The role of BLNK, DOK 3 DIP in BCR signaling 1

ABBREVIATIONS ψL Surrogate light chain Abl Abelson Murine Leukemia Bam32 B cell adaptor molecule of 32 kD BCAP B cell adaptor protein BCR + FcR B cell antigen receptor and FcγRIIB recept

THE ROLE OF BLNK, DOK-3 & DIP IN BCR SIGNALING

JOY EN-LIN TAN

(B.Biotech.(Hons.))

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE

2004

suggestions and discussion I am also grateful to Assist Prof Tang Bor Luen, who so kindly provided invaluable suggestions and gave immense support and

encouragement

Special thanks goes to my past and present colleagues in the Molecular and Cellular Immunology Laboratory of the Institute of Molecular and Cell Biology (IMCB) In particular, Dr Xu Shengli, who generated the BLNK-deficient mice, together with his advice, friendship and constant encouragements Dr Wong Siew Cheng, who taught

me how to perform western analysis and gel-shift experiments and provided valuable suggestions and discussions Mr Chew Weng Keong, Mr Lee Koon Guan, Ms Tan Su-Li, Ms Tan Ai-Tee for their excellent help through the various years in

maintaining the laboratory Fellow comrades in the laboratory Mr Ng Chee Hoe, Mr Andy Tan, Ms Valerie Chew for making the laboratory a friendlier place to be in I

would also like to acknowledge the staff of the In Vivo Model Systems Unit of IMCB for taking care of the mice

I would like to thank my friends at IMCB especially Darren, Boon Tin, Zhihong and Esther for those fun times studying and working together

I am also grateful to the Administrative Staff, the COMIT Staff, the Lab-Supply Staff, the Maintenance Staff and the Glass-Ware Staff who tirelessly helped made my work much easier at IMCB

I would like to thank my family for their constant support I am greatly indebted to

my parents who helped me each day with encouragements and advices and my

brother who never stop believing in me Above all, my husband, John who

encouraged me to persevere on even though there were times when things were tough Thank you for your constant love, unfailing support and everlasting

understanding

Lastly, I would like to attribute all glory to God for this was only made possible because of Him

I dedicate this thesis to my mother and father

Joy En-Lin Tan

July 2004

TABLE OF CONTENTS

ACKNOWLEDGMENT I TABLE OF CONTENTS III LIST OF FIGURES IX ABBREVIATIONS XI SUMMARY XIII LIST OF PUBLICATIONS XVI

CHAPTER 1: INTRODUCTION 1

1.1 THE IMMUNE SYSTEM 2

1.2 DEVELOPMENT OF B LYMPHOCYTES 4

1.2.1 Immunoglobulin gene rearrangement 5

1.3 MATURATION OF B LYMPHOCYTE REGULATED BY BCR AND THEIR SURROGATES 6

1.3.1 Development of pro- and pre-B lymphocytes 8

1.3.2 Immature B lymphocytes undergo negative selection 9

1.3.3 Transitional B lymphocytes develop into mature B lymphocytes 11

1.3.4 Functional maturation of periphery B lymphocytes 12

1.3.4.1 T cell-dependent immune response 13

1.3.4.2 T cell-independent immune response 14

1.4 ANTIGEN RECEPTORS SIGNALING IN B LYMPHOCYTES 15

1.4.1 Structure of B cell receptor 15

1.4.2 Signal transduction through the B cell receptor 17

1.4.2.1 Protein tyrosine kinases 18

1.4.2.1.1 Activation of src protein tyrosine kinases 18

1.4.2.1.2 Membrane recruitment and activation of syk 20

1.4.2.1.3 Activation of Bruton’s tec kinase 21

1.4.2.2 Protein tyrosine phosphatases 22

1.4.2.2.1 CD45 required for BCR signaling 22

1.4.2.2.2 Lipid phosphatase 5’ inositol phosphatase (SHIP-1) 23

1.4.3 Structure of co-BCR receptor FcγRIIB 23

1.4.3.1 FcγRIIB mediated inhibition 24

1.4.4 Major downstream signaling pathways of BCR 26

1.4.4.1 Activation of the PI3-K pathway 26

1.4.4.2 Activation of phospholipase C (PLCγ) pathway 30

1.4.4.3 Activation of the Rho-family GTPase 32

1.4.4.4 Activation of the Ras signaling pathway 33

1.5 LIPID RAFTS IN BCR SIGNALING 34

1.6 REGULATION OF BCR SIGNALING BY ADAPTOR PROTEINS 37

1.6.1 Domains and motifs found in adaptor proteins 37

1.6.2 Adaptor proteins in BCR signaling 38

1.6.2.1 Adaptors involved in positive regulation of BCR signaling 39

1.6.2.1.1 Bam32 39

1.6.2.1.2 BCAP 40

1.6.2.1.3 BLNK 41

1.6.2.2 Adaptors involved in negative regulation of BCR signaling 43

1.6.2.2.1 c-Cbl 43

1.6.2.2.2 Dok 43

1.7 APOPTOSIS IN B-LYMPHOCYTE DEVELOPMENT 45

1.8 AIMS AND RATIONALE OF CURRENT RESEARCH 47

CHAPTER 2: MATERIAL AND METHODS 48

2.1 LIST OF ANTIBODIES 49

2.2 LIST OF PRIMERS 51

2.3 RNA/DNA METHODOLOGY 53

2.3.1 Extraction of RNA 53

2.3.2 Northern analysis 54

2.3.3 First strand cDNA synthesis 55

2.3.4 Amplification of cDNA 56

2.3.5 Polymerase chain reaction (PCR) 57

2.3.6 Agarose gel electrophoresis 59

2.3.7 Elution of DNA from agarose gel 59

2.3.8 Restriction enzymes (RE) digestion of plasmid DNA 60

2.3.9 Dephosphorylation of plasmid DNA 60

2.3.10 Ligation 61

2.3.11 Preparation of competent cells, DH5α 61

2.3.12 Bacterial transformation by the heat shock protocol 62

2.3.13 Mini-preparation of plasmid DNA by alkaline lysis 62

2.3.14 Maxi-preparation of plasmid DNA 63

2.3.15 Sequencing of DNA 64

2.4 PROTEIN METHODOLOGY 65

2.4.1 Yeast-two-hybrid screen 65

2.4.1.1 Synthetic dropout (SD) solution 65

2.4.1.2 Small-scale yeast transformation using LiAc 66

2.4.1.3 Preparation of DNA-BD-GAL4 bait for mating 68

2.4.1.4 Mating of pre-transformed library with bait 68

2.4.1.5 β-galactosidase colony-lift filter assay 69

2.4.2 Protein concentration determination by BCA protein assay 69

2.4.3 Western blot analysis 70

2.4.4 Alkaline phosphatase assay 71

2.4.5 Immunoprecipitation 71

2.4.6 Indirect immunofluorescent labelling 71

2.4.6 Transient transfection methods 72

2.4.6.1 Lipofectamine transfection 72

2.4.6.2 Effectene transfection 73

2.4.6.3 Amaxa transfection 73

2.4.7 Lipid raft separation 74

2.4.8 NF-κB assays 74

2.4.8.1 Stimulation of cells for NF-κB assays 74

2.4.8.2 Preparation of nuclear extracts 75

2.4.8.3 Electrophoretic mobility shift assays 75

2.5 MAMMALIAN CELL CULTURE AND ASSAYS 76

2.5.1 Cell culture 76

2.5.2 Apoptosis assay 76

2.5.3 Preparation of primary B cells from mouse spleen 77

2.5.4 Cell proliferation assay 77

2.5.5 Cell cycle and cell death analyses 78

SURVIVAL IN B LYMPHOCYTES 79 3.1 INTRODUCTION 80 3.2 ANTI-IGM STIMULATED BLNK-/-B CELLS FAIL TO ENTER THE

CELL CYCLE 81

IN ANTI-IGM STIMULATED BLNK-/-B CELLS 86 3.4 BLNK-/-B CELLS DO NOT EXPRESS BCL-X L UPON ANTI-IGM

BUT THAT OF PLC- γ2 IS IMPAIRED IN BCR-STIMULATED

BLNK-/-B CELLS 102 3.9 DISCUSSION 105

REGULATION OF BCR SIGNALING 113 4.1 INTRODUCTION 114

4.2 PHOSPHORYLATION OF DOK-3 UPON BCR AND FCγRIIB CO

-LIGATION 117 4.3 LIPID RAFT LOCALIZATION OF DOK-3 UPON BCR + FCγRIIB

CO-LIGATION 119 4.4 LIPID RAFT LOCALIZATION OF DOK-3 IS PERTURBED BY

BLOCKING FCγRIIB 126 4.5 DISCUSSION 126

DOK-3-INTERACTING PROTEIN, DIP 130 5.1 INTRODUCTION 131

OF A DOK-3-INTERACTING PROTEIN, DIP 132 5.3 INTERACTION OF DOK-3 AND DIP ANALYSED BY CO-

TRANSFECTION STUDIES 135 5.4 TISSUE EXPRESSION PROFILE OF DIP 139

ACTIVATED CELLS 143 5.6 CO-LOCALIZATION OF DIP AND DOK-3 TO LIPID RAFTS UPON

BCR + FCR CO-LIGATION 146 5.7 DIP BINDS TO DOK-3 THROUGH ITS C-TERMINAL DOMAIN IN A

PHOSPHORYLATION-INDEPENDENT MANNER 150

FAMILY ADAPTORS 151

MECHANISM 154

5.10 DOK-3 AND DOK-1 INHIBITS DIP-MEDIATED APOPTOSIS 159 5.11 DISCUSSION 162

BCR SIGNALING 167

SIGNALING 168 REFERENCES 170 PUBLICATIONS 199

LIST OF FIGURES

Figure 1.1 B lymphocyte development 7

Figure 1.2 Structure of the BCR complex 16

Figure 1.3 Activation of proximal PTKs upon BCR engagement 19

Figure 1.4 Signaling of B lymphocyte inhibitory receptor, Fc γRIIB 25

Figure 1.5 Major downstream signaling pathways of BCR 28

Figure 1.6 A schematic view of BLNK protein domains 42

Figure 3.1 Defective proliferation of BLNK -/- B cells in response to anti-IgM but not LPS stimulation 83

Figure 3.2 Anti-IgM-stimulated BLNK -/- B cells failed to enter the cell cycle 84

Figure 3.3 Lack of induction of cell cycle regulatory proteins in anti-IgM-stimulated BLNK -/- B cells 87

Figure 3.4 Absence of Bcl-x L expression in anti-IgM-stimulated BLNK -/- B cells 90

Figure 3.5 The expression of Bcl-2 is not altered in wild-type and BLNK -/- B cells treated with various stimuli 91

Figure 3.6 High rate of spontaneous apoptosis of BLNK -/- B cells in culture.93 Figure 3.7 BLNK -/- B cells exhibited normal activation of MAPKs and Akt upon BCR engagement 95

Figure 3.8 Imparied NF- κB activation in BCR-stimulated BLNK -/- B cells.100 Figure 3.9 Expression and activation of Btk and PLC γ2 in anti-IgM-stimulated BLNK -/- B cells 104

Figure 3.10 A model for the BCR-induced activation of NF- κB 112

Figure 4.1 Phosphorylation of Dok-3 upon BCR and BCR+FcR co-ligation 118

Figure 4.2 PLC γ2 and BCR are present in the lipid raft upon BCR engagement 120

Figure 4.3 Dok-3 is recruited to the lipid rafts only upon BCR + FcR co-ligation 122

Figure 4.4 Association of Dok-3 with lipid rafts seen upon BCR + FcR co-ligation 124

Figure 4.5 Localization of Dok-3 to lipid rafts upon BCR + FcR co-ligation 125

Figure 4.6 Inhibiting Fc γRIIB signaling prevents Dok-3 localization to lipid

rafts 127

Figure 5.1 A schematic view of Dok-3 protein domains 133

Figure 5.2 Sequence analysis of DIP 137

Figure 5.3 Interaction of DIP and Dok-3 through overexpression studies 138

Figure 5.4 Expression of DIP in different tissues and at various stages of B and T cell development 141

Figure 5.5 Subcellular localization of DIP in resting and activated cells 144

Figure 5.6 Interaction of DIP with specific domains of Dok-3 148

Figure 5.7 Interaction of DIP with Dok-1 152

Figure 5.8 DIP causes apoptosis of mammalian cells via a caspase 3-dependent mechanism 157

Figure 5.9 Inhibition of DIP-mediated apoptosis by overexpression of Dok-1 and Dok-3 160

ABBREVIATIONS

ψL Surrogate light chain

Abl Abelson Murine Leukemia

Bam32 B cell adaptor molecule of 32 kD

BCAP B cell adaptor protein

BCR + FcR B cell antigen receptor and FcγRIIB receptor

BCR B cell antigen receptor

BLNK B cell linker protein

BrdU 5-bromo-2-deoxyuridine

Btk Bruton’s tyrosine kinase

cbl Casitas B-lineage lymphoma protein

Csk COOH-terminal Src tyrosine kinase

CTB Cholera toxin B

DAG Diacylglycerol

DIP Dok-3-interacting protein

Dok Downstream of tyrosine kinase

EGFP Enhanced green fluorescent protein

ERK Extracellular-signal-regulated kinase

FcR FcγRIIB receptor

FCS Fetal calf serum

FITC Fluorescein isothiocyanate

IgH Immunoglobulin heavy chain

IgL Immunoglobulin light chain

IP3 Inositol 3,4,5-trisphosphate

ITAM Immunoreceptor tyrosine-based activation motif

ITIM Immunoreceptor tyrosine-based inhibitory motif

JNK c-Jun amino-terminal kinase

LAT Linker of activated T cells

LPS Lipo-polysaccharide

Lyn Lck/yes- related novel tyrosine kinase

MAPK Mitogen activated protein kinase

MHC Major histocompatibility complex

MTT Tetrazolium salt 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide NF-AT Nuclear factor of activated T cells

NF-κB Nuclear factor κB

PH Pleckstrin-homology

PI(3,4)P2 Phosphatidylinositol 3,4-bisphosphate

PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate

PI3-K Phosphatidylinositol 3-kinase

PKB/Akt Protein Kinase B

PKC Protein kinase C

PLC Phospholipase C

PMA Phorbol myristate acetate

PTB Phosphotyrosine-binding

PTK Protein tyrosine kinase

RAG Recombination activating gene

RT-PCR Reverse transcribed polymerase chain reaction

SH Src-homology domain

SHIP 5’ Inositol phosphatase

Syk Spleen tyrosine kinase

Xid X-linked immunodeficiency

SUMMARY

Signaling through the B cell receptor (BCR) has been shown to be vital for B

lymphocyte development and function Engagement of the BCR can trigger proximal protein tyrosine kinases leading to the activation of an array of downstream signaling pathways, which ultimately determine the cellular responses of B lymphocytes, such

as differentiation, proliferation, activation or apoptosis Adaptor proteins have been demonstrated to be responsible for bridging these upstream proximal PTKs with downstream effector molecules, transducing the signals and translating them into downstream functional events in B lymphocytes Two different adaptor proteins found in B lymphocytes, a positive regulator adaptor protein of BCR signaling, BLNK and a negative regulator adaptor protein of BCR signaling, Dok-3, the

constitute focus of this dissertation

BLNK-deficient B cells were unable to proliferate in response to BCR engagement This defect was due to the failure of BLNK-/- B cells to enter the cell cycle upon BCR stimulation At the molecular level, BLNK-/- B cells upon BCR stimulation failed to express cell cycle regulatory proteins, cyclin D2 and cdk4, which are necessary for the progression of cell cycle beyond the G0/G1 phase Furthermore, anti-IgM

treatment of BLNK-/- B cells also failed to induce the expression of the pro-survival protein Bcl-xL

Upon examining the role of BLNK in pathways involved in cell proliferation and survival, we found that BLNK-/- B cells exhibited normal phosphorylation of Akt and MAPKs, indicating normal activation of these pathways upon BCR engagement However, activation of PLCγ2 pathway leading to the increase in Ca2+, resulting in the activation of NF-κB, was disrupted Phosphorylation of PLCγ2 was impaired in BLNK-deficient B cells upon BCR stimulation Furthermore, these mutant cells were not able to engage the NF-κB pathway upon BCR engagement

Dok-3, a negative regulation adaptor protein in BCR signaling, has been shown to interact with signaling intermediates such as SHIP, Csk and Abl Furthermore, over-expression of Dok-3 was shown to inhibit B cell function by diminishing IL-2

production and NF-AT activity Here we demonstrate that Dok-3 may act in an inhibitory manner through the FcγRIIB signaling pathway Dok-3 was found to localize to lipid rafts only after treatment of whole intact Ig (which triggers the BCR together with FcγRIIB, but not with F(ab’)2 fragment Ig (which triggers the BCR alone) SHIP and FcγRIIB were also found in the lipid rafts with Dok-3 upon whole

Ig treatment, suggesting that Dok-3 may play a role in the FcγRIIB inhibitory

pathway

To further examine the role of Dok-3 in cellular signaling, we have screened for interacting partners of Dok-3 and identified a novel protein which we termed Dok-3-interatcing protein (DIP) DIP was found to be ubiquitously expressed and possesses

cells induces in apoptosis in a caspase 3-dependent manner, which can be prevented with treatment of caspase inhibitors z-VAD and DEVD DIP-mediated cell death can also be partially overcome by the expression of the anti-apoptotic protein Bcl-xL Interestingly, DIP is also able to interact with Dok-3 homologue, Dok-1 and both Dok-3 and Dok-1 are able to inhibit DIP-mediated cell death in a dose-dependent manner The discovery of DIP therefore links Dok-3 to possible roles in B cell

elimination by apoptosis during development

LIST OF PUBLICATIONS

1 Xu, S., Tan, J E-L., Wong, E P-Y., Manickam, A., Ponniah, S and Lam,

K-P (2000) B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice Int Immunol 12: 397-

404

2 Tan, J E-L., Wong, S-C., Gan, S K-E., Xu, S and Lam K-P (2001) The

adaptor protein BLNK is required for B cell antigen receptor-induced activation of nuclear factor-κB and cell cycle entry and survival of B

lymphocytes J Biol Chem 276:20055-20063

3 Wong, S-C., Chew, W-K., Tan J E-L., Melendez, A J., Francis, F and Lam,

K-P (2002) Peritoneal CD5+ B-1 cells have signaling properties similar to

tolerant B cells J Biol Chem 277(34):30707-15

4 Tan, J E-L., Chua, B-T and Lam, K-P (2004) A Novel Dok-Interacting

Protein, DIP that Causes Mammalian Cell Death and Differentially Expressed during Lymphocyte Development (submitted)

CHAPTER 1: INTRODUCTION

1.1 The immune system

Immunity is derived from the Latin term immunis, which means “to exempt from”

Historically, immunity means the protection of an organism from foreign agents or diseases The immune system constitutes a collection of cells and molecules responsible for immunity The collective and coordinated responses of the immune system to the introduction of foreign substances is thus defined as the immune response

The immune system can defend the organism from foreign invasions but may be also harmful to the host While an insufficient reaction to infection may allow the pathogen to gain foothold and overpower the individual, an overreaction can also lead

to dire consequences The immune system is, by necessity, a highly complex system capable of responding to most challenges It also requires constant self-monitoring and self-regulation to ensure that any immune response does not become detrimental

to the individual It has been suggested that our immune systems of self-regulation may gradually break down with increase age, and may also lead to the development

of autoimmune responses

There exist two main arms of immunity, innate and adaptive Innate immunity uses the genetic memory of germline-encoded receptors to recognize the molecular patterns of common pathogens Invading pathogens first encounter this barrier and must overcome it in order to have any chance of successful infection In the event an invader gets past this first line of defense, the immune system then calls upon

evolved defense mechanism It is first activated by an initial exposure to infectious agents and increases in magnitude and defensive capabilities with each successive exposure It is unlike the innate immune system, where the resistance to infection is not improved by repeated infection

There are two major branches of adaptive immune response, the cell-mediated immunity and the humoral immunity Cell-mediated immunity involves the production of cytotoxic T lymphocytes, innate immunity, and associated cytokines in response to an antigen exposure It functions mainly to eliminate intracellular microbes such as viruses and some bacteria that survive in phagocytes and other host cells In contrast, humoral immunity is predominantly mediated by antibodies that are produced exclusively by B lymphocytes in defence against extracellular microbes and their toxins Humoral immune responses can be further classified into T cell-dependent immune response or T cell-independent immune response, based on their dependence on T helper cells The T cell-dependent immune response is mainly elicited by protein antigens, while the T cell-independent immune response is mainly directed against non-protein antigens, such as polysaccharides and lipids

B lymphocytes are an exclusive cell type that are able to secrete antibodies or immunoglobulins The abbreviation "B" is derived from the Bursa of Fabricius, an organ unique to birds and where avian B cells mature In mammals however, B lymphocytes are produced in the bone marrow and require bone marrow stromal cells and their cytokines for maturation During its development, each B lymphocyte becomes genetically programmed through a series of gene-rearrangement processes

that would allow it to produce an antibody molecule with a unique specificity, capable of binding a specific epitope of an antigen Antibodies are found in two forms, on the surface of B lymphocytes where they are expressed as integral membrane proteins or secreted forms found in the plasma, mucosal secretions or the interstitial fluid of tissues The membrane bound antibody together with the signaling subunit Igα/Igβ constitutes the B lymphocyte antigen receptor complex (BCR) Engagement of the BCR is essential for the activation and development of B-lymphocytes Secreted forms of antibodies enter the blood or mucosal secretion and circulate to sites where antigens are located to neutralize the infectivity of microbes and target them for elimination by various effector mechanisms

1.2 Development of B lymphocytes

Pluripotent hematopoietic stem cells (HSCs) found in the bone marrow are the precursors of lymphocytes and other blood cells such as granulocytes, monocytes, erythrocytes and platelets However, the committed lymphoid progenitors give rise to lymphocytes and not other blood cells

A committed B lymphocyte progenitor differentiate into mature B lymphocyte through

a series of stages Cells at each stage of development can be distinguished by their cell-surface markers, their differential expression of intracellular genes, and the status

of their Ig heavy-chain (IgH) and light-chain (IgL) gene rearrangements An array of transcription factors, such as PU.1, IKAROS, E2A as well as BSAP (PAX5), are

development is arrested at a very early stage in mice deficient in any of these transcription factors (Bain et al., 1994; McKercher et al., 1996; Nutt et al., 1997; Scott

et al., 1994; Urbanek et al., 1994; Wang et al., 1996; Zhuang et al., 1994) Moreover, the specialized cellular microenvironment in fetal liver and bone marrow is also essential for B lineage commitment

In humans and mice, B cell development can be broadly and anatomically divided into two phases The initial phase takes place mainly in the primary lymphoid organs (e.g fetal liver and adult bone marrow) where B cell progenitors rearrange their IgH and later IgL genes and in an attempt to express a functional pre-BCR and subsequently, BCRs on their cell surfaces In so doing, they become immature B cells Selected immature B cells then migrate to the secondary lymphoid organs (spleen and lymph nodes) The second phase of B cell development occurs mainly in the spleen, where the immature B cells further differentiate into mature B cells Nạve mature B cells encounter specific antigens and undergo clonal expansion and functional maturation They differentiate into either long-lived memory B cells or antibody-secreting plasma cells During this antigen-dependent phase of development, B cells can generate antibodies with higher affinity for foreign antigens or with different effector functions through the processes of somatic hyper-mutation and IgH chain class switching (Rajewsky, 1996)

1.2.1 Immunoglobulin gene rearrangement

According to the clonal selection theory enunciated by Burnet, every individual

contains numerous clonally derived B lymphocytes Each clone arised from a single precursor and is able to recognize and respond to a distinct antigenic determinant (Burnet, 1957) The specificity of each antibody is determined by the variable (V) regions of the IgH and IgL chains The genes encoding V regions are assembled

during B cell development from gene segments termed V (variable), D (diversity) and

J (joining) for the IgH locus, or V and J for the IgL locus, through a process of

site-specific recombination or ‘joining’ (Alt et al., 1987; Brack et al., 1978; Tonegawa,

1983) In humans and mice, there are numerous functional V, D and J gene segments

present in the IgH and the two IgL loci (Igκ and Igλ) The recombination process involves the introduction of double-strand DNA breaks at specific recognition signal

sequence (RSS) adjacent to V, D and J elements by the V(D)J recombinases encoded

by recombination activating gene (RAG-1) and RAG-2 (McBlane et al., 1995; Oettinger et al., 1990; Schatz et al., 1989), followed by double-strand repair (Roth et

al., 1995) Each B cell clone utilizes a unique set of VDJ gene segments and therefore

displays a distinct BCR specificity, so that different clones of B cell express different

B cell receptors (Rajewsky, 1996)

1.3 Maturation of B lymphocyte regulated by BCR and their

surrogates

B cell differentiation is a tightly regulated, multi-step process (Figure 1.1) The expression of cell surface B cell antigen receptors and their surrogates (pro-BCR and pre-BCR) is critical for B cell maturation, even prior to their encountering foreign

Figure 1.1 B lymphocyte development

This schematic figure shows the development of B cells from HSC in the mouse bone marrow through pro-B cells, to pre-B and immature B cells Before leaving the bone marrow, immature B cells first express IgM Subsequently, they leave the bone marrow to seed secondary lymphoid organs such as the spleen, where they further mature and differentiate to memory B cells, or antibodies secreting plasma cells upon their encounter with antigens Stages of B cell development can be distinguished by the expression of surface markers listed on the left

Pro-B

HSC Pre-Pro-B Late Pro-B Large

pre-B

Igµ ψL

Igµ Igκ or Igλ

Small Pre-B Immature

Igµ Igκ or Igλ

Small Pre-B Immature

antigens (Rajewsky, 1996) Furthermore, signal transduction from the BCR and their surrogates is also required for the formation of a diverse repertoire of B cells

1.3.1 Development of pro- and pre-B lymphocytes

The first descendant of the lymphoid stem cells that is committed to the B-lineage is termed a pre-pro B cell From this stage onwards, the cell begins to express B-lineage specific genes and undergo a series of ordered somatic gene rearrangements of their IgH and IgL genes They can be distinguished from progenitors of other lineages by a series of cell surface markers (Hardy et al., 1982) Pre-pro-B cells further differentiate into pro-B cells that express the pro-B cell receptor (pro-BCR) on their cell surfaces Pro-BCR is composed of immunoglobulin α (Igα), and β (Igβ) chains and calnexin (Lassoued et al., 1996) Pro-BCR is a functional receptor, as the cross-linking of pro-BCR by anti-Igβ antibodies induces pro-B cells to acquire some cell surface markers

of pre-B cells (Nagata et al., 1997) It is at this stage of B cell development that the

rearrangement of IgH chain V(D)J genes is initiated (Hardy RR, 1991) The gene rearrangement begins with D H to J H segment recombination at the heavy chain locus

(Alt et al., 1984; Ehlich et al., 1994; ten Boekel et al., 1995) After D H J H

rearrangement, V H genes become accessible to the V(D)J recombination machinery

and V H to D H J H rearrangement then occurs (Alt et al., 1984; Tonegawa, 1983) If the

assembled V H D H J H is in frame, a heavy chain of class µ is expressed The heavy chain together with Igα and Igβ chains and a surrogate light chain (ψLC), whose proteins are encoded by the Vpre-B, λ5 (mouse) and 14.1 (human) genes are subsequently

functional pre-BCR cannot progress in development and are eliminated (Rajewsky, 1996)

The expression of the pre-BCR marks the transition of the pro-B to the pre-B cell stage The pre-BCR thus functions as a key checkpoint regulator by triggering Ig heavy chain allelic exclusion Therefore, once a functional pre-BCR is expressed on the cell surface of pre-B cells, the signals transduced by the pre-BCR inhibit further

V H (D H )J H rearrangement at the second heavy chain allele, although its D H J H

recombination is already completed Following that, the pre-B cells undergo clonal

expansion and differentiation to become small pre-B cells At this stage, RAG genes

are re-expressed, together with the newly expressed Igκ germline transcripts, thereby activating IgL gene rearrangement (Maki et al., 2000; Reth et al., 1987; Schlissel and

Baltimore, 1989) Successful V L J L rearrangement at the IgL gene locus results in the expression of conventional light chains Igκ or Igλ, thereby leading to the assembly of the B cell receptor (BCR) Consequently, the pre-B cells progress into the immature

B cell stage

1.3.2 Immature B lymphocytes undergo negative selection

Phenotypically, immature B cells within the bone marrow are IgMhi and IgDneg/low and CD24hi These cells remain in the immature compartment for an average of 3.5 days (Osmond, 1993) Immature B cells are the first to express the prototypic form of BCR

on their surface Consequently, they are the first B-lineage cells to recognize and respond to antigens The immature B cell stage is therefore of critical importance because from this stage, antigen-specific positive and negative selection events can be

initiated Such events exert a profound influence on the generation of the peripheral mature B cell repertoire In order to maintain tolerance to self-antigens, immature B cells undergo negative selection to eliminate those cells with high-affinity for self-antigens There are three mechanisms underlying this negative selection process: Clonal deletion, Receptor editing and Anergy

Clonal deletion occurs with the B cells undergoing apoptosis Although both immature and mature B cells express BCR, immature B cells are more susceptible to BCR-induced apoptosis (Sandel and Monroe, 1999) Studies with transgenic mice demonstrated that recognition of self-antigen by BCRs leads to rapid apoptotic death

of immature B cells (Chen et al., 1995; Hartley et al., 1991; Nemazee and Burki, 1989; Okamoto et al., 1992) and the extent of elimination of autoreactive B cells is determined by the strength of the receptor cross-linking (Hartley et al., 1991; Okamoto

et al., 1992)

Receptor editing is a process whereby an additional IgL V to J recombination occurs

and a new Ig L chain is produced, allowing the immature B cells to re-express a new

‘innocuous’ BCR (Gay et al., 1993; Hertz and Nemazee, 1997; Radic et al., 1993; Tiegs et al., 1993)

Anergy is another mechanism in which these autoreactive immature B cells are abrogated of their normal B cell function and these fail to proliferate upon mitogen stimulation (Pike et al., 1982) The BCRs on anergic B cells remain capable of

binding antigens, but BCR signaling is down regulated implying the importance of BCR signaling in the induction of B cell anergy (Cyster and Goodnow, 1995; Cyster et al., 1996; Healy et al., 1997; Inaoki et al., 1997; Prodeus et al., 1998; Rathmell et al., 1996)

The differentiation of the immature B cells with non-autoreactive BCRs is dependent

on optimal BCR signaling Mutations in the cytoplasmic domain of the BCR signaling subunit Igα have drastic effects on immature B cell differentiation (Torres et al., 1996) Disruption of proximal BCR signaling components such as Btk (Hardy et al., 1983; Khan et al., 1995), Lyn (Hibbs et al., 1995) as well as BCR modulating co-receptors such as CD19 (Engel et al., 1995; Rickert et al., 1995) and CD22 (O'Keefe et al., 1996; Sato et al., 1996) also interferes with immature B cell development

1.3.3 Transitional B lymphocytes develop into mature B lymphocytes

Functional and non-autoreactive immature B cells migrate from bone marrow to the peripheral lymphoid organs In the peripheral organs, immature B cells are referred to

as transitional B cells and they can be distinguished from mature B cells by a series of cell surface markers (Carsetti et al., 1995; Fulop et al., 1983; Loder et al., 1999; Rolink et al., 1998) The majority of transitional B cells die after reaching the spleen, with death occurs probably in the red pulp area of the spleen Only a small fraction of transitional B cells are selected to join the long-lived IgMlowIgDhigh mature B cell pool The mechanism underlying the selection of newly generated transitional B cells to the long-lived mature B cell compartment is still poorly understood, but BCR signaling

appears to be an important determinant in this selection process Inducible ablation of BCR expression leads to the depletion of peripheral B cell pool, indicating that transitional B cells fail to develop into mature B cells in the absence of BCR expression (Lam et al., 1997) Moreover, insufficient BCR signaling due to the functional inactivation of Igα (Torres et al., 1996), Syk (Turner et al., 1995), Btk (Hardy et al., 1983; Khan et al., 1995), Vav (Tarakhovsky et al., 1995; Zhang et al., 1995) and Lyn (Chan et al., 1997; Wang et al., 1996) also affects the differentiation of transitional B cells into mature B cells (Meffre et al., 2000)

1.3.4 Functional maturation of periphery B lymphocytes

Upon encountering specific foreign antigens in the periphery, the IgM+IgD+ naive B cells become activated Activated B cells proliferate and differentiate in germinal centers to either plasma cells that secrete antibodies (Liu and Arpin, 1997; MacLennan, 1994) or membrane Ig-expressing memory B cells Memory B cells actively re-circulate through blood, lymph and lymphoid organs and are responsible for the rapid and efficient production of antibodies upon re-stimulation by previously encountered antigens (Rajewsky, 1996)

As mentioned before in section 1.1, antibody responses can be divided into T dependent and T cell-independent immune responses In both cases, BCR ligation by the antigens is required for activation

cell-1.3.4.1 T cell-dependent immune response

T cell-dependent immune responses are elicited by protein antigens and require T cell help B cells take up protein antigens via their BCRs and process them into peptides that can be presented by MHC class II molecules on B cell surface to T helper cells (Lanzavecchia, 1985; Watts, 1997) The helper T cells recognize the peptide-MHC II complex via their TCR and CD4 co-receptor Activated B cells also upregulate their costimulatory molecules such as B7.1 (CD80) and B7.2 (CD86), which deliver a second signal to further activate T cells by interacting with CD28 molecule on the T cell surface (Lenschow et al., 1996) The fully activated T cells upregulate the surface expression of CD40L, which in turn activate B cells through CD40-CD40L interaction (Schultz and Coffman, 1991; Stavnezer, 2000) These interactions lead to the full activation of B cells and consequently results in antibody secretion, Ig heavy chain class switching and affinity maturation of the antibody repertoire (Rajewsky, 1996)

Class switching is a mechanism by which the rearranged V H D H J H gene segment recombines with a downstream constant region (C) gene and with the intervening DNA sequence deleted (Coffman et al., 1993; Stavnezer, 1996) This results in antibodies with different heavy chains with either of the γ, α, or ε isotypes Affinity maturation occurs when the activated B cell enters the germinal centers and are subjected to somatic hypermutation This is a process whereby single base pair mutations are introduced into their Ig variable region genes to diversify the antibody repertoire Only B cells that express a BCR with high affinity for the specific antigens are positively selected After somatic hypermutation and selection, the activated

germinal center B cells can further differentiate into either plasma cells or memory B cells (Calame et al., 2003; Guzman-Rojas et al., 2002)

1.3.4.2 T cell-independent immune response

In contrast, T cell-independent immune responses (TI) occur without T cell help Most TI antigens are polysaccharides, glycolipids and nucleic acids These antigens are multivalent or multimeric and they have multiple identical antigenic epitopes Both conventional B (B-2) and B-1 cells participate in T-cell independent immune responses, which mainly involve IgM and IgG3 antibodies in mouse These antibodies are of low affinity and they do not show significant affinity maturation (Forster and Rajewsky, 1987; Thomas-Vaslin et al., 1992)

LPS, a major component of the cell walls of gram-negative bacteria is capable of modulating T1-2 responses Besides being recognized by the BCR as a foreign antigen, it can form a cell surface complex with LPS-binding protein CD14 and TLR-

4 (Toll-like receptor 4) in mouse Signaling through the intracellular domain of

TLR-4 engages MyD88 adapter protein to recruit interleukin-1 receptor associated kinase This kinase subsequently autophosphorylates, dissociates from MyD88 and induces the nuclear factor (NF)-κB pathway (Vos et al., 2000)

Therefore, B cell maturation is a process that requires stringent regulation and the expression and optimal signaling of the BCR is essential for B cell differentiation to

1.4 Antigen receptors signaling in B lymphocytes

Antigen receptors and other receptors on B Lymphocytes play a central role in immune regulation by transmitting signals that positively or negatively regulate B cell function Recent findings have shown that different external stimuli could affect the magnitude and duration of signaling in B cells Receptors on lymphocyte have the potential to transmit crucial activating signals for initiating immune responses or to discharge equally potent inactivating signals to abort or inhibit self-reactive clones It

is thus clear that the termination and attenuation of many immune responses is not simply due to the loss of activating signals, but rather the activation of negative or suppressive signals Here, we examine the BCR with an inhibitory IgG Fc gamma receptor II (FcγRIIB) and the various signal transduction pathways associated with these

1.4.1 Structure of B cell receptor

The BCR is characterized by a complex hetero-oligomeric structure in which binding and signal transduction capabilities are compartmentalized into distinct receptor subunits The ligand-binding portion consists of the membrane bound immunoglobulin (mIg) molecule that mediates the binding of specific antigens, and the signal transduction component which comprises of the mIg-associated Igα/Igβ (CD79a/CD79b) heterodimer that functions as the signaling subunit of BCR to mediate intracellular signal transduction (Figure1-2) mIg is a tetrameric complex that

ligand-is composed of two Ig heavy (IgH) chains and two Ig light (IgL) chains Both IgH and IgL chains consist of N-terminal variable (V) regions and C-terminal constant (C)

Figure 1.2 Structure of the BCR complex

The BCR complex consists of the IgH and IgL chains, joined together by disulphide bonds The signaling subunits Igα and Igβ with their ITAMs motifs are found associated with the BCR to form a heterodimer

ITAM Immune receptor

Tyrosine-based Activation Motif

IgH

IgL Antigen

ITAM Immune receptor

Tyrosine-based Activation Motif

IgH

IgL Antigen

regions, with the variable regions of IgH and IgL chains forming the antigen-binding site As the cytoplasmic region of the IgH chain of mIg is short and in the case of IgM, consisting of only three amino acids (lysine, valine, and lysine), it is unlikely that the IgH chain is capable of mediating signaling from BCR

It has been demonstrated that the signal transduction from the BCR is mediated by the Igα/Igβ subunit Both Igα and Igβ have one Ig domain at their N-terminus and they are disulfide-linked to form a heterodimer In contrast to the short cytoplasmic tail of mIg, Igα and Igβ have 61 and 48 amino acid residues in their respective cytoplasmic domains, which are non-covalently associated with the cytoplasmic tail of mIg Within the cytoplasmic regions of Igα and Igβ is the tyrosine-rich ITAM (immunoreceptor tyrosine-based activation motif), which is characterized by six conserved amino acids (D/Ex7D/Ex2Yx2L/Ix7Yx2L/I) (Reth, 1989; Reth et al., 1991) Delineation of the function of ITAM through chimeric and mutational studies revealed that it is essential for BCR signaling (Reth and Wienands, 1997; Sanchez et al., 1993)

1.4.2 Signal transduction through the B cell receptor

As the BCR has no intrinsic protein tyrosine kinases (PTKs) activity, it utilizes several distinct families of PTKs and protein tyrosine phosphatases (PTPases) Signaling events upon engagement of the BCR involves the activation of an array of intracellular PTKs and PTPases (Figure 1-3)

1.4.2.1 Protein tyrosine kinases

There are three distinct families of PTKs found to be activated upon BCR engagement- the Src family (Lyn, Fyn, Blk and to a lesser extend fgr and hck that are found in B cells), the Syk/ZAP70 family (mainly Syk in B cells) and the Tec family (Bruton's agammaglobulinemia tyrosine kinase (Btk) being a major player in B cells) These PTKs are required for the tyrosine phosphorylation and activation of a multitude of cellular proteins that regulate a variety of distinct downstream signaling pathways (Satterthwaite and Witte, 1996) The combinational acitivty of these various PTKs determine the quality and quantity of BCR signaling Time course studies implicate temporal activation of these proteins Src family kinases are activated first (5-10 seconds) This is followed by activation of Btk (2-5 minutes) and then Syk family of kinases (10-60 minutes) (Saouaf et al., 1994) This indicates a downstream role for Btk and Syk kinases in a signaling pathway which is initiated by the Src kinases

Upon BCR ligation, Src PTKs are the first to become phosphorylated The activated Src PTKs subsequently phosphorylate the tyrosine residues within the ITAMs of Igα and Igβ However, the mechanism underlying the activation of Src PTKs is unclear

In vitro, Src PTKs undergo autophosphorylationin their kinase domain The extent of phosphorylation of the autophosphorylationsites in vivo correlates with their activity

Figure 1.3 Activation of proximal PTKs upon BCR engagement

Upon BCR engagement, Lyn (a Src-Kinase that constitutively resides in Lipid Rafts)

is activated and phosphorylates the ITAM motif found in Igα and Igβ Syk kinase is subsequently recruited to the phosphorylated ITAM and becomes activated The activation of Btk is further dependent on the activation of both Lyn and Syk Ligation

of the BCR also results in the localization of the BCR complex into lipid rafts

Phospholipid Cholesterol

Phosporylation of ITAMs Lipid

and appears to be required for maximum catalytic activity (Cooper and Howell, 1993)

PTK Csk and PTP CD45 regulates the phosphorylation status of Tyr572 at the terminal of Lyn This is demonstrated by the dephosphorylation and hyperphosphorylation of Tyr 572 in Csk and CD45-deficient DT40 B cells correlating with the hyperactivation and hypoactivation of Lyn, respectively (Hata et al., 1994;

C-Yanagi et al., 1996) In addition, in vitro binding studies also demonstrated that the

SH2 domain of Src PTKs can bind to phosphorylated ITAM sequences and this binding stimulates the catalytic activity of the Src-family PTKs, probably by competing off the binding of negative regulatory tyrosine Tyr572 (Clark et al., 1994; Flaswinkel and Reth, 1994)

The phosphorylation of ITAMs by Src PTKs allows the recruitment of the tandem Src homology 2 (SH2) domain-containing PTKs Syk and ZAP-70 Subsequently, the membrane bound Syk becomes activated by Src-PTKs This was shown by the study

of Lyn-deficient DT40 B cells, where the BCR-induced activation of Syk was dramatically reduced (Kurosaki et al., 1994) The autophosphorylation of Y520 in the activation loop of Syk is the key to its enzymatic activation, while phosphorylation of Y317 in the linker domain inhibits the function of Syk (Kurosaki, 1997; Latour and Veillette, 2001; van Oers and Weiss, 1995) The importance of Syk for both B cell development and activation is also demonstrated by studies of mutant

mice and cells lacking Syk In Syk-/- mice, B cell development is arrested at the

pro-B to pre-pro-B cell transition stage (Cheng et al., 1995; Turner et al., 1995) In SykDT40 B cells, BCR signaling is shown to be severely compromised (Hata et al., 1994)

The Btk family of PTKs includes Btk, Itk, Tec, Bmx and Rlk/Txk (Lewis et al., 2001; Yang et al., 2000) Btk is tyrosine phosphorylated and activated in response to BCR cross-linking The mechanism by which the Src kinases regulate Btk activity is not known in detail Src kinases Blk, Fyn, Lyn and Hck may regulate Btk through an indirect mechanism, in which autophosphorylation of Btk Y551 in the activation is required for Btk activity (Mahajan et al., 1995) after which Btk autophorphorylates Y223 of the SH3 domain (Park et al., 1996; Rawlings et al., 1996) This observation

is further supported by the interaction of Btk Tec Homology (TH) domain and Src family SH3 domains (Yang et al., 1995) For BCR signaling, both phosphorylation of Y551 and Y223 in Btk are essential (Kurosaki, 1997) Furthermore, genetic studies with DT40 B cells deficient in Syk and Lyn showed that the phosphorylation of Btk is significantly reduced in both Syk and Lyn single mutant cells; whereas in the double mutant cells the phosphorylation of Btk is almost completely abolished This suggests that the phosphorylation and activation of Btk occurs through the concerted action of Src and Syk PTKs (Kurosaki, 1997) The critical role of Btk in B cell development and activation is highlighted by the studies of Btk-deficient DT40 B cells and mice BCR signaling is compromised in Btk-/- DT40 B cells (Takata and

Kurosaki, 1996), and B cell development is severely arrested at the transition of immature B to mature B cell stage in Btk-/- mice (Kerner et al., 1995; Khan et al., 1995)

1.4.2.2 Protein tyrosine phosphatases

Regulation of BCR signal transduction by phosphorylation is a dynamic process that also requires dephosphorylation of kinase substrates The phenotypes of mice deficient in PTPs emphasize the importance of PTKs and PTPs in B cell development and function

CD45 is a transmembrane PTP expressed on all nucleated heamopoietic cells It possesses an extracellular domain with variable composition and structure, due to alternative splicing of several exons and differential glycosylation (Daeron, 1997; Kane et al., 2000; Turner and Kinet, 1999) The activity of CD45 is regulated by dimerization of its extracellular domain (Desai et al., 1993; Majeti et al., 1998) and this results in a strong inhibition of its PTP activity CD45 plays a stimulatory role in BCR signaling by dephosphorylating the negative regulatory tyrosine of src family kinases (Mustelin et al., 1989; Mustelin et al., 1992; Ostergaard et al., 1989) as well

as that of Igα and Igβ (Justement et al., 1991) This was demonstrated in both CD45 deficient DT40 B cells and mice, which exhibit impaired BCR signaling (Justement et al., 1991; Kawauchi et al., 1994; Kim et al., 1993; Ogimoto et al., 1994) However

CD45 is also able to play an inhibitory role by activating Src kinases Moreover, some B cells lacking CD45 exhibit elevated baseline levels of phosphotyrosine and hyperactive Src kinases (Ashwell and D'Oro, 1999; Thomas and Brown, 1999)

SHIP-1 is a 145 kDa polypeptide expressed in all hemaopoietic cells It has an amino-terminal SH2 domain , a central lipid phosphatase domain and a long carboxyl-terminal region carrying tyrosine phosphorylation sites and several proline rich regions (Damen et al., 1996; Lioubin et al., 1996; Rohrschneider et al., 2000) SHIP-1 specifically dephosphorylates lipids at the 5’ position of the inositol ring, thus

it acts on PI(3,4,5)P3 and I(1,3,4,5,)P4 The importance of SHIP-1 was characterized

in SHIP-1 deficient mice and it was shown that SHIP-1 is an important negative regulator of several processes in hemopoietic cells, and B cell maturation is defective

in these mice (Brauweiler et al., 2000; Helgason et al., 2000; Liu et al., 1998)

In order to maintain a balance immune response, positive signals through the BCR has to be modulated by counteraction from negative signals Some of these signals are generated through the activation of negative molecules, while others are through negative signaling receptors, e.g CD72 and FcγRIIB in B cells