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.

Single-cell Profiles and Prognostic Impact of Tumor-Infiltrating Lymphocytes Coexpressing CD39, CD103, and PD-1 in Ovarian Cancer.

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.

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.

Impact of CD39 expression on CD4+ T lymphocytes and 6q deletion on outcome of patients with chronic lymphocytic leukemia.

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.

Activity and expression of E-NTPDase is altered in peripheral lymphocytes of systemic lupus erythematosus patients.

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.

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.

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.

The Differential Effect of Apyrase Treatment and hCD39 Overexpression on Chronic Renal Fibrosis After Ischemia-Reperfusion Injury.

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.

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.

Overexpression of CD39 in hepatocellular carcinoma is an independent indicator of poor outcome after radical resection.

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.

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.

Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis.

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.

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.

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.

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.

CTLA-4⁺ Regulatory T Cells Increased in Cetuximab-Treated Head and Neck Cancer Patients Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis.

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 TGFß. 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 TGFß 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.

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.