University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, USA.
J Biol Chem. 2010 Sep 3;285(36):27571-80. doi: 10.1074/jbc.M110.127100. Epub 2010 Jun 17.
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 EP(2) receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE(4) 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.
适应性调节性 T 细胞（Tr1）在外周遇到同源抗原时被诱导产生。在癌症中，它们的频率增加；然而，Tr1 介导的抑制机制尚未确定。在这里，我们评估了细胞外核苷酸酶（CD39/CD73）和环氧化酶 2（COX-2）同时参与 Tr1 介导的抑制的情况。如前所述，从外周血单核细胞衍生的、分选的 CD4(+)CD25(-)T 细胞中生成人 Tr1 细胞，并与自体未成熟树突状细胞、辐照 COX-2(+)或 COX-2(-)肿瘤细胞以及 IL-2、IL-10 和 IL-15（每种细胞因子均为 10-15IU/ml）孵育 10 天（Bergmann，C.，Strauss，L.，Zeidler，R.，Lang，S.，and Whiteside，T. L.（2007）Cancer Immunol. Immunother. 56，1429-1442）。通过多色流式细胞术对 Tr1 进行表型分析，并通过基于羧基荧光素二乙酸琥珀酰亚胺酯的测定评估增殖反应细胞的抑制情况。使用荧光素酶检测法测量 ATP 水解，通过质谱或 ELISA 分别分析细胞上清液中的腺苷或前列腺素 E2（PGE2）水平。通过酶免疫测定法测量细胞内 cAMP 水平。COX-2(+)肿瘤诱导的 Tr1 数量多于 COX-2(-)肿瘤（p < 0.05）。COX-2(+)肿瘤诱导的 Tr1 更具抑制作用，水解更多的外源性 ATP（p < 0.05），并产生更高水平的腺苷和 PGE2（p < 0.05）比 COX-2(-)肿瘤诱导的 Tr1。细胞外核苷酸酶活性的抑制剂、A(2A)和 EP(2)受体拮抗剂或 PKA 型 I 的抑制剂降低了 Tr1 介导的抑制作用（p < 0.05），而 PDE4 抑制剂 rolipram 增加了反应细胞中的细胞内 cAMP 水平及其对 Tr1 介导的抑制的敏感性。存在于头颈部鳞状细胞癌患者肿瘤或外周血中的 Tr1 共同表达 COX-2、CD39 和 CD73。通过共同的细胞内 cAMP 途径同时抑制 PGE2 和腺苷可能是改善癌症免疫治疗效果的新方法。
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