Exploring the role of CCR6/CCL20 axis in B cell migration ... · autoimmune encephalomyelitis (EAE), the animal model for MS [6]. Whether this CCR6/CCL20 axis plays a role in B cell

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

ii

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

1

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.

2

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).

3

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*


Primary*progressive*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.

5

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

6

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.

7

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

8

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

9

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

10

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

12

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-/-


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


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


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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Exploring the role of CCR6/CCL20 axis in B cell migration ... · autoimmune encephalomyelitis (EAE), the animal model for MS [6]. Whether this CCR6/CCL20 axis plays a role in B cell