021 WHO Classification of CNS tumors incorporated molecular profiling to better define the characteristics and prognosis of tumor types and subtypes. These recent advances in diagnosis have not yet resulted in breakthrough therapies capable of shifting the treatment paradigm. NT5E/CD73 is a cell surface enzyme that participates in a complex purinergic pathway in synergy with ENTPD1/CD39 producing extracellular adenosine (ADO) from ATP. ADO promotes tumor progression by inducing immunosuppression, stimulating adhesion, invasion, and angiogenesis. In this study, we performed an in silico analysis of 156 human glioblastoma samples in an unexplored public database to investigate the transcriptional levels of NT5E and ENTPD1. The analysis revealed a significant increase in transcription levels of the genes under study in GB samples versus non-tumor brain tissue samples, in concordance with previous studies. High transcriptional levels of NT5E or ENTPD1 were independently related to a decrease in overall survival (p = 5.4e-04; 1.1e-05), irrespective of the IDH mutation status. NT5E transcriptional levels were significantly higher in GB IDH wild-type patients compared to GB IDH-mutant; however, ENTPD1 levels showed no significant difference, p ≤ 0.001. This in silico study indicates the need for a deeper understanding of the purinergic pathway relation to GB development, also inspiring future population studies that could explore ENTPD1 and NT5E not only as prognostic markers but also as potential therapeutic targets., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39 + Tregs increased with disease severity. In contrast, CD39 + Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases., (© 2022 Wiley Periodicals LLC.)

Purpose: Tumor-infiltrating lymphocytes (TIL) are strongly associated with survival in most cancers; however, the tumor-reactive subset that drives this prognostic effect remains poorly defined. CD39, CD103, and PD-1 have been independently proposed as markers of tumor-reactive CD8 + TIL in various cancers. We evaluated the phenotype, clonality, and prognostic significance of TIL expressing various combinations of these markers in high-grade serous ovarian cancer (HGSC), a malignancy in need of more effective immunotherapeutic approaches., Experimental Design: Expression of CD39, CD103, PD-1, and other immune markers was assessed by high-dimensional flow cytometry, single-cell sequencing, and multiplex immunofluorescence of primary and matched pre/post-chemotherapy HGSC specimens., Results: Coexpression of CD39, CD103, and PD-1 ("triple-positive" phenotype) demarcated subsets of CD8 + TIL and CD4 + regulatory T cells (Treg) with a highly activated/exhausted phenotype. Triple-positive CD8 + TIL exhibited reduced T-cell receptor (TCR) diversity and expressed genes involved in both cytolytic and humoral immunity. Triple-positive Tregs exhibited higher TCR diversity and a tumor-resident phenotype. Triple-positive TIL showed superior prognostic impact relative to TIL expressing other combinations of these markers. TIGIT was uniquely upregulated on triple-positive CD8 + effector cells relative to their CD4 + Treg counterparts., Conclusions: Coexpression of CD39, CD103, and PD-1 demarcates highly activated CD8 + and CD4 + TIL with inferred roles in cytolytic, humoral, and regulatory immune functions. Triple-positive TIL demonstrate exceptional prognostic significance and express compelling targets for combination immunotherapy, including PD-1, CD39, and TIGIT., (©2021 American Association for Cancer Research.)

Background: During viral infection, inhibitory receptors play a key role in regulating CD8 T-cell activity. The objective of this research was to investigate programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), and CD39 exhaustion markers in CD8 T cells of new coronavirus disease-2019 (COVID-19) patients., Methods: A total of 44 patients with COVID-19 (17 subjects in a critical group and 27 patients in a non-critical group) and 14 healthy controls, who were admitted to Hospitals in Babol, were recruited to the study. In subjects' peripheral blood mononuclear cells (PBMCs), we compared the phenotype of CD8 T lymphocytes, expressing PD-1, TIM-3, or CD39, both alone and in various combinations., Results: The findings showed that the percentage of CD8 + cells was significantly lower in patients. Critical and non-critical patients were more likely than healthy controls to have an escalated frequency of CD8 + TIM-3 + , CD8 + CD39 + , and CD8 + TIM-3 + CD39 + cells. No significant differences were observed between all groups in the CD8 + PD-1 + cell counts. There was also no difference between three groups regarding the counts of CD8 + TIM-3 + PD-1 + , CD8 + PD-1 + CD39 + , and CD8 + TIM-3 + PD-1 + CD39 + cells. The counts of non-exhausted cells were significantly lower in critical and non-critical individuals compared to the healthy individuals' value., Conclusion: Patients, infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), altered exhausted CD8 T lymphocytes with CD39 and TIM-3 exhaustion markers, which may account the dysregulated immune response found in COVID-19., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Natural killer (NK) cells, a predominant innate lymphocyte subset, mediates eradicating malignant cells. Purinergic signaling by ectonucleotidase CD39 can suppress T-cell response in caner. However, the role of CD39 in NK cells has not been fully elucidated. Here, we characterized CD39 expression on NK cells and its clinical relevance in esophageal squamous cell carcinoma (ESCC). Peripheral blood and tissue samples were collected from 36 ESCC patients. We observed that the proportion of NK cells significantly decreased but CD39 was obviously up-regulated on NK cells from cancerous tissues compared to paired peripheral blood in ESCC patients. Furthermore, tumor-infiltrating NK cells with high CD39 expression exhibited a phenotype of functional impairment. In vitro, conditioned media of ESCC cell lines could induce CD39 expression on peripheral NK cells from healthy donors. IL-6 was identified as the major cytokine produced by ESCC cell lines and also elevated in both tumor tissues and blood serum from ESCC patients. Recombinant IL-6 significantly induced surface CD39 expression in human NK cells, while IL-6-receptor antagonist tocilizumab prevented this effect. Finally, tumor-infiltrating CD39 + NK cells were correlated with poor prognosis in ESCC patients. Thus, tumor-derived IL-6 might impair NK cell functions through induction of CD39 expression. CD39 + NK cells may serve as a potential prognostic biomarker for ESCC patients.

Objective/background: Chronic lymphocytic leukemia is one of the commonest leukemias affecting adults. CD39 inhibits T-cell and Natural killer (NK) cell responses by hydrolyzing adenosine triphosphate and adenosine diphosphate, suppressing the immune system. We investigated expression of CD39 on CD4+ T Lymphocytes in chronic lymphocytic leukemia (CLL) patients and its relationship with deletion 6q, its association with disease stage and survival., Methods: Thirty CLL patients and 20 matched controls were included in the study. Bone marrow studies with immunophenotyping, CD39, CD38, and ZAP-70, and detection of del 6q by FISH were performed., Results: CD39+ CD4+ T helper cells in CLL patients were significantly expressed compared with the controls (p < .001). Levels of CD39+ CD4+ T cells were significantly expressed in high risk CLL patients. Del 6q was detected in 63.3% of patients and it correlated with CD39, CD38, and ZAP-70, and advanced stage disease. There was a significant relation between response to treatment and CD39 expression and del 6q, also there was a significant difference in overall survival (OS) between patients with and without Del 6q., Conclusion: CD39 expression on CD4+ Tcells and del 6q act as prognostic markers in CLL. Blocking or inhibition of CD39 may be a target for new immune therapy for CLL., (Copyright © 2018 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.)

Background: Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease, where there is irreversible breakdown of immunological self-tolerance. Extracellular adenosine triphosphate (ATP) and adenosine are signaling molecules that play an important part in the immune response. During inflammation and the immune response, a group of enzymes control these molecules, including ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA). We determined the activity and expression of E-NTPDase, the expression of E-5'-nucleotidase, the activity of E-ADA in lymphocytes and serum of SLE patients., Methods: This study involved 35 patients with SLE and 30 healthy subjects as a control group. E-NTPDase activity and expression were increased in lymphocytes from SLE patients (31% and 37% for activity and expression, respectively) compared with the control group., Results: An approximately 42% increase in E-ADA activity in lymphocytes was observed in SLE patients compared with the control group, in serum the ADA activity was decreased by 57% in SLE patients. Expression of E-5'-nucleotidase was not changed in SLE patients., Conclusions: E-NTPDase and E-ADA perform key functions in the modulation of the immune and inflammatory response in SLE., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Extracellular adenosine triphosphate (ATP) is up-regulated in the airways of patients with chronic obstructive pulmonary disease (COPD), resulting in increased inflammation, bronchoconstriction, and cough. Although extracellular ATP levels are tightly controlled by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; also known as CD39) in the lungs, the role of CD39 in the pathology of COPD is unknown. We hypothesized that alterations in the expression and activity of CD39 could be part of the mechanisms for initiating and perpetuating the disease., Methods: We analyzed CD39 gene and protein expression as well as ATPase enzyme activity in lung tissue samples of patients with COPD (n = 17), non-obstructed smokers (NOS) (n = 16), and never smokers (NS) (n = 13). Morphometry studies were performed to analyze pulmonary vascular remodeling., Results: There was significantly decreased CD39 gene expression in the lungs of the COPD group (1.17 [0.85-1.81]) compared with the NOS group (1.88 [1.35-4.41]) and NS group (3.32 [1.23-5.39]) (p = 0.037). This attenuation correlated with higher systemic inflammation and intimal thickening of muscular pulmonary arteries in the COPD group. Lung CD39 protein levels were also lower in the COPD group (0.34 [0.22-0.92]) compared with the NOS group (0.67 [0.32-1.06]) and NS group (0.95 [0.4-1.1) (p = 0.133). Immunohistochemistry showed that CD39 was downregulated in lung parenchyma, epithelial bronchial cells, and the endothelial cells of pulmonary muscular arteries in the COPD group. ATPase activity in human pulmonary structures was reduced in the lungs of patients with COPD., Conclusion: An attenuation of CD39 expression and activity is presented in lung tissue of stable COPD patients, which could lead to pulmonary ATP accumulation, favoring the development of pulmonary inflammation and emphysema. This may be a mechanism underlying the development of COPD.

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

Background: Renal ischemia-reperfusion injury (IRI) leads to acute kidney injury and renal fibrosis. CD39 is a key purinergic enzyme in the hydrolysis of adenosine triphosphate (ATP) and increased CD39 enzymatic activity protects from acute IRI but its effect on renal fibrosis is not known., Methods: Using a mouse model of unilateral renal IRI, the effects of increased CD39 activity (using soluble apyrase and mice expressing human CD39 transgene) on acute and chronic renal outcomes were examined. Nucleotide (ATP, adenosine diphosphate, adenosine monophosphate) and nucleoside (adenosine and inosine) levels were quantified by high-performance liquid chromatography. Soluble apyrase reduced acute renal injury at 24 hours and renal fibrosis at 4 weeks post-IRI, compared with vehicle-treated mice., Results: Soluble apyrase reduced renal ATP, adenosine diphosphate, and adenosine monophosphate, but not adenosine levels, during ischemia. In comparison with wild-type littermates, hCD39 transgenic mice were protected from acute renal injury at 24 hours, but had increased renal fibrosis at 4 weeks post-IRI. hCD39 transgene expression was localized to the vascular endothelium at baseline and did not affect total renal nucleotide and nucleoside levels during ischemia. However, hCD39 transgene was more widespread at 4 weeks post-IRI and was associated with higher renal adenosine levels at 4 weeks post-IRI compared with wild-type littermates., Conclusions: A single dose of apyrase administration before IRI protects from both acute and chronic renal injuries and may have clinical application in protection from ischemic-induced renal injury. Furthermore, transgenic expression of hCD39 is associated with increased renal fibrosis after ischemia.

Background: Cerebral tissue damage after an ischemic event can be exacerbated by inflammation and thrombosis. Elevated extracellular ATP and ADP levels are associated with cellular injury, inflammation, and thrombosis. Ectonucleoside triphosphate diphosphohydrolase-1 (CD39), an enzyme expressed on the plasmalemma of leukocytes and endothelial cells, suppresses platelet activation and leukocyte infiltration by phosphohydrolyzing ATP/ADP. To investigate the effects of increased CD39 in an in vivo cerebral ischemia model, we developed a transgenic mouse expressing human CD39 (hCD39)., Methods: A floxed-stop sequence was inserted between the promoter and the hCD39 transcriptional start site, generating a mouse in which the expression of hCD39 can be controlled tissue-specifically using Cre recombinase mice. We generated mice that express hCD39 globally or in myeloid-lineage cells only. Cerebral ischemia was induced by middle cerebral artery occlusion. Infarct volumes were quantified by MRI after 48 hours., Results: Both global and transgenic hCD39- and myeloid lineage CD39-overexpressing mice (transgenic, n=9; myeloid lineage, n=6) demonstrated significantly smaller cerebral infarct volumes compared with wild-type mice. Leukocytes from ischemic and contralateral hemispheres were analyzed by flow cytometry. Although contralateral hemispheres had equal numbers of macrophages and neutrophils, ischemic hemispheres from transgenic mice had less infiltration (n=4). Transgenic mice showed less neurological deficit compared with wild-type mice (n=6)., Conclusions: This is the first report of transgenic overexpression of CD39 in mice imparting a protective phenotype after stroke, with reduced leukocyte infiltration, smaller infarct volumes, and decreased neurological deficit. CD39 overexpression, either globally or in myeloid lineage cells, quenches postischemic leukosequestration and reduces stroke-induced neurological injury., (© 2017 American Heart Association, Inc.)

Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Nucleoside triphosphate diphosphohydrolase-1 (ENTPD1/CD39) is the rate-limiting enzyme in a cascade leading to the generation of immunosuppressive adenosine and plays an important role in tumor progression. This study aimed to evaluate the expression of CD39 and CD39Foxp3 regulatory T cells (Tregs) and to determine their prognostic role in patients with hepatocellular carcinoma (HCC) after radical resection.Immunohistochemistry (IHC) and double IHC were used to analyze CD39 expression or the expression of CD39 and Foxp3 in a cohort of 324 HCC patients who underwent curative resection. The quantification of CD39 expression levels was determined using a computerized image analysis system and was evaluated by mean optical density (MOD), which corresponded to the positive staining intensity of CD39. The number of positive Foxp3 cells and both CD39 and Foxp3 positive cells in each 1-mm-diameter cylinder were counted under high-power magnification (×400). The "minimum P value" approach was used to obtain the optimal cutoff value for the best separation between groups of patients in relation to time to recurrence (TTR) or overall survival (OS). The expression of CD39 in HCC cell lines with stepwise metastatic potential and in human umbilical vein endothelial cells was determined by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence. The SPSS 17.0 statistical package was used for statistics.CD39 was principally expressed on vascular endothelial cells, macrophagocytes, Tregs, and tumor cells in HCC. Compared with paired peritumoral tissues, tumoral tissues had a significantly higher expression level of CD39 (P < 0.0001). Overexpression of tumoral CD39 was related to increased tumor recurrence and shortened overall survival. Furthermore, the expression level of peritumoral CD39 showed a prognostic role in TTR and OS. Double IHC showed that tumoral tissues had significantly higher Foxp3Tregs and CD39Foxp3Tregs count per 1 mm core (14.1659 vs 4.9877, P = 0.001; 11.5254 vs 3.3930, P < 0.001) and a higher CD39Foxp3/Foxp3 ratio compared with paired peritumoral tissues. CD39Foxp3Tregs were a better prognosticator than CD39Tregs for TTR.Overexpression of CD39 protein in HCC was an independent predictor of poor outcome after radical resection. The CD39Foxp3Tregs count added prognostic power to Foxp3Tregs, providing a potential target for tumor immunotherapy., Competing Interests: The authors report no conflicts of interest.

Obesity-induced inflammation is conducted by a metabolic pathway, which eventually causes activation of specialized immune cells and leads to an unresolved inflammatory response within the tissue. For this reason, it is critically important to determine how hypertrophic fat tissue alters T cell balance to drive inflammation. In this study, we identify the purinergic signaling as a novel mechanism driving the adaptive Th17 response in human visceral adipose tissue (VAT) of metabolically unhealthy obese patients. We demonstrate that ATP acting via the P2X7 receptor pathway promotes a Th17 polarizing microenvironment with high levels of IL-1β, IL-6, and IL-17 in VAT explants from lean donors. Moreover, in vitro blockade of the P2X7 receptor abrogates the levels of these cytokines. These findings are consistent with a greater frequency of Th17 cells in tissue from metabolically unhealthy obese donors, revealed not only by the presence of a baseline Th17-promoting milieu, but also by the higher expression of steadily recognized Th17 markers, such as RORC, IL-17 cytokine, and IL-23R, in comparison with metabolically healthy obese and lean donors. In addition, we demonstrate that CD39 expression on CD4(+)effector T cells represents a novel Th17 marker in the inflamed VAT, which also confers protection against ATP-induced cell death. The manipulation of the purinergic signaling might represent a new therapeutic target to shift the CD4(+)T cell balance under inflammatory conditions., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Mesenchymal stem cells (MSCs) are population of adult stem cells and attractive candidates for cartilage repair due to their chondrogenic potential. Purinergic compounds (purinergic receptors and ecto-enzymes metabolizing nucleotides), together with nucleotides/nucleosides present in the extracellular environment, are known to play a key role in controlling the stem cells biological potential to proliferate and differentiate. Despite the available literature pointing to the importance of purinergic signaling in controlling the fate of MSCs, the research results linking nucleotides and ecto-nucleotidases with MSCs chondrogenic differentiation are indigent. Therefore, the aim of presented study was the characterization of the ecto-nucleotides hydrolysis profile and ecto-enzymes expression in human umbilical cord-derived MSCs and chondrogenically induced MSCs. We described substantial changes of ecto-nucleotides metabolism and ecto-enzymes expression profiles resulting from chondrogenic differentiation of human umbilical cord-derived MSCs. The increased rate of ADP hydrolysis, measured by ecto-nucleotidases activity, plays a pivotal role in the regulation of cartilage formation and resorption. Despite the increased level of NTPDase1 and NTPDase3 mRNA expression in chondrogenically induced MSCs, their activity toward ATP remains quite low. Supported by the literature data, we hypothesize that structure-function relationships in chondrogenic lineage dictate the direction of nucleotides metabolism. In early neocartilage tissue, the beneficial role of ATP in improving biomechanical properties of cartilage does not necessitate the high rate of enzymatic ATP degradation., (© 2015 Wiley Periodicals, Inc.)

The ectonucleotidase CD39 is pivotal in the conversion of immunostimulatory adenosine triphosphate (ATP) into immunosuppressive adenosine which potently inhibits host immune responses against cancer. This study investigated the expression level and prognostic significance of CD39 in human rectal adenocarcinoma. Our data demonstrated that CD39 staining strongly marked malignant epithelial cells where the protein and messenger RNA (mRNA) expression levels of CD39 were significantly increased compared with paracancerous controls. In addition to primary tumors, CD39 was also abundantly expressed in liver metastases and tumor-draining lymph nodes from metastatic rectal adenocarcinoma. Although patients with higher CD39 density in tumor cells were more likely to have favorable characteristics (early TNM and N stages) and overall survival, the singular parameter cannot be used as an independent factor for predicting patients' prognosis. Intriguingly, combined analysis of CD39 and CD73 expression was more efficient to foretell patient's outcome where patients with increased CD73 but decreased CD39 levels displayed a worst prognosis. Taken together, the current study revealed that malignant epithelial cells of human rectal adenocarcinoma strongly express CD39 that may play a potential role in the tumor invasion and metastasis. Although high expression of CD39 in tumor cells is correlated with favorable clinical outcome, the combination of CD39 and CD73 expression may have a better prognostic value.

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39(+)/CD8(+) ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39(+)/CD8(+) ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.

The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E-deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39+/-Apoe-/- mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe-/- mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress.

Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The EGFR-targeted antibody cetuximab is effective against head and neck cancer (HNSCC), but in only 15% to 20% of patients, and the variability and extent of cetuximab-mediated cellular immunity is not fully understood. We hypothesized that regulatory T cells (Treg) may exert a functional and clinical impact on antitumor immunity in cetuximab-treated individuals. The frequency, immunosuppressive phenotype, and activation status of Treg and natural killer (NK) cells were analyzed in the circulation and tumor microenvironment of cetuximab-treated patients with HNSCC enrolled in a novel neoadjuvant, single-agent cetuximab clinical trial. Notably, cetuximab treatment increased the frequency of CD4(+)FOXP3(+) intratumoral Treg expressing CTLA-4, CD39, and TGFβ. These Treg suppressed cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and their presence correlated with poor clinical outcome in two prospective clinical trial cohorts. Cetuximab expanded CTLA-4(+)FOXP3(+) Treg in vitro, in part, by inducing dendritic cell maturation, in combination with TGFβ and T-cell receptor triggering. Importantly, cetuximab-activated NK cells selectively eliminated intratumoral Treg but preserved effector T cells. In ex vivo assays, ipilimumab targeted CTLA-4(+) Treg and restored cytolytic functions of NK cells mediating ADCC. Taken together, our results argue that differences in Treg-mediated suppression contribute to the clinical response to cetuximab treatment, suggesting its improvement by adding ipilimumab or other strategies of Treg ablation to promote antitumor immunity., (©2015 American Association for Cancer Research.)

Endothelial cell (EC) dysfunction plays a role in the pathobiology of occlusive vasculopathy in pulmonary arterial hypertension (PAH). Purinergic signaling pathways, which consist of extracellular nucleotide and nucleoside-mediated cell signaling through specific receptors, are known to be important regulators of vascular tone and remodeling. Therefore, we hypothesized that abnormalities in the vascular purinergic microenvironment are associated with PAH. Enzymatic clearance is crucial to terminate unnecessary cell activation; one of the most abundantly expressed enzymes on the EC surface is E-NTPDase1/CD39, which hydrolyzes ATP and ADP to AMP. we used histological samples from patients and healthy donors, radioisotope-labeled substrates to measure ectoenzyme activity, and a variety of in vitro approaches to study the role of CD39 in PAH. Immunohistochemistry on human idiopathic PAH (IPAH) patients' lungs demonstrated that CD39 was significantly downregulated in the endothelium of diseased small arteries. Similarly, CD39 expression and activity were decreased in cultured pulmonary ECs from IPAH patients. Suppression of CD39 in vitro resulted in EC phenotypic switch that gave rise to apoptosis-resistant pulmonary arterial endothelial cells and promoted a microenvironment that induced vascular smooth muscle cell migration. we also identified that the ATP receptor P2Y11 is essential for ATP-mediated EC survival. Furthermore, we report that apelin, a known regulator of pulmonary vascular homeostasis, can potentiate the activity of CD39 both in vitro and in vivo. we conclude that sustained attenuation of CD39 activity through ATP accumulation is tightly linked to vascular dysfunction and remodeling in PAH and could represent a novel target for therapy., (Copyright © 2015 the American Physiological Society.)

Background: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown., Methods: On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon., Results: This preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed., Conclusions: Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

T cells of the T helper (Th)17 subset offer promise in adoptive T-cell therapy for cancer. However, current protocols for ex vivo programming of Th17 cells, which include TGFβ exposure, increase the expression of CD39 and CD73, two cell surface ATP ectonucleotidases that reduce T-cell effector functions and promote immunosuppression. Here, we report that ATP-mediated suppression of IFNγ production by Th17 cells can be overcome by genetic ablation of CD73 or by using IL1β instead of TGFβ to program Th17 cells ex vivo. Th17 cells cultured in IL1β were also highly polyfunctional, expressing high levels of effector molecules and exhibiting superior short-term control of melanoma in mice, despite reduced stem cell-like properties. TGFβ addition at low doses that did not upregulate CD73 expression but induced stemness properties drastically improved the antitumor effects of IL1β-cultured Th17 cells. Effector properties of IL1β-dependent Th17 cells were likely related to their high glycolytic capacity, since ex vivo programming in pyruvate impaired glycolysis and antitumor effects. Overall, we show that including TGFβ in ex vivo cultures used to program Th17 cells blunts their immunotherapeutic potential and demonstrate how this potential can be more fully realized for adoptive T-cell therapy., (©2014 American Association for Cancer Research.)

Treg cells are important for the maintenance of self-tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP-hydrolyzing enzymes CD39 and CD73 and the resulting anti-inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3(+) Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3(+) Treg cells displayed high expression levels of rate-limiting CD39, whereas CD73 was diminished. FOXP3(+) CD39(+) Treg cells were also abundant in synovial tissue. Furthermore, FOXP3(+) CD39(+) Treg cells did not secrete the proinflammatory cytokines IFN-γ and TNF after in vitro stimulation in contrast to FOXP3(+) CD39(-) T cells. FOXP3(+) CD39(+) Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg-specific demethylated region and coculture assays confirmed that CD25(+) CD39(+) T cells have suppressive capacity, while their CD39(-) counterparts do not. Overall, our data show that FOXP3(+) CD39(+) Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T-cell functions including production of IFN-γ, TNF, and IL-17F but do not limit IL-17A secretion., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-β type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-β signaling. In this study, we tested the hypothesis that TGF-β drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-β receptor in mammary epithelium, an increased level of TGF-β protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFβRII(WT) control tumors with intact TGF-β signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-β signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-β signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-β signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment., (Copyright © 2014 by The American Association of Immunologists, Inc.)

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

Nucleoside transporter (NT) and nucleic-related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1-3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide., (© 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Société Française de Pharmacologie et de Thérapeutique.)

The role of ecto-5'-nucleotidase (CD73), an enzyme providing interstitial adenosine, was investigated in B16F10 melanoma progression. Chemical inhibition of CD73 decreased adherence of cells to extracellular matrix proteins in vitro and led to enhanced migration and invasion. Both processes were reversed by adenosine receptor agonists. In CD73‑deficient mice, tumor growth was decreased in comparison with that of wild-type animals. Additionally, the vasculature of CD73-inhibited tumors was impaired and neoangiogenesis in Matrigel plugs was reduced. It is, therefore, proposed that although CD73 shows anti-invasive and antimigratory function in B16F10 melanoma cells, its proangiogenic action is prevalent in vivo and may contribute to increased tumor growth.

Unlabelled: Hepatic innate immune cells, in particular, interstitial dendritic cells (DCs), regulate inflammatory responses and may promote inherent liver tolerogenicity. After tissue injury, adenosine triphosphate (ATP) is released and acts as a damage-associated molecular pattern that activates innate immune cells by pattern recognition receptors. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) rapidly hydrolyzes extracellular ATP to maintain physiological levels. We hypothesized that CD39 expression on liver DCs might contribute to regulation of their innate immune functions. Mouse liver conventional myeloid DCs (mDCs) were hyporesponsive to ATP, compared with their splenic counterparts. This disparity was ascribed to more efficient hydrolysis of ATP by higher expression of CD39 on liver mDCs. Human liver mDCs expressed greater levels of CD39 than those from peripheral blood. The comparatively high expression of CD39 on liver mDCs correlated strongly with both ATP hydrolysis and adenosine production. Notably, CD39(-/-) mouse liver mDCs exhibited a more mature phenotype, greater responsiveness to Toll-like receptor 4 ligation, and stronger proinflammatory and immunostimulatory activity than wild-type (WT) liver mDCs. To investigate the role of CD39 on liver mDCs in vivo, we performed orthotopic liver transplantation with extended cold preservation using CD39(-/-) or WT donor mouse livers. Compared to WT liver grafts, CD39(-/-) grafts exhibited enhanced interstitial DC activation, elevated proinflammatory cytokine levels, and more-severe tissue injury. Moreover, portal venous delivery of WT, but not CD39(-/-) liver mDCs, to donor livers immediately post-transplant exerted a protective effect against graft injury in CD39(-/-) to CD39(-/-) liver transplantation., Conclusions: These data reveal that CD39 expression on conventional liver mDCs limits their proinflammatory activity and confers protective properties on these important innate immune cells against liver transplant ischemia/reperfusion injury., (© 2013 by the American Association for the Study of Liver Diseases.)

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2(+/-) mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2(+/-) mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2(+/-) mice, kindlin-2(+/-) endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5'-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5'-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73) were increased twofold to threefold on kindlin-2(+/-) ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2(+/-) mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis.

The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39-. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.

Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders.

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

Unlabelled: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs., Control: 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of this study is to investigate the expression of P2X7R, IL-1beta and the ATP activity modulating ecto-apyrase CD39 on peripheral blood monocytes of MS patients and to observe the possible effects of Glatiramer Acetate (GA) on such expression. Twelve RR treatment-free MS patients were selected and peripheral blood monocytes were obtained. The expression of P2X7R, IL-1beta and CD39 on monocytes was investigated by qrt-PCR. The in vitro effects of GA on the expression of monocytes stimulated with BzATP (a potent P2X7R agonist)-were evaluated. Ten healthy donors (HDs) were similarly studied. Finally, 5 MS patients were given GA therapy and the monocytes obtained before treatment, after 3 and 12 months of GA treatment were similarly investigated. No differences were found in P2X7R, IL-1beta and CD39 expression between patients and controls. In MS Bz-ATP stimulated monocytes, GA pre-conditioning clearly downregulated P2X7R (p=0.003) but IL-1beta expression also showed a decreasing trend (p=0.07). Conversely, CD39 showed an increasing trend (p=0.07). Similar evidence was found in HDs. GA in vivo treatment induced a reduction in the expression that was clear for P2X7R and CD39 (p<0.05) but only not significant for IL-1beta after 12 months of treatment. Monocytes from both MS and control subjects express P2X7R, IL-1beta and CD39, and GA seems to interfere with such expression., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, percentages of CD39(+) Treg and Th17 cells were compared between relapsing-remitting MS patients and controls and were related to the vitamin D status. The Th17 cell population was expanded in about 40% of the MS patients. In MS patients in remission, but not during relapse, a positive association was found between Th17 cell and CD39(+) Treg percentages (r=0.468, p=0.007). Since CD39(+) Tregs have been shown to have Th17 suppressive capacities, we propose that a dysregulated Th17/CD39(+) Treg balance might contribute to disease exacerbation. A clear role for vitamin D in this regulation could not be established., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-β. To identify the responsible regulatory CD4(+) T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39(+)CD4(+) T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39(+)CD4(+) T cells into CIA mice conferred complete protection, whereas CD39(-)CD4(+) T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39(+) T cells were composed of Foxp3-GFP(-) and Foxp3-GFP(+) subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced Foxp3(-) and Foxp3(+)CD39(+)CD4(+) T cells could protect against CIA, each subset was not as efficacious as total CD39(+)CD4(+) T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3(-) CD39(+)CD4(+) T cells produced TGF-β, and Foxp3(+)CD39(+)CD4(+) T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP(-) CD4(+) T cells converted to Foxp3-GFP(+) CD39(+)CD4(+) T cells in the recipients, showing plasticity of these regulatory T cells. TGF-β was found to be essential for protection because in vivo TGF-β neutralization reversed activation of CREB and reduced the development of CD39(+)CD4(+) T cells. Thus, CD39 apyrase-expressing CD4(+) T cells stimulated by Salmonella-CFA/I are composed of TGF-β-producing Foxp3(-) CD39(+)CD4(+) T cells and support the stimulation of IL-10-producing Foxp3(+) CD39(+)CD4(+) T cells.

Levels of anti-inflammatory extracellular adenosine are controlled by the sequential action of the ectonucleotidases CD39 and CD73, whose expression in CD4(+) T cells has been associated with natural regulatory T cells (nTregs). We here show that CD73 expression on activated murine CD4(+) T cells is induced by TGF-β independently of Foxp3 expression, operates at the transcriptional level and translates into gain of functional capacity to generate adenosine. In the presence of AMP, CD73 induced by TGF-β generates adenosine able to suppress proliferation of activated CD4(+) T cells in vitro. These effects are contextual and opposed by proinflammatory cytokines. CD73 is also upregulated by TGF-β in CD8(+) T cells, DCs and macrophages, so providing an amplification mechanism for adenosine generation in tissue microenvironments. Together, these findings expose a novel anti-inflammatory role for TGF-β., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Regulatory T cells (Treg) diminish immune responses to microbial infection, which may contribute to preventing inflammation-related local tissue damage and autoimmunity but may also contribute to chronicity of infection. Nasopharyngeal carriage of pneumococcus is common in young children and can persist for long periods but it is unknown whether the presence of Treg in the nasopharynx contributes to this persistence. We have investigated the numbers and activities of Foxp3+Treg in adenoidal tissues and their association with pneumococcal carriage in children. Expression of Treg cell-related markers including Foxp3, CD25, CD39, CD127 and CLTA4 were analysed by flow-cytometry in adenoidal mononuclear cells (MNC) and PBMC from children. Unfractionated MNC or Treg-depleted MNC were stimulated with a pneumococcal whole cell antigen (WCA) and T cell proliferation measured. Cytokine production by MNC was measured using a cytometric bead array. Higher numbers of CD25(high)Foxp3(high) Treg expressing higher CD39 and CTLA4 were found in adenoidal MNC than in PBMC. Children with pneumococcus positive nasopharyngeal cultures had higher proportions of Treg and expressed higher levels of CD39 and CTLA-4 than those who were culture negative (-). WCA induced adenoidal Treg proliferation which produce IL10 but not IL17, and CD4 T cell proliferation in Treg-depleted MNC was greater in pneumococcal culture positive than negative children. Significant numbers of Treg with an effector/memory phenotype which possess a potent inhibitory effect, exist in adenoidal tissue. The association of pneumococcal carriage with an increased frequency of adenoidal Treg suggests that Treg in nasal-associated lymphoid tissue (NALT) may contribute to the persistence of pneumococcus in children. Further studies to determine what component and mechanisms are involved in the promotion of Treg in NALT may lead to novel therapeutic or vaccination strategy against upper respiratory infection.

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.

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.

Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Once released, norepinephrine is removed from cardiac synapses via reuptake into sympathetic nerves, whereas transmitter ATP is catabolized by ecto-NTP diphosphohydrolase 1 (E-NTPDase1)/CD39, an ecto-ATPase. Because ATP is known to modulate neurotransmitter release at prejunctional sites, we questioned whether this action may be ultimately controlled by the expression of E-NTPDase1/CD39 at sympathetic nerve terminals. Accordingly, we silenced E-NTPDase1/CD39 expression in nerve growth factor-differentiated PC12 cells, a cellular model of sympathetic neuron, in which dopamine is the predominant catecholamine. We report that E-NTPDase1/CD39 deletion markedly increases depolarization-induced exocytosis of ATP and dopamine and increases ATP-induced dopamine release. Moreover, overexpression of E-NTPDase1/CD39 resulted in enhanced removal of exogenous ATP, a marked decrease in exocytosis of ATP and dopamine, and a large decrease in ATP-induced dopamine release. Administration of a recombinant form of E-NTPDase1/CD39 reproduced the effects of E-NTPDase1/CD39 overexpression. Exposure of PC12 cells to simulated ischemia elicited a release of ATP and dopamine that was markedly increased in E-NTPDase1/CD39-silenced cells and decreased in E-NTPDase1/CD39-overexpressing cells. Therefore, transmitter ATP acts in an autocrine manner to promote its own release and that of dopamine, an action that is controlled by the level of E-NTPDase1/CD39 expression. Because ATP availability greatly increases in myocardial ischemia, recombinant E-NTPDase1/CD39 therapeutically used may offer a novel approach to reduce cardiac dysfunctions caused by excessive catecholamine release.

Regulatory T cells (Tregs) act by suppressing the activation and effector functions of innate and adaptive immune responses. HIV infection impacts Treg proportion and phenotype, although discrepant results have been reported depending on the patient population and the way Tregs were characterized. The effects of highly active antiretroviral therapy (HAART) on Treg frequency have not been thoroughly documented. We performed a detailed longitudinal analysis of Treg frequency and phenotype in 11 HIV-infected individuals enrolled in a single, prospective clinical trial, in which all patients underwent the same treatment protocol and were sampled at the same time points. Tregs were characterized for their expression of molecules associated with activation, cell cycle, apoptosis, or function, and compared to circulating Tregs from a group of age-matched healthy individuals.Our results revealed increased proportions, but reduced absolute numbers of circulating CD3(+)CD4(+)FOXP3(+) Tregs in chronically infected HIV-infected patients. Treg frequency was largely normalized by HAART. Importantly, we show that similar conclusions were drawn regardless of the combination of markers used to define Tregs. Our results also showed increased expression of cell cycle markers (Ki67 and cyclin B) in Tregs from untreated infected individuals, which were decreased by HAART. However, the Treg phenotype in untreated patients was not consistent with a higher level of generalized activation, as they expressed very low levels of CD69, slightly elevated levels of HLA-DR and similar levels of GARP compared to Tregs from uninfected donors. Moreover, none of these markers was significantly changed by HAART. Treg expression of CTLA-4 and cytotoxic molecules was identical between patients and controls. The most striking difference in terms of functional molecules was the high expression of CD39 by Tregs in untreated patients, which HAART only partially controlled.

The vascular ectonucleotidases CD39[ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1), EC 3.6.1.5] and CD73[EC 3.1.3.5] generate adenosine from extracellular nucleotides. CD39 activity is critical in determining the response to ischemia-reperfusion injury (IRI), and CD39 null mice exhibit heightened sensitivity to renal IRI. Adenosine has multiple mechanisms of action in the vasculature including direct endothelial protection, antiinflammatory and antithrombotic effects and is protective in several models of IRI. Mice transgenic for human CD39 (hCD39) have increased capacity to generate adenosine. We therefore hypothesized that hCD39 transgenic mice would be protected from renal IRI. The overexpression of hCD39 conferred protection in a model of warm renal IRI, with reduced histological injury, less apoptosis and preserved serum creatinine and urea levels. Benefit was abrogated by pretreatment with an adenosine A2A receptor antagonist. Adoptive transfer experiments showed that expression of hCD39 on either the vasculature or circulating cells mitigated IRI. Furthermore, hCD39 transgenic kidneys transplanted into syngeneic recipients after prolonged cold storage performed significantly better and exhibited less histological injury than wild-type control grafts. Thus, systemic or local strategies to promote adenosine generation and signaling may have beneficial effects on warm and cold renal IRI, with implications for therapeutic application in clinical renal transplantation., (©2010 The Authors Journal compilation©2010 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The mechanisms that lead to loss of tolerance in autoimmune disease have remained both elusive and diverse, including both genetic predisposition and generic dysregulation of critical mononuclear cell subsets. In primary biliary cirrhosis (PBC), patients exhibit a multilineage response to the E2 component of pyruvate dehydrogenase involving antibody as well as autoreactive CD4 and CD8 responses. Recent data from murine models of PBC have suggested that a critical mechanism of biliary destruction is mediated by liver-infiltrating CD8 cells. Further, the number of autoreactive liver-infiltrating CD4 and CD8 cells is significantly higher in liver than blood in patients with PBC. Based on this data, we have studied the frequencies and phenotypic characterization of both CD4 and CD8 regulatory T cell components in both patients with PBC and age-sex matched controls. Our data is striking and indicate that CD8 Treg populations from PBC patients, but not controls, have significant phenotypic alterations, including increased expression of CD127 and reduced CD39. Furthermore, in vitro induction of CD8 Tregs by incubation with IL10 is significantly reduced in PBC patients. Importantly, the frequencies of circulating CD4+CD25+ and CD8+ and CD28- T cell subpopulations are not significantly different between patients and controls. In conclusion, these data identify the CD8 Treg subset as a regulatory T cell subpopulation altered in patients with PBC., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+ Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+ CD25+ CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+ CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+ CD25+ CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection., (©2010 The Authors Journal compilation©2010 The American Society of Transplantation and the American Society of Transplant Surgeons.)