Adenosine and prostaglandin E2 cooperate in the suppression of immune responses mediated by adaptive regulatory T cells

Abstract

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.

FIGURE 1.

Phenotypic characterization of IVA-generated Tr1. A, representative fluorescence-activated cell sorting plots for marker expression in Tr1 cells generated in the presence of either COX-2⁺ or COX-2⁻ tumor cells. B, flow cytometry analysis of Tr1 cells generated in the presence of a COX-2⁺ or COX-2⁻ tumor cell line. CD4⁺CD25⁺ T cells cultured for 10 days in the presence of 150 IU/ml IL-2 but in the absence of tumor cells and dendritic cells served as reference cells for IVA co-cultures here and in all other experiments. C, representative fluorescence-activated cell sorting plots for co-expression of COX-2, CD39, and CD73 in Tr1 cells generated in IVA (Tr1/COX-2⁺). D, phenotypic analysis of Tr1 generated in the presence of COX-2⁺ tumor cells with or without indomethacin. Data represent 10 independent experiments. Asterisks indicate a significant difference at p < 0.05. Bars indicate ±S.D.

FIGURE 2.

Ectonucleotidase and COX-2 in Tr1. A, hydrolysis of exogenous ATP is shown by CD39⁺ Tr1 cells generated in IVA that contained COX-2⁺ or COX-2⁻ tumor cells. In some experiments, ARL67156, a CD39 inhibitor, was added. B, adenosine production by Tr1 cells generated in the presence of COX-2⁺ or COX-2⁻ tumor cells was determined by mass spectrometry. C, in some experiments, α,β-methylene ADP, a CD73 inhibitor, was added. D, the production of PGE₂ by Tr1/COX-2⁺ or Tr1/COX-2⁻ was measured by ELISA. A–D, in each panel, data represent three independent experiments. Asterisks indicate significant differences at p < 0.05. Bars indicate ±S.D.

FIGURE 3.

Suppression of RC proliferation mediated by Tr1/COX-2⁺ and Tr1/COX-2⁻ in presence and absence of various enzymatic inhibitors. MACS-sorted CD4⁺CD25⁻ cells (RC) were CFSE-labeled and stimulated with plate-bound OKT-3 and soluble anti-CD28 in the presence of Tr1/COX-2⁺ or Tr1/COX-2⁻ and 150 IU/ml IL-2 for 5 days. Cells were analyzed by flow cytometry gating on CD4⁺CFSE⁺ T cell subsets and further analyzed using the ModFit program. A, suppression of CD4⁺CD25⁻ cell proliferation mediated by Tr1/COX-2⁺ or Tr1/COX-2⁻ at various S/RC ratios. B and C, to selected wells, ARL67156, a CD39 inhibitor; α,β-methylene ADP, a CD73 inhibitor; or ZM241385, a selective A_2aR and A_2bR antagonist, was added. Data are means ± S.D. from three individual experiments.

FIGURE 4.

Suppression of RC proliferation mediated by Tr1/COX-2⁺ in presence and absence of various adenosine and prostaglandin receptor antagonists. MACS-sorted CD4⁺CD25⁻ cells (RC) were CFSE-labeled, stimulated, and cultured as described in the legend to Fig. 3. A and B, suppression of CD4⁺CD25⁻ cell proliferation mediated by Tr1 cells at various S/RC ratios in the absence or presence of indomethacin, a nonselective COX-1 and COX-2 inhibitor; α,β-methylene ADP, a CD73 inhibitor; AH6809, a EP₂R antagonist; ZM241385, a selective A_2AR and A_2BR antagonist; or a combination. Data are means ± S.D. from three independent experiments.

FIGURE 5.

Modulation of intracellular cAMP levels in different T cell subsets. IVA-generated Tr1/COX-2⁺, nTreg, or RC (100,000 cells/well) were incubated in 96-well plates in the absence or presence of various reagents. Intracellular cAMP levels were measured by enzyme immunoassay after lysing the cells. Data are means ± S.D. from three independent experiments. IBMX, isobutylmethylxanthine; CADO, chloroadenosine.

FIGURE 6.

Impact of intracellular cAMP signaling on Tr1- or nTreg-mediated suppression. MACS-sorted CD4⁺CD25⁻ cells (RC) were CFSE-labeled, stimulated, and cultured as described in the legend to Fig. 3. A and B, suppression of CD4⁺CD25⁻ cell (RC) proliferation mediated by Tr1/COX-2⁺or CD25^high nTreg cell subsets at various S/RC ratios in the absence or presence of rolipram, a PDE₄ inhibitor, or (R_p)-8-Br-cAMPS, an inhibitor of the regulatory subunit of the cAMP-dependent PKA. The inhibitors were added to the Tr1 co-cultures on days 0, 3, 6, and 9. Data are means ± S.D. from three independent experiments.

FIGURE 7.

Phenotypic analysis of different Tr1 subsets in peripheral blood and tumor tissues of HNSCC patients. A, PBMC from NC and patients with AD or NED were isolated from whole blood and stained for different Tr1 markers. Cells were analyzed by flow cytometry, gating on the CD3⁺CD4⁺ subset. Data represent five individuals in each cohort. Asterisks indicate a significant difference at p < 0.05. B, representative dot blots for NC, AD, and NED show COX-2, CD39, and CD73 expression by flow cytometry and co-expression with IL-10 and TGFβ₁. C, D, and E, CD4⁺CD132⁺COX-2⁺, CD4⁺CD132⁺CD39⁺, and CD4⁺CD39⁺COX-2⁺ T cells shown in sections of a representative tumor of five examined (magnification, ×400; inset magnification, ×800). C, sections stained for CD4, CD132, and COX-2. CD4⁺ cells are green, CD132⁺ cells are red, CD4⁺CD132⁺ cells are yellow, and CD4⁺CD132⁺COX-2⁺ cells are violet. D, sections stained for CD4, CD132, and CD39. CD4⁺ cells appear in green, CD132⁺ cells are red, CD4⁺CD39⁺ cells are yellow, and CD4⁺CD132⁺CD39⁺ cells are violet. E, sections stained for CD4, CD39, and COX-2. CD4⁺ cells are visible in green, CD39⁺ cells are red, CD4⁺CD39⁺ cells are yellow, and CD4⁺CD39⁺COX-2⁺ cells are violet. Co-expression of CD39 and COX-2 on Tr1 cells in situ is observed.