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Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint., Introduction Nucleotide metabolism controls immune cell development and function through diverse mechanisms (1). The purine nucleoside adenosine is sensed by A2a/b receptors, which mediate immunosuppressive effects in various immune cell [...]

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair., Introduction Myocardial infarction (MI) is a leading cause of systolic heart failure (1). After MI, different cell types are recruited to the heart in a spatiotemporally regulated manner to contribute [...]

Abstract In June 2023, the Purine and Pyrimidine Society (PPS) organized the 20th biennial symposium on Purine and Pyrimidine metabolism (PP23). The symposium was organized in Los Angeles, California, USA, by Pr Caius Radu affiliated to UCLA. The scientific program covered various topics such as inborn errors, cancer, immunity, enzymatic reactions, drug development etc and was presented at 9 sessions over three days. The current issue of Nucleosides, Nucleotides & Nucleic Acids is a special issue covering proceedings from PP23-presentations and other PPS-related manuscripts, and in this editorial, we will give an overview of the scientific program of the meeting. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is an autoimmune disease driven by lymphocyte activation against myelin autoantigens in the central nervous system leading to demyelination and neurodegeneration. The deoxyribonucleoside salvage pathway with the rate‐limiting enzyme deoxycytidine kinase (dCK) captures extracellular deoxyribonucleosides for use in intracellular deoxyribonucleotide metabolism. Previous studies have shown that deoxyribonucleoside salvage activity is enriched in lymphocytes and required for early lymphocyte development. However, specific roles for the deoxyribonucleoside salvage pathway and dCK in autoimmune diseases such as MS are unknown. Here we demonstrate that dCK activity is necessary for the development of clinical symptoms in the MOG35–55 and MOG1–125 experimental autoimmune encephalomyelitis (EAE) mouse models of MS. During EAE disease, deoxyribonucleoside salvage activity is elevated in the spleen and lymph nodes. Targeting dCK with the small molecule dCK inhibitor TRE‐515 limits disease severity when treatments are started at disease induction or when symptoms first appear. EAE mice treated with TRE‐515 have significantly fewer infiltrating leukocytes in the spinal cord, and TRE‐515 blocks activation‐induced B and T cell proliferation and MOG35–55‐specific T cell expansion without affecting innate immune cells or naïve T and B cell populations. Our results demonstrate that targeting dCK limits symptoms in EAE mice and suggest that dCK activity is required for MOG35–55‐specific lymphocyte activation‐induced proliferation. [ABSTRACT FROM AUTHOR]

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (~1 x [10.sup.4] cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment., In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions (1-8). A homeostatic makeup of these functional phenotypes dictates the overarching effect of [...]

Clinical tools that measure changes in immune cell metabolism would improve the diagnosis and treatment of immune dysfunction. PET, utilizing probes for specific metabolic processes, detects regions of immune activation in vivo. In this study we investigated the immune cell specificity of PET probes for two different metabolic pathways: [[.sup.18]F]-2-fluorodeoxyglucose ([[.sup.18]F]-FDG) for glycolysis and [[.sup.18]F]-2-fluoro-D-(arabinofu ranosyl)cytosine ([[.sup.18]F]-FAC) for deoxycytidine salvage. We isolated innate and adaptive immune cells from tissues of mice challenged with a retrovirus-induced sarcoma and measured their ability to accumulate FDG and FAC. We determined that the two probes had distinct patterns of accumulation: FDG accumulated to the highest levels in innate immune cells, while FAC accumulated predominantly in [CD8.sup.+] T cells in a manner that correlated with cellular proliferation. This study demonstrates that innate and adaptive cell types differ in glycolytic and deoxycytidine salvage demands during an immune response and that these differential metabolic requirements can be detected with specific PET probes. Our findings have implications for the interpretation of clinical PET scans that use [[.sup.18]F]-FDG or [[.sup.18]F]-FAC to assess immune function in vivo and suggest potential applications of metabolic PET to monitor the effects of targeted immune modulation., Introduction Innate and adaptive immune cells modulate the accumulation and metabolism of specific substrates to meet functional demands during a response. Macrophages and granulocytes, for example, increase expression of glucose [...]

Monitoring immune function with molecular imaging could have a considerable impact on the diagnosis and treatment evaluation of immunological disorders and therapeutic immune responses. Positron emission tomography (PET) is a molecular imaging modality with applications in cancer and other diseases. PET studies of immune function have been limited by a lack of specialized probes. We identified [[sup.18]F]FAC (1-(2'-deoxy-2-[[sup.18]F]fluoroarabinofuranosyl) cytosine) by differential screening as a new PET probe for the deoxyribonucleotide salvage pathway. [[sup.18]F]FAC enabled visualization of lymphoid organs and was sensitive to localized immune activation in a mouse model of antitumor immunity. [[sup.18]F]FAC micro-PET also detected early changes in lymphoid mass in systemic autoimmunity and allowed evaluation of immunosuppressive therapy. These data support the use of [[sup.18]F]FAC PET for immune monitoring and suggest a wide range of clinical applications in immune disorders and in certain types of cancer., Imaging of the immune system in living subjects can define the spatiotemporal programming of innate and adaptive immunity. PET enables noninvasive, quantitative and tomography assays of biological processes by using [...]

2-[[sup.18]F]Fluoro-2-deoxy-D-glucose positron emission tomography ([[sup.18]FDG PET) detection of the up-regulated glycolysis associated with malignant transformation is a noninvasive imaging technique used extensively in cancer diagnosis. Although striking similarities exist in glucose transport and metabolism between tumor cells and activated immune cells, the potential use of [[sup.18]F]FDG PET for the diagnosis and evaluation of autoimmune disorders has not been systematically investigated. Here we ask whether [[sup.18]F]FDG PET in conjunction with computed tomography (CT) could be used to monitor a complex autoimmune disorder such as murine experimental autoimmune encephalomyelitis (EAE) and whether this approach is sensitive enough to evaluate therapeutic interventions. We found that (i) coregistration of metabolic (i.e., microPET) and high-resolution anatomical (i.e., CT) images allows serial quantification of glycolysis with [[sup.18]F]FDG in various spinal column segments; (ii) [[sup.18]F]FDG PET/CT can detect the increased glycolysis associated with paralysis-causing inflammatory infiltrates in the spinal cord; and (iii) the [[sup.18]F]FDG measure of glycolysis in the spinal cord is sensitive to systemic immunosuppressive therapy. These results highlight the potential use of serial [[sup.18]F]FDG PET/CT imaging to monitor neuroinflammation in EAE and suggest that similar approaches could be applied to the diagnosis and evaluation of other autoimmune and inflammatory disorders in animal models and in humans. 2-[[sup.18]F]fluoro-2-deoxy-D-glucose | glycolysis | activated | lymphocytes

Current methodologies that monitor immune responses rely on invasive techniques that sample tissues at a given point in time. New technologies are needed to elucidate the temporal patterns of immune responses and the spatial distribution of immune cells on a whole-body scale. We describe a noninvasive, quantitative, and tomographic approach to visualize a primary anti-tumor immune response by using positron emission tomography (PET). Bone marrow chimeric mice were generated by engraftment of hematopoietic stem and progenitor cells transduced with a trifusion reporter gene encoding synthetic Renilla luciferase (hRluc), EGFP, and Herpes virus thymidine kinase (sr39TK). Mice were challenged with the Moloney murine sarcoma and leukemia virus complex (M-MSV/M-MuLV), and the induced immune response was monitored by using PET. Hematopoietic cells were visualized by using 9-[4-[[18.sup.F]]fluoro-3-(hydroxymethyl)butyl]guanine ([[18.sup.F]]FHBG), a radioactive substrate specific for the sr39TK PET reporter protein. Immune cell localization and expansion were seen at the tumor and draining lymph nodes (DLNs). 2-[[18.sup.F]]fluoro-2-deoxy-o-glucose ([[sup.18F]]FDG), which is sequestered in metabolically active cells, was used to follow tumor growth and regression. Elevated glucose metabolism was also seen in activated lymphocytes in the DLNs by using the [[sup.18F]]FDG probe. When M-MSV/M-MuLV-challenged mice were treated with the immunosuppressive drug dexamethasone, activation and expansion of immune cell populations in the DLNs could no longer be detected with PET imaging. The method we describe can be used to kinetically measure the induction and therapeutic modulations of cell-mediated immune responses. immunosuppressive therapy | lymphocytes

Type I interferons (IFNs) are critical effectors of emerging cancer immunotherapies designed to activate pattern recognition receptors (PRRs). A challenge in the clinical translation of these agents is the lack of noninvasive pharmacodynamic biomarkers that indicate increased intratumoral IFN signaling following PRR activation. Positron emission tomography (PET) imaging enables the visualization of tissue metabolic activity, but whether IFN signaling-induced alterations in tumor cell metabolism can be detected using PET has not been investigated. We found that IFN signaling augments pancreatic ductal adenocarcinoma (PDAC) cell nucleotide metabolism via transcriptional induction of metabolism-associated genes including thymidine phosphorylase (TYMP). TYMP catalyzes the first step in the catabolism of thymidine, which competitively inhibits intratumoral accumulation of the nucleoside analog PET probe 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT). Accordingly, IFN treatment up-regulates cancer cell [18F]FLT uptake in the presence of thymidine, and this effect is dependent upon TYMP expression. In vivo, genetic activation of stimulator of interferon genes (STING), a PRR highly expressed in PDAC, enhances the [18F]FLT avidity of xenograft tumors. Additionally, small molecule STING agonists trigger IFN signaling-dependent TYMP expression in PDAC cells and increase tumor [18F]FLT uptake in vivo following systemic treatment. These findings indicate that [18F]FLT accumulation in tumors is sensitive to IFN signaling and that [18F]FLT PET may serve as a pharmacodynamic biomarker for STING agonist-based therapies in PDAC and possibly other malignancies characterized by elevated STING expression. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that intratumoral interferon (IFN) signaling can trigger targetable vulnerabilities. A hallmark of pancreatic ductal adenocarcinoma (PDAC) is its extensively reprogrammed metabolic network, in which nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, are critical cofactors. Here, we show that IFN signaling, present in a subset of PDAC tumors, substantially lowers NAD(H) levels through up-regulating the expression of NAD-consuming enzymes PARP9, PARP10, and PARP14. Their individual contributions to this mechanism in PDAC have not been previously delineated. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD salvage pathway, a dominant source of NAD in cancer cells. We found that IFN-induced NAD consumption increased dependence upon NAMPT for its role in recycling NAM to salvage NAD pools, thus sensitizing PDAC cells to pharmacologic NAMPT inhibition. Their combination decreased PDAC cell proliferation and invasion in vitro and suppressed orthotopic tumor growth and liver metastases in vivo. [ABSTRACT FROM AUTHOR]