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...

Myeloid-derived suppressor cells (MDSCs IntroductionA major barrier to effective cancer immunotherapy is immune suppression, and the accumulation of myeloid-derived suppressor cells (MDSCs) has recently been recognized as a major mechanism to promote immune suppression (1, 2). MDSCs comprise a mixture of myeloid cells reflecting various stages of differentiation, and in mouse models, these cells are typically distinguished from other inhibitory myeloid populations based on their unique coexpression of macrophage (CD11b) and granulocyte (Gr-1) markers (1).Tumor-induced MDSCs are further dichotomized into monocytic and granulocytic subsets based on the differential expression of the Ly6G and Ly6C epitopes (3, 4). Intriguingly, granulocytic MDSCs outnumber monocytic MDSCs in numerous mouse tumor models (3, 5), although the basis for this subset dichotomy remains unclear. The phenotypes in humans are more complex and vary with tumor type. However, there is general agreement that a common lineage-negative MDSC subset observed among a range of human cancers bears the core phenotype CD33 + HLA-DR -(6-11). Interestingly, this subset resembles promyelocytes, a granulocytic population reflecting an early stage of differentiation (6, 7).Although many studies have been dedicated to the phenotypic characterization of MDSCs and unraveling mechanisms by which these cells mediate tumor progression, a large gap remains in our understanding of the mechanisms that initiate their development. It is known, however, that MDSC subsets emerge in response to tumor-derived factors (TDFs) and the signaling pathways these molecules engage. As a number of TDFs engage the STAT3 or STAT5 signaling pathway, STAT3 or STAT5 activation has been associated with various stages in MDSC biology (1,(12)(13)(14)(15)(16)(17)(18)(19).

CD8+ CTL play important roles against malignancy in both active and passive immunotherapy. Nonetheless, the success of antitumor CTL responses may be improved by additional therapeutic modalities. Radiotherapy, which has a long-standing use in treating neoplastic disease, has been found to induce unique biologic alterations in cancer cells affecting Fas gene expression, which, consequently, may influence the overall lytic efficiency of CTL. Here, in a mouse adenocarcinoma cell model, we examined whether exposure of these tumor cells to sublethal doses of irradiation 1) enhances Fas expression, leading to more efficient CTL killing via Fas-dependent mechanisms in vitro; and 2) improves antitumor activity in vivo by adoptive transfer of these Ag-specific CTL. Treatment of carcinoembryonic Ag-expressing MC38 adenocarcinoma cells with irradiation (20 Gy) in vitro enhanced Fas expression at molecular, phenotypic, and functional levels. Furthermore, irradiation sensitized these targets to Ag-specific CTL killing via the Fas/Fas ligand pathway. We examined the effect of localized irradiation of s.c. growing tumors on the efficiency of CTL adoptive immunotherapy. Irradiation caused up-regulation of Fas by these tumor cells in situ, based on immunohistochemistry. Moreover, localized irradiation of the tumor significantly potentiated tumor rejection by these carcinoembryonic Ag-specific CTL. Overall, these results showed for the first time that 1) regulation of the Fas pathway in tumor cells by irradiation plays an important role in their sensitization to Ag-specific CTL; and 2) a combination regimen of tumor-targeted irradiation and CTL promotes more effective antitumor responses in vivo, which may have implications for the combination of immunotherapy and radiation therapy.