Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4(+)CD25(-) T cells and incubated with autologous immature dendritic cells, irradiated COX-2(+) or COX-2(-) tumor cells, and IL-2, IL-10, and IL-15 (each at 10-15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429-1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E-2 (PGE(2)) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2(+) tumor induced a greater number of Tr1 than COX-2(-) tumor (p < 0.05). Tr1 induced by COX-2(+) tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE(2) (p < 0.05) than Tr1 induced by COX-2(-) tumor. Inhibitors of ectonucleotidase activity, A(2A) and EP2 receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE4 inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE(2) and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.