A2A adenosine receptor protects tumors from antitumor T cells

Abstract

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 we confirm this hypothesis by showing that genetic deletion of A2AR in the host resulted in rejection of established immunogenic tumors in approximately 60% of A2AR-deficient mice with no rejection observed in control WT mice. The use of antagonists, including caffeine, or targeting the A2 receptors by siRNA pretreatment of T cells improved the inhibition of tumor growth, destruction of metastases, and prevention of neovascularization by antitumor T cells. The data suggest that effects of A2AR are T cell autonomous. The inhibition of antitumor T cells via their A2AR in the adenosine-rich tumor microenvironment may explain the paradoxical coexistence of tumors and antitumor immune cells in some cancer patients (the "Hellstrom paradox"). We propose to target the hypoxia-->adenosine-->A2AR pathway as a cancer immunotherapy strategy to prevent the inhibition of antitumor T cells in the tumor microenvironment. The same strategy may prevent the premature termination of immune response and improve the vaccine-induced development of antitumor and antiviral T cells. The observations of autoimmunity during melanoma rejection in A2AR-deficient mice suggest that A2AR in T cells is also important in preventing autoimmunity. Thus, although using the hypoxia-->adenosine-->A2AR pathway inhibitors may improve antitumor immunity, the recruitment of this pathway by selective drugs is expected to attenuate the autoimmune tissue damage.

Fig. 1.

Overview of experimental strategy to test the hypothetical mechanism of tumor protection. (a) It is assumed that adenosine and A2AR, which inhibit overactive immune cells to protect normal tissues (18), may protect malignant tissues from antitumor T cells. The transient or chronic hypoxia in the tumor microenvironment (19, 20) could be conducive to accumulation of adenosine (15, 27), which then may inhibit antitumor CD8⁺ T cells by increasing their immunosuppressive intracellular cAMP levels (–17). Genetic targeting of A2AR may deinhibit CD8⁺ T cells and thereby facilitate their antitumor effector functions, as was shown in models of T cell-dependent viral and autoimmune hepatitis (18). A similar outcome could be accomplished by using A2AR antagonists, e.g., ZM241,385, which were shown to prevent adenosine-triggered cAMP elevation (26), reverse the adenosine-mediated inhibition of activated CD8⁺ T cells (24) in vitro, and deinhibit activated immune cells in vivo (18). Evidence for such a tumor-protecting mechanism may also be interpreted as proof of principle for the feasibility of a novel strategy of tumor destruction, where the interruption of hypoxia→adenosine→A2AR signaling in tumors may rescue the antitumor immune response from inhibition in the hostile tumor environment. (b) Demonstration of a gradient of increased levels of extracellular adenosine and cAMP in a solid tumor environment using an equilibrium microdialysis probe (see Supporting Text). (c) Expression of functional A2AR and/or A2BR on tumor-specific CD8⁺ T cells. CMS4 sarcoma-specific CD8⁺ T cells and anti-gp33 CD8⁺ T cells were used in the experiments of Figs. 4 and 11. The levels of A2AR-selective agonist CGS21680-induced cAMP reflect the expression of A2AR, and the adenosine- or 5′-(N-ethylcarboxamido) adenosine (NECA)-induced cAMP represents the sum of signaling by both A2AR and A2BR. The difference between adenosine/NECA and CGS21680 provides an indication as to the relative contribution of A2BR.

Fig. 2.

Genetic deficiency of A2AR may lead to complete rejection of established CL8-1 melanoma and to survival of tumor-bearing mice. (a) A2AR inactivation by genetic mutation may lead to complete tumor rejection. The A2AR^−/− mice or WT mice were inoculated s.c. with 3 × 10⁶ CL8-1 melanoma cells. The mouse faces on the graph indicate complete tumor rejection and mouse survival, whereas the cross indicates that the mouse had to be euthanized according to an animal care protocol when tumors reached ≈2 cm in diameter. The question mark in the lowest graph of tumor rejection by A2AR^−/− mice indicates that this mouse would likely have completely rejected tumor and survived; even though the tumor itself was being destroyed by CD8⁺ T cells, the “wounded” tissue was ≈2 cm in diameter, and mice had to be euthanized according to the animal care protocol. Shown are representative results of two experiments. (b) Genetic evidence that inactivation of A2AR may lead to survival of mice with inoculated CL8-1 melanoma (the same experiment as in a). (c) A2AR inactivation by genetic mutation is not sufficient to ensure tumor rejection and survival of mice with inoculated nonimmunogenic B16 melanoma. Groups of A2AR^−/− mice or WT mice were inoculated s.c. with 5 × 10⁴ B16 melanoma cells, and tumor growth was monitored. No rejection of B16 has been observed in any mice, and no differences in survival were observed between WT C57BL/6 and A2AR^−/− mice. Shown are representative results of two experiments. (d) Photographs of a typical tumor’s wound-healing process during and after an immune attack by anti-CL8-1 CD8⁺ T cells in A2AR^−/− mice. (e) Unusual appearance of mice with CL8-1 melanoma tumor, which grew to a large size and then was rejected. Two mice with such a phenotype were observed among five A2AR^−/− survivors of CL8-1 melanoma.

Fig. 3.

A2AR inactivation by genetic mutation may lead to a complete rejection of established RMA T lymphoma by antitumor CD8⁺ T cells, ensuring survival of tumor-bearing mice. (a and b) A2AR inactivation by genetic mutation may lead to complete RMA T lymphoma tumor rejection. The A2AR^−/− mice (n = 9) or WT mice (n = 11) were inoculated s.c. with 2 × 10⁵ RMA T lymphoma cells. Representative results of two experiments are shown. (c) A2AR inactivation by genetic mutation may lead to survival of mice with inoculated RMA lymphoma. Shown are representative results of two experiments.

Fig. 4.

Treatment of mice with A2AR/A2BR antagonist enhanced the destruction of established tumors by tumor antigen-specific CD8⁺ T cells. (a) The antagonist ZM241,385 improved destruction of CMS4 lung metastasis by antitumor CD8⁺ T cells. (b) Enhancement of destruction of CMS4 lung metastasis by adoptively transferred CD8⁺ T cells in mice that consumed caffeine in drinking water. Based on the aggregation of the data points in the control “only tumor” and “caffeine alone” groups, an arbitrary cutoff value (a more than ≈20% decrease in the number of metastatic nodules) was assigned to illustrate a potential therapeutic efficacy threshold (shown as the percentage of mice with decreased number of metastases) for those groups of mice that were treated with both CD8⁺ T cells and A2 receptor antagonists. (c) ZM241,385, an antagonist of A2AR and A2BR, enhances CD8⁺ T cell-mediated antitumor immune response in mice with established s.c. solid CL8-1 melanoma. Data represent mean ± SEM.

Fig. 5.

Treatment with caffeine inhibits neovascularization and increases apoptosis of CL8-1 melanoma in mice. (a) Genetic evidence that growth of inoculated CL8-1 melanoma cells depends on functional IFN-γ receptors (IFN-γR). The IFN-γR^−/− or WT C57BL/6 mice (8–10 mice per group) were inoculated s.c. with 3 × 10⁶ CL8-1 melanoma cells. All data represent mean ± SEM. (b) Extracellular adenosine inhibits up-regulated IFN-γ gene transcription in activated antitumor T cells, and this inhibition is prevented by adenosine receptor antagonists ZM241,385 (ZM) and caffeine. mRNA levels were analyzed by an RNase protection assay. (c) Immunohistochemical demonstration of increased tumor destruction and inhibition of angiogenesis in tumors in mice treated with caffeine. Mice were inoculated with CL8-1 and treated with caffeine. CL8-1 melanomas growing in C57BL/6 mice were removed at day 50 from control (Lower Left) and caffeine-treated (Lower Right) mice. (d) The degree of angiogenesis was determined as the percentage of CD31⁺ staining to the total field. The y axis (percentage of fields positive for CD31) reflects the percentage of area occupied by blood vessels in comparison to the total tumor area. The summary of measurements of two tumors per group is presented. Methods for the RNase protection assay and immunohistochemistry are provided in Supporting Text.

Fig. 6.

Suppression of A2AR/A2BR expression in T cells improved adoptive immunotherapy. (a) siRNA against A2AR and A2BR was transfected into anti-CMS4 CD8⁺ T cells before adoptive transfer (see Supporting Text). Lung metastasis was examined 11 days after injection of T cells (1 × 10⁶ cells per mouse) as described in Methods. T cell-specific knockdown of A2AR/A2BR facilitated inhibition of lung metastasis by antitumor T cells (∗, P = 0.0116 by ANOVA). (b) Survival of RMA tumor-bearing WT mice was improved by the transfer of A2AR/A2BR siRNA-pretreated T cells (P < 0.05 by log-rank test). Anti-RMA T cells were obtained from WT mice immunized with RMA tumor cells. Unseparated spleen and lymph node cells (5 × 10⁷) were transfected with either control or A2AR and A2BR siRNA and injected into tumor-bearing mice 10 days after inoculation of RMA cells (2 × 10⁵).