Research Report 2011 2012 - Institut für Immunologie
Transcription
Research Report 2011 2012 - Institut für Immunologie
___________________________________________ Institut für Immunologie Campus Kiel Universitätsklinikum Schleswig-Holstein ______________________________________________________ Research Report 2011/2012 Contents Preface 3 Scientific Staff 5 Research Groups 1. Prof. Dr. Dieter Adam 7 2. Prof. Dr. Ottmar Janßen 17 2.1 Dr. Markus Lettau 29 3. Prof. Dr. Dietrich Kabelitz 33 4. Prof. Dr. Stefan Schütze 43 5. PD Dr. Daniela Wesch 57 6. Associated Group 71 7. Diagnostic Group 77 8. Outpatient Service 81 Appendix 1. Institute Seminars 2011 and 2012 - Invited Speakers 83 2. Scientific Events organized by Institute Members 84 3. Completed MD and PhD Theses 2011 and 2012 85 4. Awards 85 5. Additional Scientific Activities 86 6. Impactfactors and Grants (Summary 2011 and 2012) 88 7. Publications 2011 and 2012 88 1 Contact Institute of Immunology University Hospital Schleswig-Holstein Christian-Albrechts-Universität zu Kiel Michaelisstrasse 5 D-24105 Kiel Postal address: Institute of Immunology UKSH, Campus Kiel Arnold-Heller-Str. 3, Haus 17 24105 Kiel Germany phone: +49 (0) 431/597-3341 fax: +49 (0) 431/597-3335 email: office@immunologie.uni-kiel.de http: www.immunologie-kiel.uk-sh.de 2 Preface The 6th bi-annual Research Report of the Institute of Immunology covers the years 2011 and 2012. As of January 1st 2011, a major reorganization of the diagnostic laboratories of the University Hospital Schleswig-Holstein (UKSH) became effective, with major consequences for our institute. Except for the outpatient service for couples with infertility (where a lymphocyte immune therapy is performed in selected cases), all diagnostic services of the institute were outsourced to other institutions, including the tissue typing laboratory which in fact has been among the first HLA labs established in Germany. While these measures had a dramatic impact on the organization of our institute (including a drastic decrease of the number of “immunologists”), a major drawback is the displacement from direct contact with clinicians through the abrogation of diagnostic routine labs. This notwithstanding, the institute continues to contribute to major research alliances in the fields of inflammation research and oncology. We are major players in the CRC 877 “proteolysis as a regulatory event in pathophysiology” (speaker: Prof. S. Rose-John, Biochemical Institute) and the DFG-funded “Pancreas Cancer Consortium Kiel”. Further third party funding comes from the DFG-funded Clinical Research Group KFO170 “Wegener’s granulomatosis”, the Wilhelm-SanderStiftung, The German Cancer Foundation (Mildred-Scheel-Stiftung), and several private foundations. Furthermore, a number of individual DFG research grants have been aquired by institute members. The major research topics of the institute continue to be in the areas of signal transduction (with a focus on death receptors), molecular analysis of distinct cell death pathways, and characterization of the enigmatic γδ T-lymphocytes. The institute is responsible for teaching immunology to medical students and bachelor/master students in Biology, Biochemistry and Medical Life Sciences. This Research Report summarizes our scientific activities in the years 2011 and 2012. Please visit our homepage (www.immunologie-kiel.uk-sh.de) for further information, and do not hesitate to contact us if you have any further question. We always offer motivated master, MD and PhD student positions to work with us – just ask us. Last not least, I would like to thank again Birgit Schlenga for preparing this Report. Kiel, October 2013 Dieter Kabelitz 3 4 Scientific Staff Members 2011/2012 Scientists: Adam, Dieter, Prof. Dr. rer. nat. Adam, Sabine, Prof. Dr. rer. nat. Bertsch, Uwe, Dr. rer. nat. (DFG) Both, Christiane, Ärztin Edelmann, Bärbel, Dr. rer. nat. (DFG) Fritsch, Jürgen, Dr. rer. nat. (DFG) Heidebrecht, Hans-Jürgen, Dr. rer. nat. (associated) Janßen, Ottmar, Prof. Dr. rer. biol. hum. – Head of Molecular Immunology Kabelitz, Dietrich, Prof. Dr. med. - Director Kling, Christiane, Dr. med. Leptin, Tatiana, Ärztin Lettau, Marcus, Dr. rer. nat. (DFG) Marget, Matthias, Dr. rer. nat. Oberg, Hans-Heinrich, Dr. sc. hum. (DFG) Philipp, Stefan Dr. rer. nat. (DFG) Quabius, Elgar Susanne, Dr. phil. II (associated) Schütze, Stefan, Prof. Dr. rer. nat. – Vice Director Tchikov, Vladimir, Dr. rer. nat. (DFG) Wesch, Daniela, PD Dr. rer. nat. Winoto-Morbach, Supandi, Dr. rer. nat. Guest Scientists: Guranda Chitadze National Medical University Tbilisi, Tiflis, Georgia (DAAD, IRTG SFB 877) Shirin Kalyan University of British Columbia, Vancouver, Kanada, USA (Alexander-von-Humboldt Fellowship) 5 PhD Students: Bhat, Jaydeep, M.Sc. (DFG) Duygu, Disci (associated Technical Faculty, CAU) Fazio, Juliane, Dipl.-Biol. (DFG) Koop, Anja, Dipl.-Biochem. (Wilhelm-Sander-Foundation) Marischen, Lothar, Dipl.-Biochem. (DFG) Meyer, Tim, Dipl.-Biol. (DFG & Medical Faculty) Oberdörster, Henriette, Dipl.-Biol. (DFG) Peters, Christian, Dipl.-Biol. (Medical Faculty, BMWi ZIM) Puchert, Malte, Dipl.-Biol. (DFG) Philipp, Stephan, M.Sc. (Medical Faculty) Schönbeck, Benjamin, M.Sc. (DFG) Stephan, Mario, Dipl.-Biochem. (DFG) Sosna, Justyna, M.Sc. (DAAD) Voigt, Susann, M.Sc. (DFG) MD Students:s Chitadze, Guranda Jurike, Matthias Falkenstern, Valentina Klawitter, Jan Hecht, Annika Reinicke, Maike Hellmich, Isabel Welte, Stefan Diploma Students: Master Students: Lepenies, Inga Krause, Sarah Maric, Nenad Kammel, Anne Schröder, Alexandra Luzius, Anne Tartanikova, Anastasia Trittner, Katharina 6 1. Research Group D. Adam A Group Leader: B Lab Members: Prof. Dr. rer. nat. Dieter Adam Scientists: Dr. rer. nat. Stephan Philipp (Deutsche Krebshilfe, from 07/2012) Ph. D. Students: Dipl.-Biol. Malte Puchert (DFG, until 12/2011) Susann Voigt, M. Sc. (DFG) Justyna Sosna, M. Sc. (DAAD) Diploma Student: Katharina Trittner (until 08/2011) M.Sc. Student: Anne Luzius (02/2011 – 08/2011) Medical Student: Annika Hecht Technicians: Sabine Mathieu (DFG) Parvin Davarnia Visiting Researcher: Duygu Disci-Zayed (from 02/2012) C Research Report: C. 1. Signal transduction through the 55 kDa TNF receptor: molecular and functional characterization of components of the N-SMase pathway (DFG) Recently, we have identified the Polycomb Group Protein EED as the first known interaction partner of nSMase2, a phospholipase which has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage or combined pituitary hormone deficiency (CPHD). Although activation of nSMase2 by the pro-inflammatory cytokine tumor necrosis factor (TNF) has been described almost two decades ago, the underlying signaling pathway had remained unresolved. We have demonstrated that in yeast, the N-terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize/physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. 7 After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2 and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1. Continuing these studies, we have shown that integrins are essential components of the N-SMase pathway, isolated 31 additional potential interaction partners of nSMase2 and analyzed six of these candidates in more detail for a function within the N-SMase pathway or downstream of nSMase2. The colocalization of LAMR1, one of these candidates with nSMase2 is exemplarily shown in Fig. 1. Fig. 1. Colocalisation of endogenous LAMR1 and endogenous nSMase2 in TNF-treated Jurkat cells. Jurkat cells were treated with 100 ng/ml TNF for the indicated times. Colocalization of LAMR1 (green) and nSMase2 (red) is visible in the overlay (yellow). Nuclei were stained with DAPI (blue). Scale bar: 10 µm. 8 Independently, we had found that another component of the N-SMase pathway, the protein FAN, has as yet unknown functions in the regulation of lysosome size. We have continued our cooperations with several groups to elucidate this phenomenon at the molecular level and with regard to infection as well as the functionality of immune cells. C. 2. Proteolysis in the regulation of caspase-independent programmed cell death (DFG, SFB877, together with S. Schütze) In caspase-dependent apoptosis, proteolysis is the primary mechanism for initiation and execution of programmed cell death (PCD). In caspase-independent PCD (ciPCD), proteolysis appears to be equally important but the underlying mechanisms are only marginally understood. We therefore have continued our work to further define the role of proteolysis in the regulation of ciPCD by employing the system of death receptor-mediated programmed necrotic ciPCD (“necroptosis”) that we have been studying for the last years, in combination with immunomagnetic sorting and proteomic approaches. So far, we have detected several signalling components involved in the proteolytic regulation of necrotic ciPCD, identified a serine protease that appears to be crucial in this pathway (Fig. 2), and isolated as well as characterized some of their substrates. Fig. 2. Identification of a serine protease involved in ciPCD via 2D-PAGE separation. ciPCD was induced with TNF in combination with zVAD-fmk for 5h. Proteins were labeled with the fluorescent protease inhibitor FFCK. Red spots represent proteins after flamingo pink staining and green spots are the corresponding proteins labeled with FFCK. Green spots were picked and further analysed via MALDI-TOF/TOF. Out of the identified candidates, subsequent analyses have confirmed a serine protease as a crucial component of ciPCD. In a complementary approach, we have performed a small interfering (si)RNA screen of all 566 proteases encoded by the human genome and identified 16 proteases whose downregulation modulates ciPCD . As a further goal, we are currently clarifying how acid sphingomyelinase undergoes proteolytic maturation/activation during ciPCD. In future work, we will further characterize the identified candidates and define their precise role in ciPCD. 9 C. 3. Functions of ceramide as a mediator of caspase-independent programmed cell death (DAAD) In previous work, we had demonstrated that the sphingolipid ceramide represents a previously unknown key mediator in ciPCD elicited by death receptors such as TNF-R1 and mTRAILR2. In this project, we have further explored the associated signaling pathways, with a focus on the virtually uninvestigated mechanisms by which mTRAIL-R2 mediates ciPCD. The comparison between TNF- and TRAIL-induced ciPCD has revealed several differences between both cytokines. Most importantly, the lysosomal compartment is apparently not as central in TRAIL-induced ciPCD as it is in ciPCD induced by TNF. While mitochondrial factors like ROS (specifically H2O2 and O2•–), or early loss of ∆ψm do not play a role in either TRAIL- or TNF-mediated ciPCD, the anti-apoptotic protein Bcl-2 selectively protects from ciPCD mediated by TNF, but not by TRAIL. Furthermore, a rapid externalization of PS was not essential for ciPCD induced by TRAIL. We have shown that calpain or cathepsin proteases do not participate in TNF- or TRAIL-induced ciPCD. A comparative analysis of protease inhibitors in TRAIL- and TNF-induced ciPCD revealed that inhibition of TPCKdependent serine proteases under physiological conditions is responsible for suppression of both TNF- and TRAIL-induced ciPCD. The observed differences in ciPCD induced by TRAIL or by TNF suggest that both cytokines induce ciPCD by pathways that are initially similar, but which diverge further downstream. In the long term, a better understanding how (ceramide-mediated) ciPCD is elicited by death receptors may prove beneficial not only for the therapy of tumors, but also for other illnesses which arise from deregulated cell death (e. g. autoimmunity or degenerative diseases). C. 4. Caspase-independent programmed cell death as a novel approach to eliminate tumor cells (Deutsche Krebshilfe, together with H. Kalthoff, Institute for Experimental Cancer Research, UKSH) The cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, the efficacy of treatment is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Here, ciPCD represents an alternative, molecularly distinct mode of programmed cell death that can likewise be elicited by TRAIL. The potential of TRAIL-induced ciPCD in tumor therapy is, however, almost completely uncharacterized. We have therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin. In our experiments, eight out of 14 tumor cell 10 lines were spontaneously sensitive to TRAIL-induced ciPCD. Furthermore, clonogenic survival was reduced in five sensitive and one resistant cell line that we tested. Moreover, TRAIL synergized with chemotherapeutics in killing tumor cell lines by ciPCD, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. At the molecular level, susceptibility/resistance of the investigated tumor cell lines to ciPCD was not determined by the cell surface expression of TRAIL receptors or by expression of the kinase RIPK1. Rather, our data point to expression of the related kinase RIPK3 as a primary determinant for resistance or susceptibility of the analyzed tumor cells, revealing RIPK3 as a potential predictive marker. Furthermore, our results extend the previously established role of ceramide as a pivotal downstream mediator of death receptor-induced ciPCD also to the tumor cell lines investigated in this project. C. 5. Analyses of the in-vivo function of the proinflammatory lipid ceramide in septic and hyperacute shock and in the lung (cooperation with S. Uhlig, Institute of Pharmacology and Toxicology, RWTH Aachen) As a consequence of a systemic inflammation reaction which is mostly caused by bacterial infection, septic shock is responsible for 40,000 fatalities in Germany alone and - being the third-most cause of death - represents a major problem for the public health care systems. As a central mediator of inflammation, the cytokine TNF is discussed as a causative factor in the pathogenesis of septic shock. In in-vivo experiments, we have previously observed an increased resistance of mice with genetic deficiencies in ceramide metabolism against hyperacute shock and also against bacterially induced sepsis. We have jointly investigated the pulmonary effects of TNF in mechanically ventilated wildtype and acid sphingomyelinase (ASMase)-deficient mice. Blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability were examined. TNF decreased blood pressure and increased heart rate in wild type mice. ASMase-/- mice were protected from the cardiovascular effects, but caspase inhibition had no influence. Although high levels of TNF were detected in the lung, no severe pulmonary inflammation or alteration of lung functions were found, suggesting that septic shock caused by high circulating levels of TNF is partially mediated by A-SMase. However, TNF alone is not sufficient to cause acute lung injury in ventilated mice. We have also investigated the role of A-SMase-deficiency in a murine model of allergic asthma. Mice deficient for A-SMase showed the typical characteristics of a lipid storage disease, called Niemann-Pick-disease, but their allergic inflammation was similar to that of 11 wild type mice, except for an increased neutrophil/eosinophil ratio. The major observation was reduced edema formation in A-SMase-deficient mice. We conclude that A-SMase contributes to allergic edema formation, a key feature of asthma. As an additional joint result, we have shown that lung endothelial Ca2+ and the permeability response to platelet-activating factor is mediated by ceramide generated by A-SMase and the ion channel TRPC6. C. 6. Identification of new interaction partners of coronin 1 (cooperation with N. Föger, Forschungszentrum Borstel) Coronins are an evolutionarily conserved family of actin-binding proteins with postulated functions in the regulation of actin-mediated cellular functions such as cell migration, cytokinesis, and cell growth. The WD-repeat protein coronin 1 is a leukocyte-specific member of the coronin protein family and essential for the survival of naïve T cells, most likely by acting as a central regulator of Ca2+-independent signaling. To obtain further insight into the molecular mechanisms of coronin 1-signaling, we have utilized the yeast two hybrid system and identified several novel interaction partners of coronin 1. C. 7. Nanoparticles in cancer research (cooperation with M. Elbahri, Institute for Materials Science, CAU Kiel) The main idea of this joint research project is to use the multifunctionality of nanoparticles to create an alternative cancer treatment which reduces damage to healthy tissue and destroys the cancerous clusters. Due to their high surface area/high activity, nanoparticles in their bare form can trigger cell death by inducing reactive oxygen species (ROS). ROS are important for biological functions and they are responsible for the regulation of many metabolic activities, however at elevated levels, they cause cellular damage and death. Along with their high metabolic action, cancer cells frequently have higher ROS levels, so any further increase in ROS will preferentially damage malignant cells. Independently, nanoparticles can also be used as carriers for conventional drugs. This way, therapeutics are transmitted to the preferred organ by active targeting of nanoparticles and encapsulated/attached chemotherapeutics can exert their cytotoxic effects precisely at the target location. Nanoparticle synthesis can be done either by "top down" approaches, e.g. lithography, milling or "bottom up" strategies including chemo-physical production techniques. High yield ligand-free green synthesis of nanoparticles at low cost is an objective in cancer nanotechnology. Therefore, a wet chemical synthesis is superior compared to other chemo-physical techniques. Here, an adapted wet 12 chemical approach based on the Leidenfrost phenomenon was used to generate diverse morphologies and different sizes of nanoparticles (Fig. 3). Fig. 3. Metal oxide nanoparticles at different morphologies and sizes. This was done because the activity of nanoparticles in tumor and nonmalignant cells varies with size and morphology (e.g. toxicity levels are inversely correlated to particle size, and spherical or rod shaped particles are more prone to interact with the biological media). First investigations with different sizes of spherical ZnO and ZnO2 nanoparticles have shown that the toxicity levels for both nonmalignant and tumor cells are associated with the particle size. Also, all cell lines used were found to be more sensitive to ZnO nanoparticles. In these initial experiments, we have achieved to destroy more than 80% of the tumor cells while preserving 70% of the nonmalignant cells even in the absence of any surface functionalization. Based on these promising, yet preliminary results, we will continue or joint studies using nanoparticles with alternative morphologies and further surface modifications in order to increase their efficiency. C. 8. Adhesion forces of Jurkat T lymphocytes on fibronectin measured by Atomic Force Microscopy (cooperation with C. Selhuber-Unkel, Institute for Materials Science, CAU Kiel) Force microscopy is a highly versatile method for characterizing the adhesion forces of living cells from the level of single molecule interactions and adhesion clusters to complete cells. In this project, we will jointly investigate T lymphocyte adhesion to endothelial cells. This adhesion is a crucial step in the mammalian inflammatory response, and it is still not understood how the fibronectin layer on endothelial cells guides and participates in T 13 lymphocyte adhesion. In order to obtain further insight into this process, we will use atomic force microscopy (Fig. 4) to measure the adhesion strength and dynamics of T-lymphocytes (Jurkat E6-1) to fibronectin coated surfaces under various conditions of stimulation. Fig. 4. Atomic Force Microscopy. Left panel: Sketch of the AFM measurement principle. A cell is attached to a micron-sized cantilever and brought into contact with a biofunctionalized surface. The cantilever is illuminated by a laser and the reflected laser light is detected by a quadrant photodiode. The deflection of the laser is related to the deformation of the cantilever and therefore also to the applied force. Right panel: Jurkat cell (arrow) attached to the cantilever. Scale bar: 50 µM. D Publications 2011 Nguyen XH*, Lang PA*, Lang KS*, Adam D*, Fattakhova G*, Föger N, Kamal MA, Prilla P, Mathieu S, Wagner C, Mak T, Chan AC, Lee KH. Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination. Blood 118: 598-608, 2011. *equal contribution. 2012 Durchfort N, Verhoef S, Vaughn MB, Shrestha R, Adam D, Kaplan J, Ward DM. The enlarged lysosomes in beige j cells result from decreased lysosome fission and not increased lysosome fusion. Traffic 13:108-119, 2012. Nguyen XH, Fattakhova G, Lang PA, Lang KS, Adam D, Föger N, Lee KH. Antiapoptotic function of Toso (Faim3) in death receptor signaling. Blood 119:1790-1791, 2012. Samapati R, Yang Y, Yin J, Stoerger C, Arenz C, Dietrich A, Gudermann T, Adam D, Wu S, Freichel M, Flockerzi V, Uhlig S, Kuebler WM. Lung endothelial Ca2+ and permeability response to platelet-activating factor is mediated by acid sphingomyelinase and transient receptor potential classical 6. Am. J. Respir. Crit. Care Med.185:160-170, 2012. 14 E Grants E.1. Signaltransduktion durch den 55 kDa TNF-Rezeptor: Molekulare und funktionelle Charakterisierung von Komponenten des N-SMase Signalwegs, DFG, AD 133/4-1 166.750 € (2008-2012) E.2. Proteolysis in the regulation of caspase-independent programmed cell death DFG, SFB877/B2 (Adam, Schütze) 461.856 € (2010-2014) E.3. Caspase-unabhängiger nicht-apoptotischer programmierter Zelltod zur Eliminierung von Apoptose-resistenten Tumorzellen durch Kombinationstherapien mit TRAIL Deutsche Krebshilfe (Adam, Kalthoff) 302.800 € (2012-2015) 15 16 2. Research Group Molecular Immunology A Group Leader: Prof. Dr. rer. biol. hum. Ottmar Janßen B Lab Members: Scientists: Dr. rer. nat. Marcus Lettau (associated since 8/2011) Dr. rer. nat. Hans-Jürgen Heidebrecht (associated) Ph.D. Students: M.Sc. (Biotechnology) Stephan Phillip (until 06/2012) Dipl.-Biochem. Anja Koop (until 11/2012) M.Sc. (Biology) Benjamin J. Schönbeck (since 09/2010) Dipl.-Biol. Henriette Oberdoerster (since 09/2010) Diploma Students: Alexandra Schröder (until 11/2011) Nenad Maric (until 7/2012) Technicians: Alyn Beyer Gudrun Scherer Melanie Nebendahl Signe Valentin C Research Report C.1. Fas/FasL-costimulation in resting T cells Maren Paulsen (PhD student until 2010) had found earlier that FasL ligation by plate-bound but not soluble fusion proteins or monoclonal antibodies interferes with T cell activation at an early stage of signal induction (Paulsen et al., Int. Immunol. 2009). She also observed that Fas costimulation results in either a block or an enhancement of primary T cell activation. The simultaneous ligation of Fas by FasL-Fc fusion proteins or anti-Fas antibodies at high concentrations abrogated T cell activation by CD3/CD28 stimulation whereas ligation by agonistic anti-APO-1 antibodies at lower concentrations augmented proliferation. The inhibitory branch of Fas costimulation has meanwhile also been reported by Gudrun Strauss and colleagues. Maren Paulsen thus concentrated on the activatory branch of Fas costimulation and described the role of receptor (co-)internalisation, sustained MAP kinase activation, non-apoptotic caspase activation and the upregulation of anti-apoptotic proteins, cell cycle regulators and characteristic activation markers (Paulsen et al., Cell Death Differ, 17 2011). This novel aspect of a dose-dependent CD95 signal initiation was also put in the context of pro- and anti-apoptotic CD95 signaling in naïve and activated T lymphocytes (Fig. 1, Paulsen and Janssen, Cell Communication and Signaling, 2011). Fig. 1: Modulation of T cell responses through CD95 in naïve versus activated T cells. The activation state of a given T cell (population) defines the signal threshold for pro- or non-apoptotic CD95 signaling. At the next level, the signal strength passing through CD95 determines whether signal initiation results in cell death, survival, cell cycle arrest or enhanced proliferation. In naïve CD95-resistant T cells, CD95 acts as a potent costimulatory receptor that can transduce activatory or inhibitory signals, depending on the dose of CD95 agonists to modulate TCR/CD3-signal induction. Activated T cells are CD95-sensitive and undergo apoptosis when exposed to high concentrations of CD95L. In contrast, a weak CD95 stimulus (again below a certain threshold level) might induce survival signaling in the absence of detectable cell death (from Paulsen and Janssen, Cell Communication and Signaling, 2011). C.2. Proteome analysis of secretory lysosomes from T and NK cells For the intracellular storage and release of cytotoxic effector molecules, T and NK cells utilize a special lysosomal compartment that combines properties of conventional lysosomes and exocytotic vesicles. It was supposed that these “secretory lysosomes” contain soluble and membrane-associated cytotoxic effector molecules including perforin and granzymes, and carry the death factor FasL as an integral transmembrane component and marker protein. Previously, Hendrik Schmidt and Melanie Nebendahl established a protocol for the enrichment of such effector vesicles from in vitro expanded T and NK cell (Schmidt et al., BMC Immunology, 2009). With the help of Dr. Christoph Gelhaus (Zoological Institute of the CAU), they defined the protein content of organelles from leukemic NK cell lines and IL-2expanded healthy NK cells using 2D-DIGE technology (Schmidt et al., Proteomics, 2008). In addition, Hendrik Schmidt started to investigate T cell lysosomes using the same approach. The luminal proteome of FasL-containing secretory lysosomes of T cell blasts was 18 deciphered, presenting almost 400 different proteins (Schmidt et al., Cell Communication and Signaling, 2011). Surprisingly, it turned out that several of the characteristic effector molecules (i.e. granzymes, perforin, mature granulysin) were prominently associated with a distinct lysosomal entity that differed in density and morphology (Fig. 2) and therefore segregated to a distinct fraction of the density gradient. We also analyzed the proteome of this “granzyme fraction” and compared the two lysosomal fractions directly by 2D-DIGE and Western blotting. With this study, we provided first direct biochemical evidence for the existence of distinct effector vesicle populations in T lymphoblasts (Schmidt et al., J Proteome Res, 2011). Based on imaging analyses by different groups, our present hypothesis is that the different lysosomal entities somehow associate in larger multivesicular bodies. Fig. 2: Two distinct species of effector lysosomes in T cells. (A) Different abundance of individual proteins in individual fractions. Selected proteins with a higher abundance in fraction 2 are displayed as 3D images. (B) The results were verified by Western blotting for two different donors. (C) Other proteins were enriched in fraction 6 lysosomes. (D) Respective Western blot verification for fraction 6-associated proteins. Flotilin-1 was unchanged and therefore served as a loading control). (E, F) Organelle characteristics of fraction 2 and fraction 6 lysosomes analyzed by transmission electron microscopical inspection (modified from Schmidt et al. Journal of Proteome Research 2011) C.3. Characterization of the cancer/testis antigen CT45 Ki-A10, a monoclonal antibody generated by immunizing mice with lysates of the Hodgkin's lymphoma-derived cell line L428, detects CT45, a nuclear antigen of the CT-X (cancer testis antigen-X-chromosome-linked) family. Due to their restricted expression in male germ cells and certain tumors, CT antigens are regarded as promising targets for tumor therapy. CT45 was associated with a severe disease score in Hodgkin’s lymphoma and poor prognosis in 19 multiple myeloma. Sponsored by the Wilhelm-Sander-Foundation, Anja Koop and HansJürgen Heidebrecht down-regulated CT45 in Hodgkin’s lymphoma (L428) and fibrosarcoma (HT1080) cells using RNA interference. Having confirmed an efficient CT45 knock-down by immunofluorescence staining and Western blotting, the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells was addressed. Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, Hans-Jürgen Heidebrecht initially noted alterations in cell adherence after CT45 down-regulation and observed changes in the distribution of cytoskeleton-associated proteins by confocal imaging. Anja Koop was able to record changes in cell adherence using the xCelligence system and visualized altered migratory and invasive capacity of CT45 siRNA-treated cells in 3D migration and invasion assays (Fig. 3). She also found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. Taken together, these data provided first evidence for a cell biological function of CT45. Enhanced motility and invasiveness of CT45-positive cells could contribute to the higher degree of malignancy that has been associated with CT45-positive tumor cells. Fig. 3: CT45 affects cell invasion and migration. (A-C) Cell migration and cell invasion capacities of scrRNAor CT45 siRNA-treated HT1080 or L428 cells were analyzed in colorimetric or fluorometric transwell systems (A, D: HT1080 migration, B, E: HT1080 invasion, C: L428 migration). As a control, cell motility was inhibited with 2 µM Cytochalasin D. The data analysis revealed that cell migration of siRNA-treated HT1080 cells was down-regulated by 23%. Staining of cells collected from the lower chamber revealed that less siRNA-transfected and only very few cytochalasin D-treated cells had passed the membrane (D). The cell invasion assay of HT1080 (B, E) and the migration assay for L428 cells (C) revealed similar results. (from Koop et al., Cell Communication and Signaling, 2013). 20 C.4. Proteome analysis of cell adherence-mediating plasma membrane proteins on P. falciparum-infected human erythrocytes By exporting plasmodial proteins, the intraerythrocytic stage of Plasmodium falciparum massively alters the characteristics of its host cell. Although the physical, chemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action were not completely known. In cooperation with Prof. Dr. Matthias Leippe and Dr. Christoph Gelhaus from the Zoological Institute and initially funded be the Medical Faculty of the CAU, Stephan Philipp developed strategies and methods to determine and quantitate the proteome of this interphase compartment. He enriched membranes and membrane-associated proteins from uninfected and infected erythrocyte ghosts and separated the associated proteins by non-conventional 2D electrophoresis (16-BAC/SDS or CTAB/SDS) to directly identify the plasmodial proteins from membranes of infected erythrocyte (Fig. 4). Protein spots were analyzed by MALDITOF/TOF MS and annotated in respective 2-D master gels. By using this alternative 2-D approach, characteristic host cell membrane proteins and, more importantly, membraneassociated and exported plasmodial proteins were identified that might play a role in parasiteinduced host cell modulation (Philipp et al. Electrophoresis 2012). Fig. 4: Master gels derived from three independent experiments using membranes of saponin-treated non-infected RBC or of infected RBC that were freed from the intracellular parasite. The gels were stained by Flamingo Pink fluorescent stain. (A) RBC, 2-D-16-BAC/SDS-PAGE (B) RBC, 2-D-CTAB/SDSPAGE; (C) iRBC, 2-D-16-BAC/SDS-PAGE (D) iRBC, 2-D-CTAB/SDS-PAGE (Philipp et al. Electrophoresis 2012). The erythrocytic life cycle of P. falciparum is highly associated with the severe clinical symptoms of malaria. As mentioned, the parasite develops within human erythrocytes leading 21 to the disruption of the infected red blood cell prior to the start of a new cycle of erythrocyte infection. For the analysis of direct effects of Plasmodium for the host cells and for further analysis of the plasmodial proteins, the isolation of different blood stages would be favorable. Unfortunately, however, early stages could hardly be separated from late stages and noninfected red blood cells using conventional methods. Stephan Philipp stained iRBCs with different DNA-binding dyes and Hans-Heinrich Oberg analyzed these cells by flowcytometry (Fig. 5). Indeed, they were able to discriminate early stage and late-stage iRBCs from non-infected erythrocytes. In addition, they could isolate highly purified (> 98%) early stage iRBCs by FACS. In essence, the new method allows the isolation of viable ring stages of the malarial agent P. falciparum, and thus provides the basis for a detailed molecular investigation of different developmental stages of the parasite (Philipp et al. Cytometry Part A 2012). Fig. 5: Isolation of early stages of P. falciparum-infected erythrocytes (iRBCs). Cells were stained with Vybrant1 DyeCycleTM Violet stain and sorted on a FACSAria flow cytometer. (A) The histogram shows the fluorescence distribution of 100,000 analyzed cells. For sorting of early stage parasites, the initial gate G1 was subdivided into three smaller gates. (B) For each re-sort of the gates G1a,b,c, histogram and corresponding forward/side scatter dot plots for 5,000 cells are shown. (C) Cells that were sorted in G1c were analyzed by Giemsa staining (from Philipp et al. Cytometry Part A 2012). C.5. Proteolytic processing of FasL – Role of ADAM10 and associated molecules In project B4 of the CRC 877 (Proteolysis as a Regulatory Event in Pathophysiology), we followed up on our earlier observation that the “A Desintegrin and Metalloproteinase” 10 (ADAM10) regulates the surface appearance and function of FasL. The release of soluble FasL by ADAM10 shedding leaves N-terminal fragments (NTFs) in the cell membrane that are subsequently processed by intramembrane proteolysis through the signal peptide peptidase like protein 2a (SPPL2a) to release short intracellular domains (ICDs). In both cases, the 22 regulation of proteolysis and the fate and function of the generated fragments is only poorly understood (Fig. 6). Fig. 6: FasL (CD95L) processing by proteolysis. Upon release of soluble FasL by ADAM10 shedding Nterminal fragments (NTF) are left in the cell membrane. These membrane stubs are further processed by intramembrane proteolysis through SPPL2a to generate intracellular domains (ICD) that might be degraded or exert unknown functions within the cell (e.g. move to the nucleus to regulate transcription. To date, the regulation of proteolysis and the fate and function of the generated FasL fragments is only poorly understood. The same hold true for the role of FasL- and ADAM10-interacting SH3 domain proteins in this context. In order to get more information about the regulation of ADAM10 activity, Henriette Oberdoerster used two different approaches to identify proteins that might bind to the intracellular C-terminus of ADAM10: she performed pull-down analyses using the intracellular region of ADAM10 expressed as a GST fusion protein and a SH3 domain phage display screen (from Geneart) (Fig.7). Fig. 7: Putative ADAM10-interacting SH3 domains identified by phage display screening. Potential ADAM10 binding partners were precipitated with the intracellular region of human ADAM10 (aa 697-748) coupled to GST. Respective phage clones were isolated and sequenced according to the manufacterer’s protocol. Listed are only SH3 domains that did not interact with GST. 23 Precipitated proteins from pull-down experiments were identified by mass spectrometry in cooperation with Christoph Gelhaus from the Zoological Institute. SH3 domains binding to the ADAM10 intracellular region were identified by sequencing of respective phage clones. Although most of the putative interactors still have to be verified, we found several candidate binding partners that are in line with studies on related ADAM proteases. These include Src kinases (e.g. Lck) and adapter proteins such as Grb2 or PACSINs. The partial overlap of binding partners for ADAM10 and FasL could indicate a co-regulation of transport and surface appearance. To get insight into the regulation of ADAM proteases, in her diploma work, Alexandra Schröder analyzed the constitutive and inducible surface appearance of ADAM10 and ADAM17 on a variety of T cell and tumor cell lines. She demonstrated that ADAM10 is constitutively present at comparably high levels on the majority of the tested cell types(Fig. 8, left panel). Stimulation with phorbolester and calcium ionophore does not significantly alter the amount of surface ADAM10, except for a slight down-regulation on some T cell lines. Fig. 8: Activation-dependend surface presence of ADAM10 (left) and ADAM17 (right). Surface ADAMs were analyzed by flow cytometry. Constitutive and rather high surface ADAM10 on T cells and tumor cell lines is not significantly altered over time upon stimulation with TPA and ionomycin. In contrast, ADAM17 is induced on PHA blasts by re-stimulation. The ADAM17 transport to the cell surface is apparently depending on reorganization of the actin cytoskeleton since it can be inhibited by latrunculin and cytochalasin (red lines). Using FasL shedding as a readout for ADAM10 activity, Alexandra Schröder and Henriette Oberdoerster could aalso show that both PKC activation an calcium mobilization are required for the production of soluble FasL. In contrast to ADAM10, the closely related ADAM17 was 24 detected at fairly low levels on unstimulated cells. However, ADAM17 surface appearance on T cell blasts was rapidely induced by stimulation (Fig. 8, right panel). Since the inducible mobilization of ADAM17 was sensitive to inhibitors of actin filament formation, our findings suggest that ADAM17 but not ADAM10 might be prestored in a subcellular compartment that is transported to the cell surface in an activation- and actin-dependent manner. Interestingly, Henriette Oberdoerster meanwhile detected both ADAM proteases in lysosomal fractions prepared as described in C.2. Thus, it might be possible that ADAM proteases are stored intracellularly in a vesicular compartment. A so far unanswered question of CRC project is when and where ADAM10 and or SPPL2a meet their substrate, FasL. To address this, Henriette Oberdoerster and Gudrun Scherer prepared cellular fractions according to different protocols for the preparation of lipid rafts. To cut a long story short – from numerous experiments with mixed results due to different kits and protocols, the conclusion would be that ADAM10 and ADAM17 are not present in lipid rafts, whereas FasL is at least in part raft-associated. The localization of SPPL2a in raft or non-raft areas has not yet been addressed. The analyses are hampered by the fact that only a few antibodies are available and that it turned out to be rather impossible to detect endogenous proteins in preparations of activated T cells. C.6. Sheddome analysis for ADAM proteases In a cooperative project with Athena Chalaris and Stefan Rose-John (CRC 877, project A1), Benjamin Schönbeck is trying to decipher the ADAM17 sheddome as part of the Z2-project of the CRC. He is using a 2D-DIGE approach to compare murine embryonic fibroblasts (MEF) from wt and ADAM17 hypomorphic (ADAM17ex/ex) mice. However, the analysis of shed proteins in supernatants of the two MEF populations proved to be more difficult than initially anticipated. Most of the differentially abundant proteins were cytosolic proteins and not known or putative ADAM17 substrates. In a proof-of-principle experiment, we therefore compared wt and ADAM17ex/ex MEF cells that were both transfected with TNFalpha, the prototypic substrate of ADAM17 (=TACE for TNFalpha-converting enzyme), and we could detect the soluble TNF only in wt supernatants (Fig. 9). In addition, Benjamin Schönbeck adapted the CTAB-PAGE for membrane proteins and he is using biotinylation and WGA precipitation to enrich for surface or glycosylated proteins that might be shed by the protease. Besides using the MEF populations, Benjamin also compares human and murine cell populations (e.g. T cell blasts) with or without ADAM17 knock-down. Although the 25 identifications of putative ADAM17 substrates was so far rather limited, we consistently found several interesting proteins to be differentially abundant in wt and ADAM17ex/ex MEF cells. Several of these proteins (i.e. galectins, thioredoxins, peredoxins) have been implicated in the regulation of ADA17 activity or expression and are now subject of forther analyses. Fig. 9: TNFalpha is shed from wt but not from ADAM17ex/ex MEF. 2D-DIGE analysis of supernatants from wt or ADAM17ex/ex MEF cells transfected with TNFalpha. Soluble TNF is more or less exclusively (14,78 fold) present in wt supernatants. Thus, the 2D-DIGE approach is suitable to detect shed proteins in supernatants of suited cell populations. C.7. Cooperative 2D-DIGE projects The 2D-DIGE platform was established in 2006 as an integral part of the Molecular Immunology laboratory group. Several successful collaborations were completed by Dr. Hendrik Schmidt or Benjamin Schönbeck with the expert technical assistance of Melanie Nebendahl. The mass spectrometrical analyses for the published projects were performed in collaboration with Dr. Christoph Gelhaus and Prof. Dr. Matthias Leippe from the Zoological Institute of the CAU. Since most of the projects have meanwhile been published, we refer to the respective publications for more detailed information. • The natural anti-cancer compounds Rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2. (Polier G, et al. Chem Biol. 2012) 26 • Breakpoint Characterization of der(19)t(11;19)(q13;p13) in the Ovarian Cancer Cell Line SKOV-3. (Onkes W et al. Genes Chromosomes and Cancer 2013, published online: 30 JAN 2013 DOI: 10.1002/gcc.22048) D Publications 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Lucius R, Leippe M, Kabelitz D, Janssen O, Effector granules in human T lymphocytes: The luminal proteome of secretory lysosomes from human T cells. Cell Commun Signal 9:4, 2011 Paulsen M, Valentin S, Mathew B, Adam-Klages S, Bertsch U, Lavrik I, Krammer PH, Kabelitz D, Janssen O. Modulation of CD4+ T cell activation by CD95 costimulation. Cell Death Diff 18:619-631, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Leippe M, Lucius R, Janssen O. Effector granules in human T lymphocytes: Proteomic evidence for two distinct species of cytotoxic effector vesicles. J Proteome Res 10:1603–1620, 2011 Marischen L, Wesch D, Oberg H-H, Rosenstiel P, Trad A, Shomali M, Grötzinger J, Janssen O, Tchikov V, Schütze S, Kabelitz D. Functional expression of NOD2 in freshly isolated human peripheral blood γδ T-cells. Scand J Immunol 74:126-134, 2011 Lettau M, Paulsen M, Schmidt H, Janssen O. Insights into the molecular regulation of FasL (CD178) biology. Eur J Cell Biol 90:456-466, 2011 Paulsen M, Janssen O. Pro- and anti-apoptotic CD95 signaling in T cells. Cell Commun Signal, 9:7, 2011 2012 Louis-Dit-Sully C, Kubatzky KF, Lindquist JA, Blattner,C, Janssen O, Schamel WW. Signal transduction meets systems biology Cell Commun Signal, 10:11, 2012 Deng X, Hahne T, Schröder S, Redweik S, Nebija D, Schmidt H, Janssen O, Lachmann B, Wätzig H. The challenge to quantify proteins with charge trains due to isoforms or conformers. Electrophoresis 33:263-269, 2012 Philipp S, Jakoby T, Tholey A, Janssen O, Leippe M, Gelhaus C. Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2-D gel electrophoresis. Electrophoresis 33:1120-1128, 2012 Polier G, Neumann J, Thuaud F, Ribeiro N, Gelhaus C, Schmidt H, Giaisi M, Köhler R, Müller WW, Proksch P, Leippe M, Janssen O, Désaubry L, Krammer PH, Li-Weber M. The natural anti-cancer compounds Rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2. Chem Biol 19:1093-1104, 2012 Philipp S, Oberg H-H, Janssen O, Leippe M, Gelhaus C. Isolation of erythrocytes infected with viable early stages of Plasmodium falciparum by flow cytometry. Cytometry A 81:1048-1054, 2012 27 E Grants E.1. Function and prognostic relevance of the cancer/testis antigen CT45. Wilhelm-Sander Stiftung, Project 2008.055.1 (H. J. Heidebrecht, O. Janssen) 1x BAT IIa/50%, 20.000 € p.a (2009-2011) E.2. PCH proteins in lymphocytes. Medical Faculty, (O. Janssen) 65.000 € (2010-2011) E.3. Proteolysis-dependent posttranslational modifications in control of the death factor CD95L. DFG SFB877, Project B4, 1x BAT IIa (65%), 1x BATVb, 20.000 €/year (2010-2014) E.4. Proteome platform for the analysis of proteolysis-dependent protein modifications. DFG SFB877, Project Z2, (O. Janssen, M. Leippe, A. Tholey), 1x BAT IIa (50%, 50% basic support CAU), 2x BAT IIa (65%), 1 BAT IVb (basic support CAU), 30.000 €/year (2010-2014), 130.000 € equipment 28 2.1 Associated Research Project “Nck interaction partners” (Project leader M. Lettau) Research Report The prototypical adapter proteins Nck1 and Nck2 link receptor-associated tyrosine kinases with proteins regulating the actin cytoskeleton and are functionally associated with a plethora of different processes including the development and maintenance of tissue integrity, the activation and effector function of immune cells, but also malignant transformation and invasivity of tumour cells. Importantly, all these processes rely on changes of cell polarity, morphology and migration and can be regulated by Nck-associated protein complexes. In previous work of our group (diploma thesis of J. Pieper), we identified the adapter proteins ADAP Y595/651F/Nck1-EGFP ADAP Y651F/Nck1-EGFP ADAP Y595F/Nck1-EGFP ADAP WT/Nck1-EGFP ADAP WT/EGFP - ADAP Y595/651F/Nck1-EGFP ADAP Y651F/Nck1-EGFP ADAP Y595F/Nck1-EGFP ADAP WT/Nck1-EGFP ADAP WT/EGFP - ADAP and HS1 as Nck-interacting proteins. α-ADAP α-Nck IP: α-ADAP IP: α-Nck Fig. 1: The interaction of Nck with ADAP relies on the phosphorylation of Y595 and Y651. 293T cells were transiently transfected with EGFP-tagged Nck1, WT ADAP as well as point-mutated ADAP variants (Y595F, Y651F, Y595/651F). 18 hr after transfection the cells were treated with pervanadate, lysed and subjected to immuprecipitation with anti-Nck and anti-ADAP antibodies. Western blot of the immunoprecipitates with antiNck and anti-ADAP antibodies revealed that Nck coprecipitates with ADAP. Mutation of a single tyrosine residue dramatically decreases the association. The interaction is completely lost upon mutation of both Y595 and Y651. 29 Moreover, with the heterodimeric splicing factor SFPQ/NONO, we also described a nuclear binding partner for Nck. Further analyses revealed that endogenous Nck1/2 constitutively associates with ADAP in primary human T cells. Interestingly, tyrosine phosphorylation of ADAP is slightly enhanced upon T cell receptor stimulation and the association with Nck1/2 is increased concomitantly. In this scenario, the Nck1/2 SH2 domain selectively binds to the phosphorylated tyrosines Y595 and Y651 of ADAP (Fig. 1). Ongoing studies now address the functional relevance of this interaction in human T cells. We also verified the interaction of Nck1/2 with the adapter protein HS1 in primary human T cells. Again, the interaction is direct and mediated by the Nck SH2 domain and thus strictly relies on the phoyphorylation of HS1. Present analyses focus on the regulation of HS1 phosphorylation, the physiological role of HS1 in T cells and the function of its interaction with Nck. First analyses revealed that HS1 is recruited to the immunological synapse upon target cell encounter (Fig. 2). Interestingly, synapse-associated HS1 is phosphorylated on tyrosines Y378 and Y397 and both tyrosines display the consensus sequence for Nck SH2 binding. F-actin F-actin F-actin HS1 HS1 pY378 HS1 pY397 Overlay Overlay Overlay Fig. 2: HS1 is phosphorylated and recruited to the immunological synapse upon target cell encounter. Human T cell blasts were cultivated with superantigen-loaded B lymphoblastic cells for 30 min, fixed permeabilized and stained with anti-HS1 mAb and phospho-specific anti-HS1 Y378 and anti-HS1 Y397 mAb and Alexa Fluor 488-conjugated secondary Ab. F-Actin was labeled with Alexa Fluor 555-conjugated phalloidin and nuclei were visualized with DAPI. Scale bars represent 10 µm. 30 Of note, Nck1 and Nck2 are highly homologous especially within their SH interaction domains. Consequently, commercially available antibodies usually recognize both Nck1 and Nck2 and thus Nck-associated functions and interactions have not been clearly attributed to an individual Nck variant in most published studies. Thus, it is still an open question whether Nck1 and Nck2 functions and binding partners widely overlap or rather diverge. Given the diversity of published functions and interaction partners for Nck, the analysis of individual Nck variants is urgently needed. We thus generated Nck1 and Nck2-specific monoclonal antibodies raised against peptides corresponding to a heterogeneous linker region between Nck SH domains. We show that the antibodies are suitable and specific in IP/Western blot and immunofluoresce analysis (Fig. 3). Employing these new tools we now started to analyse the expression and functions of the individual Nck variants in human T cells. Nck1-EGFP Nck2-EGFP Nck1-EGFP Nck2-EGFP Nck1-EGFP Nck2-EGFP 71814 71814 9B3 9B3 280C10 280C10 Overlay Overlay Overlay Overlay Overlay Overlay Fig. 3: Nck variant specific antibodies selectively detect Nck1 and Nck2, respectively. 293T cells were transiently transfected with EGFP-tagged Nck1 or Nck2. 18 hr after transfection the cells were fixed, permeabilized and stained with the Nck1-specific mAb 71814, the Nck2-specific mAb 9B3 or the mAb 280C10 recognizing both Nck1 and Nck2 and Alexa Fluor 555-conjugated secondary antibodies. Nuclei were visualized with DAPI. Scale bars represent 10 µm. Publications Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Lucius R, Leippe M, Kabelitz D, Janssen O. Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells. Cell Commun Signal 9:4, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Lucius R, Leippe M, Kabelitz D, Janssen O. Effector granules in human T lymphocytes: proteomic evidence for two distinct species of cytotoxic effector vesicles. J Proteome Res 10:1603-20, 2011 31 Lettau M, Paulsen M, Schmidt H, Janssen O. Insights into the molecular regulation of FasL (CD178) biology (Review). Eur J Cell Biol 90:456-66, 2011 E Grants E.1. Bonus grant based on the junior project grant „Nck-Interaktionspartner“ (30.000 €, 01.01.2008- 31.12.2009, Med. Fak. CAU Kiel) Medical Faculty CAU 2012 20.000 € E.2. „Nck interaction partners: Molecular interactions and functional consequences“ Project DFG LE 2571/3-1 272.250 € 01.09.2011 – 31.08.2014 32 3. Research Group Kabelitz A Group Leader: B Lab Members: Prof. Dr. med. Dieter Kabelitz Scientists: Dr. rer. nat. Marcus Lettau (SFB877, until 11/2011) Dr. Shirin Kalyan (AvH Fellow) Ph.D. students: M.Sc. Jaydeep Bhat (SFB877) Dipl.-Biol. Juliane Fazio (DFG KFO170) Dipl.-Biol. Christian Peters (BMWF ZIM) Medical Students: Guranda Chitadze (DAAD, SFB877 IRTG) Valentina Falkenstern Technicians: Monika Kunz Ina Martens Signe Valentin (SFB877) C Research Report C 1. Shedding of NKG2D ligands (SFB877, project A7) Natural Killer Cell Group 2 member D (NKG2D) is an activating receptor that is expressed on NK cells, γδ T-cells and further subsets of αβ T-cells. Binding of corresponding NKG2D ligands triggers cytotoxic functions in killer cells and co-stimulates T-cell activation, but can also deliver inhibitory signals (Figure 1). In humans, NKG2D ligands include the MHC class I related chain A and B molecules (MICA/B) and 6 members of the UL16-binding protein family (ULBP1-6). NKG2D ligands occur as trans-membrane molecules or as GPI-anchored molecules (Figure 1). Figure 1: The human NKG2D/NKG2D ligand system (from Chitadze et al, Scand J Immunol 2013) 33 While NKG2D ligands are not expressed on “normal” cells, they are up-regulated in response to stress signals and are constitutively expressed on many tumor cells where they can serve as ligands for NKG2D-mediated immune attack. Importantly, however, some NKG2D ligands are shed from the cell surface by members of the “A disintegrin and metalloproteinase” family, notably ADAM10 and ADAM17. In addition, some NKG2D ligands can be secreted in exosomes, and others are released through phospholipase C. Soluble NKG2D ligands can neutralize/inhibit NKG2D signaling and, therefore, shedding is considered as an important tumor immune escape mechanism (Figure 2). Figure 2: release of soluble NKG2D ligands via proteolytic cleavage (ADAM preoteases), exosome secretion, or phospholipase C-mediated release (from Chitadze et al, Scand J Immunol 2013) In this project we study the contribution of various ADAM proteases to the shedding of MICA/B and ULBPs in a variety of tumor cell lines, using siRNA-mediated down-regulation of ADAM10 and/or ADAM17 as an experimental approach. Comparing different tumor cell lines representing various cancer entities, we found that the contribution of ADAM10 versus ADAM17 to the shedding of NKG2D ligands varies considerably among tumor cells. In addition, some allelic variants of MICA (notably MICA*008) are not shed by proteolysis but rather are secreted in exosomes. Therefore, so soluble MICA is detected in supernatants of MICA*0008-positive tumor cells such as the prostatic cell line PC-3 (see Figure 3). 34 Figure 3: siRNA interference reveals differential roles of ADAM10/17 in the shedding of MICA=B molecules. (a) Cells were transfected with siRNAs targeting ADAM10 (light gray columns), ADAM17 (open columns) or with scrambled RNA as a control (dark gray columns). 72 hr after transfection, the medium was changed and the amount of soluble MICA (left) and MICB (right) released into the supernatants was assessed by ELISA after additional 24 hr. The amount of protein released from control RNA-transfected cells was set to 100%. (b) The effective silencing of ADAM10=17 was confirmed by flow cytometry. Data in (a) are represented as mean values of three to four independent experiments 6 SEM. Statistical significance is displayed as ***p < 0.001, **p < 0.01 and *p < 0.05. ND: not detectable. From Chitadze et al, 2013 On-going studies in this project address the role of additional ADAM proteases (notably ADAM9,15,28) and the functional consequences of shed and exosome-expressed NKG2D ligands on NK cells and tumor-reactive γδ T-cells. C 2. γδ T-cells in granulomatosis with polyangiitis (Wegener’s granulomatosis) Granulomatosis with polyangiitis (GPA, previously Wegener’s granulomatosis) is a systemic vasulitis characterized by anti-neutroophil cytoplasmic antibodies (ANCA) and alterations in the T-cell compartment. In this project we study the expression and function of γδ T-cells in GPA patients. We observed a selective reduction of Vδ2 γδ T-cells in the blood of GPA patients when compared to age-matched control donors (Figure 4). 35 Figure 4: γδ T cell counts and subset distribution in the blood of GPA patients and healthy donors (HD). Bars indicate median values. Statistical analysis was done using the Mann–Whitney test (*p ≤ 0.05; ***p ≤ 0.0001). GPA n = 42; HD n = 17. From Fazio et al, 2013 Although numerically reduced, the residual Vδ2 γδ T-cells responded normally to selective stimulation with so-called phosphoantigens (BrHPP) or aminobisphosphonate zoledronic acid (Figure 5). The underlying mechanism of the selective Vδ2 T-cell reduction in GPA patients is presently unknown. It is possible, however, that the continuous exposure to microbial phosphoantigens might play a role. Chronic and persistent infections with microbes such as staphylococcus aureus have been repeatedly hypothesized to be involved in the pathogenesis of GPA. Figure 5: In vitro expansion of Vδ2 T cells in response to γδ T cell-selective antigens. PBMC (1 × 105 per well) were cultured in medium, with BrHPP or zoledronic acid in the presence of exogenous IL-2. The absolute number of Vδ2/Vγ9 T cells was determined after 7 days by flow cytometry. Six GPA patients (indicated by different filled symbols) and 3 HD (indicated by different open symbols) were analyzed. The bars represent the mean values. The proportion of Vδ2 T cells within PBMC before culture ranged from 0.4 to 1.6% in GPA patients and from 0.76 to 4.2% in HD. 36 C 3. Modulation of immune cell functions by aminobisphosphonates Aminobisphosphonates (n-BP) are the most widely prescribed drugs for the treatment of bone diseases such as osteoporosis. Inaddition to their effects of bone resorption, they also have immunomodulatory effects. N-BP inhibit farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. Inhibition of FPPS results in intracellular accumulation of the isoprenoid isopentenyl pyrophosphate (IPP), an endogenous ligand for the Vδ2 T-cell receptor. As a consequence, n-BP such as zoledronic acid (Zometa) are potent activators of human (but not mouse) γδ T-cells. In view of the potent anti-tumor activity of γδ T-cells, a major burst of interest to explore n-BP-based immunotherapy in certain types of cancer has recently been seen. However, published studies indicate that the γδ T-cell-stimulating activity of n-BP when given therapeutically might be transient. In this project, we have analyzed the numbers and phenotype of γδ T-cells longitudinally in a cohort of osteoporosis patients before and after initiation of oral or intravenous n-BP therapy, as well as in some patients suffering from a rare but serious side effect of n-BP treatment, osteonecrosis of the jaw. Figure 6: Proportion of Vγ9Vδ2 T cells, total T cells, monocytes, and granulocytes present in patients with osteoporosis in relation to the length of time on n-BP) therapy. (A) In patients on intravenous (iv) treatment, a significant loss of peripheral blood Vδ2 T cells (p<0.0001) is evident within 18 months of continuous therapy. (B) In patients on oral therapy, a decline in Vδ2 T cells is observed (p < 0.03), but it is much more gradual, taking seemingly almost 4 years to materialize. (C) There was no observable difference in any of the other white blood cells assessed in relation to the length of time on intravenous (iv) treatment or (D) oral treatment. T cells are shown as black circles, monocytes as squares, and granulocytes as gray diamonds. 37 Interestingly, we observed a steady decline of Vδ2 T-cells following initiation of n-BP therapy, notably in the group on intravenous n-BP (Figure 6). In contrast to γδ T-cells, numbers of total T-cells, granulocytes and monocytes did not change upon prolonged n-BP therapy (Figure 6C,D). These observations may help to explain the lack of γδ T-cell-based anti-tumor activity upon repeated n-BP application. In order to investigate the effects of nBP on immune cells in more detail, we have generated in cooperation with the Institute of Organic Chemistry (Prof. Lindhorst, Vijay Anand) a CFSE-coupled variant (FluorZOL) of zoledronic acid (ZOL, the most potent n-BP) to study the uptake and intracellular fate of ZO by flow cytometry and laserscan microscopy. Results so far indicate that both monocytes and granulocytes within peripheral blood leukocytes readily take up FluorZOL (Figure 7). Figure 7: Up-take of FluorZOL by subsets of leukocytes in peripheral blood. EDTA- blood from a healthy donor was subjected to red blood cell lysis. Subsequently, all leukocytes were incubated for 4 and 24 rs with FluorZOL, and uptake was measured by flow cytometry. Leukocyte subsets /granulocytes, monocytes, lymphocytes) were identified on the basis of the side scatter (SSC) properties. Kalyan et al, unpublished Although granulocytes and monocytes take up FluorZOL to comparable levels, the functional consequences are quite different. Preliminary experiments suggest that following FluorZOL (or ZOL) treatment, granulocytes acquire a suppressive functional phenotype and can in fact inhibit γδ T-cell expansion in response to selective antigens. Ongoing studies address the regulatory interplay between granulocytes, monocytes and T-cells in more detail. These issues are highly relevant for the future development of γδ T-cell based immunotherapeutic strategies. C 4. Determining T-cell responses in patients allergic to TNF blocker therapeutics TNF blockers such as Remicade® (infliximab) are highly efficient therapeutics for chronic inflammatory conditions such as Morbus Crohn or rheumatoid arthritis. In rare instances, patients develop allergic symptoms when injected with TNF blockers, and severe allergic 38 reactions may occur. In this joint project with groups at FZ Borstel (Prof. Jappe) and Institute CCCC Hamburg (PD Dr. Kromminga), we aim to characterize the allergenic epitopes of the TNF blockers and to develop a predictive assay which would allow to identify patients at risk before starting the anti-TNF therapy. To this end, we develop a short-term stimulation assay based on whole blood to determine IL-4 and IFNγ-secreting T-cells before and after exposure to allergenic structures to be characterized by Prof. Jappe. This is supplements by a whole blood-based immunophenotyping of regulatory T-cells and γδ T-cells before and after initiation of anti-TNF therapy. An example of the whole blood FACS analysis is shown in Figure 8. Figure 8: Immunophenotyping of whole blood leukocytes. Treg are identified as CD4+CD127low (a) and CD4+CD25high (b). Subsets of Vδ1 (X-axis) and Vδ2 cells (Y-axis9 within γδ T-cells are detected following gating on CD45+panγδ+ cells (c). The intracellular detection of IL-4 and IFNγ (Figure 9) has been adapted to short term stimulation of RBC-lysed whole leukocytes. These assay systems are now ready to be applied for the analysis of patients before and following treatment with TNF blockers. Figure 9: CD4+ T cells were stimulated under Th-1 priming (IL-12, anti-IL4) or Th2 priming (anti-IL12, IL4) conditions. Intracellular cytokines were detected by flow cytometry following standard protocols. 39 D Publications 2011 Paulsen M, Mathew B, Valentin S, Adam-Klages S, Bertsch U, Lavrik I, Krammer PH, Kabelitz D, Janssen O. Modulation of CD4+ T cell activation by CD95 co-stimulation. Cell Death Differ 18: 619-631, 2011 Edelmann B, Bertsch U, Tchikov V, Winoto-Morbach S, Perrotta C, Jakob M, Adam-Klages S, Kabelitz D, Schütze S. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1-receptosomes. EMBO J 30: 379-394, 2011 Friedrichs B, Siegel S, Reimer R, Barsoum A, Coggin Jr. J, Kabelitz D, Heidorn K, Schulte C, Schmitz N, Zeis M. High expression of the immature laminin receptor protein correlates with mutated IGVH status and predicts a favourable prognosis in B-cell chronic lymphocytic leukemia. Leukemia Res 35: 721-729, 2011 Koop A, Lepenies I, Braum O, Davarnia P, Scherer G, Fickenscher, Kabelitz D, AdamKlages S. Novel splice variants of human IKKε negatively regulate IKKε-induced IRF-3 and NF-κB activation. Eur J Immunol 41: 224-234, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Lucius R, Leippe M, Kabelitz D, Janssen O. Effector granules in human T lymphocytes : The luminal proteome of secretory lysosomes from human T cells. Cell Commun Signal 9: 4, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Lucius R, Leippe M, Kabelitz D, Janssen O. Effector granules in human T lymphocytes: Proteomic evidence for two distinct species of cytotoxic effector vesicles. J Proteome Res 10: 1603-1620, 2011 Marischen L, Wesch D, Oberg HH, Rosenstiel P, Trad A, Shomali M, Grötzinger J, Janssen O, Tchikov V, Schütze S, Kabelitz D. Functional expression of NOD2 in freshly isolated human peripheral blood γδ T cells. Scand J Immunol 74 : 126-134, 2011 Hutchinson JA, Riquelme P, Sawitzki B, Tomiuk S, Miqueu P, Zuhayra M, Oberg HH, Pascher A, Lützen U, Janssen U, Broichhauesen C, Renders L, Thaiss F, Scheuermann E, Henze E, Volk HD, Chatenoud L, Lechler RI, Wood KJ, Kabelitz D, Schlitt HJ, Geissler EK, Fändrich F. Cutting edge : Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol 187: 2072-2078, 2011 Oberg HH, Juricke M, Kabelitz D, Wesch D. Regulation of T cell activation by TLR ligands. Eur J Cell Biol 90: 582-592, 2011 Kabelitz D. γδ T-cells: cross-talk betwen innate and adaptive immunity. Cell Mol Life Sci 68; 2331-2333, 2011 Wesch D, Peters C, Oberg HH, Pietschmann K, Kabelitz D. Modulation of γδ T cell responses by TLR ligands. Cell Mol Life Sci 68: 2357-2370, 2011 Lamprecht P, Kabelitz D. T-Zellen bei ANCA-assoziierten Vaskulitiden. Z Rheumatol 70: 698-700, 2011 Kling C, Kabelitz D. Impfen – von der Empirie zur Immunologie. Biol. Unserer Zeit 41: 375-383, 2011 Kalyan S, Wesch D, Kabelitz D. Aminobisphosphonates and Toll-like receptor ligands: recruiting Vγ9Vδ2 T cells for the treatment of hematologic malignancy. Curr Med Chem 18: 5206-5216, 2011 40 Kabelitz, D. Editorial: The German Collaborative Research Center 415 „Specificity and Pathophysiology of Signal Transduction Pathways“. Eur J Cell Biol 90: 449, 2011 2012 Meyer T, Oberg HH, Peters C, Martens I, Adam-Klages S, Kabelitz D, Wesch D. poly(I:C) costimulatation induces a stronger antiviral chemokine and granzyme B release in human CD4 T cells than CD28 costimulation. J Leukoc Biol 92 : 765-774, 2012 Kabelitz D, He W. The multifunctionality of human Vγ9Vδ2 T cells: clonal plasticity or distinct subsets? Scand J Immunol 76: 213-222, 2012 Kabelitz D. Toll-like Rezeptoren: Erkennungsstrukturen des angeborenen Immunsystems und therapeutische Zielstrukturen. Med Monatsschr Pharm 35: 238-244, 2012 Kabelitz D. CD277 takes the lead in human γδ T-cell activation. Blood 120: 2158-2161, 2012 E Grants E.1. DFG SFB 877 Project A7 «Mechanisms and implications of NKG2D ligand shedding», 2 PhD positions, 20.000 € consumables/year E.2. DFG SFB 877 IRTG : fellowship for 1 year for Guranda Chitadze, 12.000 € E.3. DFG KFO170, project 3 «T-cell subsets in Wegener’s granulomatosis», together with Prof. Lamprecht/Lübeck) ; 1 PhD, 17.500 € consumables /year E.4. DFG Pancreas Cancer Consortium Kiel, together with PD Dr. Wesch, 1 Post-Doc, 20.000 € consumables/year E.5. BMWF ZIM «Entwicklung von Testsystemen zum Nachweis allergenspezifischer IgE- Antikörper gegen therapeutische monoklonale Antikörper Evaluierung von Parametern zur Risikoabschätzung für Typ I-allergische Reaktionen bei Empfängern therapeutischer Antikörper ; Evaluierung von Parametern für die Regulation von T-Zellen und Entwicklung von Parametern für den TReg-Status eines Patienten », together with Prof. Jappe/FZ Borstel and PD Dr. Kromminga/ Hamburg, 1 PhD, 15.000 € consumables/year E.6. Alexander-von-Humboldt Foundation, 1 Post-Doc position (Dr. Kalyan), 10.000 € consumables 41 42 4. Research Group Schütze A Group Leader: B Lab Members: Prof. Dr. rer. nat. Stefan Schütze Scientists: Dr. rer. nat. Vladimir Tchikov (DFG) Dr. rer. nat. Uwe Bertsch (DFG) Dr. rer. nat Jürgen Fritsch (DFG) Dr. rer. nat Supandi Winoto-Morbach Dr. rer. nat Bärbel Edelmann (DFG) (since march 2012) Ph.D. Students: M. Sci. Bärbel Edelmann (DFG) (until february 2012) Dipl.-Biochem. Mario Stephan (DFG) Medical Students: Isabel Hellmich Jan Klawitter Technicians: Andrea Hethke (DFG) Fereshteh Ebrahim (DFG) Casimir Malanda C Research Report C 1. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes. We previously demonstrated that Tumor Necrosis Factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signaling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation (see Schütze, S., Tchikov, V., Schneider-Brachert, W. (2008): Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 655662). The mechanism of TNF-mediated A-SMase activation within the endo-lysosomal compartment, however, was poorly defined. In this project, funded by the DFG priority program 1267 “Sphingolipids- Signal and Disease” we showed that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression (Edelmann et al., 2011). The investigation of A-SMase activation after TNF-stimulation by western blotting revealed the appearance of a 57kDa proteolytic cleavage fragment which correlates with an increase ASMase activity (Figure 1). This observation suggest that A-SMase is activated by proteolytic cleavage of the pro-A-SMase molecule. 43 Figure 1: A-SMase cleavage and activation after TNF-stimulation. HeLa cells were stimulated with TNF for indicated times and cell lysates were analysed for endogenous ASMase by western blotting using anti-A-SMase antibodies that detect the 72-kDa pro-A-SMase and a 57-kDa cleavage product. A-SMase processing is accompanied by an increase of ASMase activity. Previous experiments using a caspase-8 inhibitor suggested a functional link between caspase8 and A-SMase. But since we observed, that caspase-8 is unable to activate purified pro-ASMase, we searched for caspase-8 downstream proteases and found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72 kDa proA-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule (Figure 2). A C B Figure 2: Cleavage and activation of pro-A-SMase in cell lysates and immunoprecipitated material after caspase incubation. Western blot analysis with an anti-GFP antibody and A-SMase activity assay was performed on (A) whole-cell lysate of pro-A-SMase-EGFP-transfected cells incubated with caspase-7, and (B) immunoprecipitated pro-ASMase-EGFP incubated with active caspase-7 for the indicated times. Caspase-7 cleaves A-SMase in whole cell lysates as well as the immunprecipitated A-SMase-EGFP. (C) Western blot analysis of immunoprecipitated endogenous A-SMase from wild-type HeLa cells incubated with active caspase-7 and A-SMase activity assay. Both the kinetics of cleavage and the activation of A-SMase run in parallel. Confocal Laser Scan-analysis revealed that after TNF-stimulation caspase-7 colocalizes with the TNF-R1 as well as with A-SMase (Figure 3A-C). These data indicated a possible interaction of caspase-7 with A-SMase in the same intracellular compartment. 44 Figure 3: Partial colocalization of active caspase-7 with TNF receptosomes and pro-A-SMaseEGFP as well as with endogenous A-SMase. Merged confocal microscopic images of (A) HeLa cells fluorescence labeled with biotin-TNF/avidin-FITC complexes (green) and anti-cleaved capase-7 antibody (red) after 30 min of TNF-receptor internalization. (B) HeLa cells labeled with anti-cleaved capase-7 antibody (red) and EGFP-tagged A-SMase (green) 20 min after TNF-receptor internalization, (C) HeLa cells labeled with anti-capase-7 antibody (green) and antibodies against endogenous A-SMase at various time points after TNF-receptor internalization. Colocalization of the respective fluorescently labeled molecules is indicated by yellow color (marked with arrows). In order to confirm a direct colocalization of caspase-8, caspase-7 and pro-A-SMase in TNFR1 containing membrane compartments with an independent method, we isolated TNF-R1receptosomes from wild-type HeLa cells as well as from pro-A-SMase-EGFP transfected HeLa cells using our immunomagnetic separation system. Purified TNF-receptosomes and the remaining non-magnetic lysate-fractions were analyzed by Western blotting. Already after 3 min pro-caspase-8 is activated within TNF-receptosomes as evident from the appearance of the 41/43 kDa and the 18 kDa mature caspase-8 molecules. Neither caspase-3 nor cleaved caspase-3 are present in receptosomes. The generation of the 18 kDa active caspase-8 fragment in TNF-receptosomes appears slightly in advance of cleaved caspase-7 in the magnetic fractions, primarily the 31 kDa fragment. The TNF-induced caspase-7 activation 45 seems to take place exclusively within TNF-receptosomes, since the corresponding nonmagnetic fractions did not display any caspase-7 activation. The appearance of the enzymatically active 31 kDa form of caspase-7 in TNF-receptosomes is paralleled by the generation of the active 57 kDa fragment of endogenous A-SMase in wild-type HeLa cells and also by the generation of the 82 kDa A-SMase-EGFP fragment in the A-SMase-EGFPtransfected cells. In both cases, this processing of pro-A-SMases is paralleled by a concomittant increase in the respective A-SMase activities within the isolated TNFreceptosomes (not shown). Figure 4: Cleavage and activation of caspase-8, caspase-7 and pro-A-SMase in magnetically isolated TNF receptosomes and non-magnetic fractions. Time course of intracellular TNF-R1 receptosome trafficking (left panel) and corresponding non-magnetic fractions (right panel) in HeLa wild-type and pro-A-SMase-EGFP transfected HeLa cells. Total cell lysate, magnetic and non-magnetic fractions derived after indicated times of TNF-receptor internalization were analysed for cleavage of pro-caspase-8 (54/52 kDa) to the active form of 18 kDa, cleavage of procaspase-3 to the active 17 kDa form, cleavage of procaspase-7 (35 kDa) to the active forms of 31 and 20 kDa, cleavage of endogenous pro-A-SMase (72 kDa) to the more active 57 kDa form, and cleavage of recombinant pro-A-SMase-EGFP (98 kDa) to the more active 82 kDa form. The functional link between caspase-7 and A-SMase activation was demonstrated by down regulation of caspase-7 using siRNA or with caspase-7 deficient mouse embryonic fibroblasts (MEFs) in comparison to wild type cells. Down modulation of caspase-7 blocked the TNF46 induced A-SMase activation and cleavage (not shown). Furthermore, ceramide generation was blocked in caspase-7 deficient MEF compared to the wild type as well as no CTSD activation was detectable. The caspase-7 cleavage site within the A-SMase mmolecule was identified by pointmutation of aspartate 253 to alanin which blocked the processing and activation of A-SMase (not shown). Taken together, our data suggest a signaling cascade within TNF-receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase (see Edelmann et al., 2011). Figure 5: Compartmentalization of TNF-R1 signaling and A-SMase activation. Binding of TNF ligand to TNF-R1 initializes the clathrin dependent endocytosis and the recruitment of adaptor proteins TRADD, FADD and caspase-8. Within the receptosome-bound death-inducing signaling complex (DISC), caspase-8 is activated. Along the endocytotic pathway, TNF receptosomes fuse with trans-Golgi vesicles containing pro-acid sphingomyelinase (pro-A-SMase) and pre-pro-cathepsin D (pre-pro-CTSD) to form multivesicular organelles. There, activation of caspase-7 by active caspase-8 leads to the cleavage and activation of pro-A-SMase. Activated A-SMase generates ceramide, which activates CTSD and mediates its translocation from the late endosome to the cytosol. In this compartment, the proapoptotic protein Bid is cleaved by CTSD to tBid resulting in release of cytochrome C from mitochondria and activation of caspase-9 and caspase-3 leading to apoptotic cell death (see Edelmann et al. (2011)). 47 C 2. Lipid-labeling facilitates the magnetic isolation and characterization of pathogen- containing phagosomes Pathogenic microorganisms actively modulate the host cell to prevent detection and degradation by the immune system. Some of the most vicious pathogens reside in intracellular compartments, which are highly diverse and dynamic vacuoles. The detailed molecular composition of many of these vacuoles remains insufficiently defined due to technical limitations in accessing these compartments in a purified form. In a collaborative project with the group of Norbert Reiling (FZ Borstel), funded by the DFG priority program SPP 1580, we developed a novel, rapid and versatile method for the isolation of pathogen-containing phagosomes from primary cells. We used a new lipid-based procedure to label bacterial surfaces and engineered a rapid immunomagnetic technique using a strong magnetic field in a novel free-flow system (HOKImag, now produced by Hoock GmbH, Kiel, Germany) to isolate intact bacteria-containing compartments which can be characterized by electron microscopy, Western blot and mass spectrometry (Figures 6-9) (see Steinhäuser et al., 2012). Figure 6: Scheme of the phagosome isolation protocol. Bacteria are magnetically labeled using Lipobiotin and SA magnetic beads (A) . Host cells are infected with magnetically labeled bacteria for different lengths of time (fraction ‘lysate’) (B). Stop of intracellular trafficking at 4◦C. (C) Disruption of the host cell membrane is achieved by repeated sonication and centrifugation steps (C). Post nuclear supernatants (fraction ‘PNS’) are collected and loaded into the tube adjusted between two magnets in the magnetic chamber (D). During incubation in the magnetic field, magnetic particles migrate to the tube walls. Tube is rinsed with buffer and the non-magnetic fraction is collected (E). The tube is released from the magnetic field and the magnetic, phagosome-containing fraction is collected (F). 48 Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. Figure 7: Electron microscopic images of magnetically labeled mycobacteria (taken by C. Steinhäuser, FZ Borstel). A) TEM analysis of magnetically labeled Mycobacterium avium. Magnetic beads are visible as electron dense dots in close apposition to the bacterium (indicated by arrows). B) SEM images. The images of the secondary electron image and the backscattered electron image are merged. Magnetic beads are visible as dots (red pseudocolor) in close opposition to the bacterium (indicated by arrows). C) Scheme of magnetic labeling procedure: LB is added to the bacteria and incubated over night. Then, cells are washed and subsequently SA magnetic beads are added. The bacterial surface is labeled with LB/magnetic beads. Figure 8: Infection of BMDM with magnetically labeled Mycobacterium avium and isolation of phagosomes. The kinetic analysis shows the maturation process of the phagosomes. (A–F) TEM analysis of BMDM infected with magnetically labeled M. avium (A, C,E) and isolated, intact M. avium phagosomes (B, D, F). (A+B) 15min 49 post infection (p.i.), (C+D) 60min p.i., (E + F) 120min p.i.. Magnetic beads are visible as electron dense dots (indicated by arrows). (G) Western blot analysis of the magnetic fraction (magn.) compared to PNS. BMDM were infected with magnetically labeled M. avium for 15, 60 and 120min. Figure 9: Isolation of intact Listeria monocytogenes (Dhly)-containing phagosomes from untreated and IFN-γγ treated J774 macrophages (U. Heigl., W. Schneider-Brachert, Regensburg). (A–F) TEM analysis of J774 macrophages infected with magnetically labeled L. monocytogenes (1hly) (A–C) and the magnetic fraction containing intact L. monocytogenes (1hly) phagosomes (D–F). (D) Overview of magnetic fraction containing L. monocytogenes (1hly) phagosomes, (E + F) single L. monocytogenes (1hly) phagosomes. Magnetic beads are visible as electron dense dots (indicated by arrows). (G+H) Western blot analysis of the magnetic fraction (magn.) compared to post nuclear supernantants (PNS). J774 macrophages were incubated with recombinant IFN-γ (500U/mL) overnight (H) or left untreated (G) prior infection with magnetically labeled L. monocytogenes (1hly) for 15, 60 and 120min. Listeria antigens were detected by a L. monocytogenes specific antiserum; Host cell proteins: Rab5, Rab7, LAMP1, CTSD, LRG47/IRGM1, Nucleoporin p62 (Nu p62) and Glycogen synthase kinase 3beta (GSK3beta). The newly developed method of magnetic isolation of intact pathogen-containing phagosomes is a powerful tool for the detailed molecular analysis of the intracellular host-pathogen interface, facilitates comparative studies across the kingdom of intracellular pathogens, and should be useful for identifying microbial and host cell targets for innovative anti-infective strategies. C 3. The Adenovirus protein E3-14.7K is recruited to TNF-Receptor 1 and blocks TNF cytolysis independent from interaction with optineurin. Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. In cooperation with the group of Wulf 50 Schneider-Brachert, University Regensburg, we provided evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1 (see Klingeisen et al., 2012). We hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis. C 4. The IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model. Airway epithelial NF-kB is a key regulator of host defense in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (A-SMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defense as well as in pathologic processes of acute lung injury. In cooperation with the group of Martin Krause, Childrens Hospital, University of Kiel, the influence of NF-kB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation was investigated (see von Bismarck et al., 2012). In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS, lung tissue concentrations of TNF-alpha, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to A-SMase activity and ceramide and S1-P was analyzed. Airway epithelial NF-kB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of A-SMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-kB translocation. This confirms the critical role of airway epithelial NF-kB pathway for the 51 inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defense mechanisms. C 5. Inositol-trisphosphate reduces alveolar apoptosis and improves lung function in neonatal lung injury. D-myo-Inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-Inositol-1,4,5-trisphosphate and exerts anti-inflammatory and antiedematous effects in the lung. Myo-Inositol (Inos) is a component of IP3 and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung inury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (A-SMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. Again in cooperation with the group of Martin Krause, Childrens Hospital, University of Kiel, a novel clinically relevant triple-hit model of nALI was used consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into airways, every 24 hours apart (see Preuß et al., 2012). After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation, and extra-vascular lung water improved significantly by S+IP3 intervention. In pulmonary tissues we observed decreased A-SMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, reduced expression of interleukin-6, transforming growth factor-β1 and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14+/18+ cells (macrophages) into airspaces, and lower surfactant surface tensions in S+IP3 - but not in S+Inos - treated piglets. We conclude that admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by suppression of the governing enzyme A-SMase, and that this treatment option deserves clinical evaluation. C 6. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine. Again in cooperation with Martin Krause, we comparatively assessed the benefits of topical A-SMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification (see Preuß et al, 2012). In this translational study, a triple-hit acute lung injury model was used 52 that entails repeated airway lavage, injurious ventilation, and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 h. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance, and pulmonary edema. S+Imi caused systemic A-SMase suppression and ceramide reduction, while the S+PIP2 effect remained compartmentalized in the airways due to the molecule’s bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of A-SMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure. Highlights: 2011 Final examination of the MD thesis of Maike Reinicke, awarded by the Beigelsche Promotionspreis 2011 of the Medical Faculty of the CAU. 2012 D Final examination of the PhD thesis of Bärbel Edelmann (summa cum laude). Publications 2011 - 2012 2011 Marischen, L., Wesch, D., Oberg, H.H., Rosenstiel, P., Trad, A., Shomali, M., Grötzinger, J., Janssen, O., Tchikov, V., Schütze, S., Kabelitz, D. Functional expression of NOD2 in human peripheral blood γδ T-cells. Scand J Immunol 74: 126-134, 2011 Edelmann, B., Bertsch, U., Tchikov, V., Winoto-Morbach, S., Jakob, M., Adam-Klages, S., Kabelitz, D., Schütze, S. (2011). Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1-receptosomes. EMBO J 30: 379-394, 2011 53 Bertsch, U., Edelmann, B., Tchikov, V., Winoto-Morbach, S., Schütze, S. Compartmentalization of TNF-receptor 1 signaling: TNF-R 1 - associated caspase-8 mediates activation of acid sphingomyelinase in late endosomes. Adv Exp Med Biol 691: 605-16, 2011 Tchikov, V., Bertsch, U., Fritsch, J., Edelmann, B., Schütze, S. Subcellular compartmentalization of TNF receptor-1 and CD95 signaling pathways. Eur J Cell Biol 90: 467-475, 2011 2012 Steinhäuser, C., Heigl, U., Tchikov, V., Schwarz, J., Gutsmann, T., Seeger, K., Fritsch, J., Schroeder, J., Wiesmüller, K.-H., Rosenkrands, I., Pott, J., Krause, E., Ehlers, S., SchneiderBrachert, W., Schütze, S., Reiling, N. (2012). Lipid-labeling facilitates a novel magnetic isolation procedure to characterize of pathogen-containing phagosomes. Traffic, 2012 Dec. 11. Doi:101111/tra.12031. [Epub ahead of print] Preuß, S., Omam, F.D., Scheiermann, J., Stadelmann, S., Winoto-Morbach, S., von Bismarck, P., Adam-Klages, S., Knerlich-Lukoschus, F., Lex, D., Wesch, D., Held-Feindt, J., Uhlig, S., Schütze, S., Krause, M.F. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine. J Cell Mol Med, 2012 Nov. 16(11):2813-26. doi: 10.1111/j.1582-4934.2012.01618.x. [Epub ahead of print] Klingeisen, L., Ehrenschwender, M., Heigl, U., Wajant, H., Helgans, T., Schütze, S., Schneider-Brachert, W. (2012) E3-14.7K is recruited to TNF-Receptor 1 and blocks TNF cytolysis independent from interaction with optineurin. PLoS ONE 2012;7 (6): e38348 Preuß, S., Stadelmann, S., Omam, F.D., Scheiermann, J., Winoto-Morbach, S., von Bismarck, P., Knerlich-Lukoschus, F., Adam-Klages, S., Wesch, D., Held-Feindt, J., Uhlig, S., Schütze, S., Krause, M.F. (2012) Inositol-trisphosphate reduces alveolar apoptosis and improves lung function in neonatal lung injury. Am J Resp Cell Mol Biol 47: 158-169, 2012 von Bismarck, P., Winoto Mohrbach, S., Herzberg, M., Uhlig, U., Schütze, S., Lucius, R., Krause, M. F. (2012) IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model. Pulm Pharmacol Ther 25 : 228-235, 2012 E Grants E.1. DFG Schwerpunktprogramm 1267 "Sphingolipids-Signal and Disease" Project SCHU 733/92 "Topology, function and regulation of ceramide production in death receptor signalling.” E.2. DFG SFB 877, Project B1 „Role of ubiquitinylation and proteolysis in the regulation of proand antiapoptotic TNF-R1 signaling“ E.3. DFG SFB 877, B2 (together with D. Adam) „Proteolysis in the regulation of caspaseindependent programmed cell death.“ 54 E.4. DFG Pancreatic Cancer Consortium Kiel, Project B5 (together with A. Trauzold) Dissection of TRAIL-mediated signaling in pancreatic ductal adenocarcinoma cells - towards efficient antitumor therapy E.5. DFG Schwerpunktprogramm 1580 Project (together with N. Reiling) “The influence of pathogen and host cell variability on the molecular composition of phagosomes harbouring different strains of the Mycobacterium tuberculosis complex”. 55 56 5. Research Group Wesch A Group Leader PD Dr. rer. nat. Daniela Wesch B Lab Members: Scientists: PD Dr. sc. hum. Hans-Heinrich Oberg (DFG, PCC) Prof. Dr. rer. nat. Sabine Adam Ph.D. Students: Dipl.-Biol. Christian Peters [Medical Faculty (DW), BMWF ZIM (DK)] Dipl.-Biol. Tim Meyer [Medical Faculty, DFG (DK) ] Medical Student: cand. med. Matthias Juricke Dipolma/Master Students: Anne Kammel (2011) Sarah Krause (2011) Technicians: Thi Thuy Hoa Ly Sandra Ussat Kyoung-Ae Yoo-Ott C Research Report: C.1. Immunotherapy of pancreatic ductal adenocarcinoma with human γδ T lymphocytes (Pancreatic Cancer Consortium Kiel; DFG WE 3559/2-1) Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease due to a rapid progression and the absence of initial specific symptoms. In cooperation with Prof. Susanne Sebens (Institute of Experimental Medicine, UKSH, Kiel) and Prof. Christoph Röcken (Institute of Pathology, UKSH, Kiel), we observed that γδ T cells, but not αβ T cells, are localized adjacent to or within the ductal epithelium of pancreatic tumors, which is of interest in the context of γδ T cell-based immunotherapeutic strategies (Fig. 1). 57 Isotype/2. step 200x 2. step Fig. 1. PDAC-infiltrating γδ T lymphocytes. Serial paraffin embedded tissue sections of 41 PDAC patients were stained as indicated for all stainings for one representative PDAC donor and for TCR γδ expression for another one. Immunostaining of CD3 was done with the fully automated BondTM Max-System using the BondTM Polymer Refine Detection Kit. TCR γδ expression was detected after deparaffinization and antigen retrieval in DAKO antigen retrieval solution (pH 9.0) by using the anti-γδ TCR mAb clone γ3.20 or a mouse IgG1 isotype control mAb at a concentration of 3µg/ml. For detection of primary antibodies, EnVision-mouse HRP was used. The substrate reaction was performed using the AEC substrate for peroxidase. Finally, sections were stained with hemalaun and embedded in glycerine gelatine. 200x anti-pan γδ TCR (clone: γ3.20) 200x anti-CD3 400x anti-pan γδ TCR 400x As γδ T cell stimulating antigens did not optimally increase the cytotoxic activity of γδ T cells, we used two tribodies [(Her2)2xVγ9] and [(Her2)2xCD16] and a bispecific antibody [Her2xCD3] with specificities for Vγ9, CD16 or CD3 on γδ T cells, respectively, and for human epidermal growth factor receptor Her2/neu expressed on PDAC cells. The antibodies were designed by our cooperation partners PD Dr. Matthias Peipp, Dr. Christian Kellner & Prof. Martin Gramatzki (Divison of Stem Cell Transplantation and Immunotherapy, UKSH, Kiel). We demonstrated that [(Her2)2xVγ9] selectively enhanced the degranulation of γδ T cells from PDAC patients, and thereby the release of perforin and granzyme B, whereas [(Her2)2xCD16] also activated cytotoxic activity of NK cells and [Her2xCD3] also αβ T cells including regulatory T cells. Based on the measurement of an extended time-course by a Real Time Cell Analyzer, we determined that small numbers of effector γδ T cells (as it probably occurs at the tumor site) completely lysed even almost resistant PDAC cells after addition of [(Her2)2xVγ9] in comparison to γδ T cell stimulating antigens (Phosphoantigens, PAg), and that PDAC cells did not regenerate in vitro (Fig. 2). 58 Fig. 2. Enhancement of γδ T cell cytotoxicity against PDAC cells. (A) PancTu-I and (B) Colo357 cells had been cultured overnight before they were left untreated (green line) or were treated with medium (orange line), PAg BrHPP (dark blue line), [Her2xCD3] bsscFv (light blue line), [(Her2)2Vγ9] tribody (red line) or 1% TritonX-100 (black line) as indicated in the presence of γδ T cells. The black line represents the usage of 1% TritonX-100 to determine maximal lysis. Cell index values were normalized at the time of addition of the various substances. The arrow indicates the addition of γδ T cells of different healthy donors (HD) or PDAC patients (PC) with an E/T ratio of 12.5:1. Normalized cell index values were analyzed in 5 min increments as the average of triplicates with SD. These results indicate that [(Her2)2xVγ9] selectively targeting γδ T cells to tumor antigens might provide a tool to further increase γδ T cell cytotoxicity in situations where γδ T cell stimulating antigens fail due to exhaustion, anergy or depletion of γδ T cells (Oberg et al., Cancer Res, in revision). On-going studies in this project address the efficacy of human Vγ9Vδ2-bearing γδ T cells from healthy donors against PDAC in vivo after adoptive transfer of γδ T cells into SCID Beige mice together with [(Her2)2xVγ9] and low dose IL-2. Co-workers in this project: H.-H. Oberg, S. Krause, S. Adam, S. Ussat, T.T.H. Ly, D. Kabelitz, D. Wesch C.2. Regulatory activity of T cells (Medical Faculty) Thymic-derived natural regulatory T cells (nTreg) prevent autoimmunity and regulate selftolerance. nTreg strongly express CD25, cytotoxic T lymphocyte-associated antigen 4 (CLTA4/CD152) and the transcription factors forkhead box (Fox) P3 and Helios. FoxP3 expression in human inducible Treg detected with anti-FoxP3 mAb PCH101 mAb does not necessarily correlate with regulatory function, whereas FoxP3 expression detected with 259D mAb seems to be connected to suppressive function. 59 Suppressive γδ T cells To characterize the phenotype of suppressive Vδ2 γδ T cells, we analyzed putative Treg markers in/on freshly isolated γδ T cells in comparison to freshly isolated Treg and CD25- CD4+ nonTCRγδ+ responder T cells (Fig. 3). Treg (positive control) were strongly positive for all markers, while CD25- CD4+ non-TCRγδ+ responder T cells (negative control) did not express any of the tested putative Treg markers. However, freshly isolated Vδ2 γδ T cells did neither express CD25 on the cell surface nor FoxP3 intracellularly, whereas one third of the Vδ2 γδ T cells expressed intracellular Helios (Fig. 3), which was up-regulated after stimulation with anti-CD3/anti-CD28 mAb, but not after PAg-stimulation on suppressive Vδ2 γδ T cells (Peters et al., Cellular and Molecular Life Sciences, in press). Fig. 3. Phenotypic characterization suppressive γδ T cells. Purified CD25- CD4+, non-TCRγδ responder T cells (Resp), Treg or Vδ2 γδ T cells were stained with the indicated antibodies. The Helios- as well as FoxP3-expression (with two antiFoxP3 mAb) was determined intracellulary and CD25 on the cell surface using flow cytometry. Isotype controls are shown as small-sized dot blots (located in the upper left edge of the large-sized dot blots). One representative out of five experiments is shown. The numbers in the dot blots present the relative percentage. We conclude from our data that FoxP3 and Helios expression do not represent specific markers for the suppressive capacity of γδ T cells, but Helios expression seems to be involved in the differentiation of T cells with regulatory function (data not shown). Additionally, we obtained insights into the suppressive mechanism of freshly isolated Vδ2 γδ T cells. We observed an important role for the interaction of CD86 on γδ T cells and CTLA-4 on CD25- CD4+ responder T cells which induces a reduced phosphorylation of Akt and NF-κB in CD25- CD4+ responder T cells (Peters et al., Cellular and Molecular Life science, in press). Co-workers in this project: C. Peters, D. Wesch 60 Inducible suppressive αβ T cells In cooperation with the group of Prof. Kalthoff (Molecular Oncology, UKSH, Kiel), we previously observed a constitutive FoxP3 expression at the mRNA and protein level in PDAC cells, some of which are potent inhibitors of T cell proliferation in vitro. The reduction of FoxP3 expression by siRNA partially reversed the suppressive activity of PDAC cells, indicating that ectopic expression of FoxP3 might confer regulatory activity (Hinz et al., Cancer Res., 2007). To get more insights whether the unresponsiveness of CD25- CD4+ responder T cells is mediated directly by FoxP3+ PDAC cells or by an indirect effect due to a possible conversion of certain CD25- CD4+ responder T cells into inducible regulatory T cells depending on cytokines such as IL-10 or TGF-β produced in the cell culture milieu by PDAC cells, we performed further investigations. The co-culture of freshly isolated CD25- CD4+ responder T cells with PDAC cells such as Panc89 and Colo357 suppressed the proliferation after T cell receptor (TCR) stimulation with anti-CD3/anti-CD28 mAb (Oberg et al., EJCB, 2010; Fig. 4). Fig. 4. Fox3+ PDAC cells suppress T cell proliferation. CD25CD4+ responder T cells (Resp) were magnetically isolated and stained with a Cell Trace to determine cell division. Resp were cultured in medium (black bar) or co-cultured with PDAC cells Panc89 and Colo357 as indicated (grey bars) for one day. Thereafter, cultures were activated with Activation/Expander Beads coated with anti-CD3 and anti-CD28 mAb for two further days. The percentage of proliferating responder cells is shown two days after stimulation. Mean ± SD of the indicated number of experiments with healthy donors are presented. To examine a possible induction of regulatory T cells, unresponsive T cells were removed after 3 days from the co-culture with PDAC cells and were used in a second co-culture experiment with freshly isolated CFSE-labeled CD25- CD4+ T cells. The TCR-induced proliferation of CFSElabeled CD25- CD4+ T cells was inhibited in the presence of unresponsive T cells (Resp preincubated with Panc89 or Colo357) (Fig. 5). Fig. 5. Inducible suppressive T cells. Resp cells were separated from PDAC cells after 3 days of coculture (pre-incubated Resp). Freshly isolated CFSElabeled CD25- CD4+ T cells were cultured with medium or with pre-incubated Resp, which were initially cultured in medium or co-cultured with PDAC cells Panc89 or Colo357 or as a control with freshly isolated Treg. The numbers represent the proportion of proliferating cells within the CFSElabeled CD25- CD4+ T cells. 61 The induction of suppressive T cells was observed in 2 of 5 experiments and the molecular mechanism is unknown. This suggests a substantial donor-dependent variability which requires further investigation. Co-workers in this project: M. Juricke, H.-H. Oberg, D. Wesch C.3. Modulation of T cells by TLR ligands Toll-like receptors (TLR) and NOD-like receptors (NLR) are pattern recognition receptors (PRR), which recognize a broad variety of structurally conserved molecules derived from microbes. TLR and NLR are expressed by cells of the innate immunity as well as by several T cell subsets (e.g. γδ T cells as well as CD4+ CD45RO+ T cells), and the respective ligands can directly modulate their effector functions. PRR play a crucial role in inflammation and host defence, and TLR are already used as novel therapeutic agents. As summarized in Fig. 6, we observed that TLR ligands on their own (except for TLR5 ligand flagellin) are not sufficient to exert a striking effect on freshly isolated γδ T cells. A co-stimulatory effect of combined TCRand TLR1/2/6, -3 or -5 ligand stimulation is required to enhance effector function of freshly isolated human γδ T cells. Moreover, NOD-2 receptor stimulation of freshly isolated γδ T cells does not enhance effector function in the absence of TCR-crosslinking (Marischen et al., Scand J Immunol. 2011). Our results suggested that the combined TCR-TLR ligand/ NOD2 ligand stimulation of γδ T cells could be a strategy to optimize Th1-mediated immune responses as adjuvant in vaccines against viruses or bacteria or could help to improve the therapeutic potential of cancer vaccines (Wesch et al., Cellular and Molecular Life Sciences, 2011). Fig. 6. Direct co-stimulatory effects of TLR ligands on human γδ T cells. Freshly isolated human γδ T cells are activated via their TCR with PAg or antiγδTCR mAb together with a mixture of FSL-1, Pam2CSK4 and Pam3CSK4 (TLR1/2/6-ligands [L]), with poly(I:C) (TLR3-L) or flagellin (TLR5-L). Costimulation significantly enhances the expression of CD69, cytokine/ chemokine or granzyme B production, proliferation or degranulation as indicated. Cytokines presented in brackets are exclusively produced by Vδ2 γδ T cells and not Vδ1 γδ T cells. 62 To get more insights in the regulatory function of inhibitory and co-stimulatory molecules on γδ T cells, we tested their modulation by TLR ligand such as TLR2 and TLR3 ligands. These are still on-going studies. Co-workers in this project: C. Peters, A. Kammel, H.-H. Oberg, D. Wesch Pre-treatment of γδ T cells with TLR2 ligands abrogated their suppressive capacity To analyze the direct suppressive capacity of freshly isolated Vδ2 γδ T cells on CD25- CD4+ responder T cells, we used an APC-free suppression assay where both highly purified subpopulations were co-cultured and stimulated with anti-CD3/anti-CD28/anti-CD2 coated microbeads. Under these conditions, we observed a significant suppression (comparable to the activity of Treg) on CD25- CD4+ responder T cell proliferation. The pre-treatment of γδ T-cells with a mixture of TLR2 ligands (Pam2CSK4, FSL-1 and Pam3CSK4) partially abrogated the suppressive activity of Vδ2 γδ T cells on CD25- CD4+ responder T cells. The TLR2-ligand pretreatment induced an increase in phosphorylation of Akt and Stat3 and thereby an enhanced proliferation as well as an increase in Erk-2-, p38-phosphorylation and NF-κB activation. The increased activation of MAP kinases and NF-κB resulted in an enhanced release of Th1-related cytokines and chemokines by Vδ2 γδ T cells. The suppressive mechanism of Vδ2 γδ T cells was abrogated by TLR2 ligands inducing a strong Th1 response, and abolished the γδ T cellmediated inhibition of Akt and NF-κB phosphorylation in CD25- CD4+ responder T cells. In our study, we took advantage of the PhosflowTM method, which was modified to determine in parallel the phosphorylation of co-cultured Vδ2 γδ T cells and CD25- CD4+ responder T cells. 63 Fig. 7. Phosphorylation of signalling molecules. CD25CD4+ responder T cells were cultured alone or with freshly isolated γδ T cells pre-treated in medium or with a TLR2-L-mixture. Cells were cultured for 3 days after anti-CD2, antiCD3 and anti-CD28 mAb stimulation, followed by subsequently fixation and permeabilization. Phosphorylated signalling molecules were labelled with specific fluochromeconjugated Ab as indicated, and analyzed by flow cytometry. Mean values of the median fluorescence intensity of at least 4 donors are shown. Each symbol presents the data of one donor, and the black bars present the mean value for 4 different experiments. Asterisks indicate statistical significance (* = p ≤ 0.05), (** = p ≤ 0.01) and (n. s. = non-significant). Co-workers in this project: C. Peters, H.-H. Oberg, D. Wesch Comparison between TLR3 ligand and anti-CD28 mAb co-stimulation TLR agonists are used as immune enhancers with the potential to influence proliferation, survival and cytotoxicity of different immune cells, but, additionally, they exert undesirable effects such as enhancement of immunosuppressive function of regulatory T cells and differentiation towards Th2-phenotype in some instances. Therefore, we analyzed the modulation of T cell immune response by a TLR3 agonist [poly(I:C)] and to compare the TLR3 ligand-mediated co-stimulation with the co-stimulation via of the classical co-stimulatory molecule CD28. We observed discernible differences between the two co-stimulatory signals on anti-CD3 mAb activated freshly isolated human CD4 T cells. TLR3 ligand poly(I:C) as a co-stimulus 64 significantly increased NF-κB p65 phosphorylation and interferon regulatory factor (IRF) 7 transcription, thereby enhancing the production of several chemokines (IP-10, MIP1-α/β, RANTES) and granzyme B involved in anti-viral activity. This effect was stronger than costimulation by anti-CD28 mAb. In contrast to poly(I:C), anti-CD28 mAb as a co-stimulus induced a stronger pro-inflammatory response (i.e., TNF-α production) and was essential for induction of proliferation. These results indicate that CD28- and TLR3- signaling differentially influence the CD3-driven anti-viral and pro-inflammatory response in CD4 T cells (Meyer et al., JLB, 2012). Fig. 8. Effects of anti-CD28 mAb and poly(I:C) costimulation on chemokine and TNF-α α release. CD4 T cells (106) were cultured in medium alone, with 50 µg/ml poly(I:C) or with 2 µg/ml anti-CD3 mAb (250 µl/well plate bound) ± 1 µg/ml soluble anti-CD28 mAb as indicated. Poly(I:C) together with anti-CD3 mAb stimulation is represented as ++. Supernatants were collected and analyzed by BDTM Cytometric Bead Array Human Flex Set 48 hours after incubation. Each symbol represents the data from one donor, and the bars present the mean value of five different experiments. The indicated TNFα and anti-viral chemokines were determined. Significances are presented as * (p < 0.05), ** (p < 0.01) and n.s. (nonsignificant). Co-workers in this project: T. Meyer, H.-H. Oberg, S. Adam-Klages, D. Kabelitz, D. Wesch C. 4. Immune Monitoring The quantitative analysis of B cells, NK cells and "regular" CD4+ and CD8+ T cell subsets, by flow cytometry is well established and is offered by our institute as a routine test to clinicians. In contrast, a similar analysis of the rare population of γδ T cells was not established. Therefore, we customized an already existing staining protocol for the flow cytometric analysis of γδ T cells 65 and their subpopulations Vδ1-positive and Vδ2/Vγ9-positive γδ T cells ending up in a one-tube, no-wash protocol using TrueCount tubes and a FACS Canto flow cytometer. By this method, we determined simultaneously the percentage of lymphocyte subsets and the absolute cell counts per µl blood. To validate this new staining protocol, we used the blood of five healthy donors and analyzed the distribution of γδ T cells in parallel with the old method including two washing steps after the staining and the new no-wash method. A comparison of the percentage values generated is shown in Table 1. Since Vδ2/Vγ9-positive γδ T cells display strong, MHC-independent cytotoxic activity against tumor cells, adoptive transfer of ex vivo expanded Vδ2/Vγ9 γδ T cells or their selective in vivo activation substance aminobisphosphonates might be a valuable anti-tumor therapy. During such treatments of cancer patients, the continuous quantitative determination of γδ T cells is extremely important to monitor the therapeutic progress. In the course of several studies (in collaboration with PD Dr. C. Schem and Prof. C. Mundhenke, Clinic of Gynaecology and Obstetrics, UKSH, Kiel), immune monitoring was applied to analyze the presence of γδ T cells in the peripheral blood of cancer patients. First, we determined the absolute cell counts of γδ T cells in patients with mammary carcinoma, who had been treated with zoledronate. Surprisingly, we detected a reduction of Vδ2/Vγ9-positive γδ T cells compared to healthy controls (Fig. 9). Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 with without with without with without with without with without Vδ2 9.8 9.9 2.0 2.0 3.0 3.2 2.6 2.7 2.5 2.4 Vδ2 (not-Vδ1) 9.8 8.7 2.0 1.7 3.4 3.1 2.9 2.5 2.4 1.9 Vδ1 0.8 0.8 0.4 0.4 0.5 0.5 1.1 1.2 0.7 0.5 Vδ1 (not-Vδ2) 1.0 0.9 0.4 0.5 0.8 0.7 1.5 1.2 0.7 0.6 Vδ1+Vδ2 (γδ) 10.6 10.7 2.4 2.4 3.5 3.7 3.7 3.2 3.2 2.9 Vδ1+ not-Vδ1(γδ) 10.6 9.4 2.3 2.1 3.8 3.6 3.9 2.9 2.9 2.4 Vδ2 + not-Vδ2(γδ) 10.9 10.7 2.3 2.3 3.9 4.0 4.0 3.1 3.1 2.8 Table 1: Comparison of percentage of lymphocyte values for γδ T cells of five different healthy donors generated by two different flow cytometric methods with washing steps or without. 66 Fig. 9. Vδ2/Vγ9-positive γδ T cells are reduced in zoledronatetreated mammary carcinoma patients. Comparison of 50 blood samples from 36 patients with mammary carcinoma with 10 blood samples from healthy donors. γδ Vδ1 Vδ2 In a second ongoing study, we determine absolute cell counts in patients with pancreatic cancer. The data collected here will be analyzed in the context of other results on the same patients generated within the Pancreatic Cancer Consortium (PCC, DFG WE 3559/2-1), an interdisciplinary research network in Kiel. A third also still ongoing study again involves patients with mammary carcinoma. This cohort of patients includes individuals freshly diagnosed from cancer and for the first time treated with chemotherapeutics. This treatment is accompanied by two different fitness programs, either strength training or endurance training. A third control group receives chemotherapy only. Among other parameters, the absolute cell counts of αβ and γδ T cells are determined in this project (in cooperation with Prof. C. Mundhenke/ Dr. T. Schmidt, Clinic of Gynaecology and Obstetrics, UKS-H, Kiel). Co-workers in this project: S. Adam, H.-H. Oberg, K.-A. Yoo-Ott, S. Ussat, T.T.H. Ly, D. Wesch C.5 Analysis of CD14/CD18-positive cells in bronchoalveolar lavage samples from neonatal pigs after acute lung injury In collaboration with the group of Prof. M. F. Krause from the Department of Pediatrics at the UKSH, samples of bronchoalveolar lavages from neonatal pigs, which had been treated with controlled acute lung injury in the presence or absence of various substances, were analyzed for the presence of CD14/18-positive monocytes/macrophages by flow cytometry. In this project, it was discovered that treatment with D-myo-inositol-1,2,6-trisphosphate as well as phosphatidylinositol-3,5-bisphosphate reduces the migration of monocyte-derived macrophages into airways thereby contributing to reduced pulmonary edema and improved lung functions. Co-workers in this project: S. Adam, D. Wesch (in cooperation with S. Schütze) 67 C.6. Central cell sorter facility The central cell sorter facility of the Medical Faculty (FACS Aria) which is located in the Institute of immunology is operated by PD Dr. Hans-Heinrich Oberg, Sandra Ussat and KyoungAe Yoo-Ott. The sorter facility is used by institute members and by external users of the UKSH as well as by researchers from other Institutes (e.g. Municipal hospital; Dep. of Internal Medicine, Institute of Zoology, CAU, Biochemistry, CAU). Highlights of 2011: > Final examination of the MD thesis of Kathrin Pietschmann > Daniela Wesch et al.: Best abstract-price at the Joint Annual Meeting of the Italian Society of Immunology, Clinical Immunology and Allergology (SIICA) and the German Society of Immunology (DGfI) in Riccione, Italy 2012: > Final examination of the PhD thesis of Dipl.-Biol. Tim Meyer (Co-Mentoring with Prof. Dr. D. Kabelitz) > Final examination of the PhD thesis of Dipl.-Biol. Juliane Fazio (Co-Mentoring with Prof. Dr. D. Kabelitz & Prof. Dr. S. Adam) > see Research report D.Kabelitz > Habilitation Dr. sc. hum. Hans-Heinrich Oberg K. Pietschmann D D. Wesch T. Meyer J. Fazio H.-H. Oberg Publications 2011 1. Marischen L, Wesch D, Oberg HH, Rosenstiel P, Trad A, Shomali M, Grötzinger J, Janssen O, Tchikov V, Schütze S, Kabelitz D. Functional expression of NOD2 in freshly isolated human peripheral blood γδ T cells. Scand J Immunol 74: 126-34, 2011 2. Hutchinson JA, Riquelme P, Sawitzki B, Tomiuk S, Miqueu P, Zuhayra M, Oberg HH, Pascher A, Lützen U, Janssen U, Broichhausen C, Renders L, Thaiss F, Scheuermann E, Henze E, Volk HD, Chatenoud L, Lechler RI, Wood KJ, Kabelitz D, Schlitt HJ, Geissler EK, Fändrich. Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol. 187:2072-78, 2011 68 3. Koop A, Lepenies I, Braum O, Davarnia P, Scherer G, Fickenscher H, Kabelitz D, AdamKlages S. Novel splice variants of human IKKε negatively regulate IRF3 and NF-κB activation. Eur J Immunol 41: 224-34, 2011 4. Edelmann B, Bertsch U, Tchikov V, Winoto-Morbach S, Jakob M, Adam-Klages S, Kabelitz D, Schütze S. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes. EMBO J 30: 379-94, 2011 5. Paulsen M, Mathew B, Valentin S, Adam-Klages S, Bertsch U, Krammer PH, Lavrik I, Kabelitz D, Janssen O. Modulation of CD4 T cell activation by CD95 co-stimulation. Cell Death Differ 18: 619-31, 2011 6. Kalyan S, Wesch D, Kabelitz D. Aminobisphosphonates and Toll-like receptor ligands: recruiting Vγ9Vδ2 T cells for the treatment of hematologic malignancy. Curr Med Chem 18: 5206-16, 2011 7. Wesch D, Peters C, Oberg HH, Pietschmann K, Kabelitz D. Modulation of γδ T cell responses by TLR ligands. Cell Mol Life Sci 68: 2357-70, 2011. 8. Oberg HH, Juricke M, Kabelitz D, Wesch D. Regulation of T cell activation by TLR ligands. Eur J Cell Biol 90: 582-92, 2011. 2012 1. Boehm AM, Khalturin K, Anton-Erxleben F, Hemmrich G, Klostermeier UC, LopezQuintero JA, Oberg HH, Puchert M, Rosenstiel P, Wittlieb J, Bosch TC. FoxO is a critical regulator of stem cell maintenance in immortal Hydra. Proc Natl Acad Sci U S A. 109: 19697-702, 2012 2. Hemmirch G, Khalturin K, Boehm AM, Puchert M, Anton-Erxleben F, Wittlieb J, Klostermeier UC, Rosenstiel P, Oberg HH, Domazet-Loso T, Sugimoto T, Niwa H, Bosch TC. Molecular signatures of the three stem cell lineages in hydra and the emergence of stem cell function at the base of multicellularity. Mol Biol Evol, 29: 3267-80, 2012 3. Lühr I, Friedl A, Overath T, Tholey A, Kunze T, Hilpert F, Sebens S, Arnold N, Rösel F, Oberg HH, Maass N, Mundhenke C, Jonat W, Bauer M. Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracine signals. Cancer Lett. 325: 175-88, 2012 4. Preuß S, Omam FD, Scheiermann J, Stadelmann S, Winoto-Morbach S, von Bismarck P, Adam-Klages S, Knerlich-Lukoschus F, Lex D, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause MF. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine. J Cell Mol Med 16: 2813-26, 2012 5. Meyer T, Oberg HH, Peters C, Martens I, Adam-Klages S, Kabelitz D, Wesch D. poly(I:C) costimulation induces a stronger antiviral chemokine and granzyme B release in human CD4 T cells than CD28 costimulation. J Leukoc Biol 92: 765-74, 2012 69 6. Preuß S, Stadelmann S, Omam FD, Scheiermann J, Winoto-Morbach S, von Bismarck P, Knerlich-Lukoschus F, Adam-Klages S, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause MF. Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury. Am J Respir Cell Mol Biol 47: 158-69, 2012 7. Richter J, Schlesner M, Hoffmann S, Kreuz M, Leich E, Burkhardt B, Rosolowski M, Ammerpohl O, Wagener R, Bernhart SH, Lenze D, Szczepanowski M, Paulsen M, Lipinski S, Russell RB, Adam-Klages S, Apic G, Claviez A, Hasenclever D, Hovestadt V, Hornig N, Korbel JO, Kube D, Langenberger D, Lawerenz C, Lisfeld J, Meyer K, Picelli S, Pischimarov J, Radlwimmer B, Rausch T, Rohde M, Schilhabel M, Scholtysik R, Spang R, Trautmann H, Zenz T, Borkhardt A, Drexler HG, Möller P, MacLeod RA, Pott C, Schreiber S, Trümper L, Loeffler M, Stadler PF, Lichter P, Eils R, Küppers R, Hummel M, Klapper W, Rosenstiel P, Rosenwald A, Brors B, Siebert R. Recurrent mutation of the ID3 gene in Burkitt Lymphoma identified by integrated genome, exome and transcriptome sequencing. Nat Genet 44: 131620, 2012. E Grants E.1 “Immunotherapy of pancreatic ductal adenocarcinoma with human γδ T lymphocytes” Pancreas Cancer Consortium Kiel: DFG WE 3559/2-1 (Wesch/Kabelitz) 1x E 13, 0,5x E6, 21.000 €/year, from 09/2010 until 08/2013 E.2. „Steigerung der anti-Tumorreaktivität von Vδ2 γδ T Zellen durch bispezifische Antikörper im adoptiven Transfer-Modell in der SCID Maus“ Werner und Klara Kreitz Stiftung (Heiner Oberg) 2011/12; 7.000 € E.3. „Einfluss von Kraft-und Ausdauertraining auf das Immunsystem bei MammaCaPatientinnen“ Sportinterventionsstudie (Kooperation mit Prof. C. Mundhenke/ Dr. T. Schmidt) seit 2012; Anteil: 5.000 € 70 6. Associated Group Dr. phil. II Elgar Susanne Quabius Collaboration with the Department of Prosthodontics, Propaedeutics and Dental Materials, Christian-Albrechts University Kiel, Arnold Heller Str. 16 24105 Kiel, Germany (2011) Collaboration with the Department of Otorhinolaryngology, Head and Neck Surgery, Christian-Albrechts-University Kiel, Arnold Heller Str. 16 24105 Kiel, Germany (2012) During 2011 the projects listed and described in the last research report were finalised and all but the project: “Addition of Matrix Metalloproteinase inhibitors to Primer and/or Adhesive of a commercially available total-etch adhesive bonding system: Effects on dentine MMPactivity as well ass odontoblast survival, immune competence and MMP expression” resulted in publications in peer reviewed international journals (see below). Beginning of 2011 Dr Quabius started a collaboration with PD Dr Markus Hoffmann (Department of Otorhinolaryngology, Head and Neck Surgery), resulting in a co-authorship of a paper discussing the role of a possible surrogate marker for HPV infections of head and neck cancers and the successful application for a grant from the Medical Faculty of the Christian-Albrechts University given to Dr Hoffmann. A Group Leader (extern) PD Dr. Markus Hoffmann, Department of Otorhinolaryngology, Head and Neck Surgery B C Lab Members (intern) Scientist: Dr. phil. II Elgar Susanne Quabius Technician: Hilke Clasen Research Report General Introduction: Head and neck squamous cell carcinoma (HNSCC) represent 7% of all malignant diseases. They are the 6th most common malignancy in humans and account for approximately 70.000 new cases in the USA and Europe, and approximately 10.000 deaths annually. With regard to pathogenesis and clinical behaviour, the existence of two distinct HNSCC entities is generally accepted one being predominantly associated with tobacco and alcohol consumption, the 71 other being predominantly associated with human papillomavirus (HPV) infection. The high risk HPV genotype 16 plays a pivotal role in the pathogenesis of this subset of HNSCC. Despite constant improvements in tumour surgery as well as radio- and chemotherapy, survival rates of HNSCC patients remained largely unchanged over the last decades, with 5 year overall survival rates of 50%. This might partly be due to the fact that there is still no clear strategy of individualization of the conventional therapy regimens, for instance based on the biological properties of the tumour. This, however, appears to be crucial since there is growing evidence that e.g. HPV-positive and HPV-negative HNSCCs respond differently to the various treatment regimes with a clear survival advantage of patients harbouring a HPVinfection. The latter is true despite the fact, that HPV-infection in HNSCC is known to correlate with a higher disease burden of the lateral neck, the strongest negative predictor of survival in HNSCC patients. The human papillomaviruses belong to the Papillomavaviridiae family of viruses. They are capable of infecting mucosal and cutaneous epithelia in a species specific manner and inducing cell proliferation. HPVs are divided into low risk and high risk groups. Low risk HPVs cause wart-like lesions of the skin and anogenital region and the oral mucosa. High risk HPVs are aetiologically associated with cervical, anogenital and head and neck cancers. The means of viral entry into the host cell are discussed controversial, but there is growing evidence of receptor involvement, namely involvement of the receptor annexin 2. Secretory leukocyte protease inhibitor (SLPI) is an 11.7-kDa cystein-rich protein, found in saliva, seminal plasma and in the cervical, nasal, and bronchial mucus, inhibiting serine protease activity. Overexpression of SLPI was reported to promote metastasis in lung carcinoma 3LL-S cells and oral carcinoma KB cells. In addition, overexpression of SLPI in HaCat cells resulted in decreased HPV entry into these cells. This was explained by annexin 2 binding sites being blocked by their natural ligand SLPI. SLPI and p16INK4A immunhistochemistry and HPV-DNA detection for the below mentioned projects were performed in collaboration with the Institute of Pathology, UK SH Campus Kiel. Project 1, 2 and 4 were funded by the Medical Faculty of the Christian-Albrechts University. 72 C 1. Human papillomavirus infection in head and neck cancer: the role of the secretory leukocyte protease inhibitor. The aim of this project was to investigate a possible correlation between HPV-DNA and SLPI Protein expression in HNSCCs (n=54). Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non-malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPVpositive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPVpositive. A correlation between SLPI down regulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases. Fig. 1. Immunoperoxidase staining of HNSCC biopsies and clinically normal mucosa biopsies using antiSLPI antibody. (a) Primary non-metastasizing hypopharyngeal carcinoma exhibits intense cytoplasmic reactivity in contrast to primary metastasizing hypopharyngeal carcinoma (b), (c) no reactivitiy, (d) weak reactivity, (e) moderate cytoplasmic reactivity, (magnification X 400) C 2. The role of the antileukoprotease SLPI in smoking-induced human papillomavirusindependent head and neck squamous cell carcinomas This project aimed to further focus on the role of SLPI in non-HPV-driven HNSCC, investigating tumor tissue and non-neoplastic mucosa from the same patients and from non73 HNSCC patients. Gene- and protein expression of SLPI and gene expression of annexin 2, a SLPI receptor, was analyzed in 36 HNSCC patients (20 smokers; 16 non-smokers). Nonneoplastic mucosa of these HNSCC patients and normal mucosa from 38 non-HNSCC individuals (18 smokers; 20 non-smokers) was analyzed for the same parameters. Furthermore, nasal mucosa tissue of the inferior turbinate (n=10) was incubated with nicotine for SLPI and annexin 2 gene expression. SLPI gene expression in tumor tissue was 109.26±23.08 times higher in smokers versus non-smokers. Non-neoplastic mucosa of smokers showed also higher SLPI gene expression (10.49±1.89-fold increases non-HNSCC; 18.02±3.93-fold increases HNSCC patients). Annexin 2 gene expression was also increased in smokers. A nicotine dependent correlation between SLPI and annexin 2 gene expression (r2=0.18) was shown ex vivo. Five patients, showing low or no SLPI expression, were HPV16 positive. A significant correlation between smoking and SLPI expression in tumors and in mucosa of HNSCC and non-HNSCC patients was established. Together with the finding that all patients with HPV-infection showed no or low SLPI expression, these data support our intriguing hypothesis that smoking induced up-regulated SLPI prevents HPV-infections. Figure 2: Correlation between SLPI and annexin 2 gene expression. The correlations between SLPI and annexin 2 gene expression are depicted for non-neoplastic mucosa tissue from HNSCC patients (A; n=36; r2=0.24), tumor tissue of the same patients (B; n=36; r2=0.26), clinically normal mucosa of non-HNSCC individuals (C; n=38; r2= 0.31) and form ex-vivo experiments using nasal mucosa of the lower turbinate (D; n=10) incubated in 1:10 dilutions of nicotine ranging from 100µM to 0.1µM, plus nonnicotine controls (r2=0.18). Panels A and B show a clear discrimination between patients with and without smoking habit, indicated by the diagonal line; smokers being gathered on the left side, non-smokers on the right. All axis values are cycle threshold corrected for expression levels of the house keeping gene 18S RNA (∆ct). Please note: low ∆ct-values indicate high amounts of mRNA. C 3. HPV in head and neck cancers in Northern Germany - differences in prevalence rates HPV prevalence rates vary significantly depending on the anatomical site of the tumour, the methods of HPV detection applied, and apparently the geographical region the patients live in. HPV rates do not only seem to vary when different continents or countries are compared but even in-between neighbouring areas of a certain geographical region, e.g. Northern Germany. 74 Therefore, we investigated tonsillar and non-tonsillar squamous cell carcinomas from 241 patients treated in 5 different cancer centres in Northern Germany. Participating medical centres were Kiel/Lübeck (63 patients; tonsillar cancers n=23), Oldenburg (52 patients; tonsillar cancers n=24), Hamburg (49 patients; tonsillar cancers n=17), Hannover (42 patients; tonsillar cancers n=23), and Greifswald (35 patients; tonsillar cancers n=23). p16immunohistochemistry was performed and results were correlated to HPV DNA results. HPV16 as only infecting genotype could be detected in 56/241 (23.2%) cases overall, with 42.2% positives in tonsillar cancers and 7.6% in all other cases. Among the centres, prevalence rates ranged in overall cases from 14.3% to 31.4%. In tonsillar cancer cases from 23.5% to 50%, and in all other cases from 0% to 16.7% HPV positives. The highest HPV rates could be detected in Greifswald, Oldenburg and Hannover, whereas the lowest rates were found in Hamburg. Kiel/Lübeck showed 43% and 10% HPV positives in tonsillar and non-tonsillar cases, respectively, representing the median of HPV prevalence rates. 39 of 41 HPV positive cases showed strong p16 expression signals, whereas in 2 cases the p16 expression was weak or missing. The data clearly demonstrate that HPV infection rates vary between geographical regions, even when these regions are in close proximity. Reasons for this finding need to be further elucidated. Since Hamburg – one of Germany’s largest cities shows by far the lowest HPV prevalence rates socioepidemiological reasons might be of importance, however, HPV transmission as a clear consequence of an alleged riskier sexual behaviour can most likely be ruled out. C 4. Effect of nicotine on gene expression levels of the antileukoprotease SLPI on nasal mucosa - an ex vivo approach In clinical studies we have established a correlation between smoking and non-HPV driven HNSCC, with the antileukoprotease (SLPI) playing an important role. Here human nasal mucosa was incubated with bacterial lipopolysaccharide (LPS) or nicotine and gene expression of SLPI and several (pro-) inflammatory cytokines was studied. Both substances stimulate SLPI expression but employ different pathways with different physiological outcome for the tissue. In both cases SLPI was positively correlated with expression of its receptor Annexin 2. LPS, stimulates via TLR4 IL-1ß gene expression which correlates with SLPI expression. In addition, LPS can also stimulate TNF-α production leading either via NFkB to IL-1ß production or FADD mediated into apoptosis, the latter being most likely the case here, since LPS resulted in higher TNF-α expression and stronger correlation with SLPI. Nicotine is known to down-regulate TNF-α and stimulate IL-1ß, resulting in negative and 75 positive correlations with SLPI gene expression respectively. The finding that nicotine induces SLPI expression ex vivo and the strong correlation between SLPI and other nicotine regulated genes in this system, further supports our notion that SLPI plays a role in smokinginduced HPV-independent HNSCC. Studies investigating gene expression levels of e.g. TNFα and IL-1ß in biopsies obtained from HNSCC and non-HNSCC patients with known smoking habits are ongoing. Figure 4: Effect of nicotine on SLPI, IL-1 beta and TNF-alpha gene expression The correlations between SLPI and TNF-α and IL-1ß gene expression respectively, are depicted for nasal mucosa tissue incubated ex-vivo in 1:10 dilutions of nicotine ranging from 100µM to 0.1µM, plus non-nicotine controls. In the presence of nicotine SLPI and TNF-alpha are negatively correlated (r2=0.29) and SLPI and IL1beta are positively correlated (r2=0.21). All axis values are cycle threshold corrected for expression levels of the house keeping gene 18S RNA (∆ct). Note: low ∆ct-values indicate high amounts of mRNA. D Publications (2012) Harder S, Quabius ES, Ossenkop L, Mehl C, Kern M*. Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study. J Clin Periodontol 39:987-94, 2012. Hoffmann M, Tribius S, Quabius ES, Henry H, Pfannenschmidt S, Burkhardt C, Görögh T, Halec G, Hoffmann AS, Kahn T, Röcken C, Haag J, Waterboer T, Schmitt M. HPV DNA, E6*I-mRNA expression and p16INK4A immunohistochemistry in head and neck cancer how valid is p16INK4A as surrogate marker? Cancer Lett 323:88-96, 2012 Quabius ES, Ossenkop L, Harder S, Kern M*. Dental implants stimulate expression of Interleukin-8 and its receptor in human blood-an in vitro approach. J Biomed Mater Res B Appl Biomater 100:1283-8, 2012. Harder S, Quabius ES, Ossenkop L, Kern M*. Assessment of lipopolysaccharide microleakage at conical implant-abutment connections. Clin Oral Investig 16:1377-84, 2012 *First two authors contributed equally. 76 7. Diagnostic Group Prof. Dr. Sabine Adam A Group Leader: Prof. Dr. Sabine Adam (UKSH) B Lab Member: Scientist: PD Dr. Hans-Heinrich Oberg (DFG) Technicians: Kyoung Yoo-Ott (UKSH) Sandra Ussat (Medical faculty) Hoa Ly (Medical faculty) C Research Report: C. 1 Flow Cytometry In many different diseases, the quantitative determination of the immune status, i.e. the composition of the lymphocyte subpopulations within the peripheral blood, is of high diagnostic importance. For example, after tissue transplantation the amount of activated T lymphocytes can indicate begin or exacerbation of an active rejection process. In addition, the exact cell number of T helper cells in the peripheral blood from patients suffering from AIDS provides a decisive indication for therapeutic decisions. Recently, several routine diagnostic tests using flow cytometry have been re-established in our institute. Most importantly, we measure the percentage and absolute cell counts of the lymphocyte subpopulations B-cells, T-cells (divided in CD4-positive T helper and CD8positive cytotoxic T cells), and NK cells in the peripheral blood (immune status). For this test, only 50 µl of EDTA-blood are required. The use of BD Biosciences TrueCount tubes which contain a fixed number of fluorescent microbeads enables, in addition to assessing the percentage of lymphocyte values, the exact determination of cell counts per µl blood. An example for such an analysis is shown in Figure 1. Moreover, we offer the measurement of the percentages of activated T helper (CD4/CD25-double positive) and cytotoxic (CD8/HLADR-double positive) T cells. Increased numbers of activated T cells could be for example an indication for an ongoing transplant rejection. The amount of T cells in bronchoalveolar lavage (BAL), especially the ratio of CD4/CD8 T cells, is a commonly used marker for the diagnosis of sarcoidosis (ratio >3.5) and extrinsic alveolitis (ratio <0.8). Therefore, we offer the flow cytometric analysis of BAL samples for the presence of the different T cell subpopulations. 77 Figure 1: Flow cytometric analysis of whole blood indicating the immune status given as percentage of lymphocyte values and absolute cell counts as cells per µl blood. In sepsis patient, the absolute number of HLA-DR molecules expressed on the surface of monocytes is an indication for the stage of immune paralysis. The QuantiBrite system from BD Biosciences allows for an exact determination of this marker. We now provide this flow cytometric analysis in our institute. A subpopulation of T cells expressing a T cell receptor composed of a γ chain paired with a δ chain (γδ T cells) plays an important role in for example the fight against diverse infections and anti-tumor defense. Among these atypical T cells, the subgroup of Vδ2/Vγ9-expressing γδ T cells displays a marked, MHC-independent cytotoxic activity against tumor cells. In some cancers like mamma carcinoma, the absolute cell numbers of Vδ2/Vγ9 γδ T cells can be strongly reduced, which might be an indication for a diminished anti-tumor defense. Since the determination of absolute cell counts of γδ T cells using the TrueCount system was not available, we established this analysis and compared the staining of whole blood without washing with the well established procedure of the identical staining with two washing steps after the staining. An example of such a comparison is shown in Figure 2. Now, we have started the determination of absolute cell counts of γδ T cells in tumor patients in the context of several clinical studies and can offer this service for diagnostic purposes. 78 A B Figure 2: Comparison of whole blood staining for γδ T cells and subpopulations thereof using the TrueCount system without washing (A) with the conventional staining proceduce including two washing steps (B). Finally, we also provide a quantitative, flow cytometric analysis of the functional release of lysosomes and α-granula by thrombocytes from patients suffering from delayed blood coagulation with an unknown cause. D Publications 2011 Koop A, Lepenies I, Braum O, Davarnia P, Scherer G, Fickenscher H, Kabelitz D, AdamKlages S. Novel splice variants of human IKKε negatively regulate IRF3 and NF-κB activation. Eur J Immunol 41: 224-234, 2011 Edelmann B, Bertsch U, Tchikov V, Winoto-Morbach S, Jakob M, Adam-Klages S, Schütze S. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes. EMBO J 30: 379-394, 2011 Paulsen M, Mathew B, Valentin S, Adam-Klages S, Bertsch U, Krammer PH, Lavrik I, Kabelitz D, Janssen O. Modulation of CD4 T cell activation by CD95 co-stimulation. Cell Death Differ 18: 619-631, 2011 79 2012 Preuß S, Stadelmann S, Omam FD, Scheiermann J, Winoto-Morbach S, von Bismarck P, Knerlich-Lukoschus F, Adam-Klages S, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause MF. Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury. Am J Respir Cell Mol Biol 47: 158-169, 2012 Meyer T, Oberg HH, Peters C, Martens I, Adam-Klages S, Kabelitz D, Wesch D. Costimulation of anti-CD3-activated CD4 T cells with poly(I:C): TLR3 signaling induces a stronger anti-viral immune response in human CD4 T cells than CD28 signaling. J Leukocyte Biol 92: 765-774, 2012 Preuß S, Omam FD, Scheiermann J, Stadelmann S, Winoto-Morbach S, von Bismarck P, Adam-Klages S, Knerlich-Lukoschus F, Lex D, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause MF. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine. J Cell Mol Med 16: 2813-2826, 2012 Richter J, Schlesner M, Hoffmann S, Kreuz M, Leich E, Burkhardt B, Rosolowski M, Ammerpohl O, Wagener R, Bernhart SH, Lenze D, Szczepanowski M, Paulsen M, Lipinski S, Russell RB, Adam-Klages S, Apic G, Claviez A, Hasenclever D, Hovestadt V, Hornig N, Korbel JO, Kube D, Langenberger D, Lawerenz C, Lisfeld J, Meyer K, Picelli S, Pischimarov J, Radlwimmer B, Rausch T, Rohde M, Schilhabel M, Scholtysik R, Spang R, Trautmann H, Zenz T, Borkhardt A, Drexler HG, Möller P, MacLeod RA, Pott C, Schreiber S, Trümper L, Loeffler M, Stadler PF, Lichter P, Eils R, Küppers R, Hummel M, Klapper W, Rosenstiel P, Rosenwald A, Brors B, Siebert R. Recurrent mutation of the ID3 gene in Burkitt Lymphoma identified by integrated genome, exome and transcriptome sequencing. Nat Genet 44: 13161320, 2012 80 8. Outpatient Service Dr. med. Christiane Kling Outpatient Service for Couples Suffering from Infertility A Group Leader: B Group Members: Dr. med. Christiane Kling, Oberärztin Scientist: Christiane Both Secretary: Heike Ebeling Doctor´s assistant: Technician, Quality Management: Annual treatments: Kristina Dudda Andrea Iwersen 456 (2011), 402 (2012) The outpatient department is a centre for conduction of lymphocyte immunotherapy (LIT), a therapeutic approach for women suffering from recurrent abortion or repeated embryo implantation failure in the in-vitro fertilisation programme. Patients are referred to the department from gynaecological practitioners and centres specialised in reproductive medicine in Germany and abroad. The therapy is applied to selected patient groups after immunogenetic evaluation and careful exclusion of all possible genetic, anatomical, or other reasons for infertility. Only couples matching selected inclusion criteria (e.g., age, number of IVF/ICSI cycles, etc.) can be considered. The male partner has to be evaluated according to the guidelines for donor selection in transfusion medicine. The method of lymphocyte separation for therapeutical use is approved according to the guidelines of the German law of medicinal products (Arzneimittelgesetz). Background: Human reproduction is characterised by singleton pregnancies deriving from single oocyte development per menstrual cycle. Embryo implantation failure and miscarriage in the first trimester of pregnancy are fairly common events in the reproductive history of patients. Biological factors referring to maturation of oocytes, fertilisation and embryonic development are important requirements for successful implantation. Immunological preconditions in the endometrium and decidua are comparably stable factors and are taken into consideration only after recurrent implantation failure or repeated miscarriages. Immunotherapy with the partner´s lymphocytes is an approach to enhance immune tolerance towards the embryo by providing contact of the maternal peripheral immune system with paternal antigens which are inherited by the child. In recurrent implantation failure and recurrent abortion, the tolerogenic 81 regulation of the immune system obviously is impaired, but the underlying causes are only partly understood yet. The interaction of pro-inflammatory interleukin-17 producing T cells (TH17 cells) and regulatory T cells (Treg) is important to control physiological processes in immunoregulation. Women suffering from recurrent abortion show elevated levels of TH 17 cells in peripheral blood, and for pregnant women at risk of abortion relatively lower levels of Treg are documented. Mouse models showed that Treg which are induced by contact to paternal antigens are important for maintenance of pregnancy. Treg levels in peripheral blood are also positively associated with pregnancy and birth rates in women treated with IVF. Moreover, in a mouse model spontaneous abortion can be prevented by adoptive transfer of Treg. After LIT with paternal lymphocytes, the percentage of Treg rises in peripheral blood, which may contribute to improved immune regulation. Nevertheless, the principle mode of LIT action is not known. It would be desirable to identify parameters which could provide an individual prognosis for LIT in a given patient. C Ongoing Research: 1. Prospective evaluation of two-year patient outcome after recurrent abortion and lymphocyte immunotherapy (M.D. student) T. Grindel 2. Prospective evaluation of two-year patient outcome after repeated embryo implantation failure and lymphocyte immunotherapy (publication submitted) 3. Characteristics of singleton pregnancies after embryo transfer and spontaneous conception following recurrent IVF implantation failure (M.D. student) T. Leptin 4. Association of parental Killer-cell Immunoglobulin-like Receptors (KIR) and HLA-C genotypes with Recurrent Miscarriage (Collaboration with O. Chazara, L. Farrell, A. Moffett, Department of Pathology, University of Cambridge, U.K.) D Publications: - Kling C, Kabelitz D: Impfen- von der Empirie zur Immunologie. BIUZ 41:375-383, 2011 - Kling C, Carstensen A: Impfungen bei Frauen mit Kinderwunsch. Frauenarzt 53:940950, 2012 82 Appendix 1. Institute Seminars 2011 and 2012 - Invited Speakers 13.01.201 Prof. Dr. Ralf C. Bargou, Medizinische Klinik und Poliklinik II, 1 Universitätsklinikum Würzburg: Tumortherapie mit Bispecific Tumor Engager (BiTE) – Antikörpern 10. 2. 2011 Prof. Dr. Ashley Moffet, Department of Pathology, University of Cambridge: KIR repertoire in recurrent abortion 11. 05. 2011 Prof. Viktor Umansky, PhD, Clinical Cooperation Unit Dermato-Oncology (G300), German Cancer Research Center: Overcoming immunosuppressive melanoma microenvironment in the ret transgenic mouse model 19.05.2011 Prof. Dr. Wolfgang Schamel, Max Planck Institute of Immunobiology and Epigenetics, Freiburg: Activation of the alpha-beta- and the gamma-delta TCR 16. 06. 2011 Prof. Tamás Laskay, Institut für Medizinische Mikrobiologie und Hygiene, UKSH, Campus Lübeck: Inhibition of neutrophil functions by apoptotic cells and intracellular pathogens 30.06.2011 Prof. Dr. Thomas Hünig, Institut für Virologie und Immunbiologie, JuliusMaximilians-Universität Würzburg: Lessons from anti-CD28: Separation from self explains failure of circulating T-cells to predict TGN1412 induced cytokine storm 27. 10. 2011 Prof. Dr. Reinhard Voll, Abteilung für Rheumatologie und Klinische Immunologie der Medizinischen Klinik der Universität Freiburg: Therapeutische Aspekte der NFκB-Inhibition 01. 12. 2011 Dr. Eva Verjans/Kathleen Reiss, Institut für Pharmakologie und Toxikologie, Universitätsklinikum der RWTH Aachen: Pharmacology of the lung: TNF-α induced septic shock is attenuated in acid sphingomyelinase-deficient mice/ Function of cAMP response element modulator in a murine asthma model 19. 01. 2012 Prof. Dr. Ana Claudia Zenclussen, Experimental Obstetrics and Gynaecology, Medical Faculty, Otto-von-Guericke-University Magdeburg: Cellular mechanisms of tolerance acquisition and maintenance during pregnancy 23. 02. 2012 Prof. Dr. Christine Falk, Institut für Transplantationsimmunologie, Medizinische Hochschule Hannover: Tumor immunology and transplant immunology: two sides of the same coin? 83 19. 04. 2012 Prof. Dr. Henning Walczak, Tumour Immunology Unit, Division of Immunology and Inflammation, Imperial College London: Programmed cell death 26. 04. 2012 PD Dr. Kilian Eyerich/Dr. Stefanie Eyerich, Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Technische Universität München: Distinct T cell subsets orchestrate the course of psoriasis and atopic eczema/Human Th22 cells: Differentiation, signal transduction and molecular control mechanisms 03. 05. 2012 PD Dr. Immo Prinz, Institut für Immunologie, Medizinische Hochschule Hannover: IL-17 producing gamma-delta T cells ride the embryonic wave 29.05.2012 Prof. Dr. S. V. Chiplunkar, ACTREC, Tata Memorial Centre, Kharghar, Mumbai, India: Gamma delta T Lymphocytes and anti-tumor immunity: „oral cancer scenario“ 05.07.2012 Prof. Dr. Martin Mempel, Klinik für Dermatologie, Venerologie und Allergologie, Göttingen: IgE repertoire in allergic diseases 13.12.2012 Dr. Eva Verjans, Klinik für Kinder- und Jugendmedizin, Universitätsklinikum Aachen: Function of cAMP response element modulator in a murine asthma model 1a. Scientific Events - organized by institute members 2011 15. Joint Meeting ”Signal Transduction: Receptors, Mediators and Genes” 06th – 09th November 2011 in Weimar 2. Chinese-German Meeting “Immune Intervention: From Basic Research to Clinical Application” 2nd – 4th September 2011 in Beijing, China 2012 16. Joint Meeting ”Signal Transduction: Receptors, Mediators and Genes” 05th – 08th November 2012 in Weimar 84 2. Completed MD and PhD Theses 2011 and 2012 2011 Tim Meyer Dr. rer. nat. Die Rolle von Toll-like Rezeptor 3 bei der Aktivierung primärer humaner CD4+ T-Lymphozyten Malte Puchert Dr. rer. nat. Molekulare und funktionelle Charakterisierung Komponenten der N-SMase-Signaltransduktion Isabell Hellmich Dr. med. Untersuchungen zur Aktivierung von Transglutaminasen durch das Zytokin Tumor Nekrose Faktor Alpha und Cathepsin D in Fibroblasten und Keratinozyten Stephan Porsch Dr. med. Qualitätskontrolle der RNA-Gewinnung mit quantitativer Real-Time-PCR für Vakzinierungsmethoden mittels dendritischer Zellen bei Kopf-Hals-Tumoren 2012 Greta Kathrin Pietschmann Expression und funktionelle Bedeutung Dr. med. like Rezeptoren in γδ-T-Zell-Subpopulationen von von Toll- Bärbel Edelmann Dr. rer. nat. Molecular mechanism of acid sphingomyelinase activation by tumor necrosis factor receptor 1 Juliane Fazio Dr. rer. nat. Charakterisierung von T-Zellsubpopulationen Granulomatose mit Polyangiitis 3. bei Awards 2011 PD Dr. Daniela Wesch 2011 Joint Annual Meeting SIICA and DGfI (Societá Italiana Immunologia, Immunologia Clinica e Allergolog, Deutsche Gesellschaft für Immunologie) – Posterpreis Dipl.-biol. Dipl.-biochem. Justyna Sosna Einladung zum 61. Lindauer Nobelpreisträgertreffen (Stiftung Lindauer Nobelpreisträgertreffen) 2012 M.Sc. Stephan Philipp Gesellschaft für Biochemie und Molekularbiologie e.V - GBM-Innovation-Award for Young Scientists 85 4. Additional Scientific Activities D. Adam: Environmental Protection Officer of the Medical Faculty (preclinical institutes), ChristianAlbrechts-University Kiel Member Work Group Environmental Management, Christian-Albrechts-University Kiel Fachimmunologe Deutsche Gesellschaft für Immunologie (DGfI) Reviewer Boards: Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, Studienstiftung des Deutschen Volkes, Eberhard Karls University Tübingen: fortüne-Programme, University of Ulm: Habilitation-Commission, United States – Israel Binational Science Foundation Reviewer for Scientific Journals: Blood, Oncogene, Journal of Investigative Dermatology, Archives of Biochemistry and Biophysics, Acta Pharmacologica Sinica, Naunyn-Schmiedeberg's Archives of Pharmacology, Radiation Oncology, BMC Cell Biology, Experimental Cell Research O. Janßen: Vice-President (President elect) of the Signal Transduction Society Speaker of the DGfI Study Group Signal Transduction Coordinator of the DGfI Study Groups (2012-2014) Member of the Organizing Committee of the Joint Meeting “Signal Transduction-Receptors Mediators and Genes” 2011 and 2012 Editorial Board: Cell Communication and Signaling BMC/Springer Reviewer for Scientific Journals: Biological Chemistry, Blood, Cancer Immunology Immunotherapy, Cell Communication and Signaling, Cell Death and Differentiation, Clinical Cancer Research, FEBS Letters, Gastroenterology, Gut, Immunology, Immunology Letters, Infection and Immunity, International Archives of Allergy and Immunology, International Immunology, Journal of Leukocyte Biology, Journal of Proteome Research, Leukemia, Leukemia and Lymphoma, Molecular Cancer Research, Molecular Cancer Therapeutics, PLOS One, Proteomics, Oncogene, Scandinavian Journal of Immunology, Science Signaling, Viral Immunology Reviewer for Funding Organizatons: Deutsche Forschungsgemeinschaft (German Research Foundation) Fonds zur Förderung der wissenschaftlichen Forschung (FWF) German-Israeli Foundation for Scientific Research and Development Studienstiftung des Deutschen Volkes The Wellcome Trust Wilhelm-Sander-Foundation 86 D. Kabelitz: Deutsche Gesellschaft für Immunologie (DGfI); President 2011 - 2012 Member of the Council and Chairman of the Scientific Committee of the "Deutsche Gesellschaft für Autoimmun-Erkrankungen" (German Association for Autoimmune Diseases, DGfAE) Chairman, Scientific Advisory Board IZKF Würzburg Honorary Member of the Scandinavian Society for Immunology (SSI) Member of the Ethik-Kommission, Ärztekammer Schleswig-Holstein Editorial Boards: International Archives of Allergy and Immunology, Frontiers in Bioscience, Deutsche Medizinische Wochenschrift, Recent Patents on Anti-Infective Drug Discovery, Scandinavian Journal of Immunology, Immunotherapy Reviewer Boards: Bundesministerium für Bildung und Forschung (BMBF/DLR), Austrian Academy of Sciences (APART), Mildred-Scheel-Stiftung (Deutsche Krebshilfe), National Science Foundation, USA, Human Frontier Science Program, Israel Science Foundation, German-Israeli, Foundation for Scientific Research and Development, Ministero dell´Università e delle Ricerca (MIUR), Italy, Associazione Italiana per la Ricerca sol Cancro (AIRC), Italy, Vienna Science and Technology Fund (WWTF), Austria, Werner und Klara Kreitz-Stiftung S. Schütze: Reviewer Boards: Deutsche Forschungsgemeinschaft (German Research Foundation), Dutch Cancer Society Reviewer for Scientific Journals: Cell Death and Differentiation, Journal of Biological Chemistry, International Journal of Cancer, Journal of Cancer Research and Clinical Oncology, International Journal of Nanomedicine, Journal of Investigative Dermatology, BBA Molecular Cell Research, Molecular and Cellular Biology, FEBS-letters, FASEB-Journal. D. Wesch: Radiation protection officer of the Institute of Immunology, Medical Faculty, ChristianAlbrechts-University (CAU) Kiel since 2011 Coordinator of the leukocytes-concentrates distribution for all research groups of the Medical Faculty, CAU, Kiel (authorized by the Transfusion-Medicine of the Medical Faculty, CAU, Kiel) Collaboration in the academic female network of the CAU, Kiel Member of the Organizing Committee of the γδ T Cell Conference 2012 in Freiburg, Germany 87 Reviewer for Scientific Journals: Blood, Cancer Research, Current Medicinal Chemistry, European Journal of Immunology, FEMS Immunology & Medical Microbiology, Journal of Immunology, Journal of Leukocyte Biology, Mediators of Inflammation, PLOS One Reviewer for Funding Organizations: The Wellcome Trust 5. Impactfactors and Grants (Summary 2011 and 2012) Cumulated ISI Impact Factors Grant Support (peer-reviewed) in € per year Grant Support (non-reviewed, e.g. Industry, Medical Faculty) in € per year 2011 84 879.128 96.700 2012 137 721.554 20.000 6. Publications 2011 and 2012 2011 Bertsch U, Edelmann B, Tchikov V, Winoto-Morbach S, Schütze S. Compartmentalization of TNF receptor-1 signaling: TNF-R1-associated caspase-8 mediates activation of acid sphingamyelinase in late endosomes. Adv Exp Med Biol, 691: 605-16, 2011 Edelmann B, Bertsch U, Tchikov V, Winoto-Morbach S, Perrotta C, Jakob M, Adam-Klages S, Kabelitz D, Schütze S. Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNFR1 receptosomes. EMBO J, 30:379-394, 2011 Fagin U, Csernok E, Müller A, Pitann S, Fazio J, Krause K, Bremer P, Wipfler-Freissmuth E, Moosig F, Gross WL, Lamprecht P. Distinct proteinase 3-induced cytokine patterns in Wegener´s granulomatosis, Churg-Strauss syndrome, and healthy controls. Clin Exp Rheumatol. 29: 57-62, 2011 Friedrichs B, Siegel S, Reimer R, Barsoum A, Coggin jr J, Kabelitz D, Heidorn K, Schulte C, Schmitz N, Zeis M. High expression of the immature laminin receptor protein correlates with mutated IGVH status and predicts a favorable prognosis in chronic lymphocytic leukemia. Leukemia Res, 35: 721-729, 2011 88 Geissen M, Leidel F, Eiden M, Hirschberger T, Fast C, Bertsch U, Tavan P, Giese A, Kretzschmar H, Schatzl HM, Groschup MH. From high-throughput cell culture screening to mouse model: Identification of new inhibitor classes against prion disease Chem Med Chem 6: 1928-1937 Hutchinson JA, Riquelme P, Sawitzki B, Tomiuk S, Miqueu P, Zuhayra M, Oberg HH, Pascher A, Lützen U, Janssen U, Broichhauesen C, Renders L, Thaiss F, Scheuermann E, Henze E, Volk HD, Chatenoud L, Lechler RI, Wood KJ, Kabelitz D, Schlitt HJ, Geissler EK, Fändrich F. Cutting edge : Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol 187: 2072-2078, 2011 Kabelitz D, Editorial: The German Collaborative Research Center 415 „Specificity and Pathophysiology of Signal Transduction Pathways“. Eur J Cell Biol, 90:449, 2011 Kabelitz D. γδ T-cells: cross-talk betwen innate and adaptive immunity. Cell Mol Life Sci 68; 2331-2333, 2011 Kalyan S, Wesch D, Kabelitz D. Aminobisphosphonates and Toll-like receptor ligands: recruiting Vγ9Vδ2 T cells for the treatment of hematologic malignancy. Curr Med Chem 18: 5206-5216, 2011 Kling C, Kabelitz D. Impfen - von der Empirie zur Immunologie. Biol. Unserer Zeit 41: 375-383, 2011 Koop A, Lepenies I, Braum O, Davarnia P, Scherer G, Fickenscher H, Kabelitz D, Adam-Klages S. Novel splice variants of human inhibitor of κB kinase εnegatively regulate inhibitor of κB kinase ε−ιnduced IFN regulatory factor 3 and NF-κb activation. Eur J Immunol, 41 1-11, 2011 Lamprecht P, Kabelitz D. T-Zellen bei ANCA-assozierten Vaskulitiden. Z Rheumatol 70:698-700, 2011 Lettau M, Paulsen M, Schmidt H, Janssen O. Insights into the molecular regulation of FasL (CD178) biology. Eur J Cell Biol. 90:456-66, 2011 Marischen L, Wesch D, Oberg HH, Rosenstiel P, Trad A, Shomali M, Grötzinger J, Janssen O, Tchikov V, Schütze S, Kabelitz D. Functional expression of NOD2 in freshly isolated human peripheral blood γδ T cells. Scand J Immunol 74:126-134, 2011 Nguyen XH, Lang K, Adam D, Fattakhova G, Föger N, Kamal A, Prilla P, Mathieu S, Wagner C, Mak T, Chan AC, Lee KH. Toso regulates the balance between apoptotic and non-apoptotic death receptor signaling by faciliating RIP1 ubiquitination. Blood 118: 598-608, 2011 89 Oberg HH, Juricke M, Kabelitz D, Wesch D. Regulation of T cell activation by TLR ligands. Eur J Cell Biol, 90: 582, 2011 Paulsen M, Valentin S, Mathew B, Adam-Klages S, Bertsch U, Lavrik I, Krammer PH, Kabelitz D, Janssen O. Modulation of CD4+ T cell activation by CD95 costimulation. Cell Death Differ, 18: 619-631, 2011 Paulsen M, Janssen O. Pro- und anti-apoptotic CD95 signaling in T cells. Cell Commun Signal, 9: 7, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Leippe M, Lucius R, Janssen O. Effector granules in human T lymphocytes: - The luminal proteome of secretory lysosomes from human T cells. Cell Commun Signal, 9: 4, 2011 Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Leippe M, Lucius R, Janssen O. Effector granules in human T lymphocytes: Proteomic evidence for two distinct species of cytotoxic effector vesicles. J Proteome Res, 10: 1603-1620, 2011. Tchikov V, Bertsch U, Fritsch J, Edelmann B, Schütze S. Subcellular compartmentalization of TNF receptor-1 and CD95 signaling pathways. Eur J Cell Biol, 90: 467-475, 2011 Wesch D, Peters C, Oberg HH, Pietschmann K, Kabelitz D. Modulation of γδ T cell responses by TLR ligands. Cell Mol Life Sci 68: 2357-2370, 2011 2012 von Bismarck, P., Winoto Mohrbach, S., Herzberg, M., Uhlig, U., Schütze, S., Lucius, R., Krause, M. F. IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model. Pulm Pharmacol Ther, 25: 228-235, 2012 Boehm AM, Khalturin K, Anton-Erxleben F, Hemmrich G, Klostermeier UC, Lopez-Quintero JA, Oberg HH, Puchert M, Rosenstiel P, Wittlieb J, Bosch TC. FoxO is a critical regulator of stem cell maintenance in immortal Hydra. Proc Natl Acad Sci U S A, 109:19697-19702, 2012 Deng X, Hahne T, Schröder S, Redweik S, Nebija D, Schmidt H, Janssen O, Lachmann B, Wätzig H. The challenge to quantify proteins with charge trains due to isoforms or conformers. Electrophoreses, 33: 263-9, 2012 Durchfort N, Verhoef S, Vaughn MB, Shresta R, Adam D, Kaplan J, Ward DM. The enlarged lysosomes in beige j cells result from decreased lysosome fission and not increased lysosome fusion. Traffic, 13: 108-119, 2012 90 Harder S, Quabius ES, Ossenkop L, Mehl C, Kern M. Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay. A preliminary study. J. Clin. Peridontol, 39: 987-994, 2012 Hemmirch G, Khalturin K, Boehm AM, Puchert M, Anton-Erxleben F, Wittlieb J, Klostermeier UC, Rosenstiel P, Oberg HH, Domazet-Loso T, Sugimoto T, Niwa H, Bosch TC. Molecular signatures of the three stern cell lineages in hydra and the emergence of stem cell function at the base of multicellularity. Mol Biol Evol, 29:3267-3280, 2012 Hoffmann M, Tribius S, Quabius ES, Henry H, Pfannenschmidt S, Burkhardt C, Görögh T, Halec G, Hoffmann AS, Kahn T, Röcken T, Haag J, Waterboer T, Schmitt M. HPV DNA, E6*I-mRNA expression and p16 INK4A immunohistochemistry in head and neck cancer – How valid is p16 INK4A as surrogate marker? Cancer Lett, 323: 88 – 96, 2012 Kabelitz D. Toll-like Rezeptoren – Erkennungsrezeptoren des angeborenen Immunsystems und therapeutische Zielstrukturen. Med Monatsschr Pharm, 35:238-244, 2012 Kabelitz D. CD277 takes the lead in human γδ T-cell activation. Blood, 120: 2159-2161, 2012 Kabelitz D, He W. The multifunctionality of human Vγ9Vδ2 γδ T cells: clonal plasticity or distinct subsets? Scand J Immunol, 76: 213-222, 2012 Kling C, Carstensen A. Impfungen bei Frauen mit Kinderwunsch Frauenarzt, 10: 940-950, 2012 Klingseisen L, Ehrenschwender M, Heigl U, Wajant H, Hehlgans T, Schütze S, Schneider-Brachert W. E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin. PLoS ONE, 6, e38348, 2012 Lehle K, von Suesskind-Schwendi M, Diez C, Michl M, Geissler EK, Wottge HU, Schmid C, Hirt SW. Relevance of maintenance tiple-drug immunosuppression to bridle the amplification of rat cytomegalovirus infection after experimantal lung transplantation. Transpl Infect Dis, 14: 649-656, 2012 Lühr I, Friedl A, Overath T, Tholey A, Kunze T, Hilpert F, Sebens S, Arnold N, Rösel F, Oberg HH, Maass N, Mundhenke C, Jonat W, Bauer M. Mammary fibroblasts regulate morphogenesis of normal and tumorigenic breast epithelial cells by mechanical and paracine signals. Cancer Lett, 325: 175-188, 2012 Louis-Dit-Sully C, Kubatzky KF, Lindquist JA, Blattner,C, Janssen O, Schamel WW. Signal transduction meets systems biology Cell Commun Signal, 10: 11, 2012 91 Meyer T, Oberg HH, Peters C, Martens I, Adam-Klages S, Kabelitz D, Wesch D. poly(I:C) costimulation induces a stronger antiviral chemokine and granzyme B release in human CD4 T cells than CD28 costimulation. J Leukoc Biol, 92: 765-774, 2012 Nguyen X-H, Fattakhova G, Lang PA, Lang KS, Adam D, Föger N, Lee K-H. Response: antiapoptotic function of Toso (Faim3) in death receptor signaling. Blood, 119: 1790-1791, 2012 Philipp S, Jakoby T, Tholey A, Janssen O, Leippe M, Gelhaus C. Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2-D gel electrophoresis. Electrophoresis, 33: 1120-1128, 2012 Philipp S, Oberg HH, Janssen O, Leippe M, Gelhaus C. Isolation of erythrocytes infected with viable early stages of plasmodium falciparum by flow cytometry Cytometry A, 81: 1048-1054, 2012 Polier G, Neumann J, Thuaud F, Ribeiro N, Gelhaus C, Schmidt H, Giaisi M, Köhler R, Müller WW, Proksch P, Leippe M, Janssen O, Désaubry L, Krammer PH, Li-Weber M. The natural anti-cancer compounds rocaglamides inhibit the Raf-MEK-ERK pathway by targeting prohibitin 1 and 2. Chem Biol, 19: 1093-1104, 2012 Preuß S, Stadelmann S, Omam FD, Scheiermann J, Winoto-Morbach S, von Bismarck P, KnerlichLukoschus F, Adam-Klages S, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause M.F. Inositoltrisphosphate reduces alveolar apoptosis and improves lung function in neonatal lung injury. Am J Resp Cell Mol Biol, 47: 158-169, 2012 Preuß S, Omam FD, Scheiermann J, Stadelmann S, Winoto-Morbach S, von Bismarck P, AdamKlages S, Knerlich-Lukoschus F, Lex D, Wesch D, Held-Feindt J, Uhlig S, Schütze S, Krause MF. Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine. J Cell Mol Med, 16: 2813-26, 2012 Quabius ES, Ossenkop L, Harder S, Kern M. Dental implants stimulate expression of Interleukin-8 and its receptor in human blood – An in vitro approach. J Biomed Mater Res B, 100: 1283-1288 Richter J, Schlesner M, Hoffmann, S, Kreuz M, Leich E, Burkhard B, Rosolwski M, Ammerpohl O, Wagener R, Bernhart S, Lenze D, Szczepanowski M, Paulsen M, Lipinski S, Russel RB, AdamKlages S, Apic G, Claviez A, Hasenclever D, Hovestadt V, Hornig N, Korbel JO, Kube D, Langenberger D, Lawerenz C, Lisfeld J, Meyer K, Picelli S, Pischimarov J, Radlwimmer B, Rausch T, Rohde M, Schilhabel M, Scholtysik R, Spang R, Trautmann H, Zenz T, Borkhardt A, Drexler HG, Möller P, MacLeod RAF, Pott C, Schreiber S, Trümper L, Loeffler M, Stadler PF, Lichter P, Eils R, Küppers R, Hummel M, Klapper W, Rosenstiel P, Rosenwald A, Brors B, Siebert R, fort he ICGC MMML-Seq Project. Recurrant mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcription sequencing Nat Genet, 44: 160-170, 2012 92 Samapati R, Yang Y, Yin J, Stoerger C, Aren, C, Dietrich A, Gudermann T, Adam D, Wu S, Freichel M, Flockerzi V, Uhlig S, Kuebler WM. Lung endothelial Ca2+ and permeability response to PAF is mediated by acid sphingomyelinase and TRPC6. Am J Respir Crit Care Med, 185: 160-170, 2012 93