Dr. Angelique Hölzemer, Dr. Claudia Beisel, Dr. Christian Körner, Dr. Sebastian Lunemann, Dr. Gloria Martrus, Dr. Susanne Ziegler, Dr. Madeleine Bunders
The research group at the UKE and HPI investigates cellular immune responses against viruses that infection humans. "HPI/photographer: Udo Thomas"
Research projects focus on the characterization of the molecular mechanisms that enable antiviral NK cells to recognize virus-infected cells and on the impact of sex-specific factors on the development of antiviral immune responses. The working group is involved in a number of DFG and EU-funded joint projects (research networks) .
Antiviral cellular immune responses
The human immune system plays a central role in the control of viral infections. In particular, T cells and NK cells can recognize and kill virus-infected cells. The research group of Prof. Dr. Altfeld examines antiviral T cell and NK cell responses against several viruses, including HIV-1, HCV, and Influenza. In particular, we aim to characterize the pathways by which the immune system recognizes viral infections, and the mechanisms that viruses have developed to evade antiviral immunity. In addition, we have developed in vitro models to analyze the interplay of innate and adaptive immune responses during the course of a viral infection. The aim of these studies is to identify protective immune responses that can be subsequently induced through new vaccination strategies or immunotherapeutic approaches.
Virus detection and immune activation
Human pathogenic viruses can be detected by a number of receptors of the immune system. Cells of the innate immune system express receptors that can identify components of viruses as foreign, and initiate the subsequent antiviral immune response. While activation of innate immunity plays an important role in the initial control of acute viral infections, a persistent activation of the immune system in chronic viral infections (HIV -1, HCV) contributes to viral pathogenesis and pathology (CD4 T cell decline, liver fibrosis). The research group of Prof. Dr. Altfeld investigates the extracellular and intracellular receptors and signaling cascades that lead to the detection of viruses. A particular area of interest is the analysis of Toll-like Receptors (TLRs) and their influence on the pathogenesis of viral infections. Another area of research are the effects of sex hormones on antiviral immunity, and the resulting implications for gender differences in the disease manifestations of viral infections. The aim of these studies is to develop a better understanding of the molecular mechanisms of viral pathogenesis and to develop new immune-modulatory approaches that can reduce persistent immune activation during chronic viral infections.
1: M Ziegler S, Beisel C, Sutter K, Griesbeck M, Hildebrandt H, H Hagen S,Dittmer U, Altfeld M. Human pDCs display sex-specific differences in type Iinterferon subtypes and interferon α/β receptor expression. Eur J Immunol. 2017Feb;47(2):251-256. doi: 10.1002/eji.201646725. Epub 2017 Jan 3. PubMed PMID:27891600.
2: Martrus G, Niehrs A, Cornelis R, Rechtien A, García-Beltran W, Lütgehetmann M,Hoffmann C, Altfeld M. Kinetics of HIV-1 Latency Reversal Quantified on theSingle-Cell Level Using a Novel Flow-Based Technique. J Virol. 2016 Sep29;90(20):9018-28. doi: 10.1128/JVI.01448-16. Print 2016 Oct 15. PubMed PMID:27466424; PubMed Central PMCID: PMC5044848.
3: Garcia-Beltran WF, Hölzemer A, Martrus G, Chung AW, Pacheco Y, Simoneau CR,Rucevic M, Lamothe-Molina PA, Pertel T, Kim TE, Dugan H, Alter G,Dechanet-Merville J, Jost S, Carrington M, Altfeld M. Open conformers of HLA-Fare high-affinity ligands of the activating NK-cell receptor KIR3DS1. NatImmunol. 2016 Sep;17(9):1067-74. doi: 10.1038/ni.3513. Epub 2016 Jul 25. PubMedPMID: 27455421; PubMed Central PMCID: PMC4992421.
4: Schommers P, Martrus G, Matschl U, Sirignano M, Lütgehetmann M, Richert L,Hope TJ, Fätkenheuer G, Altfeld M. Changes in HIV-1 Capsid Stability Induced byCommon Cytotoxic-T-Lymphocyte-Driven Viral Sequence Mutations. J Virol. 2016 Jul27;90(16):7579-86. doi: 10.1128/JVI.00867-16. Print 2016 Aug 15. PubMed PMID:27279617; PubMed Central PMCID: PMC4984629.
5: Lunemann S, Martrus G, Hölzemer A, Chapel A, Ziegler M, Körner C, GarciaBeltran W, Carrington M, Wedemeyer H, Altfeld M. Sequence variations in HCVcore-derived epitopes alter binding of KIR2DL3 to HLA-C∗03:04 and modulate NKcell function. J Hepatol. 2016 Aug;65(2):252-8. doi: 10.1016/j.jhep.2016.03.016.Epub 2016 Apr 4. PubMed PMID: 27057987; PubMed Central PMCID: PMC4955726.
6: Hölzemer A, Thobakgale CF, Jimenez Cruz CA, Garcia-Beltran WF, Carlson JM, vanTeijlingen NH, Mann JK, Jaggernath M, Kang SG, Körner C, Chung AW, Schafer JL,Evans DT, Alter G, Walker BD, Goulder PJ, Carrington M, Hartmann P, Pertel T,Zhou R, Ndung'u T, Altfeld M. Selection of an HLA-C*03:04-Restricted HIV-1 p24Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural KillerCells: Data from an Observational Cohort in South Africa. PLoS Med. 2015 Nov17;12(11):e1001900; discussion e1001900. doi: 10.1371/journal.pmed.1001900.eCollection 2015 Nov. PubMed PMID: 26575988; PubMed Central PMCID: PMC4648589.
7: Griesbeck M, Ziegler S, Laffont S, Smith N, Chauveau L, Tomezsko P, Sharei A,Kourjian G, Porichis F, Hart M, Palmer CD, Sirignano M, Beisel C, Hildebrandt H,Cénac C, Villani AC, Diefenbach TJ, Le Gall S, Schwartz O, Herbeuval JP, AutranB, Guéry JC, Chang JJ, Altfeld M. Sex Differences in Plasmacytoid Dendritic CellLevels of IRF5 Drive Higher IFN-α Production in Women. J Immunol. 2015 Dec1;195(11):5327-36. doi: 10.4049/jimmunol.1501684. Epub 2015 Oct 30. PubMed PMID:26519527; PubMed Central PMCID: PMC4654231.
8: Altfeld M, Gale M Jr. Innate immunity against HIV-1 infection. Nat Immunol.2015 Jun;16(6):554-62. doi: 10.1038/ni.3157. Review. PubMed PMID: 25988887.
9: Körner C, Granoff ME, Amero MA, Sirignano MN, Vaidya SA, Jost S, Allen TM,Rosenberg ES, Altfeld M. Increased frequency and function of KIR2DL1-3⁺ NK cellsin primary HIV-1 infection are determined by HLA-C group haplotypes. Eur JImmunol. 2014 Oct;44(10):2938-48. doi: 10.1002/eji.201444751. Epub 2014 Aug 12.PubMed PMID: 25043727; PubMed Central PMCID: PMC4197106.
10: Jost S, Tomezsko PJ, Rands K, Toth I, Lichterfeld M, Gandhi RT, Altfeld M.CD4+ T-cell help enhances NK cell function following therapeutic HIV-1vaccination. J Virol. 2014 Aug;88(15):8349-54. doi: 10.1128/JVI.00924-14. Epub2014 May 14. PubMed PMID: 24829350; PubMed Central PMCID: PMC4135926.
11: van Teijlingen NH, Hölzemer A, Körner C, García-Beltrán WF, Schafer JL, FaddaL, Suscovich TJ, Brander C, Carrington M, Evans DT, van Baarle D, Altfeld M.Sequence variations in HIV-1 p24 Gag-derived epitopes can alter binding ofKIR2DL2 to HLA-C*03:04 and modulate primary natural killer cell function. AIDS.2014 Jun 19;28(10):1399-408. doi: 10.1097/QAD.0000000000000284. PubMed PMID:24785948; PubMed Central PMCID: PMC4453925.
12: Simmons RP, Scully EP, Groden EE, Arnold KB, Chang JJ, Lane K, Lifson J,Rosenberg E, Lauffenburger DA, Altfeld M. HIV-1 infection induces strongproduction of IP-10 through TLR7/9-dependent pathways. AIDS. 2013 Oct23;27(16):2505-17. doi: 10.1097/01.aids.0000432455.06476.bc. PubMed PMID:24096630; PubMed Central PMCID: PMC4288813.
13: Chang JJ, Woods M, Lindsay RJ, Doyle EH, Griesbeck M, Chan ES, Robbins GK,Bosch RJ, Altfeld M. Higher expression of several interferon-stimulated genes inHIV-1-infected females after adjusting for the level of viral replication. JInfect Dis. 2013 Sep 1;208(5):830-8. doi: 10.1093/infdis/jit262. Epub 2013 Jun10. PubMed PMID: 23757341; PubMed Central PMCID: PMC3733517.
14: Jost S, Altfeld M. Control of human viral infections by natural killer cells.Annu Rev Immunol. 2013;31:163-94. doi: 10.1146/annurev-immunol-032712-100001.Epub 2013 Jan 3. Review. PubMed PMID: 23298212.
15: Fadda L, Körner C, Kumar S, van Teijlingen NH, Piechocka-Trocha A, CarringtonM, Altfeld M. HLA-Cw*0102-restricted HIV-1 p24 epitope variants can modulate thebinding of the inhibitory KIR2DL2 receptor and primary NK cell function. PLoSPathog. 2012;8(7):e1002805. doi: 10.1371/journal.ppat.1002805. Epub 2012 Jul 12.PubMed PMID: 22807681; PubMed Central PMCID: PMC3395618.
16: Chang JJ, Lacas A, Lindsay RJ, Doyle EH, Axten KL, Pereyra F, Rosenberg ES,Walker BD, Allen TM, Altfeld M. Differential regulation of toll-like receptorpathways in acute and chronic HIV-1 infection. AIDS. 2012 Mar 13;26(5):533-41.doi: 10.1097/QAD.0b013e32834f3167. PubMed PMID: 22210629; PubMed Central PMCID:PMC3738004.
17: Alter G, Heckerman D, Schneidewind A, Fadda L, Kadie CM, Carlson JM,Oniangue-Ndza C, Martin M, Li B, Khakoo SI, Carrington M, Allen TM, Altfeld M.HIV-1 adaptation to NK-cell-mediated immune pressure. Nature. 2011 Aug3;476(7358):96-100. doi: 10.1038/nature10237. PubMed PMID: 21814282; PubMedCentral PMCID: PMC3194000.
18: Fadda L, O'Connor GM, Kumar S, Piechocka-Trocha A, Gardiner CM, Carrington M,McVicar DW, Altfeld M. Common HIV-1 peptide variants mediate differential bindingof KIR3DL1 to HLA-Bw4 molecules. J Virol. 2011 Jun;85(12):5970-4. doi:10.1128/JVI.00412-11. Epub 2011 Apr 6. PubMed PMID: 21471246; PubMed CentralPMCID: PMC3126328.
19: Streeck H, Kwon DS, Pyo A, Flanders M, Chevalier MF, Law K, Jülg B, Trocha K,Jolin JS, Anahtar MN, Lian J, Toth I, Brumme Z, Chang JJ, Caron T, Rodig SJ,Milner DA Jr, Piechoka-Trocha A, Kaufmann DE, Walker BD, Altfeld M. Epithelialadhesion molecules can inhibit HIV-1-specific CD8⁺ T-cell functions. Blood. 2011May 12;117(19):5112-22. doi: 10.1182/blood-2010-12-321588. Epub 2011 Mar 14.PubMed PMID: 21403126; PubMed Central PMCID: PMC3109536.
20: Jost S, Reardon J, Peterson E, Poole D, Bosch R, Alter G, Altfeld M.Expansion of 2B4+ natural killer (NK) cells and decrease in NKp46+ NK cells inresponse to influenza. Immunology. 2011 Apr;132(4):516-26. doi:10.1111/j.1365-2567.2010.03394.x. Epub 2011 Jan 7. PubMed PMID: 21214542; PubMedCentral PMCID: PMC3075505.
21: Alter G, Kavanagh D, Rihn S, Luteijn R, Brooks D, Oldstone M, van Lunzen J,Altfeld M. IL-10 induces aberrant deletion of dendritic cells by natural killercells in the context of HIV infection. J Clin Invest. 2010 Jun;120(6):1905-13.doi: 10.1172/JCI40913. Epub 2010 May 3. PubMed PMID: 20440075; PubMed CentralPMCID: PMC2877944.
22: Meier A, Chang JJ, Chan ES, Pollard RB, Sidhu HK, Kulkarni S, Wen TF, LindsayRJ, Orellana L, Mildvan D, Bazner S, Streeck H, Alter G, Lifson JD, Carrington M,Bosch RJ, Robbins GK, Altfeld M. Sex differences in the Toll-likereceptor-mediated response of plasmacytoid dendritic cells to HIV-1. Nat Med.2009 Aug;15(8):955-9. doi: 10.1038/nm.2004. Epub 2009 Jul 13. PubMed PMID:19597505; PubMed Central PMCID: PMC2821111.
23: Alter G, Rihn S, Walter K, Nolting A, Martin M, Rosenberg ES, Miller JS,Carrington M, Altfeld M. HLA class I subtype-dependent expansion of KIR3DS1+ andKIR3DL1+ NK cells during acute human immunodeficiency virus type 1 infection. JVirol. 2009 Jul;83(13):6798-805. doi: 10.1128/JVI.00256-09. Epub 2009 Apr 22.PubMed PMID: 19386717; PubMed Central PMCID: PMC2698561.
24: Streeck H, Brumme ZL, Anastario M, Cohen KW, Jolin JS, Meier A, Brumme CJ,Rosenberg ES, Alter G, Allen TM, Walker BD, Altfeld M. Antigen load and viralsequence diversification determine the functional profile of HIV-1-specific CD8+T cells. PLoS Med. 2008 May 6;5(5):e100. doi: 10.1371/journal.pmed.0050100.PubMed PMID: 18462013; PubMed Central PMCID: PMC2365971.
25: Alter G, Martin MP, Teigen N, Carr WH, Suscovich TJ, Schneidewind A, StreeckH, Waring M, Meier A, Brander C, Lifson JD, Allen TM, Carrington M, Altfeld M.Differential natural killer cell-mediated inhibition of HIV-1 replication basedon distinct KIR/HLA subtypes. J Exp Med. 2007 Nov 26;204(12):3027-36. Epub 2007Nov 19. PubMed PMID: 18025129; PubMed Central PMCID: PMC2118524.
26: Altfeld M, Kalife ET, Qi Y, Streeck H, Lichterfeld M, Johnston MN, Burgett N,Swartz ME, Yang A, Alter G, Yu XG, Meier A, Rockstroh JK, Allen TM, Jessen H,Rosenberg ES, Carrington M, Walker BD. HLA Alleles Associated with DelayedProgression to AIDS Contribute Strongly to the Initial CD8(+) T Cell Responseagainst HIV-1. PLoS Med. 2006 Oct;3(10):e403. PubMed PMID: 17076553; PubMedCentral PMCID: PMC1626551.
27: Allen TM, Yu XG, Kalife ET, Reyor LL, Lichterfeld M, John M, Cheng M,Allgaier RL, Mui S, Frahm N, Alter G, Brown NV, Johnston MN, Rosenberg ES, MallalSA, Brander C, Walker BD, Altfeld M. De novo generation of escapevariant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape inchronic human immunodeficiency virus type 1 infection. J Virol. 2005Oct;79(20):12952-60. PubMed PMID: 16188997; PubMed Central PMCID: PMC1235830.
28: Alter G, Teigen N, Davis BT, Addo MM, Suscovich TJ, Waring MT, Streeck H,Johnston MN, Staller KD, Zaman MT, Yu XG, Lichterfeld M, Basgoz N, Rosenberg ES,Altfeld M. Sequential deregulation of NK cell subset distribution and functionstarting in acute HIV-1 infection. Blood. 2005 Nov 15;106(10):3366-9. Epub 2005Jul 7. PubMed PMID: 16002429.
29: Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for theidentification of natural killer cell activity. J Immunol Methods. 2004Nov;294(1-2):15-22. PubMed PMID: 15604012.
30: Lichterfeld M, Kaufmann DE, Yu XG, Mui SK, Addo MM, Johnston MN, Cohen D,Robbins GK, Pae E, Alter G, Wurcel A, Stone D, Rosenberg ES, Walker BD, AltfeldM. Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infectionand restoration by vaccine-induced HIV-1-specific CD4+ T cells. J Exp Med. 2004Sep 20;200(6):701-12. PubMed PMID: 15381726; PubMed Central PMCID: PMC2211961.
31: Lichterfeld M, Yu XG, Waring MT, Mui SK, Johnston MN, Cohen D, Addo MM,Zaunders J, Alter G, Pae E, Strick D, Allen TM, Rosenberg ES, Walker BD, AltfeldM. HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8(+) Tcells producing both interferon-gamma and tumor necrosis factor-alpha. Blood.2004 Jul 15;104(2):487-94. Epub 2004 Apr 1. PubMed PMID: 15059848.
32: Altfeld M, Allen TM, Yu XG, Johnston MN, Agrawal D, Korber BT, Montefiori DC,O'Connor DH, Davis BT, Lee PK, Maier EL, Harlow J, Goulder PJ, Brander C,Rosenberg ES, Walker BD. HIV-1 superinfection despite broad CD8+ T-cell responsescontaining replication of the primary virus. Nature. 2002 Nov 28;420(6914):434-9.PubMed PMID: 12459786.
33: Altfeld M, van Lunzen J, Frahm N, Yu XG, Schneider C, Eldridge RL, Feeney ME,Meyer-Olson D, Stellbrink HJ, Walker BD. Expansion of pre-existing, lymphnode-localized CD8+ T cells during supervised treatment interruptions in chronicHIV-1 infection. J Clin Invest. 2002 Mar;109(6):837-43. PubMed PMID: 11901192;PubMed Central PMCID: PMC150914.
34: Yu XG, Addo MM, Rosenberg ES, Rodriguez WR, Lee PK, Fitzpatrick CA, JohnstonMN, Strick D, Goulder PJ, Walker BD, Altfeld M. Consistent patterns in thedevelopment and immunodominance of human immunodeficiency virus type 1(HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection. J Virol.2002 Sep;76(17):8690-701. PubMed PMID: 12163589; PubMed Central PMCID: PMC136975.
35: Altfeld M, Rosenberg ES, Shankarappa R, Mukherjee JS, Hecht FM, Eldridge RL,Addo MM, Poon SH, Phillips MN, Robbins GK, Sax PE, Boswell S, Kahn JO, Brander C,Goulder PJ, Levy JA, Mullins JI, Walker BD. Cellular immune responses and viraldiversity in individuals treated during acute and early HIV-1 infection. J ExpMed. 2001 Jan 15;193(2):169-80. PubMed PMID: 11148221; PubMed Central PMCID:PMC2193337.
DFG: KFO 296 Feto-maternal immune cross talk: Consequences for maternal and offspring's (P3: Hormonal modulation of the Type I Interferon response during pregnancy: implications for maternal health and disease)
DFG: KFO 306 Primary sclerosing cholangitis
DFG: SFB 841 Liver inflammation: Infection, immune regulation and consequences (A7: HCV infection: Mechanisms of NK-cell-mediated control)
DFG: SPP 1923 Innate Sensing and Restriction of Retroviruses (Consequences of CTL-mediated immune pressure for HIV-1 capsid stability and innate sensing)
LFF-FV 45 Sex polymorphisms in the immune system
EHVA European HIV Alliance
DZIF German Center for Infection Research
Prof. Friedrich Koch-Nolte
Deputy Director (since 1997)
Phone: +49 (0) 40 7410 53612
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)
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)
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.
Fig. 1. Purinergic signaling: During inflammation, injured cells release the nucleotides NAD and ATP as danger signals which alert immune cells 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.
Fig. 2. Antibodies induced by genetic immunization recognize native proteins, antibodies induced by peptide immunization recognize denatured proteins.
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.
Fig. 3. Schematic diagram of conventional antibodies and of heavy chain only antibodies made by llamas, dromedaries and other camelids.
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.
Fig. 4. Schematic diagram of the engineered bacterial artificial chromose (BAC-V03) used to generate nanobody-producing transgenic mice.
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.
Fig. 5. ADP-ribosylation is a reversible posttranslational modification regulating protein functions.
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.
Nanobodies as modulators of inflammation: potential applications for acute brain injury.
Rissiek B, Koch-Nolte F, Magnus T.
Front Cell Neurosci. 2014 Oct 21;8:344.
The art of blocking ARTs: Nanobodies as experimental and therapeutic tools to block mammalian and toxin ADP-ribosyltransferases.
Menzel S, Rissiek B, Haag F, Goldbaum F, Koch-Nolte F.
FEBS J. 2013 Aug;280(15):3543-50.
Extracellular NAD(+): a danger signal hindering regulatory T cells.
Adriouch S, Haag F, Boyer O, Seman M, Koch-Nolte F.
Microbes Infect. 2012 Nov;14(14):1284-92.
Compartmentation of NAD+-dependent signalling.
Koch-Nolte F, Fischer S, Haag F, Ziegler M.
FEBS Lett. 2011 Jun 6;585(11):1651-6.
Toward a unified nomenclature for mammalian ADP-ribosyltransferases.
Hottiger MO, Hassa PO, Lüscher B, Schüler H, Koch-Nolte F.
Trends Biochem Sci. 2010 Apr;35(4):208-19.
Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.
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.
Med Microbiol Immunol. 2009 Aug;198(3):157-74.
Emerging roles of NAD+ and its metabolites in cell signaling.
Koch-Nolte F, Haag F, Guse AH, Lund F, Ziegler M.
Sci Signal. 2009 Feb 10;2(57):mr1.
Mammalian ADP-ribosyltransferases and ADP-ribosylhydrolases.
Koch-Nolte F, Kernstock S, Mueller-Dieckmann C, Weiss MS, Haag F.
Front Biosci. 2008 May 1;13:6716-29.
Extracellular NAD and ATP: Partners in immune cell modulation.
Haag F, Adriouch S, Braß A, Jung C, Möller S, Scheuplein F, Bannas P, Seman M, Koch-Nolte F.
Purinergic Signal. 2007 Mar;3(1-2):71-81.
Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation.
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.
Sci Transl Med. 2016 Nov 23;8(366):366ra162.
Immuno-targeting the multifunctional CD38 using nanobody.
Li T, Qi S, Unger M, Hou YN, Deng QW, Liu J, Lam CM, Wang XW, Xin D, Zhang P, Koch-Nolte F, Hao Q, Zhang H, Lee HC, Zhao YJ.
Sci Rep. 2016 Jun 2;6:27055.
Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto-ADP-Ribosyltransferase ARTC2.2.
Menzel S, Rissiek B, Bannas P, Jakoby T, Miksiewicz M, Schwarz N, Nissen M, Haag F, Tholey A, Koch-Nolte F.
J Immunol. 2015 Sep 1;195(5):2057-66.
Tuning IL-2 signaling by ADP-ribosylation of CD25.
Teege S, Hann A, Miksiewicz M, MacMillan C, Rissiek B, Buck F, Menzel S, Nissen M, Bannas P, Haag F, Boyer O, Seman M, Adriouch S, Koch-Nolte F.
Sci Rep. 2015 Mar 10;5:8959.
Selection of Nanobodies that Block the Enzymatic and Cytotoxic Activities of the Binary Clostridium Difficile Toxin CDT.
Unger M, Eichhoff AM, Schumacher L, Strysio M, Menzel S, Schwan C, Alzogaray V, Zylberman V, Seman M, Brandner J, Rohde H, Zhu K, Haag F, Mittrücker HW, Goldbaum F, Aktories K, Koch-Nolte F.
Sci Rep. 2015 Jan 19;5:7850.
Technical Advance: A new cell preparation strategy that greatly improves the yield of vital and functional Tregs and NKT cells.
Rissiek B, Danquah W, Haag F, Koch-Nolte F.
J Leukoc Biol. 2014 Mar;95(3):543-9.
Alternative splicing of the N-terminal cytosolic and transmembrane domains of P2X7 controls gating of the ion channel by ADP-ribosylation.
Schwarz N, Drouot L, Nicke A, Fliegert R, Boyer O, Guse AH, Haag F, Adriouch S, Koch-Nolte F.
PLoS One. 2012;7(7):e41269.
Single-domain llama antibodies as specific intracellular inhibitors of SpvB, the actin ADP-ribosylating toxin of Salmonella typhimurium.
Alzogaray V, Danquah W, Aguirre A, Urrutia M, Berguer P, García Véscovi E, Haag F, Koch-Nolte F, Goldbaum FA.
FASEB J. 2011 Feb;25(2):526-34.
Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway.
Hubert S, Rissiek B, Klages K, Huehn J, Sparwasser T, Haag F, Koch-Nolte F, Boyer O, Seman M, Adriouch S.
J Exp Med. 2010 Nov 22;207(12):2561-8.
NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells.
Scheuplein F, Schwarz N, Adriouch S, Krebs C, Bannas P, Rissiek B, Seman M, Haag F, Koch-Nolte F.
J Immunol. 2009 Mar 1;182(5):2898-908.
Activation of the P2X7 ion channel by soluble and covalently bound ligands.
Schwarz N, Fliegert R, Adriouch S, Seman M, Guse AH, Haag F, Koch-Nolte F.
Purinergic Signal. 2009 Jun;5(2):139-49.
ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site.
Adriouch S, Bannas P, Schwarz N, Fliegert R, Guse AH, Seman M, Haag F, Koch-Nolte F.
FASEB J. 2008 Mar;22(3):861-9.
Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix.
Niere M, Kernstock S, Koch-Nolte F, Ziegler M.
Mol Cell Biol. 2008 Jan;28(2):814-24.
Single domain antibodies from llama effectively and specifically block T cell ecto-ADP-ribosyltransferase ART2.2 in vivo.
Koch-Nolte F, Reyelt J, Schössow B, Schwarz N, Scheuplein F, Rothenburg S, Haag F, Alzogaray V, Cauerhff A, Goldbaum FA.
FASEB J. 2007 Nov;21(13):3490-8.
Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation.
Möller S, Jung C, Adriouch S, Dubberke G, Seyfried F, Seman M, Haag F, Koch-Nolte F.
Purinergic Signal. 2007 Sep;3(4):359-66.
NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo.
Adriouch S, Hubert S, Pechberty S, Koch-Nolte F, Haag F, Seman M.
J Immunol. 2007 Jul 1;179(1):186-94.
The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation.
Mueller-Dieckmann C, Kernstock S, Lisurek M, von Kries JP, Haag F, Weiss MS, Koch-Nolte F.
Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):15026-31.
Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation.
Koch-Nolte F, Glowacki G, Bannas P, Braasch F, Dubberke G, Ortolan E, Funaro A, Malavasi F, Haag F.
Cell Immunol. 2005 Jul-Aug;236(1-2):66-71.
In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs).
Otto H, Reche PA, Bazan F, Dittmar K, Haag F, Koch-Nolte F.
BMC Genomics. 2005 Oct 4;6:139.
Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization.
Adriouch S, Dubberke G, Diessenbacher P, Rassendren F, Seman M, Haag F, Koch-Nolte F.
Cell Immunol. 2005 Jul-Aug;236(1-2):72-7.
CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins.
Krebs C, Adriouch S, Braasch F, Koestner W, Leiter EH, Seman M, Lund FE, Oppenheimer N, Haag F, Koch-Nolte F.
J Immunol. 2005 Mar 15;174(6):3298-305.
Activity and specificity of toxin-related mouse T cell ecto-ADP-ribosyltransferase ART2.2 depends on its association with lipid rafts.
Bannas P, Adriouch S, Kahl S, Braasch F, Haag F, Koch-Nolte F.
Blood. 2005 May 1;105(9):3663-70.
NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor.
Seman M, Adriouch S, Scheuplein F, Krebs C, Freese D, Glowacki G, Deterre P, Haag F, Koch-Nolte F.
Immunity. 2003 Oct;19(4):571-82.
Landesforschungsförderung Hamburg: Konsortium „ReAd Me! Regulatorische Adeninnukleotide auf Membranoberflächen: quantitative Bestimmung und Modulation von Zellfunktionen“ (Project 2: Prof. F. Nolte and Prof. F.Haag)
DFG: Moleculare mechanisms and function of P2X7 ion-channel gating in T cells. (Prof. F. Koch-Nolte/Prof. E. Tolosa)
DFG: SFB 1192 Immune-mediated glomerular diseases (Project B5: “Purinergic receptors and nanobody-based treatment strategies in glomerulonephritis”)
Prof. Eva Tolosa, Anna Gieras, Anne Rissiek, Arnau Serracant-Prat, Christina Gehbauer, Kati Tillack, Laura Glau, Manuela Kolster, Nora Kersten, Romy Hackbusch
The impact of pre- and postnatal medical interventions on the developing immune system
The thymus, a primary lymphatic organ, is essential for T cell development and establishment of tolerance in order to avoid autoimmunity later in life. Around birth, thymic function is at its peak and therefore, events affecting the thymus during this sensitive period may have serious consequences for the child’s immunity.
Recent findings suggest that impairment of the thymus function during this highly vulnerable time around birth might increase the risk for autoimmune and chronic diseases like type I diabetes, allergies or asthma, later in life. Interestingly, the prevalence of autoimmune and chronic diseases increased dramatically during the past decades in industrialized countries. This trend can’t be explained exclusively by genetic factors, indicating that early-life environmental factors play a crucial role in shaping immune maturation.
Our research activities focus on the thymus and the development of the immune system, particularly the impact of early-life medical interventions (e.g. antenatal corticosteroid treatment and thymectomy) on the development of autoimmunity and other chronic diseases.
Research project: The impact of pre- and postnatal medical interventions on the developing immune system
Characterization of the adenine-metabolising ectoenzymes and purinergic rezeptors
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 on different cell types.
High pericellular concentrations of ATP trigger P2X receptor activation. Binding of ATP to the nucleotide-gated ion channel P2X7 results in influx of Ca2+, leading to inflammasome activation and release of IL-1β and IL-18, activation of metalloproteases and cell death, altogether contributing to an 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 plan to use 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.
Metabolism of extracellular ATP to Adenosin
Effector and regulatory subsets
Interleukin 17, Produced by γδ T Cells, Contributes to Hepatic Inflammation in a Mouse Model of Biliary Atresia and Is Increased in Livers of Patients. Klemann C, Schröder A, Dreier A, Möhn N, Dippel S, Winterberg T, Wilde A, Yu Y, Thorenz A, Gueler F, Jörns A, Tolosa E, Leonhardt J, Haas JD, Prinz I, Vieten G, Petersen C, Kuebler JF. Gastroenterology. 2015 Sep 25. pii: S0016-5085(15)01352-9.
The expression of CD39 on regulatory T cells is genetically driven and further upregulated at sites of inflammation. Rissiek A, Baumann I, Cuapio A, Mautner A, Kolster M, Arck PC, Dodge-Khatami A, Mittrücker HW, Koch-Nolte F, Haag F, Tolosa E. J Autoimmun. 2015 Apr;58:12-20.
Babies Galore; or recent findings and future perspectives of pregnancy cohorts with a focus on immunity. Hartwig I, Diemert A, Tolosa E, Hecher K, Arck P. J Reprod Immunol. 2015 Apr;108:6-11.
Mannose 6 phosphorylation of lysosomal enzymes controls B cell functions. Otomo T, Schweizer M, Kollmann K, Schumacher V, Muschol N, Tolosa E, Mittrücker HW, Braulke T. J Cell Biol. 2015 Jan 19;208(2):171-80.
Rissiek A*, Schulze C*, Bacher U*, Schieferdecker A, Thiele B, Jacholkowski A, Flammiger A, Horn C, Haag F, Tiegs G, Zirlik K, Trepel M, Tolosa E, Binder M. Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia. Int J Cancer. 2014 Nov 15;135(10):2370-9.
Diepenbruck I, Much CC, Krumbholz A, Kolster M, Thieme R, Thieme D, Solano ME, Arck PC, Tolosa E. Effect of prenatal steroid treatment on the developing immune system. J Mol Med (Berl). 2013; 91:1293-1302.
Swaminathan B, Cuapio A, Alloza I, Matesanz F, Alcina A, García-Barcina M, Fedetz M, Fernández O, Izquierdo I, Órpez T, Pinto-Medel MJ, Otaegui D, Olascoaga J, Urcelay E, Ortiz MA, Arroyo R, Oksen-berg JR, Antigüedad A, Tolosa E, Vandenbroeck K. Multiple sclerosis-associated CD6 haplotype R225W-A257V modifies both CD6 expression in CD4+ and CD8+ naïve T cells and IFN-g production. PLoS One 2013 Apr 24;8(4):e62376.
Gelderblom M, Weymar A, Bernreuther C, Velden J, Arunachalam P, Steinbach K, Orthey E, Arumugam TV, Leypoldt F, Simova O, Thom V, Friese MA, Prinz I, Hölscher C, Glatzel M, Korn T, Gerloff C, Tolosa E, Magnus T. Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke. Blood. 2012; 120:3793-802.
Brucklacher-Waldert V, Stuerner K, Kolster M, Wolthausen J, Tolosa E. Th17 cells display high pathogenic potential and are increased in the CSF of multiple sclerosis patients during relapse. Brain 2009; 132:3329-3341 Brucklacher-Waldert V, Steinbach K, Kolster M, Lioznov M, Hoelscher C, Tolosa E. Phenotypical and functional characterization of human Th17 cells unambiguously identified by surface IL-17A expression. J Immunol. 2009; 183:5494-501.
Luther C, Stoeckle C*, Adamopoulou*, Brucklacher-Waldert V, Rosenkranz D, Stoltze L, Lauer S, Poeschel S, Melms A, Tolosa E. Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients. J Immunol 2009; 183:841-848
Gelderblom M*, Leypoldt F*, Steinbach K*, Behrens D, Choe CU, Siler D, Arumugam TV, Orthey E, Gerloff C, Tolosa E, Magnus T. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke 2009 40(5):1849-1857
Feger U*, Tolosa E*, Yu-Hwa H*, Biedermann T, Melms A, Wiendl H. HLA-G expression defines a novel regulatory T cell subset present in human peripheral blood and sites of neuroinflammation. Blood 2007; 110:568-577
Feger U, Luther C, Schreiner B, Pöschel S, Melms A, Tolosa E*, Wiendl H*.The frequency of CD4+CD25+ T regulatory cells is increased in the CSF but not in the blood of MS patients. Clin exp Immunol 2007;147:412-418.
Luther C, Poeschel S, Varga M, Melms A, Tolosa E. Decreased frequency of intrathymic regulatory T cells in patients with myasthenia-associated thymoma. J Neuroimmunol 2005;164:124-128Tolosa E, Ashwell JD. Thymus-derived glucocorticoids and the regulation of antigen-specific T- cell development. Neuroimmunomodulation 1999;6:90-96
Tolosa E, King LB, Ashwell JD. Thymocyte glucocorticoid resistance alters positive selection and inhibits autoimmunity and lymphoproliferative disease in MRL-lpr/lpr mice. Immunity 1998;8:67-76
Antigen processing and presentation
Adamopoulou E, Tenzer S, Hillen N, Quecke P, Tietz S, Gebhardt M, Stevanovic S, Rammensee H-G, Tolosa E, Melms A,
Stoeckle C. Exploring the basis of central tolerance: what do dendritic cells pre-sent in the human thymus? Nature Communications 2013; 4:2039. doi: 10.1038/ncomms3039.
Stoeckle C, Quecke P, Rückrich T, Burster T, Reich M, Weber E, Kalbacher H, Driessen C, Melms A, Tolosa E. Cathepsin S dominates autoantigen processing in human thymic dendritic cells. J Autoimmunity 2012; 38:332-343.
Stoeckle C, Rota IA, Tolosa E, Melms A, Adamopoulou E. Isolation of myeloid dendritic cells and epithelial cells from human thymus.; J Vis Exp. 2013 Sep 19;(79):e50951. doi: 10.3791/50951.
Sevenich L, Hagemann S, Stoeckle C, Tolosa E, Peters C, Reinheckel T. Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice. Biochimie 2010; 92(11):1674-1680
Stoeckle C, Tolosa E. Antigen processing and presentation in MS. In: "Molecular Basis of MS -Research Trends. Part I: The immune system". Springer Berlin / Heidelberg (publisher). Series "Results and Problems in Cell Differentiation" Martin R, Lutterotti A (eds) 2009 Jul 7.
Stoeckle C, Gouttefangeas C, Hammer M, Weber E, Melms A, Tolosa E. Cathepsin W, expressed exclusively in CD8+ and NK cells, is secreted during target cell killing but is not essential for cytotoxicity in human CTLs. Exp Hematol. 2009; 37(2):266-75.
Viken MK, Sollid HD, Joner G, Dahl-Jørgensen K, Rønningen KS, Undlien DE, Flatø B, Selvaag AM, Førre Ø, Kvien TK, Thorsby E, Melms A, Tolosa E, Lie BA.Polymorphisms in the cathepsin L2 (CTSL2) gene show association with type 1 diabetes and early-onset myasthenia gravis. Human Immunol 2007; 68:748-755
Melms A, Luther C, Stoeckle C, Poeschel S, Schroth P, Varga M, Winehold W, Tolosa E. Thymus and myasthenia gravis: antigen processing in the human thymus and the consequences for the generation of autoreactive T cells. Acta Neurol Scand Suppl. 2006;183:12-13
Burster T, Beck A, Tolosa E, Schnorrer P, Reich M, Krauss M, Kalbacher H, Häring HU, Weber E, Overkleeft H, Driessen C. Differential processing of an autoantigen in lysosomes from human monocyte-derived and peripheral blood dendritic cells. J Immunol 2005;175:5940-5949
Stoeckle C, Burster T, Gnau V, Driessen C, Kalbacher H, Melms A, Tolosa E. Processing of MBP by human thymic and peripheral antigen presenting cells. Is processing regulated in patients with multiple sclerosis? In: Editore M, ed. Immunology 2004. Vol. 3. Bologna: Medimond; 2004:285-291.
Burster T, Beck A, Tolosa E, Marin-Esteban V, Rotzschke O, Falk K, Lautwein A, Reich M, et al. Cathepsin G, and not the asparagine-specific endoprotease, controls the processing of myelin basic protein in lysosomes from human B lymphocytes. J Immunol 2004;172:5495-5503
Tolosa E, Li W, Yasuda Y, Wienhold W, Denzin LK, Lautwein A, Driessen C, Schnorrer P, Weber E, Stevanovic S, Kurek R, Melms A, Bromme D. Cathepsin V is involved in the degradation of invariant chain in human thymus and is overexpressed in myasthenia gravis. J Clin Invest 2003;112:517-526
DFG: Moleculare mechanisms and function of P2X7 ion-channel gating in T cells. (Prof. F. Koch-Nolte/Prof. E. Tolosa)
Landesforschungsförderung Hamburg: Konsortium „ReAd Me! Regulatorische Adeninnukleotide auf Membranoberflächen: quantitative Bestimmung und Modulation von Zellfunktionen“ (Project 3: Prof. H.-W. Mittrücker and Prof. E. Tolosa)
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: KFO296 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))
Andrea Mautner (Research Assistant, Evotec)
Dr. med. Angelica Cuapio (Post-Doc, Medical University of Vienna, AG Hofer)
Denise Orozco (Clinical Research Associate, Novartis)
Dr. rer. nat. Ines Diepenbruck
Dr. med. Isabell Baumann (Post-Doc, Pulmonary and Critical Care Medicine Brigham and Women’s Hospital, Boston, AG Bruce D. Levy M.D.)
Mareike Bindszus (Institute of Molecular Health Sciences, ETH Zurich, AG Manfred Kopf)
Dr. rer. nat. Verena Brucklacher-Waldert (Post-Doc, Babraham Institut Cambridge, AG Marc Veldhoen)
Dr. med. Vivien Thom (Department of Neurology, UKE)
Signal Transduction and Clinical Immunology
Prof. Friedrich Haag, Enja Schneider, Felix Scherg, Gudrun Dubberke, Klaus Eric Kaschubowski, Sana Javed, Dr. Stephan Menzel
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).
Figure 1. Extracellular ATP acts on P2X and P2Y receptors on the cell surface. (Dr. rer. nat. Stephan Menzel, 2015)
Haag F, Menzel S, Javed S, Rissiek A, Adriouch S, Tolosa E, Koch-Nolte F. The multiple roles of ATP-gated P2(X) ion channels in T lymphocytes. Messenger 2015 (in press)
Rissiek B, Haag F, Boyer O, Koch-Nolte F, Adriouch S. P2X7 on Mouse T Cells: One Channel, Many Functions. Front Immunol,6:204 (2015)
Haag F, Buck F. Identification and analysis of ADP-ribosylated proteins. Curr Top Microbiol Immunol,384:33-50 (2015)
Laing S, Unger M, Koch-Nolte F, Haag F. ADP-ribosylation of arginine. Amino Acids,41:257-269 (2011)
Laing S, Koch-Nolte F, Haag F, Buck F. Strategies for the identification of arginine ADP-ribosylation sites. J Proteomics (2011)
Haag F, Adriouch S, Brass A, Jung C, Moller S, Scheuplein F, et al. Extracellular NAD and ATP: Partners in immune cell modulation. Purinergic Signal,3:71-81 (2007)
Seman M, Adriouch S, Haag F, Koch-Nolte F. Ecto-ADP-ribosyltransferases (ARTs): emerging actors in cell communication and signaling. Curr Med Chem,11:857-872 (2004)
Thiele HG, Haag F. The RT6 system of the rat: developmental, molecular and functional aspects. Immunol Rev,184:96-108 (2001)
Rothenburg S, Koch-Nolte F, Haag F. DNA methylation and Z-DNA formation as mediators of quantitative differences in the expression of alleles. Immunol Rev,184:286-298 (2001)
Haag F, Koch-Nolte F. Endogenous relatives of ADP-ribosylating bacterial toxins in mice and men: potential regulators of immune cell function. J Biol Regul Homeost Agents,12:53-62 (1998)
Teege S, Hann A, Miksiewicz M, MacMillan C, Rissiek B, Buck F, Menzel S, Nissen M, Bannas P, Haag F, Boyer O, Seman M, Adriouch S, Koch-Nolte F. Tuning IL-2 signaling by ADP-ribosylation of CD25. Sci Rep 5: 8959 (2015)
Rissiek A, Baumann I, Cuapio A, Mautner A, Kolster M, Arck PC, Dodge-Khatami A, Mittrucker HW, Koch-Nolte F, Haag F, Tolosa E. The expression of CD39 on regulatory T cells is genetically driven and further upregulated at sites of inflammation. J Autoimmun 58: 12-20 (2015)
Menzel S, Rissiek B, Bannas P, Jakoby T, Miksiewicz M, Schwarz N, Nissen M, Haag F, Tholey A, Koch-Nolte F. Nucleotide-Induced Membrane-Proximal Proteolysis Controls the Substrate Specificity of T Cell Ecto-ADP-Ribosyltransferase ARTC2.2. J Immunol 195: 2057-66 (2015)
Rissiek B, Danquah W, Haag F, Koch-Nolte F. Technical Advance: a new cell preparation strategy that greatly improves the yield of vital and functional Tregs and NKT cells. J Leukoc Biol 95: 543-9 (2014)
Hubert S, Rissiek B, Klages K, Huehn J, Sparwasser T, Haag F, Koch-Nolte F, Boyer O, Seman M, Adriouch S. Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway. J Exp Med 207: 2561-8 (2010)
Schwarz N, Fliegert R, Adriouch S, Seman M, Guse AH, Haag F, Koch-Nolte F. Activation of the P2X7 ion channel by soluble and covalently bound ligands. Purinergic Signal 5: 139-49 (2009)
Hong S, Schwarz N, Brass A, Seman M, Haag F, Koch-Nolte F, Schilling WP, Dubyak GR. Differential regulation of P2X7 receptor activation by extracellular nicotinamide adenine dinucleotide and ecto-ADP-ribosyltransferases in murine macrophages and T cells. J Immunol 183: 578-92 (2009)
Adriouch S, Scheuplein F, Bahring R, Seman M, Boyer O, Koch-Nolte F, Haag F. Characterisation of the R276A gain-of-function mutation in the ectodomain of murine P2X7. Purinergic Signal 5: 151-61 (2009)
Seman M, Adriouch S, Scheuplein F, Krebs C, Freese D, Glowacki G, Deterre P, Haag F, Koch-Nolte F. NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor. Immunity 19: 571-82 (2003)
Adriouch S, Dox C, Welge V, Seman M, Koch-Nolte F, Haag F. Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. J Immunol 169: 4108-12 (2002)
Schmidt T, Paust HJ, Krebs CF, Turner JE, Kaffke A, Bennstein SB, Koyro T, Peters A, Velden J, Hunemorder S, Haag F, Steinmetz OM, Mittrucker HW, Stahl RA, Panzer U. Function of the Th17/interleukin-17A immune response in murine lupus nephritis. Arthritis Rheumatol 67: 475-87 (2015)
Pischke S, Polywka S, Haag F, Iking-Konert C, Sterneck M, Lutgehetmann M, Dammermann W, Luth S, Schirmer JH. Association of hepatitis E virus and essential cryoglobulinemia? J Clin Virol 67: 23-4 (2015)
Rissiek A, Schulze C, Bacher U, Schieferdecker A, Thiele B, Jacholkowski A, Flammiger A, Horn C, Haag F, Tiegs G, Zirlik K, Trepel M, Tolosa E, Binder M. Multidimensional scaling analysis identifies pathological and prognostically relevant profiles of circulating T-cells in chronic lymphocytic leukemia. Int J Cancer 135: 2370-9 (2014)
Luetkens T, Kobold S, Cao Y, Ristic M, Schilling G, Tams S, Bartels BM, Templin J, Bartels K, Hildebrandt Y, Yousef S, Marx A, Haag F, Bokemeyer C, Kroger N, Atanackovic D. Functional autoantibodies against SSX-2 and NY-ESO-1 in multiple myeloma patients after allogeneic stem cell transplantation. Cancer Immunol Immunother 63: 1151-62 (2014)
Bogner S, Bernreuther C, Matschke J, Barrera-Ocampo A, Sepulveda-Falla D, Leypoldt F, Magnus T, Haag F, Bergmann M, Bruck W, Vogelgesang S, Glatzel M. Immune activation in amyloid-beta-related angiitis correlates with decreased parenchymal amyloid-beta plaque load. Neurodegener Dis 13: 38-44 (2014)
Briem-Richter A, Leuschner A, Krieger T, Grabhorn E, Fischer L, Nashan B, Haag F, Ganschow R. Peripheral blood biomarkers for the characterization of alloimmune reactivity after pediatric liver transplantation. Pediatr Transplant 17: 757-64 (2013)
Rahlff J, Trusch M, Haag F, Bacher U, Horst A, Schluter H, Binder M. Antigen-specificity of oligoclonal abnormal protein bands in multiple myeloma after allogeneic stem cell transplantation. Cancer Immunol Immunother 61: 1639-51 (2012)
Lindqvist M, van Lunzen J, Soghoian DZ, Kuhl BD, Ranasinghe S, Kranias G, Flanders MD, Cutler S, Yudanin N, Muller MI, Davis I, Farber D, Hartjen P, Haag F, Alter G, Schulze zur Wiesch J, Streeck H. Expansion of HIV-specific T follicular helper cells in chronic HIV infection. J Clin Invest 122: 3271-80 (2012)
Panzer U, Steinmetz OM, Paust HJ, Meyer-Schwesinger C, Peters A, Turner JE, Zahner G, Heymann F, Kurts C, Hopfer H, Helmchen U, Haag F, Schneider A, Stahl RA. Chemokine receptor CXCR3 mediates T cell recruitment and tissue injury in nephrotoxic nephritis in mice. J Am Soc Nephrol 18: 2071-84 (2007)
Stellbrink HJ, van Lunzen J, Westby M, O'Sullivan E, Schneider C, Adam A, Weitner L, Kuhlmann B, Hoffmann C, Fenske S, Aries PS, Degen O, Eggers C, Schmidt T, Paust HJ, Krebs CF, Turner JE, Kaffke A, Bennstein SB, Koyro T, Peters A, Velden J, Hunemorder S, Haag F, Steinmetz OM, Mittrucker HW, Stahl RA, Panzer U. Function of the Th17/interleukin-17A immune response in murine lupus nephritis. Arthritis Rheumatol 67: 475-87 (2015)
DFG: The role of the nucleotide-gated cation channels P2X7 and TRPM2 for TCR signalling
Landesforschungsförderung Hamburg: Konsortium „ReAd Me! Regulatorische Adeninnukleotide auf Membranoberflächen: quantitative Bestimmung und Modulation von Zellfunktionen“ (Project 2: Prof. F. Nolte and Prof. F.Haag)
The section „Infection Immunology“ headed by Prof. Dr. Hans-Willi Mittrücker is focused on the regulation of immune responses against pathogens and on the function of T cells in autoimmune kidney diseases.
Prof. Dr. Hans-Willi Mittrücker, Dr. Friederike Raczkowski, Stefanie Ahrens, Karsten Lücke, Alexandra Hierweger, Daniel Reimes, Moritz Link, Johanna Schmid
CD4 and CD8 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 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.
Function and regulation of T cells during infection with intracellular bacteria
Heesch K, Raczkowski F, Schumacher V, Panzer U, Mittrücker H-W. The function of the chemokine receptor CXCR6 in the T-cell response of mice against Listeria monocytogenes. PLoS One. 2014. 9:e977019.
Mittrücker H-W, Seidel D, Bland PW, Zarzycka A, Kaufmann SHE, Visekruna A, Steinhoff U. Lack of microbiota reduces innate responses and enhances adaptive immunity against Listeria monocytogenes infection. Eur J Immunol. 2014. 44:1710-1715.
Mittrücker H-W, Steinhoff U, Köhler A, Krause M, Lazar D, Mex P, Miekley D, Kaufmann SHE. Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis. Proc Natl Acad Sci USA. 2007. 104:12434-12439.
Kursar M, Höpken UE, Koch M, Köhler A, Lipp M, Kaufmann SHE, Mittrücker H-W. Differential requirements for the chemokine receptor CCR7 in T-cell activation during Listeria monocytogenes infection. J Exp Med. 2005. 201:1447-1457.
Kursar M, Bonhagen K, Fensterle J, Köhler A, Hurwitz R, Kamradt T, Kaufmann SHE, Mittrücker H-W. Regulatory CD4+ CD25+ T cells restrict memory CD8+ T cell responses. J Exp Med. 2002. 196:1585-1592.
Mittrücker H-W, Shahinian A, Bouchard D, Kündig TM, Mak TW. Induction of unresponsiveness and impaired T cell expansion by staphylococcal enterotoxin B in CD28-deficient mice. J Exp Med. 1996. 183:2481-2488.
Interferon Regulatory Factor 4 (IRF4)
Raczkowski F, Ritter J, Heesch K, Schumacher V, Guralnik A, Höcker L, Raifer H, Klein M, Bopp T, Harb H, Kesper DA, Pfefferle PI, Grusdat M, Lang PA, Mittrücker H-W*, Huber M*. The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells. Proc Natl Acad Sci USA. 2013. 110:15019-15124. (*shared last authorship)
Mittrücker H-W, Matsuyama T, Grossman A, Kündig TM, Potter J, Shahinian A, Wakeham A, Patterson B, Ohashi PS, Mak TW. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science. 1997. 275:540-543.
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. TH1 and TH17 cells promote crescent formation in experimental autoimmune glomerulonephritis. J Pathol. 2015. 237:62-71.
Hopfer H, Hünemörder S, Treder J, Turner JE, Paust HJ, Meyer-Schwesinger C, Hopfer U, Sachs M, Peters A, Bucher-Kocaoglu B, Ahrens S, Panzer U, Mittrücker H-W. Glomerulopathy induced by immunization with a peptide derived from the Goodpasture antigen α3IV-NC1. J Immunol. 2015. 194:3646-3655.
Hopfer H, Holzer J, Hünemörder S, Paust HJ, Sachs M, Meyer-Schwesinger C, Turner JE, Panzer U, Mittrücker H-W. Characterization of the renal CD4+ T-cell response in experimental autoimmune glomerulonephritis. Kidney Int. 2012. 82:60-71.
Yan I, Schwarz J, Lücke K, Schumacher N, Schumacher V, Schmidt S, Rabe B, Saftig P, Donners M, Rose-John S, Mittrücker H-W*. Chalaris A*. ADAM17 controls IL-6 signaling by cleavage of the murine IL-6Rα from the cell surface of leukocytes during inflammatory responses. J. Leukoc. Biol. 2016. In press. (*shared last authorship)
Hoge J, Yan I, Jänner N, Schumacher V, Chalaris A, Steinmetz OM, Engel DR, Scheller J, Rose-John S, Mittrücker H-W. IL-6 controls the innate immune response against Listeria monocytogenes via classical IL-6 signaling. J Immunol. 2013. 190:703-711.
Adenosine nucleotides, ectoenzymes and purinergic receptors
Schneider M, Schumacher V, Lischke T, Lücke K, Meyer-Schwesinger C, Velden J, Koch-Nolte F, Mittrücker H-W. CD38 is expressed on inflammatory cells of the intestine and promotes intestinal inflammation. PLoS One. 2015. 10:e0126007.
Lischke T, Heesch K, Schumacher V, Schneider M, Haag F, Koch-Nolte F, Mittrücker H-W. CD38 controls the innate immune response against Listeria monocytogenes. Infect Immun. 2013. 81:4091-4099.
Lischke T, Schumacher V, Wesolowski J, Hurwitz R, Haag F, Koch-Nolte F, Mittrücker H-W. CD8β ADP-ribosylation affects CD8+ T-cell function. Eur J Immunol. 2013. 43:1828-1838.
Heiss K, Jänner N, Mähnß B, Schumacher V, Koch-Nolte F, Haag F, Mittrücker H-W. High sensitivity of intestinal CD8+ T cells to nucleotides indicates P2X7 as a regulator for intestinal T cell responses. J Immunol. 2008. 181:3861-3869.
Landesforschungsförderung Hamburg: Konsortium „ReAd Me! Regulatorische Adeninnukleotide auf Membranoberflächen: quantitative Bestimmung und Modulation von Zellfunktionen“ (Project 3: Prof. H.-W. Mittrücker and Prof. E. Tolosa)
DFG: SFB 1192 „Immune-mediated glomerular diseases” Project A4: “Autoreactive T cell responses in glomerulonephritis”.
DFG: KFO 296 „Feto-maternal immune cross talk: Consequences for maternal and offspring’s health”. Teilprojekt 2: “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 “Leberentzündung: Infektion, Regulation und Konsequenzen”. Project A3: “Regulation der T-Zellantwort im Verlauf von bakteriellen Leberinfektionen”
DFG: KFO 228 “Immunopathogenesis and therapy of glomerulonephritis” Project A3. “Role of different T cell subsets in anti-GBM-glomerulonephritis”
Internal partners (UKE)
Ulf Panzer, Oliver Steinmetz, Rolf A.K. Stahl III. Department of Internal Medicine
Petra Arck, Department of Obstetrics and Fetal Medicine
Samuel Huber, I. Department of Internal Medicine
Gisa Tiegs, Department of Experimental Immunology and Hepatology
Thomas Braulke, Department of Pediatrics
Andreas Guse, Department of Biochemistry and Molecular Cell Biology
Helmut Hopfer, Institute for Pathology, University Hospital of Basel
Thomas Jacobs, Minka Breloer, Bernhard-Nocht Institute for Tropical Medicine, Hamburg
Magdalena Huber, Ulrich Steinhoff Michael Lohoff, Institute for Microbiology, University Marburg
Athena Chalaris, Stefan Rose-John, Institute for Biochemistry, University Kiel
Stefan H.E. Kaufmann, Max Planck Institute for Infection Biology, Berlin