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Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen–specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell–based immunotherapy for the treatment of tumor growth and metastasis.

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Accumulating evidence suggests that inhibiting adenosine-generating ecto-enzymes (CD39 and CD73) and/or adenosine A2A or A2B receptors (R) stimulates antitumor immunity and limits tumor progression. Although activating A2ARs or A2BRs causes similar immunosuppressive and protumoral functions, few studies have investigated the distinct role of A2BR in cancer. Here, we showed that A2BR expression by hematopoietic cells was primarily responsible for promoting tumor growth. Deletion of A2BR profoundly enhanced anticancer T-cell immunity. Although T-cell A2BR plays an insignificant role for A2BR-mediated immunosuppression and tumor promotion, A2BR deficiency in tumor-bearing mice caused increased infiltration of myeloid and CD103+ dendritic cells, which was associated with more effective cross-priming of adoptively transferred tumor antigen–specific CD8+ T cells. A2BR deletion also intrinsically favored accumulation of myeloid and CD11bdim antigen-presenting cells (APC) in the tumor microenvironment. Both myeloid-specific or CD11c-specific conditional deletion of A2BR delayed primary tumor growth. Myeloid, but not CD11c-specific conditional, depletion delayed lung metastasis. Pharmacologic blockade of A2BR improved the antitumor effect of adoptive T-cell therapy. Overall, these results suggested that A2BR expression on myeloid cells and APCs indirectly suppressed CD8+ T-cell responses and promoted metastasis. These data provide a strong rationale to combine A2BR inhibition with T-cell–based immunotherapy for the treatment of tumor growth and metastasis.

Extracellular adenosine accumulates in tumors and causes suppression of immune cells. Suppressive adenosine signaling is achieved through adenosine A2A and A2B receptors, which are Gs coupled, and their activation elevates cAMP levels. Gs-coupled GPCR signaling causes cAMP accumulation, which plays an anti-inflammatory role in immune cells. Protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are two intracellular receptors of cAMP. In this study we showed that adenosine receptor signaling polarizes activated murine dendritic cells (DCs) into a tumor-promoting suppressive phenotype. Adenosine receptor signaling activates cAMP pathway and upregulates the negative regulators of NF-κB but does not influence phosphorylation of immediate inflammatory signaling molecules downstream of TLR signaling. Pharmacologic activation of both PKA and Epac pathways by specific cAMP analogues phenocopied the effects of adenosine signaling on murine DCs, such as suppression of proinflammatory cytokines, elevation of anti-inflammatory IL-10, increased expression of regulators of NF-κB pathway, and finally suppression of T cell activation. Inhibition of effector cytokine, IL-12p40 production, and increased immunosuppressive IL-10 production by adenosine signaling is significantly reversed only when both PKA and Epac pathways were inhibited together. Adenosine signaling increased IL-10 secretion while decreasing IL-12p40 secretion in human monocyte-derived DCs. Stimulation of both PKA and Epac pathways also caused combinatorial effects in regulation of IL-12p40 secretion in human monocyte-derived DCs. Interestingly, PKA signaling alone caused similar increase in IL-10 secretion to that of adenosine signaling in human monocyte-derived DCs. Our data suggest adenosine/cAMP signaling targets both PKA/Epac pathways to fully differentiate DCs into a suppressive phenotype.

The nucleoside adenosine accumulates extracellularly in solid tumors and inhibits CD8

The nucleoside adenosine accumulates extracellularly in solid tumors and inhibits CD8(+) T cells by activating adenosine receptors. The cytokine interleukin-7 (IL-7), which is produced by various tissues and tumors, promotes the survival and maintenance of T cells. Adenosine and IL-7 signaling are being clinically targeted separately or in combination with other therapies for solid tumor indications. Here, we found that IL-7 signaling promoted the accumulation of tumor-associated CD8(+) T cells, in part, by preventing adenosine-mediated immunosuppression. Inhibition of the transcription factor FoxO1 downstream of IL-7 receptor signaling was important for protecting CD8(+) T cells from suppression by adenosine. These findings have implications for the development of new approaches for cancer immunotherapies that target the adenosine pathway.

Inhibition of proximal TCR signals by adenosine A2A receptors (A2AR) is important for T cell development and maintaining T cell quiescence in vivo. Deletion of A2ARs causes accelerated growth of B16 and MB49 tumors, which is associated with reduced T cell accumulation in tumors and decreased CD127 expression and interleukin-7 responsiveness. Here we showed that ectopic expression of CD127 in A2AR deficient mice restores normal numbers of thymic populations and IL-7 responsiveness of T cells. Ectopic expression of CD127 in A2AR deficient mice also caused accumulation of similar numbers of T cells in the periphery as compared to A2AR proficient mice. Growth of B16 tumors in A2AR deficient mice was accelerated as compared to A2AR proficient mice. However, growth of B16 tumors was comparable between A2AR deficient or proficient mice ectopically expressing CD127. Adenosine A2A receptor deletion or ectopic CD127 expression did not change Vbeta selection suggesting changes in T cell repertoire may not be involved in increased tumor growth and reduced T cell numbers in A2AR deficient mice. Interestingly, tumor growth in A2AR and CD127 double deficient mice was slower as compared to CD127 deficient mice. Finally, adenosine preconditioning of engineered T cells significantly reduced lung colonization of B16OVA tumors and persistence of engineered T cells. Overall these results suggest that CD127 regulation by adenosine is important for T cell maintenance and functional responses in vivo and blockade of A2AR to improve anti-tumor immune responses should be further promoted by providing signals for T cell survival.

Polarization of T helper subsets into different functional phenotypes strongly affects the progression of immune-related diseases. One of the hallmarks of immune cell activation and inflammation is the elevation of extracellular adenosine and increased expression of adenosine A2A receptors in activated immune cells. Adenosine strongly inhibits activation of helper T cells by elevating cAMP concentrations, which activate PKA and EPAC proteins to regulate cellular responses. Here we showed that adenosine strongly inhibits T cell accumulation rather than differentiating into functional subsets in the presence of polarizing conditions. Different aspects of adenosine mediated T cell suppression were phenocopied by specific activation of PKA and/or EPAC pathways. Adenosine can suppress phospho-activation of Akt pathway to promote T cell quiescence and to inhibit immediate downstream events after TCR stimulation. One of the targets for Akt is Foxo1. Foxo1 is known to suppress T cell proliferation. Our results showed that adenosine receptor stimulation reduced inhibitory phosphorylation of Foxo1 downstream of Akt. Foxo1 inhibitor, AS1842856, completely reversed the inhibition of T cell accumulation by adenosine signaling. Our results suggest that adenosine by activating PKA and/or EPAC pathways and by sustaining Foxo1 activation suppresses T cell activation and accumulation. These findings have important implications to develop novel interventions to regulate helper T cell responses in different pathological conditions.

Metabolic and immune related activities converge as main triggers of adenosine accumulation in extracellular space. Adenosine by engaging adenosine A2A and A2B receptors strongly suppresses innate and adaptive immune responses. Although adenosine receptors are being targeted in preclinical and clinical studies how different danger signals are regulated by adenosine is poorly understood. Here we showed that adenosine receptor stimulation strongly inhibited inflammatory responses while sparing type-I interferon responses downstream of different danger signals in dendritic cells and macrophages. Mechanistically, danger signals associated with MyD88-dependent inflammatory pathways such as LPS and CpG but not the danger signals associated with IRF3/Type-I interferon pathways such as pA:U and cGAMP increase the expression adenosine A2A and A2B receptors. Adenosine was shown to increase the expression of NR4A nuclear hormone receptors to inhibit NF-κB activation. Although adenosine did not influence NF-κB phosphoactivation expression of anti-inflammatory NR4A1 was increased after adenosine receptor stimulation in the presence of TLR ligands known to activate MyD88 pathway but not in the presence of cGAMP and pA:U. Overall these results indicate that there is a differential modulation of danger signaling by adenosine rather than overall supression. Our results have important implications for developing combinatorial approaches to target adenosine and danger signaling pathways to cure immune-related diseases.

Activation of immune cells is associated with increased expression of adenosine receptors. Extracellular adenosine is present in primary and secondary lymphoid organs and limit the amplitude of immune responses suggesting that antagonists for adenosine receptors can be targeted to improve the efficacy of vaccines. Here we showed that vaccine adjuvant Monophosphoryl Lipid A (MPLA) strongly increased adenosine A2A and adenosine A2B receptor expression in primary macrophages and dendritic cells. Adenosine A2A but not adenosine A2B receptor blockade significantly increased both numbers and percentages of antigen specific endogenous T cells in both spleen and draining lymph nodes after primary immunizations with MPLA and ovalbumin. We observed a similar increase in both endogenous or adoptively transferred T cells in mice with myeloid-specific deletion of A2A receptors after primary immunization. Adenosine receptor blockade significantly increased the total IgG titers and IgG2c/IgG1 ratio after rechallenge with ovalbumin. These results suggest that adenosine A2A receptor blockade potentially improve vaccine efficacy by promoting both cellular and humoral immune responses and promoting Th1 type immunity. Our results also suggest that A2ARs on antigen presenting cells are important targets to improve vaccine efficacy by adenosine A2A receptor blockade. These findings have important implications for the design of novel and more efficacious vaccine formulations.

Extracellular adenosine concentrations and expression adenosine receptors are elevated after cell death, injury and inflammation. Antigen presenting cells are one of the primary targets for adenosine to suppress T and NK cell responses. However, molecular mechanisms for adenosine regulation of dendritic cells are poorly understood. Here we show that adenosine receptor stimulation strongly suppresses dendritic cell activation. Adenosine did not affect phosphoactivation of inflammatory signaling pathways such as MAPKs, NF-κB and IRF3. Adenosine receptor signaling increased intracellular cAMP accumulation. Specific cAMP analogs for EPAC and PKA pathways phenocopied the effects of adenosine on dendritic cells especially when used together. Adenosine-mediated suppression of inflammatory/effector cytokines is associated with increased expression of anti-inflammatory c-Fos and NR4A receptors. NR4A and cFos expression were also increased by EPAC and PKA-specific cAMP analogs. Addition of conditioned mediums from dendritic cells prestimulated with stable adenosine analog or PKA and EPAC specific cAMP analogs together inhibited the IFNγ production by activated T cells. Overall our data suggest that adenosine can potentially target both PKA and EPAC pathways and increase the expression of NR4A nuclear orphan receptors and cFos to regulate dendritic cell responses. Our findings have important implications for the development of novel therapies for immune related diseases such as cancer by targeting adenosine receptors.