Abstract 5168: Adenosine Formed by CD73 on Treg Inhibits T-cell Mediated Immune Response via NFkB
CD73-derived adenosine (ADO) acts as potent inhibitor of inflammation and we have recently shown that the CD73−/−/ApoE−/− double mutants show enhanced plaque formation with local accumulation of immune cells. Since regulatory T-cells (Treg) have been recently shown to express CD73 as a novel marker enzyme, the present study explored the potential role of CD73-derived adenosine on NFkB activity and cytokine release in CD4+ cells. We asked: does adenosine physiologically formed by Treg inhibit the immune response of the entire Th-cell population? Does the lack of CD73 alter the expression pattern of various enzymes of the ecto-nucleotidase cascade and ATP/adenosine receptors in Tregs? T-cells, isolated from murine spleens by magnetic micro beads, were stimulated with αCD3/αCD28 which activates the nuclear translocation of transcriptionfactor NFκB, known to be involved in the secretion of pro-inflammatory cytokines such as IL-2. Using EMSA we found activated NFκB in T-cells lacking CD73 to be significantly upregulated (WT: 4.36±0.21, CD73−/−: 6.58±0.75; n=4; p=0.029) which was paralleled by increases in release of the pro-inflammatory cytokines IL-2 and IFN-γ (ELISA). Treatment of T-cells with ADO (25 μM) caused a massive A2a ADO receptor mediated reduction of IL-2 and IFN-ã release. Similarly, AMP (50 μ M) decreased the IFN-ã release of WT CD4+ cells. Expression analysis revealed that there were no differences in the enzymes and receptors of the ecto-nucleotidase cascade in WT and CD73−/− Tregs. Upon stimulation with ãCD3/ãCD28, however, mRNA levels of the ATP receptor P2X7 - a ligand-gated ion channel that controls CD4+ activation - was significantly reduced. Expression of the ADO transporter CNT2 was also significantly reduced while the expression of the A2b ADO receptor was increased. In summary, our data show that stimulated CD73-deficient T-cells show a proinflammtory phenotype with increased levels of active NFκ B and released proinflammatory cytokines. Thus, CD73-derived ADO is a physiological inhibitor of the immune response relevant to atherogenesis. Downregulation of P2X7 on Tregs may be important to prevent apoptosis and to maintain suppressive function of Tregs by converting extracellular ATP to ADO.