• Group members

    Prof. Marcus Altfeld, , Benjamin Grünhagel, Birgitta Maurer, Dr. Maria Pujantell, Rebecca-jo Thiele, Dr. Kati Tillack

  • Group members

    Prof. Friedrich Haag, Gudrun Dubberke, Paul Düngelhoef, Samantha Eiberg, Kilian Keetz, Birgitta Maurer

    Research interest

    The focus of our research is on exploring the mechanisms by which extracellular nucleotides, such as ATP, NAD, and their derivatives, regulate the function of immune cells. Topics include - the impact of ATP-gated P2X ion channels on T cell receptor-mediated signaling. - elucidating how and when ATP is released from immune cells, and how it is metabolized to adenosine on the outside. Because of our close connection to the Clinical Immunodiagnostics Laboratory our group is also interested in evaluating and improving immunodiagnostic methods suitable for routine use. Here our focus is currently on the development of flow cytometry protocols for the characterization of immune cells from patients. We also provide assistance and immunodiagnostic services for research projects that need to measure immunological parameters.

    Extracellular nucleotides as regulators of lymphocyte function

    Besides their metabolic importance inside cells, adenosine triphosphate (ATP) and other adenine nucleotides fulfill important roles outside of cells as autocrine and paracrine signal mediators. ATP is abundant within cells (3-10 mM), but under steady state conditions it is barely detectable in the extracellular milieu (low nanomolar conentrations). However, cells can actively secrete ATP for signaling purposes, or it can be passively released from cells upon cell damage. Once released into the extracellular space, ATP acts on ionotropic P2X or the metabotropic P2Y receptors present on the cell surface. While P2Y receptors belong to the family of G protein-coupled receptors (GPCRs), P2X receptors are ATP-gated cation channels, which among other actions can mediate the influx of calcium ions (Ca2+) into the cell. Outside the cell, ATP is degraded by the combined actions of ecto-nucleoside triphosphate diphosphohydrolases (ENTPDs) such as CD39 and 5’-nucleotidases like CD73, generating adenosine (ADO), which is a ligand for another family of GPCRs, the P1 receptors. Regulating the balance between extracellular ATP and ADO concentrations is an important mechanism for the regulation of inflammation. The release of ATP is an ancient, evolutionarily conserved danger signal heralding tissue damage, and thus alerting immune cells to initiate or augment an inflammatory reaction. This pro-inflammatory signal is counteracted by the degradation of ATP to ADO. Especially, stimulation of the A2A and A2B P1 receptors acts on many immune cells as a strong inhibitory (and thus anti-inflammatory) signal. In a similar fashion, extracellular nicotinamide adenine dinucleotide (NAD) also initiates signaling cascades by serving as a substrate either for the ADP-ribosyltransferase ARTC2 or for the ecto-NADase CD38. On mouse T lymphocytes, which carry ARTC2, a prominent target for ADP-ribosylation is the P2X7 ion channel, which is activated by this modification. CD38 uses NAD and NADP to generate calcium-mobilizing second messengers such as cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (Fig. 1).

    Research networks

    DFG: SFB 1328 Adenine nucleotides in immunity and inflammation (A12 Role of the ectonucleotidases CD39 and CD73 for the modulation of the immune response in chronic human viral infections)

    DFG: SFB 1328 Adenine nucleotides in immunity and inflammation (Z02 Antibodies and nanobodies for adenine nucleotide research)

    Selected publications

    Regulation of immune functions by extracellular nucleotides

    Brauneck F, Haag F, Woost R, Wildner N, Tolosa E, Rissiek A, Vohwinkel G, Wellbrock J, Bokemeyer C, Schulze zur Wiesch J, Ackermann C, and Fiedler W. (2021) Increased frequency of TIGIT+CD73-CD8+ T cells with a TOX+ TCF-1low profile in patients with newly diagnosed and relapsed AML. OncoImmunology. 10:193039110.1080/2162402X.2021.1930391.

    Libera J, Wittner M, Kantowski M, Woost R, Eberhard JM, de Heer J, Reher D, Huber S, Haag F, and Schulze Zur Wiesch J. (2020) Decreased Frequency of Intestinal CD39(+) γδ(+) T Cells With Tissue-Resident Memory Phenotype in Inflammatory Bowel Disease. Front Immunol. 11:56747210.3389/fimmu.2020.567472.

    Haag F, and Buck F. (2015) Identification and analysis of ADP-ribosylated proteins. Curr Top Microbiol Immunol. 384:33-5010.1007/82_2014_424.

    Seman M, Adriouch S, Scheuplein F, Krebs C, Freese D, Glowacki G, Deterre P, Haag F, and Koch-Nolte F. (2003) NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor. Immunity. 19:571-58210.1016/s1074-7613(03)00266-8.

    ATP sensors

    Kaschubowski KE, Kraft AE, Nikolaev VO, and Haag F. (2020) Using FRET-Based Fluorescent Sensors to Monitor Cytosolic and Membrane-Proximal Extracellular ATP Levels. Methods Mol Biol. 2041:223-23110.1007/978-1-4939-9717-6_16.

    Johnsen B, Kaschubowski KE, Nader S, Schneider E, Nicola JA, Fliegert R, Wolf IMA, Guse AH, Nikolaev VO, Koch-Nolte F, and Haag F. (2019) P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP. Purinergic Signal. 15:155-16610.1007/s11302-019-09654-5.

    The immune response to infection and immunization against SARS-CoV-2

    Haag F. (2021) Die natürliche und adaptive Immunantwort bei der Covid-19 Erkrankung Hamburger Ärzteblatt. 75:28-30

    Rüther DF, Schaub GM, Duengelhoef PM, Haag F, Brehm TT, Fathi A, Wehmeyer M, Jahnke-Triankowski J, Mayer L, Hoffmann A, Fischer L, Addo MM, Luetgehetmann M, Lohse AW, Schulze Zur Wiesch J, and Sterneck M. (2021) SARS-CoV2-specific humoral and T-cell immune response after second vaccination in liver cirrhosis and transplant patients. Clinical Gastroenterology and Hepatology. https://doi.org/10.1016/j.cgh.2021.09.003.

    Woo MS, Haag F, Nierhaus A, Jarczak D, Roedl K, Mayer C, Brehm TT, van der Meirschen M, Hennigs A, Christopeit M, Fiedler W, Karagiannis P, Burdelski C, Schultze A, Huber S, Addo MM, Schmiedel S, Friese MA, Kluge S, and Schulze Zur Wiesch J. (2021) Multi-dimensional and longitudinal systems profiling reveals predictive pattern of severe COVID-19. iScience. 24:10275210.1016/j.isci.2021.102752.

    Ahmadi P, Hartjen P, Kohsar M, Kummer S, Schmiedel S, Bockmann JH, Fathi A, Huber S, Haag F, and Schulze Zur Wiesch J. (2020) Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73(-) Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes. Cells. 9:10.3390/cells9081750.

  • Group members

    Prof. Dr. Hans-Willi Mittrücker, Dr. Friederike Raczkowski, Peter Bradtke, Stefanie Fertig, Katrin Heider, Niels Lory, Jonas Rösch, Joanna Schmid, Leonie Voss, Dennis Yüzen

    Research interests

    T cells play a central role in immune responses against pathogens, yet these cells are also crucial for the formation of autoimmune diseases. We use different mouse models for bacterial infection and for autoimmune kidney diseases to characterise the function and regulation of T cells. Main focuses of research are:

    ● The function and regulation of CD4 and CD8 T cells during infection with intracellular bacteria. For these studies, we use infection models for Listeria monocytogenes, Staphylococcus aureus and Salmonella typhimurium.

    ● The role of the transcription factor Interferon Regulatory Factor 4 (IRF4) in activation and differentiation of T cells.

    ● The regulation of immune responses by Interleukin-6, in particular, the impact of different Interleukun-6 signalling pathways on activation and differentiation of immune cells.

    ● Autoimmune kidney diseases and the role of T cells in formation and regulation of immune response against components of the glomerulus. Studies are conducted in mouse models for anti-glomerular basement nephritis.

    ● The role of adenine nucleotides, ectoenzymes and purinegic receptors for the regulation of T cells.

    Research networks

    DFG:SFB 1328 “Adenine Nucleotides in Immunity and Inflammation” (A03: “Adenine nucleotide-modulated T cell differentiation and effector Functions”, Prof. H.-W. Mittrücker, Prof. Samuel Huber)

    DFG: SFB 1192 „Immune-mediated glomerular diseases” (A4: “Autoreactive T cell responses in glomerulonephritis”)

    DFG: KFO 296 „Feto-maternal immune cross talk: Consequences for maternal and offspring’s health” (P2 "Identification of viral determinants and maternal immune responses underlying influenza A virus disease severity during pregnancy“, Prof. G. Gabriel, Prof. H.-W. Mittrücker, Prof. P. Arck).

    DFG: SFB 841 "Liver inflammation: Infection, immune regulation und consequences" (A3 “Regulation of antibacterial immune responses in the liver”)

    Selected publications

    Harberts A, Schmidt C, Schmid J, Reimers D, Koch-Nolte F, Mittrücker H-W*, Raczkowski F*. Interferon Regulatory Factor 4 controls effector functions of CD8+ memory T cells. (2021) Proc Natl Acad Sci USA. 118:e2014553118. (*shared last authorship)

    Krebs CF, Reimers D, Zhao Y, Paust HJ, Bartsch P, Nuñez S, Rosemblatt MV, Hellmig M, Kilian C, Borchers A, Enk LUB, Zinke M, Becker M, Schmid J, Klinge S, Wong MN, Puelles VG, Schmidt C, Bertram T, Stumpf N, Hoxha E, Meyer-Schwesinger C, Lindenmeyer MT, Cohen CD, Rink M, Kurts C, Franzenburg S, Koch-Nolte F, Turner JE, Riedel JH, Huber S, Gagliani N, Huber TB, Wiech T, Rohde H, Bono MR, Bonn S, Panzer U*, Mittrücker H-W*. (2020) Pathogen-induced tissue-resident memory TH17 (TRM17) cells amplify autoimmune kidney disease. Sci Immunol. 5:eaba4163. (*shared last authorship)

    Klinge S, Yan K, Reimers D, Brede KM, Schmid J, Paust HJ, Krebs CF, Panzer U, Hopfer H, Mittrücker H-W. (2019) Role of regulatory T cells in experimental autoimmune glomerulonephritis. Am J Physiol Renal Physiol. 316:F572-F581.

    Mahnke J, Schumacher V, Ahrens S, Käding N, Feldhoff LM, Huber M, Rupp J, Raczkowski F, Mittrücker H-W. (2016) Interferon Regulatory Factor 4 controls TH1 cell effector function and metabolism. Sci Rep. 6:35521.

    Hünemörder S, Treder J, Ahrens S, Schumacher V, Paust H-J, Menter T, Matthys P, Kamradt T, Meyer-Schwesinger C, Panzer U, Hopfer H, Mittrücker H-W. (2015) TH1 and TH17 cells promote crescent formation in experimental autoimmune glomerulonephritis. J Pathol. 237:62-71.

    Raczkowski F, Ritter J, Heesch K, Schumacher V, Höcker L, Raifer H, Klein M, Bopp T, Harb H, Kesper DA, Pfefferle PI, Grusdat M, Lang PA, Mittrücker H-W*, Huber M*. (2013) The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells. Proc Natl Acad Sci USA. 110:15019-15024. (*shared last authorship)

    Mittrücker H-W, Steinhoff U, Köhler A, Krause M, Lazar D, Mex P, Miekley D, Kaufmann SHE. (2007) Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis. Proc Natl Acad Sci USA. 104:12434-12439.

    Kursar M, Höpken UE, Koch M, Köhler A, Lipp M, Kaufmann SHE, Mittrücker H-W. (2005) Differential requirements for the chemokine receptor CCR7 in T-cell activation during Listeria monocytogenes infection. J Exp Med. 201:1447-1457.

    Kursar M, Bonhagen K, Fensterle J, Köhler A, Hurwitz R, Kamradt T, Kaufmann SHE, Mittrücker H-W. (2002) Regulatory CD4+ CD25+ T cells restrict memory CD8+ T cell responses. J Exp Med. 196:1585-1592.

    Mittrücker H-W, Matsuyama T, Grossman A, Kündig TM, Potter J, Shahinian A, Wakeham A, Patterson B, Ohashi PS, Mak TW. (1997) Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science. 275:540-543.

    Collaboration partners

    University Medical Center Hamburg-Eppendorf (UKE)

    Marcus Altfeld, Friedrich Koch-Nolte, Friedrich Haag, Eva Tolosa Institute for Immunology Ulf Panzer, Christian Krebs, Oliver Steinmetz, Nicola Tomas, Elion Hoxha, Rolf Stahl III. Medical Clinic and Polyclinic Samuel Huber, Nicola Gagliani, Peter Hübener I. Medical Clinic and Polyclinic Petra Arck Experimental feto-maternal medicine Gisa Tiegs Institute of Experimental Immunology and Hepatology Andreas Guse, Björn-Philipp Dierks Institute for Biochemistry and Molecular Cell Biology Tim Magnus, Björn Rissiek Department of Neurology Cathy Meyer-Schwesinger Institute of Cellular and Integrative Physiology Manuel Friese Institute of Neuroimmunology and Multiple Sclerosis

    external partners

    Helmut Hopfer Universitätsspital Basel Minka Breloer, Hanna Lotter Bernhard Nocht Institute for Tropical Medicine, Hamburg Magdalena Huber, Ulrich Steinhoff, Michael Lohoff Philipps University of Marburg Dirk Schmidt-Arras, Stefan Rose-John Christian-Albrechts University of Kiel Santina Bruzzone University of Genova, Italy Gülsah Gabriel Leibniz Institute for Experimental Virology, Hamburg

  • The Molecular Immunology group studies membrane-bound receptors and enzymes of immune and cancer cells and develops nanobodies and antibodies against these targets.

    Group members

    Prof. Friedrich Koch-Nolte, Dr. rer. nat. Thomas Eden, Marie Eggers, Dr. hum. biol. Carolina Pinto-Espinoza, Josephine Gebhardt, Julia Hambach, Dr. hum. biol. Klaus Kaschubowski, Cerusch Khan, Josephine Krenz, Dr. rer. nat. Anna Marei Mann, Luca Pape, Waldemar Schäfer, Alessa Schaffrath, Fabienne Seyfried, Tobias Stähler, Natalie Tode

    Research networks

    DFG: SFB 1192 „Immune-mediated glomerular diseases” (B05: Nanobody-based treatment strategies in glomerulonephritis, Nicola Wanner, Friedrich Nolte)

    DFG: SFB 1328 “Adenine Nucleotides in Immunity and Inflammation” (A10: Role of adenine nucleotides and purinergic receptors in adipose tissue, Jörg Heeren, Friedrich Nolte; Z02: Antibodies and nanobodies for adenine nucleotide research Friedrich Haag, Friedrich Nolte)

    DFG: FOR 2879 "ImmunoStroke: From Immune Cells to Stroke Recovery" (A1: Role of ATP and NAD as DAMP signals in stroke recovery and as therapy targets, Tim Magnus, Friedrich Nolte)

    DFG/ANR: Evaluation of P2X7 as a therapeutic target in autoimmune encephalomyelitis and in tumor immunity (Sahil Adriouch, Univ. Rouen, Friedrich Nolte)

    DFG: Engineering the avidity of nanobody-based bispecific diabodies and heavy chain antibodies to specifically target myeloma cells that co-express two membrane proteins (Peter Bannas, Radiologie, Friedrich Nolte)

    BMBF COMMUTE Combinatorial and multidisciplinary targeting of effective gene therapy vectors (P5 Targeting glioblastoma and muscle dystrophy with Nanobody-displaying AAV, Friedrich Nolte)

    Short CV Prof. Friedrich Koch-Nolte

    Deputy Director (since 1997)

    Phone: +49 (0) 40 7410 - 53612
    Mail: nolte@uke.de

    University Degrees
    Biology, Wesleyan University 1976 (BA)
    Medicine, University of Tübingen, 1983 (MD)
    Molceular Biology, University of Hamburg, 1986 (Diploma)

    Professor associée at the University of Rouen, France (2006-2007)
    Visiting scientist at the University of California, San Francisco, CA, USA (1994)
    Visiting scientist at the DNAX Research Institute of Molecular Biology, Palo Alto, CA, USA (1997)
    Visiting scientist at The Jackson Lab, Bar Harbor, ME, USA (1991, 1999)

    Research award of the German Cancer Society (2019)

    Mentorship Award of the Simon-Claussen Foundation (2009)
    Research Award of the Werner Otto Foundation (1997)
    Research Award of the Martini Foundation (1991)

    Member of the
    Deutsche Gesellschaft für Immunologie (DGfI)
    Deutsche Gesellschaft für Biochemie und Molekularbiologie (GBM)
    Deutsche Gesellschaft für Zellbiologie (DGZ)

    Collaboration partners

    University Medical Center Hamburg-Eppendorf (UKE)

    Friedrich Haag, Eva Tolosa, Hans-Willi Mittrücker Institute for Immunology Andreas Guse, Jörg Heeren, Ralf Fliegert Institute for Biochemistry and Molecular Cell Biology Peter Bannas Department of Diagnostic and Interventional Radiology and Nuclear Medicine Tim Magnus, Björn Rissiek Department of Neurology Cathy Meyer-Schwesinger Institute of Cellular and Integrative Physiology Nicola Wanner, Nicola Tomas, Elion Hoxha, Rolf Stahl, Ulf Panzer III. Medical Clinic and Polyclinic Boris Fehse, Kristoffer Rieken Department of Stem Cell Transplantation Irm Hermanns Borgmeyer Center for Molecular Neurobiology Katja Weisel, Walter Fiedler II. Medical Clinic and Polyclinic Thomas Braulke Institute of Osteology and Biomechanics

    external partners

    Sahil Adriouch University of Rouen Normandy Michael Hottiger University of Zurich Pablo Pelegrin University of Murcia Mascha Binder University of Halle Martin Trepel University of Augsburg Dirk Grimm University of Heidelberg Annette Nicke Ludwig Maximilian University of Munich Carsten Watzl Technical University of Dortmund Florian Schmidt University of Bonn Francesco Di Virgilio, Elena Adinolfi University of Ferrara Santina Bruzzone, Paolo Malatesta University of Genoa Jan van Lier University of Amsterdam Jaime Sancho University of Granada Pablo Engel University of Barcelona Ursula Dietrich Georg-Speyer-Haus William Heath University of Melbourne William Petri University of Virginia Steven Mansoor University of Oregon Hidde Ploegh Boston Children's Hospital

    Research interests

    The focus of our laboratory is on the molecular characterization of lymphocyte membrane proteins, in particular receptors and enzymes involved in signaling by extracellular nucleotides. We generate monoclonal and single domain antibodies as new research and therapeutic tools. We are interested in ADP-ribosylation as a pathogenic mechanism of bacterial toxins and as a reversible posttranslational modification regulating protein functions. Using genetic immunization and antibody engineering, we strive to develop new tools for combating infections and for treating diseases of the immune system.

    1. Lymphocyte membrane proteins: enzymes and receptors involved in signaling by extracellular NAD and ATP

    Membrane proteins mediate the communication of cells with their environment. They function as receptors for soluble ligands and counter-receptors on other cells, as ion channels, nutrient transporters, and enzymes. The nucleotides NAD and ATP are key metabolites of energy metabolism found in cells from all kingdoms of life. The cell membrane is impermeable to these nucleotides, but they can exit cells via channels or pores gated by mechanical and/or electrical stimuli. During infection and inflammation injured cells release NAD and ATP through the damaged cell membrane. Extracellular NAD and ATP alert cells of the immune system to sites of tissue damage.

    Cells of the immune system are equipped with a variety of sensors for these nucleotides (Fig. 1), including ligand-gated ion channels (P2X purine receptors) and nucleotide-metabolizing ecto-enzymes (CD38, CD296). CD296 (ARTs) functions as NAD-sensors and relay information about the levels of extracellular NAD into ADP-ribosylation of cell surface and secreted proteins. The NAD-hydrolyzing ecto-enzyme CD38 restricts the intensity and duration of NAD-signaling in the extracellular space by hydrolyzing NAD. Opening of the P2X7 ion channel is induced by binding of the soluble ligand ATP, or by NAD-dependent ADP-ribosylation. Passage of ions through P2X7 (calcium into the cell, potassium out of the cell) triggers a cascade of downstream events, including protease activation (caspases and ADAMs), externalization of phosphatidylserine (PS), activation of the inflammasome and cell death. Mice that cannot express the mentioned purine receptors or ecto-enzymes show impaired immune responses. The receptors and enzymes of purinergic signaling, thus, present potential targets for new anti-inflammatory or immune-stimulating drugs. In mouse models, activating the ARTC2 > P2X7 axis can enhance anti-tumor responses, while blocking this axis can reduce inflammation in autoimmune diseases.

    2. Antibody engineering: genetic immunization, single domain antibodies

    Antibodies are important tools of research, diagnosis and therapy. Antibodies are raised by immunizing experimental animals with proteins, synthetic peptides, or DNA. In the case of DNA immunizations, the protein encoded by the DNA is produced in its native conformation by the cells of the immunized animal and the induced immune response yields antibodies directed against proteins in native conformation (ADAPINCs) (Fig. 2). Such antibodies are useful in many applications were anti-peptide antibodies fail, e.g. affinity purification, flow cytometry, ELISA, FACS, and functional studies. cDNA immunization is offered as a service via the antibody core facility of the UKE.

    Llamas produce unusual antibodies composed only of heavy chains. Their antigen-binding domain (VHH) is readily produced as a soluble recombinant protein, also designated nanobody or single domain antibody (sdAb) (Fig. 3). Nanobodies can form finger like extrusions that block clefts on protein surfaces, such as the active site crevice of enzymes, the ligand binding domain of a receptor, and the receptor binding domain of a virus. Nanobodies have great potential as therapeutics and as imaging agents.

    We have generated enzyme-blocking nanobodies from llamas immunized with different ARTs: the SpvB Salmonella toxin, the binary clostridium difficile toxin CDTa, and the T cell ecto-enzyme ARTC2 (CD296). These nanobodies protect cells from the cytotoxic effects of SpvB, CDT, and ARTC2. In case of ARTC2, the nanobodies effectively block ARTC2 on the cell surface of regulatory T cells and iNKT cells within 10 minutes after intravenous injection. These nanobodies provide an important tool for protecting these regulatory cells from death induced by NAD released during tissue preparation.
    We have also generated nanobodies against CD38, the major NAD-hydrolyzing ecto-enzyme. CD38 is emerging as a therapeutic target in multiple myeloma and other hematopoertic malignancies. Some of our nanobodies outperform the recently licensed CD38-specific monoclonal antibody Daratumumab (Darzalex) in cytotoxicity vs. hematopoetic cancer cell lines.
    In cooperation with Ablynx, a Belgian company devoted to developing nanobodies for human therapies, we have generated nanobodies that block or enhance gating of the P2X7 ion channel. The P2X7-blocking nanobodies ameliorated inflammation in mouse models of glomerulonephritis and allergic dermatitis. In endotoxin-treated human blood cells, they effectively blocked LPS/ATP-induced release of the potent pro-inflammatory cytokine IL-1ß.
    In order to facilitate the generation of new nanobodies, we have cloned the nanobody-encoding IgH locus from llama and successfully transferred an engineered version of this locus to transgenic mice (Fig. 4). Upon immunization, these mice produce nanobody-based heavy chain antibodies that undergo somatic hypermutation and class switch recombination. This novel platform greatly expands our capacity to generate functional nanobodies against interesting targets.

    3. Posttranslational modifications: ADP-ribosylation, glycosylation, lipid anchors

    The function of proteins can be regulated via the attachment of chemical moieties. These enzyme-catalyzed modifications, coined posttranslational modifications (PTMs), include phosphorylation, glycosylation, ADP-ribosylation, and the attachment of lipid anchors.
    ADP-ribosylation is a reversible PTM, in which ADP-ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD onto a specific amino acid side chain in a target protein and ADP-ribosylhydrolases (ARHs) remove the ADP-ribose group (Fig. 5). We have determined the 3D structures of a prototype ART and a prototype ARH. The 3D structure of rat ARTC2 resembles a pacman with a wide-open mouth crunching on the substrate NAD. The 3D structure of human ARH3 resembles a pumpkin in which four alpha helices coordinate two magnesium ions at the bottom of the active site crevice.

    ADP-ribosylation is used by pathogenic toxins such as Diphtheria, Pertusssis and Clostridial toxins to modulate host protein functions. Toxin-related ARTs are expressed by cells of the immune system. ADP-ribosylation of membrane proteins can be monitored using labeled analogues of NAD. Using 32P-NAD, ADP-ribosylation results in radiolabeling of the target protein. Using etheno-NAD, ADP-ribosylation of target proteins can be detected with a monoclonal antibody directed against etheno-adenosine. This 1G4 antibody can be used to sort cells on the basis of cell surface ART-activity and to purify etheno-ADP-ribosylated proteins.

    ARTC2 itself is posttranslationally modified, e.g. by attachment of a GPI lipid anchor (Fig. 1 above). The GPI-anchor targets ARTC2 to lipid rafts, specialized regions of the cell membrane that play an important role in signal transduction, e.g. during activation of T cells by antigen presenting cells. The association of ARTC2 with lipid rafts focuses ARTC2 onto specific target proteins and thereby may regulate the signaling function of raft-associated proteins. ADP-ribosylation of the P2X7 ion channel on arginine residue 125 activates P2X7 to form a non-selective ion-channel, permitting calcium ions to enter the cell and potassium ions to exit the cell. This induces dramatic alterations of the cell membrane, including the externalization of PS, shedding of L-selectin, and formation of membrane blebs.

    Selected publications

    reviews

    Eggers M, Rühl F, Haag F, Koch-Nolte F. (2021) Nanobodies as probes to investigate purinergic signaling. Biochem Pharmacol. 87:114394. review PMID: 33388283.

    Linden J, Koch-Nolte F, Dahl G. (2019) Purine Release, Metabolism, and Signaling in the Inflammatory Response. Annu Rev Immunol. 37:325-347. review. PMID: 30676821

    Stortelers C, Pinto-Espinoza C, Van Hoorick D, Koch-Nolte F. (2018) Modulating ion channel function with antibodies and nanobodies. Curr Opin Immunol. 52:18-26. Review PMID: 29579624

    Bannas P, Hambach J, Koch-Nolte F. (2017) Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol. 8:1603. Review PMID: 29213270

    Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, Schwarz N, Adriouch S, Boyer O, Seman M, Licea A, Serreze DV, Goldbaum FA, Haag F, Koch-Nolte F. (2009) Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 198:157-74. Review PMID: 19529959

    Koch-Nolte F, Kernstock S, Mueller-Dieckmann C, Weiss MS, Haag F. (2008) Mammalian ADP-ribosyltransferases and ADP-ribosylhydrolases. Front Biosci. 13:6716-29. PMID: 18508690

    research reports

    Hambach J, Stähler T, Eden T, Wendt D, Tode N, Haag F, Tolosa E, Altfeld M, Fathi A, Dahlke C, Addo MM, Menzel S, Koch-Nolte F. (2021) A simple, sensitive, and low-cost FACS assay for detecting antibodies against the native SARS-CoV-2 spike protein. Immun Inflamm Dis. 9:905-917. PMID: 33979020.

    Schriewer L, Schütze K, Petry K, Hambach J, Fumey W, Koenigsdorf J, Baum N, Menzel S, Rissiek B, Riecken K, Fehse B, Röckendorf JL, Schmid J, Albrecht B, Pinnschmidt H, Ayuk F, Kröger N, Binder M, Schuch G, Hansen T, Haag F, Adam G, Koch-Nolte F, Bannas P. (2020) Nanobody-based CD38-specific heavy chain antibodies induce killing of multiple myeloma and other hematological malignancies. Theranostics. 10:2645-2658. PMID: 32194826

    Eichhoff AM, Börner K, Albrecht B, Schäfer W, Baum N, Haag F, Körbelin J, Trepel M, Braren I, Grimm D, Adriouch A, Koch-Nolte F. (2019) Nanobody-enhanced targeting of AAV gene therapy vectors Mol Ther Methods Clin Dev. 15:211-220. PMID: 31687421

    Eden T, Menzel S, Wesolowski J, Bergmann P, Nissen M, Dubberke G, Seyfried F, Albrecht B, Haag F, Koch-Nolte F. (2018) A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation. Front Immunol. 8:1989. PMID: 29410663

    Danquah W, Meyer-Schwesinger C, Rissiek B, Pinto C, Serracant-Prat A, Amadi M, Iacenda D, Knop JH, Hammel A, Bergmann P, Schwarz N, Assunção J, Rotthier W, Haag F, Tolosa E, Bannas P, Boué-Grabot E, Magnus T, Laeremans T, Stortelers C, Koch-Nolte F. (2016) Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation. Sci Transl Med. 8:366ra162. PMID: 27881823

    Adriouch S, Bannas P, Schwarz N, Fliegert R, Guse AH, Seman M, Haag F, Koch-Nolte F. (2008) ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site. FASEB J. 22:861-9. PMID: 17928361

    Koch-Nolte F, Reyelt R, Schössow B, Schwarz N, Scheuplein F, Rothenburg S, Haag F, Alzogaray V, Cauerhff A, Goldbaum F. (2007) Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J. 21:3490-8. PMID: 17575259

    Mueller-Dieckmann C, Kernstock S, Lisurek M, von Kries JP, Haag F, Weiss MS, Koch-Nolte F. (2006) The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation. Proc Natl Acad Sci U S A. 103:15026-31. PMID: 17015823

    Otto H, Reche PA, Bazan F, Dittmar K, Haag F, Koch-Nolte F. (2005) In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs). BMC Genomics. 6:139. PMID: 16202152

    Bannas P, Adriouch S, Kahl S, Braasch F, Haag F, Koch-Nolte F. (2005) Activity and specifity of toxin-related mouse T cell ecto-ADP-ribosyltransferase ART2.2 depends on it association with lipid rafts. Blood. 105:3663-70. PMID: 15657180

    Seman M, Adriouch S, Scheuplein F, Krebs C, Freese D, Glowacki G, Deterre P, Haag F, Koch-Nolte F. (2003) NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor. Immunity. 19:571-82. PMID: 14563321

    Otto H, Tezcan-Merdol D, Girisch R, Haag F, Rhen M, Koch-Nolte F. (2000) The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase. Mol Microbiol. 37:1106-15. PMID: 10972829

    Koch-Nolte F, Petersen D, Balasubramanian S, Haag F, Kahlke D, Willer T, Kastelein R, Bazan F, Thiele HG. (1996) Mouse T cell membrane proteins Rt6-1 and Rt6-2 are arginine/protein mono(ADPribosyl)transferases and share secondary structure motifs with ADP-ribosylating bacterial toxins. J Biol Chem. 271:7686-93. PMID: 8631807

    Koch F, Haag F, Kashan A, Thiele HG. (1990) Primary structure of rat RT6.2, a nonglycosylated phosphatidylinositol-linked surface marker of postthymic T cells. Proc Natl Acad Sci U S A. 87:964-7. PMID: 2300588

    Koch F, Thiele HG, Low MG. (1986) Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C. J Exp Med. 164:1338-43. PMID: 3489808

  • Group members

    Prof. Eva Tolosa, Dr. rer. nat. Anna Gieras, Dr. rer. nat. Anne Rissiek, Dr. rer. nat. Enja Schneider, Dr. rer. nat. Kati Tillack, Elena Billeb, Annika Boxnick, Sarah-Jolan Bremer, Laura Glau, Romy Hackbusch, Manuela Kolster, Maili Pöls, Niklas Eich, Hannah Lorenz, Hauke Wasielewski, Riekje Winzer

    Research interest

    Effects of prenatal and postnatal medical events on immune system development

    Over the past 30 years, prenatal corticosteroid treatment (betamethasone) has been administered to thousands of women at risk of premature birth to accelerate fetal lung maturation in the preterm neonates. A remarkably higher survival-rate of premature and infants with very low birth-weight highlights the efficiency of this treatment. However, long term effects have not been analyzed in detail, especially possible effects on the immune system, considering that steroids are known to induce cell death of developing T cells.

    Development of the immune system in mammals begins before birth. The thymus is an essential organ for T cell development and establishment of tolerance. Around birth, thymic function is at its peak and therefore events affecting the thymus at this time may have consequences for the child’s immunity as immature thymocytes are extremely sensitive to steroids. Indeed, injection of physiological doses of betamethasone to pregnant mice results a dramatic reduction of the thymus volume and death of T cell precursors (Diepenbruck 2013). The lost niche is quickly replenished, and this accelerated maturation may result in errors during selection events. Of note, in vitro treatment of human thymocytes with low betamethasone doses also induces apoptotic death of the developing cells.

    Western Societies are facing an unparalleled increase in the incidence of autoimmune diseases and asthma over the past decades. In this context it is particularly noteworthy that the age at the onset of diseases like type I diabetes is decreasing, and in many occasions with more severe course, compared to a few decades ago. Recent evidence indicates that the exposure to prenatal steroids constitutes a risk factor for neonatal sepsis and for developing asthma and diabetes later in life. Given the importance of T cells in adaptive immune responses, we hypothesise that prenatal steroid treatment impairs the normal development of the offspring’s immune system and increases the risk for autoreactivity/allergy later in life.

    The main focus of our project is to study T cell development and long-term effects on the immune system in offspring whose mothers were treated with prenatal corticosteroids. To test our hypothesis we will use animal models of autoimmunity and allergy, and we will analyse in detail the immune system at birth and later in life of children whose mothers received steroids.

    Considering that half of the women receiving prenatal steroids have not delivered by the time that the beneficial effect of steroids wears out, awareness of pernicious long term effects could lead to a re-evaluation of treatment protocols.

    Image KFO project

    Characterization of adenine nucleotide metabolizing ectoenzymes and purinergic receptors

    The extracellular adenine nucleotides and –nucleosides (AN) ATP and NAD and their metabolites play key roles in the modulation of the immune response. We are interested in the regulation of the ectoenzymes involved in the generation and destruction of AN, their role in different pathways of the immune response, and in the outcome upon activation of purinergic receptors by AN.

    The cell membrane ectonucleotidase CD39 hydrolyzes ATP and ADP to AMP, acting in concert with CD73 to generate adenosine. By this process, a pro-inflammatory molecule, ATP, is degraded to generate the anti-inflammatory adenosine. In mice, regulatory T cells (Tregs) express both ectoenzymes, and use this pathway to suppress the immune response. In humans, however, only some Tregs express CD39, and most of them do not display CD73 on the cell surface. Tregs expressing CD39 are powerful suppressors of T cell proliferation and, especially, of the production of inflammatory cytokines. We have shown that expression of CD39 on Tregs is primarily genetically determined, and this may determine interindividual differences in the control of inflammatory responses. In addition, T cell activation results in further upregulation of CD39. Much less is known on the modulation of CD73 and its consequences in human disease, although adenosine tightly controls immune cell performance by binding to P1 receptors (i.e. A2AR) on different cell types.

    Extracellular ATP triggers activation of nucleotide-gated ion channels (P2X) on the cell surface of immune cells. Binding of ATP to the P2X7 results in influx of Ca2+ and efflux of K+, leading to inflammasome activation and release of IL-1β and IL-18 in macrophages, and to activation of metalloproteases and cell death i.e. of T cells. Altogether activation of P2X7 contributes to a pro-inflammatory environment. Thus, modulation of the ATP-adenosine axis constitutes an attractive intervention target. Since immune regulation must be strenghtened or reduced according to the type of disease, understanding the regulation of the ectoenzymes CD39 and CD73, and of P1 and P2 receptors is a crucial first step for the development of novel therapies. We are using chemical and biological inhibitors as well as knock-out systems for assessing the cell-specific role of these enzymes and receptors in the modulation of immunity and to evaluate the possibility for intervention.

    Abb.. 1

    Research networks

    DFG: : Molekulare Mechanismen und Funktion der Öffnung des P2X7 Ionenkanals in T Zellen mit Prof. F. Koch-Nolte

    DFG: KFO 296 Feto-maternal immune cross talk: Consequences for maternal and offspring's (P7: Impact of prenatal steroid treatment on the offsprings immune system)

    DFG: KFO 296 Feto-maternal immune cross talk: Consequences for maternal and offspring's (S1:Coordination of recruitment, tissue acquisition, data management, statistical support and methodological quality control for the prospective cohort PRenatal DetermInants of Children`s HEalth (PRINCE))

    DFG: SFB 1328 Adenine nucleotides in immunity and inflammation (A13 The role of adenine nucleotides during progression and resolution of cerebral ischemia)

    DFG: SFB 1328 Adenine nucleotides in immunity and inflammation (A14 Extra cellular ATP to adenosine axis in the biology of regulatory and effector T cells)

    DFG: FOR 2879 ImmunoStroke (C1 Linking functional immune profile and ischemic lesion characteristics in human stroke)

    Selected publications

    Schneider E#, Winzer R#, Rissiek A, .., Magnus T, Fliegert R, Müller CE, Gagliani N, Tolosa E (2021) CD73-mediated adenosine production by CD8 T cell-derived extracellular vesicles constitutes an intrinsic mechanism of immune suppression in humans. Nat Commun. 2021 8;12:5911.

    Poch T, Krause J, Casar C, Liwinski T, Glau L, …Huber S, Tolosa E#, Gagliani N#, Schramm C# (2021) Single-cell atlas of hepatic T cells reveals expansion of liver-resident naive-like CD4+ T cells in primary sclerosing cholangitis. J Hepatol; S0168-8278(21)00219-1

    Mohme M, Schliffke S, Maire CL, Runger A, Glau L, … Westphal M, Binder M#, Tolosa E#, Lamszus K# (2018) Immunophenotyping of Newly Diagnosed and Recurrent Glioblastoma Defines Distinct Immune Exhaustion Profiles in Peripheral and Tumor-infiltrating Lymphocytes. Clin Cancer Res; 24(17):4187-200

    Lessel D, Gehbauer C, Bramswig NC, Schluth-Bolard C, Venkataramanappa S, van Gassen KLI,… Agrawal PB, Britsch S, Tolosa E#, Kubisch C# (2018) BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells. Brain; 141(8):2299-311

    Thom V, Schmid S, Gelderblom M, Hackbusch R, Kolster M, .., Pless O, Bernreuther C, Glatzel M, Wegscheider K, Gerloff C, Magnus T#, Tolosa E# (2016) IL-17 production by CSF lymphocytes as a biomarker for cerebral vasculitis. Neurol Neuroimmunol Neuroinflamm; 3(2):e214.

    Rissiek A, Baumann I, Cuapio A, Mautner A, Kolster M, Arck PC, Dodge-Khatami A, Mittrucker HW, Koch-Nolte F, Haag F, Tolosa E (2015) The expression of CD39 on regulatory T cells is genetically driven and further upregulated at sites of inflammation. J Autoimmun; 58:12-20.

    Stoeckle C, Quecke P, Ruckrich T, Burster T, Reich M, Weber E, Kalbacher H, Driessen C, Melms A, Tolosa E (2012) Cathepsin S dominates autoantigen processing in human thymic dendritic cells. J Autoimmun; 38(4):332-43.

    Feger U#, Tolosa E#, Huang YH, Waschbisch A, Biedermann T, Melms A, Wiendl H (2007) HLA-G expression defines a novel regulatory T-cell subset present in human peripheral blood and sites of inflammation. Blood; 110(2):568-77.

    Tolosa E, Li W, Yasuda Y, … Driessen C, Schnorrer P, Weber E, Stevanovic S, Kurek R, Melms A, Bromme D (2003) Cathepsin V is involved in the degradation of invariant chain in human thymus and is overexpressed in myasthenia gravis. J Clin Invest; 112(4):517-26.

    Tolosa E, King LB, Ashwell JD (1998) Thymocyte glucocorticoid resistance alters positive selection and inhibits autoimmunity and lymphoproliferative disease in MRL-lpr/lpr mice. Immunity; 8(1):67-76.

    #shared authorships