These data show

that like other lymphocyte populations, including NK cells, iNKT cells are sensitive to the immunosuppressive effects of adenosine. Adenosine-related compounds cause the simultaneous engagement of Gs- and Gi-coupled adenosine receptors. We therefore asked whether ligation of the predominant high-affinity A2aR during TCR-mediated stimulation would modulate activation or effector functions, i.e. BMN 673 molecular weight cytokine production, of iNKT cells. To exclude a lack of costimulatory molecules accounting for a lack of IFN-γ secretion, we next used BMDC at day of culture, typically consisting of both immature and mature cells. To exclude responses of the BMDC to A2aR modulation, cells were fixed upon α-galactosylceramide (α-GalCer)-loading. Enriched iNKT cell preparations were thus stimulated in the presence of a specific A2aR agonist or antagonist. Comparable to the effects of the stable adenosine analogue, the exposure to A2aR agonist CGS21680 during the stimulation period inhibited the production of IFN-γ by iNKT cells. In striking contrast, CGS21680 led to a significant increase in IL-4 production. Conversely, the A2aR antagonist ZM241485 inhibited the iNKT cell-mediated MG-132 in vitro secretion of IL-4 and concomitantly increased the production of IFN-γ (Fig. 2B), markedly skewing the Th1/Th2 ratio of cytokines produced by iNKT cells toward IFN-γ. These data were corroborated

by a similar analysis of a human iNKT cell line (Fig. 2C). The requirement of A2aR signaling for IL-4 production clearly is in opposition to the effects of A2aR ligation on conventional T cells, which are inhibited non-selectively 24. These data also provide an explanation for the phenomenon Beldi et al. recently described 17, in which iNKT cells lacking the ecto-enzyme CD39, and hence unable to generate adenosine, were not able to produce IL-4 upon CD1d-mediated activation. To determine

the physiological in vivo significance of these findings, we asked whether iNKT cells in mice lacking the predominant A2aR would be functionally altered. We injected A2aR KO mice or WT mice with α-GalCer and tested the cytokine production 90 min and 5 h later, reflecting the time of appearance science in serum. The production of IL-4 and IL-10 upon α-GalCer administration can be observed early after activation, whereas IFN-γ secretion by iNKT cells requires IL-12 produced by DC upon maturation and hence are detectable later after injection. We detected increases in all four tested cytokines (IL-4, IL-10, TGF-β and IFN-γ) in the serum of α-GalCer-injected WT mice compared to un-injected mice (data not shown). Comparable with the in vitro results, iNKT cells in the absence of A2aR produced significantly lower levels of IL-4 upon α-GalCer injection (Fig. 3A). The expression of another Th2 cytokine IL-10 was also markedly decreased in the A2aR KO mice. In marked contrast, but also comparable with the in vitro results, IFN-γ was increased in the A2aR-deficient mice.