CD137 ligand induced apoptosis in peripheral blood mononuclear cells (PBMCs)

2.12.2 Analysis of intracellular superoxide anion production...652.12.3 Analysis of mitochondrial superoxide production...652.12.4 Cell specific depletion of ROS in a mixed T cell and Mo

CD137-INDUCED CELL DEATH IN PERIPHERAL

BLOOD MONONUCLEAR CELLS

NURULHUDA BINTE MUSTAFA

(B.Sc(Hons.), NUS)

A THESIS SUBMITTED

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF

SCIENCE DEPARTMENT OF PHYSIOLOGY, MEDICINE NATIONAL UNIVERSITY OF SINGAPORE

2010

ACKNOWLEDGEMENTS

Oh humble graduate student how thy toils make thee weary!

Be not for the Grace of God and thy teachers, family and friends, thou wouldst have surely perish.

To Professor Shazib Pervaiz for the rigorous development of a scientific mindthrough your intellectual contributions and critical insights, for challenging me to rise

to the occasion, for your understanding and patience, for nurturing an amazing labwhich prizes friendship and love as much as Science, it will be difficult to find abetter atmosphere to mature in, a profound Thank You

To Associate Professor Herbert Schwarz for always being there to assist and propel

me in my research, for the unique broadening of my scientific perspective whichcomes when working with a co-supervisor in a different field of research and for yourquiet encouragement, you have been an invaluable pillar of support in my progressand I am most grateful

To Dr Jayshree, Ms Kartini, Dr Andrea and Dr Alan, Thank You very much for yourguidance especially at the initial stages as a naive UROPS student and throughout thecourse of my work, for the endless cups of coffee over science, enjoyable chats andselfless assistance beyond research

To each and every member of the ROS and Tumour Biology lab and the CD137Immunotherapy Lab whom I have worked closely with, you guys know who you are

We brave not only the trials of scientific progress together, but also really care foreach other’s personal life, you guys are like family Special mention to the class of

2005, Inthrani, Sinong, Chew Hooi, Zhi Xiong and Greg for inspiring each other tocontinue marching down this difficult road and Shaqireen for being my sparringpartner in CD137 research

To mom and dad, you have been unbelievable For motivating me to always strivefor excellence, for your precious prayers, unflagging love and support, and mostimportantly for your steadfast faith in me, words of gratitude are completelyinadequate for all you have given me To my siblings Adibah, Nabil and Hazi, thankyou for inspiring me in ways that you do not know

Finally to my husband, you alone truly know the rainbow of emotions and challengesthat I have experienced in the course of my scientific research and in writing thisthesis For being willing to make any sacrifice so that I can focus on and pursue mygraduate studies, , for painstakingly putting pieces together when they fall apart and

for your dedication towards my best well being, you are my nikmah, my miraculous

blessing

The pursuit of knowledge at its core is a commitment to apply new discoveries forthe transformation and betterment of the society May this be the beginning of such

an endeavour

TABLE OF CONTENTS

I Acknowledgements ii

II Summary ix

III List of Figures xi

IV List of Abbreviations xiv

1.0 Introduction 1

1.1 Cell Death 1

1.1.1 Overview of Cell Death 1

1.1.2 Apoptotic Cell Death 1

1.1.3 Molecular Mechanisms Mediating Apoptosis 3

1.1.4 Extrinsic, Intrinsic And Granzyme B Cell Death Pathways 4

1.1.5 Caspase-Independent Cell Death 8

1.2 Apoptosis In the Immune System 10

1.2.1 Activated Induced Cell Death in T cells (AICD) 11

1.2.2 Activated Cell Autonomous Death in T cells (ACAD) 12

1.2.3 Monocyte-Dependent Cell Death in T cells (MDCD) 13

1.3 CD137/CD137 Ligand, TNFR/TNF Superfamily Members 15

1.3.1 Molecular Characteristics and Expression Patterns of CD137 17

1.3.2 Effects of CD137 Signalling 18

1.3.2.1 CD137 Signalling in T cells 18

1.3.2.2 CD137 Signalling in B cells 19

1.3.2.3 CD137 Signalling in Dendritic Cells 20

1.3.2.4 CD137 Signalling in Granulocytes and Natural Killer Cells 20

1.3.3 Clinical Significance of CD137 21

1.3.4 Molecular characteristics and expression pattern of CD137 Ligand 23

1.3.5 Effects of CD137 Ligand Signalling 24

1.3.5.1 CD137L signalling in Monocytes/Macrophages 24

1.3.5.2 CD137L signalling in Dendritic Cells 26

1.3.5.3 CD137L signalling in B cells 27

1.3.5.4 CD137/CD137L mediated inhibitory signalling 27

1.4 Reactive Oxygen Species 31

1.4.1 Major Types of ROS and their derivative species 32

1.4.2 Superoxide Anion 32

1.4.3 The family of NADPH Oxidases 33

1.4.4 Hydrogen Peroxide 38

1.4.5 Hydrogen Peroxide-Mediated Cell Death 39

1.4.6 Oxidative Stress and Endogenous Antioxidant Defence Mechanisms 41

1.4.7 Redox Signalling in Peripheral T cells 43

1.4.7.1 ROS-mediated T cell Proliferation 43

1.4.7.2 ROS-mediated T cell Death 44

1.4.7.3 Control of the Extrinsic Apoptotic Pathway by ROS-dependent Expression of FasL 44

1.4.7.4 Control of the Intrinsic Apoptotic Pathway by the ROS-dependent Suppression of Bcl-2 Expression 45

1.4.7.5 ROS-mediated effects on T cell fate by Accessory Immune Cells 49

1.5 Aim Of Study 50

2.0 Materials and Methods 51

2.1 Chemical Reagents 51

2.2 Antibodies for Immunoblotting 52

2.3 Fluorescent dyes used for Flow Cytometry 52

2.4 Induction of CD137L signalling in Target Cells 53

2.5 Cells and Cell Culture 54

2.5.1 Isolation of Peripheral Blood Mononuclear Cells 54

2.5.2 Isolation of Mice splenocytes 55

2.5.3 SGH-MM6 cell line 56

2.6 Assays for proliferation and apoptosis 56

2.6.1 MTT Proliferation Assay 56

2.6.2 Morphological and quantitative analysis of cell size 57

2.6.3 Analysis of Phosphatidyl Serine Externalisation 58

2.6.4 Determination of Mitochondrial Transmembrane Potential 58

2.6.5 Analysis of Caspase Activity 59

2.7 Determination of protein expression levels by Immunoblotting 60

2.8 Mitochondrial and Cytosolic Isolation by differential centrifugation 62

2.9 Nuclear and cytosolic extraction……… ………62

2.10 p65NF-κB Transcriptional (DNA Binding) Assay 63

2.11 TNF Enzyme –Linked Immunosorbent Assay (ELISA) 64

2.12 Assays for the Determination of Reactive Oxygen Species 64

2.12.1 Analysis of intracellular hydrogen peroxide production 64

2.12.2 Analysis of intracellular superoxide anion production 652.12.3 Analysis of mitochondrial superoxide production 652.12.4 Cell specific depletion of ROS in a mixed T cell and

Monocyte reaction 662.13 Statistics……… ……….663.0 Results……… ……….……….673.1 Induction of CD137L signalling by immobilised CD137-Fc inhibits

proliferation and stimulates apoptosis in three different cell types 673.1.1 CD137L signalling inhibits proliferation of activated human PBMCs 673.1.2 CD137L signalling also inhibits proliferation in activated mouse

splenocytes and in multiple myeloma cell line, SGH-MM6 683.1.3 CD137L mediated decrease in cell proliferation is due to

induction of apoptosis 743.2 CD137L induced cell death is caspase independent and is

mediated by the intrinsic not the extrinsic death pathway 783.2.1 CD137L signalling increases expression levels of death receptor TNFR1 and

stimulates secretion of TNF, but is not critical to cell death 783.2.2 Inhibiting TRAIL-mediated signals with DR4 and DR5 blocking

antibodies was unable to abrogate CD137-induced cell death 823.2.3 Ligation by CD137-Fc disrupts the mitochondrial transmembrane

potential, and induces translocation of Cytochrome C into the cytosol 843.2.4 CD137L downregulates Bcl-2 protein levels at early time points,

while maintaining Bim levels thus tilting the Bcl-2: Bim

ratio towards favouring apoptosis 87

3.2.5 CD137L signalling induces slight caspase-3 and-8 activity

at 24h but cell death is caspase independent 893.2.6 CD137-induced cell death is independent of casein kinase 1

(CK1) but is dependent on the MAPK pathway 923.3 CD137 induces apoptosis specifically within the CD3+T cell

subpopulation of the PBMCs and T cell death is mediated by monocytes 953.4 Cell death induced by CD137 is critically regulated by

Reactive Oxygen Species (ROS) 1003.4.1 CD137-induced cell death can be abrogated by scavengers

of ROS, Catalase and Tiron 1003.4.2 CD137 induces significant production of ROS in whole PBMCs 1043.4.3 CD137L signalling induces ROS in both T cells and monocytes 1073.5 It is ROS from T cells and not monocytes that is critical for the

induction of CD137-mediated cell death 1133.6 The source of ROS in T cells that activates the cell death program

could be the NADPH Oxidase (NOX) or the mitochondria 1153.6.1 Production of ROS critical to apoptosis is upstream of the

disruption in mitochondrial transmembrane potential 1153.6.2 ROS in T cells originates from NOX 1153.6.3 Inhibiting superoxide dismutases (SOD) with DDC

sensitised PBMCs to cell death 1223.6.4 ROS in T cells may originate from mitochondria 1284.0 Discussion 131

4.1 CD137L-induced cell death is mediated via a caspase-independent

intrinsic (mitochondrial-mediated) pathway 132

4.2 CD137L mediated cell death signalling in PBMCs is targeted to T cells 137

4.3 CD137/CD137L induced cell death is critically mediated by Reactive Oxygen Species (ROS) 141

4.3.1 ROS is the upstream molecular event crucial for stimulating the CD137-induced death signalling cascade leading to T cell apoptosis 142

4.3.2 ROS potentially mediates CD137-induced T cell apoptosis by downregulating the expression of Bcl-2 via the activity of ERK 143

4.4 Critical ‘killer’ ROS is produced by T cells not monocytes, though cell death is monocyte dependent 146

4.5 ROS species that is ultimately responsible for the induction of cell death signalling is H2O2which is produced from the activity of NOX 149

4.6 So how is CD137/CD137L induced cell death physiologically relevant? 153

4.6.1 CD137/CD137L: The missing link in monocyte regulated T cell death? 154

4.7 Summary and Conclusion 159

5.0 References 164

6.0 Appendix 185

6.1 Supplementary Figures 185

6.2 Publication and Poster 192

CD137-INDUCED CELL DEATH IN PERIPHERAL BLOOD MONONUCLEAR CELLS

Nurulhuda Mustafa

National University of Singapore, 2010

CD137/CD137L are members of the TNFR/TNF superfamily that have exhibited significant immuno-modulatory effects in healthy and pathogenic states Anti-CD137 antibodies have shown great promise in murine therapeutic models of cancer and autoimmunity Generally, CD137/CD137L bi-directional signal transduction greatly amplifies the ongoing immune response as CD137 signalling is co-stimulatory for T-cells while CD137 Ligand (CD137L) signalling is activating for antigen presenting cells (APCs).

In spite of the solid evidence for CD137-induced survival signalling in T-cells, it was found

when CD137 was knocked out of splenocytes (1-3), splenic T cells responded not by proliferation, but by hyper-proliferating instead (4) We set out to clarify this paradox and

hypo-identified that when immobilised CD137-Fc cross-links its corresponding ligand (CD137L) on PBMCs, it activates an inhibitory signal which results in a reduced proliferative response to anti-CD3 This potentially explains why deficiency in CD137 would conversely allow cells to hyper-proliferate We further determined that the lack of proliferative response is actually a function of cell death in PBMCs and that apoptosis is targeted to T cells T cell death peaks at 24h, and there is a dose-dependent increase in apoptosis with increasing concentrations of CD137-Fc added to the PBMC culture We found contrary to expectation that although there

is a CD137-mediated upregulation of death receptors, CD137-induced apoptosis is not promoted by the extrinsic pathway but by the intrinsic pathway We found a CD137-

mediated downregulation of Bcl-2 levels, while exerting no effect on Bim levels thus favouring an increase in the Bim: Bcl-2 ratio which subsequently leads to mitochondrial membrane permeabilization, the release of Cytochrome C and cell death Despite the translocation of Cytochrome C into the cytosol, we observed only low levels of caspase 9 and caspase 3 activity and confirmed with a pan-caspase inhibitor that indeed that cell death is caspase- independent.

We discovered instead that induction of cell death is critically dependent on reactive oxygen species (ROS) production We report that ROS production is not a secondary effect of mitochondrial permeabilization but an early event initiating a death signalling cascade ROS production was stimulated as early as 2h which returns to baseline at 6h but subsequently rises stably from 12h till 24h We confirm that the key ROS species involved in cell death is hydrogen peroxide (H2O2) and that H2O2 production is NADPH oxidase (NOX)-dependent Thus, superoxide produced by NOX is potentially converted to H2O2 by superoxide dismutases in the cell.

Directly ligating purified T cells with CD137-Fc does not induce cell death Concomitant studies demonstrated that CD137-mediated cell death is induced only in the presence of monocytes and increases with increasing monocyte concentrations CD137-signalling actually induces increase in ROS levels in both monocytes and T cells and by specifically depleting ROS from T cells or monocytes we ascertained that the ROS which activates T cell

in PBMCs is produced by T cells themselves.

Altogether our data show that CD137 regulates a mechanism for caspase-independent T cell apoptosis which is mediated by the intrinsic pathway and critically regulated by H2O2 Since cell death is requires the presence of CD137-activated monocytes, we propose a novel physiological role for CD137 in mediating monocyte dependent T- cell death (MDCD).

LIST OF FIGURES

Introduction

Figure A: Three pathways leading to activation of caspases, the central executor ofapoptosis

Figure B: Expression Profile of CD137 and CD137L

Figure C: NADPH Oxidase (NOX) Family Members and Regulatory Subunits

Figure D: Reactive Oxygen Species (ROS) regulates T cell Death by modulating

expression of FasL and Bcl-2

Figure 3: CD137L induces phosphatidyl serine (PS) externalisation in PBMCs

Figure 4: CD137L upregulates TNFR-1 expression in PBMCs

Figure 5: CD137L signalling significantly elevates cytokine TNF secretion from PBMCs.Figure 6: Blocking the signalling through DR4 and DR5 was unable to reverse CD137L-induced cell death

Figure 7: CD137L signalling stimulates mitochondrial depolarisation and

hyperpolarisation

Figure 8: CD137L enhances expression of Cytochrome C and induces translocation ofCytochrome C from the mitochondria into the cytosol

Figure 9: CD137L signalling in PBMCs suppresses the expression of Bcl-2 while having

no effect on Bim expression

Figure 10: CD137L signalling does not significantly activate caspase 9 and only slightlyactivates caspase 8

Figure 11: CD137L mediates slight increase in caspase 3 activity which corroborateswith slight PARP cleavage

Figure 16: CD137L-signalling through purified T cells only induces a marginal amount

of apoptosis as compared to PBMCs However, T cell death is increasingly sensitised

in T cell-monocyte co-cultures as the concentration of monocytes increases

Figure 17: Catalase was able to significantly reduce CD137L-mediated apoptosis.Figure 18: Tiron completely abrogated CD137L-induced apoptosis

Figure 19: CD137L signalling increases the population of PBMCs that contain

elevated H2O2levels

Figure 20: CD137L mediates a temporal increase in H2O2levels which precedes celldeath at 24h

Figure 21: CD137L signalling directly through purified T cells does not produce H2O2

Figure 27: Inhibiting the activity of NOX can inhibit CD137L-mediated apoptosis.Figure 28: CD137L signalling induces increase in expression of NOX subunits gp91phoxand rac1 in T cells

Figure 29: H2O2production mediated by CD137L signalling can be blocked by

catalase, superoxide scavenger Tiron and NOX inhibitor DPI

Figure 30: There is a CD137L-mediated boost in superoxide production preceding thevast amount of H2O2production at 24h

Figure 31: Inhibitor of superoxide dismutase (SOD), DDC sensitizes PBMCs to induced cell death

CD137-Figure 32: DDC is able to reverse CD137L-mediated H2O2production but amplifiedCD137L-mediated superoxide production

Figure 33: CD137L signalling does not mediate any change in MnSOD and CuZnSODexpression levels in T cells

Figure 34: Ligation of CD137L on PBMCs leads to the increase in percentage of cells

with elevated mitochondrial ROS production.

Figure 35: Mitochondrial ROS production increases in the presence of monocytes

Supplementary Figure 2: CD137L signalling significantly elevates cytokine TNF

secretion from SGH-MM6 cells

Supplementary Figure 3: CD137L-induced apoptosis in SGH-MM6 cells are independent

TNFR-Supplementary Figure 4: CD137L induces translocation of p65NF-κB from the cytosolinto the nucleus in PBMCs

Supplementary Figure 5: CD137 induces activation of p65 NF-κB DNA binding activity

LIST OF ABBREVIATIONS

AIDS Acquired Immunodeficiency Syndrome

Apaf-1 Apoptotic Protease-Activating Factor-1

Bad Bcl-2 Antagonist of cell Death

Bax Bcl-2 Associated X protein

Bcl-2 B-cell Lymphoma protein 2

Bid BH3 Interacting Domain Death Agonist

Bim Bcl-2 Interacting Mediator

C-terminus Carboxy Terminus

Caspase Cysteine-dependent aspartate-specific protease

CED Caenorhabditis elegans death genes

CD137 Cluster of Differentiation 137

CD137L Cluster of Differentiation 137 Ligand

EDTA Ethylenediaminetetraacetic acid

EGTA Ethyleneglycotetraacetic acid

ELISA Enzyme Linked Immunosorbent Assay

ERK Extracellular regulated kinase

FACS Fluorescence activated cell sorter

FADD Fas-associated death domain-containing protein

Fc region Fragment/crystallizable region

FITC Fluorescein isothiocyanate

Hepes 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

KLH keyhole limpet hemocyanin

LEHD-AFC

N-Acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethyl coumarinMAPK Mitogen activated protein kinase

MAPKK Mitogen activated protein kinase kinase

MEK Meiosis-specific serine/threonine protein kinaseMDTCD Monocyte Dependent T cell Death

MitosoxTM Hexyl triphenylphosphonium cation (TPP+)-HEMnSOD Manganese superoxide dismutase

MOMP Mitochondrial outer membrane permeabilization

MTT 3-[4,5-dimethyl-2-hiazolyl]-2,5-diphenyl

tetrazolium bromideNADPH Nicotinamide Adenine Dinucleotide phosphateNFкB Nuclear factor of kappa light polypeptide gene

RPMI 1640 Rosewell Park Memorial Institute 1640

SDS-PAGE SDS-polyacrylamide gel electrophoresisSEB staphylococcal enterotoxin B

SLE systemic lupus erythematosus

SMAC Second mitochondrial activator of caspases

TEMED N,N,N’,N’-tetramethlethylenediamine

TNFR Tumor Necrosis Factor Receptor

TRAF TNF receptor associated factor

TRAIL TNF-related apoptosis inducing factor

VDAC Voltage dependent anion channel

XIAP X-linked inhibitor of apoptosis protein

zVAD-fmk Benzyoxycarbonyl valanyl alanyl apartyl-fluromethyl

ketone

1.0 INTRODUCTION

1.1 Cell Death

1.1.1 Overview of cell death

Cell death is a basic physiological mechanism utilised by organisms to removeunwanted or excessive cells during growth and development, and to maintain tissuehomeostasis Historically, cell death mechanisms have been classified as either aregulated or an unregulated process Regulated cell death or what is commonlydefined as apoptosis, refers to a cell’s resolution to commit suicide in response tocertain signals, by activating specialized death-inducing intrinsic cellular machinery.Unregulated cell death, or necrosis, on the other hand occurs due to thebombardment of overwhelming physico-chemical stresses on the cell whichinevitably leads to the cell’s demise However, emerging evidence suggests that theclassic dichotomy between apoptosis and necrosis is an over-simplification of ahighly sophisticated mechanism Recent studies indicate that there are several non-apoptotic regulated cell death mechanisms that can occur concurrently with orindependently from apoptosis such as necroptosis and autophagy

1.1.2 Apoptotic Cell Death

Apoptosis was the first form of regulated cell death to be discovered in 1972 (6) and was characterised in Caenorhabditis elegans (C.elegans) in the early 1990s (7).

Briefly, the transcriptional upregulation of pro-apoptotic protein Bcl-2 homology 3

(BH3)-only protein, egl-1, sequesters the anti-apoptotic C.elegans death genes

(CED)-9, which relieves the inhibition CED-9 exerts on CED-4 CED-4 is now free to activate cysteine protease CED-3, thereby allowing the cleavage of specific cellular substrates which lead to induction of cell death (8).

Extensive genetic studies of apoptosis in mammalian cells have revealed that the

apoptotic mechanism in mammals is very similar to that of C.elegans, except that it is much more complex For every class of CED proteins, there exist multiple

mammalian homologues The highly conserved and uniform nature of apoptosissuggests that it is a tightly controlled process and a critical regulatory mechanism forthe normal development of living organisms Indeed, the dysregulation of apoptosiscan lead to various disease pathologies The uncontrollable proliferation of cells due

to a suppression of cellular apoptotic mechanisms can give rise to cancer Apoptoticprotease activating factor 1 (Apaf-1)-deficient mice which have evidently lower levels

of apoptosis show significant abnormalities in the development of the brain (9) On

the other hand, excessive activation of apoptosis have been identified as the primarycause of tissue injury and functional decline in a great number of acute diseases such

as stroke and chronic diseases such as diabetes (10) Due to its integral role in

maintaining tissue homeostasis, it is important for scientists to be able to correctlyidentify cells undergoing apoptosis Currently, it is generally accepted that it is best

to distinguish apoptosis morphologically, and that morphological features ofapoptosis are characterised by the rounding up of cells, reduction in cellular volume,chromatin condensation, plasma membrane blebbing and phosphatidyl-serine

externalisation (11).

1.1.3 Molecular Mechanisms Mediating Apoptosis

Central to the execution of apoptosis are the cysteine proteases known as caspases(cysteine aspartic acid specific proteases) Caspases are highly specific proteases thatcleave protein substrates containing particular tetra- or penta-peptide recognition

sequences at the aspartic residues (12) The proteolytic activity of caspases is

targeted to literally hundreds of proteins, amongst the most important is thecleavage of major structural features of the cell such as actin, tubulins and nuclearlamins which results in the rounding up of cells, cell shrinkage, membrane blebbing

and nuclear fragmentation (13-14) Due to its destructive effects, caspases are

typically present in healthy cells as inactive precursor enzymes known as zymogens

(15) Caspase activation is induced by a processing event that rearranges the

proteolysed large and small sub-units into a heterodimer which allows the catalytic

site to organised into the active conformation (15) There are currently twelve

caspases that have been identified in mammalian cells Caspases are classified aseither initiator or effector caspases Upon oligomerization and self-activation,initiator caspases such as caspase 8 ,9 and 10 mediate the cleavage and activation ofinactive pro-forms of effector caspases such as caspases 3,6,7 which are responsible

for the ultimate disassembly of the cell (15).

At each stage of caspase activation, there is an additional level of regulationprovided by caspase inhibitors such as Flice-inhibitory protein (FLIP) and c-IAP(inhibitor of apoptosis protein) which can directly inhibit caspase 8 and caspase 3

respectively (16-17).

It is worthy to note however that mammalian caspases are not only involved inapoptosis Caspase 1 for example, is activated during an innate immune responseand regulates inflammatory cytokine processing by cleaving IL-1β and IL-18 into

mature peptides (18) while deeper elucidation of the role of caspase 8 has highlighted its role in contributing to T-cell proliferation (19).

1.1.4 Extrinsic (Death Receptor), Intrinsic (Mitochondrial) and Granzyme B Cell

Death Pathways

It is commonly accepted that there are three main pathways that lead to theultimate activation of caspases during apoptosis (Fig A) In the extrinsic pathway,pro-apoptotic signals are delivered via DISC (death-inducing signalling complex)which is assembled due to oligomerization of transmembrane death receptors (DR)belonging to the TNFR superfamily such as Fas, TNFR1 (Tumour necrosis factor

receptor 1), DR4, DR5 , upon binding by their cognate ligands (20) The trimerization

of transmembrane death receptors stimulates the recruitment of adaptor proteinssuch as Fas associated death domain protein (FADD) which in turn recruits andfacilitates the dimerization of caspase 8, thereby promoting its auto- activation.Active caspase 8 can then process caspase 3 and 7 into mature forms that will trigger

further substrate proteolysis culminating in the termination of the cell (21).

Extrinsic death signals can cross-talk with the intrinsic death pathway via the caspasemediated cleavage of BID (BH3-interacting domain death agonist) Like othermembers of the BH3-only family, truncated BID is able to lead to the release of the

The intrinsic pathway is activated by stimuli that can be detected by BH-3 only proteins, thereby sequestering anti-apoptotic Bcl-2 proteins or directly activating of Bax so that it can oligomerize on the mitochondrial membrane and facilitate release of Cytochrome C into the cytosol where it can complex with caspases 9, apaf-1 and ATP to form the apoptosome which stimulates caspases 9 to cleave effector caspases.

Granzyme B is released into the target cells through the plasma membrane via perforin and cleaves BID or effector caspases to initiate cell death.

Adapted from Taylor, 2007.

mitochondrial Cytochrome C which promotes the formation of the large apoptosomecomplex consisting of Apaf-1 and caspase 9 Apoptosome formation leads to thestimulation of caspase 9 activity and subsequent processing of effector caspases 3

and 7 (22).

In contrast, the intrinsic cell death pathway is regulated by the balance of the and anti- apoptotic members of the Bcl-2 family and by mitochondria derivedmolecules such as hydrogen peroxide and pro-apoptogenic factors such asCytochrome C Within the Bcl-2 family of proteins exist three subfamiliesrepresented by archetype molecules: (i) Bcl-2-like molecules that are anti-apoptoticand contain all or most of the Bcl-2 homology (BH) domains 1–4; (ii) Bax-likemolecules that are pro-apoptotic and contain BH domains 1–3; and (iii) ‘‘BH3-only’’molecules that are pro-apoptotic and whose only homology to Bcl-2 is in their BH3domain This latter group is the largest, consisting of members that are expressed in

pro-a tissue-specific mpro-anner pro-and pro-appepro-ar to function upstrepro-am of the Bpro-ax-like molecules

(23-24) The initiation of the intrinsic cell death pathway can be mediated by avariety of stimuli (such as drugs or reactive oxygen species) that provoke cell stress

or damage resulting in the activation of one or more members of the pro-apoptoticBH-3 only protein family For example, DNA damage induced by etoposide or 5-

Flurouracil can lead to a p53-dependent transcriptional upregulation of PUMA (25).

On the other hand, cell survival mediated the epidermal growth factor receptorinvolves a mechanism which suppresses the intrinsic apoptotic pathway by

phosphorylating and inactivating BAD (26).

BH3-only proteins (for example Bid, Bim, Puma) promote apoptosis when expressed and are akin to sensors of death-inducing stimuli although they are

over-regulated in distinct ways (27-30) Upon detection of the apoptotic trigger, BH3-only

proteins can either transmit signals directly to Bax or Bak and activate them orsequester anti-apoptotic Bcl-2 family proteins The sequestration of anti-apoptoticBcl-2 family proteins by BH3-only proteins allows the translocation andoligomerization of Bax or Bak in the mitochondrial membrane, where theoligomerization of Bax or Bak forms a channel, or with its interaction with themitochondrial permeability transition pore (PTP) or the voltage dependent anionchannel (VDAC), a channel is opened which allows the efflux of pro-apoptogenic

factors from the mitochondria (31-34) Thus, BH3-only protein activation therefore

needs to reach a critical threshold whereby it is able to sequester sufficient amounts

of anti-apoptotic Bcl-2 family member proteins so that the pro-apoptotic mechanism

of the intrinsic pathway can be stimulated

Granzyme B mediated cell death is one of the modes of cytotoxicity employed bycytotoxic T cells or natural killer (NK) cells to induce cell death in target cells.Specialised cytotoxic granules containing granzymes and perforins are released fromthe immune cells and perforins assemble to form a pore on the surface of the target

cell to facilitate the entry of granzyme B (35) Upon entry into the target cell,

granzyme B, similar to caspases, cleaves its substrates after the aspartate residue.Like caspase 8, granzyme B can process BID as well as effector caspases -3 and -7 to

permit the induction of apoptosis (36-37).

1.1.5 Caspase Independent Cell death (CICD)

CICD was first demonstrated by a study in the Jurkat cell line showing that inhibition

of caspase was only able to rescue the morphological features of apoptosis, but was

unable to inhibit Bax-induced cell death (38) CICD is defined as the type of cell death

that occurs when signals which normally activate apoptosis fail to activate caspases.The morphological features of CICD show an absence of DNA fragmentation andmembrane blebbing, but is distinguished instead by the accumulation ofautophagosomes, cytoplasmic vacuolization and peripheral nuclear condensation

(39) CICD also show evidence of slower kinetics in leading to cell death compared to apoptosis (40) Nonetheless, both apoptosis and CICD share several upstream

signalling pathways including mitochondrial outer membrane permeabilization(MOMP) which is the process that inevitably commits the cell to cell death

Death receptor-induced necroptosis is one such example of CICD Induction of

necroptosis is dependent on the activity of RIP-1 (41) Activation of necroptic cell

death leads to the upregulation of phospholipase A 2 (PLA2) or activation of RIP-1kinase which appears to induce cell death by disrupting the interaction betweenadenine nucleotide translocase (ANT) and cycophilin D thus leading to mitochondrial

dysfunction and excessive oxidative stress (42-43) While death receptor induced

necroptosis is restricted to particular cell types, MOMP-induced CICD is a much moreuniversal process The ectopic expression of Bax is sufficient to induce MOMP and

subsequently cell death in the presence of caspase inhibitors (38) CICD mediated by

MOMP occurs either due to the general decline in the mitochondrial function or the

release of pro-death factors besides Cytochrome C from the inter-membranal space

such as apoptosis-inducing factor (AIF), endonuclease G (Endo G) (44).

Endo G is a mitochondrial endonuclease which upon release from the mitochondria

could translocate to the nucleus and induce DNA degradation (45) The direct role of AIF in CICD still remains unclear While various in vitro studies have shown that AIF

translocates to the nucleus and mediates chromatin condensation and eventual cell

death (46), there is a contention that the liberation of AIF from the mitochondria

actually induces cell death due to the loss of mitochondrial function as AIF has beenidentified to play a crucial role the aerobic respiration AIF deficient cells are also

defective in electron transport chain complex I/III (47-48).

1.2 Apoptosis In the Immune System

Apoptosis is the nexus upon which revolves the effective administration of theimmune system Lymphocytic cell death is a tightly regulated process that isparamount in the maintenance of self and peripheral tolerance as well as controllingthe homeostatic levels of lymphocyte populations during the course of an immuneresponse An abnormal increase in apoptosis can lead to immunodeficiency andwhile the failure to undergo apoptosis can result in autoimmunity or lymphoma

Maintenance of cell homeostasis is a complex process, ultimately regulated by cell synthesis, proliferation and apoptosis Identification of factors that control thesemolecular mechanisms is crucial to our understanding of how immunity is preservedand autoimmunity evaded More importantly, it gives us the ability to manipulate Tcells so as to allow a therapeutic intervention in disease states Within the context of

T-an ongoing immune response, induction of cell death plays two critical roles 1) theactivation of cell-mediated cytotoxicity in response to infectious agents such as that

mediated by FasL on activated CD8+ T-cells (49) and 2) the crucial contraction of

activated and highly proliferative lymphocytes at the end of an antigen inducedresponse, so as to free up the limited space in lymphoid organs and to reduce thepotential for non-specific activation of T-cells leading to autoimmunity Mice thatcarry homozygous defects in Fas (Faslpr/lpr) or FasL (Fasgld/gld ) spontaneously developlymphadenopathy and SLE(systemic lupus erythematosus)-like autoimmune diseases

(50-51)

The activation and expansion of T cells are characteristic feature of an adaptiveimmune response since a large population of antigen-specific T cell is required to

clear the antigen from the body However, as discussed above, it is imperative formost of these clonal T cells to be deleted at the end of the immune response Thus,signals leading to the clonal expansion of T cells can also sensitise to cells to celldeath

1.2.1 Activation Induced Cell Death (AICD) in T cells

Activation Induced Cell Death (AICD) is a well characterised death signalling processtriggered when previously activated and expanded T cells are re-stimulated through

the T cell receptor (TCR) in the absence of co-stimulatory signals (52-53) TCR

restimulation results in the upregulation of FasL which induces Fas-mediated cell

death either via suicide or fratricide (54-56) The increased transcription of FasL is

calcium-dependent and requires the protein kinase c θ (PKCθ)-mediated production

of reactive oxygen species (ROS) by the complex I of the mitochondrial respiratory

chain (57) The central role of Fas/FasL in AICD is made evident by observation in

mouse models where a deficiency in Fas or FasL (Faslpr/lpror Fasgld/gld) results in theproduction of autoantibodies and the development of a lymphoproliferative disease

(50-51) Interestingly, gene-specific deletion of Fas in all T cells in a mouse model

only leads to severe lymphopenia and the formation of an inflammatory pulmonary

fibrosis-like disease (58) This suggests that the lymphoproliferative phenotype is not

just dependent on Fas death signalling in T-cells but potentially requires other independent pathways to regulate AICD

Fas-1.2.2 Activated Cell Autonomous Death (ACAD) in T-cells

Activated cell autonomous death (ACAD) is the mechanism of T cell death that istriggered due to the absence of or declining levels of pro-survival stimuli This type

of cell death is often understood as T cell death that occurs at the end of theimmune response, when the antigens have been cleared, thus resulting in cytokinewithdrawal or neglect While Fas is the central executor of AICD, Bim is the principal

effector of ACAD (59) Hildeman et al (59) demonstrated that T-cells activated with SEB (staphylococcal enterotoxin B) still died in vivo even in the absence of Fas or

TNFR signalling, but not when Bcl-2 was over-expressed in T-cells or when deficient mice were exposed to the same stimulus While Bim levels remain the same

Bim-in ACAD, endogenous anti-apoptotic Bcl-2 levels (Bcl-2 and Bcl-XL) were dimBim-inished

(59-60) This increases the Bim: Bcl-2 ratio, thus freeing Bax and/or Bak to initiate the

intrinsic apoptotic pathway

Altogether, we find that both Fas and Bim play a crucial role in mediating activated Tcell death To have an insight into the relative contributions of Fas and Bim to thecontraction of an activated immune response, we examine studies which involvemice lacking the gene encoding Bim (Bcl2l11) and an inactivating mutation for the

gene encoding Fas (designated Bcl2l11 −/− Fas lpr/lpr) mice The shutdown of an acute Tcell response to herpes simplex virus is Fas-independent and involved only Bim,whereas both pathways synergized to induce the contraction of activated T cells in

chronic infection with murine γ-herpesvirus (61) Bcl2l11 −/− Fas lpr/lpr mice alsodeveloped a more rapid and lethal form of lymphadenopathy and autoimmunity

compared to mice lacking only one of these genes (61) These results identify critical

overlapping roles for Fas and Bim in T cell death in the contraction of an immuneresponse and a possible synergistic role it can play in the prevention of immuno-pathologies

1.2.3 Monocyte-Dependent T Cell Death (MDCD).

There is increasing evidence that monocytes and macrophages play an importantrole in regulating programmed cell death of T cells Freshly isolated peripheral blood

T cells are resistant to Fas-induced cell death up to several days (62), however, it

readily undergoes apoptosis within 18h of co-culture with phorbol myristate acetate(PMA) pre-treated monocytes in a PMA, ionomycin or anti-CD3 containing medium

(63) In this study, the authors also demonstrated that peripheral CD4+T cells whichwere cross-linked on CD4 molecules by gp120, the envelope glycoprotein of HIV-1,anti-gp120 and IgG underwent a considerably larger amount of cell death when co-cultured with PMA-treated monocytes than untreated ones This reveals animportant role for accessory cells such as monocytes and macrophages in priming T-cell death, and proposes a debilitating role for monocyte dependent T-cell death inHIV-infected individuals which results in the elimination of T-lymphocyte subsetsthereby leading to ineffective viral clearance and the characteristic advancement ofthe disease This model is also applicable to other virus infections where there is ahigh ratio of macrophages to T cells in the infected tissue Indeed MDCD has beenreported as the causal mechanism that mediates the pathology observed in diseasessuch as measles and chronic hepatitis C infection and interestingly, also for the hypo-

responsiveness of T cells from the peripheral blood of advanced cancer patients who

have received peripheral stem cell transplantation (66,278).

While some studies report a role for Fas and Fas L in mediating MDCD, there are

others yet that report otherwise (64-66,236) Where MDCD is independent or

partially dependent on death receptors, the over-expression of Bcl-2 or the

depletion of ROS from the cells were able to block cell death (236,311).

1.3 CD137/CD137 Ligand, TNFR/TNF Superfamily members.

Since the discovery of tumour-necrosis factor (TNF) as the molecule that induces the

lysis of certain cell subpopulations, especially tumour cells (67), there has been a

progressive discovery of other TNF and TNF receptor superfamily members includingFas, TNFR, RANK, death receptor(DR) 4, DR5, NGFR, OX40 and CD27 Thesemolecules possess unique structural features that allow them to couple directly tosignalling pathways involved in cell survival, proliferation and differentiation, makingthem potent biological forces that can be harnessed to ameliorate human diseasessuch as autoimmune disorders, cancer, atherosclerosis and viral infections

The ability to mediate cell death is one of the most significant functions thatTNF/TNFR superfamily members have evolved There are currently 8 homologousDeath Receptors(DR) amongst the TNFR superfamily amongst which at least 6 induce

cell death through caspases (68) The others that lack cytoplasmic death domains are

able to either modulate the response through other DRs or are themselves able todirectly down-regulate cell survival signalling TNFR2 for example is able to

significantly enhance TNFR1-induced cell death (69) and CD40 can increase mediated B-cell apoptosis (70)

Fas-Figure B summarises the expression pattern of CD137 and CD137L in immune cells

1.3.1 Molecular characteristics and expression pattern of CD137.

Like Fas, CD137 is also a TNFR family member It was first discovered in a screening

for receptors induced by concanavalin A stimulated T cells in mice (71-72) CD137 is a

27kDa type I transmembrane protein chromosomally located at 1p36 It contains 4cysteine rich modular ectodomains as well as 5 amino acid sequences conservedfrom mouse to human in its cytoplasmic tail suggesting that these residues may be

important in CD137 function or signalling (73) It is expressed on the cell surface as a 55kDa disulfide linked homodimer (74) Since its identification in T cells, CD137 has

been found to be expressed on a far broader variety of cell types including B cells

(75), dendritic cells (76), differentiating myeloid cells (4), granulocytes (77) activated natural killer cells (78) and non-lymphoid cells such as the endothelial cells of tumour blood vessels (79) The expression of CD137 and CD137L are primarily activation dependent (71, 74) Interestingly, CD137 is also upregulated by DNA damaging agents such as anti-cancer drugs or γ-radiation in human PBMCs (80).

CD137-deficient mice develop normally and are fertile; they make conventionalhumoral responses to vesicular stomatitis virus, demonstrate fairly suppressed anti-KLH IgG2a and IgG3 isotype responses, exhibit diminished virus-specific cytokineproduction and cytotoxic T lymphocyte (CTL) activity; and have increased turnover of

myeloid precursor cells in the peripheral blood, bone marrow, and spleen (3).

Subsequent studies also show that CD137-null mice have suboptimal NK/NKT cellsand associated functions, higher levels of LPS-induced septic shock and reduced IL-4-

dependent Th2 immune responses (78).

1.3.2 Effects of CD137 signalling

1.3.2.1 CD137 signalling in T cells

The effect of CD137 signalling has been most extensively reviewed in T cells While as

a TNFR superfamily member, it is widely accepted that Fas induces cell death inactivated T cells, CD137 on the contrary is able to provide a potent co-stimulatorysignals leading to T-cell proliferation, cytokine production and functional maturation

(81-84), with profound effect on CD8+ cells and modest effects on CD4+ cells (83) There is a requirement for CD28 signalling for increased CD137 expression (85) It

has been concluded that the presence of CD28 signalling is important in the earlyphase of T cell activation while CD137 has a major role in the intermediate and lateimmune responses involving the clonal expansion of activated T-cells and formation

of memory T-cells, consistent with the findings that CD28 expression on T-cells isquickly down-regulated after T-cell activation while in contrast, CD137 expression israpidly increased after B7-CD28 engagement There is also evidence for CD137-stimulated activation and proliferation of CD4+CD25+ antigen-specific regulatory T-

cell subsets (86) and antigen specific CD8+cd11c+T cells (87) which suppresses CD4+Tcell responses Thus, CD137 differentially regulates CD8+ and CD4+cells, favouringCD8+ induced cell mediated cytotoxicity over CD4+ mediated humoral responsesthereby accounting for its dual anti-tumorigenic and anti-inflammatory effectsrespectively

The early molecular changes induced by CD137 cross-linking in CD8+ T-cells isdefined by the enhanced tyrosine phosphorylation of TCR-signalling proteins and theredistribution of lipid raft domains to the contact area between T-cells and the

CD137L-containing antigen presenting cell (88) This is followed by recruitment of

tumour necrosis factor receptor (TRAF) 1 and TRAF2 which activates nuclear

factor-κB (NF-factor-κB), extracellular signal regulated kinase (ERK), c-Jun-N-terminal kinase (JNK),and p38 mitogen-associated protein (MAP) kinase cascades and the stimulation ofTCR signalling molecules such as SLP-70 and PKCθ leading subsequently to increase

in intracellular calcium levels and T cell activation (88,312-314).

1.3.2.2 CD137 signalling in B cells

In vitro, CD137 is expressed in B cells only after B cell receptor (BCR) stimulation and

in vivo CD137 is naturally expressed on B cells in tonssilar tissue (75) This

BCR-mediated upregulation of CD137 is enhanced by CD40/CD40L signalling and cytokine

IFN-γ but is inhibited by cytokines IL-4, IL-10, IL-21 (75) This suggests that CD137

expression on B cells is a tightly controlled mechanism strictly dependent on theantigen encounter The net effect of CD137 signalling in B cells is to promote B cellsurvival, proliferation and cytokine secretion of TNF-α and –β but not IL-4, -6 or -10

secretion(75) Ex vivo histological analysis of the tonssilar tissue showed that the

CD137+B cells are mainly found in the germinal centre (75) Taken together with the observation that CD137 ligation in vitro promotes B cell proliferation and survival,

the authors of this study proposed that the interaction of CD137 with CD137Lexpressed on APCs plays an important role during early B cell activation andexpansion in the germinal centre reactions

1.3.2.3 CD137 signalling in dendritic cells

Although CD137 ligand plays a far more substantial functional role in the mediated responses of professional APCs, monocytes/macrophages and DCs, thereare still some CD137 receptor modulated effects on APCs that are worth noting

immune-Murine dendritic cells (DCs) show evidence of CD137 expression on the cell surface

and in soluble forms (89) Stimulation of the CD137 on DCs promotes an increase in

IL-6 and IL-12 secretion and most importantly enhances the ability of DCs to induce T

cell proliferation in response to alloantigens and nominal antigens in vitro (89).

1.3.2.4 CD137 signalling in granulocytes and natural killer cells

CD137-deficient neutrophils are defective in phagocytosis and oxidative burst which

renders CD137-deficient mice unable to overcome Listeria monocytogenes infections (4) This implicates a role for CD137 in neutrophil activation and function CD137-

deficient mast cells also exhibit defective signalling and degranulation triggered by

IgE (90) In contrast, activation of CD137 inhibits GM-CSF or IL-5-mediated survival of eosinophils in patients with atopic dermatitis and extrinsic asthma (91).

CD137 is not expressed on naive, but on activated NK cells stimulated with TCR

engagement and alpha-galactosylceramide (alpha-GalCer), or with IL-2 and IL-15 (84, 92) Ligation of CD137 on NK cells triggers NKT activation and production of IL-4, IL-

13 and IFN-γ which could exacerbate airway hyper-responsiveness and lung

inflammation (92) In normal physiological conditions, CD137-mediated activation of

NK cells has been shown to provide ‘helper’ functions to CD8+ T cells, facilitating its

activation and expansion (84) The importance of this NK-cell mediated CD8+ T cell

activity is illustrated NK-depleted mice where anti-CD137 treatment abrogated

tumour-specific CTL activity against P815 tumour (84).

1.3.3 Clinical significance of CD137.

Since cross-linking of CD137 drastically enhances CD8+T cell activity, this enables theactivation and amplification of the cytotoxic T cell mediated immune responseleading advantageously to rejection of tumours or disadvantageously to the rejection

of allogeneic transplants Ligation of CD137 receptor for example, is able to reverseestablished anergy of CD8+cells in vivo thereby inhibiting advancing tumour growth caused by soluble tumour-antigen induced tolerance (93) Additional studies

showcasing the potency of the anti-tumorigenic properties of CD137 include that theadministration of anti-CD137 monoclonal antibodies is able to eliminate establishedlarge tumours including in poorly immunogenic tumours Ag104A sarcoma and the

highly tumorigenic P815 mastocytoma (315).

Intriguingly, in vivo, CD137 agonists can also perform the opposite function, that is,

depress immune response leading to the improvement in auto-immune pathologies

(94-95) CD137 has been proven to provide protection against several autoimmune

disorders including rheumatoid arthritis, systemic lupus erythematosus (SLE) and

experimental autoimmune encephalomyelitis (EAE) (87, 96-97) As autoreactive CD4+

T cells play a central role in initiation and progression of autoimmune disease, it was

a suspected target of CD137-mediated downregulation Indeed, In the in vivo model

of EAE, Sun et al identified that the reversal of EAE was brought about by