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: [email protected]
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 εnegatively 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