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Neuropathy in a mouse model of CD8+ T cell-mediated CNS demyelination Rainone, Anthony Department of Microbiology and Immunology McGill University, Montreal July 2013 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Anthony Rainone, 2013 ABSTRACT Several lines of evidence suggest that CD8+ T cells could contribute to the pathogenesis of many autoimmune diseases of the nervous system. Our group studies a mouse model (L31 mice) that spontaneously develops neurological symptoms associated with demyelinated lesions in the CNS. We have shown that the demyelinating disease in L31 mice is T cell-dependent with CD8+ T cells acting as effector T cells in the pathological process. Because L31 mice exhibit motor dysfunction indicated by hind limb clasping and difficulty walking, we wanted to determine whether the PNS was also affected. A flow cytometry approach determined that CD8+ T cells accumulate in the CNS of L31 mice from early on in life while they only appear in the PNS once mice display neurological symptoms. Furthermore, immunofluoresecent studies of the CNS and PNS of these mice recapitulate the flow cytometry data and demonstrate the large extent of demyelination in the PNS of symptomatic L31 mice, which is associated with CD8+ T cell accumulation. However, it is also evident that these CD8+ T cell clusters are associated with macrophage infiltration in the PNS of symptomatic L31 mice and that these macrophages have a mature phagocytic phenotype. We hypothesize that epitope spreading during the course of this CD8+ T cell-mediated demyelinating disease may be responsible for the PNS pathology. It will be of further interest to determine the T cell receptor specificity of CNS and PNS infiltrating lymphocytes and the role of PNS infiltrating macrophages. ii RÉSUMÉ Plusieurs études suggèrent que les cellules T CD8+ pourraient contribuer à la pathogenèse de plusieurs maladies auto-immunes du système nerveux. Notre groupe étudie un modèle murin (souris L31) qui développe spontanément des symptômes neurologiques associés à des lésions de démyélinisation dans le SNC. Cette maladie requiert la présence de lymphocytes T et ce sont la souspopulation de lymphocytes T CD8+ qui agissent comme cellules effectrices dans le processus pathologique. Parce que les souris L31 présentent des troubles moteurs tels que le serrement des pattes arrières quand les souris sont soulevées et des difficultés à marcher, nous avons voulu déterminer si le SNP est également affecté. Une approche de cytométrie en flux a déterminé que les cellules T CD8+ s'accumulent dans le SNC des souris L31 très tôt alors qu'ils n'apparaissent dans le SNP que lorsque les souris présentent des symptômes neurologiques. De plus, des études d’immunofluorescence sur coupe en congélation du SNC et du SNP récapitulent les données de cytométrie en flux et démontrent la grande étendue de la démyélinisation dans le SNP des souris L31 symptomatiques, qui est associé à l’accumulation en amas de lymphocytes T CD8+. Il est également démontré que ces amas de cellules T CD8+ sont associés à l'infiltration de macrophages dans le SNP de souris L31 symptomatiques et que ces macrophages ont un phénotype phagocytaire et mature. Nos données suggèrent que la présentation de nouveaux épitopes au cours de la demyelinisation dans le SNC pourrait être responsable de la iii pathologie qui se développe plus tard dans le SNP. Il serait important dans le futur de déterminer la spécificité du récepteur pour l’antigène des lymphocytes T CD8+ qui s’accumulent dans le SNC et le SNP et ainsi que d’étudier le rôle des macrophages infiltrant le SNP. iv ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Sylvie Fournier for the opportunity to work in her lab and her help and guidance throughout my master’s. Thank you to all the past lab members Mohamad Habbal, Yunlin Tai, and Vlad Dragan for all their help, support, suggestions, and laughter. Especially, I would like to thank Crystal Lee for all her help and accompaniment throughout my master’s. My project would truly not have been possible without her help and presence. Thank you to Tony Lim and Dr. Ji Zhang for their technical expertise and informative discussion and input. Also, I would like to extend my thanks to the department for their feedback and support. The department has been revamped since I started my degree and it all was encouraging and of benefit. Many thanks to the CIHR Neuroinflammation Training Program for their funding, travel award, and incredible network that helped me develop as a scientist and professional, it truly ameliorated my master’s experience and offered me many opportunities. Finally, I would like to thank my family, friends, and future wife, Gabriella, for all their support and companionship throughout this journey. All of the above people were all an integral part of my master’s degree and from the bottom of my heart thank you. v TABLE OF CONTENTS Page Abstract i Résumé ii Acknowledgements iv List of figures vii List of abbreviations viii Preface 1 1. Introduction 2 1.1 The B7-CD28/CTLA-4 pathway 2 1.2 L31 mice: A mouse model of spontaneous CD8+ T cell-mediated demyelinating disease 1.3 Multiple Sclerosis 6 9 1.3.1 The role of T cells in Multiple Sclerosis 11 1.3.2 CD8+ T cell involvement in Multiple Sclerosis 13 1.4 Mechanisms of demyelination in Multiple Sclerosis 17 1.5 Demyelinating diseases of the Peripheral Nervous System 19 1.6 Rationale and goal of M.Sc. project 20 2. Materials and Methods 23 2.1 Mice 23 2.2 Preparation of cell suspensions 24 2.3 Flow Cytometry 26 vi 2.4 Immunofluorescence 27 2.5 Statistical Analysis 29 3. Results 3.1 CD8+ T cells in the PNS of symptomatic L31 mice 30 30 3.2 The differences in CD8+ T cell infiltration into the CNS and PNS of L31 mice over time 31 3.3 CD8+ T cells are found in clusters and colocalize with demyelinated lesions in the CNS of symptomatic L31 mice 32 3.4 Devastating demyelination and CD8+ T cell infiltration in the PNS of symptomatic L31 mice 33 3.5 Macrophages are in close apposition to CD8+ T cells in the PNS of symptomatic L31 mice 34 3.6 Characterizing the macrophage population in the PNS of symptomatic L31 mice 35 4. Conclusion/Discussion 43 5. References 53 vii LIST OF FIGURES Page Figure 1 CD8+ T cells are found in the PNS of symptomatic L31 mice Figure 2 CD8+ T cells are found in the CNS of L31 mice early on, but only appear in the PNS of symptomatic L31 mice Figure 3 37 38 CD8+ T cells are found in the CNS of L31 mice early on, but only cause demyelination in the CNS of symptomatic L31 mice Figure 4 CD8+ T cells are only found in the PNS of symptomatic L31 mice and severe demyelination is also seen Figure 5 40 Macrophages surround CD8+ T cell clusters in the PNS of symptomatic L31 mice Figure 6 39 41 Macrophages in the PNS of symptomatic L31 mice have a more mature and phagocytic phenotype 42 viii LIST OF ABBREVIATIONS APC antigen presenting cell B6 C57BL/6 BBB blood-brain barrier CIDP chronic inflammatory demyelinating polyneuropathy CMT Charcot-Marie-Tooth disease CNS central nervous system CSF cerebrospinal fluid CTLA-4 cytotoxic T lymphocyte-associated antigen-4 DC dendritic cell EAE experimental autoimmune encephalomyelitis FBS fetal bovine serum GBS Guillain-Barré syndrome GVHD graft versus host disease HLA human leukocyte antigen IDO indoleamine-2,3-dioxygenase IFN interferon IL interleukin L31 C57BL/6 B7.2 transgenic line 31 mAb monoclonal antibody MAG myelin-associated glycoprotein MBP myelin basic protein MHC major histocompatability complex MOG myelin oligodendrocyte protein MS multiple sclerosis ix NOD non-obese diabetic NOD-B7-2-KO non-obese diabetic background B7.2 -/NOS nitric oxide species OCT Optimal Cutting Temperature OT ovalbumin-specific T cell receptor OVA ovalbumin P0 myelin protein 0 PBS phosphate buffered saline PFA paraformaldehyde PLP proteolipid protein PNS peripheral nervous system RAG recombination activating gene ROS reactive oxygen species RRMS relapsing-remitting multiple sclerosis SEM standard error of the mean SPF specific pathogen free TCR T cell receptor TNF-α tumour necrosis factor-α Tregs T regulatory cells VLA-4 very late antigen-4 x PREFACE My thesis work focused on a mouse model of CD8+ T cell-mediated demyelinating disease (L31 mice). L31 mice are unique in that they contain a costimulatory ligand (B7.2) transgene that is expressed in both T cells and microglia. Therefore, in order to fully appreciate the model and work that was conducted using the model, an understanding of the B7-CD28/CTLA-4 costimulatory pathway will be needed and thus, the introduction of my thesis will review the topic. This will be followed by an overview of the L31 mouse model and a background on neuroinflammatory diseases and mechanisms of demyelination. Finally, the rationale for my M.Sc. project will be presented. Page | 1 1. INTRODUCTION 1.1 The B7-CD28/CTLA-4 pathway The B7-CD28/CTLA4 pathway is involved in the regulation of costimulation in the process of T cell activation. T cells can be fully activated if a sufficiently potent T cell receptor (TCR) signal occurs (i.e. high avidity peptide), but costimulation is most often required to fully activate T cells and mount a response against an antigen [1]. Without costimulation, TCR activation alone may lead to an unresponsive T cell state known as anergy [2]. The B7 family consists of many costimulatory ligands, but the most rigorously documented and most relevant costimulatory ligands to L31 mice are the B7.1 and B7.2 ligands. B7.1 and B7.2 are mostly expressed on professional antigen presenting cells (APC), but can also be expressed on T cells. B7.2 is constitutively expressed at low levels on the surface of APCs and is rapidly upregulated upon activation. However, B7.1 is inducible and is expressed later than B7.2 [3]. These ligands both bind CD28 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on T cells, but with differing affinities and kinetics. CTLA-4 has the higher affinity between the two receptors and the ligand with greatest affinity is B7.1. The understanding of this pathway is even more complex as CD28 and CTLA-4 have opposing affects on T cell activation. CD28 is a positive regulator of T cell activation, whereas CTLA-4 is a negative regulator of T cell activation [4]. To help explain how both positive and negative coreceptors bind the same ligands, researchers have looked at the cell surface Page | 2 expression dynamics of these plasma membrane proteins. CTLA-4 is mainly an intracellular protein and is only upregulated on the cell surface after T cell activation, similar to B7.1 on APCs. However CD28 is constitutively expressed on the cell surface of T cells, which is similar to B7.2 on APCs. Therefore, many interpret this temporal difference as reason to suggest that B7.2 is the major CD28 (costimulatory) ligand and that B7.1 is the major CTLA-4 (inhibitory) ligand [5]. The engagement of all these ligands and receptors causes a plethora of downstream effects. The initiating event of the antigen-specific TCR encountering its antigen in the major histocompatability complex (MHC)peptide complex can vary in affinity. Some interactions between the TCR and MHC-peptide complex are of great affinity and cause a strong signal downstream of the TCR. However, others are of low affinity binding, which causes a lesser activation signal. CD28 costimulation can lower the activation signal threshold needed to cause complete activation of the T cell [6]. As well, CD28 signaling can increase cytokine production in T cells, by both augmenting transcriptional activity and stabilizing messenger RNA [7]. Of greatest interest, is the upregulation of the interleukin (IL)-2 gene by CD28 activation [8] because of the importance of IL-2 as a growth factor in T cell proliferation. Another means by which CD28 promotes T cell survival is through the upregulation of the anti-apoptotic BCL-2 family member, BCL-XL [9]. Page | 3 The inhibiting arm of the costimulatory pathway, mediated by CTLA-4, has opposing effects. CTLA-4 can inhibit T cell proliferation even when optimal T cell activation occurs [10] and this is mediated by reducing IL-2 and IL-2 receptor production, and by arresting T cells at the G1 phase of the cell cycle [11]. Moreover, mice genetically deficient in CTLA-4 expression develop a severe lymphoproliferative disease with progressive accumulation of T cells in peripheral lymphoid organs as well as in visceral organs, such as the heart, liver, and exocrine pancreas [12, 13]. Furthermore, the engagement of CD28 or CTLA-4 with B7 ligands can also signal downstream of the B7 ligand expressing APC. In B cells, the binding of B7.2 with CD28 causes a positive downstream signal that enhances IgG1 and IgE production [14, 15]. Additionally, the engagement of B7.2 on dendritic cells (DC) with CTLA-4 on T cells has been shown to induce the release of indoleamine-2,3-dioxygenase (IDO) and this event is interferon (IFN)-γ dependent [16]. IDO is an enzyme that catalyzes the degradation of tryptophan into by-products, which are inhibitory to T cell proliferation. This evidence is an example of bidirectional signalling in the B7-CD28/CTLA-4 pathway. All the literature presented above depicts B7 ligands expressed on APCs, but they can also be expressed on T cells. In mice, B7.2 is constitutively expressed at low levels on some resting T cells, however B7.1 is not expressed on resting T cells, but can be upregulated, along with B7.2, upon T cell activation [3]. This was demonstrated using a graft versus host disease (GVHD) mouse model Page | 4 [17]. In this study, the authors demonstrated that B7 expression on T cells can act as a negative regulator of immune responses and diminish GVHD mortality. The ability of the B7-CD28/CTLA-4 pathway to regulate T cell activation or the induction of anergy is crucial in immune system physiology. An exaggerated response of the immune system may lead to autoimmunity, whereas an insufficient response may lead to severe infection. This is why the costimulatory pathway is so intriguing and many groups have studied the topic to determine its roles in autoimmunity. In an experimental autoimmune encephalomyelitis (EAE) mouse model, mice were crossed with CD28 -/- mice and the development of EAE was blocked [18]. CD28 -/- mice were also highly resistant to collagen-induced arthritis [19]. Furthermore, mice that were administered a CTLA-4 fusion protein (CTLA-4-Fc) that binds B7 ligands extensively were also resistant to EAE induction [20]. Similarly, in a spontaneous model of type I diabetes, known as non-obese diabetic (NOD) mice, disease was inhibited and did not occur in mice injected early with antiB7.2 monoclonal antibody (mAb) [21]. It is therefore evident that this costimulatory pathway is implicated in the development of autoimmunity. On the other hand, blocking CTLA-4 via an anti-CTLA-4 mAb exacerbates disease in EAE mice [22]. Exacerbated disease also occurs in NOD mice as diabetic symptoms are worsened when CTLA-4 is blocked before the onset of symptoms [23]. Targeting major players in the costimulatory pathway, such as B7 ligands, CD28, and CTLA-4, is an intuitive concept as dramatic effects on T cell Page | 5 activation can be obtained through their pharmacological alterations. Nevertheless, much more work must be done to better understand the consequences of this attractive and promising target for therapies against autoimmune diseases. 1.2 L31 mice: A mouse model of spontaneous CD8+ T cell-mediated demyelinating disease Multiple mouse lines were created in order to study the role of the B7.2 costimulatory ligand on B cell homeostasis [24]. These mouse lines were created by expressing a B7.2 transgene under the control of the MHC class I H2Kb promoter and the immunoglobulin heavy chain μ enhancer. Multiple founder mice were then crossed onto the C57BL/6 (B6) background and maintained in a specific pathogen-free (SPF) environment. It was serendipitously discovered that one of these transgenic lines (L31 mice) spontaneously develops neurological symptoms at approximately 4-5 months of age. Neurological symptoms include hind limb clasping when mice are picked up by their tail, poor proprioception as depicted by hind limbs slipping through cage bars while walking, and hind limb splaying with an endpoint of hind limb paralysis. These neurological symptoms are associated with a large infiltration of effector memory (CD44+, CD62L-) CD8+ T cells into the central nervous system (CNS) and demyelinated lesions could also be seen in the spinal cord and spinal roots of these mice [25]. Cellular infiltration of the spinal cord and Page | 6 spinal roots is most prominent in the sacral and lumbar regions of the spinal cord with a decreasing gradient of infiltration at more superior portions of the CNS. Neurological symptoms are T cell-dependant as TCRβ -/- L31 mice do not develop disease. Symptom development is also dependent on the expression of the B7.2 transgene on microglia because L27 mice (derived in the same manner as L31 mice, but which only express the transgene on T and B cells and not on microglia) do not develop neurological symptoms. Furthermore, adoptive transfers of L31 T cells into TCRβ -/- L31 mice caused disease, however the adoptive transfer of L31 T cells into TCRβ -/- B6 mice did not cause disease. As well, it is important to note that although the B7.2 transgene is under the transcriptional control of the MHC class I promoter, and therefore could be expressed by any cell type, histological staining of multiple organs demonstrated that the cellular infiltration in L31 mice is specific to neurological tissue and no tissue destruction of other organs was observed [25]. It has also been shown that I-Aβ -/-, mice in which CD4+ T cells do not develop because of the absence of a functional MHC class II molecule, and CD4 -/- L31 mice develop the same neurological symptoms as L31 mice, but with quicker onset of the disease. This demonstrates that CD4+ T cells are regulatory in the L31 model and that CD8+ T cells are pathogenic and mediate disease [26]. Further evidence supporting the pathogenic role of CD8+ T cells in L31 mice is that the large majority of infiltrating leukocytes in the CNS of symptomatic L31 mice are CD8+ T cells. Page | 7 TCR specificity was then investigated to determine if the pathogenic CD8+ T cells were infiltrating due to a TCR-specific response in L31 mice. To do so, OT-I TCR L31 mice, a mouse line that solely contains CD8+ T cells that are specific for an ovalbumin (OVA) peptide and thus have a restricted CD8+ TCR repertoire, were developed. OT-I TCR L31 mice do not develop disease and approximately 95% of the CD8+ T cells in these mice were clonally positive (Vα2+, Vβ5+), therefore the onset of neurological disease is dependent on TCR specificity. To determine if a response to a common antigen was involved in the CD8+ T cell-mediate disease, CDR3 length distribution of the Vβ chain was investigated. A skew in the distribution of CDR3 lengths would reveal that an antigen-specific response is involved in the T cell proliferation. While peripheral CD8+ T cells displayed a normal Gaussian distribution for all Vβ chains similar to controls, CD8+ T cells in the CNS of non-symptomatic preclinical I-Aβ -/- L31 mice demonstrated skewed distributions in some Vβ chains, suggesting that an antigen-specific response causes CD8+ T cell proliferation and expansion early on in L31 mice. Furthermore, sequencing of cloned cDNA derived from selected Vβ families with skewed CDR3 length distribution demonstrated that a particular clonotype was predominant in all cases, further suggesting the expansion of selected T cell clones within the CNS. Moreover, microglia, the CNS-resident myeloid-derived monocytes, are activated in L31 mice at a time point as early as 3 weeks of age in L31 mice. However, microglia are not activated at this early time point in L31 mice that are of the OT-I, recombination activating gene (RAG) -/- (which causes the Page | 8 absence of mature T- and B-lymphocytes), or IFN-γ receptor -/- backgrounds [26]. All of these crossed L31 mice also do not exhibit neurological symptoms. The notion that neurological symptoms occur in L31 mice via a TCR-specific response mediated by pathogenic CD8+ T cells and that IFN-γ signalling is necessary for disease would lead to the hypothesis that a MHC class I-restricted cytotoxic attack of oligodendrocytes is a main contributor to demyelination in L31 mice. In fact, oligodendrocytes, the myelinating cells of the CNS, upregulate MHC class I early on in the preclinical stages in L31 mice [27] and increased perforin and Fas-ligand were detected in CNS myeloid cells and heightened levels of granzyme B and IFN-γ were detected in CD8+ T cells along with CD107a expression at the cell surface, depicting cell degranulation [28]. Surprisingly, perforin or Fas-ligand -/- L31 mice develop disease with a quicker onset of neurological symptoms and the predominance of infiltrating CD8+ T cells still occurs [28]. This data suggests that perforin and Fas-ligand play a regulatory role in the L31 mouse model and are not necessary for CD8+ T cell-mediated demyelination and the onset of neurological symptoms. 1.3 Multiple Sclerosis Multiple Sclerosis (MS) is a chronic inflammatory disease that affects the CNS and is characterized by multiple demyelinated lesions in the brain. The etiology of the disease is unknown, but it is widely accepted that MS is an autoimmune Page | 9 disease and that T cells play a role in the destruction of myelin sheaths throughout the duration of the disease. MS is the most common neurological disease among young Canadians and the disease typically manifests itself between the ages of 15-40 years, a time when people are in the prime of their lives, and the disease is more common in women than in men with a predominance of approximately 2:1 [29]. Symptoms of MS are quite diverse and range from sensory disturbances and limb weaknesses to difficulties concentrating and fatigue [30]. MS is also categorized into different forms. The most common form, occurring in 80% of patients, is relapsing-remitting multiple sclerosis (RRMS). In RRMS, patients develop neurological symptoms over a few days and then, spontaneously, the symptoms remit and the patient no longer experiences neurological deficits. In fact, some symptoms improve and stabilize, however, RRMS is characterized by the repeating of this cycle as the patient will have new attacks and worsening of symptoms. Accumulation of demyelinated lesion occurs after every relapse and the baseline level of function that the patient returns to after every sequential relapse worsens. Nearly all RRMS patients eventually reach the secondary progressive form of MS. In secondary progressive MS, the disease is no longer characterized by relapses and remissions, but is a progressive gradual decline in clinical course. Furthermore, 20 % of MS patients are diagnosed with primary progressive MS, which is also depicted by a progressive gradual decline in clinical course, but is not preceded Page | 10 by a RRMS phase. Overall, MS leads to a worsening debilitation in both neurocognitive and motor functions until an endpoint of death occurs. This disease is most prevalent in the developed world such as North America, Europe and Australia. It is also apparent that areas with less ultraviolet exposure (from the sun) have a higher prevalence of the disease. Geospatial analyses demonstrate the distribution of MS prevalence in a gradient fashion with areas of low sun exposure having a high prevalence of MS and areas of high sun exposure having a low prevalence of MS. In North America, the highest prevalence is located in the northern part of the continent and the prevalence decreases at more southern latitudes [31]. This observation adds to the growing amount of evidence that a decreased serum vitamin D level is a risk factor for MS patients [32]. 1.3.1 The role of T cells in Multiple Sclerosis T cells have the capacity to recognize specific antigens from foreign invaders of the human body and this allows them to help clear many pathogens a person may encounter throughout a lifetime. However, T cells can also have a TCR specific for self antigens and this may cause autoimmunity. MS is a very complex autoimmune disease, which is influenced by both genetic and environmental factors, and the exact pathogenic involvement of T cells in the disease is unknown. However, an abundance of work exists using the most widely accepted animal model of MS, EAE. EAE is induced in susceptible mice by immunizing with different myelin proteins and adjuvant. The most Page | 11 commonly used myelin proteins are myelin oligodendrocyte protein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) [33]. The symptoms of EAE range from limp tail to complete paralysis of the mouse and the symptoms in the murine model are Th1/Th17-mediated. Th1/Th17 myelinspecific T cells have been demonstrated to cause demyelination in the CNS of EAE mice and this demyelination leads to the neurological symptoms. As well, adoptively transferred activated CD4+ T cells specific for myelin antigens into naive mice can also induce the disease [34]. Furthermore, the greatest genetic risk factor in humans that predisposes them to develop MS is located in the human leukocyte antigen (HLA) class-II region. Specifically it is the HLA-DRB1*1501 allele that has been implicated and confirmed several times through genome wide studies and is the strongest risk factor for MS [35]. This isolation to a HLA class-II allele confines the genetic predisposition to an effect on CD4+ T cells. Moreover, recent human studies have also demonstrated a very convincing case for Th17 cells as being the major player in initiating new relapses and disease. This observation comes from a study in which MS patients with aggressive disease that did not respond to available immune modulating therapies were chemoablated and then reconstituted with autologous hematopoietic stem cells. These patients were completely abrogated of any new clinical relapses. Their T cell compartments were reconstituted and T cell reactivity to multiple myelin antigens could be detected. However, the responses of these T cells were perturbed, as Th1 and Th2 responses were normal, but Th17 responses were greatly diminished [36]. Page | 12 Nevertheless, not all human MS studies agree with the evidence purposed by EAE studies. A recent human study demonstrates that the myelin protein in which autoreactive T cells may be activated against to initiate MS is not any of the EAE-inducing myelin proteins. The group used a large scale proteomics approach to analyze cerebrospinal fluid (CSF) from children that had an initial episode of CNS inflammation and followed the children to see if they developed MS or not and compared the proteins present in the CSF between the two groups. The interesting finding from this study was that children that went on to be diagnosed with MS after that first presentation of CNS inflammation did not have the typical compact myelin proteins (MOG, PLP, MBP) detected as soluble proteins in their CSF. Rather, it was molecules from the node of Ranvier (an area where myelin sheaths end and saltatory conduction can occur) and the surrounding axoglia apparatus membrane that were implicated in the CSF of children that did go on to be diagnosed with MS [37]. 1.3.2 CD8+ T cell involvement in Multiple Sclerosis The evidence presented above portrays MS pathogenesis as a CD4+ T cell dependent, most likely Th17-driven phenomenon. However, strong evidence exists implicating a role for another T cell subset, the CD8+ T cells. CD8+ T cells are an essential part of the immune system and mainly serve as cytotoxic effector cells that kill infected or neoplastic cells. Therefore, autoreactive CD8+ T cells definitely have the capacity to kill and damage self if their activation is left unregulated. Page | 13 The most convincing line of evidence for the role of CD8+ T cells in MS stems from studies using post-mortem brain sections of MS patients and the discovery that CD8+ T cells can outnumber CD4+ T cells by as much as 10-fold in regions of demyelination [38, 39]. However, this has been supported by a more recent study that used cortical (gray matter) biopsies of early MS patients. It was apparent, even in this area of the brain and at an early time point, that demyelination occurs and is accompanied by CD8+ T cells [40]. The phenotype of these infiltrating CD8+ T cells still remains quite unknown, but it has been shown that (IL)-17-producing CD8+ T cells are specifically enriched in the active lesions of MS patients [41]. Furthermore, CD8+ T cells from the CSF of MS patients have an effector memory phenotype (CD62L-, CCR7-, granzymeBhi) and this aids them in crossing the blood-brain barrier (BBB). This same report demonstrated that very late antigen-4 (VLA-4) is necessary for CD8+ T cells to cross the BBB [42]. Some HLA alleles that are not restricted to class II genes also predispose people to MS and they have been studied using humanized mice [43]. This publication demonstrates how the expression of HLA-A*0301, which predisposes humans to MS, in a humanized mouse with the coexpression of a PLP-specific T cell that was derived from an MS patient causes mild CD8+ T cell-mediated CNS disease that is similar to MS. However, if HLA-A*0201, which is protective against MS, is expressed instead, then the humanized mice do not succumb to disease. Another emerging phenomenon by which CD8+ T cells could play an effector role in MS is by expressing dual TCRs. Mice with CD8+ T cells that express two Page | 14 TCRs, one specific for MBP and another specific for viral antigens, developed CNS autoimmunity after viral infection [44]. Another line of evidence that strongly supports the pathogenic role of CD8+ T cells in MS is that the current therapies used to treat the disease are only affective if both CD4+ and CD8+ T cell compartments are targeted. Fingolimod, an oral drug that broadly inhibits exit of lymphocytes from secondary lymphoid organs by modulating the sphingosine 1-phosphate receptor, is effective in treating MS patients [45]. Likewise, blocking the global entrance of T cells into the CNS through the usage of natalizumab, an alpha-4 integrin-specific antibody, greatly ameliorates disease in people with MS as shown by a significant reduction in progression, relapse rate, and number of lesions [46]. It is also interesting to note that alpha-4 integrin is a component of VLA-4 and that natalizumab strongly affects the absolute number of leukocytes in the CSF of treated MS patient. This reduction occurs in both the CD4+ and CD8+ T cell compartments [47]. In line with all this evidence supporting the notion that both T cell compartments need to be targeted in order to treat MS, a monoclonal anti-CD4 antibody treatment failed to ameliorate disease in MS patients in a phase II clinical trial [48]. Accumulating evidence for the involvement of CD8+ T cells in MS has been supported by several murine models. More recent CD8+ T cell-mediated EAE models have been reported to better recapitulate MS pathology with more inflammation of the brain as compared to the spinal cord [39]. One model, using C3H mice, induced neurological symptoms, CNS infiltration, and Page | 15 demyelination when MBP-specific CD8+ T cells from EAE mice were transferred into naїve recipient mice [49]. Another group expressed OVA as a cytosolic protein in oligodendrocytes and observed that naїve OT-I (ovalbuminspecific) TCR CD8+ T cells can be activated without the help of antigen presentation by an APC and that this lead to IFN-γ production and fulminant demyelinating EAE with lesions reminiscent of MS [50]. Brain sections from these mice were also used to study the effect of spillover of toxic molecules such as perforin and granzyme B. OT-I-specific CD8+ T cells were co-cultured with the brain slices from the cytosolic OVA-expressing mice and this caused collateral neuronal apoptosis to surrounding neurons, outside of the immunological synapse [51]. The overexpression of PLP in mice lead to myelin degeneration and the pathology in these mice was lost when crossed with RAG -/- mice. Reconstitution of these mice with CD8+ CD4- bone marrow restored the demyelinated pathology, whereas bone marrow from CD8CD4+ mice did not restore CNS pathology [52]. Further studies on these PLP overexpressing mice demonstrates that CD8+ T effector cells are activated and undergo clonal expansion in the CNS throughout the neurodegenerative disease [53]. Moreover, CD8+ T cells can also be regulatory and many studies are now demonstrating the role of CD8+ T regulatory cells (Tregs) in MS. CD8+ Tregs have been studied in the EAE mouse model and in human samples. The evidence from the EAE mouse model is two sided as CD8+ T cells can suppress, but can also autonomously cause CNS damage as mentioned above Page | 16 [39]. Although, a study in humans did demonstrate that neuro-antigen specific CD8+ T cells could function as Tregs and suppress effector T cells and this CD8+ Treg function is diminished during patient disease exacerbation [54]. 1.4 Mechanisms of demyelination in Multiple Sclerosis Studying the pathology of MS helps in understanding the different cell types and possible mechanisms involved in causing demyelinated lesions. In MS, there are four different pathology patterns each with their own differing cell types and pathogenic molecules [55]. Pattern-I lesions (found in 15% of MS patients) consist of mainly T cell inflammation, and active demyelination with many activated microglia and macrophages. This suggests a role for macrophage-associated demyelination where macrophages release toxic factors such as tumour necrosis factor α (TNF-α), and reactive oxygen species (ROS), which lead to myelin destruction. Pattern-II lesions (found in 58% of MS patients) consist of densely packed T cells and macrophages, but also immunoglobulin deposition and compliment activation. This type of lesion supports a role for autoantibodies and compliment-mediated destruction of myelin. Pattern-III lesions (found in 26% of MS patients) are defined by oligodendrocyte apoptosis, T cell inflammation, and macrophage and microglia activation. This lesion-type supports a model of oligodendrocyte apoptosis that may be mediated by the activation of different death receptors. Pattern-IV Page | 17 lesions (found in 1% of MS patients) are associated with profound nonapoptotic death of oligodendrocytes. This lesion-type is exceedingly rare and is only identified in autopsies of primary progressive MS patients. The pathogenesis occurring in these lesions is unclear. The reoccurring presence of activated microglia and macrophages does not seem like a coincidence in MS pathogenesis. An increasing amount of studies are demonstrating that activated myeloid cells secrete toxic species that play a major role in demyelination in MS patients. Two recent studies used genomewide microarray analysis to demonstrate signs of oxidative tissue damage and mitochondrial injury in these lesions. The first analyzed active MS lesions [56] and the second analyzed cortical MS lesions [57]. The mechanism of pathogenesis proposed by the authors consists of an initial attack of demyelination caused by a T cell-specific response that may also be augmented by the release of ROS and nitric oxide species (NOS) by myeloid cells. This would cause tissue damage and mitochondrial injury, which would then continue the cycle of releasing harmful free-radicals and further neurodegeneration. The CD8+ T cells found in MS lesions can also mediate TCR-specific cell death. CD8+ T cells can kill targeted cells through Fas-ligand activation of the Fas death pathway and induce apoptosis of the targeted cell or they can also induce cytolysis through the exocytosis of granules such as perforin and granzyme B [58]. Furthermore, this targeted cell death has been shown to be extremely specific and is regulated by the formation of an immunological Page | 18 synapse between the CD8+ T cell and the target cell [59]. This is evidence that myelin-antigen specific CD8+ T cells could be involved in the killing of oligodendrocytes and the cause of demyelination in MS. Moreover, T cells that cause demyelination in the CNS can indirectly cause the activation of other T cells which have a TCR-specificity to antigens or epitopes which are similar to their own TCR-specificity. This occurs when a T cell is activated by its antigen presented in a MHC and causes destruction of that protein. This will then lead to further epitopes or antigens of that same protein to be available and presented by APCs, which could activate other T cells that are specific for these newly available epitopes. This phenomenon is known as epitope spreading and has been shown to occur in MS [60]. In fact, it has been seen that a small percentage of MS patients suffer from peripheral nervous system (PNS) deficits, neuropathy, during the course of their disease and this finding was attributed to epitope spreading [61]. Most recently, it has been shown that initial demyelination in the CNS of EAE mice by CD4+ T cells lead to the activation of CD8+ T cells by a specific type of DC [62]. 1.5 Demyelinating diseases of the Peripheral Nervous System Demyelinating pathology does not only occur in MS. Other demyelinating diseases exist where the pathology of demyelination occurs in the PNS and not in CNS. One example is Guillain-Barré Syndrome (GBS) and its chronic Page | 19 counterpart chronic inflammatory demyelinating polyneuropathy (CIDP). These diseases are characterized by rapidly evolving weakness, some sensory loss, and hyporeflexia. The diseases are caused by an immune attack of peripheral nerve axons [63]. Particularly to GBS, node of Ranvier lengthening is seen early on in the disease and is then followed by complement-mediated recruitment of macrophages to the nodal regions [64]. Another example of a PNS demyelinating disease is Charcot-Marie-Tooth disease (CMT). CMT is an inherited disorder of the peripheral nerves and can be caused by many different genetic mutations. The genetic defects are mainly found in myelin proteins of Schwann cells, PNS myelinating cells, or axonal proteins and cause a decrease in conductance of peripheral nerves [65]. The decreased conductance in peripheral nerves is caused by the genetic mutations causing either demyelination of peripheral nerves, when mutations are found in Schwann cell myelin proteins, or dysfunction of peripheral nerve axons. Furthermore, there is presently no pharmacological therapy for CMT [66]. 1.6 Rationale and goal of M.Sc. project As described above, our lab works on a mouse model that spontaneously develops neurological symptoms at approximately 4-5 months of age and the neurological symptoms consist of hind limb clasping when picked up by the tail, poor proprioception as seen by hind limbs slipping through cage bars while Page | 20 walking, and hind limb splay with an endpoint of complete hind limb paralysis. This L31 mouse model overexpresses the costimulatory ligand B7.2 on T cells and microglia and the neurological symptoms are dependent on B7.2 expression on microglia. These neurological symptoms are also T cell dependant and are associated with demyelinated lesions in the CNS. CD8+ T cells colocalize with these demyelinated lesions. This line of evidence would rationally lead to a hypothesis describing myelin-autoreactive CD8+ T cells being activated because of dysregulated costimulatory expression on microglia and causing demyelination in the CNS, which would cause the clinical neurological symptoms spontaneously seen in our mice. However, the impaired motor function and limb weakness, depicted by the neurological symptoms, may suggest that an insult to the PNS is occurring. Recently, a publication from The Journal of Experimental Medicine described NOD mice that succumbed to the exact same neurological symptoms as L31 mice [67]. These mice were first described when B7.2 -/- mice were created on the NOD background (NOD-B72-KO). It was reported that demyelination occurred solely in the PNS and the CNS was not affected and the disease was CD4+ T cell mediated [68]. Furthermore, NOD-B7-2KO mice were resistant to disease when deficient in IFN-γ, but perforin or Fas deficiencies did not affect neuropathy development [69]. Louvet et al. [67] showed that T cells specific for myelin protein 0 (P0) were responsible for the neurological disease. Indeed, mice expressing a transgenic TCR specific to P0 and were bred on a RAG -/- background developed a fulminant form of peripheral neuropathy. Page | 21 Due to the motor dysfunction nature of the spontaneous neurological symptoms seen in L31 mice and the striking resemblance of their symptoms to the symptoms exhibited by NOD-B7-2-KO mice, who only suffer from PNS demyelination and cellular infiltration, it was the goal of my M.Sc. research project to determine whether cellular infiltration and demyelination was occurring in the PNS of L31 mice and, if so, determine which compartment between the PNS and CNS is first infiltrated by CD8+ T cells. Page | 22 2. MATERIALS AND METHODS 2.1 Mice L31 mice were generated by injecting a construct, consisting of the coding region of B7.2 cDNA under the transcriptional control of the MHC class I promoter, H-2Kb, and the immunoglobulin heavy chain enhancer, Igμ, into an oocyte. The oocyte was transplanted into a pseudopregnant mouse and a founder mouse was born. The founder mouse was backcrossed onto a C57BL/6 background and L31 mice are now bred and maintained in a SPF animal facility [24]. Genotyping of these mice was performed by collecting a blood sample from the tail vein into a 5 mL polystyrene round-bottom tube (BD Falcon) containing 500 μL of Alsever’s solution (114M dextrose, 27mM Na3C6H5O7•H2O, 71mM NaCl). Red blood cells were lysed by treating with ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2-EDTA) and washed with phosphate buffered saline (PBS) (137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.47mM KH2PO4) supplemented with 1% fetal bovine serum (FBS) (GIBCO) and 0.1%NaN3. Samples were blocked by using 50μL of 2.4G2 blocking antibody for 30 minutes and then stained with rat anti-mouse B7.2 antibody conjugated to phycoerythrin (PE) (eBioscience) for 30 minutes. After incubation, samples were washed using FBS and NaN3 supplemented PBS and acquired on a BD LSR-Fortessa flow cytometer running on FACS Diva 6.0 software (BD). Samples were analyzed for high expression of B7.2, which would confirm the presence of the B7.2 transgene. All animal Page | 23 procedures were in accordance with the guidelines of the Canadian Council of Animal Care, as approved by the animal care committee of McGill University. 2.2 Preparation of cell suspensions In order to isolate the CNS, brain and spinal cord, and PNS, right and left sciatic nerves, mice were deeply anesthetized using a 4-5% isofluorane (Baxter) concentration at a 1 L/min flow rate in an induction chamber. Mice were then maintained under anesthesia by the use of a mask connected to a Bain circuit. The flow rate was adjusted to 0.5 L/min and the concentration of isofluorane was also decreased to 2.5%. Depth of anesthesia was confirmed by the lack of pedal reflex when toe pads were pinched. Mice were then surgically exposed and the abdomen opened in order to sever the abdominal vein. Then the thoracic cage was quickly opened and, while the heart beat still persisted, 25mL of ice cold PBS was intracardially perfused through the left ventricle using a 30 mL syringe and a 26G1/2 needle (BD). Then the nervous tissues were dissected and maintained in complete RPMI 1640 medium (GIBCO) supplemented with 10% FBS, 50μM β-mercaptoethanol (Bioshop), 100U/mL penicillin (Sigma), and 100μg/mL streptomycin (GibcoBRL). Tissues were then placed in a 3 mL dounce homogenizer containing 1 mL of supplemented RPMI and homogenized manually using a pestle. The cell suspensions were then collected into 15 mL canonical tubes (BD Falcon) and spun down at 1200 rpm at 4°C for 5 minutes. Supernatants were decanted and pellets were Page | 24 resuspended in 10 mL of 30% Percoll (GE Healthcare) in PBS. Tubes were spun at 1500 rpm at 18°C for 30 minutes in order to separate myelin debris from cell pellets. Myelin debris and supernatants were aspirated out and cell pellets were washed with 10 mL of supplemented RPMI. After wash, cells were resuspended in 1 mL of supplemented RPMI and filtered through a 70 μm nylon mesh. Cell concentrations were estimated by using 0.4% trypan blue (Sigma), to exclude dead cells, and a haemocytometer (Hausser Scientific). For experiments involving macrophage staining, an enzymatic method was used to create cell suspensions in order to better represent the proportion of myeloid cells. The method used consisted of the same procedures for dissection and tissues were also maintained in supplemented RPMI, but then they were processed according to a previously published protocol [70]. Briefly, tissues were cut into multiple small segments and placed into 1.5 mL microtubes containing fresh 1 mL supplemented RPMI with the addition of 1.6 mg/mL of collagenase (type IV; Sigma) and 200 μg/mL of DNase I (Sigma). Sample tubes were then incubated at 37°C for 30 minutes with agitation and after which samples were gently dissociated by repeat pipetting through a 1 mL pipet and reincubated at 37°C for 25 minutes with agitation. After this incubation, samples were dissociated again to a single-cell suspension using again a 1 mL pipet and were then centrifuged at 6000 rpm for 5 minutes in a microcentrifuge. Supernatants were decanted and cells were washed twice using PBS. Cells were resuspended in 1 mL of supplemented RPMI and filtered through a 70 μm nylon mesh. Cell concentration and percent viability Page | 25 was estimated in the same manner as the previous method by using a haemocytometer and the Trypan blue cell exclusion method. 2.3 Flow cytometry All samples for the T cell infiltration kinetics experiments were stained in the following manner. 1x106 cell aliquots were pipetted into 5 mL round bottom polystyrene tubes and washed with PBS. They were first labelled with LIVE/DEAD amine-reactive violet viability marker according to the manufacturer’s protocol (Invitrogen). Cells were then blocked with 50 μL of 2.4G2 blocking antibody for 30 minutes at 4°C and then labeled with rat antimouse CD45 conjugated to fluorescein isothiocyanate (FITC) (BD Pharmingen), rat anti-mouse CD4 conjugated to allophycocyanin (APC) (BD Pharmingen), and rat anti-mouse CD8a conjugated to PE (eBioscience). Samples were washed using PBS and then acquired on a BD LSR-Fortessa flow cytometer running on FACS Diva 6.0 software. The analysis of the acquired data was also performed using the FACS Diva 6.0 software. For the macrophage characterization experiments, samples were processed in the same manner as those from the T cell infiltration kinetics experiments up until the antibody staining step. Samples used for the macrophage characterization experiments were stained with rat anti-mouse CD45 conjugated to APC (BD Pharmingen), rat anti-mouse CD11b conjugated to FITC (BD Page | 26 Pharmingen), and rat anti-mouse F4/80 conjugated to PE (Serotec). These samples were acquired and analyzed in the same manner as described above. 2.4 Immunofluorescence Mice were intracardially perfused with PBS in the same manner as described above, but were then subsequently intracardially perfused with 4% paraformaldehyde (PFA). Spinal cord tissues were dissected and then postfixed in 4% PFA for 2 hours at room temperature and then cryoprotected by allowing them to sink in a 30% sucrose solution overnight at 4°C. Spinal cord tissue was then embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek) by orienting the tissue in disposable base molds (Canemco) containing OCT and then flash-freezing the embedded tissue in liquid nitrogen. Sciatic nerve samples were from mice that were not intracardially perfused with 4% PFA and sciatic nerve tissue was not post-fixed or cryoprotected. Dissected sciatic nerve tissue was immediately placed in OCT and then oriented in disposable base molds containing OCT and flash-frozen using liquid nitrogen. Embedded tissue cassettes were then cut into 12 μm (for CNS samples) or 6 μm (for PNS samples) sections using a CM3050 S cryostat (Leica) and mounted onto Superfrost Plus microscope slides (Fisher Scientific). Slides were maintained at -20°C until stained. To stain slides, they were first thawed and desiccated for 30-60 minutes, samples were circled with a Dako Pen, and washed with PBS. Slides were then Page | 27 flooded with blocking solution (PBS with 2% normal goat serum, 2% FBS, and 0.2% Triton-X-100) for 3-5 hours at room temperature. After blocking, slides were washed with PBS and primary antibody was added in desired concentration to blocking solution and incubated overnight at 4°C. Rat antimouse CD8a (BD Pharmingen) was used at a 1/100 dilution and rabbit anti-Iba1 (Wako) was used at a 1/1000 dilution. Slides were then washed 3 times with PBS and secondary antibodies were added in desired concentrations to blocking solution and incubated for 2 hours at room temperature. Goat anti-rat IgG conjugated to Alexa Fluor 488 (Cell Signalling) was used at a 1/1000 dilution and goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen) was used at a 1/500 dilution. Slides were then washed 3 times with PBS and then myelin lipids and nuclei were stained by flooding slides with a PBS solution containing FluoroMyelin Red (Invitrogen) at a 1/200 dilution and TO-PRO-3-iodide (Invitrogen) at a 1/1000 dilution. The slides were then incubated at room temperature for 20 minutes and washed 3 times with PBS. Slides were coversliped using Vectashield mounting medium (Vector Laboratories) and Fisherfinest premium cover glass (Fisher Scientific) and slides were sealed by applying nail polish to the edges of the cover glass. Slides were imaged on an Olympus Fluoview 1000 confocal microscope running on the Olympus FV10-ASW software version 2.01 and the objectives used to capture the images were the Olympus UPlanSApo 20x/0.75 dry, Olympus UPlanSApo 40x/0.90 dry, and the Olympus PlanApo N 60x/1.42 oil. Page | 28 2.5 Statistical analysis T cell infiltration kinetics was analysed using a two-way ANOVA for the effect of mouse group and time on the results. Statistical differences between groups was analysed using an unpaired two-tailed student’s t-test. Data are expressed as the mean ± standard error of the mean (SEM) and p values ≤ 0.05 were considered to be significant. All statistical analyses were performed using GraphPad Prism 5. Page | 29 3. RESULTS 3.1 CD8+ T cells in the PNS of symptomatic L31 mice In order to determine whether CD8+ T cells were present in the PNS of L31 mice, we used a flow cytometry approach. Mononuclear cells were isolated from the sciatic nerve and analyzed by flow cytometry for the expression of CD45, CD4 and CD8. CD45 is a tyrosine phosphatase that is expressed at high levels on T lymphocytes and at intermediate levels on myeloid cells. CD4 and CD8 expression was analyzed on the CD45high cells (lymphocyte population) present in the sciatic nerve as shown in figure 1. When comparing the B6 and L31 non-symptomatic mice, it is evident that no significant CD45hi lymphocyte population was present and that the only major population was the CD45intermediate population of myeloid cells (Figures 1A and B). However, the results clearly show that a distinctively large population of infiltrating leukocytes was present in symptomatic L31 mice, as depicted by the orange population that is CD45hi (Figure 1C). This population mainly consisted of CD8a positive cells and very few cells from this population were CD4+ (Figure 1C). This data shows that a large CD8+ T cell population infiltrates into the PNS of L31 mice, once they are symptomatic. Page | 30 3.2 The differences in CD8+ T cell infiltration into the CNS and PNS of L31 mice over time It was of interest to perform a kinetic analysis to determine at what time point CD8+ T cells infiltrate into the two nervous tissue compartments, CNS and PNS. Therefore we performed the same flow cytometry analysis on mice at 1 month, 3-4 months, 5-6 months, and symptomatic mice (4-5 months) on both their CNS and the PNS. As shown in figure 2A CD8+ T cells were found in the CNS of L31 mice at an early time point of 3-4 months of age. Furthermore, CD8+ T cells were also found in the CNS of non-symptomatic L31 mice at 5-6 months of age and were at the most abundant level once the L31 mice were symptomatic. A two-way ANOVA analysis of the CNS values indicated that there was a significant interaction between the B6 and L31 groups over time (p=0.0053), which therefore allowed us to analyze the data between mouse groups and time points using a student’s t-test. The absolute number of CD8+ T cells in the CNS of L31 mice at 3-4 months of age was significantly greater than the number found in B6 control mice (p=0.0223) and this was also the case at 5-6 months of age (p=0.0497). In the PNS of L31 mice, no CD8+ T cell infiltration was detected by flow cytometry until the mice became symptomatic (Figure 2B). The absolute number of CD8+ T cells found in the PNS of L31 mice was comparable to the B6 control samples at all other time points (Figure 2B). A two-way ANOVA analysis of the PNS values concluded that there was no significant interaction between the groups over time (p=0.6872). Page | 31 This kinetic analysis revealed that no significant number of CD8+ T cells was found in the PNS of L31 mice before the onset of neurological symptoms, but that CD8+ T cells could be seen in the CNS of L31 mice as early as 3-4 months of age, before they had any clinical signs of neurological disease. 3.3 CD8+ T cells are found in clusters and colocalize with demyelinated lesions in the CNS of symptomatic L31 mice We used an immunofluorescence approach to look at the localization of the CD8+ T cells, in the CNS and PNS of L31 mice, and to evaluate the extent of demyelination. We did so by using a mAb against CD8a, FluoroMyelin Red to stain myelin lipids, and TO-PRO-3-iodide to visualize the nuclei. 3-4 months old L31 mice were used as non-symptomatic controls for CNS staining because it was the time point where the greatest absolute number of CD8+ cells was found in the CNS (Figure 2A). The immunofluorescent staining of non- symptomatic L31 CNS sections demonstrated intact myelin with no demyelinated lesions, however clusters of CD8+ T cells could be seen (Figure 3). In contrast, in the CNS of symptomatic L31 mice, large CD8+ T cell clusters could be found that colocalized with demyelinated lesions, as seen by the decreased fluorescence in the myelin staining (Figure 3 lower panels). As expected, B6 mice demonstrated no CD8+ T cell infiltration and myelin architecture was not compromised (Figure 3 upper panels). Taken together, this Page | 32 data shows that CD8+ T cells were associated with demyelinated lesions in the CNS of symptomatic L31 mice and that CD8+ T cells were found in the CNS of non-symptomatic L31 mice, however the myelin staining in the nonsymptomatic L31 CNS was not compromised. 3.4 Devastating demyelination and CD8+ T cell infiltration in the PNS of symptomatic L31 mice The PNS of symptomatic L31 mice demonstrated a pronounced loss in myelin architecture and devastating demyelination (Figure 4). The B6 mice and nonsymptomatic L31 mice showed no CD8+ T cell infiltration and the myelin staining was normal as longitudinal sections depicted Schwann cells wrapping around axons along the whole course of the nerve (Figure 4). In addition, all the nuclei stained in the B6 and non-symptomatic L31 sections where of a squamous shape. We attributed these nuclei to normal Schwann cells and therefore, we concluded that no infiltrating cells were present as no round nuclei could have been seen (Figure 4). Although, the merged image from the L31 symptomatic mice demonstrated a cluster of round nuclei that did not stain for CD8a (Figure 4 bottom panels, arrow). Taken together this data demonstrates that CD8+ T cells are only seen in the PNS of symptomatic L31 mice and not in non-symptomatic L31 mice and that Page | 33 devastating demyelination solely occurs in the PNS of symptomatic mice. However, not all infiltrating cells were CD8+ T cells in these mice. 3.5 Macrophages are in close apposition to CD8+ T cells in the PNS of symptomatic L31 mice It was of interest to determine exactly what cell type accounted for these nuclei and therefore we investigated the possibility that the unstained cells in the PNS of symptomatic L31 mice were macrophages. We hypothesized that these cells were macrophages for two reasons. First, macrophages are known to be involved in mechanisms of demyelination in MS as they can release toxic oxygen and nitric species. Second, they also have the capacity to be APCs and the devastating demyelination in the PNS of symptomatic mice would lead to the presentation of many soluble antigens by APCs and these cells would be found surrounding clusters of T cells, just like the unstained round nuclei seen in the immunofluorescent stains mentioned above. To investigate whether these cells were macrophages, we once again stained for CD8a and nuclei, but also stained using a mAb against Iba-1, which is a cytoplasmic calcium-binding adaptor protein that is found in macrophages and microglia. However, we could not stain for CD8a and Iba-1 together because the two mAbs needed different fixation preparations in order to stain well. Therefore we had to stain serial sections of the PNS from symptomatic L31 mice and the staining pattern Page | 34 for CD8a and Iba-1 expression on serial sections suggested that macrophages were in close apposition to and encompassing CD8+ T cells in the PNS of these mice (Figure 5). 3.6 Characterizing the macrophage population in the PNS of symptomatic L31 mice To further investigate the macrophage population in the PNS of symptomatic L31 mice, we isolated mononuclear cells from the sciatic nerve using an enzymatic method in order to best represent the myeloid cell population. We then used a flow cytometry approach to compare the macrophage population in the PNS of symptomatic and non-symptomatic L31 mice. We stained for CD45 and the macrophage markers CD11b and F4/80. All CD45+ cells were gated on and CD11b and F4/80 expression was analysed. As it is seen in figure 6A, the percentage of macrophages was not increased between non-symptomatic and symptomatic L31 mice, as the two macrophage markers used, CD11b and F4/80, stained for an approximate total of 10% of the cells in both experimental groups. However it is interesting to note that there was an apparent difference in the total number of CD45+ cells between B6 controls and non-symptomatic L31 mice, as seen in the representative dot plots (Figure 6A). Page | 35 Most interestingly however, is that the percentage of CD11b+ F4/80+ macrophages was greatly increased in the PNS of symptomatic L31 mice (Figure 6A). Among the cells that were CD45+ CD11b+, the percentage of F4/80+ macrophages was significantly greater in the L31 symptomatic group compared to the L31 group (Figure 6B; p=0.0016; L31=5.5% ± 3.304, n=4; L31 symptomatic=50.0% ± 5.0, n=2). This demonstrated that the phenotype of the macrophages in the PNS of symptomatic L31 mice was different when compared to those found in the PNS of non-symptomatic L31 mice. The F4/80 marker on macrophages suggested a more mature macrophage phenotype that was phagocytosing debris in the area [71]. Page | 36 Figure 1: CD8+ T cells are found in the PNS of symptomatic L31 mice. (A) Representative flow cytometry dot plot of PNS cells from age matched B6 mice. (B) Representative flow cytometry dot plot of PNS cells from age matched non-symptomatic L31 mice. (C) Representative flow cytometry dot plot of PNS cells from L31 symptomatic mice. Page | 37 Figure 2: CD8+ T cells are found in the CNS of L31 mice early on, but only appear in the PNS of symptomatic L31 mice. (A) Graphic representation of CD8+ T cell infiltration kinetics into the CNS of L31 and control B6 mice. Data shown are the mean ± SEM and are representative of three independent experiments per mouse group per time point. (B) Graphic representation of CD8+ T cell infiltration kinetics into the PNS of L31 and control B6 mice. Data shown are the mean ± SEM and are representative of three independent experiments per mouse group per time point. Only the CNS data was significant for a two-way ANOVA analysis. *p≤0.05 Page | 38 Figure 3: CD8+ T cells are found in the CNS of L31 mice early on, but only cause demyelination in the CNS of symptomatic L31 mice. Immunofluorescent staining of spinal cord sections from 5-6 months old B6, 3-4 months old L31, and L31 symptomatic mice using anti-CD8a mAb, FluoroMyelin Red, and TO-PRO-3-iodide for the nuclei. Scale bars: 200μm; lower panels 60μm Page | 39 Figure 4: CD8+ T cells are only found in the PNS of symptomatic L31 mice and severe demyelination is also seen. Immunofluorescent staining of sciatic nerve sections from 5-6 months old B6, 5-6 months old L31, and L31 symptomatic mice using anti-CD8a mAb, FluoroMyelin Red, and TO-PRO-3iodide for the nuclei. Arrow is non-CD8a stained round nuclei population. Scale bar: 60μm Page | 40 Figure 5: Macrophages surround CD8+ T cell clusters in the PNS of symptomatic L31 mice. Immunofluorescent staining of serial sciatic nerve sections 6μm apart from L31 symptomatic mice using anti-CD8a mAb or anti-Iba-1 mAb (for macrophages), and TO-PRO-3-iodide for the nuclei. Scale bar: 20μm Page | 41 Figure 6: Macrophages in the PNS of symptomatic L31 mice have a more mature and phagocytic phenotype. (A) Representative flow cytometry dot plots of PNS cells from 5-6 months old B6, 5-6 months old L31, and L31 symptomatic mice. The dot plots are gated on CD45+ cells with the exclusion of dead cells. (B) Histogram of the % F4/80+ macrophages when gated on CD45+ CD11b+ myeloid cells in the PNS. Data presented as mean ± SEM, L31=5.5% ± 3.304, n=4; L31 symptomatic=50.0% ± 5.0, n=2; p=0.0016. Page | 42 4. DISCUSSION/CONCLUSION In this thesis, we have demonstrated that CD8+ T cells infiltrated into the CNS of L31 mice at an earlier time point and immunofluorescent staining revealed that CD8+ T cell clusters could be found in the CNS of non-symptomatic L31 mice, but no demyelination was present in these mice. CD8+ T cell clusters did colocalize with demyelinated lesions in the CNS of symptomatic L31 mice. We have also clearly demonstrated that CD8+ T cells were found in the PNS of symptomatic L31 mice and that devastating demyelination occurred in these mice, whereas there was no infiltration in non-symptomatic L31 mice and the PNS myelin architecture of non-symptomatic L31 mice was normal. Furthermore, we demonstrated that the PNS of symptomatic L31 mice is infiltrated by macrophages and these macrophages seem to surround the clusters of CD8+ T cells. The macrophages in the PNS of symptomatic L31 mice also had a distinct phenotype, which was not observed in nonsymptomatic L31 mice. The data show CD8+ T cells in the CNS of L31 mice first, before any infiltration into the PNS and CD8+ T cells are only present in the PNS of L31 mice once they exhibit neurological symptoms. It has also been previously shown that CD8+ T cells go through oligoclonal expansion early on in the CNS of L31 mice [26]. This evidence leads to the hypothesis that these CD8+ T cells are activated in an epitope specific manner in the CNS and then are Page | 43 reactivated and cause demyelination in the PNS via an epitope spreading phenomenon. Although, further experiments would have to be conducted to confirm this hypothesis, as the epitope specificity of the activated CD8+ T cells in the CNS and PNS is not known. It has been known for many decades now that the composition of CNS and PNS myelin is different [72] therefore the antigen to which the CD8+ T cells are activated against is quite limited. One example of such a shared myelin protein is MBP [73]. It would be of interest to determine if lymphocytes isolated from both the CNS and PNS of symptomatic L31 mice would respond to overlapping epitopes of the MBP. Another shared CNS and PNS myelin protein is myelin-associated glycoprotein (MAG) [74], which is a periaxonal membrane protein that was also implicated as a possible initiating epitope in the large-scale CSF proteomics study on pediatric MS patients mentioned in the introduction of this thesis [37]. The mechanism by which CD8+ T cells initially infiltrate into the CNS of L31 mice is also unknown. VLA-4 has been implicated in its role of allowing T cells, particularly CD8+ ones, to cross the BBB [42, 47]. It is obviously a well rationalized hypothesis that the CD8+ T cells in L31 mice do express VLA-4, but it has yet to be experimentally proven and it would be of interest to do so. CD8+ T cells infiltrate the PNS of symptomatic L31 mice, but this is not the only infiltrating cell type. An unexpected macrophage population found in the PNS of symptomatic L31 mice sheds light on some very interesting concepts of possible mechanisms of demyelination. As it was mentioned in the introduction, histopathological analysis of MS lesions has helped identify key Page | 44 cell types and mediators that are implicated in the demyelinated lesions of this disease [55]. Some of those cell types and mediators are T cells, both CD4+ and CD8+, macrophages, with ROS and NOS present, and autoantibody and compliment deposition. It is interesting to compare the demyelinated lesions in the CNS and PNS of symptomatic L31 mice because they have striking differences and these differences could be partially explained by the cell types present, as was also the case in the histopathological studies of MS patient samples. The demyelinated lesions in the CNS of symptomatic L31 mice are very contained and colocalize with clusters of approximately the same size of CD8+ T cells. Furthermore, flow cytometry data from the lab demonstrates that no macrophage population infiltrates into the CNS of symptomatic L31 mice [28]. The absence of macrophages in the lesions present in the CNS of symptomatic L31 mice is different than what is seen in lesions from MS patients and the lesions in the PNS of symptomatic L31 mice. Due to the very local nature of the demyelinated lesions in the CNS of symptomatic L31 mice and the fact that they colocalize with CD8+ T cell clusters, we would think that this demyelination is caused by a cell-specific cytotoxic attack, but, as aforementioned, Fas-ligand and perforin -/- L31 mice develop disease with quicker onset and kinetics, suggesting that these molecules play a regulatory role in L31 disease [28]. Therefore, a different mechanism of demyelination or cell death must be occurring. One hypothesis is that the local microglia are responsible for the demyelination in the CNS and they release ROS and NOS in Page | 45 the confined area of where they are activated and this causes the local demyelination. The demyelinated lesions in the PNS of symptomatic L31 mice are much more wide spread and the demyelination is very vast. Compared to the CNS, a large macrophage population is present, along with the CD8+ T cell clusters, and this type of infiltrating cells is similar to what has been seen in MS patients. We can hypothesize about a possible mechanism of demyelination and, although we have not experimentally demonstrated that ROS and NOS are present in the PNS of symptomatic L31 mice, we can hypothesize, due to the observed nature of the demyelinated lesions and the widespread presence of macrophages, that the surrounding macrophages in the areas of the lesions are releasing a soluble molecule, like ROS or NOS, that is causing more widespread myelin destruction. This process of demyelination is most likely also mediated by IFN-γ, as IFN-γ has been shown to be produced by CD8+ T cells in L31 mice and we have reported that IFN-γ receptor -/- L31 mice do not develop disease [26]. Furthermore, NOD-B7-2-KO mice that were discussed in the introduction and who only develop PNS pathology are also resistant to disease if they are IFN-γ -/- [69]. Another interesting finding from our results is the more mature and phagocytic phenotype of the macrophages found in the PNS of symptomatic L31 mice. This is a reasonable finding as the macrophages present in these sciatic nerves would most likely be phagocytosing a large amount of myelin debris and would be activated and promoted to mature in their phenotype. What is also Page | 46 interesting to point out from the results in section 3.6 is the fact that nonsymptomatic L31 mice do seem to have a larger number of CD45+ cells when compared to B6 mice as seen from the representative dot plots in figure 6A. This is different than what is seen in figure 2B where no obvious difference can be seen in the absolute number of CD8+ T cells between B6 and nonsymptoatic L31 mice. An explanation for this discrepancy is that a different method of cell isolation was used between the two independent experiments, mechanical dissociation for the kinetic experiments (Figure 2B) and enzymatic dissociation was used for the macrophage characterization experiments (Figure 6A). This difference may have yielded more isolated cells in the macrophage characterization experiments as the enzymatic dissociation method is becoming increasingly popular as it creates cell suspensions that have a better representation of myeloid populations. Another possible explanation for this discrepancy is the small n value used for the experiments. A total of three mice per mouse group were used for the kinetics study, therefore the representation of the groups may not have been very thorough. It is possible that a difference in absolute number of CD8+ T cells in the PNS of non-symptomatic L31 mice compared to B6 could have been seen if the n value of these experiments was increased. Moreover, the presence of a greater number of CD45+ cells in the PNS of nonsymptomatic L31 mice as compared to B6 controls raises the question if there already is pathology in the PNS of L31 mice at this time point. The fact that more lymphocytes could be present in the PNS of L31 mice compared to B6 is Page | 47 not surprising however, as T cells from L31 mice have been shown to have a more effector memory phenotype (CD44+, CD62L-) [25]. This means that more T cells are present in the periphery of L31 mice and the T cells can enter visceral organs to surveille the area, however no pathology has been seen in other visceral organs in L31 mice, even though a greater number of infiltrating T cells can be observed when compared to B6 [25]. This fact can further help explain the increase of infiltrating CD45+ cells in non-symptomatic L31 PNS compared to B6 and it is also the case that no pathology was seen in the PNS of L31 mice, until they became symptomatic (figure 4). As we have shown in the CNS-infiltrating kinetic experiments in section 3.2, mice as early as 3-4 months of age have infiltrating CD8+ T cells in their CNS and they also have infiltrating CD8+ T cells in their CNS at the 5-6 months old time point. However, the results indicate that a greater absolute number of CD8+ T cells can be found in the CNS of L31 mice at 3-4 months of age (1846 ± 483, n=3) compared to the 5-6 months old time point (689 ± 221, n=3). This difference is not significant (p=0.0953), but there is an obvious drop between the two time points. One possible explanation for this fact is that the L31 mice spontaneously develop disease at any point between the ages of 3-6 months of age but, in our SPF facility, not all L31 mice become symptomatic. This means that many mice at the 3-4 months time point may be progressing to disease, but, at the 5-6 months old time point, many of these mice may never develop neurological symptoms and thus would have fewer CD8+ T cells in their CNS. Furthermore, it has been a personal observation that L31 mice are a Page | 48 heterogeneous population and that some mice will develop disease at an earlier age and develop more aggressive symptoms that leads to sudden death and other mice will develop disease later on in life and will not develop as severe symptoms and the progression of their disease would be slower. Therefore, when sacrificing mice at a 3-4 months old time point, the mice that will develop a more aggressive disease are present in the group, but when sacrificing mice at the 5-6 months old time point, none of these mice remain. The decline in absolute number of CD8+ T cells between the CNS of L31 mice at 3-4 and 5-6 months old can thus be attributed to these reasons. The concept that CD8+ T cells can mediate a neurological disease in mice is also a subject that merits thorough discussion. As it has been presented in the introduction, the most common mouse model used to study demyelinating disease in mice is the EAE model. This model normally has a Th1/17 response against a myelin antigen and this causes demyelination in the CNS. Moreover, it has been shown in Darlington et al. [36] that a diminished Th17 response in MS patients completely abrogates any RRMS disease. This definitely points towards CD4+ T cells as the initiators of CNS inflammation, but the many lines of evidence, which were discussed in the introduction, clearly demonstrate that CD8+ T cells play a role in the pathogenesis of MS. A recent report from Nature Immunology [62] may shed some light on a process that occurs that allows both T cell subsets to be involved in a demyelinating pathology. This report uses a model of EAE to describe a DC population that cross-presents a MHC class I-restricted MBP epitope that then activates CD8+ T cells and Page | 49 allows them to specifically attack oligodendrocytes. The initial demyelination in this EAE model is also mediated by CD4+ T cells. Therefore the initial CD4+ T cell-mediated demyelination leads to the activation of CD8+ T cells, which can directly attack oligodendrocytes, and cause further demyelination. The question remains however if CD8+ T cells can initiate demyelination themselves. In L31 mice, microglia constitutively express B7.2 and we have shown that this is necessary for disease onset [25]. This may explain why a CD8+ T cellmediated demyelinating disease could be initiated in L31 mice irrespective of CD4+ T cells. It is plausible that microglia in L31 mice could present a MHC class I-restricted myelin epitope that would activate local CD8+ T cells because of the constitutive expression of B7.2 and this would cause the initiating demyelination. The importance of CNS-resident myeloid cells in the EAE mouse model was demonstrated in the March 2005 issue of Nature Medicine. In this issue three articles demonstrate a myeloid lineage cell, albeit a DC, microglia or macrophage, that is very important in the course of demyelinating disease in EAE mice. The first demonstrated how EAE was repressed when microglia were abolished using ganciclovir [75]. The second paper demonstrates how it is not DCs in the secondary lymphoid organs or the mouse brain parenchyma that where necessary to permit immune invasion of the CNS, but most likely a distinct vessel-associated DC, which was also present in human MS brain tissue, that permitted CNS immune invasion [76]. The last article uses two mouse models of MS, EAE and Theiler’s murine Page | 50 encephalomyelitis virus-induced demyelinating disease, to demonstrate that T cells within the CNS of these mice become specific against different epitopes of PLP throughout the course of disease and thus epitope spreading initiates and can occur specifically within the CNS [77]. A news and views article in this same issue nicely discusses the possibility that the myeloid-lineage APCs that are presented in all these papers may very well be a very similar or even the same cell type [78]. The authors of the news views go on to state that the overall message from all the studies demonstrates that myeloid APCs play a crucial role in mediating infiltration, presenting myelin antigens, and causing neuroinflammation and pathology in animal models of MS. This is also partially true in L31 mice as the B7.2 expression on microglia is necessary for disease and therefore the microglia are likely responsible for disease initiation and to some extent a possible epitope spreading phenomenon. Taking into consideration the data presented in this thesis and our previous knowledge of the L31 mice, we propose the following model. CD8+ T cells are activated in the CNS of L31 mice early on in life due to the expression of B7.2 on microglia and this activation leads to demyelination that may not be due to a cell specific attack by CD8+ T cells. However, this demyelination causes other myelin antigens to be exposed and therefore presented by local APCs to other CD8+ T cells, which would then be activated. Some activated CD8+ T cells could be specific for a myelin protein that is also found in the PNS, such as MBP. Through this epitope spreading phenomenon, the activated CD8+ T cells would then be able to be more readily activated if they recognize their antigen Page | 51 in the PNS and continue to cause demyelination at this distant site. The recruitment of macrophages to the PNS then causes the release of ROS and NOS that causes more widespread PNS demyelination and the onset of neurological disease. The study of L31 mice has implications to both MS and PNS demyelinating diseases like GBS and CIDP. The study of L31 mice may help in understanding mechanisms by which CD8+ T cells can cause demyelination in both the CNS and PNS. As well, it demonstrates possible modes of demyelinating pathology that are exerted by CD8+ T cells in, possibly, macrophage dependant and independent manners. The importance of CNSresident APCs cannot be overlooked in MS and this fact is also supported by the L31 mouse model. Finally, this thesis work sheds light on a possible epitope spreading phenomenon that causes demyelinating diseases to spread from the CNS to the PNS, an event that does not occur frequently in MS (5% of patients) [61], but should not be ignored as it needs to be recognized as a treatable condition. Page | 52 5. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Bluestone, J.A., New Perspectives of Cd28-B7-Mediated T-Cell Costimulation. Immunity, 1995. 2(6): p. 555-559. Linsley, P.S. and J.A. Ledbetter, The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol, 1993. 11: p. 191-212. Greenwald, R.J., G.J. Freeman, and A.H. Sharpe, The B7 family revisited. Annu Rev Immunol, 2005. 23: p. 515-48. Alegre, M.L., K.A. Frauwirth, and C.B. Thompson, T-cell regulation by CD28 and CTLA-4. Nat Rev Immunol, 2001. 1(3): p. 220-8. Teft, W.A., M.G. Kirchhof, and J. Madrenas, A molecular perspective of CTLA-4 function. Annu Rev Immunol, 2006. 24: p. 6597. Itoh, Y. and R.N. Germain, Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells. J Exp Med, 1997. 186(5): p. 757-66. Lindstein, T., et al., Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science, 1989. 244(4902): p. 339-43. Fraser, J.D., et al., Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. Science, 1991. 251(4991): p. 313-6. Boise, L.H., et al., bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell, 1993. 74(4): p. 597608. Walunas, T.L., et al., CTLA-4 can function as a negative regulator of T cell activation. Immunity, 1994. 1(5): p. 405-13. Krummel, M.F. and J.P. Allison, CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med, 1995. 182(2): p. 459-65. Tivol, E.A., et al., Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity, 1995. 3(5): p. 541-7. Waterhouse, P., et al., Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science, 1995. 270(5238): p. 9858. Podojil, J.R. and V.M. Sanders, Selective regulation of mature IgG1 transcription by CD86 and beta 2-adrenergic receptor stimulation. J Immunol, 2003. 170(10): p. 5143-51. Page | 53 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Podojil, J.R., N.W. Kin, and V.M. Sanders, CD86 and beta2adrenergic receptor signaling pathways, respectively, increase Oct-2 and OCA-B Expression and binding to the 3'-IgH enhancer in B cells. J Biol Chem, 2004. 279(22): p. 23394-404. Grohmann, U., et al., CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol, 2002. 3(11): p. 1097-101. Taylor, P.A., et al., B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions [corrections]. J Immunol, 2004. 172(1): p. 34-9. Oliveira-dos-Santos, A.J., et al., CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis. J Immunol, 1999. 162(8): p. 4490-5. Tada, Y., et al., CD28-deficient mice are highly resistant to collageninduced arthritis. J Immunol, 1999. 162(1): p. 203-8. Cross, A.H., et al., Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J Clin Invest, 1995. 95(6): p. 2783-9. Lenschow, D.J., et al., Differential effects of anti-B7-1 and anti-B7-2 monoclonal antibody treatment on the development of diabetes in the nonobese diabetic mouse. J Exp Med, 1995. 181(3): p. 1145-55. Karandikar, N.J., et al., CTLA-4: a negative regulator of autoimmune disease. J Exp Med, 1996. 184(2): p. 783-8. Luhder, F., et al., Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulates the unfolding of autoimmune diabetes. J Exp Med, 1998. 187(3): p. 427-32. Fournier, S., et al., T cell-mediated elimination of B7.2 transgenic B cells. Immunity, 1997. 6(3): p. 327-39. Zehntner, S.P., et al., Constitutive expression of a costimulatory ligand on antigen-presenting cells in the nervous system drives demyelinating disease. FASEB J, 2003. 17(13): p. 1910-2. Brisebois, M., et al., A pathogenic role for CD8+ T cells in a spontaneous model of demyelinating disease. J Immunol, 2006. 177(4): p. 2403-11. Hung, H.Y., Characterization of the CD4+ CD25+ regulatory T cells in an animal model of spontaneous demyelination in the central nervous system, in McGill theses2009. Estrada Guadarrama, J.A., Mechanisms of demyelination and axonal damage in a CD8+ T cell-mediated model of spontaneous demyelinating disease, in McGill theses2010, McGill University Library,: Montreal. Duquette, P., et al., The increased susceptibility of women to multiple sclerosis. Can J Neurol Sci, 1992. 19(4): p. 466-71. Noseworthy, J.H., et al., Multiple sclerosis. N Engl J Med, 2000. 343(13): p. 938-52. Page | 54 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. Beretich, B.D. and T.M. Beretich, Explaining multiple sclerosis prevalence by ultraviolet exposure: a geospatial analysis. Mult Scler, 2009. 15(8): p. 891-8. Munger, K.L. and A. Ascherio, Prevention and treatment of MS: studying the effects of vitamin D. Mult Scler, 2011. 17(12): p. 140511. Stromnes, I.M. and J.M. Goverman, Active induction of experimental allergic encephalomyelitis. Nat Protoc, 2006. 1(4): p. 1810-9. Stromnes, I.M. and J.M. Goverman, Passive induction of experimental allergic encephalomyelitis. Nat Protoc, 2006. 1(4): p. 1952-60. Hafler, D.A., et al., Risk alleles for multiple sclerosis identified by a genomewide study. N Engl J Med, 2007. 357(9): p. 851-62. Darlington, P.J., et al., Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation. Ann Neurol, 2013. 73(3): p. 341-54. Dhaunchak, A.S., et al., Implication of perturbed axoglial apparatus in early pediatric multiple sclerosis. Ann Neurol, 2012. 71(5): p. 60113. Babbe, H., et al., Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction. J Exp Med, 2000. 192(3): p. 393-404. Friese, M.A. and L. Fugger, Autoreactive CD8+ T cells in multiple sclerosis: a new target for therapy? Brain, 2005. 128(Pt 8): p. 174763. Lucchinetti, C.F., et al., Inflammatory cortical demyelination in early multiple sclerosis. N Engl J Med, 2011. 365(23): p. 2188-97. Tzartos, J.S., et al., Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis. Am J Pathol, 2008. 172(1): p. 146-55. Ifergan, I., et al., Central nervous system recruitment of effector memory CD8+ T lymphocytes during neuroinflammation is dependent on alpha4 integrin. Brain, 2011. 134(Pt 12): p. 3560-77. Friese, M.A., et al., Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis. Nat Med, 2008. 14(11): p. 1227-35. Ji, Q., A. Perchellet, and J.M. Goverman, Viral infection triggers central nervous system autoimmunity via activation of CD8+ T cells expressing dual TCRs. Nat Immunol, 2010. 11(7): p. 628-34. Kappos, L., et al., A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med, 2010. 362(5): p. 387-401. Polman, C.H., et al., A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med, 2006. 354(9): p. 899-910. Page | 55 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. Stuve, O., et al., Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann Neurol, 2006. 59(5): p. 743-7. van Oosten, B.W., et al., Treatment of multiple sclerosis with the monoclonal anti-CD4 antibody cM-T412: results of a randomized, double-blind, placebo-controlled, MR-monitored phase II trial. Neurology, 1997. 49(2): p. 351-7. Huseby, E.S., et al., A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis. J Exp Med, 2001. 194(5): p. 669-76. Na, S.Y., et al., Naive CD8 T-cells initiate spontaneous autoimmunity to a sequestered model antigen of the central nervous system. Brain, 2008. 131(Pt 9): p. 2353-65. Gobel, K., et al., Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte-directed CD8(+) T cell attack. Glia, 2010. 58(4): p. 469-80. Ip, C.W., et al., Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes. J Neurosci, 2006. 26(31): p. 8206-16. Leder, C., et al., Clonal expansions of pathogenic CD8+ effector cells in the CNS of myelin mutant mice. Mol Cell Neurosci, 2007. 36(3): p. 416-24. Baughman, E.J., et al., Neuroantigen-specific CD8+ regulatory T-cell function is deficient during acute exacerbation of multiple sclerosis. J Autoimmun, 2011. 36(2): p. 115-24. Popescu, B.F. and C.F. Lucchinetti, Pathology of demyelinating diseases. Annu Rev Pathol, 2012. 7: p. 185-217. Fischer, M.T., et al., NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain, 2012. 135(Pt 3): p. 886-99. Fischer, M.T., et al., Disease-specific molecular events in cortical multiple sclerosis lesions. Brain, 2013. Henkart, P.A., et al., Do CTL kill target cells by inducing apoptosis? Semin Immunol, 1997. 9(2): p. 135-44. Stinchcombe, J.C. and G.M. Griffiths, The role of the secretory immunological synapse in killing by CD8+ CTL. Semin Immunol, 2003. 15(6): p. 301-5. Davies, S., et al., Spread of T lymphocyte immune responses to myelin epitopes with duration of multiple sclerosis. J Neuropathol Exp Neurol, 2005. 64(5): p. 371-7. Misawa, S., et al., Peripheral nerve demyelination in multiple sclerosis. Clin Neurophysiol, 2008. 119(8): p. 1829-33. Ji, Q., L. Castelli, and J.M. Goverman, MHC class I-restricted myelin epitopes are cross-presented by Tip-DCs that promote determinant spreading to CD8(+) T cells. Nat Immunol, 2013. 14(3): p. 254-61. Page | 56 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. Ropper, A.H., The Guillain-Barre syndrome. N Engl J Med, 1992. 326(17): p. 1130-6. Griffin, J.W., et al., Early nodal changes in the acute motor axonal neuropathy pattern of the Guillain-Barre syndrome. J Neurocytol, 1996. 25(1): p. 33-51. Chance, P.F. and K.H. Fischbeck, Molecular genetics of CharcotMarie-Tooth disease and related neuropathies. Hum Mol Genet, 1994. 3 Spec No: p. 1503-7. Siskind, C.E., et al., A Review of Genetic Counseling for Charcot Marie Tooth Disease (CMT). J Genet Couns, 2013. Louvet, C., et al., A novel myelin P0-specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy. J Exp Med, 2009. 206(3): p. 507-14. Salomon, B., et al., Development of spontaneous autoimmune peripheral polyneuropathy in B7-2-deficient NOD mice. J Exp Med, 2001. 194(5): p. 677-84. Bour-Jordan, H., H.L. Thompson, and J.A. Bluestone, Distinct effector mechanisms in the development of autoimmune neuropathy versus diabetes in nonobese diabetic mice. J Immunol, 2005. 175(9): p. 5649-55. Barrette, B., et al., Requirement of myeloid cells for axon regeneration. J Neurosci, 2008. 28(38): p. 9363-76. Gordon, S., et al., F4/80 and the related adhesion-GPCRs. Eur J Immunol, 2011. 41(9): p. 2472-6. Horrocks, L.A., Composition of myelin from peripheral and central nervous systems of the squirrel monkey. J Lipid Res, 1967. 8(6): p. 569-76. Matthieu, J.M., et al., Low myelin basic protein levels and normal myelin in peripheral nerves of myelin deficient mutant mice (MLD). Neuroscience, 1980. 5(12): p. 2315-20. Quarles, R.H., Myelin-associated glycoprotein (MAG): past, present and beyond. J Neurochem, 2007. 100(6): p. 1431-48. Heppner, F.L., et al., Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med, 2005. 11(2): p. 146-52. Greter, M., et al., Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis. Nat Med, 2005. 11(3): p. 328-34. McMahon, E.J., et al., Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nat Med, 2005. 11(3): p. 335-9. Platten, M. and L. Steinman, Multiple sclerosis: trapped in deadly glue. Nat Med, 2005. 11(3): p. 252-3. Page | 57