exploring the role of ccr6/ccl20 axis in b cell migration ... · autoimmune encephalomyelitis...
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Exploring the role of CCR6/CCL20 axis in B cell migration into the CNS during EAE
by
Jennifer Yam
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Immunology University of Toronto
©Copyright by Jennifer Yuen-Man Yam 2016
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Exploring the role of CCR6/CCL20 axis in B cell migration into the CNS during EAE
Jennifer Yuen-Man Yam Master of Science
Department of Immunology
University of Toronto
2016
Abstract
B cells have been implicated in the pathogenesis of multiple sclerosis (MS) but how they
migrate into the central nervous system (CNS) is poorly understood. Previous work
demonstrated the use of chemokine CCL20 signaling through CCR6, in driving B cell
migration during various states of inflammation. Using a transwell assay, we determined
that CCR6 expressing B cells are responsive to CCL20 in a dose-dependent manner.
During experimental autoimmune encephalomyelitis (EAE), CCR6 expression is
upregulated on CNS-infiltrated B cells and there is increased serum concentration of
CCL20. However, lack of CCR6 expression on B cells did not affect their ability to
migrate into the CNS nor did it affect the severity of disease in a B cell independent
model of EAE. Our data therefore suggest that the CCR6/CCL20 axis is not the main
driver of B cell migration into the CNS during EAE.
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Acknowledgements
I would like to thank my supervisor, Dr. Jennifer Gommerman, who gave me the
opportunity to work on this great project as well as her guidance, patience and support
throughout my time in the lab. Thank you to my committee members Dr. Shannon Dunn,
Dr. Dan Winer and Dr. Clinton Robbins for all of your helpful comments and advice.
I would also like to thank Dr. Georgina Galicia and Dr. Olga Rojas for teaching me
everything about EAE. Thanks to my labmates, for I learned something new from each of
you and Dennis Lee for helping out with some of my bigger experiments. You guys are
awesome.
Finally, thank you to my significant other, Tim Guo for always believing in me,
encouraging me and supporting me all the way. I could not have done this without you.
Love you so much!
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Table of Contents Table of Contents ……………………………………….………………………… iv
List of Figures…………………………………….……………………………….. vi
List of Abbreviations ……………………………………….…………………….. vii
1 Introduction …………………………………………….……………………... .1
1.1 Overview ………………………………………………………………….. .1
1.2 Multiple Sclerosis ……...………………………………………………….. .2
1.2.1 Overview……………………………………………………………...2
1.2.2 Clinical Presentation………………………………………………....2
1.2.3 Immunopathogenesis ………………………………………………..4
1.3 B cells and MS ……………………………………………………………..6
1.3.1 Overview …………………………………………………………….6
1.3.2 Role of B cells in MS……………………………………………….. .7
1.3.3 B cells as targets for MS therapies …………………………………. .8
1.4 Experimental Autoimmune Encephalomyelitis (EAE)…………………….10
1.4.1 Overview ……………………………………………………………10
1.4.2 B cells in EAE………………………………………………. ……....10
1.5 Leukocyte migration into the CNS…………………………………………11
1.5.1 Barriers of the CNS…………………………………………………. 11
1.5.2 Leukocyte migration ……………………………………………….. 14
1.6 Chemokine receptor expression on B cells…………………………………15
1.6.1 Overview of chemokines and chemokine receptors…………………15
1.6.2 CCR6 and CCL20 …………………………………………………...16
1.7 Thesis Objectives …………………………………………………………..18
1.7.1 Rationale……………………………………………………………. 18
1.7.2 Hypothesis………………………………………………………….. 18
1.7.3 Specific objectives………………………………………………….. 18
2 Materials and Methods ……………………………………………………….. 19
2.1 Mice ……………………………………………………….. ………………19
2.2 In vitro migration assay……………………………………………………. 19
2.3 Preparation of hrMOG protein…………………………………………….. 19
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2.4 EAE induction …………………………………………………………….. 19
2.5 Ex vivo isolation of cells from CNS………………………………………. 20
2.6 Bone Marrow Chimeras ……………………………………………………20
2.7 Enzyme-Linked Immunosorbent Assay (ELISA) for CCL20……………...21
2.8 Real-Time PCR expression analysis ……………………………………… 21
2.9 Flow cytometry……………………………………………………………. 21
2.10 Statistical analysis ………………………………………………………… 22
3 Results ………………………………………………………………………… 23
3.1 Cross-linking of the B cell receptor (BCR) upregulates CCR6 expression
on B cells and enhances CCR6-mediated chemotaxis in vitro…………….. 23
3.2 Up-regulated CCR6 expression on B cells during EAE ………………….. 23
3.3 Increased CCL20 protein expression in the serum ……………………….. 25
3.4 CCR6 expression on B cells does not alter their ability to populate the
spinal cord…………………………………………………………………. 29
3.5 EAE in CCR6 knock-out mixed bone marrow chimeras ………….…...……31
4 Discussion …………………………………………………………………….. 37
4.1 Cross-linking the BCR upregulates CCR6 expression on B cells and
enhances CCR6-mediated chemotaxis in vitro…………………………….. 37
4.2 CCR6 is not involved in B cell migration into the CNS during EAE…….. 38
4.3 Effect of B cell intrinsic CCR6 expression on clinical presentation
of EAE………………………………………………………………………40
4.4 The pleiotropic nature of CCR6…………………………………………… 41
4.5 Conclusion…………………………………………………………………. 42
5 Bibliography ………………………………………………………………….. 43
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List of Figures
Figure 1…………………………………………………………………………….. 3
Figure 2…………………………………………………………………………….. 13
Figure 3…………………………………………………………………………….. 24
Figure 4…………………………………………………………………………….. 26
Figure 5…………………………………………………………………………….. 27
Figure 6…………………………………………………………………………….. 28
Figure 7 ……………………………………………………………………………. 30
Figure 8…………………………………………………………………………….. 33
Figure 9…………………………………………………………………………….. 34
Figure 10…………………………………………………………………………… 36
List of Tables
Table 1…………………………………………………………………………….35
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List of abbreviations
APC: antigen presenting cell
BBB: blood-brain barrier
CAM: cell adhesion molecule
CCL20: chemokine (C-C motif) ligand 20
CCR6: C-C chemokine receptor 6
CFA: complete freund’s adjuvant
CNS: central nervous system
CSF: cerebral spinal fluid
CXCL: chemokine (C-X-C motif) ligand
DC: dendritic cell
EAE: experimental autoimmune encephalomyelitis
Fab: fragment antigen-binding
Fc: fragment crystallizable
FRC: fibroblastic reticular cell
GM-CSF: granulocyte/macrophage colony-stimulating factor
IFNγ: interferon-gamma
Ig- immunoglobulin
IL: interleukin
KO: knockout
LFA-1: lymphocyte function associated antigen-1
LTα: lymphotoxin –alpha
mAB: monoclonal antibody
MBP: myelin basic protein
MHC: major histocompatibility complex
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MMP: matrix metalloproteinase
MOG: myelin oligodendrocyte glycoprotein
MRI: magnetic resonance imaging
MS: multiple sclerosis
OCB: oligoclonal bands
PLP: proteolipid protein
PPMS: primary progressive multiple sclerosis
RRMS: relapsing remitting multiple sclerosis
SAS: subarachnoid space
SPMS: secondary progressive multiple sclerosis
TNF-α: Tumor necrosis factor alpha
VCAM-1: vascular cell adhesion molecule-1
VLA-4: very late antigen-4
WT: wild type
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1 Introduction 1.1 Overview Multiple sclerosis is an inflammatory autoimmune disease of the central nervous system
(CNS) that often leads to motor and cognitive dysfunction [1]. This occurs due to a
breakdown in the blood brain barrier (BBB) that normally limits the entry of large
molecules and cells into the CNS [2]. Clinical trials using B cell depleting (anti-CD20)
therapy have shown an overall decrease in clinical symptoms amongst MS patients as
well as a reduction in the number of lesions measured by magnetic resonance imaging
(MRI) [3]. If B cells are pathogenic in MS, then rather than depleting the B cells,
preventing their migration into the CNS may represent a safer alternative for treating MS
patients.
Several studies have confirmed the presence of B cells in the CNS of humans and
rodents. However, the mechanism by which they migrate into the CNS is currently
unknown. Of interest is the chemokine (C-C motif) receptor 6 (CCR6) and its ligand,
chemokine (C-C motif) ligand 20 (CCL20) in B cell migration during inflammation.
Studies have shown that the involvement of this CCR6/CCL20 axis results in the rapid
recruitment of B cells to the skin during infection and autoimmunity [4] and that CCR6
expression on B cells is required for their migration to the spleen from the blood in
response to systemic inflammation [5]. With regards to CNS inflammation, CCR6 has
been implicated in the migration of Th17 cells into the CNS to initiate experimental
autoimmune encephalomyelitis (EAE), the animal model for MS [6]. Whether this
CCR6/CCL20 axis plays a role in B cell migration during EAE has never been studied.
This thesis will focus on the role of B cell intrinsic CCR6 expression during EAE. The
introduction will give a brief overview of MS, followed by an overview of the roles of B
cells in MS and EAE, the different barriers in the CNS that leukocytes encounter whilst
attempting to migrate into the CNS, and the possible role of CCR6/CCL20 in the context
of B cell migration during EAE.
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1.2 Multiple Sclerosis 1.2.1 Overview
Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS that often leads to
sensory and cognitive dysfunction as well as severe physical disability due to
demyelination and axonal damage [1, 7]. Affecting approximately 2.5 million people
worldwide at a ratio of 3:1 females/males, MS is one of the most common causes of
neurological disability in young working adults [1, 8], with significant socioeconomic
implications. MS is heterogeneous amongst its patients in terms of its disease course and
the symptoms exhibited. The etiology of MS remains elusive but it is believed that it may
be multifactorial, involving viral or environmental triggers, genetics or other factors that
lead to a dysregulated immune system. There is currently no cure for MS but there are
drugs and therapies that can help manage symptoms and modify the disease course. Since
these drugs do not actually halt disease, the damage accumulates over time and the
resulting physical disability can become permanent.
1.2.2 Clinical presentation
There is high variability in the specific clinical manifestations among MS patients as well
as their severity and frequency. Symptoms include loss of coordination and motor
function, hyperreflexia, visual and sensory impairment and cognitive difficulties [9]. One
of the hallmarks of MS is the presence of gadolinium-enhancing lesions in the white
matter of the brain, spinal cord or optic nerves that are detected by magnetic resonance
imaging (MRI). These lesions or plaques indicate a break in the blood- brain- barrier
(BBB) and are focal areas of demyelination and inflammation. The location of these
lesions correlates to the type of symptoms exhibited by the subject, thus explaining the
clinical heterogeneity seen in MS patients. When a patient first experiences neurological
symptoms, they are diagnosed as having a clinically isolated syndrome (CIS). Clinically
definite MS is only diagnosed when the patient experiences a second episode.
Furthermore, there must be evidence of at least 2 lesions in the brain measured by MRI as
well as the presence of oligoclonal immunoglobulin (Ig) bands (OCBs) in the cerebral
spinal fluid (CSF).
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Figure 1. A summary of events leading to clinical MS
(Left) In MS, a patient typically experiences an initial episode of neurological
dysfunction (clinically isolated syndrome), followed by recurring episodes of relapses
and remissions. (Centre) As time progresses, there may be an increase in disability due to
increased axonal loss and brain atrophy with the patient ultimately developing secondary
progressive MS. (Right) With PPMS, there are no periodic episodes of disability and
recovery; instead there is a steady progression of increasing disability.
Relapsing*–remi.ng**disease**
Secondary*progressive*disease*
Primary*progressive*disease*
Disability*
Time*
Clinically;isolated**syndrome*
Brain**volume*
Figure*1*
Disability*
Time*
Relapsing*–remi.ng**disease**
Primary*progressive*disease*
Relapsing*–remi.ng**disease**
Secondary*progressive*disease*
Clinically;isolated**syndrome*
4
There are three main disease subtypes, namely relapsing- remitting MS (RRMS),
secondary progressive MS (SPMS) and primary progressive MS (PPMS) (Figure 1).
Approximately 85% of patients have RRMS where there is an initial period of
neurological dysfunction termed a clinically isolated syndrome, followed by a period of
clinical recovery or remission and then recurring episodes of relapse and remission over
time. Patients with this relapsing-remitting type of disease exhibit characteristic
gadolinium-enhancing lesions in the white matter. As time progresses, approximately 60-
80% of these patients will develop SPMS where symptoms become more severe and their
ability to recover after each relapse diminishes. At this time, the inflammation becomes
less perivascular, often localized to the meninges and sub-pial damage in the cortical grey
matter has been noted. Neurodegeneration becomes the main feature of disease as brain
atrophy and neuronal loss increases. A small subset of patients (about 10%) has a primary
progressive type of MS where there is a continuous increase in disability after the initial
episode and no periods of recovery. Other forms of MS have also been reported such as
progressive-relapsing MS and tumefactive MS, but these cases are rare.
1.2.3 Immunopathogenesis
Although the exact cause of MS is unknown, it is thought to be initiated by a breach in
self-tolerance to myelin or other CNS-derived antigens in genetically susceptible
individuals. Some speculate that such a breakdown in self-tolerance could be triggered by
an environmental antigen [9, 10]. There are 2 models of how MS develops that are still
under debate, the CNS intrinsic model and the CNS extrinsic model. In the CNS intrinsic
model, the hypothesis is that disease may develop due to an inflammatory response
against an unknown CNS-resident inflammatory trigger with immune cell infiltration
being a secondary event [1]. This is based on the theory of immune surveillance being
carried out within the CNS where it has been reported that CNS-derived antigens may be
transported in the CSF allowing them to be sampled by local meningeal macrophages and
potentially presented to T cells [11]. Furthermore, a recent study revealed the presence of
functional lymphatic vessels that line the dural sinuses in the mouse [12], a feature that
has long been thought to be absent from the CNS, implying that CNS-restricted antigens
can be transported through the lymphatic system to the draining (cervical) lymph node.
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This new discovery may help shed light on the etiology of several neurodegenerative
diseases similar to MS.
In the CNS extrinsic/peripheral model, CD4+ T cells may be activated within peripheral
lymphoid tissues (lymph nodes) by dendritic cells (DCs) presenting sequestered myelin
that has somehow escaped the CNS, myelin cross-reactive epitopes through molecular
mimicry, or through bystander activation [10]. These activated T cells then produce
cytokines, which disrupt the BBB and allow the T cells to migrate across and gain access
to the CNS. Once there, these T cells are reactivated as they encounter local major
histocompatibility complex (MHC) class II- expressing antigen presenting cells (APCs)
such as dendritic cells, macrophages and B cells presenting self-antigens. As a result,
more inflammatory cytokines and chemokines are produced, leading to additional
recruitment of inflammatory immune cells such as other CD4+ T cells, B cells and
monocytes to the CNS where they will subsequently attack and damage the myelin sheath
surrounding the neurons. Amidst the demyelination and tissue destruction, the resulting
degraded myelin proteins can be taken up by local APCs and presented to other self-
reactive T cells thus causing the phenomenon known as epitope spreading. Although the
exact process of demyelination and axonal injury remains mechanistically unclear, it has
been thought to be caused by direct injury mediated by CD4+ T cells, CD8+ T cells,
activated microglia and auto-antibodies as well as indirect injury by proinflammatory
cytokines such as tumour necrosis factor- alpha (TNFα), nitric oxide, and matrix
metalloproteinases (MMPs) [13-15].
There are two types of CD4+ TH cells involved in the immunopathogenesis of MS,
namely CD4+ T helper 1 (TH1) and TH17 cells. TH1 cells were initially thought to be the
main effector T cells as they secrete proinflammatory cytokines such as interferon-
gamma (IFNγ), interleukin-2 (IL-2) and TNFα. IFNγ and TNFα can act together to
modulate the expression level of other proinflammatory cytokines as well as the
expression level of chemokines and cell adhesion molecules on BBB endothelial cells to
increase adhesion and leukocyte migration [2]. TH1 cells also provide additional co-
stimulatory help to CD8+ T cells, which directly injure neurons and oligodendrocytes
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expressing MHC I through cell contact –mediated lysis [16-19]. However, TH17 cells are
now also appreciated as being important players in the pathogenesis of MS as studies
showed TH17 cells to be one of the first cell types to gain access into the CNS [20]. These
cells secrete proinflammatory cytokines such as IL-17 and also induce other immune
cells to produce proinflammatory IL-6 and granulocyte/macrophage colony-stimulating
factor [9]. Furthermore, IL-17 has been shown to increase the activation of matrix
metalloproteinase-3 (MMP-3), which breaks down the BBB, thus allowing more immune
cells to infiltrate the CNS [2, 19]. In addition to TH1 and TH17 cells, γδ T cells have also
been implicated in the pathogenesis of MS and EAE but their exact role is unclear.
B cells have also been shown to play a role by producing antibodies that will bind to the
myelin sheath causing injury through complement activation or antibody- mediated
phagocytosis of axons [21]. There are other roles that B cells play outside of antibody
secretion such as antigen presentation and cytokine production.
1.3 B cells and MS 1.3.1. Overview
MS has been traditionally viewed as a T cell-mediated disease but the importance of B
cells in the pathogenesis of MS is becoming more appreciated. This stems from the
surprising results of using anti-CD20 B cell-depleting monoclonal antibodies such as
rituximab [3] and ocrelizumab [22] for MS treatments where not only did patients have a
reduction in their symptoms, but the number of lesions detected via MRI were also
reduced. However, antibody levels in the CSF (oligoclonal bands) and plasma cells
remained unchanged with anti-CD20 treatment, suggesting that apart from antibody
secretion, B cells may also contribute to MS pathology through antibody-independent
mechanisms such as antigen presentation and cytokine production. There are multiple B
cell subsets present in humans and their roles may be innate or adaptive, effector or
regulatory. Since there are still concerns with the safety of widespread B cell depletion,
the use of alternative treatments such as those that prevent B cell migration into the
inflamed CNS are being investigated.
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1.3.2. Role of B cells in MS
It has been well known for over 50 years that B cells are thought to play an important role
in the pathology of MS [23]. This is based on the observation that MS patients have
characteristic OCBs of immunoglobulin present in their CSF and the presence of B cells
and plasma cells in lesions found in the CNS. The specific roles of these B cells in MS
and how they contribute to the disease remains unclear. The initial assumption was that B
cells only had one role in the pathogenesis of MS: to differentiate into plasma cells or
plasmablasts and produce injurious antibodies against myelin that, along with an
inflammatory milieu, will contribute to demyelination or axonal damage [9, 24].
However, B cells have more than just a role in autoantibody secretion and the focus has
now shifted to their antibody-independent roles such as antigen presentation and cytokine
production that may be either pathogenic or immunoregulatory.
Several studies have shown that B cells can play a pathogenic role where they can act as
antigen presentating cells to myelin- specific T cells and provide co-stimulatory signals to
T cells via CD80/86. When B cells are depleted, T cell proliferation, expression of
activation markers and TH1 and TH17 production of proinflammatory cytokines are
reduced, indicating that there is cross talk between these two cell subsets [25]. Studies in
EAE have also shown that B cells are required for the recognition of recombinant myelin-
oligodendrocyte glycoprotein (rMOG) to initiate EAE but not for the recognition of the
short encephalitogenic peptide (MOG35-55) [26]. As demonstrated by Harp et al. DCs
alone were insufficient APCs to induce EAE with hrMOG and B cells were a necessary
requirement for antigen presentation as well for maximal EAE development [27].
B cells can also produce proinflammatory cytokines that can alter T cell function or
promote recruitment of other immune cells to the site of inflammation. When B cells
from MS patients are activated, they produced more proinflammatory cytokines such as
lymphotoxin –alpha (LTα), TNFα and IL-6 compared to healthy controls [28]. These B
cell derived cytokines can promote T cell differentiation into TH1 or TH17 cells. Ectopic
follicles are sometimes found in the meninges of MS patients and these follicles contain
B cells, plasma cells and T cells over a network of fibroblastic reticular cells (FRCs) that
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may in turn support B cell activation, expansion maturation and antibody production [29,
30]. Added to this, studies have shown that the B cells found in the CSF and CNS of MS
patients are clonally inter-related and have undergone immunoglobulin (Ig) isotype class
switching to express IgG and IgA, and somatic hypermutation in the draining cervical
lymph nodes [31, 32].
On the other hand, several studies have shown that B cells may play an
immunoregulatory role in neuroinflammation, such as dampening T cell mediated CNS
inflammation. In EAE, B cells have been shown to aid in the recruitment of regulatory T
cells (Treg) into the inflamed CNS. When these B cells were depleted, the number of Treg
was also reduced [24, 33]. Furthermore, a subset of CD1dhi CD5+ regulatory B cells has
been described to produce anti-inflammatory IL-10. B cells from MS patients have a
reduced capacity to produce IL-10 compared to healthy controls [28] suggesting that
there may be a defect in their immunosuppressive ability. B cells can also produce IL-4,
which induces TH2 differentiation and TH2 cells have been shown to be protective in MS
and EAE. Indeed, interferon beta and glatiramer acetate are two approved MS therapies
that promote TH2 responses [34].
1.3.3 B cells as targets for MS therapies
Given that B cells were originally thought to be the main source of injurious antibodies in
MS, it was intuitive to develop treatments that would deplete them. There are three B cell
depleting anti-CD20 monoclonal antibodies (mAbs) that are currently undergoing
investigation as possible therapies for MS, namely rituximab, ocrelizumab and
ofatumumab [8]. They all target CD20, a surface molecule that is constitutively expressed
on B cells from the pro-B cell stage in the bone marrow to mature circulating B cells in
the periphery. CD20 expression is lost when B cells terminally differentiate into
plasmablasts or plasma cells and therefore this subset is unaffected by these depleting
agents.
Rituximab was the first mAb out of the three to be available as a B cell depletion therapy.
It is a chimeric IgG1 mAb that has murine anti-human CD20 light- and heavy-chain
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variable regions bound to human gamma 1 heavy-chain and kappa light-chain constant
regions [29]. The fragment antigen-binding (Fab) region of rituximab binds to CD20 on
the B cell and the fragment crystallizable (Fc) domain mediates B cell lysis through
processes such as complement-dependent cytotoxicity, antibody-dependent cell mediated
cytotoxicity or apoptosis [29]. B cell depletion is rapid, dramatic and often near complete.
In an open-label add-on study, RRMS patients treated with rituximab had more than 95%
of their B cells depleted, but CSF IgG levels and OCBs remained unchanged [29]. These
patients exhibited reduced clinical symptoms and relapses as well as a significant
reduction in the number of gadolinium- enhancing lesions and the appearance of new
lesions measured by MRI compared to placebo controls. Interestingly, the B cells
returning following depletion have been shown to secrete more anti-inflammatory IL-10
than before treatment, suggesting that the treatment is resetting the B cell population in
these patients. Since rituximab is a chimeric mAb, some patients developed antibodies
against it, termed human anti-chimeric antibodies that may decrease the overall efficacy
of rituximab and increase the risk of infusion reactions [29]. In order to minimize this
humanized versions of rituximab (ocrelizumab) and fully human versions (ofatumumab)
have been generated. For ocrelizumbab, phase II trials in RRMS patients showed a 96%
reduction in the number of gadolinium-enhancing lesions compared to placebo controls
and was considered superior to other MS therapies like interferon beta-1a in reducing
these lesions [22]. In addition, phase III trials in patients with PPMS, of which there is
currently no approved treatment, was recently completed and revealed a significant
reduction in the progression of clinical disability for at least 12 weeks compared to
placebo controls (http://www.roche.com/media/store/releases/med-cor-2015-10-08.htm.)
Since there are still concerns with the safety of widespread B cell depletion, rather than
depleting all B cells, perhaps preventing certain subsets such as the pathogenic
proinflammatory B cells from entering the CNS could be beneficial. Natalizumab is a
mAb that is approved for treatment of RRMS. Its clinical benefit stems from it binding to
the human α4 subunit of the integrin very late antigen-4 (VLA-4) expressed on activated
leukocytes, therefore blocking them from interacting with its endothelial ligand vascular
cell adhesion molecule-1 (VCAM-1) and hence preventing them from crossing the BBB
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into the brain parenchyma [8, 35]. Studies showed that 60% of patients receiving this
treatment were considered disease free either clinically or neurologically (using MRI as
an endpoint), with 40% of patients free of both readouts [36]. This clinical benefit was
originally thought to be due to blocking T cell migration but a study in EAE by
Lehmann-Horn et al. showed that it might also be a result of blocking B cell migration
[35]. They showed that VLA-4 was more highly expressed in B cells than T cells and
EAE mice that selectively lacked VLA-4 on B cells had impaired migration to the CNS,
which reduced the recruitment of other effector immune cells and ultimately resulted in
less severe EAE. With these encouraging results, other molecules involved in B cell
migration such as chemokine receptors and other adhesion molecules are currently under
investigation as potential new targets for MS therapies.
1.4. Experimental Autoimmune Encephalomyelitis (EAE) 1.4.1 Overview
The EAE mouse model has been widely used to study MS, as it resembles the disease in
many aspects. There are different EAE models, namely active EAE, passive EAE and
spontaneous EAE. Typical EAE symptoms include an ascending paralysis starting with a
limp tail, eventual hindlimb and forelimb paralysis and ultimately full paralysis. The
model, however, is not perfect and there are several differences such as the location of
inflammation in the CNS. In EAE, most of the inflammation is in the spinal cord whereas
in MS, inflammation occurs also in the brain. In addition, CD4+ T cells are the dominant
effector cells in EAE whereas in MS it is both CD4+ and CD8+ T cells. Overall, the EAE
model is required for modeling MS and remains an essential tool for MS research and
developing new therapeutic strategies.
1.4.2 B cells in EAE
There are several ways of inducing EAE: a) via active immunization subcutaneously with
myelin-derived peptides emulsified in complete Freund’s Adjuvant (CFA) and intra-
peritoneal injection of Bordetella pertussis toxin b) adoptive transfer (passive EAE) of
pathogenic myelin specific T cells from an actively immunized donor mouse into a naïve
mouse or c) by generating transgenic mice where their T cell repertoire is highly skewed
11
towards T-cell receptors specific for a myelin epitope, provoking the spontaneous
development EAE. For active immunization, the antigen and mouse strain chosen will
mirror different aspects of MS. For example, C57BL/6 mice immunized with the linear
MOG 35-55 peptide will develop a monophasic chronic type of disease. In contrast,
immunization with proteolipid protein (PLP)139-151 into SJL mice yields a disease
phenotype similar to RRMS, including some brain inflammation. In addition, the type of
antigen and the conformation used (peptide versus whole protein) will determine whether
the model is dependent on the presence of B cells for pathology. An example would be
mice immunized with human recombinant MOG (hrMOG) 1-120 which is the full-length
conformational protein that is found on the extracellular portion of the myelin sheath.
When B cell deficient µMT mice are immunized with rhMOG1-120, they are completely
resistant to EAE, in contrast to MOG 35-55 immunized mice [26] . Work by Kuerten et al.
also showed that EAE induction with MP4, a chimeric fusion of myelin basic protein
(MBP) and PLP, requires the presence of B cells [37] thus further emphasizing the
importance of B cells in the pathogenesis of EAE that is provoked by whole protein
immunization. According to McLaughlin and Wucherpfennig, immunization with whole
proteins may represent a more accurate picture of what actually happens in MS. Within
the MS lesions, the myelin antigens that are recognized and taken up by APCs are usually
large fragments of intact proteins as opposed to short peptides. On the other hand,
immunization with peptides such as MOG 35-55 provides an opportunity to evaluate
regulatory functions of B cells without the complications of pathogenic antibody
production. As such, a combination of models must be used in order to fully understand
the complicated processes involved in MS pathogenesis.
1.5 Leukocyte migration into the CNS 1.5.1 Barriers of the CNS
The CNS has long been thought of as an immunologically privileged site. Under normal
conditions, the presence of the BBB, which refers to the endothelial cells lining the CNS
vasculature, restricts the entry of large molecules, leukocytes and soluble mediators from
the periphery into the CNS [2, 38]. However, immune responses do occur against the
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CNS and there are instances where the BBB is breached by a variety of immune cells as
seen in different neurological disorders such as MS. The following discussion will give a
brief overview of CNS anatomy and describe the different CNS barriers leukocytes may
encounter during migration, namely the blood to CSF barrier, blood to subarachnoid
space barrier and finally blood to parenchymal perivascular space.
The brain and spinal cord are surrounded by a three-layer membrane called the meninges
and it is comprised of the outermost dural membrane that lies beneath the skull, the
middle arachnoid membrane and the innermost pial membrane (Figure 2). In between the
arachnoid and pial membranes is the subarachnoid space (SAS), which is filled with CSF
produced from arterial blood by the epithelia cells of the choroid plexus and traversed by
arterial blood vessels. These blood vessels can penetrate into the CNS parenchyma;
giving rise to cerebral perivascular spaces called Virchow-Robin spaces. Surrounding the
external surface of the brain and spinal cord is a network of astrocytic foot processes and
parenchymal basement membrane that form the glia limitans.
As mentioned, leukocytes can gain access to the CNS through different routes. The first
route is from blood to CSF through the choroid plexus. Leukocytes first migrate across
the endothelium of the blood vessel and into the stroma of the choroid plexus. Once
there, they move through the stroma towards the basolateral side of the choroid plexus
epithelium where they will finally cross the epithelial monolayer into the CSF.
This route is supported by studies conducted in healthy mice where fluorescently labeled
lymphocytes were injected intravenously and were found in the choroid plexus stroma
and meninges 2 hours later [39]. In humans, T cells make up approximately 80-90% of
the total cells that may have entered the CSF through the choroid plexus, along with 5%
B cells, 5% monocytes and less than 1% DCs [11].
The second route is from the blood into the SAS. Leukocytes can move across the porous
endothelium of postcapillary venules near the pial membrane and into the SAS and
Virchow-Robin space. It is in these 2 areas that leukocytes may encounter local
meningeal and perivascular macrophages that are capable of antigen presentation to these
13
Figure 2. Barriers of the brain and the possible routes of leukocyte entry
Leukocytes may enter the brain by migrating from the blood through the stroma of the
choroid plexus and into the SAS (1). Leukocytes may also gain access to the SAS by
migrating across the meningeal blood vessels near the pial membrane (2). Leukocytes
may also cross the BBB through the post-capillary venules and gain direct access to the
brain parenchyma (3).
Adapted from Goverman, J., Autoimmune T cell responses in the central nervous system.
Nat Rev Immunol, 2009. 9(6): p. 393-407; Ransohoff, R.M and Engelhardt, B. The
anatomical and cellular basis of immune surveillance in the central nervous system. Nat
Rev Immunol, 2012. 12(9): p. 623-635.
14
patrolling leukocytes, thus making them a site for immune surveillance. Studies in mice
have shown that CD4+ T cells accumulate in the SAS early on in the disease course
where they interact with local APCs before they enter the spinal cord parenchyma [40].
The third and final route of entry is from the blood to the brain parenchyma. Leukocytes
can move across the endothelial basement membrane of postcapillary venules and glia
limitans and directly gain access to the brain parenchyma. This area is also thought to be
a site of antigen presentation due to the presence of microglial cells, which are deemed
the most potent APC in the CNS parenchyma.
1.5.2 Leukocyte migration
Several studies have confirmed the presence of B cells in MS lesions and in rodents but
how B cells migrate into the CNS is still poorly understood. In general, cellular migration
is a tightly regulated process and involves interacting pairs of endothelial selectins and
their carbohydrate ligands, integrins and their corresponding cell adhesion molecules
(CAMs) and chemokines and their chemokine receptors. During inflammation, E-selectin
and P-selectin are induced on the endothelial surface leading to the capture of leukocytes
via binding to P-selectin glycoprotein ligand 1 (PSGL1) and subsequent rolling along the
apical surface of endothelial cells [41]. Chemokines that are produced by the endothelium
or by immune cells are deposited on the endothelial cell surface and bind to their cognate
chemokine receptor on the leukocyte, triggering downstream G-protein-coupled receptor
signaling. This leads to the activation of integrins expressed on the leukocyte cell surface,
such as lymphocyte function associated antigen-1 (LFA-1) and VLA-4. Integrin
activation results in a conformation change from a low-affinity bent structure to a high
affinity upright extended structure. In addition, integrin activation results in clustering at
the membrane thereby increasing their avidity in order to achieve firm adhesion with
their cognate ligands intercellular adhesion molecule-1 (ICAM-1) and VCAM-1
respectively (ICAM-1/VCAM-1 are also induced by the inflammation). At this point, the
leukocytes stop rolling and begin to flatten themselves along the endothelial cell surface.
They crawl along the surface typically guided by a chemokine gradient until they reach
an exit site, which is a transiently opened endothelial cell junction. Here, they will
migrate through the endothelium either trans-cellularly or para-cellularly in a step called
15
diapedesis with the aid of platelet endothelial cell adhesion molecule-1 (PECAM-1),
junctional adhesion molecules (JAMs), endothelial cell-selective adhesion molecule
(ESAM) and other CAMs. Chemokine-chemokine receptor interactions also aid in the
process of diapededis as the cell extends its chemokine receptor enriched processes
through the exit site and migrates across the endothelium along the chemokine gradient
[42].
Within the CNS, studies have shown the importance of P-selectin in leukocyte trafficking
as it is expressed on the choroid plexus stroma and meningeal blood vessels. Blocking P-
selectin or PSGL1 with antibodies showed a decrease in rolling and adhesion and overall
less migration into the CNS [11, 43]. BBB endothelial cells also express a wide range of
CAMs on their surface such as ICAM-1 and VCAM-1 [44]. As mentioned, of most
importance is the interaction between VLA-4 and VCAM-1 since the use of VLA-4
specific blocking antibodies (natalizumab) prevented the migration of lymphocytes
across the BBB and selectively suppressed the accumulation of CD4+ T cells in the CSF
in MS patients. BBB endothelial cells are also a source of pro-inflammatory chemokines
such as CCL2, CCL5, CXCL10 and many others that promote leukocyte migration into
the inflamed CNS [44]. In particular, the CCR6/CCL20 axis has been implicated in
migration of TH17 cells from blood to CSF. Of interest is whether this CCR6/ CCL20
axis applies to B cell migration into the CNS during EAE.
1.6 Chemokine receptor expression on B cells 1.6.1. Overview of chemokines and chemokine receptors
Chemokines are small, secreted proteins that provide guidance cues for lymphocyte
migration. They typically fall under two categories: homeostatic and inflammatory. The
first group of chemokines is constitutively expressed at a basal level and is involved in
lymphoid organ development, leukocyte trafficking and leukocyte homing, while the
latter group is induced by pathogenic or inflammatory stimuli, triggering immune cell
recruitment to the site of inflammation and promoting tumorigenesis or metastasis [42,
45]. Chemokines bind to their cognate chemokine receptors that are also either
16
constitutively expressed or induced on target cells. These chemokines exert their function
through G-protein-coupled receptor signaling, where the αβγ G-protein heterotrimer
dissociates into its α and βγ subunits and the latter triggers downstream signaling of
phospholipase Cγ (PLCγ), MAP kinases, or phosphatidyl inositol-3OH kinase (PI-3K)
[46]. This ultimately leads to various functional outcomes such as adhesion, polarization
or chemotaxis. Following exposure to its ligand, the chemokine receptor is typically
down-regulated by internalization either for degradation or recycling to prevent
overstimulation of the cell [42].
1.6.2. CCR6 and CCL20
B cells express a number of different chemokine receptors on their surface and they have
various roles in homeostasis and inflammation. For example, CXCL13 and CXCR5 are
required for positioning in B cell follicles [47] whereas CXCL12 and CXCR4 are
required for homing to the bone marrow [48]. The role for CCL20 and CCR6 during EAE
has been studied extensively in T cell migration but whether it plays a role in B cell
migration is not known.
CCL20 is the only known chemokine to bind to CCR6 and unlike other chemokines,
CCL20 is involved in both homeostatic and inflammatory conditions, making this an
unusual axis [49, 50]. Under homeostatic conditions, CCR6 is constitutively expressed in
both mouse and humans on a variety of leukocytes including T cells and B cells, and the
level of expression within one cell type is dependent on the subtype, maturation and/or
differentiation stage of the cell [49]. In humans, CCR6 is expressed on mature, naïve B
cells but it is transiently lost at the germinal centre B cell stage upon antigen binding
through the B cell receptor, with only the receptor transcripts and intracellular protein
being detected [49, 51]. CCR6 is re-expressed at the post- germinal centre memory B cell
stage. CCL20 is also typically expressed at a low basal level on a variety of mucosa and
lymphoid-associated tissues during homeostasis as well as by several immune cells [49].
As such, one of the functions of this CCR6/CCL20 axis in the naïve state includes the
recruitment of lymphoid and myeloid cells in the organization and development of gut-
associated lymphoid tissues [50].
17
In the inflammatory state, CCL20 production is increased creating a chemokine gradient
that will attract immune cells towards the site of inflammation. Studies have shown the
involvement of CCR6/CCL20 in lung and gut immunity by recruiting CCR6+ immature
dendritic cells [52]. More recently, it has been shown to be involved in the rapid
recruitment of B cells to chronically inflamed skin, guided by cutaneously expressed
CCL20 [4]. Added to this, another study demonstrated that during homeostasis B cells
use the CXCR5/CXCL13 to home to splenic follicles. However, during systemic
inflammation, CCR6/CCL20 is required for B cells to accumulate within the spleen [5].
With regards to MS, studies have shown elevated levels of CCL20 mRNA expression in
leukocytes from MS patients compared to healthy controls [53] and within CNS lesions,
astrocytes were found to be the main source of CCL20 [54]. CCR6 was also found to be
expressed on CSF T cells of MS patients [55] hence this axis could potentially be
responsible for immune cell infiltration into the CNS of MS patients. However, evidence
of the functionality of CCR6 and CCL20 in MS has only been so far derived from EAE
studies, of which T cells were mainly examined and not B cells. Nevertheless, based on
these studies, CCR6/CCL20 could be a potential therapeutic target on B cells.
18
1.7 Thesis Objectives
1.7.1 Rationale
For over 50 years B cells have long been thought to play a role in the
immunopathogenesis of MS due to their presence in CNS lesions and the presence of
OCBs in CSF. With the success of B cell depletion therapies in the treatment of MS,
there is now renewed interest in determining how B cells contribute to MS as well as how
they migrate into the CNS. I am particularly interested in the chemokine receptor CCR6
and its chemokine ligand CCL20 since it has been demonstrated that CCR6 is involved in
the rapid recruitment of B cells to inflamed skin and to the spleen during systemic
inflammation. With regards to inflammation in the CNS, CCR6 has been implicated in
the migration of Th17 cells guided by CCL20 during EAE. Based on these studies, I
investigated whether CCR6/CCL20 was involved in B cell migration into the CNS during
EAE by first examining CCR6/CCL20 expression during EAE and whether B cells
actually required this CCR6/CCL20 axis to migrate into the CNS.
1.7.2 Hypothesis
B cell intrinsic CCR6 expression plays a role in EAE.
1.7.3 Specific objectives
i) Determine the expression of CCR6 and CCL20 during EAE.
ii) Determine whether B cell intrinsic CCR6 expression is required for B cell migration
into the CNS.
iii) Determine if B cell intrinsic CCR6 expression affects the clinical presentation of
EAE.
19
2 Materials and Methods 2.1 Mice Wild-type (WT) C57BL/6 CD45.2, C57BL/6 CD45.1 and Ccr6-/- mice were purchased
from The Jackson Laboratory. JH -/- mice were obtained from S. Filatreau (DRFZ, Berlin,
Germany). All animals were housed in specific pathogen-free conditions and used at 6-8
weeks of age. All experiments were performed according to animal use protocols
approved by the animal care committee of the Division of Comparative Medicine at the
University of Toronto.
2.2 In vitro migration assay Spleens were harvested from WT and Ccr6-/- mice and mashed through a 70µM nylon
filter in PBS. The cell suspension was washed with PBS and B cells were sorted through
negative selection. B cells were stimulated for 24 hrs with anti-IgM F(ab’)2 (10µg/ml) at
37°C and 5% CO2. The migration assay was conducted in 24-well plates (Costar)
carrying Transwell-permeable supports with a 6-µm polycarbonate membrane. Different
concentrations of recombinant CCL20 were placed in the lower chamber (0ng/ml,
50ng/ml, 100ng/ml, 150ng/ml, 250ng/ml and 500ng/ml). 5x105 B cells were placed in the
upper chamber and were incubated for 3 hrs at 37°C and 5% CO2. The total number of
migrated B cells in the lower chamber was enumerated using a hemocytometer.
2.3 Preparation of hrMOG protein Human rMOG (1-‐120) expressing Escherichia coli was obtained from Drs. Chris
Linington and Nancy Ruddle. hrMOG was expressed and then purified from the bacterial
supernatant using a Ni2+-His-bind resin column (Novagen). The purified rhMOG protein
was analyzed by SDS-PAGE using a 15% polyacrylamide gel stained with Coomassie
blue and confirmed to be pure and of the appropriate molecular weight.
2.4 EAE induction Active EAE was induced by immunizing 6-8 week old mice s.c. with 100µg hrMOG
emulsified in CFA (Sigma) containing 4mg/ml heat-killed Mycobateria tuberculosis
20
(H37RA) (Difco) accompanied by two i.p. injections of 200 ng Bordetella pertussis toxin
(List Biological Laboratories) the day of immunization and 48hrs later. Sham immunized
mice were immunized with CFA only and given the same pertussis treatment. Animals
were observed daily for clinical signs and the EAE severity was scored based on a
modified 16-point scale derived from Giuliani and colleagues: 0-2 for tail paralysis with
0 assigned for no symptoms, 1 for partial paralysis of the tail and 2 for full paralysis of
the tail (limp tail); 0-3 for each of the hindlimbs and forelimbs with 0 assigned for no
symptoms, 1 for weakness and abnormal walk, 2 for dragging of limbs but still
movement and 3 for full paralysis; 0-2 for righting reflex with 0 assigned for normal
righting reflex, 1 for slow righting reflex and 2 for a delay of more than 5 seconds for
righting reflex. The modified scale therefore ranges from 0 (no symptoms) to 16 (fully
quadriplegic mouse with limp tail and significantly delayed righting reflex).
2.5 Ex vivo isolation of cells from CNS Mice were harvested at the indicated time points and perfused with cold PBS. Brains and
spinal cords were dissected out and mashed through a 70µM nylon filter (BD Falcon) in
digestion buffer (HBSS supplemented with 10mM HEPES, 150mM NaCl, 1mM MgCl2,
5mM KCl and 1.8mM CaCl2) and incubated at 37°C with 1mg/ml Collagenase D (Roche
Diagnostics) and 60µg/ml DNase I (Roche Diagnostics) for 30 min. The cell suspension
was gently mixed by pipetting and incubated at 37°C for an additional 15 min before
adding 1mM EDTA and incubating at room temperature for 10 min. The cell suspension
was washed with PBS before re-suspending in 30% Percoll and centrifuged at 2000 rpm
for 20 min without brakes. The fat was aspirated and the cell suspension washed with
PBS before re-suspending in FACS buffer (10% FBS, 0.02% NaN3 PBS) for flow
cytometry.
2.6 Bone Marrow Chimeras For the generation of bone marrow chimeras, mice were irradiated with two doses of
550rad 4-5 hours apart using a MDS-Nordion Gammacell 40 irradiator and subsequently
reconstituted by i.v. injection with 2X106 BM cells from sex-matched donors. For 80:20
mixed chimeras, mice received a 4:1 mixture of either JH -/- and Ccr6-/- BM or a 4:1
21
mixture of WT and Ccr6-/- BM. For 50:50 mixed chimeras, mice received a 1:1 mixture
of either WT and Ccr6-/- BM, or a 1:1 mixture of WT and WT BM. Mice were provided
2mg/ml neomycin-sulfate (Sigma-Aldrich)-supplemented drinking water for 2 weeks
post-irradiation. Mice were used for experiments after 6-8 weeks post-reconstitution.
2.7 Enzyme-Linked Immunosorbent Assay (ELISA) for CCL20 Blood and tissues were harvested at the indicated time-points. Blood was allowed to
coagulate at room temperature and centrifuged, and the sera collected. Harvested tissues
were mashed through a 70µM nylon filter (BD Falcon) in 5ml PBS, pelleted by
centrifugation and the supernatants were collected. The concentration of CCL20 in the
sera and tissue grind were determined by ELISA, according to the manufacturer’s
recommendations (Duoset; R&D Systems).
2.8 Real-Time PCR expression analysis CNS tissues from indicated time points were harvested and stabilized in RNAlater
stabilization solution (QIAGEN). Total RNAs were purified with the RNeasy system
(QIAGEN) followed by DNase treatment with TURBO DNase (Ambion) to eliminate
endogenous DNA according to the manufacturer’s instrucions. RNA (0.5 to 1µg) was
reverse transcribed to cDNA using SuperScript IV Reverse Transcriptase (Invitrogen)
and real-time PCR reactions were performed in triplicate with SYBYR-Green PCR assay
as follows: 10 minutes at 95°C, followed by 40 amplification cycles with 15 seconds at
95°C and 1 minute at 60°C. Primers used were: mouse β-actin forward 5’-
GGCTGTATTCCCCTCCATCG-3’, reverse 5’- CCAGTTGGTAACAATGCCATGT-3’
and specific mouse primer sets for Ccl20 were purchased from QUIAGEN. Expression
fold change was calculated using the 2-(ΔΔCt) method.
2.9 Flow cytometry Cells were washed with ice-cold PBS twice before adding LIVE/DEAD Fixable Aqua
Dead Cell stain for 30mins at 4°C. The cells were then washed twice with PBS before
adding pre-determined concentrations of fluorochrome-labelled Abs in a total volume of
50µl of FACS buffer. The cell suspension was thoroughly mixed and incubated for
22
30mins at 4°C. The following antibodies were used: CCR6 - Pe, CD86 – APC-eFluor780,
CD19 - PercPCy5.5, B220 - BV605, CD45.1- FITC and CD45.2 – APC.
2.10 Statistical Analysis Statistical analysis was performed using GraphPad Prism software with either Mann-
Whitney or ANOVA. Results are reported as mean ± SD with p value of < 0.05
representing significance.
23
3 Results
3.1 Cross-linking of the B cell receptor (BCR) upregulates CCR6
expression on B cells and enhances CCR6-mediated chemotaxis in
vitro Multiple studies have demonstrated the presence of B cells within the CNS during EAE
but the mechanism(s) involved in their migration into the CNS remains unknown. Th17
cells have been shown to utilize the CCR6/CCL20 axis for migration [6]. I therefore
examined whether B cells also use CCR6 to migrate towards CCL20 by using a
Transwell assay. To test this, B cells were first purified from the spleens of WT and KO
mice by magnetic negative selection and cultured with or without anti-IgM for 24 hours.
As expected, only WT B cells upregulated CCR6 expression upon stimulation as shown
in Figure 3A. Both WT and KO B cells were, however, capable of being stimulated by
the anti-IgM as shown by the comparable upregulation of the activation marker, CD86.
Activated WT versus Ccr6-/- B cells were then exposed to increasing concentrations of
CCL20 or media alone as a control. As shown in Figure 3B, activated WT B cells
exhibited the greatest migration towards CCL20 in a dose-dependent manner. Naïve WT
B cells exhibited a slightly lower migration response but were able to migrate
nonetheless, especially at higher concentrations of CCL20. B cells that lacked CCR6,
regardless of whether they were activated or not, were unable to migrate towards CCL20.
Therefore CCR6 is expressed and is functional on the surface of WT B cells.
3.2 Up-regulated CCR6 expression on B cells during EAE Given the in vitro data that indicated a functional CCR6/CCL20 axis on B cells, I next
examined the CCR6/CCL20 axis in the context of EAE. To better understand the
expression levels of CCR6 on CNS-infiltrated B cells, I set up a kinetics experiment
where I immunized wild-type (WT) B6 mice with hrMOG1-120 and harvested the CNS
prior to clinical onset, at both the disease peak and during the chronic stages of disease,
using unimmunized mice as a control group (Figure 4A). With the gating strategy shown
in Figure 4B, I first examined the presence of CNS-infiltrating B cells at the indicated
24
Figure 3. Cellular activation up-regulates CCR6 expression on B cells and CCL20
preferentially induces chemotaxis of activated B cells.
Purified WT and KO B cells were cultured with or without anti-IgM F(ab’)2 for 24 hrs.
A) Stimulated (black line) cells and unstimulated (grey solid) cells were stained for
CCR6 and CD86. B) 5x105 B cells from A were loaded into the upper chambers of a 24-
well Transwell plate with 0, 50, 100, 150, 250 and 500 ng/ml of CCL20 in the lower
chamber, and the cells migrating into the lower chamber were counted following a 3hr
incubation. The values shown are the means from triplicate wells along with SDs. Data is
representative of 3 independent experiments. 2-way ANOVA, ** and *** denotes
significance [(P<0.01) and (P<0.001) respectively] compared to WT unstimulated.
B)!
Figure 3. Cellular activation up-regulates CCR6 expression on B cells and CCL20 preferentially induces chemotaxis of activated B cells. Purified WT and KO B cells were cultured with or without anti-IgM F(ab’)2 for 24 hrs. A) Stimulated (black line) cells and unstimulated (grey solid) cells were stained for CCR6 and CD86. B) 5x105 B cells from A were loaded into the upper chambers of a 24-well Transwell plate with 0, 50, 100, 150, 250 and 500 ng/ml of CCL20 in the lower chamber, and the cells migrating into the lower chamber were counted following a 3hr incubation. The values shown are the means from triplicate wells along with SDs. Data is representative of 3 independent experiments. 2-way ANOVA, ** and *** denotes significance [(P<0.01) and (P<0.001) respectively] compared to WT unstimulated .
Media 50 10
015
025
050
00
2×104
4×104
6×104
8×104
1×105
CCL20 concentration (ng/ml)
Num
ber o
f Mig
rate
d B
cells
WT unstimWT stimCCR6-/- unstimCCR6-/- stim
**
*** ***
N.S.
*
***
***
N.S.
**
CCR6!
WT ! CCR6-/-!
B220+ CD19+!
CD86!
A)!
0 102 103 104 1050
20
40
60
80
100
85
0 102 103 104 1050
20
40
60
80
100
87.5
0 102 103 104 1050
20
40
60
80
100
3.35
0 102 103 104 1050
20
40
60
80
100
68
87.5! 85!
68! 3.35!
25
time-points. As the disease progressed, there was a trend towards an increase in the
representation of B cells among live leukocytes in the spinal cord compared to naïve mice
however this did not reach significance (Figure 4C). In terms of absolute numbers, there
was a significant increase in the number of B cells in the brain at the peak of disease and
a trend towards an increase in the number of B cells in the spinal cord during the chronic
stages of disease (Figure 4D). I next examined CCR6 expression on CNS-resident B
cells. I found that CCR6 was induced on the surface of the B cells in the brain (Figure
5A) and spinal cord (Figure 5B) of immunized mice compared to umimmunized mice in
terms of percentage and mean fluorescence intensity (MFI) as shown in Figure 5C. Thus,
EAE induction results in an increase in the number of B cells infiltrating the brain and
these B cells have upregulate their CCR6 expression.
3.3 Increased CCL20 protein expression in the serum CCL20 is the only chemokine ligand known to bind to CCR6. As several reports have
indicated, CCL20 is produced by several cell types within the CNS namely astrocytes,
microglia and epithelial cells of the choroid plexus. Since there was an induction of
CCR6 on the B cells infiltrating the CNS, I examined whether there was also an induction
of its ligand within the CNS. I first analyzed the serum by ELISA and I found that there
was increased CCL20 expression in the immunized mice compared to unimmunized
controls (Figure 6A). When I analyzed the supernatants of mashed CNS-tissue, there
were no significant changes in CCL20 expression at the protein level course of the
disease (Figure 6B). However, real-time PCR analysis of these CNS tissues showed an
increase in CCL20 mRNA levels in both the brain and in the spinal cord (Figure 6C).
These data indicate that EAE induction triggers production of CCL20 that is detectable in
the blood.
26
Figure 4. B cell infiltration into the CNS
A) Clinical scores of immunized mice with naïve mice as a control. Blue arrows indicate
harvest time-points. B) Gating strategy for B cells from the CNS. C) Percentage of
B220+CD19+ B cells from live cell gate for brain and spinal cord. D) Absolute numbers
of B cells for brain and spinal cord. n=5-6. Each column represents means with SDs and
data are representative of 3 independent experiments. ANOVA, * denotes significance
(P<0.05) when compared to naïve group.
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC-H
96.6
0 102 103 104 105
<V525/50-A>: AQUA
0
50K
100K
150K
200K
250K
SS
C-H
65.2
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B53
0/30
-A>:
FIT
C C
D19 0.0954
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A 30.1
A)! B)!
0 2 4 6 8 10 12 14 16 18 20 22 24 26 280
2
4
6
8
10
12
14
16
Days post-immunization
Clin
ical
sco
re
ImmunizedNaive
Pre-onset
Peak Chronic
C)!
D)!
Naive
Pre-o
nset
Peak
Chronic
0.0
0.5
1.0
1.5
2.0
2.5
% o
f B c
ells
from
live
gat
e
Brain
Naive
Pre-o
nset
Peak
Chronic
0.0
5.0×103
1.0×104
1.5×104
cell
num
ber
from
tota
l lym
phoc
ytes
Brain
*
Naive
Pre-o
nset
Peak
Chronic
0.0
0.5
1.0
1.5
2.0
% o
f B c
ells
from
live
gat
e
Spinal Cord
Naive
Pre-o
nset
Peak
Chronic
0
1×103
2×103
3×103
4×103
cell
num
ber
from
tota
l lym
phoc
ytes
Spinal Cord
FSC-A!
SSC-A!
SSC-W!
SSC-H!
Aqua!
SSC-H!
B220!
CD19!
27
Figure 5. Increased CCR6 expression on CNS-infiltrating B cells
Percentage of CCR6+ B220+ CD19+ B cells in the brain A) and spinal cord B) at various
time-points. C) MFI of CCR6 compared to sham immunized and Ccr6-/- mice. n=3-6.
Each column represents means with SDs and data are representative of 3 independent
experiments. ANOVA, * and ** denotes significance [(P<0.05) and (P<0.01)
respectively] compared to naïve group
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 2.05
97.40
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105<B
576/
26-A
>: P
E C
CR
60 0
1000
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 4.66
95.30
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 7.82
92.20
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 10.6
89.40
CCR6 KO Naïve Pre-onset Peak Chronic !
CD19!
CCR6
!
A) Brain!
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 0
1010
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105<B
576/
26-A
>: P
E C
CR
60 2.63
97.40
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 8.33
91.70
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 17
830
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 1.85
98.10
CCR6 KO Naïve Pre-onset Peak Chronic!
CD19!
CCR6
!
B) Spinal cord!
0" 2.05" 4.66" 7.82" 10.6"
0" 1.85" 2.63" 8.33" 17"
KONaive
Pre-onset
Peak
Chronic
0
50
100
150
200
250
MFI
Brain
***
KONaive
Pre-onset
Peak
Chronic
0
50
100
150
200
250
300
MFI
Spinal Cord
*
*
KONaive
Pre-onset
Peak
Chronic
0
50
100
150
200
250
MFI
Brain
***
KONaive
Pre-onset
Peak
Chronic
0
50
100
150
200
250
300
MFI
Spinal Cord
*
*
C)!
28
C
Figure 6. CCL20 expression during EAE A) CCL20 concentrations determined by ELISA. B) CCL20 levels in the brain and spinal
cord normalized to per mg of total protein. Each column represents means with SDs and
data are representative of 3 independent experiments. C) Real-time PCR measuring Ccl20
mRNA levels in brain (n=3-5) and spinal cord (pooled from 3 independent experiments).
ANOVA, * denotes significance [(P<0.05)] compared to naïve group.
A
B
Naive
Pre-onset
Peak
Chronic
0
100
200
300
400
CCL2
0 pg
/ml
Serum
*
Naive
Peak
Chronic
0
1
2
3
4
Rel
ativ
e fo
ld d
iffere
nce
Brain
**
Naive
Peak
Chronic
0
20
40
60
80
100
Rel
ativ
e fo
ld d
iffere
nce
Spinal cord
Naive
Pre-onset
Peak
Chronic
0
100
200
300
400
CCL2
0 pg
/mg
of to
tal p
rote
in
BrainN.S.
Naive
Pre-onset
Peak
Chronic
0
100
200
300
400
CCL2
0 pg
/mg
of to
tal p
rote
in
Spinal cordN.S.
29
3.4 CCR6 expression on B cells does not alter their ability to populate
the spinal cord Having shown that B cells require CCR6 to migrate towards CCL20 in vitro, I next
wanted to determine whether B cells require the CCR6/CCL20 axis for entry into the
CNS during EAE. A competitive bone marrow chimera assay was set up where a 1:1
ratio of congenically marked WT vs WT and WT vs Ccr6-/- bone marrow cells were
intravenously transferred into irradiated WT recipients (Figure 7A). After 8 weeks of
reconstitution, the mice were immunized with hrMOG1-120 and there was no difference in
the incidence or severity of disease between the two groups (Figure 7B). Using the gating
strategy shown in Figure 7C, I assessed the relative fitness of Ccr6-/- B cells compared to
WT B cells for entry into the CNS at different time-points over the course of disease, as
indicated in Figure 7B. I found that there was no competitive advantage for WT B cells to
migrate into the CNS compared to Ccr6-/- B cells at any of the 3 indicated time-points
(data shown in Figure 7D represents the chronic stage of disease). Using the congenic
markers, I also examined the entry of T cells into the CNS since Th17 cells have been
shown to use CCR6 to enter the CNS. However, I did not observe a difference in the
relative fitness of Ccr6-/- T cells in their ability to enter the CNS (data shown in Figure
7D represents the chronic stage of disease). This data suggests that B cell and T cell
entry into the inflamed CNS during EAE does not appear to require CCR6 since those
that lacked the chemokine receptor were able to populate the CNS to similar frequencies
as WT cells.
30
Figure 7. CCR6 is not required for B cells and T cells to enter the CNS.
A) CD45.1 WT and CD45.2 Ccr6-/- B bone marrow were mixed in a 1:1 ratio and
transferred into irradiated mice. CD45.1 WT and CD45.2 WT bone marrow was also
mixed 1:1 and injected into separate irradiated mice as a control. B) Clinical scores after
immunization with hrMOG1-120. Blue arrows indicate time-points at which experiment
was performed. Data shown are from the chronic stages of disease. C) Gating strategy
used for determining the relative migration fitness. D) Relative migration fitness of WT
vs Ccr6-/- B cells and T cells. (WT:WT n=3, WT: Ccr6-/- n=5). Ratios of relative
migration fitness were determined by dividing the percentage of CD45.1 by the
percentage of CD45.2 and then normalizing to the ratios obtained from the spleen.
CD45.1 WT : CD45.2 CCR6-/- !1:1!
CD45.1 WT : CD45.2 WT !!
1:1!
VS!
CD45.1 WT! CD45.1 WT!
A) !
0 2 4 6 8 10 12 14 16 18 20 22 240
2
4
6
8
10
12
14
16WT+ WT WT+ CCR6-/-
Days post-immunization
Clin
ical s
core
s
D7
Onset
Chronic
B)!
D)!
WT +WT
WT + CCR6-/
-0.0
0.5
1.0
1.5
2.0
Rel
ative
mig
ratio
n fit
ness
of T
cel
ls
CD4+ T cellsB220+CD19+ B cells
WT +WT
WT +CCR6-/
-0.0
0.5
1.0
1.5
2.0
Rel
ative
mig
ratio
n fit
ness
of B
cel
ls
B) !
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SSC-A
51.9
0 50K 100K 150K 200K 250KSSC-W
0
50K
100K
150K
200K
250K
SSC-H
95.8
0 102 103 104 105
<V525/50-A>: AQUA
0
50K
100K
150K
200K
250K
SS
C-H 93.6
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B69
5/40
-A>:
PE
RC
P C
D19 29.2
0 102 103 104 105
<R660/20-A>: APC CD45-2
0
102
103
104
105
<B53
0/30
-A>:
FIT
C C
D45
-1
41.3 0.771
57.40.526
FSC-A!
SSC
-A!
SSC-W!
SSC
-H!
Aqua!SS
C-H!
B220!
CD
19!
CD45.2!
CD
45.1!
C)!
31
3.5 EAE in CCR6 knock-out mixed bone marrow chimeras To study whether the expression of CCR6 on B cells has an impact on overall EAE
incidence, onset and severity, I generated mixed bone marrow chimeric mice whereby B
cell deficient (JH-/-) bone marrow was mixed with Ccr6-/- bone marrow at a ratio of 80:20
respectively. These chimeric mice will lack CCR6 expression on all B cells as well as
lacking CCR6 on 20% of all other hematopoietic cells (B-CCR6 KO – Figure 8A). As a
control WT bone marrow was also mixed at the same ratio with Ccr6-/- bone marrow
(Control- Figure 8A) and in these mice the majority of B cells express CCR6. The
resulting chimerism at 6-8 weeks post-reconstitution is shown in Figure 8B.
We first examined whether immunization of mixed chimeric mice with linear MOG
peptides had an impact on the clinical presentation of EAE. Accordingly, at 6-8 weeks
post-reconstitution, chimeric mice were immunized with MOG35-55. Following
immunization, I observed no difference in disease severity between the 2 groups (Figure
9A). Furthermore, there were no statistically significant differences between the 2 groups
for the day of disease onset or peak clinical scores (Figure 9B). Since B cells have been
shown to play a regulatory role (but not a pathogenic role) during MOG35-55EAE, these
data indicate that B cell-intrinsic expression of CCR6 is not required for B-regulatory
function in this setting.
In contrast to MOG35-55EAE, B cells play a pathogenic role during rhMOG1-120 -induced
EAE. To test whether expression of CCR6 on B cells is required for pro-inflammatory B
cell function during rhMOG1-120 induced EAE, I immunized CCR6 mixed BM chimeric
mice as in Figure X with rhMOG1-120. Overall, while day of onset between both groups of
mice was roughly equivalent, the incidence of disease in B-CCR6 KO mice was lower
than control mice when examined over 6 independent experiments. However, in terms of
the severity of disease, the average score of B-CCR6 KO mice was variable. For
example, severity of disease in B-CCR6 KO mice was higher compared to control mice
in experiments 1 and 2 but in 4 subsequent experiments, B-CCR6 KO mice exhibited
either no difference or reduced severity of disease.
32
Since pathogenic class switched antibodies are produced during in response to rhMOG1-
120 immunization, serum anti-rhMOG1-120 IgG1 titers were measured in the final 2
experiments. In these cases where there was lower disease severity in B-CCR6 KO mice,
I found that their titres were also lower than control mice (Figure 10A).
In conclusion, expression of CCR6 by B cells had a variable effect on EAE induced by
immunization with rhMOG1-120, however preliminary data suggests that B cell intrinsic
CCR6 expression may be required for the generation of normal levels of anti-rhMOG1-120
IgG1 titers.
33
Figure 8. Generation of mixed bone marrow chimeras
A) Chimera set up where Ccr6 deficient bone marrow was mixed with either WT or B
cell deficient bone marrow at a ratio of 80:20. 2x106 cells were transferred i.v. into
irradiated WT recipients. B) Flow cytometric analysis of blood post reconstitution
showing CCR6+ frequencies within CD19+B220+ B cells prior to immunization.
Groups! 80%! 20%!B – CCR6 KO!
Jh-/-! CCR6-/-!
Control ! WT! CCR6-/-!
DONORS!B cell!
T cell!
CCR6!
No B cells!express CCR6!
RECIPIENTS!
WT!
WT*
Majority of B cells!express CCR6!
A)!
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B69
5/40
-A>:
PE
RC
P C
D19
36.5
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 0.4
99.60
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B69
5/40
-A>:
PE
RC
P C
D19
48.1
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 20.3
79.70
B220!
CD19!
CD19!
CCR6
!
B220!
CD19!
CD19!
CCR6
!
0.4*
20.3*
36.5*
48.1*
B;CCR6*KO*
Control*
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B69
5/40
-A>:
PE
RC
P C
D19
36.5
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 0.4
99.60
0 102 103 104 105
<V 605/20-A>: BV605 B220
0
102
103
104
105
<B69
5/40
-A>:
PE
RC
P C
D19
48.1
0 102 103 104 105
<B695/40-A>: PERCP CD19
0
102
103
104
105
<B57
6/26
-A>:
PE
CC
R6
0 20.3
79.70
B220!
CD19!
CD19!
CCR6
!
B220!
CD19!
CD19!
CCR6
!0.4!
20.3!
36.5!
48.1!
B-CCR6 KO!
Control!
B)!
34
A)
B)
Figure 9. Lack of CCR6 expression on B cells has no effect on EAE severity when
immunized with MOG peptide. A) Graph showing clinical scores following immunization with MOG35-55. B) Graphs
showing day of disease onset and peak clinical scores. Data is representative of 2
independent experiments. (B-CCR6 KO: n=7, Control: n=7)
0 2 4 6 8 10 12 14 16 18 20 22 240
2
4
6
8
10
12
14
16
Days post-immunization
Clin
ical s
core
s
B-CCR6 KOControl
Control
B-CCR6 K
O0
5
10
15
20
Day
of d
isea
se o
nset
Day of disease onset
Control
B-CCR6 K
O0
5
10
15
20
Dise
ase
peak
sco
res
Disease peak scoresC)! D)!
Control
B-CCR6 K
O0
5
10
15
20
Day
of d
isea
se o
nset
Day of disease onset
Control
B-CCR6 K
O0
5
10
15
20Di
seas
e pe
ak s
core
sDisease peak scores
N.S. N.S.
35
Table 1: Summary of EAE incidence, average score and day of onset in 80:20 mixed BM chimeras immunized with rhMOG protein.
Table summary of EAE in B-CCR6 KO and Control mice Values shown for disease
incidence are presented as diseased mice/total mice; average scores and day of onset are
shown as mean ± SD. Data shown are from 6 independent experiments with n=7-9 per
group. Mann-Whitney, * and ** denotes significance (P<0.05) and (P<0.01) respectively.
Expt. No.
Disease incidence Average score Day of Onset
B-CCR6 KO
Control B-CCR6
KO Control
B-CCR6 KO
Control
1 8/8
(100%)
8/8
(100%) 6.6 ± 1.3 4.8 ± 0.7
* 10.8 ± 1.3 11.3 ± 2
2 8/9
(89%)
8/9
(89%) 4.1 ± 1.7 3.5 ± 2.1 13.5 ± 2.3 15.4 ± 3.8
3 7/7
(100%)
7/7
(100%) 5.4 ± 0.9 5.7 ± 1.1 11.0 ± 0.6 11.0 ± 0.6
4 4/7
(57%)
6/7
(86%) 2.2 ± 2.8 5.9 ± 1.1
* 12.4 ± 2.8 10.0 ± 1.1
5 6/8
(75%)
8/8
(100%) 2.4 ± 1.2 3.8 ± 1.6
* 14.4 ± 1.8 13.0 ± 1.8
6 8/8
(100%)
8/8
(100%) 3.4 ± 2 4.1 ± 0.9 14.5 ± 1.3 11.9 ± 1.6
**
Average 86.8% 95.8% 4.0 ± 1.6 4.6 ± 0.9 12.8 ±1.5 12.1 ± 1.7
36
Figure 10. Lower anti-rhMOG1-120 IgG1 levels in B-CCR6 KO mice.
A) Graph showing anti-rhMOG1-120 IgG1 levels from serum at indicated time points
during disease. Data shown are pooled from 2 independent experiments (B-CCR6 KO:
n=8, Control: n=8). 2way ANOVA, * and **** denotes significance (P<0.05) and
(P<0.0001) respectively.
D7D12 D16 D22
0
100
200
300
400
Days post-immunization
IgG
1 (µ
g/m
l)
B CCR6 KOCONTROL
*
****
ns
37
4 Discussion This study focused on exploring the role of the CCR6/CCL20 axis in facilitating B cell
migration into the CNS during EAE, and whether lack of CCR6 expression has an affect
on EAE. To summarize my findings, the in vitro data showed CCR6 protein was
expressed on the surface of B cells, and that B cell intrinsic CCR6 is functionally
responsive to a CCL20 chemokine gradient. However, my in vivo data showed that while
I did observe an up-regulation of CCR6 on CNS-infiltrated B cells, there were no
changes in its ligand CCL20 within the CNS when I looked at protein expression in the
brain and spinal cord. When WT B cells were compared head to head with CCR6 KO B
cells in the context of 50:50 competitive bone marrow chimeras, the expression of CCR6
made no difference in the ability of B cells to migrate to the CNS before onset of disease,
at onset and at chronic time points. This was also true for T cells at onset and the chronic
time point. Lastly, to determine whether CCR6 deficiency on B cells affected EAE, mice
lacking CCR6 specifically on B cells were immunized with MOG35-55 and they did not
exhibit any differences in disease severity. Overall, the data suggests that CCR6/CCL20
may not be a dominant player in driving B cell migration into the CNS during EAE.
4.1 Cross-linking the BCR upregulates CCR6 expression on B cells
and enhances CCR6-mediated chemotaxis in vitro Previous EAE studies have shown that Th17 cells rely on CCR6 to migrate and enter the
CNS in a CCL20-dependent manner [6]. In terms of B cells, the role of this chemokine
axis has never been examined in EAE. Instead there have only been studies involving
systemic inflammation and locally inflamed skin [4, 5]. In these studies, they show that
CCL20 expression was up-regulated during inflammation and B cells used CCR6 to
migrate towards it. The transwell experiment confirmed this concept where I observed
activated B cells exhibiting higher CCR6 expression and migrated towards CCL20 in a
dose dependent manner as shown in Figure 3B. This is consistent with previous reports
where only activated B cells responded well to CCL20 [56]. Furthermore, migration was
dependent on CCR6 expression as B cells that lacked CCR6 were unable to migrate.
38
Naïve B cells migrated only towards higher concentrations of CCL20, perhaps due to the
basal level of CCR6 expression on their surface [57].
4.2 CCR6 is not involved in B cell migration into the CNS during
EAE We next wanted to investigate the role of CCR6/CCL20 during EAE, the animal model
of MS. From my results, B cells that were found in the CNS had higher CCR6
expression. However, I did not observe any changes in CCL20 protein expression in the
CNS during EAE. One reason could be that by the peak stage of disease CCR6
expressing immune cells may have bound and internalized CCL20 thus lowering its
expression. Assuming CCR6 positive cells need to migrate to the CNS to initiate disease,
looking at disease onset (Day 9-10) could be a better time-point to measure CCL20 levels
when cells are still on the move towards their target sites. However, if CCL20 was
internalized, we should also see a down-regulation of its receptor CCR6 but instead we
see the opposite as shown in Figure 5 where CCR6 expression increased. Another reason
that could explain why I did not see any changes in CCL20 is that the ELISA assay was
not sensitive enough since I was able to detect increased CCL20 mRNA levels in the
CNS tissue.
With the competitive chimeras, B cells lacking CCR6 expression were still able to
infiltrate the CNS. This suggests that CCR6 is not required for B cell migration into the
CNS during EAE, even though this is the opposite from what I saw in the transwell
experiment. In addition, even if CCL20 was induced in the CNS, it could be possible that
CCR6/CCL20 is not the main driving force of B cell migration in this EAE model and
some other chemotactic pathway may be drawing the B cells into the CNS (ie,
redundancy). There are three possible reasons that could explain this. Firstly, with the
transwell assay, the B cells were only exposed to one chemokine (CCL20), and without
any other competing chemotactic cues present, these B cells can readily move towards
CCL20. During EAE, however, the B cells are exposed to other chemokines. Astrocytes
are known producers of CCL2 and CXCL10 during EAE and leukocytes found in the
perivascular space also express CCL3, CCL4 and CCL5 to name a few [58]. Stromal
39
cells in the meninges have also been shown to produce CXCL13 during EAE and
inhibition of lymphotoxin β receptor (LTβR) signaling reduced both CXCL13 and B cell
accumulation in the meninges [30]. It is therefore possible that other chemokines are
playing a role in driving B cell migration into the CNS during EAE. Secondly, we did not
examine specific B cell subsets within the CNS in the competitive bone marrow
chimeras. It is possible that only certain subsets of B cells are reliant on CCR6 for
migration. Thirdly, the anatomical localization of WT versus Ccr6-/- B cells in the CNS
may differ. As reported by Reboldi et al., T cells in Ccr6-deficient mice were found
trapped between the epithelial and endothelial basement membranes of the choroid
plexus [6]. Thus, it could be possible that B cells do not require CCR6 to reach the
meninges and/or Vichrow Robbin's space, but need CCR6 to enter into the parenchyma.
Hence it would be worth studying the localization of WT versus Ccr6-/- B cells using
congenic markers and identifying the location of these B cells via immunofluorescence.
From my results, I saw no difference in the migration potential of T cells lacking CCR6
at the peak and chronic time-points, however additional markers are needed to
specifically determine if Th17 cells require CCR6 for accumulation in the CNS.
Therefore, it would be important to look at the specific migration potential of Th17 cells
to ensure that our competitive bone marrow chimeras support what is already being
shown in the literature. Specifically, Reboldi et. al showed that Th17 cells were
dependent on CCR6 to enter the CNS and initiate disease onset. In the Reboldi paper,
they go on to show that EAE is a two-step process where the first wave of T cell entry
into the CNS is CCR6-dependent followed by a second wave of T cell and other immune
cell entry that is CCR6-independent [6]. When they transferred WT naïve T cells into
Ccr6 deficient mice, the WT T cells were the dominant cells in the CNS during the
asymptomatic stage of disease in the CNS but later on during active disease the
parenchymal infiltrates were mostly endogenous Ccr6-/- T cells. They reasoned that
following the first wave of Th17 cells these Th17 cells become activated by local APCS
presenting self-antigen and begin producing cytokines and chemokines that will activate
the BBB. This will then allow the entry of other inflammatory immune cells into the CNS
that is independent of CCR6 expression. Hence this may be the reason why we see equal
40
ratios of both WT and Ccr6-/- B cells in the CNS of our chimeras. The WT B cells could
be part of the first wave of immune cell infiltration into the CNS followed by a second
wave of B cell infiltration that is not dependent on CCR6 expression. Further experiments
are therefore needed to further elucidate this concept.
4.3 Effect of B cell intrinsic CCR6 expression on clinical presentation
of EAE We next wanted to determine whether CCR6 deficiency on B cells had an effect on the
course of EAE in terms of day of onset, incidence and disease severity. When these mice
were immunized with MOG35-55, we found that they have similar disease scores with
mice that have WT B cells. In this EAE model, immunization with MOG35-55 results in a
B cell-independent model where B cells can play more of a regulatory role instead of a
pathogenic role. They can produce anti-inflammatory cytokines such as IL-10 instead of
pathogenic antibodies [59]. Since I did not see any differences in disease course, it
suggests that the B cell regulation of pro-encephalitogenic T cell responses is not affected
by the lack of CCR6 expression in B cells, otherwise the mice that lacked CCR6 on B
cells would have exhibited more severe EAE compared to mice with WT B cells.
However, following immunization with rhMOG1-120, B-CCR6 KO exhibited variable
presentation of clinical disease compared to depending on the experiment. This
variability in clinical presentation could be due different batches of rhMOG1-120, and
indeed the first 2 experiments used an entirely different batch of rhMOG1-120 compared to
the final 3 experiments. It is possible that some batches of rhMOG1-120 the protein is too
compacted during the re-folding dialysis thus obscuring the epitope required for uptake
by APCs and leading to milder disease. Further experimentation is required to determine
the source of variability in these experiments.
I also found that anti- rhMOG1-120 IgG levels were lower in the serum of B-CCR6 KO
mice compared to control mice. Of interest is the recent finding that B cells require CCR6
for correct positioning within the subepithelial dome of the Peyer’s patches to interact
with DCs and produce an optimal IgA response [60]. Perhaps this could also be true in
41
the germinal centers of lymph nodes where in B-CCR6 KO mice B cells lacking CCR6
expression are unable to migrate into the germinal center/correct position within the
germinal center for optimal interaction with the DCs. The location of these CCR6-
deficient B cells may differ compared to WT B cells within the germinal centers and this
remains to be investigated. Moreover, the affinity of these antibodies to rhMOG1-120 was
not measured which could be tested using existing serum.
4.4 The pleiotropic nature of CCR6 Chemokines and chemokine receptors are promising therapeutic targets but they can be
risky to target since they can have roles in both the homeostatic state and the disease
state. In addition they can have functions that promote inflammatory or anti-
inflammatory responses or in some cases both. An example would be the CCR6/CCL20
axis where studies have shown its involvement in both promoting EAE through CCR6
expressing Th17 cells and inhibiting EAE through regulatory T cells which also express
CCR6 [61]. On one hand, blocking this chemokine receptor could be beneficial in some
MS patients where CCR6 expressing Th17 cells predominate but it could be detrimental
to others that have a more dominant CCR6+ Treg response [50]. Hence depending on the
individual, the stage and type of disease, CCR6/CCL20 may play a regulatory,
pathogenic or redundant role [50]. Furthermore, CCR6 has been shown to play other
roles besides mediating leuokocyte chemotaxis such as mediating cell arrest on
endothelial cells. Meissner et al. showed that CCL20 partially controls adhesion of naïve
CCR6 expressing B cells to activated endothelial cells [62]. Another function is that
CCR6 is required on B cells for an optimal germinal centre (GC) response in secondary
lymphoid organs [56]. In this study, the authors show that when B cells lacked CCR6
expression, the number of GC increased but the antibodies produced by these B cells
were of lower affinity. Overall, further experiments are needed to elucidate the exact role
of this CCR6/CCL20 axis on B cells in both MS and EAE in order to rationalize whether
this is in fact a good therapeutic target for treatment.
42
4.5 Conclusion With the promising results of anti- CD20 B cell depletion therapies, there has been
renewed interest in the different roles B cells may play in MS and how we can develop
new therapies to target them. An alternative to B cell depletion is to prevent B cell
migration into the CNS by blocking chemokine receptors necessary for cell trafficking.
The CCR6/CCL20 pathway has been of interest and based on my studies, that CCR6 is
induced on activated B cells and functional in an in vitro setting where CCR6 expression
is required for B cells to migrate towards its ligand CCL20. During EAE, CCR6 is
upregulated on CNS infiltrating B cells and CCL20 is induced in the serum of immunized
mice. However, competitive chimeras show there is no advantage for CCR6+/+ B cells
over CCR6-/- B cells as both populations were able to populate the CNS at equal ratios.
This combined data suggest that the CCR6/CCL20 axis may not be the main chemokine
pathway that is driving B cell migration into the CNS during EAE. In the MOG35-55
induced mouse model, lack of CCR6 expression specifically on B cells did not alter the
severity of EAE indicating no defect in B cell regulation of pro-encephalitogenic T cells.
However, in the rhMOG1-120 induced mouse model where the B cells play a pathogenic
role, the clinical presentation of EAE was variable but preliminary data suggests that
CCR6 expression on B cells may be important in generating normal anti-rhMOG1-120
IgG1 levels. More research is needed to further investigate the potential role of CCR6 on
B cells during EAE.
43
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