XXIX Meeting of the Swiss Immunology PhD students
April 2 – April 4 2012
Schloss Wolfsberg
Generously sponsored by:
The Swiss Society for Allergology and Immunology, the Union Bank of
Switzerland and the “Kontaktgruppe für Forschungsfragen” (Roche, Novartis,
Merck Serono)
WOLFSBERG MEETING
April 2 – April 4, 2012
Bus transfer
Monday, April 2, 2012
from Weinfelden railway station to Schloss Wolfsberg
train departure Zurich at 12.07 h
arrival Weinfelden at 12.58 h
Wednesday, April 4, 2012
from Schloss Wolfsberg to Weinfelden railway station
departing Schloss Wolfsberg at 13.15 h
train departure to Zurich at 13.59 h
CONFERENCE PROGRAM
Sw iss Immunology PhD Meeting (2012)
Monday, April 2
13:00-14:00 Check-in
14:00-14:05 Welcome
Session 1
14:05-15:15
1° and 2° lymphoid organs, cell migration Chair: Christoph Müller
14:05-14:19 Angela Bosch - Uni Basel
The role of continued Foxn1 expression in thymus organogenesis and maintenance
14:19-14:33 Chen-Ying Yang - Uni. de Lausanne
What happens to the network of T zone fibroblastic reticular cells during lymph node swelling?
14:33-14:47 Qian Chai - Institute of Immunobiology
Fibroblastic Reticular Cells coordinate structural dynamics of Lymph Nodes during viral infection
14:47-15:01 Romain BALLET - CMU
The Junctional Adhesion Molecule C (JAM-C) regulates dendritic cell migration in inflammation.
15:01-15:15 Amiel Olivos - CHUV
REGULATION OF DENDRITIC CELL MIGRATION BY MACROPHAGHE MIGRATION INHIBITOR FACTOR
Session 2
15:15-15:45
Innate immunity, inflammation Chair: Christoph Müller
15:15-15:29 Jeremy Loyau - Novimmune
Structure-function relationship between 15C1 and TLR4
15:29-15:43 Sebastian Heer - ETHZ
Target gene(s) of the transcription factor Nrf2 mediate inflammasome activation.
15:45-16:00 Coffee break
Session 3
16:00-16:50
Innate immunity, inflammation (2) Chair: Christoph Müller
16:00-16:12 Pascal Ziltener - ETH Zürich
The role of innate immunity in control of Legionella pneumophila lung infection
16:12-16:24 deepa mohanan - USZ
Inhibition of inflammasomes allows BCG to escape host immune responses by decreased phagosome
maturation.
16:24-16:36 Patrycja Konieczna - SIAF Swiss Institute of Allergy and Asthma Research
Plasmacytoid dendritic cells induce Foxp3 regulatory T cells in response to Bifidobacterium infantis
16:36-16:48 Md Mahbubul Morshed - University of Bern
Thymic stromal lymphopoietin (TSLP) stimulates eosinophil extracellular trap release
Session 4
16:50-17:40
Allergy Chair: Ton Rolink
16:50-17:02 Tess Brodie - Institute for Research in Biomedicine
Distribution of allergen specific cells in diverse T cell subsets for seasonal and non seasonal allergens
17:02-17:14 Eva Gollwitzer - CHUV-UniL
Influence of Respiratory Syncytial Virus infection on allergic airway responses depends on age
17:14-17:26 Helga Pawelski - ETH - Integrative Biology
IL-21 promotes Th2 allergic airway responses by inhibition of Foxp3+ T regulatory cells
17:26-17:38 Kerstin Wanke - SIAF
Regulation of Bronchial Epithelium Integrity and Tight Junctions by Regulatory T cells in allergic airway
disease
17:45-18:30 UBS Presentation
18:45-20:00 Dinner
Session 5
20:10-21:30
Allergy (2) Chair: Ton Rolink
20:10-20:23 Mattia Garbani - Swiss Institute for Allergy and Asthma Research
Dendritic cells targeting and improved cell penetration in allergen-specific immunotherapy
20:23-20:36 Moira Prati - SIAF
Targeting the Extracellular Membrane-Proximal Domain of IgE memory B cell
20:36-20:49 Susanne Hoesli - University of Bern
Cytosolic vacuolization of eosinophils: Involvement of CD11b
20:49-21:02 Ursina Gurzeler - Institute of Pharmacology
A System For Quantitative Production Of Murine Basophils Ex Vivo
21:02-21:15 patrick buschor - University Institute of Immunologie
An Fc-engineered anti-IgE-DARPin fusion protein shows increased inhibition of ongoing allergic reactions
21:15-21:28 Stephan Lloyd Watkins - InselSpital-University of Bern
Computational, Biochemical and Cellular Elucidation of Non-hapten Pharmaceutical Interactions (p-i) in
Adverse Drug Hypersensitivity
Tuesday, April 3
07:00-08:00 Breakfast
Session 6
08:00-10:00
Adaptive immunity in infection Chair: Annette Oxenius
08:00-08:13 Josh Crouse - ETH Zurich
Direct type I IFN signaling on CD8 T cells protects them from NK cell mediated killing
08:13-08:26 Sandra Kallert - University of Geneva
Shaping of the immune response in persistent viral infections
08:26-08:39 Mai Matsushita - ETH-Molecular Health Science
Glutathione Peroxidase 4 is pivotal for maintenance of peripheral CD8+T cell pool and anti-viral CD8+ T
cell responses
08:39-08:52 Iwana Schmitz - Institute of Integrative Biology
IL-21 restricts virus-driven Treg cell proliferation to prevent T cell exhaustion and viral persistence in
chronic LCMV infection
08:52-09:05 Christoph Schneider - ETH Zürich
IL-9 is a potent suppressor of antiviral T cell responses
09:05-09:18 Daniel Utzschneider - CHUV
Recovery of functional T-cells from LCMV clone 13 infected mice
09:18-09:31 Gregor Bedenikovic - ETH Zürich
THE ROLE OF “T HELP” DURING SECONDARY CD8+ T CELL RESPONSES IN ACUTE VIRAL INFECTIONS
09:31-09:44 Jovana Cupovic - Kantonal Hospital St. Gallen
Tuning TCR avidity of virus-specific CD8+ T cells
09:44-09:57 Stéphanie Longet - Centre Hospitalier Universitaire Vaudois
A murine genital-challenge model is a sensitive measure of protective antibodies against human
papillomavirus infection
10:00-10:20 Coffee break
Session 7
10:20-11:00
Adaptive immunity to infection (2) Chair: Annette Oxenius
10:20-10:34 Kerstin Weidner - Microbiology/ETH
Antigen presenting cells involved in Th17 cell priming during Candidiasis
10:34-10:48 Sonia Liliana Bastidas Patino - ETH Zürich
THE IMPACT OF HIV-1 REPLICATION ON THE ACTIVATION OF CD8+ T CELLS WITH SPECIFICITIES FOR
DIFFERENT ANTIGENS
10:48-11:02 Kathrin Zimmermann - ETH Zürich
Co-stimulatory effect of the HIV-1 envelope glycoprotein on CD4+ T cells during chronic HIV-1 infection
Session 8
11:00-12:00
Selection and autoimmunity Chair: Maries van den Broek
11:00-11:14 Carlos Mayer - Universität Basel
About the Role of Dicer-dependent miRNA in the Thymus
11:14-11:28 Anja Nusser - University of Basel
The amount of self-antigen determines the effector function of murine T cells escaping negative selection
11:28-11:42 Sarah Enouz - CHUV
Characterizing autoreactive CD8+ T cells that bypass negative selection.
11:42-11:56 Stanislav Pantelyushin - UZH
Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice
12:00-13:00 Lunch
13:15-15:40 Excursion
15:40-16:00 Coffee break
Session 9
16:00-18:30
Poster Chair: no chair
18:30-20:00 Dinner
20:00-23:20 After dinner sports and other activities
Wednesday, April 4
07:00-08:00 Breakfast
Session 10
08:00-09:30
T cell activation / polarization Chair: Walter Reith
08:00-08:13 Jean Enno Charton - Université de Lausanne
Malt1 and Caspase-10: Proteases Essential for Lymphocyte Activation
08:13-08:26 Camilla Basso - Institute for research in Biomedicine
Cervical lymph nodes and the pivotal role in the CCR6+ T helper cell priming.
08:26-08:39 simone becattini - institute for research in biomedicine
Priming of polarized human T cells by microbes
08:39-08:52 Junhua Wang - Institute of parasitology
Course of the Th-1, Th-2 and Th-17 Cytokines and of Associated Chemokines Involved in the Periparasitic
Recruitment of Cells in Echinococcus multilocularis Infection
08:52-09:05 Peter Bretscher - ETH - Integrative Biology
Oxidized phospholipid signaling in microbial infection and inflammatory disease
09:05-09:18 Natascha Wuillemin - University Hospital Bern
Mechanisms of T cell stimulation by flucloxacillin in HLA-B*5701+ flucloxacillin-naïve individuals Natascha
Wuillemin1, Jacqueline Adam1, Stefano Fontana2, Werner J. Pichler1, Daniel Yerly1 1 Clinic for Rheumatology
and Clinical Immunology / Allergology, University Hospital of Bern, 3010 Bern, Switzerland 2 Regional Blood
Transfusion Service of the Swiss Red Cross, 3008 Bern, Switzerland
09:18-09:31 Federico Mele - Institute for research in biomedicine
Phenotype and function of pathogen-specific human CD4+ memory T cells
Session 11
09:30-10:00
Immune regulation Chair: Walter Reith
09:30-09:43 Ilaria Mosconi - EPFL
Role of Thymic stromal lymphopoietin (TSLP) signaling in intestinal immune homeostasis
09:43-09:56 Nina Suhartha - Pharmacology
IFN-α modulates the expression of the gut-homing receptor α4β7
10:00-10:20 Coffee break
Session 12
10:20-10:40
Immune regulation (2) Chair: Walter Reith
10:20-10:33 Franziska Zabel - University Hospital Zurich
Primary and memory B cell responses to Qβ-VLP in mice
Session 13
10:40-12:00
Tumor immunity Chair: Maries van den Broek
10:40-10:53 Ali Bransi - USZ
A novel model for inducible autochthonous cancer to study local immune subversion
10:53-11:06 Sara Burkhard - Experimental Immunology
Impact of innate lymphocytes on the tumor microenvironment
11:06-11:19 Sergio Haller - University of Lausanne
Augmentation of antitumor immunity applying a semi-allogeneic dendritic cell line
11:19-11:32 Christian Hagedorn - University Hospital Zurich
Tumour therapy by redirected Fibroblast Activation Protein-a specific CD8+ T cells
11:32-11:45 Cristina Riccadonna - Geneva University Hospitals and University of Geneva
Modulation of astrocytoma-cytotoxic T lymphocyte interactions by chemotherapy
12:00-13:00 Lunch
13:15-13:15 Departure
Last name
Al Sayed
First name
Mohamad
Institution
DKF
Department
Tumor Immunology
Address
Murtenstrasse 35
Zip code
3010
City
Bern
Email
mohamad.alsayed@dkf.unibe.ch
BALLET
Romain
CMU
Pathology Immunology
Rue Michel Servet, 1
1211
Geneva
ballet.r@gmail.com
Basso
Camilla
Cellular Immunology
Via Vincenzo Vela 6
6500
Bellinzona
camilla.basso@irb.unisi.ch
Bastidas Patino
Sonia Liliana
Institute
for
research in
ETH Zürich
sonia.bastidas@micro.biol.ethz.ch
simone
bellinzona
simone.becattini@irb.unisi.ch
Bedenikovic
Gregor
institute
for
research in
ETH Zürich
ETH Hönggerberg, HCI G 8093
405 Wolfgang-Pauli Str.
via vincenzo vela 6
6500
Zurich
becattini
Institute
of
Microbiology, D-BIOL
immune regulation lab
Bosch
Angela
Uni Basel
Botelho
Natalia
Bransi
Ali
Bretscher
Peter
Brodie
Tess
Burkhard
Sara
buschor
patrick
Chai
Qian
Charton
Jean Enno
Coutaz
Manuel
Crouse
Josh
Cupovic
Jovana
Enouz
Sarah
Fertig
Marina
Institute of Microbiology Wolfgang-Pauli-Strasse
10, HCI G405
Biomedicine
Mattenstrasse 28
8093
Zürich
gregor.bedenikovic@micro.biol.ethz.ch
4058
Basel
angela.bosch@unibas.ch
Ludwig Institute
for Cancer
USZ
University of Lausanne
Ch de Boveresses 155
1066
Epalinges
natalia.botelho@unil.ch
Oncology
Wagistrasse 14
8952
Schlieren
ali.bransi@hispeed.ch
ETH - Integrative
Biology
Institute
for
Research in
Experimental
Immunology
University
Institute of
Institute
of
Immunobiology
Université de
Lausanne
University of
Lausanne
ETH Zurich
d-UWIS
Wagistrasse 27
8952
Schlieren
peter.bretscher@env.ethz.ch
Cellular Immunology
Via Vincenzo Vela 6
6500
Bellinzona
tess.brodie@irb.usi.ch
Pathology
Winterthurerstrasse 190 8057
Zürich
sara.burkhard@neuroimm.uzh.ch
DOLS
Sahlihaus 2
3010
Bern
buschor@students.unibe.ch
Kantonsspital St.Gallen
Institut
für
Immunbiologie Haus 09
Chemin des Boveresses
155
F512A
Ch.
des
Boveresses 155
Wolfgang-Pauli str. 10
9007
St Galeen
qian.chai@kssg.ch
1066
Epalinges
jeanenno.charton@unil.ch
1066
Epalinges
manuel.coutaz@unil.ch
8093
Zurich
josh.crouse@micro.biol.ethz.ch
St. Gallen
jovana.cupovic
Epalinges
sarah.enouz@chuv.ch
Fribourg
marina.fertig@unifr.ch
Departmement
Biochimie
Department
Biochemistry
Microbiology
de
of
Kantonal Hospital Institut
of
Rorschacherstrasse 95
9007
St. Gallen
Immunobiology IMMBIO
CHUV
IAL
Chemin
des
1066
Boveresses,155
University of
Department of Medicine Chemin du Musée 5 2nd 1700
Fribourg
/ MED3
floor/ room 2.105
Garbani
Mattia
Gehrig
Jasmine
Gollwitzer
Eva
Govender
Lerisa
Gurzeler
Ursina
Hagedorn
Christian
Haller
Sergio
Hartley
Mary-Anne
Heer
Sebastian
Hoesli
Susanne
Kallert
Sandra
Konieczna
Patrycja
Kovtonyuk
Larisa
Longet
Stéphanie
Loyau
Jeremy
Mager
Lukas
Matsushita
Mai
Mayer
Swiss Institute for Molecular Allergology
Allergy and
Ludwig Institute LUDWIG
for Cancer
CHUV-UniL
Pneumologie
Oberestr. 22
7270
Davos Platz
mattia.garbani@siaf.uzh.ch
1066
Lausanne
jasmine.gehrig@unil.ch
1011
Lausanne
eva.gollwitzer@chuv.ch
Transplantation
Immunopathology
faculty of medicine
Ch. des Boveresses 155
CH-1066 Epalinges
CHUV Laboratoire de
Pneumologie BH19.105
Institut universitaire de
pathologie - IUP Bureau
Friedbühlstrasse 49
1011
Lausanne
lerisag@hotmail.com
3010
Bern
ursina.gurzeler@pki.unibe.ch
Oncology
Sternwartsrasse 14
8091
Zurich
chagedor@student.ethz.ch
Chemin de Boveresses
155
Chemin des Boveresses
155
ETHZ
D-UWIS
Molecular Biomedicine
Wagistrasse 27
University of Bern Institute of Pharmacolocy Friedbühlstrasse 49
1066
Epalinges
sergio.haller@unil.ch
1066
Epalinges
mary-anne.hartley@unil.ch
8952
Schlieren
sebastian.heer@env.ethz.ch
3010
Bern
susanne.hoesli@pki.unibe.ch
University of
Geneva
SIAF
Swiss
Institute of Allergy
University
Hospital Zurich
Centre Hospitalier
Universitaire
Novimmune
Pathology
and
Immunology
Molecular Immunology
1211
Geneva
sandra.kallert@unige.ch
7270
Davos
patrycja.konieczna@siaf.uzh.ch
Ramistrasse 100 G PATH 8091
37
Service of Immunology Vieux-Moulin 8
1018
and Allergy/Service of
Antibody Engineering
14 chemin des Aulx
1228
Zurich
larisa.kovtonyuk@usz.ch
Lausanne
stephanie.longet@unil.ch
Plan Les Ouates
jloyau@novimmune.com
Experimetal
Immunopathology
D-BIOL
Murtenstrasse 31 3010 3010
Bern Postfach 62
Wagistrasse 27
8952
Bern
lukas.mager@pathology.unibe.ch
Schlieren
mai.matsushita@env.ethz.ch
Carlos
Insitute
of
Pathology
ETH-Molecular
Health Science
Universität Basel
Mattenstrasse 28
Basel
carlos.mayer@unibas.ch
Meister
Fabian
ETH
Department
Biomedicine
Micro
asdfasfs
fabian.meister@micro.biol.ethz.ch
Mele
Federico
Bellinzona
federico.mele@irb.usi.ch
mohanan
deepa
Institute
for
research in
USZ
safasdfasdfasdf sdfsdfsd 34234
sadfsdfsdf
Via Vincenzo vela 6
6500
F14, Dermatologische
Klinik Gloriastrasse 31
zurich
deepa.mohanan@hotmail.com
University of
Lausanne
Institute
of
Pharmacology
University
Hospital Zurich
University of
Lausanne
UNIL
Department
Biochemistry
Biochemistry
of
Centre
Medicale
Universitaire 1, rue
Obere Strasse 22
Haematologie
of
Cellular Immunology
Dermatology
4058
8091
Morshed
Md Mahbubul
University of Bern Institute
of
Pharmacology
EPFL
Global Health Institute
Bern
mahbubul.morshed@pki.unibe.ch
Mosconi
Ilaria
Lausanne
ilaria.mosconi@epfl.c
Nueesch
Ursina
Nussbaum
Kathrin
Nusser
Anja
University
Children's Hospital
University of
Zurich
University of Basel
Zürich
ursina.nueesch@kispi.uzh.ch
8045
Zürich
nussbaum@immunology.uzh.ch
4058
Basel
anja.nusser@unibas.ch
Olivos
Amiel
CHUV
1011
lAUSANNE
amiel.olivos-ortiz@chuv.ch
Oxenius
Annette
ETH Zurich
Pantelyushin
Stanislav
Pawelski
Helga
Prati
Moira
Institute of Microbiology Wolfgang-Pauli-Str. 10
HCI G401
UZH
Experimental
Winterthurerstrasse 190
Immunology
Y44J42
ETH - Integrative D-UWIS
Wagistrasse 27
Biology
SIAF
Molecular Allergology
Obere Strasse 22
8093
Zürich
oxenius@micro.biol.ethz.ch
8057
Zurich
pante@neuroimm.uzh.ch
8952
Schlieren
helga.pawelski@env.ethz.ch
7270
Davos
moira.prati@siaf.uzh.ch
Presotto
Danilo
CHUV - UNIL
Biopôle 3, Route de la 1066
Corniche 9B
rue
Gabrielle-Perret- 1211
Gentil 4
Wagistr.27
8952
Epalinges
danilo.presotto@unil.ch
Riccadonna
Cristina
Schmitz
Iwana
Schneider
Christoph
Geneva University Oncology
Hospitals and
Institute
of
D-UWIS
Integrative Biology
ETH Zürich
D-UWIS
geneva
riccadonny@libero.it
Schlieren-Zurich
iwana.schmitz@env.ethz.ch
Wagistrasse 27
8952
Schlieren
christoph.schneider@env.ethz.ch
Schuster
Steffen
Department
of
Chemin des Boveresses 1066
Biochemistry, WHO-IRTC 155
Medicine
ch. du musée 5
1700
Epalinges
steffen.schuster@unil.ch
Fribourg
thibaud.spinetti@unifr.ch
Nina
University of
Lausanne
University of
Fribourg
Pharmacology
Spinetti
Thibaud
Suhartha
Medicine
1700
Fribourg
nina.suhartha@unifr.ch
Thom
Jenny
ETH Zurich
8093
Zurich
jenny.thom@micro.biol.ethz.ch
Utzschneider
Daniel
CHUV
1066
Epalinges
daniel.utzschneider@chuv.ch
Wang
Junhua
3001
Bern
Wanke
Kerstin
Institute
of
parasitology
SIAF
Institute of Microbiology Wolfgang-Pauli-Strasse
10
SVRI
Biopole 3 Ch. des
Boveresses 155
University of Bern
Laenggass-Strasse 122
Cellular Immunology
7270
Davos Platz
wangjh28@163.com;bruno.gottstein@vetsuis
se.unibe.ch
k_wanke@gmx.de
Immunology
Friedbühlstrasse 49
EPFL SV GHI UPHARRIS 1015
SV 3820 (Bâtiment SV)
Steinwiesstrasse 75
8032
Institute of Experimental Winterthurerstr. 190
Immunology
Department
of
Mattenstrasse 28
Biomedicine
MIN
RUE DU BUGNON 46
Immunology
3010
ch. du Musée 5
Obere Strasse 22
Watkins
Stephan Lloyd
InselSpitalRIA-Immunology
University of Bern
Microbiology/ETH D-Biol
BERN
jasongrey333@yahoo.com
Zürich
kerstin.weidner@me.com
Bern
natascha.wuillemin@allergy.unibe.ch
ZURICH
chakradhar465@gmail.com
Chen-Ying
University
Hospital Bern
University
Hospital, Zurich
Uni. de Lausanne
Inselspital Dept. RIA 3011
Sahlihaus 2 RM 43
HCI G435 Wolfgang- 8093
Pauli-Str. 10
Clinic for Rheumatology Sahli-Haus 1
3010
and Clinical
Experimental
Ramistrasse 100, G- 8091
Hematology
PATH 37
Biochemistry
Ch. des Boveresses 155 1066
Weidner
Kerstin
Wuillemin
Natascha
Yakkala
Chakradhar
Yang
Epalinges
irvineyang@gmail.com
Yun
James
Inselspital
Zabel
Franziska
Ziltener
Pascal
University
Hospital Zurich
ETH Zürich
Department
of
Rheumatology, Clinical
Dermatology
Zimmermann
Kathrin
ETH Zürich
Strandweg 9A
3400
Burgdorf
james.yun@allergy.unibe.ch
Gloriastrasse 31
8091
Zurich
franziska.zabel@usz.ch
8093
Zurich
pascal.ziltener@micro.biol.ethz.ch
8093
Zürich
kathrin.zimmermann@micro.biol.ethz.ch
Institute of Microbiology Wolfgang-Pauli-Str. 10
ETH Hongerberg HCI
Institute of Microbiology Wolfgang-Pauli-Str. 10
Wolfsberg 2012
Particpating chairs
Name
Hans Hengartner
Christoph Müller
Annette Oxenius
Walter Reith
Antonius Rolink
Maries van den Broek
Institution
University Hospital Zürich
University of Berne
ETHZ
University of Geneva
University of Basel
University Hospital Zürich
email
hans.hengartner@usz.ch
christoph.mueller@pathology.unibe.ch
oxenius@micro.biol.ethz.ch
Walter.Reith@medecine.unige.ch
antonius.rolink@unibas.ch
maries@van-den-broek.ch
Presentation abstracts
Session 1
1° and 2° lymphoid organs, cell migration
The role of continued Foxn1 expression in thymus organogenesis and
maintenance
Angela Bosch1, Thomas Barthlott1, Saulius Zuklys1 and Georg A. Holländer1,2
1
Pediatric Immunology, Department of Biomedicine, University of Basel and Basel University
Children’s Hospital (UKBB), Basel, 2Department of Paediatrics, University of Oxford, Oxford, United
Kingdom
Introduction: The transcription factor Foxn1 is essential for thymic epithelial cell
(TEC) differentiation and its loss causes athymia. The goal of this study is to define
the temporal and quantitative requirements of Foxn1 for the development,
differentiation and maintenance of TEC and hence a regular thymic
microenvironment.
Methods: Two mouse models have been generated to test the role of Foxn1 expression
for thymus organogenesis and maintenance. The first model allows to study the
consequences of a loss of Foxn1 expression after thymus organogenesis has been
initiated (embryonic day 12). In contrast, the second model expresses a
hypomorphic Foxn1 allele that results in a reduction of Foxn1 expression enabling the
analysis of a gene dosage effect.
Results: The thymus of both mouse models was significantly reduced in size,
displayed cysts and had lost its regular microarchitecture when compared to wild type
mice. Blood-borne T-cell precursors homed - albeit at reduced frequencies - to the
altered thymic microenvironment, but were functionally unimpaired. However, in situ
they gave rise to only few double positive cells generating a reduced number of
peripheral T-cells with partial functionality. Regulatory T cells were also generated
although their suppressive potential was reduced.
Conclusion: TEC differentiation and maintenance is dependant on continuous and
sufficient Foxn1 expression. A short term presence or low amounts of Foxn1 do not
support regular thymus development and function whilst homing of T-cell precursors
to the postnatal thymus and minimal T-cell differentiation is observed in the absence
of regular Foxn1 expression.
What happens to the network of T zone fibroblastic reticular cells during
lymph node swelling?
Chen-Ying Yang1,2, Tobias K. Vogt1,2 and Sanjiv A. Luther1
1
Department of Biochemistry, University of Lausanne, Switzerland
2
contributed equally
Abstract: (max. 250)
Efficient primary T cell immune responses to lymph-born antigens are mainly
initiated in the T cell-rich zone of lymph node (LN). Throughout the T zone, T
zone fibroblastic reticular cells (TRCs) form a sponge-like network that serves
as scaffold for adhesion of antigen-carrying dendritic cells (DCs) and for
migration of T cells thereby facilitating the activation of the rare antigenspecific T cells. In addition TRCs produce CCL19, CCL21 and IL-7 to support
the trafficking and homeostasis of T cells during homeostasis. TRC also
produce the matrix scaffold responsible for organ stability. However, despite
of their critical role in regulating the homeostasis and activation of T
lymphocytes, the response of the TRC network to immune response and LN
swelling in particular is poorly understood.
To study this process we set up a LN swelling model in which the mice receive
first low numbers of ovalbumin (OVA)-specific CD4 (OTII) and CD8 (OTI) T
cells followed by subcutaneous immunization with OVA in Montanide adjuvant.
Immunofluorescence microscopy demonstrated that the TRC network simply
grows in size while preserving its original organization thereby allowing a
greater number of T cells to localize there. Flow cytometric analysis further
showed that the growth of the network is due to the increase in both
the size and the number of TRC, which is mainly due to extensive
proliferation. Therefore, the TRC network adapts rapidly to LN swelling while
keeping its integrity. We are currently investigating the mechanism that
triggers the rapid proliferation of TRC during immune response.
Fibroblastic Reticular Cells coordinate structural dynamics of Lymph Nodes during viral infection
Qian Chai1*, Lucas Onder1*, Elke Scandella1, Renzo Danuser 6, Tim Sparwasser2, Sanjiv Luther3, Volker Thiel1,
Thomas Rülicke4, Thomas Hehlgans5, Jens V. Stein6 and Burkhard Ludewig1
1
Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen, Switzerland; 2Institute of Infection
Immunology, TWINCORE, Hannover Medical School, Germany; 3EPFL, Lausanne, Switzerland; 4Institute of
Laboratory Animal Science and Biomodels Austria, University of Veterinary Medicine Vienna, Vienna, Austria;
5
University of Regensburg, Regensburg, Germany; 6Theodor Kocher Institute, Bern, Switzerland
Abstract
The paracortex of lymph nodes (LNs) harbors a sponge-like scaffold of stromal cells known as
fibroblastic reticular cells (FRCs). A major function of FRCs is to build and enwrap the conduit
system of collagen fibers that directs interstitial fluids from the afferent lymph through the T-cell
zone to high endothelial venules. Furthermore, FRCs regulate T-cell migration and survival by
producing the chemokines CCL19 and the serine isoform of CCL21. To gain further knowledge
on the biology of FRCs and possibly other stromal cell subsets, we have generated a bacterial
artificial chromosome (BAC)-transgenic mouse model that utilizes the CCL19 promoter to direct
the Cre-recombinase to LN stromal cells. Crossing of CCL19-Cre mice to the R26-EYFP
reporter mice revealed that transgene expression in LNs was almost exclusively confined to
podoplanin+CD31- cells. Likewise, transgene activity in spleens and Peyer’s patches of CCL19Cre mice was largely restricted to FRC-like cells, i.e. stromal cells within the T cell zone and the
T-B border. Selective ablation of the lymphotoxin-beta receptor on CCL19-Cre-positive cells
resulted in profound changes in the development and organization of secondary lymphoid
organs. Taken together, stromal CCL19-Cre expression is well-suited (i) to characterize the
development of LN FRCs in vivo, (ii) to molecularly dissect the contribution of stromal cells to
lymphoid organogenesis, and (iii) to address the function of LN FRCs in the generation of innate
and adaptive immune responses.
The Junctional Adhesion Molecule C (JAM-C) regulates dendritic cell migration
in inflammation.
Authors: Romain Ballet, Mélanie Charmoy, Fabienne Tacchini-Cottier, and Beat
Imhof
During infections, dendritic cells (DCs) are recruited to sites of infection where they
process antigens and start to migrate to the draining lymph node in order to trigger
specific T cell responses. Here we showed that JAM-C, a tight junction protein
expressed on both vascular and lymphatic endothelial cells regulates migration of
DCs in inflammation. In vivo, using the Leishmania major experimental model of
Leishmaniasis, we found that blocking JAM-C in endothelial cell-cell contacts
enhances the recruitment of dendritic cell precursors from blood vessels to the site of
infection. In addition, blocking JAM-C increases dendritic cell migration from the
infected site to the draining lymph node. In order to decipher the mechanism, we will
apply vascular specific JAM-C k.o. animals, perform transmigration assays and vital
microscopy. Overall, blocking JAM-C could represent a therapy leading to an
increased number of matured Antigen Presenting Cells in draining lymph node,
potentially improving the protective T cell response against L. major.
REGULATION OF DENDRITIC
MIGRATION INHIBITOR FACTOR
CELL
MIGRATION
BY
MACROPHAGHE
Amiel Olivos-Ortiz1, Thierry Roger1 and Thierry Calandra1. 1Division of Infectious Diseases,
Centre Hospitalier Universitaire Vaudoise, Lausanne, Switzerland, 1011.
Inflammation process involves a multistep cascade of interactions between leukocyte
receptors and soluble mediators present in the inflammatory milieu. In order to be
recruited to the inflammation site and to produce an effective immune response,
leukocytes need to coordinate the action of multiple signaling molecules that bind to
specific receptors expressed on the leukocyte membrane and the intracellular
signaling pathways triggered as a result of these interactions, such as PI3K-Akt,
MAPK and Rho GTPase pathways. Cytokines are soluble mediators and main
players in the development of many inflammatory processes. The macrophage
migration Inhibitor factor (MIF) is a pleiotropic proinflammatory cytokine that has been
found to contribute to many inflammatory and autoimmune diseases. MIF effects
include boosting of cytokine production, TLR-4 up-regulation, promotion of cell cycle
progression and proliferation, augment of tumorigenesis and apoptosis inhibition.
Evidence suggests that MIF also plays an important role during the migration of
epithelial and tumoral cells, as well, during leukocyte recruitment in several
pathological conditions. However, the mechanisms by which MIF is able to regulate
all this processes are still unclear. In this study we show that in dendritic cells, MIF
deficiency decreases the spontaneous migration and the chemokine-induced
migration in response to CCL21 and CCL19. These effects depend on p38, PI3K and
mTOR, but are independent of ERK 1/2 activation. Nevertheless, the molecular finetuning by which MIF is able to regulate the migration of dendritic cells remains to be
determined.
Session 2
Innate immunity, inflammation
Structure-function relationship between 15C1 and TLR4
Loyan
The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment
and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram negative
bacteria and induces a signaling cascade that, when exaggerated, has been associated with
inflammatory disorders such as acute lung inflammation, septic shock and even arthritis. A
monoclonal antibody directed against TLR4, the 15C1, was developed to block its activity.
Interestingly, it was demonstrated that the antibody’s inhibitory effect is partially Fc-dependant and
mediated by binding to Fc gamma receptors, in particular FcγRI/CD64 and FcγRIIA/CD32A. This
mechanism of action has been described in the literature for other cell surface proteins yet is poorly
understood. The main goals of my PhD project are to understand the structural and functional basis of
15C1’s inhibitory effect. The study of the structural basis of the 15C1-TLR4 interaction will be
presented.
Key words: TLR4, monoclonal antibody, phage display, structure-function relationship
Target gene(s) of the transcription factor Nrf2 mediate inflammasome activation.
Sebastian M. Heer, Esther Rosenwald, Abdijapar T. Shamshiev, Jan Kisielow &
Manfred Kopf
The transcription factor Nrf2 (nuclear factor, erythroid derived 2, like 2) is described
to control expression of genes involved in the antioxidant responses during
oxidative/electrophilic stress. We investigated the effects of Nrf2 in inflammatory
responses using Nrf2-deficient (Nrf2-/-) mice. Stimulation with TLR ligands triggered
enhanced production of inflammatory cytokines, such as IL-6 and IL-12 in
macrophages and dendritic cells (DCs) derived from Nrf2-/- mice. In contrast,
secretion of IL-1β, another pro-inflammatory cytokine, was reduced. Secretion of
mature IL-1ß requires cleavage of intracellular proIL-1ß by multimeric protein
complexes called inflammasomes. When Nrf2-deficient cells were treated with
inflammasome inducers like ATP or urea crystals,
they showed reduced
inflammasome activation and IL-1β secretion. Interestingly, reactive oxygen species
(ROS) have been thought to play a key role in inflammasome activation. Expectedly,
we found that ROS breakdown was delayed in Nrf2-/- DCs. Yet, manipulation of
ROS production using various ROS inhibitors or stimulators did not rescue IL-1β
production by Nrf2-deficient DCs, indicating little if any role for altered ROS.
Expression of various mutated Nrf2 proteins in Nrf2-deficient cells showed that
transcriptional activity of Nrf2 is required for inflammasome activation. Thus one or
several target genes of Nrf2, and not the Nrf2 protein itself, are needed for efficient
inflammasome induction. However, retrovirus-driven expression of several Nrf2target genes including thioredoxin reductase or peroxiredoxin could not rescue
inflammasome activation in Nrf2-deficient bone marrow DCs. Taken together, these
findings demonstrate Nrf2-target gene(s) control of inflammasome activation
independent of ROS levels.
Keywords: Nrf2, inflammasome, reactive oxygen species
The role of innate immunity in control of Legionella pneumophila lung
infection
Pascal Ziltener, Stefan Weber and Annette Oxenius
Institute for Microbiology, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich,
Switzerland.
Though single cell amoeba are the environmental niche for Legionella pneumophila,
the bacteria can also replicate in alveolar macrophages upon transmission to the
lung via aerosols, and have the potential to cause a severe form of pneumonia
known as Legionnaires’ Disease. The intracellular replication of L. pneumophila
depends on the injection of multiple effector molecules into the host cell cytoplasm
via a type IV secretion system (T4SS). These effectors promote phagocytosis, inhibit
fusion of the phagosome with lysosomes and thus allow the bacteria to establish a
replication permissive compartment. Alveolar macrophages are able to detect the
activity of the T4SS with cytosolic pattern recognition receptors such as NLRC4,
which in combination with MyD88-dependent signals leads to inflammatory secretion
of IL-1β. This innate immune response is believed to be important for the control of
primary L. pneumophila infection, characterized by IL-1β dependent induction of
CXCL1 production by type I alveolar epithelial cells, resulting in the recruitment of
neutrophils and subsequent clearance of bacteria.
Though reduced L. pneumophila clearance has been shown in mice lacking various
innate immune effector molecules, including NLRC4-/-, Caspase-1-/- and IFNγR-/mice, we show here that the bacterial load in the lung five days post intranasal
infection is increased 10-fold more in mice lacking TNF production in macrophages,
monocytes and neutrophils (mTNF mice), and in MyD88-/- mice. Furthermore,
macrophages lacking TNF production are as permissive for intracellular L.
pneumophila replication as NLRC4 deficient macrophages. Further investigation of
the in vivo immune effector mechanisms mediated by TNF, inlcluding the source of
TNF, the mode of action of TNF in restricting L. pneumophila growth and the cell
type in which it restricts L. pneumophila growth, will enhance our understanding of
how L. pneumophila infection is controlled by the innate immune system.
Inhibition of inflammasomes allows BCG to escape host immune responses by decreased
phagosome maturation.
Mohanan Deepa1, Fettelschoss Antonia1 , Kündig Thomas M.1, Sander Peter2, Böttger Erik
C.2, Johansen Pål1
1
Unit for Experimental Immunotherapy, Department of Dermatology, University Hospital of
Zurich, Switzerland
2
Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
Tuberculosis (TB) is a highly contagious and persistent infection caused by Mycobacterium
tuberculosis and Mycobacterium bovis and has the highest mortality rate than any other
infectious disease. .Although there is a vaccine (BCG), its efficacy is poor and there is a great
need to improve it. A typical feature of M. tuberculosis infection is that the bacterium resists
the fusion of the phagosomes it resides in with the lysosomes. Phagolysosomes are rich in
machinery that help in efficient MHC class II antigen presentation and inhibition of the fusion
allows the bacterium to dampen the immune response. In a previously reported study, a
putative mycobacterial zinc metalloprotease, Zmp1, was shown to play an important role in
disease pathogenesis by interfering with caspase-1 activation leading to decreased phagosome
maturation. Hence this study’s aim is to dissect the role of inflammasomes in the increased
immunogencity of this mutant.
Plasmacytoid dendritic cells induce Foxp3 regulatory T cells in response to
Bifidobacterium infantis
Konieczna Patrycja, Ziegler Mario,Frei Remo, Ferstl Ruth, O’Mahony Liam
Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos,
Switzerland
The commensal microbiota is required for optimal immune development and for
ongoing intestinal homeostasis, which involves an inter-dependence between
microbes and immunity. Bifidobacterium infantis is a commensal bacterium which has
been previously shown to protect against inflammatory diseases in murine models
e.g. colitis and respiratory allergy. The protective host immune response in murine
models was demonstrated to include the induction of T regulatory cells. The aims of
this study are: (1) To evaluate the plasmacytoid dendritic cell response to
Bifidobacterium infantis; (2) To determine the effect of activated plasmacytoid
dendritic cells on naive T cell priming. Firstly, plasmacytoid dendritic cells were
stimulated with bacteria and cytokine secretion was measured by luminex. Moreover
gene expression was quantified using qRT-PCR. Pattern recognition receptors were
blocked using antibodies or oligonucleotides. Bacterial binding was visualized using
multispectral flow cytometric imaging. Bifidobacterium infantis stimulated
plasmacytoid dendritic cells expressed IL-10 and indoleamine 2, 3-dioxygenase
(IDO), with low levels of IL-12p70 and IFN-alpha. The cytokine response was TLR-9,
but not TLR-2, dependent. Bacterial activated plasmacytoid dendritic cells were
cultured with autologous CD4+ T cells and Foxp3 expression was analysed by flow
cytometry. Co-culture of naïve T cells with bacterial primed plasmacytoid dendritic
cells resulted in enhanced expression of Foxp3 in CD4+CD25+ cells. Inhibition of
IDO activity with 1-methyl-tryptophan blocked induction of T regulatory cells. These
results demonstrate that Bifidobacterium infantis-stimulated plasmacytoid dendritic
cells induce an immunoregulatory response, suggesting that this microbe may have
therapeutic utility in patients with inflammatory disease.
Thymic stromallymphopoietin (TSLP) stimulates eosinophil
extracellular trap release
Mahbubul Morshed 1• 2, Shida Yousefi1, Christina Merz-Stockle1, Hans-Uwe Simon 1,
Dagmar Simon2
Institute of Pharmacology1 and Department of Dermatology, lnselspitaf, University of
Bem
Background: Eosinophils play an important role in host defense, immunoregulation
and tissue remodelling. They may release mitochondrial DNA in association with
toxic granule proteins forming eosinophil extracellular traps (EETs). EETs are potent
in killing bacteria. They have been observed in infectious and non-infectious
diseases. TSLP is a cytokine produced by epithelial cells at barrier surfaces.
Elevated TSLP expression has been found in atopic diseases in which tissue
eosinophilia is a characteristic finding.
Aim: We sought to investigate TSLP receptor (TSLPR) expression, the effect of TSLP
on eosinophils to release EETs and the extra- and involved intracellular mechanisms
in vitro and in vivo.
Methods: Human eosinophils were isolated from peripheral blood by negative
magnetic selection method. For DNA release assay, cells were taken on cover slip,
stimulated with TSLP at different concentrations, fixed and analyzed by confocal
microscopy. TSLPR expression on eosinophils in vitro and in vivo was measured by
immunofluorescent staining, Western blot and flow cytometry. Viability of TSLPstimulated eosinophils was measured by FACS and calcein-ethidium live cell
staining. The role of ROS and adhesion was assessed by pharmacological inhibition
of the NADPH oxidase and antibodies to integrins, respectively. Bacterial killing
assay was analyzed in overnight cultured agar plate and by confocal microscopy.
Results: TSLP stimulated eosinophils to release DNA in association with eosinophil
cationic protein (ECP) in a time- and concentration-dependent manner. This DNA
release was dependent on adhesion and ROS production by eosinophils. Expression
of both, TSLPR and IL-7Ra, was observed on eosinophils. Eosinophil viability was
not affected by TSLP stimulation. TSLP mediated EETs also shown bacterial killing
property. Eosinophils bearing the TSLPR were also detected in lesional skin of AD,
eosinophilic cellulitis and bullous pemphigoid together with TSLPR positive dendritic
and mast cells and in association with TSLP expression by keratinocytes.
Conclusion: These results demonstrate that TSLP may directly activate eosinophils to
generate EETs consisting of mitochondrial DNA in association with granule proteins
suggesting a possible role of eosinophils in early defense mechanism against
environmental pathogens.
Session 3
Allergy
XXIV Meeting of the Swiss Immunology PhD students
Wolfsberg, 2-4 April 2019
Distribution of allergen specific cells in diverse T cell subsets for seasonal and non
seasonal allergens
Tess Brodie1, Elena Brenna1, Ravi V. Kolla2, Alessandro Sette2, Antonio Lanzavecchia1, Federica
Sallusto1
1
Institute for Research in Biomedicine, Via Vincenza Vela 6, 6500 Bellinzona, Switzerland; 2La
Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA.
Tess.brodie@irb.usi.ch
Phenotypic and functional heterogeneity is the hallmark of effector and memory T cells. Upon
antigenic stimulation, naïve CD4+ T cells make choices to become TH1, TH2, or TH17 cells, or
even regulatory T cells. In addition to differences in cytokine repertoire, memory CD4+ T cells
exhibit diversity in homing: central memory cells patrol lymphoid organs while effector memory
cells act as sentinels in peripheral tissues such as the skin and the gut. To link the phenotype and
function of the different memory T cell subsets with their antigen specificity, we have developed
a high throughput cellular screening method and are applying this method, called T cell library, to
dissect the T cell response to allergens. We will report data on the distribution and
responsiveness of allergen-specific T cells in the different subsets in allergic and non-allergic
individuals and how the response changes with season. Understanding the dynamics of allergenspecific T cells in naïve, effector and memory Th2 subsets can give insight to the mechanisms of
pathology for allergic individuals and tolerance for non-allergic individuals.
Influence of Respiratory Syncytial Virus infection on allergic airway
responses depends on age
Eva S. Gollwitzer1,2, Aurélien Trompette1, Koshika Yadava1,2, Anke K.
Sichelstiel1,2, Robert Driscoll1, and Benjamin J. Marsland1,2
1
Service de Pneumologie, CHUV, Lausanne, Switzerland; 2Faculty of Biology
and Medicine, University of Lausanne, Switzerland
Respiratory Syncytial Virus (RSV) is linked to severe lower respiratory tract
infections in infants and young children. It infects nearly all children at least
once by the age of three and the age of the initial infection is thought to have
a strong influence on the outcome of the disease. A correlation between
severe RSV infections in infants and the development of asthma later in life
has been established. Nevertheless, little is known about the mechanisms
underlying this phenomenon. A predominant theory suggests that a TH2
response is mounted upon RSV infection in infancy as a default mechanism
due to an immaturity in TH1 priming. This TH2 bias then influences the
development of unrelated allergic responses. However, definitive evidence
supporting or challenging this concept has yet to be found.
In order to elucidate the mechanisms underlying the impact of RSV infection
on subsequent allergic airway responses in vivo, neonatal as well as adult
murine models have been established. Mice that were neonatally infected with
RSV show increased eosinophilic infiltration into the airways, accompanied by
increased mucus production and elevated proliferation of T cells following
challenge with an unrelated allergen in adulthood. In contrast, adult RSV
infection elicits a protective effect against subsequent allergic airway
responses, as characterised by decreased airway eosinophilia, a decrease in
allergen-specific T-cell proliferation as well as abrogated TH2 cytokine
production.
These findings illustrate, that the cellular responses and consequent sequelae
following RSV infection differ depending on the age of the initial infection.
IL-21 promotes Th2 allergic airway responses by inhibition of Foxp3+
regulatory T cells
Helga Pawelski1, Sanchaita Sonar1 , Luigi Tortola1, Koshika Yadava1,
Christoph Schneider1, Tim Sparwasser2 and Manfred Kopf1
1
2
Molecular Biomedicine, ETH Zurich, Switzerland
Twincore, Institut für Infektionsimmunologie, Hannover, Germany.
IL-21 plays an important role in the development of Th2-driven airway
inflammation. Previous studies performed in IL-21 receptor deficient (IL-21R-/-)
mice revealed a key role of IL-21 in TH2 cell driven inflammatory responses
such as lung eosinophilia, airway hyperresponsiveness, blood basophils and
type 2 granuloma in asthma and nematode infection. In this study we sought
to identify IL-21 dependent mechanisms in the course of allergic asthma. IL21 has been previously shown to inhibit development of inducible T regulatory
cells (iTregs) in vitro. We found a significantly increased population of Tregs
in the lung of IL-21R-/- as compared to WT mice upon airway challenge with
OVA. To further elucidate the role of Treg cells in asthma we utilized DEREG
mice carrying a BAC transgene encoding for the diphtheria toxin receptor
(DTR) under the Foxp3 promoter. Here specific depletion of Tregs by
administration of diptheria toxin (DT) is possible. Depletion of Tregs led to an
enhanced influx of Th2 cells into the lung and exaggerated features of asthma
in both IL-21R+/+xDEREG and IL21R-/-xDEREG mice, indicating that Tregs are
potent suppressors of asthma. To identify the time window when Tregs are
imperative for the outcome of asthma, administration of DT was limited to
either the sensitization or the challenge phase. Tregs were seen to be
essential in both phases since depletion in either phase affected the outcome
of asthma, suggesting an additive effect of Treg action. Taken together, these
results suggest that IL-21R signaling negatively regulates Treg cell population
and consequently the severity of OVA-induced lung inflammation.
Regulation of Bronchial Epithelium Integrity and Tight Junctions by
Regulatory T cells in allergic airway disease
Kerstin Wanke, Ruth Ferstl, Zsolt I. Komlosi, Jeanette I. Kast, Maciej
Chalubinski, Paulina Wawrzyniak, Michael Soyka, Stefan Söllner, Liam
O’Mahony, Cezmi A. Akdis
Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich,
Davos, Switzerland,
kerstin.wanke@siaf.uzh.ch
Background: Bronchial epithelial cells represent a physical barrier that
confers protection and plays a key role in asthma-associated lung
inflammation. Tight junction (TJ) complexes are responsible for the integrity of
the epithelial barrier. They form close cell-cell contacts and control cellular
integrity, polarity and paracellular flow of molecules and cells. The interplay
between epithelium integrity and resident T-cells has been unknown so far.
Aim: To investigate the control of TJ and epithelium integrity by regulatory Tcells (Tregs).
Methods: Pre-activated CD4+CD25+FoxP3+ Tregs were transferred
systemically or intratracheally into BALB/c mice with OVA-induced allergic
airway disease. Hallmarks of allergic airway disease such as differential cell
count in BAL, cytokine production in lung cells and lung inflammation were
assessed. Bronchial and TJ integrity was analyzed by total protein content in
BAL supernatants, qPCR and confocal microscopy.
Results: A wide range of TJ proteins was expressed in bronchial epithelial
cells and had the tendency to be downregulated in lung inflammation. The
adoptive transfer of Tregs decreased peribronchial inflammation, and
prevented the down-regulation of TJ proteins. The expression of TJ proteins
correlated strongly with the expression of the Treg cytokines IL-35 and
TGFbeta, while they didn’t correlate with Th2 cytokines and negatively
correlated with IFN-g. Further, intratracheal administration of Tregs seemed to
be more effective than systemic administration in supressing hallmarks of
allergic airway inflammation. This lead to a strong recovery in epithelial
integrity including TJ expression.
Conclusion: We provide here evidence for a key role of Tregs in controlling
TJ and bronchial epithelium integrity.
Dendritic cells targeting and improved cell penetration in allergen-specific
immunotherapy
Mattia Garbani, Claudio Rhyner, Reto Crameri
Swiss Institute for Allergy and Asthma Research (SIAF), University of Zürich,
Oberestr. 22, Davos, Switzerland
Todays therapies for allergic diseases aim to induce a switch towards a T H 1/T reg
dominated immune response by challenging the patient with increasing doses of
allergen. This approach has two major disadvantages: the duration of the treatment
and the anaphylactic side-effects.
The Modular Antigen Translocation (MAT) Vaccine consist of a cell penetration
peptide linked to a part of invariant-Chain (Li). In the cell Li targets the immunogenic
protein directly to MHC-II, therefore i) less antigen is needed and ii) less injections are
necessary. MAT vaccines are potentially internalized in every cell and do not
specifically target antigen presenting cells. Moreover they show the undesired feature
of migrating to the nucleus. The aim of the project is to create a new generation of
vaccines where the TAT peptide is substituted or supplemented by newly discovered
peptides, lacking nuclear localization sequence or able to specifically target dendritic
cells.
By confocal microscopy and flow cytometry it was shown that, when added
exogenously and fused to a protein of interest, CPP512 triggers the internalization of
the construct into HeLa cells without driving it towards the nucleus. Additionally, using
Green Fluorescent Protein fusions, we were able to confirm the specific targeting of
the DC-specific peptide pep3. Pep3 constructs were specifically binding to DCs, but
not to other investigated cell types like CaCo-2 and THP1.
In the next stage the efficacy of the modified versions of the MAT vaccine will be
tested in vivo in mouse models of allergy.
Targeting the Extracellular Membrane-Proximal Domain of IgE memory B cell
Prati Moira, Rhyner Claudio, Crameri Reto
Swiss Institute for Allergy and Asthma Research (SIAF), CH-7270 Davos, Switzerland
E-mail: moira.prati@siaf.uzh.ch
Background: In industrialized countries approximately 30% of the population is
affected by IgE-mediated allergies, including asthma, atopic dermatitis or allergic
rhinoconjunctivitis. Allergen-specific IgE is the key molecule in allergic diseases and it
can be expressed either as secreted IgE (sIgE) or as membrane-bound form (mIgE),
which contains two additional domains termed M1 and M2. The M1 region codes for
an extracellular membrane-proximal domain (EMPD) and for the transmembrane
domain, whereas the M2 region codes for the cytoplasmic domain expressed only in
memory B cells as integral part of the B cell receptor (BCR).
Aims: We think that specific targeting of mIgE on memory B cells is a useful strategy
for prophylactic therapeutic interventions. With this study we aim therefore to
demonstrate that anti-EMPD mAbs can be used to isolate IgE-switched memory B
cells from blood of allergic patients.
Results: One anti-murine and three anti-human hybridomas, were selected for
further characterization and for production and purification of the corresponding
monoclonal antibodies. The mAb specific for the human EMPD domain with higher
specificity and affinity (KD ~ 10-10), was used to sort IgE+ memory B cell from allergic
patients’ PBMC’s. Usually the frequency of IgE+ B cells is very low. Preliminary
results have shown that about 0.3 % of the total B cell can be targeted with the
monoclonal hAbC20.
Conclusions: With this approach we want to generate clones producing human
allergen-specific mAbs of the IgE isotype by immortalization in order to elucidate IgEbinding epitopes by co-crystallization of allergens and allergen-specific human Fab
fragments. These initial studies can facilitate the discovery and development of new
therapeutics, vaccines, and diagnostics, thus providing new insights into allergyrelated disorders.
Cytosolic vacuolization of eosinophils:
Involvement of CD11b
Susanne Hoesli1 , Dagmar Simon 1,3, Beata Styp-Rekowska2 , Ruslan Hlushchek 2, Shida
Yousefi 1, Hans-Uwe Simon 1
Institutes ofPharmacology 1 and Anatoml, University of Bern, and Clinic ofDermatology3 , Inselspital,
University Hospital Bern
Eosinophils are a subset of granulocytes involved in the pathogenesis of different diseases,
including parasitic infections and allergies. An eosinophil infiltration has been observed in
skin biopsies from patients suffering from a broad range of skin disorders of allergic,
autoimmune, or neoplastic genesis. In samples derived from these patients, a fraction of
eosinophils shows the distinct morphology of cytosolic vacuolization. Here, we performed in
vitro experiments aiming to understand the molecular mechanisms and functional
consequences of vacuole formation in eosinophils.Cytosolic vacuole formation was associated
with activation and degranulation of eosinophils. Moreover, vacuolization was dependent on
CD11b, an integrin expressed on eosinophils mainly upon activation and transmigration
through endothelial cells. Firm adhesive processes, in which CDllb is involved, have been
linked to a process called cytolysis, a form of degranulation leading to the loss of nuclear and
cellular integrity and consequent cell death. We demonstrate that the progression of cytosolic
vacuolization leads to eosinophil death characterized by loss of nuclear and cellular integrity.
We thus suggest a morphological progression of cytosolic vacuolization to cytolysis.
A System For Quantitative Production Of Murine Basophils Ex Vivo
Ursina Gurzeler1, Tatiana Rabachini1, Clemens Dahinden2 and Thomas Kaufmann1
1
2
Institute of Pharmacology, University of Bern, Bern, Switzerland
Institute of Immunology, University of Bern, Bern, Switzerland
Basophils are the least common granulocytes in the circulation. They have recently
been recognized as important immunomodulatory cells and play a major role as
effector cells in allergic inflammation. However, functional and biochemical assays
on basophils have long been hampered due to their extremely low number in the
blood (<1% of peripheral blood leukocytes in the mouse). Consequently, very little is
known about the regulation of apoptotic signalling pathway in basophils. In addition,
basophils are short-lived cells with an expected half-life of approximately a few days.
For these reasons, we established a system for quantitative production of murine
basophils in vitro.
We modified the method published by Wang et al. (Nat Meth 2006), where they
described a method for the quantitative production of macrophages or neutrophils
using conditional Hoxb8. Besides different cytokine cocktails, we use a 4hydroxytamoxifen-inducible lentiviral system for conditional Hoxb8 expression
(Vince et al. Cell 2008). In order to generate Hoxb8-immortalised, basophilcommitted myeloid progenitor cell lines, we infect an enriched fraction of bone
marrow-derived multipotent cells with a conditional Hoxb8 expression construct. This
progenitor cell line is genetically stable (immortalised but not transformed), can be
maintained in culture for a long time and progenitors can be differentiated into mature
basophils in near unrestricted number upon shutdown of Hoxb8 expression
Importantly, after differentiation, these cells exhibit many characteristics of mature
murine basophils. They stop cycling, have multilobed nuclei, exhibit mouse basophil
specific surface markers (c-kit -/FcεRIhi), express high-levels of basophil specific mast
cell proteases 8 (Mcpt8) and MCP-11, degranulate in response to IgE crosslinking and
start to produce IL4.
To our knowledge, this is the first report presenting an in vitro model to produce large
numbers of mouse basophils. This system is important not only to study the molecular
mechanisms of basophil differentiation, but also because of the possibility of using
genetically modified mice to further investigate basophil biology. In terms of
apoptosis signalling, we have started to look into the role of BH3-only proteins (Bid,
Bim) in death receptor-induced and mitochondrial apoptosis.
Patrick Buschor1, Fabian Zellweger1, Alexander Eggel1, Michael Stumpp2, Beda M.
Stadler1, Monique Vogel1
1
University Institute of Immunology, Inselspital, CH-3010 Bern, Switzerland
Molecular Partners AG, Wagistrasse 14, 8952 Zürich-Schlieren, Switzerland
2
The high affinity receptor for human IgE (FcεRI) is expressed on the surface
of mast cells and basophils. Allergen-induced cross-linking of FcεRI via
receptor bound IgE stimulates the degranulation of pro- inflammatory
mediators that induce the allergic symptoms. These effector cells also
express the low affinity IgG receptor FcγRIIb that is known to inhibit allergeninduced cell degranulation when cross-linked with FcεRI. Considering the
inhibitory function of FcγRIIb, we previously developed a bispecific molecule
to efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of
basophils. The fusion protein is based on an IgE reactive DARPin (designed
ankyrin repeat protein) that was linked to the Fc-part of a human IgG1
antibody for binding to FcγRIIb. This protein was not anaphylactogenic and
inhibited allergen-induced basophil activation in whole blood assays. In order
to increase the inhibitory effect the Fc part of the fusion protein was
engineered by site directed mutagenesis. Through a single point mutation
within the IgG1 CH2 domain (S267A) the binding affinity to FcγRIIb was
improved 10-100 times, whereas the binding to IgE was not affected.
Interestingly, inhibition of allergen-induced basophil activation in whole blood
assays was more efficient with the altered construct. A long incubation time
prior the allergen stimulation did not seem to be crucial for the inhibition.
Furthermore the inhibitory effect was still present when the construct was
applied after allergen exposure. The effect of the molecule to inhibit de novo
synthesis of lipid mediators (e.g. Leukotriene) and cytokines is currently
tested in purified human basophils.
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Computational, Biochemical and Cellular Elucidation of Non-hapten Pharmaceutical Interactions (p-i)
Adverse Drug Hypersensitivity
in
Watkins
Our study began with computational screening of 10 T Cell Clones (TCC) isolated from a patient showing
macular papular exanthema caused by sulfamethoxazol (SMX), using AutoDock and varied Docking software.
All of these clones had been shown to produce some proliferative response to SMX stimulation prior to our
initial computational analysis, which was dependent on the HLA DRB10 from the patient.
All TCC T Cell
Receptors (TCR) had been sequenced and models were generated from these. Initial DOCKing screens with
SMX showed 2 hapten restricted clones, and 2 clones that gave binding sites for the compound at nonpeptide groove interacting sites. From these we chose 1 hapten restricted (clone 1) and one p-i clone
(clone 2) to further characterize interaction mechanisms and kinetics. Our initial screening was
expanded to 11 other SMX derivatives, showing a cross reactive profile for the p-i clone and a non-cross
reactive profile for the hapten restricted clone. We were however able to demonstrate a competitive
inhibition in Ca2++ assays to the clone 1, but not clone 2. From the original generated models, we began
computational screening of peptides recognized by the respective TCR, and were able to generate a
molecular dynamics simulation for clone 2 based on these results. Initial results from these runs give
an estimated binding affinity and reaction mechanisms in the presence and absence of SMX. Together,
these data indicate that Adverse Hypersensitivity reactions may not be caused solely by hapten restricted
clones but may include a immix of other p-i interactions which yield the same outcomes. We also show
that haptens may not be necessary for reactions to occur to specific drugs when a person has already been
primed and generated a TCR against a hapten. The problem now remains to determine which of these
reactions are the root cause of the adverse reactions, weather it is a combined effect of both
interactions within an allergic individual, or whether it can be either type of molecular interaction.
Session 4
Adaptive immunity in infection
Direct type I IFN signaling on CD8 T cells protects them from NK cell mediated
killing
Josh Crouse and Annette Oxenius
Institute of Microbiology, ETH Zurich
josh.crouse@micro.biol.ethz.ch
Direct type I IFN signaling on CD8 T cells is necessary for the proper expansion,
differentiation and survival of responding CD8 T cells following certain viral infections
such as during lymphocytic choriomeningitis virus (LCMV) infection. The reasons for
the reduced survival of CD8 T cells lacking the type I IFN receptor (IFNAR-/-)
following an acute LCMV infection remain unclear. Using an adoptive co-transfer
system of WT and IFNAR-/- CD8 T cells followed by an acute LCMV-W E infection
we have found that the ability to sense type I IFN by CD8 T cells protects them from
natural killer (NK) cell mediated killing. In an NK cell sufficient environment IFNAR-/CD8 T cells exhibit reduced survival compared to WT CD8 T cells, but depletion of
NK cells prior to LCMV-WE infection restores the expansion potential of IFNAR-/CD8 T cells up to day 4 post infection. This immunoregulatory role of NK cells is
mediated in a perforin dependent manner and requires the activation of CD8 T cells.
Therefore, following an infection associated with early and abundant levels of type I
IFN, direct sensing of the proinflammatory cytokine by CD8 T cells protects them
from NK cell mediated killing. This may serve as an immunoregulatory mechanism
where improperly activated CD8 T cells are eliminated.
XXIV Meeting of the Swiss Immunology PhD students
Wolfsberg, 2-4 April 2012
Shaping of the immune response in persistent viral infections
Sandra Kallert 1,2 and Daniel D. Pinschewer 1,2
1
2
Department of Pathology and Immunology, University of Geneva, 1 rue Michel Servet, 1211 Geneva 4, Switzerland
World Health Organization Collaborating Center for Vaccine Immunology, University of Geneva, Switzerland
Persistent infection of mice with lymphocytic choriomeningitis virus causes direct
immunopathology and generalized immunosuppression. Mice acquiring LCMV
infection at birth, however, develop a healthy life-long carrier state with impaired
innate but intact adaptive immune responses. During this study we intend to analyze
the molecular mechanisms by which chronic LCMV infection can shape the immune
system. We are particularly interested in the influence of LCMV infection on
dendritic cells (DCs) and stromal fibroblastic reticular cells (FRCs). DCs are known
main players in anti-viral immunity whereas recent studies also emphasize the
importance of FRCs in this context. Here we aim at analyzing how LCMV alters the
gene expression profile of infected cells, resulting in modified immunocompetence
and pathology. We can track infected cells using different viral reporter systems,
which allow us to isolate infected and uninfected cells from the same animal. Whole
genome cDNA expression analysis of infected and uninfected DCs and stromal FRCs
will subsequently provide us with candidate host genes accounting for viral
immunosuppression in chronic LCMV infection. Taken together this approach should
give us new insights into how chronic viral infections can imprint host cells of both
hematopoietic and non-hematopoietic origin leading to suppressed immune responses.
Such knowledge may ultimately lead to new strategies and molecular targets to
counteract viral persistence and virus-induced immunosuppression.
Glutathione Peroxidase 4 is pivotal for maintenance of peripheral CD8+T
cell pool and anti-viral CD8+ T cell responses
Mai Matsushita1, Peter Bretscher1, Christoph Schneider1, Stefan Freigang1,
Jan Kisielow1, Marcus Conrad2, Georg Bornkamm2, Manfred Kopf1
1
Molecular Biomedicine, Institute of Integrative Biology, ETH Zurich,
Schlieren, Switzerland
2
Helmholtz Center Munich, Institute of Clinical Molecular Biology and Tumor
Genetics, Germany
The selenoenzyme glutathione peroxidase 4 (Gpx4) is regarded as a major
scavenger of phospholipid hydroperoxides and thus represents a key
component of the reactive oxygen species (ROS) scavenging network. In our
studies, we study on the role of Gpx4 T cells, dendritic cells and macrophages
using conditional knockouts, as the underlying molecular mechanisms still
remain undefined. Focusing on T cells, our results from CD4cre-Gpx4lox/lox
mice have revealed that despite normal thymic T cell development, CD8+ T
cells failed to maintain a competitive pool size in the peripheral lymphoid
organs resulting in homeostatic imbalance of naïve CD8+ T cells.
Reconstitution analyses from mixed bone marrow chimera have further
confirmed an intrinsic defect in CD8+ T cell survival in the periphery. CD4creGpx4lox/lox thymocytes have revealed the ability to expand properly yet failed
to sustain survival upon transfer into RAG deficient mice. Furthermore, in vitro
culture of both CD4+ and CD8+ T cells equally displayed rapid cell death,
which was reverted upon addition of antioxidant Vitamin E. Under
inflammatory conditions, Gpx4 plays a vital role in proper CD8+ T cell function
and viral clearance as CD4cre-Gpx4lox/lox mice failed to mount efficient
antiviral CD8+ T cell responses upon infection with LCMV and exhibited
delayed viral clearance. However, the impaired virus-specific CD8+ T cell
response was partially rescued upon diet supplementation with high
concentrations of Vitamin E, thereby indicating the increased toxicity of
accumulating phospholipid hydroperoxides as a mechanism.
IL-21 restricts virus-driven Treg cell proliferation to prevent T cell
exhaustion and viral persistence in chronic LCMV infection
Iwana Schmitz1, Anja Fröhlich1, Helge Frebel2, Tim Sparwasser3, Annette
Oxenius2, Stefan Freigang1,4, and Manfred Kopf1,4
1) Molecular Biomedicine, Institute of Molecular Health Sciences, Department of Biology,
ETH Zurich, Switzerland
2) Institute of Microbiology, Department of Biology, ETH Zurich, Switzerland
3) Institute of Infection Immunology, TWINCORE/Centre for Experimental and Clinical
Infection Research, Hannover, Germany
4) These authors contributed equally to this work.
Foxp3+ regulatory T (Treg) cells are essential for the maintenance of immune
homeostasis and tolerance; however, their impact on infectious diseases can
be a double-edged sword and remains to be defined. During viral infections,
Treg cells limit the immunopathology resulting from excessive inflammation,
yet potentially inhibit effective antiviral T cell responses and promote virus
persistence. We report here that LCMV Docile triggers a massive expansion
of the Treg population that directly correlates with the size of the virus
inoculum and its tendency to establish a chronic infection. This Treg cell
proliferation was greatly enhanced in IL-21R-/- mice, suggesting that IL-21
restrains the virus-induced Treg cell expansion. Moreover, experimental
augmentation of Treg cells drastically impaired the functionality of the antiviral
T cell response and impeded virus clearance. As a consequence, mice
became highly susceptible to chronic infection, even with a viral isolate that
otherwise fails to establish persistence in immunocompetent hosts. These
findings reveal virus-driven Treg cell proliferation as an immunoevasive
strategy that facilitates T cell exhaustion and virus persistence. Furthermore,
they suggest a dual role for IL-21 in chronic infections by providing survival
signals to antiviral CD8+ T cells, but also by restricting the suppressive
activity of infection-induced Treg cells.
IL-9 is a potent suppressor of antiviral T cell responses
Christoph Schneider 1, Jan Kisielow 1, Jean-Christophe Renauld 2, Manfred
Kopf 1
1
2
Molecular Biomedicine, ETH Zürich
Ludwig Institute for Cancer Research and Experimental Medicine Unit,
Université Catholique de Louvain, Brussels, Belgium
Interleukin-9 (IL-9) belongs to the common gamma-chain (γc) receptor
cytokine family. Members of this family such as IL-2, IL-4, IL-7, IL-15 and IL21 are important regulators of T cell immunity. IL-9 has been primarily
associated with Th2-type immune responses. More recently, IL-9 was
proposed to enhance the function of regulatory T cells and facilitate the
expansion of Th17 cells. However, the direct effects on T cell effector
functions are not well understood. To investigate the role of IL-9 in the
regulation of T cell responses, we studied IL-9 in the context of LCMV
infection in mice. Using mice deficient in the IL-9 receptor, we could show that
IL-9 signaling is a potent suppressor of LCMV-specific T cell responses. Upon
infection with LCMV docile, IL-9R knockout mice show a more activated
phenotype, produce more effector cytokines and eventually clear an otherwise
persistent LCMV infection within 2 weeks. Moreover, IL-9 supplementation
during infection results in the suppression of the antiviral CD8 T cell response
and prolonged presence of the virus in the mice. These results provide
evidence for a new function of IL-9 in regulating T cells by suppressing their
effector function.
3 Key Words: IL-9, LCMV, T cell regulation
Recovery of functional T-cells from LCMV clone 13 infected mice
Daniel Utzschneider, Lucie Carrie, and Dietmar Zehn
Swiss Vaccine Research Institue, IAL, Centre Hospitalier Universitaire Vaudois, Lausanne,
Switzerland
Persisting viral infections go along with the appearance of antigen-specific T-cells that show
different degrees of functional impairments. These cells up-regulate inhibitory molecules such
as PD-1, Lag-3, and Tim-3 and given their reduced ability to proliferate and produce
cytokines in response to antigen stimulation they are often considered as exhausted T-cells.
However, T-cell exhaustion progresses to different levels resulting in a very heterogeneous
population of virus-specific T-cells.
So far, it remained unclear to what extent exhausted T-cells might be able to regain
functionality and if such a recovery potential could be associated with different levels of
exhaustion. We address this question by transferring exhausted LCMV-specific CD8 T-cells
into naïve mice and re-challenge with LCMV strains causing a chronic or an acute infection.
We observed that the exhausted T-cell population robustly expands after a second antigen
challenge. Moreover, we noticed that the extent of this re-expansion varies for different
LCMV epitopes. By using modified LCMV Clone 13 strains, which differ in the affinity
towards P14 transgenic CD8 T-cells, we were able to show that the functional avidity of a
pMHC-TCR interaction impacts the level of T-cell exhaustion suggesting that differences in
the strength of stimulation could be responsible for differences in the recovery potential.
Thus, our data show that the fate of antigen-specific T-cells in a chronic infection is not
finally terminated and that the population of exhausted T-cells comprises a substantial
recovery potential. These results obtained by us broaden our basic knowledge of T-cell
dynamics in persistent infections.
THE ROLE OF “T HELP” DURING SECONDARY CD8+ T CELL RESPONSES IN
ACUTE VIRAL INFECTIONS
Gregor Bedenikovic, Melanie Wiesel, Annette Oxenius
Swiss Federal Institute of Technology Zurich, Institute of Microbiology, gregor.bedenikovic@micro.biol.ethz.ch
CD8 T cells represent an important part of the adaptive immune system as they are essential for
the eradication of intracellular pathogens. They have the capacity to differentiate into long-lived
memory cells, which allows them to resolve subsequent infect ions with the same pathogen. Upon
re-exposure to their cognate antigen, these memory cells quickly expand to high numbers of
effector T cells that control secondary infections faster and more efficient ly than CD8 T cells
during the primary response.
It was shown that CD4 T cells play a crucial role during primary CD8 T cell responses and also
during the generation and recall responses of memory CD8 T cells. However, the timing and
mechanisms by which these cells and other factors influence the mentioned pro cesses may differ
between primary and secondary responses and they may even differ between different
experimental models. Thus, the function of CD4 T cells and the role of "T help" during recall
responses remains incompletely defined.
Therefore, the goal of this project is to further examine the in vivo role of CD4 T cells and “T
help” during secondary CD8 T cell responses, in the setting of acute viral infections. Special
emphasis will be given to unravel the molecular mechanisms and timing of T help that support
CD8 T cell recall responses (in comparison to the situation during primary CD8 T cell
responses). In addition the contribution of APCs, different soluble mediators (such as IL-2, IL12, IL-15 or Type I IFN) and other cellular components (such as CD40/CD40L signalling, OX40 or PD-1) for CD8 recall responses will be defined in more detail.
Tuning TCR avidity of virus-specific CD8+ T cells
Three key-Words: Cytotoxic T cells, Mouse hepatitis virus, T cell receptor
Jovana Cupovic1, Veronika Nindl1, Lilian Staerck 2, Wolfgang Uckert 2, Thomas Rülicke3 Volker Thiel1,
Burkhard Ludewig1
1
Institute of Immunobiology, Kantonal Hospital St. Gallen, Switzerland
Max Delbrück Center for Molecular Medicine Berlin, Campus Buch, Germany
3
Institute of Laboratory Animal Science, University of Veterinary Medicine, Vienna, Austria
2
Molecularly well-defined infection models can contribute to a better understanding of the basic
mechanisms underlying interactions between the virus and the host immune system. The
mouse hepatitis virus (MHV) is a cytopathic mouse pathogen able to establish infection in liver
and CNS whereby CD8 + T cells are crucial for the control of acute MHV infection. To allow
comprehensive studies of the role and behavior of MHV A59-specific CD8+ T cells, T cell
receptor (TCR) retrogenic and transgenic mice were generated. Retrogenic TCR technology
facilitated expression of variable numbers of TCRs on CD8+ T cells. According to TCR densities,
retrogenic cells were classified as low, intermediate and high avidity T cells. Assessment of
proliferative responses (judged by CFSE dilution) revealed faster onset of proliferation in high
avidity retrogenic TCR T cells and a higher sensitivity of these cells to antigen. However,
differences of TCR numbers had no influence on minimal dose of antigen needed for the
initiation of proliferation. Using adoptive transfer of virus-specific CD8+ T cells to C57BL/6 mice
revealed that both optimal avidity retrogenic and transgenic CD8+ T cells protected the
recipients from virus-induced liver disease. However, during MHV CNS infection adoptively
transferred TCR retrogenic and transgenic MHV-specific CD8+ T cells exacerbated virusinduced CNS disease. Altering the avidity of virus-specific CD8+ T cells will facilitate detailed
analyses of CD8+ T cell activation thresholds and differentiation patterns during systemic and
CNS virus infection.
A murine genital-challenge model is a sensitive measure of protective antibodies against
human papillomavirus infection
Stéphanie Longet1, Martine Bobst1, John Schiller2, Patrice Jichlinski1, Denise NardelliHaefliger1
Department of Urology, CHUV and University of Lausanne, Lausanne, Switzerland1 ;
National Cancer Institute, NIH, Bethesda, MD, USA2.
The available virus-like particle (VLP)-based prophylactic vaccines against specific HPV
types afford close to 100% protection against the type-associated lesions and disease. Based
on papillomavirus animal models, it is likely that protection against genital lesions in humans
is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the
sites of genital infection. However, a correlate of protection was not established in the clinical
trials because few disease cases occurred, and true incident infection could not be reliably
distinguished from the emergence or reactivation of prevalent infection. In addition, the
current assays for measuring vaccine-induced antibodies, even the gold standard HPV
pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the
minimum level of antibodies needed for protection. Here, we characterize the recently
developed model of genital challenge with HPV PsV and determine the minimal amounts of
VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo
after transfer into recipient mice. Our data clearly demonstrate that, remarkably, the in vivo
assay is substantially more sensitive than in vitro PsV neutralization and thus may be better
suited for studies to establish correlates of protection.
Antigen presenting cells involved in Th17 cell priming during Candidiasis
Kerstin Weidner, André Gladiator and Salomé LeibundGut-Landmann
Institute for Microbiology ETH Zürich, Wolfgang-Pauli-Strasse 10, HCI G435, 8093
Zürich, Switzerland
kerstin.weidner@micro.biol.ethz.ch
The fungus Candida albicans lives as part of the normal microflora in healthy
individuals without triggering any harmful effects. However, it can cause severe
disease in immunocompromised individuals. The increased prevalence of mycoses
such as orophangyngeal candidiasis (OPC) in patients with profound T cell
deficiencies provides evidence for CD4+ T cells playing a key role in protection from
fungal diseases. C. albicans-specific CD4+ T cells are primed efficiently in response
to oropharyngeal infection in mice and these T cells produce high levels of IL-17A
and other Th17-type cytokines. However, it remains unclear how the differentiation of
Th17 cells during OPC is controlled. Here we thus address the question which
subset(s) of antigen presenting cells (APCs) mediate this process. Among others,
inflammatory monocytes infiltrate the draining lymph nodes in high numbers during
infection, suggesting that they mediate the priming of Candida-specific Th17 cells,
either directly or in cooperation with lymph node-resident APCs. Inflammatory
monocytes indeed present Candida-derived antigens in the draining lymph nodes of
infected mice as they are able to activate Candida-specific T cell hybridoma ex vivo.
The role of inflammatory monocytes for the activation of Candida-specific T cells
during OPC in vivo is being confirmed. Together, this study shall illuminate and
extend the prominent function of inflammatory monocytes as activators of the
adaptive immune system that has recently been attributed to them in various tissues,
including the lung, but not yet in the oral cavity, which is another prominent site of
pathogen entry.
THE IMPACT OF HIV-1 REPLICATION ON THE ACTIVATION OF CD8+ T CELLS
WITH SPECIFICITIES FOR DIFFERENT ANTIGENS
Sonia Bastidas, Huldrych Guenthard and Annette Oxenius
Institute for Microbiology, Zürich, ETH Zürich, sonia.bastidas@micro.biol.ethz.ch
Continuous loss of CD4+ T lymphocytes and chronic immune activation are
hallmarks of chronic HIV-1 infection. It is suggested that apoptosis (activation
induced cell death) of these abnormally activated cells is one of the main causes of
T-cell decline. Interestingly, T cells with an activated phenotype are neither
necessarily HIV-specific nor HIV-infected. Usually 1-2% of all circulating CD8+ T
cells during chronic infection are HIV specific whereas the level of CD8+ T cells
exhibiting activated phenotypes (as judged by CD38 and HLA-DR expression) is in
the range of 60%, indicating that immune activation is not restricted to HIV-specific
CD8+ T cells. Several hypotheses have been forwarded to explain the mechanism of
CD8+ T cell activation. It is believed that most of these cells are activated by
nonspecific mechanisms, including cross-reactivity and cytokine-driven activation, a
phenomenon referred to as bystander activation. The mechanisms underlying this
phenomenon are not well defined. Additionally, little to nothing is known about the
antigen-specificity of bystander activated CD4+ and CD8+ T-cells.
In this study we analyzed in a cohort of patients if cessation of antiretroviral therapy
(ART) specifically leads to activation of CD8+ T cells with specificities for different
antigens such as EBV, CMV, Adenovirus and Flu. Based on our data we propose
that HIV-driven activation occurs for CD8+ T cells, and that they are getting activated
in an antigen independent manner. These cells are likely to represent a large part of
activated CD8+ T cells in untreated HIV infection and thereby significantly contribute
to chronic immune activation.
XXIV Meeting of the Swiss Immunology PhD students
Wolfsberg, 2- 4 April 2012
Co-stimulatory effect of the HIV-1 envelope glycoprotein on CD4+ T cells
during chronic HIV-1 infection
Kathrin Zimmermann, Manuela Rehr, Anna Haas and Annette Oxenius
kathrin.zimmermann@micro.biol.ethz.ch
Institute of Microbiology, ETH Zürich
Continuous loss of CD4+ T lymphocytes and systemic immune activation are the two
major hallmarks of chronic HIV-1 infection. Chronic immune activation is associated
with hyperactivation of T cells, reflected by the acquisition of an activated T cell
phenotype and enhanced T cell turnover. Since the majority of activated CD4+ T
cells is neither HIV-specific nor HIV-infected, it is currently believed that non-specific
activation of CD4+ T cells occurs in the context of chronic HIV-1 infection. So far,
mechanisms of non-specific T cell activation are poorly investigated and are
currently attributed to pro-inflammatory cytokines and Toll-like receptor ligands which
are both supposed to be elevated in the context of HIV-1 infection.
In this study, we suggest a new mechanism of virus mediated CD4+ T cell costimulation during HIV-1 infection. Our in vitro experiments demonstrate that HIV-1
envelope glycoprotein gp120 (HIV-1 gp120) binding to the CD4 receptor of CD4+ T
cells in temporal proximity of suboptimal T cell receptor (TCR) stimulation enhances
their activation. We observe up-regulation of early T cell activation and exhaustion
markers as well as increased levels of proliferation. We are currently investigating
how HIV-1 gp120 ligation of the CD4 receptor affects proximal TCR signaling by
reverse phase protein array technology. In order to validate the HIV-1 gp120 costimulation on a cellular level we aim to establish an assay in which HIV-1 gp120anti-gp120 immune complexes bound to dendritic cells might co-stimulate CD4+ T
cells in the presence of suboptimal TCR stimulation.
Session 5
Selection and autoimmunity
About the Role of Dicer-dependent miRNA in the Thymus
Carlos Mayer1, Saulius Zuklys1, Saule Zhanybekova1, Alberto PascualMontano2 and Georg Holländer1,3
1
Pediatric Immunology, Department of Biomedicine, University of Basel and
University Children’s Hospital Basel (UKBB), Switzerland
2
Functional Bioinformatics Group, National Center for Biotechnology-CSIC,
Universidad Autónoma de Madrid, Spain
3
Developmental Immunology, Department of Pediatrics, University of Oxford,
United Kingdom
The thymus stromal microenvironment is mainly comprised of two, namely
cortical and medullary, subsets of thymic epithelial cells (TEC) that in
aggregate provide immature T progenitor cells with the cell bound and soluble
factors required for their commitment and maturation to fully functional T-cells.
MicroRNA (miRNA) are small, non-coding nucleotides that have been
implicated in many different aspects of biology, including morphogenesis and
tissue maintenance. To investigate the role of miRNA in TEC development
and function, we established a mouse model with a thymus epithelial targeted,
conditional deletion of the miRNA-processing enzyme Dicer. Mice exclusively
deficient for Dicer expression in TECs revealed severe defects in the
composition and structure of their thymus microenvironment as demonstrated
by an early and progressive loss of cortical and medullary thymus epithelial
and significant changes in their phenotype. Consequently, these alterations
compromised the commitment of T cell precursors to a T-cell fate and affected
positive and negative thymocyte selection already at a stage when a physical
loss of TECs was not yet detected. As a result, central tolerance became
severely affected allowing for the generation of a repertoire of mature T-cells
able to mediate overt autoimmunity. With age, the thymus of gene-targeted
mice gained the structural features typical of secondary lymphoid tissues and
supported in situ B cell development. In summary, our results demonstrate the
importance of miRNA for TEC survival and function.
The amount of self-antigen determines the effector function of murine T cells escaping
negative selection
Lee Kim Swee*,2,3, Anja Nusser*,2, Maurus Curti*,4, Matthias Kreuzaler*, Hannie Rolink*, Luigi
Terracciano†, Fritz Melchers§, Jan Andersson* and Antonius Rolink*
*
Developmental and Molecular Immunology, Department of Biomedicine, University of Basel,
Basel, Switzerland
†
Department of Pathology, University Hospital of Basel, Basel, Switzerland
§
Max Planck Institute for Infection Biology, Berlin, Germany
2
These authors contributed equally
3
Current address: Whitehead Institute, Cambridge, MA, USA
4
Current address: Viollier AG, Basel, Switzerland
Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate
a harmful immune response against self. However, factors which influence the initiation and
progression of an autoimmune response remain incompletely understood. By establishing a
double transgenic mouse system in which dendritic cells express different amounts of a cellsurface neo-self antigen we demonstrate that antigen dose dramatically influences T cell
tolerance mechanisms. Moderate antigen expression by dendritic cells favors the development of
antigen-specific regulatory T cells and the establishment of a tolerogenic environment. In
marked contrast, high dose of antigen expression by dendritic cells results in poor development
of antigen-specific regulatory T cells and the early onset of anemia and splenomegaly and the
late development of arthritis and high titers of IgG auto-antibodies. Disease is initiated by
autoreactive T cells, which escape negative selection by expressing a second non-autoreactive T
cell receptor. Transfer of antigen-specific regulatory T cells ameliorates the early onset signs of
disease but does not prevent the development of long-term chronic pathologies.
XXIV Meeting of the Swiss Immunology PhD students Wolfsberg, 2- 4 April
2012
Characterizing autoreactive CD8+ T cells that bypass negative selection
Enouz S, Carrié L, Zehn D.
Swiss Vaccine Research Institute, Centre Hospitalier Universitaire Vaudois, Service of
Immunology and Allergy, Lausanne, Switzerland
Central and peripheral tolerance mechanisms efficiently remove autoreactive T cells.
However it has been shown that autoreactive CD8 + T cells that weakly react with selfantigen can bypass negative selection in the thymus and can be found in the periphery.
Our aim is to precisely characterize these low avidity CD8+ T cells, study their selfdamaging potential, and their ability to circumvent central and peripheral tolerance. To
answer these points we generated a new transgenic mouse model designated as OT-3
TCR transgenic mouse. Importantly, we can demonstrate that the OT-3 resemble the
low avidity T cells found in the periphery of Rip-mOva mice and that the OT-3 can
induce autoimmune diabetes. This indicates that we have generated a model that
allows us to study the properties of auto-reactive T cells that bypass negative selection.
OT-3 cells can also be recruited and activated when pathogens that encode crossreactive epitopes that are of lower affinity than the native ovalbumin antigen are
introduced. These data define a threshold for activating T cells in the periphery that is
below the threshold for negative selection. If these cells can be activated in the
periphery upon low affinity epitopes highlights the existence of an affinity danger
zone involved in the development of autoimmunity.
XXIV Meeting of the Swiss Immunology PhD students Wolfsberg, 2- 4 April 2012
Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation
in mice
S. Pantelyushin1*, S. Haak1*, B. Ingold2*, P. Kulig1 , Frank L. Heppner2, A. Navarini3,
B. Becher1
1 – Institute of Experimental Immunology, University of Zürich, Switzerland
2 – Institute of Pathology, Charité University Hospital, Berlin, Germany
3 – Department of Dermatology,University Hospital Zürich, Switzerland
4 – Current address: Columbia University Medical Center, Dept. Microbiology &
Immunology, 701 West 168th Street, New York, NY-10032, USA
* - equal contribution
Abstract:
Psoriasis is a common, chronic, relapsing inflammatory skin disease characterized by
erythematous scaly plaques. Histologically its manifestations are: keratinocyte
hyperproliferation, elongated rete ridges and inflammatory infiltrates consisting of T
cells, macrophages, dendritic cells and neutrophils. Despite the availability of new
effective drugs to treat psoriasis, there are still many unknowns regarding disease
pathogenesis. Recent studies have shown that Aldara cream (5% Imiquimod), used to
treat benign skin abnormalities, triggers psoriasis-like disease in humans and mice. In
both cases the IL-23/IL-17 pathway is implicated in the disease pathogenesis, with
TH17 cells thought to be the main players. Using this model ofAldara psoriasiform
plaque formation in mice, we could establish a predominant role of the TH17
signature cytokines, IL-17A, IL-17F and IL-22, in this process. Loss of either of these
cytokines in gene-targeted mice strongly reduced disease severity. The main source of
these cytokines, however, was not TH17 cells but a skin-invading population of γδ T
cells and innate lymphocytes. Furthermore, our findings establish that RORγt+ innate
lymphocytes are sufficient and necessary for psoriatic plaque formation in an
experimental disease model that closely resembles early human psoriasis.
Session 6
T cell activation / polarization
Malt1 and Caspase-10: Proteases Essential for Lymphocyte Activation
Jean Enno Charton, Fabien Rebeaud and Margot Thome
Departement de Biochimie, Université de Lausanne, CH-1066 Epalinges,
Switzerland.
T cell activation via the T cell receptor (TCR) through antigen recognition is one of the key steps to
initiate the adaptive immune response. The mechanisms of TCR-induced nuclear factor κB (NF-κB)
and Activator Protein 1 (AP-1) are the subject of intense research since deregulated signaling in
lymphocytes can lead to immunodeficiency, autoimmunity or lymphomas. In T lymphocytes, a complex
composed of Carma1, Bcl-10 and Malt1 transfers signals from TCR proximal events to NF-κB
activation. Gene translocations of the BCL10 or MALT1 genes or oncogenic mutations affecting
CARMA1 result in constitutive NF-κB activation and are related to the development of certain forms of
lymphomas.
Here we report a cleavage of certain Caspase-10 isoforms by Malt1 in T and B cells after antigen
receptor stimulation. Caspase-10 has so far only been shown to be involved in the induction of
apoptosis and is closely related to Caspase-8. W e see a crucial role of the Caspase-10, but not
Caspase-8, in T cell activation after T cell receptor stimulation in an IKK independent manner. Lack of
Caspase-10 leads to abrogation of AP-1 activation, reduced NF-κB activation and a severe defect in
IL-2 production, and thus drastically reduces T cell activation. W hether the cleavage is important for
NF-κB or AP-1 activation and how Caspase-10 is involved in the signaling is the matter of ongoing
research. Taken together, these results give new insights into lymphocyte activation and could lead to
the development of new drugs modulating the immune response or inhibiting lymphoma formation.
Cervical lymph nodes and the pivotal role in the CCR6+ T helper cell priming.
Basso Camilla, Lanzavecchia Antonio and Sallusto Federica.
Institute for Research in Biomedicine, Bellinzona, Switzerland.
To perform their function, effector and memory T cells migrate to sites of antigen
challenge. Previous studies have shown that CCR9 is expressed by T cells that migrate
to the gut, while CCR10 is expressed on T cells that migrate to the skin. These
receptors are induced in T cells by dendritic cells (DCs) that process vitamin A and D,
which are selectively produced in the gut and skin, respectively, to their active
metabolites for presentation with antigen to lymphocytes. Using the experimental
autoimmune encephalomyelitis model, we have shown that pathogenic Th17 cells
upregulate expression of CCR6 and use this receptor to cross the choroid plexus
epithelium, which constitutively express the CCR6 ligand CCL20, to enter the CNS.
Based on these data we hypothesized that CCR6 regulates constitutive migration of
lymphocytes in the CNS and that this receptor may be induced in cervical lymph nodes
(CLNs). Consistent with this hypothesis, we found that naive CD4+ T cells primed by
antigens draining in the CLNs selectively upregulate CCR6 expression. In vitro
experiments showed that CD4+ T cells cultured with cells isolated from CLNs also
upregulate CCR6 expression. Migratory DCs seem to be responsible for CCR6
upregulation on T cells, since CCR6 was not induced in CLNs of CCR7 deficient
animals which lack this DC subset. We are performing experiments to understand what
are the signals required for induction of CCR6 expression in T cells primed in the CLNs
and to define the cytokine profile of these CCR6+ CLN primed T cells.
Priming of polarized human T cells by microbes
Simone Becattini, Laurent Perez, Antonio Lanzavecchia, Federica Sallusto
Institute for Research in Biomedicine, Via Vincenzo Vela 6, 6500 Bellinzona
The innate immune system is endowed with invariant receptors that recognize pathogenassociated or danger-associated molecular patterns (PAMPS and DAMPS, respectively). These
pattern-recognition receptors (PRRs) are highly expressed in antigen presenting cells (APCs)
where they control the level of MHC and costimulatory molecules and the production of
cytokines that contribute to T cell differentiation, such as IL-1β, IL-6, IL-12, and IL-23. Thus, it
is thought that the combination of PRRs triggered by a given pathogen determines the class of
CD4+ T cells elicited, i.e. Th1, Th2, or Th17. While most studies in humans have focused on the
effect of single microbial components, much fewer dealt with the effect of whole microbes on
APCs, especially on T cell priming. We therefore developed a combined in vitro/ex vivo
approach to study the mechanisms of microbe-induced T cell priming and identify the PAMPS
and the antigens associated with a given polarized T cell response using monocytes as APCs.
Monocytes exposed to microbes prime in vitro naïve CD4+ T cells, leading to expansion of
antigen-specific T cell populations with various phenotypes, despite the homogeneity of the
culture conditions. The chemokine receptor pattern of in vitro primed T cells allows enrichment
of specific T cell subsets, as it is the case for ex vivo analyzed memory T cells. Specific
microbial components responsible for induction of a characteristic T cell response can be
identified; in particular we found that C. albicans mannoprotein is a key component for
induction of a Th17 response.
Course of the Th-1, Th-2 and Th-17 Cytokines and of Associated C hemokines Involved in the
Periparasitic Recruitment of Cells in Echinococcus multilocularis Infection
Junhua Wang, Bruno Gottstein*
Institute of Parasitology, University o f Bern, Bern, Switzer land
Methodology/Principal Findings: Hepatic gene expression profiles were assessed using
DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis
metacestode into the liver of susceptible mice. Quantitative RT-PCR was used to describe the
changes in the expression of 10 selected cytokine and chemokine genes from 2 days to 12
mo nths. Microarray analyses showed that CCL8, CCL12 and CCL17 were up -regulated 30fo ld, 6-fo ld and 3- fold at 1 month, respectively. CXC L-9 was up-regulated 3- fold at 6 months.
Among genes o f cytokine receptors, only those for IL-1 (IL-R1 like) and IL-7 (at 1 month,
2.92 and 2.25 fold respectively), and IL-13 (IL-13 Rα1) and IL-17 (IL-17 R) were upregulated (2.39 at 3 months and 2.90 at 6 months respectively). Real time RT-PCR data
showed that a Th1 immune response was predominant in the early stage, but shifted to Th2 in
the middle/late stage. IL-17 mRN A increased from ~2.41-fold at day 2 to 6.58-fold at month
6 with a maximum expressio n at month 6. In the periparasitic infiltrate, positive
immunostainings for IL17 were observed fro m day 8 to mo nth 12, and highly correlated with
CD4 (r=0.864), C D8 (r=0.684) and CD4/8 ratio (r=0.524).
Conclusions: Our data confirm that Th1, then later Th1/Th2 profiles characterize the local
immune response during E. multilocularis infection at the early and middle/late stage,
respectively; in addition, the marked expression of IL-17 and chemokines suggest that they
may p lay an important role in cell homing to inflammatory reaction sites around the parasite
vesicles.
Peter Bretscher, Julian Egger, Erick M. Carreira, Manfred Kopf, and Stefan
Freigang
Oxidized phospholipid signaling in microbial infection and
inflammatory disease
Phospholipids are major constituents of cellular membranes that ensure
structural integrity and provide a barrier to maintain the membrane potential.
However, accumulating evidence also documents their important role in
cellular signaling processes. In particular, oxidatively modified phospholipids
(OxPL) that are generated from unsaturated phospholipids by reactive oxygen
species exert a multitude of biological effects. OxPL are present at sites of
inflammation in vivo and have been implicated in acute and chronic
inflammatory conditions, such as microbial infections, acute lung injury,
atherosclerosis and rheumatoid arthritis. However, the biologically active lipid
species as well as the pathways that mediate their signaling still remain
elusive.
Here we have characterized the complex mixture of OxPL species generated
during the non-enzymatic oxidation of the common phospholipid 1-palmitoyl2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and identified the
isoprostane containing phospholipids 1-palmitoyl-2-(5,6)-epoxyisoprostane
E2-sn-glycero-3-phosphocholine (PEIPC) and 1-palmitoyl-2-[(5R,6R,7E,12S)5,6-epoxyisoprostane A2]-sn-glycero-3-phosphocholine (PECPC) as the main
components mediating the biological activities of OxPAPC. Using synthetic
compounds we show that PEICP and PECPC modulate the production of proinflammatory cytokines by dendritic cells and macrophages. Furthermore,
dendritic cells treated with these OxPL become licensed to polarize naïve
CD4+ T cells towards Th2 differentiation. These effects directly correlated
with the ability of OxPL to activate nuclear factor erythroid 2-related factor 2
(Nrf2), the transcriptional master regulator of the antioxidant response.
Moreover, we demonstrate that structurally related endogenous lipids, e.g. the
prostaglandin J2 (15d-PGJ2), also signal via this OxPL/Nrf2 axis, suggesting
that OxPL induce their biological effects by mimicking the activity of
endogenous signaling lipids.
24th Meeting of the Swiss Immunology PhD students, Wolfsberg, April 2 - April 4, 2012
Mechanisms of T cell stimulation by flucloxacillin in HLA-B*5701+ flucloxacillin-naïve
individuals
Natascha Wuillemin1, Jacqueline Adam1, Stefano Fontana2, Werner J. Pichler1, Daniel Yerly1
1
Clinic for Rheumatology and Clinical Immunology / Allergology, University Hospital of Bern, 3010 Bern,
Switzerland
2
Regional Blood Transfusion Service of the Swiss Red Cross, 3008 Bern, Switzerland
Purpose: Genetic studies identified HLA-B*5701 as risk factor for flucloxacillin (FLUX)
induced liver injury. To better understand this side effect, we established an in vitro model for
FLUX induced T cell activation.
Methodology: Peripheral blood mononuclear cells from FLUX-naïve, HLA-B*5701+ healthy
donors were cultured by iterative in vitro restimulations with FLUX. FLUX-specific T cells
(FLUX-Tc) were characterized by flow cytometry (IFNγ, CD107a) and cytotoxicity assays.
Results: FLUX-Tc could be generated in 7/7 healthy HLA-B*5701+ donors and were mainly
CD8+. They react by IFNγ secretion and CD107a upregulation upon drug stimulation and
exert cytotoxicity against autologous antigen presenting cells (APC). In most cases, FLUXpulsed APC were not able to activate FLUX-Tc. In fact, the addition of FLUX in solution was
sufficient to fully activate FLUX-Tc even in absence of autologous APC. Moreover
proteasome inhibition by lactacystin and bortezomib did not affect the Tc reactivity.
Nevertheless, in a minority of FLUX-Tc the reactivity upon FLUX stimulation was reduced
when APC were not pulsed with flucloxacillin in advance or when APC were not present at
all.
Conclusions: The observations that FLUX in solution can activate FLUX-Tc do not support
the classical hapten theory, explaining the immunogenicity of drug through covalent binding
to macromolecules. In contrast, FLUX-Tc reactivity is best explained by a direct interaction
of FLUX with the restricting HLA-B*5701 molecule. Different ways of drug presentation
may lead to distinct clinical manifestations of FLUX allergy and will further be investigated.
Phenotype and function of pathogen-specific human CD4+ memory T cells
Federico Mele1, Christina E. Zielinski1, Dominik Aschebrenner1, Silvia Monticelli1, Antonio
Lanzavecchia1,2 and Federica Sallusto1
1
Institute for Research in Biomedicine, Bellinzona, and 2ETH Zurich, Switzerland
Using high throughput cellular screening methods we are performing an analysis of the
functional properties of human memory T cells specific for different pathogens. To do this
we established libraries of human memory T cell subsets isolated from blood samples of
immune donors according to the expression of CD45RO and chemokine receptors. The
libraries were interrogated for reactivity against a wide panel of antigens from viruses,
bacteria and fungi. We found that i) virus-specific T cells are present mainly in
CXCR3+CCR6+ and CXCR3+CCR6– Th1 subsets; ii) T cells specific for bacteria are mainly
present in the CXCR3+CCR6+ Th1 and CCR6+CCR4+ Th17 subsets; iii) T cells specific for
Mycobacterium tuberculosis are almost exclusively present in the CXCR3+CCR6+ Th1
subset; iv) T cells specific for Candida albicans are present in the CCR6+CCR4+ Th17
subset and in some donors, are also present in the CCR4+ Th2 subset.
C. albicans-specific Th17 clones were isolated and analyzed for their capacity to produce
IL-17 at different time points following restimulation. We found that while resting clones
produced high amounts of IL-17, day 5-activated clones strongly downregulated IL-17
production and then gradually regained the capacity to produce IL-17 as the cells reverted
to the resting state. The analysis of transcription factors showed that on day 2 and 5
following restimulation, Th17 clones downregulated RORC mRNA expression,
concomitant with downregulation of IL17 mRNA. Overexpression of RORt significantly
restored IL-17 production in activated Th17 clones, and restimulation in the presence of a
STAT5 inhibitor rescued RORC mRNA expression and IL-17 production in a proportion of
clones. These data suggest that decreased RORt expression and increased pSTAT5
levels, contribute to the transient downregulation of IL-17 production in activated human
memory Th17 cells. Taken together our findings reveal a novel type of self-regulatory
mechanism and suppressive function of IL-2.
Session 7
Immune regulation
Role of Thymic stromal lymphopoietin (TSLP) signaling in intestinal immune
homeostasis
Ilaria Mosconi1, Markus B. Geuking2, Joanna C. Massacand1, Kathy D. McCoy2,
Nicola L. Harris1
1) Laboratory of Intestinal Immunology, School of Life Sciences, EPF Lausanne, Switzerland.
2) Mucosal Immunology Lab, Maurice Müller Laboratories, University Clinic for Visceral Surgery and
Medicine, University of Bern, Switzerland
TSLP is a cytokine constitutively expressed by intestinal epithelial cells (IECs) which
has been hypothesized to regulate intestinal inflammation and homeostasis. To
dissect its role in modulating intestinal immunity we utilized TSLP receptor (TSLPR)
deficient mice. We observed significant changes in immune cell populations of both
the lymphoid tissues and intestinal lamina propria (LP) of TSLPR-/- mice: we found a
higher proportion of interferon-γ (IFNγ) producing-CD8+ T cells and of IFNγ and
interleukin (IL)-17 producing-CD4+ T cells. To determine whether dysregulated
cytokine production was occurring as a result of pathogen infection or was in
response to commensal intestinal bacteria we repeated our experiments in germ-free
mice colonized with a minimal, and entirely commensal, bacterial community called
the altered Schaedler flora (ASF). Following recolonization of wildtype mice a
mutualistic TLR-dependent Treg response occurs in wild type mice, leading to a
successful immune adaptation and establishment of host-microbial mutualism
(Geuking et al, 2011). However, in presence of a defective Treg cell compartment,
colonization causes an immune deviation to Th1 and Th17 cell responses.
Consistent with our earlier experiments TSLPR-/- mice re-colonized with ASF
bacteria exhibited an increased percentage of CD4+ T cells producing IFNg or IL-17.
They also exhibited a lower percentage of Helios- Foxp3+ Tregs, indicating a defect
in the expansion of inducible Tregs. Taken together, these data indicate that the
mechanism by which TSLP impacts on colonic inflammation may involve a role for
this cytokine in the activation or maintenance of Tregs in response to the commensal
intestinal bacteria.
IFN-a modulates the expression of the gut-homing receptor a 417
1,2
2
2
2
2
Nina Suhartha , Simon Heidegger , Nadja Sandholzer , Nicolas Stephan , David Anz , Stefan
2
Endres , Carole Bourquin
1
1
Chair of Pharmacology, Department Medicine, University of Fribourg, Switzerland
2
Department of Medicine, Division of Clinical Pharmacology, Ludwig-Maximilians-Unversity of Munich,
Germany
a417 is an integrin that is expressed at low levels on naïve T cells. Through
interaction with its ligand MAdCAM-1, a4 17 enables naïve T cells to enter the gutassociated lymphoid organs (GALT) such as mesenteric lymph nodes and Peyer’s
patches. Upon T cell activation in the GALT, a417 expression on T cells is
upregulated allowing the T cells to migrate into the gut.
Previous in vitro finding in our lab showed that Toll-like receptor (TLR) agonists
decrease a417 expression on T cells. Similarly, I found that when poly I:C, a TLR3
ligand, is injected into mice, a417 expression on CD8+ T cells is reduced in all
secondary lymphoid organs. This reduction was abolished when poly I:C was
injected into IFNAR-/- knockout mice which lack the type I interferon receptor. When
splenocytes are incubated with recombinant mouse IFN-a, a417 expression on T cells
is significantly reduced. Likewise, when mice are treated with IFN-a, a417 expression
on T cells in secondary lymphoid organs is downregulated. When IFN-a-treated
splenocytes are transferred into mice, these cells show clearly an impaired migration
into the GALT in comparison to the untreated splenocytes. Furthermore, splenocytes
infected with the Sendai, EMCV or VSV viruses, show a decrease of a417 expression
on the T cells.
In conclusion, this project has revealed that IFN-a decreases a417 expression on
naïve CD8+ T cells and blocks their migration to GALT. This may be a mechanism to
prevent activated non-cognate T cells from initiating an overwhelming immune
response in the gut.
Primary and memory B cell responses to Qβ-VLP in mice
Franziska Zabel, Alexander Link, Juliana Bessa, Philippe Saudan, Martin F.
Bachmann
Department of Dermatology, Zurich, University Hospital Zurich
Long-lived humoral memory is induced by the interaction of B cells with their cognate
antigen in the presence of T cell help through both cell bound (e.g. CD40L) and
secreted molecules (e.g. IL-4, IFNγ or IL-21) which promote isotype-switching, B-cell
proliferation and differentiation. In contrast to the well understood differentiation of
naïve B cells into memory and/or plasma cells, relatively little is known about the fate
of memory B cells upon exposure to antigen.
To address this question, we used virus-like particles (VLPs) derived from the
bacteriophage Qβ, which are highly immunogenic due to their structure. To trace
memory B cells, congenic Ly5.1+ C57BL/6 mice were immunized with Qβ, splenic
memory B cell isolated and transferred intravenously into naïve Ly5.2+ C57BL/6. 24
hours later recipient mice were challenged with Qβ.
The transferred memory B cells homed to the spleen but did not proliferate as
efficiently as naïve B cells after immunization not did they contribute effectively to the
germinal centre reaction. In contrast, they differentiated more quickly into plasma
cells and secreted antibodies at a higher level than plasma cells derived from naïve B
cells. Thus, memory B cells are likely the main source of antibodies during secondary
B cell responses.
Session 8
Tumor immunity
Ali Bransi, Alexander Knuth, Maries van den Broek
University Hospital Zurich, Wagistrasse 14, 8952 Schlieren, Switzerland
Ali.Bransi@usz.ch
The tumour environment delivers suppressive signals to the immune system, which impair
the effector function of tumour-infiltrating lymphocytes. The identity of these signals, their
targets and their mode of action, however, are largely unresolved.
Using mouse models of inducible cancer in a tissue specific fashion, we aim to identify
mechanisms that locally suppress the function of tumour-specific T cells. We are
establishing an inducible Cre/LoxP-based model system to express the immunologically
well-characterized oncogene SV40 large T antigen with the luciferase in a tissue specific
fashion. While waiting for the birth of the new transgenic mice, pilot studies were realized
using the already available TRAMP mice. These mice develop progressive forms of
prostate cancer with an early onset of the disease (12 weeks). The carcinogenesis is
driven by the SV40 large T antigen (SV40LT) under the androgen-regulated rat probasin
promoter to limit expression to the prostate.
We observed rapid local and peripheral immune subversion of adoptively transferred
tumour-specific CD8 T cells in tumour-bearing animals. Homing and proliferation were not
affected, but effector functions of these cells were greatly reduced at all sites compared to
controls. This immunosuppression was also more severe inside the tumour than in the
periphery. In addition, we observed a striking difference in tolerance of CD8 T cells
towards different epitopes of SV40LT.
This project will give new knowledge about molecules and pathways that are crucial in
deviating tumour-specific immunity.
Impact of innate lymphocytes on the tumor microenvironment
Sara Burkhard, Maya Eisenring, Burkhard Becher
University of Zurich, Institute of Experimental Immunology
The potent tumoricidal activity of interleukin 12 (IL-12) has been described in numerous studies.
Previously our group has identified the mechanism by which IL-12 mediates tumor suppression in the
subcutaneous layer. It was found that the IL-12-mediated repression of tumor growth within the skin
surprisingly functions completely independent of T lymphocytes or NK cells. In contrast, by
systematically analyzing the IL-12 mediated anti-tumor immunity in a series of mouse mutants, they
discovered that the IL-12 responsive tumor-invading cell type is an innate, lineage negative leukocyte
expressing the transcription factor RORγT. This was demonstrated with a subcutaneously inoculated
melanoma cell line and attributes a novel function to this cell type in tumor immunology. The
presence of these innate lymphoid cells (ILC) leads to the upregulation of the adhesion molecules
ICAM-1 and VCAM-1 in the tumor vasculature and increased leukocyte invasion. To unravel the
mechanism of action, the endothelial cell line MS-1 was cocultured with YFP+ RORγT+ ILCs,
obtained from RORc-Cre x Rosa26-stop-eYFP mice deficient in Rag1. The in vitro adhesion molecule
upregulation by the IL-12 administered ILCs was found to be dependent on IFNγ. Furthermore timelapse imaging of tumor cell lines and tumor explants will be performed. B16 melanoma cells stably
transfected with tdTomatoPEST will be cocultured with YFP+ RORγT+ ILCs, which enables us to
visualize tumor as well as effector cells.
Augmentation of antitumor immunity applying a semi-allogeneic
dendritic cell line
Sergio Haller and Hans Acha-Orbea: Department of Biochemistry, University of Lausanne,
Switzerland
Dendritic cells (DCs) are master regulators of adaptive immune responses and tolerance. They
efficiently capture, process and present to T cells, non- self and self-antigens such as
pathogens and cancerous cells respectively. Although various approaches using DCs as
therapeutic cancer vaccines have been established in murine models, they have met only
limited success in clinical trials. Present publications are predominantly restricted monocyte
or bone marrow-derived DCs which are limited by cell number, homogeneity and phenotype
of the applied cells.
A stable murine DC line has been established in our group previously. They were derived
from CD11c promoter driven large T oncogene expressing transgenic mice. This allows the
preparation of large batches of homogenous CD8α+ DCs and its application as a more
standardized vaccine in terms of phenotype and maturation. This will give us a better insight
of the fate and function of injected DCs within a tumor bearing host.
We first increased the immunogenicity of the DC line using two different approaches: The
cell line was modified in order to express MHC-I H-2Kd. We then demonstrated that in vitro
these DCs are able to present tumor antigens to CD8+ lymphocytes also on BALB/c
background and induce a Th1 response. Preliminary in vivo experiments indicate delayed
tumor growth upon the injection of semi-allogeneic H-2Kd+ DCs when compared to
autologous or full-allogeneic vaccination. We further transduced the DC lines to secrete the
pro-inflammatory cytokines IL-2, IL-12 or IL-15. The combination of vaccination and local
delivery of pro-inflammatory cytokines should induce a more effective anti-tumor immunity.
Tumour therapy by redirected Fibroblast Activation Protein- specific CD8+ T cells
Christian Hagedorn1, Alex Soltermann2, Maries van den Broek1, Chrisoph Renner1, and Ulf
Petrausch3
1
Department of Oncology, University Hospital Zurich, 8091 Zurich, Switzerland
Institute for Surgical Pathology, University Hospital Zurich, 8091 Zurich, Switzerland
3
Departent of Immunology, University Hospital Zurich, 8091 Zurich, Switzerland
2
Through introduction of a chimeric antigen receptor (CAR), T cells can be redirected to
recognize tumour-associated antigens (TAAs). CARs consist of an antibody-derived domain
(scFv) specific for a particular TAA plus intracellular signaling moieties of CD3 and CD28.
Engagement of the CAR with the TAA will thus result in bona fide T cell activation.
We aim to redirect T cells against Fibroblast Activation Protein-FAP- FAP- is a
surface protein expressed by tumour stroma of the majority of human carcinomas as well as
by sarcomas. In addition, in particular malignancies, tumour cells themselves express FAP-.
In healthy adult tissue FAP-is only expressed by pancreatic islet cells, which makes
targeting FAP- an attractive therapy for the treatment of cancer.
Using a phage library, we selected two FAP- specific scFvs and generated CARs with
thoseIn contrast with the well-known and clinically tested human FAP- specific F19
antibody, both newly selected scFvs (ESC11 and ESC14) recognized both human and murine
FAP-. We expressed F19-, ESC11- and ESC14-CARs in human primary CD8+ T cells by
retroviral transduction. Redirected human T cells against FAP-specifically lysed FAP-+
tumour cells in vitro. In addition, redirected FAP- specific human CD8+ T cells produced
IFN- in vitro in a FAP- specific fashion.
Next, we will test the therapeutic efficacy of FAP- specific redirected T cells using
xenografted FAP-+ human tumours in NOD.SCID.c-/- mice. In addition, we will generate
murine CARs with ESC11 and ESC14 and test their therapeutic potential in syngeneic mouse
models in which the tumour stroma is FAP-+.
XXIV Meeting of the Swiss Immunology PhD students Wolfsberg, 2- 4 April 2012
Modulation of astrocytoma-cytotoxic T lymphocyte interactions by chemotherapy
Cristina Riccadonna and Paul R. Walker
Geneva University Hospitals and Geneva University, Centre of Oncology
Introduction
Malignant astrocytoma is an aggressive brain tumour with poor prognosis, despite treatment
(surgical resection, radiotherapy and chemotherapy). It is infiltrated by immune cells,
including cytotoxic T lymphocytes (CTLs). The majority of astrocytoma cells are
differentiated, whereas a small fraction (defined as astrocytoma initiating stem-like cells), is
de-differentiated, reported to be chemoresistant, and is likely to be the cause of tumour
recurrence. Here, we explore the potential of immunotherapy for astrocytoma.
Aims
1) Can chemotherapy impact on astrocytoma-CTL interactions?
2) Can death pathways induced in astrocytoma by immune and chemotherapy mediators be
synergistically combined?
Astrocytoma model and preliminary results
GL261 murine astrocytoma grown as adherent cultures are differentiated cells, whereas
GL261 neurospheres are astrocytoma initiating stem-like cells. Neurospheres showed
enhanced resistance to two different chemotherapeutic drugs: temozolomide, an alkylating
agent, and paclitaxel, which stabilises microtubules and interferes with mitosis. Both adherent
cells and neurospheres express MHCI, essential for recognition by CTL, and cytotoxicity tests
confirmed sensitivity to CTL-mediated lysis.
Perspectives
CTL-tumour cell adhesion as well as subsequent death pathway events will dictate sensitivity
to immune-mediated killing, and will be characterised in the presence of chemotherapy
treatment. Granzyme uptake by tumour cells, proposed to be modulated by some drugs, will
be explored for astrocytoma using confocal microscopy. Synergistic immune and
chemotherapy approaches will ultimately be validated using stringent in vivo models.
Poster Abstracts
The Role of ICOS/ICOSL in anti-tumour immune response.
Natalia Botelho, Rachel Perret, Alena Donda, Pedro Romero
Ludwig Center for Cancer Research of the University of Lausanne
ICOS is a costimulatory molecule expressed on T cells upon cell activation. Its ligand,
ICOSL, belongs to the B7 family and is constitutively expressed on APCs as well as
nonhematopoietic tissues, such as endothelial cells, brain, liver, lung and others. ICOS
ligation by ICOSL provides costimulatory signals that promote T cell proliferation,
differentiation and increase in cytokines secretion. There is conflicting evidence in the
literature as to whether ICOS/ICOSL ligation in a cancer setting is beneficial or detrimental
to antitumour immune responses. Some murine studies show that ICOSL- tumours get less
rejected by the immune system than ICOSL+ tumours, while others have shown that ICOSL+
melanoma promote Treg survival and expansion, thus dampening antitumour immunity. We
hypothesize that the functional outcome of ICOS ligation on T cells is context dependent.
The stimulatory or regulatory outcome of the ICOS-ICOSL connection would depend on
whether the ICOSL signal is provided by immature DCs (or nonprofessional APCs), leading
to a regulatory outcome, or by mature DCs, or in the presence of a strong inflammatory
environment, leading to a Th1/Tc1 polarity.
The Sanroque mouse line is a strain that carries a mutation in the roquin gene, which leads to
overexpression of ICOS, even in naïve T cells. Unexpectedly, upon human gp100 lentivirus
immunisation, specific CD8+ T cell expansion in the circulating lymphocyte compartment
was markedly reduced in Sanroque mice as compared to that measured in the wild type mice.
We do not know whether this is due to the ICOS overexpression in T cells or to other cell
extrinsic mechanism in these mice. A possible mechanism could result from ICOS induced
downregulation of ICOSL in dendritic cells that would lead to a reduced ability to prime a
CD8 T cell response. Alternatively, the role of ICOS overexpressing Tregs in Sanroque mice
will be evaluated, as they might limit T cell priming.
Wolfsberg Meeting 2012
Manuel Coutaz1, Floriane Auderset1, Stéphanie Corgnac2, Pedro Romero2, Fabienne Tacchini-Cottier 1
1
Department of Biochemistry, WHO Immunology Research and Training Center, University of
Lausanne, Epalinges, Switzerland
2
Ludwig Institute for Cancer Research Ltd, Lausanne Branch, University of Lausanne, Epalinges,
Switzerland.
TH17 cells have been described as an effector CD4+ T helper subset on the basis of their
secretion of specific cytokines (IL-17A, IL-17F) and their dependence of specific transcription
factors (RORγt, RORα). Notch signalling is a highly evolutionary conserved pathway
regulating cell lineage and cell differentiation fates in many organs and tissues. Notch
receptors regulate cell differentiation during development but their role in the differentiation of
CD4+ T helper subsets remains unclear. Of the four Notch receptors, only Notch1 (N1) and
Notch2 (N2) are expressed on activated CD4+ T cells. In contrast to its documented role in
TH1 and TH2 cell differentiation, only a few studies have demonstrated that Notch signalling
might be involved TH17 cell differentiation. To investigate this, initial experiments were
performed in attempt to define the role of Notch in TH17 differentiation in vitro. Naive CD4 T
cells isolated from mice carrying a T cell-specific gene ablation of Notch receptors
(N1N2ΔCD4Cre) were differentiated in TH17 condition. The absence of Notch receptors
specifically decreased IL-17A secretion and mRNA expression but not the mRNA expression
of RORγt. We then investigate the role of Notch signalling in Th17 differentiation in vivo,
using the experimental subcutaneous melanoma. An increased percentage of IL-17A+ in
CD4+ T cells was measured in the TDLNs in N1N2ΔCD4Cre mice. These results suggest that
Notch signalling pathway may have an impact on IL-17A cytokine release correlating with
delayed tumor growth observed in B16 N1N2ΔCD4Cre mice.
Marina Fertig, Carole Bourquin
Chair of Pharmacology, Department of Medicine, Faculty of Science, University of Fribourg
Immunotherapy of gastric cancer: enhancing T cell recruitment into tumors
Gastric cancer is one of the most common types of cancer in the world. So far, there
is no effective therapeutic treatment for stomach cancer which is characterized by its
high mortality rate. One promising approach to treat gastric tumors is
immunotherapy.
Previous studies have shown that intratumoral T cell infiltration positively correlates
with the survival of patients with gastric cancer. Thus, T cell recruitment into the
tumor is one of the important goals of cancer immunotherapy. However, the
immunosuppressive tumor microenvironment can impair the efficiency of T cell
infiltration.
The gastric tumor microenvironment and its role in affecting lymphocyte infiltration
are the main interests of this project. To examine the efficacy of the immunotherapy,
we are using the CEA424-SV40 T antigen mouse model. These mice develop
spontaneous gastric tumors in the pyloric region of the stomach at an early age and
this model reflects the development of gastric cancer in humans.
Previous in vivo findings in our lab revealed that autochthonous gastric tumors do not
respond to T cell-based therapy, in contrast to subcutaneous tumors that are induced
by injection of a stomach tumor-derived cell line. In the present project, we are
comparing both autochthonous and subcutaneous tumors in the same mice in order
to investigate the factors that control the T cell recruitment.
The results of this study may provide an important contribution to the development of
additional therapeutic strategies to target gastric tumors.
Strength and limitations of regulatory T cells for immunotherapy in transplantation
Lerisa Govender1 and Dr Dela Golshayan (MD PhD) 2
1
University of Lausanne, PhD Cancer and Immunology Program, Transplantation Immunopathology
Laboratory,CHUV
2
Dpt of Medicine, Nephrology and Transplantation Centre, Transplantation Immunopathology Laboratory,
CHUV
In many experimental models, CD4+CD25+Foxp3+ regulatory T cells (nTreg) have been
identified as key players in promoting peripheral transplantation (Tx) tolerance. We are
focusing on therapies based on antigen-specific nTreg that control effector T cells (Teff) and
prevent allograft rejection. The use of nTreg in immunotherapeutic protocols for solid organ
Tx is however limited by their overall low numbers (2-10% of CD4+ T cells).
In this study, we aimed to expand Treg directly in vivo and determine their suppressive
function, efficacy and stability in promoting donor-specific tolerance in a stringent murine Tx
model. Our data suggest that IL-2-based therapies lead to a significant increase of Treg in
vivo. The expanded Treg suppressed Teff proliferation and allowed prolonged graft survival
of major MHC-mismatched skin grafts in wild-type non-lymphopenic recipients. The
expanded Treg alone were however not sufficient to induce tolerance in stringent
experimental conditions. Rapamycin reduced the frequency of Teff but did not impede
expansion of Treg. Pro-inflammatory stimuli hindered the expansion of Treg and resulted in
an increase in the frequency of CD4+IFN-γ+ and CD4+IL17+ T cells.
We propose that IL-2-based treatments would be an efficient method for expanding functional
Treg in vivo, thereby favorably shifting the pool of alloreactive T cells towards regulation in
response to an allograft. However, we also highlight some potential limitations of Treg
expansion such as concomitant inflammatory events.
TITLE:
Infectious metastasis of mucocutaneous leishmaniasis:
An IL-17-mediated
response to leishmania RNA virus
PRESENTED BY: Mary-Anne Hartley
AFFLIATION:
University of Lausanne, Department of Biochemistry, Laboratory of Professor
Nicolas Fasel (Co-supervision: Dr. Catherine Ronet)
Leishmania guyanensis (L.g) parasites have been plaguing humankind for
centuries, provoking a deleterious inflammatory response, destroying host
tissue and forming the ulcerating lesions, which typify most forms of the
disease. About 15% of patients infected with a certain subtype of these
parasites develop secondary lesions in the mouth and nose, where parasites
metastasise to mucocutaneous tissues. We have recently linked disease
severity in these infections to a virus infecting this parasite subtype:
Leishmania RNA virus (LRV).
Using L.g. clones, which are either naturally infected by LRV (V+) or depleted
in it (V-), we set out to characterise the immune phenotype in a murine model
of infection. Here, we found that V+ parasites potently induced the production
of IL-17A as compared to its V- equivalent, thus insinuating that this proinflammatory cytokine plays a destructive role in the devolution and severity of
metastatic leishmaniasis. Indeed, IL-17A-/- mice infected with V+ parasites
had a significantly reduced disease severity compared to wild type and in vitro
studies revealed that IL-17A directly increased the parasitism of macrophages
while simultaneously decreasing their ability to clear the infection. Although IL17 has a promiscuous role in immunity, stimulating both the innate and
adaptive immune systems (as well as a plethora of non-immune cells), a
major shared goal of the IL-17 pathways is its self-propagation, where downstream effects converge to create a hyper-inflammatory feedback loop. This
study will hopefully open a door to the development of a simple
immunotherapeutic solution to a devastating and neglected disease.
Intrinsic and extrinsic change in HSC behaviour upon aging
Larisa Kovtonyuk, Ramin Radpour, Markus G. Manz and Hitoshi Takizawa
Division of Hematology, University Hospital Zürich, Rämistrasse 100, CH-8091 Zürich,
Larisa.kovtonyuk@usz.ch
Hematopoiesis is an active ongoing process in bone marrow (BM) in which life-­ long self-­­
renewing hematopoietic stem cells (HSCs) replenish short-­ lived mature blood cells each day.
Aging has an impact on homeostasis and regeneration of many tissues and their
respective stem cells. Upon aging, HSCs also change their function including BM­ homing ability, differentiation and self renewal capacity. Our group demonstrated with
in vivo single HSC divisional tracking that at any given time HSC consists of actively
cycling and dormant pools with fluctuating hematopoietic contribution. HSCs with high
proliferative history during physiological aging or serial transplantation tend to be
dormant in a permissive environment. We hypothesized that cell-­ intrinsic drive toward
dormancy is imprinted on HSCs through extensive divisions. The project aims to answer
the questions on how the extrinsic and intrinsic factors determine HSC behaviour upon
hematopoietic stress and aging, and what their relative contribution and molecular signature
are on HSC cell fate decision.
Preliminary data have demonstrated that aged HSCs increased dormancy when
transplanted into non-­ irradiated young animals, suggesting cell-­ intrinsic regulation of
dormancy. Fast-­ cycling aged HSCs showed balanced lineage repopulation as do both
fast-­­ cycling and dormant young HSCs, while aged HSCs contributed to more
myelopoiesis than lymphopoiesis, indicating the link between HSC lineage choice and cell
cycle activity. Similar biology as in aged HSCs was observed in HSCs with extensive
divisions during serial transplantation. It was shown, that both young and aged HSCs
proliferated faster in an aged than in a young environment. In addition, hematopoietic
stress as bleeding and infection, drives dormant HSCs into division, suggesting the
extrinsic drive of HSC division. To dissect aging-­ associated intrinsic and extrinsic factors,
we performed antibody based protein arrays with young versus aged BM cell lysates
followed by bioinformatical analysis. The result revealed that expression levels of some
secreted factors and cell surface receptors were altered in aged BM.
Our findings suggest that extensive proliferative history imprints on HSCs intrinsic programs
for dormancy and myeloid-­­biased differentiation, and that the dormant HSC can be
activated possibly via age-­­and hematopoietic stress-­ related protein(s). Understanding of the
molecular signature of intrinsic and extrinsic factors will provide new knowledge of
regulatory mechanisms of stem cell-­­sustained organ systems, and open new avenues to
control normal HSC cell cycle and function and to potentially allow specific eradication
of pre-­­malignant stem cells in aged individuals.
Title: Triaka, a novel mouse model for intestinal disease.
Authors: Lukas Mager1, Philippe Krebs1
1
Division of Experimental Pathology, Institute of Pathology, University of Bern, Bern,
Switzerland
Abstract:
Inflammatory Bowel Disease (IBD) is of multifactorial origin, encompassing genetic
predispositions, intestinal microbiota and immunopathologic responses to them. The
intestinal epithelial barrier plays a vital role in gut homeostasis and its integrity is
altered in intestinal disorders such as IBD and colorectal cancer (CRC). Gut epithelial
cells express genes like occludin and claudin but also immuno-modulatory
molecules, among them IL-10, TGF-b and IL-8, depicting their importance for
controlling the intestinal flora via interaction with the immune system. Here, we
investigated a novel mouse model called Triaka that is characterized by a mutation in
a conserved region of the Epithelial Splicing Regulator Protein 1 (Esrp1) gene. Esrp1
is highly expressed in the gut epithelium and Esrp1Triaka mice were found to be more
susceptible to the dextran sodium sulfate (DSS) model of acute colitis. Histological
sections of colon of DSS treated Esrp1Triaka mice showed massive loss of intestinal
epithelial architecture, exacerbated inflammation, leucocyte infiltration, which are all
features that can be found in IBD patients. Therefore, we hypothesize that the
mutation leads to an alteration in the alternative splicing pattern of Esrp1 target
genes, such as CD44, FGFR2, that are critical for the homeostatic properties of the
gut epithelium. We are currently in the process of identifying these target genes that
may become prognostic markers of IBD susceptibility. Also, since there is an
undeniable relationship between IBD and CRC, the role of the Triaka mutation for
carcinogenesis will be studied in a model of CRC.
Clinical and histological features of flaky skin (fsn) mice
Ursina Nüesch, Reinhard Seger, Jana Pachlopnik Schmid
Division of Immunology/Haematology/Bone marrow transplantation, University Children’s Hospital Zürich,
Steinwiesstrasse 75, 8032 Zürich
Mice homozygous for the flaky skin (fsn) mutation in the gene encoding
tetratricopeptide repeat protein 7 (TTC7) show a complex phenotype including
inflammation and autoimmunity. The aim of our study was to perform a clinical and
histopathological examination of the fsn/fsn mice mandatory for our future
immunological investigations. We observed decreased body weight in fsn/fsn mice
compared to their littermates. At the age of 8 weeks, spleens, axillary lymph nodes
and livers were significantly increased in size in the fsn/fsn mice, while the thymus
was smaller in fsn/fsn mice compared to the one in littermates. Histological analysis
showed a disturbed splenic and lymph node architecture. At the age of 8 weeks, we
could not find any deposits or inflammatory infiltrates in the kidneys of fsn/fsn mice.
However, in 6 of 9 fsn/fsn mice we observed diarrhea. Inflammatory infiltrates and
apoptotic cells were detected in the intestine. Our observations of lymphadenopathy
and splenomegaly are consistent with previous publications. In contrast, we did not
see any histopathological changes in the kidneys but this might be due to the
younger age at examination in our study (8 vs 12 weeks). Interestingly, we observe
an intestinal pathology that has not been described before. In order to better
characterize the infiltrating cell subpopulations, we intend to perform flow cytometric
analyses and immunohistochemistry.
key words: flaky skin mouse, TTC7, inflammation
1
Kathrin Nussbaum
Group of Prof. Dr. Burkhard Becher
University of Zurich
Institute for Experimental Immunology
Winterthurerstrasse 190
CH-8057 Zürich
Abstract
Molecular mechanisms of innate lymphoid cells on IL12-mediated tumor suppression in
the skin
Malignant melanoma is a particularly aggressive tumor due to its metastasizing propensity
and is the cause of most skin cancer-related deaths. Interleukin-12 (IL12) has consistently
been demonstrated to possess potent tumor suppressing activity in murine melanoma models,
however systemic administration in melanoma patients led to limited responses and severe
adverse effects. Our lab showed that local instead of systemic IL12 delivery likewise results
in strong anti-tumor suppressive activity mediated by innate lymphoid cells (ILCs). Although
the molecular underpinnings of this phenomenon remain elusive we hypothesize that IL12
initiates tumor suppression through ILCs by changing the tumor microenvironment to be
more conducive to subsequent immune invasion. Our overall goal is to determine how the
immune system can be utilized to recognize and eliminate cancer cells. Therefore, we
specifically aims to (1) systematically elucidate the impact of local IL12 treatment and
combinatorial therapies using co-inhibiting immunosuppressive molecules in prophylactic and
therapeutic settings in distinct murine melanoma models and (2) to determine the molecular
mechanism underlying the
IL12
mediated tumor
suppression
by
identifying and
characterizing molecular targets of ILCs. A better understanding of the role of IL12 and ILC
function in tumors suppression and may lead to new immunotherapeutic applications taking
advantages of the specific ability of cytokine-mediated changes to the tumor
microenvironment.
1
PD-1 and SHP-1 expression finely tunes TCR-affinity-mediated cell activation
and function in human tumor reactive T lymphocytes
Danilo Presotto1, Michael Hebeisen1, Lukas Baitsch2, Petra Baumgaertner2, Olivier
Michielin2,3, Daniel E. Speiser2 and Nathalie Rufer1,2
1
Department of Research, Lausanne University Hospital Center and University of
Lausanne, Lausanne, Switzerland
2
Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland
3
Multidisciplinary Oncology Center (CePO), University Hospital Center, Lausanne,
Switzerland
Adaptive immune responses rely on TCR-dependent recognition of antigenic peptides
displayed by self-MHC molecules, and the strength of this interaction is a major
correlate of disease protection. Because of central tolerance, T cells bearing TCRs
against (self)-tumor antigens have lower affinity/avidity than pathogen-specific ones
and elicite weaker immune responses. One strategy to overcome this limitation is to
increase TCR affinity for tumor Ags. We generated a panel of optimized TCR
variants having gradual increases in affinity for the melanoma cancer-germline Ag
NY-ESO-1. We found that optimal mRNA expression and T cell function were
achieved for CD8+ T cells expressing TCR with upper physiological affinities (KD 1-5
mM). Unexpectedly, a drastic functional decline occurred in T cells with TCRs of
supraphysiological affinities (< 1 mM). Biochemical characterization of the TCR
activation pathway in these cells showed reduced phosphorylation levels of both
ZAP-70 and ERK 1/2 compared to the WT, with a concurrent increase in the
expression levels of PD-1 and its associated phosphatase SHP-1. Importantly, PD-1
and SHP-1 blockade could partially restore T cell function in T cells of highest
affinity. Collectively, these results indicate that SHP-1 and PD-1 act very proximally
in the TCR pathway and could serve as negative regulators to control cell activation,
signaling and function in T cells bearing self TCRs of supra-physiological affinities.
Such studies are essential to identify putative targets to increase T cell function,
allowing further promoting of therapeutic immune interventions like adoptive T cell
therapy.
XXIV Meeting of the Swiss Immunology PhD students
Wolfsberg, 2- 4 April 2012
_
Abstract
CD27-CD70 costimulatory axis
Mohamad Al Sayeda and Adrian Ochsenbeina, b
a. University of Bern; Department of Clinical Research, Tumor immunology
Mohamad.Alsayed@dkf.unibe.ch
b. University Hospital Bern; Institute for Medical Oncology Adrian.Ochsenbein@insel.ch
T cell activation and survival are critical processes for an efficient immune response against various
infections and cancer. Besides T cell receptor stimulation, another signal through a co-stimulatory
molecule is required for efficient T cell activation. The co-stimulatory molecule CD27, a member of the
TNF receptor superfamily, is expressed on subsets of T, B and NK cells. CD27 promotes expansion,
survival and memory formation of T cells. To date, two isoforms of CD27 have been identified, but the
differences in expression and function of these two isoforms in different immune cells have not been
addressed. In this study, we identified a third isoform of CD27 which we characterized on the RNA level.
We also studied the expression of the three isoforms in different immune cell populations. Further
characterization of CD27 isoforms, at the protein level, is required. On the other hand, CD70, the unique
ligand of CD27, which is expressed on activated T cells and antigen-presenting cells, has been shown to
have a reverse signaling activity on B cells. Our preliminary results shows a possible CD70 reverse
signalling activity on T cells. Taken all together, CD27-CD70 co-stimulatory axis seems to have both
negative and positive regulatory role during immune response, and more investigation about CD27
isoforms and their ligand may reveal the underlying mechanisms of such regulatory effects.
1
Controlling the recruitment of myeloid-derived suppressor cells into the tumor
microenvironment.
Thibaud Spinetti, Carole Bourquin
Chair of Pharmacology, Department of Medicine, University of Fribourg, Switzerland
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of
immature myeloid cells with the capacity to inhibit immunological responses. During
cancer progression, MDSC are recruited to the tumor sites and secondary lymphoid
organs. This recruitment leads to the suppression of the anti-tumor function of T and
natural killer cells. Factors released by tumor cells or their microenvironment such as
GM-CSF or IL1b lead to the migration of MDSC into the tumor.
Previous studies in the Bourquin group show that treatment by different TLR ligands
in tumor-bearing mice leads to a change in the phenotype and their capacity to
suppress the T cell response. The aim of this project is to identify effects of these
different TLR ligands on the migration patterns of MDSC. Investigation into the
regulation of chemokines and their receptors as well as homing molecules such as
selectins or integrins after TLR challenge are ongoing. For a global understanding of
the mechanisms involved, in vitro and in vivo experiments are planned and analysis
by different methods like flow cytometry or RT-PCR are used to identify the
modulation of protein expression.
In conclusion, this project should give new information about the impact of innate
immune stimulation on MDSC migration in tumor-bearing mice. Considering the
suppressive activity of MDSC in anti-tumor immunity, identification of new strategies
to control trafficking of these cells may improve immune responses against cancer.
XXIV Meeting of the Swiss Immunology PhD students Wolfsberg, 2- 4 April 2012
Immune Control of MCMV in the Salivary Gland
Jenny Thom, Annette Oxenius
ETH Zürich - Institute of Microbiology
Wolfgang-Pauli-Strasse 10
CH-8093 Zürich
Jenny.thom@micro.biol.ethz.ch
Cytomegaloviruses (CMVs) are ubiquitous β-herpesviruses that establish lifelong
infection and latency. Prolonged secretion of virus in saliva represents a major route
of CMV transmission with acinar glandular epithelial cells propagating virus for
months after virus replication is controlled in all other organs. While CD8 T cells are
sufficient to control viral replication in visceral organs, they fail to do so in the salivary
glands where virus is eventually controlled by IFNg producing CD4 T cells. Using
murine CMV (MCMV) our group showed that despite MCMV-specific CD8 T cells
being recruited to the salivary glands, their activation is prevented as MCMV
encoded immune evasion genes impair MHC I expression on infected acinar
glandular epithelial cells. Moreover, our data indicate that CD4 T cells in the salivary
glands are activated by non-infected antigen presenting cells (APCs) which present
internalized antigen via MHC II but are unable to cross-present on MHC I, thus failing
to activate CD8 T cells. Our aim is to further analyze the mechanisms leading to CD4
T cell mediated control of lytic viral replication in the salivary glands. To this end, we
will characterize salivary gland resident APCs and compare their phenotypes and
functions with APCs derived from lymphoid tissue and from other secretory organs.
Furthermore, we aim to visualize MCMV spread, T cell migration, antigen
recognition, and finally control of lytic viral replication via intravital microscopy of the
salivary glands to understand the dynamic processes involved in creating the unique
resistance towards CD8 T cell mediated viral clearance.
Flt3-mediated regulation of dendritic cell homeostasis
Chakradhar Yakkala, Dior Kingston, Hitoshi Takizawa, Yasuyuki Saito, Tim Sparwasser,
Markus G Manz
Division of Hematology, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich
Chakradhar.Yakkala@usz.ch
Dendritic cells (DC) are the antigen presenting cells that act as pedagogues for the generation of an
immune response. Because of their short life span, they need to be continuously replenished by the
upstream hematopoietic stem and progenitor cells. FMS like tyrosine kinase 3 (Flt3) is a class III
membrane bound tyrosine kinase receptor that is expressed on early hematopoietic progenitors, DC
precursors and DCs, and has been shown to instruct the differentiation of hematopoietic progenitors
towards DC lineage. However, the regeneration kinetics of DC lineage cells via their respective
progenitors and functional dynamics of DC precursors in steady state and inflammation remain unclear.
To address these questions, our group has generated a reporter-depleter mouse model, in
which human diphtheria toxin receptor (DTR) fused with green fluorescent protein (GFP) is expressed
under the control of murine Flt3 promoter and thus, allows to transiently deplete Flt3 expressing cells
upon diphtheria toxin (DT) administration. The flow cytometric analysis showed that GFP signal is well
correlated with Flt3 expression in various hematopoietic compartments, ranging from early progenitors to
mature cells in non-hematopoietic and hematopoietic tissues. The DT dose was successfully titrated to
ensure
a
complete
depletion
of
GFP+
cells
and
no
obvious
toxicity
in
vivo.
Given the importance of DC in the context of infection, cancer and autoimmunity,
acquiring the knowledge of DC homeostasis under physiological and inflammatory conditions will be
highly valuable for the development of new drug and design of therapeutic strategy.
Deciphering Allopurinol Hypersensitivity – Characterisation of risk factors for
allopurinol hypersensitivity in vitro.
Authors:
JamesYun, Daniel Yerly, Werner Pichler
Affiliation:
Division of Allergology, Clinic for Rheumatology and Clinical Immunology/Allergy,
University of Bern, Inselspital, Bern, Switzerland
Background:
Allopurinol hypersensitivity is a major cause of severe cutaneous adverse reactions
(SCAR) and it is strongly association with HLA-B*58:01 allele. We took advantage of
this association to investigate the immune mechanism of allopurinol-induced SCAR.
Method:
Allopurinol-specific T cell lines (TCL) were generated from PBMC isolated from
allopurinol allergic individuals, allopurinol-naïve HLA-B*58:01 positive donors and
allopurinol-naïve HLA-B*58:01 negative donors. Autologous Epstein-Barr virustransformed lymphoblastoid B cell lines (EBV-BLCL) were used in some experiments as
antigen presenting cells (APC). The specificity of TCL was assessed by FACS analysis.
Results:
In allopurinol allergic patients (2), allopurinol-specific TCL could be generated within 4
weeks but this was not possible in all allopurinol-naïve individuals (8). However, in
allopurinol-naïve HLA-B*58:01 positive individuals (5), allopurinol-specific TCL could
be generated after 4 weeks with multiple restimulations. In allopurinol-naïve HLAB*58:01 negative individuals (3), allopurinol-specific TCL could not be generated at all.
However, when EBV-BLCL were used as APC for TCL induction in these HLAB*58:01 negative individuals (3), allopurinol-specific TCL could also be generated.
There were no obvious phenotypic differences between these groups.
Conclusion:
Our in vitro data suggest that HLA-B*58:01 molecule is likely involved in the
pathogenesis of allopurinol hypersensitivity as it facilitates reactivity to allopurinol in
allopurinol naïve individuals. However, this allele is not an absolute requirement for the
development of allopurinol hypersensitivity as the in-vitro induction occurred when
EBV-BLCL were used as APC. This implies that in the presence of a co-factor, such as
viral infection, the threshold for the development of allopurinol hypersensitivity may be
lowered.