The Role of CD40L and CD40 in the Pathogenesis of

Kawasaki Disease

by

Parnian Arjmand

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Immunology

University of Toronto

© Copyright by Parnian Arjmand 2011

ii

The role of CD40L and CD40 in the Pathogenesis of Kawasaki Disease

Parnian Arjmand

Master of Science

Department of Immunology

University of Toronto

2011

Abstract

Kawasaki Disease (KD) is a childhood disease leading to coronary arteritis. Elevated numbers of

CD40L+

platelets in circulation is correlated with risk of heart damage. CD40L is a tumor

necrosis family member that binds to CD40 and αIIbβ3, receptors which are also expressed on

platelets. A single injection of Lactobacillus casei Cell Wall Extract (LCWE) induces a disease

similar to KD in mice, where LCWE superantigen (SAg) reactive T-cells persist in the coronary

artery. This phenotype is inconsistent with the fate of SAg-stimulated cells and is likely mediated

by co-stimulation. This work shows that stimulation with a SAg induces platelet activation and

CD40L expression in vitro. Furthermore, enhanced survival of SAg-reactive T-cells is

demonstrated following antibody-mediated CD40L cross-linking. This effect is mediated via

inhibition of the extrinsic apoptosis pathway. In addition, CD40 cross-linking is also reported to

enhance SAg-reactive T-cell survival by enhancing CD86 expression on APCs and CD28 co-

stimulation.

iii

Acknowledgments

First and foremost, I thank my supervisor, mentor and advisor, Dr. Rae Yeung, for accepting me

as a trainee and showing me the path throughout the past two years.

I am indebted to my supervisory committee members, Dr. Michael Ratcliffe, Dr. Margaret Rand

and Dr. Joan Wither for their guidance and advice throughout my project.

I also thank Dr. Trang Duong for patiently orienting me at the lab and helping me trouble shoot

obstacles in my experiments.

In addition, I extend my deep gratitude to the supportive and collegial members of the Yeung

lab:

Our early lunch club members: Ken Little (you‟ve been a true friend), Aaron Wong and Dr. Matt

Gronski; ladies of the late lunch club: Jaimie Wardinger, Fiona Almeida, Lisa Liang and

Suzanne Tam; and those, of course, who don‟t ascribe to such clubs: the always graceful Reena

Riarh. Especially, I thank Dr. Vahid Khajooe for patiently listening to my career dilemmas and

for his contribution to my culinary skills.

I thank my friends, Daniel Bertrand, Blerta Green, Sepideh Sarachi, Milu Jauregi and Devin

Anderson without whose comradeship this thesis would not have happened. Lastly, I thank my

loving mom, dad and sister who have supported me in every decision that I have made in my life

thus far.

Thank you!

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Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ............................................................................................................................... vii

List of Abbreviations ..................................................................................................................... ix

1 Introduction ............................................................................................................................. 1

1.1 Kawasaki Disease ............................................................................................................. 1

1.1.1 Clinical Features ....................................................................................................... 1

1.1.2 Etiology ..................................................................................................................... 2

1.1.3 Coronary Arteritis and Aneurysm Formation ........................................................... 3

1.2 Superantigens ................................................................................................................... 4

1.3 Superantigens and Apoptosis ........................................................................................... 6

1.4 Superantigens and Anergy ............................................................................................... 9

1.5 Co-stimulation ................................................................................................................ 10

1.5.1 CD40L Protein Structure and Expression ............................................................... 12

1.5.2 CD40: The Classical CD40L Receptor ................................................................... 14

1.5.3 CD40 – CD40L Forward Signaling ........................................................................ 15

1.5.4 CD40L Reverse-Signaling ...................................................................................... 16

v

1.6 Platelets .......................................................................................................................... 18

1.6.1. Platelet Histology ........................................................................................................ 18

1.6.2. Platelets and Immunity ............................................................................................... 20

1.6.3. Platelets and Kawasaki Disease .................................................................................. 22

1.7 Animal Models of Kawasaki Disease ............................................................................ 22

1.7.1 Lactobacillus casei Cell Wall Extract (LCWE) Model of Kawasaki Disease ........ 23

1.8 Rationale......................................................................................................................... 26

1.9 Hypothesis ...................................................................................................................... 26

2 Methods................................................................................................................................. 27

2.1 Mice ................................................................................................................................ 27

2.2 Blood Collection ............................................................................................................ 27

2.3 Platelet Stimulation ........................................................................................................ 28

2.4 Cell Culture .................................................................................................................... 28

2.5 Annexin-AV Apoptosis Assay ....................................................................................... 29

2.6 Flow-Cytometry ............................................................................................................. 29

2.7 Caspase Staining ............................................................................................................ 30

2.8 Immunoblotting .............................................................................................................. 30

2.9 Statistical Analysis ......................................................................................................... 31

3 Results ................................................................................................................................... 32

3.1 SEB induces Platelet De-granulation and αIIbβ3 Receptor Activation ......................... 32

vi

3.2 Kinetics of CD40L Expression on SEB Stimulated Vβ8+

T-cells ................................... 35

3.3 CD40L Cross-linking Leads to Rescue of SEB-Reactive T-cells from Apoptosis ........ 37

3.4 Caspase-3 Staining Confirms Annexin-AV Gating Strategy ......................................... 40

3.5 Pro-survival Effects of αCD40L mAb are mediated through T-cell CD40L Cross-

linking ....................................................................................................................................... 42

3.6 CD40L Cross-linking Decreases Caspase-3 Activation in SEB Reactive Splenocytes . 44

3.7 CD40L Cross-linking Does Not Change Intracellular Bcl-2 Expression in SEB-Reactive

T-cell Populations ..................................................................................................................... 46

3.8 CD40L Cross-linking Lowers Caspase-8, but not Caspase-9 Activity in SEB-Reactive

T-cells ....................................................................................................................................... 48

3.9 CD40 Co-stimulation with SEB Stimulation Enhances B-cell CD86 Expression ......... 50

3.10 CD40 Cross-linking Enhances SEB-Reactive T-cell Survival ................................... 52

3.11 Pro-survival Effects of CD40 Stimulation on T-cells are CD86 – CD28 Dependent 54

3.12 CD40 Cross-linking Leads to Enhanced Bcl-2 Expression in SEB-Reactive T-cells 56

4 Discussion ............................................................................................................................. 58

4.1 Limitations and Future Directions.................................................................................. 65

4.2 Conclusions and Proposed Model .................................................................................. 66

5 Bibliography ......................................................................................................................... 68

vii

List of Figures

Figure 1. T-cell activation with a superantigen versus a conventional peptide antigen………..…6

Figure 2. Apoptotic Signaling pathways…………………………………………………….……8

Figure 3. CD40 and CD40L are expressed on a variety hematopoietic and non-hematopoietic cell

types…………………………………………………………………………………………...…15

Figure 4. SEB stimulates platelet de-granulation and induces αIIbβ3 receptor activation in

vitro…………………………………………………………………………………………………………..34

Figure 5. Kinetics of surface CD40L expression on Vβ8+

T-cells………………………..………36

Figure 6. CD40L cross-linking leads to rescue of SEB-reactive Vβ8+

T-cells from apoptosis….38

Figure 7. Compiled results of repeated apoptosis assays with anti-CD40L mAb…...………..…39

Figure 8. Active caspase-3 staining confirms annexin-AV gating strategy…...…………...….....41

Figure 9. Mixing experiments to elucidate the cellular source of CD40L leading to enhanced T-

cell survival following SEB stimulation…………………………………………………………43

Figure 10. Immunoblot and flow-cytometry analysis of caspase-3 activity following CD40L

cross-linking…………………………………………………………...…………………………45

Figure 11. CD40L cross-linking does not affect intracellular Bcl-2 expression in SEB-reactive T-

cell populations…………………………………………………………………………..………47

Figure 12. CD40L cross-linking lowers caspase-8 but not caspase-9 activity in SEB-reactive T-

cells………………………………………………………………………………………………49

Figure 13. Kinetics of CD86 Expression on CD19+ splenocytes (B-cells) following CD40 cross-

linking……………………………………………………………………………………………51

Figure 14. Enhanced Survival of SEB-reactive T-cell Populations Following CD40

Stimulation………………………………………………………………………………………53

viii

Figure 15. Compiled results of multiple apoptosis assays demonstrating the pro-survival effects

of CD40 and the importance of CD86 and CD28 in T-cell survival following SEB stimulation.55

Figure 16. CD40 Cross-linking Enhances Intracellular Bcl-2 Protein Expression in Vβ8+ T-

cells………………………………………………………………………………………………57

Figure 17. Model of Platelet-CD40-CD40L interaction in Enhanced Superantigen-Reactive T-

cell Survival……………………………………………………………………………….……..67

ix

List of Abbreviations

AA – Amino Acid

ADP – Adenosine Di-Phosphate

AICD – Activation Induced Cell Death

Apaf-1 – Apoptotic Protease Activating Factor

APC – Antigen Presenting Cell

Bcl-2 – B-cell Lymphoma-2

BH – Bcl-2 Homology

CD40L – CD40 Ligand/ CD154

c-FLIP – Cellular FLICE Inhibitory Protein

CR3 – Complement Receptor 3

Cyt-c – Cytochrome-c

DD – Death Domain

DISC – Death-Inducing Signaling Complex

DNA – Deoxyribonucleic Acid

EC – Endothelial Cell

FADD – Fas Associated Death Domain

FasL – Fas Ligand

FMO – Fluorescence Minus One

x

GP – Glycoprotein

HIGM – Hyper Immunoglobulin-M Syndrome

HIV – Human Immunodeficiency Virus

IL – Interleukin

IP3 – Inositol Tris Phosphate

IVIG – Intravenous Immunoglobulin

KD – Kawasaki („s) Disease

LCWE – Lactobacillus casei Cell Wall Extract

mAb – Monoclonal Antibody

MFI – Mean Fluorescent Intensity

MHC – Major Histocompatibility Complex

MK – Megakaryocyte

MLI – Mean Luminescence Intensity

MMP-9 – Matrix Metalloprotease-9

PKC – Protein Kinase C

PLC – Phospholipase C

PMA – Phorbol Myristic Acetate

PMP – Platelet-Derived Microparticle

PS – Phosphatidyl Serine

SAg - Superantigen

xi

sCD40L – Soluble CD40 Ligand

SEB – Staphylococcal Enterotoxin B

SMC – Smooth Muscle Cell

TCR – T-cell Receptor

TGF – Transforming Growth Factor

TLR – Toll-Like Receptor

TNF – Tumour Necrosis Factor

TNFR- TNF Receptor

TNFSF – Tumour Necrosis Factor Super Family

TPO - Thrombopoietin

WT – Wild Type

α – Anti

1

1 Introduction

1.1 Kawasaki Disease

1.1.1 Clinical Features

Originally described by Dr. Tomasaku Kawasaki in 1967 as an acute febrile mucotaneous lymph

node syndrome (1), Kawasaki Disease (KD) is now recognized as the most common cause of

acquired heart disease in children in the industrialized world (2). KD is an acute, self-limiting,

multisystem vasculitis of childhood affecting small and medium sized vessels. Inflammation may

herald localized cardiovascular sequelae ranging from asymptomatic to giant coronary artery

aneurysms, with thrombosis, myocardial infarction, and sudden death (3-5).

In the absence of a common clinical feature or diagnostic test, KD is diagnosed with

presentation of prolonged fever (at least 5 days) and a minimum 4 of 5 symptoms:

“polymorphous skin rash, oral mucosal changes, cervical adenopathy, bilateral conjunctival

injection and extremity changes” (i.e. redness, swelling of the hands and feet). High platelet

numbers (thrombocytosis) particularly late in the course of disease is another feature of KD (6)

(see section 1.6.3).

There is a large genetic and age-biased component contributing to the incidence of KD. Children

of Asian ethnicity are more commonly affected than Africans, Hispanics and Caucasians.

Ethnicity-based variance is particularly evident from Japanese data (7-10).

2

1.1.2 Etiology

Despite extensive studies to elucidate the causative agent of KD, its etiology is still debated. KD

fits anywhere between an infectious to an autoimmune disease, with a pathogenic trigger leading

to sustained autoimmune response (11).

Certain epidemiologic and immunogenic features of KD suggest an infectious trigger of disease.

KD is an endemic disease occurring in seasonal pandemics in geographical clusters with similar

clinical features to a viral/ bacterial infection: fever, rash and erythema. Furthermore, antigen-

specific IgA antibodies and CD8+ T-cell infiltration of the coronary arteries of KD patients

suggest cellular and mucosal immune responses to an infectious pathogen (12-15).

With each outbreak of disease, a pathogenic agent has been identified as an etiologic trigger; yet,

a consistent correlation between viruses and bacteria isolated from KD patients and disease

recurrence has not been established (16-24). A feature common to most of these pathogens as

well as agents used to trigger disease in animal models is the presence of superantigen activity

(25). This has led some to the theory that KD is triggered by a superantigen agent.

Further support for a superantigen (SAg) trigger of disease lies in the nature of the immune

response and the signature T-cell expansion in KD. Systemic inflammation, cytokine storm and a

characteristic expansion of certain families of T-cells are similar to other SAg-mediated diseases,

such as toxic shock syndrome (3, 25-26). T-cell V beta skewing is a feature of SAg-mediated

responses: certain families of T-cells are preferentially activated by SAgs and consequently

deleted in the periphery (27) (see section 1.2). In KD, T-cell V beta 2+ and V beta 8

+ families are

preferentially expressed in peripheral blood (28-30). In addition, IgM antibodies against

3

streptococcal and staphylococcal SAgs have been detected in sera of KD patients during the

acute phase (31).

1.1.3 Coronary Arteritis and Aneurysm Formation

While the acute inflammatory response in KD is found in medium and small vessels throughout

the body, coronary arteries are the most common site of persistent inflammation and end organ

damage (32). Biopsy studies of KD patients indicate that coronary artery inflammation begins in

the surrounding tissue and the inner lining of small coronary arteries and microvessels, followed

by transmural inflammation. The infiltrating cells in the coronary artery are initially neutrophils,

followed by large mononuclear cells, dendritic cells and T lymphocytes (12, 15, 33). The latter

are indeed the most common cell infiltrates contributing to coronary inflammation and are

believed to play a key role in disease pathogenesis.

The cytokine profile of KD patients is also one of inflammation. Inflammatory cytokines such as

IL-6, TNF-α, and IFN-γ are upregulated in the acute phase. Moreover, IL-2, IL-6, and IL-8 are

elevated throughout the course of disease and are implicated in coronary lesion formation in KD

patients (34-36) In mediating inflammation, these cytokines activate endothelial cells of the

artery as seen by an over expression of HLA-DR, I-CAM1 and adhesion molecules, E- and P-

selectin, which recruit leukocytes (37-38). In conjunction with cytokines, endothelial cells, in

turn, have the capacity to activate and recruit platelets cells to the site of inflammation.

The effects of coronary artery inflammation may lead to vessel wall destruction and aneurysm

formation in KD affected children; up to 25% of untreated and 5% of those treated in the acute

phase develop coronary artery lesions (5, 39).

4

The two major cellular and extra-cellular processes leading to aneurysm formation are Smooth

Muscle Cell (SMC) apoptosis and loss of Extracellular Matrix (ECM), scaffolding that supports

and surrounds the blood vessel wall. These events are triggered by persistent infiltration of

inflammatory T-cells as well as the action of matrix proteases. Elastin breakdown, which is a key

feature of aneurysm formation, was previously shown by our laboratory to be largely induced by

Matrix Metalloprotease-9 (MMP-9) activity. This protein is secreted by SMCs as well as de-

granulating activated platelet cells (40-42) (for more on platelets and KD, see section 1.6).

1.2 Superantigens

Originally described in 1989, superantigens are a family of proteins that elicit a dramatic T-cell

dependent immune response. SAgs are grouped in two categories; foreign or self. Foreign SAgs

are soluble intermediate-sized proteins that are produced by gram positive and some gram

negative bacteria (27). Endogenous SAgs are cell-bound and are likely traces of genome-

incorporated retroviral sequences common in mice (43).

Unlike conventional antigens, SAgs bind as intact protein directly to the variable region of T-cell

receptor β chain (TCR Vβ) and outside of peptide-binding pocket of the MHC II molecule on the

antigen presenting cell (APC) (Figure 1) (a minority of SAg binds to the Vα region instead of Vβ

on the TCR). As such, SAg-mediated T-cell activation occurs without antigen processing by the

APC regardless of the specificity of the T-cell receptor for any particular antigen. Thus, SAgs

can activate up to 30% of an individual‟s T-cell repertoire (1:5 to 1:30 versus 1:104

– 1:106

with a

conventional antigens). Such polyclonal activation leads to signature expansion of responsive

TCR Vβ families (~50 families exist in humans) known as Vβ skewing, and a massive systemic

production of inflammatory cytokines.

5

A signature TCR Vβ2+ and Vβ8+ skewing of T-cells and elevated concentration of IL-1, IL-2,

IL-6 and TNFα cytokines are reported in KD patients. Systemic T-cell expansion and

inflammatory cytokine storm, in turn, lead to development of fever and vascular leakiness (39,

44).

Staphylococcal Enterotoxin B, SEB, is the prototypic SAg that is secreted from the gram positive

bacterium, Staphylococcus aureus. SEB activates T-cells bearing Vβ 3, 7, 8 and 17 receptors in

mice and Vβ 3, 12, 14 and 17 in humans. Following this clonal expansion, up to 50% of

activated T-cell undergo apoptosis (45) and the remaining Vβ8+ T cells demonstrate an anergic

phenotype (46). Anergy describes a state in which T-cells do not respond to stimuli, but remain

alive for an extended period in a hypo-responsive state (47). Following in vivo stimulation of T-

cells with SEB, a subset of T-cells become unresponsive to further stimulation with either T-cell

mitogens or anti-TCR mAbs (46, 48). Interestingly, SEB-induced anergized T-cell populations

do respond to PMA/Ionomycin stimulation; suggesting an anergy induced block in signal

transduction from stimulation of the T-cell receptors to Protein Kinase C (PKC) activation and

calcium mobilization (49). This post-activation scenario has been reported with many other

superantigens, signifying a similar interaction and mechanism of action amongst SAgs despite

varying structure (27, 50). For a more detailed discussion on SAgs and T-cell fate, see below.

6

Figure 1. T-cell activation with a superantigen versus a conventional peptide antigen.

Adapted from (25).

1.3 Superantigens and Apoptosis

Homeostasis in the immune system is achieved by a balance between the generation,

proliferation and differentiation of lymphocytes and apoptosis. Apoptosis, also called

programmed cell death is characterized by morphological changes of the dying cell: blebbing

and cell shrinkage, chromatin condensation, DNA fragmentation, phosphatidyl serine (PS)

externalization, and proteolytic cleavage of a number of intracellular substrates (51). In general,

two types of signaling pathways lead to apoptosis: the extrinsic pathway which is activated in

response to external death signals, and the intrinsic or mitochondrial pathway which is turned on

in response to cell stress such as withdrawal of growth factors or cytokines and irradiation.

7

The extrinsic pathway is mediated by death receptors of the TNFR family. The Fas receptor

(CD95, APO-1) is the most extensively studied death receptor in this family. Upon Fas Ligand

(FasL, CD178) binding, the Fas forms an intracellular death-inducing signaling complex (DISC)

through its cytoplasmic carboxy terminus “death domains” (DD) (52). Next, Fas-associated

death domain (FADD) adaptor protein is recruited to the receptor‟s DD, which in turn recruits

procaspase-8 (and procaspase-10) proenzyme. These proenzymes catalyze the formation of

active caspase-8/10 to trigger the apoptotic caspase cascade leading to DNA fragmentation and

cell death (Figure 2). Constitutive Fas expression has been reported in the cells of thymus, liver,

heart and ovaries, and its receptor, FasL, has been found in the testes, bone marrow, eyes, uterus,

spleen and on activated lymphocytes (53). Mice deficient in either Fas or FasL acquire

lymphoproliferative disease and humoral autoimmunity (54).

Mitochondria also play an essential role in the intrinsic apoptosis pathway by releasing

apoptogenic factors, such as cytochrome-c (cyt-c) into the cytoplasm (55). Mitochondrial

changes are predominantly prevented by antiapoptotic members of the Bcl-2 family of proteins

which contain 1- 4 conserved motifs known as Bcl-2 homology domains (BH1-BH4). These

molecules can be divided into 3 categories: antiapoptotic members (Bcl-2, Bcl-XL, Bcl-w, Mc1-1

and A1), proapoptotic members (such as Bax, Bak and Bok) and BH3-only proapoptotic proteins

(Bid, Bad and Bim) (56). Cyt-c is released in the cytoplasm during apoptosis where it initiates

caspase activation (56). While the antiapoptotic molecules work to prevent cyt-c release from the

mitochondrial inner membrane, proapoptotic members of the Bcl-2 family induce this event. In

the cytoplasm, cyt-c binds to and induces the oligomerization of apoptotic protease activating

factor – 1 (Apaf-1). This complex recruits an initiator caspase, procaspase – 9, which becomes

activated through this interaction. The cyt-c – Apaf-1 – Caspase-9 complex is termed the

8

apoptosome. When formed in the cytoplasm, the apoptosome recruits procaspase-3, which is

cleaved and activated by caspase-9 to caspase-3 and induces apoptosis (57).

Figure 2- Apoptotic Signaling Pathways - cellular stress leads to the activation of the intrinsic

mitochondrial pathway, while ligand binding to death receptors (i.e. Fas), leads to the caspase

cascade of the extrinsic pathway. Adapted from (51).

9

The mechanism of apoptosis following SEB stimulation has been controversial. Mice deficient

for Fas (lpr) showed impaired or no deletion of SEB-reactive CD4+ T-cells and CD8+ T-cells,

respectively, following a single dose of SEB injection (58). In vitro, Miethke et al. have also

reported that proliferating Vβ8+ T-cells are sensitive to Fas mediated death using a Fas receptor

cross-linking antibody (59). In contrast, a single dose injection of SEB reduces auto-immunity

and T-cell proliferation in lpr mice (60). SEB-activated T-cells have also been shown to undergo

apoptosis in mice deficient for Fas and TNFRs, but are resistant to apoptosis in mice over-

expressing the Bcl-2 protein (61). Another study also reported SAg-induced cell death as a result

of IL-2 deprivation, suggesting a role for the intrinsic apoptosis pathway (62). It is possible that

while the Fas pathway drives T-cell death following SAg-induced activation, the role of Bcl-2

dependent pathway is a result of antigen withdrawal and cytokine deprivation following

activation (61, 63) (for a discussion on co-stimulation and apoptosis see section 1.5 and

Discussion).

1.4 Superantigens and Anergy

As mentioned above, following SAg stimulation, most activated T-cells undergo apoptosis, but a

small subset of cells persist with minimal functionality and become anergized. T-cell anergy is a

state of intrinsic unresponsiveness that is usually characterized by limited proliferation,

differentiation and effector function as well as an extended life-span before apoptosis. While

anergic and apoptotic cells may share several biochemical signaling pathways, caspase activation

has been dissociated from the induction of anergy (64-65). Anergy may be reversible (in the case

of clonal anergy with IL-2 stimulation), or may require persistent antigen presentation (in the

case of adaptive tolerance). While clonal anergy is induced in previously activated T-cells due to

10

limited antigen stimulation, adaptive tolerance (or in vivo anergy) is induced in the absence of

proper co-stimulation or due to strong co-inhibition in naïve T-cells. SAg-induced anergy has

characteristics of both clonal and “in vivo” anergy (47). Following SAg stimulation in vivo, SAg

reactive T-cells fail to respond to re-stimulation with superantigen or TCR stimulating mAbs, but

their response to IL-2 re-stimulation remains intact (66). Anergy and apoptosis as SAg-induced

cell fates compared to activation-induced cell death (AICD) following activation with a

conventional peptide may be due to the differential signaling of SAg-stimulated T-cells that

occurs independently of Lck tyrosine kinase, but via a G-coupled protein/ PLC dependent

signaling pathway (50).

Work by McCormack et al has shown that re-stimulation of transgenic SEB stimulated T-cells

with a TCR specific peptide in vivo can rescue a subset of these cells from apoptosis (67).

Schwartz also suggests that SAg anergized T-cells retain full functionality upon re-stimulation

with a strong peptide or co-stimulation with CD28 (47). Preliminary work in our lab further

demonstrates that co-stimulation of SEB-activated T-cells with anti-CD28 or anti-4-1-BB mAbs

enhances T-cell survival and prevents apoptosis following SEB stimulation (68) (see below).

1.5 Co-stimulation

As mentioned above, T-cell activation with a conventional peptide requires two signals, one

provided by the peptide antigen and the other via co-stimulation. The CD28 – B7 pathway is the

best characterized co-stimulatory pathway, and is considered to be most important in the initial

activation of T cells (69). CD28 is constitutively expressed on almost all T-cells and binds to

B7.1 (CD80) and B7.2 (CD86), which are expressed on professional APCs (70-72). CD86 is a

monomeric ligand that is constitutively expressed and its expression is further increased upon

11

CD28 engagement. A synergistic effect on CD86 expression also occurs with the developing

CD40L – CD40 pathway upon T-cell activation (see section 1.5.1-1.5.4). In contrast, the

expression of CD80, a more potent dimeric ligand for CD28, is induced upon APC activation

with slower kinetics (73). Studies have shown that the monomeric binding of CD86 to CD28 is

essential for preventing T-cell activation in the absence of TCR stimulation (74).

In addition to its role in enhancing the delivery of signal to TCR and inducing the formation of

lipid rafts at the site of TCR engagement, CD28 signaling induces T-cell proliferation,

prevention of anergy, cytokine production and survival (75-77). CD28 signaling prevents CD3-

mediated induction of FasL gene expression and thus inhibits AICD (78-79). Ohashi and others

have also shown that CD28 signaling directly inhibits the Fas-signaling pathway by inducing the

expression of a negative regulator of this pathway, c-FLIP, which inhibits caspase-8 signaling

(80-81). In contrast, other studies have demonstrated that CD28 signaling promotes T-cell

survival by inducing the expression of anti-apoptotic protein, Bcl-XL, which inhibits cyt-c

release and mitochondrial induced cell death (82-83). Thus, CD28-mediated co-stimulation

inhibits T-cell apoptosis via interference with both extrinsic and intrinsic apoptosis inducing

pathways.

Mice deficient for CD28 (CD28 -/-

) mount a normal cytotoxic T-cell response to antigenic

stimulation with reduced T helper (Th) cell and B-cell activity in vivo. In vitro, CD28- /-

T cells

can initiate but not maintain a normal antigen-specific immune response – as measured by IL-2

production and proliferative capacity (77). Studies using CD28-/- mice have also demonstrated

the importance of CD28 signaling in IL-2Rα expression, humoral immune responses, germinal

centre formation and SAg-induced responses. These findings also suggest that although

prototypic as a co-stimulatory molecule, CD28 is not necessary for all T-cell functions (76).

12

It is now well-known that co-stimulation can be delivered via a number of other distinct

molecular interactions between the T-cell and APC. From a structural standpoint, these

molecular interactions can be grouped in two categories: the CD28 – B7 and the Tumour

Necrosis Factor (TNF) – TNF – receptor (TNFR) family of molecules (84). Stimulation of SAg-

reactive T-cells with other TNFSF members, namely 4-1BBL and TNFα, has previously been

shown to enhance survival in a subset of activated T-cells. Here, I focus on two other members

of the TNF – TNFR family, namely CD40 Ligand (CD40L, CD154, gp39, T-BAM, or TRAP)

and its receptor CD40, and examine their role in T-cell survival following SAg stimulation.

1.5.1 CD40L Protein Structure and Expression

CD40L is a 33 kDa type II membrane protein consisting of a 22- amino acid (AA) cytoplasmic

domain, a 24 – AA transmembrane domain and a 214-AA extracellular domain (TNF – like

region) (85). Like other members of the TNF superfamily, CD40L is mainly expressed on the

cell surface as a trimeric complex or shed in a soluble form (sCD40L). This soluble form retains

the ability to form trimers, bind to receptors and deliver biological signals. Murine CD40L lacks

an amino-terminal signal peptide and presents an extracellular carboxy terminus. Sequencing

studies predict a 78% homology between murine and human CD40L; with 75% and 81% identity

between the extracellular and cytoplasmic domains, respectively (85).

CD40L expression is mainly restricted to mature activated T-cells. Its expression can be induced

on Th0, Th1, and Th2 cells and is primarily confined to CD4+ T cells. A small number of CD8+

T cells and CD4- CD8- γδ TCR+ T-cells also express CD40L (86). In addition, CD40L has been

detected on mast cells, basophils, eosinophils, and under some conditions on B-cells, NK cells,

monocytes/ macrophages and DCs (Figure 2). More recently, platelets were found to contain

13

intracellular stores of CD40L which is expressed on the cell surface and shed as sCD40L upon

activation. Platelet-bound CD40L demonstrates biological functionality and induces expression

of chemokines, adhesion molecules and tissue factor (87). CD40L has also been detected on non-

hematopoietic cells such as endothelial cells (ECs), SMCs and fibroblasts (86).

In vitro, multiple modes of activation have been shown to induce CD40L expression on CD4+

T-cells; stimulation with phorbol myristate acetate (PMA) plus ionomycin, SAg stimulation, and

anti-CD3 antibodies. In addition, co-stimulation through CD28 further augments and stabilizes

anti-CD3-induced CD40L expression on CD4+ T-cells. The expression of CD40L on activated T

cells is transient and tightly regulated. Early expression from preformed CD40L is reported as

early as 5-15 minutes on anti-CD3 stimulated CD4+ CD45RO

+ T cells. A second wave of

CD40L expression following protein synthesis can be detected on T-cells as early as 1-2 hours,

peaking at 6-8 hours and gradually decreasing. Apart from this intrinsic transient expression,

CD40L expression is regulated by CD40 mediated CD40L endocytosis and degradation,

proteolytic cleavage to sCD40L and CD40L mRNA down-regulation (85).

On platelets, classic agonists such as thrombin and ADP as well as pathogenic agents can induce

platelet activation and CD40L release as early as 1 minute (88). Platelet expression of CD40L

highlights a previously unappreciated role for these cells in immunological functions. Similarly,

the description of new receptors for CD40L besides CD40, namely, the integrins αIIbβ3, α5β1

and Mac-1 on non-hematopoietic cells, has established a role for CD40L in vasculature and

atherosclerosis (89) (for more details on platelets and CD40L, see section 1.6 and Discussion).

14

1.5.2 CD40: The Classical CD40L Receptor

CD40 is a 45-50 kDa member of the TNFR superfamily and the classic receptor for CD40L (90).

CD40 expression was originally described on B-cells, fluctuating with progression through B

cell cycle, differentiation and survival upon ligation with CD40L (91). Besides B-cells, it is now

known that CD40 is expressed on a variety of other cell types: platelets, neutrophils, monocytes

and macrophages, immature and mature DCs, eosinophils and basophils (92-98). CD40

expression has also been described on non-hematopoietic cells: ECs, SMCs, fibroblasts and

keratinocytes (99-100).

Although constitutively expressed on most cell types, CD40 expression can be regulated. CD40

expression on other professional APCs and non-lymphoid APCs (ECs and SMCs) is upregulated

by cytokines and chemokines such as IL-3, IL-4, IL-1, IFN-γ and TNFα (101-104). Inwald et al.

have demonstrated constitutive expression of functional CD40 on platelets (105).

15

Figure 3. CD40 and CD40L are expressed on a variety hematopoietic and non-

hematopoietic cell types. Adapted from (86).

1.5.3 CD40 – CD40L Forward Signaling

The pivotal role of CD40L – CD40 signaling in T-cell – dependent B-cell responses was

highlighted by the finding that patients suffering from X-linked hyper-IgM syndrome (HIGM)

had mutations in the CD40L gene (106). Similar defects were found in mice with CD40L/ CD40

deficiencies. Indeed, it is now known that CD40 cross-linking guides B-cell differentiation

through rescue from apoptosis, localization to germinal centres, and selection and maturation

into memory cells (85). In addition, CD40 signaling leads to B-cell proliferation, isotype

switching, cytokine (TNFα, IL-10, IL-6) production and expression of co-stimulatory molecules,

CD80 and CD86 (107).

16

T cell dependent B-cell responses are impaired in the absence of CD28 – B7 signaling. Lumsden

et al. have demonstrated that the role of CD40 – CD40L and CD28 – B7 pathways are non-

redundant and distinct: the CD28 – B7 pathway is required for proper T-cell activation and

CD40L expression, which in turn cross-links B-cell CD40. CD40 signaling then triggers isotype

switching and B – cell CD80 and increased CD86 expression for further T-cell co-stimulation

(108).

CD40 cross-linking on monocytes and DCs also triggers increased CD80/ 86 expression,

survival, cytokine secretion and nitric oxide (NO) production (109-110). Indeed, CD40 signaling

is critical for DC maturation and proper antigen presentation and enhances the ability of

monocytic DCs in cross-priming CD8+ T cells. Increased CD40 expression on non-

hematopoietic cells has been reported in vitro following stimulation with IL-1/ IFN-γ. However,

the relevance of CD40 signaling on these cells is relatively less understood. It is more likely that

CD40 – CD40L signaling on non-hematopoietic cells has an important role in inflammation (86).

CD40 signaling on these cells leads to increased expression of adhesion molecules and release of

pro-inflammatory cytokines. In addition, many inflammatory diseases such as allograft rejection,

atherosclerosis, lupus, HIV infection, and Alzheimer‟s disease are associated with elevated

expression of endothelial and epithelial CD40 (86).

1.5.4 CD40L Reverse-Signaling

While CD40 signaling and function in humoral and cellular responses has been extensively

studied, CD40L (reverse-) signaling and its effect on activated T-cells is relatively less

understood (111). Work on CD40L reverse-signaling suggests an important role for this

molecule in T-cell co-stimulation and development of T-cell helper function. Van Essen et al.

17

showed that sCD40 can initiate germinal centre formation in CD40-/-

mice. In addition, they

demonstrated that T-cells primed in the absence of CD40 are not able to provide help to WT B-

cells (112). Similarly, Cayabyab et al. reported that interaction between human small, resting T-

cells and CD40 transfected murine cells substantively enhances αCD3-induced T-cell

proliferation particularly in CD4+ T-cells (113). Another group showed that CD40L cross-

linking in conjunction with αCD3 + αCD28 stimulation provides co-stimulation to CD4+ T-cells

leading to enhanced IL-4 synthesis (114).

Reverse-signalling downstream of CD40L also leads to phosphorylation of tyrosine kinases with

localization to lipid-rafts as a crucial outcome of this effect. Brenner et al. used anti-CD40L

mAbs to induce CD40L cross-linking on T-cells and showed that CD40L reverse-signaling

induces phosphorylation of Lck tyrosine kinase, JNK and p38 signaling proteins and leads to

PLC-γ activation (115-116). More recently, cross-linking of CD40L in Jurkat cell lines with

sCD40 was shown to induce PKC activation, calcium mobilization and phosphorylation of MAP

kinases ERK1/2. El-Fakhry et al. demonstrated that CD40L reverse-signaling also leads to

recruitment of this molecule into lipid-rafts, providing more support for the role of CD40L

signaling on T-cell co-stimulation (111).

In the context of SAg stimulation, El-Fakhry et al. used sCD40 to show that CD40L reverse-

signaling acts as an effective co-stimulator for IL-2 production in SAg-stimulated T-cell

populations (111). A role for CD40L reverse-signaling in T-cell co-stimulation is significant

both in the context of B-cell/ T-cell interactions, as well as T-cell cross-talk with non-

hematopoetic cells and platelets expressing CD40 and other cross-linking receptors of CD40L.

18

1.6 Platelets

1.6.1. Platelet Histology

Platelets are small, discoid anucleate granular cells that are formed in the bone marrow as a

result of megakaryocyte (MK) cytoplasmic reorganization. Primarily, platelets function to

maintain homeostasis by forming clots in response to tissue and vascular damage and

inflammation. Platelet numbers also increase during inflammation (thrombocytosis) through IL-6

induction of hepatic thrombopoietin (TPO) production- a regulator of thrombopoiesis.

Platelets contain three types of granules: lysosomes, dense (δ) granules and α-granules.

Lysosomes are involved in intracellular protein degradation and thrombus dissolution. Dense

granules are rich in mediators of vascular tone: serotonin, 5‟-diphosphase (ADP), calcium and

phosphate. In contrast, α-granules store and release contents relevant to haemostatic functions,

including adhesion (fibrinogen, thrombospondin, vitronectin, von Willebrand factor and

laminin), coagulation (e.g. plasminogen), endothelial cell repair [e.g. platelet derived growth

factor, permeability factor, transforming growth factors α and β (TGFs)], and innate and adaptive

immune functions (MMP release, response to bacteria and pathogens, and CD40L expression)

(117) (for more on platelets and adaptive immunity see below).

The platelet plasma membrane contains glycoprotein (GP) receptors that determine platelet

reactivity in homeostasis and arterial thrombosis. These GP receptors either facilitate platelet

adhesion (by binding to adhesive protein) and/or respond to chemical agonists. The GP receptors

that respond to chemical agonists are primarily comprised of integrins: a major class of adhesive

molecules composed of non-covalently linked heterodimers of α- and β- subunits. Of these,

19

GPIIbIIIa/ αIIbβ3 is the most prevalent receptor (>80,000 copies per platelet), and mediates

platelet aggregation and adherence to the endothelium. The integrin αIIbβ3 (GPIIbIIIa) is

formed via calcium-dependent association of gpIIb (CD41) and gpIIIa (CD61) glycoproteins.

Platelet adhesion at the site of vessel wall damage initiates activation that results in platelet

aggregation. Upon activation, platelets undergo a dramatic shape change to increase surface

membrane with multiple filipodia. Activation also leads to secretion of platelet granular contents

(i.e. degranulation) to increase the concentration of effector molecules at the site of injury. A

structure unique to platelets is a surface-connected canalicular system which weaves throughout

the cell and is continuous with the plasma membrane. During degranulation, platelet granules

“coalesce at the centre of the platelet, fuse with the canalicular system or plasma membrane and

secrete their contents”. P-selectin (CD62P), a classic marker of platelet activation, is one of the

molecules exposed on the platelet surface membrane upon degranulation. P-selectin acts as an

adhesion molecule and attracts leukocytes to the site of injury (117).

Another outcome of platelet activation is the scrambling of PS molecules on the outer surface of

platelet plasma membrane. This event leads to the formation of small platelet-derived particles,

known as platelet-microparticles (PMPs). PMPs express many of the same surface receptors as

platelets, and their formation is detected by annexin-AV binding to surface PS.

Platelet activation ultimately leads to aggregation which is characterized by a conversion of low

affinity αIIbβ3 receptor to a high affinity activated state capable of binding to fibrinogen, von

Willebrand factor and CD40L. “Outside-in signaling” by this activated receptor also leads to

stabilization of platelet aggregates (117). Thus, αIIbβ3 is a receptor crucial to many platelet

functions.

20

1.6.2. Platelets and Immunity

Thirty years ago, platelet function was simplified to maintaining homeostasis and inflammation

through clot formation and leukocyte recruitment at sites of inflammation. Today, platelets are

also known as important mediators of innate and adaptive immunity with an array of anti-

bacterial and anti-viral functions (118).

Platelets interact directly and indirectly with microbial pathogens and contribute to pathogen

clearance. Clawson and White originally demonstrated that bacterial pathogens could adhere to,

aggregate, activate and prompt degranulation of platelets (119). Streptococcal and staphylococcal

microorganisms have most extensively been studied in their interaction with and aggregation of

platelets (118). Platelets have also been shown to bind to and internalize viruses (e.g. HIV,

Rotaviruses, adenoviruses, Ebola virus, etc.) through their surface integrin receptors (120).

Internalization of these particles induces changes in platelets such as surface P-selectin

expression which enables macrophage mediated clearance of these pathogens (120). Platelets

also express receptors common to monocytes: FcγRII (121), FcεRI (122), C-reactive protein

receptor, thrombospondin receptor CD36 (123), complement receptor 3 (CR3) and receptors for

C3a and C5a (124), products of complement fixation. The expression of Toll-like receptors has

also been described on platelets, particularly upon activation (118, 125). This is particularly

interesting since the extract used to induce KD in an animal model has TLR-2 agonistic activity,

and coronary arteritis cannot be induced in mice deficient in TLR-2 (126) (for more on KD

disease model, see section 1.7). In addition, platelets express functional chemokine receptors,

CCR1, CCR3, CCR4 and CXCR4, and respond to TNFα, IL-1 and IL-6 cytokines in a manner

similar to leukocytes (127-129).

21

Platelets mediate many adaptive B and T cell responses involved in pathogen clearance and

autoimmune inflammatory conditions such as arthritis, atherosclerosis and systemic lupus

erythematosus (130). Kroczek et al. originally described the expression of functional CD40L on

activated platelets, which is able to induce endothelial cell activation and leukocyte recruitment

in a manner similar to IL-1 and TNFα (131). Its receptor, CD40 was also shown to be

constitutively expressed on platelet cells (105). It was then found that platelet CD40L enables

regulation of B-cells through interaction with DC CD40 (132), and enhances T-cell mediated

responses (132). Further support for the role of platelet CD40L in modulation of T-cell responses

comes from studies demonstrating reduced CD8+ lytic function in platelet depleted mice (133).

Others have reported that platelet soluble CD40L mediates cytokine and chemokine secretion by

CD40 receptor bearing cells (134). In addition, studies using CD40L-/- mice injected with

CD40L+ platelets have established a role for platelet CD40L in B-cell responses (132).

Platelets interact with many other cells besides DCs and lymphocytes, including other platelets

and non-professional APCs through CD40L-CD40 interactions. In fact, platelet CD40L can

activate other platelets by binding to their surface CD40 and αIIbβ3 receptors. CD40L binding to

the β3 portion of the αIIbβ3 receptor induces its phosphorylation and CD40L cross-linking (105,

135); an interaction important in human thrombus stabilization.

22

1.6.3. Platelets and Kawasaki Disease

Platelets are the earliest cells to appear at sites of vascular inflammation and have been

implicated in vasculitis, atherosclerosis and plaque formation. In Kawasaki Disease, platelet

numbers are elevated to > 450,000, and peak platelet number correlates with subsequent risk of

coronary artery aneurysm formation (136). Since then, many studies have reported a link

between platelet numbers, aggregation and stimulation, defective apoptosis and risk of coronary

artery complications in KD (137-140). Increased levels of CD40L expression on activated

platelets and CD4+ T-cells from blood samples of KD patients are also correlated with risk of

aneurysm formation (137). These reports as well as studies establishing platelet-leukocyte cross-

talk through CD40-CD40L interactions suggest an important role for platelets in the

pathogenesis of KD. Indeed, anti-platelet therapeutics, such as aspirin, are commonly used in KD

patients with more powerful antiplatelet agents such as abciximab (anti-αIIbβ3) in the presence

of intra-mural thrombus; though their efficacy and therapeutic mechanism of action remain

elusive (141-142).

1.7 Animal Models of Kawasaki Disease

The study of KD in human subjects is not feasible in part due to inaccessibility of diseased

coronary artery tissue from KD patients (15, 143). Thus, several animal models of disease have

been developed, which although imperfect, mimic human KD in some or most aspects.

A canine juvenile polyarthritis syndrome was first described in 1992 as a model for KD (144-

145), in which spontaneous vasculitis leads to immunological abnormalities such as increased

serum IgA, a decrease in total peripheral T-cells and increased monocyte/ macrophage

23

activation. Use of canines and the spontaneous nature of disease made this model impractical

however. In another model, horse serum infusion leads to coronary artery aneurysms in weanling

rabbits, but coronary infiltration is minimal and vasculitis is not specific to the coronary arteries

(146). Infusion of horse serum has also been used to develop immune complex vasculitis in

swine, characterized by open skin rashes and coronary artery dilation (147). Like the rabbit

model, arteritis is not specific to vessels of the heart in this model.

In 1985, Lehman et al. described an inflammatory coronary arteritis disease similar to KD in

various strains of inbred mice that was induced by a single intraperitoneal (I. P.) injection of

group B Lactobacillus casei cell wall fragments (148).

1.7.1 Lactobacillus casei Cell Wall Extract (LCWE) Model of Kawasaki

Disease

L. casei is a common component of the normal enteric flora of both humans and rodents (149).

Although non-pathogenic, L. casei has been studied due to its similarities with group A and

group C streptococci – which are known causative agents of post-infectious arthritis in both

humans and mice. Like these arthritic agents, L. casei has a high cell wall rhamnose content and

is resistant to degradation by macrophages and lysozymes (150-151).

Work at our laboratory has described a novel SAg in Lactobacillus casei Cell Wall Extract

(LCWE). It was shown that the LCWE model has many features of a SAg-mediated disease:

polyclonal proliferation of T-cells followed by peripheral deletion, non-clonal MHC restriction,

an allotype-dependent hierarchy in the efficiency of antigen presentation by MHC II molecules,

requirement for antigen presentation but not processing, and a characteristic Vβ skewing (Vβ 2,

4, 6, and 14). In addition, Duong et al. demonstrated a correlation between LCWE SAg activity

and its ability to induce coronary arteritis (152).

24

Progression of inflammation in the LCWE model of coronary arteritis closely mimics KD in

humans. Inflammation and infiltration of the coronary artery is visible as early as 3 days

following LCWE injection. By days 7-14, immune cells infiltrate the entire vessel wall and

maximal immune cell infiltration is seen throughout the coronary artery vessel wall on day 28

post LCWE injection. This time point correlates approximately with the acute phase of KD (see

1.1.1). Although aneurysms and myocarditis can be detected as early as day 14, elastin

breakdown is visibly detectable in the coronary arteries by day 42. This time point corresponds

to the subacute phase of KD in children when aneurysms are most commonly detected. In

addition, studies indicate that young mice of 5-7 weeks are most susceptible to LCWE-induced

coronary arteritis, and that mice deficient in T-cells do not develop disease (152).

Histological changes are also comparable between the LCWE model of KD and human KD, and

LCWE-injected mice are responsive to IVIG therapy (153), the most common treatment for KD.

Thus, the LCWE model of KD resembles human KD in histopathological changes, time-course

of disease and therapeutic response (152, 154).

Work at our lab has characterized several immune features of the LCWE model of KD. Lau et al.

have shown that TNFR deficient mice do not develop disease and TNFα is necessary for

coronary pathogenesis following LCWE injection. TNFα secretion was shown to activate SMCS

and lead to MMP-9 secretion; an enzyme that is also stored in platelet cell granules and is

released upon platelet activation (see section 1.6) (41, 155-157). The role of platelets in MMP-9

release leading to aneurysms in this model remains to be investigated.

The most important feature of this model is T-cell infiltration and persistence in the coronary

artery (152). Sequencing studies have characterized the population of T-cells that persist in the

25

coronary artery following deletion of activated T-cells to be an oligoclonal SAg-reactive Vβ6+ T-

cell population (158). Extended survival and persistence of SAg activated T-cells is not

consistent with the established outcomes of SAg stimulation: anergy or apoptosis (see sections

1.3 and 1.4).

26

1.8 Rationale

Kawasaki Disease is an inflammatory disease of childhood characterized by coronary arteritis

that may lead to aneurysm formation. Numerous pieces of evidence suggest a superantigenic –

mediated trigger of disease. SAg activity also induces disease in an LCWE murine model of

disease. Normally, T-cells undergo apoptosis following SAg stimulation; thus T – cell

persistence in the KD heart cannot be solely explained by SAg stimulation. Previous work

demonstrates that stimulation of SAg – reactive T-cells with a peptide or co-stimulation via

CD28 and 4-1BB rescues a subset of these cells from apoptosis. CD40L, another member of the

TNF superfamily of molecules, is also important in T-cell co-stimulation. Elevated numbers of

activated platelets, which also express CD40L receptors, CD40 and αIIbβ3, and CD4+ T-cells

expressing surface CD40L have been linked with aneurysm formation in KD patients. In addition

to CD40L, activated platelets also store and release MMP-9 – an enzyme that is critical for

aneurysm formation in the LCWE model of disease.

1.9 Hypothesis

Co-stimulatory molecules such as CD40L and CD40, and cell subsets such as platelets may

contribute to enhanced survival of SAg-reactive T-cells in an LCWE model of Kawasaki

Disease.

27

2 Methods

2.1 Mice

C57BL/6 mice were purchased from Charles River Laboratory (Wilmington, MA) or the Center

for Phenogenomics (TCP). CD40L-/-

, CD86-/-

and CD28-/-

mice from Jackson Laboratory (Bar

Harbor, ME). Animals were housed under specific pathogen-free conditions at TCP or the

Hospital for Sick Children. All animal procedures were approved by the Animal Care Committee

at the Hospital for Sick Children and at TCP.

2.2 Blood Collection

For tail-vein bleeds, each animal was heated under a heat-lamp for 20 minutes, placed in a

whole-body restrainer and its tail was swabbed with alcohol. A sharp surgical blade was then

used to make an incision over the tail vein located approximately 2-3 cms from the tip of the tail.

No pressure was applied to the tail. Blood was collected using heparinized micro-hematocrit

capillary tubes (Fisher Scientific, Pittsburgh, PA) with a CAPTROL III microdispenser

(Drummond, Broomall, PA). For retro-orbital bleeds, animals were anesthetized by inhaled

isofluorane. A heparinized micro-hematocrit capillary tube was inserted into the retro-orbital

sinus venous plexus, and blood was collected by capillary action and dispensed using the

CAPTROL III microdispender. As previously described (159), 50 µl of blood was dispensed in

1.5 ml plastic tubes containing 200 µl of Tris-Buffered Saline (TBS) with heparin: 20 mM

Tris/HCl (Omnipur, Gibbstown, NJ), 137 mM NaCl (Bioshop, Burlington, ON), pH 7.3,

containing 20 U/ ml heparin (Pharmaceutical Partners of Canada, Richmondhill, ON). Whole

blood was further diluted in modified Tyrode Albumin Hepes (TAH) buffer, pH 7.4: 137 mM

28

NaCl; 2.68 mM KCl (Bioshop), 11.9 nM NaHCO3 (Sigma-Aldrich, Saint Louis, MO), 0.4 mM

NaH2PO4 (Bioshop), 2 mM CaCl2 (BPH Inc., Toronto, ON), 5 mM Hepes (Omnipur), 5.55 mM

D-Glucose Anhydrous (Bioshop) and 0.35% Bovine Serum Albumin Fraction V (Omnipur). For

flow-cytometry, blood was further diluted 1:2000.

2.3 Platelet Stimulation

Diluted whole blood (25 µl) in modified THA buffer was aliquoted to 1.5 ml plastic tubes and

incubated with 10 µg/ml ADP (Sigma-Aldrich), collagen, AYPGKF (Par4 peptide) (gifts from

Dr. Margaret Rand, Hospital for Sick Children, Toronto, ON) or 10 µg of partially purified SEB

(Toxin Technology Inc., Sarasota, FL). Samples were simultaneously incubated with titrated

concentrations of antibodies, where indicated, for 30 min at 37 °C and fixed in 1%

paraformaldehyde (PFA).

2.4 Cell Culture

For apoptotic assays, ex-vivo splenocytes from 5-7 week old mice were cultured in 12-well plates

(106 cells/ ml) in complete Iscove‟s medium supplemented with 10% heat-inactivated fetal

bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen, Burlington, ON), 1mM sodium

pyruvate (Invitrogen), 0.1mM non-essential amino acids (Invitrogen), 50 M trypan blue and 10

mM HEPES (Invitrogen), and stimulated with highly purified SEB (0.3 µg/mL). Where

indicated, 12-well plates were pre-coated for a minimum of 3 hours at 37°C with stimulatory

antibodies and appropriate isotype controls in 500 µl/ well Phosphate Buffer Saline (PBS, pH

7.2) as follows: murine anti-CD40L (clone MR1, Armenian hamster anti-mouse IgG), anti-CD40

(clone 1C10. Rat anti-mouse IgG2aĸ) and anti-CD28 (Golden Syrian hamster anti-mouse IgG,

29

clone 37.51). For CD40L kinetics assay, splenocytes were stimulated with 10 ng/ml and 5 µg/ml

PMA plus ionomycin respectively, and harvested at 6 hours post-stimulation. For T-cell isolation

and mixing assays, a Pan T-Isolation Kit II (Miltenyi Biotec, Germany) was used and cells were

isolated according to manufacturer‟s protocol. Unless otherwise noted, all antibodies were

purchased from eBioscience.

2.5 Annexin-AV Apoptosis Assay

Live cell numbers were counted using Trypan Blue exclusion dye, and surface stained for Vβ8+

(clone F23.1) TCR. Death was measured using annexin-AV binding and 7-ADD exclusion

according to manufacturer‟s protocol (Annexin-AV FITC kit, BDbioscience). Relative

percentage of cells undergoing apoptosis and absolute numbers of live cells were calculated as

follows:

% cells undergoing apoptosis = (%7-ADD-ve

, annexin-AV+ve

, Vβ8+ve

cells)/

(%7-ADD-ve

, Vβ8+ve

cells)

Absolute # live cells = live cell counts X (%7-ADD-ve

, annexin-AV-ve

, Vβ8+ve

cells)

2.6 Flow-Cytometry

Cells were stained with 1 µg/ 106

cells eFluor-450 (eBioscience) fixable viability marker,

permeabilized and intracellularly stained using Bcl-2 staining kit (BDBioscience). The following

antibodies were used to stain for B-cell CD80/86, T-cell CD40L expression and platelet

activation: anti-CD40L-APC/ biotin (MR1), anti-CD40-PE (1C10), anti-mouse activated αIIbβ3

(JON/A, Emfret Analytics, Wurzburg, Germany), anti-CD80-FITC (16-10A1), anti-CD86-FITC

30

(GL1), anti-CD19-PE (1D3), anti-CD41-PE-Cy7 (MWReg30), anti-CD178(FasL)-PE (MFL3,

BDbioscience). Unless otherwise indicated, all antibodies were purchased from eBioscience.

10,000 – 100,000 cell events were collected on LSR II (BDbioscience) and FACSCanto II

(BDbioscience) flow cytometers, and analyzed on FlowJo (version v 9.1, Tree Star, Ashland,

OR).

2.7 Caspase Staining

For active caspase flow-cytometry, harvested splenocytes (106 cells in 500 µl medium) were

incubated for 1 hour at 37° C with 1 mg/mL FITC-conjugated catalytic peptide-specific

inhibitors of active caspases-3, -8 and -8 (Abcam, Cambridge, MA), washed twice with wash

buffer provided with the staining kit, and surface stained as described above for flow-cytometry.

2.8 Immunoblotting

Splenocytes stimulated with cross-linking antibodies (2.4.) were harvested on day 5 and

homogenized in ice-cold lysis buffer (50 mM Tris pH 7.6, 2% SDS, 10µg/mL leupeptin, 0.1 mM

phenylmethylsulfonyl fluoride). Samples were sonicated for 5 seconds and total protein

concentration was determined using a Lowry-based method according to manufacturor‟s

protocol (DC-Protein Assay, BioRad, Mississauga, ON). Equal amounts of total protein (10 µg)

were diluted in lysis buffer and reducing sample buffer (containing 2-mercaptoethanol). Proteins

were loaded on a 15% polyacrylamide gel, and electrophoresed through SDS running buffer at a

voltage of 135V for 2.5 hours. Gels were wet-transferred to a methanol-activated polyvinylidene

fluoride membrane at 70V for 45 minutes. Membranes were blocked over night at 4°C in

blocking buffer (5% BSA, 0.1% Tween-20/ TBS), followed by incubation in blocking buffer

containing an optimal concentration of rabbit anti-mouse cleaved caspase-3 primary antibody

31

(Asp175, clone 23, Cell Signaling, Boston, MA) for 1 hour at room temperature. Membranes

were washed 5 times (5 minutes each) in wash buffer, and incubated with an HRP-conjugated

goat anti-rabbit secondary antibody for 1 hour at room temperature. After further washes, an

enhanced chemiluminescent luminal substrate was applied to the membrane according to

manufacturer‟s recommendation (PerkinElmer, Woodbridge, ON). Photographic films was

exposed to luminal-treated membrane in the dark (2-10 minutes) and developed. Membranes

were further stripped of protein per manufacturer‟s protocol (Restore Western Stripping Buffer,

Pierce, Rockford, IL), and re-probed for the housekeepining gene GAPDH used as a loading

control. Mean luminescence intensity (MLI) of bands was measured using Adobe Photoshop

CS5 and expressed as MLI ratio of caspase-3 protein to GAPDH.

2.9 Statistical Analysis

Statistical significance was calculated where applicable by paired, two-tailed Student‟s t-tests. P

values of <0.05 were considered significant. Where indicated, error bars indicate mean ±

standard deviation (SD).

32

3 Results

3.1 SEB induces Platelet De-granulation and αIIbβ3 Receptor

Activation

Many bacteria and viruses have been shown to activate platelets and induce de-granulation and

aggregation both in vitro and in vivo (118, 160). However, the effect of superantigenic

stimulation on platelet de-granulation is not known. Initially, platelet activation was measured

by surface P-selectin, CD40L and activated αIIbβ3 receptor expression using classic platelet

agonists, including collagen, ADP and AYGPKF peptide. Tail-vein blood was collected for these

experiments. However, subsequent experiments showed that retro-orbital blood was a superior

method for platelet flow-cytometry as it avoided basal platelet activation through contact with

tissue factor.

I aimed to establish platelet de-granulation and activation, as measured by surface CD40L

expression and αIIbβ3 receptor activation, respectively, upon stimulation with SEB in vitro.

Retro-orbital blood was stimulated with SEB and analyzed for surface expression of various

markers by flow-cytometry. Platelet single file events were identified based on light-scatter

properties and CD41 positivity (Figure 4a). P-selectin was chosen as a classic marker of platelet

de-granulation. CD40L and CD40 were chosen due to their importance in platelet – lymphocyte

interactions. In addition, the activated conformation of αIIbβ3, which is able to bind to and cross-

link CD40L, was detected using a conformation-specific antibody. SEB was found to induce

platelet de-granulation (increased P-selectin and CD40L surface expression) and to activate the

αIIbβ3 receptor on platelet single-file events, compared to unstimulated controls (Figure 4b-d).

In contrast, CD40 was constitutively expressed on platelets in both unstimulated and SEB

stimulated samples (Figure 4e).

33

The Fluorescence Minus One (FMO) control stains of the unstimulated samples are shown in

grey. FMO controls for stimulated samples were also analyzed to control for enhanced cell auto-

fluorescence following SEB stimulation. No difference between FMO stains on SEB stimulated

and SEB unstimulated samples were observed (not shown).

34

Figure 4. SEB stimulates platelet de-

granulation and induces αIIbβ3

receptor activation in vitro. a. CD41+

positive single-file platelet events in

whole blood were gated based on light

scatter properties with a CD41

positivity threshold. b, c. SEB induces

platelet degranulation as measured by

P-selectin and CD40L expression. d.

SEB stimulation also induces integrin

receptor αIIbβ3 activation. e. CD40 is

constitutively expressed on platelets

regardless of activation state. f – i. Bar

graphs showing compiled results of 3

independent experiments. Black and

white bars show unstimulated and SEB-

stimulated samples respectively. * and

** indicate P values of <0.05 and

<0.001, respectively, as measured by

Student‟s two-tailed paired t-test (n=3).

Error bars denote standard deviation

from the mean.

35

3.2 Kinetics of CD40L Expression on SEB Stimulated Vβ8+

T-cells

Platelet CD40 and αIIbβ3 receptors are capable of binding to and cross-linking the CD40L

receptor on T-cells (132, 161). In Figure 4, I determined platelet expression of CD40L and its

cross-linking receptors (αIIbβ3 and CD40) in response to SEB stimulation, thus we proceeded to

examine the interaction of CD40L with its receptors and its effect on T-cell survival following

SEB stimulation. As previously mentioned, SAg – reactive T-cells persist in the coronary artery

and survive apoptosis in the LCWE-model of KD. We have previously shown that co-stimulation

via CD28, 4-1BB and TNFα rescues a subset of SAg reactive T-cells from apoptosis (162-163).

To investigate the role of CD40L (reverse-) signaling in co-stimulation for SAg-reactive T-cells,

I determined the kinetics of CD40L expression following SEB stimulation on Vβ8+

T-cell

populations. C57BL/6 WT splenocytes were cultured with 0.3 µg SEB (Figure 5a) or in medium

alone (Figure 5b) and CD40L expression on Vβ8+

T-cells was measured using flow-cytometry. A

combination of PMA plus ionomycin, which is known to activate T-cells and induce rapid

CD40L expression in a Vβ – nonspecific manner, was included as a positive control (Figure 5c).

T-cell CD40L was detectable by 48 hours post-SEB stimulation. Expression levels were

maximal by 72 hours in the Vβ8+

population, and started to decline by 96 hours post stimulation.

These results suggest that CD40L cross-linking is most important in the first 72 hours post-SEB

stimulation in vitro.

36

Figure 5. Kinetics of surface CD40L expression on Vβ8+

T-cells. Splenocytes from C57BL/6

mice were stimulated with SEB, PMA/Ionomycin or in media alone and CD40L surface

expression on Vβ8+ T-cells was assessed by flow cytometry at 6, 24, 48, 72 and 96 hours post-

stimulation. a. CD40L expression on Vβ8+ T-cells gradually increases until 72 hours post-SEB

stimulation, after which it begins to decline (96 hours). b. CD40L expression does not increase in

the absence of SEB stimulation in media alone (negative control). c. PMA/Ionomycin

stimulation upregulates CD40L expression at 6 hours post-stimulation in a Vβ8 non-specific

manner (positive control). These results are representative of two independent experiments.

37

3.3 CD40L Cross-linking Leads to Rescue of SEB-Reactive T-cells

from Apoptosis

To investigate the effect of CD40L – cross-linking on T-cell survival following SEB stimulation,

splenocytes from C57BL/6 and CD40L-/-

mice were stimulated with SEB plus αCD28 and

αCD40L cross-linking mAbs and analyzed 5 days later for cell death using flow-cytometry. Cell

death was measured by several viability dye markers: trypan blue and 7-ADD to count and gate

on live cells respectively; and annexin-AV to identify live T-cell populations undergoing

apoptosis. Following SEB stimulation, T-cell proliferation as measured by [3H] –thymidine

incorporation, declines by day 5 (not shown). Optimization experiments indicated that this time-

point coincides with the period when a significant proportion of T-cells are still alive but are

undergoing apoptosis. These results indicate that CD40L cross-linking reduces T-cell death as

measured by lower relative numbers of Vβ8+ T-cells undergoing apoptosis compared to isotype

treated controls (Figure 6d). This difference, although small, was observed across 3 independent

experiments, and is statistically significant (Figure 7b). Absolute numbers of live cells,

calculated as described in methodology, were also significantly higher amongst the αCD40L

treated samples compared to isotype treated controls (Figure 7e). Similar trends were observed in

CD28 co-stimulated splenocytes (used as a positive control in all rescue assays).

To confirm that the αCD40L mAb effect on survival is indeed CD40L mediated, splenocytes

from CD40L-/-

mice were assayed for cell death. Indeed, in the absence of CD40L, no significant

difference in apoptosis or absolute numbers of live cells was observed between stimulated and

control samples (Figure 7c, f) (n = 6). These results suggest that co-stimulation via CD40L cross-

linking has a positive effect on overall survival and rescue from apoptosis of SEB-reactive Vβ8+

T-cells populations.

38

Figure 6. CD40L cross-linking leads to rescue of SEB-reactive Vβ8+ T-cells from apoptosis.

WT splenocytes were stimulated with αCD40L (or isotype specific antibody), harvested on day 5

post-stimulation, and analyzed for cell death. a. Live splenocytes were gated based on 7-ADD

dye exclusion (top left). b. Annexin-AV and Vβ8 positive events were gated based on

fluorescence minus one (FMO) and unstained controls, respectively, from left to right:

splenocytes stained with annexin-AV + 7-ADD , Vβ8 + 7-ADD or unstained. Relative

percentage of Vβ8+ T-cells undergoing apoptosis was calculated by dividing annexin-AV

+ Vβ8

+

events over total percentage of Vβ8+ events. b – c. CD28 (positive control) and CD40L cross-

linking leads to lower percentage of cells undergoing apoptosis compared to appropriate isotype

treated controls. These experiments are representative of 3 independent experiments with 1-3

mice per experiment (n=9).

39

Figure 7. Compiled results of repeated apoptosis assays with anti-CD40L mAb. a, b. Co-

stimulation, delivered via CD28 or CD40L cross-linking reduces the relative numbers of SEB-

reactive Vβ8+ T-cells undergoing apoptosis c. No significant difference is observed in relative

numbers of CD40L vs. isotype stimulated SEB-reactive splenocytes from CD40L-/-

mice. d, e.

CD40L cross-linking (and CD28 cross-linking) also leads to increased absolute numbers of live

(7-ADD- Annexin-AV

- Vβ8

+ T-cells following SEB stimulation. f. this effect is abrogated in the

absence of endogenous CD40L. * and ** indicate P values of <0.05 and <0.001 respectively, as

measured by Student‟s paired t-test. Each line represents splenocytes from individual mice in 3

independent experiments, with 3 to 1 mice per experiment.

40

3.4 Caspase-3 Staining Confirms Annexin-AV Gating Strategy

Since Annexin-AV binding does not result in two obvious positive and negative peaks, I sought

to confirm our apoptosis assay gating strategy using caspase-3 activity as another marker of

apoptosis. The protocol used to analyze apoptosis was followed and ~7% fewer cells were found

to undergo apoptosis (annexin-AV, Vβ8+) when cross-linked with αCD40L mAb compared to

the isotype control as reported previously. Splenocytes positive or negative for active caspase-3

were expected to map within the annexin-AV positive and negative regions, respectively, used to

analyze apoptosis. Figure 8 shows that this was indeed the case. While a majority of active

caspase-3 positive cell events were annexin-AV (and Vβ8) positive (37.8%), a mere 2.43 % of

active-caspase-3 negative Vβ8 T-cells showed annexin-AV binding (Figure 8c). Thus, the

annexin-AV/ Vβ8 gating strategy that was set using FMO and isotype controls to analyze T-cell

apoptosis is a valid means of identifying relative and absolute numbers of T-cells undergoing

apoptosis.

41

Figure 8. Active caspase-3 staining confirms annexin-AV gating strategy. Splenocytes were

stimulated with SEB + αCD40L/ isotype and analyze for apoptosis as measured by annexin-AV

binding and caspase-3 staining. a. live (efluor450-ve) caspase-3 positive and negative

splenocytes were gated based on a negative (FMO, blue) and positive control (SEB stimulated

cells, red). b. CD40L cross-linking rescues Vβ8+ T-cells from apoptosis as measured by lower

annexin-AV binding compared to the isotype control. c. caspase-3 positive and negative gated

splenocytes map to the annexin-AV positive and negative quadrants respectively. d. FMO

staining controls used originally for gating annexin-AV+/-

populations.

42

3.5 Pro-survival Effects of αCD40L mAb are mediated through T-cell

CD40L Cross-linking

Although mainly confined to activated CD4+

T-cells, CD40L is also expressed on some APCs

and non-hematopoietic cells as well as activated platelets. To dissect the source of CD40L (T-

cells or APCs) leading to increased T-cell survival in whole splenocytes, isolated WT and

CD40L-/-

T-cells and APCs were mixed at a ratio of 25:75 (T-cell: APC) in various combinations

(Figure 9) and assayed for apoptosis. Figure 9 shows that when WT T-cells are mixed with WT

or CD40L-/-

APCs and stimulated with SEB, CD40L cross-linking leads to increased survival

compared to isotype treated controls. In contrast, in the absence of T-cell CD40L (WT APC +

CD40L-/-

T-cells), this trend is abrogated. Although these results do not reach statistical

significance (p= 0.0534 and 0.0865 for WT T-cells mixed with CD40L-/-

APCs, vs. p = 0.4190

for CD40L-/-

T-cells mixed with WT APC), a replicable trend exists in experiments where T-cell

survival was induced. The significance of this trend could be further determined via a power

analysis to see how many times the experiment would have to be performed to reach statistical

significance without committing a type I error. These results suggest a role for T-cell CD40L

cross-linking (and not APCs) in pro-survival effects of this molecule following SEB stimulation.

43

Figure 9. Mixing experiments to elucidate the cellular source of CD40L leading to

enhanced T-cell survival following SEB stimulation. T-cells and APCs from WT C57BL/6 or

CD40L-/-

splenocytes were isolated and mixed in various combinations, stimulated with SEB

and αCD40L (or isotype), and analyzed for Vβ8+

T-cell apoptosis on day 5 post-stimulation using

flow cytometry. a. CD40L cross-linking on WT APCs and WT T-cells lowers the relative

numbers of SEB reactive T-cells undergoing apoptosis compared to the isotype treated condition.

b. Similar relative percentages of Vβ8+

T-cells undergo apoptosis following CD40L cross-linking

compared to isotype treated WT APCs + CD40L-/-

T-cells. c. In the absence of APC CD40L (WT

T-cell + CD40L-/-

APC), CD40L cross-linking leads to lower percentage of Vβ8+

T-cells

undergoing apoptosis compared to the isotype-treated condition. d-f. CD40L on T-cells is

necessary for the prosurvival effects of CD40L cross-linking as measured by absolute numbers

of live Vβ8+

T-cells (n=5; each line indicates an independent experiment).

44

3.6 CD40L Cross-linking Decreases Caspase-3 Activation in SEB

Reactive Splenocytes

Caspase-3 is activated downstream of both the intrinsic and extrinsic apoptosis caspase cascades,

and was used as a final common marker of apoptosis to further investigate the effect of CD40L

cross-linking on T-cell survival following SEB stimulation. I used immunoblotting as well as

flow-cytometry to detect active caspase-3 levels in splenocytes stimulated with SEB + αCD40L

or the appropriate isotype control. As previously noted, αCD28 stimulation was used as a

positive control. Figure 10a shows the western blot of a representative experiment where

stimulation with either αCD40L or αCD28 (plus SEB) led to lower levels of cleaved caspase

(p17 subunit of cleaved caspase-3) protein concentration in whole splenocyte lysates. Results of

4 independent experiments are plotted in Figure 10b as ratio of caspase-3 to GAPDH (loading

control) housekeeping gene MLIs. Although not statistically significant by Student‟s paired t-

test, there is a reproducible trend for decreased caspase-3 protein expression amongst anti-

CD40L cross-linked samples as compared to isotype treated controls. Flow cytometry also

showed significantly lower levels of caspase-3 activity in SEB-reactive T-lymphocytes (Vβ8+)

following CD40L cross-linking on day 5 post-SEB stimulation (Figure 10c-d). A significant

decrease in caspase-3 activity was not observed on days 1-4 following CD40L + SEB

stimulation (data not shown).

45

Figure 10. Immunoblot and flow-cytometry analysis of caspase-3 activity following CD40L

cross-linking. a. WT whole splenocytes stimulated with αCD40L/ αCD28 (or isotype controls) +

SEB (or SEB alone) were lysed on day 5 post-stimulation and analyzed for cleaved caspase-3

levels (p17 subunit). b. MLI ratio of caspase-3 to GAPDH loading control from 4 independent

experiments shows a visible pattern in lower caspase-3 activity in 3 out of 4 experiments in

whole splenocytes following CD40L cross-linking. c. WT Splenocytes were stimulated with

SEB + αCD40L (red) or SEB + isotype (blue) and harvested on day 5. Vβ8+ events were gated by

flow-cytometry and analyzed for intracellular active caspase-3 expression. d. Compiled results of

3 independent experiments with 2 mice/ experiment show a significant decrease in caspase-3

activity 5 days post-SEB stimulation and CD40L cross-linking [Each line represents an

individual mouse. * indicates statistical significance as measured by Student‟s paired t-test, p <

0.05, FMO: fluorescent minus one (grey)].

46

3.7 CD40L Cross-linking Does Not Change Intracellular Bcl-2

Expression in SEB-Reactive T-cell Populations

To measure the effect of CD40L cross-linking on SEB-reactive T-cell populations, intracellular

concentrations of anti-apoptotic protein, Bcl-2, were measured on days 1-5 post SEB-

stimulation. No significant difference (or consistent trend) in Bcl-2 expression in Vβ8+ T-cells

was observed at any time point following stimulation (days 1-4 not shown). Figure 11 shows a

representative histrogram of intracellular Bcl-2 expression in live Vβ8+ T-cells treated with

αCD40L antibody + SEB (red), isotype + SEB (blue) or unstimulated (green) splenocytes on day

5 (a). Compiled results of 5 independent experiments are plotted in Figure 11b. No statistical

significance (Student‟s paired t-test) or visible trend in MFI of intracellular Bcl-2 expression is

observed in αCD40L treated samples versus the isotype treated controls. These results suggest

that the pro-survival effects of CD40L reverse-signaling are not mediated via inhibition of the

Bcl-2 protein. In addition, I did not see any difference in intracellular Bcl-XL protein expression

either amongst αCD40L + SEB or isotype + SEB treated controls (preliminary data not shown).

47

Figure 11. CD40L cross-linking does not affect intracellular Bcl-2 expression in SEB-

reactive T-cell populations. a. Splenocytes from C57BL/6 mice were cultured in media + SEB,

cross-linked with anti-CD40L/ isotype mAbs as described previously, and harvested on day 5

post-stimulation. No difference in intracellular Bcl-2 protein expression is seen amongst live

Vβ8+ T-cell populations. b. Compiled results of 5 independent experiments indicate no visible

trend or statistical significance (paired t-test) in MFI of αCD40L vs. isotype treated SEB-reactive

T-cells (MFI: mean fluorescent intensity, red: antiCD40L + SEB, blue: isotype + SEB, green:

media alone, grey: isotype control).

48

3.8 CD40L Cross-linking Lowers Caspase-8, but not Caspase-9

Activity in SEB-Reactive T-cells

To further elucidate how CD40L reverse signaling enhances T-cell survival following SEB

stimulation, αCD40L stimulated splenocytes were incubated with a cell-membrane permeable

fluorophore-conjugated substrate inhibitor of activated caspase-8 or caspase-9 prior to Vβ8

staining and analyzed at multiple time-points post SEB activation using flow-cytometry.

Figure 12 shows that caspase-8 activation is substantially lower in Vβ8+ T-cells treated with anti-

CD40L cross-linking mAb + SEB on days 4 and 5 post-stimulation as measured by percentage of

Vβ8+ T-cells positive for active caspase-8 and MFI of active caspase-8 (day 5 shown for latter).

In contrast, caspase-9 activity does not change significantly in CD40L cross-linked Vβ8+ T-cells

at either time-point following SEB stimulation. No visible difference in activity of either

caspases was observed on days 1-3 post-SEB stimulation by CD40L cross-linking (data not

known). These results suggest that the pro-survival effects of CD40L reverse-signaling are likely

mediated by inhibition of caspase-8 activity and thus the extrinsic apoptosis pathway.

49

Figure 12. CD40L

cross-linking lowers

caspase-8 but not

caspase-9 activity in

SEB-reactive T-

cells. C57BL/6

Splenocytes were

cultured with SEB +

αCD40L cross-

linking mAb or

isotype, harvested on

days 4 and 5 post-

stimulation, and Vβ8+

T-cells were analyzed

for active caspase-8

and -9 expression. a.

Sample gating

strategy showing live

Vβ8+ /-

populations

(red: SEB stimulated,

gray: FMO). b, c.

Representative plots

of active caspase-8/-9

expression in SEB +

αCD40L (red) or SEB

+ isotype (blue)

treated splenocytes on

days 4 and 5. d, e.

Compiled results of

caspase-8/-9 activity

in Vβ8+ T-cells from

two independent

experiments (D: day;

* and ** indicate p

values of <0.05 and

<0.005 respectively

as measured by

Student‟s paired t-

test; FMO:

fluorescent minus

one; MFI: Mean

Fluorescent Intensity;

each line represents

an individual mouse).

50

3.9 CD40 Co-stimulation with SEB Stimulation Enhances B-cell

CD86 Expression

Platelet CD40L is also capable of binding to and cross-linking its receptor, CD40, on APCs and

on non-hematopoietic cells (132). CD40 signaling on APCs has been shown to enhance the

expression of B7 family molecules which co-stimulate activated T-cells via CD28 signaling.

Since CD28-mediated co-stimulation has also been shown to enhance T-cell survival following

SEB stimulation, I sought to investigate the effect of B-cell CD40 cross-linking on rescue from

apoptosis and survival of SEB-reactive T-cells.

Initially, I confirmed the effect of CD40 signaling on enhanced B-cell expression of CD86;

splenocytes were stimulated with SEB alone or SEB + αCD40 cross-linking antibody (or the

isotype control) and CD19+ B-cells were analyzed for CD86 surface expression. I also measured

CD86 expression following SEB + αCD40 stimulation of CD40L-/-

splenocytes to exclude the

effects of endogenous CD40L mediated CD40 cross-linking. Figure 13 shows CD86 expression

kinetics on WT and CD40L-/-

B-cells. CD86 expression was most enhanced after 48 hours post

CD40 stimulation + SEB (green), as compared to the isotype + SEB (orange) or SEB alone

(green) stimulated conditions.

51

Figure 13. Kinetics of CD86 Expression on CD19+ splenocytes (B-cells) following CD40

cross-linking. WT or CD40L-/-

splenocytes were stimulated with SEB + αCD40 (green), SEB +

isotype specific antibody (orange), or with SEB alone (red), and CD19+ events were analyzed for

CD86 expression. a. CD40 cross-linking enhanced CD86 expression at 48 hours post SEB

stimulation compared to control conditions. b. Enhanced CD86 expression following mAb-

mediated CD40 cross-linking was more considerable compared to control conditions in the

absence of endogenous CD40L at 48 hours post-stimulation (CD40L-/-

). These results were

replicated across multiple repeated experiments. Unstimulated, isotype stained and fluorescent-

minus-one (FMO) controls are shown in blue, brown and tinted grey respectively.

52

3.10 CD40 Cross-linking Enhances SEB-Reactive T-cell Survival

After confirming the effect of CD40 cross-linking on enhanced B-cell CD86 expression, I sought

to investigate T-cell rescue from apoptosis following splenocyte SEB stimulation and cross-

linking with αCD40 mAb. WT splenocytes were stimulated with SEB + αCD40 and apoptosis

was measured on day 5 post stimulation on SEB-reactive T-cell populations.

Fewer Vβ8+

T-cell populations undergo apoptosis as measured by 7-ADD exclusion and annexin-

AV binding following SEB stimulation and αCD40 cross-linking compared to splenocytes

stimulated with SEB plus the isotype specific antibody (Figures 14, 15a). Similarly, higher

absolute numbers of live cells survive in αCD40 treated versus control conditions (figure 15d).

These results suggest that CD40 signaling (on APCs) leads to enhanced T-cell survival following

SEB stimulation.

53

Figure 14. Enhanced Survival of SEB-reactive T-cell Populations Following CD40

Stimulation. C57BL/6 mouse splenocytes were stimulated with SEB plus αCD40 mAb or the

isotype specific antibody and assayed for apoptosis on day 5 post-stimulation using flow

cytometry. a. Live splenocytes were gated based on 7-ADD dye exclusion (top left). b. panel

depicting the gating strategy based on isotype and FMO controls. c. CD28 co-stimulation

reduces the relative percentage of Vβ8+ T-cells undergoing apoptosis (positive control). d. CD40

co-stimulation with SEB also lowers the relative numbers of Vβ8+ T-cells undergoing apoptosis

as measured by annexin-AV incorporation. These results are representative of 3 independent

experiments.

54

3.11 Pro-survival Effects of CD40 Stimulation on T-cells are CD86 –

CD28 Dependent

CD40 cross-linking leads to enhanced B-cell CD86 expression and T-cell survival following

SEB stimulation (Figure 13). To test whether the pro-survival effects of CD40 signaling are

mediated by enhanced stimulation of the B7 – CD28 pathway, I used CD86 -/-

and CD28-/-

splenocytes to study αCD40 –mediated rescue from apoptosis on SEB-reactive T-cells. Figure

15b, c. shows that in the absence of CD86 or CD28, similar relative percentages of SEB-reactive

T-cell undergo apoptosis. Similarly, absolute numbers of live cells are not significantly different

in CD86 -/-

or CD28-/-

Vβ8+

T-cell populations stimulated with αCD40 (Figure 15e,f). These

results suggest that the effect of CD40 cross-linking on enhanced T-cell survival following SEB

stimulation are dependent on co-stimulatory molecules CD86 and CD28, indicating an indirect

role for B-cells (and other professional APCs expressing CD86 following CD40 stimulation) in

CD40 mediated rescue from apoptosis of Vβ8+ T-cells.

55

Figure 15. Compiled results of multiple apoptosis assays demonstrating the pro-survival

effects of CD40 and the importance of CD86 and CD28 in T-cell survival following SEB

stimulation. a-c. CD40-mediated co-stimulation lowers the relative percentage of Vβ8+ T-cells

undergoing apoptosis in C57BL/6 WT but not CD28-/-

or CD86-/-

splenocytes respectively. d.

Higher absolute numbers of Vβ8+ T-cells survive following SEB stimulation when stimulated

with αCD40 mAb versus the isotype specific antibody. e, f. This effect is abrogated in the

absence of CD28 or CD86 (n = 7 for WT, n = 6 for CD28-/-

and n = 3 for CD86-/-

); each line

indicates an independent experiment). * indicates statistical significance as determined by

Student‟s paired two-tailed t-test (p < 0.05). Each line represents splenocytes from an individual

mouse repeated 3 (a) and 2 times (b,c).

56

3.12 CD40 Cross-linking Leads to Enhanced Bcl-2 Expression in SEB-

Reactive T-cells

To further confirm the effect of CD40 cross-linking on SEB-reactive T-cell survival, I measured

intracellular concentrations of pro-apoptotic molecule Bcl-2 on day 5 post-SEB stimulation in

Vβ8+ T-cell populations. C57BL/6 splenocytes were cultured with SEB in plates coated with anti-

CD40, anti-CD28 (positive control) or anti-isotype mAbs. Cells were harvested on day 5 post-

stimulation and analyzed for intracellular Bcl-2 expression by flow-cytometry. Figure 16 shows

that co-stimulation with anti-CD28 or anti-CD40 enhances T-cell Bcl-2 protein expression

following SEB stimulation. This effect was consistent across multiple independent experiments

(n=5, p=0.0475).

57

Figure 16. CD40 Cross-linking Enhances Intracellular Bcl-2 Protein Expression in Vβ8+ T-

cells. a. Representative graph of intracellular Bcl-2 protein expression in live Vβ8+ gated

splenocytes following SEB + αCD40 (red), SEB + isotype (blue) stimulation for 5 days; isotype

control (gray). b. compiled results of 5 experiments show enhanced Bcl-2 expression in anti-

CD40 treated splenocytes compared to the isotype-treated controls. * indicates p < 0.05 as

measured by Student‟s paired t-test. Each line represents an individual mouse in an independent

experiment.

58

4 Discussion

Several pieces of evidence indicate an important role for CD40L and platelets in KD. First, KD

patients have elevated numbers of platelets – many of which are activated – in the acute and

subacute phases of disease (137, 164). Second, co-stimulation enhances T-cell survival and

persistence in the coronary artery in a murine model of disease (162-163). Third, platelets

express an important co-stimulatory molecule, CD40L, as well as its receptors, CD40 and

αIIbβ3, in activated and resting states respectively.

SEB, a prototypic superantigen, has previously been shown to reduce and inhibit human platelet

aggregation and adhesion respectively (165-166). In these studies, aggregation and adhesion

have been assessed by indirect methods; namely, optical density readings and enzymatic activity

following fibrinogen binding. In addition, Tran et al. show that SEB induces increased platelet

metabolism using high performance liquid chromatography (165). However, to our knowledge,

no studies have yet investigated platelet surface expression of classic activation markers (e.g. P-

selectin) or CD40L following SEB stimulation. Moreover, SEB-mediated inhibition of platelet

aggregation has not been investigated directly via αIIbβ3 mAb binding.

Here, I show that ex-vivo stimulation of mouse platelets with SEB in whole blood induces

enhanced surface P-selectin and CD40L expression. In addition, I demonstrate that SEB induces

αIIbβ3 receptor activation on platelets (Figure 4). Whether this effect is directly mediated

through platelet receptor binding or is indirect via T-cell cytokine production remains unknown.

Nonetheless, some evidence suggests that SEB activates platelets independently of leukocytes;

Lisman et al. have shown that Staphylococcal superantigen-like 5 directly binds to the αIIbβ3

integrin receptor and activates platelets (167). Our preliminary work using platelet rich plasma

59

(PRP) and SEB also indicates enhanced expression of these markers on platelets in the absence

of lymphocytes (not shown). Whether SEB activation of mouse platelets is direct or is mediated

via secondary effectors, platelet expression of CD40L following SEB stimulation suggests an

important role for these cells in T-cell co-stimulation, survival, and prevention of apoptosis in

our animal model of KD. Activation of platelets by a prototypic SAg is also significant because

many of the pathogens isolated from KD patients contain SAg-activity and activate platelets in

vitro or in vivo (168-193); though a correlation between the two has not previously been

established.

Coronary arteritis is a hallmark of KD in the subacute phase with an inflammatory infiltrate

consisting mainly of T-lymphocytes; a phenomenon that is inconsistent with the fate of SAg

reactive T-cells: anergy or apoptosis. Antigen re-stimulation or co-stimulation with αCD28 or

TNFSF members, TNFα and 4-1BBL, enhances T-cell survival (162-163, 194) and rescues SEB-

reactive from apoptosis following SAg stimulation; suggesting a secondary theme at play in

persistence of inflammatory cells in the coronary artery. These molecules share a common

property: they execute effector functions via forward signalling to their respective receptors. In

addition, secretion of TNFα and expression of 4-1BBL are both dependent on activated T-cells

which are also undergoing apoptosis in response to SAg stimulation. Thus, in order for these co-

stimulatory molecules to have an effect on enhanced T-cell survival, a balance must be struck

between co-stimulation and the rate at which T-cells undergo apoptosis following SAg

stimulation.

Alternatively, another cell subset that persists in circulation despite SAg stimulation could

provide the “rescue” signal to activated T-cells. The platelet cell, expressing both CD40L and its

cross-linking receptors, CD40 and αIIbβ3, is a good candidate for this role. While CD40L can

60

mediate forward signalling through CD40 and lead to enhanced co-stimulation via other cells,

CD40 and αIIbβ3 on platelets can cross-link CD40L, leading to CD40L reverse-signaling and

direct T-cell stimulation (195-196). In addition, SAg stimulation enhances the expression of

platelet CD40L (Figure 4), further hinting to a potential role for this molecule and its receptors in

enhancing T-cell survival in a SAg model of KD.

I demonstrated that CD40L stimulation with a cross-linking mAb in vitro enhances SEB-reactive

T-cell survival as measured by several markers of apoptosis. Since CD40L expression on Vβ8+

T-

cells following SEB stimulation peaks at 72 hours post-stimulation (Figure 5), we chose day 5 as

an end-point to measure apoptosis. This choice was confirmed by optimization assays measuring

apoptosis on days 4, 5 and 7 (optimization data not shown).

I examined relative numbers of Vβ8+

T-cells undergoing apoptosis (annexin-AV binding) as well

as absolute numbers of live cells (trypan blue, 7-ADD and annexin-AV exclusion) as two cohorts

of cell survival following CD40L cross-linking; increase in the latter marker indeed recapitulated

a decrease in the former. In the absence of endogenous CD40L, I did not expect the mAb to

have any effect on T-cell survival; this was indeed the case (Figures 6, 7).

While CD28 co-stimulation has been reported to further enhance T-cell CD40L expression

(197), I did not see an additive effect on SEB-reactive T-cell survival following CD28 + CD40L

cross-linking (preliminary data not shown). Furthermore, I confirmed the crucial role of T-cell

CD40L compared to APC CD40L on T-cell rescue from apoptosis (Figure 9). Although

compiled results of multiple experiments in mixing assays were not statistically significant, there

was a trend for enhanced T-cell survival with WT T-cells and APCs or WT T-cells and CD40L-/-

APCs, which was abrogated in the absence of T-cell CD40L.

61

In this work, I did not differentiate between proliferation and rescue from apoptosis of SAg-

reactive T-cells – two non-mutually exclusive outcomes of co-stimulation (82, 198-199). Instead,

global read-out of enhanced survival following co-stimulation was measured. Indeed, it is likely

that lower percentages of annexin-AV+

cells and higher absolute numbers of live cells are a

secondary outcome of increased T-cell proliferation following CD40L cross-linking.

One of the challenges of characterizing apoptosis via annexin-AV staining by flow-cytometry

was the ambiguous nature of this dye: annexin-AV positive and negative T-cell cohorts were not

readily identifiable but only seen as slight shifts in mean fluorescence intensity. Despite the use

of appropriate fluorescence-minus-one and isotype controls, the gating strategy remained

contentious due to the nature of the annexin staining profile. Thus, I showed that live (efluor-450

negative) T-cells that were positive and negative for active caspase-3 mapped to annexin-AV

positive and negative gates, respectively, set using FMO and isotype controls (figure 8). This is

in accordance with the fact that live active caspase-3 positive cells are undergoing apoptosis and

are thus annexin-AV positive (200).

SEB-induced T-cell apoptosis is mediated by both the extrinsic and the intrinsic pathways (see

section 1.3). Co-stimulation via CD28 has been shown to enhance T-cell survival by enhancing

the expression of Bcl-XL anti-apoptotic protein as well as c-flip, inhibitor of caspase-8 activation

(80-82, 201). Similarly, other TNF superfamily molecules, 4-1BBL and TNFα, have been shown

to enhance T-cell survival by inhibiting both the extrinsic and intrinsic apoptosis pathways (61,

194, 202-205). Unpublished work at our lab has also demonstrated enhancement of Bcl-2

expression and interference with the mitochondrial apoptosis pathway following TNFα + SEB

stimulation of T-cells. Less is known about the effects of reverse-signaling by TNFSF proteins

on T-cell survival. Reverse-signaling via membrane-bound TNF-α (206) , OX40L and FasL

62

(207) have been shown to enhance T-cell survival, but the exact mechanism of this effect is not

known (208-209). Blair et al. have demonstrated that in vivo administration of humanized anti-

CD40L in non-human primate models prevents renal allograft rejection. In vitro, co-stimulation

of human anti-CD3 stimulated CD4+ T-cells with anti-CD40L mAb was shown to induce short-

term CD4+ T-cell effector responses followed by rapid apoptosis (210). However, to our

knowledge, the effect of CD40L reverse-signaling on SAg-reactive T-cell survival has not

previously been described.

I showed that CD40L cross-linking delayed the activation of caspase-3, a caspase downstream of

both the extrinsic and intrinsic apoptosis pathways. These results were also replicated by flow-

cytometry (statistically significant) in SEB-reactive T-cells by day 5 post SEB stimulation

(Figure 10). Lower caspase-3 activation indicates a later or inhibited onset of apoptosis following

CD40L cross-linking and SAg stimulation. Furthermore, CD40L reverse-signaling was shown to

interfere with the extrinsic pathway as measured by limited activation of caspases-8 in αCD40L

+ SEB stimulated splenocytes (Figure 12). The kinetics of caspase-8 and caspase-3 activation –

lowered by days 4 and 5 respectively – provide further support for an inhibitory effect of CD40L

reverse-signaling on the extrinsic apoptotic pathway, as early activation of caspase-8 leads to

downstream cleavage and systemic activation of caspase-3 (211).

The most striking feature of CD40L reverse-signaling is perhaps the lack of interference of this

pathway following SEB stimulation with the intrinsic apoptosis pathway. While CD40L cross-

linking lowered activation of caspases-3 and -8 in SEB-reactive T-cell subsets, there was no

significant difference in the intracellular expression of active caspase-9 following CD40L

reverse-signaling (Figure 11) (a slight trend in lower % but not MFI of active caspase-9 in

αCD40L stimulated samples was seen). Moreover, Bcl-2, an anti-apoptotic molecule shown to be

63

enhanced in expression following co-stimulation via CD28, 4-1BBL, TNFα and CD40 (see

below), was not upregulated following CD40L cross-linking (Figure 12). Although not in a SAg

context of T-cell activation, these data are consistent with previous work reporting no effect on

Bcl-2 or Bcl-xL protein expression following in vitro co-stimulation of αCD3 activated human T-

cells with αCD40L (210). A plausible explanation for these observations is that CD40L reverse-

signaling and co-stimulation of T-cell mediates cell survival via an alternate pathway compared

to traditional forward signaling via CD28 and other co-stimulatory molecules.

The question of how CD40L reverse-signaling leads to interference with the caspase cascade is

an interesting one. Since the intracellular signalling domain of the CD40L protein lacks any

signalling motifs (111), it is likely that the pro-survival effects of CD40L reverse-signaling are

mediated indirectly via adaptor proteins. El-Fakhry et al. demonstrated that a Src family kinase

protein mediates CD40L signalling within T-cells lipid-rafts, leading to cytokine secretion and

activation induced proliferation of T-cells (111). In addition, Cursi et al. have shown that the

tyrosine kinase, Src, phosphorylates caspase-8 on the Tyr380 residue, leading to downregulation

of caspase-8 function (212). Thus, it is possible that CD40L cross-linking on SEB-reactive T-

cells leads to increased Src kinases activity and procaspase-8 phosphorylation, inducing lower

caspase-8 activity and T-cell survival.

T-cells are not the only type of cells platelets interact with. While platelet-mediated CD40L

cross-linking may indeed lead to enhanced T-cell survival and coronary arteritis in KD, it is

possible that T-cell survival following SAg stimulation is further enhanced by indirect platelet-

mediated CD86-CD28 co-stimulation via platelet CD40L binding to CD40 on APCs. I confirmed

the effect of SEB stimulation and CD40 stimulation on B-cell CD86 expression (213), and

demonstrated that the effect of SEB stimulation alone on enhanced B-cell CD86 is likely

64

mediated by T-cell CD40L expression. As expected, this effect was abrogated in CD40L-/-

splenocytes (Figure 12). In addition, while most studies have used soluble CD40L to achieve

CD40 cross-linking (107-108, 214), I used plate-bound anti-CD40 mAb to cross-link and

stimulate B-cell CD40 signaling.

I report that CD40 cross-linking results in enhanced T-cell survival and rescue from apoptosis

likely through an indirect mechanism (Figures 13-14). While SEB stimulation leads to maximal

T-cell CD40L expression by 72 hours post stimulation, CD40 cross-linking facilitates B-cell

CD86 expression by 48 hours - yet, cross-linking of either molecule enhanced T-cell survival by

day 5 post-stimulation. Furthermore, the pro-survival effect of CD40 stimulation on SEB-

reactive T-cells is abrogated in the absence of endogenous CD28 or CD86 (Figure 15), lending

more support for the role of B-cells (or other professional APCs) in CD40 mediated T-cell rescue

from apoptosis.

Another piece of evidence for the role of B-cell CD40 signaling and T-cell survival following

SAg stimulation mediated by CD86- CD28 signaling comes from enhanced expression of anti-

apoptotic molecule, Bcl-2, in αCD40 + SEB stimulated splenocytes. Though these results do not

clearly elucidate inhibition of which apoptosis pathway leads to enhanced CD40 mediated SAg-

reactive T-cell survival, they suggest a similar mechanism to CD28-mediated co-stimulation and

rescue from apoptosis (82, 163). While previous studies have shown that CD28 co-stimulation

enhances Bcl-XL, but not Bcl-2, protein expression (82, 215) , our results suggest that CD28

enhances the latter but not the former in SAg-stimulated cells (Bcl-XL data not shown). This

discrepancy may be explained by a differential trigger of apoptosis and survival by SAg +

αCD28 stimulation vs. TCR cross-linking with mAbs as done in mentioned studies. Since CD28-

mediated co-stimulation enhances T-cell survival by inhibiting both the extrinsic and intrinsic

65

apoptotic pathways (see Section 1.5), it is expected that CD40 cross-linking would also enhance

caspase-3, caspase-9 and caspase-8 activity.

Platelet modulation of immune responses has been widely reported in the literature. Sprague et

al. have reported that platelet-derived membrane vesicles are sufficient to deliver CD40L to

stimulate antigen-specific B-cell IgG production and modulate germinal center formation

through cooperation with responses elicited by CD4(+) T cells (216). In addition, soluble platelet

CD40L has long been known to induce endothelial cell activation and production of

inflammatory cyotkines by cross-linking CD40 on these cells (217).

4.1 Limitations and Future Directions

In this work, SEB was shown to induce platelet activation in whole blood in vitro, however, the

mechanism of SEB interaction with platelets, directly or indirectly via other cells/ soluble

factors, was not dissected. Showing that SEB induces the same activation profile on washed

platelets would help address this question. In addition, platelets were not used in culture to

directly stimulate CD40L or CD40 signaling on T-cells and APCs respectively. The next logical

step to assess the role of platelet-derived CD40L/CD40/αIIbβ3 in enhanced SEB-reactive T-cell

survival is to incubate washed SEB activated or unstimulated platelets cells with isolated T-cells/

APCs and assess T-cell apoptosis at appropriate time points by the cohorts of apoptosis that were

established in this study; namely trypan blue and 7-ADD exclusion, annexin-AV binding and

active intracellular caspases-3, -8 and -9 expression. In addition, in order to apply these findings

to the in vivo disease model, it must be shown that LCWE stimulation induces similar effects to

SEB on platelet activation and T-cell survival in vitro. Mixing assays of CD28-/- and CD86-/- T-

cells with APCs in αCD40 + SEB stimulation assays would provide further support for the

conclusion that CD40 stimulation enhances SEB-reactive T-cell survival indirectly via APCs. In

66

vivo, adoptive transfer of T-cell that survive apoptosis and persist in the coronary artery to naïve

WT or CD40/ CD40L-/-

, and platelet depleted mice could further shed light on our understanding

of platelet SAg responses and CD40L/CD40 cross-talk with lymphocytes. Furthermore, infusion

of previously activated platelets with LCWE in naïve or LCWE injected mice to induce or

exacerbate disease would provide further support for the specific role of these cells in relation to

our disease model.

4.2 Conclusions and Proposed Model

There is a body of information suggesting a SAg trigger for KD, yet the exact etiology and

pathogenesis of disease remains unknown. Persistent inflammation due to T-cell infiltration of

the heart, or coronary arteritis, may lead to acquired heart damage. Thus, understanding the

mechanism of T-cell survival, particularly from a SAg stimulant perspective, is important. There

is compelling evidence that stimulation with a prototypic SAg, SEB, activates platelets, leading

to platelet surface expression of CD40L and its cross-linking receptors. Furthermore, it was

shown that CD40L cross-linking enhances SEB-reactive T-cell survival, and that this is likely

mediated by inhibition of the extrinsic apoptosis pathway. In addition, platelet may mediate

enhanced T-cell survival indirectly via B-cell CD40 cross-linking – which was shown to enhance

CD86 expression and T-cell survival in a CD86-CD28 dependent manner (Figure 17). I conclude

that CD40L and CD40, expressed on platelets and other cells, may play a previously

underappreciated role in the pathogenesis of KD, calling attention to the potential for anti-

CD40/40L therapy or anti-platelet therapy targeting these molecules in children with coronary

arteritis.

67

Figure 17. Model of Platelet-CD40-CD40L interaction in Enhanced Superantigen-Reactive

T-cell Survival. 1. Superantigen stimulation induces platelet de-granulation, CD40L surface

expression and αIIbβ3 integrin receptor activation. 2. Platelet CD40 binds to and cross-linsk

CD40L on T-cells. 3. Platelet integrin receptor, αIIbβ3, binds to and cross-links CD40L on T-

cells. T-cell CD40L reverse-signaling enhances cell survival following SAg stimulation. 4.

Platelet CD40L may also cross-link B-cell CD40, enhancing B-cell CD86 expression. 5. B-cell

CD86 signals to T-cell CD28, enhancing T-cell survival following SAg stimulation.

68

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