Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

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.

Pronounce expression of Tim-3 and CD39 but not PD1 defines CD8 T cells in critical Covid-19 patients.

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.

IL-6-induced CD39 expression on tumor-infiltrating NK cells predicts poor prognosis in esophageal squamous cell carcinoma.

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.

Ischemic Cerebroprotection Conferred by Myeloid Lineage-Restricted or Global CD39 Transgene Expression.

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.

Is the purinergic pathway involved in the pathology of COPD? Decreased lung CD39 expression at initial stages of COPD.

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.

Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension.

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)-1ß 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.

ATP-Induced Inflammation Drives Tissue-Resident Th17 Cells in Metabolically Unhealthy Obesity.

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-1ß, 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.

The expression of NTPDase1 and -2 of Leishmania infantum chagasi in bacterial and mammalian cells: Comparative expression, refolding and nucleotidase characterization.

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.

CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

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.

Role of TGF-ß signaling in generation of CD39+CD73+ myeloid cells in tumors.

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-ß type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-ß signaling. In this study, we tested the hypothesis that TGF-ß 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-ß receptor in mammary epithelium, an increased level of TGF-ß protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFßRII(WT) control tumors with intact TGF-ß signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-ß 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-ß 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-ß 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.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells Results in Substantial Changes of Ecto-Nucleotides Metabolism.

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.

Suppression of endothelial CD39/ENTPD1 is associated with pulmonary vascular remodeling in pulmonary arterial hypertension.

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.

Surface expression of CD39 identifies an enriched Treg-cell subset in the rheumatic joint, which does not suppress IL-17A secretion.

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.

Regulatory T cells negatively affect IL-2 production of effector T cells through CD39/adenosine pathway in HIV infection.

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.

Kindlin-2 regulates hemostasis by controlling endothelial cell-surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism.

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.

High expression of CD39/ENTPD1 in malignant epithelial cells of human rectal adenocarcinoma.

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.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

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 ß-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.

CD39 expression by hepatic myeloid dendritic cells attenuates inflammation in liver transplant ischemia-reperfusion injury in mice.

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.

Overexpression of CD39 in mouse airways promotes bacteria-induced inflammation.

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.

Characterisation of regulatory T cells in nasal associated lymphoid tissue in children: relationships with pneumococcal colonization.

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.