NAD+-consuming enzymes in immune defense against viral infection.

The COVID-19 pandemic reminds us that in spite of the scientific progress in the past century, there is a lack of general antiviral strategies. In analogy to broad-spectrum antibiotics as antibacterial agents, developing broad spectrum antiviral agents would buy us time for the development of vaccines and treatments for future viral infections. In addition to targeting viral factors, a possible strategy is to understand host immune defense mechanisms and develop methods to boost the antiviral immune response. Here we summarize the role of NAD+-consuming enzymes in the immune defense against viral infections, with the hope that a better understanding of this process could help to develop better antiviral therapeutics targeting these enzymes. These NAD+-consuming enzymes include PARPs, sirtuins, CD38, and SARM1. Among these, the antiviral function of PARPs is particularly important and will be a focus of this review. Interestingly, NAD+ biosynthetic enzymes are also implicated in immune responses. In addition, many viruses, including SARS-CoV-2 contain a macrodomain-containing protein (NSP3 in SARS-CoV-2), which serves to counteract the antiviral function of host PARPs. Therefore, NAD+ and NAD+-consuming enzymes play crucial roles in immune responses against viral infections and detailed mechanistic understandings in the future will likely facilitate the development of general antiviral strategies.

The Extracellular NADome Modulates Immune Responses.

The term NADome refers to the intricate network of intracellular and extracellular enzymes that regulate the synthesis or degradation of nicotinamide adenine dinucleotide (NAD) and to the receptors that engage it. Traditionally, NAD was linked to intracellular energy production through shuffling electrons between oxidized and reduced forms. However, recent data indicate that NAD, along with its biosynthetic and degrading enzymes, has a life outside of cells, possibly linked to immuno-modulating non-enzymatic activities. Extracellular NAD can engage puriginergic receptors triggering an inflammatory response, similar - to a certain extent - to what described for adenosine triphosphate (ATP). Likewise, NAD biosynthetic and degrading enzymes have been amply reported in the extracellular space, where they possess both enzymatic and non-enzymatic functions. Modulation of these enzymes has been described in several acute and chronic conditions, including obesity, cancer, inflammatory bowel diseases and sepsis. In this review, the role of the extracellular NADome will be discussed, focusing on its proposed role in immunomodulation, together with the different strategies for its targeting and their potential therapeutic impact.

CD4+ T-cell differentiation and function: Unifying glycolysis, fatty acid oxidation, polyamines NAD mitochondria.

The progression through different steps of T-cell development, activation, and effector function is tightly bound to specific cellular metabolic processes. Previous studies established that T-effector cells have a metabolic bias toward aerobic glycolysis, whereas naive and regulatory T cells mainly rely on oxidative phosphorylation. More recently, the field of immunometabolism has drifted away from the notion that mitochondrial metabolism holds little importance in T-cell activation and function. Of note, T cells possess metabolic promiscuity, which allows them to adapt their nutritional requirements according to the tissue environment. Altogether, the integration of these metabolic pathways culminates in the generation of not only energy but also intermediates, which can regulate epigenetic programs, leading to changes in T-cell fate. In this review, we discuss the recent literature on how glycolysis, amino acid catabolism, and fatty acid oxidation work together with the tricarboxylic acid cycle in the mitochondrion. We also emphasize the importance of the electron transport chain for T-cell immunity. We also discuss novel findings highlighting the role of key enzymes, accessory pathways, and posttranslational protein modifications that distinctively regulate T-cell function and might represent prominent candidates for therapeutic purposes.

NAD+ metabolism, stemness, the immune response, and cancer.

NAD+ was discovered during yeast fermentation, and since its discovery, its important roles in redox metabolism, aging, and longevity, the immune system and DNA repair have been highlighted. A deregulation of the NAD+ levels has been associated with metabolic diseases and aging-related diseases, including neurodegeneration, defective immune responses, and cancer. NAD+ acts as a cofactor through its interplay with NADH, playing an essential role in many enzymatic reactions of energy metabolism, such as glycolysis, oxidative phosphorylation, fatty acid oxidation, and the TCA cycle. NAD+ also plays a role in deacetylation by sirtuins and ADP ribosylation during DNA damage/repair by PARP proteins. Finally, different NAD hydrolase proteins also consume NAD+ while converting it into ADP-ribose or its cyclic counterpart. Some of these proteins, such as CD38, seem to be extensively involved in the immune response. Since NAD cannot be taken directly from food, NAD metabolism is essential, and NAMPT is the key enzyme recovering NAD from nicotinamide and generating most of the NAD cellular pools. Because of the complex network of pathways in which NAD+ is essential, the important role of NAD+ and its key generating enzyme, NAMPT, in cancer is understandable. In the present work, we review the role of NAD+ and NAMPT in the ways that they may influence cancer metabolism, the immune system, stemness, aging, and cancer. Finally, we review some ongoing research on therapeutic approaches.

Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation.

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1ß but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1ß expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

NAD+ Metabolism Maintains Inducible PD-L1 Expression to Drive Tumor Immune Evasion.

NAD+ metabolism is implicated in aging and cancer. However, its role in immune checkpoint regulation and immune evasion remains unclear. Here, we find nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ biogenesis, drives interferon γ (IFNγ)-induced PD-L1 expression in multiple types of tumors and governs tumor immune evasion in a CD8+ T cell-dependent manner. Mechanistically, NAD+ metabolism maintains activity and expression of methylcytosine dioxygenase Tet1 via α-ketoglutarate (α-KG). IFNγ-activated Stat1 facilitates Tet1 binding to Irf1 to regulate Irf1 demethylation, leading to downstream PD-L1 expression on tumors. Importantly, high NAMPT-expressing tumors are more sensitive to anti-PD-L1 treatment and NAD+ augmentation enhances the efficacy of anti-PD-L1 antibody in immunotherapy-resistant tumors. Collectively, these data delineate an NAD+ metabolism-dependent epigenetic mechanism contributing to tumor immune evasion, and NAD+ replenishment combined with PD-(L)1 antibody provides a promising therapeutic strategy for immunotherapy-resistant tumors.

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity.

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+ Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

Influence of NAD+ as an ageing-related immunomodulator on COVID 19 infection: A hypothesis.

The aging-associated decline of biological functions represents an important contributor to the increase in morbidity and mortality of human beings. Of these biological functions deterioration; there is a significant decline in the heart function, impairments in the lungs gas exchange, and impairments in the immune function. Many alterations in the body humeral and cellular immune response were observed with ageing process: The circulating pro-inflammatory cytokines are increased, the naive lymphocytes are decreased, the numbers of the antigen-presenting cells areelevated and the overall response is impaired. In addition, ageing is associated with a progressive restriction in the telomere length. Telomeres are located at chromosomes ends and play an essential role in preserving chromosome stability. Also, telomere length is very important to the immune system, because of the high sensitivity of the immune cells to the shortening of telomeres. Telomeres shortening adversely affect the immune cells' function and developments. These adverse changes increased the susceptibility for severe infection, risk of hospitalization, and even death. Elderly COVID-19 patients are at a real risk of complications due to impaired immune function, cytokine storm and defective respiratory function. Administration of anti-ageing immunomodulation factors like Nicotinamide Adenine Dinucleotide NAD+ can minimize these changes through its potent immunomodulation and longevity effects. NAD+ has a direct inhibitory effect on PARP-1 and can prevent pro-inflammatory cytokines over-activation. Increasing the NAD+ level will also result in stabilizing telomeres and this has a positive impact on immune cells function.

Switch of NAD Salvage to de novo Biosynthesis Sustains SIRT1-RelB-Dependent Inflammatory Tolerance.

A typical inflammatory response sequentially progresses from pro-inflammatory, immune suppressive to inflammatory repairing phases. Although the physiological inflammatory response resolves in time, severe acute inflammation usually sustains immune tolerance and leads to high mortality, yet the underlying mechanism is not completely understood. Here, using the leukemia-derived THP-1 human monocytes, healthy and septic human peripheral blood mononuclear cells (PBMC), we report that endotoxin dose-dependent switch of nicotinamide adenine dinucleotide (NAD) biosynthesis pathways sustain immune tolerant status. Low dose endotoxin triggered nicotinamide phosphoribosyltransferase (NAMPT)-dependent NAD salvage activity to adapt pro-inflammation. In contrast, high dose endotoxin drove a shift of NAD synthesis pathway from early NAMPT-dependent NAD salvage to late indoleamine 2,3-dioxygenase-1 (IDO1)-dependent NAD de novo biosynthesis, leading to persistent immune suppression. This is resulted from the IDO1-dependent expansion of nuclear NAD pool and nuclear NAD-dependent prolongation of sirtuin1 (SIRT1)-directed epigenetics of immune tolerance. Inhibition of IDO1 activity predominantly decreased nuclear NAD level, which promoted sequential dissociations of immunosuppressive SIRT1 and RelB from the promoter of pro-inflammatory TNF-α gene and broke endotoxin tolerance. Thus, NAMPT-NAD-SIRT1 axis adapts pro-inflammation, but IDO1-NAD-SIRT1-RelB axis sustains endotoxin tolerance during acute inflammatory response. Remarkably, in contrast to the prevention of sepsis death of animal model by IDO1 inhibition before sepsis initiation, we demonstrated that the combination therapy of IDO1 inhibition by 1-methyl-D-tryptophan (1-MT) and tryptophan supplementation rather than 1-MT administration alone after sepsis onset rescued sepsis animals, highlighting the translational significance of tryptophan restoration in IDO1 targeting therapy of severe inflammatory diseases like sepsis.

Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase/BAK1 complex.

Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant immunity induced by mobile signals produced in the local leaves where the initial infection occurs. Although multiple structurally unrelated signals have been proposed, the mechanisms responsible for perception of these signals in the systemic leaves are unknown. Here, we show that exogenously applied nicotinamide adenine dinucleotide (NAD+) moves systemically and induces systemic immunity. We demonstrate that the lectin receptor kinase (LecRK), LecRK-VI.2, is a potential receptor for extracellular NAD+ (eNAD+) and NAD+ phosphate (eNADP+) and plays a central role in biological induction of SAR. LecRK-VI.2 constitutively associates with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in vivo. Furthermore, BAK1 and its homolog BAK1-LIKE1 are required for eNAD(P)+ signaling and SAR, and the kinase activities of LecR-VI.2 and BAK1 are indispensable to their function in SAR. Our results indicate that eNAD+ is a putative mobile signal, which triggers SAR through its receptor complex LecRK-VI.2/BAK1 in Arabidopsis thaliana.

Host NAD+ metabolism and infections: therapeutic implications.

Nicotinamide adenine dinucleotide (NAD+) is both a crucial coenzyme and a cosubstrate for various metabolic reactions in all living cells. Maintenance of NAD+ levels is essential for cell energy homeostasis, survival, proliferation and function. Mounting evidence points to NAD+ as one of the major modulators of immuno-metabolic circuits, thus regulating immune responses and functions. Recent studies delineate impaired host NAD+ metabolism during chronic infections and inflammation, suggesting NAD+ replenishment as an avenue to ameliorate deleterious inflammatory responses. Here, we discuss aspects of NAD+ biosynthesis and consumption, NAD+ biology during infections and how NAD+ metabolism can be intervened with pharmacologically to enhance the host's immunological fitness against pathogens.

A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Rho GTPase effectors and NAD metabolism in cancer immune suppression.

INTRODUCTION: Sustained proliferative signaling and de-regulated cellular bioenergetics are two of the chief hallmarks of cancer. Alterations in the Ras pathway and its downstream effectors are among the major drivers for uncontrolled cell growth in many cancers. The GTPases are one of the signaling molecules that activate crucial signal transducing pathways downstream of Ras through several effector proteins. The GTPases (GTP bound) interact with several effectors and modulate a number of different biological pathways including those that regulate cytoskeleton, cellular motility, cytokinesis, proliferation, apoptosis, transcription and nuclear signaling. Similarly, the altered glycolytic pathway, the so-called 'Warburg effect', rewires tumor cell metabolism to support the biosynthetic requirements of uncontrolled proliferation. There exists strong evidence for the critical role of the glycolytic pathway's rate limiting enzymes in promoting immunosuppression. Areas covered: We review the emerging roles of GTPase effector proteins particularly the p21 activated kinase 4 (PAK4) and nicotinamide biosynthetic pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) as signaling molecules in immune surveillance and the immune response. Expert opinion: In this expert opinion article we highlight the recent information on the role of GTPases and the metabolic enzymes on the immune microenvironment and propose some unique immune therapeutic opportunities.

Evolution of the metabolome in response to selection for increased immunity in populations of Drosophila melanogaster.

We used NMR-based metabolomics to test two hypotheses-(i) there will be evolved differences in the metabolome of selected and control populations even under un-infected conditions and (ii) post infection, the metabolomes of the selected and control populations will respond differently. We selected replicate populations of Drosophila melanogaster for increased survivorship (I) against a gram-negative pathogen. We subjected the selected (I) and their control populations (S) to three different treatments: (1) infected with heat-killed bacteria (i), (2) sham infected (s), and (3) untreated (u). We performed 1D and 2D NMR experiments to identify the metabolic differences. Multivariate analysis of the metabolic profiles of the untreated (Iu and Su) flies yielded higher concentrations of lipids, organic acids, sugars, amino acids, NAD and AMP in the Iu treatment as compared to the Su treatment, showing that even in the absence of infection, the metabolome of the I and S regimes was different. In the S and I regimes, post infection/injury, concentration of metabolites directly or indirectly associated with energy related pathways (lipids, organic acids, sugars) declined while the concentration of metabolites that are probably associated with immune response (amino acids) increased. However, in most cases, the I regime flies had a higher concentration of such metabolites even under un-infected conditions. The change in the metabolite concentration upon infection/injury was not always comparable between I and S regimes (in case of lactate, alanine, leucine, lysine, threonine) indicating that the I and S regimes had evolved to respond differentially to infection and to injury.

Use of immunoproteomics to identify immunogenic proteins in a rat model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a common and life­threatening clinical syndrome, and seeking biomarkers of ARDS has been an area of continuing research. The present study hypothesized that alterations to certain immunogenic substances occur in injured lungs and are able to specifically bind with corresponding proteins in the blood, and that these proteins may be readily detected. To investigate this hypothesis, a rat model of ARDS was established by cecal ligation and puncture surgery, and an immunoproteomics approach, using serum as the primary antibody in a western blot analysis, was used with the aim of identifying immunogenic proteins in the injured lungs. Ingenuity Pathway Analysis (IPA) was used for bioinformatics analysis, and mass spectrometric analysis was used to identify a total of 38 differentially expressed immunogenic proteins. Bioinformatics analysis revealed that the top canonical pathways in which the identified proteins may be involved were gluconeogenesis I, glycolysis I, choline degradation I, NADH repair and heme degradation. IPA Biomarker Filter analysis with the terms 'acute respiratory distress syndrome/acute lung injury' was used to screen 13 proteins as candidate biomarkers. These proteins were described as antigens, and suggested that paired antibodies may be detected in the plasma of patients at high risk of ARDS. Analysis of these identified proteins may provide novel insights into the potential pathological mechanisms of ARDS.

Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP.

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.Several metabolic factors affect cellular glucose metabolism as well as the innate inflammatory response. Here, the authors show that glucose metabolism regulates pro-inflammatory responses through effects on the cytosolic NADH:NAD+ ratio and the NAD(H)-sensitive transcription co-repressor CtBP.

Targeting NAMPT for Therapeutic Intervention in Cancer and Inflammation: Structure-Based Drug Design and Biological Screening.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate limiting enzyme that plays an important role in the synthesis of nicotinamide adenine dinucleotide (NAD) via a salvage pathway. Along with a role in bioenergetics, NAMPT regulates the activity of proteins such as SIRT-1 that utilize NAD as a cofactor. As NAD metabolism is usually high in diseased conditions, it has been hypothesized and proven that NAMPT is over expressed in various cancers and inflammatory disorders. Inhibitors targeting NAMPT could therefore be useful in treating disorders arising from aberrant NAMPT signalling. In this study, inhibitors against NAMPT were designed using an energy-based pharmacophore strategy and evaluated for efficacy in cellular assays. Besides reducing cellular pools of NAD and NMN, NAMPT inhibitors decreased concentrations of reactive oxygen species as well as mRNA levels of TNFα and IL6, thereby implicating their potential in alleviating the inflammatory process. In addition, reduced NAD levels corroborated with an induction of apoptosis in prostate cancer cell lines.

Nicotinamide phosphoribosyltransferase (NAMPT) activity is essential for survival of resting lymphocytes.

NAD biosynthesis is emerging as a key regulator of immune cell functions. Accordingly, inhibitors of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) have anti-inflammatory effects, counteract hematological malignancies and are being tested in clinical trials. Still, their effect on different cell types still waits to be fully investigated. Here we show that the NAMPT inhibitor FK866 induces NAD depletion in various mouse organs but selectively causes dramatic atrophy of the spleen red pulp. Accordingly, in cultured mouse lymphocytes exposed to FK866, NAD contents drop to 50% of basal values within 2 days, a condition sufficient to prompt complete cell death. Cultures of human lymphocytes are more resistant to FK866 and sustain a 50% NAD reduction for 5 days before dying. Death of both cell types can be prevented by different NAD precursors, indicating critical NAD homeostasis in lymphocytes. Indeed, inhibition of the NAD-consuming enzyme poly(ADP-ribose) polimerase-1 suffices to prevent FK866-induced NAD depletion and death of both lymphocyte types. Poly(ADP-ribose) polymerase-1-null lymphocytes also undergo lower NAD depletion and reduced cell death when exposed to the drug. At variance with other cell types, neither apoptosis nor autophagy are exclusively responsible for lymphocyte death by FK866, consistent with a general impairment of lymphocyte homeostasis following NAD depletion. Data demonstrate a unique sensitivity of resting lymphocytes to NAD-depleting agents, providing new hints of relevance to lymphocyte biology and therapeutic interventions with NAMPT inhibitors.

CD8-ß ADP-ribosylation affects CD8(+) T-cell function.

The CD8αß coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-ß on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-ß mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-ß. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-ß occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-ß can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .