Composition and efficacy of cytotoxic T-cell responses determine virus elimination and immunopathology after virus infections [Elektronische Ressource] / vorgelegt von Birthe Jessen

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ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG IM BREISGAU Composition and efficacy of cytotoxic T cell responses determine virus elimination and immunopathology after virus infections INAUGURAL-DISSERTATION zur Erlangung der Doktorwürde der Fakultät für Biologie und der Fakultät für Medizin der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Birthe Jessen aus Bad Neustadt/Saale September 2010 Dekan der Biologischen Fakultät: Prof. Dr. rer. nat. Ad Aertsen Dekan der Medizinischen Fakultät: Prof. Dr. med. Christoph Peters Betreuer der Arbeit/Doktorvater: Prof. Dr. Hanspeter Pircher Betreuer der Arbeit: Prof. Dr. Stephan Ehl Koreferent: Prof. Dr. Peter Stäheli Promotionsvorsitzender: Prof. Dr. Samuel Rossel Tag der Verkündigung des Prüfungsergebnisses: 30.11.2010 Diese Arbeit wurde am Centrum für Chronische Immundefizienz (CCI) des Universitätsklinikums Freiburg - Albert-Ludwigs-Universität Freiburg - erstellt. ‘Nothing shocks me. I'm a scientist.’ - Harrison Ford as Indiana Jones Contents 4 Contents............................................................................................................................... 4 Abstract ....................................................................................................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 23
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Source : D-NB.INFO/100939438X/34
Nombre de pages : 97
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ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG IM BREISGAU


Composition and efficacy of cytotoxic
T cell responses determine virus elimination
and immunopathology after virus infections





INAUGURAL-DISSERTATION
zur Erlangung der Doktorwürde der Fakultät für
Biologie und der Fakultät für Medizin
der Albert-Ludwigs-Universität
Freiburg im Breisgau


vorgelegt von
Birthe Jessen
aus Bad Neustadt/Saale

September 2010


Dekan der Biologischen Fakultät: Prof. Dr. rer. nat. Ad Aertsen

Dekan der Medizinischen Fakultät: Prof. Dr. med. Christoph Peters

Betreuer der Arbeit/Doktorvater: Prof. Dr. Hanspeter Pircher

Betreuer der Arbeit: Prof. Dr. Stephan Ehl

Koreferent: Prof. Dr. Peter Stäheli

Promotionsvorsitzender: Prof. Dr. Samuel Rossel



Tag der Verkündigung des Prüfungsergebnisses: 30.11.2010









Diese Arbeit wurde am Centrum für Chronische Immundefizienz (CCI) des
Universitätsklinikums Freiburg - Albert-Ludwigs-Universität Freiburg - erstellt.




























‘Nothing shocks me. I'm a scientist.’

- Harrison Ford as Indiana Jones Contents 4
Contents............................................................................................................................... 4
Abstract ............................................................................................................................... 7
Abbreviations ...................................................................................................................... 8

1 Introduction ..................................................................................................... 10

1.1 Immune system...................................................................................................10
1.1.1 Innate immune system ................................................................................................ 10
1.1.2 Adaptive immune response......................................................................................... 11
1.1.3 Antiviral immune responses ........................................................................................ 12
1.2 T cell-mediated immunopathology following RSV infection............................13
1.3 Control of immune homeostasis by T cells ......................................................14
1.4 Cell death induced by cytotoxic lymphocytes ..................................................15
1.4.1 Ligation of death receptors.......................................................................................... 15
1.4.2 Exocytosis of lytic granules ......................................................................................... 16
1.5 Hemophagocytic Lymphohistiocytosis.............................................................19
1.5.1 Genetic defects affecting lymphocyte cytotoxicity....................................................... 19
1.5.1.1 Familiar hemophagocytic lymphohistiocytosis (FHL).............................................. 21
1.5.1.2 Chèdiak-Higashi syndrome..................................................................................... 22
1.5.1.3 Griscelli syndome type II......................................................................................... 23
1.5.1.4 Hermansky-Pudlak syndrome type II ...................................................................... 24
1.5.2 Diagnostic criteria........................................................................................................ 25
1.5.3 Treatment .................................................................................................................... 26
1.5.4 Open questions in disease pathogenesis ................................................................... 26
1.6 Aims of the study................................................................................................29

2 Materials and Methods.................................................................................... 30

2.1 Mice, Viruses and Materials ...............................................................................30
2.1.1 Mice ............................................................................................................................. 30
2.1.2 Viruses......................................................................................................................... 30
2.1.3 Cells............................................................................................................................. 31
2.1.4 Narcotics...................................................................................................................... 31
2.1.5 Cell culture media........................................................................................................ 31
2.1.6 Synthetic peptides ....................................................................................................... 32
2.1.7 Antibodies.................................................................................................................... 32
2.1.8 Primer .......................................................................................................................... 33
2.1.9 Kits............................................................................................................................... 34
2.1.10 Enzymes...................................................................................................................... 35 Contents 5
2.1.11 Chemicals, buffers and solutions ................................................................................ 35
2.1.12 Plastic materials .......................................................................................................... 37
2.1.13 Instruments.................................................................................................................. 38
2.2 Methods...............................................................................................................39
2.2.1 Viruses......................................................................................................................... 39
2.2.2 Hybridoma ................................................................................................................... 39
2.2.3 Mice ............................................................................................................................. 39
2.2.5 Treatment of mice........................................................................................................ 43
2.2.6 Preparation of mice ..................................................................................................... 44
2.2.7 In vitro activation of T cells .......................................................................................... 44
2.2.8 Determination of virus titers......................................................................................... 45
2.2.9 Flow cytometry ............................................................................................................ 46
2.2.10 Magnetic Activated Cell Separation ............................................................................ 47
2.2.11 Blood count.................................................................................................................. 47
2.2.12 Proliferation assay....................................................................................................... 47
2.2.13 Cytotoxicity Assay ....................................................................................................... 47
2.2.14 Determination of cytokine levels.................................................................................. 48
2.2.15 Analysis of liver enzymes, triglycerides and ferritin serum levels ............................... 49
2.2.16 Histology...................................................................................................................... 49
2.2.17 Statistical analysis ....................................................................................................... 49

3 Results ............................................................................................................. 50

3.1 Strain-specific disease susceptibility after RSV infection in the mouse is
determined by MHC dependent CTL responsiveness ......................................50
3.1.1 The MHC haplotype is an important determinant of disease susceptibility following
RSV infection. .............................................................................................................. 50
3.1.2 RSV induced disease is not determined by peak virus titers or virus elimination
kinetics. ........................................................................................................................ 51
3.1.3 The pulmonary CTL response is of similar magnitude in MHC congenic mice........... 52
+3.1.4 Neither regulatory nor IL-17-producing CD4 T cells influence the different outcomes
of disease..................................................................................................................... 53
d3.1.5 Vβ skewing of pulmonary CTL is more pronounced in BALB/c and C57BL/6- H-2
mice than in C57BL/6 mice.......................................................................................... 53
d3.1.6 The epitope-specific pulmonary CTL response is more focused in H-2 mice
bcompared with H-2 mice............................................................................................. 54
b d3.1.7 H-2 -restricted M187-specific CTLs have a higher avidity than H-2 -restricted M2-1
82-specific CTLs. ......................................................................................................... 56
+3.1.8 The Vβ 8.2 M2-1 82-specific CTL response is responsible for the RSV-nduced
ddisease in H-2 mice .................................................................................................... 56 Contents 6
3.2 Hermansky-Pudlak Syndrome Type II confers a risk for hemophagocytic
lymphohistiocytosis ...........................................................................................58
3.2.1 Pearl mice develop transient HLH following LCMV infection. ..................................... 58
3.2.2 Pearl mice show a delay in virus control after LCMV WE infection. ........................... 61
3.2.3 Pearl CTLs have a reduced capacity to proliferate in vitro and in vivo ....................... 62
3.2.4 Pearl CTLs have a defect in degranulation and cytotoxicity. ...................................... 63
3.2.5 The cytotoxicity defect of pearl CTLs is relevant for virus control in vivo.................... 65
3.2.6 An additional heterozygous Rab27a mutation does not influence the outcome of
disease in pearl mice. .................................................................................................. 66

3.3 Impact of viral and host parameters on the pathogenesis of hemophagocytic
syndrome in beige mice - a mouse model of Chèdiak-Higashi Syndrome .....68
3.3.1 Beige mice carry a mutation in the WD40 domain of the Lyst protein. ....................... 68
3.3.2 Beige mice do not develop HLH after low dose LCMV WE infection.......................... 69
3.3.3 An increased T cell frequency does not change the outcome of disease in beige mice.
..................................................................................................................................... 70
3.3.4 Changing virus dose induces transient disease in beige mice. .................................. 71
3.3.5 The souris mutation confers a risk for developing severe HLH following low dose
LCMV WE infection...................................................................................................... 73
3.3.6 HLH in souris mice is associated with virus persistence............................................. 74
3.3.7 CTL activity rather than NK cell activity determines the risk for HLH.......................... 74

4 Discussion ....................................................................................................... 77

4.1 Disease susceptibility after RSV infection is favored by a highly focused, low
avidity, MHC-dependent CTL response.............................................................77

4.2 HLH in mouse models for Hermansky-Pudlak syndrome type II and the
Chèdiak-Higashi syndrome................................................................................79
4.2.1 Hermansky-Pudlak syndrome type II confers a risk for HLH ...................................... 80
4.2.2 AP-3 deficiency - more than a defect in cytotoxicity.................................................... 81
4.2.3 Geno-phenotype correlation in the mouse models for CHS ....................................... 82
4.2.4 HLH in mice is associated with antigen persistence ................................................... 84
4.2.5 CTL rather than NK cell cytotoxicity determines the risk for HLH. .............................. 85

5 References....................................................................................................... 87

Acknowledgement/Danksagung.........................................................................................97
Abstract 7
Abstract

+This study addresses different aspects of antiviral CD8 T cell-mediated immunopathology
using two mouse models of human diseases. After pulmonary infection with respiratory
syncytial virus (RSV), disease is mainly mediated by cytotoxic T cells (CTL). We used
+MHC congenic mice to address the question to what extent the MHC-determined CD8 T
dcell response contributes to the different outcomes after RSV infection of BALB/c (H-2 )
band C57BL/6 (H-2 ) mice. The two investigated MHC alleles had no impact on virus
elimination, but influenced weight loss and pulmonary inflammation. The overall
dmagnitude of the virus-specific CTL response was similar. However, the H-2 restricted
CTL response was less diverse and of lower avidity, thereby probably contributing to
delayed elimination of stimulating APCs followed by prolonged CTL activation and
cytokine-mediated immunopathology. The concept of CTL-mediated disease due to
prolonged antigen stimulation is well illustrated in another disease called hemophagocytic
lymphohistiocytosis (HLH). This disease is due to defects in cytotoxicity and can be
modelled by LCMV infection of perforin-deficient mice. In this study, we analyzed two
mouse models of partially impaired cytotoxicity. LCMV infection of pearl mice (a mouse
model for Hermansky-Pudlak syndrome type II - HPSII) induced a transient HLH, which
was related to a delayed virus control. In addition, pearl CTLs showed a proliferation
defect indicating that defective APCs also contributed to the phenotype. Since the only
human HPSII patient who developed lethal HLH also had a RAB27A mutation, we also
analyzed pearl mice heterozygous for a Rab27a mutation. This did not aggravate the
cytotoxicity defect or the disease phenotype, suggesting that in that patient, HPSII was
sufficient for the disease. LCMV infection of beige and souris mice (mouse models for
Chèdiak-Higashi syndrome with different mutations in the Lyst gene) was used to
investigate the impact of viral, T cell and host genetic factors on the threshold of HLH
induction. After LCMV infection, beige mice developed disease that did not fulfil all criteria
of HLH. The phenotype was slightly aggravated by higher virus doses, but not by
increasing the precursor frequency of antiviral CTLs. However, severe disease was
observed when souris mice were infected. Both beige and souris mice showed a
comparable defect in NK cell cytotoxicity and degranulation. In contrast, the defect in CTL
cytotoxicity and degranulation was more pronounced in souris mice. These mice failed to
eliminate the virus leading to prolonged CTL activation and development of the cytokine-
mediated immunopathology of HLH.
This study shows that genetically determined subtle differences in the efficacy of the
antiviral T cell response and their impact on the kinetic of virus and APC elimination can
determine the outcome of a viral infection. Abbreviations 8
Abbreviations

AP adaptor protein
APC antigen presenting cell
ALPS autoimmune lymphoproliferative syndrome
BAL(F) bronchoalveolar lavage (fluid)
BCR B cell receptor
bp base pair
CHS Chédiak-Higashi syndrome
CTL cytotoxic T lymphocyte
DNA desoxyribonucleic acid
EBV Epstein-Barr virus
ELISA enzyme-linked immunosorbent assay
FACS fluorescence activated cell sorter
FADD FAS associated via death domain
FCS fetal calf serum
FHL familial hemophagocytic lymphohistiocytosis
GLDH glutamate dehydrogenase
GPT gutamate pyruvate transaminase
GSII Griscelli syndrome type II
HBV hepatitis B virus
HIV human immunodeficiency virus
HPSII Hermansky-Pudlak syndrome type 2
HRS hepatocyte growth factor-regulated tyrosine kinase substrate
HSCT hematopoietic stem cell transplantation
HVH herpesvirus hominis (herpes simplex virus)
ICAM intracellular adhesion molecule
IFN interferon
IL interleukin
ILT immunoglobulin like transcripts
i.n. intranasal
i.p. intraperitoneal
i.v. intraveneous
KIR killer immunoglobulin-like receptors
LAMP lysosom associated membrane protein
LAT linker for activation of T cells
LCMV lymphocytic choriomeningitis virus
LDH lactate dehydrogenase
LFA lymphocyte function-associated antigen
LILR leukocyte immunoglobulin-like receptors
MCP macrophage chemoattractant protein
MHC major histocompatibility complex
MIP macrophage inflammatory protein
moi multiplicity of infection
MTOC microtubule-reorganization center
NK Natural Killer
pfu plaque forming units
RANTES regulated upon activation, normal T cell expressed and secreted
RNA ribonucleic acid
RSV respiratory syncytial virus
RT room temperature
SD standard deviation
SLP Scr homology 2 domain containing leukocyte protein
SNAP soluble N-ethylmaleimide sensitive factor attachment protein
SNARE SNAP receptor
TCR T cell receptor Abbreviations 9
T T helper H
TNF(R) tumor necrosis factor (receptor)
Treg T regulatory
TRAIL TNF-related apoptosis inducing ligand
ZAP zeta-associated protein





























Nomenclature of genes and proteins:
Gene symbols are italicized. Human origin is indicated by using uppercase letters and for
murine genes only the first letter is capitalized. Protein designations follow the same rules as
gene symbols, but protein symbols are not italicized. Introduction 10
1 Introduction

1.1 Immune system

The human organism is challenged daily by microorganisms that only occasionally cause
disease. Most of them are detected and destroyed by the immune system within hours after
entering the body while the clearance of others requires more time. The immune system can
be divided into two parts: the innate and the adaptive immune system.

1.1.1 Innate immune system
The first contact between a microorganism and its host usually occurs at epithelial surfaces
followed by colonization or penetration before replication occurs. At this stage the innate
immune system responds to invading pathogens while the adaptive immune response
requires several days to develop [1-3].
After breaking the epithelial barrier and replicating in the tissue, the first line of defense
consists of macrophages and granulocytes, especially neutrophils [4]. Cells of the innate
immune system use a variety of germline-encoded receptors to discriminate between normal
or uninfected and transformed or infected cells. These pattern-recognition receptors
recognize highly conserved motifs that are shared by many pathogens, the pathogen-
associated molecular patterns [5].
Macrophages carry e.g. mannose-receptors, scavenger receptors, and CD14 [6-8]. After
recognition of the pathogen via one of these receptors, it is ingested through phagocytosis
and destroyed intracellularly. Macrophages and neutrophils have a variety of toxic
mechanisms to kill microorganism. Following phagocytosis, the phagosome fuses with
lysosomes and the pathogen is killed by toxic substances (nitric oxide, reactive oxygen
species) and degraded by various enzymes. In addition, macrophages release
proinflammatory cytokines (e.g. IL-1β, IL-6, IL-12, TNF-α, MCP-1, MIP-1β) and chemokines
(e.g. CXCL8) and other mediators (e.g. complement components, prostaglandins,
leukotriene, platelet-activating factor) to recruit inflammatory cells to the site of infection and
induce expression of co-stimulatory molecules on macrophages and dendritic cells [9].
The secretion of type I IFNs by infected cells and cyokines derived from macrophages (e.g.
IL-12) leads to the activation of Natural Killer (NK) cells. NK cells exhibit a wide range of
inhibitory and activating surface receptors that have been grouped in: C-type lectin receptors
(CD94 and NKG2D family), immunoglobulin like transcripts (ILTs) or leukocyte

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