Interestingly, we also noted TGF-β secretion, which was lost in A2aR KO mice, suggesting that TGF-β may be produced by iNKT cells and enhanced through adenosine stimulating A2aR. However, TGF-β production has not been described in iNKT cells and could have been indirectly from other cells. We therefore activated sorted iNKT cells with

plate-bound CD1d molecules and assessed their TGF-β production. As Fig. 3B shows, iNKT cells directly produced TGF-β in the active form in response to CD1d-mediated activation. To further confirm selleckchem that the cytokines observed in sera were from NKT cells, we injected WT and A2aR KO mice with α-GalCer and tested NKT and NK cells for their intracellular cytokine content. NKT cells from A2aR KO mice produced significantly more IFN-γ compared to stimulated WT counterparts. Additionally, NK cells known to be transactivated by NKT cells produced significantly more IFN-γ in the absence of an A2aR (Fig. 3C, bottom), however, no IL-4 could be detected in these cells (data not shown). Supporting the serum data

(Fig. 3A), we observed a clear trend to a lower IL-4 production in A2aR−/− NKT cells, although not reaching statistical significance (n=3). Collectively, our data suggest that the secretion of type-2 cytokines IL-4, IL-10 and Cilomilast cost TGF-β by iNKT cells requires signaling through the A2aR since blocking or genetic ablation of this receptor efficiently abrogates Buspirone HCl their secretion. In contrast, ligation of the same receptor abrogates the production of IFN-γ. Pharmacological ligation of the high-affinity A2aR might reflect the situation in vivo with low

adenosine concentrations skewing the cytokine production of iNKT cells toward a Th2-type phenotype. Increased levels of adenosine, such as found in tumors might then additionally ligate the low-affinity A2bR and thus inhibit the activation of iNKT cells, comparable to other cell types. Conceivably, the manipulation of the A2aR on iNKT cells might control their activation and support host defense and immunotherapeutic approaches in both malignancy and tolerance. C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, MA, USA). Mice deficient the A2aR were previously described and backcrossed to C57BL/6 background 8. Mice were housed under specific pathogen-free conditions. Animal experiments were performed in accordance to protocols approved by Institutional Animal Care and Use Committee. Six- to eight-week-old C57BL/6J mice were used for experiments. PBS57-loaded or empty CD1d monomers and tetramers were provided by the NIH tetramer facility (Emory Vaccine Center, Atlanta, GA, USA). CADO, CGS21680, and ZM241485 were purchased from Tocris (Ellisville, MO, USA). Cells were cultured in RPMI-1640 supplemented with penicillin, streptomycin (Mediatech, Manassas, VA, USA) and 5% FBS (Hyclone, Logan, UT, USA). DC were generated from mouse BM in the presence of GM-CSF as described in 25 with modifications.