Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia–A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor–β (TGF-β), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell– and natural killer cell–dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

Cancerous tissue protection from tumor-recognizing CD8 + and CD4 + T cells (antitumor T cells) limits the therapeutic potential of immunotherapies.We propose that tumor protection is to a large extent due to (a) inhibition of antitumor T cells by hypoxia-driven accumulation of extracellular adenosine in local tumor microenvironment and due to (b) Tregulatory cell-produced extracellular adenosine. The adenosine triggers the immunosuppressive signaling via intracellular cyclic AMP^elevating A2A adenosine receptors (A2AR) on antitumorTcells. In addition, the activated antitumor T cells in hypoxic tumor microenvironment could be inhibited by elevated levels of immunosuppressive hypoxia-inducible factor-1a. Complete rejection or tumor growth retardation was observed when A2AR has been genetically eliminated or antagonized with synthetic drug or with natural A2AR antagonist 1,3,7-trimethylxanthine (caffeine).The promising strategy may be in combining the anti-hypoxia-adenosinergic treatment that prevents inhibition of antitumor T cells by tumor-produced and T regulatory cell-produced adenosine with targeting of other negative regulators, such as CTL antigen-4 blockade. Observations of tumor rejection in mice and massive prospective epidemiologic studies support the feasibility of anti-hypoxia-adenosinergic combined immunotherapy.

Intratumoral hypoxia and Hypoxia Inducible Factor-1α (HIF-1α)-dependent CD39/CD73 ecto-enzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through the A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60% O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomics studies in mice. Importantly, oxygenation also down-regulated the expression of adenosine-generating ecto-enzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the up-regulation of antigen-presenting MHC-class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60% oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer.

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.

Tumor hypoxia-driven accumulation of extracellular adenosine was shown to facilitate tumor evasion by engaging the immunosuppressive, intracellular cAMP-elevating A adenosine receptors (AR) on tumor-reactive effector T cells, but there remains a need for careful evaluation of the limiting factors and properties of AR blockade-enabled antitumor immunity. In studies of AR and/or AR gene-deficient mice, we found that AR deletion-but not AR deletion-liberates endogenous CD8 T cell antitumor immunity against weakly immunogenic MCA205 sarcomas. Studies of adoptively transferred AR, AR, or AR/AR tumor-reactive T cells confirmed that immunosuppression in the tumor microenvironment was mediated by AR on CD8 T cells. Treatment with AR antagonist mimicked AR gene deletion in adoptive T cell immunotherapy. This therapeutic benefit of targeting AR was independent of the anatomical location of tumor growth. The enhanced antitumor reactivity also led to the eradication of established intracranial tumors, which was associated with mouse survival and the maintenance of long-lasting, tumor-specific immunological memory. The blockade of the AR on adoptively transferred T cells by synthetic AR antagonist led to higher levels of IFN-γ secretion by tumor-infiltrating CD8 T cells. These data clarify the mechanism of hypoxia-driven immunosuppression in the tumor microenvironment by AR on tumor-reactive CD8 T cells and show that selective AR antagonists can be effective in improving the outcomes of T cell-based immunotherapies. Demonstration of the T cell dose dependency of tumor rejection points to a major limitation of current cancer immunotherapies, in which the presence of sufficient numbers of tumor-reactive T cells in a patient is not known.