direct sequencing. Oligonucleotide sequences and PCR conditions are available on request. Northern and in situ hybridizationsNorthern and in situ hybridization to sectioned tissue were done as described 3,11 . The 738-nucleotide edaradd probe contained the entire 627-nucleotide ORF plus 76 nucleotides of the 59 and 35 nucleotides of the 39 untranslated region. For whole-mount in situ hybridization 3 , we used full-length edar and Shh riboprobes. Protein interaction and NF-kB reporter assaysGST fusion constructs were prepared by cloning cDNAs into pGEX4T-1 (Amersham Pharmacia). The intracellular portion of Edar (EdarIC) contained amino acids 212±448; the Edar death domain deletion (EdarICDDD) contained amino acids 212±322; EdaraddDDD contained amino acids 1±124. Deletion of the seven amino acids Tyr-Pro-IleGln-Asp-Thr-Gly from wild-type Edaradd created EdaraddD34-40. RANKIC is amino acids 235±625 and p75NTRIC amino acids 264±417 of mouse RANK and mouse p75NTR, respectively. We carried out in vitro transcription/translation using the TNT Coupled Reticulocyte Lysate system (Promega) in the presence of [ 35 S]methionine (Amersham Pharmacia). We then expressed the GST fusion proteins in Escherichia coli strain BL21. Bacteria were lysed by sonication in PBS plus 0.5% Triton X-100. We used 2 ml of in vitro transcription/translation reaction for each pull-down assay. Protein mixtures were incubated at 4 8C for 2 h with 15 ml of glutathione±sepharose beads (Amersham Pharmacia). The beads were washed four times with PBS plus 0.1% Triton X-100 and bound proteins were eluted with 20 ml of 50 mM glutathione.For immunoprecipitations, individual cDNAs were cloned into pSG5 (Stratagene) downstream of two copies of a Flag or haemagglutinin A (HA) epitope tag. HEK293T cells grown in 25-cm 2¯a sks were transfected using Lipofectamine2000 (Lifetech). Whole-cell extracts were prepared 30 h after transfection by adding 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 0.5 % NP-40, protease inhibitors). Cell lysates (850 ml) were added to 40 ml of anti-Flag M2 af®nity gel (Sigma) and rotated at 4 8C overnight. The af®nity gels were washed with lysis buffer ®ve times and eluted with 20 ml of 0.1 M glycine (pH 3.1); the eluted proteins were assayed on western blots using a 1:1,250 dilution of rat anti-HA primary antibody (3F10; Roche) followed by a 1:3,500 dilution of HRP conjugated rabbit anti-rat secondary antibody (Zymed) and detection by enhanced chemiluminescence (Amersham).For NF-kB activation assays, HEK293T cells grown in 24-well plates were transfected using Lipofectamine2000 (Lifetech). Each well received 0.2 mg of pNF-kBLuc luciferase reporter (Clontech), cDNA(s) for expression in pSG5±HA, and empty pSG5±HA vector to give 1.0 mg of DNA per well. We lysed the cells 28 h after transfection and measured luciferase activities (Tropix; Promega) using a luminometer. Each transfection was performed in quadruplicate. To compare relative protein expression levels, transfections were done as in the reporter assays. After 28 h, each well...

The A2A adenosine receptor (A2AR) has been shown to be a critical and nonredundant negative regulator of immune cells in protecting normal tissues from inflammatory damage. We hypothesized that A2AR also protects cancerous tissues by inhibiting incoming antitumor T lymphocytes. Here autoimmunity ͉ cancer ͉ therapy ͉ hypoxia ͉ inflammation T he coexistence of tumors and antitumor immune cells is currently explained by the inhibition of immune cells in a poorly understood ''hostile'' tumor microenvironment (1-3). This unidentified immunosuppressive mechanism limits promising cancer therapies using antitumor T cells (4-14). We hypothesized that cancerous tissues are protected from antitumor T cells because of immunosuppressive signaling via T cell A2A adenosine receptor (A2AR) (15-17) activated by extracellular adenosine produced from hypoxic tumor (Fig. 1a). Indeed, hypoxic cancerous tissues may be protected by the same hypoxia3adenosine3A2AR pathway that was recently shown to be critical and nonredundant in preventing excessive damage of normal tissues by overactive immune cells in vivo (18). It is well established that some areas of solid tumors often have transient or chronic hypoxia (19,20), which is conducive to extracellular adenosine accumulation (21). Hypoxia has been implicated in mechanisms of tumor protection against ionizing radiation and some chemotherapeutic agents (19) and is associated with poor prognosis (20).T cells, including antitumor T cells, do predominantly express cAMP-elevating Gs protein-coupled high-affinity A2AR and͞or low-affinity A2B adenosine receptors (A2BR) (16,17,(22)(23)(24); the number of A2AR per T cell may determine the intensity of maximal T cell response to adenosine (25, 26). Whereas we focused on A2AR, others have discounted A2 receptors and suggested the A3 adenosine receptors as responsible for inhibition of antitumor killer T cells (27,28). Here we report that genetic deletion of A2AR accomplishes the complete rejection of immunogenic tumors by antitumor CD8 ϩ T cells in the majority (Ϸ60%) of mice, whereas the antagonists of A2 receptors facilitate CD8 ϩ T cell-mediated retardation of tumor growth. Results The Gradient of T Cell-Inhibiting Extracellular Adenosine in Tumors.It was important to confirm the presence of elevated extracellular adenosine levels in cancerous tissues using a reliable method (29). The HPLC analysis and the use of equilibrium dialysis probes demonstrated higher levels of extracellular adenosine (Fig. 1b), increased adenosine metabolism, and the concomitant increase in cAMP (29) in a solid tumor microenvironment (Fig. 7, which is published as supporting information on the PNAS web site). We also confirmed that antitumor CD8 ϩ T cells used in this study do express the cAMP-elevating functional A2AR and A2BR (Fig. 1c). To directly test whether A2AR inhibit antitumor T cells in vivo, we studied the effects of A2AR gene deletion or competitive antagonists on tumor growth in mice using different CD8 ϩ T celldependent cancer immunosurveillance and ad...