Cytotoxic NKG2C + CD4 T cells target oligodendrocytes in Multiple Sclerosis

Fatma Zaguia

Microbiology and Immunology McGill University, Montreal December 2012

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master’s in Science

© Fatma Zaguia, 2012

Table of Contents ACKNOWLEDGEMENTS ...... 4

CONTRIBUTIONS OF AUTHORS ...... 8

ABSTRACT ...... 9

RÉSUMÉ ...... 10

INTRODUCTION AND LITTERATURE REVIEW ...... 12

Multiple Sclerosis ...... 12 Disease Overview ...... 12 Clinical Features ...... 13 Pathological Features ...... 13 Diagnosis and disease modulating therapies ...... 14 Immune-mediated damage to the CNS ...... 15 Evidence of autoimmunity in the pathogenesis of MS ...... 15 Immune mediators involved in disease pathogenesis ...... 16 CD8 T cells ...... 17 Natural Killer cells ...... 17 B cells ...... 18 Gamma delta T cells ...... 19 Macrophages ...... 20 Cytokines ...... 20 Role of CD4 T cells in MS pathogenesis ...... 23 Implication in MS and EAE model ...... 23 Autoreactive CD4 T cells ...... 25 Direct targeting of oligodendrocytes ...... 26

Signaling through NKG2 receptors and the HLA-E ligand...... 28 HLA-E ...... 28 NKG2A and NKG2C ...... 30 Signaling through NKG2A ...... 32 Signaling through NKG2C ...... 33

FINDINGS ...... 36

Cytotoxic NKG2C + CD4 T cells target oligodendrocytes in Multiple Sclerosis.... 36 Abstract ...... 37 Introduction...... 38 Results and Discussion ...... 40 Materials and Methods ...... 49 Bibliography ...... 56 Figure legends ...... 60

2 DISCUSSION ...... 70

CONCLUSION AND FUTUR DIRECTIONS ...... 75

REFERENCES ...... 77

3 ACKNOWLEDGEMENTS

First and foremost I would like to thank my supervisors, Dr. Jack Antel and Dr. Nathalie Arbour. Dr. Antel, it has been an immense pleasure to have the opportunity to work in your laboratory. You have been an amazing mentor, teacher and a constant source of encouragement. No matter how busy your day was, you never hesitated to lean back in your chair, put your feet up on the table and tell me “OK…. Let’s take the time to discuss what’s next”. When times were tough, you always knew how to tell me to take a step back, and to remember the big picture and the story we want to tell. Science aside, you are one of the kindest people I have had the chance of meeting; thank you for always being so attentive and enthusiastic about whichever direction the project took. I can only start to show my gratitude by helping you from time to time navigate your Mac laptop. You have most definitely set an example as to the kind of clinician and researcher I aspire to become one day.

My experience as a Master’s student would not h ave been complete without my co-supervisor, Dr. Nathalie Arbour. Nathalie, from the first day you sat me down to explain what a CD8 T cell was, I knew there was an encyclopedia of knowledge that I could gain from you. You really trained me from scratch, helped me overcome my fear of the FACS machine, and taught me the rigor and discipline required for “good science”. You have been incredibly generous with your time and attention throughout the years; hours were spent helping me with writing, presentations, understanding concepts – all this with incredible kindness and laughter. Any organizational skills that I possess today, I mostly have you to thank for (although my lab desk will probably always be just a little too messy for your liking…).

Thank you both for an incredible MSc. experience!

Next, I would like to thank members from the MNI laboratory. First, a big thank you to Manon Blain and Ellie McCrea, our two technicians and the “real

4 bosses” of the lab. You both make the environment and life in the lab so enjoyable and pleasant. Ellie, thank you for the hours spent helping me generate the T cell lines. We definitely had our shares of laughs and hair- tearing moments and no one activates those T cells better than you! Manon, “la maman du labo”, we would al l be lost without you. From day one I knew that when times were tough, there would always be someone to turn to for help or just a pep talk.

A big thank you to Philippe Saikali, who started this project and who took time in the beginning of my MSc. to explain everything and anything; you were always an email away when I had questions and I am so grateful for that!

Thank you Veronica Miron-Lafrance, Caroline Lambert and Qiao-Ling Cui for teaching me how to do the adults preps. I wish our paths had crossed more often; I know there is much to learn from you.

Thank you Trina Johnson for your presence in lab meetings. You always say it as it is and some of the best constructive criticism has come from you. Big thanks also for hosting the greatest parties!

And last but not least, I am eternally indebted towards Bryce Durafourt. Who knew that walking into the basement of the MNI on that first day I would be meeting one of my best friends and future classmates. Thank you for being an amazing friend throughout the years; you constantly pushed me, didn’t let me give up (and god knows there were times where that’s all I wanted to do!), took care of my traveling documents, put up with my hysterical moments (missing prereqs?!), corrected my statements (apparently I cannot use “whilst”), shared great Top40 music with me, and the list goes on….I can only hope that I’ve been half as valuable as a friend as you’ve been to me. Can’t wait until the days we “consult and discuss patient files while using our pagers”.

5 Now on to my lab at the CHUM. Thank you Diane Beauseigle for your kindness and help throughout the years.

A big thank you to Alma Mohebiany. You were my first friend in the lab and here in Montreal; I am so happy that our friendship hasn’t wavered despite that you are now across the Atlantic Ocean. Thank you for sharing my love of music, baked sweets and large coffee mugs! One day we will find that perfect cup…Late days in the lab wouldn’t have been the same without you.

Camille Pittet, thank you for being a great lab and life friend. Your organized, and well-written lab books made my life so easy on more than one occasion. You always had time for all my questions especially the eternal “what concentration did you use?”. And then we would proceed to go have lunch and talk about shoes; what more could I ask for?

Thank you Raphael Schneider, for your help throughout my time in the lab. You always had a kind comment to say or a funny joke to crack; working together on the Journal Club paper was a pleasure.

I would like to also thank Dr. Alexandre Prat and his laboratory, for being great neighbors. A special thank you to Hania Kebir; we have shared great moments together and your friendship is invaluable to me. Hours were spent discussing, drinking coffee, laughing; you are brilliant and it was always such a pleasure to get your input. Thank you Igal Ifergan, for being my go-to person for anything FACS related; you are an encyclopedia! You are one of the kindest people I know, always available to help me, or to just for a latte and a talk…Thank you Jorge Ivan Alvarez for the hours spent with me at the confocal microscope; your insight on anything, whether it is science or life, is inspiring. You always push to try new things (ie. chicharron) and they have been some of my best memories.

6 Thank you to the pathologists Dr. Samuel Ludwin and Dr. Jia Newcombe for their contributions to the project. Thank you to Dr. Jeffrey Hall and Dr. André Olivier for their collaboration by providing us with the surgical samples.

To my family, without whom I would be lost, I owe you the biggest thank you of all. To my wonderful parents and my beautiful sisters, thank you for your unconditional support throughout everything. Thank you to all the other people in my life for simply always being there: Fredj, Rami, Georges, Lydia, Katherine, Mel, Ammayas and Haitham.

7 CONTRIBUTIONS OF AUTHORS

The manuscript included in the thesis is entitled “ Cytotoxic NKG2C + CD4 T cells target oligodendrocytes in Multiple Sclerosis”. I conducted most of the experimental work in this paper. Dr. Philippe Saikali started the project prior to my arrival. Diane Beauseigle stained the tissue sections for immunohistochemistry that were provided by Dr. Jia Newcombe and Dr. Samuel Ludwin. Ellie McCrea helped generate the myelin-specific T cell lines. Dr. Alexandre Prat provided us with antibodies and access to the flow cytometry machine, as well as scientific input. The writing of the paper was done collaboratively between Drs. Nathalie Arbour, Jack Antel and myself. At the time of the submission of this thesis, this paper was being reviewed at the Journal of Immunology.

8 ABSTRACT

The mechanisms whereby immune cells infiltrating the central nervous system in multiple sclerosis (MS) contribute to tissue injury remain to be defined. CD4 T cells are implicated as key mediators of the deleterious inflammation observed in MS. Myelin reactive CD4 T cells expressing CD56, an NK cell marker, were previously shown to be cytotoxic to human oligodendrocytes in vitro . We sought to determine whether myelin reactive

CD4 T cells could also express and utilize other NK associated markers to mediate oligodendrocyte directed cytotoxicity. We observed that myelin reactive CD4 T cell lines, as well as short term PHA-activated CD4 T cells can express NKG2C, the activating receptor that interacts with HLA-E, a non- classical MHC class I molecule. These cells co-expressed CD56, had elevated levels of cytotoxic molecules FasL, granzyme B and perforin compared to their NKG2C-negative counterparts and mediated significant in vitr o cytotoxicity towards human oligodendrocytes induced to express HLA-E with pro-inflammatory cytokines. A significantly elevated proportion of ex-vivo peripheral blood CD4 T cells from MS patients expressed NKG2C compared to controls. Our immunohistochemical analysis demonstrated that MS tissue sections displayed HLA-E+ oligodendrocytes and NKG2C + CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4T cells could contribute to tissue injury in MS.

9 RÉSUMÉ

La sclérose en plaques (SEP) est caractérisée par l ’infiltration de cellules immunes au sein du système nerveux central (SNC). Les mécanismes par lesquels ces cellules contribuent à la formation des lésions typiques de cette pathologie restent à élucider. Les cellules T CD4 jouent un rôle important dans l'inflammation observée en SEP. Des travaux antérieurs ont démontré que des cellules T CD4 spécifiques à la myéline et exprimant le CD56, un marqueur des cellules NK, ont une activité cytotoxique in vitro dirigée contre les oligodendrocytes humains. Nous avons déterminé si les cellules T CD4 réactives à la myéline expriment et utilisent d'autres marqueurs NK impliqué dans cette cytotoxicité contre les oligodendrocytes. Nous avons observé que des lignées de cellules T CD4 spécifiques pour la myéline, ainsi que des cellules T CD4 activées par la PHA peuvent exprimer le NKG2C, le récepteur activateur qui interagit avec le HLA-E, une molécule non-classique du CMH de classe I. Les cellules T CD4 positives pour le NKG2C co-expriment le CD56, des niveaux élevés de molécules cytotoxiques tels que le FasL, le granzyme B et la perforine par rapport à leurs homologues n’exprimant pas le NKG2C.

Nous avons aussi montré que ces cellules sont cytotoxiques envers des oligodendrocytes humains, qui expriment le HLA-E suite à un traitement avec des cytokines des inflammatoires. La proportion des cellules T CD4 exprimant le NKG2C est significativement plus élevée dans les échantillons de sang ex-vivo provenant de patients atteints de la SEP par rapport à ceux des témoins. Nous avons observé par immunohistochimie sur des coupes de

10 tissus de patients atteints de la SEP la présence d ’oligodendrocytes exprimant le HLA-E ainsi que des cellules T CD4 arborant le NKG2C. Nos résultats révèlent un nouveau mécanisme par lequel les cellules T CD4 pourraient contribuer directement à la formation des lésions tissulaires observées dans le SNC des patients atteints de la SEP.

11 INTRODUCTION AND LITTERATURE REVIEW

Multiple Sclerosis

Disease Overview

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), characterized by injury to oligodendrocytes, which are responsible for the synthesis of myelin, and axonal loss[1]. MS is the most common neurological disease affecting young adults in Canada, with an age of onset that varies between 15 and 40. Women are more than three times as likely to develop MS then men[2]. The prevalence of MS varies widely around the world. The prevalence is highest in northern Europe, southern Australia, and North America. There has also been a trend toward an increasing prevalence and incidence in specific regions such as southern Europe. While the etiology of MS is still unclear, both environmental and genetic factors have been suggested to play a role [2].

The disease susceptibility has been linked to genetic background. For example, identical twins have a concordance of MS of 30% supporting the notion that genes significantly contribute but do not completely explain susceptibility. Candidate gene approaches revealed multiple possible candidates that contribute to disease susceptibility, severity or disease course. However, the strongest and unequivocal evidence for an association with MS is established for certain alleles of the human histocompatibility leukocyte antigen class II (HLA-DR) region[3].

12 Clinical Features Symptoms of MS are unpredictable and vary greatly from person to person.

Clinical manifestations can involve motor, sensory, visual, and symptoms such as chronic fatigue, dizziness, bladder dysfunction as well as cognitive impairment[4]. Few of the clinical features are disease-specific, but particularly indicative ones are Lhermitte's sign (an electrical sensation running down the spine or limbs on neck flexion) and the Uhthoff ’s phenomenon (transient worsening of symptoms and signs when core body temperature increases, such as after exercise or a hot bath)[5]. The pattern of progression of MS can also differ from patient to patient.

Eighty percent of patients present with the relapsing –remitting form of MS, where symptoms and signs typically evolve over a period of several days, stabilize, and then often improve, within weeks. Persistent signs of CNS dysfunction may develop after a relapse, and the disease may progress between relapses as well as in absence of relapses; it is then termed secondary progressive MS. Twenty percent of affected patients have primary progressive MS, which is characterized by a gradually progressive clinical course with no recovery periods[2].

Pathological Features Pathologically, MS lesions are characterized by perivascular infiltrates of CD4 and CD8 T cells and macrophages. T cells as well as macrophages/microglia are found toward the periphery of the lesion and in the normal appearing white matter[6]. These lesions typically develop in white matter, where the

13 primary targets are the myelin sheath and the myelinating cell, the oligodendrocyte. However, gray-matter lesions, are known to occur[7].

Furthermore, differences in pathology have also been documented for chronic versus acute lesions. Active lesions are characterized by an indistinct margin, intense perivascular infiltration by lymphocytes, loss of myelin and oligodendrocytes, widespread axonal damage, myelin-laden macrophages, hypertrophic astrocytes, and little or no astroglial scarring. Areas of tentative remyelination can be observed. In contrast, with acute lesions, chronic lesions display sharp edges, few infiltrating leukocytes, naked axons embedded in a matrix of fibrous, scarring astrocytes and virtually no oligodendrocytes[7].

Diagnosis and disease modulating therapies

Diagnosis is usually established through clinical evidence, supplemented by laboratory investigations such as Magnetic Resonance Imaging (MRI) and cerebral spinal fluid (CSF) analysis. For diagnosis purposes, the principle is to establish the existence of disease activity consistent with focal demyelination affecting more than one part of the CNS and on more than one occasion[4, 5].

The concept of MS as an autoimmune inflammatory disease is supported by the response to immunomodulatory and immunosuppressive treatments.

While no therapies are known to repair the deleterious effects of the disease to the CNS, available treatments are known to decrease the severity, duration and quantity of relapses, thus slowing accumulation of disability. Therapies include interferon-beta (an immunomodulatory agent), glatiramer acetate

14 (injection of a mix of four amino acids that is thought to shift T cells towards an anti-inflammatory profile), natalizumab (an antibody that blocks entry of lymphocytes into organs) and FTY-720 (a sphingosine analog that sequesters immune cells in lymph nodes)[8]. Unfortunately, these therapies have only been shown to modulate disease in patients with relapsing-remitting MS; there are much fewer treatment options for patients with primary or secondary progressive MS.

Immune-mediated damage to the CNS

Evidence of autoimmunity in the pathogenesis of MS

The ability to discriminate between a foreign pathogen and self-tissue is a key feature of the immune system. Breakdown of self –tolerance leads to the development of autoimmune diseases characterized by deleterious attack of self (cells, structures, etc.) by the immune system. Evidence seems to point to the classification of MS as an autoimmune pathogenesis of the CNS. These include: l) pronounced B-cell responses within the CNS reflected in the CSF by increased immunoglobulin’s reflected by oligoclonal bands [9] 2) accumulation of T cells and plasma cells and of major histocompatibility

(MHC) class II-positive microglial/macrophage cells in MS brain lesions [10]

3) an elevation systemically and further enrichment within the CSF of interferon- gamma (IFN-γ) -secreting T-helper type 1 (Th 1)-like cells as well as interleukin 17 (IL-17)-producing T-helper type 17(Th17) cells. Impact of

15 immunomodulatory therapies is also evidence that MS is immune-mediated.

Studies of animal models demonstrating that autoreactive T cells (CD4 or

CD8) can result in inflammatory demyelination of the central nervous system further support the theory that MS is an immune-mediated disorder involving one or more antigens located in the myelin of the central nervous system. If myelin-specific lymphocytes are present in the cells that infiltrate the CNS, they may trigger a cascade of events resulting in the formation of an acute inflammatory, demyelinating lesion[7]. In general, an optimal balance between these T-cell subsets and their cytokine production is necessary for a successful immune response, whereas a disturbed balance may lead to disease, as is thought to be the case in MS.

Immune mediators involved in disease pathogenesis

Evidence from animal studies as well as human studies identify CD4 T cells as key effector cells in the pathogenesis of MS. Adoptive transfer of activated myelin reactive CD4 T cells trigger disease in naive animal recipients [11].

However, the role of CD8 T cells in disease development has come to be recognized. Activated effector memory CD8 T cells are enriched in the CNS and the cerebrospinal fluid (CSF) of MS patients [12]. This subset normally resides in tissues and can quickly and efficiently mediate effector function upon antigen recognition.

16 CD8 T cells

In studies with human cells, it has been shown that CD8 T cells can kill oligodendrocytes and neurons. Neurons and oligodendrocytes can express

MHC-I in MS lesions and CD8 T cells can attack neurites in an antigen-specific manner, leading to axonal damage similar to that seen in MS brain lesions[13]. Furthermore, it has been shown that by micromanipulation and single-cell polymerase chain reaction, the occurrence of clonal T cell receptor

V-beta (TCRBV) expansion in CNS lesions of two MS patients mainly involved

CD8 T cells[14] suggesting the local intra-CNS activation of these immune cells.

Experimental autoimmune encephalomyelitis (EAE) has been one of the most valuable autoimmune models, providing insights into many mechanisms during physiological and pathological immune responses. Classical EAE is induced in susceptible animal strains by immunization with myelin antigens and Freund's adjuvant. The disease is usually monophasic with spontaneous remission[15]. The role of CD8 T cells in MS has been further stimulated by findings in animal models where EAE was induced by purified myelin- specific CD8 T cells[16]. Similarly, the adoptive transfer of CD8 T cell clones specific for a myelin basic protein peptide (MBP) also induced EAE [17].

Natural Killer cells

An association between decreased Natural Killer (NK) cell activity and MS was first reported in the 1980’s and later studies expanded on this topic,

17 although findings remain controversial [18, 19]. Potential explanations are disease heterogeneity among patient groups and fluctuations of NK activity and number during the disease course. Furthermore, NK deficiencies exist in peripheral blood, lesions, and CSF of MS patients[20]. Studies have suggested that NK cell depletion in two different EAE models exacerbate disease whereas the transfer of in vitro generated NK cells decreases autoimmunity.

NK cells could suppress autoimmunity by cytokine production or by the induction of target lysis via perforin- and/or TRAIL dependent

Mechanisms, with the target being autoreactive T cells. These combined observations indicate that NK cells may exert important immunoregulatory functions in MS [21]. In addition, recent work shows that the MS therapy daclizumab (humanized antibody against anti-CD25) can expand CD56 bright

NK cells [22]. CD56 bright NK cells express a high surface density of CD56 on flow cytometry and have been shown to play an immunoregulatory role by regulating T-cell responses[22].

B cells

It has long been recognized that intrathecal synthesis of immunoglobulins is increased in patients with MS, as evidenced by the presence of oligoclonal bands on agarose-gel electrophoresis and an increased IgG index or synthesis rate compared to the autologous serum. The demonstration in the CSF fluid of B-cell proliferation and increased mutations in B-cell receptors suggest that a B-cell response to a specific antigen is occurring in the CNS, whereas corresponding clones are absent from the peripheral circulation [23].

18 Examination of these B-cell clones also indicated that some B cells had undergone a process called receptor revision, or editing, in which these cells recognize the body's misguided capability to manufacture autoantibodies and subsequently remove this capacity[7]. Finally, with the success of therapies targeting the B-cell component of the immune response (ex:

Rituximab) its been suggested that B cells play a crucial role in immune regulation that is not solely antibody mediated [7].

Gamma delta T cells

Gamma-delta ( γδ ) T cells represent another distinct lymphocyte population that mediates host defense and immunoregulatory functions. With respect to

γδ T cells, these cells are known to possess potent cytotoxic activity in vitro , including toxicity towards oligodendrocytes via release of perforin, and to be a major source of proinflammatory cytokines, particularly IFN γ [24]. γδ T cells have been shown to be present in MS lesions, as well as in the CSF, and

PCR analysis has shown that the major subsets present in MS lesions differ from those in the CSF, suggesting specific functions for these cells in lesion development[25]. Several functional studies have suggested that factors within the lesion result in the expression of proteins on the surface of stressed host cells (ex: MICA/B) that activate γδ T cells and promote further tissue damage[26]. In a virally-infected (coronavirus) demyelinating mouse model, γδ T cells also mediate injury through receptors such as NKG2D and production of IFNy [27] .

19 Macrophages

In immune responses mediated by CD4 T cells (which is thought to be the case in MS), recruitment and activation of professional antigen presenting cells (APC) plays a central role. To display full effector function, T cells need to be reactivated in the CNS by the APC. Consistent with this is the observation that deletion of macrophages can protect animals against EAE, and that the presence of chemokines involved in macrophage recruitment is also linked to disease pathogenesis[28]. Amongst the numerous inflammatory mediators produced by activated macrophages TNF and IL-I are abundantly present in MS lesions [29]. Both pro-inflammatory cytokines are thought to contribute to immune-mediated tissue injury by initiating inflammation, by activating other leukocytes to produce cytokines, and by acting synergistically with cytokines such as IFN g. In MS lesions, IL-1 and

TNF are prominently expressed by macrophages in lesions [26]. The role of pro-inflammatory cytokines in MS is discussed below.

Cytokines

To maintain homeostasis, a dynamic balance between pro- and anti- inflammatory cytokines is required. Cytokines are critical components of the immune inflammatory process. They are implicated in oligodendrocyte cell death, axonal degeneration [30] and neuronal dysfunction, which are all key features in MS pathology [31] and the inducers of irreversible deficits.

Cerebrospinal fluid (CSF) from MS patients carries soluble mediators that have been shown to induce axonal damage and neuronal apoptosis in vitro

20 [32]. In the context of MS, in very broad terms, Th1 (e.g. IFNγ ) and Th17 cytokines (e.g. IL-17, IL-22) are thought to contribute to disease pathogenesis, while cytokines (such as IL-4 and IL-10) produced by T helper type 2 (Th2) cells are believed to play a protective role. Th1 cytokines are predominantly found in the brains of MS patients, while a paucity of Th2 cytokines, in particular TGF -β, and IL-10 was observed[33]. IL -6, IFN -γ, and

TNF are expressed by cells located in the perivascular cuffs, suggesting that in acute MS lesions, these soluble mediators could be driving a detrimental inflammatory response. IFN-γ is a key player in MS inflammation and is produced by T cells and NK cells. Its functions include activation of mononuclear cells, differentiation of T cells to a Th1 phenotype, induction of

MHC I and MHC II expression, B cell class switching, and apoptosis of T and other cell types[34]. It is typically produced by Th1 cells and is the principal marker of a Th1 response. IFN-γ is expressed in the CNS at the onset of EAE, its expression increases during the peak of disease and decreases during disease remission, thus levels correlate with disease progression. IFN- γ was demonstrated to induce oligodendrocyte death by apoptosis, underlining its potentially damaging role in MS. It is important to note that IFN-γ is detected in MS lesions[7]. Furthermore, MS patients have high levels of circulating

MBP- and PLP-reactive IFN-γ expressing CD4 T cells [35]. In a randomized study, treatment of MS by IV IFN-γ was shown to exacerbate disease, fur ther confirming the deleterious effect of this inflammatory cytokine in the context of MS[36].

21 TNF production is associated with a Th1 response and classically induces activation of a variety of cell types and expression of adhesion molecules, chemokines, and cytokines. The expression of TNF in the CNS parallels the disease course in EAE [37]. Injections of TNF result in a significant prolongation of clinical EAE and more severe cellular infiltration in the spinal cord.[38] Further evidence for TNF involvement in MS has been presented by identification of TNF in astrocytes, microglia, and endothelial cells, preferentially in acute and chronic active MS brain lesions[10].

However, it is important to note the surprising results of trials aiming to block TNF. A randomized, placebo-controlled study by the Lenercept Multiple

Sclerosis Study Group and the University of British Columbia MS/MRI

Analysis Group showed that blocking TNF exacerbated disease in MS patients, while it was shown to be beneficial in other inflammatory diseases such as rheumatoid arthritis[39].

Another important cytokine that we used in our studies is IL-1β. IL -

1β, alone or in combination with other factors, may be important in neuronal and axonal damage in the CNS. Increased levels of IL-1β are also fou nd in the

CNS of MS patients.

Studies using RNA microarrays in MS brains at autopsy found increased transcripts of genes encoding for IL -6, IL -17, and IFN -γ [40], yet again supporting a role for both Th1 and Th17 cells in proinflammatory responses in MS. Studies examining Th17 cell activity in MS found that dendritic cells from MS patients secrete elevated amounts of IL -23 and

22 express increased levels of IL -23p19 mRNA, and are associated with increased T -cell production of IL -17 [41].

Role of CD4 T cells in MS pathogenesis

Implication in MS and EAE model

Evidence of CD4 T cell implication in MS has been shown time and time again in human and mouse studies. The strongest genetic susceptibility to develop

MS is conferred by MHC-II alleles, supporting a crucial role for CD4 T cells in disease pathogenesis, since CD4 TCR recognizes peptides presented by MHC-

II. Furthermore, multiple studies have demonstrated the presence of CD4 T cells in MS lesions, arguing for a central role in MS pathogenesis[42]. EAE has been extensively characterized during the past two decades providing evidence that it can be induced by adoptive transfer of activated myelin- specific CD4 T cells into naïve recipients [43]. In the development of tissue damage in the CNS in MS, studies with EAE have suggested the following series of events: peripherally activated myelin-reactive CD4 T cells enter the

CNS where they encounter antigen in the context of MHC-II. These T cells, and the associated antigen presenting cells, then release proinflammatory cytokines and chemotactic factors that lead to activation of the endothelium and resident glial cells, resulting in the recruitment of additional monocytes and lymphocytes to sites of inflammation. These recruited inflammatory cells are then activated by cytokines to release toxic factors that lead initially to the destruction of myelin and activation of local glial elements, and

23 subsequently to the loss of oligodendrocytes and sometimes, in areas of severe inflammation, axonal loss as well. One theory of the initial break of tolerance and etiology of MS is that of “molecular mimicry” whereby CD4 T cells activated by a foreign antigen cross -react with myelin antigens.

Activation of myelin reactive CD4 T cells and/or increased presentation of myelin peptides by APCs is also thought to occur in the context of various infections or subsequent to an initial abnormality in CNS cells. Upon reactivation in the CNS, CD4 T cells can yet again produce cytokines that amplify the immune response or directly mediate damage by yet undefined mechanisms[44].

Upon activation and in response to specific environmental cues, Th cells differentiate into specific subsets, each associated with distinct features

(transcription factors, cytokines, etc)[45]. Whereas Th1 cells produce interferon-gamma (IFN-g), IL-2 and TNF; Th2 cells secrete interleukin-4 (IL-

4) IL-5, IL-10, and IL-13. Th17 cells produce cytokines such as interleukin

(IL)-17, IL-6, IL-21, IL-22, IL-23 and TNF, which have proinflammatory functions[46]. Classification of CD4 T cells into distinct populations is extremely complex in part due to the heterogeneity and plasticity of different

Th subsets[45].

Numerous observations have demonstrated that both Th1 and Th17 cells can be involved in the initiation and progression of MS in humans and EAE models[47-49]. Th1 (IFN-g) and Th17 (IL-17, IL-22) cytokines are detected in

24 cerebrospinal fluid (CSF) and CNS lesions of MS patients[48-51]. Perivascular

CD4 T cell infiltration is observed in MS lesions[52] and some CD4 T are detected in close contact with dying neurons in these lesions[53]. Activated

Th1 and Th17 CD4 T cells can kill human oligodendrocytes and/or neurons in vitro [49, 54]. Moreover, adoptive transfer of myelin specific Th1 or Th17 cells is sufficient to induce EAE in naïve recipient mice[47, 55].

Perforin is produced preferentially by cytotoxic T cells and NK cells and is a key effector molecule for T- and NK-cell-mediated cytolysis[56]. It has cytotoxic effects on oligodendrocytes in vitro and elevated numbers of MBP- reactive perforin mRNA-expressing T cells have been detected in MS patients’ blood[35]. Perforin could be one of several factors, which, in concert, cause the demyelination in MS.

Identification of T helper type 17 (Th17) cells led to breaking the dichotomy of Th1 ⁄ Th2 axis in MS and EAE.

Autoreactive CD4 T cells

The elusive search for the autoantigen in MS has lead to considerable interest being devoted on myelin proteins and, in particular, Myelin Basic Protein

(MBP) and the role of T-cell responses to MBP. Autoreactive T cells are also frequently part of the mature immune repertoire of healthy humans. Thus, humans often show comparable frequencies of myelin reactive T cells in their blood whether they are MS patients or healthy controls [57-60]. These findings suggest that the more important factor in disease development may

25 be activation; that is the frequency of activated myelin-reactive cells in MS patients as compared to healthy individuals [61]. Activated myelin -reactive

CD4 T cells are present in the blood and CSF of MS patients; in contrast, only non-activated myelin -reactive T cells are present in the blood of controls

[44]. MBP -reactive T cells isolated from the CSF of MS patients display increased expression of the IL -2 receptor, consistent with a previously activated or memory phenotype[62]. In MS patients, but not in healthy controls, these cells can be activated in the absence of CD28 -B7 costimulation, implying that they have been previously activated in vivo [63].

MBP -reactive T cells from MS patients were found to be less responsive to

CTLA -4 blockade compared to those from healthy controls, signifying that in

MS patients these T cells are not subject to the normal regulatory mechanisms[64]. Several studies support this concept as (MBP)-specific cells derived from MS patients undergo in vivo activation and expansion in blood, and accumulate in the CSF of these patients [61, 65-67]. In addition, an important proportion of autoreactive cells derived from MS patien ts’ peripheral blood and CSF secrete pro-inflammatory cytokines such as IL-2,

IFN-g, TNF [68, 69]. Although the specific mechanisms by which myelin- reactive T cells are involved in MS pathogenesis are unknown, their differential phenotype and costimulatory requirements indicate a memory and potentially dysregulated cell population.

Direct targeting of oligodendrocytes

The cytotoxic activity of CD4 T cells is relatively poorly understood compared

26 with that of CD8 T cells. In addition to the release of diffusible toxic mediators (i.e. cytokines), CD4 T lymphocytes may also directly lyse their targets. One mechanism by which CD4 T cells could mediate such injury is via the Fas-Fas Ligand interaction. By Fas-FasL ligation the “cell death“ domain on the Fas antigen is activated, resulting in death of the cell. There seems to be upregulated surface expression of this pro-apoptotic molecule on oligodendrocytes along the edge of chronic-active lesions. In contrast, in normal CNS tissue Fas and FasL expression is low or absent[26]. This seems to suggest a cytotoxic mechanism that would be enhanced in the inflamed brain of MS patients. While CD4 T cells are generally believed to mediate cytoxicity through cytokines, the perforin/granzyme pathway appears to be a more important mechanism of toxicity in the direct targeting of oligodendrocytes[70]. Similarly, MBP-specific CD4 T cells mediate both perforin/granzyme and Fas/Fas-L-mediated cytotoxicity of MBP or MBP peptide –pulsed targets[71]. The mechanisms involved in this direct lysis has been unclear for a long time since neither oligodendrocytes nor neurons express MHC-II [72]. A subtype of MBP-specific CD4+ TCR αβ + T cells expresses the neural cell adhesion molecule family member CD56 (also a marker for NK cells) and is capable of lysing CD56+ target cells via homotypic

CD56-CD56 interactions independent of HLA restriction. A number of CNS cells, including oligodendrocytes, express CD56, and CD4+ CD56+ T cells can indeed lyse oligodendrocytes in an HLA-unrestricted fashion[21]. However, blocking CD56 did not abrogate the CD4 T cell cytotoxicity, suggesting that

27 other concurrently expressed molecules should be targeted[73].

We have examined the contribution of a novel mechanism by which this subset of pathogenic CD56+ CD4 T cells could mediate direct injury to oligodendrocytes, namely through the molecule NKG2C and its ligand HLA-E.

In the next part, we will describe the mechanism and its contribution to the observed tissue damage in MS.

Signaling through NKG2 receptors and the HLA-E ligand HLA-E Major histocompatibility complex (MHC), human leukocyte antigens (HLA) in human, is a highly polymorphic system. The complex of genes encoding the

HLA system is found on the sixth chromosome and contains over a hundred genes. Its primary function is antigen binding and presentation. HLA molecules are divided into two classes: I and II. Class I contains Ia classical molecules (HLA-A, HLA-B and HLA-C) and Ib non-classical molecules (HLA-E,

HLA-F, HLA-G, MICA and MICB). Class II is similarly divided into classical

(HLA-DP, HLA-DQ, HLA-DR) and non-classical (HLA-DM, HLA-DO) molecules .

Highly polymorphic HLA class Ia genes are expressed in most tissues, while relatively conserved class Ib genes have restricted tissue distribution[74].

Among class Ib genes one of the better studied genes is HLA-E. It has relatively wide tissue distribution, yet its expression level is still lower than the expression levels of most of the HLA class Ia genes.

28 In contrast to the classical HLA class I molecules which present a wide range of peptides from intracellular proteins, HLA-E molecules normally present a highly restricted peptide content. The leader peptide of HLA class I proteins is cut by signal peptidase .during protein translocation to the cell membrane [75]. The hydrophilic oligopeptide is processed further by proteasome, resulting in leader peptide. In the endoplasmic reticulum (ER)

HLA-E molecules are formed from β2 microglobulin and heavy chain.

Transporters associated with antigen processing (TAP) carry leader peptides to the ER, where they can bind HLA-E molecules, allowing surface expression of these proteins[76]. HLA-E–peptide complex is then transported from ER via the Golgi apparatus to the cell surface and interacts with its receptor. As the availability of a peptide for HLA-E is dependent on the expression of other MHC-I proteins and functioning TAP, cell surface expression of HLA-E is indicative of a cell that has functional antigen processing machinery and normal MHC-I expression. It is important to note that a variety of peptides other than MHC-I were shown to bind to HLA-E and modify its affinity to

NKG2 receptors; peptides include, amongst others, leader sequences of heath shock protein 60[77], HIV, hepatitis C virus[78] and many others. Thus, when a cell is under a “stressful” state (following infection, inflammation, etc), activity of these mechanism can be changed, inducing a change of HLA-E expression. Changes in HLA-E expression can modulate the immune response of T cells and NK cells that express the receptors for HLA-E.[79]

29 NKG2A and NKG2C HLA-E was implicated in immune regulation when it was shown to be recognized by CD94/NKG2 receptors expressed by subsets of immune cells, especially NK cells. These receptors are members of the C-type lectin superfamily and consist of an invariant CD94 subunit disulfide-linked to a member of the NKG2 family. The NKG2 family comprises of inhibitory (2A) and activating (2C) isoforms and when in complex with CD94 are capable of transducing opposing signals upon ligation with HLA-E. The inhibitory isoform CD94/NKG2A contains ITIM motifs in the cytoplasmic tail, and its engagement leads to recruitment and activation of Src homology 2 domain- bearing tyrosine phosphatase 1 (SHP-1). In contrast, the activating isoform

CD94/NKG2C contains a lysine residue in the transmembrane regions, which associates with the ITAM containing adaptor molecule DAP-12[82].

For simplicity, CD94/NKG2A will be referred to as NKG2A and CD94/NKG2C will be referred to NKG2C. NKG2A and NKG2C signaling have a large impact on immune cell functions. Depending on which of the NKG2 receptors is expressed by the effector cell, binding of HLA-E will trigger either an inhibitory response (if the cell expresses NKG2A) or an activating response

(if the immune cell expresses NKG2C). Immune cells are known to express either one of the other receptor; only a minor population of NK cells have been shown to express both NKG2C and NKG2A following short-term activation in vitro[83]. Levels of NKG2A and NKG2C can be modulated by cytokines such as IL-12 and IL-15[84]. Tissue-specific factors in vivo are

30 thought to shape the receptor expression on immune cells. These factors include cytokines and chemokines that are unique to the organ and to the type of response mounted[85].

Extensive research has been done on the expression of CD4/NKG2 receptors on NK cells and CD8 T cells, yet little is known about the expression of these receptors on CD4 T cells. Groups have correlated CD4/NKG2 expression on

Th1 CD4 T cells, as well as in vitro activated CD4 T cells, while they were absent on naïve or Th2 CD4 T cells[86, 87]. Expression of these markers on

NK cells and CD8 T cells has been studied. NKG2A + NK cells are much more prominent than their NKG2C + counterparts. NKG2A - HLA-E interactions are a surveillance mechanism for NK cells. HLA-E levels on a cell have been shown to determine if a cell will be killed or not; a constant supplement of peptide is required to keep HLA-E on the cell surface, this providing an updated status on the status of the cell. A “healthy” cell that expresses HLA -E will be spared due to negative feedback provided by the NKG2A receptor on the NK cell[88]. While NKG2C + NK cells represent only approximately 3% of the total NK population in a healthy individual, in NK cells from patients infected with HIV or HCMV are much more elevated[89].

Similarly to NK cells, NKG2A + CD8 T cells are much more present than

NKG2C + CD8 T cells. NKG2A is expressed solely on effector or memory CD8 T cells, and more specifically following TCR triggering. It is thought to be part of a negative feedback loop that dampens signaling downstream of TCR[90].

It is unclear if NKG2A expression on CD8 T cells is a protective mechanism

31 (that minimizes exaggerated immune responses) or a detrimental factor that compromises response efficiency. In mice, NKG2A is induced on antigen- specific CD8 T cells following infection with LCMV or Listeria monocytogenes

[91]. On the other hand, NKG2C + CD8 T cells are restricted to terminally differentiated CD8 T cells. As in NK cells, HCMV infection induces expression of NKG2C on CD8 T cells[92].

Signaling through NKG2A

The best-described interaction is that of HLA-E with the inhibitory NKG2A, which acts as a crucial checkpoint in NK cell surveillance. Hence, the interaction of NKG2A with HLA-E transduces inhibitory signals to NK cella.

Events such as viral infection or transformation that can effect supply of

MHC-I-derived leader sequence peptides either directly through downregulation of class I expression or indirectly through inhibition of TAP function can therefore impact on the cell surface expression of HLA-E rendering a cell more susceptible to lysis by NK cells [82]. NKG2A has been shown to be functional in CD8 T cell populations with an effector/memory phenotype[90]. NKG2A + T cells expand in the course of some infections in mice, such as polyovirus and herpes simplex-2 virus [93] and have been reported to be increased in HIV + patients[94]. Several reports indicate that

NKG2A expression and triggering on antigen-specific CD8 T cells can minimize secondary damages caused by anti-viral responses in certain contexts. NKG2A expression by influenza-specific CD8 T cells reduces the

32 cytotoxic lymphocyte response, minimizing the extent of the immunopathology they cause in the lungs[95]. On the other hand, NKG2A expression can restrain CD8 effector functions and compromise the efficiency of their response. Resistance to the polyoma virus was associated to decreased expression of NKG2A + polyoma-specific CD8 T cells[96]. It is plausible that this inhibitory receptor may contribute to prevent T cell mediated autoreactivity by decreasing T cell cytotoxicity in general [97] and counterbalance the action of stimulatory NK receptors (i.e. NKG2D,

CD94/NKG2C) expressed by other immune subsets [98].

In the EAE model, the expression of Qa.1 (the mouse homolog of HLA-E) has not been assessed in the CNS of mice. Instead, the paradigm presented is different. It has been shown that low but measurable levels of Qa.1 are expressed on a subset of regulatory CD8 T cells. Thus, during disease, these autoregulatory T cells are being inhibited by NK cells that express the inhibitory NKG2A receptor. Studies have shown that disruption of the

NKG2A/Qa.1 interaction (either by a circulating anti-NKG2A antibody or knock-out mouse whose NK cells do not express NKG2A) completely abolishes the development of EAE in immunized mice[80, 81].

Signaling through NKG2C

Although interactions through the activating NKG2C receptor have been less well documented, their role in pathological/autoimmune situations has been suggested. In individuals seropositive for human cytomegalovirus (HCMV),

33 NKG2C + NK and T cells were increased in peripheral blood [92]. In the context of autoimmunity and tissue destruction, CD8 T cells infiltrating the inflamed gut of patients with celiac disease highly upregulate their NKG2C expression. Indeed, NKG2C can mediate the expansion of infiltrating cytotoxic T cells in active celiac patients, and celiac enterocytes in these patients express upregulated levels of HLA-E, the cognate ligand [99].

Cytotoxic CD4 T cells expressing NKG2C and other activating NK-associated markers have been detected at the inflammation site of other pathologies such as the inflamed intestine of patients with Crohn’s disease [100], the synovial tissue of rheumatoid arthritis patients [101] and blister infiltrates of patients with Steven-Johnson cutaneous diseases and toxic epidermal necrolysis [102]; in each case they have been associated with tissue destruction. Interestingly, and very relevant to our studies, NKG2C is detected on a small subset of ex-vivo human T cells [103], concurrently with other NK-receptors (NKRs) such as CD56 and NKG2D [104]. T cells with high expression of CD56 have been associated with the acquisition of cytolytic effector functions. Moreover, these cells express high levels of NKRs and showed NK-like cytotoxic activity. Growing interest has been shown about these T cells exhibiting NK-like activity that have been named NK-CTLs, and are characterized as T cells expressing CD56 and NKRs.

While the NKG2C-expressing T cells and their interaction with HLA-E have been implicated in tissue damage in other disease pathologies, whether the interaction between NKG2C and its ligand HLA-E plays a role in the

34 pathogenesis of MS has not been studied. Furthermore, while a subset of cytotoxic myelin-reactive CD4 T cells have been shown to express CD56, it is unclear which other concurrently expressed molecules mediates this cytotoxicity. We sought to determine whether myelin reactive CD4 T cells as well as short-term activated CD4 T cells could also express and utilize NKG2C and other NK-associated markers to mediate oliogodendrocyte-directed cytotoxicity.

35 FINDINGS

Cytotoxic NKG2C + CD4 T cells target oligodendrocytes in Multiple Sclerosis

Fatma Zaguia 1, 2 , Philippe Saikali 1,2 , Samuel Ludwin 3, Jia Newcombe 4, Diane

Beauseigle 1, Ellie McCrea 2, Pierre Duquette 5, Alexandre Prat 1, 5 , Jack P. Antel 2, and Nathalie Arbour 1

1Department of Medicine, Université de Montréal, CRCHUM-Notre-Dame Hospital, Pavilion

JA de Sève, 1560 Sherbrooke E, Montreal, QC, Canada, H2L 4M1.

2 Neuroimmunology Unit, Montreal Neurological Institute, McGill University, 3801 University

Street, Montreal, QC, Canada, H3A 2B4.

3 Department of Neuropathology, Queen’s University, 88 Stuart Street, Kingston, ON, Canada,

K7L 3N6.

4 NeuroResource, UCL Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, England

5Multiple Sclerosis Clinic, Department of Neurology, Faculty of Medicine, CHUM-Notre-Dame

Hospital, Montréal, QC, H2L 4M1, Canada

Address correspondence :

Nathalie Arbour Ph.D.

Department of Medicine, Université de Montréal

CRCHUM-Notre-Dame Hospital (Y-3609)

1560 Sherbrooke E

Montreal, QC, Canada, H2L 4M1

Phone: 1-514-890-8000, ext: 25112 Fax: 1-514-412-7602 email: [email protected]

36 Abstract The mechanisms whereby immune cells infiltrating the central nervous system in multiple sclerosis contribute to tissue injury remain to be defined. CD4 T cells are implicated as initiators of the inflammation. Myelin reactive CD4 T cells expressing CD56, an NK cell marker, were previously shown to be cytotoxic to human oligodendrocytes in vitro . We sought to determine whether myelin reactive

CD4 T cells could also express and utilize other NK associated markers to mediate oliogodendrocyte directed cytotoxicity. We observed that myelin reactive

CD4 T cell lines, as well as short term PHA-activated CD4 T cells can express

NKG2C, the activating receptor that interacts with HLA-E, a non-classical MHC class I molecule. These cells co-expressed CD56, had elevated levels of cytotoxic molecules FasL, granzyme B and perforin compared to their NKG2C-negative counterparts and mediated significant in vitro cytotoxicity towards human oligodendrocytes induced to express HLA-E with pro-inflammatory cytokines. A significantly elevated proportion of ex-vivo peripheral blood CD4 T cells from

MS patients expressed NKG2C compared to controls. Immunohistochemical analysis demonstrated that MS tissue sections displayed HLA-E+ oligodendrocytes and NKG2C + CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4T cells could contribute to tissue injury in MS.

37 Introduction Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS) characterized by immunological attacks on oligodendrocytes and their myelin membranes. The crucial role of CD4 T cells in disease pathogenesis is documented in both human studies and animal models of MS [105]. However, the mechanisms whereby CD4 T cells directly target oligodendrocytes have not been completely elucidated. We and others have shown that a subset of activated myelin-specific CD4 T cells expressing CD56, a marker associated with NK cells, exhibits cytotoxic properties [54, 106]. CD56 + myelin specific CD4 T cells show elevated cytotoxicity towards human oligodendrocytes that is non-MHC restricted and non-CD56 mediated, strongly supporting that other molecules are implicated

[54].

Activated T cells can acquire NK cell receptors (NKR) including members of the NKG2 family. NKG2A and NKG2C form heterodimers with

CD94; both heterodimers (NKG2A/CD94 and NKG2C/CD94) recognize HLA-

E, a non-classical MHC class I molecule. Binding to the NKG2C/CD94 complex conveys a stimulatory signal whereas binding to the NKG2A/CD94 transmits an inhibitory signal. NKG2C/CD94 is detected on a small subset of ex-vivo human T cells [103], concurrently with other NKR such as CD56 and NKG2D

[104]. NKG2C/CD94 expressing T cells are elevated under pathologic conditions such as infection [107, 108] and inflammation [99]. Indeed,

NKG2C can mediate the expansion of infiltrating cytotoxic T cells in active celiac patients, and celiac enterocytes in these patients express upregulated

38 levels of HLA-E, the cognate ligand [99]. Whether the interaction between

NKG2C and its ligand HLA-E plays a role in the pathogenesis of MS has not been studied.

We provide evidence that NKG2C is expressed by myelin specific and short-term PHA activated CD4 T cells; this is associated with simultaneous expression of CD56 and additional molecules involved in cytotoxicity. We found that in response to pro-inflammatory cytokines human oligodendrocytes up-regulate HLA-E, the cognate ligand, and that cytotoxicity mediated by myelin-specific CD4 T cell towards oligodendrocytes is in part mediated via NKG2C. We further show that CD4 T cells expressing NKG2C and other activating NKR are enriched in peripheral blood of MS patients compared to healthy controls. Finally, we establish that NKG2C +CD4 T cells and HLA-E expressing oligodendrocytes are indeed present in the CNS of MS patients.

39 Results and Discussion We and others [54, 106] have shown that human myelin specific CD4

T cells expressing CD56 display elevated cytotoxicity towards oligodendrocytes compared to their CD56 negative counterparts. However, anti-CD56 blocking antibodies (Ab) do not inhibit the CD56 +CD4 T cell mediated cytotoxicity suggesting that other receptors are functionally required [54]. Therefore, we sought to assess whether myelin specific CD4 T cells could gain receptors associated with cytotoxicity. We focused our analysis on NKG2C since it has been detected concurrently with CD56 on human T cells [104]. We analyzed 9 myelin basic protein (MBP)-specific CD4

T cell lines expanded in vitro according to our published protocol [109] and established that within each MBP-specific CD4 T cell line an average of 13% of these cells expressed CD56 (Fig. 1B). Most ( >85%) of these CD56 +CD4 T cells displayed NKG2C but did not express NKG2A (Fig. 1B).

We determined whether other activation conditions could induce

NKG2C on CD4 T cells. We assessed NKG2A and NKG2C expression on CD4 T cells under resting conditions and following a 5d activation with either cytokines (IL-15, IL-12, IL-2), TCR stimulation (α -CD3+α -CD28) or PHA. We observed a minimal expression of NKG2C and NKG2A on ex vivo and untreated CD4 T cells obtained from healthy controls and these levels were not altered by cytokines or TCR triggering (Fig. 1C). However, PHA activation induced a significantly greater proportion of CD4 T cells expressing NKG2C

(average 33%) for all tested donors (Fig. 1C). We analyzed both MBP-specific

40 CD4 T cell lines and short-term PHA-activated CD4 T cells for additional NK- associated markers (Fig. 1D). We compared NKG2C + (top row) and NKG2C -

(bottom row) cells and observed that most NKG2C + cells did not have NKG2A on their surface. In contrast, elevated proportions of NKG2C + cells expressed

NKp46, NKG2D, CD56, FasL, granzyme B and perforin compared to their

NKG2C - counterparts (Fig. 1D and Fig 1E).

We investigated whether oligodendrocytes express HLA-E, the ligand of NKG2C and NKG2A, under basal or inflammatory conditions mimicking the inflamed environment observed in the CNS of MS patients. Primary cultures of human oligodendrocytes under basal conditions (nil) expressed detectable but low levels of HLA-E (Fig. 2A) as measured by flow cytometry. IFN-γ or IL -

1β increased those HLA -E levels, however combined cytokines were more potent at inducing a significant upregulation of HLA-E (*p<0.05 vs. nil) (Fig.

2A). We observed that, TNF either alone or added to IL-1β or IFN -γ had no impact on HLA-E expression (data not shown). We similarly observed by immunocytochemistry that HLA-E was barely detectable on untreated oligodendrocytes but elevated following IFN-γ and IL -1β treatment co - localizing with the oligodendrocyte-specific marker Nogo-A (Fig. 2B). Our results demonstrate that oligodendrocytes enhance their low basal levels of

HLA-E in response to cytokines suggesting that immune mediators present in

MS lesions [110, 111] enhance such expression.

We then determined whether activated NKG2C +CD4 T cells could target human oligodendrocytes expressing HLA-E. We used the CD107a

41 mobilization flow cytometry based assay which correlates with lytic Cr 51 release assay [112]; CD107a is transiently detected on the cell surface of degranulating cytolytic cells. Using this assay we could analyze simultaneously NKG2C + and NKG2C - cells. To impede the interaction between

NKG2C + CD4 T cells and HLA-E on oligodendrocytes, we applied a commercially available blocking Ab targeting CD94, the co-receptor of

NKG2A and NKG2C as used by others [113]. Since CD4 T cells in PHA- activated cells or MBP-specific lines showed undetectable or very low levels of NKG2A, the a-CD94 blocking Ab interacts with the NKG2C/CD94 heterodimers on these effector CD4 T cells.

CD4 T cells purified from PHA-activated PBMC were pre-incubated with either an isotype control (Fig. 2C, first column) or a-CD94 (Fig. 2C, middle column), or a-NKG2D (Fig. 2C last column) blocking Ab and then added to inflamed human oligodendrocytes, in the presence of anti-CD107a

Ab or an isotype. After 8h, CD4 T cells were harvested and analyzed by flow cytometry for NKG2C, CD4, and CD107a. We gated either on NKG2C+ (first row) or NKG2C - (second row) CD4 T cells and assessed the expression of

CD107a (Fig. 2C). CD4 T cells in the absence of target cells did not significantly degranulate with only 1% expressing CD107a. In contrast, we detected a significant level of CD107a expression (19%) on activated NKG2C + but not NKG2C - CD4 T cells added onto oligodendrocytes in the presence of an isotype control. Since we have previously shown that inflamed oligodendrocytes are susceptible to NKG2D-mediated cytotoxocity as they

42 express the cognate ligands [114], we also evaluated the contribution of this receptor. We observed that blocking NKG2C via a-CD94 led to a significant decrease in CD107a-expression by NKG2C + cells (9%) and similarly when effector CD4 T cells were pre-incubated with an a-NKG2D blocking Ab (Fig.

2C) CD017a expression diminished (11%) in the NKG2C + population.

Similarly, MBP-specific CD4 T cells pre-incubated with a-CD94 blocking Ab

(Fig. 2D) before being added to inflamed oligodendrocytes did significantly degranulate less (22%) than their counterparts put in the presence of an isotype control (33%). CD4 T cells in the absence of target cells did not significantly display CD107a (Fig. 2D, left panel). The significant impact of blocking the NKG2C/CD94 complex on MBP-specific CD4 T cells was confirmed using 6 distinct lines expanded from 2 donors (Fig. 2D). Overall, we established that blocking NKG2C interaction with its ligand inhibited CD4

T cell-mediated killing of human oligodendrocytes (Fig. 2C-D). As NKG2C or

NKG2D blockade did not completely abrogate CD4 T cell degranulation, additional activating receptors could play a role in the observed cytotoxicity.

Our phenotyping results suggest that other activating receptors such as

NKp46, although the cognate ligand remains unknown, could play a role in

CD4 T cell-mediated cytotoxicity.

To evaluate the physiological relevance of NKG2C, we investigated

NKG2A and NKG2C expression on ex vivo PBMCs from MS patients and healthy controls by flow cytometry, using specific cell markers for CD4

(CD3 +) and CD8 (CD3 +) T cells, and NK cells (CD3 -CD56 +). Representative dot

43 plots from one MS and one healthy control for NKG2C expression on gated

CD4 T cells, CD8 T cells or NK cells demonstrate that NKG2C was detected only on small subsets in healthy controls (Fig. 3). In regards to the inhibitory

NKG2A receptor, only NK cells expressed significant levels of this receptor

(60% of NK cells were NKG2A+, data not shown). In contrast, a greater proportion of CD4 T cells expressed NKG2C in MS patients, although the proportion of CD8 T cells and NK cells positive for NKG2C was similarly low in patients and normal controls (Fig. 3A, B); results from 17 MS patients and

12 normal controls show a significantly higher percentage of NKG2C + CD4 T cells in MS patients (Fig. 3B). These increased proportions in MS patients were observed regardless of their clinical status. NKG2A was not detected on ex-vivo T cells but only on a small subset of NK cells and no difference was observed between MS and healthy controls (data not shown).

We compared NKG2C + and NKG2C - CD4 T cells obtained from MS patients for the expression of numerous immune molecules. Representative dot plots are illustrated (Fig. 3C) and demonstrate the important differences between these subsets. A minor or undetectable population expressed

NKG2A in both subpopulations. However, a greater proportion of

NKG2C +CD4 T cells expressed NKp46, NKG2D, CD56, FasL, granzyme B and perforin. We confirm the strong cytotoxic profile of NKG2C +CD4 T cells compared to the NKG2C - counterparts on ex-vivo blood samples from 3 MS patients (Fig. 3D). We observed that the ΔMFI (~1300) for NKG2C on ex-vivo

CD4 T cells from MS patients was similar to those we detected on short-term

44 PHA-activated CD4 T cells from healthy donors. Similarly to the MBP-specific or PHA-activated CD4 T cells, ex vivo NKG2C +CD4 T cells from MS patients also displayed elevated levels of FasL. Human oligodendrocytes express elevated levels of Fas in MS lesions [115], therefore infiltration of these

NKG2C +CD4 T cells in the CNS could potentially be even more deleterious.

Indeed, others have shown that MBP-specific CD4 T cells can mediate their cytotoxicity through Fas-FasL interactions and perforin release [116].

We determined whether the HLA-ENKG2C interaction takes place in the CNS of MS patients. We observed in paraffin-embedded CNS tissues obtained from MS patients that cells of oligodendrocyte lineage (O4 + cells) can express HLA-E in areas from lesions involving both white matter and cortex (Fig 4A-C). Hence, we can conclude that human oligodendrocytes both in vitro (Fig. 2) and in situ (Fig. 4) express HLA-E especially in inflammatory conditions. Conversely, we performed immunohistochemistry on frozen human post-mortem brain tissues obtained from individuals affected with

MS and control donors for NKG2C and CD4. We found a subpopulation of CD4

T cells (12%) within MS tissues both in perivascular and parenchymal areas that expressed NKG2C (Fig. 4B orange arrowheads) similarly to what we observed in the peripheral blood (9.6 + 1.3%) of untreated MS patients. We also observed NKG2C +CD4 - cells with morphology suggestive of infiltrating leukocytes, suggesting that other infiltrating immune cells express NKG2C

(Fig. 4B white arrows). NKG2C +CD4 T cells are present in the CNS of MS patients, while they are undetected in control sections (data not shown). We

45 were unable to successfully detect NKG2C on paraffin-embedded material and conversely HLA-E detection was unsuccessful on frozen samples, therefore it was not possible to co-localize NKG2C and HLA-E on the same tissues. Cytotoxic CD4 T cells expressing activating NK-associated markers have been detected at the inflammation site of other pathologies such as the inflamed intestine of patients with Crohn’s disease [100], the synovial tissue of rheumatoid arthritis patients [101] and blister infiltrates of patients with

Steven-Johnson cutaneous diseases and toxic epidermal necrolysis [102]; in each case they have been associated with tissue destruction.

In the present study, we identify a novel mechanism by which CD4 T cells in MS patients could directly interact and kill oligodendrocytes. MS patients carry elevated proportions of NKG2C +CD4 T cells (9.6 + 1.3%) in their peripheral blood compared to healthy controls (3.0 + 0.6%). Moreover,

NKG2C +CD56 +CD4 T cells observed in PHA-activated cells or in MBP specific

CD4 T cell lines display enhanced effector functions by producing higher levels of lytic enzymes and increased expression of activating receptors such as NKG2D, NKp46 and FasL compared to their NKG2C - counterparts.

Furthermore, blocking NKG2C or NKG2D on these cells decreased the targeting of human oligodendrocytes. Our results have also provided the mechanism behind the previously observed cytotoxicity of CD56 + MBP- specific CD4 T cells towards human oligodendrocytes [54]; such cytotoxicity is in part through NKG2C, expressed concomitantly with CD56 (Fig. 2).

Finally, we detected NKG2C +CD4 T cells in MS lesions, supporting the notion

46 that these cytotoxic cells get access to the inflamed CNS of these patients.

Increased HLA-E expression on cells following exposure to inflammatory cytokines is thought to imply that these cells can tentatively protect themselves in the midst of inflammation from immune-mediated injury by cells that express the inhibitory NKG2A. In mice, Qa-1+ neurons are protected from cytotoxicity mediated by NKG2A +CD8 T cells [117].

Furthermore, trophoblast cells that surround a developing embryo and in the protective layer of the eye [118] express HLA-E and are thus protected from

NKG2A + NK cells. Indeed, the expression of HLA-E on human oligodendrocytes was sufficient to decrease NK cell-mediated cytotoxicity

(data not shown). Our in vitro results show that human oligodendrocytes sufficiently express HLA-E adult to allow cytotoxic interactions with

NKG2C +CD4 T cells. NKG2C has been implicated in detrimental autoimmune responses in other inflammatory disorders such as celiac disease, where

NKG2C + T cells are induced in an inflamed bowel, proving to be a contributing mechanism to the targeting of intestinal enterocytes [99].

Furthermore, an increase in circulating NKG2C + T cells has been reported in other detrimental autoimmune responses such as Stevens-Johnson syndrome and celiac disease [99, 102]. Our studies show a contributing mechanism by which pathogenic CD4 T cells can directly target human oligodendrocytes in the context of a demyelinating disease. Moreover, the development of therapies targeting NKG2C warrant further investigations since this receptor is specifically expressed by a subset of cytotoxic CD4 T cells in MS patients

47 and therefore other cell types (others CD4 T cells, CD8 T cells, NK cells) would not be affected by such therapy.

48 Materials and Methods Donors

Seventeen patients with clinically definite MS according to McDonald’s criteria

[119] and characterized by a relapse-remitting (n=7), secondary progressive (n=6) or primary progressive (n=4) disease course and twelve healthy volunteers were included in the study. None of the patients had received immunosuppressive, immunomodulatory, or steroid therapy for at least six months prior to blood collection.

Informed consent was obtained from all donors according to the local ethics committees (Centre Hospitalier de l’Université de Montréal or McGill University ethical boards).

Myelin specific CD4 T cell lines

CD4 T cell lines specific for myelin basic protein (MBP) were expanded as previously published [109] using whole human MBP (40ug/ml) as antigen.

CD4 T cell lines were stimulated with autologous antigen-loaded B cells every

10-14 days and recombinant human interleukin-2 (IL-2) (Roche, Nutley, NJ) was added (25 U/ml) every 3 days between each round of stimulation. CD4 T cell lines were considered specific when the amount of IFN-g secreted after

24-36h (ELISA from BD Biosciences) in the presence of specific peptide loaded autologous B cells was at least 1.5 the amount secreted in the presence of irrelevant peptide loaded B cells. This method of generating myelin-specific CD4 T cells has been previously validated and published

[109].

49 Isolation and activation of human CD4 T cells

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient using Ficoll-Paque™ PLUS (GE Healthcare). PBMC were either stained for flow cytometry analysis or put in culture for 5 days at 2x10 6 cells/ml in the presence of a-CD3 (0.9 ug/ml, in house purified OKT3 clone) and a-CD28 (1 ug/ml; BD Biosciences) antibodies, PHA (2ug/ml; Sigma-

Aldrich), IL-2 (1000 U/ml, Roche), IL-12 (10 ng/ml, Biosource), or IL-15 (10 ng/ml, R&D Systems) in RPMI supplemented with 10% FBS, glutamine and antibiotics (complete RPMI). CD4 T cells were purified from PHA-activated

PBMC using CD4 Microbeads (Miltenyi Biotec), according to the manufacturer’s instructions. Purity was typi cally >90% as assessed by flow cytometry.

Isolation and activation of adult human oligodendrocytes

CNS tissue was obtained from surgical resections performed for the treatment of non tumor-related epilepsy, in accordance with the guidelines set by the Biomedical Ethics Unit of McGill University. Oligodendrocytes were isolated as previously published [115] and maintained them under basal culture conditions for 1 wk, allowing cells to recover from any stress induced by the isolation.. Oligodendrocytes were either left untreated or treated for

24h with IFN-γ (1000U/ml; Pierce Endogen), IL -1β ( 10ng/ml; Invitrogen), and/or TNF (200U/ml; Invitrogen).

Cytotoxicity assays

50 Oligodendrocytes were treated with IFN-g (1000U/ml) and IL-1b (10ng/ml) for 24h, and then washed prior to the addition of CD4 T cells (2x10 6/well for a 10:1 E:T ratio). Anti-CD94 (eBiosciences) or anti-NKG2D (M585 clone, kindly provided by Amgen) blocking antibodies (25ug/ml) or mouse IgG1 were added to CD4 T cells 1h prior to their addition to target cells in the presence of 2mM monensin (Sigma-Aldrich) and anti-CD107a Ab (BD

Biosciences). After 8h, effector cells were harvested and stained for NKG2C and CD4.

Flow cytometry

Oligodendrocytes were detached using 1mM EDTA-PBS whereas immune cells were washed prior to being stained for surface and/or intracellular proteins as previously published [114]. Mouse monoclonal antibodies directed at human protein and conjugated to Alexa Fluor® 488, Alexa Fluor®

700, PE, PE-Cy7, Pacific Blue, or allophycocyanin were used. Surface stainings targeted: HLA-E, CD3, CD4, CD8, CD56, NKG2D, NKG2A, NKp46 (all from BD

Biosciences), NKG2C, NKG2A (both from R & D Systems), FasL (BioLegend).

To identify oligodendrocytes, cells were labeled with rabbit anti-Nogo A

(Millipore) antibodies followed by Alexa Fluor® 488-conjugated goat anti- rabbit antibodies (Invitrogen). Intracellular staining targeted: Perforin

(Abcam) and Granzyme B (Invitrogen). Isotypes matched for concentration of primary antibodies were used for all stainings. All results were acquired on a

LSRII (BD Biosciences) and analyzed with FlowJo Software (Treestar). Δ

51 Median fluorescence intensity (MFI) was calculated by subtracting the fluorescence of the isotype from that of the stain.

Immunostaining of human oligodendrocytes

Oligodendrocytes were fixed with 2% paraformaldehyde, blocked as previously published [120] and then incubated overnight with a mouse anti- human HLA-E Ab (10ug/ml; eBioscience). Biotinylated goat-anti-mouse antibodies were added for 2 hours, followed by Cy3-conjugated streptavidin concomitantly with the cell-specific rabbit anti-Nogo-A Ab (10ug/ml) for 1h, and finally Alexa Fluro®-488 conjugated goat anti-rabbit (10ug/ml) antibodies were added. Cells were finally incubated with Hoechst 33258 nuclear stain (Invitrogen), and mounted in Gelvatol. Isotypes matched for concentration of primary antibodies were used for all stainings. Slides were observed using a Leica DM6000B fluorescent microscope using the OpenLab software.

Immunostaining of human brain samples

Immunostaining for NKG2C and CD4 was performed on snap frozen sections whereas HLA-E and O4 detection was performed on paraffin-embedded sections. Postmortem frozen brain sections from three patients diagnosed clinically and confirmed by neuropathological examination as having MS and one control were obtained from the NeuroResource tissue bank, UCL

Institute of Neurology, London, UK. Tissues were donated to the tissue bank with informed consent following ethical review by the London Research

Ethics Committee, UK. Snap-frozen sections (10 µm thick) were air-dried,

52 fixed in paraformaldehyde 4% for 10 min, and blocked for non-specific binding for 1h and then a mouse anti-human NKG2C Ab (20μg/ml, R&D

Systems) was incubated 1h at room temperature and then overnight at 4°C.

Sections were then incubated for 1h with a biotinalyted goat anti-mouse antibodies (Dako), followed by Cy3-conjugated streptavidin (Jackson

ImmunoResearch) concomitantly with rat anti-human CD4 Ab (20 ug/ml,

Lifespan Biosciences) followed by Alexa Fluor 488-conjugated donkey anti- rat antibodies. Paraffin-embedded blocks from two patients diagnosed clinically and confirmed by neuropathological examination as having MS were cut into 7 mm thick sections, and stained with Hematoxylin Eosin

(H&E), and Luxol Fast Blue (LFB), for histopathological examination and lesion localization. For immunostaining, the sections were de-paraffinized in toluene twice 5 minutes. Antigen retrieval was done in sodium citrate

(100ºC, 30 min). Sections were blocked for 1h and then incubated overnight with a mouse IgG1 anti-HLA-E Ab (20 ug/ml; R&D Systems) and a mouse IgM anti-O4 Ab (10ug/ml; Chemicon) followed by 1h incubation with secondary antibodies (Alexa488-conjugated goat anti-mouse IgG1 and Cy3-conjugated goat-anti-mouse IgM, Jackson ImmunoResearch). Finally, all sections

(paraffin and snap frozen) were incubated with a nuclear stain TO-PRO®-3 iodide (Invitrogen), treated with Sudan Black and mounted as previously described [120]. Controls were concurrently carried out on adjacent sections using appropriate primary isotype controls at the same concentrations.

Slides were observed using a SP5 Leica confocal microscope. Confocal images

53 were acquired simultaneously in different channels throughout 4-8mm z- stack every 0.2-0.5 mm using the LAS AF software. We validated staining specificity by lack of signal only when the corresponding laser was turned off but not when others were still on. Images were subsequently merged using

Adobe Photophop software. Moreover, we confirmed the absence of bleed- through by re-examining selected sections using sequential scanning.

Statistical analyses

Statistical analyses were performed using PRISM Graphpad™ software.

54 ACKNOWLEDGEMENTS

The authors are grateful for the technical assistance of Manon Blain. The authors acknowledge the contribution of Drs. André Oliver and Jeffrey Hall for providing adult human CNS tissues for the in vitro studies and Dr. Yves Lapierre for facilitating access to some MS patients.

This work was supported by grants from the Multiple Sclerosis Society of Canada

(MSSC) to NA and JPA. FZ holds a studentship from the MSSC and P.S. was supported by a Canadian Graduate Scholarships doctoral research award from the

Canadian Institutes of Health Research. NA and AP hold Donald Paty Career

Development Award from MSSC and are Research Scholars from the Fonds de la

Recherche en Santé du Québec.

The authors have no conflicting financial interests.

PS Current address: Experimental Immunology, Department of

Rheumatology and Clinical Immunology, Charité –University Medicine Berlin,

Berlin, Germany.

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59

Figure legends

Figure 1: Activated NKG2C +CD4 T cells display a cytotoxic profile

(A-B) CD56, NKG2A and NKG2C expression by human MBP specific CD4 T cell lines was assessed; representative FACS dot plots (A) and individual T cell lines obtained from 2 donors are shown (B). Most CD56 + MBP-specific CD4 T cells express NKG2C. (C) Representative FACS dot plots of PBMC gated on

CD3 +CD4 T cells either ex vivo or following a short in vitro activation. Cells were either untreated (Nil) or treated with different stimuli as indicated: α -

CD3+α -CD28, PHA, IL-2, IL-12 or IL-15. Only PHA-activation induced NKG2C expression on CD4 T cells. (D-E) Expression of molecules involved in cytotoxicity was assessed in 4 distinct MBP specific CD4 T cells lines and

PHA-activated CD4 T cells from 3 donors. Representative FACS dot plots of

NKG2C + (top row) and NKG2C - (bottom row) gated cells (D) and percentage of NKG2C + and NKG2C - PHA-activated CD4 T cells expressing NKG2D, CD56,

NKp46, FasL and lytic enzymes. Data are presented as means + SEM.

Student’s t -test n=3; NKG2C+ vs. NKG2C- : * P<0.05, ** P<0.01.

60 Figure 2: NKG2C +CD4 T cells target HLA-E expressing human oligodendrocytes

(A) The expression of HLA-E was determined by flow cytometry on human oligodendrocytes either untreated or cultured in the presence of IFN-γ, IL-1β, or IFN-g+IL-1b. Representative histograms of HLA-E expression on NogoA- gated oligodendrocytes are illustrated . Fold increase of the HLA-E ΔMFI of oligodendrocytes from 3 different donors are illustrated (mean + SEM); treatment with either IFN-g (x p= 0.0623) or IFN-g+IL-1b (* p<0.05) consistently elevated HLA-E expression by oligodendrocytes. (B, C) Cytotoxic potential of PHA-activated CD4 T cells and MBP-specific CD4 T cells toward

IFN-γ+IL -1β -treated oligodendrocytes was evaluated using the CD107a assay after 8h co-culture. (B) Representative dot plots of CD107a expression on

PHA-activated CD4 T cells, pre-incubated with either an isotype control, an a-

CD94 or an a-NKG2D blocking Ab prior to their addition to oligodendrocytes are shown. Flow cytometry events were gated on CD3 +CD4+ either NKG2C + or

NKG2C - cells. Data obtained from 3-6 donors of PHA-activated CD4 T cells are shown as mean + SEM; the percentage of CD107a expressing cells for effector cells alone, pre-incubated with either an isotype control, an a-CD94, an a-

NKG2D, or both a-CD94 and a-NKG2D blocking Abs. (C) Oligodendrocytes that were incubated in the absence (no effector) or in the presence of PHA- activated CD4 T cells pre-incubated with either an isotype control (Isotype) or a-CD94+ a- NKG2D blocking Abs were fixed and then stained for Nogo-A.

Pictures are representative of 5 fields from killing assays performed with 2

61 distinct CD4 T cell donors. Scale= 10 micron. (D) Representative dot plots of

CD107a expression on MBP-specific CD4 T cells, pre-incubated with either an isotype control, an a-CD94 or alone prior to their addition to oligodendrocytes are shown. Flow cytometry events were gated on

CD3 +CD4 + cells. Data obtained from 6 different MBP-specific CD4 T cell lines generated from 2 donors are shown as mean + SEM. (B, D) isotype vs. a-CD94 or a- NKG2D or a-CD94+ a- NKG2D * P<0.05, ** P<0.01.

62 Figure 3: NKG2C + CD4 T cells are more abundant in MS patients and exhibit a highly cytotoxic profile.

Ex-vivo PBMC obtained from untreated MS patients (MS) or normal controls

(NC) were analyzed for immune molecules. (A-B) Representative dot plots

(A) of NKG2C expression on CD4 +CD3 + T cells, CD8 +CD3 + T cells, and (CD3 -

CD56 +) NK cells in 17 MS patients and 12 normal controls and data from individual donors (B) are shown. (C-D) Representative dot plots (C) of CD4 T cells from untreated MS patients gated on NKG2C + (top row) or NKG2C -

(bottom row) cells for the expression of NKG2A, NKG2D, CD56, NKp46, FasL, granzyme B and perforin, pool data obtained from 3 untreated MS patients are shown (D). Data are presented as means + SEM . Student’s t -test n=3,

NKG2C+ vs. NKG2C- : * P<0.05, ** P<0.01.

63 Figure 4: HLA-E and NKG2C are expressed in MS brain tissues

(A) A representative staining of paraffin-embedded sections with luxol-fast- blue for myelin; cortex (C) and white matter (WM) are identified; within the white matter well demarcated MS lesions (L) are seen as well as diffuse areas of demyelination and tissue damage, known as dirty appearing white matter

(DAWM). The areas of cortex designated (CL) contain cortical demyelinated lesions. (B) Hematoxylin/Eosin-stained section of the area of DAWM depicted in A; tissue is rarefied due to loss of myelin, reactive astrocytes (black arrows) are seen throughout the area and a mononuclear infiltrate (blue arrows) in the perivascular space around a blood vessel. (C) Paraffin embedded brain sections from MS patients were immunostained for HLA-E

(green), O4 (red) and nuclear stain (blue). Fluorescent microscopy shows the presence of oligodendrocytes (O4 +) expressing HLA-E. Orange arrowheads indicate examples of oligodendrocytes expressing HLA-E; white arrows indicate example of an HLA-E expressing cell that is not of oligodendrocyte lineage. Areas in white boxes are shown enlarge in right column as well as corresponding isotype controls. Data shown are representative of 15 fields from 3 distinct MS tissue blocks. Scale = 25 mm. (D) Frozen brain tissues from patients with MS were immunostained for CD4 (green), NKG2C (red) and nucleus (blue). Fluorescent microscopy reveals the presence of CD4 T cells and NKG2C positive cells. Orange arrowheads indicate examples of NKG2C expressing CD4 T cells whereas white arrows indicate example of a NKG2C positive cell that is not a CD4 T cell and pink arrows indicate examples of CD4

64 T cells that do not express NKG2C. Isotype controls and enlarged areas in white boxes are shown in last column. Three distinct fields obtained from 2

MS brain specimens are shown and are representative of 4 distinct MS brain samples.

65

66 67

68

69 DISCUSSION

This study pursues the as yet unresolved mechanisms whereby inflammation in the CNS in MS can result in the characteristic oligodendrocyte/myelin injury. We have focused this study on CD4 T cells given the evidence from animal model studies of their central role in initiating the neuroinflammatory process. We aimed to identify novel mechanisms of injury by which these cells could directly mediate injury towards oligodendrocytes. Vergelli et al [116] initially reported that MBP- specific CD4 T cell lines acquiring expression of the NK cell associated marker CD56 could mediate non MHC restricted toxicity; however the effect was not inhibited with anti-CD56 antibody. We demonstrated that human oligodendrocytes were also susceptible to such injury. Our results with the short-term activated CD4 T cells and with CD4 MBP T cell lines provide a mechanism to explain the previously observed cytotoxicity of CD56 positive

CD4 T cells. Our studies show that short-term activated CD4 T cells and MBP

T cell lines induce the activating NKG2C receptor instead of NKG2A on their surface. These are the cells that co-express CD56. Expression is also linked with expression of additional NK cell markers including NKG2D, granzyme, perforin and NKp46 that the NKG2Cnegative counterpart does not. Indeed, others have previously shown that MBP-specific CD4 T cells can mediate their cytotoxicity through Fas-FasL interactions and perforin release [116].

Human adult oligodendrocytes are known to have increased levels of fas in

70 MS lesions [121], therefore infiltration of this subset of CD4 T cells in the CNS would prove to be even more deleterious.

Our in vitro results also show that the ligand for NKG2C, HLA-E, is also expressed by human primary oligodendrocytes. We used oligodendrocytes derived from non-tumoral surgical adult brain tissues and maintained under basal culture conditions for 1 week, leaving the cells to recover from any stress induced by the isolation. Our flow cytometry data shows that oligodendrocytes express low levels of HLA-E under basal conditions but such expression was upregulated on oligodendrocytes by cytokines (IFN-γ +

IL-1β) suggesting that immune mediators present in MS lesions [122, 123] can enhance such expression.

Conventionally, increased HLA-E expression on cells following exposure to inflammatory cytokines is thought to imply that these cells can tentatively protect themselves in the midst of inflammation from immune- mediated injury by cells that express the inhibitory NKG2A. In mice, Qa-1 expressed by neurons inhibits cytotoxic immune responses mediated by

NKG2A+ CD8 T cells[93]. Furthermore, trophoblast cells that surround a developing embryo and in the protective layer of the eye [118] express HLA-

E and trigger NKG2A on surrounding NK cells, inhibiting any cytotoxic response. Indeed, the expression of HLA-E on adult oligodendrocytes even at low levels was sufficient to decrease immune-targeting by NKG2A+ NK cells

(data not shown), proving that the induced HLA-E is functional. Depending on the context, NKG2A or NKG2C may modulate the immune response to its

71 advantage or deleteriously. HLA-E can interact with NKG2A and inhibit immune cells that express the receptor. Vice versa, HLA-E can interact with

NKG2C and trigger immune cells to become more cytotoxic. Therefore, the immune response towards an increased HLA-E response depends on which receptor is expressed on the infiltrating cells. Our in vitro results show that the expression of HLA-E adult oligodendrocytes was sufficient to allow cytotoxic interactions with NKG2C+ CD4 T cells. We demonstrated that blocking NKG2C interaction with its ligand inhibited CD4-mediated killing of human oligodendrocytes. As we were not able to completely abrogate CD4 degranulation, additional activating receptors could play a role in the observed cytotoxicity. Indeed, blocking NKG2D on these cells also decreased killing; further preliminary data show that blocking simultaneously NKG2C and NKG2D almost completely abrogates killing. Our phenotyping results also suggest additional cytotoxicity could be mediated via other activating receptors expressed by activated CD4 T cells such as NKp46.

Cytotoxic CD4 T cells expressing activating NK-associated markers have been documented in the inflammation site of other pathologies such as the inflamed intestine of patients with Crohn’s d isease[100], the synovial tissue of rheumatoid arthritis patients[124] and blister infiltrates of patients with Steven-Johnson cutaneous diseases and toxic epidermal necrolysis

[125]; in each case they have been associated with tissue destruction.

In our study we also compared NKG2C expression on CD4 T cells in

MS patients with sex and age matched healthy controls. We observed a

72 significantly higher proportion of circulating NKG2C+ CD4 T cells in the periphery of MS patients. These NKG2C CD4 T cells also expressed the

NK/cytotoxicity associated markers. We observed that the ΔMFI (~1300) for

NKG2C on ex-vivo CD4 T cells from MS patients was similar to those we detected on short-term PHA-activated CD4 T cells from healthy donors.

Similarly to the MBP-specific or PHA-activated CD4 T cells, ex vivo

NKG2C +CD4 T cells from MS patients also displayed elevated levels of FasL.

We determined whether the HLA-ENKG2C interaction takes place in the CNS of MS patients. Our immunohistochemical studies show that NKG2C+

CD4 T cells, are present in the CNS of MS patients, while they are undetected in control sections, as are oligodendrocytes expressing HLA-E. We observed in paraffin-embedded CNS tissues obtained from MS patients that cells of oligodendrocyte lineage (O4+ cells) can express HLA-E in areas from lesions involving both white matter and cortex. Hence, we can conclude that human oligodendrocytes both in vitro and in situ express HLA-E especially in inflammatory conditions. Conversely, we performed immunohistochemistry on frozen human post-mortem brain tissues obtained from individuals affected with MS and control donors for NKG2C and CD4. We found a subpopulation of CD4 T cells within MS tissues both in perivascular and parenchymal areas that expressed NKG2C similarly to what we observed in the peripheral blood of untreated MS patients. We also observed

NKG2C +CD4 - cells with morphology suggestive of infiltrating leukocytes, suggesting that other infiltrating immune cells express NKG2C. We were

73 unable to successfully detect NKG2C on paraffin-embedded material and conversely HLA-E detection was unsuccessful on frozen samples, therefore it was not possible to co-localize NKG2C and HLA-E on the same tissues

The role of NKG2C in detrimental autoimmune responses has been described in other inflammatory disorders such as celiac, where NKG2C+ T cells are induced in an inflamed bowel, proving to be a contributing mechanism to the targeting of intestinal enterocytes [126]. Furthermore, an increase in circulating NKG2C+ T cells has also been reported in several detrimental autoimmune responses [125, 126]. Our studies show a contributing mechanism by which pathogenic CD4 T cells can directly target adult oligodendrocytes in the context of a demyelinating disease.

74 CONCLUSION AND FUTUR DIRECTIONS

In the present study, we have identified a novel mechanism by which

CD4 T cells in MS patients could directly interact and kill oligodendrocytes in

MS patients. Our initial in vitro studies indicated that acquisition of NKG2C expression by CD4 T cells was associated to a cytotoxic phenotype.

Functional assays show that cytotoxicity was regulated by interaction of this receptor with its HLA-E ligand, whose expression could be induced on oligodendrocytes by pro-inflamamtory stimuli. MS patients carry elevated proportions of NKG2C+ expressing CD4 T cells in their peripheral blood compared to healthy controls. Finally, we document presence of NKG2C expressing CD4 T cells in MS lesions, supporting the notion that these cytotoxic cells do get access to the inflamed CNS of these patients, as well as oligodendrocytes expressing the requisite ligand (HLA-E).

Our studies show a contributing mechanism by which pathogenic CD4

T cells can directly target human oligodendrocytes in the context of a demyelinating disease. Our results have also provided the mechanism behind the previously observed cytotoxicity of CD56 + MBP-specific CD4 T cells towards human oligodendrocytes [54]; such cytotoxicity is in part through

NKG2C, expressed concomitantly with CD56. Finally, we detected

NKG2C +CD4 T cells in MS lesions, supporting the notion that these cytotoxic cells get access to the inflamed CNS.

As NKG2C or NKG2D blockade did not completely abrogate CD4 T cell

75 degranulation, additional receptors could play a role in the observed cytotoxicity. Our phenotyping results suggest that other activating receptors such as NKp46, although the cognate ligand remains unknown, could play a role in CD4 T cell-mediated cytotoxicity. It is of interest to us to determine the contributions of the other activating receptors that we have identified as being co-expressed with NKG2C. In the future, we would like to examine the

HLA-E/NKG2C interaction in vivo . It has never been documented whether mouse oligodendrocytes express Qa.1, the mouse homolog of HLA-E. Using the EAE model, we will assess whether infiltrating CD4 T cells in the CNS express NKG2C, and determine if blocking NKG2C on these cells can alleviate disease progression and/or symptoms.

Moreover, the development of therapies targeting NKG2C warrant further investigations since this receptor is specifically expressed by a subset of cytotoxic CD4 T cells in MS patients and therefore other cell types (others

CD4 T cells, CD8 T cells, NK cells) would not be affected by such therapy.

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