Friedrich Haag

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

NAD-Induced T Cell Death: ADP-Ribosylation of Cell Surface Proteins by ART2 Activates the Cytolytic P2X7 Purinoceptor

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.

In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs)

BackgroundADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.ResultsUsing a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.ConclusionThe pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.

Cutting Edge: A Natural P451L Mutation in the Cytoplasmic Domain Impairs the Function of the Mouse P2X7 Receptor

The P2X7 receptor (P2X7R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X7R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X7R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X7R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X7R. These P2X7R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.

The family of toxin‐related ecto‐ADP‐ribosyltransferases in humans and the mouse

ADP‐ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP‐ribose onto a critical amino acid residue. Cross‐species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP‐ribosylating toxins in humans and the mouse. The human genome contains four functional toxin‐related ADP‐ribosyltransferase genes (ARTs) and two related intron‐containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R‐S‐EXE) typical of arginine‐specific ADP‐ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position‐sensitive iterative database search program PSI‐BLAST connected the mammalian ARTs with most known bacterial ADP‐ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP‐ribosylhydrolases, the enzymes that reverse protein ADP‐ribosylation. This suggests that the two enzyme families that catalyze reversible mono‐ADP‐ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.

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)5CCGT(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.

Effects of interleukin-2 plus highly active antiretroviral therapy on HIV-1 replication and proviral DNA (COSMIC trial).

BackgroundThe effect of interleukin-2 (IL-2) in combination with antiretroviral therapy on HIV-1 replication and reservoirs was investigated. MethodsIn a prospective, open-label trial, 56 asymptomatic HIV-1-infected subjects (CD4 T cell count > 350 × 106 cells/l) were randomized to highly active antiretroviral therapy (HAART: stavudine, lamivudine, nelfinavir, saquinavir) with or without IL-2 (9 megaunits daily for 5 days in 6-weekly intervals for a total of eight cycles). Productive and latent infection were analysed in peripheral blood, and residual virus replication in the lymphoid tissue and in the cerebrospinal fluid. The influence of IL-2 on viral rebound after treatment discontinuation was studied. ResultsVirus replication was detected in 21 of 31 on-treatment lymph nodes despite undetectable plasma viraemia. Viral RNA was found in resting as well as in proliferating cells. RNA-negative patients tended towards more rapid proviral DNA elimination. Supplementary IL-2 led to a greater increase in CD4 T cell counts than HAART alone (P < 0.001), resulting in normalization in ~90% of IL-2-treated patients compared with ~50% HAART-only subjects. IL-2 had no beneficial effect on virus replication and on proviral DNA in peripheral blood. ConclusionsViral persistence during HAART is partly a result of continued low-level replication, calling for more active regimens. IL-2 accelerates the normalization of CD4 T cell counts but does not impact on virus production or latency.

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.

Ecto-ADP-Ribosyltransferases (ARTs): Emerging Actors in Cell Communication and Signaling

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.

Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2–P2X7 pathway

Extracellular NAD+ affects the survival and function of regulatory T cells, and NAD-mediated depletion of regulatory T cells promotes anti-tumor responses in mice.