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The role of germ-line immunoglobulins in autoimmunity and B cell activation Cho, Chin-wen C. 2007

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The role of germ-line immunoglobulins in autoimmunity and B cell activation by Chin-wen C. Cho B.Sc: Simon Fraser University, 2003 A THESIS S U B M I T T E D IN P A R T I A L F U L T U I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Experimental Medicine) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A July, 2007 © Chin-wen C. Cho, 2007 ABSTRACT The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus ( H C M V ) is dominated by a family of closely related somatically mutated antibodies that are all derived from the same V H and V K germline genes. Our laboratory has previously reconstructed the putative germ-line-based ancestor of a somatically hypermutated antibody (8F9) against the AD-2S1 and characterized and compared the binding of the germ-line-based ancestral immunoglobulin and 8F9 (Igs) to the AD-2S1 epitope. I g G l versions of these antibodies bound to AD-2S1 by E L I S A and glycoprotein B (gB) by flow cytometry. Here, I show that 8F9 neutralizes viral infectivity whereas the germ-line Igs do not. However, the germ-line Igs bind to an unidentified, phylogenetically conserved auto-antigen. Small changes in the binding site of the germ-line Igs abolished binding to the auto-antigen. Even though mechanisms do exist during B cell development to eliminate self-reactive B cells, these observations suggest that autoreactivity is not necessarily a barrier for immature B cells to further develop into follicular B cells that subsequently undergo antigen-driven affinity maturation. Our observations that the hypermutated high affinity 8F9 antibody was derived from a primary immunoglobulin that was reactive with both an auto-antigen and AD-2S1 raise important questions including 1) the threshold of affinity required for triggering the clonal expansion and somatic mutation of new B cells, and, 2) the role of low-affinity cross-reactivity with ubiquitous antigens (eg self-antigens), in rescuing B lymphocytes with primary receptors for important pathogens from "death by neglect". To further understand the initial primary B cell receptor (BCR) response to A D - 2 S 1 , we expressed the membrane I g M forms of the 8F9 and primary germ-line Igs. In our experiments, the human germ-line Igs and 8F9 were expressed in a mouse cell-line as membrane IgM. I showed that cross-linking this human I g M triggered calcium release and tyrosine phosphorylation of intra-cellular signalling molecules. Morever, the membrane I g M bound A D -2S1. Thus, I demonstrated that the human IgM expressed in a murine cell line is capable of activating many B cell responses and can serve as an in vitro model to look at affinity threshold. i i I also constructed lentiviral vectors to enable infection of R A G - 1 knockout stem cells and reconstitution of R A G - 1 knockout mice with B cells expressing human germ-line IgMs. The in vitro and in vivo systems w i l l be useful in studying the affinity threshold needed to initiate B cell activation and other questions that are important in B cell immunity. i i i T A B L E O F CONTENTS Page A B S T R A C T i i T A B L E O F C O N T E N T S iv L I S T O F T A B L E S : v i i L I S T O F F I G U R E S v i i i L I S T O F S Y M B O L S / A B B R E V I A T I O N ix A C K N O W L E D G E M E N T S x i i 1. I N T R O D U C T I O N 1 1.1 Overview o f human immune response 2 1.1.1 Innate immune response 2 1.1.2 Adaptive immune response 3 1.2 B cell development and humoral response 3 1.2.1 B cell repertoire diversity 4 1.2.2 B cell fates 4 1.2.3 B cell subsets 5 1.2.4 T cell independent (TI) and T cell dependent (TD) B cell responses 6 1.2.5 Somatic hypermutation, affinity maturation & class switch recombination..7 1.2.6 The Germinal Centre (GC) Reaction 7 1.2.7 Signaling by B cell receptors 8 1.3 Human Cytomegalovirus 10 1.3.1 Epidemiology and disease 10 1.3.2 Virus structure.... 10 1.3.3 Viral tropism and latency 10 1.3.4 Course of infection 11 1.4 Mechanism of host defense against virus 12 1.4.1 Innate and adaptive immunity against viral invasion 12 iv 1.4.2 Humoral response during H C M V infection 13 1.5 Purpose of this study 14 1.5.1 Previous studies on H C M V antibodies against gB 14 1.5.2 Proposed study 15 2. A U T O R E A C T I V I T Y OF H U M A N A N T I - H C M V A N T I B O D I E S 17 2.1 Rationale 17 2.2 Materials and methods 18 2.3 Results 21 2.3.1 Relative antigen binding of germ-line primary antibodies and somatically hypermutated antibodies 21 2.3.2 Virus neutralization by human IgG 22 2.3.3 Autoreactivity of primary lg 24 2.3.4 Human primary Igs display fine specificity for the AD-2S1 epitope from H C M V 27 2.4 Discussion and conclusion 29 3. E X P R E S S I N G A N T I - H C M V A N T I B O D I E S A S M E M B R A N E IgMs 31 3.1 Rationale 31 3.2 Materials and Methods 32 3.3 Results 37 3.3.1 Igoc and IgP expression in murine B cell lines 37 3.3.2 Expression o f human membrane I g M on murine cell line 38 3.3.3 Functional analysis of human membrane I g M on A20 cells 42 3.3.4 Chimeric analysis 45 3.3.5 Antigen-specific interaction of human I g M expressing murine cells 46 3.3.6 In vivo human IgMs expression 51 3.3.7 Bone marrow infection and B cell reconstitution 54 3.4 Discussion and conclusion 55 4. C O N C L U S I O N 57 5. B I B L I O G R A P H Y 58 6. A P P E N D I X 67 vi LIST O F TABLES Page Table 1: Use of common germ-line elements o f human a n t i - H C M V antibodies 15 Table 2: High affinity S H M antibodies and their germline ancestors 21 Table 3: Constructs for human germline and somatic hypermutated I g M 38 vii LIST OF FIGURES Figure Title Pag Figure 1.1: B cell fates at different developmental stages 5 Figure 1.2: B C R signaling 9 Figure 1.3: Structure and neutralizing antibody bidning site of glycoprotein B 13 Figure 2.1: Human C M V antibodies in H C M V neutralization assay 23 Figure 2.2: Primary human I g G l bind to the nucleus of HepG2 and H F F cells; dependence on L-chains 25 Figure 2.3: AD-2S1 peptides and human I g G l binding by E L I S A 28 Figure 3.1: R T - P C R of lgcc and IgP in murine B cell lines 38 Figure 3.2: Immunoblot Analysis of human K and m expression in A20 drug resistant clones 40 Figure 3.3: Surface expression of human I g M on murine cell line A20 41 Figure 3.4: In vitro tyrosine phosphorylation in response to surface B C R aggregation.... 43 Figure 3.5: Calcium release in response to surface B C R aggregation 44 Figure 3.6: In vitro tyrosine phosphorylation and E R K activation in response to chimeric surface B C R aggregation 46 Figure 3.7: Antigen specificity of A20 clones binding to A D - 2 peptides 47 Figure 3.8: Antigen-specific interaction of human IgM expressing murine cells 49 Figure 3.9: Human H C M V I g M stimulation by human A D - 2 coated on a surface.......... 50 Figure 3.10: Human K and p expression in 293T by lentiviral gene delivery and in N I H 3T3 by viral infection 52 Figure 3.11: Viral infection of human DG75 B cell line 53 Figure 3.12: Lentiviral infected R A G - / - b o n e marrow cells... 54 viii LIST OF SYMBOLS/ABBREVIATIONS Abbreviation Meaning A 405 nm absorbance at 405 nm Ab/Abs antibody AD-2S1 antigenic determinant 2 site one A g antigen A I D activation-induced cytidine deaminase A I D S acquired immune deficiency syndrome A N A anti-nuclear antibodies B a C M V baboon cytomegalovirus B C R B cell receptor P-Me beta-mercaptoethanol B S A bovine serum albumin B C A bicinchoninic acid protein assay C a 2 + calcium ion C D R 3 complementarity-determining region 3 C C M V chimpanzee cytomegalovirus chlgG chimpanzee IgG C M V cytomegalovirus C S R class switch recombination D A P I 4 . 6-Diamidino-2-phenylindole D M E M Dulbecco's Modified Eagle's Medium E C L electrochemiluminescence E G F R epidermal growth factor receptor E L I S A enzyme linked immunosorbent assay E R K extracellular signal-regulated protein kinase F B S fetal bovine serum F C S fetal calf serum FITC fluorescein isothiocyanate gB glycoprotein B ix G C germinal centre H B S S Hank's Buffered Salt Solution H C M V Human cytomegalovirus H E K human embryonic kidney cell line HepG2 human hepatocellular liver carcinoma cell line H F F human foreskin fibroblast H R P horseradish peroxidase G F P green fluorescent protein Ig/Igs immunoglobulins IgG Immunoglobulin G I g M Immunoglobulin M IP3 inositol 1,4,5-trisphosphate I T A M immunoreceptor tyrosine-based activation motif J H heavy chain joining region K immunoglobulin kappa chain K D kilodalton M A P K mitogen-activated protein kinase M O I multiplicity of infection P immunoglobulin membrane I g M heavy chain O D optical density P A G E polyacrylamide gel electrophoresis P B S phosphate buffered saline P F U plaque forming unit PI3K phosphoinositide-3 kinase P L C - y phospholipase C gamma R h C M V rhesus cytomegalovirus SDS sodium dodecyl-sulsphate S H M somatic hypermutation slgM/sIg surface IgM/surface l g S L A M Selected Lymphocyte Antibody Method T C tissue culture x TdT terminal deoxynucleotidyl transferase Tris tris(hydroxymethyl) aminomethane u M immunoglobulin membrane mu-chain V H heavy chain variable region V K kappa light chain variable region V S V Vesicular Stomatitis Virus xi ACKNOWLEDGEMENT I want to express my sincerely thanks to Dr. John Schrader and Dr. Gary McLean for giving me guidance and support while pursuing the degree. Dr. K e l l y McNagny, Dr. Linda Matsuuchi and Dr. Vince Duronio, for contributing great suggestions and helping with this accomplishment. I also want to thank everyone in the Biomedical Research Centre, especially Poh Tan, Eunice Yao and Kaan Biron. Xll 1. I N T R O D U C T I O N 1.1 General Overview of the Human Humoral Response The primary function o f the immune system is to protect the host from infectious microbial agents in its environment. Environmental pathogens threaten the host wi th a large spectrum o f pathological mechanisms. The immune response therefore uses a complex array o f protective mechanisms to control and usually eliminate these organisms. A l l o f these mechanisms rely on detecting structural features o f the pathogens that mark them as foreign from host cells. Such host-pathogen discrimination is essential to permit the host to eliminate the pathogen without excessive damage to its own tissues. Host mechanisms for recognition o f microbial structures are o f two general classes: (1) innate immune responses that are encoded i n the host's germ-line and recognize molecular patterns that are shared by many microbes but are not present in the mammalian host and (2) adaptive immune responses that are encoded by gene elements that somatically rearrange to assemble novel genes that encode antigen-binding molecules with exquisite specificity for individual unique microbial and environmental structures. The recognition molecules used by the innate immune response are broadly expressed on a large number o f cells, this system is poised to act rapidly after an invading pathogen is encountered. Thus, it provides the init ial host response. The adaptive immune system init ial ly produces only small numbers o f cells with specificity for any individual pathogen, cells that encounter and recognize a pathogen must proliferate to attain sufficient numbers to mount an effective response. A key feature o f the adaptive response is that it produces long-lived cells that persist i n an apparently dormant state but and can re-express effector functions rapidly when they encounter their cognate antigen for a second time. The innate and adaptive systems usually act together, wi th the innate response representing the first line o f host defense until the adaptive response becomes mature after several days as antigen-specific T and B cells have undergone clonal expansion. Furthermore, the antigen-specific cells amplify their response by recruiting innate effector mechanisms to bring about the complete control o f invading microbes. 1 Thus although the innate and adaptive immune responses are fundamentally different in their mechanisms of action, synergy between them is essential for an efficient host immune defense. 1.1.1 Innate Immune Response The innate immune system includes all defense mechanisms that are encoded in the germ-line genes of the host. These innate defense components are presented on cell surface or in soluble form, ready to respond upon encountering antigens. Cell surface receptors that bind molecular patterns expressed on the surfaces of invading microbes are essential to initiate cellular response against invading pathogens (Pulendran et al., 2001 and 2001). The humoral immunity of naive animals consists of B cells secreting "natural" antibodies of the primary repertoire. These circulating Igs belong to the IgM class without antigen-induced affinity alteration and thus usually have low affinity and may be poly-reactive (Reviewed in Carroll, 1998; Duan and Morel, 2006). Natural antibodies may facilitate antigen uptake, processing, and presentation by B lymphocytes via complement and Fc receptors, may induce or prevent autoimmune disease, and they may clear lipopolysaccharide (Ochsenbein et ai, 1999). 1.1.2 Adaptive Immune Response The adaptive immune response comprises antigen-specific lymphocytes and development of immunological memory. The adaptive immune system manifests fine specificity for its target antigens by virtue of the antigen-specific receptors expressed on the surface of T and B lymphocytes (Janeway et al, 2001). The genes encoding antigen-specific receptors of the adaptive response are assembled by means of somatic rearrangement of germ-line gene elements to form intact T-cell receptor (TCR) and immunoglobulin (lg) genes (Muljo and Schlissel, 2000). The focus of this thesis is on B cells, and the initial interaction and response of viral antigen with antigen receptor on B cells. Although the T cell components are essential for the complete effect of adaptive immune response, it will not be addressed in this thesis. 2 1.2 B Cell Development and Humoral Response B cells are one of the two major types of lymphocytes. They express Igs either on the cell surface or they secrete Abs. The development of B lymphocytes from pluripotent hematopoietic stem cells is a tightly regulated process that originates in the fetal liver and maintained in the adult bone marrow and further proceeds in the secondary lymphoid organs. Although there are many classifications of B cell subsets, in general, B cells in the periphery can be divided into the B l extra-follicular subset and the conventional follicular B2 subset that undergoes somatic hypermutation (SHM) and/or class-switch recombination (CSR). 1.2.1 B Cell Repertoire Diversity The B cell immune system has the potential to recognize an enormous number of antigens from infectious agents, environmental particulates and self-antigens using limited elements -a few hundred lg variable gene segments by four somatic D N A alterations that are not encoded in the germ-line. With the help of recombinase-activating genes 1 and 2 (RAG1 and RAG2), V(D)J recombination assembles two or three pieces of germ-line segments (variable segment, joining segment and/or diversity segment) to form a germ-line-based antigen receptor on the surface of each naive B cell in the bone marrow (Grawunder et al, 1998). Another enzyme, terminal deoxynucleotidyl transferase (TdT) is an enzyme that contributes to the diversity of the lg repertoire by adding N-nucleotides at the joints between rearranged heavy chain gene segments. After leaving the bone marrow, the immature B cells with the rearranged primary germ-line heavy and light chains encounter their cognate antigen. Immature follicular B cells undergo further genetic changes in the peripheral lymphoid organs to improve their BCRs ' specificities and affinities to the antigen, fn humans and mice, affinity maturation of the antibody response is mediated by S H M of the lg genes followed by selection of B cell clones with higher affinity B C R against the particular antigen. S H M introduces a massive number of point mutations and occasional deletions and insertions at rates 1 million times higher than that of spontaneous mutations at a defined region between the V H promoter and the intronic enhancer (Martin and Schraff, 2002). The fourth somatic gene alteration in B cell repertoire is class-switch recombination. The B C R can undergo CSR in which it switches the constant heavy (CH) region of antibodies to produce lg with the same specificity but a different isotype 3 thereby changing the effector functions of the antibodies (Besmer et al., 2004). Each isotype determines how the captured antigens are eliminated. Even though SHM usually occurs before CSR, neither is a requirement for the other. These two processes expand and refine the humoral immune response; however, not every B cell has the equal ability to do so. 1.2.2 B Cell Fates The stochastic nature of the recombination processes ensures a diverse repertoire of binding specificities but it also generates Igs that react with self, and thus pose the threat of autoimmune disease. The production of autoreactive B cells in mice and in humans, is limited by a variety of mechanisms that include receptor editing, the deletion of autoreactive cells and induction of anergy (Goodnow et al., 1998). Upon engagement of the BCR, mature, peripheral B cells enter the cell cycle and proliferate. Within the bone marrow, however, immature B cells become anergic or are deleted in response to antigen encounter, processes which silence autoreactive B cells (Goodnow et al., 1991; Hartley et al., 1993). In mice, those B cells that express self-reactive BCRs that do survive the bone marrow selection processes undergo anergy or, under the influence of signals triggered by their autoreactive receptors, differentiate into BI or marginal zone B cells, rather than the follicular (B2) lymphocytes that populate germinal centers and undergo SHM and affinity maturation. 4 Figure 1.1: B cell fates at different developmental stages. The pre-B cells receive signals via their pre-BCR in order to survive or differentiate into immature B cells (a). If antigen encounter occurs at immature B cell stage, the cell wi l l undergo apoptosis and be deleted (b) or become anergic and therefore silenced (e). Mature B cells wil l be activated and proliferate after antigen stimulation (d). Pre-B cell Immature B cell Mature B cell Anergic B cell • • • Apoptosis Activation N o response Adapted from: Pierce S K . (2002) Nature Reviews Immunology 2, 96-105. 1.2.3 B Cell Subsets Although a matter still under great debate, it is believed that B cells can be divided into different subsets by developmental lineage and the characteristics of their antibody repertoire (Reviewed in Hardy and Hayakawa, 2001, Sagaert and Wolf-Peeters, 2003). The phenotypic distinction between the two main B cell lineages, B l and B2, such as anatomical locations, surface marker expressions, antibody repertoires and growth properties is useful but not definitive. Another emerging subset, marginal zone B cells, is phenotypically similar to B l subsets; but some believe they are derived from the B2 lineage (Reviewed in Viau and Zouali, 2005). The characterization of B cell subsets responsible for the first line of defense in mouse is thought to be the C D 5 + B1 -a B cells that originated from fetal liver. However, in human, the functionally lg equivalent subset is less well-defined, but it is believed to be the memory IgM cells that originated from the bone marrow and developed in the periphery (Reviewed in Carsetti etal.). 5 BI B cells and marginal zone (MZ) B cells constitute the first line of defense against blood-borne microorganisms, viruses and toxins in the spleen. The BI BCR repertoire is dominated by unmutated IgM. The fast and efficient protective antibody responses of MZ B cells are well characterized; however, much less is known of their interactions with other cell types during immune responses. Recent work (Kearney et al., 2004; Lopes-Carvalho et al., 2005) has demonstrated that MZ B cells in mouse can directly activate T cells. MZ B cells also interact with other antigen presenting cells and transport and concentrate antigen during the course of T-dependent and T-independent immune responses. In humans, the origin and characterization of the B cell subset that perform the same function as mouse B I B cells (CD5 positive) are not well-defined and understood. The transitional B cells in humans migrate from the bone marrow to the spleen and develop into mature B cells. The B cells that are functionally equivalent to the B-la mouse B cells are the memory IgM cells that are of low affinity but effective against encapsulated bacteria infection (Ghia et al., 1996). 1.2.4 T Cell Independent (TI) and T Cell Dependent (TD) B cell response B cells are activated in different ways in vivo. Antigens such as proteins or sheep red blood cells induce a B cell response only if B cells bind the antigen in the presence of specific T cell help, such as CD40 ligand co-stimulation and IL-4 (Cyster et al, 1999). One of the most important co-stimulatory signals on B cells is delivered by CD40. Interaction of B cell receptors with TD antigens leads to the activation of a cascade of protein kinases and to an increase in intracellular calcium concentrations (Rubier, 2001). However, neither proliferation nor antibody production occurs unless the same B cells receive co-stimulatory signals. Ligation of CD40 promotes long-term proliferation of B-cells, germinal centre formation, lg class-switching, IL-5 production and up-regulation of molecules such as LFA-1 and CD23 (Erickson et al, 2001). CD40 co-stimulation also suppresses the induction of apoptosis induced by cross-linking the BCR on immature B cells. This interaction leads to release of cytokines that induce B cell proliferation, antibody production, and in a later phase, germinal centre CSR and/or SHM (Parker, 1993). 6 In contrast, multivalent antigens such as bacterial lipopolysaccharide do not need accessory signals provided by cognate T helper cells to induce B cell responses. B - l and MZ B cells have been described as cells with "natural memory" because they have a repertoire that recognizes T-cell independent type 2 (TI-2) antigens. TI-2 antigens contain multiple identical epitopes that are capable of crosslinking BCRs. BI and MZ cells are the first response to foreign antigens. Upon activation, these cells migrate to the spleen and form foci of plasmablasts (Lopes-Carvalho and Keraney, 2004), and mature into short-lived antibody-secreting plasma cells (Bikah et al, 1996; Hippen et al, 2000). 1.2.5 Somatic Hypermutation , Affinity Maturation and Class Switch Recombination SHM introduces single base pair substitutions, with rare insertions and deletions into the variable regions of antibody genes. Somatic hypermutation of lg genes is required for affinity maturation of the humoral responses to foreign antigens (reviewed in Martin, 2002). Many recent publications indicate that activation-induced cytidine deaminase (AID) is required for SHM and CSR (Dickerson et al, 2003; Shinkura et al, 2004). AID is specifically expressed in the germinal centres where affinity maturation and class switching occurs (Reynaud et al, 2003). Approximately one mutation is introduced into the BCR with each cell division. The variant BCR are then expressed on the surface of non-cycling centrocytes which are selected on the basis of the affinity of the BCR binding to antigen (Reviewed in Rajewsky, 1996). Igs can undergo CSR in which the constant heavy chain region of antibodies is switched to that of another lg class. This results in the production of Igs with the same specificity but as a different isotype and thus different effector function (Besmer et al, 2004). 1.2.6 The Germinal Centre (GC) Reaction The GC reaction depends on cycles of cellular activity and molecular changes that regulate antigen-specific clonal evolution during the development of B cell memory (reviewed in McHeyzer-Williams et al, 1993). Germinal centers form around follicular dendritic cell networks when activated B cells migrate into lymphoid follicles (Janeway et al, 2001). Polarization of secondary follicles into a T cell-proximal zone of expanding centroblasts (the dark zone) and a T cell-distal zone of non-cycling centrocytes (the light zone) signifies the beginning of the GC reaction. Clonal expansion followed by BCR diversification and 7 affinity-based selection results in either re-entry into a second GC cycle of events, or exit from the GC. These B cells either entry into the pool of long-lived memory B cell that circulate around the body to provide humoral immunity memory, or into the pool of plasma cells that home to the marrow and synthesize circulating antibodies (Reviewed Wolniak et al, 2004). 1.2.7 Signaling by B cell receptors The BCR is composed of an lg molecule which is involved in recognition and binding of antigen and two associated chains, Iga and Ig(3, each containing ITAMs. They become tyrosine phosphorylated upon antigen binding and recruit downstream signaling molecules, such as Syk, to the receptor complex. The Ras/MAPK pathway, PI3K and PLC-y are also activated after ligation of the BCR (Kumar et al, 1995; reviewed in Gold and Matsuuchi, 2000). BCR activation leads to rapid stimulation of phosphoinositol metabolism and generation of multiple second messengers (Rameh and Cantley, 1999). PLC-y is rapidly recruited by an activated BCR and hydrolyzes its substrate to form two second messengers, diacylglycerol and IP3. By binding to specific intracellular receptors, IP3 stimulates Ca 2 + release from intracellular stores. Ca 2 + then binds to calmodulin, which in turn activates a family of Ca /calmodulin-dependent protein kinases. Upon encounter with antigen, signals are transduced from the extracellular environment, through the plasma membrane and to the nucleus, eventually resulting in the transcription of new genes. In summary, activation of lymphocytes refers to a complex process associated with generation of cytoplasmic second messengers, elevations in RNA and protein synthesis, changes in gene expression, and initiation of processes leading to the ability to elicit and receive secondary signals from T cells, culminating with the growth and proliferation of the B cell. 8 Many recent studies suggest that the "strength" of antigen recognition, such as number of receptors present and level of affinity between antigens and receptors, regulates the choice between extra-follicular plasma cell and germinal center B cell differentiation (Paus et al, 2006). In germinal centers, responding B cells collaborate with follicular T helper and dendritic cells to undergo S H M with mutant clones that recognize the antigen with increase affinity being selectively propagated (Berek et al, 1991). Figure 1.2: BCR Signaling. B cell receptor signal transduction initiates at the cell membrane by antigen binding induce aggregation of the surface immunoglobulin. Since surface immunoglobulins do not have intrinsic phosphorylation motif, associated signal transducing elements Igoc and Ig(3 are required. The initial phosporylation activates the subsequent signal pathways such as PI3 kinases and M A P kinases etc. Signals are propagated by means of protein phosphorylations and interactions. Several signaling pathways work in network to translate external stimulus into expression of desired genes. Adapted from Dal Porto etal. (2004). Molecular Immunology 41:5 99-613 9 1.3. Human Cytomegalovirus 1.3.1 Epidemiology and Disease Human cytomegalovirus causes wide-spread and persistent human infection. HCMV is found throughout the world and its prevalence can vary from 50 to 90%, mainly depend on the socio-economic status of the population. Transmission of HCMV occurs from person to person, through close contact with body fluids (urine, saliva, breast milk, blood, tears, semen, and vaginal fluids). In HCMV, the co-evolution of virus and host has resulted in a balanced survival of both. The virus usually persists over the lifetime of the host and is frequently transmitted from carrier mothers to their children through the placenta before birth or at the time of birth. The primary infection usually causes minimal clinical manifestation and the virus remains latent throughout the lifetime of the host. If sporadic reactivation occurs, it is generally well-controlled by cell-mediated immunosurveillance. However, primary infection or reactivation of HCMV in immunocompromised individuals such as pregnant women, transplant recipients and AIDS patients can be a severe health threat, even leading to death. Primary and sometimes secondary HCMV infection in pregnant women is the leading cause of congenital defects such as hearing loss, mental retardation, and brain and organ damage. 1.3.2 Virus Structure HCMV is a P-herpesvirus. Several strains of C M V infects human but serological tests do not define different specific serotypes. The virus has a large genome, with a double-stranded DNA genome, coding for 200 proteins, including 35 structural proteins and glycoproteins, enclosed in an icosahedral capsid. It is an intranuclear virus and the core is assembled in the nucleus of the host and exits from affected cells by the secretory pathway (Ohnoy & Diav-Citrin, 2006). 1.3.3 Viral Tropism and Latency HCMV can infect most cells; HCMV latency defined as the persistence of the viral genome in the absence of production of infectious virions. HCMV gene products can be found in monocyte/macrophages, endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, stromal cells, neuronal cells, neutrophils and hepatocytes (Compton, 2004). Many research 10 groups have demonstrated that the cells of myeloid lineage are an important reservoir of latent H C M V (Taylor-Wiedeman et al, 1991, Larsson et al, 1998). 1.3.4 Course o f In fec t ion The viral replication cycle can be separated into three stages, initiation/entry, gene replication and the release of newly synthesized virus. The ability of H C M V to infect an extensive range of cell-types makes it complicated to identify cellular receptors. However, recent studies indicate that glycoprotein B (gB) is essential for viral attachment and penetration of host cells (Lopper and Compton, 2004; Reviewed in Compton, 2004) 11 1.4 Mechanism of Host Defense Against Virus 1.4.1 Innate and Adaptive Immunity against viral invasion The humoral response acts to prevent entry of virus by neutralizing its ability to bind to the cellular receptor required for its entry. Antibodies can also facilitate fixation of complement on the virion and aid in its destruction. The early production of neutralizing antibodies is essential for protection against many cytopathic viruses (such as polio, influenza, and rabies viruses) and limits virus spread and confers protection from a lethal challenge (Reddehase et al, 1994). During the initial stages of primary encounter with a virus, the body lacks antigen-specific defenses and innate immunity is the first line of defense. Although cellular immunity may be responsible for recovery from HCMV infection, humoral immunity is likely more important in protection against primary infection (Speckner et al, 1999). It has been demonstrated that antibodies can limit virus spread and confer protection from a lethal challenge (Reviewed in Johjic et al, 1994). Low-affinity and polyreactive antibodies that bind many antigens are present at low titres in the blood of naive individuals. The poly-reactive specificity of natural antibodies has been attributed to conformational flexibility within the CDR3 region of the lg heavy chain, which allows these antibodies to bind at low affinity to a broad range of antigens, including proteins, nucleotides, polysaccharides and lipids (Adib-Conquy et al, 1997; Wardemann et al, 2003). Despite their low affinity, if present as a decavalent IgM, the avidity of these primary antibodies may be able to effectively defend against viral infection or facilitate the development of an adaptive immune response. Recent reviews have summarized evidence that natural antibodies provide an important link between the innate and adaptive immune systems by restricting initial viral dissemination (Ochsenbein et al., 1999; Diamond et al, 2003). Moreover, natural antibodies can neutralize viruses directly (Reviewed in Hangartner, 2006). Binding or low affinity antibodies of the primary repertoire to viruses can and activate complement system. Viral proteins are recognized as foreign, and antibodies against many viral proteins are raised following infection. However, only a small fraction of these newly generated antibodies have direct anti-viral or neutralizing activity. Neutralizing activity requires the antibody to be of 12 relatively high affinity and/or avidity for exposed structures on the surface of the virus (Bachmann et al, 1997; Roost et al, 1995). Such antibodies render virions non-infectious by interfering with receptor binding or cell entry. While pre-existing germ-line encoded antibodies can help to combat the primary infection; the antigen-specific activation of B cells under the influence of helper T cells triggers the process of affinity maturation in the germinal centre. In some cases, S H M and affinity maturation can account for more than 100-fold increased neutralizing capacity in vitro. This is accompanied by abrogation of the polyreactivity (Oppezzo et al, 2004) of the germ-line antibodies. 1.4.2 Humoral Response during H C M V infections Glycoprotein B (gB) is a major target of neutralizing antibodies that develop after natural infection with H C M V (Marshall et al, 1992 and Wagner et al, 1992). The mechanism by which gB-specific antibodies interfere with the complex processes leading to loss of viral infectivity has not be clearly defined, but these neutralizing antibodies appear to block viral attachment and viral membrane fusion with host cells (Tugizov et al, 1994; Gicklhorn et al, 2003). The generation of antigen-specific antibodies in B cell immunity is the focus of the thesis. Figure 1.3: Structure and neutralizing antibody binding site of glycoprotein B. Glycoprotein B (gB) is a major target of neutralizing antibodies that develop after natural infection. The antibody-binding sites have been mapped on the gB molecule and the extracellular immunodominant antigenic domain 2 (AD-2S1) is located between amino acids 50-86 at the N-terminus. 13 1.5 Purpose of this study The immunological effector functions that control HCMV infections are difficult to analyze since the virus is strictly species-specific and no experimental animal model system is available. Our proposed study provides us with means to study viral infection and antibody response to a human pathogen that is otherwise unavailable. Although there are some differences in mouse and human, the in vitro setting allows us to investigate the affinity required of the BCR for antigen to initiate B cell response. Future in vivo studies will hopefully provide insights into the effects on activation of B cells with low and high affinity BCR to a viral antigen. 1.5.1 Previous study on H C M V antibodies against gB The principal target of the humoral immune response to HCMV is the envelope protein glycoprotein B (gB), which contains several dominant antigenic determinants. Antigenic determinant 2 site 1 (AD-2S1) is a linear epitope encoded by residues 69-78 of the N-terminal surface fragment of gB, gp 116. Abs targeting the AD-2S1 epitope neutralize viral infectivity, probably by blocking a critical function of gp 116 in attachment and fusion of virus to the cell. Our laboratory has cloned and characterized anti-AD-2Sl antibodies using the selected lymphocyte antibody method (SLAM; Babcook et al., 1996) and found that a series of human Abs specific for AD-2S1 were all derived from primary Igs encoded by single V H (VH 3-30), JH (JH 4), and V K (VK 3-11) genetic elements (Table 1), suggesting that these primary Igs might have been able to bind HCMV. Moreover, an SGL motif in the CDR3 of heavy chain added by random nucleotide incorporation by TdT is found to be involved in fine specificity against AD-2S1 epitope, and single amino acid replacements in this motif significantly affected binding to AD-2 and a longer sequence of the N-terminus of gB (gB-NT). As VH3-30 and V K 3-11 occur in all humans, they potentially confer an innate capability to generate primary Igs that readily bind a key component on a ubiquitous, potentially pathogenic virus. 14 Table 1: Use of common germ-line elements of human anti-HCMV antibodies Source v„ l i t J . . ifkddition in CDR3 V K J * ' J r . ' . KE5 3-30 4 3-22 SGLI 3-11 5 8F9 3-30 4 2-15 SGLL 3-11 1 Kalinke et al. (2000) showed that some germ-line precursors of a hypermutated antibody bound and neutralized vesicular stomatitis virus (VSV). Roost et al. (1995) also demonstrated that early anti-viral IgG neutralizes VSV with high-affinity and SHM did not significantly improve overall affinity. Given that these germ-line antibodies show AD-2S1 binding and are ancestral to the somatic hypermutated 8F9, we suspected that these primary Igs would, when expressed as the receptors on naive B lymphocytes, bind gB with sufficient avidity to initiate the process of clonal expansion, affinity maturation, and somatic mutation. 1.5.2 Proposed Study Our study on anti-gB germ-line and somatic hypermutated IgG indicate that there is some innate basis for an adaptive immune response against viral antigens. Antibodies of the primary repertoire are often polyreactive and the signal strength required to initiate antigen-induced B cell activation is still debated (Batista and Neuberger, 1997 and 2000; Inaoki et al., 1997). Our previous data are derived from studies on serum IgGs. IgG antibodies are the product of CSR which occurs in the germinal centre, along with affinity maturation. In contrast, the initial contact with antigen that initiates the response is mediated by germ-line-based primary Igs in the form of surface IgM. After the activation, the slgM expressing B cells will migrate to germinal centers and undergo class switching and affinity maturation. In order to study the generation of anti-HCMV germ-line and somatic hypermutated antibodies as surface IgMs, Dr. McLean and Dr. Watt in the lab had cloned these primary V-regions of anti-HCMV Abs into p constructs with a trans-membrane sequence and leader sequence in order to express them on the cell surface. 15 This thesis aims to examine the hypothesis that the primary that these B cell repertoire is dominated by promiscuous low-affinity antibody-antigen interactions and forms the basis o f adaptive responses by initiating B cel l responses and affinity maturation. In the next chapter, we present evidence o f the autoreactivity o f a germ-line antibody that gives rise to a high-affinity antibody against another antigen, gB o f H C M V . The third chapter o f the thesis attempts to address (1) What is the affinity threshold required to activate a B cel l into affinity maturation? (2) What are the mechanisms (competition for antigen by secreted antibodies or by other B cells) that result i n domination o f the repertoire o f B cells responding to one epitope o f one or two clones? 16 2. AUTOREACTIVITY OF HUMAN ANTI-HCMV ANTIBODIES Noted: The data in this section is a summary of "Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV" (McLean, Cho and Schrader, 2006) and modified to fit the scope of the thesis. 2.1 Rationale McLean et al. (2005) indicated that human neutralizing, hypermutated IgG Abs that bind to the short, linear AD-2S1 epitope of HCMV gB are derived from a structural subfamily of primary Igs exhibiting restricted V gene usage. The study demonstrated that the germ-line lg of anti-HCMV antibodies bind AD-2S1 and one of the primary germ-line-based antibodies (J5 IgG) binds this antigen with high affinity. This line of evidence suggests that these common V genes with junctional diversity make up for an innate anti-HCMV antibody foundation for generation of high-affinity neutralizing antibodies to HCMV. A large proportion of the precursors of human B lymphocytes express primary human Igs that display polyreactivity or self-reactivity (Wardemann et al, 2003). Many self-reactive B lymphocytes are eliminated during development to avoid the risk of auto-immune diseases. Of the two primary germline-based antibodies made in the laboratory, both of which had given rise to high affinity antibodies against HCMV, one appears to bind HCMV neutralizing epitope efficiently (J5 IgG) and the other only weakly (Jl IgG) (See Table 2). One possibility is that these germ-line antibodies may have reacted also with other antigens, including self-antigens. 17 2.2 Materials and Methods Expression vectors Expression vectors (pLC-huCK, pHC-huCYl) used for the production of IgGs were described previously (McLean et al, 2000). Production and purification of chimeric IgG Human embryonic kidney (HEK) 293 cells were transiently transfected with plasmids encoding L-chain and various H-chains to produce chlgGl. Briefly, 3 ug of each plasmid was mixed with lipofectamine reagent (Invitrogen, Carlsbad, CA) and added to a monolayer of 4 x 106 HEK 293 cells for 6 h at 37 °C. Complete media (DMEM including 10% FCS) was then added, harvested and re-added every 24 h for up to 5 days. Recombinant IgGf were purified from cell supernatants using a protein-G affinity column (Amersham Biosciences, Uppsala, Sweden). Following elution with 100 mM glycine pH 2.5, the purified chlgG was dialyzed for two days versus three changes of PBS. Preparations were quantitated for IgG concentration by BCA protein assay (Pierce Biotechnology, Rockford, IL). Peptides Peptides were made by solid-phase synthesis (Phil Owen, The Biomedical Research Centre, Vancouver, BC, Canada). The 25mer peptide corresponding to amino acids 64-88 of HCMV gB that contains the AD-2S1 epitope has been described previously (Babcook et al, 1996; McLean et. al, 2003). For comparative analyses, 14mer peptides containing the AD-2S1 epitope of CMV gB from human (HCMV) (Davison et al, 2003), chimpanzee (CCMV) (Davidson et al., 2003), baboon (BaCMV) (Blewett et al, 2001), and rhesus monkey (RhCMV) (Kravitz et al, 1997) were made. The HCMV peptide corresponds to amino acids 64-7 of gB (SHRANETITYNTTLKY) (A03782 accession #), the CCMV peptide to amino acids 61-75 of gB (TTNGTEYIFNTTLRP) NC_003521 accession #), the BaCMV peptide to amino acids 58-72 of gB (NTTTGALIENTTLRT) (AF324835 accession #), and the RhCMV peptide to amino acids 39-53 of gB (ENTTGPLIENTTLRT). 18 ELISA A l l Abs .were from Southern Biotechnology Associates. Coating with peptides or anti-human IgG or anti-human k (in PBS) and blocking (2% B S A in PBS) of plates was performed overnight at 4°C. Dilutions of Abs (in PBS containing 1% BSA) were added and plates were left either overnight at 4°C for an hour at 37°C. Similar results were obtained under both conditions, suggesting that equilibrium was reached. Washing (with PBS-0.05% Tween 20) was performed quickly using the SkanWasher 300 (Skatron). The OD at 405 nm was read in a SpectraMax 190 spectrophotometer (Molecular Devices). Flow Cytometry on HCMV antibodies binding to N-terminus gB (NT-gB) on NSO cells. Parental NSO cells and NSO cells engineered to stably express the AD-2S1 epitope on the cell surface were suspended at ~10 6/ml in PBS containing 0.5% FCS and the indicated Igs for 45 min on ice. They were washed once with ice-cold PBS-0.5% B S A and then stained with anti-human light chain FITC conjugate (Southern Biotechnology Associates). Fluorescence was analyzed using a FACScan (BD Biosciences) and the data were analyzed using CellQuest software. Virus Neutralization Assay Human foreskin fibroblast (HFF) cells were seeded in 24-well culture plates at 20,000 cells/well. Recombinant H C M V (RC 2924) Towne strain that expresses an ie2-GFP fusion protein upon cell infection (Abate et al., 2004), was generously provided by Dr. J. X u and Dr. E. Mocarski ( Stanford University School of Medicine, San Francisco, USA) and was used at a low multiplicity of infection (MOI=<l PFU/cell). Virus was produced and stored as described (Greaves and Mocarski, 1998) and the titre (ID5o=10~8'6) was determined using the Reed-Muench formula (Reed and Muench, 1938). Dilutions of IgG were pre-incubated with the appropriate dilution of R C 2924 for 30 min at 37°C before adding the entire mixture to a monolayer of HFF cells. Levels of H C M V infection were assessed by measuring cells that express GFP by FACS analysis 2-7 days later. GFP was analyzed using a FACScan (BD Biosciences) and the data were analyzed using CellQuest software. 19 Immunofluorescence Human hepatoma (HepG2) and human foreskin fibroblast (HFF) cells were grown to 50-75% confluence in D M E M with 10% FCS in chamber slides (Lab-Tek II, Nalge Nunc International). Cells were treated with 4% paraformaldehyde and stored for a maximum of 4 days in PBS at 4°C. Binding to fixed and permeabilized cells was investigated by allowing dilutions of human IgGl in PBS containing 4% FCS and 0.2% Triton-X-100 (IF buffer) to react with cells on microscope slides for 1 hour at room temperature followed by washing with IF buffer. Bound IgGl was detected using anti-human IgG Alexa 594 conjugate (Molecular Probes). Nuclei were stained with DAPI (0.2 ug/ml). Images were obtained and analyzed as described (Noseda et al, 2004). 20 2.3 Results 2.3.1 Relative antigen binding of germ-line-based primary antibodies and somatically hypermutated antibodies against AD-2 epitope of HCMV. Analysis of the anti-AD-2Sl high affinity somatically mutated IgGs (KE5 and 8F9) identified the germline gene elements used in their primary ancestors. Although the same V H , JH, and V K gene segments are used, junctional differences contributed by DH, JK and random N-nucleotide addition also appeared to be involved in coding for the antigen binding specificity. McLean et al. (2006) cloned both SHM antibodies and their ancestral primary ancestors and expressed them as IgGl (See Table 2). ELISA and FACS analysis showed that both KE5 and 8F9 bind AD-2 and gB-NT with high affinity. The primary immunoglobulin composed of the combination of the M l heavy chain and the J5 light chain (M1-J5) also binds relatively well. However, the ancestor of 8F9, M2-J1, bound less well. Comparisons of the sequences of these two germ-line antibodies, suggested that a bulky tryptophan encoded by the JIK appears to play a large role in the ineffective binding. Replacing the tryptophan with hydrophobic residues with smaller side chains greatly improve binding to AD-2 peptide (McLean et al, 2006). Table 2: High affinity SHM antibodies and their germline ancestors. Somatic hypermutated antibody gL Heavy Chain gL K KE5 M l J5 8F9 M2 Jl The two high affinity SHM antibodies KE5 and 8F9 were sequenced and the germ-line components of their heavy and light chains were identified (Table 1). It can be seen that KE5 is a combination of somatically mutated M l and J5 chains and 8F9 a combination of mutated M2 and Jl chains. 21 2.3.2 Virus Neutralization by Human IgG The hypermutated 8F9 IgGl has been previously demonstrated to neutralize HCMV (McLean et al, 2005). However, no neutralization assay had been performed on M1-J5. Therefore, having demonstrated the relatively high affinity of AD-2/gB binding of M1-J5, we tested its ability to neutralize HCMV. The neutralization assay was performed by incubating constant amounts of infectious virus incubated with serially diluted antibodies prior to addition of the mixture to susceptible cells. Neutralizing titres are defined as the dilution that reduces infectivity in cell cultures by at least 50%. We used a recombinant Towne strain of HCMV that expresses a fusion protein of GFP and an immediate-early viral protein (ie2-GFP), enabling the detection and enumeration of infected cells by flow cytometry. The hypermutated 8F9 efficiently neutralized HCMV, however, M1-J5 and M2-J1 did not neutralize HCMV infectivity. The fact that the M1-J5 and M2-J1 germ-line antibodies did not neutralize viral infectivity at the doses used does not exclude the likelihood that they bound HCMV with sufficient affinities to induce B cell activation and trigger their development into hypermutated antibodies that neutralize at lower concentration. Although M1-J5 IgGl failed to neutralize HCMV infection in this assay, these results do not exclude the possibility that it can function in neutralization as in multi-valent form as an IgM antibodies. Also, it does not exclude the likelihood that the affinity of Mi-J5 for gB was sufficient to initiate BCR activation, expansion, and affinity maturation that lead to its high affinity progeny KE5. 22 Figure 2.1: Human CMV antibodies in HCMV neutralization assay. no virus 1:100000 1:50000 1:25000 <§ 800' 1 8 600-•p 400" 1 200-/ 0.1% // / 4.2% M 711.8% 1 10 100 1 10 100 1 10 100 1 10 100 GFP expression a) H C M V titre on HFF cells without neutralizing antibodies. Twenty thousand cells/well HFF cells were seeded in 24 well TC plate. A titration of virus stock was made and the G F P + cells were measured by flow cytometry 5 days later. Increasing virus concentration results in increasing virus mediated GFP expression in the HFF cells (gated population). It is interesting to note that with high virus concentration (1:25000), a third population appears showing higher GFP expression. This might indicate higher degree of viral infection in these HFF cells. M2-J1 M2-J5 8F9 0 J . T 0 1 T P T T T 1 (B) 10 IgGl (ng/ml) 100 b) Neutralization of H C M V by somatically mutated but not primary human Igs. 8F9 IgGl (closed circle), M2-J5 IgGl (open square), and M2-J1 IgGl (open triangle). 23 c) Virus Neutralization by anti-HCMV antibodies (5 (ig/ml). Incubation of 8F9 IgGs prior to viral infection showed significantly low level of virus-associated GFP expression by the HFF cells. However, incubation of the M2-J5 and M2-J1 IgGs showed similar GFP level as cells without any antibody added. Magnification 40X. (Bar = 50 urn) 2.3.3 Autoreactivity of primary lg We next proceeded to determine whether or not the primary ancestors of 8F9 and KE5 were autoreactive and polyreactive to self and foreign antigens. We performed a standard test for autoreactive anti-nuclear Abs (ANA) that bound to HepG2 cells in an immunofluorescence assay. This revealed that while S H M 8F9 did not react with antigens presented in the nucleus of HepG2, its ancester, M2-J1 bound strongly to nuclear antigens. To test whether or not this binding was specific to HepG2, we performed similar immunofluorescence experiments on human fibroblasts (HFF), mouse fibroblasts (NIH 3T3), and human embryonic kidney ( H E K 293) cells. In all cases, the anti-nuclear activity of M2-J1 was observed (Figure 2.2 A) . Because this same pattern of A N A immnuofluorescence was observed in multiple cell types of murine and human origin, it suggests that the antigen involved is phylogenetically conserved. 24 Figure 2.2: Primary human IgGl bind to the nucleus of HepG2 and HFF cells; dependence on L-chains. (A) Indirect immunofluorescence of fixed and permeablized HepG2 cells stained with 10 mg/ml of 8F9, M2-J1 or M2-J5 and detected wit 8F9 ary antibody anti-human IgG-alexa 594. DAPI staining was used to visualize cell nuclei. Magnification 40X. (B) Increase magnification (100X) and photomerging of Dapi-stained HepG2 nuclei showing the speckled and nucleolar-excluded pattern of staining with M2-J5. (Bar = 20 urn). McLean, Cho & Schroder, Molecular Immunology, 2006. 25 (D) L-chain J K Sequence A N A 8F9 V T F G Q G T K L E I K no J l W T F G Q G T K V E I K yes W96V V T F G Q G T K V E I K yes (C) Indirect immunofluorescence staining of HFF cells with 10 mg/ml of 8F9, 8-J1, 8-W96V, 8-J5, M2-8F, M2-J1, M2-W96V or M2-J5. Staining with Dapi was used to visualize cell nuclei and with anti-human IgG-alexa 594 to detect human IgGl . Magnification 40X. (Bar = 50 um). (D) Comparison of JK sequences of L-chains with amino acid differences shown in bold. Summary of binding to nuclei is also shown ( A N A = anti-nuclear antibody). Note that the J l L-chain displays nuclear binding only when paired with primary, unmutated H-chains. McLean, Cho & Schroder, Molecular Immunology, 2006. 26 Thus somatically mutated 8F9 IgG shows no reactivity to HepG2 and other cell lines, but its ancestor M2-J1 demonstrates anti-nuclear binding. A chimera of the mutated 8F9 H-chain with the germ-line unmutated L-chains of its ancestor M2-J1 did not show ANA activity. However, a chimera with the light chain of M1-J5 or the M2-J1 L-chain in which tryptophan 96 had been replaced with a valine (W96V) showed nuclear staining pattern. Pairing of the germline H-chain of M2-J1 with the mutated L-chain of 8F9 failed to show nuclear reactivity. However, chimeric IgG in which the germline H-chain of M2-J1 was paired with the other L-chain (Jlk, J5k and W96V) clearly reacted with HepG2 nuclei. The results suggest an important contribution of the germ-line Ji light chain to autoreactivity. 2.3.4 Human primary Igs display fine specificity for the AD-2S1 epitope from H C M V . One explanation for the autoreactivity we observed was that the relevant lg combining sites were highly promiscuous or "polyreactive." Indeed there is evidence that the majority of primary human Igs expressed by early immature B cells exhibit polyreactivity (Wardemann et al, 2003). This ability to bind multiple antigens is thought to reflect the flexibility of their combining sites (Notkins, 2004). However, our anti-HCMV antibodies were able to discriminate between closely related antigens. A core sequence of the AD-2S1 epitope NTTL(K/R), is highly conserved in CMV gB from human (HCMV), chimpanzee (CCMV), baboon (BaCv) and rhesus monkeys (RhCMV) (Figure 2.3 A). These peptides were synthesized and we tested the ability of the human hypermutated Ab (8F9), its putative primary lg ancestor, or related predicted (combination of different heavy and light chains) primary Igs to bind them in ELISA experiments. We consistently observed weak binding to the CCMV peptide with high concentrations of 8F9. However, with that exception, the hypermutated 8F9 and its precursor Igs, as well as chimeras of primary and somatically mutated H- and L-chains, failed to bind to the homologs of AD:2S1 in CCMV, BaCMV or RhCMV gB, although they clearly bound to HCMV Ad-2S 1 (Figure 2.3 B). 27 Figure 2.3: AD-2S1 peptides and human IgGl binding by ELISA. Human ETIYNTTLKY Chimpanzee EYIFNTTLRP Baboon ALIENTTLRT (A) Rhesus Monkey PLIENTTLRT (A) Alignments of the homologous AD-2 SI regions of C M V gB proteins from human, chimpanzee, baboon and rhesus monkey. The core consensus sequence is shown in bold. (B) Human IgGl consisting of 8F9 H-chains (open symbols) or primary H-chains (closed symbols) and 8F9 L-chains (circles), W96V L-chains (squares), J l L-chains (up triangles), J5 L-chains (down triangles) binding to AD-2 SI peptides by ELISA. 28 2.4 Discussion & Conclusion Although cellular immunity may be responsible for recovery from H C M V infection, humoral immunity is likely to be more important in protection against primary infection (Speckner et al, 1999). It has been demonstrated that antibodies can limit virus spread and confer protection from a lethal dose. The primary antibody repertoire consists of germ-line-encoded antibodies that have not undergone antigen specific affinity maturation; it is likely that many of these antibodies are poly-reactive and bind weakly to multiple antigens. During the development of B cells in the bone marrow, checkpoints exist to eliminate many autoreactive B cells. In immature B cells, strong BCR-mediated signals leads to cell death while in mature B cells they lead to activation and clonal expansion. However, for a B C R signaling-dependent checkpoint in the immature B cell pool to result in elimination of autoreactive B cells, the self-antigens must be presented during the selection and development processes. For example self-antigens that are intra-cellular may not be effectively presented to B cells. Autoreactive B cells that survive negative selection do occur and do not necessarily cause autoimmune diseases. Our evidence is consistent with the idea that although autoreactive primary antibodies may react with intracellular self-antigens, they may also be able to bind foreign antigens with sufficient affinity to initiate B cell activation and affinity maturation. Our study shows that one of the putative ancestral primary Igs of these two high-affinity anti-viral antibodies, but not the other, recognized autoantigens in addition to recognizing AD-2S1. In contrast, as expected, the hypermutated derivatives did not demonstrate autoreactivity. If the self-reactive primary lg was truly ancestral to the mutated higher affinity anti-viral antibody, these findings indicate that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation. It is noteworthy that only minor structural changes in primary Igs were sufficient to generate or abolish reactivity to autoantigens. Moreover, ELISA analysis revealed that these primary Igs fail to bind AD-2 epitopes of other closely related primate species. Thus they showed specificity rather than polyreactivity (Figure 2.3). These findings suggest that the poly-reactivity of primary fgs do not reflect a generic stickiness for proteins, but results from 29 the multiplicative effect of many weak but specific interactions. Our data also show that the very small differences (single amino acid changes) in the L-chain could destroy autoreactivity in germ-line lg . In summary, we have demonstrated that the human germ-line repertoire encodes the capacity to generate primary Igs that bind a key site on H C M V . One of these primary Igs that bind H C M V gB but not the other bound to self-antigens. Our data are consistent with the idea that primary Igs have flexible combining sites and that minor changes in amino acid seqences can confer changes in binding specificities. Interestingly these two particular V genes are very frequently used in human antibodies, suggesting that they may be very versatile in binding to different antigens. Our demonstration that high affinity anti-HCMV that were derived from an autoreactive ancestor can dominate the humoral response to an important viral epitope, show that, even though mechanisms do exist to edit these or delete self-reactive B cells, not all autoreactive B cells are precluded from differentiation to hypermutated B2 lymphocytes. Thus autoreactivity to intracellular epitopes is not necessarily a barrier for development into follicular B cells that undergo antigen-driven affinity maturation. 30 E X P R E S S I N G A N T I - H C M V A N T I B O D I E S A S M E M B R A N E IgM's 3.1 Rationale The inability of M1-J5 to neutralize HCMV, although it exhibited binding to the gB-NT and AD-2S1 peptide at concentrations that are close to that of 8F9, may reflect the complexity of antibody-mediated neutralization of viral infectivity (Knossow et al., 2002). Moreover, it is possible that when originally expressed as a secreted decavalent IgM, with much greater avidity for the virion than the bivalent IgGl molecules, these primary lg may have had some ability to neutralize HCMV infectivity. In order to generate high-affinity antigen-specific antibodies, a responsive B cell clone must be activated by specific antigen and stimulated into clonal expansion and affinity maturation. Surface IgMs (slgM) are part of the antigen receptor expressed on B cells and a series of signaling events are necessary to drive the subsequent B cell responses that result in Ab secreting plasma cells. In the periphery, insufficient B cell signaling strength will lead to anergy while strong signaling leads to clonal expansion. To investigate the process in which germ-line antibodies trigger B cell activation, we constructed the membrane bound (slgM) version of germ-line or mutated antibodies against HCMV. We planned to study BCR activation upon viral antigenic stimulation by expressing various human IgMs with known relative antigen-binding strength as slgM in murine B cell lines, we could study the signal transduction threshold required for B cell activation and the initiation of a specific anti-viral antibody response. 31 3.2 Materials and Methods Cell Culture A20 (Kim et al, 1979), B J A B , J558L and Ramos cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 m M glutamine, 1 m M pyruvate, 50 u M p-ME, non-essential amino acids, penicillin and streptomycin. 293T and NSO cells were grown in D M E M (Invitrogen Gibco, Burlington, ON) supplemented with 10% heat-inactivated FBS, 2 m M glutamine, 1 m M pyruvate, non-essential amino acids, penicillian and streptomycin. RT-PCR amplification oflg Vregions R N A extraction of A20, B J A B , and J558L cell lines using TRIZOL reagent was according to manufacturer's guidelines (Invitrogen Gibco, Burlington, ON). The first strand c D N A synthesis of murine B cell R N A from uniform cell cultures using Superscriptll according to manufacturer's guidelines (Invitrogen Gibco, Burlington, ON). The PCR amplifications, the Igct and IgP sequences were amplified (35 cycles, 94°C, 30 seconds; 50°C, 30 seconds; 72°C, 1 minute) with Taq polymerase (Invitrogen) and 250uM of each dNTP (Invitrogen). (Iga sense p r i m e r , T A G C T A G C C A C C A T G C C A G G G G G T C ; anti-sense primer, T A C T C G A G T C A T G G C T T T T C C A G . Igp sense primer, T A G C T A G C C A C C A T G G C C A C A C T G G ; anti-sense primer, T A C T C G A G T C A T T C C T G G C C T G G ) . Transfection and selection of Human IgMexpressing A20 cells. Human K and u c D N A were cloned into p L C or pHC vectors (McLean et al). 25 x 106 A20 Parental cells were transfected with 10 ug of human K or human p or both using electroporation (Biorad). Electroporated A20 cells were cultured in 96-well tissue culture plates with 600 ug/ml puromycin (K) or 1.5 mg/ml geneticin (u) or both. Single cell colonies were expanded and tested for human K expression by screening supernatants in ELISA tests or surface human u M expression by FACS analysis. The electroporation conditions were as following for each cell-line: A20: 25million cells/600 pi, 400V, 975 pF J558L: 15 million cells/600 pi, 240V, 950 uF 32 NSO: 10 million cells/600 ul, 200V, 955 uF ELISA Previously described in Chapter two. Flow cytometry analysis Parental A20 cells and A20 cells stably expressing germline or somatic hypermutated human IgM on the cell surface were suspended at ~10 6/ml in PBS containing 0.5% FCS and the indicated biotinlyated anti-human IgM (Southern Biotechnology) for 45 min on ice. They were washed three times and then stained with Avidin-FITC conjugate (Pierce). Fluorescence was analyzed using a FACScan (BD Biosciences) and the data were analyzed using Flowjo software. Western Blot analysis One hundred milligrams of whole cell lysates determined by B C A method were loaded to 12.5% gel and separated on a 12.5% SDS-polyacrylamide gel under reducing conditions. Proteins were electroblotted onto nitrocellulose membranes (Bio-Rad, Richmond, CA) and the efficiency of protein transfer was determined by staining the blots with Ponceau S (Sigma, St. Louis, MO). Membranes were washed briefly in TBST to remove the Ponceau S stain, blocked in TBST containing 5% skimmed milk at 4°C overnight and incubated at room temperature for 1 h then incubate with goat anti-human IgM or goat-anti-human k polyclonal antibody (1:1000 dilution). Following three 20 min washes in TBS with 1% Tween20, the membranes were incubated for 1 h with HRP-conjugated rabbit-anti-goat IgG antibody (1: 8000). After washing in TBST, membranes were developed with the enhanced chemiluminescent (ECL) reagent (Amarsham Biosciences) as described by the manufacturer. Cross-linking Experiments Cells were washed in PBS and resuspended at 2 x l0 6 /m l in RPMI 1640 and 20 m M HEPES, pH 7.2. After 2-4 hours of serum starvation, cells were stimulated with 5 ug anti-human IgM unlabelled antibody (Jackson Laboratory) for 3 minutes and immediately centrifuged at 4°C and lysed in 0.5% NP40 in PBS. Cells stimulated with anti-mouse IgG served as a positive control of receptor cross-linking on A20 parental cells. In western blots of 33 cross-linked samples, membrane were probed with either anti-phospho ERK antibody or anti-phosphotyrosine 4G10 antibodies. Calcium Release Experiments Cells were washed twice with HBSS and resuspend cells in HBSS at 5 x lOVml. 2 ul of 1 mM Fluo-4 (detects Ca 2 + , Molecular probes) was added first, then 2 ul of 2 mM SNARF-1 (pH sensitive dye, Molecular probes) and the cells were incubated at 37°C for 25 minutes before washing twice with HBSS. Cells were resuspended to 1 x 106 /ml and kept at room temperature and away from light. Cells were stimulated with ct-mouse IgG, a-human IgM or ionomycin for 15 seconds before detecting Fluo-4/SNARF-l ratio by FACS analysis. Biotinlyated peptides Peptides were made by solid-phase synthesis (Phil Owen, The BRC, Vancouver, Canada). The 15mer biotinlyated peptide of HCMV gB that contains the AD-2S1 epitope from human and rhesus monkey (RhCMV) (Kravitz et al., 1997) were made. AD-2 Peptide Binding Experiment Biotinlyated human or rhesus AD-2 peptides were incubated with Avidin-FITC at equal molar concentrations for 30 minutes at 37oC. Cells were washed in cold PBS twice and peptide-avidin-FITC complexes were added and incubated for 30 minutes on ice. Cells were then washed twice in cold FACS buffer and fluorescence was detected by FACS analysis. B cell Activation by AD-2 peptides Six well plates were coated with avidin overnight at 4°C then blocked with 2% BSA. Human or rhesus AD-2 biotinlyated peptides were added and incubated at RT for 1 hour before 2 washes with PBS. Serum starved A20 parental or slgM expressing cells are then added to the plate, centrifuge at 800 rpm for 1 min, 3 min or 5 min. Plates were immediately placed on ice and cells were collected and lysed in 1% NP40 for phosphotyrosine western blot analysis (detection of phosphorylated protein by anti-phosphorylated tyrosine antibody, 4G10). 34 Immunofluorescence A20 cells and NSO cells were plated on poly-L-lysine coated glass slides and cells were fixed with 100% methanol. Binding to fixed cells was investigated by allowing dilutions of goat-anti-human IgM in PBS containing 4% FCS to react with cells on microscope slides for 1 hour at room temperature followed by washing with 4% FCS in PBS. Bound goat-anti-human IgM was detected using anti-goat IgG-alexa 594 conjugate (Molecular Probes). Nuclei were stained with DAPI (0.2 g/ml). Images were obtained and analyzed as described (Noseda et al, 2004). Lentiviral gene delivery Expression vectors Expression vectors (pLC-huCK, pHC-huCyl) used for the production of chlgGs were exactly the same as described previously (McLean et al, 2000). The lg V domains for the murine monoclonal antibody 18B7 (Casadevall et al, 1998) were used to construct mouse-human chlgGs; these sequences have been described previously (McLean et al, 2002). Cell Culture HEK 293T were cultured in DMEM media and supplemented with 10%o heat-inactivated fetal bovine serum (Invitrogen Gibco, Burlington, ON), 2 mM glutamine, 1 mM pyruvate, non-essential amino acids, penicillin and streptomycin. NIH 3T3 cells were cultured in the same media except 10%) bovine serum (Invitrogen Gibco, Burlington, ON) was substitute for fetal bovine serum. Production of Virus Transfection of 8F9 heavy chain and light chain carrying lentiviral constructs (pLenti) and packaging constructs into 293T packing cell line was by calcium chloride method. Plate 293T cells and transfect at ~80%> confluence. At time of transfection, add the following solutions sequentially: 2M CaCl 2, ddH20, and 2x HBS (1:4:5 ratio) to the mixture of lentiviral and packaging constructs and finally add the final mixture to the 293T cells. Virus is collected in the supernatant between 36-48 hour post-infection. Transfection efficiency was measured by GFP expression using flow cytometry. 35 Infection of STS Cells and Infection of Human B Cell Line Virus containing 293T supernatant was added to cells. GFP expressing cells were measured by flow cytometry 48 hours post-infection. Bone marrow infection and transplantation Harvested murine bone marrow cells were cultured in 10% stem cell factor, 1% IL-3 and 1% IL-6 for 4 hours at 37°C and 5% CO2. Bone marrow cells is then transferred to virus-coated tissue culture plates for 24 hours and washed in PBS before introduction in sub-lethally irradiated RAG 7" mice intravenously. Reconstitution of human Igs is monitored by collecting blood 8 weeks post-infection and checked for GFP positive cells by flow cytometry. 36 3.3 Results: 3.3.1 Iga and Igp expression in Murine B cell lines Antigen binding, of a functional BCR activates a cascade of signaling events to stimulate B cell responses, such as release of calcium, activation of various pathways and leading to gene expression. Antigen-induced BCR aggregation results in tyrosine phosphorylation of specific protein-signaling motifs called ITAMs present in the cytoplasmic regions of both Iga and Igp. The cytoplasmic portion of the lg molecules lack phosphorylation sites. ITAM phosphorylation initiates recruitment of cytoplasmic signaling proteins and formation of a stable signaling complex that functions to link Ag binding to B cell activation (Williams et al, 1994). A transmembrane sequence on the lg molecules is required. However, surface expression itself is not the only requirement for a functional BCR, as the BCR has to be able to translate the external signal into cellular response by activating signal transduction. The Igct/p heterodimer is also required for BCR surface expression (Matsuuchi et al, 1992). Murine B cell lines previously used in studying the function of BCR include A20 and J558L cells. A20 cells carry both Iga and IgP and express mouse IgG on the surface (Justement et al, 1989). J558L plasmacytoma cells express IgP only as well as endogenous X light chain. Our laboratory also has another plasmacytoma line, NSO, which was used to express the N-terminal fragment of gB (described in McLean et al., 2006). RT-PCR confirmed IgP expression in all 3 B cell lines, but not Iga (Figure 3.1). Even though RT-PCR was unable to confirm Iga expression in A20, J558L, and NSO cells, subsequent slgM expression and slgM-mediated signal transduction experiments demonstrated that Iga was expressed in A20 cell line. 37 Figure 3.1: RT-PCR of Iga and Igfi in murine B cell lines, a: RT-PCR with mouse Iga primers. B: RT-PCR with mouse IgB primers. Positive control: RT-PCR of mouse Iga and IgB constructs. The RT-PCR reveals that all 3 mouse B cell lines NSO, J558L and A20 contain endogenous IgB (Lane 2, 4, 6). However, Iga expressions were not confirmed. Positive NSO J558L A20 control a B a p a B a p Mil m Lane 1 2 3 4 5 6 7 8 -300 bp 3.3.2 Expression of Human Surface IgM on Murine Cell Line A series of constructs were made for the expression of the 8F9 as a transmembrane u chain of 8F9 or its germ-line ancestor H-chain variable regions u-chain. These were transfected into A20, J558L and NSO cells by electroporation, together with constructs for the expression of the 8F9 L-chains or of its germ-line ancestor J1K or J5K L-chain. The electroporation parameters are described in the materials and methods and the combination of K and |o, are summarized in Table 3. Table 3: Constructs for human germline and somatic hypermutated IgM K li J l IgM J l K J5 IgM J5K 8F9 IgM 8F9K 8F9^ I 38 J558L: Attempts were made to make human slgM in J558L by transfecting exogenous Iga, human p-chain and K-chain; while immunoblotting demonstrated p-chain and K-chain protein expression, surface human IgM was not detected by flow cytometry. We were not able to demonstrate expression of the Iga protein expression in the Iga deficient plasmacytoma J558L by immunoblotting. Thus although human p-chain and K-chain proteins were being synthesized intra-cellularly, J558L cells likely lack the components to express the proteins on the cell membrane. NSO: Human p-chain and K-chain in NSO were also detected by immunoblotting. However, once again, no surface human K and p were detected by FACS analysis since the plasmacytoma cell-line lacks Iga. Thus, as with J558L cells, surface expression of human K and p by electroporation was unsuccessful. A20: Human p. and K cDNAs were electroporated into Iga/IgP expressing A20 murine cells. After 2-3 weeks of puromycin and G418 selection, single cell colonies were expanded. Secretion of human K into culture supernatants was confirmed by ELISA. Expression of surface p-chain was confirmed by flow cytometry. Positive clones were identified, expanded further and characterized. Expression of human K and p chains was further confirmed by making whole cell lysates and subjecting them to SDS-PAGE and immunoblotting with anti-human lg antibodies. 39 Figure 3.2: Immunoblot Analysis of human K and ju expression in A20 drug-resistant clones. Western blot analysis confirmed the expression of H-chain or L-chain. Lane 1, 2, 3: human K expression in the clones transfected with human L-chain alone; lane 4, 8: human p chain clones transfected with H-chain alone; Lane 5, 6, 7: Clones transfected with both human H-chain and L-chain. The level of protein expression were not equivalent in all clones studied. Note that different ratios of human K and p protein levels are observed in different IgM clones (Lane 5, 6, 7). Human (I Human K 1) A20J1K 6)A20JlIgM 2) A20J5K 7)A20J5IgM 3) A20 8F9K 8)A20gLu 4) A20 8F9U. 9) A20 Parental 5) A20 8F9IgM 10) BJAB Immunoblotting of human-K and human p protein in A20 slgM clones revealed differences in their expression levels. While there is little variation among J1K, J5K and 8F9K human-K chain clones (Figure 3.2: lane 1, 2, 3), the differences were greater among the p clones (Lane 4, 8). The fact that staining with anti-p antibodies was more intense than staining for K chains could be due to differences in sensitivity of the different detection reagents and may not be a true reflection of expression levels between heavy and light chains. Immunofluorescence analysis also showed similar differences in the ratio of surface K and p expressed. 40 Flow cytometric analysis demonstrated that different drug-resistant clones showed variations in surface expression. The subsequent analysis and characterization was only performed in the clones that expressed the highest expression of human | i chains on the surface as determined by flow cytometry. Figure 3.3: Surface expression of human IgM on the Murine cell UneA20. FACS analysis of human u M surface expression on A20 surface IgM clones. A) J l IgM, B) J5 IgM,. C) 8F9 IgM. Each panel shows clones with varying surface IgM expression. Clones expressing IgM were selected and the ones with highest levels were expanded and used for experiments. Each color peak represents an A20 clone expressing human IgM. 41 3.3.3 Functional Analysis of Human Membrane IgM on A20 cells Once surface human IgM expression was confirmed, the next step was to determine whether it would transduce an activation signal. Surface IgM by itself has no phosphorylation sites and therefore signaling requires help from other molecules. There are the ITAM-containing Iga/p heterodimers which are bound to slgM and work in concert in B cell activation and BCR-mediated signal transduction. BCR aggregation activates several signal transduction pathways including tyrosine phosphorylation events, activation of MAP kinases and Ca 2 + release. We used anti-BCR antibodies to crosslink the human surface receptor in our initial approach to studying the ability of the human slgM to activate signal transduction. We monitored the response using phospho-tyrosine immunoblotting and a calcium release assay. While cross-linking of the endogenous mouse BCR with anti-mouse IgG and cross-linking of exogenous human slgM with anti-human-p induced tyrosine phosphorylation in A20 8F9 IgM cells as expected, there are some difference in phosphorylation pattern signaling cascades. However, the fact that the p cross-linking of the human p chain induced tyrosine phosphorylation suggested that the exogenous human BCR is functional in murine cells and the mouse Iga/IgP is capable of transducing activation signals from the p chain of human slgMs. BCR activation can also lead to Ca 2 + release through the activation of PLC-y leading to calcium release from intracellular stores. To test whether or not ligation of human slgM on A20 cells could induce calcium release, A20 parental and 8F9 slgM cells were treated with either anti-mouse IgG or anti-human p. While in A20 parental cells calcium release was only induced by anti-mouse IgG, in A20 8F9 IgM cells, calcium release was stimulated by both the anti-mouse IgG and the anti-human p antibodies (Figure 3.6). These results indicate likewise, that the mouse Iga/p is able to form a functional BCR complex with the human slgM molecules. 42 Figure 3.4: In vitro tyrosine phosphorylation in response to surface BCR aggregation. A20 cells were washed and serum-starved for 2-4 hours, B C R were aggregated by addition of either anti-mouse IgG or anti-human p (with amount indicated below) (n=4). Immunoblotting analysis with the anti-phosphotyrosine mAb, 4G10 was employed to monitor protein tyrosine phosphorylation. 6 7 8 9 10 A) A20 parental 1) Unstimulated 2) 15 pg a-mouse IgG 3) 25 pg a-mouse IgG 4) 50 pg a-mouse IgG 5) 25 pg of a-human u B) A20 8F9 IgM 6) Unstimulated 7) 15 pg a-human p 8) 25 pg a-human p 9) 50 pg a-human p 10) 25 pg of a-mouse IgG A) A20 parental cells (positive for endogenous surface mouse IgG). Stimulation by 25 pg of anti-human p antibodies (Lane 5) resulted in no increase in the baseline phosphorylation profile (Lane 1) since the parental cells lacks human slgM. A20 parental cells stimulated by anti-mouse IgGs have different phosphorylation profile (Lane 2, 3, 4). B) A20 8F9 IgM cells (positive for both surface mouse IgG and human IgM). Cells stimulated with different amount of anti-human p show upregulation of phosphorylation (lane 7, 8, 9) compared to unstimulated cells (Lane 6). There was reduced tyrosine phosphorylation in cells stimulated by anti-mouse IgG (lane 10). 43 Figure 3.5: Calcium release in response to surface BCR aggregation. Calcium release assay in A20 parental (mouse slgG positive cells) and A20 8F9 IgM cells. A baseline of cellular calcium level was first established (panel a and e). Crosslinking anti-mouse lg antibodies were added as positive controls (panel d and h). A20 parental cells released C a 2 + only after addition of anti-mouse IgG while anti-human \x antibodies induced no effect (panel b and c, respectively). However, in A20 8F9 IgM cells both mouse and human cross-linking antibodies stimulate C a 2 + release (panels f and g). A20 Parental a) i b) i c) . \ 2 t l p « n - n b l u n - t i m u L i h O 0 *> MP A30 p * r m L i l -ittMni.iii-i! h% i .i— i | 4 — . — . . . . . . d) w - X3> p M v n t a l < 4 i m u l A k i l b y j n h h u n u n I g M < 1 * u*» ITO -um-V -> tt If lO 150 3» l i t " , t .™ . 1.1 a » -A20 p a r m t i l s l r m u L i t r \ J K » « « m » n n < l u l l I » limrt'tlZMtwi' ) A20 8F9IgM e) 7 i w i \ .V * f * * 4 M r . m i - . t i m i i l j l . i l J n i < -• » W D -i* 111 i j ,—,—, \2P I t f * 4 M * » - t t n i u b M J bv j n t t n w i u w Iff.. . , . , , , 1 ' " —— 1 ' ' 1 I- I • ' • I Wl • . I 4 4 3.3.4. Chimeric Analysis To explore whether the human and mouse H- and L-chains could form chimeras, we investigated stable A 2 0 cells that expressed either human p or human K alone. In these clones, if surface expression of human p or K chains are detected that it implies pairing with endogenous mouse K or mouse y lg. We confirmed that we could detect both surface human K and p expression by flow cytometry (data not shown). Cross-linking and calcium release assays were repeated with these cells. As shown in Figure 3.6, cross-linking surface human lg p induced tyrosine phosphorylation and phospho-ERK in A 2 0 cells expressing two types of human p chains, gL p and 8 F 9 p. This confirms that the human p associated functionally with mouse Iga/p but also shows that human p chains could associate with the endogenous mouse K chain. The demonstration of human-mouse chimeric BCR did not affect the results of our pure human BCR signaling experiments as we used antibodies to human p to ligate these receptors. Whether these p chain will associate with mouse or human light chains would not affect our conclusion that p chains functionally associate with mouse Iga/p. However, pairing of mouse light chain with human p chains and vice versa would complicate analysis of antigen binding. Several attempts were made to estimate the relative expression of mouse and human lg proteins or their surface expression. Immunoblotting of the human U/K and mouse IgG/K level from A 2 0 parental and human lg expression clones was inconclusive since different primary and secondary antibodies were used. Similar considerations also apply to attempts to compare levels of mouse and human sig or chimeric sig in cells by flow cytometry. 45 Figure 3.6: In vitro tyrosine phosphorylation and ERK activation in response to chimeric surface BCR aggregation. Treatment A: unstimulated, B: stimulated with 10 pg of anti-mouse IgG, C: stimulated with 10 pg of a-human p. In 8F9 IgM clone, treatment B and C yielded expected tyrosine phosphorylation and p-ERK upregulation as the result of surface receptor aggregation. However, in both p chain only clones, gL p and 8F9 p, similar responses were observed. This suggests that introduction of p chains, only results pairing with endogenous A20 mouse light chains to form surface receptors that are able to transduce signals downstream. The human slgM positive cell, BJAB, served as positive control. 3.3.5 Antigen-specific Interaction of Human I g M expressing Murine Cells. Having shown that exogenous human Igs form functional BCR in A20 cells, we next investigated whether they were able to interact with the specific antigen, AD-2S1. In order to detect the interaction of anti-HCMV IgM and AD-2S1, AD-2S1 was biotinlyated and incubated with avidin conjugated with FITC. The peptide-avidin-FITC complex was then added to A20 clones and incubated on ice for 30 minutes. After thorough washing, FITC levels on the cell were assayed by flow cytometry. 46 Binding of AD-2 peptide to A20 clones expressing either gL K, gL p, 8F9K or 8F9 p were tested. As expected, cells expressing J1K appeared to have the weakest binding, with those expressing J5K binding slightly more and 8F9Kbest with AD-2S1 binding. Of cells expressing just the human p chains, those expressing gL p showed some binding to AD-2S1 peptide but not as much as those expressing 8F9 p (Figure 3.7). These experiments suggest that expressing either the H-chain or L-chain of the human antibodies wassufficient for detecting binding of the AD-2-avidin complex. Figure 3.7: Antigen Specificity of A20 clones binding to AD-2 peptides. Biotinylated human AD-2S1 peptide and rhesus AD-2S1 peptides were incubated with avidin-FITC and applied to the individual clones of cells expressing human lg chains. A l l clones appeared to weakly but selectively bind human AD-2S1 peptide but not rhesus AD-2S1. 47 The mutated and the germ-line-based antibodies have higher affinity to a fragment of gB expressed on cells than to the short AD-2 peptide. This is because the epitope is probably folded (McLean et al, 2005). Therefore, we purified the membranes of NSO cells expressing gB-NT using sucrose gradients. The membrane fraction was dialyzed in PBS before adding it to the various clones of A20 cells. We then used immunoblotting to look for evidence that the gB fragment had induced cross-linking of BCR's. As judged by phosphorylation of ERK, neither membrane preparation stimulated the BCR when added to the solution. In contrast, the positive control cells stimulated with cross-linking antibodies in solution shown increased ERK expression. Soluble antigen does not effectively stimulate BCR signal transduction, and in nature gB is arrayed on the virion as a multimer fashion. We exploited the biotinlyated AD-2S1 peptides to array the AD-2S1 epitope on TC plates coated with avidin. Cells were serum starved in the same fashion as in cross-linking assay and then applied to the AD-2S1 peptide coated surface for 5 minutes. Contacts with the peptides were ensured by centrifiiging cells onto the plastic. The cells were then lysed on ice. Immunoblotting with 4G10 revealed no differences between the effects of contact with human AD-2S1 and the negative control, rhesus AD-2S1 which the antibodies have very low affinity for at all three times (Figure 3.9). The ability of these cells to be activated was confirmed by cross-linking human p chains in solution. Thus, although we could detect AD-2 specific binding, presentation of short peptides did not seem to effectively crosslink surface receptor and transduce signals downstream. The difficulty in demonstrating antigen-specific BCR activation could be due to insufficient expression of human slgM, for example due to competition for the human K chain by mouse y chain and of the human p chain by mouse K chain. To increase expression of the human chains, I tried increasing the concentration of the selective drug and double sorting human K and human p high expressing A20 cells. However, no significant improvements in expression were observed. Another approach to expressing more human IgM on the surface would be to utilizing siRNA to knockdown endogenous mouse lg expression; however, this approach was beyond the scope of this thesis. 48 Figure 3.8: Antigen-specific Interaction of Human IgM expressing Murine Cells. Stimulation of 8F9 IgM cells by membrane preparation of NSO parental and NSO-NT-gB cells. Stimulation by crude membrane extract of NSO parental cells (Lane 2, 3, 4) and NSO-NT-gB (Lane 5 6, 7) resulted in similar phosphorylation profile. Increased phosphorylation of E R K was observed only where cells were stimulated by crossing human p chain or mouse IgG (Lane 2 to 9). Show on the left ware immunoblots made with the anti-phospho-tyrosine antibody 4G10. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 A ) 4G10 B ) p-ERK 1) Unstimulated 6) 50 pg of NSO-gBNT 2) 25ug of NSO 7) 100 pg of NSO-gBNT 3) 50 Mg of NSO 8) 15 jig of a-human u 4) 100 ug of NSO 9) 15 pg of a-mouse IgG 5) 25 ug of NSO-gBNT 49 Figure 3.9: Human a-HCMV IgM stimulation by human AD-2S1 coated on a surface. A20 8F9 IgM cells were stimulated with two peptides coated on a tissue culture plate, the human AD-2 peptide from H C M V that bind 8F9 IgMs and the ortholog peptides from rhesus C M V that does not. There were no differences in tyrosine phosphorylation between unstimulated cells (Lane 1) and cells stimulated with the two different AD-2 peptides (Lane 4 to 9). Lane 2 and 3 are positive and negative controls (respectively) with reactive or non-reactive cross-linking antibodies presented in solution. 98 64 50 36 22 MW KDa 1 2 8 9 1 min 3 min 1) Unstimulated 2) 15 ug a-human u 3) 15 ug a-mouse u 4) Human AD-2 B (1 min) 5) Rhesus AD-2 B (1 min) 5 min 6) Human AD-2 B (3 min) 7) Rhesus AD-2 B (3 min) 8) Human AD-2 B (5 min) 9) Rhesus AD-2 B (5 min) 50 3.3.6 In vivo Human IgMs Expression Experiments were initiated towards an in vivo system for expressing human IgM in murine B cells. My goal was to reconstitute B cells in Rag"'" deficient mice with human slgM. RAG1 deficiencies in these mice results in an inability to generate heavy or light chain genes. As a result, these mice lack B adaptive immune responses and are prone to bacterial and viral infections. By introducing rearranged lg molecules in stem cells or pro-B cells, B cell development should continue and lead to development of B cells that express human slgM. First human p and K cDNAs were cloned into the lentiviral vector P114.0. Then attempts were made to produce virus in 293T packaging cell line. Lentiviral constructs were transfected into packaging cell-line 293T by a calcium chloride method. The P114.0 lentiviral constructs is a third generation viral vector that requires co-transfection of additional packaging plasmids into 293T cells. A 95% transfection efficiency was observed by expression of GFP encoded by the construct. Expression of human K and human p chain proteins was confirmed by immunoblotting. Virus titres were also examined by infecting a mouse fibroblast cell line, NIH 3T3. Human K and p were also expressed in this cell line, demonstrating the infectivity of the virus and its ability to transduces expression of the human lg chains. The infectivity of the lentivirus was also tested on a human B cell line, DG75 (Figure 3.11). Although initially these B cells were only faintly GFP positive, 25% of the cells were infected, with the cells becoming more GFP positive with time. The A20 murine B cell line was also infected, but the infection was less than 2%. 51 Figure 3.10: Human K and ju expression in 293 T by lentiviral gene delivery and in NIH 3T3 by viral infection. A) Packaging cell line 293T effectively incorporated packaging constructs and viral constructs carrying human K and p chains at high efficiency. Cells were washed and lysed and blotted with anti-human K and p antibodies. B) Virus produced by packaging 293 T cells were collected in the media and were used to infect NIH 3T3 cells to test infectivity and the ability of viruses to transducer expression of human K and p chains. (a) 1 2 3 4 5 M W (b) 1 2 3 4 K D a 98 64 50 36 24 A . 293T B . 3T3 1) PI14.0 vector alone 1) P114.0 vector alone 2) P114.0 8F9K 2)Pll4.0 8F9p 3) P114.0 8F9 p 3) P114.0 8F9 IgM 4) P114.0 8P9 IgM 4) BJAB (positive control) 5) BJAB (positive control) 52 Figure 3.11: Viral infection of human DG75 B cell line. P114.0 lentiviral constructs produces active virus as demonstrated by GFP expression. P114.0 vector alone, and lentiviral vector encoding 8F9K, 8F9 p and 8F9 IgM all appear to infect a human cell line, though in this experiment, the pll4.0 8F9K virus was significantly less efficient than the others. (Bar = 50 pm). No auto fluorescence of uninfected cells was observed. 53 Figure 3.12: Lentiviral infected RAG-/- bone marrow cells. Bone marrow cells extracted from RAG-/- mice were pooled and cultured in presence of virus coated tissue culture plates for 48 hours. Positively infected GFP expressing cells were screened by flow cytometry, a) uninfected, b) vector alone and c) 8F9 IgM infected cells. .0= •3: Uninfected a) 13' I — I F L 1 - H : G F P b) VA only 8 F 9 K and 8 F 9 u F L 1 - H : G F P 1 0 ' 1 0 s F L 1 - H : G F P 3.3.7 Bone marrow infection and B cell reconstitution After demonstrating the infectivity of these lentiviral vectors and their ability to transduce expression of H- and L-chains of the HCMV antibodies, bone marrow was infected. Stem cells from Rag"7" mice were used these to reconstitute B cells in vivo. Bone marrow cells of Rag"" mice were harvested and cultured with virus for 2 hours at 4°C. Virus-infected bone-marrow cells were then either kept in a 37°C incubator or transplanted back to RAG"" recipients by intravenous injection. Fourty-eight hours post-infection, the bone marrow cells in culture were analyzed by flow cytometry to evaluate infection efficiency by GFP expression. Based on the FACS profile, the virus infected the bone marrow cells efficiently. However, eight-week post-reconstitution, no GFP positive population was identified in the vector alone or 8F9 IgM co-transfected recipients. To confirm this finding, we also obtained the serum from the blood and tested for the presence of human K-chain by ELISA. However, the results were negative. 54 3.4 Discussion and Conclusion The objective of this project was to study the anti-viral B cell responses against a human pathogen by express H C M V antibodies as surface IgM molecule (slgM) in murine B cells as a model system. To this end, I first attempted to express the germ-line and S H M slgM in three murine B cell lines, A20, J558L and NSO. Only A20 cells demonstrated surface human lg expression, likely because it alone expressed the Iga/p heterodimer. In contrast, the other two cell lines only expressed IgP. We attempted to express exogenous Iga, but were unable to monitor Iga expression. Once we obtained A20 human slgM clones, we decided to focus our experiments on this cell line and characterize the s lgM functionality. Ig molecules do not have any phosphorylation sites to transduce signals into the cell, requiring the help of the ITAM-containing Iga/IgP to pass the signal to the downstream signaling molecules. Expressing human Ig in a mouse cell line expressing Iga/p may potentially fail to fully initiate or show partial activation of B cell activation due to lack of the correct interaction with Iga/p. We performed specific cross-linking of human p. chain to examine the process of tyrosine phosphorylations. We showed that tyrosine phosphorylation occurs in response to ligation of anti-human p. and that there is a difference in phosphorylation patterns when compared to the ligation of the endogenous mouse IgG. The mouse-human chimeric receptors were present, as we demonstrated by flow cytometry, should not contribute to the observed difference in phosphorylation as we believe that anti-p. would only ligate homodimers of u chains, and the nature of the light chains used should not affect signaling. As with the tyrosine-phosphorylation assays, in A20 parental cells Ca release was only induced by the anti-mouse IgG antibody. In contrast, in cells expressing human p chains, C a 2 + release was induced by both the anti-human u chain antibodies and the anti-mouse Ig antibodies. After we demonstrated that human s lgM could signal in murine cells, it was important to demonstrate whether the AD-2S1 peptide or gBNT could induce antigen-specific B cell activation. Several strategies were employed to stimulate the A20 clones. However, we were not able to show antigen-specific activation in any of the clones by either soluble complexes of AD-2S1 peptides or AD-2S1 peptides arrayed on plastic. Crude membrane preparation of NSO-gBNT also failed to activate B cells antigen-specifically. Even though 55 we did not demonstrate antigen-specific activation, we did observe antigen-specific interaction with AD-2S1. Thus I saw binding to A20 clones expressing a-AD-2 germ-line and the hypermutated 8F9 antibodies. The antigen specificity could be inferred by the fact that human AD-2 SI peptide did not bind to the A20 parental cells and (or to another B cell line), and that none of the A20 clones with human Ig chains bound the homologous rhesus AD-2S1 peptide. However, it was surprising that we observed binding to A20 cells expressing Jl IgM and J1K since the binding of an IgGl version of Jl IgM and Jl was very weak. Moreover, these results suggested that chimeric binding sites that had a contribution from either the heavy or the light chain for the human antibodies and the endogenous mouse chains were able to bind the peptide. It would be interesting to express these chimeric Ig as soluble proteins to examine in detail the interaction with AD-2S1. In our in vivo attempt to reconstitute Rag-l"/_ B cells with human slgM, we successfully expressed human 8F9K and 8F9 p in the packaging cell line, 293T and demonstrated viral infection in NIH 3T3 cells. While DG75 cells tend to settle down in tissue culture plates, A20 parental cells clump in suspension. Cell attachment was not the factor behind increasing infection efficiency of DG75 cells, cells were centrifuged during the viral infection, but no improvement was observed. The only attempt at bone marrow infection and transplantation did not result in reconstitution B cells in RAG_ /" mice. We speculate that consistent production of virus was achieved. However, concentration of virus using ultracentrifuge has not been optimized and may be needed to ensure high infectivity of the BM cells. While we did not successfully reconstitute the RAG_ /" mice with B cells expressing human IgM, some bone marrow cells were clearly successfully infected. Further optimization should improve the transduction efficiency and ensure equal ratios of heavy chain and light chain in the bone marrow cells. These in vivo experiments are beyond the scope and the devotion for a Masters degree. 56 4. CONCLUSION Our laboratory has studied antibodies against A D - 2 S 1 epitope o f glycoprotein B o f human cytomegalovirus. This project set out to explore the origin o f these high affinity antibodies. The evidence suggests that the human antibody response to the A D - 2 S 1 epitope is dominated by a family o f closely related somatically mutated antibodies that have common motifs and common usage o f germ-line elements. Our data showing that the primary ancestor o f one family o f these antibodies was auto-reactive, suggests that even though self-reactivity is generally eliminated during development, it is not an absolute barrier for immature B cells to further develop into high affinity somatically mutated B cells. We have made significant progress towards developing a system where human surface IgMs that are able to transduce signals can be expressed in murine cells. I showed that crosslinking human s l g M expressed i n a mouse cell line could trigger intracellular signals such as phosphorylation events and calcium release. Moreover, I have shown that these human a n t i - A D - 2 S l surface IgMs expressed on mouse cells bind the A D - 2 peptides specifically. I have also have successfully produced viruses that are able to transduce the expression o f human antibodies in both mouse and human cells. 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A P P E N D I X The animal work involved in this thesis was performed under Animal Care Certificate (Protocol number: A04-0390) 67 U B C THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A04-0390 Investigator or Course Director: John W. Schrader Department: Biomedical Research Centre (BRC) Animals: Mice C57BL/6 1200 j | Mice Rag-1 null 300 j Rabbits 24 j Mice M-Ras null 600 | Mice smg-GDS null 100 i Mice Caprin-1 null 600 ) Mice Balb/c600 ! I Mice G3BP-1 null 600 j Start Date: October 1,2001 Funding Sources: Approval Date: April 20,2007 Funding Agency: Funding Title: Canadian Institutes of Health Research (CIHR) Molecular regulation of hemopoiesis Unfunded title: N / A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the C C A C and some granting agencies. 1 of 2 6/21/07 3:09 P M 


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