The ectonucleotidases CD39 and CD73 hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)-cell responses, thereby suppressing the immune system. The generation of adenosine via the CD39/CD73 pathway is recognized as a major mechanism of regulatory T cell (Treg) inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39-and adenosine-dependent manner. Treatment with a CD39 inhibitor or blocking antibody alleviated the tumor-induced inhibition of CD4 and CD8 T-cell proliferation and increased CTL-and NK cell-mediated cytotoxicity. In conclusion, interfering with the CD39-adenosine pathway may represent a novel immunotherapeutic strategy for inhibiting tumor cell-mediated immunosuppression.

<p>Supplementary material and methods and supplementary figure legends.</p>

<p>Validation of CD39 IHC.</p>

<p>Representative IHC staining of CD39 expression in human normal and tumor tissues (clone HPA014067).</p>

<p>Validation of CD39 IHC.</p>

<div>Abstract<p>The ectonucleotidases CD39 and CD73 hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)–cell responses, thereby suppressing the immune system. The generation of adenosine via the CD39/CD73 pathway is recognized as a major mechanism of regulatory T cell (Treg) immunosuppressive function. The number of CD39⁺ Tregs is increased in some human cancers, and the importance of CD39⁺ Tregs in promoting tumor growth and metastasis has been demonstrated using several <i>in vivo</i> models. Here, we addressed whether CD39 is expressed by tumor cells and whether CD39⁺ tumor cells mediate immunosuppression via the adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 expression is significantly higher in several types of human cancer than in normal tissues. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, the tumor stroma, and tumor cells. Furthermore, the expression of CD39 at the cell surface of tumor cells was directly demonstrated via flow cytometry of human cancer cell lines. CD39 in cancer cells displays ATPase activity and, together with CD73, generates adenosine. CD39⁺CD73⁺ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner. Treatment with a CD39 inhibitor or blocking antibody alleviated the tumor-induced inhibition of CD4 and CD8 T-cell proliferation and increased CTL- and NK cell–mediated cytotoxicity. In conclusion, interfering with the CD39–adenosine pathway may represent a novel immunotherapeutic strategy for inhibiting tumor cell–mediated immunosuppression. <i>Cancer Immunol Res; 3(3); 254–65. ©2014 AACR</i>.</p></div>

<p>CD39 ATPase activity can be assessed by measuring exogenous ATP consumption or phosphate release.</p>

<p>Representative IHC staining of CD39 expression in vascular endothelial cells and lymphocytes from various normal tissues (clone 22A9).</p>