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Abstract: Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD+ stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals. [Copyright &y& Elsevier]

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2 a2 and Art2 b2 . Monoclonal antibody Gy12/61 specifically reacted with Art2 a2 but not Art2 a1 lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2 a but not the Art2 b alleles in all rats studied. Analysis of Art2 a1 and Art2 b2 genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection. [ABSTRACT FROM AUTHOR]

RT6 is a developmentally regulated cell-surface membrane adenosine 5′-diphosphate-ribosyltransferase/nicotinamide adenine dinucleotide-glycohydrolase inserted within the membrane by a glycophosphatidylinositol anchor. In the rat it is restricted to mature T lymphocytes and a subpopulation of natural killer cells. With respect to the data now available, three aspects concerning the function of RT6 are discussed: first, the meaning of the marked polymorphisms; second, its enzymatic activity; third, its possible role concerning T-cell survival. The observation that the rat RT6 gene contains two transcription start sites suggests their different use by distinct subpopulations of T cells. The fact that the expression of RT6 is defective in lymphopenic diabetes prone (DP-BB) rats, although the RT6 gene is structurally not grossly altered in these animals, makes this rat strain a promising model to study the biological meaning of RT6. While it mostly is believed that the RT6 expression defect of the DP-BB rat is a consequence of the lymphopenia, the present paper discusses the possibility that the RT6 expression defect is causally involved in the lymphopenia, and that a normal expression of RT6 may protect the recent thymic emigrants from apoptosis. [ABSTRACT FROM AUTHOR]

Outcomes of pediatric liver transplantation have constantly improved in the last decade. Living-related liver transplantation does not seem to improve long-term outcomes following liver transplantation, but few studies have evaluated immunological parameters of the alloimmune response after living vs. deceased donor organ transplantation. We analyzed numbers of regulatory T cells, lymphocyte subsets, and serum cytokine concentrations in 12 pediatric recipients of living-related liver transplants and in 28 pediatric recipients of deceased donor organs during their annual follow-ups. Transplant recipients who underwent living donor organ transplantation had significantly higher numbers of regulatory T cells and IL-4 serum concentrations than recipients of deceased donor organs; both of these factors are associated with beneficial outcomes and transplantation tolerance. Living-related liver transplantation may have potentially beneficial immunological aspects, although long-term outcomes do not seem to be better in recipients of living donor organs than in recipients of deceased donor organs. Further studies are needed to compare immunological aspects of the two transplant procedures. [ABSTRACT FROM AUTHOR]

With draft copies of several model genomes available in the near future, attention is turning towards the genetic mechanisms that determine differences between individuals. While mutations in protein coding regions affect the structure of gene products, polymorphisms outside such regions may cause quantitative differences in gene expression. Here we summarize observations indicating that such differences may be mediated by allele-specific alterations in the modification or structure of DNA. Mono-allelic expression of the rat T-cell differentiation marker RT6 in a subpopulation of cells is associated with allele-specific differences in DNA methylation in the RT6 promoter. In contrast to previously described examples of mono-allelic expression, these are determined neither stochastically nor by parental origin, but by cis-acting elements within the alleles. An attractive candidate is a rodent identifier (ID) element exclusively present in the RT6α allele. In the case of the rat nucleolin gene, a polymorphic dinucleotide repeat in the 5′ region modulates promoter strength and form left-handed Z-DNA in vivo. Models explaining putative effects of DNA formation on transcription are presented. These observations suggest novel mechanisms by which repetitive DNA, an abundant source of polymorphism in the mammalian genome, may exert quantitative effects on gene expression. [ABSTRACT FROM AUTHOR]

The ectoenzymes CD39 and CD73 degrade extracellular ATP to adenosine. ATP is released by stressed or damaged cells and provides pro-inflammatory signals to immune cells through P2 receptors. Adenosine, on the other hand, suppresses immune cells by stimulating P1 receptors. Thus, CD39 and CD73 can shape the quality of immune responses. Here we demonstrate that upregulation of CD39 is a consistent feature of activated conventional CD4+ and CD8+ T cells. Following stimulation in vitro, CD4+ and CD8+ T cells from human blood gained surface expression of CD39 but displayed only low levels of CD73. Activated human T cells from inflamed joints largely presented with a CD39+CD73— phenotype. In line, in spleens of mice with acute Listeria monocytogenes, listeria-specific CD4+ and CD8+ T cells acquired a CD39+CD73— phenotype. To test the function of CD39 in control of bacterial infection, CD39-deficient (CD39-/-) mice were infected with L. monocytogenes. CD39-/- mice showed better initial control of L. monocytogenes, which was associated with enhanced production of inflammatory cytokines. In the late stage of infection, CD39-/- mice accumulated more listeria-specific CD8+ T cells in the spleen than wildtype animals suggesting that CD39 attenuates the CD8+ T-cell response to infection. In conclusion, our results demonstrate that CD39 is upregulated on conventional CD4+ and CD8+ T cells at sites of acute infection and inflammation, and that CD39 dampens responses to bacterial infection. [ABSTRACT FROM AUTHOR]

• Dental pulp cells (DPC) possess potent immunomodulatory capacity in vitro. • The accessibility and abundance of DPC make them a valuable source for cell-based immunomodulatory treatments. • DPC represent an attractive alternative to bone marrow-derived mesenchymal stem cells. • The enzymatic activity of CD73 is critical for the generation of adenosine (ADO) by DPC. • Overall, our results suggest a contribution of ADO signaling in DPC-mediated immunosuppression. The pulp of human teeth contains a population of self-renewing stem cells that can regulate the functions of immune cells. When applied to patients, these cells can protect tissues from damage by excessive inflammation. We confirm that dental pulp cells effectively inhibit the proliferation and activation of cytotoxic T cells in vitro , and show that they carry high levels of CD73, a key enzyme in the conversion of pro-inflammatory extracellular ATP to immunosuppressive adenosine. Given their accessibility and abundance, as well as their potential for allogeneic administration, dental pulp cells provide a valuable source for immunomodulatory therapy. [ABSTRACT FROM AUTHOR]

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD+ released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7- triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD+ increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio-ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotideinduced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2. The Journal of Immunology, 2015, 195: 2057-2066. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) use different mechanisms to exert their suppressive function, among them the conversion of ATP to adenosine initiated by the ectonucleotidase CD39. In humans, the expression of CD39 on Tregs shows a high interindividual variation, and is especially high at sites of inflammation, like the synovia of patients with arthritis. How CD39 expression is regulated, and the functional consequences of different levels of CD39 expression is not known. We show here that stimulation of CD39 − Tregs results in a modest upregulation of CD39, which cannot explain the high levels observed in many donors. Moreover, CD39 + Tregs are present in naïve compartments such as cord blood and thymus, and the individual frequency of CD39 + Tregs remains stable over time, suggesting inherent regulation of CD39 expression. Indeed, we show that a single nucleotide polymorphism in the CD39 gene determines expression levels in Tregs. CD39 + Tregs suppress T cell proliferation and inflammatory cytokine production more efficiently than CD39 − Tregs. Accordingly, Tregs from donors with the GG (high CD39) genotype have a higher capacity to suppress IFN-γ and IL-17 production by effector cells than Tregs from AA (low CD39) donors. Our study demonstrates that the expression of CD39 in Tregs is primarily genetically driven, and this may determine interindividual differences in the control of inflammatory responses. [ABSTRACT FROM AUTHOR]

Objective The CD4+ T cell immune response plays a pivotal role in the immunopathogenesis of human and experimental lupus nephritis, but the contribution of the Th17/interleukin-17 (IL-17) immune pathway to renal tissue injury in systemic lupus erythematosus (SLE) remains to be elucidated. The aim of this study was to characterize the function of the Th17/IL-17A immune response in 2 murine models of lupus nephritis. Methods IL-17A-deficient MRL/MPJ-Fas lpr/2J (MRL/ lpr) mice were generated, and the clinical course of nephritis was monitored by assessing the levels of albuminuria, extent of renal tissue injury, and functional parameters. In addition, lupus-prone (NZB × NZW)F1 (NZB/NZW) mice were treated with anti-IL-17A and anti-interferon-γ (anti-IFNγ) antibodies, and their effects on the clinical course of lupus nephritis were assessed. Results Characterization of renal IL-17A-producing and IFNγ-producing T cells in MRL/ lpr and NZB/NZW mice revealed low numbers of infiltrating CD3+IL-17A+ cells. Renal IL-17A was mainly produced by CD4/CD8 double-negative CD3+ T cells and CD4+ Th17 cells. In contrast, the number of renal CD3+IFNγ+ cells continuously increased over time and largely consisted of typical CD4+ Th1 cells. IL-17A deficiency did not affect the morphologic or functional parameters in MRL/ lpr mice with lupus nephritis, nor did IL-17A neutralization affect the clinical course of nephritis in NZB/NZW mice, but anti-IFNγ treatment attenuated the severity of the disease. Conclusion The Th17/IL-17A immune response plays no major role in the immunopathogenesis of lupus nephritis in MRL/ lpr and NZB/NZW mice. Thus, the results of this study do not support the hypothesis that IL-17A targeting could be an intriguing new therapeutic approach for the management of proliferative lupus nephritis in SLE patients. [ABSTRACT FROM AUTHOR]

Individualization of immunosuppressive medications is an important objective in transplantation medicine. Reliable biomarkers to distinguish between patients dependent from intensive immunosuppressive therapy and those where therapy can be minimized among pediatric transplant recipients receiving immunosuppressive medications are still not established. We evaluated the potential of cross-sectional quantification of regulatory T cells, lymphocyte subsets, and cytokine concentrations as biomarkers in 60 pediatric liver transplant recipients with AR, CR, or normal graft function and in 11 non-transplanted patients. Transplant recipients presenting with AR had significantly higher CD8+ T-cell counts, significantly higher concentrations of IL-2, and increased levels of IFN-γ compared with asymptomatic patients or controls. Regulatory T-cell numbers did not differ between children with rejection and children with good graft function. A tendency toward increased concentrations of IL-4 and TGF-β was detected in transplant recipients with good graft function. Cross-sectional parameters of peripheral regulatory T cells in pediatric liver transplant recipients do not seem to be valuable biomarkers for individualizing immunosuppressive therapy prior to the weaning process. Lymphocyte subsets, IL-2, IFN-γ, IL-4, and TGF-β serum concentrations may be helpful to identify children in whom immunosuppression can be reduced or discontinued. [ABSTRACT FROM AUTHOR]

Abstract: T cells constitutively express low amounts of a toxin-related ADP-ribosylating ecto-enzyme, ART2.2. In inflammatory settings, cells release NAD, the substrate for ART2.2. The ART2.2 catalyzed ADP-ribosylation of cell surface proteins induces cell death. However, the low expression levels of ART2.2 have hampered analysis of ART2.2 in physiological settings. Here we report the generation of transgenic mice over-expressing ART2.2 under the control of the H2K promoter and Igμ enhancer. ART2.2 transgenic mice were healthy and fertile and exhibited normal development of the major lymphocyte subsets. Most T cells and a small subpopulation of B cells from transgenic mice showed more than 10-fold higher levels of ART2.2 expression than their wild-type counterparts. Exposure of ART2.2-transgenic T cells to low, submicromolar concentrations of NAD caused cell membrane alterations including uptake of propidium iodide, externalization of phosphatidylserine, and shedding of CD62L, while ART2.2-transgenic B cells were resistant to NAD. The ART2.2-overexpressing animals described here confirm that ART2.2 is an essential component for the regulation of T-cell functions by extracellular NAD and provide a useful tool to further elucidate the function of ART2.2 in vivo. [Copyright &y& Elsevier]

The objective of this study was to quantify enzymatic activity on the surface of T cells by magnetic resonance imaging (MRI) using R2 and R2* relaxometry. Lymphoma cells expressing adenosine diphosphate (ADP)-ribosyltransferase 2 (ART2) were incubated with increasing doses of its substrate etheno-nicotinamide adenine dinucleotide (NAD), resulting in increasing amounts of surface protein ADP-ribosylation. Etheno-ADP-ribosylated proteins were detected with monoclonal antibody 1G4 and superparamagnetic iron oxide conjugated secondary antibodies (Ab-SPIO). Labeling efficiency was determined with R2 and R2* relaxometry on a clinical 3.0 T scanner. Parallel aliquots of cells were analyzed by flow cytometry. Cell-bound SPIO conjugates were detected by immunofluorescence and electron microscopy and quantified by atomic absorption spectroscopy. To mimic an inflammatory site in vivo, Ab-SPIOlabeled cells were injected subcutaneously in mice and analyzed by MRI. Immunofluorescence and electron microscopy confirmed cell-surface localization of Ab-SPIO. MRI of Ab-SPIO-labeled cells showed a corresponding signal reduction. Increases in R2 and R2* determined by magnetic resonance relaxometry correlated linearly with the expression level of ART2 and the concentration of the ART2 substrate etheno-NAD. R2 and R2* increases correlated linearly with the results from flow cytometry and atomic absorption spectroscopy analyses. Quantitative R2 and R2* mapping enable noninvasive determination of enzymatic activity on T cells and holds promise for characterization of inflammatory sites in vivo by MRI. [ABSTRACT FROM AUTHOR]

The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2 x 7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection, the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection). Our findings constitute a proof of principle that opens up a new avenue for targeting leukocyte ecto-enzymes in vivo and that can serve as a model also for developing new antidotes against ADP-ribosylating toxins. [ABSTRACT FROM AUTHOR]

T cells express a toxin-related ADP-ribosylating ectoenzyme, ART2. Exposure of mature T cells to NAD, the substrate for ADP-ribosylation, induces cell death. ART2-catalyzed ADP-ribosylation activates the cytolytic P2X7 purinoceptor, causing calcium flux, pore formation, phosphatidylserine exposure, shedding of CD62L, cell shrinkage, and propidium iodide uptake. Interestingly, much lower NAD than ATP concentrations are required to activate P2X7. NAD-induced cell death (NICD) operates with endogenous sources of NAD released upon cell lysis. These findings identify P2X7 as a key effector of NICD and demonstrate that P2X7 can be activated by an endogenous ligand other than ATP. Our results delineate an alternative mechanism for inducing T cell death and set an interesting precedent for immunoregulation via crosstalk between NAD-dependent ADP-ribosyltransferases and purinoceptors. [Copyright &y& Elsevier]

The ectonucleotidases CD39 and CD73 regulate immune responses by balancing extracellular ATP and adenosine in inflammation and are likely to be involved in the pathophysiology of COVID-19. Here, we analyzed CD39 and CD73 on different lymphocyte populations in a small cohort of COVID-19 patients and in healthy individuals. We describe a significantly lower level of expression of CD73 on cytotoxic lymphocyte populations, including CD8+ T, natural killer T (NKT), and natural killer (NK) cells, during COVID-19. Interestingly, the decrease of CD73 on CD8+ T cells and NKT cells correlated with serum ferritin levels. Furthermore, we observed distinct functional differences between the CD73+ and CD73- subsets of CD8+ T cells and NKT cells with regard to cytokine/toxin secretion. In COVID-19 patients, the majority of the CD73-CD8+ T cells were capable of secreting granzyme B, perforin, tumor necrosis factor (TNF-α) or interferon-gamma (IFN-γ). To conclude, in this first study of CD39 and CD73 expression of lymphocytes in COVID-19, we show that CD8+ T cells and NKT cells lacking CD73 possess a significantly higher cytotoxic effector functionality compared to their CD73+ counterparts. Future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies. [ABSTRACT FROM AUTHOR]