Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets
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Regulatory T cells that express CD39 (CD39 Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39 Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39 Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4 T cells, CD4 CD25 T cells and CD4 CD25 CD39 T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39 Treg cells and higher levels of ART1 expression in CD4 CD39 T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39 Treg or CD39- Treg cells was observed by 1mM ATP or 60. μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300. μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39 Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD. © 2015 Elsevier GmbH.
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Regulatory T cells that express CD39 (CD39%2b Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39%2b Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39%2b Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4%2b T cells, CD4%2b CD25%2b T cells and CD4%2b CD25%2b CD39%2b T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39%2b Treg cells and higher levels of ART1 expression in CD4%2b CD39%2b T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39%2b Treg or CD39- Treg cells was observed by 1mM ATP or 60. μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300. μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39%2b Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD. © 2015 Elsevier GmbH.
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ART1; ATP; CD39; Immune regulation; NAD; P2X7 receptor adenosine A2a receptor agonist; adenosine A2a receptor antagonist; adenosine triphosphate; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 1; purinergic P2X7 receptor; 2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; 4 [2 [7 amino 2 (2 furyl) 1,2,4 triazolo[2,3 a][1,3,5]triazin 5 ylamino]ethyl]phenol; adenosine; adenosine A2 receptor; adenosine A2 receptor agonist; adenosine A2 receptor antagonist; adenosine triphosphate; apyrase; ART1 protein, human; CD39 antigen; glycosylphosphatidylinositol anchored protein; L selectin; leukocyte antigen; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase; P2XR receptor, human; phenethylamine derivative; purinergic P2X7 receptor; triazine derivative; triazole derivative; animal cell; Article; CD39 regulatory T lymphocyte; CD39- regulatory T lymphocyte; CD4 CD25 CD39 T lymphocyte; CD4 CD25 T lymphocyte; CD4 T lymphocyte; cell death; cell protection; controlled study; flow cytometry; human; human cell; immunomodulation; nonhuman; peripheral blood mononuclear cell; priority journal; protein expression; real time polymerase chain reaction; regulatory T lymphocyte; T cell depletion; T lymphocyte; adolescent; adult; analogs and derivatives; cell proliferation; cytology; drug effects; female; gene expression regulation; genetics; immunology; immunophenotyping; male; primary cell culture; signal transduction; T lymphocyte subpopulation; Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine Triphosphate; Adolescent; ADP Ribose Transferases; Adult; Antigens, CD; Apyrase; Cell Death; Cell Proliferation; Female; Gene Expression Regulation; GPI-Linked Proteins; Humans; Immunophenotyping; L-Selectin; Male; NAD; Phenethylamines; Primary Cell Culture; Receptors, Adenosine A2; Receptors, Purinergic P2X7; Signal Transduction; T-Lymphocyte Subsets; Triazines; Triazoles
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ART1; ATP; CD39; Immune regulation; NAD; P2X7 receptor adenosine A2a receptor agonist; adenosine A2a receptor antagonist; adenosine triphosphate; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase 1; purinergic P2X7 receptor; 2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; 4 [2 [7 amino 2 (2 furyl) 1,2,4 triazolo[2,3 a][1,3,5]triazin 5 ylamino]ethyl]phenol; adenosine; adenosine A2 receptor; adenosine A2 receptor agonist; adenosine A2 receptor antagonist; adenosine triphosphate; apyrase; ART1 protein, human; CD39 antigen; glycosylphosphatidylinositol anchored protein; L selectin; leukocyte antigen; nicotinamide adenine dinucleotide; nicotinamide adenine dinucleotide adenosine diphosphate ribosyltransferase; P2XR receptor, human; phenethylamine derivative; purinergic P2X7 receptor; triazine derivative; triazole derivative; animal cell; Article; CD39+ regulatory T lymphocyte; CD39- regulatory T lymphocyte; CD4+ CD25+ CD39+ T lymphocyte; CD4+ CD25+ T lymphocyte; CD4+ T lymphocyte; cell death; cell protection; controlled study; flow cytometry; human; human cell; immunomodulation; nonhuman; peripheral blood mononuclear cell; priority journal; protein expression; real time polymerase chain reaction; regulatory T lymphocyte; T cell depletion; T lymphocyte; adolescent; adult; analogs and derivatives; cell proliferation; cytology; drug effects; female; gene expression regulation; genetics; immunology; immunophenotyping; male; primary cell culture; signal transduction; T lymphocyte subpopulation; Adenosine; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine Triphosphate; Adolescent; ADP Ribose Transferases; Adult; Antigens, CD; Apyrase; Cell Death; Cell Proliferation; Female; Gene Expression Regulation; GPI-Linked Proteins; Humans; Immunophenotyping; L-Selectin; Male; NAD; Phenethylamines; Primary Cell Culture; Receptors, Adenosine A2; Receptors, Purinergic P2X7; Signal Transduction; T-Lymphocyte Subsets; Triazines; Triazoles
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