‘Targeting ectonucleotidases to treat inflammation and halt cancer development in the gut’

Abstract

CD39 and CD73 control cell immunity by hydrolyzing proinflammatory ATP and ADP (CD39) into AMP, subsequently converted into anti-inflammatory adenosine (CD73). By regulating the balance between effector and regulatory cells, these ectonucleotidases promote immune homeostasis in acute and chronic inflammation; while also appearing to limit antitumor effector immunity in gut cancer. This manuscript focuses on the pivotal role of CD39 and CD73 ectonucleotidase function in shaping immune responses in the gut. We focus on those mechanisms deployed by these critical and pivotal ectoenzymes and the regulation in the setting of gastrointestinal tract infections, inflammatory bowel disease and tumors of the gastrointestinal tract. We will highlight translational and clinical implications of the latest and most innovative basic research discoveries of these important players of the purinergic signaling. Immunotherapeutic strategies that have been developed to either boost or control ectonucleotidase expression and activity in important disease settings are also reviewed and the in vivo effects discussed.

1. Introduction

Adenosine triphosphate (ATP) is not only the energy currency of all cells, but is critical for multiple biological functions and pathways, upon which life critically depends. In addition, when released from cells or platelets, as in the setting of inflammation, ATP and derivative nucleotides and nucleosides signal to cells through networks of receptors to modulate these essential processes [1].

This review on has been written on the regulatory aspects of purinergic signaling in the gut and liver and prepared for this special issue of Biochemical Pharmacology to mark the dual legacy of Geoffrey Burnstock, on his recent passing in Melbourne, Australia.

Dr. Burnstock made seminal discoveries in both areas of neuronal cotransmission and purinergic signaling. He developed a large body of work to show that extracellular ATP can serve as a cotransmitter with classical others in nerves in the peripheral nervous and central nervous systems [2]. Indeed, it was his work to first show that extracellular ATP was released from sympathetic nerves supplying the guinea-pig tenia coli [3]. Later work indicated that ATP and related nucleotides were also co-transmitter substances released by non-adrenergic and other nerves of the gut and this resulted in the further development of the purinergic hypothesis [4].

It has now been shown that purinergic responses develop in response to cellular activation or of tissue disturbing events like inflammation, hypoxia or ischemic conditions [1], in the gut and elsewhere. Extracellular ATP and other released nucleotides, e.g. UTP pyrimidines, mediate a plethora of biologic and inflammatory events upon binding to cell surface type 2 purinergic receptors (P2) [5]: namely ligand-gated ion channel receptors (P2XR) and G-protein coupled receptors (P2YR). ATP and other nucleotides are then hydrolyzed to adenosine diphosphate (ADP) and monophosphate (AMP), and are ultimately converted into adenosine derivatives, which have largely anti-inflammatory and immunosuppressive properties.

This extracellular phosphohydrolysis of ATP and other nucleotides is mediated by ectonucleotidases (ectoenzymes), inclusive of: the ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases/CD39 family), the ecto-5’-nucleotidase (NT5E)/CD73, the ecto-nucleotide pyrophosphate phosphodiesterases (E-NPPs), NAD glycohydrolases, the CD38/NADase, alkaline phosphatase, adenylate kinase, the nucleoside diphosphate kinase; and the ecto-F1-F0 ATP synthases [6].

The family of ENTPDases includes NTPDases 1, 2, 3 and 8, which are located on the cell surface and are a primary focus of our laboratory. ENTPD1 or CD39 catalyzes the conversion of ATP and ADP rapidly into AMP, whereas NTPDase 3 and 8 also have ATP and other nucleoside triphosphates as the preferential substrate generating levels of ADP and nucleoside diphosphates en route to AMP; and NTPDase 2 mainly hydrolyzes ATP to ADP [7]. NTPDase 4, 5 and 6 are located inside the cells but NTPDase 5 and 6 are secreted upon heterologous expression.

The AMPase NT5E/CD73 is the ectoenzyme catalyzing the conversion of AMP into adenosine, which is then converted into inosine by membrane-bound adenosine deaminase. CD73 has been described as GPI-anchored protein or soluble enzyme [8, 9], and albeit ubiquitous is also derived from lymphocyte shedding [8].

ENTPD1/CD39 is present in several cell types including endothelial cells and immune cells where it is expressed in more than 90% of B cells and monocytes/macrophages, 20–30% of CD4 cells, 2–5% of NK cells and less than 5% of CD8 lymphocytes. CD73 is present in the majority of B lymphocytes, 50% of CD8 T cells, 10% of CD4 T cells and 2–5% of NK cells [10]. ENTPD2 and ENTPD3 can be expressed by cells of the enteric nervous system [11–13]. ENTPD2 is also expressed on liver portal fibroblasts [14] on vascular smooth muscle cells [15] and can be induced by hypoxia inducible factor 1alpha (HIF-1α) in hepatocellular carcinoma (HCC) tumor cells [16]. ENTPD3 is abundantly expressed by pancreatic β cells [17] and likely noted on hepatic nervous elements and stellate cells [18].

In this review, we will focus on the role of ectonucleotidases in the regulation of immune responses in the context of gastrointestinal inflammatory conditions, namely infections of the GI tract, inflammatory bowel disease (IBD) and cancer. We also discuss the implications of ectonucleotidase targeting for immunotherapy and novel approaches to this.

2. ENTPD1/CD39

ENTPD1/CD39 is the prototype member of the ENTPDase family that is constitutively expressed on the vasculature as well as on various adaptive and innate immune cells, chiefly B lymphocytes, monocytes/macrophages, CD4 cells including effector and regulatory subsets, CD8 lymphocytes and NK cells. Amongst CD4 T lymphocytes, CD39 is noted on T regulatory cells [19] and can be also present on long lived memory T cells [20]. These latter cells are characterized by a T helper type 1 (Th1), Th2 and Th17 phenotype and display heightened alloreactivity by mediating MHC-mismatched skin rejection in mice [20]. In this subset, CD39 confers protection from ATP-induced apoptosis [20]. CD39 can be also expressed by a distinct population of human CD4 cells with memory effector phenotype [21]. Expression of CD39 was also found to increase on effector CD4 cells that undergo metabolic stress and are paradoxically highly susceptible to apoptosis [22].

CD39 can be also expressed on a subset of “regulatory” human Th17 cells that, at variance with bona fide effector Th17 lymphocytes, display immunosuppressive properties and effectively generate AMP and adenosine [23]. In a cancer study, CD39⁺ Th17 cells were found capable of suppressing CD4 and CD8 T cell activation [24] while expressing both FOXP3 and RORγt and secreting Th17 related cytokines [24].

Expression of CD39 by murine and human regulatory T cells (Tregs) is driven, at least in part, by Foxp3 [25] and is linked to these cells’ suppressive function upon ATP catalysis [19, 25]. Importantly, Tregs from Cd39^−/− mice are functionally impaired and fail to block allograft rejection in vivo [19].

In humans, CD39⁺ Treg block both IFNγ and IL-17 production by effector cells, at variance with CD39⁻ Tregs that suppress IFNγ production only [26]. Further, CD39 expression by human CD4⁺CD25⁺ cells also identifies a lymphocyte subset with regulatory memory cell phenotype, which is decreased in the peripheral blood of patients with renal allograft rejection [21]. The presence of CD39 on Tregs is further associated with suppression of anti-tumor immunity mediated by NK cells in vitro and in vivo [27].

Based on their specificity for viral or gluten epitopes, CD39⁺ Tregs can display a polyclonal or oligoclonal TCR repertoire [28]. Within the regulatory cell compartment, CD39 promotes the differentiation of T regulatory type 1 (Tr1) cells, which predominate during protracted or chronic inflammation [29] and contributes to these cells’ suppressor activity, at least in part through the ultimate generation of adenosine [30].

When considering CD8 cells, expression of CD39 has been previously linked with specific killer activity [31], as well as with terminal exhaustion [32, 33]. High levels of CD39 could result from peripheral tolerance induction in vivo, and this was accompanied by IL-2 and IL-10 release and enhanced by an A2A receptor agonist [34].

CD39 can be potently regulated at the genetic level, as demonstrated by the presence of a single nucleotide polymorphism (SNP) tagging low CD39 mRNA levels and found to be associated with increased susceptibility to Crohn’s disease and inflammatory bowel disease [35]. Additional studies have shown that most cell populations with high heritability links for autoimmunity are positive for CD39 [36] and that quantity of CD39 expression is under genetic control in Treg and non-Treg lymphocytes [37]. Treg CD39 levels can be determined by specific SNPs; in this regard, Tregs obtained from donors of GG genotype, which is associated with high CD39 levels are more effective at suppressing pro-inflammatory cytokine production, when compared to Tregs obtained from donors of AA genotype, which is associated with low CD39 levels [38].

ENTPD1 is further regulated at the transcriptional level upon engagement of aryl hydrocarbon receptor (AHR), a toxin or xenobiotic mediator, important in modulating adaptive and innate immune responses [39–41]. Unconjugated bilirubin (UCB) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), two endogenous AHR ligands, also induce CD39 expression in human and murine Th17 and Treg cells [42, 43].

Exposure of healthy donor derived Th17 cells to UCB results in enhanced regulatory properties of these cells that upregulate FOXP3, IL-10 and display suppressor activity in vitro [42]. Blockade of AHR using CH223191 limits in part these effects [42].

Analysis of the human CD39 promoter has revealed also a critical role for specificity protein 1 (Sp-1), a transcriptional factor upregulated during hypoxia, in the induction of CD39 [44]. These findings were further corroborated by the evidence that ischemic preconditioning resulted in CD39 induction and improved outcome of ischemia reperfusion injury in the liver; these beneficial effects being contained upon treatment with Sp1 specific siRNA and mimicked upon intraperitoneal administration of soluble apyrase that has ectoenzymatic activity comparable to CD39 [45]. Further evidence has indicated that hypoxia/HIF-1α impairs AHR levels limiting the differentiation of Tr1 cells [30] as well as AHR signaling in Th17 cells [46].

Regulation of CD39 at the transcriptional level might also be impacted upon exposure to statins, the lipid lowering class of drug, as there is evidence that thrombin treated endothelial cells preserve CD39/ENTPDase activity and expression, following exposure to simvastatin or cerivastatin [47]. Other factors involved in CD39 upregulation are Stat-3 and Gfi-1, transcription factors that were found to support and repress the expression of CD39 in Th17 cells, upon binding to the CD39 promoter [48]. TGF-β and IL-6 that favor Th17 cell differentiation in vitro were also found to increase CD39 in tumor infiltrating Th17 cells [24]. Additional cytokines that are involved in CD39 upregulation in CD4 cells include IL-35 [49] and IL-27, which drives CD39 expression in Tregs [50]. ENTPD1 on T cells is also closely positively regulated by cJUN [51] and down regulated by IL-7 [52], which binds to the CD127 receptor on naive, memory, and activated memory Tregs. IL-7 particularly has impacts on memory Treg and also upregulates P2X7 on these cells.

CD39 levels are also regulated by mechanisms operating at the post-transcriptional level. These include inhibition of phosphodiesterase 3 (PDE3) [53], antisense oligonucleotides [54] as well as antisense to LMP1, a gene that plays a role in the survival of B cells after EBV infection [55]. We have recently also reported that CD39 is also regulated by an endogenous antisense RNA that is present at the 3’ end of the human ENTPD1/CD39 gene and is upregulated in both Tregs and Th17 cells derived from Crohn’s disease patients [56]. Importantly, silencing of this antisense RNA results in increased CD39 expression levels in both cell types and in ameliorated Treg function in Crohn’s disease samples [56].

3. CD73

CD73 can be expressed on both murine Tregs and uncommitted primed precursors Th cells that secrete IL-2 but are not suppressive [57]. Unlike murine Tregs, only a small percentage of human Tregs express CD73 suggesting that generation of adenosine in human Tregs depends on paracrine interactions with CD73 expressed on adjacent cells (i.e. CD4 or other effectors). CD73 can be also expressed on a subset of Th17 cells [23] that have immunosuppressive properties, including ability to generate adenosine [23]. Early studies reported expression of CD73 on CD28⁺ cells [58]. Amongst T cells, CD73 is mainly present on CD8 lymphocytes of naïve phenotype [59], whereas fewer memory CD8 are expressing this molecule [59]. Subsequent investigations conducted in the cancer setting revealed that the protective effects of CD73 deficiency on primary tumors are mainly associated with increased frequency of antigen-specific CD8 cells in both peripheral blood and tumors and with increased production of IFNγ [60].

IL-17 producing CD8 cells (Tc17), which are generated in the presence of TGF-β display also high CD73 levels and produce adenosine [61] although they do not suppress CD4 T cell proliferation. In this study, CD73 was found to be highly expressed in CD62L⁺CD127⁺CD8⁺ memory T cells while being downregulated in GZMB⁺KLRG1⁺ terminally differentiated CD8 T cells [61]. Akin to CD39, CD73 can be upregulated by TGF-β on CD8 T cells [62], supported by Stat-3 in Th17 cells [48] and further regulated by HIF-1α [63].

The following sections review the role of CD39 and CD73 in luminal gastrointestinal disease with a focus on infections, IBD and cancer. Interested readers wishing to read more on the role of ectonucleotidases of the CD39 family in liver disease are requested to refer to prior work published together with Dr. Burnstock and others [64, 65].

4. Ectonucleotidases in gastrointestinal infections

A wealth of studies has provided evidence about the role of CD39 and CD73 in the modulation of immune responses during infections of the gastrointestinal tract. In the context of Helicobacter felis (H. felis)-induced gastritis and colonization, CD4 T cells isolated from the blood and the gastric mucosa co-express CD39 and CD73 [66]. Increased levels of proinflammatory cytokines and worse disease outcome were noted in H. felis-infected Nt5e/Cd73^−/− mice; whereas Nt5e/Cd73^−/− Tregs were unable to inhibit gastritis [66], this strengthening the notion that CD73 plays an important role in Treg mechanism of function. In a subsequent study by the same group in the context of clinically relevant model of Helicobacter pylori (H. pylori) gastritis, administration of an A2A adenosine receptor (A2AR) agonist, namely ATL313, to infected IL-10 immunodeficient mice, reduced gastritis and proinflammatory cytokine responses; whereas infection of Adora2a^−/− mice enhanced gastritis [67]. Expression of CD73 has been reported being downregulated in both conventional and Treg lymphocytes, obtained from the intestine of naïve mice during Toxoplasma gondii acute infection [68], this resulting in inability to generate adenosine [68].

In the setting of pathogenic simian immunodeficiency virus infection, an expansion of CD8 Tregs endowed with suppressive function and expressing the CD39 ectonucleotidase has been found in the colorectal mucosa and associated with antiviral T cell responses [69]. This impairment could derive from increased ATP degradation by enhanced CD39 levels and consequent inhibition of effector cell proliferation. In a study on intestinal tuberculosis, Banerjee et al have also reported that CD73 expression on mesenchymal cells enables Mycobacterium Tuberculosis to evade host immunity [70].

In a rat model of herpes simplex virus 1 infection, alterations of adenosinergic responses were found. These included impairment in adenosine mediated contractile responses by A1 and A2A receptors and upregulation of both CD73 and adenosine deaminase in ileal smooth muscle layers and myenteric plexus during latent infection [71]. A role for CD73 ectoenzyme in the regulation of intestinal colonization and infection by non-typhoidal Salmonella was shown, as intestinal epithelial cells (IEC)-specific CD73 knockout mice (CD73^f/f/Villin^Cre) displayed a considerably higher colonization rate, when compared to controls [72], postulating a role for CD73 and adenosinergic signaling as modulators of host-microbe interactions.

There has been much written of the gut microbiome and of its modification in disease states as with Clostridium difficile or the dysbiosis noted in IBD, as in next section, and genetic links to purinergic signaling and fecal microbial therapy (stool transplants) [73, 74]. The gut microbiome is further known to critically influence the response of cancer patients to the new checkpoint inhibitor drugs, which can restore immunological responses of the host [75]. As we note later, recent interest has targeted CD39 and CD73 as immune check points in cancer immunotherapy [76]. Recent work has examined the role of the microbiome and metabolism of purines in modulation of anticancer modalities and led to some really interesting findings concerning purines and inosine generated by gut bacteria that will drive further research.

5. Ectonucleotidases in IBD

IBD is a chronic inflammatory condition, which results from altered interactions between the gut microbiota and the immune system in a genetically predisposed individual. IBD most frequent manifestations include Crohn’s disease and ulcerative colitis, which are diagnosed based on the location of tissue inflammation and clinical symptoms [77–79]. Imbalance between Tregs and effector Th17 cells has been documented in both human and experimental in vivo models and reported as one of the factors permitting perpetration of tissue damage. A large number of studies have demonstrated a pivotal role of CD39 in the regulation of immune responses in models of experimental colitis. In the setting of colitis induced by dextran-sulfate-sodium (DSS), mice globally deficient for CD39 undergo a more severe course of the disease, as compared to their wild type (WT) counterpart [35]. Administration of soluble apyrase, which has ectoenzymatic activity comparable to CD39, prevents inflammation in Cd39^−/− mice exposed to DSS and attenuates colitis in Balbc mice [80]. The immunoregulatory properties of CD39 were further emphasized in subsequent studies showing that the salutary effects of UCB, administered to DSS colitic mice, are dependent on CD39 expression [42]. These findings were corroborated by additional investigations showing that human CD39 overexpression in transgenic mice confers beneficial effects in DSS colitis, as reflected by decreased disease activity index, reduced histology score and higher colon length, when compared to WT mice [81].

When considering experimental colitis induced by trinitrobenzene-sulfonic-acid (TNBS), a chemical compound with haptenic properties [82], Goettel et al. reported that mice transgenic for human HLA-DR2⁺, reconstituted with CD4 cells derived from an HLA-DR2⁺ donor and exposed to TNBS, develop severe colitis that can be attenuated upon administration of the AHR ligand ITE [43]. ITE beneficial properties were found to depend on CD39 induction [43]. In C57BL6 mice, however, global deficiency of CD39, was reported being a protective factor in the development of TNBS colitis, postulating that genetic background could also impact immune response to TNBS, therefore leading to a different clinical outcome [83]. Protective effects of CD39 in the context of experimental colitis have been also shown in an adoptive transfer model where Tregs obtained from Cd39^−/− mice are less effective at controlling disease activity than their WT counterpart [84]. On the other hand, Tregs obtained from human CD39 transgenic mice are superior at dampening inflammation induced by adoptive transfer of CD45RB^high lymphocytes, when contrasted with WT Tregs [81].

Besides animal studies, a wealth of investigations conducted on IBD patients’ samples has supported a crucial role of the CD39 ectoenzyme in shaping Treg/Th17 cell balance in this condition. Therefore, Crohn’s disease patients display decreases in the proportion of supTh17 cells, defined as CD4⁺IL-17⁺FOXP3⁺ and CD39⁺ cells, as compared to normal controls [23]. Exposure of bona fide Th17 cells to endogenous AHR ligands like UCB boosts the immunosuppressive properties of these cells, as reflected by increased CD39, FOXP3 and IL-10 levels, in healthy individuals. Upregulation of CD39 upon UCB exposure is markedly impaired in both peripheral blood and lamina propria derived Th17 cells of Crohn’s disease patients [42]. This impairment derives in part from high Th17 cell levels of HIF-1α that is upregulated during chronic inflammatory conditions and induces expression of multidrug-resistant-1 (MDR1) and multidrug-resistant-protein-4 (MRP4), two ATP-binding cassette drug transporters that favor exit of immunometabolites, including UCB, out of cells [46]. Molecular blockade of HIF-1α restores response of Th17 cells to AHR ligation, as reflected by increase in the levels of CD39 and FOXP3, in Crohn’s disease samples, also manifesting under hypoxic conditions [46].

CD39⁺ Th17 cells have been also shown to express CD161, which promotes effector phenotypes and IL-17 expression through acid sphingomyelinase catalytic bioactivity. Increases in these cells are noted both in the peripheral blood and lamina propria in IBD [80]. Similarly, CD3/CD28 stimulation of IFNγ producing cells not only boosted IFNγ production by these effector cells but also promoted reactive oxygen species and CD39 expression levels [80]. In another, recent study, Huang and colleagues reported various cellular immune defects in pediatric patients with IBD and these alterations include impaired cyclic-AMP response signaling, infiltration of phosphodiesterase 4B and TNF-expressing macrophages, platelet aggregation and decrease in CD39 expressing T cells [85]. A further study from Noble et al. reported defective CD39 levels in γδ T cells obtained from the intraepithelial lymphocytes of patients with Crohn’s disease and ulcerative colitis, this suggesting impaired regulatory function [34].

Increase in the frequencies of CD4⁺CD73⁺ cells within Th17 lymphocytes were found in the circulation and lamina propria of active IBD patients, postulating a role for CD73 as a biomarker to monitor disease activity over time [86].

The pivotal role of CD39 in governing immune responses during gut inflammation has prompted strategies aimed at boosting levels and ectoenzymatic activity, either directly upon administration of compounds mimicking CD39 functional properties or by targeting/regulating pathways that induce or inhibit CD39.

As discussed above, high levels of inflammation-induced HIF-1α may have inhibiting properties over CD39 upon induction of the drug transporters MDR1 and MRP4 that favor exit of AHR immunometabolites from the cells [46]. In vitro exposure of Th17 cells to ritonavir, an anti-retroviral drug used in HIV treatment that also serves as a non-specific antagonist to MDR1 and MRP4, restores the ability of Crohn’s derived Th17 cells to upregulate CD39 and FOXP3 and to suppress effector cell proliferation [46].

Beneficial properties of soluble apyrase have been demonstrated in mouse models of experimental colitis [35, 87]. Subsequent studies have proposed the use of APT102, the extracellular domain with improved ADPase activity of human nucleoside-triphosphate-diphosphohydrolase-3 (CD39L3), a member of the CD39 family, in experimental colitis induced upon DSS administration and found that the compound augments UCB salutary properties in this model. In addition to improving the disease activity score during recovery, the combinatorial APT102 and UCB treatment boosts the proportion of CD4⁺FOXP3⁺, CD4⁺LAG-3⁺ and CD4⁺CD39⁺ cells in the lamina propria. We will also study newly developed CD39-CD73 fusion proteins [88] in these models.

Of high translational relevance, addition of APT102 enhances the effects of AHR activation in Treg and Tr1 cells obtained from the peripheral blood of Crohn’s disease patients, as shown by increased levels of FOXP3, LAG-3 and CD39. When considering cells obtained from the lamina propria of non-inflamed biopsied areas, combinatorial treatment of UCB and APT102 enhanced CD49b, LAG-3 and CD39 levels in Tr1 cells [81]. These results indicate that exogenous apyrase has beneficial effects in experimental IBD and IBD patient samples by boosting regulatory properties derived from the activation of AHR signaling [81].

In addition to CD73 and CD39, there are other ectonucleotidases namely ENTPD2 and ENTPD3 that are known having a protecting role during gut inflammation. Previous studies have shown that both Entpd2^−/− and Entpd3^−/− mice develop more severe disease during DSS colitis and macrophages isolated from Entpd2^−/− colitic mice display a more proinflammatory phenotype compared to their WT counterpart [12]. Importantly, Crohn’s patients with Harvey-Bradshaw-Index of 4 or higher show diminished polyioxometalate-6 (POM-6) sensitive ADPase activity as compared with patients with milder disease activity. As POM-6 inhibits both ENTPD2 and ENTPD3, it is plausible to conclude that impaired ADPase activity in plasma samples from Crohn’s disease patients mainly results from diminished ENTPD2 and ENTPD3 activity [12].

6. Ectonucleotidases in gastrointestinal tract tumors

In this section we will review and discuss the role of CD39 and CD73 ectonucleotidases in tumors of the gastrointestinal tract, with a focus on gastric and colorectal cancers, although some work has been also done in pancreatic and liver tumors [89, 90].

High levels of infiltration of A2AR⁺ CD8 T cells have been noted in gastric cancer tissues, when contrasted to peritumoral “normal tissues”. This was found to significantly correlate with the tumor/node/metastasis stage, lymph node and distant metastasis [91]. Further, Tregs isolated from the peripheral blood of gastric cancer patients are capable of hydrolyzing ATP into adenosine and suppress CD8 T cell proliferation. These effects are inhibited upon treatment with A2AR antagonists or a combination of A2AR and A2BR antagonists [91].

The role of CD39 in favoring tumor growth was supported by the evidence that the hepatic growth of melanoma metastatic tumors was strongly inhibited in CD39 immunodeficient mice or in WT mice repopulated with Cd39^−/− bone marrow derived cells. Importantly, inhibition of tumor immunity was mediated by NK cells and inhibited by Cd39^−/− Tregs.

In a subsequent study on colorectal cancer induced upon injection of MC-26 cells, the derived hepatic metastases grew significantly faster in CD39 transgenic mice, when compared with Cd39^−/− mice [92]. When examining clinical samples, lower levels of CD39 mRNA in malignant colorectal tissues was associated with longer duration of survival and with diminished tumor invasiveness [92]. A study on human rectal cancer indicated high CD39 levels in both primary and metastatic tumors, although it was proposed that determination of both CD39 and CD73 could have a better prognostic value [63].

Additional investigations conducted by our group highlighted that hyperthermia (i.e. 40° C) enhanced ATP-mediated cytotoxicity on MCA38 colon cancer cells [93] and that combination of hyperthermia with cisplatin or mitomycin C further enhanced cancer cell death [93].

The role of the CD73 ectoenzyme in dampening antitumor immunity has been highlighted by Stagg et al., who have demonstrated the beneficial effects of CD73 ablation on the growth of ovalbumin-expressing MC38 colon cancer [60]. This protective effect was dependent on IFNγ producing CD8 T lymphocytes. In the same study, evidence was provided that the protumorigenic effect of Tregs was dependent on CD73 [60].

Because of the role of CD39 and CD73 ectoenzymes in curbing antitumor immunity, targeted blockade of these two molecules has been considered to reduce the detrimental effects derived from ATP hydrolysis in the tumor microenvironment. In this regard, inhibition of CD39 ectoenzymatic activity using polyoxometalate-1 (POM-1), a pharmacologic inhibitor of nucleoside triphosphate diphosphohydrolase activity, was shown to significantly inhibit tumor growth in colonic tumor-bearing WT mice [27]. More recent advances have been made in development of monoclonal antibodies to CD39 and testing has been done in vivo and in evolving clinical studies [76, 94, 95].

In parallel, CD73 has been also considered a checkpoint inhibitor, linked to CD39 [76]. Hence, decreases in tumor size and lung metastases have been noted upon administration of CD73 inhibitors or anti-CD73 monoclonal antibody in mice injected with EG7 or B16F10 cells [60]. In subsequent investigations, anti-CD73 monoclonal antibody was found to enhance the activity of both anti-CTLA-4 and anti-PD1 monoclonal antibodies against MC38-OVA colon cancer. Recently, Tsukui et al. have shown that intraperitoneal administration of anti-CD73 antibody in combination with radiotherapy suppresses lung metastasis of JuM-1, a highly metastatic murine colon cancer [96]. Importantly, the splenocytes of mice subjected to radiotherapy and administered anti-CD73 monoclonal antibody showed enhanced IFNγ production and cytotoxicity compared to controls.

Recent investigations have reported the generation of two antibodies, namely IPH5201 and IPH5301, targeting human membrane associated and soluble forms of CD39 and CD73 respectively. These antibodies could boost antitumor immunity by stimulating dendritic cells and macrophages and by restoring activated T cells obtained from cancer patients [97]. This indicates that control of both CD39 and CD73 might be effective at boosting antitumor immunity and postulates the role of these antibodies in combination with chemotherapy or immune checkpoint inhibitors [97].

One theoretical concern is that prolonged targeting of CD39, or related downstream targets such as CD73 or ADORA2A, might have deleterious off target impacts. These include altering blood clotting, platelet reactivity, insulin resistance and promoting development of other tumors as with the spontaneous or increases in induced liver cancers noted in aged Entpd1 null mice [90]. Another consideration relates to the fact that often colon cancer occurs as a complication of longstanding inflammation resulting from IBD, which in the instance of Crohn disease may be characterized by low CD39/ENTPD1 mRNA levels. In this specific context, targeting of CD39 may result in further impairment of systemic ectonucleotidase activation with further perturbation of Treg/effector cell imbalance and disease progression.

However, overall the published studies provide evidence that blockade of CD39 and CD73 might result in beneficial effects by boosting anticancer effects, as mediated by ATP-P2X7 mediated effects on the inflammasome [98–100], by limiting generation of adenosine and restoring effector responses in the tumor microenvironment.

7. Concluding remarks

CD39 and CD73 ectonucleotidases have important modulatory influences on purinergic cellular immunity by hydrolyzing the extracellular “danger molecules” ATP and ADP (CD39) into AMP and then converting it into immunosuppressive adenosine (CD73). This ectoenzymatic cascade results in enhanced regulatory over effector T cell immunity and consequent beneficial effects in the context of acute and chronic inflammation. In contrast, CD39 and CD73 check point regulatory functions are boosted in cancer and have detrimental effects due to inhibition of tumor specific immunity.

In this review, we have briefly discussed the role of CD39 and CD73 in gastrointestinal pathology and highlighted the mechanisms by which these ectonucleotidases regulate cell immunity in inflammatory conditions. Immunotherapeutic approaches based on boosting or inhibiting ectonucleotidase expression and activity during inflammation or cancer have been reviewed, and potential effects on immune responses and disease course discussed.

Evidence provided by a growing number of studies, collectively identifies CD39, CD73 and purinergic signaling as one of the major targets for re-establishing immunotolerance as well as restoring homeostatic regulatory - effector cell interactions in human malignant diseases. Further studies are underway to expand the current mechanistic knowledge on how exactly purinergic signaling is operational and how this could be effectively modulated by immunotherapy.

8. Dedication to Dr. G. Burnstock

All three authors have been inspired by Dr. Geoffrey Burnstock and of his innovative and ground breaking work in the discovery of neuronal cotransmission and in formulating the purinergic hypothesis, around 50 years ago. His legacy is remarkable and he was a wonderful and empathetic colleague. This special issue in Biochemical Pharmacology is a worthy testament to how his formative work has developed to impact many distinct areas and clinical disciplines.

We will all miss his physical presence at conferences, when these start up again, and in editorial meetings of the journal “ Purinergic Signalling” (MSL and SCR). One of us was honored to deliver the Third Burnstock Lecture in Copenhagen at the PURINES2008 meeting, where the focus was on “CD39/ENTPD1: At the interface between innate and adaptive immunity”, and we are pleased to further develop this area in this review in honor of Dr. Burnstock and his family.

His was a life well lived with so many remarkable achievements, and he always seemed to have fun doing the science and work. It was clearly a great joy for him to realize this wonderful scientific legacy and inspire so many others.

Figure 1. CD39 and CD73 expression in immune cells.

CD39 catalyzes ATP and ADP into AMP, which is then converted into immunosuppressive adenosine by CD73, the ectoenzyme that works in tandem with CD39. CD39 and CD73 expression is depicted across myeloid and lymphoid cell lineages. Both myeloid and lymphoid derived cells can express both CD39 and CD73, as in the case of monocytes, macrophages, B cells, CD4 and CD8 T lymphocytes and NK cells. Amongst T cells, Th17 cells and murine Tregs express both ectoenzymes; whereas human Tregs preferentially express CD39 only.

Figure 2. CD39 and CD73 antithetical role in inflammatory bowel disease and gastrointestinal tract tumors.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition, which results from altered interactions between the gut microbiota and the immune system in genetically predisposed individuals. Initiation and progression of tissue damage is favored by imbalance between pathogenic Th17 cells and Tregs. In IBD defective immune regulation is linked to low CD39 levels and activity in both Tregs and supTh17 cells, a subset of Th17 cells endowed with immunosuppressive properties. There are a number of strategies that might aid in re-establishing CD39 levels in these subsets: 1) ritonavir, a non-specific antagonist of MDR1 and MRP4 drug transporters, which favor exit of immunomodulatory metabolites like unconjugated bilirubin (UCB) out of Th17 cells; 2) soluble apyrase that displays ectoenzymatic activity comparable to CD39; and 3) APT102 that boosts the immunosuppressive properties of UCB in Tregs. Imbalance between regulatory and effector CD8 cells is present in the context of gastrointestinal tract tumor, where tumor invasiveness has been associated with increased CD39 and CD73 expression in Tregs. Inhibition of CD39 ectoenzymatic activity via POM-1, use of anti-CD73 monoclonal antibody - alone or in association with anti-CTLA4/PD-1 monoclonal antibody or radiotherapy – or IPH5201 and IPH5301 to target both CD39 and CD73 are amongst the strategies proposed to restore anti-tumor immunity in the GI cancer setting.

Abbreviations:

ATP

adenosine triphosphate

AMP

adenosine monophosphate

ENTPDase

ecto-nucleoside triphosphate diphosphohydrolase

NT5E

ecto-5’-nucleotidase

E-NPP

ecto-nucleotide pyrophosphate phosphodiesterase

HIF-1α

hypoxia inducible factor 1-alpha

HCC

hepatocellular carcinoma

IBD

inflammatory bowel disease

Th1

T helper type 1

Tr1

T regulatory type 1

SNP

single nucleotide polymorphism

AHR

aryl hydrocarbon receptor

UCB

unconjugated bilirubin

ITE

2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester

Sp-1

specificity protein 1

Tregs

regulatory T cells

IEC

intestinal epithelial cells

DSS

dextran-sulfate-sodium

TNBS

trinitro-benzene-sulfonic-acid

MDR1

multidrug-resistant-1

MRP4

multidrug-resistant-protein-4

CD39L3

nucleoside-triphosphate-diphosphohydrolase-3

POM-6

polyioxometalate-6

POM-1

polyioxometalate-1