Composition and efficacy of cytotoxic T cell responses

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

3.1.5 Vβ skewing of pulmonary CTL is more pronounced in BALB/c and C57BL/6- H-2d

mice than in C57BL/6 mice.......................................................................................... 53

3.1.6 The epitope-specific pulmonary CTL response is more focused in H-2d mice

compared with H-2b mice............................................................................................. 54

3.1.7 H-2b-restricted M187-specific CTLs have a higher avidity than H-2d-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

disease in H-2d 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

cell response contributes to the different outcomes after RSV infection of BALB/c (H-2d)

and C57BL/6 (H-2b) mice. The two investigated MHC alleles had no impact on virus

elimination, but influenced weight loss and pulmonary inflammation. The overall

magnitude of the virus-specific CTL response was similar. However, the H-2d 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

TH T helper 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

Introduction 11

immunoglobulin-like receptors (LILRs) and Killer immunoglobulin-like receptors (KIRs) [10,

11]. Using these receptors, NK cells can detect reduced or absent expression of classical

and non classical MHC class I molecules on target cells and destroy them. NK cells kill

infected cells via release of cytotoxic granules. In addition, they produce IFN-γ upon

stimulation with IL-12 and TNF-α. IFN-γ then activates macrophages to kill intracellular

pathogens [2, 12].

This innate immune response can prevent an infection from being established or can prevent

its spreading until the adaptive immune response has developed.

1.1.2 Adaptive immune response

To induce an adaptive immune response, activated professional antigen-presenting cells

(APCs) that reside in most tissues leave the site of infection to enter peripheral lymphoid

tissues such as draining lymph nodes, where they get in contact with naïve CD4+ and CD8+ T

cells [3, 13]. These T cells can specifically recognize processed peptide antigens derived

from pathogens in context of MHC molecules using their specific antigen-receptors (T cell

receptor, TCR). During T cell development, gene segments (V(D)J) of the α-, β-, γ-, and δ-

chains of the TCRs are rearranged to create genes that encode for the huge variety of

antigen-specific receptors carried by the available pool of T lymphocytes. The αβ T cells

account for most of the T cells and only a small proportion of the T lymphocytes express the

γδ TCR that recognizes antigen in the context of non-classical MHC molecules [13].

Following activation, T cells begin to proliferate and mature into effector T cells that migrate

back to the site of infection [14]. Proliferation and differentiation are driven by IL-2 that is

produced by activated T cells themselves. The differentiation of CD4 T cells into TH1 or TH2

cells determines whether a humoral or cell-mediated immunity will predominate. Under the

influence of IL-12, IFN-γ, and the T-box expressed in T cells transcription factor (T-bet), naïve

CD4 T cells differentiate into TH1 cells with the capacity to produce IL-2 and IFN-γ. In

contrast, the development of TH2 cells that can produce IL-4, IL-5, IL-10, and IL-13, is driven

by IL-4 and the transcription factor GATA-3.[15]

While cytotoxic CD8 T cells (CTL) have to leave the lymphoid organs in order to exert their

effector function in the inflamed tissue, the most important function of T helper cells

(predominantly TH2 cells) is to interact with B cells in the lymphoid tissues. Following binding

of antigen with their antigen-specific receptor (B cell receptor, BCR) and co-stimulation, B

cells are activated. They proliferate and migrate to the primary follicles to form germinal

centers [16]. Here B cells interact with helper T cells to undergo isotype switching and affinity

maturation of their BCR before becoming either memory cells or antibody-secreting plasma

cells that leave the germinal center. Depending on the infectious agent T cells -cytotoxic and

Introduction 12

helper T cells- together with specific antibodies produced by B cells are able to clear an

infection and build up immunological memory to prevent reinfection [13].

1.1.3 Antiviral immune responses

Primary antiviral immune responses are mainly mediated by T cells. CD8+ T cells play a key

role in the elimination of certain viruses [17, 18]. In this study two viruses were used to

induce local or systemic infections in order to analyze different aspects of the T cell-

dependent immune response and T cell-mediated immunopathology in the mouse model.

The virus used for a local infection is the human Respiratory Syncytial Virus A2 (RSV)

belonging to the genus Pneumovirus of the family of Paramyxoviridae. RSV is a negative-

sense, single-stranded RNA virus that upon intranasal application infects and predominantly

replicates in the epithelial cells of the respiratory tract. BALB/c mice and C57BL/6 mice

exhibit different susceptibilities to infection and virus-induced pathology, but the reason for

these differences is unknown [19, 20]. To establish a systemic infection, mice were infected

intravenously with Lymphocytic choriomeningitis virus WE (LCMV WE). LCMV is an

Arenavirus with a bisegmented negative-strand RNA genome and is a natural pathogen of

mice [21].

Following infection, both viruses replicate to maximal titers four days after infection [19, 21]

(Fig. 1). During this time NK cells are activated and reach their maximal activity about three

days after infection [22-24]. NK cells are not sufficient for virus control but contribute to the

induction of T cell responses. After NK cell activation in the lymph node, T cells are activated,

proliferate and reach their maximum in numbers and activity seven to eight days after

infection (Fig. 1). Both CD4 and CD8 T cells contribute to virus clearance, but CD8+ cytotoxic

T cells play the major role in eliminating the virus [18]. In LCMV infected mice, CTLs

massively expand until -at the peak of expansion- more than 80% of spleen cells are LCMV-

specific [17, 25]. In contrast, following RSV infection only about 20% of total pulmonary CTL

are specific for RSV [26, 27]. Following RSV infection the virus is cleared until day seven

after infection. The kinetics for virus replication and T cell expansion are very similar in LCMV

and RSV infection but after infection LCMV is cleared until day ten [21] (Fig. 1).

Introduction 13

Fig. 1: Scheme of virus kinetics and kinetics of the immune response after LCMV WE infection.

1.2 T cell-mediated immunopathology following RSV infection

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in

infants but also causes significant morbidity and mortality in immunocompromised adults

and the elderly. The nature and severity of disease vary widely between infected

individuals. Viral, host and environmental factors probably all contribute to this variable

disease expression, but the relative role of these factors in human RSV disease remains

difficult to evaluate. A series of recent genetic association studies have suggested that

polymorphisms in a range of genes encoding cytokines [28-30], chemokines, surfactant

proteins [31] or toll-like receptors [32] influence the disease phenotype in humans.

However, it remains unclear, how and to which extent these factors contribute to disease

pathogenesis [33].

Both in humans and in mice, cytotoxic T cells (CTL) have been identified as important

mediators of virus control and disease. Infants with congenital T cell deficiencies can not

eliminate RSV [34-36] and depletion of T cells leads to persistent infection in BALB/c mice

[18]. Adoptive transfer of RSV-specific T cells can eliminate RSV from infected mice, but

also aggravates disease [37]. In immunodeficient human infants persistently infected with

RSV undergoing bone marrow transplantation, virus control has been observed in parallel

to donor T cell reconstitution and this was associated with significant deterioration of lung

disease [34]. Both CD4+ and CD8+ T cells can eliminate virus and cause

immunopathology independently, but CD8+ T cells appear to be more effective.

LCMV

NK cells

CD4 T cells

0 4 8 12

time after infection [d]

CD8 T cells

LCMV

NK cells

CD4 T cells

0 4 8 12


CD8 T cells

LCMV

NK cells

CD4 T cells

0 4 8 12


0 4 8 120 4 8 12


CD8 T cells

Introduction 14

The strength and composition of the CTL response to viruses is determined by the MHC

haplotype. An important contribution of the MHC to susceptibility to viral infections has

been documented in murine HVH-1 [38] and LCMV infection [39] and has been linked to

the impact of the MHC on the T cell repertoire and T cell avidity [38]. In murine RSV

infection a varying permissiveness to viral replication was shown in different mouse

strains [19]. Analysis of virus titers and disease susceptibility revealed that “resistant”

mice, e.g. C57BL/6 mice, exhibited low permissiveness to viral replication and no disease.

In contrast, for “susceptible” mouse strains, such as AKR, 129P3 and BALB/c mice, higher

virus load and a more pronounced disease was reported [20, 40]. The differences in viral

replication and weight loss between BALB/c and C57BL/6 mice were confirmed in further

studies [41, 42], although airway obstruction and airway hyperresponsiveness were

similar [41]. Higher viral replication is a reasonable explanation for the different outcomes

of infection in the two strains, but this has not been formally proven and may not be the

only relevant factor. Furthermore, the contribution of MHC to disease susceptibility in RSV

infection is not very well characterized by now and is further analyzed in this study.

1.3 Control of immune homeostasis by T cells

Once an infection is cleared, control mechanisms are required to maintain immune

homeostasis to avoid unnecessary inflammatory responses and damage of healthy tissues.

At the end of an antiviral immune response especially the activity of CTLs needs to be strictly

controlled. Defects in the control mechanisms may result in overactivation of CTLs and

immunopathology caused by inflammatory cytokines and uncontrolled CTL-mediated effector

functions [43]. The termination of immune responses to viruses is mainly mediated by

cytotoxic T cells. The first mechanism for termination of the immune response involves

programmed cell death induced by death receptors. CTLs express both FAS ligand and its

receptor FAS. Ligation of the FAS receptor by FAS ligand on neighboring T cells (“fratricide”)

is thought to contribute to contraction of the CTL pool at the end of an immune response [44].

Defects in FAS or the FAS receptor lead to a lymphoproliferative syndrome accompanied by

severe autoimmunity, i.e. the autoimmune lymphoproliferative syndrome (ALPS) [45].

Expansion of CTLs can also be limited by CTL-mediated perforin-dependent killing of

neighboring T cells. During the killing of target cells CTLs are in close contact with the target

cell membrane at the immunological synapse. Here CTLs can transiently acquire

peptide:MHCI complexes from target cells [46-48] making them susceptible to killing by other

CTLs. This self-regulating mechanism for downregulation of the immune response can also

contribute to contraction of the CTL pool.

Introduction 15

The third mechanism is dependent on perforin-mediated lysis of stimulating APCs. CTLs are

activated via APCs, proliferate and acquire effector function. They migrate out into infected

tissues to exert their antiviral activity. At the same time, they also re-encounter APCs, which

are now recognized as targets and are therefore killed in a perforin-dependent manner. This

terminates CTL activation and is therefore a negative feedback mechanism to avoid

overstimulation of the CTL response [49]. Defects in perforin or in the lytic granule exocytosis

pathway cause hemophagocytic lymphohistiocytosis (HLH). In this syndrome CTLs are not

able to terminate the stimulation via APCs [50-52]. Data from perforin-deficient mice confirm

this concept by showing increased activation of CTLs and therefore an increased IFN-γ

production as a result of elevated or prolonged antigen presentation [49]. This prolonged

contact between CTLs and APCs may also rescue activated CTLs from undergoing

apoptosis. The pathogenetic concepts of hemophagocytic lymphohistiocytosis are further

explained in chapter 1.5.

Another manifestation of the failure to control immune responses is immunopathology [53]

following virus infections. This occurs e.g. after liver infection with non-cytopathic viruses

(e.g. hepatitis B virus, HBV [54]) or after lung infection with respiratory viruses (e.g.

respiratory syncytial virus, RSV [55]). Here the pathology is not exclusively caused by virus-

induced tissue damage but also by a not properly controlled CTL response. CTLs not only

destroy infected cells but can also cause damage in the surrounding healthy tissue by

attracting additional inflammatory cells.

1.4 Cell death induced by cytotoxic lymphocytes

Cytotoxic lymphocytes -NK cells and CTLs- detect and destroy infected and transformed

cells to eliminate pathogens and tumor cells and to maintain immune homeostasis. The two

cell populations differ in the way of recognizing their target cells and in activation. However,

their cytotoxic effector function is carried out in the same way with the final consequence of

inducing apoptosis of the target cells [44, 56]. To eliminate virus-infected and transformed

cells, CTLs and NK cells are able to induce programmed cell death via two major pathways:

ligation of death receptors and exocytosis of lytic granules [57].

1.4.1 Ligation of death receptors

One mechanism of CTLs and NK cells to induce apoptosis in target cell is the ligation of

death receptors expressed in the membrane of target cells. Aggregation of death receptors -

FAS, TNF receptor (tumor necrosis factor) and TRAIL (TNF-related apoptosis inducing

ligand) receptor- on target cells through binding of their cognate ligands -FAS, TNF and

Introduction 16

TRAIL- that are expressed on the cytotoxic effector cells- leads to an intracellular signaling

cascade finally inducing target cell death [56]. Interaction of FAS ligand with the FAS

receptor represents the most important death receptor system, but TNF and TRAIL and their

receptors (TNFR-I and TRAILR) can act in a similar fashion.

FAS and its ligand must aggregate in trimers to become biologically active. Binding of the

homotrimeric FAS ligand to the receptor induces conformational changes of the trimeric

receptor. Adaptor proteins like FADD (FAS associated via death domain) can bind to the

clustered death domains in the cytoplasmic tails of the receptors. FADD interacts via a

second death domain with caspase-8. Caspases are cysteine proteases that upon activation

cleave protein chains on the C-terminal side of aspartic acid residues. Clustered caspase-8

can activate and cleave capase-8 in trans so that an active domain is released leading to a

cascade of cleaving and activating downstream caspases. At the end of this cascade a

caspase-activated DNase is released from its inhibitory protein, enters the nucleus and

cleaves DNA. This DNA fragmentation is characteristic for apoptosis. [44]

Congenital defects in these pathways cause dysregulation of immune homeostasis and

autoimmunity rather than increased susceptibility to infection [44]. These clinical

observations support the idea that the function of FAS-FAS ligand interactions rather serve to

terminate the immune response after elimination of the initiating pathogen than to contribute

to pathogen elimination.

1.4.2 Exocytosis of lytic granules

A major pathway of CTL- and NK cell-mediated killing involves the exocytosis of lytic

granules into the immunological synapse, which is the contact site between effector and

target cell [58]. These granules are modified lysosomes that contain distinct cytotoxic effector

proteins like perforin, granzymes and granulysin. In the lysosomal membrane, proteins such

as LAMP-1 (lysosom associated membrane protein-1; CD107a), LAMP-2 and LAMP-3 [59]

are expressed. This pathway accounts for most of the cytotoxic activity of CD8 effector cells

as shown by the loss of most of the killing activity in perforin-deficient mice [60, 61].

1.4.2.1 Perforin

Perforin is a pore forming protein of ~67kDa that is found in a soluble monomeric form within

lytic granules. After its release it is anchored in the target cell membrane and begins to

polymerize in the presence of Ca2+ to form cylindric pores. Currently three mechanisms are

discussed by which perforin support granzymes in entering the target cell. The first

mechanism is based on the theory that perforin forms pores into target cell membrane so

that granzymes can diffuse from the immunological synapse into the target cell [62, 63]. An

Introduction 17

alternative theory suggests that perforin and granzymes bind to the target cell membrane via

electrostatic adhesion or receptor interactions, and then both are taken up by endocytosis.

Perforin is then thought to disrupt the endosomal membrane thereby releasing granzymes

into the cytosol of the target cell [63]. The most recent theory is a combination of both. It is

thought that perforin introduced pores into the target cell membrane causing an ion flux

(Ca2+). During an attempt to repair such lesions, perforin and granzymes are internalized [43,

44, 56]. When discussing these concepts, it is important to keep in mind that perforin is

needed for granzyme-dependent cytotoxicity as shown in perforin-deficient mice and

perforin-deficient patients [43, 64-66].

1.4.2.2 Granzymes

Granzymes belong to a family of serine proteases. Granzymes A-G, K, L, M, and N are found

in lytic granules of mice, while CTL and NK cell granules of humans only contain granzymes

A, B, H, K, and M. Both in humans and mice granzymes A and B are the most abundant [67].

Granzyme B preferentially cleaves substrates after aspartic residues. There are two main

pathways of granzyme B-dependent killing, one involving the direct activation of caspases

while the other pathway is mediated through promotion of mitochondrial permeabilization.

Here, granzyme B indirectly promotes caspase activation via the cytochrome c/Apaf-1

pathway in which the BH-3 only protein BID is activated by granzyme B, leading to the

opening of the BAX/BAK channels in the outer membrane of mitochondria. Cytochrome c

released into the cytosol binds and activates the so called apoptosome that promotes

downstream caspase activation and cell death [68]. Granzyme A can induce apoptosis via

multiple pathways. The main mechanism involves proteolysis of components of the

endoplasmatic reticulum-associated SET complex [69]. The cleaved components translocate

to the nucleus where they activate a nuclease causing multiple DNA nicks [43, 70]. Recent

work implicates other roles for granzymes beside cytotoxicity, in particular a role as

proinflammatory extracellular mediators. [62]

1.4.2.3 Granulysin

Granulysin is a cytolytic and proinflammatory protein expressed in human CTLs and NK cells

[71, 72] and until now no mouse homologue has been found. It is synthesized in a 15-kDa

form that partially is cleaved into a 9-kDa form. Both forms of granulysin exist in equal

amounts in CTLs and NK cells with only the 9-kDa form being sequestered in lytic granules.

The 15-kDa form is secreted constitutively but until now the function of this form is unknown.

The 9-kDa form binds to the cell surface without a specific receptor. There it causes

membrane disruption allowing ion fluxes and finally inducing apoptosis of the target cell.

Introduction 18

Granulysin also acts as a chemoattractant for T cells and monocytes and activates the

expression of cytokines like RANTES (regulated upon activation, normal T cell expressed

and secreted), MCP-1 (macrophage chemoattractant protein 1), MCP-3, MIP-1α

(macrophage inflammatory protein), interleukin (IL)-10, IL-1,IL -6 and IFN-α [71].

1.4.2.4 Lytic granule exocytosis

Once an armed effector CD8+ T cells recognizes its target cell via specific binding of the TCR

to the appropriate peptide:MHC-I complex, the supramolecular adhesion complex (SMAC) is

formed at the site of cell-cell interaction. Initial stability of this complex is achieved by

recruitment of the integrin LFA-1 (lymphocyte function-associated antigen 1) which binds to

its ligand ICAM-1 (intracellular adhesion molecule 1) on the target cell. This clustering

induces signaling and a local reorientation of the actin and microtubule cytoskeleton towards

the target cell followed by polarization of the microtubule-reorganization center (MTOC) and

the golgi apparatus. Experiments using TCR cross-linking showed that signaling molecules

like Zap-70 (zeta-associated protein-70), LAT (linker for activation of T cells), SLP-76 (Scr

homology 2 domain containing leukocyte protein-76), and calcium are essential for MTOC

polarization [59, 73]. Following this polarization, lytic granules are directed specifically into

the immunological synapse [74]. In CTLs the granules are synthesized only after activation

through the specific antigen. In contrast, NK cells are already equipped with the lytic granules

during development [44, 63, 75-77]. Before they undergo exocytosis, these vesicles

(granules) have to follow a series of transportation steps that eventually localize them to the

target membrane. Following docking vesicle priming occurs with the help of SNARE (soluble

N-ethylmaleimide sensitive factor attachment protein (SNAP) receptor) complexes expressed

on both the vesicle and target membrane. Thereafter, the vesicle membrane fuses with the

target membrane and the content of the vesicles is released into the immunological synapse

[78, 79].

Introduction 19

Figure 2: Scheme of the immunological synapse between a CTL and a target cell. Stabilization of the immunological synapse via LFA-1 and ICAM following T cell activation via TCR-peptide:MHC binding is shown. After reorientation and polarization of the MTOC and the microtubule cytoskeleton, vesicles are transported to the immunological synapse, degranulate and release perforin, granzymes and granulysin into the cleft between effector and target cells. Modified from [43]

1.5 Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a severe and life-threatening disorder due to a

failure of regulation of the immune response [80, 81]. There are primary (familial) and

secondary (acquired) forms of the disease [82]. In both forms, the clinical syndrome is

usually triggered in the context of strong immune reactions induced by infections, tumors or

severe autoimmune diseases. Many pathogens like EBV, HIV, intracellular bacteria (e.g.

mycobacteria) or parasites (e.g. leishmania) have been identified as a trigger for the disease.

In about 50-60% of primary HLH, no pathogen can be detected [83]. However, it is likely that

in these cases as yet uncharacterized infectious agents are causing the syndrome. This

concept is strengthened by data from mouse models for primary HLH. Thus, perforin-

deficient mice do not develop HLH spontaneously but do so after infection with viruses

strongly activating the immune system, like LCMV [49, 84]. Typically, the manifestation of the

disease is in early childhood and is fatal when untreated [85-87].

1.5.1 Genetic defects affecting lymphocyte cytotoxicity

Defects in seven genes associated with the perforin-dependent granule exocytosis pathway

have been associated with the familial forms of HLH (Tab. I).

MTOC

TCR MHC I complex

LFA-1ICAM

CTL

target cell

perforingranzymegranulysin

MTOC

TCR MHC I complex

LFA-1ICAM

CTL

target cell

perforingranzymegranulysin

Introduction 20

Genetic Forms of HLH Gene Protein Mouse

FHL-1 unknown unknown

FHL-2 PRF1 Perforin PKO [49]

FHL-3 UNC13D MUNC13-4 jinx [88]

FHL-4 STX11 Syntaxin-11

FHL-5 STXBP2 MUNC18-2

associated with albinism

Griscelli syndrome II RAB27A RAB27A ashen [84]

Chèdiak-Higashi syndrome CHS1/LYST LYST beige [89]

Hermansky-Pudlak syndrome II AP3B1 AP-3 pearl [90]

Table I: Genetic forms of HLH with affected genes, proteins and mouse models.

The familial hemophagocytic lymphohistiocytosis 2 (FHL-2) is caused by mutations in the

perforin gene (PRF1) itself, while other primary forms of HLH are caused by defects in

proteins needed for exocytosis of lytic granules (Fig. 3). In some cases of FHL (~10 %) the

genetic defects have not yet been identified [91, 92]. Another group of genetic diseases

predisposing to HLH is also associated with albinism and other characteristic clinical features

in hematopoetic and other tissues (Griscelli syndrome type II, Chédiak-Higashi syndrome,

Hermansky-Pudlak syndrome type II).

Introduction 21

Figure 3: Proteins involved in the lytic granule exocytosis of NK cells and CTLs. The exact function of LYST is still unknown [93] but a possible role in maturation of lysosomes into fusion-competent postlysosomal vesicles is discussed [94, 95]. AP-3 was shown to be needed for polarization of the vesicles. Perforin induces apoptosis in the target cell [96] [97]. RAB27A is involved in the docking of granules at the membrane and interacts with MUNC13-4 [98, 99] that is responsible for granule priming at the immunological synapse. Synaxin-11 is also involved in the priming step and defective in about 20% of patients with Turkish origin with HLH [100-102].

1.5.1.1 Familiar hemophagocytic lymphohistiocytosis (FHL)

The first gene identified causing Familiar Hemophagocytic Lymphohistiocytosis (FHL)

encodes perforin (PFR1) [103, 104]. Perforin mutations account for about 30% of the FHL

cases [105-108]. Most of the mutations found in FHL-2 patients leading to an undetectable

expression of perforin in lytic granules. The age of onset of disease in patients with FHL-2 is

mostly very early in life (first 3 months) [91] with cases described where disease even

developed in utero [109]. Depending on the residual function/expression of the protein, later

onsets can be observed that were linked to missense mutations in PRF1. HLH can also be

induced in a perforin-deficient mouse model through infection with a virus strongly triggering

an antiviral CTL response (LCMV) [49].

FHL-3 is caused by mutations in the UNC13D gene encoding for MUNC13-4 [110, 111]

[112]. MUNC13-4-deficient cells can properly get in contact with target cells, from an

immunological synapse and polarize the lytic granule machinery. Secretory vesicles are

closely associated with the plasmamembrane at the immunological synapse but the priming

of vesicle is impaired. MUNC13-4 was shown to be an effector of RAB27A and the MUNC13-

4/RAB27A complex is important for the regulation of secretory granule fusion with the plasma

membrane in hematopoietic cells [99].

CTL/NK cell

Target cell

Maturation (LYST?)

Polarization (AP-3)

Docking (RAB27A)

Priming (MUNC13-4)

Apoptosis (Perforin)

MTOC

Fusion (Syntaxin-11; MUNC18-2)

perforin

granzyme

granulysin

microtubule

CTL/NK cell

Target cell

Maturation (LYST?)

Polarization (AP-3)

Docking (RAB27A)

Priming (MUNC13-4)


MTOC


CTL/NK cell

Target cell

Maturation (LYST?)

Polarization (AP-3)

Docking (RAB27A)

Priming (MUNC13-4)


MTOC


perforin

granzyme

granulysin

microtubule

perforin

granzyme

granulysin

microtubule

Introduction 22

FHL-4 is caused by mutations in the STX11 gene [102, 113]. Syntaxin-11 is expressed in

resting T cells and NK cells. The t-SNARE syntaxin-11 interacts with vesical SNARES (v-

SNARE) [113] and this interaction is needed for the docking and fusion process of vesicles.

FHL-5 is due to mutations in the syntaxin binding protein 2 (STXBP2) also called MUNC18-2

[114, 115]. MUNC18-2 interacts with the SNARE proteins syntaxin-11 and syntaxin-3 and

this interaction was shown to be disturbed in patients with FHL-5 causing instability of both

proteins. [116].

1.5.1.2 Chèdiak-Higashi syndrome

Chèdaik-Higashi syndrome (CHS) -first described by Beguez-Cesar in 1943 followed by

Steinbrinck (1948) and Chèdiak (1952) and Higashi (1954)- is a rare autosomal recessive

disorder characterized by hypopigmentation and a defective cytotoxicity of NK and T cells

[117] with a high risk for developing HLH.

A striking characteristic of CHS is the presence of giant granules in the cytoplasm of

melanocytes and hematopoietic cells as a result of excessive fusion and inability to

degranulate. Most of the patients (~85%) are diagnosed in early childhood (during the first 10

years of life) and present with severe clinical manifestations [118]. Those children suffer from

recurrent life-threatening infections as a result of a pronounced neutropenia and a defective

NK cell activity. Patients with the childhood form of CHS have a high risk to develop HLH that

is usually fatal unless they are bone marrow transplanted. About 10-15% of patients with

CHS show a much milder clinical phenotype [118]. They have no or very few severe

infections and do not develop HLH. Patients with this adult form of CHS survive to adulthood

but they are predisposed to develop peripheral neuropathy as adults. The minority of patients

with CHS (~5%) have an intermediate phenotype with recurrent severe infection in childhood

[118]. Those patients do not develop HLH and by adolescence they have few or no severe

infections anymore.

The defective protein causing CHS (and the beige mouse phenotype) is the 419kDa

CHS1/LYST protein [89, 119] with the length of 13.5 kb. The exact function of the protein is

still unknown. In patients with CHS, a missorting of proteins from the trans-golgi or the early

endosomes to the late endosomes was observed [120]. Additionally the LYST protein is

thought to be involved in the regulation of organelle fusion [121]. One of the identified

partners of the CHS/LYST protein is the HRS (hepatocyte growth factor-regulated tyrosine

kinase substrate) that inhibits exocytosis by its interaction with the t-SNARE SNAP-25,

supporting a role for LYST in the regulation of transmembrane interactions. The giant

granules observed in CTLs do not polarize at the immunological synapse leading to their

impaired secretion [122, 123]. In patients with the classical (childhood) form of CHS

nonsense and frameshift mutations have been identified leading to an early truncation of the

Introduction 23

protein. Patients with milder (adolescent or adult) forms of CHS more often showed

missense mutations in LYST [118, 124]. So far, no studies have been performed correlating

the genetic and clinical findings with immunological studies of lymphocyte degranulation and

cytotoxicity.

The mouse model for CHS is the beige mouse. By now there a several strains of beige mice

carrying different mutations in the Lyst gene, but a common feature of those mice is the

diluted coat color and the giant granule formation that can be observed in hematopoietic cells

and melanocytes. Beige mice are often used as a model for defective NK cell activity.

However, so far these mice have not been used for the study of the pathogenesis of HLH.

1.5.1.3 Griscelli syndome type II

Griscelli syndrome type II (GSII) -first described by Griscelli et al. 1978 [125]- is an

autosomal recessive disorder characterized by a pigmentary dilution with silver gray sheen of

the hair that can be observed under the light microscope as an uneven distribution of

pigment with large pigment granules [126]. GSII is frequently associated with neurological

manifestations cause by infiltration of lymphocytes and macrophages in the CNS. GSII

results from mutations in the RAB27A gene located in the 15q21 chromosomal region [126,

127]. RAB27A belongs to a family of small GTPases that cycle between the cytosol and the

membrane and are inactive in the GDP-bound form and active when GTP is bound. Its active

form is able to bind its specific effectors [128].

Patients with a mutation in RAB27A show an impaired cytotoxicity of NK and T cells.

RAB27A-deficient T cells can form stable complexes with target cells and polarize their

granule exocytosis machinery, but the granules are unable to dock to the plasma membrane

at the immunological synapse. A direct interaction of RAB27A and MUNC13-4 has been

demonstrated [129] in platelets indicating that RAB27A may also be involved in the priming

Figure 4: Mouse model for Chèdiak-Higashi syndrome: the beige mouse. In contrast to wildtype C57BL/6 mice (left panel), beige mice (right panel) show a diluted coat color associated with a disturbed pigment contribution in the hair shaft that can be observed via light microscopy. In blood smears, hematopoietic cells of beige mice -especially neutrophils- display giant granule formation in the cytoplasm, characteristic for CHS.

Introduction 24

step of lytic granules [130]. Patients with Griscelli syndrome type II have a high risk of

developing HLH.

The mouse model for Griscelli Syndrome II is the ashen mouse. Cells from ashen mice have

been used as models for the analysis of the cell biology of lysosomal transport. An impaired

CTL cytotoxicity has been demonstrated and recent studies have described that ashen mice

develop all clinical features of HLH following LCMV infection [84]. However, the phenotype is

not as pronounced as in PKO mice, since the clinical course of LCMV-infected ashen mice is

not lethal, at least after low dose infection.

1.5.1.4 Hermansky-Pudlak syndrome type II

Hermansky-Pudlak syndrome type II (Hermansky and Pudlak 1959 [131]) is a rare

autosomal recessive disorder caused by mutations in the gene encoding the β3A subunit of

the adaptor protein 3 (AP-3) complex. The AP-3 complex is a heterotetramer containing a δ

subunit shared with other AP complexes and AP-3 specific β3-, µ3-, σ3 subunits. There are

two different AP-3 complexes –one expressed ubiquitously [132] comprising δ-, β3A-, µ3A-,

σ3 (A or B) subunits and another brain-specific complex containing δ-, β3B-, µ3B-, σ3 (A or

B) subunits mostly expressed in cells of neuronal origin. [133-136] The AP-3 complex is

involved in the biogenesis of lysosome-related organelles and in protein trafficking to

lysosomes or to specialized endosomal-lysosomal organelles such as pigment granules,

melanosomes, and platelet dense granules [137, 138]. The AP-3 complex was described to

interact with the scaffolding protein clathrin but the physiological relevance is still discussed

[139]. In CTLs AP-3 was shown to be essential for polarized secretion of lytic granules [140].

Patients with HPSII show oculocutaneous albinism and platelet defects. They also suffer

from immunodeficiency with an increased susceptibility to infections due to congenital

neutropenia [141]. By now, only one of the eleven described HPSII patients developed the

full clinical picture of HLH with a lethal course [142]. A severe cytotoxicity defect was noted in

that patient. However, since he also carried a heterozygous mutation in RAB27A, it remains

unclear whether the cytotoxicity defect presumably leading to the lethal HLH in this particular

patient can be explained by the AP-3 mutations alone or by an additional contribution of the

RAB27A mutation. This is of obvious clinical importance because preemptive stem cell

transplantation is an important consideration in patients with a high risk of developing HLH.

Meanwhile, a second HPSII patient has been described who developed transient HLH [143],

but data on cytotoxicity were variable and no possible additional genetic lesions has been

presented.

The mouse model for HPSII is the pearl mouse that also carries a mutation in the β3A

subunit of the AP-3 complex and shows a diluted coat color (Fig. 5). Again, the mouse has

Introduction 25

mostly been studied in a cell biological context. Decreased CTL cytotoxicity has been shown,

but the mouse has not been analyzed in a disease model of HLH.

1.5.2 Diagnostic criteria

HLH is characterized by an uncontrolled activation of T cells and macrophages accompanied

by an excessive cytokine production, massive infiltration of tissues and hemophagocytosis.

Histological demonstration of hemophagocytosis is one of the cardinal clinical features of the

syndrome. Because the initial signs of the syndrome are unspecific and are difficult to

differentiate from those of infectious diseases, diagnostic criteria for HLH have been

proposed [80, 81, 144]. These include prolonged fever, cytopenia, hepatosplenomegaly,

elevated serum levels of ferritin and soluble IL2-receptor α chain (sCD25), elevated serum

levels of triglycerides or reduced levels of fibrinogen, histological demonstration of

hemophagocytosis and reduced or absent NK cell cytotoxicity. At least 5 of these 8 criteria

must be fulfilled in order to establish the clinical diagnosis. In some cases, central nervous

system lesions are observed [145] that can support the diagnosis. Moreover, elevated

transaminases, bilirubin and LDH [80] are frequently observed as a sign of liver damage.

Prolonged fever is a consequence of the massive cytokine production, especially the

interleukins (e.g. IL-1). The cytopenia is attributed to high levels of cytokines like IFN-γ and

TNF-α and partially to bone marrow infiltration by highly activated phagocytosing

macrophages. Hepatosplenomegaly is also due to infiltrating macrophages and lymphocytes.

Elevated levels of triglycerides result from increased levels of TNF-α leading to an increased

activity of the lipoprotein lipase. Ferritin is secreted by activated macrophages, whereas

activated T cells massively secrete the soluble IL2-receptor α chain (sCD25). In FHL, the

reduced or absent NK cell cytotoxicity is due to mutations affecting perforin or parts of the

lytic granule exocytosis machinery [81, 146].

Figure 5: Mouse model of Hermansky-Pudlak Syndrome Type II: pearl mice. Compared to wildtype C57BL/6 mice (left panel), pearl mice (right panel) have a diluted coat color with pigment unevenly distributed in hair shafts that can be observed under the light microscope.

Introduction 26

1.5.3 Treatment

HLH is a life-threatening disease and requires intensive immunological treatment. If a

microbial trigger can be identified, antimicrobial therapy is crucial to remove the antigenic

trigger. However, in most cases immunosupression is required, usually as a combination of

dexamethasone, etoposide, and cyclosporin A or anti-thymocyte globulin. While this

treatment can induce stable remission in most secondary forms of the disease, it is not

enough to induce long-term remission in patients with genetic disease. In these cases,

allogenic hematopoietic stem cell transplantation (HSCT) is needed. [144, 147-150]

1.5.4 Open questions in disease pathogenesis

The current model of HLH pathogenesis is based on the concept that a defect in the negative

“feedback” provided by the killing of APCs by CTLs is the cause for the development of the

disease. It is postulated, that one major function of CTLs apart from the killing of virus-

infected target cells is the killing of APCs. This mechanism evolved to avoid overactivation of

CTLs by reducing the antigenic stimulus. If this negative feedback loop is reduced or absent,

CTLs are continuously stimulated and expand and produce massive amounts of IFN-γ and

other proinflammatory cytokines (Fig. 6). This ongoing cytokine release leads to continuous

macrophage activation leading to additional cytokine production. These highly activated

lymphocytes and macrophages infiltrate multiple organs tissues causing tissue damage. The

organ infiltration together with the overproduction of multiple cytokines, such as IFN-γ, TNF-

α, IL-1β, IL-6 and IL-18 [151-155], are likely responsible for symptoms observed in patients

with HLH.

Introduction 27

APC CTL

Activation

Perforin-dependentlysis

Expansion

IFN-γγγγ production

Antigen

Antigen

Macrophage activation

Massive cytokine production

(e.g. TNF-αααα, IL-1, IL-6)

Tissue infiltration

Hemophagocytosis

APC CTL

Activation


Expansion


Antigen

APC CTL

Activation


Expansion


Antigen

Antigen

Macrophage activation

Massive cytokine production

(e.g. TNF-αααα, IL-1, IL-6)

Tissue infiltration

Hemophagocytosis

APC CTL

Activation

Perforin-dependent lysis

Expansion


Cytotoxic activity

Antigen

Apoptosis

Contraction

APC CTL

Activation

Perforin-dependent lysis

Expansion


Cytotoxic activity

Antigen

Apoptosis

Contraction

B

A

Figure 6: Feedback between CTLs and APCs. (A)The negative feedback between CTLs and APCs is shown leading to a perforin-dependent killing of APCs after CTLs are activated and gained their effector functions. (B) In hemophagocytic lymphohistiocytosis lysis of APCs is disturbed leading to further CTL expansion and macrophage activation with massive cytokine production finally resulting in tissue infiltration and hemophagocytosis.

Introduction 28

Despite recent advances that have led to the elaboration of that concept, many questions

remain unanswered. It Is clear that genetic defects leading to defects in lymphocyte

cytotoxicity are causally linked to the disease. Furthermore, it is likely, that an antigenic

trigger is needed to kick off the pathogenetic sequence of events leading to HLH. This is

particularly well illustrated in mice with perforin deficiency which remain perfectly healthy

unless infected with LCMV. Once infected with LCMV, the mice can not control the virus and

develop the full picture of HLH. It is not clear at the moment, whether -once triggered-

ongoing disease follows an autonomous course or whether antigen (in particular viral)

persistence is needed for development of the lethal course of the disease. This also raises

the question, to which extent an alteration of viral parameters (infection doses, replicative

capacity) can influence disease susceptibility. Experiments with perforin-deficient mice have

shown that CTLs and not NK cells are the critical mediators of disease in the LCMV model.

However, whether this is also true for other microbial triggers of the disease is unknown.

Given the key role of CTLs, it is likely, that increased CTL responsiveness may also

contribute to a lower threshold for disease. However, this has not yet been addressed

experimentally.

Aims of the study 29

1.6 Aims of the study

This study addressed different aspects of CD8 T cell-mediated pathology. The first part of

this study used a model of respiratory virus infection, in which cytotoxic T cells are crucial

for virus elimination following infection, but also augment immunopathology. In the second

part of the study two different mouse models for hemophagocytic lymphohistiocytosis

were analyzed, in which disease is mediated by an uncontrolled CTL activation

accompanied by hypercytokinemia and overactivation of macrophages. The following

issues were investigated:

1. RSV-induced immunopathology is observed following infection of BALB/c mice but

not after infection of C57BL/6 mice. To evaluate the role of the MHC in this

pathology, MHC congenic C57BL/6 mice carrying the H-2d haplotype were

analyzed. Because CD8 T cells are known to be the main mediators of RSV-

induced disease, we particularly focused on different parameters of the CTL

response following RSV infection.

2. LCMV infection is eliminated within 10 days after infection and causes little

pathology in wild-type mice, while it causes chronic infection and severe CD8 T

cell-mediated immunopathology in mice with defects in perforin-dependent

cytotoxicity. We used two different mouse models of partially impaired cytotoxicity,

pearl mice (a model for Hermansky-Pudlak syndrome type II) and beige mice (a

model for Chèdiak-Higashi syndrome) to address the following questions:

2.1 Do pearl mice and beige mice develop HLH after LCMV infection?

2.2 How does the extent of the cytotoxicity defect correlate with susceptibility to HLH?

2.3 What is the impact of different virus doses on disease induction?

2.4 What is the impact of variations in the CTL precursor frequency on disease

induction?

2.5 How do genetic factors (different mutations) influence the susceptibility to HLH?

Material and Methods 30

2 Materials and Methods

2.1 Mice, Viruses and Materials

2.1.1 Mice

Strain referred to as provided from

C57BL/6NCrl C57BL/6 Charles River, Sulzfeld, Germany

BALB/cAnNCrl BALB/c Charles River, Sulzfeld, Germany

B6.C-H2d/bByJ C57BL/6-H-2d Jackson Laboratory, Bar Harbor, USA

C57BL/6-H-2dxb C57BL/6-H-2dxb F1 generation of breeding of

B6.C-H2d/bByJ and C57BL/6NCrl

C57BL/6J-Lystbg-J/J beige Jackson Laboratory, Bar Harbor, USA

C57BL/6-Lystbg-Btlr/Mmcd souris Mutant Mouse Regional Resource Center,

University of California, Davis, USA

B6Pin.C3-Ap3b1pe/J pearl Jackson Laboratory, Bar Harbor, USA

Perforin 0/0 PKO H. Hengartner (Zürich, Switzerland) [96]

C57BL/6J-Rab27aash/J ashen G. de Saint Basile (Paris, France) [84]

318 318 H. Pircher (Freiburg, Germany) [156]

318 C57BL/6J-Lystbg-J/J 318 beige mating of 318 and C57BL/6J-Lystbg-J/J

318 B6Pin.C3-Ap3b1pe/J 318 pearl mating of 318 and B6Pin.C3-Ap3b1pe/J

2.1.2 Viruses

Name originally from:

RSV A2 P. Openshaw, Imperial College, London, UK

rRSV 8A C. Krempl, Institute of Virology and Immunobiology,

Würzburg, Germany [157]

LCMV WE H. Pircher, IMMH Freiburg, Germany


2.1.3 Cells

2.1.3.1 Cell culture

Designation ATCC number origin

RAW309Cr1 TIB-69 mouse macrophage (H-2bxd)

EL-4 TIB-39 mouse lymphoma (H-2b)

P815 TIB-64 mouse mastocytoma (H-2d)

HEp-2 CCL-23 human larynx carcinoma

MC57G CRL-2295 mouse fibrosarcoma (H-2b)

YAC-1 TIB-160 mouse lymphoma

2.1.3.2 Hybridomas

Designation provided from

F23.2 H. Pircher, IMMH, Freiburg, Germany

KJ16 H. Pircher, IMMH, Freiburg, Germany

2.1.4 Narcotics

Trading name active substance provided from

Ketamine 10% Ketaminehydrochloride Intervet, Unterschleißheim, Germany

used in a final concentration of 0.4 % (BALB/c) to 1 % (C57BL/6)

Rompun 2% Xylazinehydrochloride Bayer HealthCare, Leverkusen, Germany

used in a final concentration of 0.03 % (BALB/c) to 0.05 % (C57BL/6)

Thiopental 0.5g Thiopental-Natrium Inresa Arzneimittel, Freiburg, Germany

used in a final concentration of 50mg/ml

2.1.5 Cell culture media

Designation provided from

Iscove's Modified Dulbecco's Medium (IMDM) Gibco Invitrogen, Darmstadt, Germany

RPMI 1640 Biochrome AG, Berlin, Germany

Eagle's minimal essential medium (EMEM) Biochrome AG, Berlin, Germany

PFHM-II (Protein-Free Hybridoma Medium) Invitrogen, Karlsruhe, Germany

5 % to 10 % FCS PAN Biotech, Aidenbach, Germany

1 % L-Glutamine Invitrogen, Karlsruhe, Germany

1 % Penicillin/ Streptomycin Invitrogen, Karlsruhe, Germany


2.1.6 Synthetic peptides

proteins amino acid sequence MHC reference

RSV

M2-1 82-90 SYIGSINNI H-2Kd [158-160]

M 187-195 NAITNAKII H-2Db [161]

LCMV

Gp 33- 41 KAVYNFATM H-2Db [162]

All peptides were obtained from PolyPeptide, Strasbourg, France.

2.1.7 Antibodies

2.1.7.1 Flow cytometry

Specificity α-mouse fluorochrome clone provided from

CD3e APC/FITC/purified 145-2C11 Ebioscience

CD8a PE-Cy5 53-6.7 BD

CD107a FITC/PE 1D4B Ebioscience

H-2Db PE CTDb Serotec

H-2Kd FITC SF1-1.1 BD

IgG1 FITC A85-1 BD

IgG1, rat, κ isotype PE R3-43 BD

IFN-γ PE XMG1.2 BD

NK1.1 PE/APC PK136 BD

Thy-1.1 FITC/PE Ox-7 BD

Vα2 TCR PE B20.1 Ebioscience

Vβ panel FITC BD

anti-rat Ig FITC/PE polyclonal BD

2.1.7.2 Depletion antibodies

Specificity α-mouse clone reference

Vβ 8.2 F23.2 [163]

Vβ 8.1/8.2 KJ16 [164]


2.1.7.3 Antibodies for virus detection

Specificity provided from

goat-anti RSV Biotin AbD Serotec, Düsseldorf, Germany

rat anti-LCMV-NP monoclonal Ab (VL-4) H. Pircher, IMMH, Freiburg, Germany

Peroxidase-conjugated goat anti-rat IgG Dianova, Hamburg, Germany

2.1.7.4 IFN-γγγγ ELISA

Specificity provided from

purified rat α-mouse IFN-γ BD Pharmingen, Heidelberg, Germany

biotinylated rat α-mouse IFN-γ BD Pharmingen, Heidelberg, Germany

2.1.8 Primer

All primers were obtained from biomers, Ulm, Germany Table II: Primer pairs for mutation analysis on cDNA of beige mice.

Primer Sequence 5´-3´ PCR

Products (bp)

Position 5´-3´ cDNA

Annealing temperature

(°C)* Ref.

fFR279 rFR280

GGAGGTGAAGCCTTATGCTG TTCGGAGTCAGAGGTGGAG 605 85-689 48 [165]

fFR348 rFR282

AAGTTTGCAAAAATCAACTCAGG TTTGAAGCTGCACTCTGAAGAC 930 517-1446 47 [165]

fFR281 rFR284

GGTTCAACGGATGCTCTTTC AGGTAGGACCAGCACCACA 1028 1147-2174 48 [165]

fFR283 rFR287

GTCTGTGATCGCCCCTTTAC ACACTTGGAAACCACCAAGC 1199 1948-3146 48 [165]

fFR511 rFR512

GTCAGCCTAGGGGAACAACAGAAAG TACTCGAAGCAGGGCATCAAATAAAG 1008 2654-3661 57 #

fFR521 rFR522

AAGGCAGGGAGAAATGAGTAGAAATGAA TAAACTCGGGGTATGCAGGAAAGGAA 1165 3211-4375 58 #

fFR531 rFR532

ATGAAGCGGATAGTGAAAG GAGGATGCTGAATATAGGTAAGTA 1149 3822-4970 42 #

fFR288 rFR289

CGAGAGTGCTGCAGAAAGG CCTGGATGGCTTTACATTCC 946 4624-5570 48 [165]

fFR352 rFR353

GGGCCAAGTGAAAACTCAGC GCTGCAGTAAAGGCAGATGG 953 5452-6405 48 [165]

fFR354 rFR355

AGTGCAGTCACTCGCGTACC CTCCCAGTCATCAGCATTCC 666 6307-6973 48 #


fFR91 rFR92

CTACGCACTGAAAACAAGCAAAGAGG CGAGAACGGGAATGACAGACCACT 836 6607-7442 58 #

fFR101 rFR102

TCGACCGATTGGCCTGGATGA AGGTCTTGCTTTGGGATGTATTTTCTGG 821 7354-8174 58 #

fFR111 rFR112

CAGTGGCCAGCGATGAG AGCCTGGGTGATGTCTGC 845 7974-8818 48 #

fFR121 rFR122

GTCTTAGCCCCTCCCCACAACA GAAAATGATGCCGGCTCTAACTCC 859 8478-9336 56 #

fFR131 rFR132

TGCTCAGGCCCCCACTCT GACGCCTTCCCCTTTTGCTTGTAG 1162 9090-10251 56 #

fFR141 rFR142

CACAACTTGGCGCCTCTCCTCCTT GCTCACGCTTATCATCACGCTGTA 1078 9967-11044 58 #

fFR151 rFR152

CTGGGAGCAAGTGTGGTGT AGGGCAGGGCTTTACTCTCA 821 10884-11704 48 #

* calculated from Tm of primers given by biomers # determined with DNA Star Primer Select 5.06 software

Table III: Primer pair for mutation confirmation on genomic DNA of beige mice.

Table IV: Primer pair for genotyping ashen mice.

fRab rRab

CTTTCTCTTCCACCATACTT AAGCGAGTTCCAGGGCAG 534 4 +

Table V: Primer pairs for genotyping souris mice.

fSouris rSouris

TCACCCTTCATGTAGACCAGGAACC TGCAGGGCCAAAGCCATATCTAAAC 1228 56 [166]

fSouris_seq rSouris_seq

CAGAAGTCAATGTGCCTTACTTGC GATGACCTTGAAATTCTGACCATCC 778 54 [166]

* calculated from Tm of primers given by biomers # determined with DNA Star Primer Select 5.06 software + sequence provided by G. de Saint Basile

2.1.9 Kits

Kit provided from

QIAquick Gel Extraction Kit Qiagen, Hilden, Germany

SuperScriptTM Frist-Strand Synthesis System for RT-PCR Invitrogen, Karlsruhe, Germany

Taq Polymerase Qiagen, Hilden, Germany

Primer Sequence 5´-3´ PCR

Products (bp)

Annealing temperature

(°C)* Ref.

fMut rMut

CTCCCGGTCCTGGCTCAC TGGGTTTTAAAGGCAAGTCTGTA 810 51 #


Mouse Inflammation Kit (CBA) BD Pharmingen, Heidelberg, Germany

Mouse IL-2R alpha (sCD25) DuoSet R&D Systems, Wiesbaden, Germany

MACS CD8a+ T Cell Isolation Kit II Miltenyi, Bergisch Gladbach, Germany

2.1.10 Enzymes

Enzyme provided from

RsaI Fermentas, St.Leon-Roth, Germany

ProteinaseK Qiagen, Hilden, Germany

DNase I Fermentas, St.Leon-Roth, Germany

RNase H Invitrogen, Karlsruhe, Germany

2.1.11 Chemicals, buffers and solutions

2.1.11.1 Chemicals and reagents

Chemical or reagents provided from

Agarose peqLab, Erlangen, Germany

Albumin, Bovine (BSA) Sigma-Aldrich, Steinheim, Germany

CFSE (Carboxyfluorescein succinimidyl ester) Sigma-Aldrich, Steinheim, Germany 51Chromium Perkin Elmer, Rodgau, Germany

DAB (3,3´ diamino-benzidine-tetrahydrochloride) Merck, Darmstadt, Germany

Desoxy nucleotides (dATP, dTTP, dGTP, dCTP) Fermentas, St. Leon-Roth, Germany

EDTA Serva, Heidelberg, Germany

Ethanol Merck, Darmstadt, Germany

Formaldehyde solution, 4% VWR Int., Bruchsal, Germany

GelRedTM Nucleic Acid Gel Stain Biotrend Chemikalien, Köln, Germany

Gel loading Dye Blue NewEngland Biolabs, Frankfurt, Germany

HCl Merck, Darmstadt, Germany

Hydrogen peroxide (30% H2O2) Merck, Darmstadt, Germany

IL-2 (Proleukin) Chiron, Munich, Germany

Isopropanol Merck, Darmstadt, Germany

L-Glutamine Invitrogen, Karlsruhe, Germany

Lidocaine Sigma-Aldrich, Steinheim, Germany

Methanol Merck, Darmstadt, Germany

Methylcellulose Sigma-Aldrich, Steinheim, Germany

Milipore H2O IMMH, Freiburg, Germany

Nuclease free H2O Qiagen, Hilden, Germany

Paraformaldehyd Merck, Darmstadt, Germany


Polyinosinic–polycytidylic acid (poly (I:C)) Sigma-Aldrich, Steinheim, Germany

Streptavidin-Horseradish Peroxidase BD Pharmingen, Heidelberg, Germany

SigmaFast® OPD Sigma-Aldrich, Steinheim, Germany

TRIS Sigma-Aldrich, Steinheim, Germany

Trizol ® Invitrogen, Karlsruhe, Germany

Triton X -100 Sigma-Aldrich, Steinheim, Germany

Trypan blue Bayer, Leverkusen, Germany

Trypsin IMMH, Freiburg, Germany

TWEEN® 20 Sigma-Aldrich, Steinheim, Germany

2.1.11.2 Buffers and solutions

Buffers or solutions provided from

PBS (phosphate buffered saline) IMMH, Freiburg, Germany

PBS without Mg2+ and Ca2+ IMMH, Freiburg, Germany

MACS buffer (PBS, 0.5% BSA, 2mM EDTA)

Monensin (BD GolgiStop) BD Pharmingen, Heidelberg, Germany

Permeabilizing buffer (Perm WashTM) BD Pharmingen, Heidelberg, Germany

Lysis Buffer (BD FACS lysing solution) BD Pharmingen, Heidelberg, Germany

FACS buffer (BD FACSflow) BD Pharmingen, Heidelberg, Germany

2.1.11.3 Gel electrophoresis

Tris-acetate-EDTA buffer (TAE) 50x 242 g Tris

57.1 ml acetic acid

100 ml 0.5 EDTA (pH 8.0)

add H2O to a final volume of 1 l

Agarose gel (1% to 2%) 100 ml TAE buffer

1 to 2 g agarose

10 µl Gel Red

Gel Loading Dye Blue NewEngland Biolabs, Frankfurt, Germany

1 kb ladder NewEngland Biolabs, Frankfurt, Germany


2.1.12 Plastic materials

Plastic materials provided from

Plates

96-well V bottom Greiner Bio-One, Frickenhausen, Germany

96-well U bottom Greiner Bio-One, Frickenhausen, Germany

96-well flat bottom BD Bioscience, Heidelberg, Germany

96-well flat bottom (ELISA) Greiner Bio-One, Frickenhausen, Germany

24-well flat bottom Corning Inc., Wiesbaden, Germany

Tubes

0,6 ml (γ-counter) Greiner Bio-One, Frickenhausen, Germany

1,8 ml (cryo-tubes) Nunc, Wiesbaden, Germany

6 ml (FACS) BD Bioscience, Heidelberg, Germany

15 ml BD Bioscience, Heidelberg, Germany

50 ml BD Bioscience, Heidelberg, Germany

0.5 ml and 1.5 tubes Eppendorf, Hamburg, Germany

PCR tubes peqLab, Erlangen, Germany

Eppendorf UVette Eppendorf, Hamburg, Germany

Cell culture

25 cm2 flask Corning Inc., Wiesbaden, Germany



CELLline CL350 Integra Bioscience, Chur, Swiss

Cell scraper Corning Inc., Wiesbaden, Germany

Tissue preparation

Petri dishes 94 mm Greiner Bio-One, Frickenhausen, Germany

Cell strainer 100 µm BD Bioscience, Heidelberg, Germany

Cell strainer 100 µm BD Falcon, Heidelberg, Germany

20µl Capillaries (Sysmex KX21) Sysmex GmbH, Norderstedt, Germany

Ceramic spheres (FastPrep 24) MP Biomedicals, Heidelberg, Germany

MACS separation columns (LS) Miltenyi Biotec, Bergisch Gladbach, Germany


2.1.13 Instruments

Instruments provided from

CO2 incubator Hereaus Instruments, Hanau, Germany

Cobra II auto-gamma Perkin Elmer, Waltham, USA

Eppendorf Centrifuge 5417R Eppendorf, Hamburg, Germany

Eppendorf BioPhotometer Eppendorf, Hamburg, Germany

FACSsort BD Bioscience, Heidelberg, Germany

FastPrep 24 MP Biomedicals, Heidelberg, Germany

LaminAIR HB2448 Hereaus Instruments, Hanau, Germany

Microflow® Biological Safety Cabinets Bioquell, Andover, UK

Mastercycler ep gradient S Eppendorf, Hamburg, Germany

Multifuge 1S-R Hereaus, Hanau, Germany

Microscope Carl Zeiss, Jena, Germany

Roche Modular Analytics Evo Roche Diagnostics, Mannheim, Germany

Sysmex KX21 Sysmex GmbH, Norderstedt, Germany

Titertek Mulatiskan MCC/340 Berthold Detection System, Pforzheim, Germany

ThermoScan IRT 4520 Braun, Kronberg, Germany

Venti-rack BioZone, Kent, UK


2.2 Methods

2.2.1 Viruses

The Lymphocytic choriomeningitis virus WE (LCMV WE) was kindly provided by Hanspeter

Pircher (IMMH Freiburg, Germany) and stored at -80°C until use.

Human RSV A2 strain was kindly provided by P. Openshaw (Imperial College, London, UK),

grown on HEp-2 cells and stored at -80°C until use. The recombinant RSV - rRSV 8A - with a

single amino acid exchange (Asn� Ala) at position 8 of the immunodominant epitope M2-1

82-90 was generated and kindly provided by Christine Krempl (Institute of Virology and

Immunobiology, Würzburg, Germany, [157]).

2.2.2 Hybridoma

F23.2 and KJ16 cells were cultivated in IMDM supplemented with 10% FCS and 1%

Penicillin/Streptomycin and 1% Glutamine. To adapt the cells to the serum-free and protein-

free Hybridoma medium (PFHM II), the direct adaptation protocol was used according to the

manufacturer’s protocol. Cells were directly transferred into PFHM II and cell growth was

monitored until a cell density of 1x106/ml was reached. 1-2x105/ml cells were subcultured in

fresh serum free medium for 5 passages. After this, the CELLline CL 350 bioreactor was

used to cultivate cells and gain high protein expression levels in the supernatant. 8x106 cells

were inoculated into the cell compartment and cultivated with 350ml medium in the medium

compartment. While monitoring the culture every 3 days, cells were harvested once every

week, splitted 1:5 back into the culture flask and supernatant was stored at -20°C. Protein

concentration in the supernatant was determined with the Bio-Rad Protein Assay based on

the method of Bradford, which uses a differential color change after adding an acidic dye to

the protein solution. Absorbance was measured at 595 nm using the eppendorf

BioPhotometer.

2.2.3 Mice

C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Sulzfeld,

Germany) and the MHC congenic mouse strain B6.C-H2d/bByJ (C57BL/6-H-2d) from Jackson

Laboratory (Bar Harbor, USA). C57BL/6-H-2dxb mice were obtained by mating C57BL/6 mice

(H-2b) with C57BL/6-H-2d mice. Mice were kept in an individual ventilated cage (IVC) unit

(BioZone, Kent, UK) and infected intranasally with RSV at the age of 6-12 weeks.

Peforin deficient (PKO) and 318 mice were kindly provided by H. Pircher (IMMH, Freiburg,

Germany). Beige and pearl mice were purchased from Jackson Laboratory (Ben Harbor,


USA). Souris mice were obtained from the Mutant Mouse Regional Resource Center

(University of California, Davis, USA) and ashen mice were kindly provided by G. de Saint

Basile (Paris, France).

Homozygous beige, souris, pearl and ashen mice were detected by their diluted coat color.

Heterozygous ashen and souris mice were typed by PCR and enzyme restriction or

sequencing.

2.2.3.1 Genotyping of ashen mice

DNA was extracted from mouse tails (0.5cm cut with a scalpel) by incubating the tail tissue in

lysis buffer (100mM Tris-HCl pH 8.5, 5mM EDTA, 200mM NaCl, 0.2% SDS) containing

proteinase K (0.1mg/ml) over night at 56°C.

Samples were centrifuged (13000rpm, 5min) and DNA containing supernatant was mixed

with an equal amount of isopropanol. Precipitated DNA was transferred into elution buffer

and solubilized for 1h at 56°C. Thereafter polymerase chain reaction (PCR) was performed

using the following primers: forward: 5’ CTTTCTCTTCCACCATACTT 3’ and reverse: 5’

AAGCGAGTTCCAGGGCAG 3’.

Table VI: PCR protocol and conditions for genotyping ashen mice

reagent Volume (µl)

10x PCR reaction buffer 5

desoxynucleotides (each 10 mM) 1

forward primer (10 µM) 1

reverse primer (10 µM) 1

Taq polymerase (2U) 0.5

template DNA (concentration) 2 µl

H2O milipore add to 50 µl

The presence of the Rab27a-mutation was verified by restriction site analysis of PCR

products. For this, PCR products were purified by gel extraction and digested by RsaI for 4h

at 37°C following the manufacturer’s instruction. Following separation on a 2%-agarose gel

DNA from homozygous ashen mice showed a single band of 534 base pairs (bp), DNA from

temperature time repeats

denaturation 94°C 10 min

denaturation 94°C 30 s

annealing 42°C 30 s x35

elongation 72°C 35 s

72°C 10 min


wildtype mice produced a band of 242 bp and another band of 291 bp whereas DNA from

heterozygous mice resulted in 3 bands of 534, 291 and 242 bp, respectively.

2.2.3.2 Genotyping of souris mice

Souris genotyping is performed by amplifying the region containing the mutation using PCR

with the following primers: forward: 5’- TCACCCTTCATGTAGACCAGGAACC -3’ and

reverse: 5’- TGCAGGGCCAAAGCCATATCTAAAC -3’. PCR was performed using the

protocol described in Table VI and the conditions described in Table VII.

Table VII: PCR conditions for genotyping souris mice

temperature time repeats

denaturation 94°C 2 min


annealing 56°C 30 s x30

elongation 72°C 1 min

72°C 7 min

Thereafter, the 1228 nt PCR product was purified via gel extraction and sequenced to detect

the single nucleotide change with the primers for sequencing as described in Tab. V.

Sequencing was done by GATC Biotech (Konstanz, Germany) on an ABI 3730xl.

2.2.4 Mutation analysis of beige mice

To identify the mutation of beige mice, complete cellular RNA was isolated from spleens of

mice with TrizolTM reagent based on the method developed by Chomczynski and Sacchi

[167]. The obtained RNA pellet was dissolved in 30µl nuclease free water. Prior to reverse

transcription of RNA DNA was digested using following protocol for 30 min at 37°C:

Table VIII: DNA-digestion protocol

component amount

RNA 1µg

10x reaction buffer 1

nuclease free water to 9µl

DNase I 1µL (1U)

To stop digestion, 1µl of 25mM EDTA was added and the mix was incubated at 65°C for

10min. Afterwards the RNA was directly used as template for reverse transcription.


To obtain first-strand cDNA, RNA was transcribed with SuperScriptTM II Reverse

Transcriptase according to the protocol for OligodT primers (Tab. IX).

Table IX: Protocol for RT PCR

component amount time

RNA (DNA digested) 10 µl

dNTP mix (10 mM) 1 µl

OligodTprimer (0.5µg/µl) 1 µl

5 min at 65°C

1 min on ice

5x RT buffer 4 µl

25 mM MgCl2 4 µl

0.1 M DTT 2 µl

RNase out 1 µl

2 min at 42°C

SuperScriptTM

II RT 50 U 50 min at 42°C

Termination 15 min at 70°C

1 min on ice

RNase H 1 µl 20 min at 37°C

The cDNA obtained by this procedure was used as template for searching the mutation in the

Lyst gene of beige mice. For this, primer pairs as listed in Tab. II were used. PCRs were

performed following the protocol described in Tab. VI and the following conditions:

Table X: PCR conditions for genotyping beige mice

PCR fragments were purified via gel extraction and sequenced with either forward primer,

reverse primer or both. Sequencing was done by GATC Biotech (Konstanz, Germany).

To confirm the mutation on a genomic level, DNA was extracted from mouse tails using the

same conditions and protocol as described in 2.2.3.1. PCR and sequencing were performed

using the primers in Tab. III.

temperature time

94°C 2 min


annealing see Tab. II 30 s

elongation 72°C 30 s per 500 bp

x 35

72°C 7 min


2.2.5 Treatment of mice

2.2.5.1 Virus infection

For infection with LCMV WE, mice were injected intravenously (i.v.) with either 200 pfu (low

dose), 1x104 or with 2 x 104 pfu (intermediate dose).

For infection with RSV A2 or the recombinant RSV 8A, mice were anesthetized

intraperitoneally (i.p.) with ketamin and rompun and inoculated with 1-2x106 pfu intranasally

(i.n.).

2.2.5.2 Poly (I:C) treatment

To activated NK cells without virus infection, mice were injected i.p. with 100-200µg of

poly(I:C) 24 hrs prior to the experiment.

2.2.5.3 Depletion of T cell populations

To selectively deplete T cells bearing TCRs with the Vβ 8.2 (F23.2 [163]) or Vβ 8.1/8.2 (KJ16

[164]) chains, mice were injected i.p. with 100µg of F23.2 or KJ16 in 200 µl PBS one day

prior to and two and five days after infection [168]. Efficiency of depletion was determined by

flow cytometry.

2.2.5.4 Adoptive transfer of T cells

Spleen cells from either transgenic 318 donor mice (Thy1.1+) -expressing an LCMV gp33-

specific TCR on about 50% of their CTLs (P14 T cells)- or d8 spleen cells of LCMV-infected

mice were used. Spleen cells were isolated and counted. Naïve P14 T cells were stained for

T cell populations expressing a TCR specific for the LCMV gp33 epitope using αCD3, αCD8,

αThy1.1, and αVα2 antibodies. In some experiments CTLs obtained from donor mice 8 days

after LCMV infection were purified via MACS separation.

Either defined numbers of total splenocytes or calculated T cell populations were transfused

in a volume up to 300µl of IMDM i.v. into sex-matched naïve recipients or recipients that

were infected with 1x104 pfu LCMV 10h before.


2.2.6 Preparation of mice

2.2.6.1 LCMV-infected mice

Mice were anesthetized with ketamin and rompun for blood sampling from the retro-orbital

plexus followed by sacrificing mice by cervical dislocation. To isolate splenocytes, spleens

were removed and dissociated into single cell suspensions by mashing them through a cell

strainer. Cell debris was removed by short centrifugation.

Kidneys, lungs, and parts of spleen and liver were removed and kept in PBS with 5% FCS at

-80°C for virus titer determination or in 4% formaldehyde solution at room temperature for

histological analysis.

2.2.6.2 RSV-infected mice

Mice were injected i.p. with 10mg thiopental and exsanguinated via the femoral arteries. The

diaphragm was removed to open the thorax without injuring the lung. The trachea was

exposed in order to make a small incision allowing passage of a lavage tube into the trachea.

This lavage tube was attached directly to a syringe filled with 1ml PBS containing 0.3%

Lidocain. PBS was injected and aspirated from the animals’ lungs five times. The procedure

was repeated with IMDM three times and the obtained fluids were pooled.

For analysing cytokines, BAL fluid yielded during the first of the 5 washing steps was stored

in a separate tube, centrifuged and analysis was performed by taking an aliquot of the

supernatant.

For virus titer determination, lungs were removed and kept in 1.5ml SF RPMI in liquid

nitrogen until further analysis.

2.2.7 In vitro activation of T cells

Spleen cells from 318 mice containing 4x105 TCR transgenic T cells (P14) were seeded into

a 24 well plate in 1ml medium with or without recombinant human interleukin-2 (IL-2). GP33

peptide was added to a final concentration 10-7M and cells were kept in culture for 72 to 96h.


2.2.8 Determination of virus titers

2.2.8.1 Quantification of RSV with an immunological focus assay

Lungs kept in 1.5ml SF RPMI were homogenized manually and after a short centrifugation,

3.3-fold serial dilutions from supernatants were made using SF RPMI medium in a 96-well

plate. From each dilution an aliquot was transferred to a ~90% confluent monolayer of Hep-2

cells in a 96-well flat bottom plate which had been seeded 24 hours before. Virus was

allowed to infect the cells for 3h at 37°C prior to washing and incubating the cell layer for

additional 24h with RPMI containing 10% FCS. After this incubation period, plates were

washed and fixed with 100% methanol containing 2% H2O2 for 20min at room temperature

(RT). After washing, fixed cells were incubated with RSV goat-anti mouse biotin polyclonal

antibody (1:400 in PBS with 1%FCS) for 30min at RT. Streptavidin HRP was added (1:2000)

for 30min and finally, to visualize RSV infected cells, a DAB solution (each tablet solved in

10ml H2O with 10µl H2O2) was added for another 20min at RT. Plates were washed twice

with PBS between each step. Viral titers were determined by counting visible infected cell

foci under the light microscope.

2.2.8.2 Quantification of LCMV with an immunological focus assay [169]

Organs from LCMV infected mice were homogenized in 1ml PBS with 5% FCS with ceramic

spheres using a FastPrep machine. Organ debris was removed by centrifugation for 5min at

10000rpm and supernatant was used to detect virus titers. Ten fold serial dilutions were

made and mixed with 8x105 MC57 cells per well in a 24 well plate in a total volume of 400µl.

After letting the virus infect the cells for 5h, cells were incubated for 48h under a

methylcellulose overlay. After this, cell monolayers were fixed with 4% formaldehyde for

20min and permeabilized with 0.5% Triton X-100 in PBS for 30min. After washing with PBS

and blocking with 10% FCS in PBS, cells were stained with a monoclonal rat anti-LCMV-NP

antibody (VL-4) followed by washing with PBS and staining with a peroxidase-labeled second

stage anti-rat IgG antibody. Virus infected cell were detected by adding OPD until foci

became visible. Viral titers were determined by macroscopically counting stained foci.


2.2.9 Flow cytometry

Numbers of live cells were determined by counting cells under the light microscope excluding

dead cells by trypan blue staining. For determination of absolute cell counts of particular cell

populations, live cell counts as determined by microscopy were put equal with cells identified

in the live gate in forward and side scatter as determined by flow cytometry.

Cells were analysed on a FACSort cytometer using CellquestPro v4.02 software.

2.2.9.1 Surface Staining

For surface staining, 1x105 to 1x106 cells were incubated with the appropriate antibodies for

30min at 4°C.

2.2.9.2 Intracellular cytokine staining

To determine the frequency of IFN-γ producing CTLs, 1-5x105 lymphocytes were

restimulated in vitro with the respective peptides or medium as a control for 3h at 37°C in a

96 well V-bottom plate in the presence of monensin in a total volume of 150µl. Peptides were

added to a final concentration of 10-6M. Following surface staining, cells were fixed with 4%

PFA and permeabilized and stained either with an anti-IFN-γ antibody or an IgG isotype

control diluted in Perm/Wash buffer.

In some experiments 1x105 cells were restimulated with 4x104 RAW macrophages that had

been infected with RSV at a moi of 5 24h prior to the experiment.

2.2.9.3 Degranulation

To test the ability of CTLs to degranulate, 5x105 spleen cells were restimulated with the

respective peptides in the presence of a fluorescence labelled anti-CD107a antibody for one

hour without monensin. After this incubation period, monensin was added and restimulation

was continued for additional 3h. Thereafter, cells were treated as described in 2.2.9.3.

NK cell degranulation was determined by incubating 5x105 spleen cells with 5x105 YAC-1 cell

for 2h in the presence of anti-CD107a without monensin. Following restimulation, cells were

stained with anti-CD3, anti-NK 1.1 and anti-CD107a antibodies and analyzed by flow

cytometry.


2.2.10 Magnetic Activated Cell Separation

To isolate CD8 T cells, a MACS CD8a+ T cell isolation Kit was used following manufacturer's

instructions. Non CD8+ T cells were labelled by using a cocktail of biotin-labelled antibodies

against CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC-class II,

and Ter-119. Thereafter, cells were incubated with anti-biotin micro-beads and magnetic cell

separation was performed using LS MACS columns. The effluent contained enriched CD8+ T

cells. Cell purity achieved was more than 90 %.

2.2.11 Blood count

White blood cell counts, haemoglobin, and thrombocyte counts were determined by taking a

blood sample of 20µl with a capillary from mice via retro-orbital bleeding. Samples were

diluted in 500µl EDTA and measured using a Sysmex KX-21 hematology analyzer.

2.2.12 Proliferation assay

T cell proliferation was quantified in a carboxyfluorescein diacetate succinimidyl ester (CFSE)

proliferation assay. Membranes of cells were stained with CFSE dye (0.5µM in PBS) for

10min at 37°C. Thereafter FCS was added to a final concentration of 5% to stop staining and

cells were washed twice with ice cold PBS. Stained spleen cells from 318 mice containing

4x105 TCR transgenic T cells were seeded into a 24 well plate in 1ml medium. GP33 peptide

was added to a final concentration 10-7M and cells were kept in culture for 72h. Proliferation

was determined by flow cytometry.

2.2.13 Cytotoxicity Assay

2.2.13.1 “Mini Killer” conditions for BAL cells

To determine the cytotoxicity of T cells, P815, EL-4 or RAW cells were loaded with peptide

(2x10-5M to 2x10-13) and 51chromium for 2h at 37°C. In some experiments RAW cells were

loaded with 51chromium that had been infected with RSV at a moi of 5 for 24h.

Target cells were incubated with BAL cells at different effector to target ratios starting with

25:1 (1x104 effector cells, 2x103 target cells) in a total volume of 100µl followed by five 2-fold

serial dilutions of effector cells for 5h at 37°C. After this, 50µl of the supernatant was

removed and the radioactivity released to the supernatant during the incubation period was

measured in an γ-counter. Chromium spontaneously released from target cells incubated

with medium was measured (S) and total release (T) was determined by lysing target cells

with 2N HCl.


2.2.13.2 “Maxi Killer” conditions for spleen cells

Cytotoxicity of spleen cells was determined by incubating peptide-loaded (gp33) chromium-

labeled target cells with spleen cells starting with an effector to target ratio of 100:1 (1x106

effector cells, 1x104 target cells) in a total volume of 150µl followed by five 3-fold serial

dilutions of effector cells. After 5h incubation at 37°C, 70µl of supernatant was removed and

analyzed in a γ-counter. Spontaneous and total release was determined as described above.

In some experiments LCMV-infected MC57G cells were used as target cells. The MC57G

cells were infected before with LCMV WE at a moi of 0.01 for 48h.

For analyzing NK cell cytotoxicity YAC-1 cells were labeled with 51Cr and spleen cells were

incubated with target cells at an initial effector to target ratio of 200:1.

In all experiments percentage of specific lysis was determined as follows:

specific lysis = [(cpmE-cpmS) x100] / [cpmT-cpmS]

cpm= counts per minute; E = effector release; S = spontaneous release; T = total release

Spontaneous 51Cr release was below 20% in all experiments.

2.2.14 Determination of cytokine levels

2.2.14.1 ELISA

IFN-γ serum levels were determined by ELISA. For this, 96-well plates were coated with

purified anti-IFN-γ antibody (1µg/ml) overnight at 4°C. After washing with PBS containing

0.05% Tween, unspecific binding was blocked by incubating the plates with 10%FCS in PBS

at RT for 1h. Serum samples were diluted 1:2 to 1:10 and a serial dilution of the IFN-γ

standard was made from 200ng/ml to 0,09 ng/ml. Samples were incubated on the plate for

2.5h. Then, a biotin labelled anti-IFN-γ antibody (0.5µg/ml) was added for 1h followed by

Streptavidin-Peroxidase incubation for 30min. The plate was washed after each step. The

bound Streptavidin-Peroxidase was detected by adding OPD. To stop the reaction 2N HCl

was added and absorbance was measured using the Titertek Multiskan MCC/340 plate

reader at 492 nm.

To determine serum levels of sCD25, the Mouse IL-2 R alpha DuoSet kit was used. 96-well

plates were coated with the capture antibody (0.4µg/ml in PBS) overnight. Plates were

washed as recommended between each step. Blocking was performed by using 1% BSA in

PBS. A serial dilution of the sCD25 standard was made starting from 4ng/ml to 0,03 ng/ml.


Samples diluted 1:2 to 1:10 and standards were incubated on the plate for 2h at RT followed

by the addition of a detection antibody (0.4µg/ml) for additional 2h. Finally Streptavidin-HRP

was added and the bound Streptavidin-HRP was detected by incubating with OPD. Reaction

was stopped by adding 2N HCl and absorption was measured using the Titertek Multiskan

MCC/340 plate reader at 492 nm.

2.2.14.2 Cytometric bead array – mouse inflammation kit

To analyze cytokine levels in BAL, fluid samples were treated as described in 2.2.6.2. 50µl of

BAL supernatants were used for analysis of levels of IL-6, IL-10, MCP-1, IFN-γ, TNF-α and

IL12p70, using the CBA Mouse Inflammation Kit following the manufacturer’s instructions.

Data were analysed by flow cytometry using CBA software.

2.2.15 Analysis of liver enzymes, triglycerides and ferritin serum levels

Serum levels of LDH (lactate dehydrogenase), GLDH (glutamate dehydrogenase), GPT

(glutamate pyruvate transaminase or alanine transaminase), triglycerides and ferritin were

determined by the central laboratory of the University Hospital Freiburg. Analysis was

performed with Roche diagnostic reagents using the Roche Modular Analytics Evo.

2.2.16 Histology

Organs were kept in 4% formaldehyde until they were embedded in paraffin. Sections were

made using a standard microtome and stained with hematoxylin and eosin (HE). Tissue

processing and staining was performed at the Institute of Pathology, University Hospital

Freiburg, and analysis and of the sections was done by A. Schmitt-Gräff.

2.2.17 Statistical analysis

Data were analysed by using GaphPad InStat software version 3.06.

The comparison between data was evaluated with Student`s t test if data were sampled from

populations that followed Gaussian distribution and had equal standard deviations (SD). If

the difference between SDs was significant, the alternate (Welch) t test was used. To

compare more than two groups a one-way ANOVA (ANalysis Of Variance) with post test was

used. Differences were considered being significant at a p value below 0.05.

Results 50

3 Results

3.1 Strain-specific disease susceptibility after RSV infection in the mouse is determined by MHC dependent CTL responsiveness

RSV infected BALB/c mice develop an inflammatory pneumonia that is clinically associated

with signs of illness like weight loss, ruffled fur and inactivity. These signs of illness can not

be observed after RSV infection of C57BL/6 mice. In order to analyze, to which extent these

differences are determined by the MHC locus, we compared these disease parameters after

RSV infection in BALB/c (H-2d), C57BL/6 (H-2b), MHC congenic C57BL/6-H-2d mice and the

F1 backcross to C57BL/6 (H-2bxd).

3.1.1 The MHC haplotype is an important determinant of disease susceptibility following RSV infection.

BALB/c (H-2d), C57BL/6 (H-2b), MHC congenic C57BL/6 (H-2d) mice and an F1 backcross

(H-2dxb) of these mice to C57BL/6 mice were infected intranasally with 106 pfu RSV and

weight was monitored daily. As expected, C57BL/6 mice did not show any signs of illness,

while BALB/c mice significantly lost weight from day 5 to day 7 after infection (Fig. 7A and

7B).


0 1 2 3 4 5 6 7 8 9

bod

y w

eig

ht [

%]

80

90

100

110A

n.s.

∗∗ ∗ ∗∗ ∗ ∗

∗

∗ ∗ ∗B

BALB/c

C57BL/6

C57BL/6-H-2d

C57BL/6-H-2dxb

60

70

80

90

100

110

120

d6 b

ody

wei

ght [

%]


0 1 2 3 4 5 6 7 8 9

bod

y w

eig

ht [

%]

80

90

100

110A

n.s.

∗∗ ∗ ∗∗ ∗ ∗

∗

∗ ∗ ∗B

BALB/c

C57BL/6

C57BL/6-H-2d

C57BL/6-H-2dxb

60

70

80

90

100

110

120

d6 b

ody

wei

ght [

%]

Figure 7: Weight loss after RSV infection is more pronounced in mice carrying the H-2d

MHC haplotype. Mice were infected intranasally (i.n.) with 106 pfu RSV and weight was monitored for seven days. (A) The data show mean and SD from mice of one experiment representative for 6 independent experiments. (B) Each box represents the weight on d6 after infection in 12 independent experiments for BALB/c and C57BL/6 mice (n = 44). In six of those experiments C57BL/6-H-2d and C57BL/6-H-2bxd mice were included (n = 18). *p < 0.05; ***p < 0.001; n.s. (not significant) p > 0.05

Results 51

C57BL/6-H-2d mice lost weight similar to BALB/c mice, but recovered earlier (Fig. 7A),

while C57BL/6-H-2dxb did not loose weight after RSV infection (Fig. 7B). The results

indicated that disease susceptibility can in part be transferred with the H-2d MHC allele

and that the protective effect of the H-2b allele is dominant.

To determine whether these differences in weight loss were also reflected by the extent of

inflammation in the infected lungs, we determined a panel of inflammatory cytokines in the

BAL supernatant (BAL fluid, BALF) on d7 after infection. BALB/c mice showed higher

levels of IFN-γ, IL-6 and MCP-1 compared to C57BL/6 mice (Fig. 8A-C), while no

differences were found in the levels of TNF-α, IL-10 and IL-12p40 (data not shown).

C57BL/6 mice carrying the H-2d haplotype had higher levels of these cytokines than those

with the H-2b haplotype, but the pulmonary inflammatory response was less pronounced

compared to BALB/c mice (Fig. 8A-C). These data support a correlation between weight

loss and inflammatory changes in the infected lungs.

3.1.2 RSV induced disease is not determined by peak virus titers or virus elimination kinetics.

RSV replicates to higher titers in lungs of BALB/c mice compared to C57BL/6 mice [19,

20]. To determine whether the disease observed in C57BL/6-H-2d mice is due to

differences in peak virus titers or to a different kinetic of viral elimination, RSV lung titers

at d4 and d6 after infection were compared in the four mouse strains. As expected, at the

peak of viral replication BALB/c mice displayed 10-fold higher pulmonary virus titers

10

100

1000

10000

pg/m

l

A B C

BALB/c C57BL/6 C57BL/6-H-2d C57BL/6-H-2dxb

IFN-γγγγ IL-6 MCP-1

10

100

1000

10000

pg/m

l

10

100

1000

10000

pg/m

l

∗

∗

∗ ∗ ∗

∗ ∗

∗ ∗ ∗

∗

10

100

1000

10000

pg/m

l

A B C


IFN-γγγγ IL-6 MCP-1

10

100

1000

10000

pg/m

l

10

100

1000

10000

pg/m

l

∗

∗

∗ ∗ ∗

∗ ∗

∗ ∗ ∗

∗

Figure 8: Different cytokine patterns in BALF of BALB/c, C57BL/6, C57BL/6-H-2d and

C57BL/6-H-2dxb

mice. Mice were infected with 106 pfu RSV and the indicated cytokines were determined in BAL fluid (BALF) seven days after infection by cytometric bead array. Results of two independent experiments are shown (n= 5-9). *p < 0.05; **p < 0.01; ***p < 0.001; nothing indicated p > 0.05

Results 52

compared to C57BL/6 mice, while the virus load in lungs of C57BL/6-H-2d and C57BL/6-

H-2bxd mice was similar to C57BL/6 mice (Fig. 9A). At d6 after infection all groups

displayed comparable virus titers (Fig. 9B) and at d7 virus was eliminated below detection

limit in all strains (data not shown). The poor correlation between signs of illness and viral

titers suggests that of the different replication kinetic of the virus is not responsible for the

different infection outcomes in C57BL/6 and MHC congenic C57BL/6-H-2d mice.

3.1.3 The pulmonary CTL response is of similar magnitude in MHC congenic mice.

Differences in MHC result in a different extent and composition of T cell responses. Since

T cells and in particular CD8+ cytotoxic T cells play a major role in RSV-induced pathology

in BALB/c mice [18], we compared the CTL response to RSV infection in the different

mouse strains. Seven days after RSV infection, cells recruited to the airways were eluted

via bronchoalveolar lavage (BAL) and restimulated with RSV-infected RAW309Cr.1 cells

expressing both MHC haplotypes, H-2d and H-2b. The percentage of IFN-γ producing cells

among CD8+ T cells was determined by intracellular cytokine staining. The absolute

numbers of CTLs eluted by BAL were comparable in all groups. Moreover, the fraction of

virus-specific CTLs among total BAL CTLs was similar in all 4 experimental groups (Fig

10A and B).

101

102

103

104

105

106

RS

V lo

ad d

4 [p

fu/lu

ng]

A B


101

102

103

104

105

106

RS

V lo

ad d

6 [p

fu/lu

ng]

∗ ∗ ∗

∗ ∗ ∗

101

102

103

104

105

106

RS

V lo

ad d

4 [p

fu/lu

ng]

A B

BALB/c C57BL/6 C57BL/6-H-2d C57BL/6-H-2dxbBALB/c C57BL/6 C57BL/6-H-2d C57BL/6-H-2dxb

101

102

103

104

105

106

RS

V lo

ad d

6 [p

fu/lu

ng]

∗ ∗ ∗

∗ ∗ ∗

Figure 9: Virus load and elimination kinetic in C57BL/6-H-2d mice is

comparable to C57BL/6 mice. Mice were infected i.n. with 106 pfu RSV. Pulmonary virus load was determined at d4 (A) and d6 (B) after infection. Data were pooled from two independent experiments with 3-5 mice per group and time point. Dashed lines indicate detection limit. ***p < 0.001; nothing indicated p> 0.05

Results 53

Figure 10: Total antiviral CTL response is comparable in all groups. Seven days after i.n. RSV infection, BAL CTLs were analyzed for IFN-γ production after restimulation with RSV-infected RAW macrophages. (A) Representative FACS plots of BALB/c mice gated on CD3

+ T

cells are shown. (B) Pooled data from 6 independent experiments with 3-5 mice/group for BALB/c and C57BL/6 mice are shown and in three of those experiments C57BL/6-H-2

d and

C57BL/6-H-2bxd

were included. n.s. p > 0.05.

3.1.4 Neither regulatory nor IL-17-producing CD4+ T cells influence the different outcomes of disease.

Since pulmonary inflammatory responses may also be influenced by regulatory or

inflammatory helper T cell responses, we determined the number and percentage of

FOXP3-expressing and IL-17-expressing CD4+ T cells in the BAL in the four mouse

strains. On d7 after RSV infection, all strains showed a similar number and percentage of

BAL CD4+ T cells expressing IL-17 or FOXP3 (data not shown).

3.1.5 Vββββ skewing of pulmonary CTL is more pronounced in BALB/c and C57BL/6-H-2d mice than in C57BL/6 mice.

Although the MHC differences did not lead to a difference in the overall number of

pulmonary virus-specific CTLs, their composition and quality could be influenced by the

MHC. To address this issue, the Vβ chain usage of pulmonary CTLs was measured and

compared with the Vβ usage of CTLs obtained from the spleens of naïve mice from the

same strain. Seven days after RSV infection, there was a significant increase in

Vβ8.1/8.2-expressing CTLs in the BAL of BALB/c and of C57BL/6-H-2d mice, while there

were no significant changes in the frequency of CTLs using other chains (Fig. 11A).

C57BL/6 mice showed a relative increase in the dominant Vβ9-expressing population, but

this was less pronounced than the Vβ8 peak in BABL/c mice (Fig. 11B). In C57BL/6-H-2dxb

0

10

20

30

40

IFN

- γ+ o

f C

D8+

T c

ells

[%]

n.s. n.s.

IFN

-γ

CD8

RAWuninfected

RAWRSV infected

n.s.

A

B


0

10

20

30

40

IFN

- γ+ o

f C

D8+

T c

ells

[%]

n.s. n.s.

IFN

-γ

CD8

RAWuninfected

RAWRSV infected

n.s.

A

B


Results 54

mice, the expansion of the Vβ8.1/8.2-expressing population was attenuated and there was

only a slight increase of the Vβ9-expressing population (Fig. 11C). Overall, the response

was highly oligoclonal in the H-2d strains while this was significantly less pronounced in

the strains carrying H-2b alleles.

Figure 11: TCR repertoire of BALB/c, C57BL/6, C57BL/6-H-2

d

and C57BL/6-H-2bxd

mice after RSV infection. Mice were infected i.n. with 106 pfu RSV. Seven days later, BAL cells were stained with antibodies against CD3, CD8 and the indicated Vβ chains and analyzed by flow cytometry. The mean percentages of the indicated Vβ

+ CD8+ T cells from

BAL of RSV infected BALB/c and C57BL/6-H-2d (A), C57BL/6 (B) and C57BL/6-H-2bxd (B) mice (n= 4) are shown, normalized by subtraction of the mean values from spleens of naïve control mice. The experiments were repeated with similar results.

3.1.6 The epitope-specific pulmonary CTL response is more focused in H-2d mice compared with H-2b mice.

To further investigate the T cell repertoire in the different mouse strains, defined virus-

derived CTL responses were analyzed. After RSV infection of BALB/c mice, the majority

-10

0

10

20

30

diff

eren

ce t

o na

ive

[%]

-10

0

10

20

30

diff

eren

ce t

o na

ive

[%]

2 3 4

5.1/5

.2 6 7

8.1/8

.2 8.3 9 10 11 12 13 14

-10

0

10

20

30

Vβ chain

diff

eren

ce t

o na

ive

[%]

BALB/cand C57BL/6-H-2d

C57BL/6

C57BL/6-H2dxb

A

B

C

-10

0

10

20

30

diff

eren

ce t

o na

ive

[%]

-10

0

10

20

30

diff

eren

ce t

o na

ive

[%]

2 3 4

5.1/5

.2 6 7

8.1/8

.2 8.3 9 10 11 12 13 14

-10

0

10

20

30

Vβ chain

diff

eren

ce t

o na

ive

[%]

BALB/cand C57BL/6-H-2d

C57BL/6

C57BL/6-H2dxb

A

B

C

Results 55

of the RSV-specific CTL response is directed against the immunodominant CTL epitope

M2-1 82–90 (M2-1 82) presented by H-2Kd [26, 159] while in C57BL/6 mice the

immunodominant epitope is M187-195 (M187) presented by H-2Db [161]. On d7 after RSV

infection, in BALB/c and C57BL/6-H-2d mice about 20% of the pulmonary CTLs

(representing more than 80% of the virus-specific CTLs) were specific for M2-1 82 (Fig.

12A). This was confirmed by tetramer staining (data not shown). In contrast, only about

10% of the BAL CTLs of C57BL/6 mice were specific for the immunodominant epitope

M187 (Fig. 12C). Analyzing the Vβ usage of these immunodominant populations revealed

that in BALB/c and C57BL/6-H-2d mice about 75% of the M2-1 82-specific CTLs carried

the dominant Vβ8.1/8.2 chain (Fig. 12B), mostly consisting of the Vβ8.2 chain (data not

shown). In contrast, only about 40% of the M187-specific CTL of C57BL/6 mice carried

the dominant Vβ9 chain (Fig. 12D). These data revealed significant differences in the

composition of the virus-specific CTL repertoire with a highly focused response in H-2d

mice and a broader, less focused response in H-2b mice. Of note, the co-expression of the

H-2b allele, which protected from RSV-induced weight loss, also led to a significant

reduction in the dominant H-2d restricted CTL population and to a more diverse CTL

response.

Figure 12: CTL response against the immunodominant peptide epitopes of BALB/c and C57BL/6 mice. Seven days after intranasal inoculation with 106 pfu RSV, IFN-γ production of BAL CTLs were quantified by flow cytometry after restimulation with the indicated peptides – (A) H-2Kd epitope M2-1 82-90; (C) H-2Db epitope M187-195. The percentage of Vβ 8.1/8.2+ (B) and Vβ9

+ (D) cells among the IFN-γ+

cells is shown. **p < 0.01; ***p < 0.001; n.s. p > 0.05.

0

25

50

75

100

Vβ 9

+ of

IF

N- γ

+ ce

lls [

%]

0

25

50

75

100

Vβ 8

.1/8

.2+ o

f IF

N- γ

+ cel

ls [

%]

0

10

20

30

IFN

- γ+

of C

D8+

T c

ells

[%]

0

10

20

30

IFN

- γ+

of C

D8+

T c

ells

[%]

M 187M2-1 82

∗ ∗ ∗

n.s.

n.s.∗ ∗

A

B

C

D


n.s.

∗

0

25

50

75

100

Vβ 9

+ of

IF

N- γ

+ ce

lls [

%]

0

25

50

75

100

Vβ 8

.1/8

.2+ o

f IF

N- γ

+ cel

ls [

%]

0

10

20

30

IFN

- γ+

of C

D8+

T c

ells

[%]

0

10

20

30

IFN

- γ+

of C

D8+

T c

ells

[%]

M 187M2-1 82

∗ ∗ ∗

n.s.

n.s.∗ ∗

A

B

C

D


n.s.n.s.

∗

Results 56

3.1.7 H-2b-restricted M187-specific CTLs have a higher avidity than H-2d-restricted M2-1 82-specific CTLs.

In order to compare the functional properties of the immunodominant CTL populations,

their ability to lyse peptide loaded target cells was tested. For this, RAWdxb target cells

were loaded with decreasing concentrations of the M2-1 82 and the M187 peptides and

incubated with BAL cells obtained 7 days after RSV infection. Since among the BAL cells

BALB/c mice have about two times more peptide-specific CTLs than C57BL/6 mice (Fig.

12 A and C), an overall BAL cell effector to target (E:T) ratio of 25:1 for C57BL/6 mice was

compared with an E:T ratio of 12.5:1 for BALB/c mice. While the specific lysis of both

groups was comparable at high peptide concentrations, CTLs of C57BL/6 mice were able

to lyse their target cells more efficiently at lower peptide concentrations (Fig. 13). These

data indicate that the dominant M2-1 82-specific CTL population in H-2d mice has a lower

avidity for their cognate antigen/MHC complex than the M187-specific CTL population in

H-2b mice. This may contribute to their increased potential to cause disease.

Figure 13: C57BL/6 CTLs have a higher avidity to M187 than BALB/c CTLs to M2-1 82. The specific lysis of target cells loaded with the M2-1 82 and M187 peptides in decreasing concentrations is shown for d7 BAL CTLs of BALB/c and C57BL/6 mice. For BALB/c mice an effector to target (E:T) ratio of 12.5:1 is compared with an E:T ratio of 25:1 for C57BL/6 mice.

3.1.8 The Vββββ 8.2+ M2-1 82-specific CTL response is responsible for the RSV-induced disease in H-2d mice

The obtained results suggested that the focussed M2-1 82-specific CTL response in H-2d

mice was a significant determinant of RSV-induced disease. This would predict that failure

to activate this T cell population or depletion of Vβ8.2 cells from infected mice should

attenuate the disease. In order to test this prediction, C57BL/6-H-2d mice were infected

either with RSV or with RSV8A, carrying a loss-of-recognition point mutation in the

immunodominant M2-1 82 epitope. Indeed, - as we showed it previously for BALB/c mice

[157]- weight loss was significantly reduced in mice infected with the mutant virus (Fig.

14A), despite the fact that the virus grows to similar titers in vivo [157]. In a second set of

experiments, Vβ8.2 cells were depleted by injecting an anti-Vβ8.2 depleting antibody one

day before and 2 and 5 days after RSV infection of BALB/c mice. This treatment depleted

peptide concentration [2x10xM]

-7 -8 -9 -10 -11 -12 -13

targ

et c

ell l

ysis

[%]

0

20

40

60

80BALB/c (E:T 12,5 : 1)C57BL/6 (E:T 25:1)

Results 57

about 90% of the Vβ8.2+ T cells (Fig. 14B) and about 70% of the M2-1 82 specific CTLs

(Fig. 14C) in the BAL on d7 after infection compared to mice injected with PBS as a

control. This partial depletion of M2-1 82-specific CTLs significantly attenuated weight loss

after RSV infection of BALB/c mice. These data indicate that the M2-1 82-specific CTLs

make a major contribution to RSV-induced disease in mice carrying the H-2d haplotype.

Figure 14: Reduced weight loss after infection of C57BL/6-H-2d with an RSV strain carrying

loss-of-recognition mutation and after depletion of the Vβ 8.2+ CTL population in BALB/c mice.

(A) C57BL/6-H-2d mice were infected i.n. with 106 pfu of RSV or RSV 8A. Weight was monitored for 7 days. Data show mean and SD of 6 mice per group obtained in 2 independent experiments. (B-D) BALB/c mice were treated with the hybridoma supernatant (F23.2; d-1, d2, d5) i.p. and infected i.n. with 106 pfu RSV. (B) Efficacy of depletion was monitored by flow cytometry of BAL CTLs at d7 after infection. (C) After in vitro restimulation with M2-1 82 the percentage of IFN-γ+ CD8+ T cells is shown. (D) Percent body weight on d6 after infection is shown for depleted and PBS-treated mice. Results are shown from one of two independent experiments with 4 mice per group. *p < 0.05; ***p < 0.001


0 1 2 3 4 5 6 7

body

wei

ght [

%]

80

90

100

110

rRSV 8ArRSV wt

C57BL/6-H-2d

∗∗ ∗ ∗ ∗ ∗ ∗

M2-1 82DB C

A

0

10

20

30

40

50

60

70

Vβ 8

.2+

of C

D8+

cells

[%

]

0

10

20

30

40

50

IFN

- γ+

of C

D8+

T c

ells

[%]

70

80

90

100d6

bod

y w

eigh

t [%

]

αVβ8.2 PBS


0 1 2 3 4 5 6 7

body

wei

ght [

%]

80

90

100

110

rRSV 8ArRSV wt

C57BL/6-H-2d

∗∗ ∗ ∗ ∗ ∗ ∗

M2-1 82DB C

A

0

10

20

30

40

50

60

70

Vβ 8

.2+

of C

D8+

cells

[%

]

0

10

20

30

40

50

IFN

- γ+

of C

D8+

T c

ells

[%]

70

80

90

100d6

bod

y w

eigh

t [%

]

αVβ8.2 PBS

Results 58

3.2 Hermansky-Pudlak Syndrome Type II confers a risk for hemophagocytic lymphohistiocytosis

As mentioned above, we have previously described a patient with HPSII, who developed

hemophagocytic lymphohistiocytosis (HLH). Since this was the only one of 11 described

HPSII patients who developed lethal HLH and since he also carried a heterozygous RAB27A

mutation, it remained unclear, whether Hermansky-Pudlak syndrome type II confers a risk for

HLH or if the lethal HLH observed in the patient was due to the additional heterozygous

RAB27A mutation.

Perforin deficient (PKO) and Rab27a-deficient mice (ashen) do not develop HLH

spontaneously, but after infection with the lymphocytic choriomeningitis virus (LCMV WE).

Therefore the mouse model for HPSII -the pearl mouse strain- was examined following

LCMV infection and the PKO strain was used as a positive control for disease induction. In

order to remain close to the human disease, we established assays that allowed evaluating

all diagnostic criteria in mice that are usually used for the diagnosis of the disease in humans

as described in chapter 1.5.2.

3.2.1 Pearl mice develop transient HLH following LCMV infection.

Pearl mice, wildtype C57BL/6 (negative control) and perforin-deficient mice (positive

controls) were infected intravenously with 200 pfu LCMV WE. Body weight and ear

temperature were monitored daily for 12 days after infection (Fig. 15 A and B). While no

changes in body weight and temperature were observed in wildtype C57BL/6 mice, perforin-

deficient mice showed a significant decrease in body weight starting at d6 and in body

temperature starting at d8 after infection remaining low until d12. Pearl mice displayed weight

loss from day 6 to 9 after infection but recovered thereafter (Fig. 15A). At the time point of

maximal weight loss (d8-9) they also showed a significant reduction in ear temperature (Fig

15B). On d8 after infection, mice were anesthetized and bleed retroorbitally to determine

blood counts. While C57BL/6 mice had developed a leukocytosis, pearl and PKO mice

showed a leukopenia that was more pronounced in PKO mice (Fig. 15C). Both pearl and

PKO mice showed a mild anemia and a more pronounced thrombocytopenia on d8 after

LCMV infection (Fig. 15C). The thrombocytopenia was also observed in C57BL/6 mice.

While white blood counts returned to almost normal values at d12, anemia and

thrombocytopenia persisted in the two mutant strains, while the thrombocytopenia returned

to preinfection values in C57BL/6 mice (data not shown).

Results 59

At d8 after infection liver and spleens were removed for histological analysis. While in spleen

and liver of wildtype mice only very few hemophagocyting macrophages could be observed,

hemophagocytosis was highly evident in spleens and livers of pearl and PKO mice (Fig.

16A). The liver damage was also reflected by highly elevated liver enzymes and LDH in the

serum of pearl and PKO mice compared to C57BL/6 mice at d8 after infection (Fig. 16B). As

expected, splenomegaly was observed not only in the two mutant groups, but also in

C57BL/6 mice due to the strongly induced T cell response (data not shown).

Because elevated serum levels of ferritin and sCD25 are additional diagnostic criteria for

HLH, they were determined in d8 sera. Both ferritin and sCD25 were found to be increased in

pearl mice compared to C57BL/6 mice but they did not reach the high levels of PKO mice

(Fig. 16C and D). Serum levels of triglycerides showed comparable values in pearl and

C57BL/6 mice and only a slight increase in infected PKO mice (data not shown). IFN-γ serum

levels are no diagnostic criteria for HLH, but have been described as the key cytokine

Figure 15: Pearl mice develop weight loss, decrease in temperature and cytopenia following LCMV infection. C57BL/6, pearl and PKO mice were infected i.v. with 200 pfu LCMV WE and body weight (A) and temperature (B) was monitored daily. Blood counts were analyzed on d8 after infection (C: white blood counts left panel, hemoglobin middle panel, platelet counts right panel). Gray bars indicate the range of values of naïve C57BL/6 mice. Black dots indicated single values of C57BL/6 mice, grey dots of pearl and black triangles of PKO mice. Dashed lines indicate detection limit. Graphs show pooled data of 2 independent experiments with at least 3 mice per group.


0 2 4 6 8 10 12

body

wei

ght [

%]

70

80

90

100

110


0 2 4 6 8 10 12

34

36

38

40

tem

pera

ture

[°C

]

34

36

38

40

34

36

38

40

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

HG

B [

g/d

l]

0

10

20

30

40

WB

C c

ou

nts

[x 1

03/µ

l]

0

200

400

600

800

1000

1200P

LT

co

un

ts [

x 1

03/µ

l]

A B

C

naive d8 LCMV

C57BL/6 PKOpearl

naive d8 LCMV naive d8 LCMV


0 2 4 6 8 10 12

body

wei

ght [

%]

70

80

90

100

110


0 2 4 6 8 10 12

34

36

38

40

tem

pera

ture

[°C

]

34

36

38

40

34

36

38

40

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

HG

B [

g/d

l]

0

10

20

30

40

WB

C c

ou

nts

[x 1

03/µ

l]

0

200

400

600

800

1000

1200P

LT

co

un

ts [

x 1

03/µ

l]

A B

C

naive d8 LCMV

C57BL/6 PKOpearl



0 2 4 6 8 10 12

body

wei

ght [

%]

70

80

90

100

110


0 2 4 6 8 10 12

34

36

38

40

tem

pera

ture

[°C

]

34

36

38

40

34

36

38

40

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

HG

B [

g/d

l]

0

10

20

30

40

WB

C c

ou

nts

[x 1

03/µ

l]

0

200

400

600

800

1000

1200P

LT

co

un

ts [

x 1

03/µ

l]

A B

C

naive d8 LCMVnaive d8 LCMV

C57BL/6 PKOpearl

naive d8 LCMVnaive d8 LCMV naive d8 LCMVnaive d8 LCMV

Results 60

alteration in HLH mice [49, 170]. IFN-γ serum levels were elevated in pearl mice (Fig. 16E),

but did not reach the levels observed in infected PKO mice (Fig. 16E). At d12 after infection

the amount of IFN-γ was slightly reduced in PKO mice and had decreased to undetectable

levels in pearl mice (data not shown).

NK cells of patients with HPSII were shown to exhibit a reduced or absent NK cell activity.

Therefore the NK cell activity of pearl mice was determined using two different experimental

approaches. Mice were injected i.p. with 100 or 200µg of poly(I:C) and 24h later cytotoxicity

and degranulation of splenic NK cells in response to NK-sensitive YAC-1 targets were

analyzed. Although great variations were observed among individual mice, target cell lysis

mediated by NK cells of pearl mice was significantly reduced compared to wildtype mice (Fig

17A and B). NK cell degranulation was measured by CD107a surface expression after in

vitro restimulation with YAC-1 target cells. Similar to the cytotoxicity data, pearl NK cells

GLDH

0

50

100

150

LDH

0

1000

2000

3000

GPT

0

50

100

150

200

250

300

350

U/l

C57BL/6

PKO

pearl

B

4886

Ferritin

0

500

1000

1500

2000

2500

ng

/ml

C1580

sCD25

0

200

400

600

800

1000

1200

pg

/ml

D100 – 230

IFN-γγγγ

0

10

20

30

ng

/ml

E

PKOpearlC57BL/6A

10 µm 10 µm10 µm

GLDH

0

50

100

150

GLDH

0

50

100

150

LDH

0

1000

2000

3000

LDH

0

1000

2000

3000

GPT

0

50

100

150

200

250

300

350

U/l

GPT

0

50

100

150

200

250

300

350

U/l

C57BL/6

PKO

pearl

C57BL/6

PKO

pearl

B

4886

Ferritin

0

500

1000

1500

2000

2500

ng

/ml

C4886

Ferritin

0

500

1000

1500

2000

2500

ng

/ml

C1580

sCD25

0

200

400

600

800

1000

1200

pg

/ml

D1580

sCD25

0

200

400

600

800

1000

1200

pg

/ml

D100 – 230

IFN-γγγγ

0

10

20

30

ng

/ml

E100 – 230

IFN-γγγγ

0

10

20

30

ng

/ml

E

PKOpearlC57BL/6A

10 µm 10 µm10 µm

PKOpearlC57BL/6A

10 µm10 µm 10 µm10 µm10 µm10 µm

Figure 16: Pearl mice develop liver disease and increased serum levels of ferritin, sCD25

and IFN-γγγγ following LCMV infection. Mice were infected with 200 pfu LCMV WE and eight days later the liver was histologically analyzed for hemophagocytosis (A). Arrows indicated hemophagocyting macrophages. Serum levels of (B) GPT, LDH GLDH, (C) ferritin, (D) sCD25 and (E) IFN-γ were determined on d8 after infection. Black dots indicate single values of C57BL/6 mice, grey dots of pearl and black triangles of PKO mice. Graphs show pooled data from two independent experiments with 3 mice per group (B-E).

Results 61

showed a significantly reduced capacity to degranulate compared to wildtype NK cells (Fig.

17C and D).

Taken together, pearl mice fulfilled 7 of the 8 criteria for HLH on d8 after LCMV infection and

thus clearly developed the disease. However, disease manifestations were transient and did

not progress to a lethal disorder as observed in PKO mice.

3.2.2 Pearl mice show a delay in virus control after LCMV WE infection.

HLH manifestations in PKO and ashen mice were shown to be associated with virus

persistence. We therefore analyzed the virus elimination kinetics in the different strains.

LCMV titers were analyzed in spleen, liver and lung at different time points after infection.

Viral titers were comparable in spleens of pearl, C57BL/6 and PKO on day 4 after infection

and no spreading to peripheral organs was detectable (Fig. 18B). While C57BL/6 mice had

significantly reduced the virus titers in the spleen on d8 after infection, pearl mice still had

significant titers although they were about 1.5 log lower than in PKO mice (Fig. 18A). At this

time point virus spread to lungs and livers was observed both in PKO as well as in pearl mice

(Fig. 18A). However, in contrast to PKO mice that show long-term virus persistence, pearl

mice were able to reduce LCMV titers below detection limit until d12 in all investigated

organs (Fig. 18C).

Figure 17: Pearl mice have a defect in NK cell cytotoxicity and degranulation. NK cell cytotoxicity was determined in a 5h chromium release assay on YAC-1 target cells. C57BL/6 and pearl mice were injected i.p. with 100µg (A) or 200µg (B-D) poly(I:C) and 24h later cytotoxicity of splenic NK cells was analyzed (A,B). NK to target ratio was determined by calculating percentage of NK cells among total spleen cells by FACS analysis. NK cell degranulation was measured after 2h restimulation with YAC-1 cells. (C) Representative FACS plots are shown for degranulation of NK cells after restimuation with medium or YAC-1 cells. (D) Degranulation is shown as ∆CD107

+ in

percent representing the differences between values of restimulation with YAC-1 cells compared to medium control. Black dots indicated single values of C57BL/6 mice and grey dots of pearl mice. Data shown are representative for 2 independent experiments.

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

CD107a

NK

1.1

C57BL/6 pearl

med

ium

YA

C-1

A B

C D

C57BL/6 pearl

0

10

20

30

40

∆∆ ∆∆C

D107

+ [

%]

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

CD107a

NK

1.1

C57BL/6 pearl

med

ium

YA

C-1

A B

C D

C57BL/6 pearl

0

10

20

30

40

∆∆ ∆∆C

D107

+ [

%]

Results 62

3.2.3 Pearl CTLs have a reduced capacity to proliferate in vitro and in vivo

The above findings suggested that a delay in virus control contributed to the transient

disease manifestations in pearl mice. Because LCMV elimination is dependent on antiviral

CTLs, we analyzed the CD8+ T cell compartment in pearl mice. On d8 after infection the

absolute numbers of CTLs in the spleen of pearl mice was reduced 2-fold when compared to

wildtype mice (Fig. 19B). To analyse whether this reduction is due to a defect in proliferation

and expansion, we generated 318 pearl mice expressing an LCMV gp33-specific TCR on

about 50% of their CTLs (P14). Spleen cells from 318 and 318 pearl mice containing the

same number of P14 CTLs were cultured for 72h with the gp33 peptide. In this assay, pearl

CTLs showed a reduced proliferation capacity when compared to C57BL/6 CTLs (Fig. 19A

and C). This proliferation defect could not be corrected by the addition of IL-2 to the culture

(Fig. 19C). In a second experimental approach, we compared the proliferation capacity of

P14 and P14 pearl CTL in vivo. For this, spleen cells containing 104 P14 cells were

Figure 18: LCMV titers in spleens, lungs and livers of C57BL/6, pearl and PKO mice. C57BL/6, pearl and PKO mice were infected with 200 pfu LCMV WE and virus titer in spleens, lungs and livers were determined on d4 (B), d8 (A) and d12 (C) after infection. Black dots indicated values of C57BL/6 mice, grey dots of pearl and black triangles of PKO mice. Graphs show pooled data of 2 independent experiments with at least 3 mice per group. Dashed line indicates detection limit.

C57BL/6

PKO

pearld4 d12B

splee

nlun

gliv

er101

102

103

104

105

106

107

108

pfu

per

org

an

splee

nlun

gliv

er101

102

103

104

105

106

107

108

pfu

per

org

an

101

102

103

104

105

106

107

108

pfu

per

org

an

d8

spleen lung liver

A

C

d8

C57BL/6

PKO

pearl

C57BL/6

PKO

pearld4 d12B

splee

nlun

gliv

er101

102

103

104

105

106

107

108

pfu

per

org

an

splee

nlun

gliv

er101

102

103

104

105

106

107

108

pfu

per

org

an

101

102

103

104

105

106

107

108

pfu

per

org

an

d8

spleen lung liver

A

C

d8

Results 63

adoptively transferred into either pearl or wildtype mice one day prior to infection with 200 pfu

LCMV WE. Eight days later, the frequency of P14 cells was determined in spleens of

recipient mice. While massive expansion was observed after transfer of P14 wildtype cells

into wildtype mice, the expansion of pearl P14 cells transferred into wildtype mice was

significantly reduced (Fig. 19D). Of note, the expansion was even more reduced following

transfer of P14 pearl cells into pearl mice (Fig. 19D), suggesting that both an intrinsic

proliferation defect of the CTL as well as an additional defect in the pearl APC compartment

contributed to the impaired in vivo proliferation.

3.2.4 Pearl CTLs have a defect in degranulation and cytotoxicity.

Since a twofold difference in CTL numbers is not likely to account completely for the loss of

virus control in pearl mice, we performed further experiments to detect functional

abnormalities in these cells. First, we analyzed the production of intracellular IFN-γ upon

gp33 stimulation of spleen cells obtained on d8 after LCMV infection. Fig. 20 shows that the

Figure 19: Pearl CTL show a reduced capacity to proliferate and expand. Wildtype (black) and pearl (grey) P14 T cells were CFSE stained and cultured for 72h in the presence of the gp33 peptide. Proliferation is shown for one representative pearl and wildtype mouse (A) and expansion in culture with or without IL-2 is shown by the frequency of P14 T cells among living cells in culture (C). (B) On d8 after infection with 200 pfu LCMV absolute CTL numbers in spleens of C57BL/6 and pearl mice were determined. (D) The expansion of adoptively transferred P14 T cells in recipient mice is shown on d8 after LCMV infection. P14 T cells are determined via Thy1.1, Vα2 and CD8 staining by FACS analysis. Pooled data from 2 (C and D) and 3 (B) independent experiments are shown with 3 mice per group.

C57BL/6

pearl

w/o IL-2 w/ IL-2

A

B C

C57BL/6 pearl

0

10

20

30

40

P14 T

cell

s [

%]

D

100 101 102 103 104

CFSE

coun

ts

318 C57BL/6

318 pearl

318 pearl

donor

C57BL/6 pearl

recipient

0

25

50

75

Th

y1.1

+V

αα αα2

+ o

f C

D8

+

T c

ell

s [

%]

0

5.0×107

1.0×108

1.5×108

CD

8+

T c

ell

s p

er

sp

leen

C57BL/6

pearl

w/o IL-2 w/ IL-2

A

B C

C57BL/6 pearl

0

10

20

30

40

P14 T

cell

s [

%]

D

100 101 102 103 104

CFSE

coun

ts

100 101 102 103 104

CFSE

coun

ts

318 C57BL/6

318 pearl

318 pearl

donor

C57BL/6 pearl

recipient

0

25

50

75

Th

y1.1

+V

αα αα2

+ o

f C

D8

+

T c

ell

s [

%]

0

5.0×107

1.0×108

1.5×108

CD

8+

T c

ell

s p

er

sp

leen

Results 64

frequency of gp33-specfic CTLs was similar in spleens of wildtype, PKO and pearl mice (Fig.

20A and B). Interestingly, although the total frequency of IFN-γ producing CTLs was similar,

the relative frequency of IFN-γhigh CD8+ T cells was lower in pearl and PKO mice (squares in

Fig. 20A). This probably reflected virus persistence, since at d12, the IFN-γ staining pattern

returned to normal in pearl mice -that had by then eliminated the virus-, but not in PKO mice -

where virus still persisted (data not shown).

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

89,2 76,0

***

CD107a

IFN

-γ

C57BL/6 pearl

med

ium

gp33

E F

0

25

50

75

100

CD

107a

+ o

f IF

N- γγ γγ

+C

D8

+

cell

s [

%]

C

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

D

B

0

10

20

30

40

IFN

- γγ γγ+ o

f C

D8

+T

cell

s[%

]

C57BL/6 pearl

PKO

CD8

IFN

-γ

A

C57BL/6

PKO

pearl

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

89,2 76,0

***

CD107a

IFN

-γ

C57BL/6 pearl

med

ium

gp33

E F

0

25

50

75

100

CD

107a

+ o

f IF

N- γγ γγ

+C

D8

+

cell

s [

%]

C

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

D

B

0

10

20

30

40

IFN

- γγ γγ+ o

f C

D8

+T

cell

s[%

]

C57BL/6 pearl

PKO

CD8

IFN

-γ

A

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

89,2 76,0

***

CD107a

IFN

-γ

C57BL/6 pearl

med

ium

gp33

E F

0

25

50

75

100

CD

107a

+ o

f IF

N- γγ γγ

+C

D8

+

cell

s [

%]

C

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

D

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

89,2 76,076,0

***

CD107a

IFN

-γ

C57BL/6 pearl

med

ium

gp33

E F

0

25

50

75

100

CD

107a

+ o

f IF

N- γγ γγ

+C

D8

+

cell

s [

%]

C

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

D

B

0

10

20

30

40

IFN

- γγ γγ+ o

f C

D8

+T

cell

s[%

]

C57BL/6 pearl

PKO

CD8

IFN

-γ

A

C57BL/6

PKO

pearl

C57BL/6

PKO

pearl

Figure 20: Pearl CTLs have a defect in degranulation and cytotoxicity. Wildtype and pearl CTL were analyzed 8 days after infection with 200 pfu LCMV WE. The frequencies of IFN-γ producing CTLs were determined by intracellular cytokine staining after in vitro restimulation with the gp33 peptide. Representative FACS plots (A) and pooled data from 2 independent experiments (B) are shown. IFN-γhi population is indicated in squares (A). Ex vivo cytotoxicity was tested in a 5h 51cr-release assay on (C) either target cells (MC57) infected with LCMV WE 2 days before or (D) target cells (EL-4) loaded with a gp33 peptide concentration of 10

-10M. Degranulation -measured by CD107a

surface expression of IFN-γ+ CTLs- was examined following 4h of in vitro restimulation with gp33 peptide. (E) Representative FACS plots and (F) percentage of degranulating cells among gp33-specific CTLs are shown. Black dots indicated single values of C57BL/6, grey dots of pearl and black triangles of PKO mice. Data from one representative experiment are shown in C and D and pooled data from 3 independent experiments are shown in F. ***p < 0.001

Results 65

We then analyzed CTL cytotoxicity and degranulation. On d8 after infection, spleen cells

from pearl mice showed a 3-fold reduced ex vivo cytotoxicity on LCMV-infected target cells

when compared to wildtype mice (Fig. 20C). This defect was more pronounced, when CTL

activity was analyzed on target cells loaded with a limiting concentration of gp33 peptide

(10-10M) (Fig. 20D). The partial defect in cytotoxicity was also reflected by the impaired ability

of pearl CTL to degranulate. After in vitro restimulation with the gp33 peptide, pearl CTL

showed a significantly reduced degranulation when compared to C57BL/6 mice (Fig. 20E

and F). Thus, pearl CTL have a defect in degranulation and cytotoxicity, but not in cytokine

production.

3.2.5 The cytotoxicity defect of pearl CTLs is relevant for virus control in vivo.

To analyze whether the degranulation and cytotoxicity defect observed in pearl CTL is

relevant in vivo, we performed adoptive transfer protection experiments that have previously

been shown to reflect perforin-dependent cytotoxicity [171]. For this, splenic CTLs of wildtype

and pearl mice were isolated via MACS separation 8 days after LCMV infection. 2x106

isolated CD8 T cells were adoptively transferred into C57BL/6 mice that had been infected

with 104 pfu LCMV WE 10h previously (Fig. 21A). After additional 18h viral titers in the spleen

were analyzed. In this experimental setting, identical numbers of LCMV-specific CTL were

transfused and equal homing to the spleen was documented by CFSE labelling (data not

shown). While transfusion of activated wild-type CTLs could significantly control the infection

in this short-term assay, pearl CTLs were not able to prevent virus replication as efficiently

(Fig 21B).

C57BL/6

pearl

donor

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

BA200 pfu LCMV

8dspleen

10 h

MACS:

CD8+

18 h LCMV titers

in spleen

104 pfu LCMV

C57BL/6

pearl

donor

C57BL/6

pearl

donor

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

BA200 pfu LCMV

8dspleen

10 h

MACS:

CD8+

18 h LCMV titers

in spleen

104 pfu LCMV

Figure 21: Pearl CTL show a defect in short term virus elimination in an adoptive transfer system. Splenic CD8 T cells isolated from spleens of C57BL/6 or pearl mice on d8 after infection with 200 pfu LCMV were MACS purified and adoptively transfused into C57BL/6 mice infected with 104 pfu LCMV 10h before (A). Viral titers in spleens were determined after additional 18h (B). Data are shown for one experiment being representative for two independent experiments.

Results 66

Since the pearl CTLs were generated in a pearl environment, the defect observed in this

assay is probably due to both, an intrinsic cytotoxicity defect of the CTLs and a defect in the

APC compartment contributing to the generation of the CTLs in a pearl environment.

However, these data confirm, that pearl CTLs have a defect that significantly impairs virus

control in vivo.

3.2.6 An additional heterozygous Rab27a mutation does not influence the outcome of disease in pearl mice.

To examine the influence of a heterozygous Rab27a mutation on CTL cytotoxicity and

disease in HPSII, pearl mice were crossed with ashen mice. Pearl mice additionally carrying

a heterozygous ashen mutation (pearl ashen+/-) were infected with 200 pfu LCMV WE and

HLH disease parameters were analyzed in comparison to pearl and ashen mice.

Figure 22: An additional ashen mutation does not change the outcome of disease in pearl mice.

Pearl, pearl ashen+/- and ashen mice were infected with 200 pfu LCMV WE. On d8 after infection blood counts (A) and serum levels of sCD25 (B) and IFN-γ (C) were analyzed. Gray bars indicate the range of blood count values of naïve C57BL/6 mice (A). (D) CTL cytotoxicity was tested on LCMV-infected target cells (MC57) in a 5h 51

chromium-release assay. (E) Virus titers in spleens and livers were determined on d12 after infection. Pooled data from 2 independent experiments are shown. Dots in dark grey indicated single values of pearl, white dots of pearl ashen+/- and grey dots of ashen mice.

0

5

10

15

HG

B [

g/d

l]

0

10

20

30

WB

C c

ou

nts

[x

10

3/µ

l]

0

200

400

600

800

1000

1200

PL

T c

ou

nts

[x 1

03/µ

l]

0

25

50

75

100

IFN

- γγ γγ [

ng

/ml]

0

500

1000

1500

sC

D25 [

pg

/ml]

1

2

3

4

5

6

7

8

101

102

103

104

105

106

107

108

pfu

per

org

an

d12

spleen liver

A

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

CB

D E

132 – 250

pearl ashenpearl ashen+/-

0

5

10

15

HG

B [

g/d

l]

0

10

20

30

WB

C c

ou

nts

[x

10

3/µ

l]

0

200

400

600

800

1000

1200

PL

T c

ou

nts

[x 1

03/µ

l]

0

25

50

75

100

IFN

- γγ γγ [

ng

/ml]

0

500

1000

1500

sC

D25 [

pg

/ml]

1

2

3

4

5

6

7

8

101

102

103

104

105

106

107

108

pfu

per

org

an

d12

spleen liver

A

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

CB

D E

132 – 250

pearl ashenpearl ashen+/-

Results 67

While ashen mice displayed a significant persistent weight loss and decrease in ear

temperature, pearl ashen+/- mice showed no differences in body weight or temperature when

compared to pearl mice (data not shown). Analysis of blood counts on d8 after infection also

did not reveal any difference between pearl mice with and without the heterozygous ashen

mutation, while ashen mice displayed a more pronounced leukopenia and anemia than the

two other strains (Fig. 22A). As described, serum levels of IFN-γ and sCD25 were highly

elevated in ashen mice, while they were lower and similar in pearl ashen+/- and pearl mice

(Fig. 22B and C). The additional heterozygous ashen mutation did also not influence the CTL

cytotoxicity measured on LCMV-infected target cells whereas ashen mice showed a

dramatically reduced CTL cytotoxicity (Fig. 22D). This was also reflected in the virus load in

livers and spleens twelve days after LCMV infection. While pearl and pearl ashen+/- mice

were able to eliminate the virus until d12, ashen mice still showed high virus titers (Fig. 22E).

These data show that a heterozygous ashen mutation, that in homozygous form leads to a

significant reduction in cytotoxicity and virus control and predisposes to HLH, does not

influence the outcome of disease in pearl mice. Altogether this suggests that the pearl

mutation alone confers a certain risk for HLH which is not significantly increase by a

heterozygous Rab27a mutation. .

Results 68

3.3 Impact of viral and host parameters on the pathogenesis of hemophagocytic syndrome in beige mice - a mouse model of Chèdiak-Higashi Syndrome

Current pathogenetic concepts on HLH postulate that three major factors determine the

threshold for disease induction. First, even in the presence of a genetic predisposition, a

trigger is required to induce the disease, in most cases an infection. Thus, it is likely, that

changes in infection parameters such as the pathogen itself, but also the infection dose or

the replication behavior of the viral isolate can influence the disease threshold. Second, the

key cells mediating disease are CTLs and therefore a difference in CTL responsiveness

should influence the disease threshold. And third, different mutations in genes required for

cytotoxicity cause different degrees of impairment. This may have an impact on virus control

and on the disease threshold. To investigate the relative role of these parameters in the

pathogenesis of HLH, we analyzed two different mouse strains of Chèdiak-Higashi

syndrome. Initial experiments showed that C57BL/6J-Lystbg-J mice, hereafter referred to as

beige mice, show a partial cytotoxicity defect, but remain below the threshold of HLH

induction in the LCMV model. We therefore used this strain and the C57BL/6-Lystbg-Btlr strain,

referred to as souris mice, in order to study the role of different viral and host parameters in

HLH induction.

3.3.1 Beige mice carry a mutation in the WD40 domain of the Lyst protein.

The mutation in the Lyst gene of souris mice was described as a donor splice site mutation in

intron 27 with a T to A transversion. The mutation is predicted to cause skipping of exon 27,

thereby destroying the reading frame. This finally creates a premature stop codon that would

truncate the protein after amino acid 2482 (Fig. 23A and B).The exact mutation of the beige

mouse was not known when this study was initiated and therefore mutation analysis was

performed via primer walking using cDNA from beige and wildtype C57BL/6 mice (also

compared with NCBI Reference Sequence: NM_010748.2) and sequencing. Only the PCR

product using of primers fFR151 and rFR152 (Tab. II) revealed differences in the cDNA

sequence of beige and wildtype mice. Here, a deletion of 3 nucleotides was found leading to

an inframe deletion of one amino acid -isoleucin- at position 3741 in the WD40 domain (Fig

23A and C) [172]. This 3 nucleotide deletion was confirmed by sequencing of the appropriate

region on genomic DNA of beige mice. The WD40 domain is thought to mediate protein-

protein interactions that might be disturbed by this mutation.

Results 69

Figure 23: Mutations of different CHS mouse strains. (A) Scheme of the Lyst protein with its different domains and indicated site of mutations (* souris; x beige). (B) Nucleotide sequence of donor splice site of intron 27 and the T to A transversion in souris mice. (C) Result of mutation analysis of beige mice with a 3 nucleotide deletion resulting in deletion of one amino acid, I3741del.

3.3.2 Beige mice do not develop HLH after low dose LCMV WE infection.

In order to analyze whether the beige mutation confers a risk for HLH, beige mice, C57BL/6

mice (negative control) and PKO mice (positive control) were infected intravenously with a

low dose of LCMV WE (200pfu) and twelve days later HLH disease parameters were

analyzed. When compared to C57BL/6 mice, beige mice showed a decrease in body weight

until day 7 to 8 after infection and recovered thereafter (Fig. 24A). During this time no

changes in the ear temperature could be observed in contrast to perforin-deficient mice that

showed a dramatic decrease in ear temperature starting at about day 8 after infection (data

not shown). All 3 groups of mice developed splenomegaly on day 12 after infection (data not

shown). While beige mice showed leukocytosis on day 12 after infection, PKO mice

developed leukopenia (data not shown). Similar to control mice, beige mice developed a mild

anemia and thrombocytopenia, which was much more severe in PKO mice (Fig. 24B). Liver

enzymes and LDH serum levels on d12 were similar in beige and C57BL/6 mice (Fig. 24C).

In addition, serum levels of ferritin (Fig 24D), sCD25 (Fig. 24E), IFN-γ (Fig. 24F), and

triglycerides (data not shown) were not elevated in beige sera compared to sera of wildtype

mice, while all of these values were significantly elevated in PKO mice. In summary, these

data illustrated that beige mice remain below the threshold and do not fulfill the criteria for

HLH after low dose LCMV WE infection.

ARM/HEAT repeats BEACHPH

3738 - Pro - Ile - Ile - Ser - 3743C57BL/6„wt“

C57BL/6J-Lystbg-J

„beige“

3738 - Pro - Ile - Ser - 3742

3009 - 3098

3118 - 3408

3463 - 3764

W D repeats

beige

*

souris

intron 27

*

A

B C

C57BL/6-Lystbg-Btlr

„souris“

ARM/HEAT repeats BEACHPH

3738 - Pro - Ile - Ile - Ser - 3743C57BL/6„wt“

C57BL/6J-Lystbg-J

„beige“

3738 - Pro - Ile - Ser - 3742

3009 - 3098

3118 - 3408

3463 - 3764

W D repeats

beige

*

souris

intron 27

*

A

B C

C57BL/6-Lystbg-Btlr

„souris“

Results 70

Figure 24: Low dose LCMV WE infection of beige mice does not induce HLH. C57BL/6, beige and PKO mice were infected i.v. with 200 pfu LCMV WE and body weight was monitored for 12 days (A). Blood counts were analyzed on d12 after infection (B: hemoglobin left panel, thrombocyte counts right panel). Gray bars indicate the range of values of naïve C57BL/6 mice (B). Serum levels of GLDH and LDH (C) and ferritin (D), sCD25 (E) and IFN-γ (F) were determined 12 days after infection. Black dots indicated single values of C57BL/6 mice, grey dots of beige and black triangles of PKO mice. Graphs show pooled data of 2 independent experiments with at least 3 mice per group.

3.3.3 An increased T cell frequency does not change the outcome of disease in beige mice.

Because CTLs were shown to play a major role in disease pathogenesis of HLH, the T cell

precursor frequency was increased in beige mice by using adoptive transfer of spleen cells

from 318 beige mice. Different amounts of naïve LCMV gp33-specific P14 beige CTLs were

adoptively transfused into beige mice one day prior to infection with 200 pfu LCMV WE (Fig.

25A) and twelve days later HLH disease parameters were analyzed. Transfer of 105 P14

beige cells resulted in the highest increase in gp33-specific CTLs on d12 after infection. A 3-

4-fold increase in gp33-specific CTLs was achieved as determined by intracellular IFN-γ

production after restimulation with the gp33 peptide (Fig. 25B). Increasing the CTL precursor

frequency did not lead to changes in the weight curve in comparison with non-transfused

beige mice (Fig. 25C). In addition, blood count values like hemoglobin and platelet counts

were not influenced by the increased number of LCMV-reactive CTLs (Fig. 25D). Finally,

serum levels of GLDH, LDH (Fig. 25E) and IFN-γ (Fig. 25F) also did not show any changes

compared to beige mice without T cell transfer.


0 2 4 6 8 10 12

body

wei

ght [

%]

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C57BL/6 PKObeige


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D E F

C57BL/6 PKObeige

Results 71

Thus, increasing frequency of virus-responsive CTLs by a factor of 3-4 did not influence the

disease outcome in beige mice and therefore was not able to cross the threshold for

induction of HLH.

Figure 25: Increase of CTL precursor frequency does not induce HLH in low dose LCMV WE infection. Beige mice were transferred with 104 to 106 P14 CTLs one day prior to i.v. infection with 200 pfu LCMV WE and 12 day days later HLH disease parameters were analyzed (A). (B) Increase in gp33-specific CTLs determined by intracellular IFN-γ staining is shown for transfer of 105 P14 T cells compared with untransfused beige mice. Weight was monitored for 12 days (C). On d12 after infection blood counts were analyzed (D: hemoglobin left panel, platelet counts right panel; gray bars indicate the range of values of naïve C57BL/6 mice) and serum levels of GLDH, LDH (E) and IFN-γ (F) were determined. Grey dots indicate single values of untransferred beige mice and grey triangles indicate single values of beige mice adoptively transfused with 105 naïve beige P14 CTLs. Pooled data from two independent experiments are shown with 3-4 mice per group.

3.3.4 Changing virus dose induces transient disease in beige mice.

Next, we analyzed the impact of changing viral parameters on disease outcome in beige

mice. For this, mice were infected with a 100 fold higher dose of LCMV WE (2x104). Under

these conditions, beige mice showed a more pronounced weight loss (Fig. 26A). In beige

mice 12 days after infection a mild anemia and a more pronounced thrombocytopenia was

observed in contrast to C57BL/6 mice (Fig. 26B). On d12 after infection liver enzymes (like

GLDH) and LDH were slightly elevated in beige mice infected with the high dose of LCMV

0.0

2.5

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HG

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g/d

l]

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nts

[x 1

03/µ

l]

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IFN

- γγ γγ+ o

f C

D8

+ T

cell

s[%

]

D

A B

E 105 P14.beige in beige

beige

318 beige

HLH disease parameter

beigeLCMV

0

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U/l

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10

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U/l

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IFN

- γγ γγ [

ng

/ml]

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timer after infection [d]

0 2 4 6 8 10 12

body

wei

ght

[%]

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110C

GLDH LDH IFN-γγγγ

104 - 106 P14 cells

0.0

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HG

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l]

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T c

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nts

[x 1

03/µ

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IFN

- γγ γγ+ o

f C

D8

+ T

cell

s[%

]

D

A B

E 105 P14.beige in beige

beige

318 beige

HLH disease parameter

beigeLCMV

0

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U/l

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U/l

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IFN

- γγ γγ [

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/ml]

F

timer after infection [d]

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body

wei

ght

[%]

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110C

GLDH LDH IFN-γγγγ

104 - 106 P14 cells

Results 72

WE (Fig. 26C). Considering elevated serum levels of ferritin (Fig. 26D) and histological

evidence of hemophagocytosis (data not shown), beige mice fulfilled some criteria for HLH

after high dose LCMV WE infection.

Figure 26: Infection with 2x104 pfu of LCMV WE induced transient disease in beige mice.

C57BL/6 and beige mice were infected i.v. with 2x104 pfu LCMV WE. Body weight was monitored for 12 days (A). Hemoglobin concentration (B, left panel, gray bars indicate the range of values of naïve C57BL/6 mice) and platelet counts (B, right panel) were analyzed 12 days after infection and serum levels of GLDH and LDH (C), and ferritin (D) were determined. Splenic virus titers were analyzed on d8 and d12 after infection (E). C57BL/6 mice are shown in black dots and beige mice in grey dots. Pooled data from two independent experiments with 3 mice per group are shown.

However, infection with a high dose of LCMV WE did not result in increased IFN-γ serum

levels (data not shown). Moreover the disease was transient and self limiting in beige mice.

Of note, on d8 after infection -the time point of the most pronounced disease in beige mice-

beige mice displayed slightly elevated virus titers in spleens (Fig. 26E). Nevertheless, virus

was eliminated by d12 after infection (Fig. 26E). These data suggest that a change in viral

parameters can have an impact on HLH induction, but disease remains limited if virus control

is maintained.

101

102

103

104

105

106

107

pfu

per

sp

leen

GLDH LDH

B

C D

C57BL/6

beige

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

HG

B [

g/d

l]

0

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PL

T c

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nts

[x 1

03/µ

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ght [

%]

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110A

0

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/ml

ferritin Ed8

0

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U/l

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U/l

d12

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106

107

pfu

per

sp

leen

101

102

103

104

105

106

107

pfu

per

sp

leen

GLDH LDH

B

C D

C57BL/6

beige

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

HG

B [

g/d

l]

0

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PL

T c

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nts

[x 1

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Results 73

3.3.5 The souris mutation confers a risk for developing severe HLH following low dose LCMV WE infection.

We then varied the genetic defect and compared the effects of LCMV WE low dose infection

in beige and souris mice. Intravenous infection of souris mice with 200 pfu LCMV WE

induced significant weight loss starting at d7 after infection and the mice did not recover until

d12 (Fig. 27A). In addition, souris mice -in contrast to wildtype and beige mice- displayed

many features of HLH on d12 after infection, such as a severe anemia and thrombocytopenia

(Fig. 27B), elevated GLDH and LDH serum levels (Fig. 27C), highly elevated serum levels of

ferritin (Fig. 27D), sCD25 (Fig. 27E) and IFN-γ (Fig. 27F) and slightly increased serum levels

of triglycerides (data not shown). Thus, souris mice developed severe HLH following LCMV

WE infection, even though the phenotype was not quite as pronounced as in PKO mice.

Figure 27: Souris mice develop HLH following low dose LCMV WE infection. C57BL/6, souris and beige mice were infected i.v. with 200 pfu LCMV WE and body weight was monitored for 12 days (A). Blood counts were analyzed 12 days after infection (B: hemoglobin left panel, platelet counts right panel). Gray bars indicate the range of values of naïve C57BL/6 mice. Serum levels of GLDH and LDH (C) and ferritin (D), sCD25 (E) and IFN-γ (F) were determined at the end of the experiment (d12). Black dots indicated single values of C57BL/6 mice, grey dots of beige and white dots of souris mice. Graphs show pooled data of 2 independent experiments with 3 mice/ group.

0.0

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HG

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l]

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LT

co

un

ts [

x 1

03/µ

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/ml

B

FD EC ferritin

0

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pg

/ml

sCD25

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/ml

IFN-γγγγ

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U/l

GLDH LDH


0 2 4 6 8 10 12

body

wei

ght [

%]

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souris

beige

C57BL/6

0.0

2.5

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HG

B [

g/d

l]

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LT

co

un

ts [

x 1

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l]

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ng

/ml

B

FD EC ferritin

0

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pg

/ml

sCD25

0

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pg

/ml

sCD25

0

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ng

/ml

IFN-γγγγ

0

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ng

/ml

IFN-γγγγ

1

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U/l

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U/l

GLDH LDH


0 2 4 6 8 10 12

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ght [

%]

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souris

beige

C57BL/6

Results 74

3.3.6 HLH in souris mice is associated with virus persistence.

One characteristic feature of HLH in LCMV-infected PKO mice is lack of virus control. We

therefore determined viral titers on different time points after infection (Fig. 28). While

comparable virus titers were detected in spleens at d4 after infection (data not shown),

analysis of d8 virus titer revealed significant differences. Beige mice showed similar virus

elimination kinetics as C57BL/6 mice with reduced virus titers at d8 after infection and

cleared the infection until d12. In contrast, virus load in spleens of souris mice more

resembled the titers of PKO mice, which were still high at d8 and d12 after infection. Souris

mice were not only unable to eliminate the virus but also could not prevent spreading of the

infection to peripheral organs such as liver, lung and kidney (data not shown) as we also

observed in PKO mice.

Figure 28: LCMV WE titers in spleens of beige and souris mice. C57BL/6, souris, beige and PKO mice were infected i.v. with 200 pfu LCMV WE and virus titer in spleen were determined on d8 and d12 after infection. Black dots indicated single values of C57BL/6 mice, white dots of souris, grey dots of beige and black triangles of PKO mice. Graphs show pooled data of 2 independent experiments with at least 3 mice per group. Dashed line indicates the detection limit.

3.3.7 CTL activity rather than NK cell activity determines the risk for HLH.

To determine the reason for the different disease outcomes in beige and souris mice, we

analyzed lymphocyte cytotoxicity in these two mouse strains. Because reduced or absent NK

cell cytotoxicity is one of the diagnostic criteria for HLH, we first compared NK cell activity of

beige and souris mice. NK cells were activated in two different ways: via poly(I:C) injection

(Fig. 29A) and via LCMV WE infection (Fig. 29B-D). With both experimental approaches,

souris and beige mice showed a significant decrease in NK cell cytotoxicity on YAC-1 target

cells (Fig 29 A and B). Moreover, after in vitro restimulation with YAC-1 cells, degranulation

of NK cells measured by CD107a expression was severely impaired in beige mice 24h after

poly(I:C) treatment (data not shown) and three days after LCMV infection (Fig. 29C and D).

Because no significant differences in NK cell cytotoxicity and degranulation were found

between souris and beige mice that could account for the difference in the pathology, we

analyzed CTL cytotoxicity. Splenic CTLs obtained 8 days after LCMV infection were tested

d8 d12

souris PKObeigeC57BL/6

101

102

103

104

105

106

107

108

pfu

pe

r s

ple

en

d8 d12

souris PKObeigeC57BL/6

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106

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pfu

pe

r s

ple

en

Results 75

on 51Cr-labelled MC57G target cells that had been infected with LCMV WE two days

previously. While no differences were observed between beige and C57BL/6 mice, souris

CTLs showed a significantly impaired cytotoxicity (Fig. 30A). This defect was more

pronounced when CTL activity was analyzed on target cells loaded with a limiting

concentration of gp33 peptide (10-10M) (Fig. 30B). In that situation, a partial defect was also

observed in beige CTLs. In addition, CTL degranulation was impaired in CTLs from both

strains, but this was more pronounced in souris than in beige mice. (Fig. 30C and D)

Figure 29: Defective NK cell cytotoxicity and degranulation of beige and souris mice. NK cell cytotoxicity was determined in a 5h chromium-release assay on YAC-1 target cells. C57BL/6, souris and beige mice were injected with 200µg of poly(I:C) i.p. and 24h later cytotoxicity of splenic NK cells was analyzed (A). Activity of splenic NK cells was examined 3 days after i.v. infection with 200pfu LCMV WE (B-D). NK to target ratio was determined by calculating percentage of NK cells among total spleen cells by FACS analysis. NK cell degranulation was measured after 2h restimulation with YAC-1 cells. (C) Representative FACS plots are shown for degranulation of NK cells after restimulation with medium or YAC-1 cells. (D) Degranulation is shown as ∆CD107

+ in percent representing the

difference between values of restimulation with YAC-1 cells compared to medium control. Black dots indicated single values of C57BL/6 mice, grey dots of beige and white dots of souris mice. Data shown are representative for 2 independent experiments.

To determine whether the observed differences in CTL degranulation and cytotoxicity were

relevant for the differences in virus control in vivo, we performed adoptive transfer protection

experiments. CD8 T cells from the spleens of beige and souris mice obtained 8 days after

LCMV infection were MACS purified and 2x106 cells were adoptively transfused into C57BL/6

recipients that had been infected with LCMV WE 10 hours before. Eighteen hours later, viral

titers in the spleen were analyzed. Fig. 30E shows that while transfer of beige CTLs had a

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100LCMVPoly (I:C)A B

C D

0

10

20

30

40

50

∆∆ ∆∆C

D107a

+ [

%]

CD107a

NK

1.1

C57BL/6 souris

med

ium

YA

C-1

beige

souris

beige

C57BL/6NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

10

20

30

40

50

NK:T ratio

0,010,1110

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100LCMVPoly (I:C)A B

C D

0

10

20

30

40

50

∆∆ ∆∆C

D107a

+ [

%]

CD107a

NK

1.1

C57BL/6 souris

med

ium

YA

C-1

beige

CD107a

NK

1.1

C57BL/6 souris

med

ium

YA

C-1

beige

souris

beige

C57BL/6

souris

beige

C57BL/6

Results 76

significant antiviral effect, this was not obtained with souris CTLs. These findings suggest

that the more pronounced CTL cytotoxicity defect of souris mice leads to a loss of virus

control and subsequently to the clinical features of HLH.

Figure 30: Defective CTL cytotoxicity and degranulation in souris and beige mice. CTLs of C57BL/6, souris and beige mice were analyzed 8 days after infection with 200 pfu LCMV WE. Ex vivo cytotoxicity was tested in a 5h chromium-release assay on either (A) target cells (MC57) infected with LCMV WE 2 days before or (B) target cells (EL-4) loaded with a gp33 peptide concentration of 10-10 M. Degranulation -measured by CD107a surface expression of IFN-γ producing CTLs- was examined following 4h of in vitro restimulation with gp33 peptide. (C) Representative FACS plots are shown and (D) percentage of degranulating cells among gp33-specific CTLs is shown. (E) Splenic CD8 T cells isolated from spleens of C57BL/6, beige or souris mice on d8 after infection with 200 pfu LCMV were MACS purified and adoptively transfused into C57BL/6 mice infected with 104 pfu LCMV WE 10h before. Viral titers in spleens were determined after additional 18h. Black dots indicated single values of C57BL/6 mice, white dots of souris and grey dots of beige mice. Data from one representative experiment are shown in A, B and E and pooled data from 2 independent experiments are shown in D. ***p < 0.001

0

25

50

75

100

CD

10

7a

+ o

f IF

N- γγ γγ

+C

D8

+

ce

lls

[%

]

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

D

CD107a

IFN

-γ

C57BL/6 souris

med

ium

gp33

Cbeige

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

BA

***

E

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

sourisbeige

donor

nil

souris

beige

C57BL/6

0

25

50

75

100

CD

10

7a

+ o

f IF

N- γγ γγ

+C

D8

+

ce

lls

[%

]

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%

]0

20

40

60

80

100

D

CD107a

IFN

-γ

C57BL/6 souris

med

ium

gp33

Cbeige

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

BA

***

E

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

sourisbeige

donor

nil

0

25

50

75

100

CD

10

7a

+ o

f IF

N- γγ γγ

+C

D8

+

ce

lls

[%

]

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%

]0

20

40

60

80

100

D

CD107a

IFN

-γ

C57BL/6 souris

med

ium

gp33

Cbeige

CTL:T ratio

0,1110100

targ

et c

ell l

ysis

[%]

0

20

40

60

80

100

BA

***

E

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

sourisbeige

donor

nil

101

102

103

104

105

106

107

pfu

per

sp

leen

C57BL/6

recipient

sourisbeige

donor

nil

sourisbeige

donor

nil

souris

beige

C57BL/6

souris

beige

C57BL/6

Discussion 77

4 Discussion

This study used two different experimental models to show 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.

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

Our experiments in the mouse model of RSV infection showed that the MHC-dependent

determination of the antiviral CTL repertoire can contribute to disease severity after a

respiratory viral infection. Starting from the observation that RSV-induced weight loss and the

pulmonary inflammatory response is more pronounced in BALB/c (H-2d) versus C57BL/6 (H-

2b) mice, the current study analyzed the contribution of the MHC and therefore the CTL

response to the differences in disease susceptibility.

The critical contribution of the MHC to disease susceptibility (mainly assessed by weight

loss) was determined in MHC congenic mice. The disease observed in BALB/c mice was in

part transferred with the H-2d allele on the C57BL/6 background. In contrast, C57BL/6 mice

carrying both MHC haplotypes -H-2d and H-2b- were found to be less susceptible to disease,

indicating that the H-2b haplotype confers resistance to RSV-induced disease. Disease

observed in BALB/c mice was accompanied by increased levels of IFN-γ, IL-6 and MCP-1

levels in the BAL fluids. While IFN-γ was shown before to play a critical role in mediating

immunopathology in the mouse model [173], serum level of IL-6 was described as a marker

for severity in patients with lower respiratory tract infections caused by RSV [174, 175]. The

absence of IL-10 was recently correlated with an increased protection against influenza

infection [176]. IL-10 levels in the BAL fluids of RSV infected mice were low and comparable

in all groups (data not shown), rendering a significant contribution of IL-10 to disease unlikely

in this model.

Because RSV replicates to higher titers in BALB/c mice compared with C57BL/6 mice [20,

41, 42], we analyzed peak virus titers and virus elimination kinetics as causes for RSV-

induced pathology. While BALB/c mice exhibited higher virus titers than C57BL/6 mice at the

peak of replication at day 4 after infection, the pulmonary virus load of C57BL/6-H-2d mice

was comparable to C57BL/6 mice, suggesting that peak virus titers were not the only

determinant of disease. Furthermore all groups showed similar virus elimination kinetics.

T cells -especially CD8 T cells- were shown to be the main mediators of immunopathology in

BALB/c mice [18, 177]. However, differences in MHC will not only lead to differences in the

CTL response, but also in the T helper cell response. We therefore also addressed the

Discussion 78

contribution of CD4 T cells to disease development. Overall CD4+ T cell numbers recruited to

the lung were similar in all strains. Recently, regulatory T cells were shown to promote an

early pulmonary CD8 T cell influx and therefore to limit immunopathology following RSV

infection [178, 179]. In our experiments, we could not detect a difference in Treg numbers in

the lungs of BALB/c and C57BL/6 mice (data not shown). In influenza infection a critical role

for IL-17 and IL-17RA in disease development was observed [180] and CD8+ IL-17 producing

T cells -Tc17 cells- were described to protect against a lethal influenza infection [181]. In our

RSV model, we found comparable amounts of pulmonary IL-17-producing CD4 and CD8 T

cells (data not shown) in BALB/c and C57BL/6 mice, rendering a key role for IL-17 in RSV-

induced pathology unlikely.

We then analyzed the RSV-specific CTL response in H-2d versus H-2b mice on the same

C57BL/6 background in order to address the question, whether differences in the CTL

response could account for the differences in disease susceptibility. No significant

differences in numbers of total CD8 T cells (data not shown) and in the RSV-specific CTL

responses were observed. However, analysis of the immunodominant CTL populations

revealed several differences in H-2d versus H-2b mice. Among RSV-specific CTLs in BALB/c

mice, there was a particular dominance of M2-1 82-specific cells, showing a Vβ usage that

was strongly skewed to Vβ8.2. Analysis of C57BL/6 mice also revealed an oligoclonal CTL

response to RSV, but it was less pronounced. The dominant M187-specific population

represented a smaller part of the total RSV-specific response and the Vβ usage of these

cells was more variable. In particular, while about 80% of the total RSV-specific CTLs were

M2-1 82-specific, Vβ8.2+ T cells in BALB/c mice, about 30% of the total RSV-specific CTLs

were Vβ9+ T cells specific for M187. The immunodominant CTL population carrying the

Vβ8.2 chain was responsible for the RSV-induced disease in H-2d mice as shown in two

different experimental setups. First, infection with a mutant virus carrying a loss-of-

recognition mutation in the immunodominant epitope failed to induce pathology in C57BL/6-

H-2d mice as it was shown before for BALB/c mice [157]. Second, deletion of the Vβ8.2+ CTL

population also resulted in a markedly reduced pathology in BALB/c mice.

These observations suggest that the dominant M2-1 82-specific Vβ8.2+ CTL population

makes a major contribution to disease in H-2d mice. It is not completely clear, how this T cell

population contributes to disease and what makes it different from the M187-specific CTL

response. Interestingly, the ability to lyse target cells at lower, more physiological peptide

concentrations was reduced in M2-1 82-specific CTLs compared to M187-specific CTLs.

These data indicate a lower avidity of the TCR specific for the M2-1 82-H-2d complex

compared to the TCR specific for the M187-H-2b complex, leading to the following

hypothesis:

Discussion 79

The less efficient target cell lysis may result in a prolonged stimulation of CTLs via APCs as it

was shown for LCMV-infected PKO mice [49]. As it will be discussed in the following

chapters, this reduced efficiency in target cell lysis does not necessarily impair virus

clearance. This is also illustrated by the fact, that BALB/c mice can eliminate RSV infection

despite the lack of the immunodominant CTL population (comprising about 80% of the total

RSV-specific CTL response), although virus clearance was slightly delayed [157]. Inefficient

target cell lysis can contribute to disease e.g. a prolonged stimulation of virus-specific CTLs

by their APCs with subsequent inflammatory cytokine production, in particular IFN-γ, and

disease [49, 173]. This concept is also well illustrated by a disease called hemophagocytic

lymphohistiocytosis (HLH) that will be further discussed in the next chapter.

In how far this study has implications for human RSV infection, remains to be determined as

infection of mice with high doses of a non species-conform pathogen may not mirror the

human disease. Murine RSV infection may thus rather represent a model situation, in which

easily accessible parameters –such as weight loss and the increase in pulmonary cytokines–

reflecting some pathological consequences of a respiratory viral infection can be monitored

under controlled conditions rather than an optimal model for a human disease.

4.2 HLH in mouse models for Hermansky-Pudlak syndrome type II and the Chèdiak-Higashi syndrome

In the second part of the study, we analyzed two mouse models of partially impaired

cytotoxicity with two different genetic diseases predisposing to HLH: the Hermansky-Pudlak

syndrome type II and the Chèdiak-Higashi syndrome. Mice were tested for developing

hemophagocytic lymphohistiocytosis following LCMV infection according to the human

diagnostic criteria for HLH [80, 81, 144]: fever, splenomegaly, cytopenia, histological proof of

hemophagocytosis, elevated serum levels of ferritin, triglycerides, and sCD25, and reduced

or absent NK cell cytotoxicity. As proposed for the diagnosis of HLH in humans, mice were

considered to have HLH if they fulfilled 5 out of the 8 criteria. Furthermore, the cytotoxic

activity of NK cells and cytotoxic T cells was characterized and the ability of mice to control

and eliminate the LCMV infection was determined. LCMV WE was used as an infectious

trigger, because this virus is a potent inducer of disease in different mouse models for HLH:

perforin-deficient mice (a mouse model for FHL-2) [49], ashen mice (a mouse model for

Griscelli syndrome 2) [84] and jinx mice (a mouse model for FHL-3) [88].

We used pearl mice, carrying mutations in the gene encoding Ap3b1, as a model for HPSII,

and beige and souris mice, carrying different mutations in the gene encoding Lyst, as two

different models for CHS.

Discussion 80

4.2.1 Hermansky-Pudlak syndrome type II confers a risk for HLH

The mouse model of HPSII -the pearl mice- were found to develop HLH following LCMV

infection defined by: weight loss, decrease in temperature measured in the ear, histological

proof of hemophagocytosis in liver and spleen, splenomegaly, and elevated ferritin and

sCD25 serum levels. The disease was transient and mice recovered eventually. At the time

point of the most obvious disease, additional features underlining the diagnosis of HLH were

found. IFN-γ serum levels, resembling the hallmark cytokine of HLH in humans [151, 153,

154, 182, 183] and mice [49, 84, 88, 184], were highly elevated after infection. In addition,

liver damage was detectable by increased serum levels of GPT, GLDH and LDH.

These data indicate that HPSII confers a risk for HLH. However, disease was transient in this

mouse model. One of the 11 patients so far described with HPSII (Tab. XI) also developed a

transient disease with two episodes of hemophagocytic syndrome following common viral

infections [143]. Only one HPSII patient so far developed a fulminant lethal HLH. He carried

an additional heterozygous mutation in RAB27A [142]. Homozygous mutations in RAB27A

cause Griscelli Syndrome type II (GSII) with a cytotoxicity defect predisposing to HLH [126,

127]. However, the functional influence of this additional mutation was not evaluated.

In this study, we analyzed the influence of a heterozygous ashen (GSII) mutation in pearl

mice (HPSII). Pearl ashen+/- mice developed a transient disease in which all parameters

tested resembled those of pearl mice. This indicated that an additional GSII mutation that

causes a severe cytotoxicity defect in a homozygous state does not enhance the cytotoxicity

defect or influence susceptibility to disease in pearl mice.

The pearl mice used in this study carry a mutation in the Ap3b1 gene, encoding the β3A

subunit of the AP-3 complex. Pearl mice were shown to have a tandem duplication of 793 bp

starting at nucleotide 2135 in the Ap3b1 gene that alters the reading frame. In consequence,

a stop codon develops at the duplication junction and therefore the protein is predicted to be

truncated 130 amino acids from the C-terminus [185, 186]. Though there are conflicting data

about residual expression of the β3A subunit in pearl mice [185-189], it is very likely that

there is residual expression of the truncated β3A protein missing the C-terminal end

containing the so called “ear” domain [190]. This domain was described to interact with

proteins such as scaffolding proteins, proteins of the cytoskeleton or proteins involved in the

vesicle fusion machinery [190, 191]. Peden et al. described a possible formation of the AP-3

complex lacking the ear domain of the β3A subunit. This complex is probably degraded

faster but may account for residual function of the AP-3 complex [187]. Thus, the AP-3

mutation in the pearl mice used in this study is probably rather a hypomorphic mutation than

a functional null mutation.

Discussion 81

Table XI: Overview of patients with HPSII described by now.

So far, due to the limited number of patients reported (Tab. XI) [140-143, 192-194], a clear

genotype-phenotype correlation with respect to the risk for developing HLH could not be

established in HPSII patients. Nevertheless, in both patients that developed HLH, the β3A

protein is predicted to be truncated very early [142, 143]. Analysis of mutations in more

HPSII patients developing HLH will be needed before better conclusions can be drawn.

Overall the results in pearl mice in combination with the reported observations in patients

suggest that HPSII on its own confers a certain risk for HLH. The combination of the

particular HPSII and GSII mutations analyzed in this study did not further increase the risk for

developing HLH. Therefore we believe that the disease in the patient, who developed lethal

HLH, was not due to the additional GSII mutation, but due to his HPSII disease. This implies

an increased risk for HLH for HPSII patients in general.

4.2.2 AP-3 deficiency - more than a defect in cytotoxicity

NK cell cytotoxicity was analyzed in three [142, 193] and CTL cytotoxicity in four [140, 142,

143] of the described HPSII patients (Tab. XI). While cytotoxicity of freshly isolated NK cells

was almost absent, it was partially restorable after in vitro stimulation with IL-2. Clark et al.

described a severely reduced CTL activity of CTL clones of one patient [140], while the

activity of CTL clones described by Wenham et al. showed a reduced cytotoxicity but with

values highly varying between different experiments [143]. PHA blasts analyzed by Enders et

al. [142] also displayed a defect in cytotoxicity that was less pronounced compared to the

patients data from Clark et al. [140]. Unfortunately, no clinical details have been reported

from the latter patient. From these human data, no clear correlation between cytotoxicity

defect and risk for HLH can be found. It would be important to analyze CTL and NK cell

cytotoxicity, genotype and clinical phenotype in more patients.

(+)-?+-?--Hemophago-

cytosis

+?+?+??CTL defect

??++???NK defect

++++?++Neutropenia

+-+/-++?++Recurrent infections

c.del153-156p.E52fsX11

g.del180242-180866

p.E693fsX13

Del491-550Del491-550

R302XR302X

Q355fsX360I597fsX608

IVS1416T>CA541fsX24

R509X,E659X

Del390-410L580R

AP3B1mutation

232115274?52520Age of

diagnosis (years)

Reference

Wenhamet al.

Junget al.

Enderset al.

Fontanaet al.

Clarket al.

Huizinget al.

Shotelersuket al.

(+)-?+-?--Hemophago-

cytosis

+?+?+??CTL defect

??++???NK defect

++++?++Neutropenia

+-+/-++?++Recurrent infections

c.del153-156p.E52fsX11

g.del180242-180866

p.E693fsX13

Del491-550Del491-550

R302XR302X

Q355fsX360I597fsX608

IVS1416T>CA541fsX24

R509X,E659X

Del390-410L580R

AP3B1mutation

232115274?52520Age of

diagnosis (years)

Reference

Wenhamet al.

Junget al.

Enderset al.

Fontanaet al.

Clarket al.

Huizinget al.

Shotelersuket al.

[192] [194] [140] [193] [142] [141] [143]

Discussion 82

Even though mice with Ap3b1 null alleles exist, neither cytotoxicity nor HLH development in

those mice were analyzed by now [195]. As described later in more detail, our experiments

with pearl mice with a presumably hypomorphic mutation revealed both a reduction in NK cell

and CTL cytotoxicity. This is in contrast to previous reports describing normal NK cell

cytotoxicity in pearl mice but here tests were performed without previous activation of NK

cells [196]. Importantly, the influence on the immune system of the defective AP-3 complex is

not restricted to a disturbed cytotoxicity. Pearl mice were shown to develop lung pathology

with pulmonary inflammatory dysregulation and constitutive alveolar macrophage activation

[197-199]. Furthermore, trafficking and antigen presentation by CD1 was shown to be

defective and therefore NKT cell development was disturbed [200-202]. Reduced numbers of

NKT cells were also observed in one patient with HPSII [141]. Although it was shown that

MHC class II traffics normally in AP-3 deficient cells [202], our results suggest, that AP-3

deficiency also leads to an APC problem. Thus, we observed a proliferation defect in pearl

CTLs that was obvious in combination with wildtype APCs but most pronounced in

combination with pearl APCs.

In conclusion, AP-3 deficiency leads to a complex phenotype that is not limited to cytotoxicity

defects. Further studies are needed to understand the contribution of the multiple defects to

the phenotype of pearl mice.

4.2.3 Geno-phenotype correlation in the mouse models for CHS

Analysis of beige and souris mice as models for Chèdiak-Higashi syndrome revealed an

apparent geno-phenotype correlation. The nature of the mutation influenced the CTL and NK

cell cytotoxicity and therefore determined the risk for HLH in CHS.

Souris mice developed severe disease following low dose LCMV infection fulfilling all

diagnostic criteria for HLH. In contrast, low dose LCMV infection of beige mice did not induce

disease. Both mice carry a mutation in the Lyst gene, the homologue of the human

CHS1/LYST gene and both mice show the typical inclusion bodies on blood cell morphology

and dilution of coat color that is slightly more pronounced in souris mice. While the mutation

of souris mice was described to truncate the protein after amino acid 2482 and therefore the

PH, BEACH and WD domains are missing, mutation analysis of the beige mice revealed an

inframe deletion of one amino acid at position 3741 in the WD40 domain. These genetic

differences had important consequences for disease susceptibility.

Discussion 83

Figure 31: Parameters possibly influencing the development of HLH.

Beige mice that did not develop HLH after low dose LCMV infection showed a transient

disease with some features of HLH after increasing the infection dose and therefore the virus

load during the course of infection. Nevertheless, mice were still able to clear the infection

and recovered eventually. In contrast, an increase in the precursor frequency of CD8 T cells -

that were shown to be the major mediators of disease [49]- did not change the outcome of

disease in beige mice. Thus, the extent of T cell activation does not appear to be a crucial

variable for disease development (Fig. 31).

LCMV is the major pathogen inducing disease in all HLH mouse models ([49, 84, 88] and our

data from pearl, beige and souris mice). Most of the mouse models such as jinx [88], beige

[203-205], PKO [206], souris, pearl and ashen (data from the Beutler Lab [207]) were also

shown to have a higher susceptibility to MCMV infection compared to wildtype mice. While in

perforin-deficient mice MCMV infection is described to induce a HLH-like syndrome [206],

MCMV was not able to induce HLH in the mouse model for FHL-3 (jinx) [88] in contrast to

LCMV infection. For beige mice (bgJ/bgJ) it was shown that mice were susceptible to fatal

disease after MCMV infection depending on the genetic background. It was not further

analyzed whether this disease resembles HLH, but some parameters described like normal

blood counts are not consistent with the diagnosis of HLH [203-205]. Furthermore, the

influence of the beige mutation on the susceptibility to several pathogens other than LCMV

and MCMV has been analyzed. This included pyogenic infections such as Escherichia coli,

Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae [208]

infection with fungi such as Candida albicans, and infection with parasites like Leishmania

donovani [209]. Beige mice showed an increased susceptibility and mortality. However, HLH

development was not analyzed.

In summary, only a few pathogens are able to induce HLH in the mouse model which is in

line with data from human patients. While most patients have many episodes of infections,

the pathogenetic sequence of HLH is only rarely triggered. Even though in most cases of

patients developing HLH no causing infectious agent was found, it is very likely that

pathogens are involved in triggering the immune system to overcome the critical threshold for

HLH

Pathogen parameters

- infectiouse agent

- infection dose

- ?

Host parameters

- genetic defect

- T cell frequency

- ?

Discussion 84

developing HLH. It is not clear whether, once established HLH requires the presence of an

infectious agent. In support of this concept, our data obtained with LCMV in a variety of

model situations showed that disease was in all cases associated with failure to eliminate the

pathogen.

In conclusion, pathogen parameters as well as host parameters have an influence on

disease development. The data from this study using the strongest HLH trigger in mice -

LCMV- indicate that in CHS the nature of the mutation plays the critical role in determining

the risk for HLH. Both mouse models for CHS analyzed in this study conferred a risk for

developing HLH, but with different thresholds. While a low dose LCMV infection is sufficient

to overcome the threshold for souris mice and PKO mice, beige mice only showed transient

features of HLH even after increasing the infection dose.

Geno-phenotype studies of patients with CHS revealed a correlation between the nature of

the mutation and the severity of the clinical picture [118]. While most of the patients with CHS

(~85%) show a severe and often fatal phenotype in early childhood, there are some cases

(~15%) that do not develop HLH and survive to adulthood but with progressive and often

fatal neurological dysfunction. A minority of CHS patients show an intermediate phenotype

with recurrent infections in childhood without developing HLH. Mutation analysis revealed

that functionally null -especially nonsense- mutations are predominantly found in the severe

childhood forms. Missense mutations, probably allowing residual function of the LYST protein

were found to be the cause of the milder adulthood form of CHS [118, 124, 210, 211].

Beige mice -analyzed in this study- displayed a high threshold for developing disease and

were also observed to develop neurological problems over time [212] and could therefore

resemble a model for the adult form of HLH. Another mouse model for CHS carrying a

missense mutation in the same protein domain as beige mice, the WD40 domain, displayed

a predominant neurodegenerative phenotype and lacked severe impairment of the immune

system [213]. In contrast, souris mice with a mutation resulting in an early truncation of the

Lyst protein could resemble a mouse model for the severe childhood form of CHS early

developing severe HLH.

4.2.4 HLH in mice is associated with antigen persistence

As described above, souris mice did not develop HLH spontaneously but after infection with

LCMV as was previously shown for perforin-deficient and ashen mice [49, 84]. In this study,

we observed that HLH in mice was invariably associated with impaired virus elimination.

Souris mice -like PKO and ashen mice- were not able to eliminate the virus at all. In beige

mice, a transient disease was only induced by an increased infection dose and therefore an

increased virus load. Pearl mice showed virus persistence at the time point of most severe

Discussion 85

disease and disease symptoms waned when virus was eventually eliminated. Moreover mice

with features of disease also showed a spread of virus to peripheral organs that was never

observed in wildtype mice. These data lead to the conclusion that in the mouse model, HLH

pathology is directly linked to virus, i.e. antigen persistence. Presumably, the continued

stimulation of CTLs is the basis for maintenance of the disease.

4.2.5 CTL rather than NK cell cytotoxicity determines the risk for HLH

Patients with FHL either show mutations in the gene encoding for perforin or in genes

involved in the exocytosis pathway of lytic granules containing cytotoxic proteins like perforin

and granzymes. As a consequence, cytotoxicity of NK cells is affected. In fact, defective NK

cell cytotoxicity is a diagnostic criterion of HLH.

We found that NK cell cytotoxicity and degranulation are significantly reduced in HPSII mice

compared to wildtype mice. Both CHS mouse strains showed a comparable, almost absent

NK cell activity. However, while souris mice developed HLH, beige mice did not. Hence, the

NK cell function does not seem to be the major factor determining the development of HLH in

mice.

Cytotoxic CD8 T cells are crucial for the elimination of LCMV infection [17, 214] but they

were also shown to be the main mediators of HLH in perforin-deficient mice [49]. The virus

control in mice in this study ranged from unaffected virus elimination in beige mice and a

delayed virus control in pearl mice to an absent virus control in souris and ashen mice. We

found that these differences in virus control correlated well with the activity of CTLs

determined by degranulation and cytotoxicity. Pearl CTLs showed a reduced cytotoxicity on

LCMV-infected target cells and on target cells loaded with limiting peptide concentrations that

was also associated with a significant decrease in CTL degranulation. In CHS mice, the

difference in CTL activity probably explains the differences between those two mouse strains

in virus control and development of HLH. Souris CTLs showed a significantly reduced

cytotoxicity on LCMV-infected target cells while beige CTLs showed no defect in this

experimental setup. Using limiting peptide concentrations, lysis of target cells was obviously

defective in beige CTLs and almost absent in souris CTLs. Furthermore, ashen mice -

susceptible to HLH- also displayed an almost absent CTL cytotoxicity in both experimental

settings.

The physiological relevance of these defects was reflected in the LCMV elimination. The

direct link between CTL defects and the failure to control the infection was evidenced in our T

cell adoptive transfer model. Virus control in that model was previously shown to be perforin

dependent [171]. It was impressive to see that small differences in cytotoxicity in vitro (in

particular under saturating peptide conditions) led to such large differences in virus control in

Discussion 86

vivo. These results clearly demonstrate the following: First, the degree of the CTL cytotoxicity

defect determines virus control. Small in vitro differences lead to large effects in vivo.

Second, the more pronounced the defect in CTL cytotoxicity, the higher is the risk for

developing severe HLH. These findings emphasize that the methods how to assess

cytotoxicity in vitro are crucial. They must be sensitive enough to detect subtle differences. In

our experimental setup, none of the cytotoxicity defects were visible when target cells were

loaded with normally used saturating peptide concentrations (data not shown). These

methodological considerations can also explain some of the discrepancies in the literature.

While beige NK cell activity was described to be defective in all setting such as poly(I:C)

activation [215, 216], infection with VSV [215], LCMV WE and Armstrong [217-222], MHV

[217] and MCMV [218], data for CTL activity were not as consistent. Beige CTLs were

reported to mediate normal cytotoxicity following VSV infection [215] or in vivo

alloimmunisation [216, 219, 220], while other groups reported an impaired CTL cytotoxicity

following LCMV infection [23, 223] or in vivo alloimmunization [221, 222]. In contrast,

cytotoxicity of CTLs from CHS patients were consistently described to be reduced [117, 211,

224-226]. This discrepancy is probably in part due to different sensitivity of the cytotoxicity

assays.

Overall, the results of this study show that rather CTL than NK cell cytotoxicity determines

the risk for HLH and therefore should be studied when performing diagnostic evaluations for

HLH. Further investigations are required to evaluate the optimal in vitro assay conditions, in

particular for humans. Data obtained in larger cohorts of patients with severe and mild forms

of HLH may help to validate the predictive value of these assays concerning the risk for HLH

development.

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Danksagung 97

Acknowledgement/Danksagung Mein besonderer Dank gilt Prof. Dr. Stephan Ehl für die Bereitstellung dieses Themas und

die tolle Betreuung während meiner Doktorarbeit auch in stressigen Zeiten.

Außerdem möchte ich mich bei Prof. Dr. Hanspeter Pircher bedanken, der nicht nur meine

Arbeit betreut hat, sondern unserer kleinen Maus-Arbeitsgruppe auch ein neues Zuhause

gegeben hat.

Ich möchte mich bei ihm, allen in seiner Arbeitsgruppe und Dr. Peter Aichlele mit Gruppe

danken für die Diskussionen und Anregungen in Seminaren und die Zusammenarbeit und

Hilfsbereitschaft im 4. Stock.

Prof. Dr. Anette Schmitt-Gräff möchte ich für die Hilfe bei der Histologie danken.

Mein Dank gilt auch allen aus der AG Ehl, insbesondere Jan Rohr, Carsten Speckmann,

Andrea Maul-Pavicic, Nadja Goos und auch den ehemaligen Kollegen Stefanie Frey und

Simone Faller.

Den Mitarbeitern des Mausstalls möchte ich für die gute Pflege unserer Versuchstiere und

gute Laune im Mausstall danken.

Meiner Familie und meinen Freunden danke ich für die tolle Unterstützung, ohne die diese

Arbeit niemals möglich gewesen wäre, insbesondere Maike, die immer bereit war mit mir

über meine Arbeit zu diskutieren.

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Composition and efficacy of cytotoxic T cell responses