No abstract

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.

The hypoxia-driven and A2A or A2B adenosine receptors (A2AR/A2BR)-mediated (“Hypoxia-A2-Adenosinergic”) and T cell autonomous immunosuppression was first recognized as critical and non-redundant in protection of normal tissues from inflammatory damage and autoimmunity. However, this immunosuppressive mechanism is high-jacked by bacteria and tumors to misguidedly protect pathogens and cancerous tissues. The inhibitors of Hypoxia-A2-Adenosinergic pathway represent the conceptually novel type of immunological co-adjuvants to be combined with cancer vaccines, adoptive cell transfer and/or blockade of immunological negative regulators in order to further prolong survival and minimize side effects. In support of this approach are preclinical studies and findings that some human cancers are resistant to chemotherapies and immunotherapies due to the tumor-generated extracellular adenosine and intracellular cAMP-elevating A2AR and A2BR on anti-tumor T and NK cells. Among co-adjuvants are i) antagonists of A2AR/A2BR; ii) extracellular adenosine-degrading drugs; iii) inhibitors of adenosine generation by CD39/CD73 ecto-enzymes and iv) inhibitors of the hypoxia-HIF-1 alpha signaling. It is emphasized that even after the multi-combinatorial blockade of immunological negative regulators the anti-tumor T and NK cells would be still vulnerable to inhibition by hypoxia and A2AR and A2BR. The advantage of combining these co-adjuvants with the blockade of the CTLA4-A and/or PD-1 is in expectations of additive or even synergistic effects of targeting both immunological and physiological tumor-protecting mechanisms. Yet to be tested is the potential capacity of co-adjuvants to minimize the side effects of blockade of CTLA-4 and/or PD1 by decreasing the dose of blocking antibodies or by eliminating the need in dual blockade.

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.

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high affinity, class switched antibodies. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper (TFH) cells and plasmacytes, as well as lower expression of ICOS on TFH. Importantly, this reversal of GC hypoxia decreased antigen-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the antigen specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.