Molecular and cellular regulation of inflammatory T cell

function in the experimental autoimmune

encephalomyelitis model of multiple sclerosis

Thèse

Irshad Akbar Irshad Akbar

Doctorat en médecine moléculaire

Philosophiæ doctor (Ph. D.)

Québec, Canada

© Irshad Akbar Irshad Akbar, 2025

Molecular and cellular regulation of inflammatory
T cell function in the experimental autoimmune
encephalomyelitis model of multiple sclerosis

Thèse

Irshad Akbar

Sous la direction de :

Manu Rangachari, directeur de recherche

Résumé
La sclérose en plaques (SEP), une maladie auto-immune et neurodégénérative du système
nerveux central (SNC), affecte près de 2 millions de personnes dans le monde. Ce désordre
est provoqué par les cellules immunitaires qui ciblent et attaquent de façon spécifique la
myéline du SNC. L’encéphalomyélite auto-immune expérimentale (EAE) est un modèle
animal répandu et bien caractérisé dans le domaine de la SEP. Il a permis d’étudier de
nombreux éléments de la cascade pathogénique de la maladie. Il est connu que les cellules
Th1 ont un rôle central dans la pathogénèse de l’EAE. Des résultats récents indiquent que
l’inhibiteur de la sérine protéase clade E1 (Serpine1) est un modulateur négatif de la
production de cytokines par les cellules Th1 et en particulier, de l’interféron-gamma (IFN-
γ). La fonction de Serpine1 dans le contexte de l’auto-immunité reste toutefois à préciser.
Dans notre étude, nous avons démontré que les souris Serpine1 knockout (KO) présentent
une EAE exacerbée en comparaison avec les souris contrôles (WT) en protocole
d’immunisation active ce qui met en évidence le rôle protecteur de Serpine1 dans le modèle.
Notamment, la surexpression de Serpine1 réduit efficacement la pathogénicité des cellules
Th1, tandis que les cellules Serpine1-KO 2D2 provoquent une EAE de plus grande sévérité
que les cellules WT. Il est intéressant de noter que la polarisation des cellules Serpine1-KO
en Th1 montrent une expression retardée du récepteur inhibiteur Tim-3 suggérant une
altération de cette voie de régulation. Lorsque les souris Serpine1-KO sont génétiquement
modifiées afin de surexprimer Tim-3 (Serpine1-KO : Tim-3-Tg), les cellules Th1 qui
proviennent de ces souris dévoilent une augmentation des niveaux d’expression de l’IFN-γ
et une diminution des molécules checkpoint inhibitrices Lag-3 et de PD-1 en comparaison
avec les cellules Th1 WT Tim3-Tg. De plus, l’absence de Serpine1 rétablit le phénotype EAE
des souris Tim-3-Tg qui normalement, développent une maladie de faible gravité. Ces
résultats soutiennent la participation de Serpine1 dans les réponses immunitaires des cellules
Th1.

De façon complémentaire, notre recherche cible également l’implication des cellules T CD8+
dans l’immuno-pathogénèse de la SEP. En utilisant des lymphocytes T de souris
transgéniques 1C6 dans le background NOD, souris dont le récepteur transgénique (TcR-Tg)
cible spécifiquement la glycoprotéine oligodendrocyte de la myéline (MOG), nous avons

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étudié la fonctionnalité et le potentiel pathogénique des cellules T CD8+. Nos résultats
démontrent que si les lymphocytes T

CD8+ 1C6 présentent une faible prolifération dans des conditions de différenciation Tc1 et
Tc17 en réponse au MOG, ils répondent fortement à la stimulation avec les anticorps anti-
CD3 et anti-CD28 indiquant l’absence de défaut intrinsèque quant à leur capacité à proliférer.
Le transfert adoptif des cellules Tc17 dans des souris immunosupprimées [Link]
provoquent une EAE de progression sévère marquée par l’infiltration substantielle des
lymphocytes T CD8+ dans le SNC. Les cellules T CD8+ infiltrées sont caractérisées par
l’expression de l’IL-17, IFN-γ et le TNF-α. Lors de ces transferts adoptifs, nous avons
curieusement observé une expansion in vivo prononcée des cellules T CD4+ dans la rate et
le SNC des souris receveuses et ce, malgré la purification des cellules CD8+ réalisée au
préalable. L’expansion des cellules T CD4+ a d’ailleurs été corroboré lors de l’administration
d’un anticorps anti-CD4 chez les souris receveuses [Link] de cellules Tc17. Le blocage
des cellules CD4+ chez les souris [Link] en protocole adoptif de cellules Tc17 atténue
efficacement la progression de la maladie.

Finalement, l’ensemble de nos travaux exposent une coopération entre les lymphocytes T
CD8+ réactifs au peptide MOG et les lymphocytes T auxiliaires CD4+ dans l’étiologie de
l’EAE, soulignant l’interaction complexe des différents sous-groupes de cellules T dans les
processus auto-immunitaires. Cette recherche fournit d’importantes informations sur les
mécanismes de régulation des lymphocytes T dans le contexte de l’EAE et identifie des cibles
thérapeutiques potentielles telles que Serpine1 et Tim-3, des collaborateurs essentiels dans la
réponse auto-immune des cellules T.

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Abstract
Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease of the central
nervous system (CNS) that affects nearly 2 million people worldwide, characterized by an
adaptive immune cell-mediated attack against CNS myelin constituents. Exp erimental
autoimmune encephalomyelitis (EAE) is a well-accepted animal disease for the study of MS,
as it permits the study of numerous elements of the MS pathogenic cascade. In experimental
autoimmune encephalomyelitis (EAE), Th1 cells play a pivotal role in driving the disease's
pathogenesis. Recent findings indicate that the serine protease inhibitor clade E1 (Serpine1)
serves as a negative regulator of Th1 cytokine production, particularly interferon-gamma
(IFN-γ), although its specific function in autoimmune contexts remains inadequately
understood. In our study, we demonstrate that Serpine1 knockout (KO) mice exhibit a
markedly exacerbated EAE phenotype compared to wild-type (WT) controls, highlighting
Serpine1's protective role in this model. Notably, Serpine1 overexpression effectively
suppresses Th1 cell pathogenicity, while Serpine1-KO 2D2 Th1 cells induce more severe
EAE than their WT counterparts. Interestingly, polarized Serpine1-KO Th1 cells show
delayed expression of the inhibitory receptor Tim-3, suggesting an altered regulatory
pathway in these cells. When Serpine1-KO mice were genetically modified to overexpress
Tim-3 (Serpine1-KO:Tim-3 Tg), these Th1 cells exhibited increased levels of IFN-γ along
with decreased expression of other checkpoint molecules such as LAG-3 and PD-1 compared
to WT Tim-3-Tg Th1 cells. Moreover, the absence of Serpine1 restored the EAE phenotype
in Tim-3-Tg mice, which typically present with milder disease, thereby reaffirming
Serpine1's role in modulating Th1 immune responses.

Complementing these findings, our research also emphasizes the significance of CD8+ T cells
in the immunopathogenesis of multiple sclerosis (MS). Utilizing 1C6 T cell receptor
transgenic (TcR-Tg) mice on a nonobese diabetic (NOD) background, we explored the
functionality and pathogenic potential of CD8+ T cells specific to myelin oligodendrocyte
glycoprotein (MOG[35-55]). Our observations revealed that while 1C6 CD8+ T cells
displayed poor proliferation under Tc1 and Tc17 differentiation conditions in response to
MOG, they strongly responded to anti-CD3 and anti-CD28 stimulation, indicating no
intrinsic defects in their proliferative capacity. The adoptive transfer of Tc17 cells into

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[Link] mice led to severe progressive EAE, marked by substantial infiltration of CD8+ T
cells expressing IL-17, IFN-γ, and TNF into the central nervous system (CNS). Intriguingly,
a pronounced in vivo expansion of CD4+ T cells was observed in both the spleen and CNS
of recipient mice, despite prior purification of Tc17 cells based on CD8 expression. This
CD4+ T cell expansion was further corroborated when Tc17 cells were transferred into
lymphocyte-sufficient hosts, with anti-CD4 T cell blockade effectively mitigating disease
progression in Tc17 recipient [Link] mice.

Together, these findings delineate a collaborative interaction between MOG-reactive CD8+

T cells and CD4+ T helper cells in the etiology of EAE, underscoring the complex interplay
of different T cell subsets in autoimmune processes. This research provides valuable insights
into the regulatory mechanisms of T cell responses in EAE and identifies potential
therapeutic targets, such as Serpine1 and Tim-3, to modulate autoimmune T cell activity
effectively.

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Table of contents
RÉSUMÉ ....................................................................................................................................................... II
ABSTRACT ................................................................................................................................................... IV
TABLE OF CONTENTS ................................................................................................................................... VI
LIST OF FIGURES .......................................................................................................................................... IX
LIST OF ABBREVIATIONS .............................................................................................................................. X
ACKNOWLEDGEMENTS .............................................................................................................................XIV
PREFACE ...................................................................................................................................................XVI
INTRODUCTION ........................................................................................................................................... 1
1.1 INNATE AND ADAPTIVE IMMUNE RESPONSE ............................................................................................ 1
1.1.1 Innate Immune Response .................................................................................................................. 1
[Link] Physical Barrier ............................................................................................................................................ 1
[Link] Cellular innate immunity .............................................................................................................................. 2
[Link].1 Pathogen Recognition Receptors (PRRs) .............................................................................................. 2
1.1.2 Adaptative immune response ............................................................................................................ 4
[Link] T lymphocytes .............................................................................................................................................. 5
[Link].1 T cell development ............................................................................................................................... 6
[Link].2 Thymic selection of the T cell repertoire .............................................................................................. 9
[Link].2.1 Positive selection........................................................................................................................... 9
[Link].2.2 Negative selection ......................................................................................................................... 9
[Link].2.3 Immunological ignorance ............................................................................................................ 11
[Link].2.4 T cell anergy ................................................................................................................................ 12
[Link].2.5 Regulatory T cell-mediated tolerance ......................................................................................... 12
[Link].3 T cell activation ................................................................................................................................... 13
[Link].3.1 TCR signalling .............................................................................................................................. 13
[Link].3.2 Antigen Recognition .................................................................................................................... 16
[Link].3.2.1 Antigen processing and presentation .................................................................................. 16
[Link].3.2.2 Antigen recognition by T cells .............................................................................................. 18
[Link].3.3 Co-stimulation ............................................................................................................................. 19
[Link].3.4 Role of cytokines in T cell activation and differentiation ............................................................ 20
[Link].3.5 T cell subsets ............................................................................................................................... 22
[Link] B lymphocytes ............................................................................................................................................ 25
1.2 AUTOIMMUNITY AND NEURO AUTOIMMUNITY .................................................................................... 28
1.2.1 Thymic/Central Mechanisms ........................................................................................................... 29
1.2.2 Peripheral Mechanisms ................................................................................................................... 29
1.3 MULTIPLE SCLEROSIS .............................................................................................................................. 30
1.3.1 Prevalence and pathogenesis .......................................................................................................... 32
1.4 SUBTYPES OF MULTIPLE SCLEROSIS ........................................................................................................ 33
1.4.1 Relapsing-remitting multiple sclerosis ............................................................................................. 33
1.4.2 Secondary Progressive Multiple Sclerosis ........................................................................................ 34
1.4.3 Primary Progressive Multiple Sclerosis ............................................................................................ 35
1.5 RISK FACTORS IN MS ............................................................................................................................... 36
1.5.1 Genetic Factors ................................................................................................................................ 37
1.5.2 Biological sex ................................................................................................................................... 37
1.5.3 Environmental Factors ..................................................................................................................... 38

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 [Link]. Vitamin D ..................................................................................................................................... 39
[Link] Smoking ........................................................................................................................................ 39
1.5.6 Viruses ............................................................................................................................................. 40
1.5.7 Microbiome...................................................................................................................................... 41
1.6 MECHANISMS OF BLOOD BRAIN BARRIER DISRUPTION IN MS ............................................................... 42
1.7 EAE AS AN ANIMAL MODEL FOR MS........................................................................................................ 43
1.7.1 Active Immunization ........................................................................................................................ 44
1.7.2 T cell adoptive transfer .................................................................................................................... 44
1.7.3 Spontaneous EAE ............................................................................................................................. 46
1.7.4 Toxin-induced demyelinating models .............................................................................................. 47
1.7.5 Cuprizone model .............................................................................................................................. 47
1.7.6 Lysophosphatidylcholine (LPC)-induced demyelination ................................................................... 48
1.8 KEY ROLES OF CD8 T CELLS IN AUTOIMMUNE DISEASE ........................................................................... 49
1.9 PATHOPHYSIOLOGY OF CD8+ T CELLS IN MULTIPLE SCLEROSIS ............................................................... 50
1.10 EAE MODELS OF CD8+ T CELLS ............................................................................................................... 53
1.10.1 MBP[79-87]–specific CD8+ T cell model ......................................................................................... 53
1.10.2 Met+ CD8+ T cell model................................................................................................................... 54
1.10.3 GFAP specific CD8+ T cell model ..................................................................................................... 54
1.11 CD8+T CELLS IN OTHER AUTOIMMUNE DISEASES .............................................................................................. 56
1.12 SERPINE1 AND ITS ROLE IN INFLAMMATION ........................................................................................ 57
1.12.1 Role of Serpine1 in CNS inflammation ........................................................................................... 59
1.13 T CELL IMMUNE CHECKPOINTS AND THEIR ROLE IN T CELL EXHAUSTION ............................................. 60
1.13.1 Tim-3 .............................................................................................................................................. 61
1.13.2 PD-1 ............................................................................................................................................... 63
1.13.3 Lymphocyte activation gene-3 (Lag-3) .......................................................................................... 64
1.14 HYPOTHESIS AND OBJECTIVES .............................................................................................................. 65
1.14.1 Hypothesis 1 .................................................................................................................................. 65
1.14.2 Hypothesis 2 .................................................................................................................................. 66
CHAPTER 1. SERPINE1 NEGATIVELY REGULATES TH1 CELL RESPONSES IN EXPERIMENTAL AUTOIMMUNE 69
1.1 Résumé ............................................................................................................................................... 70
1.2 Abstract .............................................................................................................................................. 71
1.3 Introduction ........................................................................................................................................ 72
1.4 Results ................................................................................................................................................ 72
1.4.1 Serpine1 inhibits the severity of EAE ............................................................................................................ 72
1.4.2 Serpine1 suppresses Th1 cell function in vitro and in vivo ........................................................................... 76
1.4.3 Loss of Serpine1 function or expression exacerbates Th1 responses ........................................................... 79
1.4.4 Serpine1 promotes Tim-3 expression and inhibits Tim-3 Th1 cell inflammatory responses ......................... 82
1.5 Materials and Methods ...................................................................................................................... 86
1.5.1 Mice .............................................................................................................................................................. 86
1.5.2 Th cell differentiation .................................................................................................................................... 86
1.5.3 EAE model ..................................................................................................................................................... 87
1.5.4 Flow cytometry ............................................................................................................................................. 87
1.5.5 Ex vivo assessment of T cell function ............................................................................................................ 88
1.5.6 Statistical analysis ......................................................................................................................................... 88
1.6 Data availability.................................................................................................................................. 88
1.7 Acknowledgments .............................................................................................................................. 88
1.8 Disclosures .......................................................................................................................................... 88
1.9 References .......................................................................................................................................... 89

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CHAPTER 2. CD4+ T CELLS ARE IMPORTANT FOR TC17 MEDIATED PROGRESSIVE EAE IN AN ADOPTIVE
TRANSFER EAE MODEL............................................................................................................................... 92
2.1 Résumé ............................................................................................................................................... 93
2.2 Abstract .............................................................................................................................................. 94
2.3 Introduction ........................................................................................................................................ 95
2.4 Results ................................................................................................................................................ 96
2.4.1 1C6 CD8+ T cells respond suboptimally to MOG35-55 peptide but show no intrinsic proliferative defect ..... 96
2.4.2 Assessment of cytokine production from 1C6 effector T cells .................................................................... 100
2.4.3 1C6 Tc1 and Tc17 cells adoptively transfer EAE .......................................................................................... 103
2.4.4 Male and female 1C6 Tc17 cells induce EAE of similar onset and severity ................................................. 107
2.4.5 Ex vivo analysis of T cells from Tc17 recipients ........................................................................................... 110
2.4.6 CD4+ T cells arise in [Link] recipients of 1C6 Tc cells ............................................................................. 113
2.4.7 CD4+ T cells are essential to 1C6 Tc17-mediated EAE ................................................................................. 116
2.6 Materials and Methods .................................................................................................................... 123
2.6.1 Experimental animals and ethics ................................................................................................................ 123
2.6.2 Effector Tc and Th culture and differentiation ............................................................................................ 123
2.6.3 Flow Cytometry ........................................................................................................................................... 123
2.6.4 Adoptive transfer EAE ................................................................................................................................. 124
2.6.5 CNS Mononuclear Cell Isolation .................................................................................................................. 124
2.6.6 CD4 blockade .............................................................................................................................................. 125
2.6.7 Statistics ...................................................................................................................................................... 125

CHAPTER 3. DISCUSSION .......................................................................................................................... 126

3.1 Overexpression of Serpine1 in 2D2 cells induces disease of lessened severity ................................. 127
3.2 Knockout of Serpine1 inTim3-Tg Th1 cells increase their pathogenicity and reduce T cell inhibition
molecules. ............................................................................................................................................... 128
3.3 CD4+ T cells appear in Tc1/Tc17 recipient [Link] mice and blocking of CD4 + T cells decreased CD8+
T cells in CNS ........................................................................................................................................... 129
3.4 CD4+ and CD8+ T cells induce highly severe disease when transferred together. ............................. 130
CONCLUSION AND FUTURE PERSPECTIVES ............................................................................................... 132
BIBLIOGRAPHY ......................................................................................................................................... 133

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List of figures
FIGURE 1.1 INNATE IMMUNE COMPONENTS. ............................................................................................................... 3
FIGURE 1.2 T CELL DEVELOPMENT IN THYMUS .............................................................................................................. 8
FIGURE 1.3 POSITIVE AND NEGATIVE SELECTION OF T CELLS ........................................................................................... 10
FIGURE 1.4 TCR SIGNALLING .................................................................................................................................. 15
FIGURE 1.5 ANTIGEN PRESENTATION ....................................................................................................................... 18
FIGURE 1.6 THREE SIGNALS FOR T CELL ACTIVATION .................................................................................................... 21
FIGURE 1.7 CD4+ T CELL SUBSETS ........................................................................................................................... 23
FIGURE 1.8 CD8+ T CELL SUBSETS ........................................................................................................................... 24
FIGURE 1.9 FIVE CLASSES OF B CELL IMMUNOGLOBULINS .............................................................................................. 27
FIGURE 1.10 SYMPTOMS OF MULTIPLE SCLEROSIS....................................................................................................... 32
FIGURE 1.11 TYPES OF MULTIPLE SCLEROSIS .............................................................................................................. 36
FIGURE 1.1. SERPINE1 INHIBITS THE SEVERITY OF EAE.................................................................................................. 75
Figure 1.2. Serpine1 inhibits the severity of EAE……………………………………………………………………………………..……75
FIGURE 1.3 LOSS OF SERPINE1 FUNCTION OR EXPRESSION EXACERBATES TH1 RESPONSES.................................................... 81
FIGURE 1.4 SERPINE1 PROMOTES TIM-3 EXPRESSION AND INHIBITS TIM-31 TH1 CELL INFLAMMATORY RESPONSES. ................ 85
FIGURE 2.1 1C6 CD8+ T CELLS RESPOND SUBOPTIMALLY TO MOG[35-55] PEPTIDE BUT SHOW NO INTRINSIC PROLIFERATIVE
DEFECT. ....................................................................................................................................................... 99
FIGURE 2.2 ASSESSMENT OF CYTOKINE PRODUCTION FROM 1C6 EFFECTOR T CELLS. ........................................................ 102
FIGURE 2.3 1C6 TC1 AND TC17 CELLS ADOPTIVELY TRANSFER EAE. ............................................................................. 106
FIGURE 2.4 MALE AND FEMALE 1C6 TC17 CELLS INDUCE EAE OF SIMILAR ONSET AND SEVERITY. ....................................... 109
FIGURE 2.5 EX VIVO ANALYSIS OF T CELLS FROM TC17 RECIPIENTS. ............................................................................... 112
FIGURE 2.6 CD4+ T CELLS ARISE IN [Link] RECIPIENTS OF 1C6 TC CELLS. ................................................................. 115
FIGURE 2.7 CD4+ T CELLS ARE ESSENTIAL TO 1C6 TC17-MEDIATED EAE ...................................................................... 118

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List of abbreviations

Ag – Antigen
AIRE – Autoimmune Regulator
anti-CD4 – Antibody targeting CD4
AP-1 – Activator Protein 1
APC – Antigen-Presenting Cell
APECED – Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy
BAT3 – HLA-B-associated Transcript 3
BBB – Blood-Brain Barrier
Bcl-xL – B-cell lymphoma-extra large
BCR – B Cell Receptor
CaM – Calmodulin
cAMP – Cyclic Adenosine Monophosphate
CCL – C–C Chemokine Ligand
CD – Cluster of Differentiation
CFA – Complete Freund's Adjuvant
CLIP – Class II-associated Invariant Chain Peptides
CLR – C-type Lectin Receptor
CMV – Cytomegalovirus
CNS – Central Nervous System
COAD-READ – Colon Adenocarcinoma and Rectum Adenocarcinoma
CRAC – Calcium Release-Activated Calcium Channels
cTEC – Cortical Thymic Epithelial Cell
CTLA-4 – Cytotoxic T Lymphocyte Antigen 4
CYP – Cytochrome P450
DAMP – Danger-Associated Molecular Pattern
DC – Dendritic Cell
DMTs – Disease Modifying Therapies
DN – Double Negative
DP – Double Positive
EAE – Experimental Autoimmune Encephalomyelitis
EBV – Epstein-Barr Virus
EDSS – Expanded Disability Status Scale
ER – Endoplasmic Reticulum
FAS – FS-7 associated surface antigen
Fc – Fragment crystallizable
Fc receptor – Fragment crystallizable receptor
Foxp3 – Forkhead Box P3
GBM-LGG – Glioblastoma Multiforme and Lower-grade Glioma
GFAP – Glial Fibrillary Acidic Protein

x
GWAS – Genome-Wide Association Studies
HCV – Hepatitis C Virus
HGF – Hepatocyte Growth Factor
HHV – Human Herpesvirus
HLA – Human Leukocyte Antigen
HLA-DM – Human Leukocyte Antigen DM
HSC – Hematopoietic Stem Cells
ICIs – Immune Checkpoint Inhibitors
ICOS – Inducible T Cell Co-Stimulator
ICOS-L – ICOS Ligand
IDO – Indoleamine 2,3-Dioxygenase
IFN – Interferon
Ig – Immunoglobulin
IKK – IκB Kinase
IL – Interleukin
IL-17 – Interleukin 17
IP3 – Inositol Triphosphate
IP-30 – IFN-γ Inducible Lysosomal Thiol Reductase
IRF – Interferon Regulatory Factor
J chain – Joining chain
JAK-STAT – Janus Kinase-Signal Transducer and Activator of Transcription
JCV – John Cunningham Virus
KO – Knockout
LAG-3 – Lymphocyte Activation Gene 3
LAT – Linker for Activation of T Cells
LCK – Lymphocyte-Specific Protein Tyrosine Kinase
LPS – Lipopolysaccharide
mAb – Monoclonal Antibody
MAPK – Mitogen-Activated Protein Kinase
MBP – Myelin Basic Protein
MHC – Major Histocompatibility Complex
mRNA – Messenger RNA
MS – Multiple Sclerosis
NFAT – Nuclear Factor of Activated T Cells
NfL – Neurofilament Light Chain
NF-κB – Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells
NK – Natural Killer
NLR – NOD-like Receptor
NOD-Scid IL2Rγc−/− – Non-Obese Diabetic Severe Combined Immunodeficient
Interleukin- 2 Receptor Gamma Chain Deficient
OVA – Ovalbumin
OX40 – Tumor Necrosis Factor Receptor Superfamily Member 4
OX40L – OX40 Ligand

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PAAD – Pancreatic Adenocarcinoma
PAMP – Pathogen-Associated Molecular Pattern
PD-1 – Programmed Cell Death-1
PD-L1 – Programmed Death Ligand 1
PD-L2 – Programmed Death Ligand 2
PLCγ1 – Phospholipase C-gamma1
PP – Primary Progressive
PPMS – Primary Progressive Multiple Sclerosis
PRR – Pattern Recognition Receptor
PTX – Pertussis Toxin
pwMS – People with MS
RAG – Recombination Activating Gene
Rag1 – Recombination Activation Gene 1
RLR – RIG-I-like Receptor
RNA – Ribonucleic Acid
ROS – Reactive Oxygen Species
RRMS – Relapsing-Remitting Multiple Sclerosis
RV – Retrovirus
S1-WT – Serpine1 Wild Type
SAS – Subarachnoid Space
Serpine1 – Serine Protease Inhibitor E1 (also known as Serpin E1)
SP – Secondary-Progressive
SP – Single Positive
SPMS – Secondary Progressive Multiple Sclerosis
T1D – Type 1 Diabetes
TAP – Transporter Associated with Antigen Processing
Tc – Cytotoxic T Cells
TCR – T Cell Receptor
Tg – Transgenic
Th – T Helper Cell
Th1 – T Helper 1
Th17 – T Helper 17
Tim-3 – T cell Immunoglobulin and Mucin Domain-containing Protein 3
TLR – Toll-Like Receptor
TNF – Tumor Necrosis Factor
Tregs – Regulatory T Cells
TSA – Tissue-Specific Antigen
UVM – Uveal Melanoma
V(D)J – Variable, Diversity, and Joining region
VAV-1 – Guanine Nucleotide Exchange Factor Vav protein 1
Vβ – Variable Beta
WT – Wild Type
ZAP70 – Zeta-Chain-Associated Protein Kinase 70

xii
β2M – Beta-2 Microglobulin
κ chain – Kappa light chain
λ chain – Lambda light chain

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 Acknowledgements
I would like to express my deepest gratitude to my supervisor, Dr. Manu Rangachari, for the
invaluable opportunity to pursue my PhD under his supervision. His guidance, constant
support and dedication has been crucial in my development both as a researcher and as an
individual. Dr. Rangachari has always been supportive in every stage of the project, from
breakthroughs to challenges. His extensive knowledge and experience have helped in the
successful completion of the project. His encouragement to continuously develop our skills
and expand our expertise has been invaluable, as has his commitment to showcasing his
students’ work in meetings, conferences, and beyond. I am particularly thankful for his
insights and the opportunities he extended, which have enriched both my scientific
perspective and my career aspirations.

I am also sincerely thankful to my predoctoral committee members, Dr. Martin Pelletier, Dr.
Jean Philippe Lambert and Dr. Stephane Gobeil, for their invaluable suggestions and
insightful discussions which have been immensely helpful. I am also thankful to the jury
members Dr. Martin Pelletier, Dr. Luc Vallières, Dr. Deepak Kaushik and Dr. Stephane
Gobeil for accepting to be the evaluators of my thesis.

I extend my warmest thanks to the foundational girls of the lab “Joanie Baillargeon, Prenitha
Mercy and Asmita Yeola” for their guidance and support both scientifically and personally.
My sincere thanks to all the lab members Mohammad Umair, Reda Fazazi, Alexia Falle and
Vahid Safdari. Each of them has contributed to my experience in the lab, assisting with
experimental design, data analysis, and during life’s highs and lows. I would like to extend a
heartfelt thank you to everyone in the Axis Neurosciences team of block T at past and present.
My gratitude also goes to the staff of the animal care unit, whose diligent work in caring for
our mice was essential to this research. I am especially thankful to Vincent Desrosiers for his
excellent technical support and help in learning the flow cytometry. Additionally, I am
grateful to the outstanding teams of Dr. Luc Vallières, Dr. David Gosselin, Dr. Serge Rivest
and Dr. Steve Lacroix for generously sharing their expertise, equipment, and reagents.

xiv
My journey at Laval University has been an enriching experience, both intellectually and
personally. This journey wouldn’t have been amazing without my friends who made it truly
memorable. I would like to extend my gratitude to my friends Mohmmad Jabbar, Talal Asif
and Ishfaq Mir for their friendship and encouragement along the way. Special thanks to Ali
Riaz who has been very caring and always encouraged me to do the best. A big thanks to my
first mentor, Sriparna Mukherjee who has introduced me to the field of research and has
always been a big support and guide throughout my career.

Finally, I would like to extend my heartfelt gratitude to my parents for their prayers and
especially to my dad who I lost during my PhD. Although he never had the chance to attend
the school, but he put all his efforts to make sure that I never miss a single day of my
education. I special thanks goes to my brothers Suliman and Irfan who put all their efforts
for my education. I wouldn’t miss to thank my sisters Amira and Nuzhat for the care and
guidance they offered since my childhood.

I acknowledge MS Canada for providing the support of endMS doctoral studentship

throughout my PhD. This support has been instrumental, motivating me to deepen my skills
and expand my knowledge in the field of MS research.

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Preface
The research presented in this thesis is conducted by me, Irshad Akbar, during my doctoral
studies under the mentorship of Dr. Manu Rangachari. The studies presented in this thesis
reflect a combining approach to understand T cell function and pathogenicity in autoimmune
neuroinflammation, with a focus on Multiple Sclerosis, using EAE mice model.

The introduction section provides essential background on T cell immunology, which

includes T cell development, signalling mechanism and subsets of CD4+ and CD8+ T cells.
It also includes an overview of MS, describing its pathogenesis, subtypes and disease models,
followed by the role of CD8+ T cells in MS pathogenesis, and EAE models of CD8+ T cells.
The last part of the introduction describes the role of Serpine-1 in CNS inflammation and T
cell inhibitory receptors. These concepts led to our main hypothesis and the hypothesis were
evaluated by me as a first author in Chapter 1 and Chapter 2.

Chapter 1 details on the role of Serpine-1 in modulating T cell autoimmunity, presented in

the manuscript “Serpine1 Negatively Regulates Th1 Cell Responses in Experimental
Autoimmune Encephalomyelitis”. In this study we found that Serpine-1 plays a critical role
in mediating Th1 inflammation. Overexpression of Serpine-1 decreases IFN-γ expression
and pathogenicity of Th1 cells, while knockout of Serpine-1 in Th1 cells induces more severe
EAE. In Tim-3 transgenic Th1 cells, Serpine-1 KO leads to reduced expression of T cell
inhibitory molecules (PD-1 and Lag-3) and increased expression of IFN-γ, confirming the
inhibitory role of Serpine-1 in Th1-driven autoimmunity. This work is published in The
Journal of Immunology([Link] .

Chapter 2 investigates the function and pathogenicity of MOG35-55 specific 1C6 CD8+ T
cells and their need for the CD4+ T cell in inducing CNS inflammation. This study is
presented in the manuscript “CD4+ T cells are important for Tc17 mediated progressive EAE
in an adoptive transfer EAE model”. In this part of the thesis, we explored the response of
CD8+ T cells to the antigenic peptide (MOG35-55), their proliferative capacity and expression
of proinflammatory cytokines. We also explored the combined impact of CD4+ and CD8+ T
cell on the disease burden when they are adoptively transferred together. We found that CD4+

xvi
T cells are important for the infiltration of CD8+ T cells to the CNS, and transfer of only
CD8+ T cells give rise to the CD4+ T cells in [Link] mice spleen and CNS.

Chapter 3 provides an overall discussion and conclusion related to the findings in this thesis.
Based on these findings several research directions are proposed.

xvii
Introduction

1.1 INNATE AND ADAPTIVE IMMUNE RESPONSE

The immune response is a complex and highly regulated system. As our body is continuously
exposed to disease-causing pathogens in our environment, we need continuous surveillance
to protect ourselves from these pathogens. To protect ourselves from the infectious agents
encountered, our body has a defence system, the immune system, which provides different
lines of protection and adapts as we encounter new pathogens. However, the power of the
immune system requires the tight regulation of its function; dysregulated immune response
can lead to inappropriate responses and, potentially, to autoimmune diseases [1], [2].

Broadly speaking, the immune system consists of two main arms. The first line of defence is
the innate immune system, which is rapidly engaged when the body encounters a pathogen.
The adaptive immune system comprises the second line of the defence system. It takes longer
to engage but is more specifically tailored to individual pathogens and is responsible for
immunological memory. This latter characteristic is very important for the rapid clearance of
the pathogens that the organisms have previously encountered [3], [4]

1.1.1 Innate Immune Response

The innate immune response has three key components, which are as follows:

[Link] Physical Barrier

The physical barrier includes the skin and the mucus membranes. The skin acts as a physical
barrier between the environment and our body, preventing the entry of pathogens. The mucus
membranes lining the respiratory, gastrointestinal, and urogenital tract help in trapping the
pathogens and expelling them from the body [1], [2]. The complement system complements
or helps the body’s immune cells to fight the infection. Once the complement system gets
activated, it starts a cascade of events that leads to the killing of pathogens like bacteria (Fig
1.1).

1
[Link] Cellular innate immunity

Innate immune cells are so named because they do not require fine specificity tuning prior to
their activation. Key innate immune cells include phagocytic cells - macrophages, dendritic
cells and neutrophils that engulf and degrade pathogens. Macrophages and dendritic cells
have the additional ability to generate short pathogen-derived peptide sequences, called
antigens or epitopes, that they present on their surface to invoke a T cell-dependent adaptive
immune response. It is important to note that not all innate immune cells can phagocytose
pathogens. For example, natural killer (NK) cells kill virally infected or transformed cells by
releasing cytotoxic granules. Other cells, such as mast cells, basophils and eosinophils, also
play an important role in mediating innate immune response despite not being adept at
presenting antigen [1], [2]. In this thesis, however, I will focus principally on antigen-
presenting innate immune cells as they are most pertinent to T cell activation (section
[Link].3.2)

[Link].1 Pathogen Recognition Receptors (PRRs)

PRRs are a specialized set of receptors present on the phagocytes that recognize pathogen-
associated molecular patterns (PAMPs) that are specific to pathogens [5], [6]. Each PRR
recognizes a defined subset of PAMPs; thus, while innate immune cells do not show the
exquisite specificity to unique pathogens that is characteristic of adaptive immunity, they do
broadly distinguish between different classes of pathogens.

There are five major groups of PRRs: TLRs, RLRs, CLRs, NLRs and AIMLRs. Toll-like
receptors (TLRs) recognize the PAMPs based on the expression of TLRs within the cell.
Some of the TLRs e.g: TLR 1,2,4,5,6 and 11 are expressed on the extracellular surface while
other TLRs like TLR 3,7,8 and 9 are expressed in the endosomal compartment. TLRs
recognize different types of PAMPs e.g: TLR2 recognizes lipoproteins and glycolipids;
TLR4, lipopolysaccharide; TLR3, double-stranded RNA; TLR5, flagellin; TLR7, chemical
compounds (imidazoquinolines); TLR9, CpG sequences and TLR11 recognizes
uropathogenic bacteria [7]. Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs)
recognizes double stranded RNA generated during viral replication [8], C-type lectins

2
(CLRs) recognize viruses by binding to their carbohydrate moieties, using carbohydrate
recognition domains [9], and NOD like receptors (NLRs), and absent in melanoma (AIM)-
like receptors recognizes double-stranded DNA. Upon recognizing PAMPs, PRRs activate
inflammatory signalling pathways such as NF-κB, MAPK, IRF, NLRP3 inflammasome
activation, JAK-STAT and AP-1, that further leads to the activation of other immune cells
and thus helps in the clearance of the pathogens [10]. Intriguingly, PRRs have been described
as also recognizing endogenous motifs. These danger-associated molecular patterns
(DAMPs) are released during apoptosis or other conditions that cause tissue damage like
trauma, ischemia and cancer. These molecular patterns (DAMPs) are also called alarmins.

1. Anatomical barriers
• Physical barriers
• Chemical barriers
2 2. Cells
• Phagocytic cells
• Dendritic cells
• NK cells, ILC
3. Soluble proteins
• Complement proteins
• Cytokines, chemokines
• Microbial substances

Figure 1.1 Innate Immune Components

([Link]

The innate immune response is composed of several key components (1) physical and
chemical barriers; which include skin and mucosal layers that serve as body’s first line of

3
defense (2) cellular components, including natural killer (NK) cells, macrophages and
dendritic cells (DCs)which actively participate in phagocytosis, inflammation and acute
phase protein synthesis. (3) soluble proteins such as complement and cytokines, which
further support immune activation and pathogen clearance. The primary role of the innate
immune system is to directly engage and kill pathogens while also signalling and priming the
adaptive immune system for targeted immune response.

While the rapidity of the innate immune response is crucial to controlling pathogens, its
effectiveness is limited by its ability to recognize only broadly conserved molecular patterns.
The inherent variability in the antigenic repertoire of pathogens and their ability to mutate to
evade the host immune response requires activation of the adaptive immune response. In
addition to its early role in clearing pathogens, the innate immune system plays crucial
functions in activating the adaptive immune system by releasing cytokines and presenting
antigens to the T and B cells of the adaptive immune system [11]. The coordinated interaction
between the innate and adaptive immune response makes it a firm and effective defence
system against pathogens.

1.1.2 Adaptative immune response

The adaptive immune response differs from the innate immune response in its ability to
mount a highly specific and potent immune attack to a given pathogen. The adaptive immune
response may be classified as humoral or cell-mediated. Humoral immunity is dependent on
the secretion of antibodies by B cells. By contrast, cell-mediated immunity is primarily driven
by the recognition by T cells of their specific cognate antigen on antigen presenting cells
(APCs), followed by the cytokine-dependent recruitment of immune effector cells to sites of
infection and inflammation.

As adaptive immune cells, T and B cells possess antigenic specificity. This is encoded post-
somatically during the development of these cells via a process known as V(D)J
recombination. This genetic rearrangement occurs during the early stages of development in

4
these immune cells and is essential for the diversity of receptors that can recognize a wide
range of antigens [2].

In mammals, the development and maturation of B and T cells begin with the hematopoietic
stem cells (HSCs) in the fetal liver and later in the bone marrow, where HSCs differentiate
into multipotent progenitors. A subset of multipotent progenitors initiates the transcription
of recombination activating gene 1 and 2 (RAG1 and RAG2) and become lymphoid-primed
multipotent progenitors. To develop into fully mature and functional T cells, these lymphoid
progenitor cells enter the thymus for gradual reprogramming. The initial stages of the B cell
development occur in complex microenvironments created by the stromal cells of the bone
marrow, which provide the stimulus and factors required to initiate a series of cell signals
[2].

After their development in the bone marrow and thymus, respectively, T and B lymphocytes
migrate to the secondary lymphoid organs (lymph nodes or spleen), where they encounter
circulating antigens from blood and lymph. Both B and T cells recognize their cognate
antigen via the antigen-specific receptors present on their surfaces: the B cell receptor (BCR)
and T cell receptor (TCR).

[Link] T lymphocytes

T lymphocytes develop in the thymus as CD4+ T helper (Th) or CD8+ T cytotoxic (Tc) cells.
CD4+ T cells recognize antigens that are conjugated to Major Histocompatibility Complex
(MHC) class II molecules on the surface of APCs (discussed in detail in section [Link].2).
Upon their activation, CD4+ T cells differentiate into specific effector cell subsets (Th1, Th2,
Th17, Treg, among others) that release cytokines and recruit other immune cells. By placing
the recruitment of immune effector cells downstream of the antigen-MHC-II dependent
activation of CD4+ T cells, it is assured that the potent inflammatory response is mediated in
an antigen-specific manner. In contrast, the activation of cytotoxic T cells takes place upon
recognition of the antigen presented on the surface of cells in the context of MHC class I
(discussed in detail in section [Link].2). Cytotoxic T cells directly kill cells that express their
cognate antigen via the release of cytotoxic granules (perforin and granzyme), which induces

5
the apoptosis of the target cell. CD8+ T cells also eliminate their target cells (virus-infected
or tumour cells) by expressing FAS ligand that binds to the FAS receptor present in a wide
variety of cells, including immune cells, epithelial cells and some tumour cells. The binding
of the extracellular FAS domain to its FAS ligand transmits the apoptotic signal via the
intracellular death domain of the target cell [12], [13]. Notably, CD8+ T cells can also
differentiate into effector subsets such as Tc1 or Tc17 [1], [2]. As CD8+ T cells are vital for
the elimination of infected cells and as any cell can, in theory, be infected, most nucleated
cells have the capacity to express MHC class I. On the other hand, MHC class II expression
is largely restricted to “professional” APCs such as dendritic cells, macrophages and B cells.

[Link].1 T cell development

T cells play a central role in orchestrating the adaptive immune response, exhibiting a
remarkable capacity to differentiate into diverse subpopulations with distinct effector
functions in response to signals from innate immune cells. This thesis delves into the
exploration of various facets of the T cell response within the context of experimental
autoimmune encephalomyelitis (EAE), an established animal model designed to simulate
central nervous system (CNS) autoimmune disorders, including the clinically relevant
multiple sclerosis (MS).

T cell development starts in the bone marrow, where CD34+ hematopoietic stem cells
transform into common lymphoid and myeloid progenitors. While myeloid progenitors
diversify into various cell types, such as erythroid, megakaryocytic, and monocytic lineages,
lymphoid progenitors can become natural killer (NK), T, and B cells. B cell maturation
mostly occurs in the bone marrow microenvironment, but T cell progenitors, known as
thymocytes, take a different route. They migrate from the bone marrow to the thymus, where
they undergo a series of maturation steps without expressing signature T cell markers such
as CD3, CD4, or CD8. These thymocytes are termed "double negative" (DN) due to their
lack of CD4 and CD8. The DN population further subdivides into four early stages (DN1-
DN4) based on the expression of CD44 and CD25 [14]. As DN1 cells enter the thymic cortex,
they start proliferating and expressing CD25 (becoming CD44+CD25+). At the DN2 stage,
cells migrate towards the subcapsular zone, committing more to the T-cell lineage and

6
expressing genes needed for TCR rearrangements, such as RAG1 and RAG2 [15]. The DN3
stage is further divided into DN3a and DN3b, with regulatory changes in the markers
associated with checkpoint arrest, γδ selection and β-selection [16].

At the DN3a stage, the cell cycle halts, allowing the rearrangement of TCRβ genes through
V(D)J recombination. This leads to the formation of the pre-TCR complex, involving the β
chain pairing with the pre-TCR α-chain and CD3 signalling molecule [17]. β-selection causes
downregulation of CD25, and DN3 cells quickly progress to the transient DN4 stage
(CD44−CD25−). CD25 downregulation increases the expression of both CD4 and CD8,
yielding double-positive (DP) cells. DP cells rapidly proliferate and successfully rearrange
their TCR α-chain, producing the αβTCR-CD3 complex. Before leaving the thymus, DP cells
must undergo positive and negative selection [18]. Approximately 2% of DP thymocytes
survive these selection stages and mature into single-positive (SP) T cells, which then migrate
from the thymus to the periphery (Fig 1.2) [19]

7
Figure 1.2 T cell development in the thymus
[14][Link]
22/T-cell-development-in-the-thymus

Double-negative thymocytes enter the thymus in the subcapsular region. These DN cells then
migrate towards the thymic capsule, where they differentiate into DP αβ thymocytes,
expressing both CD4 and CD8 coreceptors. In the positive selection, where the T cells
interact with the cortical epithelial cells, the double-positive cells mature into CD4+ or CD8+
T cells. Finally, in the medulla, where the negative selection takes place, the self-tolerance
of the single positive cells is checked, and only the cells that are tolerant to the self-antigens
are allowed to go into the periphery.

8
[Link].2 Thymic selection of the T cell repertoire

T cells respond to pathogens upon recognizing specific pathogenic determinants (antigenic

epitopes) presented on MHC molecules on the surface of APCs. As MHC molecules are
multiallelic, it is essential that a T cell can interact with MHC molecules present in the given
individual. Further, to avoid autoimmune reactivity, the T cell should not react to peptides
derived from self-tissue. To ensure that both requirements are satisfied, thymocytes undergo
two overlapping selection processes in the thymus [20].

[Link].2.1 Positive selection

Positive selection, which occurs in the thymic cortical region, ensures that the αβ TCR
expressed by a DP thymocyte can effectively interact with self-MHC, a crucial factor for the
resulting T cell's capability to mount an effective response to specific antigens [21]. Cortical
thymic epithelial cells express either Class I or Class II MHC and present self-peptides to DP
thymocytes. Those that fail to bind to these peptides undergo apoptosis, leading to their
elimination. However, if the TCR successfully binds to the MHC complexes on thymic cells,
these thymocytes receive survival signals via the TCR signalling. Survival signals can also
be provided by cytokines such as IL-2, IL-4, IL-7, IL-15 and IL-21 [22], [23]. Whether a
thymocyte will commit to the CD4+ or CD8+ T cell lineage is determined by positive
selection (Fig 1.3). Double positive thymocyte precursors that bind well with MHC class II
commit to a CD4+ T cell fate, whereas those with effective interaction with MHC class I are
directed towards CD8+ T cell lineage [24].

[Link].2.2 Negative selection

SP CD4+ or CD8+ thymocytes migrate from the thymic cortex to the medulla, where they
undergo negative selection. This crucial step ensures that mature self-reactive T cells do not
initiate undesired responses to self-antigens. Medullary thymic epithelial cells express tissue-
specific antigens (TSAs) facilitated by the transcriptional derepressor, autoimmune regulator
(AIRE). This allows the presentation of a broad range of self-peptide-MHC complexes to SP

9
cells [25]. If a TCR exhibits excessive affinity toward these self-antigens, the cells undergo
apoptosis (Fig 1.3). Negative selection plays a critical role in eliminating potentially self-
reactive T cells, ensuring maturation and emigration of only those T cells that are less prone
to triggering autoimmune responses in the periphery [26].

Figure 1.3 Positive and negative selection of T cells

([Link]/474x/31/82/04/[Link])

10
Thymic selection processes ensure that mature T cells are both self-MHC restricted as well
as tolerant to self-peptide antigens. Immature thymocytes move from bone marrow to the
thymus for maturation (1) Within the thymus, T cells whose T cell receptors (TCRs) exhibit
moderate binding affinity to MHC complexes presenting self-antigens receive survival
signals, allowing them to mature (2), thymyocytes that survive positive selection become
double positive and exhibit both CD4+and CD8+ receptor (3). T cells with TCRs that bind
too strongly to these MHC-self antigen complexes undergo apoptosis through negative
selection (4), resulting in the elimination of autoreactive cells and promoting self-tolerance
(5).

Upon positive and negative selection, the surviving T cells are, in theory, able to recognize
foreign, but not self-derived, epitopes on the surface of self-MHC. It largely ensures that the
adaptive immune response is directed towards external threats and not towards destructive
self-reactive responses. Nevertheless, despite these mechanisms, some self-reactive T cells
manage to escape negative selection and circulate in the peripheral tissues [27]. Various
mechanisms of peripheral tolerance have evolved to control their potentially harmful
reactivity against self-tissues. These mechanisms include immunological ignorance, T cell
anergy, and the generation of regulatory T cells (Tregs), each of which is discussed below
[27]

[Link].2.3 Immunological ignorance

T cell ignorance is a condition where autoreactive T cells, which have the potential to target
self-antigens, remain in the body without responding to these antigens [28]. This
unresponsiveness of T cells occurs because many self-proteins are expressed at very low
levels, insufficient to trigger an immune response. Consequently, the TCRs on these
autoreactive T cells bind to self-peptides with such low affinity that no signalling pathway is
activated, leaving these T cells in a state of immunological ignorance[1]. The phenomenon
of immunological ignorance can also be explained by the advent of transgenic mice models,
where a foreign antigen is inserted into the genome of mice to be expressed as a self-antigen

11
in a particular tissue. Effector T cells for this antigen would be either deleted, regulated,
anergic or ignorant [29].

[Link].2.4 T cell anergy

Normally, T cells are activated when their TCRs recognize an antigen presented by MHC
molecules on APCs [30]. For full activation, T cells require two signals: i) the binding of the
TCR to the MHC-antigen complex; ii) the binding of the costimulatory receptor CD28 on the
T cell to its ligands CD80/CD86 on the APC [31]. When a T cell receives the first signal
without the second, it enters a state of anergy, in which T cells are unable to proliferate,
secrete cytokines, or differentiate in response to an antigen. This typically happens when T
cells encounter self-antigens without proper co-stimulation, leading to reduced production of
interleukin-2 (IL-2) and poor proliferation [32]. Anergy can also be induced by inhibitory
signals from receptors such as PD-1 (programmed cell death-1) and CTLA-4 (cytotoxic T
lymphocyte antigen-4). PD-1 binds to its ligands PD-L1 and PD-L2 on APCs, while CTLA-
4 interacts with CD80/CD86 on APCs, sending inhibitory signals that prevent full T cell
activation[33].

[Link].2.5 Regulatory T cell-mediated tolerance

Treg cells are essential for maintaining immunological tolerance, preventing the immune
system from attacking the body's own tissues [34]. These CD4+ T cells express high levels
of CD25 and are governed by the transcription factor Fork head Box P3 (Foxp3) [35]. Tregs
modulate the activity of autoreactive T cells by downregulating costimulatory signals on
dendritic cells (DCs). A key feature of Tregs is the constitutive expression of CTLA-4, which
becomes active upon stimulation and plays a crucial role in suppressing autoimmune
responses. Disruption of CTLA-4 function can lead to loss of self-tolerance and immune
imbalance [35].

Tregs employ multiple mechanisms to exert their suppressive effects. Upon antigenic
stimulation, they reduce the expression of CD80/CD86 on APCs and may also promote the
catabolism of tryptophan via indoleamine 2,3-dioxygenase, the depletion of tryptophan

12
leading to local immunosuppression by reducing the proliferation of effector T cells [36],
[37]. Tregs downregulate CD80/CD86 on DCs through CTLA4; the depletion of CD80 on
the surface of DCs may increase the expression of PD-L1 [38]. Thymic Tregs have also been
shown to inhibit CD70/CD27 pathway, which is required for T cell priming by DCs [39].
Tregs also inhibit the production of pro-inflammatory cytokines such as IL-6 and TNF from
DCs and decrease IL-2 levels in the APC environment [39]. This modulation results in the
generation of tolerogenic DCs, which makes the T cells nonresponsive by either preventing
their activation, inducing anergy due to insufficient co-stimulation or driving them to undergo
apoptosis. Tregs may also secrete immunosuppressive molecules such as IL-10, TGF-
β(transforming growth factor beta), IL-35, and galectin-1, and can kill APCs or responder T
cells via perforin/granzymes [40].

A breakdown in immunological tolerance can lead to autoimmunity, where the immune

system mistakenly attacks self-tissues. Autoimmune diseases arise from the activation of
autoreactive T and B cells or the production of autoantibodies against self-molecules, causing
inflammation and tissue damage. The onset and progression of autoimmunity are influenced
by a combination of genetic, immunological, and environmental factors, contributing to the
complex pathogenesis of these conditions [41].

[Link].3 T cell activation

For effective immune responses, T cells need three distinct signals from APCs of the innate
immune system: antigenic recognition via the TCR, costimulatory molecule activation and
soluble cytokine signalling. Before detailing these three signals, it’s essential to first
understand the mechanism of TCR signalling.

[Link].3.1 TCR signalling

Antigen recognition and signalling through the TCR are fundamental processes that enable
T cells to specifically target and respond to threats. The signalling pathways initiated by the

13
TCR are crucial for T cell activation and function. In this section, I will outline the basic T
cell signalling pathway, highlighting key molecules involved in CD4+ T cell activation.

The TCR is noncovalently associated with the CD3 complex, which consists of the ε, δ, γ,
and ζ chains. These CD3 chains contain immunoreceptor tyrosine-based activation motifs
(ITAMs), which are critical for signal transduction. When the TCR recognizes an antigen
presented by the MHC class II molecule, it triggers the phosphorylation of ITAMs on the
CD3 ζ chains. This event sets off a cascade of intracellular signalling pathways [42]. The
phosphorylation of ITAMs leads to the activation of Src-family kinases, such as Lck
(lymphocyte-specific protein tyrosine kinase) and Fyn, as well as ZAP70 (zeta-chain-
associated protein kinase 70). Lck plays a dual role, being activated by CD28 co-stimulation
and dephosphorylation via CD45. ZAP70, in turn, activates LAT (linker for activation of T
cells), which stimulates the phospholipase C-gamma1 (PLCγ1) pathway through
intermediaries such as SOS (son of sevenless) and GRB2 (growth factor receptor-bound
protein 2[43]. The activation of PLCγ1 leads to the production of the secondary messenger
diacylglycerol (DAG), which interacts with protein kinase C theta (PKCθ). This interaction
initiates the mitogen-activated protein kinase (MAPK) pathway, resulting in the activation
of IκB kinase (IKK). IKK phosphorylates inhibitory molecules like IκB, freeing the
transcription factor NF-κB to translocate to the nucleus and promote inflammatory gene
expression. PKCθ also influences the activity of the transcription factor AP-1 (Activator
protein 1), independent of JNK-dependent activation (c-Jun N-terminal kinase), thus linking
NF-κB and AP-1 activities [44], [45]. Additionally, ZAP70 and LAT-associated molecules
phosphorylate VAV-1, a guanine nucleotide exchange factor. VAV-1 activates a series of
mitogen-activated protein kinase kinases (MKKs), leading to the activation of MAPK family
members such as p38 and JNK. These kinases directly phosphorylate components of the AP-
1 transcription factor complex, further driving the expression of inflammatory genes [46].
An important aspect of this signalling cascade is increased intracellular calcium (Ca2+),
initially triggered by PLCγ1-mediated inositol triphosphate (IP3) action on endoplasmic
reticulum receptors. This rise in Ca2+ facilitates the influx of extracellular calcium through
membrane calcium release-activated calcium channels (CaCns). Elevated Ca2+ levels
counteract the inhibitory effects of calmodulin (CaM) on calcineurin, a phosphatase.

14
Calcineurin dephosphorylates the transcription factor NFAT (nuclear factor of activated T
cells), allowing it to enter the nucleus and activate gene transcription, completing the
signalling process essential for T cell activation (Fig 1.4) [47], [48].

Figure 1.4 TCR signalling [49]Binding of peptide-MHC complexes present on APCs to the TCRs
starts the TCR activation. Binding of the TCR to the MHC leads to the recruitment of LCK,
which phosphorylates ITAMS on CD3 chains. This binding leads to the activation and
recruitment of ZAP70; after this, the assembly of the signalling complex occurs (the LAT
signalosome) when ZAP70 phosphorylates LAT. This complex activates three main
pathways: Ca2+–calcineurin, MAPK, and NF-κB. These pathways drive T cell proliferation,
migration, cytokine production, and effector functions by the nuclear translocation of
transcription factors (NFAT, NF-κB, AP-1).

15
[Link].3.2 Antigen Recognition

[Link].3.2.1 Antigen processing and presentation

The immune system comprises three main types of professional APCs: dendritic cells (DCs),
macrophages, and B cells [1], [2], [50]. These cell types are principally responsible for the
processing and the presentation of antigenic epitopes: the proteolytic degradation of foreign
proteins into shorter peptides and their subsequent presentation on MHC class I or class II
molecules (Fig 1.5). Antigen presentation is obligatory for the recognition of cognate
antigens by T cells and, thus, for the initiation of an appropriate adaptive immune response.

Antigen processing and presentation rely on two primary cellular pathways, each dedicated
to presenting antigens to distinct T-cell populations. The classical MHC I pathway processes
endogenous and cytosolic antigens, which include self-proteins and foreign proteins such as
viral antigens synthesized within the infected host cells. For this reason, the MHC class I
pathway can be activated by all nucleated cells. Endogenous proteins are broken down into
antigenic peptides by the 26S proteasome, a large proteolytic complex that cleaves proteins
into smaller antigenic peptides suitable for MHC class I binding. These peptides are then
transported from the cytosol into the endoplasmic reticulum (ER) via Transporter associated
with antigen processing 1 and 2 (TAP1 and TAP2). MHC class I molecules bind to the TAP
transporter through the TAP-binding protein, Tapasin. The MHC class I heavy chains
associate with the ER chaperone calnexin. After folding, they dissociate from calnexin, bind
to β-2 microglobulin, ER chaperone calreticulin, and thiol oxidoreductase PDIA3 (Protein
disulfide-isomerase A3). Subsequently, the formation of MHC class I-peptide complex takes
place after the MHC class I molecule bind the antigenic peptide. This complex is presented
to the CD8+ T cells after being transported on the cells surface of APCs [51].

In contrast, the MHC II pathway is responsible for processing exogenous antigens. Unlike
class I, the class II pathway is largely restricted to professional APCs. Exogenous proteins
are taken up by the cell through endocytosis and are then broken down by proteases,

16
including legumain, Cathepsin L, and Cathepsin S, located within endosomes and lysosomes
[52]. IFN-γ inducible lysosomal thiol reductase (IP-30) facilitate the enzymatic cleavage of
disulphide bonds, enabling the degradation of endocytosed antigens in compartments
containing MHC class II [52]. Assembly of MHC class II molecules occurs in the ER with
the assistance of the invariant chain CD74, which transports these complexes to endosomal
compartments. Legumain initiates CD74 cleavage, followed by late-stage cleavage by
cathepsin S and cathepsin L [53]. HLA-DM, a membrane protein in endocytic vesicles,
facilitates the removal of CD74 from MHC class II molecules, including the final fragment
CLIP (class II-associated invariant chain (Ii) peptide). Subsequently, MHC class II molecules
become capable of binding antigens, and the antigen loading preferentially occurs in late
endosomal and lysosomal compartments. The MHC class II-antigen complex is then
transported to the cell surface via vesicular transport mechanisms for presentation to CD4+ T
cells, which recognize these complexes through their T cell receptors [54].

Importantly, the MHC I pathway can also process exogenous antigens through an alternative
pathway known as cross-presentation [55]. Dendritic cells and macrophages are the main cell
types that can present the exogenous antigens via MHC-I. Cross-presentation plays a key role
in how dendritic cells can collect the antigens from malignant and virus-infected cells and
present them to CD8 T cells [56]. The delivery of antigens for a cross-presentation takes
place by phagocytic machinery, presumably because of the large quantity of antigen ingested
and the presence of cross-presenting machinery in phagosomes. To generate the peptides for
cross-presentation by the phagosome to cytosol pathway, antigens are transferred to the
cytosol, hydrolyzed by proteosomes and the resulting peptides are transported to MHC-I
molecules in ER or phagosomes [56].

17
 Figure 1.5 Antigen Presentation [57]

This figure shows the presentation of antigens to CD4+ and CD8+ T cells. CD8+ T cells are
presented by the endogenous antigens once the cell gets infected with viruses. While CD4+ T
cells are presented with exogenous antigens by the APCs, once they engulf any foreign
pathogen, they process it and present it to the CD4+ T cells

[Link].3.2.2 Antigen recognition by T cells

Mature T cells are considered immunologically "naïve" until they encounter their cognate
antigen in secondary lymphoid organs [58]. Naïve T cells express the surface adhesion
molecule CD62L (L-selectin) and the chemokine receptor CCR7, which interact with
glycosylated peripheral node addressins and CCL21(chemokine ligand 21) [59],

18
respectively. These interactions facilitate T-cell migration into lymph nodes. Recognition of
peptide-MHC by the TCR is critical to T cell activation and differentiation. TCR activation
initiates several signalling cascades that ultimately determine cell fate through processes such
as cytokine production, cell survival, proliferation, and differentiation.

[Link].3.3 Co-stimulation

Naïve T cells, in addition to recognizing the MHC-antigen complex by the TCR, require
signals from costimulatory molecules, which can either positively or negatively regulate T-
cell activation. [60]. Co-stimulation, also known as the second signal, is essential for properly
activating T cells. This signal occurs when co-stimulatory molecules on T cells bind to their
corresponding ligands on APCs [61]. Without this co-stimulation, T cells may become
anergic or undergo apoptosis, thereby preventing an effective immune response. Co-
stimulation also plays a critical role in preventing autoimmunity by ensuring that T cells are
fully activated only in the presence of a real threat, such as an infection. Without co-
stimulation, a T cell will not achieve full activation even if the TCR recognizes a self-antigen
[62]. For a robust immune response, co-stimulation is crucial because it facilitates the
proliferation and survival of activated T cells. Additionally, it supports the differentiation of
T cells into effector cells, which directly combat antigens, and memory T cells, which provide
long-lasting immunity. Co-stimulation also boosts cytokine production by T cells, which in
turn recruits other immune cells and helps regulate inflammation [63].

The principal costimulatory molecules are:

CD28: Identified as the first costimulatory receptor, CD28 is consistently expressed on naïve
T cells and acts as a positive regulator for T cell activation. Blocking CD28 or its ligands
(CD80 and CD86 on APCs) during T cell-APC interactions leads to T cell anergy and
unresponsiveness. CD28 is crucial for optimal production of the T cell growth factor IL-2,
induces cytokines like IL-4 and IL-5, and promotes the anti-apoptotic factor Bcl-xL,
ultimately facilitating T-cell expansion and migration into inflammatory sites [64].

19
CTLA-4: Expressed by both CD4+ and CD8+ T cells, CTLA-4 shares homology with CD28.
Despite the similarities, CTLA-4 competes with CD28 for binding on CD80 and CD86 with
higher affinity. This competitive blocking delivers inhibitory signals to the T cell, leading to
a reduction in T cell proliferation, cytokine production and activation markers. One of the
direct outcomes of CTLA-4 signalling is the downregulation of IL-2 and IL-2R expression,
thus blocking T-cell activation [65].

ICOS: Inducible costimulatory is a member of the CD28 subfamily, ICOS lacks the B7
ligand-binding motif present in CD28. Expressed on activated and resting memory T cells,
ICOS binds to the ICOS-L (B7-H2 or CD275) on the APCs and provides co-stimulatory
signals, leading to T cell proliferation, cytokine secretion (IL-4 and IL-10) and upregulation
of early activation markers. ICOS also plays a role in the differentiation of T cells to Th2 and
Th17, thus influencing the production of IL-4 and IL-17 [66]. It also provides essential
signals for the differentiation and proliferation of T follicular helper cells, which play an
important role in the formation of germinal centers and effectively help with antibody
secretion by B cells [67].

OX40: A member of the TNF receptor superfamily, OX40 is expressed on activated T cells.
This costimulatory receptor is integral to the survival, homeostasis and long-term function of
effector and memory T cells. OX40 binds to its ligand (OX40L), expressed on professional
APCs, including dendritic cells, macrophages and B cells and enhances T-cell functions such
as T-cell expansion, cytokine production and survival [68].

The interaction between these costimulatory molecules and their respective ligands
influences the outcome of T-cell activation, determining the nature and strength of the
immune response.

[Link].3.4 Role of cytokines in T cell activation and differentiation

The initiation of proliferation in naïve T cells by the TCR and costimulatory signals is crucial,
but for these cells to develop optimal effector functions, survive effectively, and establish a

20
responsive memory population, the presence of soluble cytokine signals is essential [69].
This cytokine signal, alongside TCR (signal 1) and costimulatory signals (signal 2), is termed
'signal 3' (Fig 1.6). [70]. The nature of this cytokine signal determines the type of effector the
T cell will become. For helper T cells, it directs them into distinct subsets such as T helper 1
(Th1) when exposed to the cytokine IL-12, Th2 with IL-4, or Th17 with a combination of
IL-6, TGFβ, IL-1, IL-21, and IL-23. Each effector subset plays a specific role in the tissue
and contributes to further immune responses [71].

Figure 1.6 Three signals for T cell activation [71]

For T cells to be fully activated and differentiated, three signals are needed: Signal 1 involves
the recognition of a specific peptide antigen presented by MHC class I or II molecules on
APCs. Signal 2 is provided through costimulatory molecules, primarily by membrane-bound
receptors CD80 and CD86. Signal 3 consists of cytokine-mediated polarization signals,
which direct the differentiation of activated T cells towards a particular lineage based on the
cytokine environment.

21
[Link].3.5 T cell subsets

CD4+ T lymphocytes are central to immune function, representing the most abundant
lymphocyte subset in the peripheral blood and serving as primary initiators of immunological
responses. They contribute significantly to both humoral and cell-mediated immune
responses by recruiting other immune cells. Upon antigen exposure presented by APCs, naïve
CD4+ T cells differentiate into distinct effector subsets with specific immunological
functions. This allows them to mount an appropriate immune response against invading
pathogens at the infection site, contributing to host defence [72].

In the peripheral lymphoid microenvironment, CD4+ T cells can differentiate into various
subsets, including Th1, Th2, Th17, regulatory T cells (Treg), T follicular helper cells (Tfh),
and T helper 9 (Th9), depending on the cytokines produced by APCs [73]. This
differentiation is regulated by signal transducer and activator of transcription (STAT)
proteins, which play key roles in defining T cell lineage. Upon stimulation of cytokine
receptors on T cells, activated STAT proteins promote the expression of lineage-specific
transcription factors and control cytokine production by each effector T cell lineage [74]. For
instance, IL-12 drives Th1 differentiation, while IL-4 and IL-2 are crucial for Th2
differentiation. Transforming growth factor (TGF-β) is crucial for Th17 differentiation,
particularly in the presence of IL-6 (with IL-1 and IL-21 also playing supporting roles).
Additionally, TGF-β can promote Treg cell differentiation in an environment rich in IL-2 but
lacking IL-6. The essential transcription factors for Th lineages are T-bet/STAT4 (Th1),
GATA-3/STAT6 (Th2), RORγt/STAT3 (Th17), and Foxp3/STAT5 (Treg) (Fig 1.7) [75].

22
 Figure 1.7 CD4+ T cell subsets [76]

Exposure of naïve CD4+ T cells to antigens in the presence of polarizing cytokines leads to
the activation and differentiation of naïve CD4+ T cells into various T cell lineages. This
figure shows the regulation of the Th cell fate by the given differentiation cytokines,
transcriptional factors, and functional cytokine effects.

Each CD4+ T cell subset releases a unique profile of cytokines that mediate either pro- or
anti-inflammatory effects. Th1 cells primarily produce IL-2 and IFN-γ, Th2 cells secrete IL-
4, IL-5, and IL-13, Th17 cells secrete IL-17A and IL-17F, Tfh cells produce IL-21, which
drives B cell proliferation, and Th9 cells are defined by their expression of IL-9 [77]. While
much of our understanding of these T cell subsets comes from in vitro differentiation studies,
it is important to note that, in vivo, these subsets demonstrate considerable plasticity. Th17
cells, in particular, are highly adaptable and may express non-canonical cytokines, such as
IFN-γ, depending on the immunological context, thus revealing a more dynamic range of
functions [77].

CD8+ T cells, which play an important role in fighting against pathogens by removing the
infected cells and also in killing tumor cells, have also been shown to be differentiated into

23
IFN-γ-producing Tc1, IL-4-producing Tc2, IL-9-producing Tc9, IL-17-producing Tc17 and
IL-22 producing Tc22 cells. One important characteristic of Tc17 cells is that they express
both IFN-γ and IL-17. Tc17 do not rely on granzyme B and perforin for their effector function
in contrast to Tc1 cells [78]. Tc1 cells produce IFN-γ, TNF, IL-2, perforin and granzyme B,
while Tc17 cells produce IFN-γ, TNF and IL-17. The other Tc subsets that have been reported
and play a role in different diseases are Tc2, Tc9, Tc22, Tfcs and regulatory CD8+Foxp3+
Tregs [79]. In in vitro conditions, CD8+ T cells can also be activated and differentiated into
different subsets by using similar conditions that are used for CD4+ T cell activation and
differentiation (Fig 1.8) [80].

Figure 1.8 CD8+ T cell subsets [81]

Upon initial exposure to antigen in the presence of polarizing cytokines, naïve CD8+T cells
are activated and differentiate into various T cell lineages. The Tc cell fate and function are
regulated by the differentiation cytokines, transcriptional factors, and functional cytokine
effects, as shown in this figure.

24
[Link] B lymphocytes

The primary function of B cells is to produce antibodies, though it has become appreciated
that they can also act as APCs to T cells. Antibody molecules, also known as
immunoglobulins (Ig), exist in two forms: 1) the membrane-bound glycosylated protein
molecule present on the surface of the B cells known as the BCR that serves as an antigenic
receptor, allowing the B cells to recognize and bind to the antigens; 2) soluble antibodies that
are secreted into the extracellular space, where they can bind to the pathogens, block their
harmful effects and prevent them from infecting the host cells. Soluble antibodies can also
act as opsonins by binding to the pathogens and marking them for recognition and engulfment
by phagocytic cells. Another function of the soluble antibodies is to activate the complement
system. Antibodies exist in five isotypic forms (IgM, IgD, IgA, IgG and IgE) and consist of
two heavy and two light chains (Fig 1.9) [82].

IgM: It is the primary antibody produced by developing B cells, characterized by five

monomeric units connected by a joining (J) chain. Each monomeric unit consists of two
heavy (µ chains) and two light chains (κ or λ). IgM is important in the early stages of immune
response and is primarily found in the blood and lymph [83]. IgM is effective in activating
the complement system, which helps to remove pathogens from the body. In response to
infection, IgM is the first antibody produced by B cells, which makes it an important
component of primary immune response before the other classes of antibodies like IgG are
produced [82].

IgD: Present on the surface of mature naïve B cells, IgD comprises two heavy (delta) chains
and two light chains (κ or λ). IgD antibodies are typically present in monomeric forms, and
their exact function is not fully understood, but they are believed to play a role in the
activation of B cells. IgD antibodies are secreted only in small amounts, and they act mainly
as cell surface receptors for antigens [84].

IgG: The predominant class of antibody present in the blood is IgG, which is a four-chain
monomer predominantly produced during secondary immune responses. In addition to its
role in complement activation, the Fc (fragment crystallizable) region of an IgG molecule

25
binds to specific receptors located on macrophages and neutrophils. Through these Fc
receptors, these phagocytic cells can bind to, ingest, and destroy microorganisms that have
been opsonized with IgG antibodies generated in response to an infection [85].

Notably, IgG molecules are unique among antibodies in their ability to cross from the mother
to the fetus via the placenta. Placental cells in direct contact with maternal blood possess Fc
receptors that specifically bind to blood-borne IgG molecules, facilitating their transfer to the
fetus. This transfer occurs through receptor-mediated endocytosis, followed by transcytosis,
whereby the antibody molecules are internalized into placental cells, transported across these
cells within vesicles, and subsequently released into the fetal circulation via exocytosis. This
selective transport mechanism is exclusive to IgG, as other antibody classes do not interact
with these specific Fc receptors and, therefore, cannot cross the placental barrier.
Furthermore, IgG is secreted into maternal milk and can be absorbed from the neonate's
gastrointestinal tract into the bloodstream, thereby conferring passive immunity to the infant
and offering protection against infections [86].

IgA: Immunoglobulin A is the principal antibody class found in secretions such as saliva,
tears, milk, and respiratory and intestinal fluids. In the blood, IgA exists as a four-chain
monomer, but in secretions, it forms an eight-chain dimer. This dimeric form is transported
through secretory epithelial cells from the extracellular fluid into the secreted fluid via a
specialized Fc receptor that is unique to secretory epithelia. This Fc receptor can also
transport IgM into secretions, albeit less efficiently. Consequently, individuals with selective
IgA deficiency, the most common form of antibody deficiency, tend to experience only mild
symptoms, likely due to the compensatory role of IgM in secretions [1], [87].

IgE: Immunoglobulin E is crucial in the body's immune response, particularly in allergic

reactions and defence against parasitic infections. Structurally, IgE is a four-chain monomer,
like other immunoglobulins, but it is distinguished by its specific functions and interactions
with immune cells. The tail region of the IgE antibody binds with exceptionally high affinity
to a distinct class of Fc receptors. These receptors are located on the surface of mast cells in
tissues and basophils in the blood [88]. The IgE molecules bound to these receptors function
as passively acquired receptors for antigens. When an antigen binds to the IgE, it triggers the

26
mast cell or basophil to release various cytokines and biologically active amines, particularly
histamine [1]. These substances cause blood vessels to dilate and become permeable,
facilitating the entry of white blood cells, antibodies, and complement components into sites
of infection. IgE antibodies are also primarily responsible for the symptoms of allergic
reactions such as hay fever, asthma, and hives. Additionally, mast cells release factors that
attract and activate eosinophils. Eosinophils also have Fc receptors that bind IgE molecules
and can kill various types of parasites, especially those coated with IgE antibodies [89].

Figure 1.9 Five classes of B cell immunoglobulins.

([Link]
[Link])

The five main classes of antibodies, and their structure and function are shown in the figure.

27
1.2 AUTOIMMUNITY AND NEURO AUTOIMMUNITY

Autoimmunity arises from the breakdown of immune tolerance, whereby the immune system
mistakenly recognizes the body’s own tissues as foreign. This misguided immune response
triggers chronic inflammation. Autoimmune disorders affect approximately 3-5% of
populations worldwide, putting a substantial burden on global health [90].

The adaptive immune system has the remarkable capacity to generate antigen-specific
receptors that can detect and neutralize foreign pathogens. Inevitably some receptors
recognize components of our own body, which can lead to the development of autoimmunity
[2]. Autoimmunity arises through a complex interplay of maladaptive molecular and cellular
mechanisms, often involving a genetic predisposition. This predisposition is typically
determined by specific alleles that regulate antigen presentation by antigen-presenting cells,
which is crucial for T-cell recognition. In some cases, an autoimmune response may be
triggered following an infection, where a pathogen's proteins share structural similarities with
the host's proteins. This molecular mimicry can result in the production of antibodies that,
while targeting the pathogen, also cross-react with self-proteins, leading to the formation of
autoantibodies. These autoantibodies can perpetuate the autoimmune response by
continuously stimulating the immune system against the autoantigen [91]. Loss of self-
tolerance plays an important role in developing autoimmunity, and some common
mechanisms that contribute to it include impaired deletion or heightened activation of
autoreactive CD4+ T-helper (Th) lymphocytes. This process is often compounded by
defective immunomodulation, particularly by CD4+ regulatory (Treg) and CD8+CD28-

28
suppressor T lymphocytes (Ts), which fail to exert adequate control over immune responses.
Additionally, dysregulated signalling pathways can shift the balance toward pro-
inflammatory cytokine production, further promoting autoimmunity [92].

To avoid the development of autoimmunity, the immune system has a mechanism of negative
selection to remove lymphocytes bearing self-antigen-specific receptors. Nevertheless, self-
reactive lymphocytes can evade negative selection through several mechanisms, which can
be divided into thymic and peripheral.

1.2.1 Thymic/Central Mechanisms

Incomplete thymic deletion of self-reactive cells: Negative selection is primarily mediated in

the thymus, where tissue-specific antigens are presented to thymocytes by the thymic
epithelial cells or DCs [93]. Defects in the presentation of tissue-specific antigens can lead
to the evasion of some self-reactive thymocytes, which can lead to autoimmunity. For
example, mutations in the AIRE gene can lead to the development of APECED (Autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy), a syndrome characterized by
widespread autoimmunity due to defective negative selection [94].

Low affinity for self-antigens: Some T cells bear TCR with low affinity for self-antigens in
the thymus, which may avoid their deletion during negative selection [95]. These T cells can
survive and enter the peripheral repertoire because their affinity for the self-antigens may not
cross the threshold necessary to trigger apoptosis. While in the periphery, encountering
similar peptides or inflammatory signals may occur at higher levels, which may reduce their
activation threshold, leading to their activation and participation in autoimmunity [95].

1.2.2 Peripheral Mechanisms

Impaired function of regulatory T cells: Some thymocytes with low affinity for self antigens
are directed into the regulatory T cell development pathway [96]. However, these Tregs may
fail to control self-reactive T cells in the periphery if they are defective in function, like
mutations in the FOXP3 gene that are linked with an autoimmune condition where the
regulatory function of Tregs is severely impaired, IPEX syndrome (immune dysregulation,

29
polyendocrinopathy, enteropathy, and X-linked syndrome) [97]. CD25 and CTLA4 also play
a key role in the proper functioning of Tregs; mutations in these molecules can also lead to
autoimmunity [98]. Depending on the inflammatory milieu, Tregs have been reported to
produce IL-17 and lose the Foxp3 expression; these Tregs can induce severe damage to joints
and have been found within the inflamed joints of rheumatoid arthritis patients [99].

Impaired expression of inhibitory receptors: Peripheral tolerance plays an important role in

restraining autoimmunity: a study has shown that around 30% of CD4+Foxp3- T cells react
to self-peptides in a healthy mouse, but their autoreactivity is constrained by continuous
suppression of Tregs and the expression of inhibitory receptors [100]. Peripheral tolerance is
mediated by inhibitory receptors by antagonizing TCR signalling and resulting in a
dysfunctional state [101]. T-cell function is also inhibited by the upregulation of inhibitory
receptors under chronic disease conditions [101]. In chronic viral infections, which may also
lead to autoimmune disease, the state of T cell exhaustion has also been defined by the
increased and continuous expression of inhibitory receptors like PD-1, Lag-3, 2B4, Tim-3
and CTLA4 [102]. In chronic viral infection, tumour and autoimmunity mouse models, the
loss of one inhibitory receptor PD-1 has been associated with a compensatory increase of the
other receptor Tim-3 [103], [104]. In this thesis, we also observed that Serpine1 represses
IFN-γ expression in a Tim-3-dependent manner and the expression of the co-inhibitory
molecules (Lag3, PD-1) was also reduced in Serpine1-/- Tim3-Tg Th1 cells [105].

This thesis focuses on autoimmune neurodegenerative diseases and, in particular multiple

sclerosis. In these disorders, the immune system of our body mounts an unwanted immune
response against the components of the CNS, contributing to the loss of neural tissue,
including neurons, myelin and other important cells that form the brain and spinal cord [106].
This category of disorders includes several conditions such as multiple sclerosis, transverse
myelitis, optic neuritis, neuromyelitis optica and acute disseminated encephalomyelitis, each
of which is distinguished by different pathophysiological mechanisms, clinical
manifestations and affected CNS regions [107].

1.3 MULTIPLE SCLEROSIS

30
Multiple sclerosis is a chronic autoimmune disorder of the CNS characterized by the
infiltration of immune cells, demyelination, and axonal damage. Unfortunately, as of now,
there is no cure for MS. The clinical spectrum of MS is broad, reflecting the diverse impact
of CNS damage on neurological function: vision impairment, loss of coordination and
balance, fatigue, pain, bladder dysfunction, cognitive impairment, numbness, weakness, and
mood changes are all observed (Fig 1.10) [108]. The etiology of MS is multifactorial,
involving a complex interplay between genetic susceptibility and environmental factors.
Traditionally, MS has been primarily considered as a T-cell-mediated disease, a theoretical
framework drawn from observations in the experimental autoimmune encephalomyelitis
(EAE) animal model. EAE has served as a valuable tool for understanding the immunological
aspects of MS, especially the involvement of T cells in CNS pathology [109]. As knowledge
about both MS and the mechanism of immune responses governing autoimmunity has
advanced, however, there has been a shift towards understanding the pathogenesis of MS to
include other cell types such as B cells, innate immune cells, and endothelial cells as players
and even drivers of the immune dysregulation in MS.

31
 Figure 1.10 Symptoms of Multiple Sclerosis
([Link]

1.3.1 Prevalence and pathogenesis

MS affects approximately 2.5 million individuals worldwide. The onset of MS typically

occurs between the ages of 20 and 40. About 80% of MS-affected individuals first show a
form of the disease characterized by a sequence of attacks followed by clinical remission
(referred to as relapse-onset or relapsing-remitting, RR). While estimates vary, 30-60% of
individuals with RR-MS will transition to a chronically worsening secondary progressive
(SP) form, characterized by a slow yet unrelenting accumulation of disability. A further 20%
of individuals present a disease that is progressive from its outset [110]. There are >10
disease-modifying therapies (DMTs) for RRMS that reduce the frequency and severity of
relapses. However, progressive forms of MS remain challenging to treat, and thus, research
into the pathophysiology of MS is still a high priority. Notably, sex-based differences in MS
incidence and severity are linked to these different clinical courses. Women are three times

32
more susceptible to developing RR-MS; however, men are proportionally more likely to
develop progressive forms and exhibit a faster trajectory of disability accumulation [111].

1.4 SUBTYPES OF MULTIPLE SCLEROSIS

1.4.1 Relapsing-remitting multiple sclerosis

MS manifests in diverse clinical patterns. The most prevalent subtype is relapsing-remitting

MS (RRMS), affecting approximately 85% of patients [112]. RRMS follows a recurrent
acute inflammatory attack, leading to relapses marked by a worsening of symptoms. These
relapses can occur weeks, months or even years apart, with patients experiencing 1-2 relapses
annually on average (Fig 1.11). Relapses are unpredictable and do not follow a regular
pattern; the frequency of relapses also depends on the treatment, environmental triggers and
overall disease progression [113] Relapses are followed by complete or partial recovery,
termed "remissions," which is the period when the disease is not active, and patients will
experience stable neurological functions [114]. Like relapses, the length of remission can
also vary between patients, and it can also vary between different relapses of the same
individual. The remission period can last for several weeks to months or even years,
depending on the effectiveness of the treatment, the overall health of a patient and disease
progression [115], [116]. Onset typically transpires between 20 and 40 years, with a female
prevalence of approximately 2:1 [117]. Clinical manifestations of RRMS include sensory
disturbances, fatigue, unilateral optic neuritis, diplopia, limb weakness, gait ataxia, and
paraesthesia, with bladder dysfunction occurring as the disease advances. In advanced stages,
cognitive impairments such as memory loss and impaired attention may manifest [118]. The
first line of treatment for acute relapses is corticosteroid therapy using high doses of
methylprednisolone (MP) [119]. For those patients with steroid resistance exacerbations
(worsening EDSS; expanded disability status scale) , plasmapheresis is the second line of
treatment [120]If the patients are showing minimal response after five plasmapheresis
exchanges, then a third line therapy is started, which includes cytotoxic therapy with
myeloablative agents such as cyclophosphamide [121], an initial high loading dose of

33
rituximab [122] or a single dose of natalizumab to stop the crossing of the blood-brain barrier
by lymphocytes [123]

1.4.2 Secondary Progressive Multiple Sclerosis

With each remission, the accumulation of axonal damage diminishes the degree of recovery,
resulting in increased neurological disability and disease progression [124]. Between 30-60%
of people with RRMS will convert to secondary progressive MS (SPMS), which is
characterized by the steady accumulation of disability, independent of clinically-apparent
relapses [125]. The transition of RRMS to SPMS has been linked to cortical demyelination
and diffuse white matter injury; these features increase with disease duration and become
more prominent in patients with SPMS [126]. Although brain and spinal cord inflammation
are present in both RRMS and SPMS, it appears that compartmentalized immune reactions
within the CNS are more relevant to SPMS pathology than peripheral immune reactions
[127]. Intriguingly, cortical lesions in SPMS are characterized by dominant microglial
populations and the absence of macrophage and leukocyte inflammatory infiltrates [128].

The persistent inflammation in SPMS patients also drives the formation of meningeal
lymphoid aggregates. These are organized structures in the meninges that contain distinct
zones for B and T cells, similar to the secondary lymphoid organs like lymph nodes.
Follicular dendritic cells and high endothelial venules that are important for lymphocyte
recruitment and retention are also present inside these structures. They are also thought to
exacerbate neuronal damage by the local production of autoantibodies and proinflammatory
cytokines [129].

While we cannot currently predict which RRMS-affected individuals will transition to

SPMS, several risk factors are associated with this possibility. These include older age at the
onset of MS, male sex, a history of high relapse frequency in the early stages, prolonged
disease duration, elevated baseline EDSS score, significant early increases in the EDSS score,
heightened T2 lesion burden in MRI scan, the involvement of the spinal cord, and reduced
brain volume [130]. Studies on the neurofilament light chain (NfL) have shown it to be a
promising reliable biomarker for MS progression, and the levels of NfL in blood correlate

34
with the progression of neurodegeneration [131]. Patients with RRMS who progressed in
EDSS after 5 years had been reported to have significantly higher CSF NfL levels than those
who did not. Moreover, a correlation between high serum NfL levels and volume loss in the
brain and spinal cord has been found in two other studies [132], [133]. Other than NfL, serum
GFAP (glial fibrillary acidic protein) levels have also been reported to have a positive
correlation with the disease severity and MRI lesion count in progressive MS patients [134].

1.4.3 Primary Progressive Multiple Sclerosis

Primary progressive MS (PPMS), accounting for approximately 10% of cases, differs from
other MS types by displaying a progressive onset without relapses (Fig 1.11). This course of
disease lacks a sex-based incidence difference and predominantly affects the spinal cord,
occasionally involving the optic nerve, cerebrum, or cerebellum. PPMS frequently manifests
as an upper motor neuron syndrome affecting the legs, progressively worsening over time
and leading to a range of complications, including cognitive decline, quadriparesis, vision
impairment, and dysfunctions in bladder and sexual function. [135]. A minority of patients,
around 5%, experience steadily worsening disease with a progressive onset and relapses,
termed progressive-relapsing MS [136].

35
 Figure 1.11 Types of Multiple Sclerosis
([Link]

This graph shows the progression of the disease with time in different types of MS. Relapsing
Remmiting MS episodes of acute relapses followed by partial or complete recovery,
Secondary progressive MS worsens slowly and steadily with few or no relapses in RRMS
patients, Primary progressive MS steady worsening from the onset without relapses

1.5 RISK FACTORS IN MS

The etiology of MS is complex. The literature indicates that both genetic and environmental
factors contribute to disease onset. Below, I outline the known triggers and risk factors
associated with the development of MS.

36
1.5.1 Genetic Factors

Familial history and ethnicity-related analyses have uncovered important observations

regarding the genetic underpinnings of MS [137]. While the general population has a roughly
1 in 1,000 chance of developing MS, this risk increases significantly to approximately 1 in 4
for identical twins when one twin is affected. A pivotal genetic factor in MS susceptibility is
the human leukocyte antigen (HLA)/MHC gene cluster on chromosome 6, accounting for an
estimated 10%-60% of the genetic risk. The HLA-DRB1*15:01 allele, part of the MHC class
II gene cluster, is the most significant genetic risk factor identified for MS. This allele is
associated with approximately a threefold increase in MS susceptibility compared to
individuals without this variant. This allele contributes substantially to genetic predisposition
for MS, particularly among Caucasian populations, where it is most prevalent
[138].Extensive genome-wide association studies (GWAS) have significantly advanced our
understanding of the genetic landscape of MS identifying over 200 genetic variants that
contribute to MS risk. These variants include genes involved in immune regulation, such as
the IL-7 receptor (IL7RA), IL-2 receptor (IL2RA), tumour necrosis factor receptor
superfamily member 1A (TNFRSF1A), C-type lectin-domain family 16 member A
(CLEC16A), interferon regulatory factor 8 (IRF8), CD58, STAT3, and CD6 [139], [140].
Notably, variations within the HLA gene complex, particularly within the MHC class II
region, are considered to have the strongest influence on MS risk, as they are critical in
shaping the T cell repertoire that recognizes self and non-self-antigens. By contrast, non-
HLA genetic variants tend to influence broader immune functions. They modulate immune
response sensitivity and contribute to the regulation of immune cell activation. This
functional diversity among MS-related variants underscores a complex interplay between
genetic factors that govern T cell functions and other immune pathways, ultimately shaping
the likelihood of an autoimmune response within the CNS [141], [142]

1.5.2 Biological sex

37
MS exhibits a notable impact of biological sex on its prevalence, development, and
progression. The condition disproportionately affects women, occurring three times more
frequently than in men. Intriguingly, despite the higher incidence in women, men often
experience a more severe form of MS with a poorer prognosis, reaching severe disability at
a faster rate [143]. Female (estrogen, progesterone) and male (androgen) sex hormones play
pivotal roles in these sex differences [144]. For instance, the risk of MS increases after female
puberty, while during pregnancy, disease severity tends to decrease. Estrogen, particularly
17-β estradiol (E2), has complex immunomodulatory effects, exhibiting both pro-
inflammatory and anti-inflammatory actions by altering cytokine production levels [145].
Progesterone, another sex hormone, plays a primarily anti-inflammatory role, promoting the
differentiation of Treg cells, which helps modulate immune responses. Meanwhile,
androgens like testosterone are thought to offer protective effects against autoimmune
diseases, possibly contributing to the lower rates of multiple sclerosis observed in men. These
hormonal influences underscore the interplay between sex hormones and immune regulation,
potentially affecting susceptibility to autoimmune conditions such as MS[146]

The interplay of sex hormones in MS is not solely responsible for sex differences; sex
chromosomes may also play a crucial role. The Four Core Genotype (FCG) model, which
involves deleting the Sex-determining region Y (Sry) gene from the Y chromosome, has
emerged as a significant tool for studying the role of sex chromosome complement (XX vs.
XY) in sex differences [147] In recent investigations by our research group, we unveiled
intriguing insights into the pathogenicity of Th17 cells in the context of MS. Utilizing the
1C6 mouse model, a TCR transgenic model that bears anti MOG35-55 peptide specific CD4+
and CD8+ T cells, we observed that Th17 cells derived from male 1C6 mice, upon adoptive
transfer into [Link] mice, induced a more severe form of the disease compared to
recipients of female Th17 cells, regardless of the gender of the recipient [148]. This insight
underscores the multifaceted nature of sex differences in MS, involving both hormonal and
chromosomal influences in disease pathogenesis and prevalence.

1.5.3 Environmental Factors

38
When people move from areas with low MS risk to those with high risk, or vice versa, the
prevalence of MS is higher than expected, highlighting the significance of environmental
factors in MS development [149]. Consequently, numerous environmental agents have been
examined as potential contributors to MS onset.

Geographically, the prevalence of MS follows a latitude gradient, with lower incidence near
the equator and increasing prevalence with greater distance from the equator in both
hemispheres [150]. High-prevalence areas, including Canada, northern United States,
northern Europe, New Zealand, and southeastern Australia, are situated at extreme northern
or southern latitudes [151]. Reports evaluating the incidence of MS cases at the provincial
level within Canada further support the nation's status as a high-prevalence area [152]. This
geographical variation underscores the importance of considering environmental factors in
addition to genetic and hormonal influences when exploring the pathogenesis of MS.

[Link] Vitamin D

Higher vitamin D levels appear to provide protection against MS [153]. As sunlight exposure
is the principal environmental factor for vitamin D, this is thought to explain why MS is more
common in countries farther from the equator. In one study, a reduction in the relapse rate
was observed by treating MS-affected individuals vitamin D supplements concomitant with
MS therapy [154]. In another cohort study supplemental vitamin D levels along with the
treatment of IFN-β and fingolimod (oral sphingosine-1-phosphate receptor modulator) were
associated with 30-50% reduction in relapses or new inflammatory lesions on MRI [155].

[Link] Smoking

Smoking is an important aggravating factor for MS incidence. Among individuals affected

with RR-MS who had quit smoking, each smoke-free year was associated with a lower risk
of attaining an EDSS of 6.0 [156] Smoking have also been found to correlate with known
HLA-risk genes [157]. For instance, smokers with specific HLA-risk alleles, such as the
HLA-DRB1*15 allele, are at a markedly higher risk of developing MS compared to non-
smokers with the same genetic predisposition. This connection is thought to involve immune-

39
mediated mechanisms, potentially leading to increased proinflammatory responses [157].
Smoking has been shown to activate cytochrome P450 (CYP1A2), because CYP enzymes
generate reactive oxygen species (ROS) and ROS-mediated damages can alter the peptides
presented to T cells [158]. This modification could increase the likelihood of T cells
recognizing these altered peptides, which may have evaded clonal deletion processes in the
thymus, thereby rendering them more prone to autoreactivity.

1.5.6 Viruses

The epidemiology of MS suggests that Epstein Bar virus (EBV) and Human Herpesvirus
6(HHV-6) may influence the clinical course and progression of the disease, with EBV being
strongly associated with MS pathogenesis [159]. Indeed, several other human demyelinating
diseases appear to have viral triggers: these include progressive multifocal
leukoencephalopathy triggered by the John Cunningham virus (JCV), post-infectious
encephalitis, and subacute sclerosing panencephalitis caused by measles virus, and HIV-
induced encephalopathy [160]. However while viruses such as measles, rubella, mumps, and
herpes viruses (HSV-1 and 2, varicella-zoster virus, HHV-6), have been posited as potential
contributors to MS, a definite casual relationship with these pathogens have not been
confirmed [161].

The EBV has emerged as the most compelling viral link to MS, largely due to its lifelong
infection of B cells, which is observed in nearly all individuals with MS [162]. This
connection is particularly evident in pediatric MS, where nearly every child diagnosed with
MS has undergone EBV seroconversion, a rate significantly higher than in children without
MS [163]. Furthermore, another study has reported an unusual accumulation of EBV-
infected B cells in the brains of individuals with MS [164]. A longitudinal study was
conducted using a cohort of 10 million active-duty US personnel to investigate the potential
causality between EBV infection and MS. This population was followed over 20 years, and
among the 955 documented MS incidents, 801 were assessed with EBV infection. Elevated
serum antibody titers against EBV nuclear antigens and the presence of EBV in some MS
demyelinating lesions was also found in MS patients [165]. These findings collectively

40
highlight EBV as a critical factor for MS etiology, which warrants further research on it’s
role in MS pathogenesis.

1.5.7 Microbiome

Recently, the microbiome has garnered increasing attention as a potential influencer of MS

susceptibility [166]. Our gastrointestinal tract hosts trillions of microbes capable of
modulating the immune system, regulating both innate and adaptive immune responses to
maintain homeostasis [167]. As a result, dysbiosis appears to be associated with a range of
conditions, including MS [168]. A study on MS patients has shown that two bacterial groups
(Acinetobacter and Akkermansia) were four times more common in people with MS
compared to controls. Also, mice that were transferred with some of the gut bacteria from
MS patients developed severe brain swelling, but when the gut bacteria were transferred from
healthy individuals to the mice with MS-like disease, milder symptoms were observed [169].
In another study, 34 sets of identical twins were recruited, where only one twin has MS, and
their gut bacterial profile was examined. It was confirmed that the twin with MS had
significantly higher levels of Akkermansia group of bacteria. The gut bacteria of each group
of twins were transferred to mice with MS-like disease, the mice who received gut bacteria
from the twin with MS developed brain swelling three times more often than mice who
received bacteria from the non-MS twin. It was also discovered that gut bacteria from the
twin with MS seemed to inhibit the production of anti-swelling molecules [170].
Nonetheless, the precise functional role of the microbiome in MS remains to be fully
understood. Research has yet to conclusively determine whether the identified dysbiosis is a
contributing factor to the disease or merely a secondary effect of the pathological processes
involved in MS.

41
1.6 MECHANISMS OF BLOOD BRAIN BARRIER DISRUPTION IN MS

Early studies showed that transplantation of tissue into the brain parenchyma was less likely
to cause immunologic rejection than transplantation in other anatomical sites; this led to the
concept of the CNS being immune-privileged [171]. To this day, the CNS has been
considered as an "immune-privileged" area in the body, with features exhibiting low levels
of MHC molecules [172]. The immune privilege of the CNS is largely attributed to its unique
anatomical features that protect it from cellular infiltration and regulate substance transport
between the blood and the brain [173]. Specialized barriers, including the blood-brain barrier
(BBB), the blood-cerebrospinal fluid (CSF) barrier, and the blood-meningeal barrier,
separate the CNS parenchyma from the circulation [174]. The BBB is composed of tight
junctions between endothelial cells, a specialized basement membrane, astrocyte end-feet,
and microglia. The blood-CSF barrier, formed by tight junctions between epithelial cells,
surrounds the choroid plexus and meningeal venules [174]. It is now recognized that the
immune privilege of the CNS is not absolute; recent studies have demonstrated that activated
and memory T cells, under certain circumstances, can express the necessary adhesion
molecules and chemokine receptors to cross the BBB and conduct immune surveillance in
the CNS [175]. The blood brain and the blood-CSF barriers play an important role in
regulating T cell trafficking into the CNS, with the blood-CSF barrier emerging as a key site
for the entry of T cells by expressing adhesion molecules and selectins [176].

T cells making their way across the blood-CSF barrier enter the subarachnoid space (SAS)
that lies between the pial and arachnoid membranes [176]. The SAS serves as the initial point
of entry for CD4+ T cells into the CNS, where selectins and adhesion molecules are
constitutively expressed during EAE, the mouse model mirroring immune aspects of MS
[177], [178]. While within the SAS, T cells undergo restimulation by local antigen-presenting
cells (APCs), leading to the proliferation and activation of endothelial cells. The endothelial
cells express CCL20, which is the ligand for CCR6 expressed by a subset of pathogenic T
cells, leading to the activation and recruitment of other immune cells, contributing to
inflammatory damage in the SAS. This damage might activate distant microglial cells,
promoting the upregulation of adhesion molecules on parenchymal vessels and facilitating
the recruitment of additional immune cells through the BBB [179], [180]. Activated T cells,

42
traversing BBB endothelial cells, must break down the subendothelial basal lamina
containing type IV collagen to reach the brain parenchyma. Matrix metalloproteinases
(MMPs), particularly MMP-2 and MMP-9, are crucial in this process, as they specifically
target and degrade type IV collagen. Elevated levels of these MMPs have been found in the
perivascular regions of MS lesions and the cerebrospinal fluid of individuals with MS [181],
[182].

A crucial molecule controlling the transmigration of CD4+ T cells into the CNS is the integrin
very late antigen-4 (VLA-4; α4β1; CD49d/CD29). In acute MS lesions, VLA-4 has been
identified in perivascular T cells [183]. Upon ligation of VLA-4 to vascular cell adhesion
molecule (VCAM)-1, the firm binding of T cells to the inflamed brain endothelium is
facilitated in RR-MS patients [184]. The interaction between VLA-4 and VCAM-1 triggers
the Rac-1 signalling pathway, which drives immune cell transmigration across the
endothelium by destabilizing endothelial junctions [185]. Astrocytes and pericytes have also
been shown to express VCAM-1, which enables their interaction with VLA-4+ T cells, thus
explaining the role played by BBB in regulating T-cell transmigration [186]. Once the blood-
CSF and blood-brain barriers are compromised, immune cells, including T cells, B cells,
macrophages, antigen-presenting cells (APCs), and mast cells, can diffuse into the white
matter of the CNS. These cells recognize and target components of the myelin sheath, leading
to the demyelination and axonal injury characteristic of MS lesions. This immune-mediated
assault on myelin disrupts signal transmission along nerves, contributing to the progressive
neurological deficits observed in MS patients [187].

1.7 EAE AS AN ANIMAL MODEL FOR MS

MS is a complex disease with a diverse clinical presentation, making it challenging to

replicate its pathogenesis using a single in vivo model. Consequently, various models have
been developed to simulate specific stages of the disease. EAE, primarily studied in rodents,
serves as an animal model that reproduces key clinical and pathological features of MS,
including inflammation, CNS damage, neuronal loss, and axonal damage [188]. Notably,

43
EAE is initiated by an autoimmune attack on CNS tissues, typically involving antigens
derived from myelin, and has been instrumental in advancing our understanding of MS
pathogenesis and identifying therapeutic targets. The preclinical efficacy of therapeutic
targets that this model is used for includes; demyelination, remyelination, neuroprotection,
neuroinflammation, peripheral inflammation and autoimmunity [189]. EAE encompasses
various animal models that mirror different MS phenotypes, highlighting the distinct roles of
various lymphocyte classes [190]. Despite their differences, these models share a
commonality in initiating disease processes through an autoimmune response against CNS-
derived antigens. The primary approaches taken to induce EAE are described below:

1.7.1 Active Immunization

Active immunization is the process of activating the host immune system against a foreign
antigen by injecting the actual peptide antigen inside the body of the host. In susceptible
mouse genetic backgrounds, EAE can be induced by subcutaneous immunization with
myelin-derived antigens such as myelin oligodendrocyte glycoprotein (MOG35-55, MOG92-
106,), myelin basic protein (MBP 84-104), and proteolipid protein (PLP 139-151 or PLP178-191).
The peptides are administered subcutaneously in an emulsion combined with complete
Freund's adjuvant (CFA), which is a blend of Mycobacterium tuberculosis extract and
mineral oil to enhance the immune response [191]. This is followed by an intraperitoneal
(i.p.) injection of pertussis toxin (PTX), which is believed to facilitate disease onset by
disrupting the integrity of the blood-brain barrier, allowing immune cells to access the CNS
[192]. Although these steps primarily induce a T cell response, the coordinated activity of
various immune cells ultimately results in the demyelination, mirroring mechanisms seen in
neuroinflammatory conditions. Disease onset typically starts from the 2nd week after
immunization, with symptoms presenting as ascending paralysis, beginning at the tail and
progressing to the hind and upper limbs[193] .

1.7.2 T cell adoptive transfer

EAE can also be triggered by transferring encephalitogenic T cells from an immunized

animal to a naïve one of a genetically similar background [192]. In this approach, T cells are

44
extracted from the spleens and lymph nodes of immunized mice before symptoms emerge.
These cells are then stimulated outside the body (ex vivo) with the specific antigen of interest,
often in the presence of recombinant T cell growth factors that promote the development of
effector T cells. Afterward, these primed T cells are injected into naïve animals. These T
cells, primed with CNS antigens, express integrins on their surface that facilitate their passage
across the BBB [194]. Once inside the CNS, they are reactivated by resident APCs, leading
to the expression of pro-inflammatory cytokines like IFN-γ, IL-17, TNF, and GM-CSF. Once
these cytokines are released, they induce the expression of various chemokines which attracts
the other immune cells, such as B cells, macrophages, monocytes, and neutrophils to the CNS
[195], [196].

EAE can also be achieved by the adoptive transfer of transgenic T or B cells with specificity
for myelin into an immunocompromised host. In this method of disease induction, the T cells
of the donor mice express a myelin-specific transgenic T-cell receptor that evades negative
selection mechanisms and promotes an immune response against myelin peptides. Since the
recipient mice lack T cells or B cells, this method enables the study of the individual or
combined roles of encephalitogenic T and B cells [197]. Our lab employs two crucial T cell
receptor transgenic strains, namely 2D2 (T cells from these mice express a MOG specific
TCR) [198] and 1C6 (CD4+ and CD8+ T cells from thismice express a MOG35-55 specific
TCR) [199].

TCR transgenic strains offer a significant advantage by enabling EAE induction without the
need for active immunization with peptide and adjuvant, it also enables a more precise
investigation of the role played by T cells in disease pathogenesis. Naïve T cells purified
from these mice can be cultured in vitro typically for 5 days under T cell culturing conditions,
in the presence of cytokines and blocking antibodies. This culture setup facilitates the
differentiation of naïve T cells into various T cell subsets, such as Th1, Th2, and Th17, each
with distinct immunological profiles [199]. By using this method, researchers can assess the
pathogenic capabilities of specific T cell subsets, such as Th1, Th17, and Th9, and their
unique roles in mediating inflammation and tissue damage within the CNS [200].

45
The 1C6 mouse model was generated by isolating a CD4+ T cell clone from a NOD
background mouse immunized with MOG35-55. 1C6 TCR is composed of Vβ7 and Vα5, and
both CD4+ and CD8+ T cells from the 1C6 progeny express Vβ7. 1C6 CD8+ T cells also
express rearranged Vα5; thus, they are specific for MOG35-55 like 1C6 CD4+ T cells. The ratio
of CD4+ and CD8+ T cells in 1C6 spleen and lymph nodes is equal. Splenocytes from 1C6
Tg mice respond well to the MOG35-55 and rMOG by expressing proinflammatory cytokines
(IFN-γ, IL-17 and TNF). Upon checking the response of 1C6 Tg CD4+ and CD8+ T cells to
the MOG35-55 peptide, the response of CD8+ T cells was lower in magnitude compared to
CD4+ T cells, but CD8+ T cells were found to recognize MOG35-55 in the context of both
MHC-I and MHC-II. Adoptive transfer of 1C6 Tg CD4+ or CD8+ T cells after activating and
differentiating them in T cell culture conditions to the [Link] mice induced a progressive
EAE. 1C6 Tg CD8+ T cells induced a delayed disease compared to the CD4+ T cells in
[Link] mice. Active immunization of 1C6 Tg mice with a low dose of MOG35-55 led to
the EAE induction by day 9. Initially, the mice developed mild disease, which was resolved
completely, followed by a relapse of greater severity, which did not resolve completely and
further, a chronic phase of disease was developed in 1C6 Tg mice. Upon examination of the
CNS of the mice with EAE, both CD4+ and CD8+ T cells were present, while CD8+ T cells
were outnumbered by CD4+ T cells by 30-fold. CD4+ T cells in the CNS expressed IFN-γ,
IL-17, and the CD8+ T cells mostly expressed IFN-γ [201].

1.7.3 Spontaneous EAE

In some cases, TCR-transgenic lines specific to myelin antigens can exhibit spontaneous
development of EAE. These models offer valuable insights into the interaction of T and B
cells in pathophysiology.

In the first model, double-transgenic animals on the C57BL/6 background were created by
crossing IgHMOG and TCRMOG transgenic strains to obtain TCRMOGxIgHMOG, known as OSE,
from opticospinal EAE [202]. Consequently, these mice carry transgenic T cells specific for
MOG35-55 and transgenic B cells binding to the native MOG antigen [203]. The disease in
these mice clinically resembles neuromyelitis optica (NMO), characterized by the lesions
present only in the optic nerve and spinal cord [204]. At approximately 6 weeks of age, half

46
of the OSE mice develop spontaneous disease. OSE typically exhibits a chronic progressive
disease course without remission or relapses [205].

An alternative mouse model has been established on the SJL/J background, expressing a
specific TCR for the mouse/rat MOG92-106 peptide in the context of the IAs construct, termed
TCR1640. This model, often referred to as the RR EAE model, consists of single-transgenic
SJL/J mice expressing MOG-specific TCRs [206]. In this model, the MOG-specific T cells
effectively engage with endogenous MOG-specific B cells, triggering a robust immune
response. Remarkably, over 90% of RR mice spontaneously undergo a relapsing-remitting
course of EAE within two to three months of age. The resulting lesions are dispersed
throughout the CNS, including the spinal cord, optic nerve, brainstem, and cerebellum,
mirroring some features of the relapsing-remitting form of multiple sclerosis [207].

1.7.4 Toxin-induced demyelinating models

Toxin-induced experimental models of demyelination have emerged as invaluable tools in

unravelling the intricate mechanisms governing both demyelination and remyelination
processes. These models, while not always manifesting clinically visible symptoms of
paralysis, have significantly contributed to our understanding of the underlying
immunological and molecular intricacies associated with myelin disruption and repair. By
employing toxins to induce demyelination, researchers can dissect the intricate cellular and
molecular events that orchestrate these processes. Despite the absence of overt clinical
symptoms, these models provide a controlled and manipulable environment for investigating
the immune responses and molecular pathways involved in demyelination and subsequent
remyelination[208]. Such insights are crucial for advancing our comprehension of the
pathophysiology of demyelinating disorders and may inform the development of targeted
therapeutic interventions.

1.7.5 Cuprizone model

Cuprizone, a copper ion chelator, exerts its effects by inhibiting copper-dependent

mitochondrial enzymes, including cytochrome oxidase and monoamine oxidase. This

47
interference leads to copper deficiency in oligodendrocytes, resulting in mitochondrial
dysfunction, disturbance of energy metabolism, oxidative stress and activation of the
apoptotic process, which leads to oligodendrocyte death, culminating in demyelination
within the corpus callosum[209].

The administration of cuprizone induces demyelination primarily in the corpus callosum

[210], hippocampus, cerebellar peduncles, and cerebellar cortex after 4–6 weeks of treatment
[211]. Notably, the BBB remains intact in the cuprizone model, setting it apart from the
classical EAE model [212]. T- and B-cells do not play a crucial role in cuprizone-induced
demyelination [213]. Instead, it serves primarily to replicate certain neuropathological
features characteristic of RR-MS, particularly due to the extensive demyelination that
develops within 5-6 weeks of oral administration of cuprizone, correlates with significant
microgliosis, astrogliosis and axonal damage. The removal of cuprizone from the diet allows
for significant remyelination, making this model particularly valuable for studying glial
reactions and interactions during remyelination in the absence of peripheral immune cells
[214]. It is crucial to acknowledge that cuprizone-induced demyelination predominantly
occurs in the brain, limiting its applicability for investigating demyelination or remyelination
processes in the spinal cord [215]. Nonetheless, the cuprizone model provides a controlled
environment to scrutinize the intricate cellular and molecular events associated with CNS
demyelination and subsequent repair.

1.7.6 Lysophosphatidylcholine (LPC)-induced demyelination

LPC, a specific toxin for myelin proteins, serves as a potent tool for inducing focal
demyelination in experimental models. The administration of LPC involves a stereotactic
injection directly into the white matter tracts of the CNS. This precise delivery allows
researchers to study localized demyelination phenomena. LPC exhibits a pronounced affinity
for myelin, leading to the reduction in the size of myelin lamellae, their fusion into spherical
vesicles, and eventual phagocytosis [216]. Oligodendrocyte death is induced by LPC, while
neuronal axons remain unaffected. In the acute phase of this model, infiltration of T cells, B
cells, and macrophages is observed at the lesion site [217]. Immediate demyelination is

48
observed following lysolecithin injection, with substantial remyelination becoming apparent
21 days post-lesion formation [218].

In addition to its role in inducing demyelination in experimental models, LPC is a naturally

occurring lysophospholipid widely present in blood plasma and is strongly associated with
several pathological conditions, including obesity and coronary artery disease, and its levels
rise in the brain after ischemic events [218], in the spinal cord after injury [219] and in the
CSF of MS patients [220]. The advantages of this model lie in its precise temporal control,
targeted anatomical location, and consistency in inducing demyelination. This experimental
paradigm allows for a comprehensive exploration of the complex dynamics of demyelination
and remyelination, shedding light on potential therapeutic targets and underlying cellular and
molecular events [221].

1.8 KEY ROLES OF CD8 T CELLS IN AUTOIMMUNE DISEASE

CD8+ T cells, traditionally associated with cytotoxicity, play significant roles in T-cell-
mediated autoimmune disorders such as MS and Type 1 diabetes (T1D). In T1D, CD8+ T
cells infiltrate the pancreas, targeting pancreatic beta cells. While high-avidity CD8+ T cells
are pathogenic, recent research suggests low-avidity CD8+ T cells may possess immune
regulatory properties, protecting against diabetes [222]. Similarly, in MS, CD8+ T cells
contribute to CNS lesions [223]. The role of CD8+ T cells in autoimmunity is complex,
involving both pathogenic and regulatory functions. Checkpoint molecules, such as CTLA-
4 and PD-1/PD-L1, modulate CD8+ T cell activation [224], while CD8+ T cell subsets such
as Qa-1-restricted CD8+ T cells, CD8+ CD122+ T cells, and CD8+ CD28− T cells, exhibit
regulatory properties [138]. Intriguingly, emerging evidence suggests that epigenetic
modifications may contribute to CD8+ T cell dysregulation and activation in autoimmune
diseases [225].

CD8+ T cells are predominant in acute and chronic MS lesions, outnumbering CD4+ T cells
and contributing to oligoclonal expansions in the CNS, blood, and cerebrospinal fluid of MS

49
patients [226]. Autoreactive CD8+ T cells can recognize myelin antigens, neuronal proteins
and viral antigens on MHC I molecules of resident CNS cells and thus leading to
demyelination [227]. In the EBV-induced autoimmune disease, CD8+ T cells play a role in a
process called epitope spreading, where they recruit autoreactive CD4+ T cells and B cells,
thus amplifying the immune response that leads to disease exacerbation. Since EBV infection
has been linked to MS pathogenesis in different studies, it is possible that CD8+ T cells might
play a similar role in MS pathogenesis [228]. Another study demonstrated that, after EBV
infection, humanized NOD-Scid IL2 receptor γ-chain-deficient mice reconstituted with
HLA-DR15-positive donors immune cells exhibited higher CD8+ T-cell expansion and
activation compared to HLA-DR15-negative cell recipients [229]. In RRMS patients, no
effect was observed on B cells or CD4+ T cells after 12 months of treatment with
teriflunomide (DMT for RRMS) but the CD8+ T cells were significantly affected,
characterized by decreased proliferation and reduced proinflammatory cytokine TNF and
IFN-γ production [230]. A substantial proportion of CNS-infiltrating CD8+ T cells were
found to express granzyme B and perforin supported by lytic granule polarization, the
expression of degranulation marker CD107a [231] and adjacent cells expressing cleaved
caspase 3 was also shown by in situ cytotoxic activity [232] . CD8+ T cells in MS exhibit
distinct DNA methylation profiles. DNA hypermethylation at CpG sites in genes such as
APC2, HOXA2, HRNBP3, HEXDC, and NTRK3 may inhibit CD8+ T cell functions,
potentially contributing to MS pathogenesis [233]. Overall, the complex interplay of
cytokines, checkpoint molecules and epigenetic factors describes the regulatory and
pathogenic role of CD8+ T cells in autoimmune diseases.

1.9 PATHOPHYSIOLOGY OF CD8+ T CELLS IN MULTIPLE SCLEROSIS

In MS, CD8+ T cells are notably enriched in the perivascular space and leading edge of white
matter lesions, surpassing the numbers of CD4+ T cells, and are also present in the majority
of cortical lesions [232] . Importantly, CD8+ T cells in MS lesions display clonal expansion,
indicating a proliferation driven by antigens [234]. Common clonal CD8+ T cell populations
are identified within both the lesions and normal-appearing white matter in individual MS

50
patients [235]. The role of major MHC I alleles in MS susceptibility is evident, with alleles
such as HLA-A02:01 (HLA-A2) and HLA-A03:01 (HLA-A3) known to influence the
disease [236]. Furthermore, MHC I is specifically upregulated in microglia, macrophages,
astrocytes, and oligodendrocytes in acute MS lesions [237]. The close proximity of CD8+ T
cells to oligodendrocytes in MS lesions suggests a potential direct contribution to
demyelinating pathology [238].

A study was conducted to identify and validate myelin-specific CD8+ T cell epitopes and
assess their frequencies and phenotypes in MS patients compared to healthy controls. By
using a comprehensive approach involving myelin peptide:MHC I (pMHC I) tetramers, the
researchers validated five myelin CD8+ T cell epitopes, including two not previously reported
in humans. Despite detecting myelin specific CD8+ T cells in both MS patients and controls,
the frequencies did not significantly differ. However, memory myelin-specific CD8+ T cells
were increased in MS patients, indicating prior exposure to antigen. The phenotypic
characteristics of these CD8+ T cell populations were also explored. Memory myelin-specific
CD8+ T cells in MS patients exhibited an activated phenotype, with increased expression of
CD20, a marker associated with activated T cells. Treatment with anti-CD20 monoclonal
antibody (mAb) led to a preferential reduction in CD20-expressing myelin-specific CD8+ T
cells. Anti-CD20 mAb therapies (rituximab and ocrelizumab) that have been used for B cell
depletion have been very effective in MS treatment, CD8+ T cells can express CD20, it is
possible that they may also have been depleted simultaneously [239]. CD8+ T cells that target
MBP have been identified in both individuals with MS and healthy subjects. These CD8+ T
cell clones exhibit a pro-inflammatory profile, secreting cytokines such as (IFN-γ) and TNF.
Notably, these MBP-specific CD8+ T cell clones demonstrate a remarkable capability to
selectively lyse freshly isolated human oligodendrocytes that match the HLA type, all
without the addition of external peptides [240].

Another study suggests that IL-17-producing CD8 T cells (Tc17) may play a role in the
development of MS. The researchers found that in the areas around blood vessels in active
MS lesions, most of the CD4+ and CD8+ T cells were producing IL-17, seen in
immunohistochemistry and in-situ hybridization. In contrast, in inactive lesions, there were

51
only a few of these IL-17-producing T cells [241]. An increase in effector memory CD8+ T
cells in the CSF of 52 RRMS patients at the disease's onset was observed [242].

This finding, along with the enrichment of granzyme B-expressing CD8+ T cells, has been
consistently confirmed in subsequent studies involving 17 other RR patients Notably, these
effector memory CD8+ T cells exhibited enhanced migration through a model of the BBB in
vitro. This migration was particularly notable in cells producing granzyme B, perforin, IFN-
γ, and IL-17. The relevance of these findings was further supported by a mouse model of
EAE [230],

Studying the transmigration of CD8+ T cells into the CNS has also shed some light on their
role in the progression of MS. In MOG35-55-immunized mice, the blockade of α4 integrin
resulted in a reduced number of infiltrating CD8+ T cells and a lower EAE score, mirroring
a similar effect observed in CD4+ T cells [243]. Recently, melanoma cell adhesion molecule
(MCAM), expressed by a subset of human effector CD8+ T cells, was found to be upregulated
during MS relapse compared to controls [244]. Intriguingly, blocking MCAM prevented the
transmigration of human CD8+ T cells across a BBB model and led to decreased EAE scores
in active, transfer, and spontaneous models [245]. The ability of MCAM to bind to both itself
and laminin 411, both expressed by endothelial cells, adds complexity to its mode of action
[246]. P-glycoprotein, known for its role in drug efflux and cytokine/chemokine secretion,
has also emerged as crucial for CD8+ T cell trafficking into the brain during the disease. Mice
lacking Mdr1a/b, the genes encoding P-glycoprotein, exhibited significantly reduced EAE
[247]. Silencing P-glycoprotein specifically decreased CD8+ T cell infiltration into the brain
without affecting CD4+ T cells, with P-glycoprotein's control of endothelial CCL2 secretion
playing a key role. EAE mice lacking P-glycoprotein or CCL2 displayed a substantial
reduction in CD8+ T cell migration into the brain. Moreover, elevated CCL2 transcripts were
found in six MS lesions compared to six controls [248]The upregulation of miR-16, miR-
155, and miR-142-3p in CD8+ T cells from MS patients targets FOXP3, IRF2BP2, and
FOXO1, respectively, activating CD8+ T cells and mediating autoimmune inflammation
[249]. In summary, CD8+ T cells are significantly involved in MS pathogenesis, as evidenced
by their enrichment in specific regions of lesions, clonal expansion, potential interaction with
key cell types in the CNS and their ability for transmigration into the CNS.

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1.10 EAE MODELS OF CD8+ T CELLS

While EAE models have traditionally focused on the involvement of CD4+ T cells, there is a
growing interest in understanding the contribution of CD8+ T cells to the pathogenesis of
neuroinflammatory diseases. In the context of EAE models, CD8+ T cells have been shown
to play a role in both the initiation and progression of the disease.

1.10.1 MBP[79-87]–specific CD8+ T cell model

In this CD8+ T cell model presence and impact of MBP-specific CD8+ T cells in C3Hwild-
type mice was investigated. It was demonstrated that MHC-I restricted, MBP-specific CD8+
T cells can be isolated from [Link] (MBP-deficient) mice primed with recombinant virus
encoding MBP. The findings revealed that these CD8+ T cells, specific for the MBP79-87
epitope associated with the Kk MHC molecule, circulate in the periphery of mice on the C3H
genetic background. Interestingly, MBP79-87–specific T cells in wild-type mice appeared to
undergo substantial tolerance induction, similar to certain T cells specific for MHC class II–
restricted myelin epitopes [250]. To assess whether some MBP-specific CTLs escape
tolerance, another study was conducted by the same group in which mice were immunized
with the MBP79-87 peptide, and T cells were cloned from the draining lymph nodes. MBP-
specific CD8+ T cell clones from both C3H wild-type and MBP-deficient mice were
established. These CD8+ T cell clones from wild-type mice exhibited cytotoxic activity
against H-2Kk-expressing, MHC-II-deficient target cells infected with recombinant vaccinia
virus encoding MBP. The cytotoxicity was also observed against target cells coated with
MBP79-87 in a dose-dependent manner, similar to T cell clones from MBP-deficient mice.
Furthermore, the study explored the ability of MBP-specific CD8+ T cells to induce CNS
autoimmunity. When activated MBP-specific CD8+ T cell clones from C3H wild-type mice
were transferred to C3H wild-type and [Link] mice, both C3H wild type and [Link]
mice displayed pronounced neurological disease. The clinical symptoms included upper
motor neuron impairment, weight loss, and, in some cases, mortality. Histological analysis
revealed distinct lesions in the brain, involving small blood vessels and surrounding nervous
tissue, differing from the classic EAE pattern. The effector mechanisms driving this

53
autoimmune response were associated with IFN-γ, as neutralizing this cytokine reduced the
severity of the disease [251].

1.10.2 Met+ CD8+ T cell model

In Met+CD8+ T cell model a subpopulation of CD8+ T cells expressing the c-Met receptor
was identified. These c-Met+CD8+ T cells demonstrated enhanced cytolytic activities
compared to their c-Met− counterparts both in vitro and in vivo. The researchers investigated
the expression of c-Met during the course of EAE and found a significant increase in c-Met
expression on CD8+ T cells at the peak of the disease. Further analysis revealed that c-Met+
CD8+ T cells displayed increased activation levels and proliferated more when exposed to
MOG35–55 and MOG37–50 peptides compared to c-Met−CD8+ T cells. Additionally, these c-
Met+CD8+ T cells exhibited higher levels of granzyme B and IFN-γ secretion upon
stimulation with the MOG37–50 peptide, indicating their potential involvement in the immune
response during EAE. The study also explored the impact of hepatocyte growth factor (HGF)
on c-Met+ CD8+ T cells. A treatment with HGF resulted in the down-modulation of the
cytolytic function of c-Met+CD8+ T cells, as evidenced by a decrease in granzyme B
production, which suggests that HGF may regulate the effector functions of these cells in the
context of CNS demyelination [252].

1.10.3 GFAP specific CD8+ T cell model

GFAP specific CD8+ T cell model was used to understand the CNS antigen reactive CD8++
T cells that avoid negative selection in the thymus and contribute to autoimmune responses.
Mice were infected with viruses expressing CNS antigens, including GFAP, MAG, MBP,
MOG, and PLP, and CD8+ T cell clones specific for certain antigens were isolated. It was
found that C57BL/6 mice have CD8+ T cells reactive to multiple CNS antigens with varying
avidities [253]. GFAP-specific CD8+ T cells exhibited a strong response, while MAG- and
MOG-specific CD8+ T cells showed weaker responses. The stability of peptide/MHC
complexes differed among antigens. To investigate how the strong avidity of CD8+ T cells
reactive to GFAP avoids tolerance, the researchers created TCR transgenic mice expressing
the TCR chains from a GFAP-specific CD8+ T cell clone (BG1) [254]. They observed that

54
BG1 CD8+ T cells could develop without undergoing overt thymic deletion or
unresponsiveness. These cells spontaneously entered the brain and spinal cord, expressing
TCR signaling-dependent proteins and activation markers within the CNS. BG1 mice
developed spontaneous RRCNS autoimmunity. Histological analysis revealed that BG1
CD8+ T cells in spontaneous disease targeted the cerebellum, mid-brain, and spinal cord
early. The CNS lesions showed signs of apoptosis, necrosis, glial cell responses, and
demyelination, as shown in another study after viral infection of the CNS [255]. The role of
B cells in regulating CD8+ T cell-mediated CNS autoimmunity was also highlighted in BG1
mice. Mice lacking B cells showed increased susceptibility to spontaneous CNS disease,
which induced severe ataxia and lethargy, like disease patterns observed with activated MBP-
specific CD8+ T cells [256].

While these models highlight the pathogenic role of CD8+ T cells, other studies demonstrate
the regulatory role of CD8+ T cells. It was shown that Myelin reactive CD8+ T cells (PLP-
CD8) induce regulatory changes in conventional dendritic cells (cDCs) both in vitro and in
vivo. Adoptive transfer of PLP-CD8+ T cells induced a mature and regulatory phenotype in
cDC subsets, characterized by an anti-inflammatory cytokine profile and reduced capacity to
support CD4+ T cell proliferation [257]. Previous studies from the same research group
observed that adoptive transfer of PLP-CD8+ T cell prevents EAE induction and supress the
ongoing disease in recipient mice through the engagement of MHC-I [258]. Upon
investigating the interaction of CD4+ and CD8+ T cells and their effect on EAE, it was
observed that CD8+ T cells do not exhibit a pathogenic role, by showing no signs of EAE
when the CD8+ T cells were present in the CNS of the mice. It was also observed that CD8+
T cells doesn’t inhibit CD4+ T cell induced EAE, by showing no difference in the disease
when both CD4+ and CD8+ T cells were present in the CNS compared to when the CD4+ T
cells were present alone[259].

In conclusion, CD8+ T cells can exhibit both pathogenic as well as regulatory functions in
EAE, with their role being influenced by different factors like activation of the CD8+ T cells,
the model of EAE and the antigenic peptide used to induce the disease.

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1.11 CD8+T cells in other autoimmune diseases

Type 1 Diabetes (T1D) involves the destruction of pancreatic beta cells by CD8+ T cells.
Autoimmune CD8+ T cells recognize the β cell antigen on APCs and rapidly transform into
effector cells [260]. Preproinsulin-reactive CD8+ T cells were more common among
autoreactive CD8+ T cells in T1D patients than healthy donors [261]. Secretion of Fas, TNF
and IFN-γ has also been found as the promoting factor for the development of T1D by CD8+
T cells [262]. In a murine model, downregulated miR-29b reduced the cytolytic activity of
CD8+ T cells, while miRNAs like miR-23b, miR-98, and miR-590-5p could modify disease
susceptibility by targeting apoptotic molecules [263]. Understanding these molecular
mechanisms may offer insights into potential therapeutic strategies.

Systemic Lupus Erythematosus (SLE) is a multi-system autoimmune disease characterized

by antinuclear autoantibody production. CD8+ T cells in SLE exhibit overexpression of PRF1
due to DNA hypomethylation, contributing to monocyte/macrophage killing [264].
Dysregulation of mTOR activity in CD8+ T cells is implicated in SLE pathology, and
oxidative stress, originating from mitochondrial hyperpolarization, activates mTOR and
contributes to DNA hypomethylation. N-acetylcysteine, an oxidative stress inhibitor, can
block mTORC1 activity in lupus T cells [265].

Systemic Sclerosis (SSc) is a life-threatening autoimmune disease characterized by fibrosis

in various tissues and organs. CD8+ T cells are upregulated in the peripheral blood of SSc
patients, secreting high levels of IL-13, associated with skin fibrosis [266], [267]. Epigenetic
changes in CD8+ T cells include global hypomethylation of type I IFN signalling pathway-
associated genes, such as IFI44L and IFITM1. IFI44L, a potential biomarker in SLE, and
IFITM1, linked to Wnt signalling, may play roles in SSc pathogenesis [268].

Graves' Disease (GD) is an autoimmune thyroid disorder characterized by the production of

autoantibodies that stimulate the thyroid hormone receptor, leading to goitre and

56
hyperthyroidism [269]. Thyroid autoreactive CD8+ T cells are crucial in GD pathogenesis
[270]. Limbach et al. found differential DNA methylation in CD8+ T cells of GD patients,
impacting genes involved in T cell activation, including LCK, CD247, ZAP70, CTLA4, and
CD8A. Hypermethylation of these genes may influence CD8+ T cell function. Additionally,
decreased H3K4me3 and H3K27ac signals in CD8+ T cells of GD patients suggest potential
impairment in T cell receptor signalling [271]. miRNA-200a reduction in CD8+ T cells could
contribute to developing thyroid-specific antibodies, leading to the destruction of thyroid
tissue [272].

Severe Aplastic Anemia (SAA) is characterized by CD8+ T cell-mediated destruction of

hematopoietic cells. Hypomethylation of the linker for activation of T cells (LAT) promoter
in SAA enhances CD8+ T cell cytotoxicity, potentially contributing to hematopoietic failure
[273]. Histone H3 acetylation in CD8+ T cells negatively correlates with immune status and
hematopoietic function in SAA patients, suggesting its role in hyperfunction and disease
exacerbation [274]. These findings highlight the intricate interplay between epigenetic
modifications and CD8+ T cell function in various autoimmune diseases, providing potential
avenues for therapeutic interventions.

1.12 SERPINE1 AND ITS ROLE IN INFLAMMATION

The Serpine1 gene encodes for the plasminogen activator and inhibitor 1 (PAI-1) protein.
PAI-1 is a serine protease inhibitor that is crucial in regulating fibrinolysis. PAI-1 specifically
inhibits tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator
(uPA), which are key enzymes involved in the conversion of plasminogen to plasmin [275].
PAI-1 is synthesized by various cell types, including endothelial cells, liver cells, and
immune cells. PAI-1 concentrations exhibit variations influenced by genetics, race/ethnicity,
gender, and circadian rhythms [276]. Genetic variations, such as the promoter −675 4G/5G
polymorphism, have been linked to PAI-1 concentrations, although the relationship is still
under scrutiny [277], [278].

PAI-1 is widely expressed and plays a key role in a number of diseases. In triple-negative
breast cancer, Serpine1 expression was elevated in paclitaxel-resistant breast cancer cells;

57
suppressing Serpine1 led to increased sensitivity to Paclitaxel and enhanced apoptosis.
Importantly, in vivo studies demonstrated that tumours with suppressed Serpine1 were
smaller in size [279]. Another study on colorectal cancer identified 1191 upregulated genes
that were associated with stromal and immune cell invasion: after combining the 100 genes
with the most nodes with the top 24 genes ranked by p-value in the univariate COX
regression. Finally, two genes, TGFβ1 and Serpine1 was identified. Further analysis of
Serpine1 showed its significant differential expression in colorectal cancer compared to
normal tissue and exhibited distinct expression patterns in different cancer stages and
classification [280]. A third study exploring the association of Serpine1 with immune
response and tumour characteristics by using data from various cancers, such as
glioblastoma multiforme and lower-grade glioma (GBM-LGG) Uveal Melanoma (UVM),
Pancreatic adenocarcinoma (PAAD), and Colon adenocarcinoma and rectum
adenocarcinoma (COAD-READ). The results indicated that high Serpine1 expression was
linked to immune-related pathways across different cancers. Notably, it showed positive
associations with inhibitory immune checkpoints in most cancers, indicating its potential role
in immune regulation [281].

To check the correlation of PAI-1 with the cytokines, a study was conducted where the
cytokine levels were checked in the plasma of PAI-1-/- and WT mice after lipopolysaccharide
(LPS) and Staphylococcal enterotoxin B (SEB) injection. IL-6, IL-12p70 and IFN-γ levels
were significantly increased after LPS injection, while SEB injection significantly increased
IL-6, MCP-1 and IFN-γ in the plasma of PAI-1-/- mice. Ex vivo stimulation of splenocytes
with LPS or SEB also showed significantly higher expression IFN-γ from PAI-1-/-
splenocytes. A further effect of PAI-1-/- was checked on T cells after stimulation of
splenocytes with SEB, and both CD4+ and CD8+ T cells from PAI-1-/- mice were found to
express significantly higher expression of IFN-γ compared to T cells from WT mice [282].
Another study was conducted to show the importance of PAI-1 in immune responses. In this
study, nasal allergy was induced in WT (C57BL/6J) and PAI-1-/- mice by intranasal challenge
of OVA after sensitization with the i.p injection of OVA. To check the presence of cytokines
in the nasal cavity during the development of allergy, nasal lavage fluid (NLF) was collected,
and it was found that IFN-γ was significantly higher in NLF of PAI-1-/--OVA mice compared

58
to WT-OVA mice. Cytokine expression was also checked in the supernatant of lymphocytes
after OVA stimulation. IL-4 and IL-5 were significantly higher in the supernatant of WT-
OVA lymphocytes. Conversely, significantly higher levels of IFN-γ were found in the PAI-
1-/- OVA lymphocyte supernatant. This shows that PAI-1 not only plays a role in
thrombolysis but also in immune response, and it was found that PAI-1-/- shifts the immune
response from T helper 2 response to T helper 1 response, with increased IFN-γ production
[283].

The increased IFN-γ production in PAI-1-/- mice, with IFN-γ being the key cytokine of Th1
cells, coupled with the role of Th1 cells in inducing EAE led us to investigate the functional
role of Serpine-1 in Th1 cells.

1.12.1 Role of Serpine1 in CNS inflammation

The role(s) played by Serpine1 in CNS autoimmunity are complex. Using the EAE model by
active immunization of C57BL/6 female mice with MOG35-55, it was shown that mice lacking
the uPA and its receptor (uPAR) in neurodegeneration showed more severe neurological
symptoms and delayed recovery compared to normal mice. Neuropathological analysis
revealed increased inflammation and axonal loss in the spinal cords of uPA−/− and uPAR−/−
mice during the chronic phase of the disease, and these mice also had heightened activation
of microglia. Interestingly, the absence of uPA and uPAR reduced T-cell reactivity to the
immunizing antigen (MOG35-55). As Serpine1 negatively regulates uPA, this provided
indirect evidence that it may repress CNS autoimmunity. Indeed, a significant reduction in
disease severity and T-cell activity was observed upon injecting a peptide derived from PAI-
1 [284].

Serpine1-deficient mice were then examineddirectly using a chronic relapsing EAE model in
which the PAI-/- and WT mice were immunized with whole spinal cord homogenate. PAI-/-
had a reduced incidence, delayed onset and less frequent relapses as compared to WT mice.
Histopathologically, fewer inflammatory cells and perivascular cuffs were revealed in
Serpine-deficient mice, and demyelination was less evident. Mechanistically, it was observed
that WT spinal cord lysate showed impaired fibrinolytic capacity when harvested at disease

59
relapse; this function was relatively preserved in lysate from Serpine1-deficient mice [285].
Thus, the apparently paradoxical finding that Serpine1 promotes CNS autoimmunity in this
model might be explained by its effect on non-immune cells.

Another study investigated the effect of tissue-specific inhibition of PAI-1 by using TM5484,
a PAI-1 inhibitor designed to effectively penetrate the BBB. When administered to an EAE
mice model, TM5484 significantly alleviated motor paralysis and preserved motor
coordination. The compound also demonstrated anti-inflammatory effects by reducing the
expression of pro-inflammatory markers in the spinal cord and spleen. TM5484 further
showed protective effects against demyelination, axonal degeneration, and collagen IV
deposition in the spinal cord of EAE mice. Interestingly, TM5484 did not affect fibrin
deposition, suggesting a specific impact on inflammatory cell infiltration. Additionally,
TM5484 delayed the onset of paralysis and had an additive benefit when combined with the
MS drug fingolimod. The compound was also effective in a rat model of EAE. These findings
suggest that TM5484, a PAI-1 inhibitor with enhanced CNS penetration, holds promise as a
potential therapeutic agent for MS [286].

1.13 T CELL IMMUNE CHECKPOINTS AND THEIR ROLE IN T CELL

EXHAUSTION

The activation and expansion of T cells are crucial for a strong immune response. However,
regulatory mechanisms are important to prevent the immune system from becoming too
active and causing autoimmune diseases. The regulation of T cell activation is a complex
process involving various signalling pathways. The CD28 family of receptors on the cell
surface plays a key role in controlling T-cell activation. Even though these receptors share a
similar structure, they have different functions. For instance, when CD28 and ICOS are
engaged, they promote T cell activation. On the other hand, when CTLA-4, programmed
death 1 (PD-1), and B and T lymphocyte attenuator (BTLA) are involved, they inhibit T cell
activation [287]. This balance ensures that the immune system responds effectively without
causing harm. Immune checkpoint inhibitors (ICIs), particularly those targeting CTLA-4 and
PD-1, have demonstrated significant success in enhancing T cell responses and treating
various cancers [288]. However, challenges such as immune-related adverse events (IRAEs)

60
and acquired resistance have prompted investigations into combination therapies and the
exploration of alternative inhibitory receptors.

1.13.1 Tim-3

The T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a crucial

immune checkpoint receptor that plays a significant role in regulating T cell responses. TIM-
3 is a member of the TIM family and is encoded by the HAVCR2 gene. The TIM family
genes are located on chromosome band 5q33.2 in humans and chromosome band 11B1.1 in
mice [289]. This genomic region is associated with allergy and asthma [290]. TIM-3 has
received significant attention among the TIM family due to its association with regulating
immune responses in autoimmunity and cancer. TIM-3 is expressed by various cell types,
including CD4+ and CD8+ T cells, Treg cells, myeloid cells, NK cells, and mast cells
[291][292], [293]. Therapeutic targeting of TIM-3 is under investigation in clinical trials for
cancer treatment, often in combination with other checkpoint receptors such as LAG3 and
TIGIT [294].

The molecular structure of TIM-3 includes an amino-terminal immunoglobulin variable

domain (V domain), a mucin stalk, a transmembrane domain, and a cytoplasmic tail. TIM-3
lacks known inhibitory signalling motifs in its cytoplasmic tail, distinguishing it from classic
checkpoint receptors like PD1 and TIGIT [291]. The cytoplasmic tail contains five tyrosines,
including Tyr256 and Tyr263, allowing interactions with HLA-B-associated transcript 3
(BAT3) and the tyrosine kinase FYN [295]. The molecular mechanism of TIM-3 involves its
recruitment to the immunological synapse upon T-cell activation, where it interacts with
BAT3 and the tyrosine kinase LCK [296]. Galectin-9 and carcinoembryonic antigen-related
cell adhesion molecule 1 (CEACAM1) are ligands for TIM-3 [291]. Engagement of Tim-3
with its ligands, such as Galectin-9, can lead to the inhibition of T-cell proliferation and
cytokine production. Its role in T cells is multifaceted, particularly in the context of immune
regulation and exhaustion. Tim-3 is involved in the regulation of immune responses. It acts
as a negative regulator of T-cell activation, helping to prevent excessive immune reactions
[297].

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Tim-3 is a pivotal player in immune regulation across diverse contexts, initially recognized
as a marker for IFN-γ-secreting effector CD4+ Th1 cells and CD8+ type 1 cytotoxic T cells
[298]. The Th1 cell transcription factor T-bet, in cooperation with NFIL3 [299], PRDM1,
and MAF [291] , regulate TIM-3 expression, impacting its role in limiting IFN-γ-driven
inflammation. Tim-3 deficiency leads to dysregulation of Th1 cells in EAE exacerbating the
disease [300]. Interestingly, Tim-3 expression on T cells is diminished in patients with
autoimmune diseases, such as ulcerative colitis, multiple sclerosis, rheumatoid arthritis, and
psoriasis, reflecting its potential involvement in regulating inflammatory responses [301],
[302].

Tim-3 is prominently associated with T cell exhaustion, a state of dysfunctionality that occurs
during chronic infections and cancer. Recent findings show Tim-3 expression on virus-
specific T cells during HIV-1 and HCV infections, correlating with CD8+T cell exhaustion.
Blocking Tim-3 in vitro improved the responsiveness of exhausted T cells, suggesting its role
as another inhibitory marker in chronic viral infections [303], [304]. Investigating the
expression and functional implications of Tim-3 on CD8+ T cells during both acute and
chronic LCMV infection, Tim-3 expression on LCMV-specific CD8+ T cells increased
during acute infection but rapidly decreased after the infection was cleared. However, during
chronic infection, Tim-3 expression remained elevated for an extended period. Importantly,
the co-expression of Tim-3 and PD-1 was more prominent during chronic infection,
suggesting a potential role in CD8+ T cell exhaustion. The Tim-3+ PD-1+ subset was
particularly dominant, indicating a distinct population of exhausted CD8+ T cells. Upon
assessing the phenotypic and functional profiles of Tim-3+ PD-1+ and Tim-3−PD-1+ CD8+ T
cell subsets, the Tim-3+PD-1+ population exhibited higher levels of inhibitory receptors,
lower expression of activation markers, and a more severe exhaustion phenotype compared
to Tim-3−PD-1+ cells. Functionally, Tim-3+ PD-1+ CD8+ T cells showed reduced proliferation
and cytokine production, indicating a deeper state of exhaustion, with Tim-3+ PD-1+ subset
also expressing IL-10. In vivo blockade was also conducted by using Tim-3-Ig fusion protein
and an anti-PD-L1 antibody either alone or in combination. Dual blockade significantly
increased the quantity and functionality of virus-specific CD8+ T cells compared to single
blockade or control groups [305].

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Another study showed elevated expression of Tim-3 on both CD4+ and CD8+ T cells in
chronic HCV-infected patients compared to normal controls. HCV-specific CTLs showed
higher Tim-3 levels than CMV-specific CTLs, indicating a more exhausted phenotype in
persistent HCV infection. Co-expression of Tim-3 and PD-1 was examined, revealing a
higher percentage of dual-positive T cells in chronic HCV patients, especially within the
intrahepatic compartment. HCV-specific CTLs expressing both Tim-3 and PD-1 were more
pronounced, suggesting a distinct exhausted T cell population in chronic infection. Tim-3
expression also correlated with a higher proportion of central memory CD8+ T cells and
increased levels of the senescence marker CD57. Tim-3+CD8+ T cells also exhibited lower
expression of CD127, associated with effector CTL survival. Functionally, Tim-3-positive T
cells displayed impaired production of Th1/Tc1 cytokines. The blockade of Tim-3 with an
anti-Tim-3 antibody enhanced proliferation and cytokine production in HCV-specific CD4+
and CD8+ T cells, indicating a role for Tim-3 in T cell dysfunction during chronic HCV
infection [306]. The expression of Tim-3 also increases in conditions where T cells are
persistently stimulated, such as in neurodegenerative diseases. This upregulation is often
linked to a loss of effector functions, such as reduced cytokine production and cytotoxicity.
T cells expressing Tim-3 can infiltrate the CNS and contribute to the inflammatory milieu,
potentially impacting disease progression [291].

1.13.2 PD-1

Programmed Cell Death Protein-1 (PD-1) is expressed on developing thymocytes and on

various hematopoietic cells in the immune periphery in an inducible manner. In the thymus,
PD-1 is found on immature double-negative (CD4-CD8-) thymocytes during TCRβ
rearrangement [307]. Once in the periphery, PD-1 is inducibly expressed on CD4+ and CD8+
T cells, natural killer T (NKT) cells, B cells, monocytes, and certain DCs subsets upon
activation [308]. Estrogens have also been identified as a stimulator of PD-1 expression on
both T cells and APCs [309]. PD-1 expression is triggered by TCR or BCR signalling and
persists in the presence of prolonged antigen stimulation, irrespective of whether the
stimulation is foreign or self-derived. Notably, PD-1 is highly expressed in non-functional
exhausted T cells during chronic viral infections [310]. Key cytokines like IL-2, IL-7, IL-15,

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and IL-21, crucial for T-cell expansion and survival, also induce PD-1 expression in T cells
[311].

PD-1 is a pivotal immune checkpoint receptor belonging to the immunoglobulin superfamily

(Ig-SF) with a transmembrane structure. Its cytoplasmic domain encompasses crucial
inhibitory motifs, including an immunoreceptor tyrosine-based inhibition motif (ITIM) and
an immunoreceptor tyrosine-based switch motif (ITSM) [312]. Following TCR-ligation, the
phosphatase SHP-2 associates with PD-1 via these motifs, indicating its role in T cell
regulation [312]. PD-1 exerts its inhibitory function by co-localizing with the TCR-CD3
complex or CD28 [313]. PD-L1 and PD-L2, members of the B7 family, serve as ligands for
PD-1, with PD-L2 expressed mainly on professional APCs and PD-L1 broadly expressed on
hematopoietic cells, including activated T cells [314]. PD-1 is vital in maintaining central
and peripheral tolerance, contributing to Treg induction and regulating PD-ligand expression
during inflammation [315]. However, it also limits productive T-cell immunity against
persistent antigens, leading to T-cell exhaustion [316].

1.13.3 Lymphocyte activation gene-3 (Lag-3)

Lag-3, is a crucial player in constraining T cell activity, controlling their growth, function,
and homeostasis within the immune system [317], [318]. It also plays a significant role in
ensuring the optimal function of Tregs, whether they occur naturally or are induced [319].
Interestingly, LAG-3 shares a significant homology with CD4 at the genomic level and both
are located on the same chromosome, hinting that LAG-3 might have emerged from a gene
duplication event [320]. LAG-3 binds to MHC class II molecules with strong affinity [321].
In terms of expression, previous studies suggested that LAG-3 in humans is principally found
in activated T and NK cells [322].

In mice, LAG-3 expression is again restricted to T and NK cells. While approximately 18%
of TCRγδ T cells and 10% of NK cells are LAG-3 positive, only a small percentage (about
1-2%) of resting TCRαβ T cells in the spleen and thymus show LAG-3 on their surface.
However, virtually all T cells rapidly upregulate LAG-3 upon activation [323].

64
Despite lacking classical motifs for inhibitory phosphatase recruitment in its cytoplasmic tail,
LAG-3 functions as an intrinsic negative regulator of CD4+ and CD8+ T cells, as well as
contributing to Treg-mediated inhibition [324]. Notably, LAG-3's unique feature of ligation
to MHC class II molecules distinguishes it from other co-inhibitory receptors, and its large
extracellular domain sets it apart from immune checkpoints such as PD-1, BTLA, and CTLA-
4. However, the precise mode of action and signalling pathways involved in LAG-3-mediated
inhibition remain incompletely understood, emphasizing the need for further research to
unravel its distinct regulatory mechanisms [325].

1.14 HYPOTHESIS AND OBJECTIVES

Multiple sclerosis is a chronic autoimmune neurodegenerative disease of CNS in which the

T cell response plays a critical role. Both CD4+ and CD8+ T cells have been observed in the
CNS of MS patients during the disease course. A vast number of studies have been conducted
to investigate the mechanism behind the pathogenicity of CD4+ and CD8+ T cells. Using the
EAE mouse model, I worked on two projects. In the 1st project, the effect of Serpine1 on
theTh1 cell immune response was studied. In the 2nd project, I focussed on the role of CD8+
T cells in inducing the disease pathogenicity in an adoptive transfer EAE mice model.

1.14.1 Hypothesis 1

Serpine1 gene encodes for the formation of a protein called plasminogen activator and
inhibitor 1 (PAI-1). Utilizing both the adoptive transfer model and the active immunization
model in our lab, we aim to study the role of Serpine-1 deficiency (Serpine-1-/-) in general
and its effects on Th1 cells. Knowing that Tim-3 is expressed on IFN-γ-producing Th1 cells
and binding of Tim-3 to its ligand terminates the Th1 immunity, we hypothesized that
Serpine-1 inhibits Th1 immune response in a Tim-3-dependent manner.

Evaluation of the hypothesis:

1. Effect of Serpine-1-/- on disease: Based on a previous study, where PAI-1

deficient mice were shown to have increased IFN-γ expression in response to
LPS and Staphylococcal enterotoxin B (17114493). We first checked the

65
 effect of Serpine-1-/- on EAE progression after active immunization of the
mice with MOG35-55 peptide.
2. Pathogenicity of immune cells: After observing higher disease progression in
Serpine1-/- mice in Exp #1, it was important to check if the increased disease
in Serpine1-/- mice was because of the increased pathogenicity of immune
cells. To validate that, splenocytes isolated from immunized Serpine-1-/- mice
were restimulated and adoptively transferred to WT mice.
3. Direct assessment of Serpine-1 in Th1/Th17 cells: The functional role of
Serpine-1 has never been directly assessed in differentiated bona fide Th1,
Th17 cells, and the adoptive transfer of splenocytes in Exp #2 could not
directly answer that question. To answer this, we overexpressed the Serpine-
1 in 2D2 TCR transgenic Th1 and Th17 cells by RV transduction and we also
transferred the Serpine-1 OE 2D2 Th1 cells to the Rag1-/- mice to check the
effect of Serpine-1 OE in Th1 disease pathogenicity.
4. Role of Tim-3: As we observed that Serpine-1 inhibits Th-driven
inflammation, we decided to look at the expression of Tim-3 in Serpine1 KO
Th1 cells. To check if Serpine-1 mediates its effect through Tim3, we
generated Th1 cells after crossing Serpine-1 KO mice to the Tim-3-Tg mice
and checked the expression of IFN-γ, PD-1 and Lag3 in Serpine-1 KO:Tim-
3-Tg Th1 cells.
5. Combined effect of Serpine-1 and Tim-3: Tim-3-Tg mice develop EAE of
attenuated severity [326] to check the combined effect of Serpine-1 and Tim-
3; WT Tim-3-Tg and Serpine-1 KO:Tim-3-Tg mice were actively immunized,
and the disease was compared between two groups.

1.14.2 Hypothesis 2

In the 2nd part of the thesis, I investigated the role of CD8+ T cells in inducing the progressive
form of EAE. Our hypothesis was that MOG35-55 specific CD8+ T cells contribute to the
development and progression of CNS autoimmunity in an adoptive transfer EAE model. In
our lab, I used the 1C6 TCR transgenic mouse model, in which mice have both CD4+ and

66
CD8+ T cells that recognize the MOG35-55 peptide. The CD8+ T cells from these mice were
isolated and cultured under T cell conditions. The differentiated and activated T cells were
adoptively transferred into the [Link] mice and the mice were observed for the disease
course.

Evaluation of the hypothesis:

1. Reactivity of 1C6 CD4+ and CD8+ T cells against MOG35-55: Atigenic CD4+
and CD8+ T cells were cultured with the actual antigenic peptide (MOG35-55)
and APCs and compared with the CD4+ and CD8+ T cells cultured under the
plate-bound anti-CD3/CD28 antibody conditions.
2. Proinflammatory cytokine production: The expression of proinflammatory
cytokines was checked after culturing 1C6 CD4+ and CD8+ T cells under T
cell culture conditions.
3. Effect of sex on disease progression: Our lab has investigated the varied effect
of sex on disease progression after the adoptive transfer of CD4+ T cells in
[Link] mice [148]. Here we assessed the disease course induced after the
transfer of sex matched 1C6 Tc17 cells in male and female [Link] mice.
4. Tracking CD4+ T cell emergence: Observing the rise of CD4+ T cells after the
adoptive transfer of CD8+ T cells was an interesting finding, so we looked at
the time frame of CD4+ T cell rise after Tc17 cell transfer at day 7, day 14 and
day 21.
5. Role of CD4+ T cells in CD8-induced EAE: Since both CD8+ T cells and the
CD8+ T cell generated CD4+ T cells appeared to play an important role in
developing EAE, we evaluated their contribution in the disease induction by
blocking one or the other after the CD8+ T cell adoptive transfer.
6. Combined effect of CD4+ and CD8+ T cells on EAE: In an EAE disease
course, the disease induction and progression are very important. In
experiment #3, we observed the progressive disease when the percentage of
CD4+ and CD8+ T cells were almost equal. So, we decided to check the effect
on the disease progression by transferring an equal number of CD4+ and CD8+

67
T cells together and comparing them with only CD4+ T cells and only CD8+
T cell transfers.

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Chapter 1. Serpine1 negatively regulates Th1 cell
responses in experimental autoimmune

Irshad Akbar*, #, Ruihan Tang†, Joanie Baillargeon*, Andrée-Pascale Roy*, Prenitha Mercy
Ignatius Arokia Doss*, Chen Zhu†, Vijay K. Kuchroo†,‡,§,||, Manu Rangachari*,¶, ||, **,††, ‡‡

*Axe Neurosciences, Centre de recherche du CHU de Québec-Université Laval, Québec

QC Canada;† Evergrande Center for Immunologic Diseases, Harvard Medical School,

Brigham and Women’s Hospital, Boston MA USA;‡Ann Romney Center for Neurologic

Diseases, Harvard Medical School, Brigham and Women’s Hospital, Boston MA USA;§

Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge MA USA;¶

Department of Molecular Medicine, Université Laval, Québec, QC, Canada;|| Address

correspondence and reprint request to Dr. Manu Rangachari or Dr. Vijay K. Kuchroo; Axe
Neurosciences, Centre de recherche du CHU de Québec-Université Laval, 2705 boul
Laurier, Québec QC G1V 4G2 (M.R) or Brigham and Women’s Hospital, Hale Building for
Transformative Research, 60 Fenwood Rd, HBTM 10016F, Boston MA 02115.

#funding to I.A., MS Society of Canada Doctoral Studentship

**funding to M.R., MS Society of Canada Discovery Grant #3781

††funding to M.R., Canadian Institutes of Health Research (CIHR) Project Grant #159713

‡‡funding to M.R., Senior scholar award, Fonds de recherche de Québec – Santé #313330

Contact information for corresponding authors:

[Link]@[Link], vkuchroo@[Link]
Phone (M.R): +1-418-525-4444 x 46461
Fax (M.R): +1-418-654-2298

69
1.1 Résumé

Les cellules Th1 ont un rôle essentiel dans le modèle de l’encéphalomyélite auto-immune
expérimentale (EAE). Il est connu que l’inhibiteur de la sérine protéase clade E1 (Serpine1)
réduit l’expression de l’interféron-gamma (IFN-γ) dans les lymphocytes T. Toutefois, son
rôle dans l’auto-immunité n’est pas défini. Dans cette étude, nous avons démontré que les
souris Serpine1 knockout (KO) développent une EAE plus sévère par rapport aux souris
contrôles (WT). La surexpression de Serpine1 dans les cellules Th1 diminue la production
de cytokines ainsi que la pathogénicité de ces mêmes cellules. Lors de protocoles de transfert
adoptif, les cellules Th1 de souris Serpine1-KO:2D2 déclenchent une EAE de plus grande
sévérité que l’EAE induite par les cellules Th1 de souris 2D2 WT. De plus, lors de la
polarisation des cellules Th1 de souris Serpine1-KO, nous constatons une expression retardée
du récepteγur inhibiteur Tim-3 (T cell immunoglobulin and mucin domain-containing-3), un
récepteur Th1-spécifique. La comparaison des cellules Th1 Tim-3-Tg WT et des cellules Th1
Serpine1-KO:Tim3-Tg qui se caractérisent par la surexpression du transgène Tim-3,
démontrent une augmentation de l’expression de l’IFN-γ et une expression réduite de Lag-
3 et PD-1, des molécules checkpoint inhibitrices. De plus, le déficit en Serpine1 restaure le
phénotype EAE des souris Tim3-Tg en protocole qui, normalement, développent une maladie
de faible gravité. Finalement, l’ensemble de ces résultats confirment que Serpine1 est un
régulateur inhibiteur des cellules Th1.

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1.2 Abstract

Th1 cells are critical in experimental autoimmune encephalomyelitis (EAE). Serine protease
inhibitor clade E1 (Serpine1) has been posited as an inhibitor of IFN-γ from T cells, although
its role in autoimmunity remains unclear. In this study, we show that Serpine1 knockout (KO)
mice develop EAE of enhanced severity relative to wild-type (WT) controls. Serpine1
overexpression represses Th1 cell cytokine production and pathogenicity, whereas Serpine1-
KO:2D2 Th1 cells transfer EAE of increased severity in comparison with WT 2D2 Th1 cells.
Notably, polarized Serpine1-KO Th1 cells display delayed expression of the Th1-specific
inhibitory receptor, Tim-3 (T cell Ig and mucin domain containing-3). Serpine1-KO:Tim-3-
Tg Th1 cells, which transgenically overexpress Tim-3, showed increased expression of IFN-
γ and reduced expression of the checkpoint molecules Lag-3 and PD-1 relative to WT Tim-
3-Tg counterparts. Furthermore, Serpine1 deficiency restored the EAE phenotype of Tim-3-
Tg mice that normally develop mild disease. Taken together, we identify Serpine1 as a
negative regulator of Th1 cells.

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1.3 Introduction

Effector CD4 Th1 cells are potent initiators and propagators of autoimmune diseases, such
as in the T cell-driven experimental autoimmune encephalomyelitis (EAE) model of multiple
sclerosis (1). Th1 cells express a lineage-specific inhibitory receptor, Tim-3 (T cell Ig and
mucin domain containing-3), that resolves inflammatory responses when triggered at sites of
inflammation (2, 4). The Tim-3 pathway is crucial to repressing EAE pathology (2, 5, 6).
Understanding the processes regulating Tim-3 expression and function might permit us to
develop strategies to curb Th1-mediated inflammation in self-tissue. Serine protease inhibitor
clade E1 (Serpine1), or plasminogen activation inhibitor 1, represses the conversion of the
plasminogen proenzyme into mature plasmin and was previously suggested to regulate IFN-
γ-driven T cell responses (7, 8). In this study, we show that Serpine1 restrains EAE
pathogenicity via its inhibitory effects on Th1 cells and that Serpine1 is required for optimal
expression of Tim-3 by Th1 cells.

1.4 Results

1.4.1 Serpine1 inhibits the severity of EAE

Upon immunization with MOG35_55 Serpine1-KO mice developed EAE of significantly

greater severity than did WT controls (Fig. 1.1A). Notably, Ag-specific proliferation
responses to MOG35_55 were enhanced in peripheral Serpine1-KO CD41 T cells prior to
clinical onset (Fig. 1B), as was secretion of IFN-γ and IL-2, but not IL-17 (Fig. 1.1C).
Serpine1 regulates CNS fibrinolysis (11) and thus heightened EAE severity in Serpine1-KO
mice might be due to its absence in a non-T cell compartment. We thus crossed Serpine1-
KO mice to the 2D2-Tg strain, which bears a MOG35_55-specific TCR (12), and immunized
WT 2D2 and Serpine1-KO:2D2 mice. Prior to disease onset, we isolated splenocytes and
restimulated them with Ag plus IL-12 and IL-23. Serpine1-KO:2D2 Ag-restimulated blasts
induced EAE of significantly greater severity relative to WT 2D2 blasts upon adoptive
transfer (Fig. 1.1D). Previous EAE studies that directly targeted Serpine1 in vivo revealed

72
conflicting results, possibly due to opposing effects on T cells and CNS repair (13, 14).
Notably, in the chronic/relapsing Biozzi model of EAE, Serpine1-KO mice developed EAE
of delayed onset and milder severity, due to superior CNS fibrinolytic capacity compared
with controls (11); it is possible that the fibrogenic, pathogenic properties of Serpine1
outweigh its anti- inflammatory function in that model. In this study, we show that Serpine1
represses Ag- specific T cell-driven CNS autoimmunity in a T cell-intrinsic manner.

73
74
Figure 1.1. Serpine1 inhibits the severity of EAE. (A) Left, Representative EAE curve of
MOG35_55-immunized WT (n = 9) and Serpine1-KO (n =7; abbreviated S1-KO) female mice.
Middle, Linear regression curves of the representative disease courses. The dashed lines
indicate the 95% confidence intervals for each curve. Right, Area under the curve (AUC)
comparison of mice pooled from three experiments; female mice (n =14; WT, n = 12,
Serpine1-KO); *, male mice (n =10; WT, n = 10, Serpine1-KO). Incidence of EAE over all
experiments was 24 of 25 WT, 22 of 24 Serpine1-KO. **p < 0.01 by two way-ANOVA
analysis of genotype as a variable. (B) MOG35_55-immunized female WT and Serpine1-KO
mice (n = 3 each) were sacrificed 10 d postimmunization, and lymph node cells were
stimulated (or not) with 10 ug/ml MOG35_55. Proliferation was assessed at 48 h. *p < 0.05 by
t test. (C) Splenocytes from immunized female WT and Serpine1-KO mice (n 5 3 each) were
restimulated (or not) with 10 ug/ml MOG35_55 for 48 (IL-2) or 72 h (IFN-γ, IL-17), and
secretion of the indicated cytokines was measured by ELISA. In (B) and (C), each data point
represents an individual animal. *p < 0.05 by t test. (D) Splenocytes from female 2D2 and
Serpine1-KO:2D2 mice were restimulated with MOG35_55 in the presence of IL-12 and IL-
23 and were transferred to WT mice (n = 5 each condition; one transfer) that were monitored
for signs of EAE. Right, Linear regression analysis with 95% confidence intervals. ****p <
0.0001.

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1.4.2 Serpine1 suppresses Th1 cell function in vitro and in vivo

We next found that Serpine1 secretion was sharply upregulated in both Th1 (Fig.1.2A) and
Th17 cells (Fig.1.2B) upon a second round of in vitro differentiation. Serpine1-deficient T
cells can produce increased levels of IFN-γ relative to WT cells in vivo (7, 8); however, the
functional role of Serpine1 in differentiated bona fide Th1 and Th17 cells has never been
directly assessed. Upon retroviral (RV)overexpression (OE) of Serpine1 in Th1 cells, we
observed a downregulation in expression of IFN-γ and a trend toward reduced TNF-a relative
to control-transduced cells. In contrast, OE of Serpine1 in Th17 cells did not impact
expression of either IL-17 or TNF-a (Fig.1.2C). To determine whether enforced expression
of Serpine1 altered Th1 cell pathogenicity, we transduced 2D2 Th1 cells with Serpine1-OE
or control RV and adoptively transferred these cells to Rag1−/− mice (6). Interestingly,
Serpine1-OE 2D2 Th1 cells induced disease of lessened severity compared with control cells
(Fig. 1.2D), characterized by a reduced frequency of inflammatory IFN-γ +IL-2+ T cells in
vivo (Fig. 1.2E).

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77
Figure 1.2 Serpine1 is expressed in Th1 cells and suppresses Th1-driven EAE. (A and
B) Naive CD41CD62Lhi T cells were isolated from female B6 mouse spleen and were
differentiated under Th1 or Th17 conditions for two rounds of polarization. Secretion of
Serpine1 protein was measured by ELISA in supernatant from Th1 (A) or Th17 (B) by
ELISA. Data points represent four independent cultures. *p < 0.05, ***p < 0.001 by one-way
ANOVA. (C) Female B6 Th1 or Th17 cells were transduced with control or Serpine1-OE
RV. Cells were analyzed for production of the indicated cytokines after 5 d. Cells were gated
on GFP-positive live events. Quantitation represents t test analysis of three independent pairs
of control versus Serpine1-OE RV cultures. *p < 0.05. (D) Female 2D2 Th1 cells were
transduced with control (n = 5) or Serpine1- OE (n = 4) RV (one transfer). After 5 d of
stimulation, cells were adoptively transferred to Rag1 −/− recipients who were monitored for
signs of EAE. Right, Linear regression curves and 95% confidence intervals for the disease
courses.

78
1.4.3 Loss of Serpine1 function or expression exacerbates Th1 responses

We next treated Th1 cells with tiplaxtinin, a small molecule that inhibits the antiproteolytic
activity of Serpine1 toward the plasminogen activators urokinase and tissue plasminogen
activator (15, 16). Tiplaxtinin enhanced the frequency of IFN-γ +IL-2+ Th1 cells (Fig.1.3A)
suggesting that Serpine1 may downregulate Th1 responses by repressing the activation of
mature plasmin. We then generated Th1 cells from WT 2D2 and Serpine1-KO:2D2 mice and
found that the latter induced EAE of significantly greater severity upon adoptive transfer
(Fig.1.3B).

Increased IFN-γ was previously observed from Serpine1-KO CD41 and CD81 T cells upon
LPS or staphylococcal enterotoxin B treatment in vivo (7). Furthermore, Serpine1-KO mice
are resistant to nasal allergy in a Th2-mediated OVA sensitization model, with IFN-γ
production upregulated by Serpine1-KO splenocytes upon Ag recall (8). In this study, we
show that while Serpine1 is expressed by both Th1 and Th17 cells, it impacts Th1 cells
specifically by downregulating their inflammatory cytokine production and autoimmune
potential.

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80
Figure 1.3 Loss of Serpine1 function or expression exacerbates Th1 responses. (A)
Female B6 Th1 cells were treated with DMSO (control) or 25 uM tiplaxtinin for 5 d, at which
point IFN-γ and IL-2 were measured by flow cytometry. *p < 0.05 by t test analysis of three
independent pairs of control versus tiplaxtinin-treated cultures. (B) Female 2D2 and
Serpine1-KO:2D2 CD41 Th1 cells were transferred i.v. to Rag1−/− recipients (2 × 10 6
cells/mouse). 2D2, n =4; Serpine1-KO:2D2, n =5. Mice were subsequently assessed for signs
of EAE. Left, linear regression curves of the disease courses. Results are representative of
two experiments. ****p < 0.0001.

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1.4.4 Serpine1 promotes Tim-3 expression and inhibits Tim-3 Th1 cell inflammatory
responses

As Serpine1 inhibits Th1-driven inflammation, we next asked whether it could augment the
expression of inhibitory Tim-3. Repeatedly polarized Serpine1-KO Th1 cells expressed
lower Tim-3, and with delayed kinetics, relative to S1-WT controls (Fig.1.4A). Loss of
Serpine1 signalling did not increase expression of IFN-γ however, the frequency of IFN-γ+
Tim-3+ Th1 cells was strikingly lower in its absence (Fig.1.4B).

Tim-3-Tg mice ectopically overexpress Tim-3 cDNA in a T cell-restricted manner, without

the need for multiple rounds of polarization under Th1 conditions (17). Reasoning that this
might help us uncover Serpine1-dependent differences in IFN-γ expression, we crossed
Serpine1-KO mice to the Tim-3-Tg strain and generated Th1 cells from these and Tim-3-Tg
control mice. Tim-31 Th1 cells derived from Serpine1-KO:Tim-3-Tg mice were strikingly
more positive for IFN-γ than Tim-3-Tg counterparts; notably, no differences were observed
with Tim-3−Th1 cells between the strains (Fig.1.4C). This indicated that Serpine1 represses
IFN-γ expression in a Tim-3dependent manner. Interestingly, there was a concomitant
reduction in the expression of the T cell negative regulatory receptors PD-1 and Lag-3 in
Serpine1-KO:Tim-3-Tg Th1 cells when compared with Tim-3-Tg controls (Fig.1.4D).

Tim-3-Tg mice develop EAE of attenuated severity (5). To examine whether loss of Serpine1
expression could reverse this phenotype, we actively immunized WT, Tim-3-Tg, and
Serpine1:KO Tim-3-Tg mice. Whereas Tim-3-Tg mice developed disease of only mild
severity as expected, Serpine1-KO:Tim-3-Tg mice displayed EAE that was of comparable
severity to that seen in WT animals (Fig.1.4E). Our data thus support a model in which
Serpine1 is required for Tim-3-mediated repression of T cell inflammation and pathogenicity
in EAE. Tim-3 marks exhausted T cells in chronic viral infections and cancers and is
functionally tractable, as concomitant blockade of Tim-3 and PD-1 causes tumor regression
to a greater degree than that with antiPD-1 alone (20). Furthermore, depletion of Bat3, an
intracellular repressor of Tim-3 signaling, causes Th1 cells to adopt an exhausted-like
phenotype in vivo (6, 21). While Serpine1-mediated upregulation of Tim-3 may be desirable

82
in the context of autoimmunity, it remains to be seen what role, if any, Serpine1 plays in T
cell exhaustion.

Altogether, our data identify a novel Th1 cell-intrinsic regulatory mechanism. Serpine1
represses Th1 cell pathogenicity by inhibiting their production of inflammatory cytokines
and by restraining them from adopting a highly differentiated Tim-3phenotype. Strategies to
augment Serpine1 expression and function in Th1 cells could present an attractive therapeutic
option in autoimmune disease.

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84
Figure 1.4 Serpine1 promotes Tim-3 expression and inhibits Tim-31 Th1 cell
inflammatory responses. (A and B) Female WT and Serpine1-KO:T cells were subjected
to four successive rounds of Th1 polarization, and Tim-3 (A and B) and IFN-γ (B) were
assessed by flow cytometry. (A) Quantification of Tim-3 expression at the end of each round.
**p < 0.01 by one-way ANOVA. (B) Expression of Tim-3 versus IFN-γ after 20 d of total
culture (end of round 4). Cells were gated on live CD41 events. Results are representative of
four experiments. (C) Splenic T CD41 T cells from male Tim-3-Tg and Serpine1-KO:Tim-
3-Tg mice (n =6 independent cultures from each) were differentiated under Th1 conditions
for 5 d. CD4, Tim-3, and IFN-γ expression levels were assessed by flow cytometry, with
cells first gated on live CD41Tim-31 or live CD41Tim-3−. ***p < 0.001 by t test. (D) Splenic
T CD41 T cells from male Tim-3-Tg and Serpine1-KO:Tim-3-Tg mice (n 5 3 independent
cultures from each) were differentiated under Th1 conditions for 5 d. Lag-3 and PD-1
expression levels were assessed by flow cytometry. Cells were gated on live CD41Tim-31
events. *p < 0.05 by t test. (E) Female WT (n =4), WT Tim-3-Tg (n 5 5), and Serpine1-
KO:Tim-3-Tg mice (n =14) were immunized with MOG35_55 and were monitored for signs
of EAE. Right, Linear regression analysis of the disease curves with Bonferroni’s correction
applied. Results are representative of three immunizations. ****p < 0.0001.

85
1.5 Materials and Methods

1.5.1 Mice

Tim-3-Tg mice have been described (5). C57BL/6J (B6) wild-type (WT) (stock no. 000664),
Serpine1-knockout (KO) (B6.129S2-Serpine1tm1Mlg/J; no. 002507), 2D2-Tg (C57BL/6-
Tg(Tcra2D2,Tcrb2D2)1Kuch/J; no.006912), and Rag1−/− (B6.129S7-Rag1tm1Mom/J; no.
002216) mice were obtained from The Jackson Laboratory. Experimental and control
animals were cohoused at the animal facilities of Centre de Recherche du CHU de Quebec-
Universit´e Laval or Brighamand Women’s Hospital. All procedures were authorized by the
Animal Care Committee of Universit´e Laval or the Institutional Animal Care and Use
Committee of Harvard University.

1.5.2 Th cell differentiation

CD41 T cells were enriched from B6 spleens using anti-CD4 Micro Beads (Miltenyi Biotec),
purified as CD41CD62Lhi using a FACS Aria (BD Biosciences) high-speed cell sorter, and
cultured in supplemented T cell media as described (3). They were differentiated (9) for 2 d
with plate bound anti-CD3 and anti-CD28 (2 ug/ml each; Bio X Cell) into Th1 cells as
follows: 10 ng/ml recombinant mouse (rm)IL-12 (R&D Systems) plus antiIL-4 (10 ug/ml,
Bio X Cell). Th17 cells were generated with recombinant human TGF-b (3 ng/ml, Miltenyi
Biotec) 1 rmIL-6 (20 ng/ml, Miltenyi Biotec) 1 anti-IFN-γ (10 ug/ml, Bio X Cell). Cells
were then transferred to uncoated tissue culture plates and cultured for an additional 3 d, with
rmIL-2 (10 ng/ml, Miltenyi Biotec) added to Th1, and rmIL-23 (20 ng/ml, R&D Systems)
added to Th17. For multiple rounds of polarization, cells were collected at day 5 and then
restimulated for a subsequent 5-d period as above. Tiplaxtinin (25 uM, Tocris) or an
equivalent volume of DMSO was maintained for 5 d of culture where indicated. Serpine1
cDNA was cloned into pMSCV-IRESGFP (pMIG) vector containing a GFP bicistronic
reporter. Retroviral (RV) gene transduction of Th1 or Th17 cells was conducted using our
described spin-infection protocol (10).

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1.5.3 EAE model

Active immunization was induced by s.c. injection of myelin oligodendrocyte glycoprotein

aa 35-55 (MOG35-55) (CHU de Quebec) in IFA (Difco) supplemented with 5 mg/ml
Mycobacterium tuberculosis extract (Fisher Scientific). A dose of 25 ug of MOG35-55 per
mouse was used to compare WT to Serpine1-KO mice, whereas 100 mg per mouse was used
in EAE experiments involving Tim-3-Tg mice. In Fig. 1.1D, passive EAE was induced by
first immunizing Serpine1-WT 2D2 and Serpine1-KO 2D2 mice with MOG35-55. Nine days
later, splenocytes were collected and stimulated ex vivo for 48 h with MOG35-55 (20 ug/ml),
rmIL-12, and rmIL-23. Next, 20 × 106 splenocytes were injected i.p. into unimmunized B6
recipients. Adoptive transfer of WT 2D2, Serpine1-KO:2D2, or RV-transduced 2D2 Th1
cells entailed i.v. injection (2 × 106) into Rag1−/− recipients at day 5 of culture. In all EAE
experiments, mice received 200 ng of pertussis toxin (List Biological Labs) i.p. at days 0 and
2. Mice were assessed for clinical symptoms daily as previously described (9).

1.5.4 Flow cytometry

Cell surface cytometry and intracellular flow cytometry were conducted as previously
described (10). The following Abs and dyes were used: CD4, clone RM4-5, Thermo Fisher
Scientific, catalog nos. 45-0042-82 and 48-0042-82; CD62L, MEL-14, Thermo Fisher
Scientific, no. 47-0621-82; Tim-3, RMT3-23, BioLegend, no. 119706; PD-1, J43, Thermo
Fisher Scientific, no. 25-9985-82; Lag-3, eBioC9B7W, Thermo Fisher Scientific, no. 12-
2231-82; IFNc, XMG1.2, Thermo Fisher Scientific, no. 48-7311-82; TNFa, MP6-XT22,
Thermo Fisher Scientific, nos. 11-7321-82, 12-7321-41, and 17-7321-82; IL-2, JES6-5H4,
BD Biosciences, no. 560547; IL-17, TC11-18H10.1, BioLegend, no. 506922; fixable
viability dye, Thermo Fisher Scientific, no. 65-0865-14; 7-aminoactinomycin D, Thermo
Fisher Scientific, no. A1310. Data were collected using an LSR II flow cytometer (BD
Biosciences) and were analyzed with FlowJo (BD Biosciences). The following global
strategy was used: 1) singlets were selected based on forward scatter height versus forward
scatter area; and 2) live CD4+ T cells based on CD4+ viability dye−, or on CD4+ 7-
aminoactinomycin D− events. RV-transduced CD4+ T cells were further gated on GFP

87
positivity as indicated in the figure legends. In Fig. 1.4C and 1.4D, live CD4+ T cells were
gated as CD4+Tim-3+ or CD4+Tim-3−.

1.5.5 Ex vivo assessment of T cell function

Splenocyte cultures from EAE mice were stimulated (or not) with 10 mg/ml MOG35-55 for 48
h. For proliferation studies, 1.25 mCi of [3H]thymidine (PerkinElmer) was added to each
culture well for the last 16 h. Cytokine supernatant ELISAs were conducted using the
following capture/detection sets: IFNc, clones RA-6A2/XMG1.2; IL-2, JES6-1412/JES6-
5H4; IL-17, TC11-18H10.1/TC11-8H4. Serpine1 protein was measured using Serpin
E1/PAI-1 (plasminogen activation inhibitor 1) DuoSet ELISA (R&D Systems).

1.5.6 Statistical analysis

Two-tailed parametric tests were conducted using Prism (GraphPad). Comparisons of two
groups were made by a t test. Comparisons in experiments involving multiple rounds of T
cell culture were made by one-way ANOVA. In EAE studies, linear regression (6, 10) or area
under curve (10) analyses were conducted on mice with symptoms.

1.6 Data availability

The datasets generated for this study are available from the corresponding authors upon
reasonable request.

1.7 Acknowledgments

We thank Vincent Desrosiers for technical assistance, Kim Larose-Labrecque and Andrée
Brisson for animal care, and Ryder Whittaker Hawkins for critical reading of the manuscript.
Biorender was used to generate the visual abstract.

1.8 Disclosures

88
V.K.K. has an ownership stake and is a member of the Scientific Advisory Board for Tizona
Therapeutics. Furthermore, he is a cofounder of, and has an ownership stake in, Celsius
Therapeutics. In addition, he is an inventor on patents related to Th17 cell function. His
interests are reviewed and managed by the Brigham and Women’s Hospital and Partners
Healthcare in accordance with their conflict-of-interest policies. The other authors have no
financial conflicts of interest.

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J. B. Williams, M. Leclercq, C. Joly-Beauparlant, et al. 2021. Male sex chromosomal
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11. East, E., D. Gveri´c, D. Baker, G. Pryce, H. R. Lijnen, and M. L. Cuzner. 2008. Chronic
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2003. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop
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13. Gur-Wahnon, D., T. Mizrachi, F.-Y. Maaravi-Pinto, A. Lourbopoulos, N. Grigoriadis,

A.-A. R. Higazi, and T. Brenner. 2013. The plasminogen activator system: involvement in
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15. Elokdah, H., M. Abou-Gharbia, J. K. Hennan, G. McFarlane, C. P. Mugford, G.

Krishnamurthy, and D. L. Crandall. 2004. Tiplaxtinin, a novel, orally efficacious inhibitor of
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Wong, M. Satkunarajah, M. Schweneker, J. M. Chapman, et al. 2008. Tim-3 expression
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19. Jin, H.-T., A. C. Anderson, W. G. Tan, E. E. West, S.-J. Ha, K. Araki, G. J. Freeman, V.
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91
Chapter 2. CD4+ T cells are important for Tc17 mediated
progressive EAE in an adoptive transfer EAE model

Irshad Akbar1, Prenitha Mercy Ignatius Arokia Doss1, Joanie Baillargeon1, Mohamed Reda
Fazazi1, Ana C Anderson2, Manu Rangachari1,3,4

1
Axe Neurosciences, Centre de recherche du CHU de Québec-Université Laval, 2705 boul
Laurier, Québec QC CANADA, G1V 4G2; 2 Evergrande Center for Immunologic Diseases
and Ann Romney Center for Neurologic Diseases, Harvard Medical School and Brigham &
3
Women's Hospital, 60 Fenwood Road, Boston, MA 02115, USA; Faculty of Medicine,
Université Laval, 1050 ave de la Médecine, Quebec City, QC, Canada.4 corresponding author

Contact information for corresponding author:

Manu Rangachari, Ph.D.

Axe Neurosciences

Centre de recherche du CHU de Québec-Université Laval

Pavillon CHUL, T4
2705 boul Laurier, Québec, QC Canada G1V 4G2
[Link]@[Link]
Tel: 1-418-525-4444 x 46461

92
2.1 Résumé

Les lymphocytes T CD4+ et CD8+ jouent tous deux un rôle essentiel dans
l’immunopathogénèse de la sclérose en plaques. Les lymphocytes T des souris transgéniques
1C6 du background NOD possèdent un récepteur transgénique (TcR-Tg) spécifique au MOG
[35-55]. Il est restreint au complexe majeur d’histocompatibilité de classe II (MHC II) et
sélectionne à la fois les cellules CD4+ et CD8+. Nous avons récemment démontré que les
cellules 1C6 CD4+ T helper 17 (Th17) induisent une encéphalomyélite auto-immune
expérimentale (EAE) de forme progressive lors de leur transfert adoptif dans des souris
receveuses [Link]. Dans cette étude, nous avons étudié la fonction et la pathogénicité des
lymphocytes T 1C6 CD8+. Nous avons démontré qu’ils prolifèrent difficilement en présence
du peptide MOG [35-55] dans les conditions de différentiation en cellules T cytotoxiques 1
(Tc1) et 17 (Tc17). Par contre, nous avons observé que ces cellules répondent fortement à la
stimulation combinée avec l’anti-CD3 et l’anti-CD28, indiquant qu’elles n'ont pas de défaut
intrinsèque dans leur capacité à proliférer. Le transfert adoptif des cellules Tc17 dans les
souris [Link] immunosupprimées déclenche une maladie progressive sévère caractérisée
par l’infiltration de cellules T CD8+ dans le SNC et ces dernières expriment de hauts niveaux
d’IL-17, IFN-γ et de TNFα. Étonnamment, nous avons également noté une expansion in vivo
des cellules T CD4+ dans la rate et le SNC de ces mêmes souris [Link] en dépit de la
purification des cellules CD8+ qui précède leur différentiation en Tc17 et le transfert dans les
souris receveuses. Plusieurs évidences montrent l’expansion des cellules CD4+ lors du
transfert des cellules 1C6 Tc17 dans les souris [Link] déficientes en lymphocytes. De
plus, le blocage des lymphocytes T CD4+ avec un anticorps spécifique prévient la maladie
chez les souris receveuses de Tc17. L’ensemble de nos données démontrent que les cellules
effectrices T CD8+ réactives au MOG [35-55] collaborent avec les cellules T CD4+ dans le
développement de l’EAE.

93
2.2 Abstract

Both CD4+ and CD8+ T cells play critical roles in the immunopathogenesis of MS. 1C6 T
cell receptor transgenic (TcR-Tg) mice on the nonobese diabetic (NOD) background have a
MOG[35-55]-specific, MHC class II-restricted, TcR that selects for both CD4+ and CD8+ T
cells. We recently demonstrated that 1C6 CD4+ T helper 17 (Th17) cells can induce a
progressive form of experimental autoimmune encephalomyelitis (EAE) upon adoptive
transfer to [Link] recipient mice. In the current study, we wanted to assess the function
and pathogenicity of 1C6 CD8+ T cells. We found that they proliferated poorly in response
to MOG[35-55] peptide under both T cytotoxic 1 (Tc1) and Tc17 differentiation conditions.
However, they responded strongly to anti-CD3anti-CD28 stimulation, indicating that they
had no intrinsic defect in proliferative capacity. Adoptive transfer of Tc17 cells to [Link]
mice caused severe progressive disease characterized by the infiltration into the CNS of CD8+
T cells that were highly positive for IL-17, IFN-γ and TNFα. Intriguingly, we noted a striking
in vivo expansion of CD4+ T cells in the spleen and CNS of recipient mice, despite Tc17
cells being purified on CD8 expression prior to transfer and [Link] mice not having
endogenous lymphocytes. We found evidence of CD4+ expansion upon transfer of 1C6 Tc17
cells to lymphocyte-sufficient animals; further, anti-CD4+ T cell blockade abrogated disease
in Tc17 recipient mice. Together, our data show that MOG[35-55]-reactive CD8+ effector T
cells induce EAE in collaboration with CD4+ T cells.

Keywords: multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), T

cell, Tc1 cell, Tc17 cell, nonobese diabetic (NOD)

94
2.3 Introduction

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease of the central

nervous system (CNS) that affects nearly 2 million people worldwide [110]. MS is
characterized by an adaptive immune cell-mediated attack against CNS myelin constituents,
though reactivity to other CNS antigens has also been observed [327]. Approximately 80%
of people with MS (pwMS) will initially experience a relapse-onset form of the disease that
is relatively controllable by existing disease-modifying treatments. However, 30-60% of
these individuals will eventually transition to secondary-progressive (SP) MS, while 20% of
all MS patients show progressive disease from the outset (primary progressive, PP).
Treatment for progressive forms of MS is currently limited, leaving these individuals to face
decades of accumulating symptoms [328].

Experimental autoimmune encephalomyelitis (EAE) is a well-accepted animal disease for

the study of MS, as it permits the study of numerous elements of the MS pathogenic cascade
[329]. EAE can be induced either by active immunization with encephalitogenic peptides or
proteins, or by the transfer of CNS-antigen specific lymphocytes; different models of EAE
exhibit variations in clinical presentation, depending on factors such as method of induction,
target immunogen, and genetic background [330]. Notably, nonobese diabetic (NOD)
background mice can develop a chronically worsening disease pattern over the course of
several months upon either active immunization with myelin oligodendrocyte glycoprotein
35-55 (MOG35-55) peptide [331], [332], [333] or upon adoptive transfer of MOG35-55-specific
Th17 cells [148]. NOD-EAE, therefore, permits us to study mechanisms that regulate
progressive forms of CNS autoimmunity.

The majority of EAE models feature a CD4+ T cell-initiated pathology, either because they
depend on immunization with an MHC class II-restricted autoantigenic peptide or because
they require the adoptive transfer of CD4+ T cells. Nevertheless, a number of CD8+ T cell-
dependent models have been described; together, they show that EAE can arise from class I-
restricted T cell responses characterized by the influx of inflammatory CD8+ T cells into the
CNS and by lesions in both white and grey matter [256], [334]. However, the role of CD8+
T cells in driving progressive forms of EAE remains obscure.

95
NOD-background T cell receptor transgenic (TcR-Tg) 1C6 mice possess an MHC class II-
restricted TcR with specificity for MOG35-55. Surprisingly, both CD4+ and CD8+ MOG35-55-
reactive T cells are detected in the immune periphery of these animals [201]. We recently
showed that adoptive transfer of in vitro-differentiated male 1C6 T helper 17 (Th17) effector
cells caused a severe progressive disease course in more than half of recipients [148]. The
goal of the current study was to investigate the function of 1C6 T cytotoxic 1 (Tc1) and Tc17
cells, and to ascertain their capacity to induce progressive disease upon adoptive transfer.

2.4 Results

2.4.1 1C6 CD8+ T cells respond suboptimally to MOG35-55 peptide but show no intrinsic
proliferative defect

The class II-restricted 1C6 TcR is expressed by both mature CD8+ and CD4+ T cells, despite
the fact that CD8+ T cells typically possess a class I-restricted TcR. It was previously shown
that 1C6 CD8+ T cells proliferate in response to MOG35-55 and that this is abrogated upon
blockade of the NOD class II allele I-Ag7 [201]. Here, we wanted to directly compare the
proliferative capacity of 1C6 Tc1 and Tc17 cells to that of Th1 and Th17 cells. T cell
stimulation was achieved using two different stimuli: with MOG35-55-pulsed irradiated
splenocytes to test their ability to respond to cognate antigen or with plate-bound agonistic
antibodies to CD3 plus CD28 that would trigger T cell responsiveness in an antigen-
independent manner. After 5 days of culture under well-defined type-1 and type-17 culture
conditions [256], CD8+ Tc1 and Tc17 cultures showed distinctly reduced cellularity relative
to their CD4+ Th1 and Th17 counterparts when cultured with MOG35-55-pulsed irradiated
splenocytes. Interestingly, despite the low cell count of peptide-stimulated Tc1 and Tc17
cells, their proliferative index trended towards being higher than that of their Th1 and Th17
counterparts (Fig. 2.1A). We similarly assessed 1C6 CD4+ and CD8+ T cells under type-1
and type-17 conditions, but the T cells were stimulated with anti-CD3+CD28. No differences
in cellularity between Tc1, Tc17 and Th1, Th17 cultures were observed when they were
stimulated with anti-CD3+CD28 (Fig. 2.1B), demonstrating that there was no intrinsic defect
in the ability of Tc1 and Tc17 cells to differentiate. Further, there were no differences in
96
proliferative index when the cells were stimulated with anti-CD3+CD28. Overall, our data
indicated that the proliferative responses of 1C6 effector Tc1 and Tc17 cells to cognate
MOG35-55 antigen were defective, though not completely absent. Further, there was no
intrinsic defect in the capacity of 1C6 effector T cells to proliferate.

97
A MOG35-55 pulsed
APCs
CTV+ CTV+

Tc1 Tc17

Th1 Th17
% of max

Cell trace violet

✱✱✱ ns

Proliferation Index
✱ ✱✱✱ 4
1000000

3
Cell count

800000

600000
2
400000
1
200000

0 0
Th1 Tc1 Th17 Tc17 Th1 Tc1 Th17 Tc17

B Anti-CD3+anti-CD28
CTV+ CTV+
Tc1 Tc17
Th1 Th17
% of max

CellTrace Violet
ns ns
Proliferation Index

ns ns 3
400000
Cell count

300000 2

200000
1
100000

0 0
Th1 Tc1 Th17 Tc17 Th1 Tc1 Th17 Tc17

98
Figure 2.1 1C6 CD8+ T cells respond suboptimally to MOG[35-55] peptide but show no
intrinsic proliferative defect. A. 1C6 CD8+ and CD4+ T cells were stimulated with MOG35-
55 pulsed APCs under Tc1/Th1 culture conditions (top left) or under Tc17/Th17 culture
conditions (top right). 1C6 and cell proliferation was assessed by CellTrace Violet dilution
after 3 days of culture. B. 1C6 CD8+ and CD4+ T cells were stimulated with plate-bound anti-
CD3+CD28 under Tc1/Th1 culture conditions (bottom left) or under Tc17/Th17 culture
conditions (bottom right) and cell proliferation was assessed at d3. *, p<0.05; ***, p<0.001;
n.s, not significant; two-tailed t-test. Each replicate represents a culture derived from an
individual mouse. Data representative of 2 experiments.

99
2.4.2 Assessment of cytokine production from 1C6 effector T cells

We next assessed cytokine production from 1C6 effector Tc and Th cells. Upon stimulation
with MOG35-55-pulsed splenocytes, a lower frequency of Tc1 cells were positive for
production of IFN-γ and TNF relative to Th1 comparators In assessing type-17 cultures, we
found that similar frequencies of Tc17 and Th17 cells were positive for IL-17 MOG35-55-
stimulated Tc17 cells robustly generated IFN-γ; by contrast, unsurprisingly, Th17 cells were
unable to produce this cytokine. In addition, a significantly greater proportion of Tc17 cells
were positive for TNF as compared to Th17 counterparts (Fig. 2.2A)

To assess the intrinsic capacity of 1C6 Tc1 cells to generate inflammatory cytokines, we
stimulated them with plate-bound anti-CD3+CD28; under these conditions, Tc1 production
of IFN-γ and TNF exceeded that of Th1 cells. Upon plate-bound stimulation in 1C6 Tc17
and Th17 culture conditions, the percentage of IL-17+ and TNFα+ cells was no different
between the two culture conditions. Again, Tc17 cells showed stronger production of IFN-γ
than Th17 cells (Fig. 2.2B) Together, these data showed that 1C6 Tc1 and Tc17 showed no
intrinsic defects in inflammatory cytokine production. By contrast, peptide stimulation
revealed striking differences between these two effector CD8+ T cell subsets: while 1C6 Tc1
cells showed somewhat impaired cytokine production as compared to Th1 cells, Tc17 cells
were robustly cytokine-positive when compared to Th17 comparators.

100
A MOG35-55 pulsed APCs
Th1 Th17
CD4

Tc1 Tc17
CD8

IFNγ TNF IL-17 IFNγ TNF IL-17

✱✱✱ ✱✱ ns
✱ ✱✱✱ ✱✱✱✱
100 100
Th1 Th17
80 80
% of cells

% of cells
Tc1 Tc17
60 60

40 40

20 20

0 0
γ F 7

7
γ

F
N TN -1
B

-1
N

TN
IF IL
IF

IL
Anti-CD3+anti-CD28

Th1 Th17
CD4

Tc1 Tc17
CD8

IFNγ TNF IL-17 IFNγ TNF IL-17

✱✱✱ ns ns 100
100
Th1 Th17
80 ✱✱✱✱ ns ✱
% of cells
% of cells

80
Tc1 Tc17
60 60

40 40

20 20

0 0
7
Nγ

F
7
Nγ

-1
TN
-1
TN

IF
IF

IL
IL

101
Figure 2.2 Assessment of cytokine production from 1C6 effector T cells. A. Female 1C6
CD8+ and CD4+ T cells were stimulated with MOG35-55 pulsed APCs under Tc1/Th1 culture
conditions (top left) or under Tc17/Th17 culture conditions (top right). Expression of the
indicated cytokines was assessed by flow cytometry after 5 days of culture. B. Similarly,
cytokine positivity was assessed at d5 after stimulating 1C6 CD8+ and CD4+ T cells with
plate-bound anti-CD3+CD28 under Tc1/Th1 culture conditions (bottom left) or under
Tc17/Th17 culture conditions (bottom right). Gated on live CD4+ or live CD8+ T cells. *,
p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; n.s., not significant; two-tailed t-test.
Each replicate represents a culture derived from a distinct mouse. Data representative of 2
experiments.

102
2.4.3 1C6 Tc1 and Tc17 cells adoptively transfer EAE

We next wanted to ascertain whether 1C6 effector CD8+ T cells were encephalitogenic. We
had previously shown that plate-bound-stimulated (anti-CD3anti-CD28) 1C6 Th1 and Th17
cells could induce EAE upon adoptive transfer to [Link] recipient mice. As we had found
that 1C6 Tc1 and Tc17 cells showed robust activation in vitro upon the same stimulation
parameters, we used our previous experimental paradigm [148] to assess their capacity to
cause EAE (Fig. 2.3A).

We first purified CD8+CD62Lhi naïve T cells from the spleens and lymph nodes of female
1C6 mice and differentiated them under Tc1 conditions. We next adoptively transferred the
Tc1 cells (5x106 per mouse) to female [Link] recipients that we monitored for EAE
symptoms over 70 days. In parallel, we monitored recipients of an identical quantity of
female 1C6 Th1 cells that had similarly been differentiated from CD4+CD62Lhi cells, as well
as mice that received both Th1 and Tc1 cells (2.5x106 each). Tc1 recipients developed EAE
of delayed onset relative to those that received either Th1 cells alone or Th1+Tc1 cells
together (Fig. 2.3B); however, their disease burden was no less than that observed in the other
groups. Th1+Tc1 co-transfer recipients developed EAE with similar onset to Th1-alone
recipients and showed no increase in disease burden relative to the other groups.

NOD-background mice are prone to developing a chronic progressive disease course [331]
[335], [336]In our 1C6 adoptive transfer model, we had previously found that a small
frequency of female Th1 cell recipients (~20%) also display this phenotype [148]. Here, we
found that 3/10 Th1 recipients displayed chronic disease, as did 5/9 Tc1 and 6/10 Th1+Tc1
recipients (Fig. 2.3B).

We next wanted to ask whether 1C6 Tc17 cells could induce EAE. We therefore
differentiated Tc17 and Th17 cells from female CD8+CD26Lhi and CD4+CD62Lhi naïve
progenitors, respectively, and then adoptively transferred Tc17 cells alone, Th17 cells alone
and Th17+Tc17 cells together. We found that transfer of Tc17 cells alone caused disease that
was again of delayed onset, yet ultimately of similar overall severity, as compared to Th17
cells alone or of Th17 cells with Tc17 cells (Fig. 2.3C). In line with our previous data, a

103
minority (1/4) of female Th17 cell recipients developed progressive disease; of Tc17
recipients, 1/4 showed this phenotype, as did 2/3 Th17+Tc17 recipients (Fig. 2.3C).

Overall, our data indicated that 1C6 Tc1 and Tc17 cells induced EAE of comparable
severity to Th1 or Th17 counterparts, with a similar incidence of progressive disease;
however, disease onset was delayed in Tc1 or Tc17 recipients as compared to their Th
counterparts.

104
A
Type-1 or type-17
. naive differentiation
conditions
1C6
5d

naive anti-CD3+ anti-CD28

1C6

B CD4+

. Tc1 Transfer Th1 Transfer Tc1+Th1 Transfer

5 5 5

Disease Score
Disease Score
Disease Score

4 4 4

3 3 3

2 2 2

1 1 1

0 0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Days Days Days

ns ns n.s
10

Number of mice
2.0 ns ns Progressive
40 ✱✱ ✱
Th1 Th1 Non Progressive
Day of onset

Tc1 Tc1
AUC d-1

30 1.5
Th1+Tc1 Th1+Tc1 5

20 1.0

10 0.5
0
1
1+ 1
0.0 1
Th
Th Tc
Tc
0

Th17 Transfer Tc17+Th17 Transfer

C Tc17 Transfer 5 5
5
Disease Score
Disease Score

Disease Score

4 4
4

. 3 3
3

2 2
2

1 1
1

0 0
0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Days Days Days

ns
ns
5 n.s
Number of mice

40 5 ns ns
✱✱ ✱✱ Th17 Th17 Progressive
Day of onset

4
Tc17 4 Non Progressive
AUC d-1

30 Tc17
Th17+Tc17 3
3 Th17+Tc17
20 2
2
1
10
1
0
0 0
Th T 7
+T 7
7
1
17 c1
c1
Th

105
Figure 2.3 1C6 Tc1 and Tc17 cells adoptively transfer EAE. A. Schematic depicting
experimental approach used in this Figure. B. Female 1C6 Tc1 and Th1 cells were
differentiated in vitro and were injected i.v. into three groups as follows: Tc1 alone (5×10 6
cells, n=9), Th1 alone (5×106 cells, n=10), Th1+Tc1 (2.5×106 cells each, n=10). Graphs
represent EAE progression after adoptive transfer of Th1 alone, Tc1 alone or Th1+Tc1 co-
transfer; mice that reached a score of 5 were euthanized. The day the mice started to show
disease symptoms is shown in the day of onset graph, and the progressive disease graph
shows the disease burden within the groups. C. Female 1C6 Tc17 and Th17 cells
differentiated in vitro and were injected i.v. into three groups as follows: Tc17 alone (n=4),
Th17 alone (n=4) or Th17+Tc17 recipients (n=3). Graphs represent EAE progression after
adoptive transfer of Th17 alone, Tc17 alone or Th17+Tc17 co-transfer; mice that reached a
score of 5 were euthanized. The day the mice started to show disease symptoms is shown in
the day of onset graph, and the progressive disease graph shows the disease burden within
the groups. *, p<0.05; **, p<0.01; n.s, not significant; Tukey’s post-hoc test.

106
2.4.4 Male and female 1C6 Tc17 cells induce EAE of similar onset and severity

Fortuitously, we found that subjecting Tc17 cells to a second 5-day round of in vitro
differentiation (Fig.2. 4A) increased cellular yield (Fig.2. 4B). Further, it augmented the
cultures' encephalitogenic capacity by accelerating disease onset and increasing disease
burden in recipient mice relative to Tc17 cells stimulated by a single round (Fig. 2.4C). Thus,
from this point forward, we assessed Tc-driven EAE using 1C6 CD8+ T cells stimulated over
two rounds prior to adoptive transfer.

We had previously shown that adoptive transfer of male 1C6 Th17 cells caused disease of
higher overall severity relative to female Th17 counterparts; this was irrespective of the sex
of the recipients [148]. We thus wanted to ask whether 1C6 Tc17 pathogenicity was similarly
regulated by biological sex. Both female (3/6) and male (5/6) Tc17 cells could induce a
progressive disease course in sex-matched recipients. No differences were observed in either
time of onset or overall disease burden (Fig. 2.4D) between recipients of female or male Tc17
cells. Thus, unlike Th17 cell-driven EAE[148] Tc17 cells did not display sex-driven
differences in pathogenicity.

107
A One-round
B
.

Absolute cell count

Type-17 conditions
. 150000
✱✱
Round-1
Round-2
Two-round 100000

naive
50000

1C6
anti-CD3+
5d anti-CD3+
5d 0
CD8+ Tc17
anti-CD28 anti-CD28
C 5
Round-1 Round-2
Disease score

. 4

3
Disease Score 4

2 2

1 1

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Days Days
70 2.5 ✱
✱ Round 1 Round 1
Day of onset

60
Round 2
2.0 Round 2
d-1

50
1.5
AUC

40
30 1.0

20 0.5
10
0.0
0

D 5 5
Males
Disease Score

Disease score

Female
. 4 4

3 s 3

2 2

1 1

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Days Days

ns
2.5 Female
Day of onset

70 Female
60 ns
2.0 Male
d-1

50
Male
40 1.5
AUC

30 1.0
20
10 0.5
0 0.0

108
Figure 2.4 Male and female 1C6 Tc17 cells induce EAE of similar onset and severity. A.
Schematic depicting experimental approach with restimulated Tc17 cells. B. Comparison of
live CD8+ T cell yield from Tc17 cultures after one versus two rounds of stimulation. C.
Female Tc17 cells were generated using one-round and two-round stimulation protocols and
were adoptively transferred to female [Link] recipients. D. Tc17 cells were generated
from female and male 1C6 CD8+ T cells using the two-round protocol and were adoptively
transferred to sex-matched recipients. Graph showing the day of onset, represents the day
when the disease symptoms begin in the mice. Mice that reached score 5 were euthanized.
*, p<0.05; n.s., not significant; t-test.

109
2.4.5 Ex vivo analysis of T cells from Tc17 recipients

We next assessed T cell frequency and inflammatory markers from recipients of male versus
female Tc17 cells at experimental endpoints. The frequency and absolute number of CD8+ T
cells was no different between spleens of female versus male Tc17 recipients (Fig. 2.5A).
Curiously, and despite the fact that we had transferred CD8+ Tc17 cells alone, a substantial
frequency of CD4+ T cells were observed in both conditions.

To assess T cell infiltration in the target organ, we analyzed the presence of T cells in the
CNS of mice that received male or female Tc17 cells. We found a greater accumulation of T
cells in the CNS of male Tc17 recipients as compared to the CNS of female Tc17 recipients
(Fig. 2.5B), even though we had observed no differences in disease burden between the two
groups (Fig. 2.4E). Thus, while male sex did not increase the pathogenicity of Tc17 as it did
for Th17 cells [148], it did appear to facilitate the entry of T cells into the CNS, Again, a
substantial proportion of CD4+ T cells were observed from the recipients of both male and
female Tc17 cells (Fig.2.5B).

We next assessed inflammatory cytokine expression from T cells isolated ex vivo from these
adoptive transfer recipients. A substantial frequency of CD8+ T cells from both CNS and
spleen were positive for IL-17, IFN-γ and TNF, with no differences noted between the sexes
(Fig. 2.5C). As we had noted a striking proportion of CD4+ T cells in both spleen and CNS
of Tc17 recipients, we examined cytokine production from these cells in both compartments.
CD4+ T cells were robustly positive for IFN-γ and TNF but were less so for IL-17 (Fig. 2.5D).

CD8+ T cells directly damage tissue via the production of cytotoxic molecules such as
Granzyme B and perforin [337]. A modest proportion of CD8+ (Fig. 2.5E) and CD4+ (Fig.
2.5F) T cells were positive for these markers, irrespective of sex or compartment.

110
 A Female Spleen Male
.

% of CD8,CD4 T cells

absolute cell count

ns ns
80 250000
Female ns ns
Female
60 Male 200000 Male
150000
40
100000
20
50000
CD8

0 0
4 8

8
D D

CD

CD
C C
CD4

B CNS

% of CD8,CD4 T cells

absolute cell count

50 ✱ ✱ ✱ ✱
150000
Female

.
Female
40 Male Male
100000
30

20
50000

10
0
0
4 8
CD8

4 8 D D
D D C C
C C

CD4

C CD8 Spleen CD8 CNS

D CD4 Spleen
CD4 CNS
% of CD8+ T cells

.
% of CD8+ T cells

100 100 ns ns ns ns ns ns
ns ns ns

% of CD4+ T cells
% of CD4+ T cells

Female Female 80 100 ns ns ns

.
80 Male Female Female
80 Male Male
60
80 Male
60 60
60
40 40
40
40
20 20 20
20
0 0 0
γ 7 F 0
N -1
7
Nγ

TN
7
Nγ

F
-1

IF
TN

IL
-1

TN

7
Nγ

F
IF

IL

IF

-1

TN
IL

IF

IL

E ns
CD8 Spleen
ns
CD8 CNS F CD4 Spleen CD4 CNS

.
% of CD4+ T cells

ns ns

.
% of CD8+ T cells

% of CD8+ T cells

% of CD4+ T cells

20 30 20 ns ns 30
Female Female Female Female
15 Male Male 15 Male Male
20 20
10 10
ns ns
10 10
5 5

0 0 0 0
zf zf
f
zB

zB zB
Pz

f
zB

Pz

P P
G

G G
G

111
Figure 2.5 Ex vivo analysis of T cells from Tc17 recipients. A, B. Splenic (A) and CNS-
infiltrating (B) T cells were isolated from recipient animals in Figure 4 and were assessed for
expression of CD8 vs CD4. C, D. CD8+ (C) and CD4+ (D) T cells from recipient mice were
assessed for expression of the indicated cytokines. E, F. CD8+ (E) and CD4+ (F) T cells from
recipient mice were assessed for expression of Granzyme B (GzB) and perforin (Pzf). *,
p<0.05, n.s., not significant; t-test.

112
2.4.6 CD4+ T cells arise in [Link] recipients of 1C6 Tc cells

Our observation of CD4+ T cells at endpoints in Tc17 recipients was curious, as we had
transferred CD8+ T cells alone and [Link] mice lack endogenous lymphocytes. To
explore this further, we adoptively transferred Tc17 cells to [Link] recipients that we
sacrificed 7, 14 or 21 days later. In the spleen, we noted a comparable frequency of CD4+
and CD8+ T cells at day 7 and day 21, albeit with a significantly greater frequency of CD8+
T cells at d14. There were no differences in the frequency of CD4+ T cells versus CD8+ T
cells in the CNS at any timepoint (Fig. 2.6A). These data suggested that Tc17 cells give rise
to a population of CD4+ T cells in vivo. Importantly, when we compared the kinetics of CD4+
and CD8+ T cell accumulation upon transfer of 1C6 Th17 cells, the frequency of CD8+ T
cells was negligible at all timepoints (Fig. 2.6B).

The 1C6 TcR is class II-restricted and was cloned from a MOG[35-55]-specific CD4+ T cell
[79]. Further, 1C6 effector CD4+ T cells appear to expand more robustly than CD8+
counterparts in response to MOG[35-55] (Figure 1). Thus, while we purified CD8+ T cells by
high-speed cell sorting prior to Tc17 differentiation, we could not fully rule out that a small
number of CD4+ T cells were transferred in the Tc17-alone EAE group, and that these CD4+
T cells subsequently responded to cognate MOG[35-55] antigen and expanded in vivo to fill
the absent CD4 niche in lymphopenic [Link] mice. We therefore asked whether adoptive
transfer of 1C6 Tc17 cells would give rise to CD4+ T cells in lymphocyte-sufficient mice
with an intact CD4+ T cell compartment. We transferred Tc17 cells to NOD-background
BDC2.5 mice in which >95% of T cells bear a transgenic Vβ4+ TcR [338] that can be readily
distinguished from Vβ7+ 1C6 T cells by flow cytometry. After 21 days, we assessed the
presence of Vβ7+ T cells in the spleens of BDC2.5 mice that had received 1C6 Tc17 cells
versus those that had not (Fig. 2.6C). Unsurprisingly, the frequency and absolute number of
Vβ7+ CD8+ T cells was higher in BDC2.5 mice that received 1C6 Tc17 cells. Interestingly,
we noted that both the frequency and absolute number of Vβ7+ CD4+ T cells were
significantly increased in these animals relative to BDC2.5 mice that did not receive 1C6
Tc17 cells (Fig 2.6D). These data indicated that the adoptive transfer of 1C6 CD8+ Tc17 cells
could give rise to CD4+ T cells in an immunosufficient setting in vivo.

113
 A C
Spleen 1C6 Tc17 to N.S CNS 1C6 Tc17 to N.S
1C6 Tc17
. 15 CD4 15 CD4.
CD8 CD8 (Vb7+)

% of cells
% of cells

n.s
10 10
n.s
n.s *
5 5 n.s n.s 5d
0 0
7 14 21 7 14 21
ay ay ay ay ay ay
D D D D D D
Days post adoptive transfer Days post adoptive transfer
5d
B BDC2.5
(Vb4+)
. Spleen 1C6 Th17 to N.S
100
CNS 1C6 Th17 to N.S
100
****
80 **** 80
% of cells
% of cells

60 CD4 60
CD4
CD8 21d
CD8 40
40
n.s
20 n.s 20
n.s n.s

Vβ4
0 0
7

14

21
7

14

21

ay
ay

ay

ay
ay

ay

D
D

D
D

Days post adoptive transfer Days post adoptive transfer

Vβ7
D
No Tc17 Tc17 0.5
.
% of CD4+ cells

✱✱ Absolute cell count ✱

8000
0.4 No Tc17 transfer
6000 Tc17 transfer
0.3

CD4 0.2
4000

0.1 2000

0.0 0

CD4+Vb7+ CD4+Vb7+

✱✱
Absolute cell count

30
% of CD8+ cells

100000 ✱
No Tc17 transfer
20
80000 Tc17 transfer

CD8
60000

10 40000

20000
Vβ4

0 0
CD8+Vb7+ CD8+Vb7+
Vβ7

114
Figure 2.6 CD4+ T cells arise in [Link] recipients of 1C6 Tc cells. A, B. Female 1C6
Tc17 (A) or Th17 (B) cells were adoptively transferred to [Link] recipients (n=9). At d7,
d14 and d21, n=3 mice were sacrificed and the presence of CD8+ vs CD4+ T cells was
assessed by flow cytometry. *, p<0.05; ****; p<0.0001; n.s., not significant; Tukey’s post-
hoc test. C. Schematic depicting the transfer of female 1C6 Tc17 cells to BDC2.5 recipients.
D. Expression of Vβ7 vs Vβ4 on CD4+ and CD8+ splenic T cells from BDC2.5 mice
receiving, or not, 1C6 Tc17 cells. The % frequency and absolute number of CD4+Vβ7+ and
CD8+Vβ7+ T cells were quantified. *, p<0.05; **, p<0.01; t-test. Data are from two
experiments with each symbol representing a unique recipient.

115
2.4.7 CD4+ T cells are essential to 1C6 Tc17-mediated EAE

To determine whether Tc17-derived CD4+ T cells were essential to EAE, we transferred 1C6
Tc17 cells to two sets of recipients that we treated with either anti-CD4 or isotype control.
Strikingly, CD4 blockade sharply reduced disease severity in recipient mice, indicating that
the CD4+ T cells that arise upon Tc17 transfer are essential to disease outcomes (Fig. 2.7A).
We next examined the relative accumulation of CD8+ and CD4+ T cells in anti-CD4-treated
and isotype control recipients. As expected, the frequency of CD4 cells in the spleens and
CNS of anti-CD4-treated mice was greatly reduced relative to controls. Notably, CD4
blockade strongly reduced the frequency of CD8+ T cells that infiltrate the CNS, though the
frequency of CD8+ T cells in the spleen did not change (Fig. 2.7B). These data show that
CD4+ T cells are essential to Tc17-mediated disease induction and to CD8+ T cell infiltration
of the target organ.

116
 A Isotype Anti-CD4

. 5 5
Disease score

Disease score
4 4

3 3

2 2

1 1

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Days Days

✱✱ 2.0
70
✱ Isotype
Isotype 1.5 Anti-CD4
Day of onset

60

AUC d-1
50 Anti-CD4
40 1.0

30
0.5
20
10
0.0
0

Isotype Spleen Anti-CD4 Isotype CNS Anti-CD4

B
CD8

CD4
Spleen CNS
40 15
*** Isotype Isotype
* 0.067
Anti-CD4 Anti-CD4
% of cells
% of cells

30
10

20

5
10

0 0
8 4
8

D D
CD

CD

C C

117
Figure 2.7 CD4+ T cells are essential to 1C6 Tc17-mediated EAE. A. Female 1C6 Tc17
cells were differentiated using the two-round protocol and were adoptively transferred to two
sets of [Link] recipients: one that received anti-CD4 antibody 2x weekly until d35, and
another that received isotype control (both n=5). Graph showing the day of onset represents
the day when the disease symptoms begin in the mice. Mice that reached a score of 5 were
euthanized. B. CD4+ and CD8+ T cells were assessed from the spleen and CNS of recipient
animals at endpoints by flow cytometry. *, p<0.05; **, p<0.01; ****, p<0.0001; n.s., not
significant; t-test.

118
Discussion

Influenced in part by evidence from animal models [108], MS has traditionally been
considered a CD4+ T cell-mediated disease. However, several lines of evidence point to CD8+
T cells as important players in disease. They outnumber CD4+ T cells in active lesions [339]
and do so in progressive MS by as much as 50-fold [138]. Further, the presence of injured
axons in MS lesions correlates with the number of CD8+ T cells [340] and MHC class I alleles
(A3 and B7) are linked to MS incidence [341]. A better understanding of CD8+ T cell-driven
pathogenic mechanisms is therefore required, particularly given the paucity of current
treatments for progressive MS.

There have been multiple reports of MHC class II-restricted CD8+ T cells in the
context of chronic viral infection. They are present in a small proportion of HIV-1-infected
elite controllers [341] and expand in rhesus macaques vaccinated with simian
immunodeficiency virus (SIV) antigen-expressing cytomegalovirus [342]. However, a
possible role for such cells in autoimmunity is less well appreciated. In rodents, class II-
restricted CD8+ T cells have been reported, but in the context of Cd4-/- mice [343], [344] in
which unconventional CD8+ T cells may arise to compensate for the lack of CD4+ T cells.
As 1C6 mice unexpectedly possess CD8+ T cells that recognize MOG35-55 cognate antigen
in an MHC class II-dependent manner [201], this afforded us the opportunity to study the
function of these unusual cells in CNS autoimmunity.

It had previously been shown that 1C6 CD8+ T cells proliferate in response to MOG35-
55 in a class II-dependent manner. However, a head-to-head comparison of 1C6 CD8+ to
CD4+ T cells revealed that the former displayed ~3-4 fold lower [3H]-thymidine
incorporation upon MOG35-55 stimulation [201]. Here, we found that while 1C6 Tc1 and
Tc17 cultures expanded less robustly than Th1 and Th17 comparators, the cell-intrinsic
proliferative capacity of MOG35-55 1C6 Tc cells was unimpaired. Further, 1C6 Tc1 cells
capably produced the signature cytokine IFN when cultured with MOG35-55, while Tc17
cells produced both IFN and IL-17. Thus, we confirmed that 1C6 CD8+ Tc cells respond to
the cognate antigen MOG35-55.

We recently demonstrated that adoptive transfer of anti-CD3 + anti-CD28-stimulated

1C6 Th1 or Th17 cells elicited chronically worsening CNS autoimmunity in recipient
119
mice[148]. As 1C6 mice possess transgenic CD8+ T cells, we asked whether transfer of 1C6
Tc1 and Tc17 cells could induce the same disease pattern. Indeed, both Tc1 and Tc17 1C6
cells transferred disease with a similar severity and incidence of progression as Th1 and Th17
counterparts. Several models of CNS antigen-specific CD8+ T cell-mediated EAE have been
previously described. Adoptive transfer of myelin basic protein (MBP)-reactive CD8+ T cell
clones caused atypical EAE characterized clinically by ataxia, and histopathologically by
demyelination and vacuolation in the brain but not the spinal cord [256]. In another study,
mice bearing a class I-restricted transgenic TCR with specificity for the astrocytic marker
glial fibrillary acidic protein (GFAP) developed spontaneous ataxic disease characterized
with lesions in both white and grey matter; notably, inflammation in thalamic-adjacent tissue
was noted [253]. Focal thalamic lesions characterize most cases of MS [345], [346], with
signs of inflammation, demyelination and axonal damage being observed [347] .
Interestingly, we noted a large thalamic lesion with similar characteristics in a Tc1 recipient
mouse. Further work is required to fully understand the potential contribution of effector
CD8+ T cells to deep grey matter injury.

Surprisingly, we noted a robust presence of CD4+ T cells in the spleens and CNS of
[Link] mice that received 1C6 Tc effector T cells. These CD4+ T cells were essential to
Tc-mediated disease and to the infiltration of CD8+ T cells into the CNS. An elegant recent
study [348] used two different class I-restricted TcR transgenic mouse lines to show that
CD8+ T cells can cross-differentiate into CD4+ T cells in the in vivo setting. This property
was unrelated to self/non-self-specificity, as one strain (OT-I) recognized exogenous antigen,
while the other (8.3) was specific for a pancreatic self-Ag. Cross-differentiated CD4+ T cells
were observed at the highest frequency in the gut lamina propria (LP) and mesenteric lymph
nodes, and it was posited that gut microbiota are responsible for the expansion of these cells.
Nevertheless, cross-differentiated OT-I CD4+ T cells were also seen at detectable frequencies
(>1%) in the spleen. Notably, passive transfer of mature TcR-transgenic CD8+ T cells
resulted in a predominantly CD4+ T cell population in immunodeficient recipients, and
transfer of mature polyclonal CD8+ T cells also caused the expansion of a robust population
of CD4+ T cells (>20%) in mesenteric lymph nodes. These findings indicate that the capacity
of CD8+ T cells to convert to CD4+ T cells is not specific to the 1C6 line, nor to the unique
case of class II-restricted CD8+ T cells. In the future, it would be interesting to determine the

120
molecular mechanisms underpinning this observation; interestingly, we have noted that 1C6
CD8+ T cells show elevated expression of the CD4 lineage determining factor ThPOK.

Strikingly, we have shown that disease in 1C6 Tc17 recipients is nearly completely
abrogated by anti-CD4 blockade, as is the capacity of these Tc cells to access the CNS. This
indicates that the CD4+ T cells that arise in these transfer mice are critical to pathogenicity.
It was previously shown that Rag1-KO mice on the C57BL/6J background, reconstituted
with both CD4+ and CD8+ T cells, did not show increased EAE upon MOG35-55 immunization
relative to mice reconstituted with CD4+ T cells alone. Further the presence of CD8+ T cells
had no impact on CD4+ T cell motility within the CNS [259]. These data suggested that
while CD8+ T cells may proliferate in response to MOG35-55, they do not contribute to
disease. However, these data might be explained in part by the fact that MOG35-55 will
inherently favor a class II-restricted response, as immunization of C57BL/6J mice with the
shorter class I-restricted peptide MOG[37-46] elicits severe disease [349].

While CD4+ T cells are essential to 1C6 Tc17-mediated EAE, we observe no

differences in disease severity between recipients of male vs female Tc17 cells: this contrasts
with our previous observation that male 1C6 Th17 cells induce disease of greater severity
than female counterparts. These data argue against the possibility that the CD4+ T cells that
arise upon Tc17 transfer are identical to bonafide Th17 cells. Intriguingly, 1C6 Tc17 cells
are absent from the CNS upon anti-CD4 treatment. This raises the possibility that the CD4+
T cells seen in Tc17 recipients contribute to pathogenicity by facilitating Tc17 entry into the
CNS: indeed, Leuenburger and colleagues have seen that CD8+ T cell motility within the
CNS requires the presence of CD4+ T cells [259]. Further work is needed to characterize the
phenotype and contributions of CD4+ T cells to EAE in CD8+ T cell recipients.

In conclusion, we show that adoptive transfer of myelin antigen-specific effector

CD8+ T cells can induce EAE with a progressive clinical course. This disease is characterized
by inflammatory demyelinating lesions in the brain stem and thalamus that are associated
with neuronal damage. Intriguingly, Tc17 transfer gives rise to CD4+ T cells that are essential
to disease. Together, our data contribute to accumulating evidence that CD4+ and CD8+ T
cells collaborate in the pathogenesis of CNS autoimmunity.

121
2.5 Acknowledgments

We acknowledge Kim Larose-Labrecque for animal care, and Vincent Desrosiers for
assistance with flow cytometry experiments. The work was supported by a Project Grant
from the Canadian Institutes of Health Research and by a Discovery Research Grant from
the Multiple Sclerosis Society of Canada (both to M.R.). I.A. and P.M.I.A.D held Doctoral
Studentships from the Multiple Sclerosis Society of Canada. M.R. is a Senior Scholar
(«chercheur-boursier») of the Fonds de la Recherche de Québec-Santé.

122
2.6 Materials and Methods

2.6.1 Experimental animals and ethics

1C6 mice were a kind gift of Dr. Vijay Kuchroo and were maintained in our animal facility
at the CHU de Québec. NOD.CB17-PrkdcScid/NCrCrl mice were maintained in our animal
facility, and BDC2.5 TCR mice were obtained from the Jackson Laboratory. All animal
breeding and experiments were approved by the Animal Protection Committee of Université
Laval (protocols 2021-820, 2021-830).

2.6.2 Effector Tc and Th culture and differentiation

Total CD8+ and CD4+ T cells were isolated from 1C6 spleens and LNs using anti-CD8 and
anti-CD4 MicroBeads (Miltenyi), respectively. Naïve CD8+CD62Lhi and CD4+CD62Lhi T
cells were purified using a FACSAria (BD) high-speed cell sorter and were cultured in
supplemented T cell media as described [297]. For type-1 (Tc1/Th1) differentiation, T cells
were cultured with 10 ng mL-1 rmIL-12 (R&D Biosystems) plus anti-IL-4 (10 µg mL-1,
BioXcell) for 2 days, and with 10 ng mL-1 rmIL-2 (Miltenyi) for another 3 days. For type-17
(Tc17/Th17) differentiation, T cells were cultured for 2 days with 3 ng mL -1 rhTGFb
(Miltenyi) + 20 ng mL-1 rmIL-6 (Miltenyi) + 10 µg mL-1 anti-IFN-γ (BioXcell), and with 20
ng mL-1 rmIL-23 (R&D Biosystems) for an additional 3 days[350] . For plate-bound
stimulation, T cells were differentiated for 2 days on plates coated with anti-CD3 and anti-
CD28 (4 µg mL-1 for CD8 and 2ug mL-1 for CD4; BioXcell) and were subsequently
transferred to uncoated plates for an additional 3 days. For stimulation with cognate
peptide+APCs, T cells were cultured for 5 days at a 1:5 ratio with irradiated splenocytes
(2000rads/27min) pulsed with 15ug mL-1 of MOG35-55 (CHU de Québec).

2.6.3 Flow Cytometry

Cells were pre-incubated with Fc Block (BD Biosciences) to prevent non-specific antibody
binding and then stained with cell surface markers: CD8 (clone 53.6.7 ThermoFisher), CD4
(clone RM4.5 ThermoFisher), Vβ7 (clone TR310, Biolegend), Vβ4 (clone KT4 BD
Biosciences) for 20 min at 4⁰C. Cells were also stained with Fixable Viability Dye
123
(ThermoFisher) for 15 min; positive (dead) cells were gated out in all analyses. 123count
eBeads(ThermoFisher) were used for the absolute cell counting. For intracellular cytokine
staining, cells were first incubated for 4 hrs in the presence of 50 ng mL-1 phorbol 12-
myristate 13-acetate (Sigma-Aldrich), 1µM ionomycin (Sigma-Aldrich) and Golgi Stop (1
µL per mL culture; BD Biosciences) and then stained for surface antigens and live/dead
indicator. Cells were processed using Fixation and Perm/Wash buffers (eBioscience) and
were stained for intracellular antigens using the following antibodies: TNF (clone MP6-
XT22 ThermoFisher), IFN-γ (clone XMG1.2, ThermoFisher), IL-17A (clone TC11-
18H10.1, ThermoFisher), Granzyme B (clone NGZB, ThermoFisher), Perforin (clone
eBio0MAK-D, ThermoFisher). Cell proliferation was checked by incubating the cells with
CellTrace Violet (ThermoFisher) before starting the cell culture. Flow cytometry data were
collected using the FACS Canto II flow cytometer (BD Biosciences) and analyzed using
FlowJo software. Gates were set based on fluorescence minus one control.

2.6.4 Adoptive transfer EAE

1C6 Tc1, Tc17, Th1 or Th17 cells were generated by plate-bound stimulation as described
above and were administered i.v. to [Link] mice (5×106 cells per mouse). For Th+Tc co-
transfers, 2.5×106 of each cell type were transferred at the same time. In some experiments,
Tc cells were restimulated for a second 5-day round of differentiation as indicated in the
Figure legends. Recipient animals were treated i.p. with 200 ng pertussis toxin on day 0 and
day 2. They were weighed and scored daily up to 70 days using a well-established
semiquantitative scale that we have used previously [106]: 0, no disease; 1, loss of tail tone;
2, hind limb weakness or partial paralysis; 3, complete hind limb paralysis; 4, front and hind
limb paralysis; 5, ethical endpoints attained. Mice with advanced symptoms (score ≥3) were
monitored daily by trained animal technicians, in collaboration with the veterinary service of
Université Laval, to assess whether ethical endpoints had been attained. Individual disease
curves were assessed for day of symptom onset, disease burden (area under curve) and for
progression using our previously described criteria[148].

2.6.5 CNS Mononuclear Cell Isolation

124
Mice were euthanized and perfused with cold PBS administered through the left cardiac
ventricle. CNS was dissected from the skull and spinal column, respectively. CNS tissue was
homogenized using a PTFE Tissue Grinder (VWR) and was incubated at 37⁰c for 30 min in
homogenization solution (HBSS containing 4 ng mL-1 liberase and 25 ng mL-1 DNase).
Homogenate was filtered through a 70-µm cell strainer, resuspended in 35% Percoll (GE
Healthcare) and centrifuged. Mononuclear cells were collected, washed and prepared for
flow cytometric analysis.

2.6.6 CD4 blockade

Starting from day 0 of adoptive transfer of Tc17 cells, CD4+ T cells were depleted by treating
the mice with intraperitoneal injection of 400ug/mouse of anti-CD4 (clone GK1.5, BioXCell)
in PBS, twice weekly from day 0 to day 35. Anti-Keyhole limpet hemocyanin (clone LTF-2,
BioXCell) was used as an isotype control.

2.6.7 Statistics

Statistical analyses were conducted using Prism software (GraphPad). Information regarding
the specific tests used are indicated in individual Figure legends. Two-tailed tests were used
in each case. Comparisons between multiple groups or timepoints were conducted using
ANOVA followed by a post-hoc test.

125
Chapter 3. Discussion

Multiple Sclerosis has long been regarded as a prototypical CD4+ T cell-mediated

autoimmune disorder. However, the precise role of CD8+ T cells in MS and its animal
models, such as EAE, remains an area of ongoing debate. The bias toward CD4+ T cells is
largely due to the strong association between MS susceptibility and MHC class II alleles
[351], as well as the fact that CD4+ T cells are recognized as the predominant effector T cells
in animal models of MS, including EAE and Theiler’s murine encephalomyelitis virus-
induced demyelinating disease [197]. Unlike CD4+ T cells, CD8+ T cells are equipped with
cytotoxic machinery, allowing them to directly eliminate target cells by releasing lytic
granules containing perforin and granzymes. Despite this distinct functionality, the
involvement of CD8+ T cells in MS pathogenesis remains controversial. A potential
pathogenic role for CD8+ T cells has been suggested, as these cells have been detected in
inflammatory brain lesions and exhibit oligoclonal expansions in both the brain and CSF of
MS patients [236], [352]. Understanding the mechanisms by which CD4+ and CD8+ T cells
contribute to CNS autoimmunity is a complex challenge. Despite extensive studies on this
disease, the precise pathways through which the T cells instigate and perpetuate MS remain
incompletely understood.

We also investigated the role of Serpine-1 in Th1 cell-mediated immune response by using
the EAE disease model. In Chapter 2, we explored the influence of Serpine-1 on the EAE
progression, cytokine production and the expression of T-cell inhibitory molecules in Th1
cells.

126
3.1 Overexpression of Serpine1 in 2D2 cells induces disease of lessened severity.

In our recent investigation, we discerned a notable upregulation of Serpine1 secretion in both

Th1 and Th17 cells upon undergoing a secondary round of in vitro differentiation. While
prior studies have established that Serpine1-deficient T cells exhibit heightened IFN-γ
production relative to wild-type cells in vivo [282], the precise functional role of Serpine1 in
fully differentiated Th1 and Th17 cells has remained unexplored. To address this gap, we
employed retroviral overexpression of Serpine1 in Th1 cells, leading to an intriguing
outcome. The elevated expression of Serpine1 in Th1 cells resulted in a concomitant
downregulation in IFN-γ expression and a discernible trend toward reduced TNF, compared
to control-transduced cells. Conversely, the OE of Serpine1 in Th17 cells did not elicit
significant alterations in the expression levels of either IL-17 or TNF. This finding confirms
the Th1 cell-specific effect of Serpine1. As it was also shown before that the loss of Serpine1
shifts the Th2 response towards Th1 response [353], we further explored the effect of
enforced expression of Serpine1 on the pathogenicity of 2D2 Th1 cells, with Serpine1
overexpression a disease of diminished severity was induced compared to control cells. This
was characterized by a reduced frequency of inflammatory IFN-γ+ IL-2+ T cells in vivo [106].
The immunosuppressive role of Serpine1 has been demonstrated in various studies, including
cancer research, where higher Serpine1 levels have been associated with poor prognosis of
lung, neuroblastoma and colorectal cancers [354], [355], [356]. Higher expression of
Serpine1 has been shown to enhance cell migration and apoptosis resistance in head and neck
cancers, while reduced expression of Serpine1 has been associated with decreased cancer cell
migration in nasopharyngeal carcinoma [357], [358].

Collectively, findings from our research and others indicate that Serpine1 plays a complex
immunosuppressive role in both autoimmunity and cancer. Serpine1’s function could be
beneficial in autoimmune conditions by dampening excessive immune responses. However,
in cancer therapy, its immunosuppressive activity may hinder anti-tumour immunity. These
contrasting roles highlight the need for therapeutic approaches that can modulate Serpine1 in
a targeted way—either broadly in chronic conditions like cancer or with cell-specific
precision to manage T cell pathogenicity in autoimmunity.

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3.2 Knockout of Serpine1 inTim3-Tg Th1 cells increase their pathogenicity and reduce
T cell inhibition molecules.

The observation that Serpine1 inhibits Th1-driven inflammation prompted us to investigate

its potential role in augmenting the expression of the inhibitory receptor Tim-3. The link
between Serpine-1 and Tim-3 is one of the main findings in this study. Upon multiple rounds
of polarization, Serpine1-/- Th1 cells exhibited lower and delayed expression of Tim-3
compared to Serpine1 controls, which leads to prolonged Th1 activation and increased
pathogenicity. Notably, the loss of Serpine1 signalling did not result in an increase in the
expression of IFN-γ, but it significantly lowered the frequency of IFN-γ+Tim-3+ Th1 cells.
This finding aligns with the evidence from another study showing that Serpine1 miRNA acts
as a modulator of immune-related gene expression, functioning through the TRAB2 splicing
factor [359], highlighting its role by which Serpine1 influences immune cell phenotype and
cytokine production. To gain deeper insights, we utilized Tim-3 Tg mice, where Tim-3 is
overexpressed without the need for multiple rounds of polarization under Th1 conditions
[360]. Crossing Serpine1-/- mice with Tim-3-Tg mice allowed us to generate Th1 cells and
uncover potential Serpine1-dependent differences in IFN-γ expression. These findings
indicate that Serpine1 likely contributes to immune tolerance mechanisms by facilitating
Tim-3-mediated inhibition. Strikingly, Tim-3+Th1 cells derived from Serpine1-/-Tim-3-Tg
mice exhibited higher positivity for IFN-γ compared to their Serpine1 Tim-3-Tg
counterparts. Intriguingly, no differences were observed with Tim-3 negative Th1 cells
between the strains, which suggests that Serpine1 represses IFN-γ expression in a Tim-3-
dependent manner. Serpine1-KO Th1 cells that overexpress Tim-3 and IFN-γ showed a more
pronounced effect of the loss of Serpine-1 by reducing the expression of other checkpoint
molecules like PD-1 and Lag-3. This suggests a complex interplay between Serpine1 and
Tim-3 in modulating the expression of key immune regulatory molecules to enhance CNS
inflammation. Supported by the finding from another study, showing that combined
inhibition of Tim-3, PD-1 and Lag-3 enhanced the antitumor response of CTLs in the gastric
cancer T cell coculture models [361]. Tim-3, PD-1 and Lag-3 being the T cell exhaustion
markers, it will be very interesting to investigate the role of Serpine1 and its relationship with
the T cell exhaustion markers in chronic diseases. Tim3, PD-1 and Lag-3 have been

128
associated with T cell exhaustion, particularly in tumour microenvironment [362], and a
negative correlation has been demonstrated between Serpine1 miRNA expression and CD8
T cell infiltration in various cancers[359]. This inverse relationship may help to explain why
Serpine1 deficiency is associated with reduced expression of immune checkpoint markers.
This finding opens avenues for understanding the intricate molecular mechanisms
orchestrated by Serpine1 in shaping the functional properties of Th1 cells. The observed Tim-
3-dependent repression of IFN-γ and the coordinated modulation of other regulatory
receptors imply a broader regulatory role for Serpine1 in fine-tuning immune responses.

3.3 CD4+ T cells appear in Tc1/Tc17 recipient [Link] mice and blocking of CD4+ T
cells decreased CD8+ T cells in CNS

Our previously described 1C6 CD4+ Th1/Th17 adoptive transfer EAE model relied on the
differentiation of these cells with agonistic anti-CD3/CD28 [148]. We, therefore, asked
whether 1C6 Tc1 or Tc17 cells, generated in a similar manner, would induce CNS
autoimmunity. Indeed, we found that both Tc effector subpopulations transferred EAE; while
it had a delayed onset relative to that caused by 1C6 Th cells, disease severity did not differ
overall. Surprisingly, we noted a robust presence of CD4+ T cells in the spleens and CNS of
[Link] mice that received 1C6 Tc effector T cells. These CD4+ T cells were essential to
Tc-mediated disease and the infiltration of CD8+ T cells into the CNS. These findings were
also reported in another mice model, where the presence of CD4+ T cells was observed in the
CNS of CD8+ T cell-reconstituted Rag1-/- mice [259]. This suggests that CD4+ T cells may
act as facilitators of peripheral immune response, providing critical signals that enable CD8+
T cell migration to CNS. Our data from the CD4+ T cell transfers also supports this statement,
where we did not find the expansion of CD8+ T cells in the CNS and spleen of [Link]
mice that received only Th effector T cells. Another study shows that MOG37-46-specific
CD8+ T cells are proinflammatory and can induce mild EAE on their own. However, the
presence of CD4+ T cells makes the disease severe, emphasizing their role in modulating
CD8+ T cell responses [363]. These findings highlight the critical helper role of CD4+ T cells
in amplifying CNS inflammation, which is consistent with the co-localization of CD4+ and
CD8+ T cells observed in CNS lesions of MS patients.

129
To further investigate the support CD4+ T cells provide to CD8+ T cells, we blocked CD4+ T
cells after Tc17 cell transfer in [Link] mice. A milder or no disease was observed in the
CD4 blocked group, and we also noticed a significant decrease in the percentage of CD8+ T
cells in the CNS of the mice that were treated with anti-CD4 antibodies. This suggests that
CD4+ T cell presence is required for the optimal migration and pathogenicity of CD8+ T cells
to the CNS. Supporting this hypothesis, previous research has shown the reduced mobility of
CD8+ T cells to the CNS after the blockade of CD4+ T cells, and the absence of CD4+ T cells,
likely lead to suboptimal activation of CD8+ T cells, limiting their capacity to initiate CNS
inflammation [259]. However, this contrasts with the observations that CD8+ T cells move
rapidly through the CNS parenchyma when they are highly activated [364]. Therefore, in the
mice that received only CD8+ T cells, a lack of local reactivation or inflammatory signals is
indicated in those mice. Nonactivated T cells that did not find adequate migratory signals in
the CNS tissue were shown to have similar mobility patterns [365]. Further, we wanted to
validate whether any CD4+ T cell can provide help to the CD8+ T cells or only the ones that
are specific to the same antigen as CD8+ T cells. We transferred only CD8+ T cells to the
BDC2.5 mice, in which the CD4+ T cells are already present. To our surprise, we observed
the expansion of CD4+ T cells that were Vb7+, which is similar to the CD8+TCR that were
transferred in the mice. These findings support that only the CD4+ T cells that are specific
for the same antigen as CD8+ T cells may provide help to the CD8+ T cells. In essence, our
study unveils intriguing dynamics in the interplay between CD8+ and CD4+ T cells in
different host environments, shedding light on the influence of the host's immune milieu on
the behaviour of adoptively transferred T cell populations. Further examining the migratory
molecules expressed on CD8+ T cells after the blockade of CD4+ T cells may unravel the
intricacies of how blocking CD4+ T cells influence the migratory behaviour of CD8+ T cells.

3.4 CD4+ and CD8+ T cells induce highly severe disease when transferred together.

CD4+ and CD8+ T cells have been found together in the CNS lesions of MS patients [235].
Knowing this, we transferred CD4+ and CD8+ T cells together and CD4+ or CD8+ T cells
alone in [Link] mice. Transfer of CD4+ and CD8+ T cells together induced a very severe
EAE and we also observed an early disease onset compared to the single transfer of CD4+ or
CD8+ T cells. Presence of CD8+ T cells expressing IL-17A (Tc17) in the lymph nodes and
130
the CNS of mice immunized with MOG37-55 was reported in one study [366]. These Tc17
cells were later shown to play a supportive role for Th17 cells in EAE, but they needed to
express IL-17A to render CD4+ T cells pathogenic. Further they also observed that single
transferred CD8+ T cells couldn’t infiltrate CNS and transfer of small number CD4+ T cells
only does not induce EAE but when transferred with CD8+ T cells, together they induced
severe disease [367]. This suggests a synergistic interaction between CD4+ and CD8+ T cell
to induce the disease. In our study we did not only explore the disease induced by the IL-17A
expressing Th17+Tc17 but also the disease induced by IFN-γ expressing Th1+Tc1 cells, in
both the cases, co-transfer of CD4+ and CD8+ T cells induced severe disease than single
transfers. Therefore, the observations by our group and others explain that the helper T cells
and cytotoxic T cells together can induce a stronger immune response. Another group also
showed that the presence of CD4+ T cells in CD8-induced EAE, and presence of both CD4+
and CD8+ T cells together made the disease worse compared to those where EAE was
induced by CD4+ T cells only. It was also shown that presence of a small number of CD4+
T cells lead to the infiltration of very high number of CD8+ T cells to the CNS [259] This
aspect of the study provides valuable insights into the relevance of having both CD4+ and
CD8+ T cells in the CNS, mirroring observations in multiple sclerosis (MS) patients.
However, it is evident that further investigation is necessary to draw conclusive and insightful
outcomes from this complex interplay between CD4+ and CD8+ T cells in the context of EAE
progression.

131
 Conclusion and future perspectives

Elucidating the mechanism underlying the pathogenicity of CD4+ and CD8+ T cells is
important to advance our knowledge of their pathogenesis in autoimmune diseases. This
knowledge allows us to identify new therapeutic targets and develop innovative strategies of
treatment. The studies present in this thesis explore the contribution of CD4+ and CD8+ T
cells independently and in collaboration with each other in EAE.

The first part of my thesis reveals that Serpine-1 serves as a negative regulator of Th1
mediated autoimmune response in EAE and deficiency of Serpine-1 in Th1 cells leads to the
induction of more severe disease. This role of Serpine-1 was further explored by observing
higher IFN-γ expression and reduced expression of T cell inhibitory molecules such as PD-
1 and Lag-3 in Serpine-1 KO Th1 cells. The mechanism behind the reduced expression of T
cell inhibitory molecules by Serpine-1 deficiency needs to be further explored, which will
provide new immunoregulatory strategies to restrict immunopathology driven by Th1 cells.

The 2nd part of this thesis demonstrates that both CD4+ and CD8+ T cells are crucial for the
progression of the disease in a CNS autoimmune EAE model. We show that while 1C6 CD8+
T cells do not respond to MOG35-55 antigenic stimulation robustly, but they increase the
pathogenicity of the disease when co-transferred with CD4+ T cells. Their pathogenicity was
characterized by extensive CNS infiltration and proinflammatory cytokine production. A key
interaction between CD4+ and CD8+ T cells in promoting inflammation also suggested by
the expansion of CD4+ T cells in the recipient mice after the adoptive transfer of Tc17 cells
only. Overall, in this study a cooperative pathogenic role of CD4+ and CD8+ T cells is
revealed, which provides insights for therapeutic approaches targeting both CD4+ and CD8+
T cells in autoimmunity. Further detailed studies are needed for deeper understanding the
interdependent roles and mutual influence of CD4+ and CD8+ T cells in autoimmune
pathogenesis, which will help to refine the therapeutics

132
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