Increased frequency of TIGIT+CD73-CD8+ T cells with a TOX+ TCF-1low profile in patients with newly diagnosed and relapsed AML.

The inhibitory receptor TIGIT, as well as theectonucleotidases CD39 and CD73 constitute potential exhaustion markers for T cells. Detailed analysis of these markers can shed light into dysregulation of the T-cell response in acute myeloid leukemia (AML) and will help to identify potential therapeutic targets.  The phenotype and expression of transcription factors was assessed on different T-cell populations derived from peripheral blood (PB, n = 38) and bone marrow (BM, n = 43). PB and BM from patients with AML diagnosis, in remission and at relapse were compared with PB from healthy volunteers (HD) (n = 12) using multiparameter flow cytometry. An increased frequency of terminally differentiated (CD45R-CCR7-)CD8+ T cells was detected in PB and BM regardless of the disease state. Moreover, we detected an increased frequency of two distinct T-cell populations characterized by the co-expression of PD-1 or CD39 on TIGIT+CD73-CD8+ T cells in newly diagnosed and relapsed AML in comparison to HDs. In contrast to the PD-1+TIGIT+CD73-CD8+ T-cell population, the frequency of CD39+TIGIT+CD73-CD8+ T cells was normalized in remission. PD-1+- and CD39+TIGIT+CD73-CD8+ T cells exhibited additional features of exhaustion by decreased expression of CD127 and TCF-1 and increased intracellular expression of the transcription factor TOX. CD8+ T cells in AML exhibit a key signature of two subpopulations, PD-1+TOX+TIGIT+CD73-CD8+- and CD39+TOX+TIGIT+CD73-CD8+ T cells that were increased at different stages of the disease. These results provide a rationale to analyze TIGIT blockade in combination with inhibition of the purinergic signaling and depletion of TOX to improve T-cell mediated cytotoxicity in AML. Abbreviations: AML: Acute myeloid leukemia; pAML: newly diagnosed AML; rAML: relapse AML; lrAML: AML in remission; HD: healthy donor; PB: peripheral blood; BM: bone marrow; TIGIT: T-cell immunoreceptor with Ig and ITIM domains; PD-1: Programmed cell death protein 1; CD73: ecto-5'-nucleotidase; CD39: ectonucleoside triphosphate diphosphohydrolase 1; ATP: adenosine triphosphate; ADO: adenosine; CD127: interleukin-7 receptor; CAR-T cell: chimeric antigen receptor T cell; TCF-1: transcription factor T-cell factor 1; TOX: Thymocyte selection-associated high mobility group box protein; NFAT: nuclear factor of activated T cells; NA: Naïve; CM: Central Memory; EM Effector Memory; EMRA: Terminal Effector Memory cells; FMO: Fluorescence minus one; PVR: poliovirus receptor; PVRL2: poliovirus receptor-related 2; IFN-γ: Interferon-γ; IL-2: interleukin-2; MCF: multiparametric flow cytometry; TNFα: Tumornekrosefaktor α; RT: room temperature.

Validation of the inverted adsorption structure for free-base tetraphenyl porphyrin on Cu(111).

Utilising normal incidence X-ray standing waves we rigourously scrutinise the "inverted model" as the adsorption structure of free-base tetraphenyl porphyrin on Cu(111). We demonstrate that the iminic N atoms are anchored at near-bridge adsorption sites on the surface displaced laterally by 1.1 ± 0.2 Å in excellent agreement with previously published calculations.

Amino acids as possible alternative nitrogen source for growth of Euglena gracilis Z in life support systems.

In recent times Euglena gracilis Z was employed as primary producer in closed environmental life-support system (CELSS), e.g. in space research. The photosynthetic unicellular flagellate is not capable of utilizing nitrate, nitrite, and urea as nitrogen source. Therefore, ammonium is supplied as an N-source in the lab (provided as diammonium-dihydrogenphosphate, (NH4)2HPO4) to E. gracilis cultures. While nitrate exerts low toxicity to organisms, ammonium is harmful for many aquatic organisms especially, at high pH-values, which causes the ionic NH4+ (low toxicity) to be partially transformed into the highly toxic ammonia, NH3. In earlier reports, Euglena gracilis was described to grow with various amino acids as sole N-source. Our aim was to investigate alternatives for (NH4)2HPO4 as N-source with lower toxicity for organisms co-cultivated with Euglena in a CELSS. The growth kinetics of Euglena gracilis cultures was determined in the presence of different amino acids (glycine, glutamine, glutamic acid, leucine, and threonine). In addition, uptake of those amino acids by the cells was measured. Cell growth in the presence of glycine and glutamine was quite comparable to the growth in (NH4)2HPO4 containing cultures while a delay in growth was observed in the presence of leucine and threonine. Unlike, aforementioned amino acids glutamate consumption was very poor. Cell density and glutamate concentration were almost unaltered throughout the experiment and the culture reached the stationary phase within 8 days. The data are compared with earlier studies in which utilization of amino acids in Euglena gracilis was investigated. All tested amino acids (glutamate with limitations) were found to have the potential of being an alternative N-source for Euglena gracilis. Hence, these amino acids can be used as a non-toxic surrogate for (NH4)2HPO4.

Nonlinear stiffness characteristics of the annular ligament.

The annular ligament provides a compliant connection of the stapes to the oval window. To estimate the stiffness characteristics of the annular ligament, human temporal bone measurements were conducted. A force was applied sequentially at several points on the stapes footplate leading to different patterns of displacement with different amounts of translational and rotational components. The spatial displacement of the stapes footplate was measured using a laser vibrometer. The experiments were performed on several stapes with dissected chain and the force was increased stepwise, resulting in load-deflection curves for each force application point. The annular ligament exhibited a progressive stiffening characteristic in combination with an inhomogeneous stiffness distribution. When a centric force, orientated in the lateral direction, was applied to the stapes footplate, the stapes head moved laterally and in the posterior-inferior direction. Based on the load-deflection curves, a mechanical model of the annular ligament was derived. The mathematical representation of the compliance of the annular ligament results in a stiffness matrix with a nonlinear dependence on stapes displacement. This description of the nonlinear stiffness allows simulations of the sound transfer behavior of the middle ear for different preloads.

Immune activation in amyloid-ß-related angiitis correlates with decreased parenchymal amyloid-ß plaque load.

BACKGROUND: Primary angiitis of the central nervous system (PACNS) is a rare but serious condition. A fraction of patients suffering from PACNS concurrently exhibit pronounced cerebral amyloid angiopathy (CAA) which is characterized by deposits of amyloid-ß (Aß) in and around the walls of small and medium-sized arteries of the brain. PACNS with CAA has been identified as a distinct disease entity, termed Aß-related angiitis (ABRA). Evidence points to an immune reaction to vessel wall Aß as the trigger of vasculitis. OBJECTIVE: To investigate whether the inflammatory response to Aß has (1) any effect on the status of immune activation in the brain parenchyma and (2) leads to clearance of Aß from brain parenchyma. METHODS: We studied immune activation and Aß load by quantitative immunohistochemical analysis in brain parenchyma adjacent to affected vessels in 11 ABRA patients and 10 matched CAA controls. RESULTS: ABRA patients showed significantly increased immune activation and decreased Aß loads in the brain parenchyma adjacent to affected vessels. CONCLUSION: Our results are in line with the hypothesis of ABRA being the result of an excessive immune response to Aß and show that this can lead to enhanced clearance of Aß from the brain parenchyma by immune-mediated mechanisms.

Structure, chromosomal localization, and expression of the gene for mouse ecto-mono(ADP-ribosyl)transferase ART5.

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome 7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six exons spanning 8 kb of genomic DNA. The 5' end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfecting an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.

ADP-ribosyltransferases: plastic tools for inactivating protein and small molecular weight targets.

ADP-ribosyltransferases (ADPRTs) form an interesting class of enzymes with well-established roles as potent bacterial toxins and metabolic regulators. ADPRTs catalyze the transfer of the ADP-ribose moiety from NAD(+) onto specific substrates including proteins. ADP-ribosylation usually inactivates the function of the target. ADPRTs have become adapted to function in extra- and intracellular settings. Regulation of ADPRT activity can be mediated by ligand binding to associated regulatory domains, proteolytic cleavage, disulphide bond reduction, and association with other proteins. Crystallisation has revealed a conserved core set of elements that define an unusual minimal scaffold of the catalytic domain with remarkably plastic sequence requirements--only a single glutamic acid residue critical to catalytic activity is invariant. These inherent properties of ADPRTs suggest that the ADPRT catalytic fold is an attractive, malleable subject for protein design.

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice.

AIMS/HYPOTHESIS: ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. METHODS: ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. RESULTS: Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 micromol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. CONCLUSIONS: ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation.

A polymorphic dinucleotide repeat in the rat nucleolin gene forms Z-DNA and inhibits promoter activity.

Many sequences in eukaryotic genomes have the potential to adopt a left-handed Z-DNA conformation. We used a previously described assay based on the binding of a mAb to Z-DNA to inquire whether Z-DNA is formed in the rat nucleolin (Ncl) gene in metabolically active, permeabilized nuclei. Using real-time PCR to measure Z-DNA formation, the potential Z-DNA sequence element Z1 [(CA)(10)(CG)(8)] in the promoter region was found to be enriched 571- to 4,040-fold in different cell lines, whereas Z2 [AC(GC)(5)CCGT(CG)(2)] in the first intron was enriched 12- to 34-fold. Ncl promoter activity was 1.5- to 16-fold stronger than that of the simian virus 40 promoter and enhancer. This activity was further increased 36-54% when Z1 was deleted. The inhibitory effect of Z1 on Ncl promoter activity was independent of location and orientation. The Ncl Z1 element is identical to the genetic marker D9Arb5. Five allelic variants of Z1 were identified by sequence analysis of genomic DNA from various rats. The two most common alleles differed significantly (up to 27%) in their capacity to inhibit Ncl promoter activity. This finding suggests that differences in Z-DNA formation by polymorphic dinucleotide repeats may be one of the factors contributing to genetic variation.

Rapid induction of naive T cell apoptosis by ecto-nicotinamide adenine dinucleotide: requirement for mono(ADP-ribosyl)transferase 2 and a downstream effector.

Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC(50) of 3-5 microM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [(32)P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [(32)P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 x NZW)F(1) animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 x NZW)F(1) T cells. The fact that T cells of F(1) animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).

DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6.

We investigated the role of DNA methylation in gene regulation of the rat T-cell differentiation marker RT6. Analysis of the methylation status of various tissues revealed that the RT6 promoter was hypomethylated in RT6-expressing tissues, and methylated in nonexpressing ones. Remarkably, among RT6-nonexpressing tissues, the extent of methylated regions varied greatly between lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb region surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subpopulation of T cells. We now show that the RT6 alleles in these cells differed in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5-azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cells, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the control of monoallelic and tissue-specific RT6 expression.

Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB.

The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.

DNA methylation and Z-DNA formation as mediators of quantitative differences in the expression of alleles.

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 RT6a allele. In the case of the rat nucleolin gene, a polymorphic dinucleotide repeat in the 5' region modulates promoter strength and forms left-handed Z-DNA in vivo. Models explaining putative effects of Z-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.

The RT6 system of the rat: developmental, molecular and functional aspects.

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.

Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation.

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.

The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase.

A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1-ART7) with activities similar to bacterial toxins have also been cloned. We show here that PSIBLAST, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by PSIBLAST from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the PSIBLAST program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.

A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation.

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6-8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.