Download Neuropathy in a mouse model of CD8+ T cell

Neuropathy in a mouse model of CD8+ T cell-mediated CNS demyelination
Rainone, Anthony
Department of Microbiology and Immunology
McGill University, Montreal
July 2013
A thesis submitted to McGill University in partial fulfillment of the
requirements of the degree of Master of Science
© Anthony Rainone, 2013
ABSTRACT
Several lines of evidence suggest that CD8+ T cells could contribute to the
pathogenesis of many autoimmune diseases of the nervous system. Our group
studies a mouse model (L31 mice) that spontaneously develops neurological
symptoms associated with demyelinated lesions in the CNS. We have shown
that the demyelinating disease in L31 mice is T cell-dependent with CD8+ T
cells acting as effector T cells in the pathological process. Because L31 mice
exhibit motor dysfunction indicated by hind limb clasping and difficulty
walking, we wanted to determine whether the PNS was also affected. A flow
cytometry approach determined that CD8+ T cells accumulate in the CNS of
L31 mice from early on in life while they only appear in the PNS once mice
display neurological symptoms. Furthermore, immunofluoresecent studies of
the CNS and PNS of these mice recapitulate the flow cytometry data and
demonstrate the large extent of demyelination in the PNS of symptomatic L31
mice, which is associated with CD8+ T cell accumulation. However, it is also
evident that these CD8+ T cell clusters are associated with macrophage
infiltration in the PNS of symptomatic L31 mice and that these macrophages
have a mature phagocytic phenotype. We hypothesize that epitope spreading
during the course of this CD8+ T cell-mediated demyelinating disease may be
responsible for the PNS pathology. It will be of further interest to determine
the T cell receptor specificity of CNS and PNS infiltrating lymphocytes and the
role of PNS infiltrating macrophages.
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RÉSUMÉ
Plusieurs études suggèrent que les cellules T CD8+ pourraient contribuer à la
pathogenèse de plusieurs maladies auto-immunes du système nerveux. Notre
groupe étudie un modèle murin (souris L31) qui développe spontanément des
symptômes neurologiques associés à des lésions de démyélinisation dans le
SNC. Cette maladie requiert la présence de lymphocytes T et ce sont la souspopulation de lymphocytes T CD8+ qui agissent comme cellules effectrices
dans le processus pathologique.
Parce que les souris L31 présentent des
troubles moteurs tels que le serrement des pattes arrières quand les souris sont
soulevées et des difficultés à marcher, nous avons voulu déterminer si le SNP
est également affecté. Une approche de cytométrie en flux a déterminé que les
cellules T CD8+ s'accumulent dans le SNC des souris L31 très tôt alors qu'ils
n'apparaissent dans le SNP que lorsque les souris présentent des symptômes
neurologiques.
De plus, des études d’immunofluorescence sur coupe en
congélation du SNC et du SNP récapitulent les données de cytométrie en flux et
démontrent la grande étendue de la démyélinisation dans le SNP des souris L31
symptomatiques, qui est associé à l’accumulation en amas de lymphocytes T
CD8+. Il est également démontré que ces amas de cellules T CD8+ sont
associés à l'infiltration de macrophages dans le SNP de souris L31
symptomatiques et que ces macrophages ont un phénotype phagocytaire et
mature. Nos données suggèrent que la présentation de nouveaux épitopes au
cours de la demyelinisation dans le SNC pourrait être responsable de la
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pathologie qui se développe plus tard dans le SNP. Il serait important dans le
futur de déterminer la spécificité du récepteur pour l’antigène des lymphocytes
T CD8+ qui s’accumulent dans le SNC et le SNP et ainsi que d’étudier le rôle
des macrophages infiltrant le SNP.
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ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Sylvie Fournier for the opportunity to
work in her lab and her help and guidance throughout my master’s. Thank you
to all the past lab members Mohamad Habbal, Yunlin Tai, and Vlad Dragan for
all their help, support, suggestions, and laughter. Especially, I would like to
thank Crystal Lee for all her help and accompaniment throughout my master’s.
My project would truly not have been possible without her help and presence.
Thank you to Tony Lim and Dr. Ji Zhang for their technical expertise and
informative discussion and input. Also, I would like to extend my thanks to the
department for their feedback and support. The department has been revamped
since I started my degree and it all was encouraging and of benefit. Many
thanks to the CIHR Neuroinflammation Training Program for their funding,
travel award, and incredible network that helped me develop as a scientist and
professional, it truly ameliorated my master’s experience and offered me many
opportunities. Finally, I would like to thank my family, friends, and future
wife, Gabriella, for all their support and companionship throughout this
journey. All of the above people were all an integral part of my master’s degree
and from the bottom of my heart thank you.
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TABLE OF CONTENTS
Page
Abstract
i
Résumé
ii
Acknowledgements
iv
List of figures
vii
List of abbreviations
viii
Preface
1
1. Introduction
2
1.1 The B7-CD28/CTLA-4 pathway
2
1.2 L31 mice: A mouse model of spontaneous CD8+ T cell-mediated
demyelinating disease
1.3 Multiple Sclerosis
6
9
1.3.1 The role of T cells in Multiple Sclerosis
11
1.3.2 CD8+ T cell involvement in Multiple Sclerosis
13
1.4 Mechanisms of demyelination in Multiple Sclerosis
17
1.5 Demyelinating diseases of the Peripheral Nervous System
19
1.6 Rationale and goal of M.Sc. project
20
2. Materials and Methods
23
2.1 Mice
23
2.2 Preparation of cell suspensions
24
2.3 Flow Cytometry
26
vi
2.4 Immunofluorescence
27
2.5 Statistical Analysis
29
3. Results
3.1 CD8+ T cells in the PNS of symptomatic L31 mice
30
30
3.2 The differences in CD8+ T cell infiltration into the CNS and PNS
of L31 mice over time
31
3.3 CD8+ T cells are found in clusters and colocalize with demyelinated
lesions in the CNS of symptomatic L31 mice
32
3.4 Devastating demyelination and CD8+ T cell infiltration in the PNS
of symptomatic L31 mice
33
3.5 Macrophages are in close apposition to CD8+ T cells in the PNS of
symptomatic L31 mice
34
3.6 Characterizing the macrophage population in the PNS of
symptomatic L31 mice
35
4. Conclusion/Discussion
43
5. References
53
vii
LIST OF FIGURES
Page
Figure 1
CD8+ T cells are found in the PNS of symptomatic L31
mice
Figure 2
CD8+ T cells are found in the CNS of L31 mice early on,
but only appear in the PNS of symptomatic L31 mice
Figure 3
37
38
CD8+ T cells are found in the CNS of L31 mice early on,
but only cause demyelination in the CNS of symptomatic
L31 mice
Figure 4
CD8+ T cells are only found in the PNS of symptomatic
L31 mice and severe demyelination is also seen
Figure 5
40
Macrophages surround CD8+ T cell clusters in the PNS
of symptomatic L31 mice
Figure 6
39
41
Macrophages in the PNS of symptomatic L31 mice have
a more mature and phagocytic phenotype
42
viii
LIST OF ABBREVIATIONS
APC
antigen presenting cell
B6
C57BL/6
BBB
blood-brain barrier
CIDP
chronic inflammatory demyelinating polyneuropathy
CMT
Charcot-Marie-Tooth disease
CNS
central nervous system
CSF
cerebrospinal fluid
CTLA-4
cytotoxic T lymphocyte-associated antigen-4
DC
dendritic cell
EAE
experimental autoimmune encephalomyelitis
FBS
fetal bovine serum
GBS
Guillain-Barré syndrome
GVHD
graft versus host disease
HLA
human leukocyte antigen
IDO
indoleamine-2,3-dioxygenase
IFN
interferon
IL
interleukin
L31
C57BL/6 B7.2 transgenic line 31
mAb
monoclonal antibody
MAG
myelin-associated glycoprotein
MBP
myelin basic protein
MHC
major histocompatability complex
MOG
myelin oligodendrocyte protein
MS
multiple sclerosis
ix
NOD
non-obese diabetic
NOD-B7-2-KO non-obese diabetic background B7.2 -/NOS
nitric oxide species
OCT
Optimal Cutting Temperature
OT
ovalbumin-specific T cell receptor
OVA
ovalbumin
P0
myelin protein 0
PBS
phosphate buffered saline
PFA
paraformaldehyde
PLP
proteolipid protein
PNS
peripheral nervous system
RAG
recombination activating gene
ROS
reactive oxygen species
RRMS
relapsing-remitting multiple sclerosis
SEM
standard error of the mean
SPF
specific pathogen free
TCR
T cell receptor
TNF-α
tumour necrosis factor-α
Tregs
T regulatory cells
VLA-4
very late antigen-4
x
PREFACE
My thesis work focused on a mouse model of CD8+ T cell-mediated
demyelinating disease (L31 mice). L31 mice are unique in that they contain a
costimulatory ligand (B7.2) transgene that is expressed in both T cells and
microglia. Therefore, in order to fully appreciate the model and work that was
conducted using the model, an understanding of the B7-CD28/CTLA-4
costimulatory pathway will be needed and thus, the introduction of my thesis
will review the topic. This will be followed by an overview of the L31 mouse
model and a background on neuroinflammatory diseases and mechanisms of
demyelination. Finally, the rationale for my M.Sc. project will be presented.
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1. INTRODUCTION
1.1 The B7-CD28/CTLA-4 pathway
The B7-CD28/CTLA4 pathway is involved in the regulation of costimulation in
the process of T cell activation. T cells can be fully activated if a sufficiently
potent T cell receptor (TCR) signal occurs (i.e. high avidity peptide), but
costimulation is most often required to fully activate T cells and mount a
response against an antigen [1]. Without costimulation, TCR activation alone
may lead to an unresponsive T cell state known as anergy [2]. The B7 family
consists of many costimulatory ligands, but the most rigorously documented
and most relevant costimulatory ligands to L31 mice are the B7.1 and B7.2
ligands. B7.1 and B7.2 are mostly expressed on professional antigen presenting
cells (APC), but can also be expressed on T cells. B7.2 is constitutively
expressed at low levels on the surface of APCs and is rapidly upregulated upon
activation. However, B7.1 is inducible and is expressed later than B7.2 [3].
These ligands both bind CD28 and cytotoxic T lymphocyte-associated antigen
4 (CTLA-4) on T cells, but with differing affinities and kinetics. CTLA-4 has
the higher affinity between the two receptors and the ligand with greatest
affinity is B7.1. The understanding of this pathway is even more complex as
CD28 and CTLA-4 have opposing affects on T cell activation. CD28 is a
positive regulator of T cell activation, whereas CTLA-4 is a negative regulator
of T cell activation [4].
To help explain how both positive and negative
coreceptors bind the same ligands, researchers have looked at the cell surface
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expression dynamics of these plasma membrane proteins. CTLA-4 is mainly
an intracellular protein and is only upregulated on the cell surface after T cell
activation, similar to B7.1 on APCs.
However CD28 is constitutively
expressed on the cell surface of T cells, which is similar to B7.2 on APCs.
Therefore, many interpret this temporal difference as reason to suggest that
B7.2 is the major CD28 (costimulatory) ligand and that B7.1 is the major
CTLA-4 (inhibitory) ligand [5].
The engagement of all these ligands and receptors causes a plethora of
downstream effects.
The initiating event of the antigen-specific TCR
encountering its antigen in the major histocompatability complex (MHC)peptide complex can vary in affinity. Some interactions between the TCR and
MHC-peptide complex are of great affinity and cause a strong signal
downstream of the TCR. However, others are of low affinity binding, which
causes a lesser activation signal. CD28 costimulation can lower the activation
signal threshold needed to cause complete activation of the T cell [6]. As well,
CD28 signaling can increase cytokine production in T cells, by both
augmenting transcriptional activity and stabilizing messenger RNA [7]. Of
greatest interest, is the upregulation of the interleukin (IL)-2 gene by CD28
activation [8] because of the importance of IL-2 as a growth factor in T cell
proliferation. Another means by which CD28 promotes T cell survival is
through the upregulation of the anti-apoptotic BCL-2 family member, BCL-XL
[9].
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The inhibiting arm of the costimulatory pathway, mediated by CTLA-4, has
opposing effects. CTLA-4 can inhibit T cell proliferation even when optimal T
cell activation occurs [10] and this is mediated by reducing IL-2 and IL-2
receptor production, and by arresting T cells at the G1 phase of the cell cycle
[11]. Moreover, mice genetically deficient in CTLA-4 expression develop a
severe lymphoproliferative disease with progressive accumulation of T cells in
peripheral lymphoid organs as well as in visceral organs, such as the heart,
liver, and exocrine pancreas [12, 13].
Furthermore, the engagement of CD28 or CTLA-4 with B7 ligands can also
signal downstream of the B7 ligand expressing APC. In B cells, the binding of
B7.2 with CD28 causes a positive downstream signal that enhances IgG1 and
IgE production [14, 15]. Additionally, the engagement of B7.2 on dendritic
cells (DC) with CTLA-4 on T cells has been shown to induce the release of
indoleamine-2,3-dioxygenase (IDO) and this event is interferon (IFN)-γ
dependent [16]. IDO is an enzyme that catalyzes the degradation of tryptophan
into by-products, which are inhibitory to T cell proliferation. This evidence is
an example of bidirectional signalling in the B7-CD28/CTLA-4 pathway.
All the literature presented above depicts B7 ligands expressed on APCs, but
they can also be expressed on T cells. In mice, B7.2 is constitutively expressed
at low levels on some resting T cells, however B7.1 is not expressed on resting
T cells, but can be upregulated, along with B7.2, upon T cell activation [3].
This was demonstrated using a graft versus host disease (GVHD) mouse model
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[17]. In this study, the authors demonstrated that B7 expression on T cells can
act as a negative regulator of immune responses and diminish GVHD mortality.
The ability of the B7-CD28/CTLA-4 pathway to regulate T cell activation or
the induction of anergy is crucial in immune system physiology.
An
exaggerated response of the immune system may lead to autoimmunity,
whereas an insufficient response may lead to severe infection. This is why the
costimulatory pathway is so intriguing and many groups have studied the topic
to determine its roles in autoimmunity.
In an experimental autoimmune
encephalomyelitis (EAE) mouse model, mice were crossed with CD28 -/- mice
and the development of EAE was blocked [18]. CD28 -/- mice were also
highly resistant to collagen-induced arthritis [19]. Furthermore, mice that were
administered a CTLA-4 fusion protein (CTLA-4-Fc) that binds B7 ligands
extensively were also resistant to EAE induction [20].
Similarly, in a
spontaneous model of type I diabetes, known as non-obese diabetic (NOD)
mice, disease was inhibited and did not occur in mice injected early with antiB7.2 monoclonal antibody (mAb) [21].
It is therefore evident that this
costimulatory pathway is implicated in the development of autoimmunity.
On the other hand, blocking CTLA-4 via an anti-CTLA-4 mAb exacerbates
disease in EAE mice [22]. Exacerbated disease also occurs in NOD mice as
diabetic symptoms are worsened when CTLA-4 is blocked before the onset of
symptoms [23].
Targeting major players in the costimulatory pathway, such as B7 ligands,
CD28, and CTLA-4, is an intuitive concept as dramatic effects on T cell
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activation can be obtained through their pharmacological alterations.
Nevertheless, much more work must be done to better understand the
consequences of this attractive and promising target for therapies against
autoimmune diseases.
1.2 L31 mice: A mouse model of spontaneous CD8+ T cell-mediated
demyelinating disease
Multiple mouse lines were created in order to study the role of the B7.2
costimulatory ligand on B cell homeostasis [24]. These mouse lines were
created by expressing a B7.2 transgene under the control of the MHC class I H2Kb promoter and the immunoglobulin heavy chain μ enhancer.
Multiple
founder mice were then crossed onto the C57BL/6 (B6) background and
maintained in a specific pathogen-free (SPF) environment.
It was
serendipitously discovered that one of these transgenic lines (L31 mice)
spontaneously develops neurological symptoms at approximately 4-5 months of
age. Neurological symptoms include hind limb clasping when mice are picked
up by their tail, poor proprioception as depicted by hind limbs slipping through
cage bars while walking, and hind limb splaying with an endpoint of hind limb
paralysis. These neurological symptoms are associated with a large infiltration
of effector memory (CD44+, CD62L-) CD8+ T cells into the central nervous
system (CNS) and demyelinated lesions could also be seen in the spinal cord
and spinal roots of these mice [25]. Cellular infiltration of the spinal cord and
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spinal roots is most prominent in the sacral and lumbar regions of the spinal
cord with a decreasing gradient of infiltration at more superior portions of the
CNS. Neurological symptoms are T cell-dependant as TCRβ -/- L31 mice do
not develop disease.
Symptom development is also dependent on the
expression of the B7.2 transgene on microglia because L27 mice (derived in the
same manner as L31 mice, but which only express the transgene on T and B
cells and not on microglia) do not develop neurological symptoms.
Furthermore, adoptive transfers of L31 T cells into TCRβ -/- L31 mice caused
disease, however the adoptive transfer of L31 T cells into TCRβ -/- B6 mice did
not cause disease. As well, it is important to note that although the B7.2
transgene is under the transcriptional control of the MHC class I promoter, and
therefore could be expressed by any cell type, histological staining of multiple
organs demonstrated that the cellular infiltration in L31 mice is specific to
neurological tissue and no tissue destruction of other organs was observed [25].
It has also been shown that I-Aβ -/-, mice in which CD4+ T cells do not
develop because of the absence of a functional MHC class II molecule, and
CD4 -/- L31 mice develop the same neurological symptoms as L31 mice, but
with quicker onset of the disease. This demonstrates that CD4+ T cells are
regulatory in the L31 model and that CD8+ T cells are pathogenic and mediate
disease [26]. Further evidence supporting the pathogenic role of CD8+ T cells
in L31 mice is that the large majority of infiltrating leukocytes in the CNS of
symptomatic L31 mice are CD8+ T cells.
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TCR specificity was then investigated to determine if the pathogenic CD8+ T
cells were infiltrating due to a TCR-specific response in L31 mice. To do so,
OT-I TCR L31 mice, a mouse line that solely contains CD8+ T cells that are
specific for an ovalbumin (OVA) peptide and thus have a restricted CD8+ TCR
repertoire, were developed. OT-I TCR L31 mice do not develop disease and
approximately 95% of the CD8+ T cells in these mice were clonally positive
(Vα2+, Vβ5+), therefore the onset of neurological disease is dependent on TCR
specificity. To determine if a response to a common antigen was involved in
the CD8+ T cell-mediate disease, CDR3 length distribution of the Vβ chain was
investigated. A skew in the distribution of CDR3 lengths would reveal that an
antigen-specific response is involved in the T cell proliferation.
While
peripheral CD8+ T cells displayed a normal Gaussian distribution for all Vβ
chains similar to controls, CD8+ T cells in the CNS of non-symptomatic preclinical I-Aβ -/- L31 mice demonstrated skewed distributions in some Vβ
chains, suggesting that an antigen-specific response causes CD8+ T cell
proliferation and expansion early on in L31 mice. Furthermore, sequencing of
cloned cDNA derived from selected Vβ families with skewed CDR3 length
distribution demonstrated that a particular clonotype was predominant in all
cases, further suggesting the expansion of selected T cell clones within the
CNS. Moreover, microglia, the CNS-resident myeloid-derived monocytes, are
activated in L31 mice at a time point as early as 3 weeks of age in L31 mice.
However, microglia are not activated at this early time point in L31 mice that
are of the OT-I, recombination activating gene (RAG) -/- (which causes the
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absence of mature T- and B-lymphocytes), or IFN-γ receptor -/- backgrounds
[26]. All of these crossed L31 mice also do not exhibit neurological symptoms.
The notion that neurological symptoms occur in L31 mice via a TCR-specific
response mediated by pathogenic CD8+ T cells and that IFN-γ signalling is
necessary for disease would lead to the hypothesis that a MHC class I-restricted
cytotoxic attack of oligodendrocytes is a main contributor to demyelination in
L31 mice.
In fact, oligodendrocytes, the myelinating cells of the CNS,
upregulate MHC class I early on in the preclinical stages in L31 mice [27] and
increased perforin and Fas-ligand were detected in CNS myeloid cells and
heightened levels of granzyme B and IFN-γ were detected in CD8+ T cells
along with CD107a expression at the cell surface, depicting cell degranulation
[28]. Surprisingly, perforin or Fas-ligand -/- L31 mice develop disease with a
quicker onset of neurological symptoms and the predominance of infiltrating
CD8+ T cells still occurs [28]. This data suggests that perforin and Fas-ligand
play a regulatory role in the L31 mouse model and are not necessary for CD8+
T cell-mediated demyelination and the onset of neurological symptoms.
1.3 Multiple Sclerosis
Multiple Sclerosis (MS) is a chronic inflammatory disease that affects the CNS
and is characterized by multiple demyelinated lesions in the brain. The etiology
of the disease is unknown, but it is widely accepted that MS is an autoimmune
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disease and that T cells play a role in the destruction of myelin sheaths
throughout the duration of the disease. MS is the most common neurological
disease among young Canadians and the disease typically manifests itself
between the ages of 15-40 years, a time when people are in the prime of their
lives, and the disease is more common in women than in men with a
predominance of approximately 2:1 [29]. Symptoms of MS are quite diverse
and range from sensory disturbances and limb weaknesses to difficulties
concentrating and fatigue [30].
MS is also categorized into different forms. The most common form, occurring
in 80% of patients, is relapsing-remitting multiple sclerosis (RRMS).
In
RRMS, patients develop neurological symptoms over a few days and then,
spontaneously, the symptoms remit and the patient no longer experiences
neurological deficits. In fact, some symptoms improve and stabilize, however,
RRMS is characterized by the repeating of this cycle as the patient will have
new attacks and worsening of symptoms. Accumulation of demyelinated lesion
occurs after every relapse and the baseline level of function that the patient
returns to after every sequential relapse worsens. Nearly all RRMS patients
eventually reach the secondary progressive form of MS.
In secondary
progressive MS, the disease is no longer characterized by relapses and
remissions, but is a progressive gradual decline in clinical course. Furthermore,
20 % of MS patients are diagnosed with primary progressive MS, which is also
depicted by a progressive gradual decline in clinical course, but is not preceded
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by a RRMS phase. Overall, MS leads to a worsening debilitation in both
neurocognitive and motor functions until an endpoint of death occurs.
This disease is most prevalent in the developed world such as North America,
Europe and Australia.
It is also apparent that areas with less ultraviolet
exposure (from the sun) have a higher prevalence of the disease. Geospatial
analyses demonstrate the distribution of MS prevalence in a gradient fashion
with areas of low sun exposure having a high prevalence of MS and areas of
high sun exposure having a low prevalence of MS. In North America, the
highest prevalence is located in the northern part of the continent and the
prevalence decreases at more southern latitudes [31]. This observation adds to
the growing amount of evidence that a decreased serum vitamin D level is a
risk factor for MS patients [32].
1.3.1 The role of T cells in Multiple Sclerosis
T cells have the capacity to recognize specific antigens from foreign invaders of
the human body and this allows them to help clear many pathogens a person
may encounter throughout a lifetime. However, T cells can also have a TCR
specific for self antigens and this may cause autoimmunity. MS is a very
complex autoimmune disease, which is influenced by both genetic and
environmental factors, and the exact pathogenic involvement of T cells in the
disease is unknown. However, an abundance of work exists using the most
widely accepted animal model of MS, EAE. EAE is induced in susceptible
mice by immunizing with different myelin proteins and adjuvant. The most
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commonly used myelin proteins are myelin oligodendrocyte protein (MOG),
myelin basic protein (MBP), and proteolipid protein (PLP) [33]. The symptoms
of EAE range from limp tail to complete paralysis of the mouse and the
symptoms in the murine model are Th1/Th17-mediated. Th1/Th17 myelinspecific T cells have been demonstrated to cause demyelination in the CNS of
EAE mice and this demyelination leads to the neurological symptoms. As well,
adoptively transferred activated CD4+ T cells specific for myelin antigens into
naive mice can also induce the disease [34].
Furthermore, the greatest genetic risk factor in humans that predisposes them to
develop MS is located in the human leukocyte antigen (HLA) class-II region.
Specifically it is the HLA-DRB1*1501 allele that has been implicated and
confirmed several times through genome wide studies and is the strongest risk
factor for MS [35]. This isolation to a HLA class-II allele confines the genetic
predisposition to an effect on CD4+ T cells. Moreover, recent human studies
have also demonstrated a very convincing case for Th17 cells as being the
major player in initiating new relapses and disease. This observation comes
from a study in which MS patients with aggressive disease that did not respond
to available immune modulating therapies were chemoablated and then
reconstituted with autologous hematopoietic stem cells. These patients were
completely abrogated of any new clinical relapses. Their T cell compartments
were reconstituted and T cell reactivity to multiple myelin antigens could be
detected. However, the responses of these T cells were perturbed, as Th1 and
Th2 responses were normal, but Th17 responses were greatly diminished [36].
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Nevertheless, not all human MS studies agree with the evidence purposed by
EAE studies. A recent human study demonstrates that the myelin protein in
which autoreactive T cells may be activated against to initiate MS is not any of
the EAE-inducing myelin proteins. The group used a large scale proteomics
approach to analyze cerebrospinal fluid (CSF) from children that had an initial
episode of CNS inflammation and followed the children to see if they
developed MS or not and compared the proteins present in the CSF between the
two groups. The interesting finding from this study was that children that went
on to be diagnosed with MS after that first presentation of CNS inflammation
did not have the typical compact myelin proteins (MOG, PLP, MBP) detected
as soluble proteins in their CSF. Rather, it was molecules from the node of
Ranvier (an area where myelin sheaths end and saltatory conduction can occur)
and the surrounding axoglia apparatus membrane that were implicated in the
CSF of children that did go on to be diagnosed with MS [37].
1.3.2 CD8+ T cell involvement in Multiple Sclerosis
The evidence presented above portrays MS pathogenesis as a CD4+ T cell
dependent, most likely Th17-driven phenomenon. However, strong evidence
exists implicating a role for another T cell subset, the CD8+ T cells. CD8+ T
cells are an essential part of the immune system and mainly serve as cytotoxic
effector cells that kill infected or neoplastic cells.
Therefore, autoreactive
CD8+ T cells definitely have the capacity to kill and damage self if their
activation is left unregulated.
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The most convincing line of evidence for the role of CD8+ T cells in MS stems
from studies using post-mortem brain sections of MS patients and the discovery
that CD8+ T cells can outnumber CD4+ T cells by as much as 10-fold in
regions of demyelination [38, 39]. However, this has been supported by a more
recent study that used cortical (gray matter) biopsies of early MS patients. It
was apparent, even in this area of the brain and at an early time point, that
demyelination occurs and is accompanied by CD8+ T cells [40].
The
phenotype of these infiltrating CD8+ T cells still remains quite unknown, but it
has been shown that (IL)-17-producing CD8+ T cells are specifically enriched
in the active lesions of MS patients [41]. Furthermore, CD8+ T cells from the
CSF of MS patients have an effector memory phenotype (CD62L-, CCR7-,
granzymeBhi) and this aids them in crossing the blood-brain barrier (BBB).
This same report demonstrated that very late antigen-4 (VLA-4) is necessary
for CD8+ T cells to cross the BBB [42]. Some HLA alleles that are not
restricted to class II genes also predispose people to MS and they have been
studied using humanized mice [43]. This publication demonstrates how the
expression of HLA-A*0301, which predisposes humans to MS, in a humanized
mouse with the coexpression of a PLP-specific T cell that was derived from an
MS patient causes mild CD8+ T cell-mediated CNS disease that is similar to
MS. However, if HLA-A*0201, which is protective against MS, is expressed
instead, then the humanized mice do not succumb to disease.
Another
emerging phenomenon by which CD8+ T cells could play an effector role in
MS is by expressing dual TCRs. Mice with CD8+ T cells that express two
Page | 14
TCRs, one specific for MBP and another specific for viral antigens, developed
CNS autoimmunity after viral infection [44].
Another line of evidence that strongly supports the pathogenic role of CD8+ T
cells in MS is that the current therapies used to treat the disease are only
affective if both CD4+ and CD8+ T cell compartments are targeted.
Fingolimod, an oral drug that broadly inhibits exit of lymphocytes from
secondary lymphoid organs by modulating the sphingosine 1-phosphate
receptor, is effective in treating MS patients [45]. Likewise, blocking the
global entrance of T cells into the CNS through the usage of natalizumab, an
alpha-4 integrin-specific antibody, greatly ameliorates disease in people with
MS as shown by a significant reduction in progression, relapse rate, and
number of lesions [46]. It is also interesting to note that alpha-4 integrin is a
component of VLA-4 and that natalizumab strongly affects the absolute number
of leukocytes in the CSF of treated MS patient. This reduction occurs in both
the CD4+ and CD8+ T cell compartments [47]. In line with all this evidence
supporting the notion that both T cell compartments need to be targeted in order
to treat MS, a monoclonal anti-CD4 antibody treatment failed to ameliorate
disease in MS patients in a phase II clinical trial [48].
Accumulating evidence for the involvement of CD8+ T cells in MS has been
supported by several murine models. More recent CD8+ T cell-mediated EAE
models have been reported to better recapitulate MS pathology with more
inflammation of the brain as compared to the spinal cord [39]. One model,
using C3H mice, induced neurological symptoms, CNS infiltration, and
Page | 15
demyelination when MBP-specific CD8+ T cells from EAE mice were
transferred into naїve recipient mice [49]. Another group expressed OVA as a
cytosolic protein in oligodendrocytes and observed that naїve OT-I (ovalbuminspecific) TCR CD8+ T cells can be activated without the help of antigen
presentation by an APC and that this lead to IFN-γ production and fulminant
demyelinating EAE with lesions reminiscent of MS [50]. Brain sections from
these mice were also used to study the effect of spillover of toxic molecules
such as perforin and granzyme B. OT-I-specific CD8+ T cells were co-cultured
with the brain slices from the cytosolic OVA-expressing mice and this caused
collateral neuronal apoptosis to surrounding neurons, outside of the
immunological synapse [51].
The overexpression of PLP in mice lead to
myelin degeneration and the pathology in these mice was lost when crossed
with RAG -/- mice. Reconstitution of these mice with CD8+ CD4- bone
marrow restored the demyelinated pathology, whereas bone marrow from CD8CD4+ mice did not restore CNS pathology [52]. Further studies on these PLP
overexpressing mice demonstrates that CD8+ T effector cells are activated and
undergo clonal expansion in the CNS throughout the neurodegenerative disease
[53].
Moreover, CD8+ T cells can also be regulatory and many studies are now
demonstrating the role of CD8+ T regulatory cells (Tregs) in MS. CD8+ Tregs
have been studied in the EAE mouse model and in human samples. The
evidence from the EAE mouse model is two sided as CD8+ T cells can
suppress, but can also autonomously cause CNS damage as mentioned above
Page | 16
[39]. Although, a study in humans did demonstrate that neuro-antigen specific
CD8+ T cells could function as Tregs and suppress effector T cells and this
CD8+ Treg function is diminished during patient disease exacerbation [54].
1.4 Mechanisms of demyelination in Multiple Sclerosis
Studying the pathology of MS helps in understanding the different cell types
and possible mechanisms involved in causing demyelinated lesions. In MS,
there are four different pathology patterns each with their own differing cell
types and pathogenic molecules [55]. Pattern-I lesions (found in 15% of MS
patients) consist of mainly T cell inflammation, and active demyelination with
many activated microglia and macrophages.
This suggests a role for
macrophage-associated demyelination where macrophages release toxic factors
such as tumour necrosis factor α (TNF-α), and reactive oxygen species (ROS),
which lead to myelin destruction. Pattern-II lesions (found in 58% of MS
patients) consist of densely packed T cells and macrophages, but also
immunoglobulin deposition and compliment activation. This type of lesion
supports a role for autoantibodies and compliment-mediated destruction of
myelin. Pattern-III lesions (found in 26% of MS patients) are defined by
oligodendrocyte apoptosis, T cell inflammation, and macrophage and microglia
activation. This lesion-type supports a model of oligodendrocyte apoptosis that
may be mediated by the activation of different death receptors. Pattern-IV
Page | 17
lesions (found in 1% of MS patients) are associated with profound nonapoptotic
death of oligodendrocytes. This lesion-type is exceedingly rare and is only
identified in autopsies of primary progressive MS patients. The pathogenesis
occurring in these lesions is unclear.
The reoccurring presence of activated microglia and macrophages does not
seem like a coincidence in MS pathogenesis. An increasing amount of studies
are demonstrating that activated myeloid cells secrete toxic species that play a
major role in demyelination in MS patients. Two recent studies used genomewide microarray analysis to demonstrate signs of oxidative tissue damage and
mitochondrial injury in these lesions. The first analyzed active MS lesions [56]
and the second analyzed cortical MS lesions [57].
The mechanism of
pathogenesis proposed by the authors consists of an initial attack of
demyelination caused by a T cell-specific response that may also be augmented
by the release of ROS and nitric oxide species (NOS) by myeloid cells. This
would cause tissue damage and mitochondrial injury, which would then
continue
the
cycle
of
releasing
harmful
free-radicals
and
further
neurodegeneration.
The CD8+ T cells found in MS lesions can also mediate TCR-specific cell
death. CD8+ T cells can kill targeted cells through Fas-ligand activation of the
Fas death pathway and induce apoptosis of the targeted cell or they can also
induce cytolysis through the exocytosis of granules such as perforin and
granzyme B [58]. Furthermore, this targeted cell death has been shown to be
extremely specific and is regulated by the formation of an immunological
Page | 18
synapse between the CD8+ T cell and the target cell [59]. This is evidence that
myelin-antigen specific CD8+ T cells could be involved in the killing of
oligodendrocytes and the cause of demyelination in MS.
Moreover, T cells that cause demyelination in the CNS can indirectly cause the
activation of other T cells which have a TCR-specificity to antigens or epitopes
which are similar to their own TCR-specificity. This occurs when a T cell is
activated by its antigen presented in a MHC and causes destruction of that
protein. This will then lead to further epitopes or antigens of that same protein
to be available and presented by APCs, which could activate other T cells that
are specific for these newly available epitopes. This phenomenon is known as
epitope spreading and has been shown to occur in MS [60]. In fact, it has been
seen that a small percentage of MS patients suffer from peripheral nervous
system (PNS) deficits, neuropathy, during the course of their disease and this
finding was attributed to epitope spreading [61]. Most recently, it has been
shown that initial demyelination in the CNS of EAE mice by CD4+ T cells lead
to the activation of CD8+ T cells by a specific type of DC [62].
1.5 Demyelinating diseases of the Peripheral Nervous System
Demyelinating pathology does not only occur in MS. Other demyelinating
diseases exist where the pathology of demyelination occurs in the PNS and not
in CNS. One example is Guillain-Barré Syndrome (GBS) and its chronic
Page | 19
counterpart chronic inflammatory demyelinating polyneuropathy (CIDP).
These diseases are characterized by rapidly evolving weakness, some sensory
loss, and hyporeflexia.
The diseases are caused by an immune attack of
peripheral nerve axons [63]. Particularly to GBS, node of Ranvier lengthening
is seen early on in the disease and is then followed by complement-mediated
recruitment of macrophages to the nodal regions [64]. Another example of a
PNS demyelinating disease is Charcot-Marie-Tooth disease (CMT). CMT is an
inherited disorder of the peripheral nerves and can be caused by many different
genetic mutations. The genetic defects are mainly found in myelin proteins of
Schwann cells, PNS myelinating cells, or axonal proteins and cause a decrease
in conductance of peripheral nerves [65].
The decreased conductance in
peripheral nerves is caused by the genetic mutations causing either
demyelination of peripheral nerves, when mutations are found in Schwann cell
myelin proteins, or dysfunction of peripheral nerve axons. Furthermore, there
is presently no pharmacological therapy for CMT [66].
1.6 Rationale and goal of M.Sc. project
As described above, our lab works on a mouse model that spontaneously
develops neurological symptoms at approximately 4-5 months of age and the
neurological symptoms consist of hind limb clasping when picked up by the
tail, poor proprioception as seen by hind limbs slipping through cage bars while
Page | 20
walking, and hind limb splay with an endpoint of complete hind limb paralysis.
This L31 mouse model overexpresses the costimulatory ligand B7.2 on T cells
and microglia and the neurological symptoms are dependent on B7.2 expression
on microglia. These neurological symptoms are also T cell dependant and are
associated with demyelinated lesions in the CNS. CD8+ T cells colocalize with
these demyelinated lesions. This line of evidence would rationally lead to a
hypothesis describing myelin-autoreactive CD8+ T cells being activated
because of dysregulated costimulatory expression on microglia and causing
demyelination in the CNS, which would cause the clinical neurological
symptoms spontaneously seen in our mice.
However, the impaired motor
function and limb weakness, depicted by the neurological symptoms, may
suggest that an insult to the PNS is occurring. Recently, a publication from The
Journal of Experimental Medicine described NOD mice that succumbed to the
exact same neurological symptoms as L31 mice [67]. These mice were first
described when B7.2 -/- mice were created on the NOD background (NOD-B72-KO). It was reported that demyelination occurred solely in the PNS and the
CNS was not affected and the disease was CD4+ T cell mediated [68].
Furthermore, NOD-B7-2KO mice were resistant to disease when deficient in
IFN-γ, but perforin or Fas deficiencies did not affect neuropathy development
[69]. Louvet et al. [67] showed that T cells specific for myelin protein 0 (P0)
were responsible for the neurological disease. Indeed, mice expressing a
transgenic TCR specific to P0 and were bred on a RAG -/- background
developed a fulminant form of peripheral neuropathy.
Page | 21
Due to the motor dysfunction nature of the spontaneous neurological symptoms
seen in L31 mice and the striking resemblance of their symptoms to the
symptoms exhibited by NOD-B7-2-KO mice, who only suffer from PNS
demyelination and cellular infiltration, it was the goal of my M.Sc. research
project to determine whether cellular infiltration and demyelination was
occurring in the PNS of L31 mice and, if so, determine which compartment
between the PNS and CNS is first infiltrated by CD8+ T cells.
Page | 22
2. MATERIALS AND METHODS
2.1 Mice
L31 mice were generated by injecting a construct, consisting of the coding
region of B7.2 cDNA under the transcriptional control of the MHC class I
promoter, H-2Kb, and the immunoglobulin heavy chain enhancer, Igμ, into an
oocyte.
The oocyte was transplanted into a pseudopregnant mouse and a
founder mouse was born. The founder mouse was backcrossed onto a C57BL/6
background and L31 mice are now bred and maintained in a SPF animal facility
[24]. Genotyping of these mice was performed by collecting a blood sample
from the tail vein into a 5 mL polystyrene round-bottom tube (BD Falcon)
containing
500
μL
of
Alsever’s
solution
(114M
dextrose,
27mM
Na3C6H5O7•H2O, 71mM NaCl). Red blood cells were lysed by treating with
ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2-EDTA) and
washed with phosphate buffered saline (PBS) (137mM NaCl, 2.7mM KCl,
4.3mM Na2HPO4, 1.47mM KH2PO4) supplemented with 1% fetal bovine serum
(FBS) (GIBCO) and 0.1%NaN3. Samples were blocked by using 50μL of
2.4G2 blocking antibody for 30 minutes and then stained with rat anti-mouse
B7.2 antibody conjugated to phycoerythrin (PE) (eBioscience) for 30 minutes.
After incubation, samples were washed using FBS and NaN3 supplemented
PBS and acquired on a BD LSR-Fortessa flow cytometer running on FACS
Diva 6.0 software (BD). Samples were analyzed for high expression of B7.2,
which would confirm the presence of the B7.2 transgene.
All animal
Page | 23
procedures were in accordance with the guidelines of the Canadian Council of
Animal Care, as approved by the animal care committee of McGill University.
2.2 Preparation of cell suspensions
In order to isolate the CNS, brain and spinal cord, and PNS, right and left
sciatic nerves, mice were deeply anesthetized using a 4-5% isofluorane (Baxter)
concentration at a 1 L/min flow rate in an induction chamber. Mice were then
maintained under anesthesia by the use of a mask connected to a Bain circuit.
The flow rate was adjusted to 0.5 L/min and the concentration of isofluorane
was also decreased to 2.5%. Depth of anesthesia was confirmed by the lack of
pedal reflex when toe pads were pinched. Mice were then surgically exposed
and the abdomen opened in order to sever the abdominal vein. Then the
thoracic cage was quickly opened and, while the heart beat still persisted, 25mL
of ice cold PBS was intracardially perfused through the left ventricle using a 30
mL syringe and a 26G1/2 needle (BD).
Then the nervous tissues were
dissected and maintained in complete RPMI 1640 medium (GIBCO)
supplemented with 10% FBS, 50μM β-mercaptoethanol (Bioshop), 100U/mL
penicillin (Sigma), and 100μg/mL streptomycin (GibcoBRL). Tissues were
then placed in a 3 mL dounce homogenizer containing 1 mL of supplemented
RPMI and homogenized manually using a pestle. The cell suspensions were
then collected into 15 mL canonical tubes (BD Falcon) and spun down at 1200
rpm at 4°C for 5 minutes.
Supernatants were decanted and pellets were
Page | 24
resuspended in 10 mL of 30% Percoll (GE Healthcare) in PBS. Tubes were
spun at 1500 rpm at 18°C for 30 minutes in order to separate myelin debris
from cell pellets. Myelin debris and supernatants were aspirated out and cell
pellets were washed with 10 mL of supplemented RPMI. After wash, cells
were resuspended in 1 mL of supplemented RPMI and filtered through a 70 μm
nylon mesh. Cell concentrations were estimated by using 0.4% trypan blue
(Sigma), to exclude dead cells, and a haemocytometer (Hausser Scientific).
For experiments involving macrophage staining, an enzymatic method was
used to create cell suspensions in order to better represent the proportion of
myeloid cells.
The method used consisted of the same procedures for
dissection and tissues were also maintained in supplemented RPMI, but then
they were processed according to a previously published protocol [70]. Briefly,
tissues were cut into multiple small segments and placed into 1.5 mL
microtubes containing fresh 1 mL supplemented RPMI with the addition of 1.6
mg/mL of collagenase (type IV; Sigma) and 200 μg/mL of DNase I (Sigma).
Sample tubes were then incubated at 37°C for 30 minutes with agitation and
after which samples were gently dissociated by repeat pipetting through a 1 mL
pipet and reincubated at 37°C for 25 minutes with agitation.
After this
incubation, samples were dissociated again to a single-cell suspension using
again a 1 mL pipet and were then centrifuged at 6000 rpm for 5 minutes in a
microcentrifuge. Supernatants were decanted and cells were washed twice
using PBS. Cells were resuspended in 1 mL of supplemented RPMI and
filtered through a 70 μm nylon mesh. Cell concentration and percent viability
Page | 25
was estimated in the same manner as the previous method by using a
haemocytometer and the Trypan blue cell exclusion method.
2.3 Flow cytometry
All samples for the T cell infiltration kinetics experiments were stained in the
following manner. 1x106 cell aliquots were pipetted into 5 mL round bottom
polystyrene tubes and washed with PBS.
They were first labelled with
LIVE/DEAD amine-reactive violet viability marker according to the
manufacturer’s protocol (Invitrogen). Cells were then blocked with 50 μL of
2.4G2 blocking antibody for 30 minutes at 4°C and then labeled with rat antimouse
CD45
conjugated
to
fluorescein
isothiocyanate
(FITC)
(BD
Pharmingen), rat anti-mouse CD4 conjugated to allophycocyanin (APC) (BD
Pharmingen), and rat anti-mouse CD8a conjugated to PE (eBioscience).
Samples were washed using PBS and then acquired on a BD LSR-Fortessa flow
cytometer running on FACS Diva 6.0 software. The analysis of the acquired
data was also performed using the FACS Diva 6.0 software.
For the macrophage characterization experiments, samples were processed in
the same manner as those from the T cell infiltration kinetics experiments up
until the antibody staining step.
Samples used for the macrophage
characterization experiments were stained with rat anti-mouse CD45 conjugated
to APC (BD Pharmingen), rat anti-mouse CD11b conjugated to FITC (BD
Page | 26
Pharmingen), and rat anti-mouse F4/80 conjugated to PE (Serotec). These
samples were acquired and analyzed in the same manner as described above.
2.4 Immunofluorescence
Mice were intracardially perfused with PBS in the same manner as described
above, but were then subsequently intracardially perfused with 4%
paraformaldehyde (PFA). Spinal cord tissues were dissected and then postfixed in 4% PFA for 2 hours at room temperature and then cryoprotected by
allowing them to sink in a 30% sucrose solution overnight at 4°C. Spinal cord
tissue was then embedded in Optimal Cutting Temperature (OCT) compound
(Tissue-Tek) by orienting the tissue in disposable base molds (Canemco)
containing OCT and then flash-freezing the embedded tissue in liquid nitrogen.
Sciatic nerve samples were from mice that were not intracardially perfused with
4% PFA and sciatic nerve tissue was not post-fixed or cryoprotected. Dissected
sciatic nerve tissue was immediately placed in OCT and then oriented in
disposable base molds containing OCT and flash-frozen using liquid nitrogen.
Embedded tissue cassettes were then cut into 12 μm (for CNS samples) or 6 μm
(for PNS samples) sections using a CM3050 S cryostat (Leica) and mounted
onto Superfrost Plus microscope slides (Fisher Scientific).
Slides were
maintained at -20°C until stained.
To stain slides, they were first thawed and desiccated for 30-60 minutes,
samples were circled with a Dako Pen, and washed with PBS. Slides were then
Page | 27
flooded with blocking solution (PBS with 2% normal goat serum, 2% FBS, and
0.2% Triton-X-100) for 3-5 hours at room temperature. After blocking, slides
were washed with PBS and primary antibody was added in desired
concentration to blocking solution and incubated overnight at 4°C. Rat antimouse CD8a (BD Pharmingen) was used at a 1/100 dilution and rabbit anti-Iba1 (Wako) was used at a 1/1000 dilution. Slides were then washed 3 times with
PBS and secondary antibodies were added in desired concentrations to blocking
solution and incubated for 2 hours at room temperature. Goat anti-rat IgG
conjugated to Alexa Fluor 488 (Cell Signalling) was used at a 1/1000 dilution
and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen) was used at
a 1/500 dilution. Slides were then washed 3 times with PBS and then myelin
lipids and nuclei were stained by flooding slides with a PBS solution containing
FluoroMyelin Red (Invitrogen) at a 1/200 dilution and TO-PRO-3-iodide
(Invitrogen) at a 1/1000 dilution. The slides were then incubated at room
temperature for 20 minutes and washed 3 times with PBS.
Slides were
coversliped using Vectashield mounting medium (Vector Laboratories) and
Fisherfinest premium cover glass (Fisher Scientific) and slides were sealed by
applying nail polish to the edges of the cover glass.
Slides were imaged on an Olympus Fluoview 1000 confocal microscope
running on the Olympus FV10-ASW software version 2.01 and the objectives
used to capture the images were the Olympus UPlanSApo 20x/0.75 dry,
Olympus UPlanSApo 40x/0.90 dry, and the Olympus PlanApo N 60x/1.42 oil.
Page | 28
2.5 Statistical analysis
T cell infiltration kinetics was analysed using a two-way ANOVA for the effect
of mouse group and time on the results. Statistical differences between groups
was analysed using an unpaired two-tailed student’s t-test. Data are expressed
as the mean ± standard error of the mean (SEM) and p values ≤ 0.05 were
considered to be significant. All statistical analyses were performed using
GraphPad Prism 5.
Page | 29
3. RESULTS
3.1 CD8+ T cells in the PNS of symptomatic L31 mice
In order to determine whether CD8+ T cells were present in the PNS of L31
mice, we used a flow cytometry approach. Mononuclear cells were isolated
from the sciatic nerve and analyzed by flow cytometry for the expression of
CD45, CD4 and CD8. CD45 is a tyrosine phosphatase that is expressed at high
levels on T lymphocytes and at intermediate levels on myeloid cells. CD4 and
CD8 expression was analyzed on the CD45high cells (lymphocyte population)
present in the sciatic nerve as shown in figure 1.
When comparing the B6 and L31 non-symptomatic mice, it is evident that no
significant CD45hi lymphocyte population was present and that the only major
population was the CD45intermediate population of myeloid cells (Figures 1A
and B). However, the results clearly show that a distinctively large population
of infiltrating leukocytes was present in symptomatic L31 mice, as depicted by
the orange population that is CD45hi (Figure 1C). This population mainly
consisted of CD8a positive cells and very few cells from this population were
CD4+ (Figure 1C). This data shows that a large CD8+ T cell population
infiltrates into the PNS of L31 mice, once they are symptomatic.
Page | 30
3.2 The differences in CD8+ T cell infiltration into the CNS and PNS of
L31 mice over time
It was of interest to perform a kinetic analysis to determine at what time point
CD8+ T cells infiltrate into the two nervous tissue compartments, CNS and
PNS. Therefore we performed the same flow cytometry analysis on mice at 1
month, 3-4 months, 5-6 months, and symptomatic mice (4-5 months) on both
their CNS and the PNS. As shown in figure 2A CD8+ T cells were found in
the CNS of L31 mice at an early time point of 3-4 months of age. Furthermore,
CD8+ T cells were also found in the CNS of non-symptomatic L31 mice at 5-6
months of age and were at the most abundant level once the L31 mice were
symptomatic. A two-way ANOVA analysis of the CNS values indicated that
there was a significant interaction between the B6 and L31 groups over time
(p=0.0053), which therefore allowed us to analyze the data between mouse
groups and time points using a student’s t-test. The absolute number of CD8+
T cells in the CNS of L31 mice at 3-4 months of age was significantly greater
than the number found in B6 control mice (p=0.0223) and this was also the case
at 5-6 months of age (p=0.0497).
In the PNS of L31 mice, no CD8+ T cell infiltration was detected by flow
cytometry until the mice became symptomatic (Figure 2B).
The absolute
number of CD8+ T cells found in the PNS of L31 mice was comparable to the
B6 control samples at all other time points (Figure 2B). A two-way ANOVA
analysis of the PNS values concluded that there was no significant interaction
between the groups over time (p=0.6872).
Page | 31
This kinetic analysis revealed that no significant number of CD8+ T cells was
found in the PNS of L31 mice before the onset of neurological symptoms, but
that CD8+ T cells could be seen in the CNS of L31 mice as early as 3-4 months
of age, before they had any clinical signs of neurological disease.
3.3 CD8+ T cells are found in clusters and colocalize with demyelinated
lesions in the CNS of symptomatic L31 mice
We used an immunofluorescence approach to look at the localization of the
CD8+ T cells, in the CNS and PNS of L31 mice, and to evaluate the extent of
demyelination. We did so by using a mAb against CD8a, FluoroMyelin Red to
stain myelin lipids, and TO-PRO-3-iodide to visualize the nuclei. 3-4 months
old L31 mice were used as non-symptomatic controls for CNS staining because
it was the time point where the greatest absolute number of CD8+ cells was
found in the CNS (Figure 2A).
The immunofluorescent staining of non-
symptomatic L31 CNS sections demonstrated intact myelin with no
demyelinated lesions, however clusters of CD8+ T cells could be seen (Figure
3). In contrast, in the CNS of symptomatic L31 mice, large CD8+ T cell
clusters could be found that colocalized with demyelinated lesions, as seen by
the decreased fluorescence in the myelin staining (Figure 3 lower panels). As
expected, B6 mice demonstrated no CD8+ T cell infiltration and myelin
architecture was not compromised (Figure 3 upper panels). Taken together, this
Page | 32
data shows that CD8+ T cells were associated with demyelinated lesions in the
CNS of symptomatic L31 mice and that CD8+ T cells were found in the CNS
of non-symptomatic L31 mice, however the myelin staining in the nonsymptomatic L31 CNS was not compromised.
3.4 Devastating demyelination and CD8+ T cell infiltration in the PNS of
symptomatic L31 mice
The PNS of symptomatic L31 mice demonstrated a pronounced loss in myelin
architecture and devastating demyelination (Figure 4). The B6 mice and nonsymptomatic L31 mice showed no CD8+ T cell infiltration and the myelin
staining was normal as longitudinal sections depicted Schwann cells wrapping
around axons along the whole course of the nerve (Figure 4). In addition, all
the nuclei stained in the B6 and non-symptomatic L31 sections where of a
squamous shape. We attributed these nuclei to normal Schwann cells and
therefore, we concluded that no infiltrating cells were present as no round
nuclei could have been seen (Figure 4). Although, the merged image from the
L31 symptomatic mice demonstrated a cluster of round nuclei that did not stain
for CD8a (Figure 4 bottom panels, arrow).
Taken together this data demonstrates that CD8+ T cells are only seen in the
PNS of symptomatic L31 mice and not in non-symptomatic L31 mice and that
Page | 33
devastating demyelination solely occurs in the PNS of symptomatic mice.
However, not all infiltrating cells were CD8+ T cells in these mice.
3.5 Macrophages are in close apposition to CD8+ T cells in the PNS of
symptomatic L31 mice
It was of interest to determine exactly what cell type accounted for these nuclei
and therefore we investigated the possibility that the unstained cells in the PNS
of symptomatic L31 mice were macrophages. We hypothesized that these cells
were macrophages for two reasons.
First, macrophages are known to be
involved in mechanisms of demyelination in MS as they can release toxic
oxygen and nitric species. Second, they also have the capacity to be APCs and
the devastating demyelination in the PNS of symptomatic mice would lead to
the presentation of many soluble antigens by APCs and these cells would be
found surrounding clusters of T cells, just like the unstained round nuclei seen
in the immunofluorescent stains mentioned above. To investigate whether
these cells were macrophages, we once again stained for CD8a and nuclei, but
also stained using a mAb against Iba-1, which is a cytoplasmic calcium-binding
adaptor protein that is found in macrophages and microglia. However, we
could not stain for CD8a and Iba-1 together because the two mAbs needed
different fixation preparations in order to stain well. Therefore we had to stain
serial sections of the PNS from symptomatic L31 mice and the staining pattern
Page | 34
for CD8a and Iba-1 expression on serial sections suggested that macrophages
were in close apposition to and encompassing CD8+ T cells in the PNS of these
mice (Figure 5).
3.6 Characterizing the macrophage population in the PNS of symptomatic
L31 mice
To further investigate the macrophage population in the PNS of symptomatic
L31 mice, we isolated mononuclear cells from the sciatic nerve using an
enzymatic method in order to best represent the myeloid cell population. We
then used a flow cytometry approach to compare the macrophage population in
the PNS of symptomatic and non-symptomatic L31 mice. We stained for CD45
and the macrophage markers CD11b and F4/80. All CD45+ cells were gated
on and CD11b and F4/80 expression was analysed.
As it is seen in figure 6A, the percentage of macrophages was not increased
between non-symptomatic and symptomatic L31 mice, as the two macrophage
markers used, CD11b and F4/80, stained for an approximate total of 10% of the
cells in both experimental groups. However it is interesting to note that there
was an apparent difference in the total number of CD45+ cells between B6
controls and non-symptomatic L31 mice, as seen in the representative dot plots
(Figure 6A).
Page | 35
Most interestingly however, is that the percentage of CD11b+ F4/80+
macrophages was greatly increased in the PNS of symptomatic L31 mice
(Figure 6A). Among the cells that were CD45+ CD11b+, the percentage of
F4/80+ macrophages was significantly greater in the L31 symptomatic group
compared to the L31 group (Figure 6B; p=0.0016; L31=5.5% ± 3.304, n=4;
L31 symptomatic=50.0% ± 5.0, n=2). This demonstrated that the phenotype of
the macrophages in the PNS of symptomatic L31 mice was different when
compared to those found in the PNS of non-symptomatic L31 mice. The F4/80
marker on macrophages suggested a more mature macrophage phenotype that
was phagocytosing debris in the area [71].
Page | 36
Figure 1: CD8+ T cells are found in the PNS of symptomatic L31 mice.
(A) Representative flow cytometry dot plot of PNS cells from age matched
B6 mice. (B) Representative flow cytometry dot plot of PNS cells from age
matched non-symptomatic L31 mice. (C) Representative flow cytometry dot
plot of PNS cells from L31 symptomatic mice.
Page | 37
Figure 2: CD8+ T cells are found in the CNS of L31 mice early on, but
only appear in the PNS of symptomatic L31 mice. (A) Graphic
representation of CD8+ T cell infiltration kinetics into the CNS of L31 and
control B6 mice. Data shown are the mean ± SEM and are representative of
three independent experiments per mouse group per time point. (B) Graphic
representation of CD8+ T cell infiltration kinetics into the PNS of L31 and
control B6 mice. Data shown are the mean ± SEM and are representative of
three independent experiments per mouse group per time point. Only the
CNS data was significant for a two-way ANOVA analysis. *p≤0.05
Page | 38
Figure 3: CD8+ T cells are found in the CNS of L31 mice early on, but
only cause demyelination in the CNS of symptomatic L31 mice.
Immunofluorescent staining of spinal cord sections from 5-6 months old B6,
3-4 months old L31, and L31 symptomatic mice using anti-CD8a mAb,
FluoroMyelin Red, and TO-PRO-3-iodide for the nuclei. Scale bars: 200μm;
lower panels 60μm
Page | 39
Figure 4: CD8+ T cells are only found in the PNS of symptomatic L31
mice and severe demyelination is also seen. Immunofluorescent staining of
sciatic nerve sections from 5-6 months old B6, 5-6 months old L31, and L31
symptomatic mice using anti-CD8a mAb, FluoroMyelin Red, and TO-PRO-3iodide for the nuclei. Arrow is non-CD8a stained round nuclei population.
Scale bar: 60μm
Page | 40
Figure 5: Macrophages surround CD8+ T cell clusters in the PNS of
symptomatic L31 mice. Immunofluorescent staining of serial sciatic nerve
sections 6μm apart from L31 symptomatic mice using anti-CD8a mAb or
anti-Iba-1 mAb (for macrophages), and TO-PRO-3-iodide for the nuclei.
Scale bar: 20μm
Page | 41
Figure 6: Macrophages in the PNS of symptomatic L31 mice have a
more mature and phagocytic phenotype.
(A) Representative flow
cytometry dot plots of PNS cells from 5-6 months old B6, 5-6 months old
L31, and L31 symptomatic mice. The dot plots are gated on CD45+ cells
with the exclusion of dead cells. (B) Histogram of the % F4/80+
macrophages when gated on CD45+ CD11b+ myeloid cells in the PNS. Data
presented as mean ± SEM, L31=5.5% ± 3.304, n=4; L31 symptomatic=50.0%
± 5.0, n=2; p=0.0016.
Page | 42
4. DISCUSSION/CONCLUSION
In this thesis, we have demonstrated that CD8+ T cells infiltrated into the CNS
of L31 mice at an earlier time point and immunofluorescent staining revealed
that CD8+ T cell clusters could be found in the CNS of non-symptomatic L31
mice, but no demyelination was present in these mice. CD8+ T cell clusters did
colocalize with demyelinated lesions in the CNS of symptomatic L31 mice.
We have also clearly demonstrated that CD8+ T cells were found in the PNS of
symptomatic L31 mice and that devastating demyelination occurred in these
mice, whereas there was no infiltration in non-symptomatic L31 mice and the
PNS myelin architecture of non-symptomatic L31 mice was normal.
Furthermore, we demonstrated that the PNS of symptomatic L31 mice is
infiltrated by macrophages and these macrophages seem to surround the
clusters of CD8+ T cells. The macrophages in the PNS of symptomatic L31
mice also had a distinct phenotype, which was not observed in nonsymptomatic L31 mice.
The data show CD8+ T cells in the CNS of L31 mice first, before any
infiltration into the PNS and CD8+ T cells are only present in the PNS of L31
mice once they exhibit neurological symptoms. It has also been previously
shown that CD8+ T cells go through oligoclonal expansion early on in the CNS
of L31 mice [26]. This evidence leads to the hypothesis that these CD8+ T
cells are activated in an epitope specific manner in the CNS and then are
Page | 43
reactivated and cause demyelination in the PNS via an epitope spreading
phenomenon. Although, further experiments would have to be conducted to
confirm this hypothesis, as the epitope specificity of the activated CD8+ T cells
in the CNS and PNS is not known. It has been known for many decades now
that the composition of CNS and PNS myelin is different [72] therefore the
antigen to which the CD8+ T cells are activated against is quite limited. One
example of such a shared myelin protein is MBP [73]. It would be of interest to
determine if lymphocytes isolated from both the CNS and PNS of symptomatic
L31 mice would respond to overlapping epitopes of the MBP. Another shared
CNS and PNS myelin protein is myelin-associated glycoprotein (MAG) [74],
which is a periaxonal membrane protein that was also implicated as a possible
initiating epitope in the large-scale CSF proteomics study on pediatric MS
patients mentioned in the introduction of this thesis [37].
The mechanism by which CD8+ T cells initially infiltrate into the CNS of L31
mice is also unknown. VLA-4 has been implicated in its role of allowing T
cells, particularly CD8+ ones, to cross the BBB [42, 47]. It is obviously a well
rationalized hypothesis that the CD8+ T cells in L31 mice do express VLA-4,
but it has yet to be experimentally proven and it would be of interest to do so.
CD8+ T cells infiltrate the PNS of symptomatic L31 mice, but this is not the
only infiltrating cell type. An unexpected macrophage population found in the
PNS of symptomatic L31 mice sheds light on some very interesting concepts of
possible mechanisms of demyelination.
As it was mentioned in the
introduction, histopathological analysis of MS lesions has helped identify key
Page | 44
cell types and mediators that are implicated in the demyelinated lesions of this
disease [55]. Some of those cell types and mediators are T cells, both CD4+
and CD8+, macrophages, with ROS and NOS present, and autoantibody and
compliment deposition. It is interesting to compare the demyelinated lesions in
the CNS and PNS of symptomatic L31 mice because they have striking
differences and these differences could be partially explained by the cell types
present, as was also the case in the histopathological studies of MS patient
samples. The demyelinated lesions in the CNS of symptomatic L31 mice are
very contained and colocalize with clusters of approximately the same size of
CD8+ T cells. Furthermore, flow cytometry data from the lab demonstrates
that no macrophage population infiltrates into the CNS of symptomatic L31
mice [28]. The absence of macrophages in the lesions present in the CNS of
symptomatic L31 mice is different than what is seen in lesions from MS
patients and the lesions in the PNS of symptomatic L31 mice. Due to the very
local nature of the demyelinated lesions in the CNS of symptomatic L31 mice
and the fact that they colocalize with CD8+ T cell clusters, we would think that
this demyelination is caused by a cell-specific cytotoxic attack, but, as
aforementioned, Fas-ligand and perforin -/- L31 mice develop disease with
quicker onset and kinetics, suggesting that these molecules play a regulatory
role in L31 disease [28]. Therefore, a different mechanism of demyelination or
cell death must be occurring. One hypothesis is that the local microglia are
responsible for the demyelination in the CNS and they release ROS and NOS in
Page | 45
the confined area of where they are activated and this causes the local
demyelination.
The demyelinated lesions in the PNS of symptomatic L31 mice are much more
wide spread and the demyelination is very vast. Compared to the CNS, a large
macrophage population is present, along with the CD8+ T cell clusters, and this
type of infiltrating cells is similar to what has been seen in MS patients. We
can hypothesize about a possible mechanism of demyelination and, although we
have not experimentally demonstrated that ROS and NOS are present in the
PNS of symptomatic L31 mice, we can hypothesize, due to the observed nature
of the demyelinated lesions and the widespread presence of macrophages, that
the surrounding macrophages in the areas of the lesions are releasing a soluble
molecule, like ROS or NOS, that is causing more widespread myelin
destruction. This process of demyelination is most likely also mediated by
IFN-γ, as IFN-γ has been shown to be produced by CD8+ T cells in L31 mice
and we have reported that IFN-γ receptor -/- L31 mice do not develop disease
[26]. Furthermore, NOD-B7-2-KO mice that were discussed in the introduction
and who only develop PNS pathology are also resistant to disease if they are
IFN-γ -/- [69].
Another interesting finding from our results is the more mature and phagocytic
phenotype of the macrophages found in the PNS of symptomatic L31 mice.
This is a reasonable finding as the macrophages present in these sciatic nerves
would most likely be phagocytosing a large amount of myelin debris and would
be activated and promoted to mature in their phenotype.
What is also
Page | 46
interesting to point out from the results in section 3.6 is the fact that nonsymptomatic L31 mice do seem to have a larger number of CD45+ cells when
compared to B6 mice as seen from the representative dot plots in figure 6A.
This is different than what is seen in figure 2B where no obvious difference can
be seen in the absolute number of CD8+ T cells between B6 and nonsymptoatic L31 mice. An explanation for this discrepancy is that a different
method of cell isolation was used between the two independent experiments,
mechanical dissociation for the kinetic experiments (Figure 2B) and enzymatic
dissociation was used for the macrophage characterization experiments (Figure
6A). This difference may have yielded more isolated cells in the macrophage
characterization experiments as the enzymatic dissociation method is becoming
increasingly popular as it creates cell suspensions that have a better
representation of myeloid populations. Another possible explanation for this
discrepancy is the small n value used for the experiments. A total of three mice
per mouse group were used for the kinetics study, therefore the representation
of the groups may not have been very thorough. It is possible that a difference
in absolute number of CD8+ T cells in the PNS of non-symptomatic L31 mice
compared to B6 could have been seen if the n value of these experiments was
increased.
Moreover, the presence of a greater number of CD45+ cells in the PNS of nonsymptomatic L31 mice as compared to B6 controls raises the question if there
already is pathology in the PNS of L31 mice at this time point. The fact that
more lymphocytes could be present in the PNS of L31 mice compared to B6 is
Page | 47
not surprising however, as T cells from L31 mice have been shown to have a
more effector memory phenotype (CD44+, CD62L-) [25]. This means that
more T cells are present in the periphery of L31 mice and the T cells can enter
visceral organs to surveille the area, however no pathology has been seen in
other visceral organs in L31 mice, even though a greater number of infiltrating
T cells can be observed when compared to B6 [25]. This fact can further help
explain the increase of infiltrating CD45+ cells in non-symptomatic L31 PNS
compared to B6 and it is also the case that no pathology was seen in the PNS of
L31 mice, until they became symptomatic (figure 4).
As we have shown in the CNS-infiltrating kinetic experiments in section 3.2,
mice as early as 3-4 months of age have infiltrating CD8+ T cells in their CNS
and they also have infiltrating CD8+ T cells in their CNS at the 5-6 months old
time point. However, the results indicate that a greater absolute number of
CD8+ T cells can be found in the CNS of L31 mice at 3-4 months of age (1846
± 483, n=3) compared to the 5-6 months old time point (689 ± 221, n=3). This
difference is not significant (p=0.0953), but there is an obvious drop between
the two time points. One possible explanation for this fact is that the L31 mice
spontaneously develop disease at any point between the ages of 3-6 months of
age but, in our SPF facility, not all L31 mice become symptomatic. This means
that many mice at the 3-4 months time point may be progressing to disease, but,
at the 5-6 months old time point, many of these mice may never develop
neurological symptoms and thus would have fewer CD8+ T cells in their CNS.
Furthermore, it has been a personal observation that L31 mice are a
Page | 48
heterogeneous population and that some mice will develop disease at an earlier
age and develop more aggressive symptoms that leads to sudden death and
other mice will develop disease later on in life and will not develop as severe
symptoms and the progression of their disease would be slower. Therefore,
when sacrificing mice at a 3-4 months old time point, the mice that will develop
a more aggressive disease are present in the group, but when sacrificing mice at
the 5-6 months old time point, none of these mice remain. The decline in
absolute number of CD8+ T cells between the CNS of L31 mice at 3-4 and 5-6
months old can thus be attributed to these reasons.
The concept that CD8+ T cells can mediate a neurological disease in mice is
also a subject that merits thorough discussion. As it has been presented in the
introduction, the most common mouse model used to study demyelinating
disease in mice is the EAE model. This model normally has a Th1/17 response
against a myelin antigen and this causes demyelination in the CNS. Moreover,
it has been shown in Darlington et al. [36] that a diminished Th17 response in
MS patients completely abrogates any RRMS disease. This definitely points
towards CD4+ T cells as the initiators of CNS inflammation, but the many lines
of evidence, which were discussed in the introduction, clearly demonstrate that
CD8+ T cells play a role in the pathogenesis of MS. A recent report from
Nature Immunology [62] may shed some light on a process that occurs that
allows both T cell subsets to be involved in a demyelinating pathology. This
report uses a model of EAE to describe a DC population that cross-presents a
MHC class I-restricted MBP epitope that then activates CD8+ T cells and
Page | 49
allows them to specifically attack oligodendrocytes. The initial demyelination
in this EAE model is also mediated by CD4+ T cells. Therefore the initial
CD4+ T cell-mediated demyelination leads to the activation of CD8+ T cells,
which can directly attack oligodendrocytes, and cause further demyelination.
The question remains however if CD8+ T cells can initiate demyelination
themselves.
In L31 mice, microglia constitutively express B7.2 and we have shown that this
is necessary for disease onset [25]. This may explain why a CD8+ T cellmediated demyelinating disease could be initiated in L31 mice irrespective of
CD4+ T cells. It is plausible that microglia in L31 mice could present a MHC
class I-restricted myelin epitope that would activate local CD8+ T cells because
of the constitutive expression of B7.2 and this would cause the initiating
demyelination. The importance of CNS-resident myeloid cells in the EAE
mouse model was demonstrated in the March 2005 issue of Nature Medicine.
In this issue three articles demonstrate a myeloid lineage cell, albeit a DC,
microglia or macrophage, that is very important in the course of demyelinating
disease in EAE mice. The first demonstrated how EAE was repressed when
microglia were abolished using ganciclovir [75].
The second paper
demonstrates how it is not DCs in the secondary lymphoid organs or the mouse
brain parenchyma that where necessary to permit immune invasion of the CNS,
but most likely a distinct vessel-associated DC, which was also present in
human MS brain tissue, that permitted CNS immune invasion [76]. The last
article uses two mouse models of MS, EAE and Theiler’s murine
Page | 50
encephalomyelitis virus-induced demyelinating disease, to demonstrate that T
cells within the CNS of these mice become specific against different epitopes of
PLP throughout the course of disease and thus epitope spreading initiates and
can occur specifically within the CNS [77]. A news and views article in this
same issue nicely discusses the possibility that the myeloid-lineage APCs that
are presented in all these papers may very well be a very similar or even the
same cell type [78]. The authors of the news views go on to state that the
overall message from all the studies demonstrates that myeloid APCs play a
crucial role in mediating infiltration, presenting myelin antigens, and causing
neuroinflammation and pathology in animal models of MS.
This is also
partially true in L31 mice as the B7.2 expression on microglia is necessary for
disease and therefore the microglia are likely responsible for disease initiation
and to some extent a possible epitope spreading phenomenon.
Taking into consideration the data presented in this thesis and our previous
knowledge of the L31 mice, we propose the following model. CD8+ T cells are
activated in the CNS of L31 mice early on in life due to the expression of B7.2
on microglia and this activation leads to demyelination that may not be due to a
cell specific attack by CD8+ T cells. However, this demyelination causes other
myelin antigens to be exposed and therefore presented by local APCs to other
CD8+ T cells, which would then be activated. Some activated CD8+ T cells
could be specific for a myelin protein that is also found in the PNS, such as
MBP. Through this epitope spreading phenomenon, the activated CD8+ T cells
would then be able to be more readily activated if they recognize their antigen
Page | 51
in the PNS and continue to cause demyelination at this distant site.
The
recruitment of macrophages to the PNS then causes the release of ROS and
NOS that causes more widespread PNS demyelination and the onset of
neurological disease.
The study of L31 mice has implications to both MS and PNS demyelinating
diseases like GBS and CIDP.
The study of L31 mice may help in
understanding mechanisms by which CD8+ T cells can cause demyelination in
both the CNS and PNS.
As well, it demonstrates possible modes of
demyelinating pathology that are exerted by CD8+ T cells in, possibly,
macrophage dependant and independent manners. The importance of CNSresident APCs cannot be overlooked in MS and this fact is also supported by
the L31 mouse model. Finally, this thesis work sheds light on a possible
epitope spreading phenomenon that causes demyelinating diseases to spread
from the CNS to the PNS, an event that does not occur frequently in MS (5% of
patients) [61], but should not be ignored as it needs to be recognized as a
treatable condition.
Page | 52
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