Golgi-localized APYRASE 1 is critical for Arabidopsis growth by affecting cell wall integrity under boron deficiency.

Many nucleoside triphosphate-diphosphohydrolases (NTPDases/APYRASEs, APYs) play a key role in modulating extracellular nucleotide levels. However, the Golgi-localized APYs, which help control glycosylation, have rarely been studied. Here, we identified AtAPY1, a gene encoding an NTPDase in the Golgi apparatus, which is required for cell wall integrity and plant growth under boron (B) limited availability. Loss of function in AtAPY1 hindered cell elongation and division in root tips while increasing the number of cortical cell layers, leading to swelling of the root tip and abundant root hairs under low B stress. Further, expression pattern analysis revealed that B deficiency significantly induced AtAPY1, especially in the root meristem and stele. Fluorescent-labeled AtAPY1-GFP localized to the Golgi stack. Biochemical analysis showed that AtAPY1 exhibited a preference of UDP and GDP hydrolysis activities. Consequently, the loss of function in AtAPY1 might disturb the homoeostasis of NMP-driven NDP-sugar transport, which was closely related to the synthesis of cell wall polysaccharides. Further, cell wall-composition analysis showed that pectin content increased and borate-dimerized RG-II decreased in apy1 mutants, along with a decrease in cellulose content. Eventually, altered polysaccharide characteristics presumably cause growth defects in apy1 mutants under B deficiency. Altogether, these data strongly support a novel role for AtAPY1 in mediating responses to low B availability by regulating cell wall integrity.

Platelets induce CD39 expression in tumor cells to facilitate tumor metastasis.

BACKGROUND: Tumor cells continue to evolve the metastatic potential in response to signals provided by the external microenvironment during metastasis. Platelets closely interact with tumor cells during hematogenous metastasis and facilitate tumor development. However, the molecular mechanisms underlying this process are not fully understood. METHODS: RNA-sequencing was performed to screen differentially expressed genes mediated by platelets. The effects of platelet and CD39 on tumor metastasis were determined by experimental metastasis models with WT, NCG and CD39-/- mice. RESULTS: RNA-sequencing results showed that platelets significantly up-regulated CD39 expression in tumor cells. CD39 is a novel immune checkpoint molecule and a key driver of immunosuppression. Our data provided evidence that the expression of CD39 was enhanced by platelets in a platelet-tumor cell contact dependent manner. Although the role of CD39 expressed by immune cells is well established, the effect of CD39 expressed by tumor cells on tumor cell behavior, anti-tumor immunity and tumor metastasis is unclear. We found that CD39 promoted tumor cell invasion, but had no effect on proliferation and migration. Notably, we showed that the ability of platelets to prime tumor cells for metastasis depends on CD39 in the experimental tumor metastasis model. CD39 silencing resulted in fewer experimental metastasis formation, and this anti-metastasis effect was significantly reduced in platelet-depleted mice. Furthermore, overexpression of CD39 in tumor cells promoted metastasis. In order to eliminate the effect of CD39 expressed in cells other than tumor cells, we detected tumor metastasis in CD39-/- mice and obtained similar results. Moreover, overexpression of CD39 in tumor cells inhibited antitumor immunity. Finally, the data from human samples also supported our findings. CONCLUSIONS: Our study shows that direct contact with platelets induces CD39 expression in tumor cells, leading to immune suppression and promotion of metastasis.

Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX/RALF/FER growth regulatory module.

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. The cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 (APY7) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture and -composition, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/RALF/FER signaling module.

Growth regulation by apyrases: Insights from altering their expression level in different organisms.

Apyrase (APY) enzymes are nucleoside triphosphate (NTP) diphosphohydrolases that can remove the terminal phosphate from NTPs and nucleoside diphosphates but not from nucleoside monophosphates. They have conserved structures and functions in yeast, plants, and animals. Among the most studied APYs in plants are those in Arabidopsis (Arabidopsis thaliana; AtAPYs) and pea (Pisum sativum; PsAPYs), both of which have been shown to play major roles in regulating plant growth and development. Valuable insights on their functional roles have been gained by transgenically altering their transcript abundance, either by constitutively expressing or suppressing APY genes. This review focuses on recent studies that have provided insights on the mechanisms by which APY activity promotes growth in different organisms. Most of these studies have used transgenic lines that constitutively expressed APY in multiple different plants and in yeast. As APY enzymatic activity can also be changed post-translationally by chemical blockage, this review also briefly covers studies that used inhibitors to suppress APY activity in plants and fungi. It concludes by summarizing some of the main unanswered questions about how APYs regulate plant growth and proposes approaches to answering them.

Cancer CD39 drives metabolic adaption and mal-differentiation of CD4+ T cells in patients with non-small-cell lung cancer.

While ectonucleotidase CD39 is a cancer therapeutic target in clinical trials, its direct effect on T-cell differentiation in human non-small-cell lung cancer (NSCLC) remains unclear. Herein, we demonstrate that human NSCLC cells, including tumor cell lines and primary tumor cells from clinical patients, efficiently drive the metabolic adaption of human CD4+ T cells, instructing differentiation of regulatory T cells while inhibiting effector T cells. Of importance, NSCLC-induced T-cell mal-differentiation primarily depends on cancer CD39, as this can be fundamentally blocked by genetic depletion of CD39 in NSCLC. Mechanistically, NSCLC cells package CD39 into their exosomes and transfer such CD39-containing exosomes into interacting T cells, resulting in ATP insufficiency and AMPK hyperactivation. Such CD39-dependent NSCLC-T cell interaction holds well in patients-derived primary tumor cells and patient-derived organoids (PDOs). Accordingly, genetic depletion of CD39 alone or in combination with the anti-PD-1 immunotherapy efficiently rescues effector T cell differentiation, instigates anti-tumor T cell immunity, and inhibits tumor growth of PDOs. Together, targeting cancer CD39 can correct the mal-differentiation of CD4+ T cells in human NSCLC, providing in-depth insight into therapeutic CD39 inhibitors.

Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Identification, characterization of Apyrase (APY) gene family in rice (Oryza sativa) and analysis of the expression pattern under various stress conditions.

Apyrase (APY) is a nucleoside triphosphate (NTP) diphosphohydrolase (NTPDase) which is a member of the superfamily of guanosine diphosphatase 1 (GDA1)-cluster of differentiation 39 (CD39) nucleoside phosphatase. Under various circumstances like stress, cell growth, the extracellular adenosine triphosphate (eATP) level increases, causing a detrimental influence on cells such as cell growth retardation, ROS production, NO burst, and apoptosis. Apyrase hydrolyses eATP accumulated in the extracellular membrane during stress, wounds, into adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and regulates the stress-responsive pathway in plants. This study was designed for the identification, characterization, and for analysis of APY gene expression in Oryza sativa. This investigation discovered nine APYs in rice, including both endo- and ecto-apyrase. According to duplication event analysis, in the evolution of OsAPYs, a significant role is performed by segmental duplication. Their role in stress control, hormonal responsiveness, and the development of cells is supported by the corresponding cis-elements present in their promoter regions. According to expression profiling by RNA-seq data, the genes were expressed in various tissues. Upon exposure to a variety of biotic as well as abiotic stimuli, including anoxia, drought, submergence, alkali, heat, dehydration, salt, and cold, they showed a differential expression pattern. The expression analysis from the RT-qPCR data also showed expression under various abiotic stress conditions, comprising cold, salinity, cadmium, drought, submergence, and especially heat stress. This finding will pave the way for future in-vivo analysis, unveil the molecular mechanisms of APY genes in stress response, and contribute to the development of stress-tolerant rice varieties.

Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p.

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

Increased frequency of CD39+CD73+ regulatory T cells and Deltex-1 gene expression level in kidney transplant recipients with excellent long-term graft function.

BACKGROUND: The ability of regulatory T cells (Tregs) to limit inflammatory responses has been demonstrated. However, different subpopulations of this cell have varying abilities to suppress alloreactive immune responses. The primary goal of this study was to assess the frequency of CD4+FOXP3+CD39+CD73+ Tregs and Deltex-1 gene expression on long-term renal transplant function. METHODS: A total of 49 subjects were divided into 3 groups: (i) the excellent long-term graft function (ELTGF) group, (ii) the chronic graft dysfunction (CGD) group, and (iii) the healthy control (HC) group. Following sample collection, peripheral blood mononuclear cells (PBMCs) were isolated, and the Deltex-1 gene expression level and the frequency of CD4+FOXP3+CD39+CD73+ Tregs were evaluated. RESULTS: The ELTGF group had more CD4+FOXP3+ Tregs than the CGD group, but the difference was not statistically significant (P = 0.07). However, the frequency of CD4+FOXP3+CD39+CD73+ Tregs and the ratio of these cells to total CD4+ lymphocytes significantly increased in the ELTGF group than in the CGD group (P = 0.04 and P = 0.02 respectively). In addition, the expression level of the Deltex-1 gene was significantly lower in the CGD group than in the other 2 groups (P = 0.01 and P = 0.04 respectively). CONCLUSIONS: Given the increased frequency of CD4+FOXP3+CD39+CD73+ Tregs and the expression level of the Deltex-1 gene in the ELTGF group, it appears that these factors probably improved function and long-term survival of the transplanted organ through the suppression of alloreactive responses and reduction of inflammation. In other words, one of the immunological mechanisms involved in the CGD group may be a deficiency in Tregs.

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Time- and cost-efficient bacterial expression and purification of potato apyrase.

Apyrase from potato (Solanum tuberosum) is a divalent metal ion-dependent enzyme that catalyzes the hydrolysis of nucleoside di- and tri-phosphates with broad substrate specificity. The enzyme is widely used to manipulate nucleotide levels such as in the G protein-coupled receptor (GPCR) field where it is used to deplete guanine nucleotides to stabilize nucleotide-free ternary agonist-GPCR-G protein complexes. Potato apyrase is available commercially as the native enzyme purified from potatoes or as a recombinant protein, but these are prohibitively expensive for some research applications. Here, we report a relatively simple method for the bacterial production of soluble, active potato apyrase. Apyrase has several disulfide bonds, so we co-expressed the enzyme bearing a C-terminal (His)6 tag with the E. coli disulfide isomerase DsbC at low temperature (18 °C) in the oxidizing cytoplasm of E. coli Origami B (DE3). This allowed low level production of soluble apyrase. A two-step purification procedure involving Ni-affinity followed by Cibacron Blue-affinity chromatography yielded highly purified apyrase at a level of ∼0.5 mg per L of bacterial culture. The purified enzyme was functional for ATP hydrolysis in an ATPase assay and for GTP/GDP hydrolysis in a GPCR-G protein coupling assay. This methodology enables the time- and cost-efficient production of recombinant apyrase for various research applications.

Exhaustion of CD39-Expressing CD8+ T Cells in Crohn's Disease Is Linked to Clinical Outcome.

BACKGROUND & AIMS: Exhaustion of CD8 T cells has been suggested to inform different clinical outcomes in Crohn's disease, but detailed analyses are lacking. This study aimed to identify the role of exhaustion on a single-cell level and identify relevant CD8 T cell populations in Crohn's disease. METHODS: Blood and intestinal tissue from 58 patients with Crohn's disease (active disease or remission) were assessed for CD8 T cell expression of exhaustion markers and their cytokine profile by highly multiplexed flow and mass cytometry. Key disease-associated subsets were sorted and analyzed by RNA sequencing. CD39 inhibition assays were performed in vitro. RESULTS: Activated CD39+ and CD39+PD-1+ CD8 T cell subsets expressing multiple exhaustion markers were enriched at low frequency in active Crohn's disease. Their cytokine production capacity was inversely linked to the Harvey-Bradshaw Index. Subset-level protein and transcriptome profiling revealed co-existence of effector and exhaustion programs in CD39+ and CD39+ PD-1+CD8 T cells, with CD39+ cells likely originating from the intestine. CD39 enzymatic activity controlled T cell cytokine production. Importantly, transcriptional exhaustion signatures were enriched in remission in CD39-expressing subsets with up-regulation of TOX. Subset-level transcriptomics revealed a CD39-related gene module that is associated with the clinical course. CONCLUSIONS: These data showed a role for the exhaustion of peripheral CD39-expressing CD8 T cell subsets in Crohn's disease. Their low frequency illustrated the utility of single-cell cytometry methods for identification of relevant immune populations. Importantly, the link of their exhaustion status to the clinical activity and their specific gene signatures have implications for exhaustion-based personalized medicine approaches.

Genetically Driven CD39 Expression Affects Sezary Cell Viability and IL-2 Production and Detects Two Patient Subsets with Distinct Prognosis.

Sézary syndrome (SS) is a rare and aggressive variant of cutaneous T-cell lymphoma. It is characterized by the copresence of CD4+ neoplastic lymphocytes, named Sezary cells, mainly in the blood, lymph nodes, and skin where they induce chronic inflammation that in turn impairs the patient's QOL and fuels neoplastic cells. SS is not readily cured, but immunotherapy is becoming an effective option for this lymphoma. In this study, we investigated, in a large cohort of patients with SS, the expression and function of the immune checkpoint molecule CD39, which degrades proinflammatory extracellular adenosine triphosphate. We showed that the SNP rs10748643 A/G within the ENTPD1 gene coding for the CD39 protein controls its expression level. Patients carrying the A/G‒G/G genotype showed a significantly higher frequency of clonal CD4+CD39+ SS cells than those carrying the A/A genotype. Different from other cancers, high CD39 expression correlates with a better prognosis. Comparing primary G/G with A/A lymphoma cells, we observed that G/G SS cells have a higher ability to degrade adenosine triphosphate, increased apoptotic susceptibility, and upon activation, reduced IL-2 production. Accordingly, CD39 enzymatic inhibition enhances SS cell viability and IL-2 production on activation. These results strongly suggest a special caution for SS treatment with therapeutic inhibitors of CD39.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Dynamics and epigenetic signature of regulatory T-cells following antiretroviral therapy initiation in acute HIV infection.

BACKGROUND: HIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied. METHODS: Treg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology. FINDINGS: Total Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-ß1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-ß7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-ß1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-ß1)+ Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-ß1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different. INTERPRETATION: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression. FUNDING: This study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Plasmacytoid dendritic cells recruited by HIF-1α/eADO/ADORA1 signaling induce immunosuppression in hepatocellular carcinoma.

Plasmacytoid dendritic cells (pDCs) play immunosuppressive roles in the tumor microenvironment (TME). However, the molecular mechanisms underlying the recruitment and dysfunction of pDCs in the TME remain largely elusive, especially in hepatocellular carcinoma (HCC). In this study, we observed the accumulation of pDCs in the blood, tumor tissue, and ascitic fluid of HCC patients. A high density of tumor-infiltrating pDCs was correlated with poor prognosis in patients with HCC. Hypoxia-induced extracellular adenosine (eADO) significantly enhanced pDC recruitment into tumors via the adenosine A1 receptor (ADORA1). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transcriptionally upregulated the expression of the ectonucleotidases CD39 and CD73 in HCC cells, both of which are essential for the generation of eADO. Moreover, eADO-stimulated pDCs promoted the induction of regulatory T cells and suppressed proliferation and cytotoxicity of CD8+ T cells. Depletion of pDCs using a monoclonal antibody or an ADORA1 antagonist significantly improved antitumor immunity and suppressed HCC growth in the immunocompetent HCC mouse model. Thus, targeting pDC recruitment may serve as a potential adjuvant strategy for immunotherapies in HCC.