Regulatory T-cells (Treg) are known to alter the immune response of both Th1 and Th2 cells either by cell-cell contact or by the release of pro-/anti-inflammatory cytokines. Adenosine inhibits T-cell activation most likely through A2a receptors and can be formed by the extracellular degradation of adenine nucleotides involving the E-NTPDase (CD39) degrading ATP to ADP and AMP, and the ecto-5'-nucleotidase (CD73) converting AMP to adenosine. Recently it has been shown that CD39 and CD73 are marker enzymes of Treg.

The present study tested two hypotheses: Firstly, does adenosine physiologically formed by Treg inhibit the immune response of the entire Th-cell population? Secondly, can a phosphorylated A2a receptor agonist serve as prodrug, which after dephosphorylation by CD73 specifically inhibits the T-cell immune response?

T-cells, isolated from murine spleens by magnetic micro beads, were stimulated with aCD3/aCD28 which initiated the nuclear translocation of transcriptionfactor NFkB, known to be involved in the secretion of pro-inflammatory cytokines such as IL-2. Using EMSA we found activated NFkB 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-g (ELISA). Treatment of T-cells with AMP (50 mM) caused a massive reduction of IL-2 and IFN-g release which was less pronounced in CD73-/- cells. Similarly, the newly synthesised phosphorylated A2a receptor agonist Cyclo-Hexyl-Ethyl-Thio-AMP (chet-AMP) dose dependently decreased IFN-g release, the EC50 being 5mM. The EC50 of the dephosphorylated chet-AMP was 50 nM.

In summary our data show that stimulated CD73-deficient T-cells show a proinflammtory phenotype with increased levels of active NFkB. Thus, the adenosine formed by CD73 is physiological modulator of the immune response. Furthermore, the phosphorylated A2a receptor agonist chet-AMP serves as a prodrug to specifically inhibit cytokine release from T-cells, which is a novel concept with clinical potential.