Oxygen-dependent regulation of immune checkpoint mechanisms

Oxygen-dependent regulation of immune checkpoint mechanisms Abstract Immunotherapy of cancer has finally materialized following the success of immune checkpoint blockade. Since down-regulation of immune checkpoint mechanisms is beneficial in cancer treatment, it is important to ask why tumors are infamously filled with the immunosuppressive mechanisms. Indeed, immune checkpoints are physiological negative feedback mechanisms of immune activities, and the induction of such mechanisms is important in preventing excessive destruction of inflamed normal tissues. A condition commonly found in tumors and inflamed tissues is tissue hypoxia. Oxygen deprivation under hypoxic conditions by itself is immunosuppressive because proper oxygen supply could support bioenergetic demands of immune cells for optimal immune responses. However, importantly, hypoxia has been found to up-regulate a variety of immune checkpoints and to be able to drive a shift toward a more immunosuppressive environment. Moreover, extracellular adenosine, which accumulates due to tissue hypoxia, also contributes to the up-regulation of other immune checkpoints. Taken together, tissue oxygen is a key regulator of the immune response by directly affecting the energy status of immune effectors and by regulating the intensity of immunoregulatory activity in the environment. The regulators of various immune checkpoint mechanisms may represent the next focus to modulate the intensity of immune responses and to improve cancer immunotherapy. adenosine, hypoxia, immunosuppression, immunotherapy, tumor microenvironment Introduction: immunotherapy as the fourth strategy against cancer It was more than 100 years ago that inflammatory cell infiltration into tumors prompted the idea of tumor regression by immunological means. Since then, cancer immunology has been a focus of intensive research. Vital anti-tumor immunity should be beneficial in cancer prevention by eliminating abnormal cells and in clinics through suppression of cancer progression and metastatic burden. Cancer immunotherapy represents a novel therapeutic strategy that may work alone or in a complementary way to the three main conventional therapeutic strategies—surgical therapy, chemotherapy and radiotherapy. After initial efforts using non-specific immune activators saw little success, the discovery of tumor-associated antigens granted antigen specificity to cancer immunotherapy. Tumor-specific T cells have been used in adoptive therapy, and tumor-associated antigens have been inoculated as vaccines. A more recent invention of adoptive immunotherapy uses T cells with chimeric activating receptors (CAR-T cells), which may overcome the poor immunogenicity of tumor cells that have down-regulated major histocompatibility complex (MHC) expression (1). It should be noted that the long and extensive quest for practical cancer immunotherapy has been mostly focused on how to increase the total attacking potential of anti-tumor immunity, for example by transferring effector cells that have been expanded ex vivo, including CAR-T cells, and by inducing effector cells in vivo. But, what about the persistence of immune activities? In 1968, Hellström et al. questioned the paradoxical co-existence of anti-tumor immune cells in cancer patients (2). T cells that have infiltrated growing tumors are capable of recognizing tumor cells; however, they are somehow quiescent in vivo without demonstrating any significant anti-tumor activity. Later, it became apparent that the tumor microenvironment is very immunosuppressive, and incoming anti-tumor effector cells are prone to the inhibitory mechanisms in tumors (3–5). Analyses of the immunosuppressive mechanisms identified some key targets, and antibodies that block molecules such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1) have demonstrated impressive efficacy against cancer by inducing a persistent anti-tumor immune response (6, 7). The eventual success of these antibodies in human cancer is an indication that the fourth therapeutic strategy—immunotherapy—has finally become a realistic option in the treatment of cancer patients (8, 9). Since then, research has identified quite a few other negative immune regulators in the tumor microenvironment, but it is not clear how tumors acquire so many immunosuppressive mechanisms. Here, I discuss a potential role of tissue oxygen levels as a key regulator of immune responses. The first half of this review focuses on the significance of oxygen in T-cell immunity and the second part describes specific regulation of each immune checkpoints dependent on oxygen levels. Physiological down-regulation of inflammation Current practical immunotherapy targets components of potent immunosuppressive mechanisms: CTLA-4 blocks the CD28-dependent co-stimulatory signal (10, 11), and interaction of PD-1 with its ligands PD-L1 and PD-L2 can also down-regulate the T cell receptor (TCR)-mediated activating signal (12, 13). As well as these cell surface molecules, there are other negative immunoregulators that discourage effector T cells, ranging from small molecules to enzymes and to immunoregulatory cells (Table 1). Table 1. Immunosuppression by different classes of immune checkpoint mechanisms Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression BTLA, B and T lymphocyte attenuator; EP2/EP4, prostaglandin E2 receptor 2/4; HMGB-1, high-mobility group protein B-1; LAG-3, lymphocyte activation gene 3; PGE2, prostaglandin E2; TIGIT, T cell immunoglobulin and ITIM domain; TIM-3, T-cell immunoglobulin and mucin domain 3; TNFRSF14, TNFR superfamily member 14. View Large Table 1. Immunosuppression by different classes of immune checkpoint mechanisms Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression BTLA, B and T lymphocyte attenuator; EP2/EP4, prostaglandin E2 receptor 2/4; HMGB-1, high-mobility group protein B-1; LAG-3, lymphocyte activation gene 3; PGE2, prostaglandin E2; TIGIT, T cell immunoglobulin and ITIM domain; TIM-3, T-cell immunoglobulin and mucin domain 3; TNFRSF14, TNFR superfamily member 14. View Large Immune checkpoints, which have become best known as potential targets of cancer therapy, are indispensable immunoregulatory mechanisms anywhere in the body. In an episode of inflammation, a pro-inflammatory response is followed by down-regulation of inflammatory activities and subsequent tissue remodeling. Immune checkpoints are responsible for the down-regulation of inflammation. Importantly, many immune checkpoint mechanisms become available after the induction of inflammation. For example, CTLA-4 and PD-1 are normally absent in resting effector T cells but are up-regulated upon T-cell activation. Extracellular adenosine increases in response to tissue injury during inflammation. Treg cells may be also inducible after stimulating T cells in a specific cytokine milieu. Extensive up-regulation of immunosuppressive mechanisms after immune activation suggests that immune checkpoints are essentially negative feedback mechanisms to ongoing pro-inflammatory activities in order to prevent unwanted damage to the tissue. Such endogenous immunoregulation is so important that the lack of only one of the immune checkpoints causes persistent inflammatory tissue injury, which is often severe enough to be fatal (14–17). The balance between pro-inflammatory and anti-inflammatory mechanisms defines the intensity and duration of inflammation and outcome of diseases. Tumors, as a tissue that originates in the same body, take advantage of the endogenous tissue-protective mechanisms and fortify themselves against immune attack. To defeat tumors’ heavy emphasis on their anti-inflammatory potential to evade immune attack, it is important to ask about the mechanism by which tumors successfully acquire an assortment of immune checkpoints. Since normal tissues employ the same mechanisms in the protection from overactive immune responses, there might be a clue in something common in cancerous and inflamed tissues. What is commonly observed may be an imbalance of energy demand and supply. The vasculature in tumors is notoriously disorganized as represented by a number of branches, hair-pins and dead ends. Slow and unstable blood flow in tumors causes deficiencies in nutrient and oxygen delivery. Analyses of tissue oxygen levels demonstrated tissue hypoxia in tumors with a variety of origins (18, 19). In addition, aggressive proliferation of tumor cells can increase the energy demand, thereby the energy situation becomes even worse (Fig. 1). Fig. 1. View largeDownload slide Energy deficit in tumors and inflamed tissues. Disturbance of blood circulation in the tissue means a lack of nutrient and oxygen supply. Massive accumulation of inflammatory cells and aggressive proliferation of tumor cells significantly increase the energy demand in the environment. A low energy supply in spite of high demand results in an energy deficit. The insufficiency of ATP and biosynthesis attenuates immune activity in the tissue. To compensate the deficit, tissue hypoxia triggers a metabolic shift from aerobic ATP production by oxidative phosphorylation (Oxphos) to predominantly glycolytic ATP production, which does not require oxygen. In addition, hypoxia facilitates angiogenesis for a long-term solution to chronic tissue hypoxia. Fig. 1. View largeDownload slide Energy deficit in tumors and inflamed tissues. Disturbance of blood circulation in the tissue means a lack of nutrient and oxygen supply. Massive accumulation of inflammatory cells and aggressive proliferation of tumor cells significantly increase the energy demand in the environment. A low energy supply in spite of high demand results in an energy deficit. The insufficiency of ATP and biosynthesis attenuates immune activity in the tissue. To compensate the deficit, tissue hypoxia triggers a metabolic shift from aerobic ATP production by oxidative phosphorylation (Oxphos) to predominantly glycolytic ATP production, which does not require oxygen. In addition, hypoxia facilitates angiogenesis for a long-term solution to chronic tissue hypoxia. Similar to tumors, an energy deficit is almost inevitable in tissues under inflammation. Pro-inflammatory activities frequently accompany bystander damage to the surrounding healthy tissue. The damage to the surroundings involves vascular injury that decreases the blood supply in the inflamed tissue, which is often found to be hypoxic (20–22). As an example, the hypoxic area in normal colon is limited to the epithelium forming the luminal surface; however, the entire mucosa becomes highly hypoxic after the induction of inflammatory bowel disease (23, 24). Moreover, a massive accumulation of inflammatory cells can drop the energy supply-to-demand ratio even lower (Fig. 1). Tissue oxygen levels may potentially play a crucial role in regulating the immune response in both cancerous and inflamed tissues (25). Since an insufficient energy supply does not afford aggressive cellular activities, the intensity of inflammation positively correlates with oxygen availability. Mice inspiring 10% oxygen were resistant to the induction of liver inflammation (26). The diminished inflammation in mice under hypoxia accompanied a reduction of pro-inflammatory cytokine production. T-cell stimulation in mice breathing a hypoxic atmosphere largely reduced T-cell proliferation in vivo (27). In the induction of acute lung injury, exposure of mice to a hypoxic atmosphere reduced lung inflammation; however, induction of lung inflammation in a hyperoxic atmosphere exacerbated lung injury, resulting in a higher mortality of hyperoxia-treated mice (28). The exaggerated inflammation by the reversal of tissue hypoxia suggests that inflammation-associated hypoxia may trigger the spontaneous up-regulation of anti-inflammatory responses. Although alleviation of systemic hypoxemia by inspiring hyperoxic gas is crucial in the treatment of patients with lung injury, the study implied that normalization of tissue oxygen levels can break the negative feedback response and augment inflammation by unleashing previously inactive pro-inflammatory effectors (28). This concept led to application of hyperoxia to cancer treatment where vigorous anti-tumor inflammatory activities are strongly desired. Indeed, mice inspiring 60% oxygen efficiently reduced their tumor burden (29, 30). The effectiveness of 60% oxygen treatment in wild-type mice, but not in immunocompromised mice, suggested that the mechanism involves enhancement of anti-tumor immunity. Oxygen-dependent regulation of T-cell activity Since aerobic energy production fulfills a large part of cellular demand, oxygen levels in their environment draw critical attention from most cells in the body. Oxygen deficiency is an emergency situation that cells must do something about in order to survive, and hopefully adapt to, this harsh environment. Hypoxia-inducibe factor 1α (HIF-1α) is one of the key molecules to overcome the serious energy deficit under hypoxia (31–33). First of all, HIF-1α switches the energy-producing pathway from oxidative phosphorylation (oxphos) to anaerobic glycolysis. HIF-1α, as a transcription factor, induces glucose transporters and glycolytic enzymes, which promote glucose utilization by increasing its uptake and metabolism. In oxygenated conditions, pyruvate, a product of glycolytic metabolism, is converted to acetyl-CoA by pyruvate dehydrogenase and is utilized in the tricarboxylic acid cycle. However, in hypoxic conditions, HIF-1α induces pyruvate dehydrogenase kinase 1, which inhibits pyruvate dehydrogenase and reduces acetyl-CoA production. Instead, HIF-1α-dependent induction of lactate dehydrogenase increases pyruvate conversion to lactate. Thus, pyruvate metabolism is diverted to lactate production, and HIF-1α secures smooth glycolytic energy production (Fig. 1). Moreover, HIF-1α up-regulates a set of genes responsible for angiogenesis and erythropoiesis, indicating that reaction to hypoxic stress includes not only cellular adaptation to the less oxygenated environment but also efforts to improve the oxygen supply at tissue and systemic levels. With these efforts, cells can secure energy to survive hypoxic conditions. But anaerobic glycolysis can produce only 2 ATPs per 1 glucose molecule, whereas oxphos produces 36 ATPs. Even though the predominantly anaerobic glycolytic ATP production certifies the survival of cells at a quiescent state, the amount may be insufficient to support aggressive activities. Cell proliferation demands a large amount of energy for biosynthesis of every component of daughter cells (34, 35). Many of these components, such as lipids, nucleotides and amino acids, are products of oxidative metabolism. Consequently, T cells suspend proliferation under hypoxic conditions. Most in vitro studies indicate impaired T-cell proliferation as well as cytokine production when stimulated in reduced oxygen concentrations, i.e. 1–5% oxygen (27, 36–39). In vivo T-cell proliferation after the injection of an anti-CD3 monoclonal antibody (mAb) was largely inhibited in mice breathing 10% oxygen (27). Cell proliferation in the periphery is important for T-cell immunity. The T-cell population consists of numerous kinds of T cells that can cover a wide array of antigens. Since only a tiny portion of T cells can recognize a particular antigen, priming of resting T cells always starts with a small number of cells. However, the activities of T cells, especially CD8+ cytotoxic effectors, are based on direct recognition of antigen-expressing cells; therefore, antigen-specific T cells have to quickly proliferate to a significant number in order to compete with fast-growing pathogens. Naive T cells are relatively enriched with mitochondria and normally rely on oxphos, whereas activated T cells up-regulate the aerobic glycolytic pathway to support the high energy demand for proliferation (40–42). Consistent with this change, mitochondrial activities seem to be important for optimal T-cell activation (43, 44). Metabolism in mitochondria produces reactive oxygen species (ROS), which in turn promote T-cell activation (45). Manipulation to reduce mitochondrial ROS showed the importance of mitochondria in T-cell activation; mitochondrial ROS was responsible for nuclear factor of activated T cells (NF-AT) activation and subsequent T-cell proliferation (46, 47). A recent study demonstrated an increased mitochondrial content in anti-tumor T cells when PD-1 blockade therapy was applied in vivo (48). The positive correlation of mitochondrial activity in T cells with anti-tumor efficacy may suggest an advantage for oxphos in activated T cells. Moreover, treatments that increase ROS levels could further improve the anti-tumor efficacy of PD-1-blocking therapy (48). These studies indicate the significance of mitochondrial regulation for optimal activation of T cells. In contrast to T cells, ATP generation in granulocytes relies on anaerobic glycolysis, consistent with low numbers of mitochondria (49). Since granulocytes work as a first line of defense against invaded pathogens, they can migrate to any site of infection no matter what the tissue oxygen levels are. To accomplish their task even in a bleak environment lacking oxygen, aerobic energy production may not be reliable for granulocytes. HIF-1α knockdown in myeloid cells diminished in vivo inflammation led by these cells (50, 51). This finding suggests that HIF-1α-dependent glycolysis is critical for these myeloid effectors in vivo. The role of HIF-1α in T cells may not be as straightforward as in myeloid cells (52). In T cells, their activation accompanies HIF-1α stabilization even in an oxygenated environment (53). T-cell-specific deletion of HIF-1α was reported to augment numbers of IFN-γ-producing T cells (54) and to reduce eosinophilic inflammation (55), suggesting a predisposition to a Th1-type response over a Th2-type. For CD8+ T cells, in vitro studies have shown that hypoxia significantly inhibits cytotoxic T cell (CTL) development, especially in terms of the number of induced CTLs (27, 38). Mice constitutively expressing abundant HIF proteins developed CTLs with an augmented effector phenotype after viral infection in vivo (56). These results suggest the importance of adaptation to hypoxia in CTL development. When stimulated in the presence of transforming growth factor-β (TGF-β) and IL-6, HIF-1α deficiency in T cells diminished differentiation into Th17 cells and instead increased regulatory T (Treg) cells (57, 58). As a result of predisposition to Treg over Th17 cells, T-cell-specific HIF-1α knockdown diminished the intensity of inflammation. HIF-1α may intervene in the Th17/Treg balance by up-regulating RORγt while mediating degradation of FoxP3. In spite of the preferential Treg induction in the absence of HIF-1α, its role in Treg function might be a different story. Comparison of the immunosuppressive potential of Treg cells showed inferior T-cell suppression by HIF-1α-deficient Treg cells compared to wild-type Treg cells (24). Oxygen-dependent regulation of immune checkpoints As discussed in the previous section, oxygen tension directly affects the functions of immune cells. Deficits in energy production and mitochondrial metabolism can cause significant inhibition of T-cell activation and retardation of cell proliferation. Besides these direct immunosuppressive effects, hypoxia can up-regulate various immunoregulatory pathways. Targets of such regulation are not necessarily effector T cells, but the induction of negative immunoregulatory molecules in the surroundings indirectly affects the intensity of T-cell activities. The shift toward a more immunosuppressive environment by the direct and indirect effects of hypoxia is beneficial in controlling overwhelming inflammation but is detrimental in containing infection and tumor progression. Mentioned below are immunosuppressive mechanisms that are under the influence of tissue oxygen levels. Some of the components are controlled by extracellular adenosine, which is one of the immunosuppressive mechanisms induced by hypoxia, representing indirect effects of tissue hypoxia on the immune system (Fig. 2). Fig. 2. View largeDownload slide Up-regulation of immune checkpoint mechanisms by the hypoxia-adenosine pathway. Hypoxia induces quite a few immunoregulatory mechanisms. Hypoxia-dependent accumulation of extracellular adenosine, which is a potent anti-inflammatory metabolite, further triggers up-regulation of fellow immune checkpoint mechanisms. Hypoxia together with adenosine can activate the PD-1-PD-L1 system and CTLA-4-dependent immunosuppression as well as induction of immunosuppressive cytokines and immunoregulatory cells. Fig. 2. View largeDownload slide Up-regulation of immune checkpoint mechanisms by the hypoxia-adenosine pathway. Hypoxia induces quite a few immunoregulatory mechanisms. Hypoxia-dependent accumulation of extracellular adenosine, which is a potent anti-inflammatory metabolite, further triggers up-regulation of fellow immune checkpoint mechanisms. Hypoxia together with adenosine can activate the PD-1-PD-L1 system and CTLA-4-dependent immunosuppression as well as induction of immunosuppressive cytokines and immunoregulatory cells. Adenosine Extracellular adenosine is known to be a potent immunosuppressive metabolite. Of the four known adenosine receptors, the A2A receptor (A2AR) subtype is predominantly expressed in most immune cells. A2AR is a Gs protein-coupled receptor and transduces inhibitory signals via cAMP. The A2AR-mediated signal down-regulates pro-inflammatory activities in most immune cells including T cells, natural killer cells, monocytes and granulocytes (59, 60). The increase of extracellular adenosine levels is controlled by two cell surface nucleotidases—CD39 and CD73. CD39 degrades extracellular ATP to AMP and subsequently CD73 metabolizes AMP to adenosine. The massive decrease of adenosine levels in CD73-deficient mice suggests that extracellular degradation of ATP is the major adenosine-producing pathway (61, 62). Endogenously produced adenosine has been demonstrated to be indispensable in the negative regulation of immune responses. The remarkable exaggeration of inflammatory tissue injury in the absence of A2AR defined adenosine as another class of immune checkpoint molecules (52, 63, 64). Indeed, adenosine-dependent immunoregulation is noteworthy in the tumor microenvironment: the blockade of A2AR or CD73 enhanced T-cell-mediated tumor regression (65, 66). Supporting this observation, a high adenosine content has been evident in tumors (65, 67, 68). Extracellular adenosine is known to increase in hypoxic conditions, concomitantly with the up-regulation of CD39 and CD73 (69, 70). It is tissue hypoxia that is responsible for adenosine accumulation in tumors. Alleviation of hypoxia by exposing tumor-bearing mice to a hyperoxic atmosphere led to reduced adenosine levels in the tumor and enhanced anti-tumor immune responses (29). The findings suggest the biological significance of the adenosine-dependent immunoregulation, which is controlled by oxygen levels. Although the accumulation of adenosine dictates an immunosuppressive environment, hypoxia was also found to block activation of A2AR-deficient T cells, suggesting the dual involvement of adenosine-dependent and adenosine-independent mechanisms (27). The energy deprivation that is inevitable in the hypoxic condition may account for the adenosine-independent T-cell suppression. Furthermore, hypoxia can also up-regulate many other immunoregulatory mechanisms as discussed below. Cell surface checkpoint molecules Activated T cells express PD-1, and its interaction with PD-L1-expressing cells discourages T-cell activities. The mechanism of PD-1-dependent immunosuppression involves recruitment of SHP-2 tyrosine phosphatase, which down-regulates TCR signaling (13, 71). Blockade of PD-1 or PD-L1 enhances T-cell responses and promotes inflammation. The tremendous success of the PD-1-blocking strategy in cancer patients paved the way to new therapies for cancer and PD-1 became one of the best-known immune checkpoint molecules. Some studies have suggested oxygen-dependent regulation of the PD-1/PD-L1 system in the tumor microenvironment. Hypoxia induces PD-L1 in tumor cells, making them resistant to T-cell-dependent cytotoxicity. The regulation is mediated by HIF-1α, and the inhibition of HIF-1α stabilization reduced PD-L1 expression and tumor growth (72). Hypoxic induction of PD-L1 in antigen-presenting cells also reduced their T-cell stimulatory activity (73, 74). Tumor immunotherapy by anti-PD-1 or anti-PD-L1 mAbs is effectively counteracting the immunosuppressive mechanism, which might be even more important in hypoxic tumors. The effects of hypoxia on the PD-1-dependent immunoregulatory pathway may also involve an indirect mechanism through up-regulation of extracellular adenosine. Although changes in PD-L1 have not been reported, adenosine can increase PD-1 levels in T cells (75–77). Similarly, A2AR stimulation induces CTLA-4 in T cells (76, 78). The induction of PD-1 and CTLA-4 diminishes the intensity of effector T cell activities. In hypoxic conditions, PD-L1 induction in antigen-presenting cells and tumor cells among others changes the environment to an immunosuppressive one and the increase of extracellular adenosine, one of the consequences of tissue hypoxia, may enhance T-cell susceptibility to the environment. Immunosuppressive cytokines TGF-β is a potent immunosuppressive cytokine, which inhibits effector T-cell activation and the T-cell stimulatory activity of antigen-presenting cells. CD4+ T-cell activation in the presence of TGF-β induces preferential differentiation into Treg cells (79). Up-regulation of TGF-β is observed in tumor cells after culturing in hypoxic conditions (80, 81). The in vivo significance of hypoxic TGF-β induction in tumors is implicated by the diminished TGF-β expression in tumor-bearing mice that have inspired hyperoxic gas (30). Hypoxia is also reported to enhance TGF-β-induced Smad signaling and subsequent transcriptional activation (82, 83), suggesting that hypoxia may further enhance the immunosuppressive activity of TGF-β. Adenosine signaling is suggested to positively regulate TGF-β levels (84). The in vivo significance of adenosine-dependent TGF-β regulation is suggested from fibrosis studies using A2AR-deficient mice. A2AR-deficiency decreased TGF-β production and consequently prevented collagen deposition and fibrosis (85–87). Adenosine also mediates the induction of IL-10, another anti-inflammatory cytokine. A2AR stimulation of antigen-presenting cells induced IL-10 production but decreased IL-12, implicating down-regulation of cellular immune response (88, 89). Taken together, hypoxia can induce immunosuppressive cytokines in tumors directly and indirectly via adenosine production. Immunoregulatory cells The immunoregulatory activity of Treg cells is indispensable as evidenced by the severe systemic inflammation and early death of mice lacking Treg cells or their function (17, 90). Massive infiltration by Treg cells into tumors implicates their roles in the establishment of an immunosuppressive tumor microenvironment. Elimination of Treg cells may change the environment to a more immunopermissive one and improve anti-tumor immune responses (91, 92). As discussed above, the oxygen concentration may affect the local induction and immunoregulatory activity of Treg cells. However, HIF-1α-dependent regulation of Treg cells is complex, and more studies will be needed to define the precise ways in which HIF-1α-dependent regulation of Treg cells contributes to the immunosuppressive tumor microenvironment. Although the role of HIF-1α is unclear, the overall effect of hypoxia on Treg cells in tumors seems to be positive (93). Hypoxia can recruit Treg cells by inducing CCL28, a chemokine attracting peripheral Treg cells to tumor tissue (94). Alleviation of tumor tissue hypoxia by exposing mice to 60% oxygen reduced the Treg-cell population in tumors in contrast to the massive infiltration by CD8+ T cells (30). After hyperoxia treatment, intratumoral Treg cells expressed lower levels of CD39, CD73 and CTLA-4, suggesting that hypoxic conditions can enhance the immunoregulatory potential of Treg cells. Dual expression of CD39 and CD73 in Treg cells indicates their capability as independent adenosine producers, which partially accounts for the immunoregulatory activity of Treg cells (95–97). Treg cells do not just utilize extracellular adenosine as an immunosuppressive mechanisms—Treg-cell development and their immunoregulatory activity are further enhanced in adenosine-rich conditions (98). T-cell stimulation in the presence of A2AR agonists promoted expansion of Treg cells in vitro, and these Treg cells demonstrated up-regulation of immunoregulatory activity (78, 99). The biological significance of A2AR-dependent Treg-cell regulation was observed in vivo. Although adoptive transfer of Treg cells can diminish inflammation, the inferior anti-inflammatory effect of A2AR-deficient Treg cells upon transfer suggests that endogenous adenosine plays a role in the modulation of Treg activities (77). Thus, tissue hypoxia, directly or indirectly via extracellular adenosine, can promote Treg-cell-mediated immunosuppression through an increase of the local Treg population and the enhancement of immunoregulatory activities. Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) are also representative immunosuppressive immune cells in the tumor microenvironment. Local oxygen tension can regulate the immunosuppressive activities of these myeloid-cell populations. Interaction with MDSC reduces the extent of T-cell activation and, under hypoxic conditions, MDSC were found to have pronounced T-cell inhibitory effects (74). Hypoxia-induced PD-L1 up-regulation is responsible for the T-cell inhibition, since blockade of PD-L1 reversed the change and augmented the T-cell stimulatory activity of MDSC (73). HIF-1α mediates PD-L1 induction under hypoxia and is also involved in the conversion of MDSC to TAM (74). Extracellular adenosine can influence MDSC populations in tumors as well. This effect is mediated by A2B adenosine receptor (A2BR), a low-affinity receptor subtype expressed in the myeloid lineage and epithelial cells in various tissues. A2BR agonists may stimulate expansion of MDSC and increase the MDSC burden in tumors (100, 101). Prevention of endogenous adenosine binding to A2BR using a selective antagonist decreased MDSC infiltration into tumors, corresponding to slower tumor growth (102, 103). Such an effect of A2BR blockade on tumor growth may involve other cell types than MDSC. When macrophages are going to functionally differentiate, A2BR stimulation has been shown to skew them into the M2 type (104–106). M2 macrophages are regarded as tolerogenic macrophages because of their suboptimal T-cell stimulatory activity and expression of immunoregulatory molecules such as arginase, indole-2,3-deoxygenase (IDO) and TGF-β. Hypoxia impairs the expression of MHC and co-stimulatory molecules on antigen-presenting cells, thus making these cells inefficient T-cell stimulators (107, 108). Interestingly, hypoxia is also involved in the regulation of MHC expression in tumor cells. Hypoxic tumors express lower levels of MHC class I and are relatively poor targets of anti-tumor T cells. MHC down-regulation under hypoxia is mediated by HIF-1α, and reversal of hypoxia in the hyperoxic environment increased MHC levels (109). New targets to improve immunotherapy With the success of immune checkpoint blockade, we have seen cancer immunotherapy shifting its emphasis from augmentation of anti-tumor potential to neutralization of immunosuppressive barriers in tumors. Anti-tumor T cells could not be persistently functional in the tumor microenvironment, if not for the suppression of immune checkpoint mechanisms. What needs to be addressed in future is the fact that only a fraction of patients responds well to the current immune checkpoint therapy. To enhance the anti-tumor efficacy, one possibility is to combine immune checkpoint therapy with other immunotherapy, e.g. cancer vaccines and adoptive cell transfer. Blockade of multiple immune checkpoints may be additive or synergistic in cancer treatment. Some of the immune checkpoint mechanisms are so crucial that deficiency of one of these, i.e. FoxP3, CTLA-4, PD-1 or PD-L1, causes spontaneous induction of overwhelming inflammation despite all other immunoregulatory mechanisms being still functional. A2AR-deficient mice do not spontaneously develop severe autoimmune-like symptoms, but when inflammation is induced, A2AR-deficiency results in a quite exacerbated tissue injury that was not compensated for by the presence of CTLA-4 and PD-1 (63). Such a non-redundancy of each mechanism suggests that activated immune responses after the blockade of an immune checkpoint may be further enhanced by additional inactivation of other checkpoints. As in the case of co-blockade of CTLA-4 and PD-1, combined treatment with anti-adenosine approach may promote the efficacy of tumor immunotherapy by PD-1 blockade (25, 75, 110, 111). Obstacles in inducing vital anti-tumor immune responses are the deprivation of energy source and enormous activity of immune checkpoints in the tumor microenvironment. The lack of aerobic energy production in the hypoxic tissue has a direct impact on T-cell activity. Therefore, the improvement of the metabolic status in immune cells may be able to promote tumor regression induced by immune checkpoint therapy (112, 113). Improvement of mitochondrial bioenergetics by AMP-activated protein kinase is suggested to be a key regulator in the adaptation of incoming immune cells to the tumor environment where oxygen and nutrients are depleted (114). Improvement of immunological tumor regression has been reported in experiments using the AMP kinase activator metformin (115) and by the combined use of anti-PD-L1 mAb and bezafibrate, which can enhance mitochondrial activity (48). In this context, tissue hypoxia represents a potential target of immunotherapy (25, 72, 116). Tissue hypoxia and its product extracellular adenosine are not just immunosuppressive due to direct effects on T cells but are also inducers of diverse checkpoint mechanisms, including PD-1/PD-L1, CTLA-4, TGF-β, IL-10, Treg cells, MDSC and M2-type macrophages. Reversal of the hypoxic up-regulation of immune checkpoints will change the tumor microenvironment to become more immunopermissive and enhance the anti-tumor immune response. Moreover, induction of MHC expression by tissue oxygenation may increase the immunogenicity of tumor cells (107, 109). Indeed, the treatment of tumor-bearing mice with 60% oxygen alleviated tumor hypoxia and induced tumor regression (29, 30). Combined treatment with hyperoxia enhanced the anti-tumor efficacy of PD-1 blockade. Conclusions The clinical outcome of immune checkpoint blockade in cancer treatment has clearly demonstrated that attenuation of immunosuppressive activity in the tumor microenvironment is the key to the success. Although current approaches to the immune checkpoints mostly focus on the blockade of their functions, the regulatory mechanisms of immune checkpoint components may represent a new target of therapeutic intervention. Evidence indicates the uniqueness of tissue hypoxia as a negative regulator of T-cell immunity by multiple mechanisms: energy metabolism, induction of immune checkpoints and MHC regulation. 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Oxygen-dependent regulation of immune checkpoint mechanisms

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Oxford University Press
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© The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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0953-8178
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1460-2377
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10.1093/intimm/dxy038
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Abstract

Abstract Immunotherapy of cancer has finally materialized following the success of immune checkpoint blockade. Since down-regulation of immune checkpoint mechanisms is beneficial in cancer treatment, it is important to ask why tumors are infamously filled with the immunosuppressive mechanisms. Indeed, immune checkpoints are physiological negative feedback mechanisms of immune activities, and the induction of such mechanisms is important in preventing excessive destruction of inflamed normal tissues. A condition commonly found in tumors and inflamed tissues is tissue hypoxia. Oxygen deprivation under hypoxic conditions by itself is immunosuppressive because proper oxygen supply could support bioenergetic demands of immune cells for optimal immune responses. However, importantly, hypoxia has been found to up-regulate a variety of immune checkpoints and to be able to drive a shift toward a more immunosuppressive environment. Moreover, extracellular adenosine, which accumulates due to tissue hypoxia, also contributes to the up-regulation of other immune checkpoints. Taken together, tissue oxygen is a key regulator of the immune response by directly affecting the energy status of immune effectors and by regulating the intensity of immunoregulatory activity in the environment. The regulators of various immune checkpoint mechanisms may represent the next focus to modulate the intensity of immune responses and to improve cancer immunotherapy. adenosine, hypoxia, immunosuppression, immunotherapy, tumor microenvironment Introduction: immunotherapy as the fourth strategy against cancer It was more than 100 years ago that inflammatory cell infiltration into tumors prompted the idea of tumor regression by immunological means. Since then, cancer immunology has been a focus of intensive research. Vital anti-tumor immunity should be beneficial in cancer prevention by eliminating abnormal cells and in clinics through suppression of cancer progression and metastatic burden. Cancer immunotherapy represents a novel therapeutic strategy that may work alone or in a complementary way to the three main conventional therapeutic strategies—surgical therapy, chemotherapy and radiotherapy. After initial efforts using non-specific immune activators saw little success, the discovery of tumor-associated antigens granted antigen specificity to cancer immunotherapy. Tumor-specific T cells have been used in adoptive therapy, and tumor-associated antigens have been inoculated as vaccines. A more recent invention of adoptive immunotherapy uses T cells with chimeric activating receptors (CAR-T cells), which may overcome the poor immunogenicity of tumor cells that have down-regulated major histocompatibility complex (MHC) expression (1). It should be noted that the long and extensive quest for practical cancer immunotherapy has been mostly focused on how to increase the total attacking potential of anti-tumor immunity, for example by transferring effector cells that have been expanded ex vivo, including CAR-T cells, and by inducing effector cells in vivo. But, what about the persistence of immune activities? In 1968, Hellström et al. questioned the paradoxical co-existence of anti-tumor immune cells in cancer patients (2). T cells that have infiltrated growing tumors are capable of recognizing tumor cells; however, they are somehow quiescent in vivo without demonstrating any significant anti-tumor activity. Later, it became apparent that the tumor microenvironment is very immunosuppressive, and incoming anti-tumor effector cells are prone to the inhibitory mechanisms in tumors (3–5). Analyses of the immunosuppressive mechanisms identified some key targets, and antibodies that block molecules such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1) have demonstrated impressive efficacy against cancer by inducing a persistent anti-tumor immune response (6, 7). The eventual success of these antibodies in human cancer is an indication that the fourth therapeutic strategy—immunotherapy—has finally become a realistic option in the treatment of cancer patients (8, 9). Since then, research has identified quite a few other negative immune regulators in the tumor microenvironment, but it is not clear how tumors acquire so many immunosuppressive mechanisms. Here, I discuss a potential role of tissue oxygen levels as a key regulator of immune responses. The first half of this review focuses on the significance of oxygen in T-cell immunity and the second part describes specific regulation of each immune checkpoints dependent on oxygen levels. Physiological down-regulation of inflammation Current practical immunotherapy targets components of potent immunosuppressive mechanisms: CTLA-4 blocks the CD28-dependent co-stimulatory signal (10, 11), and interaction of PD-1 with its ligands PD-L1 and PD-L2 can also down-regulate the T cell receptor (TCR)-mediated activating signal (12, 13). As well as these cell surface molecules, there are other negative immunoregulators that discourage effector T cells, ranging from small molecules to enzymes and to immunoregulatory cells (Table 1). Table 1. Immunosuppression by different classes of immune checkpoint mechanisms Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression BTLA, B and T lymphocyte attenuator; EP2/EP4, prostaglandin E2 receptor 2/4; HMGB-1, high-mobility group protein B-1; LAG-3, lymphocyte activation gene 3; PGE2, prostaglandin E2; TIGIT, T cell immunoglobulin and ITIM domain; TIM-3, T-cell immunoglobulin and mucin domain 3; TNFRSF14, TNFR superfamily member 14. View Large Table 1. Immunosuppression by different classes of immune checkpoint mechanisms Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression Category Examples Mechanisms Small molecules Adenosine cAMP signal through A2AR PGE2 cAMP signal through EP2/EP4 Cytokines IL-10 IL-10 receptor signaling TGF-β TGF-β receptor signaling Enzymes IDO Tryptophan deprivation Cell surface molecules PD-1 Interaction with PD-L1/PD-L2 CTLA-4 Competition with CD28 at CD80/CD86 LAG-3 Competition with CD4 at MHC class II TIM-3 Interaction with galectin-9/ HMGB-1 BTLA Interaction with B7/TNFRSF14 TIGIT Interaction with CD155 Immunoregulatory cells Treg cells Antigen-specific immunosuppression MDSC Non-specific immunosuppression TAM Non-specific immunosuppression BTLA, B and T lymphocyte attenuator; EP2/EP4, prostaglandin E2 receptor 2/4; HMGB-1, high-mobility group protein B-1; LAG-3, lymphocyte activation gene 3; PGE2, prostaglandin E2; TIGIT, T cell immunoglobulin and ITIM domain; TIM-3, T-cell immunoglobulin and mucin domain 3; TNFRSF14, TNFR superfamily member 14. View Large Immune checkpoints, which have become best known as potential targets of cancer therapy, are indispensable immunoregulatory mechanisms anywhere in the body. In an episode of inflammation, a pro-inflammatory response is followed by down-regulation of inflammatory activities and subsequent tissue remodeling. Immune checkpoints are responsible for the down-regulation of inflammation. Importantly, many immune checkpoint mechanisms become available after the induction of inflammation. For example, CTLA-4 and PD-1 are normally absent in resting effector T cells but are up-regulated upon T-cell activation. Extracellular adenosine increases in response to tissue injury during inflammation. Treg cells may be also inducible after stimulating T cells in a specific cytokine milieu. Extensive up-regulation of immunosuppressive mechanisms after immune activation suggests that immune checkpoints are essentially negative feedback mechanisms to ongoing pro-inflammatory activities in order to prevent unwanted damage to the tissue. Such endogenous immunoregulation is so important that the lack of only one of the immune checkpoints causes persistent inflammatory tissue injury, which is often severe enough to be fatal (14–17). The balance between pro-inflammatory and anti-inflammatory mechanisms defines the intensity and duration of inflammation and outcome of diseases. Tumors, as a tissue that originates in the same body, take advantage of the endogenous tissue-protective mechanisms and fortify themselves against immune attack. To defeat tumors’ heavy emphasis on their anti-inflammatory potential to evade immune attack, it is important to ask about the mechanism by which tumors successfully acquire an assortment of immune checkpoints. Since normal tissues employ the same mechanisms in the protection from overactive immune responses, there might be a clue in something common in cancerous and inflamed tissues. What is commonly observed may be an imbalance of energy demand and supply. The vasculature in tumors is notoriously disorganized as represented by a number of branches, hair-pins and dead ends. Slow and unstable blood flow in tumors causes deficiencies in nutrient and oxygen delivery. Analyses of tissue oxygen levels demonstrated tissue hypoxia in tumors with a variety of origins (18, 19). In addition, aggressive proliferation of tumor cells can increase the energy demand, thereby the energy situation becomes even worse (Fig. 1). Fig. 1. View largeDownload slide Energy deficit in tumors and inflamed tissues. Disturbance of blood circulation in the tissue means a lack of nutrient and oxygen supply. Massive accumulation of inflammatory cells and aggressive proliferation of tumor cells significantly increase the energy demand in the environment. A low energy supply in spite of high demand results in an energy deficit. The insufficiency of ATP and biosynthesis attenuates immune activity in the tissue. To compensate the deficit, tissue hypoxia triggers a metabolic shift from aerobic ATP production by oxidative phosphorylation (Oxphos) to predominantly glycolytic ATP production, which does not require oxygen. In addition, hypoxia facilitates angiogenesis for a long-term solution to chronic tissue hypoxia. Fig. 1. View largeDownload slide Energy deficit in tumors and inflamed tissues. Disturbance of blood circulation in the tissue means a lack of nutrient and oxygen supply. Massive accumulation of inflammatory cells and aggressive proliferation of tumor cells significantly increase the energy demand in the environment. A low energy supply in spite of high demand results in an energy deficit. The insufficiency of ATP and biosynthesis attenuates immune activity in the tissue. To compensate the deficit, tissue hypoxia triggers a metabolic shift from aerobic ATP production by oxidative phosphorylation (Oxphos) to predominantly glycolytic ATP production, which does not require oxygen. In addition, hypoxia facilitates angiogenesis for a long-term solution to chronic tissue hypoxia. Similar to tumors, an energy deficit is almost inevitable in tissues under inflammation. Pro-inflammatory activities frequently accompany bystander damage to the surrounding healthy tissue. The damage to the surroundings involves vascular injury that decreases the blood supply in the inflamed tissue, which is often found to be hypoxic (20–22). As an example, the hypoxic area in normal colon is limited to the epithelium forming the luminal surface; however, the entire mucosa becomes highly hypoxic after the induction of inflammatory bowel disease (23, 24). Moreover, a massive accumulation of inflammatory cells can drop the energy supply-to-demand ratio even lower (Fig. 1). Tissue oxygen levels may potentially play a crucial role in regulating the immune response in both cancerous and inflamed tissues (25). Since an insufficient energy supply does not afford aggressive cellular activities, the intensity of inflammation positively correlates with oxygen availability. Mice inspiring 10% oxygen were resistant to the induction of liver inflammation (26). The diminished inflammation in mice under hypoxia accompanied a reduction of pro-inflammatory cytokine production. T-cell stimulation in mice breathing a hypoxic atmosphere largely reduced T-cell proliferation in vivo (27). In the induction of acute lung injury, exposure of mice to a hypoxic atmosphere reduced lung inflammation; however, induction of lung inflammation in a hyperoxic atmosphere exacerbated lung injury, resulting in a higher mortality of hyperoxia-treated mice (28). The exaggerated inflammation by the reversal of tissue hypoxia suggests that inflammation-associated hypoxia may trigger the spontaneous up-regulation of anti-inflammatory responses. Although alleviation of systemic hypoxemia by inspiring hyperoxic gas is crucial in the treatment of patients with lung injury, the study implied that normalization of tissue oxygen levels can break the negative feedback response and augment inflammation by unleashing previously inactive pro-inflammatory effectors (28). This concept led to application of hyperoxia to cancer treatment where vigorous anti-tumor inflammatory activities are strongly desired. Indeed, mice inspiring 60% oxygen efficiently reduced their tumor burden (29, 30). The effectiveness of 60% oxygen treatment in wild-type mice, but not in immunocompromised mice, suggested that the mechanism involves enhancement of anti-tumor immunity. Oxygen-dependent regulation of T-cell activity Since aerobic energy production fulfills a large part of cellular demand, oxygen levels in their environment draw critical attention from most cells in the body. Oxygen deficiency is an emergency situation that cells must do something about in order to survive, and hopefully adapt to, this harsh environment. Hypoxia-inducibe factor 1α (HIF-1α) is one of the key molecules to overcome the serious energy deficit under hypoxia (31–33). First of all, HIF-1α switches the energy-producing pathway from oxidative phosphorylation (oxphos) to anaerobic glycolysis. HIF-1α, as a transcription factor, induces glucose transporters and glycolytic enzymes, which promote glucose utilization by increasing its uptake and metabolism. In oxygenated conditions, pyruvate, a product of glycolytic metabolism, is converted to acetyl-CoA by pyruvate dehydrogenase and is utilized in the tricarboxylic acid cycle. However, in hypoxic conditions, HIF-1α induces pyruvate dehydrogenase kinase 1, which inhibits pyruvate dehydrogenase and reduces acetyl-CoA production. Instead, HIF-1α-dependent induction of lactate dehydrogenase increases pyruvate conversion to lactate. Thus, pyruvate metabolism is diverted to lactate production, and HIF-1α secures smooth glycolytic energy production (Fig. 1). Moreover, HIF-1α up-regulates a set of genes responsible for angiogenesis and erythropoiesis, indicating that reaction to hypoxic stress includes not only cellular adaptation to the less oxygenated environment but also efforts to improve the oxygen supply at tissue and systemic levels. With these efforts, cells can secure energy to survive hypoxic conditions. But anaerobic glycolysis can produce only 2 ATPs per 1 glucose molecule, whereas oxphos produces 36 ATPs. Even though the predominantly anaerobic glycolytic ATP production certifies the survival of cells at a quiescent state, the amount may be insufficient to support aggressive activities. Cell proliferation demands a large amount of energy for biosynthesis of every component of daughter cells (34, 35). Many of these components, such as lipids, nucleotides and amino acids, are products of oxidative metabolism. Consequently, T cells suspend proliferation under hypoxic conditions. Most in vitro studies indicate impaired T-cell proliferation as well as cytokine production when stimulated in reduced oxygen concentrations, i.e. 1–5% oxygen (27, 36–39). In vivo T-cell proliferation after the injection of an anti-CD3 monoclonal antibody (mAb) was largely inhibited in mice breathing 10% oxygen (27). Cell proliferation in the periphery is important for T-cell immunity. The T-cell population consists of numerous kinds of T cells that can cover a wide array of antigens. Since only a tiny portion of T cells can recognize a particular antigen, priming of resting T cells always starts with a small number of cells. However, the activities of T cells, especially CD8+ cytotoxic effectors, are based on direct recognition of antigen-expressing cells; therefore, antigen-specific T cells have to quickly proliferate to a significant number in order to compete with fast-growing pathogens. Naive T cells are relatively enriched with mitochondria and normally rely on oxphos, whereas activated T cells up-regulate the aerobic glycolytic pathway to support the high energy demand for proliferation (40–42). Consistent with this change, mitochondrial activities seem to be important for optimal T-cell activation (43, 44). Metabolism in mitochondria produces reactive oxygen species (ROS), which in turn promote T-cell activation (45). Manipulation to reduce mitochondrial ROS showed the importance of mitochondria in T-cell activation; mitochondrial ROS was responsible for nuclear factor of activated T cells (NF-AT) activation and subsequent T-cell proliferation (46, 47). A recent study demonstrated an increased mitochondrial content in anti-tumor T cells when PD-1 blockade therapy was applied in vivo (48). The positive correlation of mitochondrial activity in T cells with anti-tumor efficacy may suggest an advantage for oxphos in activated T cells. Moreover, treatments that increase ROS levels could further improve the anti-tumor efficacy of PD-1-blocking therapy (48). These studies indicate the significance of mitochondrial regulation for optimal activation of T cells. In contrast to T cells, ATP generation in granulocytes relies on anaerobic glycolysis, consistent with low numbers of mitochondria (49). Since granulocytes work as a first line of defense against invaded pathogens, they can migrate to any site of infection no matter what the tissue oxygen levels are. To accomplish their task even in a bleak environment lacking oxygen, aerobic energy production may not be reliable for granulocytes. HIF-1α knockdown in myeloid cells diminished in vivo inflammation led by these cells (50, 51). This finding suggests that HIF-1α-dependent glycolysis is critical for these myeloid effectors in vivo. The role of HIF-1α in T cells may not be as straightforward as in myeloid cells (52). In T cells, their activation accompanies HIF-1α stabilization even in an oxygenated environment (53). T-cell-specific deletion of HIF-1α was reported to augment numbers of IFN-γ-producing T cells (54) and to reduce eosinophilic inflammation (55), suggesting a predisposition to a Th1-type response over a Th2-type. For CD8+ T cells, in vitro studies have shown that hypoxia significantly inhibits cytotoxic T cell (CTL) development, especially in terms of the number of induced CTLs (27, 38). Mice constitutively expressing abundant HIF proteins developed CTLs with an augmented effector phenotype after viral infection in vivo (56). These results suggest the importance of adaptation to hypoxia in CTL development. When stimulated in the presence of transforming growth factor-β (TGF-β) and IL-6, HIF-1α deficiency in T cells diminished differentiation into Th17 cells and instead increased regulatory T (Treg) cells (57, 58). As a result of predisposition to Treg over Th17 cells, T-cell-specific HIF-1α knockdown diminished the intensity of inflammation. HIF-1α may intervene in the Th17/Treg balance by up-regulating RORγt while mediating degradation of FoxP3. In spite of the preferential Treg induction in the absence of HIF-1α, its role in Treg function might be a different story. Comparison of the immunosuppressive potential of Treg cells showed inferior T-cell suppression by HIF-1α-deficient Treg cells compared to wild-type Treg cells (24). Oxygen-dependent regulation of immune checkpoints As discussed in the previous section, oxygen tension directly affects the functions of immune cells. Deficits in energy production and mitochondrial metabolism can cause significant inhibition of T-cell activation and retardation of cell proliferation. Besides these direct immunosuppressive effects, hypoxia can up-regulate various immunoregulatory pathways. Targets of such regulation are not necessarily effector T cells, but the induction of negative immunoregulatory molecules in the surroundings indirectly affects the intensity of T-cell activities. The shift toward a more immunosuppressive environment by the direct and indirect effects of hypoxia is beneficial in controlling overwhelming inflammation but is detrimental in containing infection and tumor progression. Mentioned below are immunosuppressive mechanisms that are under the influence of tissue oxygen levels. Some of the components are controlled by extracellular adenosine, which is one of the immunosuppressive mechanisms induced by hypoxia, representing indirect effects of tissue hypoxia on the immune system (Fig. 2). Fig. 2. View largeDownload slide Up-regulation of immune checkpoint mechanisms by the hypoxia-adenosine pathway. Hypoxia induces quite a few immunoregulatory mechanisms. Hypoxia-dependent accumulation of extracellular adenosine, which is a potent anti-inflammatory metabolite, further triggers up-regulation of fellow immune checkpoint mechanisms. Hypoxia together with adenosine can activate the PD-1-PD-L1 system and CTLA-4-dependent immunosuppression as well as induction of immunosuppressive cytokines and immunoregulatory cells. Fig. 2. View largeDownload slide Up-regulation of immune checkpoint mechanisms by the hypoxia-adenosine pathway. Hypoxia induces quite a few immunoregulatory mechanisms. Hypoxia-dependent accumulation of extracellular adenosine, which is a potent anti-inflammatory metabolite, further triggers up-regulation of fellow immune checkpoint mechanisms. Hypoxia together with adenosine can activate the PD-1-PD-L1 system and CTLA-4-dependent immunosuppression as well as induction of immunosuppressive cytokines and immunoregulatory cells. Adenosine Extracellular adenosine is known to be a potent immunosuppressive metabolite. Of the four known adenosine receptors, the A2A receptor (A2AR) subtype is predominantly expressed in most immune cells. A2AR is a Gs protein-coupled receptor and transduces inhibitory signals via cAMP. The A2AR-mediated signal down-regulates pro-inflammatory activities in most immune cells including T cells, natural killer cells, monocytes and granulocytes (59, 60). The increase of extracellular adenosine levels is controlled by two cell surface nucleotidases—CD39 and CD73. CD39 degrades extracellular ATP to AMP and subsequently CD73 metabolizes AMP to adenosine. The massive decrease of adenosine levels in CD73-deficient mice suggests that extracellular degradation of ATP is the major adenosine-producing pathway (61, 62). Endogenously produced adenosine has been demonstrated to be indispensable in the negative regulation of immune responses. The remarkable exaggeration of inflammatory tissue injury in the absence of A2AR defined adenosine as another class of immune checkpoint molecules (52, 63, 64). Indeed, adenosine-dependent immunoregulation is noteworthy in the tumor microenvironment: the blockade of A2AR or CD73 enhanced T-cell-mediated tumor regression (65, 66). Supporting this observation, a high adenosine content has been evident in tumors (65, 67, 68). Extracellular adenosine is known to increase in hypoxic conditions, concomitantly with the up-regulation of CD39 and CD73 (69, 70). It is tissue hypoxia that is responsible for adenosine accumulation in tumors. Alleviation of hypoxia by exposing tumor-bearing mice to a hyperoxic atmosphere led to reduced adenosine levels in the tumor and enhanced anti-tumor immune responses (29). The findings suggest the biological significance of the adenosine-dependent immunoregulation, which is controlled by oxygen levels. Although the accumulation of adenosine dictates an immunosuppressive environment, hypoxia was also found to block activation of A2AR-deficient T cells, suggesting the dual involvement of adenosine-dependent and adenosine-independent mechanisms (27). The energy deprivation that is inevitable in the hypoxic condition may account for the adenosine-independent T-cell suppression. Furthermore, hypoxia can also up-regulate many other immunoregulatory mechanisms as discussed below. Cell surface checkpoint molecules Activated T cells express PD-1, and its interaction with PD-L1-expressing cells discourages T-cell activities. The mechanism of PD-1-dependent immunosuppression involves recruitment of SHP-2 tyrosine phosphatase, which down-regulates TCR signaling (13, 71). Blockade of PD-1 or PD-L1 enhances T-cell responses and promotes inflammation. The tremendous success of the PD-1-blocking strategy in cancer patients paved the way to new therapies for cancer and PD-1 became one of the best-known immune checkpoint molecules. Some studies have suggested oxygen-dependent regulation of the PD-1/PD-L1 system in the tumor microenvironment. Hypoxia induces PD-L1 in tumor cells, making them resistant to T-cell-dependent cytotoxicity. The regulation is mediated by HIF-1α, and the inhibition of HIF-1α stabilization reduced PD-L1 expression and tumor growth (72). Hypoxic induction of PD-L1 in antigen-presenting cells also reduced their T-cell stimulatory activity (73, 74). Tumor immunotherapy by anti-PD-1 or anti-PD-L1 mAbs is effectively counteracting the immunosuppressive mechanism, which might be even more important in hypoxic tumors. The effects of hypoxia on the PD-1-dependent immunoregulatory pathway may also involve an indirect mechanism through up-regulation of extracellular adenosine. Although changes in PD-L1 have not been reported, adenosine can increase PD-1 levels in T cells (75–77). Similarly, A2AR stimulation induces CTLA-4 in T cells (76, 78). The induction of PD-1 and CTLA-4 diminishes the intensity of effector T cell activities. In hypoxic conditions, PD-L1 induction in antigen-presenting cells and tumor cells among others changes the environment to an immunosuppressive one and the increase of extracellular adenosine, one of the consequences of tissue hypoxia, may enhance T-cell susceptibility to the environment. Immunosuppressive cytokines TGF-β is a potent immunosuppressive cytokine, which inhibits effector T-cell activation and the T-cell stimulatory activity of antigen-presenting cells. CD4+ T-cell activation in the presence of TGF-β induces preferential differentiation into Treg cells (79). Up-regulation of TGF-β is observed in tumor cells after culturing in hypoxic conditions (80, 81). The in vivo significance of hypoxic TGF-β induction in tumors is implicated by the diminished TGF-β expression in tumor-bearing mice that have inspired hyperoxic gas (30). Hypoxia is also reported to enhance TGF-β-induced Smad signaling and subsequent transcriptional activation (82, 83), suggesting that hypoxia may further enhance the immunosuppressive activity of TGF-β. Adenosine signaling is suggested to positively regulate TGF-β levels (84). The in vivo significance of adenosine-dependent TGF-β regulation is suggested from fibrosis studies using A2AR-deficient mice. A2AR-deficiency decreased TGF-β production and consequently prevented collagen deposition and fibrosis (85–87). Adenosine also mediates the induction of IL-10, another anti-inflammatory cytokine. A2AR stimulation of antigen-presenting cells induced IL-10 production but decreased IL-12, implicating down-regulation of cellular immune response (88, 89). Taken together, hypoxia can induce immunosuppressive cytokines in tumors directly and indirectly via adenosine production. Immunoregulatory cells The immunoregulatory activity of Treg cells is indispensable as evidenced by the severe systemic inflammation and early death of mice lacking Treg cells or their function (17, 90). Massive infiltration by Treg cells into tumors implicates their roles in the establishment of an immunosuppressive tumor microenvironment. Elimination of Treg cells may change the environment to a more immunopermissive one and improve anti-tumor immune responses (91, 92). As discussed above, the oxygen concentration may affect the local induction and immunoregulatory activity of Treg cells. However, HIF-1α-dependent regulation of Treg cells is complex, and more studies will be needed to define the precise ways in which HIF-1α-dependent regulation of Treg cells contributes to the immunosuppressive tumor microenvironment. Although the role of HIF-1α is unclear, the overall effect of hypoxia on Treg cells in tumors seems to be positive (93). Hypoxia can recruit Treg cells by inducing CCL28, a chemokine attracting peripheral Treg cells to tumor tissue (94). Alleviation of tumor tissue hypoxia by exposing mice to 60% oxygen reduced the Treg-cell population in tumors in contrast to the massive infiltration by CD8+ T cells (30). After hyperoxia treatment, intratumoral Treg cells expressed lower levels of CD39, CD73 and CTLA-4, suggesting that hypoxic conditions can enhance the immunoregulatory potential of Treg cells. Dual expression of CD39 and CD73 in Treg cells indicates their capability as independent adenosine producers, which partially accounts for the immunoregulatory activity of Treg cells (95–97). Treg cells do not just utilize extracellular adenosine as an immunosuppressive mechanisms—Treg-cell development and their immunoregulatory activity are further enhanced in adenosine-rich conditions (98). T-cell stimulation in the presence of A2AR agonists promoted expansion of Treg cells in vitro, and these Treg cells demonstrated up-regulation of immunoregulatory activity (78, 99). The biological significance of A2AR-dependent Treg-cell regulation was observed in vivo. Although adoptive transfer of Treg cells can diminish inflammation, the inferior anti-inflammatory effect of A2AR-deficient Treg cells upon transfer suggests that endogenous adenosine plays a role in the modulation of Treg activities (77). Thus, tissue hypoxia, directly or indirectly via extracellular adenosine, can promote Treg-cell-mediated immunosuppression through an increase of the local Treg population and the enhancement of immunoregulatory activities. Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) are also representative immunosuppressive immune cells in the tumor microenvironment. Local oxygen tension can regulate the immunosuppressive activities of these myeloid-cell populations. Interaction with MDSC reduces the extent of T-cell activation and, under hypoxic conditions, MDSC were found to have pronounced T-cell inhibitory effects (74). Hypoxia-induced PD-L1 up-regulation is responsible for the T-cell inhibition, since blockade of PD-L1 reversed the change and augmented the T-cell stimulatory activity of MDSC (73). HIF-1α mediates PD-L1 induction under hypoxia and is also involved in the conversion of MDSC to TAM (74). Extracellular adenosine can influence MDSC populations in tumors as well. This effect is mediated by A2B adenosine receptor (A2BR), a low-affinity receptor subtype expressed in the myeloid lineage and epithelial cells in various tissues. A2BR agonists may stimulate expansion of MDSC and increase the MDSC burden in tumors (100, 101). Prevention of endogenous adenosine binding to A2BR using a selective antagonist decreased MDSC infiltration into tumors, corresponding to slower tumor growth (102, 103). Such an effect of A2BR blockade on tumor growth may involve other cell types than MDSC. When macrophages are going to functionally differentiate, A2BR stimulation has been shown to skew them into the M2 type (104–106). M2 macrophages are regarded as tolerogenic macrophages because of their suboptimal T-cell stimulatory activity and expression of immunoregulatory molecules such as arginase, indole-2,3-deoxygenase (IDO) and TGF-β. Hypoxia impairs the expression of MHC and co-stimulatory molecules on antigen-presenting cells, thus making these cells inefficient T-cell stimulators (107, 108). Interestingly, hypoxia is also involved in the regulation of MHC expression in tumor cells. Hypoxic tumors express lower levels of MHC class I and are relatively poor targets of anti-tumor T cells. MHC down-regulation under hypoxia is mediated by HIF-1α, and reversal of hypoxia in the hyperoxic environment increased MHC levels (109). New targets to improve immunotherapy With the success of immune checkpoint blockade, we have seen cancer immunotherapy shifting its emphasis from augmentation of anti-tumor potential to neutralization of immunosuppressive barriers in tumors. Anti-tumor T cells could not be persistently functional in the tumor microenvironment, if not for the suppression of immune checkpoint mechanisms. What needs to be addressed in future is the fact that only a fraction of patients responds well to the current immune checkpoint therapy. To enhance the anti-tumor efficacy, one possibility is to combine immune checkpoint therapy with other immunotherapy, e.g. cancer vaccines and adoptive cell transfer. Blockade of multiple immune checkpoints may be additive or synergistic in cancer treatment. Some of the immune checkpoint mechanisms are so crucial that deficiency of one of these, i.e. FoxP3, CTLA-4, PD-1 or PD-L1, causes spontaneous induction of overwhelming inflammation despite all other immunoregulatory mechanisms being still functional. A2AR-deficient mice do not spontaneously develop severe autoimmune-like symptoms, but when inflammation is induced, A2AR-deficiency results in a quite exacerbated tissue injury that was not compensated for by the presence of CTLA-4 and PD-1 (63). Such a non-redundancy of each mechanism suggests that activated immune responses after the blockade of an immune checkpoint may be further enhanced by additional inactivation of other checkpoints. As in the case of co-blockade of CTLA-4 and PD-1, combined treatment with anti-adenosine approach may promote the efficacy of tumor immunotherapy by PD-1 blockade (25, 75, 110, 111). Obstacles in inducing vital anti-tumor immune responses are the deprivation of energy source and enormous activity of immune checkpoints in the tumor microenvironment. The lack of aerobic energy production in the hypoxic tissue has a direct impact on T-cell activity. Therefore, the improvement of the metabolic status in immune cells may be able to promote tumor regression induced by immune checkpoint therapy (112, 113). Improvement of mitochondrial bioenergetics by AMP-activated protein kinase is suggested to be a key regulator in the adaptation of incoming immune cells to the tumor environment where oxygen and nutrients are depleted (114). Improvement of immunological tumor regression has been reported in experiments using the AMP kinase activator metformin (115) and by the combined use of anti-PD-L1 mAb and bezafibrate, which can enhance mitochondrial activity (48). In this context, tissue hypoxia represents a potential target of immunotherapy (25, 72, 116). Tissue hypoxia and its product extracellular adenosine are not just immunosuppressive due to direct effects on T cells but are also inducers of diverse checkpoint mechanisms, including PD-1/PD-L1, CTLA-4, TGF-β, IL-10, Treg cells, MDSC and M2-type macrophages. Reversal of the hypoxic up-regulation of immune checkpoints will change the tumor microenvironment to become more immunopermissive and enhance the anti-tumor immune response. Moreover, induction of MHC expression by tissue oxygenation may increase the immunogenicity of tumor cells (107, 109). Indeed, the treatment of tumor-bearing mice with 60% oxygen alleviated tumor hypoxia and induced tumor regression (29, 30). Combined treatment with hyperoxia enhanced the anti-tumor efficacy of PD-1 blockade. Conclusions The clinical outcome of immune checkpoint blockade in cancer treatment has clearly demonstrated that attenuation of immunosuppressive activity in the tumor microenvironment is the key to the success. Although current approaches to the immune checkpoints mostly focus on the blockade of their functions, the regulatory mechanisms of immune checkpoint components may represent a new target of therapeutic intervention. Evidence indicates the uniqueness of tissue hypoxia as a negative regulator of T-cell immunity by multiple mechanisms: energy metabolism, induction of immune checkpoints and MHC regulation. 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International ImmunologyOxford University Press

Published: Aug 1, 2018

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