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The role of the Rap GTPases in B-cell morphology, function, and malignancy Lin, Bin Liang Kevin 2009

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THE ROLE OF THE RAP GTPASES IN B-CELL MORPHOLOGY, FUNCTION, AND MALIGNANCY  by Bin Liang (Kevin) Lin B.Sc., University of British Columbia, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Microbiology & Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2009  ! Bin Liang (Kevin) Lin, 2009  Abstract B-lymphocytes rearrange their cytoskeleton and undergo dramatic morphological changes when searching for antigens and when forming immune synapses upon contacting cells that display antigens on their surface. Although these morphological changes are essential to B cell function, the signaling pathways underlying these processes are not fully understood. The aim of this thesis is to investigate how B cell receptor (BCR) and integrin signaling regulate B cell morphological changes. The Rap GTPases (Rap) are molecular switches that regulate integrin activation, adhesion, migration in B cells and other cell types. I hypothesize that activation of the Rap GTPases is important for regulating changes in B cell morphology. Indeed, in this thesis I showed that activation of Rap is essential for B cell cytoskeletal rearrangements. I found that Rap activation is important for BCR- and lymphocyte functionassociated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens leading to B cell activation. Consistent with Rap activation being important for B cell adhesion and migration, I showed that Rap activation is important for the dissemination of B cell lymphomas in vivo. B cell lymphomas are common malignancies in which transformed B cells enter the circulation, extravasate into tissues, and form tumors in multiple organs. Lymphoma cells are thought to exit the vasculature and enter tissues via the same chemokine- and adhesion moleculedependent mechanisms as normal B cells. Using A20 murine B lymphoma cells, I showed that Rap activation is important for circulating lymphoma cells to invade tissue and form tumors in vivo in syngeneic mice. Moreover, using in vitro models I showed that Rap activation is required for these cells to extend membrane processes between vascular endothelial cells and undergo transendothelial migration. Thus, by controlling B cell morphology and cytoskeletal organization, the Rap GTPases play a key role in both malignant and normal B cell functions, and may be a potential therapeutic target for treatment of B cell-related diseases.  ii  Table of contents Abstract.................................................................................................................................... ii! Table of contents .................................................................................................................... iii! List of tables............................................................................................................................ vi! List of figures......................................................................................................................... vii! List of abbreviations .............................................................................................................. ix! Acknowledgements ................................................................................................................ xi! Dedication .............................................................................................................................. xii! Co-authorship statement ..................................................................................................... xiii! 1.! Introduction...................................................................................................................... 1! 1.1! The immune system ..................................................................................................... 1! 1.2! B lymphocytes ............................................................................................................. 2! 1.2.1! Overview ............................................................................................................... 2! 1.2.2! B cell development ................................................................................................ 5! 1.2.3! B cell subsets......................................................................................................... 7! 1.3! B cell migration............................................................................................................ 9! 1.3.1! Overview ............................................................................................................... 9! 1.3.2! Chemokines and chemokine receptors................................................................ 11! 1.3.3! Chemokines and B cell migration patterns ......................................................... 13! 1.3.4! Sphingosine 1-phosphate and B cell egress from SLOs...................................... 15! 1.3.5! Migration of marginal zone and B-1 B cells....................................................... 15! 1.3.6! Selectins and leukocyte rolling ........................................................................... 16! 1.3.7! Integrins – an overview....................................................................................... 17! 1.3.8! Integrins and slow rolling ................................................................................... 20! 1.3.9! Integrins, adhesion, and outside-in signaling..................................................... 21! 1.3.10! Lymphocytes undergo amoeboid migration........................................................ 22! 1.3.11! Cell motility and cytoskeletal remodeling........................................................... 23! 1.3.12! Lymphocyte transendothelial and interstitial migration .................................... 25! 1.4! B cells and antigen encounters................................................................................... 27! 1.4.1! Lymph node and spleen architecture .................................................................. 27! 1.4.2! Activation of B cells by antigen .......................................................................... 28! 1.4.3! Soluble antigens .................................................................................................. 29! 1.4.4! Particulate antigens ............................................................................................ 30! 1.4.5! Membrane-bound antigens presented by macrophages ..................................... 30! 1.4.6! Membrane-bound antigens presented by dendritic cells .................................... 32! 1.4.7! Antigens in fibroblastic reticular cell conduits................................................... 33! 1.5! B cell activation ......................................................................................................... 34! 1.5.1! Overview ............................................................................................................. 34! 1.5.2! Kinetic segregation ............................................................................................. 36! iii  1.5.3! BCR microclusters .............................................................................................. 37! 1.5.4! B cell receptor signaling ..................................................................................... 38! 1.5.5! B cell spreading and contraction ........................................................................ 40! 1.5.6! B cell immune synapses ...................................................................................... 41! 1.6! Rap GTPases.............................................................................................................. 42! 1.6.1! Overview ............................................................................................................. 42! 1.6.2! Overview of Rap1 functions ................................................................................ 45! 1.6.3! The mechanism of integrin activation by Rap GTPases ..................................... 45! 1.6.4! Rap GTPases modulate cytoskeletal dynamics and polarity .............................. 47! 1.7! B cell lymphomas ...................................................................................................... 49! 1.7.1! Overview ............................................................................................................. 49! 1.7.2! Genetic causes of B cell malignancy .................................................................. 51! 1.7.3! B cell lymphoma development ............................................................................ 51! 1.7.4! B cell lymphoma dissemination .......................................................................... 53! 1.8! Rationale, objectives, and hypothesis ........................................................................ 54! 1.9! References.................................................................................................................. 56! 2.! The Rap GTPases regulate B cell morphology, immune synapse formation and signaling by particulate B cell receptor ligands ................................................................. 84! 2.1! Introduction................................................................................................................ 84! 2.2! Results........................................................................................................................ 86! 2.2.1! BCR- and LFA-1-induced B cell spreading ........................................................ 86! 2.2.2! BCR- and LFA-1-induced B cell spreading depends on Rap activation ............ 91! 2.2.3! Rap activation is required for immune synapse pSMAC formation ................... 98! 2.2.4! Particulate BCR ligands induce the formation of F-actin-rich cups via Rap activation.......................................................................................................... 100! 2.2.5! Rap activation is important for BCR signaling initiated by anti-Ig beads ....... 111! 2.3! Discussion ................................................................................................................ 118! 2.4! Experimental procedures ......................................................................................... 121! 2.4.1! Cells .................................................................................................................. 121! 2.4.2! Cell spreading ................................................................................................... 121! 2.4.3! Bead:cell conjugates ......................................................................................... 121! 2.4.4! Scanning EM ..................................................................................................... 122! 2.4.5! Confocal microscopy ........................................................................................ 122! 2.4.6! Rap activation ................................................................................................... 122! 2.4.7! Intracellular staining analysis of signaling ...................................................... 122! 2.4.8! Immune synapse (IS) formation on lipid bilayers ............................................. 123! 2.4.9! Statistics ............................................................................................................ 123! 2.5! Supplemental movies ............................................................................................... 123! 2.6! Supplemental procedures ......................................................................................... 124! 2.7! References................................................................................................................ 127! 3.! The Rap GTPases regulate the migration, invasiveness, and in vivo dissemination of B-cell lymphoma.................................................................................................................. 131! 3.1! Introduction.............................................................................................................. 131! 3.2! Results and discussion ............................................................................................. 132! 3.3! References................................................................................................................ 145! iv  4.! Concluding chapter...................................................................................................... 147! 4.1! Summary of main findings ...................................................................................... 147! 4.2! Discussion and future directions.............................................................................. 148! 4.2.1! Importance of B cell morphology in B cell function and development............. 149! 4.2.2! Modulation of B cell activation by the Rap GTPases ....................................... 153! 4.2.3! Novel effectors and pathways regulated by the Rap GTPases ......................... 156! 4.3! Closing remarks ....................................................................................................... 160! 4.4! References................................................................................................................ 162! 5.! Appendix A: Rap activation is important for optimal B cell activation by soluble and particulate antigen....................................................................................................... 167! 5.1! 5.2! 5.3! 5.4!  Rationale .................................................................................................................. 167! Experimental procedure ........................................................................................... 167! Results...................................................................................................................... 168! Conclusions.............................................................................................................. 170!  6.! Appendix B: Rap activation is required to sustain phosphorylation of myosin IIa heavy chain .......................................................................................................................... 171! 6.1! 6.2! 6.3! 6.4!  Rationale .................................................................................................................. 171! Experimental procedure ........................................................................................... 171! Results...................................................................................................................... 172! Conclusions.............................................................................................................. 172!  7.! Appendix C: Particulate antigens induce activation of cofilin ............................... 173! 7.1! 7.2! 7.3! 7.4!  Rationale .................................................................................................................. 173! Experimental procedure ........................................................................................... 173! Results...................................................................................................................... 174! Conclusions.............................................................................................................. 175!  8.! Appendix D: UBC Research Ethics Board's Certificates of Approval .................. 176!  v  List of tables Table 1.1! Integrins found on B cells.................................................................................... 18! Table 1.2! B cell lymphomas ................................................................................................ 50!  vi  List of figures Figure 1.1! The B cell antigen receptor (BCR)....................................................................... 3! Figure 1.2! Stages of B cell development and expression of key molecules .......................... 6! Figure 1.3! The leukocyte adhesion cascade......................................................................... 11! Figure 1.4! Structure of the prototypic integrin LFA-1 and models of integrin avidity regulation........................................................................................................... 19! Figure 1.5! Mechanism of antigen encounter for B cells in the lymph node........................ 29! Figure 1.6! The stages of B cell response to membrane-bound antigen ............................... 35! Figure 1.7! Summary of the Rap GTPase structure and signaling pathway ......................... 44! Figure 2.1! B cells undergo F-actin-dependent spreading on immobilized anti-Ig, anti-LFA1 or ICAM-1...................................................................................................... 88! Figure 2.2! A20 cell spreading can be induced by different anti-LFA-1 mAbs ................... 90! Figure 2.3! BCR- and LFA-1-induced B cell spreading is dependent on Rap activation..... 93! Figure 2.4!  Rap1N17 inhibits B cell spreading.................................................................... 95!  Figure 2.5! BCR- and LFA-1-induced B cell spreading and F-actin reorganization is dependent on Rap activation ............................................................................. 96! Figure 2.6! Plating cells on anti-Ig or anti-LFA-1 induces Rap1 activation......................... 97! Figure 2.7! Rap activation is important for IS formation...................................................... 99! Figure 2.8! B cells form F-actin-rich cups when they contact anti-Ig- or Ag-coated beads................................................................................................................ 101! Figure 2.9! BCR accumulation and BCR signaling at the site of bead:cell contact ........... 102! Figure 2.10! Beads coated with anti-CD40 mAb do not induce cup formation .................. 103! Figure 2.11! Anti-Ig- and anti-LFA-1-coated beads induce Rap1 activation; anti-LFA-1coated beads do not induce cup formation ...................................................... 104! Figure 2.12! Rap activation is important for formation of F-actin-rich cups at contact site with anti-Ig beads ............................................................................................ 107! Figure 2.13! Formation of bead:cell conjugates does not depend on Rap activation .......... 109! Figure 2.14! Rap activation and F-actin are important for anti-Ig beads to induce phosphorylation of ERK and Akt.................................................................... 113! Figure 2.15! Rap activation is not essential for anti-Ig beads to induce protein tyrosine phosphorylation or for soluble anti-Ig to induce phosphorylation of ERK or Akt................................................................................................................... 115! Figure 2.16! Akt phosphorylation induced by anti-Ig beads is dependent on F-actin ......... 117! vii  Figure 3.1! RapGAPII expression blocks CXCL12-induced activation of Rap1 and Rap2 in A20 cells.......................................................................................................... 133! Figure 3.2! Blocking Rap activation inhibits lymphoma development in vivo................... 136! Figure 3.3! Rap activation is important for A20 cells to undergo chemokine-induced migration and transendothelial invasion. ........................................................ 138! Figure 3.4! Blocking Rap activation impairs the ability of A20 B lymphoma cells to invade the liver............................................................................................................ 141! Figure 5.1! Optimal A20 B cell activation by anti-Ig and anti-Ig-coated beads require Rap activation ......................................................................................................... 168! Figure 6.1! Rap activation is required to sustain phosphorylation of myosin IIa heavy chain following stimulation with particulate antigen................................................ 172! Figure 7.1! Anti-IgG-coated beads induces activation of cofilin........................................ 174!  viii  List of abbreviations Antibody  Ab  Antigens  Ags  Antigen presenting cells  APCs  B cell receptor  BCR  Bone marrow dendritic cells  bmDCs  Central supramolecular activation cluster  cSMAC  Complement receptor 1 and 2  CR1/CD35; CR2/CD21  Dendritic cells  DCs  Diacylglyercol  DAG  Extracellular matrix  ECM  Fibroblastic reticular cells  FRCs  Follicular  FO  Follicular dendritic cells  FDCs  Follicular T helper  TFH  G-protein-coupled receptors  GPCRs  Germinal center  GC  GTPase activating proteins  GAPs  Guanine nucleotide exchange factors  GEFs  Guanosine diphosphoate  GDP  Guanosine triphosphate  GTP  High endothelial venules  HEV  Intercellular adhesion molecule-1, 2, 3, 5  ICAM-1, 2, 3, 5  Immune synapse  IS  Immunoglobulin  Ig  Immunoreceptor tyrosine-based activation motifs  ITAMs  Inositol (1,4,5)-trisphosphate  IP3  Interferon gamma  IFN!  Interleukin-  ILix  Leukocyte adhesion deficiency I  LAD-I  Lymphocyte function-associated antigen-1  LFA-1  Major histocompatibility class II  MHC II  Marginal zone  MZ  Mucosal associated lymphoid tissue  MALT  Multiple myeloma  MM  Non-Hodgkin’s lymphoma  NHL  P-selectin glycoprotein ligand 1  PSGL-1  Pathogen-associated molecular patterns  PAMPs  Peripheral supramolecular activation complex  pSMAC  Phosphoinositide-3-OH kinase  PI3K  Phosphatidylinositol-(3,4)-bisphosphate  PIP2  Phosphatidylinositol-(3,4,5)-trisphosphate  PIP3  Phospholipase C gamma  PLC!  Pattern recognition receptors  PRRs  Secondary lymphoid organs  SLOs  Scanning electron microscopy  SEM  Somatic hypermutation  SHM  Sphingosine 1-phosphate  S1P  Src homology 2  SH2  Subcapsular sinus  SCS  T cell receptor  TCR  T helper type 2  TH2  Toll-like receptors  TLR  Transendothelial migration  TEM  Transitional 1  T1  Tumor necrosis factor alpha  TNF-"  Vascular cell-adhesion molecule-1  VCAM-1  Very late antigen-4  VLA-4  Wiskott-Aldrich Syndrome protein  WASP  x  Acknowledgements I would like to offer my gratitude to everyone in my lab who has supported me and encouraged me, especially when things were difficult. Particularly, I would like to thank Dr. Michael Gold for being a fantastic and understanding supervisor, for his guidance, support, and for the opportunity to be a student in his lab. I thank Kathy Tse, Dr. Sarah McLeod, Caylib Durand, Victor Lei, Sonja Christian, Zinaida Tebaykina, Steve Machtaler, Caren Jang, Stephanie Mancini, Marcia Lynn Graves, and May Dang-Lawson for being fantastic colleagues. I thank Spencer Freeman for his many insightful scientific comments and contributions to my work, Hayley Brugger for her dedication and contributions, and Saba Zabetian for her wonderful SEMs. I would also like to acknowledge the summer students and directed studies students who have taught me to be a better mentor and teacher. I am also indebted to the LSI Imaging Facility and UBC Flow Cytometry for their technical support with experiments. I would like to especially thank Dr. Facundo Batista and Michele Weber for their collaboration on the Immunity paper, which would not have been possible without their contributions. I would like to thank Dr. Kelly McNagny and Poh Tan for their help with the B cell lymphoma project. I owe particular thanks to Poh Tan for spending countless hours with this project, even when it was 1 AM. I am also greatly indebted to my committee members Dr. Ninan Abraham, Dr. Calvin Roskelley, and Dr. Fumio Takei for their scientific guidance, support, and encouragement. Special thanks are owed to my parents, for their love, financial and moral support, encouragement, and understanding. Also, I would like to thank my brother Darwin Lin and my very good friends Mike Chow, Christine Chow, Carman Mak, Kelly Chan, and Philip Ly. Lastly, I apologize to those that I forgot to acknowledge for their support and contributions.  xi  Dedication  I dedicate this thesis to future generations of graduate students, to inspire them to strive for excellence while pursuing a challenging, but valuable and rewarding goal.  xii  Co-authorship statement My participation in the work presented in Chapter 2: •  I performed all experiments except for: o Figure 2.2, 2.6A: performed by Victor Lei o Figure 2.3B, 2.12C: SEM was performed by Saba Zabetian o Figure 2.4, 2.9C: performed by Spencer Freeman o Figure 2.7: performed by Michele Weber as a collaboration with Dr. Facundo Batista  •  Prepared manuscript with Dr. Michael Gold  My participation in the work presented in Chapter 3: •  I performed all experiments except for: o Figure 3.2A: assisted by Poh Tan as a collaboration with Dr. Kelly McNagny  •  Prepared manuscript with Dr. Michael Gold  My participation in the work presented in Appendices: •  I performed all experiments except for: o Figure 7.3B: Total cofilin staining and data analyzed by Victor Lei  xiii  1. Introduction 1.1 The immune system The human body is exposed to, and attacked by, infectious pathogens such as bacteria, viruses, fungi, and parasites. Consequently, humans have evolved mechanisms to resist these infectious agents. This includes barriers such as the skin, and mucosal linings, as well as chemical secretions. However, when these defenses are compromised by pathogens or by injury, such that pathogens can invade the tissues, the immune system is ready to respond and eliminate this challenge (Janeway et al., 2005). The immune system is divided into two branches: innate immunity and adaptive immunity. The innate immune system includes cells that respond immediately to an infectious insult and provide the first line of defense. In most cases, innate immunity can rid the body of pathogens. Cells of the innate immune system, which include neutrophils, monocytes, macrophages, mast cells, basophils, natural killer cells, and dendritic cells, express pattern recognition receptors (PRRs) that recognize danger signals. These germ-line encoded receptors recognize conserved patterns on pathogens called pathogen-associated molecular patterns or PAMPs (Akira et al., 2006; Janeway and Medzhitov, 2002; Medzhitov, 2007). Simultaneously, the adaptive immune system or acquired immune system is activated to assist with the clearance of pathogens and to generate immunological memory. Lymphocytes are cells that comprise the adaptive immune system. They are divided into two large families with many sub-types, named aptly based on their site of development: T (thymus-derived) and B (bone marrow or bursa of Fabricius) lymphocytes. Unlike the evolutionary older innate immune system, the adaptive immune system dates back to the first jawed vertebrates (gnathostomes), although there is increasing evidence that jawless fish such as the lamprey eels and hagfish possess cells that exhibit the characteristics of lymphocytes (Cooper and Alder, 2006). In addition to the hallmark of immunological memory, the adaptive immune system is distinguished from innate immunity by its vast repertoire of antigen-specific T cell receptors 1  (TCR) and B cell receptors (BCR). Unlike innate immunity, where broad classes of molecules are recognized by genetically-programmed receptors, adaptive immunity employs somatic diversification of its antigen receptor genes (Cooper and Alder, 2006). During development, lymphocytes randomly rearrange gene segments that encode for the antigen receptor. In B cells, these genes are the variable (VH), diversity (DH), and joining (JH) segments that make up the heavy chain, and VL and JL of the light chain that assemble together to form the BCR (Brack et al., 1978). In T cells, V" and J" segments are rearranged to make the alpha subunit while V#, D#, and J# segments are rearranged to make the beta subunit; together they form the TCR (Davis and Bjorkman, 1988). Since this process is random, and there are multiple variants of each gene segment, a vast number of antigen receptors can be generated (Rajewsky, 1996). Because this process is random, it can also lead to autoimmunity if self-reactive cells are generated and not eliminated (negative selection) or silenced (anergy). As vast as the adaptive immune repertoire is, each lymphocyte has only one antigen specificity. This is because during development, a successfully generated complete antigen receptor sends signals that turn off further gene rearrangement. After development, lymphocytes enter the circulation and scan for their cognate antigen, and upon antigen encounter, lymphocytes undergo clonal expansion and differentiation into effector cells (Section 1.5).  1.2 B lymphocytes 1.2.1  Overview  B cells produce antibodies that protect against invading pathogens in multiple ways. Firstly, antibodies can neutralize pathogens and their toxins by preventing them from binding cellular receptors and infecting host cells. Secondly, innate immune cells that express Fc receptors more readily phagocytose pathogens that are coated with antibodies. Lastly, antibodies can activate the complement cascade, which results in the formation of the membrane attack complex (MAC) and subsequent lysis of bacteria, as well as opsonization since innate immune cells also have complement receptors. B cells produce antibodies when the BCR binds their cognate antigen that its binding site is complementary to. The BCR (Figure 1.1) is a complex consisting of a membrane-bound  2  immunoglobulin (Ig) and two invariant polypeptides, Ig! (CD79a) and Ig# (CD79b). Binding of antigen by the BCR induces signaling that results in proliferation and subsequent differentiation into antibody-secreting cells and memory B cells (Section 1.5). In addition to activating B cells, BCR signaling plays a crucial role in the development of B cells from progenitor stem cells in the bone marrow (Section 1.5.4) (Niiro and Clark, 2002). For effective BCR signaling, activation of co-receptors and co-stimulatory complexes is required.  Figure 1.1  The B cell antigen receptor (BCR)  The BCR is comprised of four polypeptides: immunoglobulin heavy chains that are joined to immunoglobulin light chains by a disulfide bond and a disulfide-linked heterodimer of the two invariant polypeptides Ig" (CD79a) and Ig# (CD79b), which are the signaling components of the BCR that contain immunoreceptor tyrosine-based activation motifs (ITAMs).  3  Formation of a B cell immune synapse also lowers the threshold for B cell activation (Section 1.5.5). Much like the T cell immune synapse, the B cell immune synapse consists of antigen-bound BCRs gathered into a central supramolecular activation cluster (cSMAC) that is surrounded by a ring of integrins (Carrasco and Batista, 2006b; Carrasco et al., 2004). Formation of this ring of activated integrins, called the peripheral supramolecular activation complex (pSMAC), is important for activation of naïve B cells as it prolongs adhesion to antigen-presenting cells, allowing the B cell to accumulate more antigen, thereby reducing the signaling activation threshold (Carrasco and Batista, 2006b; Carrasco et al., 2004). Furthermore, most B cells require additional co-stimulatory help from T helper cells to produce antibodies (Alam and Gorska, 2003). Antigens that require help from T helper cells to produce maximal antibody responses are referred to as thymus-dependent (TD) antigen. There are also thymus-independent (TI) antigens, in particular bacterial carbohydrates, that crosslink the BCR and are able to generate antibody responses in the absence of costimulatory help from T cells. This type of antibody response is usually mediated by innatelike subsets of B cells and may require TLR signaling as a co-stimulatory signal (Section 1.2.3). For conventional circulating B cells however, T helper co-stimulatory signals are required and this is supplied through CD154 (CD40L) on the activated TH2 or follicular T helper (TFH) cell binding to CD40 on the B cell (Grewal and Flavell, 1998), as well as ICOS on these helper T cells binding to ICOS-L on the B cell (Fazilleau et al., 2009). Engagement of these receptors is required for Ig class switching to IgG, IgA and IgE. The CD40-CD40L interaction also enhances B cell proliferation, differentiation into both memory and plasma cells, and the survival of B cells. Furthermore, T helper cells also provide other cytokines that promote B cell activation and Ig class switching. These include, but are not limited to, interleukin-4, 5, 6, and 21 (IL-4, IL-5, IL-6, IL-21), and interferon gamma (IFN!). B cells elicit co-stimulatory help by presenting peptide-major histocompatibility class II (MHC II) complexes to T helper cells (Fazilleau et al., 2009; McHeyzer-Williams et al., 2006). B cells are antigen presenting cells (APCs) that process exogenous antigen for loading onto MHC II proteins (Chen and Jensen, 2008; Rodriguez-Pinto, 2005). Briefly, after binding of antigen by surface Ig, the antigen is internalized by receptor-mediated endocytosis into endosomes, which eventually fuse with lysosomes. The internalized antigen 4  is degraded into short peptides, loaded onto MHC II proteins, and subsequently displayed on the B cell surface to T helper cells. In addition to producing antibodies and presenting peptides to T cells, there are an increasing number of additional roles ascribed to B cells. Insights into these additional functions were gleaned from mouse models in which B cells are depleted by anti-IgM Abs or genetic mutations that prevent B cell development (LeBien and Tedder, 2008). In particular, B cells have been shown to be important for the development of the immune system and for its maintenance. For example, abolishment of B cells leads to significant decrease in thymocyte numbers, defects in lymphoid tissue organogenesis, and the absence of macrophages in the marginal zone (LeBien and Tedder, 2008). Furthermore, B cells may also have a regulatory role since a specific B cell subtype, called Bregs or B10 cells, appear to limit T-cell mediated inflammatory responses by producing IL-10 (Mizoguchi et al., 2002; Yanaba et al., 2008). 1.2.2  B cell development  The development of B cells from hematopoietic stem cells (HSC) takes place in the fetal liver of embryos and in the bone marrow of adults (Hardy et al., 2007). HSC give rise to lymphoid-primed multipotent progenitor (LMPP) that differentiates into a B cell precursor commonly referred to as the common lymphoid progenitor (CLP; B220—Mac-1—GR1— Ter119—IL-7Ra+ c-kit+) that eventually develops into a B cell (Northrup and Allman, 2008). B cell development is precisely regulated by adhesion molecules, chemokines, cytokines (e.g. IL-7), and transcription factors such as EBF (Early B-cell Factor), E2A, and Pax5 (Bartholdy and Matthias, 2004; Hardy and Hayakawa, 2001; Nutt and Kee, 2007). These pathways work in concert to positively and negatively regulate the expression of genes that determine B cell fate.  5  Figure 1.2  Stages of B cell development and expression of key molecules  The progression of different developmental stages from HSC (hematopoietic stem cell) to follicular (FO) B is outlined in the above. The pink box indicates cell surface expression and the green box indicates gene expression. The intensity of the black lines represents the amount of expression of that particular protein or gene. MLP/ELP: multilineage progenitor and early lineage progenitor; CLP: common lymphoid progenitor; T1: transitional 1 B cell or newly formed B cell; FO: follicular B cell; SLC: surrogate light chain; Rag1/2: recombination activating gene 1/2; TdT: terminal deoxynucleotidyl transferase (Adapted and modified from Hardy et al., 2007).  B cell development is a sequential process marked by different surface phenotypes at each stage of development (Figure 1.2). The first step involves CLP commitment to the B cell lineage. The resulting pre-pro-B cells express B220 (CD45R) and leukosialin (CD43) (Hardy et al., 1991). Pre-pro-B cells then develop into pro-B cells, which undergo Ig heavy chain rearrangement that is facilitated by expression of the genes RAG1 and RAG2, which are responsible for the rearrangement of the various gene segments in the immunoglobulin heavy ($ chain) and light chain (% and &) loci (Hardy et al., 2000). After successful Ig heavy chain rearrangement, the $ heavy chain associates with the VpreB and &5 surrogate light chains to form the pre-BCR. Expression of the pre-BCR characterizes the next stage of development, the pre-B cell (Hardy and Hayakawa, 2001). Signaling via the pre-BCR allows the pre-B cell 6  to undergo clonal expansion and Ig light chain rearrangement (Hardy and Hayakawa, 2001; Pillai, 1999). Successful expression of surface IgM, a complex of $ chain and Ig light chains, allows the B cell to undergo negative selection in which B cells that react strongly to self-antigens are either clonally deleted or become anergic, thereby establishing B cell tolerance (Ohashi and DeFranco, 2002). B cells (B220+CD19+IgM+) that survive negative selection then undergo a final step in development with the expression of another surface immunoglobulin, IgD. The IgD molecule shares the same antigen specificity as the IgM molecule on a given cell but has a different Ig heavy chain, the ' chain. 1.2.3  B cell subsets  Immature B cells that exit the bone marrow are referred to as transitional 1 (T1) B cells. Once the T1 B cells leave the bone marrow, they travel to the spleen to complete differentiation. Most of these cells will die in the spleen within a few days post-development if they do not encounter their cognate antigen. In the spleen, T1 B cells differentiate into one of three subsets of B cells: B-1, B-2 (follicular), or marginal zone (MZ) B cells (Allman and Pillai, 2008; Chung et al., 2003). The development of T1 B cells into these subsets is governed by B cell receptor signaling strength (possibly by engagement with self-antigens), by cytokines, and by Notch signaling, which together orchestrate the expression of transcription factors such as Aiolos and c-Myb (Casola, 2007). Follicular (FO) B cells are the most common and well-studied subset of the B cells. FO B cells are constantly produced postnatally from T1 B cells that emigrate from the bone marrow but they are short-lived. FO B cells are highly motile and circulate through the body via blood and lymph to lymphoid follicles of secondary lymphoid organs (SLOs), and to the vascular sinusoids of the bone marrow (Cariappa et al., 2005). These “conventional” B cells produce mainly TD antibody responses that culminate in the production of high-affinity antibodies due to somatic hypermutation (SHM), a process where V-region DNA sequences undergoes mutations that result in the generation of variant Ig with different affinity. Moreover, FO B cells can also undergo class-switch recombination (CSR) whereby activated FO B cells switch to secrete antibodies of a different class (isotype) such as IgG, IgA, and IgE. Class switching does not alter the specificity of the antibody but does alter the effector functions that an antibody can engage. Furthermore, FO B cells are the main source of 7  memory B cells that respond more quickly and more effectively upon secondary encounter with the same antigen. MZ B cells also develop from the same TI B cell progenitor as FO B cells. Their differentiation is driven by interactions between Notch2 on the progenitor cell and the Notch ligand Deltalike-1 on endothelial cells in the marginal zone (Tan et al., 2009). As their name implies, MZ B cells are found specifically in the marginal zone of the spleen, the interface between the red and white pulps, and are ideally situated to respond to blood-borne pathogens as blood from the follicular arteriole is released into the marginal sinus and percolates through the marginal zone (Pillai et al., 2005). Unlike FO B cells, MZ B cells do not recirculate throughout the body but are sessile and remain within the spleen. MZ B cells are considered innate-like because their antigen receptors, which are mainly IgM, have a limited but polyreactive repertoire that recognizes microbial polysaccharides as well as selfantigens (Viau and Zouali, 2005). MZ B cells also express high levels of Toll-like receptors (TLR) on their surfaces and engagement of these receptors induce MZ B cells to differentiate into short-lived plasmablasts (Allman and Pillai, 2008). MZ B cells also express high levels of the MHC I-like protein CD1d, which presents lipid antigens to NKT cells. They also express high levels of CD21, which allows MZ B cells to bind immune complexes decorated with the complement component iC3b (Pillai et al., 2005). Even though MZ B cells are sessile, engagement of immune complexes by CD21 increases MZ B cell motility, allowing the cell to shuttle antigen from the marginal sinus to splenic lymphoid follicles, where they transfer antigens to follicular dendritic cells (FDCs), which can then activate FO B cells (Cinamon et al., 2008; Meyer-Bahlburg et al., 2008). B-1 cells represent about 5% of all peripheral B cells and can be found in the peritoneum, pleural cavities, spleen, and intestines (Dorshkind and Montecino-Rodriguez, 2007). Like MZ B cells, B-1 cells have a limited Ig repertoire and are considered innate-like. They respond to TI antigens that are common on pathogenic bacteria, including carbohydrates and phosphorylcholine (Montecino-Rodriguez and Dorshkind, 2006). The B-1 cell compartment can be further divided into B-1a and B-1b cells, each with their own distinct functions (Haas et al., 2005). B-1a cells develop neonatally from progenitors in the fetal liver and are selfreplenishing postnatally. B-1a cells produce IgM “natural antibodies,” antibodies that are 8  found in normal individuals in the absence of apparent exogenous antigen stimulation. These natural antibodies confer immediate protection against encapsulated bacteria such as Streptococcus pneumoniae. Conversely, B-1b cells are believed to be produced from the same progenitors as FO and MZ B cells, and they only produce antibodies after antigen stimulation. Therefore, B-1b cells are thought to aid in the clearance of bacterial infections and confer long-term protection. In terms of mobility, B-1 cells are more motile than MZ B cells but less so than FO B cells. B-1 cells can home from the peritoneum to target organs such as the mesenteric lymph nodes, intestinal lamina propria, intestines, and parathymic lymph nodes (Allman and Pillai, 2008). In addition to these well-characterized subsets, a very rare subset of B cells termed “B10 regulatory B cells” (Bregs or B10 cells) has been recently identified (Yanaba et al., 2008). Unlike other B cells that are considered positive regulators of the immune response, these regulatory B cells suppress autoimmunity and inflammation (Mizoguchi and Bhan, 2006). In particular, B10 cells suppress contact hypersensitivity by producing IL-10 (Yanaba et al., 2008). IL-10 is a potent anti-inflammatory and immunosuppressive cytokine that inhibits TH1 cytokine production, prevents TH2 responses, suppresses inflammatory cytokine production by monocytes and macrophages, and suppresses antigen presentation by professional antigen presenting cells (Bouaziz et al., 2008). The origins and trafficking patterns of B10 cells are not known.  1.3 B cell migration 1.3.1  Overview  A recurring theme in B cell development, activation, and function is the requirement for cell motility. During development, before mature B cells leave the bone marrow, hematopoietic progenitors that give rise to B cells must undergo specific migration patterns within the bone marrow, sequentially visiting specific niches. Lymphoid progenitors migrate from the subendosteal region (inner bone surface) towards the central cavity of the bone, where they can interact with the endosteum, a thin layer of cells that line the medullary cavity (Nagasawa, 2006). Afterwards, immature B cells will migrate to the spleen to complete their differentiation. FO B cells, the most abundant subset, circulate throughout the body via the  9  blood and lymph, and pass through peripheral lymphoid organs for no more than a day before re-entering the circulation (Miyasaka and Tanaka, 2004; von Andrian and Mempel, 2003). This movement into and within different lymphoid compartments allows B cells to scan for antigens and increases the likelihood that an individual B cell will come into contact with its cognate antigen. Furthermore, secondary lymphoid organs act as collecting stations for activated APCs and for soluble antigens from the periphery. Thus, homing to the SLOs is essential for immune surveillance by FO B cells. Following recognition of antigen and activation in the periphery, naïve B cells will differentiate into memory cells and antibodyproducing plasmablasts. Some of these effector cells will then migrate back to the bone marrow and enter specific niches that support their long-term survival (Moser et al., 2006). MZ B cells and B-1 cells do not display the same extent of motility as FO B cells. Nonetheless, MZ B cells are still shuttle within the spleen, transporting antigen between the marginal sinus and follicles (Cinamon et al., 2008). B-1 cells home to gut-associated lymphoid tissues where they can rapidly respond to bacteria (Montecino-Rodriguez and Dorshkind, 2006). The coordinated movement of B cells into lymphoid tissues is a multi-step process that involves three distinct steps: tethering and rolling, firm adhesion, and transmigration, also known as extravasation or diapedesis (Figure 1.3). These steps are regulated by chemokines and adhesion molecules on the surfaces of endothelial cells of the post-capillary venules, specifically high endothelial venules (HEV) in lymph nodes and Peyer’s patches (Cyster, 1999; Miyasaka and Tanaka, 2004; Moser and Loetscher, 2001; von Andrian and Mempel, 2003). The first step, tethering and rolling is mediated by selectins expressed on the surfaces of lymphocytes and endothelial cells (Section 1.3.5). Because this interaction is weak and transient, lymphocytes “roll” along the surfaces of endothelial cells as bonds are broken and reformed. Mice deficient in selectins have greatly impaired lymphocyte homing, demonstrating the importance of selectins in this process (Arbones et al., 1994; Bullard et al., 1996).  10  Figure 1.3  The leukocyte adhesion cascade  Lymphocytes exit the circulation and home into peripheral lymphoid tissues via a series of concerted steps. Tethering and rolling is mediate by selectins, allowing B cells to sample bound chemokines. Chemokines activate integrins via inside-out signaling and mediate firm arrest. The lymphocytes will then crawl until a site of least resistance for extravasation is reached. Extravasation can occur via two routes: paracellular or transcellular. Key molecules that participate at each step are listed in the gray boxes. The figure is reproduced with permission from the Nature Publishing Group (Ley et al., 2007).  After the cell begins to roll, the next step involves firm adhesion of the lymphocyte on the endothelial cells. This process is mediated by integrins on the lymphocytes binding to their respective ligands on endothelial cells (Section 1.3.7). A prerequisite for this interaction involves the activation of integrins, which is mediated by the presence of chemokines that are immobilized on the surface of the endothelial cells. Chemokines also induce cytoskeletal rearrangements that promote lymphocyte crawling (Section 1.3.10) and extravasation (Section 1.3.12) (Thelen and Stein, 2008). 1.3.2  Chemokines and chemokine receptors  The migration of B cells is orchestrated by gradients of chemoattractants. Chemokines are short peptide chemoattractant cytokines (Cyster, 1999; Proudfoot, 2002; Thelen and Stein, 2008). They are approximately 70 – 90 amino acids in length (8 – 12 kDa) and contain one or more conserved cysteines at the amino-terminus. Based on this, chemokines are classified  11  into C, CC, CXC, or CXXXC subgroups. Even though they have low sequence homology, all chemokines show remarkably similar three-dimensional structures, especially at the amino-terminus. So far, over 50 different chemokines have been identified (Thelen and Stein, 2008). Chemokines can be classified as either “inflammatory” or “homeostatic.” Inflammatory chemokines mediate the migration of leukocytes and activated lymphocytes to sites of inflammation. This is especially important for the migration of innate immune cells such as neutrophils and monocytes, as well as effector T cells. Homeostatic chemokines mediate the normal development and trafficking of lymphocytes into and within primary and secondary lymphoid organs. Endothelial cells, as well as stromal cells of SLOs, produce homeostatic chemokines such as CCL19 (ELC) and CCL21 (SLC), which direct the migration of naïve lymphocytes into SLOs where they scan for antigen (Link et al., 2007; Luther et al., 2000). Chemokine receptors are seven transmembrane G protein-coupled receptors (GPCRs). Over 20 chemokine receptors have been discovered and many have overlapping binding specificities (Bromley et al., 2008). Chemokine receptors are differentially expressed depending on the type of cell, its maturation, and the cell’s activation state. How chemokines bind and activate chemokine receptors is not fully understood (Thelen and Stein, 2008). Chemokine molecules exist as both monomers and oligomers. Both configurations are equally adept at activating chemokine receptors. Chemokine receptors can also exist as monomers, dimers, or heteromers, and their ability to form aggregates can affect downstream signaling pathways. In addition to receptor aggregation, covalent modification of chemokine receptors (such as phosphorylation), as well as compartmentalization in membrane microdomains affects GPCR signaling (Thelen and Stein, 2008). Nonetheless, signaling by chemokine receptors results in integrin activation and cell migration towards a chemokine gradient. After the activation of chemokine receptors, the G"i and G!# subunits dissociate from the receptor and from each other to transduce signals. A major pathway that is activated by these proteins is the phosphoinositide-3-OH kinase (PI3K) pathway, which results in the generation of the phospholipid phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) from phosphatidylinositol-(3,4)-bisphosphate (PIP2) (Okkenhaug and Vanhaesebroeck, 2003a, b). 12  The activation of the PI3K pathway and the role of the phosphatase PTEN in generating an intracellular gradient of PIP3 are essential for cell polarization, actin polymerization, and cell migration (Iijima and Devreotes, 2002; Reif et al., 2004; Sasaki et al., 2004). Chemokine receptor signaling also results in activation of phospholipase C gamma (PLC!), which generates inositol (1,4,5)-trisphosphate (IP3) and diacylglyercol (DAG). IP3 and DAG are second messengers that activate Rho and Rap GTPases, which are involved in the establishment of polarity, induction of cytoskeleton remodeling, and integrin activation (Kinashi, 2005; Ley et al., 2007). 1.3.3  Chemokines and B cell migration patterns  Chemokines are important for many aspects of B cell function and development. The chemokine CXCL12 (SDF-1) and its receptor CXCR4 are crucial for B cell development in the bone marrow and in the fetal liver (Egawa et al., 2001). CXCR4 signaling mediates the migration of hematopoietic stem cells into the bone marrow (Ara et al., 2003; Ma et al., 1998; Ma et al., 1999; Nagasawa et al., 1996). It is also important for retaining developing B cells in the bone marrow and the loss of CXCR4 results in the exodus of pre-B cells into the blood (Ma et al., 1999; Nie et al., 2004), reflecting the requirement for CXCL12 to attract and/or tether precursor B cells to the appropriate bone marrow niches that foster B cell development in vivo (Nagasawa, 2006; Tokoyoda et al., 2004). Immature T1 B cells express the chemokine receptor CXCR5, which facilitates their homing to the spleen in response to the chemokine CXCL13 (BLC), allowing them to complete their differentiation (Bowman et al., 2000). Most T1 B cells will differentiate into FO B cells and this is accompanied by increased expression of the CXCR5 and CCR7 receptors, which allow FO B cells to recirculate throughout the body guided by the homeostatic chemokines such as CXCL12, CCL19, and CCL21 (Okada and Cyster, 2006). The chemokines CCL19, CCL21, and CXCL12 are expressed on the lumenal surfaces of HEV in lymphoid tissues and guide circulating B cells into lymphoid tissues such as the Peyer’s Patches and lymph nodes (Okada et al., 2002). Disrupting the genes that encode these chemokines results in severe defects in B cell homing to SLO (Okada et al., 2002).  13  CCL19 and CCL21 binds to the CCR7 receptor and along with CXCL12, activate integrins, allowing B cells to adhere and transmigrate into the lymphoid organ. Once inside the lymphoid organ, B cells are directed to the T cell zone in response to CCL19 and CCL21 to scan for antigen, before migrating into the lymphoid follicles via the chemokine CXCL13. CXCL13 is produced by follicular stromal cells and is bound to the surfaces of FDCs (Ansel et al., 2000; Nolte et al., 2003). The display of CXCL13 by FDCs ensures that FO B cells will crawl on the surfaces of FDCs, which present antigens and provide survival signals for B cells (Okada and Cyster, 2006). In the absence of encountering cognate antigen during their stay in the SLO, B cells exit back into circulation by responding to sphingosine 1-phosphate (S1P), and then move into other SLOs to continue their immune surveillance role. If an FO B cell recognizes antigen and becomes activated, it will increase its expression of the CCR7 receptor by 2-3 fold while maintaining the same levels of CXCR5. This increase in CCR7 expression allows the antigen-experienced B cell to migrate to the border between the lymphoid follicle and the T cell zone to receive help from TFH cells (Okada et al., 2005; Reif et al., 2002). TFH helper cells are essential regulators of effector and memory B cell responses (Fazilleau et al., 2009; King et al., 2008). TFH cells localize to follicular borders in response to CXCL13 because of their high expression of CXCR5. TFH cells also express high levels of ICOS and CD40, which bind to ICOS-L and CD40L on B cells and provide costimulation. ICOS-ICOS-L interactions are particularly important as it regulates Ig class switching, germinal center (GC) formation, and promotes memory B cell formation (Fazilleau et al., 2009). Most of these processes are regulated by TFH-secreted cytokines such as IL-2, IL-4, IL-10, and IFN!. Fully activated B cells will differentiate into short-lived extrafollicular plasmablasts, whereas others will migrate back to the lymphoid follicle to form a germinal center where they undergo further differentiation. In the germinal center, activated B cells will differentiate into memory B cells, and upon secondary antigen encounter, differentiate into long-lived plasmablasts (Allen et al., 2007a). These plasmablasts will leave the SLO and migrate back to the bone marrow in response to CXCL12, where they will enter niches that support their long-term survival (Hargreaves et al., 2001; Kunkel and Butcher, 2003). Some plasmablasts  14  can also home to sites of inflammation such as the mucosal lining in response to chemokines such as CCL25 (TECK) and CCL28 (MEK) (Cyster, 2003; Kunkel and Butcher, 2003). 1.3.4  Sphingosine 1-phosphate and B cell egress from SLOs  Lipid chemoattractants such as S1P are potent regulators of cell trafficking that are produced by endothelial cells (Rivera et al., 2008; Rivera and Chun, 2008). There are five receptors for S1P called S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. S1PR1 and S1PR3 are the most important receptors for lymphocyte trafficking because they regulate the egress of both T and B cells from SLOs and T cells from the thymus (Allende et al., 2004; Mandala et al., 2002; Matloubian et al., 2004). Trafficking into and out of SLOs is regulated by the cyclic expression of S1P receptors. When in the blood, high levels of S1P induce the downregulation of S1P receptors on lymphocytes, allowing them to respond to gradients of homeostatic chemokines that direct the trafficking of lymphocytes into lymphoid tissues. Once within the lymphoid tissues, S1P receptors are re-expressed due to the very low levels of S1P within the tissue. This allows the lymphocytes to respond to the S1P gradient emanating from the blood, which induces the egress of the lymphocytes from the SLO back into the blood (Rivera et al., 2008). Furthermore, S1P signaling is important for positioning B cells within specific compartments of the spleen, such as the marginal zone (Cinamon et al., 2004; Lo et al., 2005). Disrupting the gene encoding the S1PR3 receptor disrupts MZ B cell positioning in the spleen (Girkontaite et al., 2004). 1.3.5  Migration of marginal zone and B-1 B cells  S1P-induced signaling via S1PR3 is important for localizing MZ B cells in the marginal zone. Even though MZ B cells are considered to be largely sessile, they do migrate towards CXCL13 in vitro (Bowman et al., 2000). MZ B cells also migrate in a CXCL13/CXCR5dependent manner towards lymphoid follicles when stimulated with lipopolysaccharide (LPS) or other microbial products (Cinamon et al., 2004; Groeneveld et al., 1985). Additionally, MZ B cells are able shuttle immune complexes from the marginal sinus to the follicles, where they transfer immune complexes to FDCs, which present these antigens to FO B cells (Cinamon et al., 2008; Ferguson et al., 2004). This process is continually ongoing, with MZ B cells in constant “recirculation” between the marginal zone and the follicles in the splenic white pulp. Every few hours MZ B cells will shuttle to the follicles in 15  response to CXCR5, and then move back to the MZ by responding to S1P (Cinamon et al., 2008). Less is known about B-1 cell migration. CXCL13 is an important mediator of B-1 cell homing to peritoneal and pleural cavities, but not to the spleen (Ansel et al., 2002). Migration into these organs is regulated by CXCL13 produced by cells in the omentum and by peritoneal macrophages (Ansel et al., 2002). CXCL13-deficient animals are deficient in natural antibodies against phosphorylcholine and demonstrate poor responses to peritoneal challenges with Streptococcus antigens, indicating a lack of B-1 cells or impaired activation (Ansel et al., 2002). CXCL12 also appears to guide B-1 cell migration into gut-associated lymphoid tissues (Berberich et al., 2007; Foussat et al., 2001), while TLR signaling following antigen stimulation is important for B-1 cell egress from the peritoneum (Ha et al., 2006). Furthermore, B-1 and B-2 B cells home to the gut lamina propria in response to S1P, where they B cells undergo class switching to IgA (Kunisawa et al., 2007). Thus, these recent insights into the migratory patterns of MZ and B-1 cells confirm that cell migration is an important requirement for B cell function. 1.3.6  Selectins and leukocyte rolling  The traditional model for leukocyte trafficking, or “leukocyte adhesion cascade,” involves three steps, tethering and rolling, adhesion, and transmigration. In light of recent data, the model has recently been refined to include new steps: slow rolling, adhesion strengthening, intralumenal crawling, and paracellular or transcellular migration (Ley et al., 2007). The selectin family of transmembrane proteins mediates the slow rolling of B cells and other leukocytes (Rosen, 2004). Selectins are a family of adhesion molecules that are C-type lectins with large extracellular domains containing an amino-terminal lectin domain (Kansas, 1996). Selectins are expressed exclusively on endothelial cells and cells that are derived from the bone marrow, such as hematopoietic cells. L-selectin is expressed on all leukocytes, E-selectin is expressed on endothelial cells, and P-selectin is expressed by platelets and endothelial cells (Rosen, 2004). Selectins bind to highly glycosylated ligands, specifically sialyl-Lewis X-like carbohydrates on glycoprotein scaffolds (sialomucin-like cell surface molecules). P-selectin glycoprotein ligand 1 (PSGL-1) is an important example of an L-selectin ligand. PSGL-1 is expressed on all leukocytes and can mediate leukocyte16  leukocyte “secondary tethering” via the interaction of L-selectin with PSGL-1 on each leukocytes (Eriksson et al., 2001). This interaction is important for increasing the number of cells that reach sites of inflammation. PSGL-1 is also expressed on endothelial cells and interacts with L-selectin on lymphocytes to mediate tethering and rolling in venules, which is important for lymphocyte entry into peripheral lymphoid organs (Rivera-Nieves et al., 2006; Sperandio et al., 2003). E-selectins on endothelial cells can also bind glycosylated CD44 and E-selectin ligand 1 (ESL1) on neutrophils to promote neutrophil rolling in blood vessels (Hidalgo et al., 2007). Since T and B cells also express CD44, E-selectin interactions with glycosylated CD44 on lymphocytes may also mediate tethering and rolling (Ley et al., 2007). The interactions between selectins and their ligands results in leukocyte rolling because of the exceptionally high on- and off-rates for their binding (Alon et al., 1995). The force of interaction between selectins and their ligands is not sufficient to overcome the shear stress (force) generated by flowing blood, and mediate firm adhesion. In fact, the shear stress is required for L- and P-selectin to support adhesion and rolling. In the absence of shear stress, cells detach and do not roll (Finger et al., 1996; Lawrence et al., 1997). This phenomenon is called “catch bonds,” a molecular bond that becomes stronger or longer lived as a tensile mechanical force is applied to it (Thomas, 2008). Conversely, “slip bonds” are those that become weaker or shorter lived as a pulling force is applied to it, which selectins demonstrate at high shear forces. 1.3.7  Integrins – an overview  Integrins are a large family of glycoproteins that act as adhesion molecules for extracellular matrix components and immunoglobulin superfamily adhesion molecules (Giancotti and Ruoslahti, 1999; Hynes, 2002). They link the extracellular environment with intracellular signaling and the cytoskeleton, which allows cells to sense and respond to their environment. Integrins are heterodimeric cell surface molecules that contain an "- and a #-subunit that are non-covalently linked (Hynes, 2002). There are 18 "-subunits and 8 #-subunits, resulting in 24 known "# pairs or 24 unique integrins. Immune cells all express integrins, but only those composed from #1, #2, or #7 subunits. Of these, #2 and #7 are exclusive to leukocytes as #1 subunits are commonly found on all cell types (Luo et al., 2007). For a list of integrins found on B cells, please refer to Table 1.1. Integrins play essential roles in B cell development, 17  trafficking from the bloodstream into SLOs, migration within SLOs and tissues, immune synapse formation, polarization, and activation (see following sections) (Luo et al., 2007). Integrin CD antigen heterodimer designation  Common name  Common ligands  LFA-1 (Lymphocyte function-associated antigen-1)  ICAM-1, 2, 3, 5 (Intercellular adhesion molecule-1, 2, 3, 5)  CD49d/ CD29  VLA-4 (Very late antigen-4)  VCAM-1 (Vascular cell-adhesion molecule-1) Fibronectin  "4#7  CD49d/ beta7  LPAM-1 (Lymphocyte Peyer’s patch adhesion molecule-1)  MAdCAM-1 (Mucosal addressin cell adhesion molecule-1) Fibronectin  "1#1  CD49a/ CD29  VLA-1 (Very late antigen-1)  Collagen Laminin  "2#1  CD49b/ CD29  VLA-2 (Very late antigen-2)  Collagen Laminin  "L#2  "4#1  Table 1.1  CD11a/ CD18  Integrins found on B cells  Heterodimer pairs are listed along with CD antigen designation, commonly referred name, and their major ligands (adapted from Luo et al., 2007)  Integrin adhesiveness is tightly regulated. Normally in a resting B cell, integrins have very low binding affinity for ligand. Integrins are activated when a B cell is stimulated by chemokines, cytokines, or antigen receptor stimulation. Signaling through these receptors activates signaling pathways that converge on the cytoplasmic domain of integrins. This is called “inside-out” signaling. Inside-out signaling induces the inactive integrin to undergo conformation changes, initiated at the integrin tails by their physical separation from each other, that result in an intermediate affinity active state. This is commonly referred to as “affinity regulation” (see Figure 1.4) and is one mechanism by which the avidity of integrins 18  is regulated (Carman and Springer, 2003; Luo et al., 2007). Mechanical forces applied via ligand binding to the integrin in its intermediate affinity state, as well as lateral pulling forces applied by the actin cytoskeleton, and shear stress generated by the blood flow convert integrins to a high affinity state (Alon et al., 1995; Astrof et al., 2006; Puklin-Faucher et al., 2006; Zhu et al., 2008).  Figure 1.4  Structure of the prototypic integrin LFA-1 and models of integrin  avidity regulation (A) Integrin avidity or total integrin binding strength for ligand is regulated by affinity regulation and valency regulation. Affinity regulation is mediated by inside-out signaling that results in conformation changes that convert the integrin from a low affinity state to an intermediate affinity state. High affinity can be induced upon ligand binding as shown here, or by shear force or tensile force generated by the actin cytoskeleton (not shown). Valency regulation is mediate by clustering of integrins. (B) Structure of LFA-1 ("L#2) integrin. Reproduced with permission from the Nature Publishing Group (Kinashi, 2005).  Signaling by chemoattractants such as CXCL12, CXCL13, CCL21, and S1P induces activation of "1, "2, and "7 integrins via inside-out signaling (Campbell et al., 1998; Chan et al., 2001; Durand et al., 2006; McLeod et al., 2002; Soede et al., 2001). BCR signaling can  19  also activate integrins following antibody-induced clustering or binding to its cognate antigen (Laudanna et al., 2002; McLeod et al., 2004). Substantial progress has been made over recent years in elucidating the mechanisms that lead to integrin activation following chemokine stimulation, TCR stimulation, and stimulation following adhesion to extracellular matrix. The Rap GTPases are key molecules that mediate integrin activation in many cell types, especially lymphocytes (see Section 1.6) (Arana et al., 2008a; Evans et al., 2009; Kinashi, 2005). Other signaling pathways and molecules are also involved in the affinity regulation of integrins, including protein kinase C (PKC), talin, cytohesin-1, "-actinin, and the kindlin family of proteins (Abram and Lowell, 2009). Another mechanism by which the avidity of integrins is increased is via the clustering of integrins, or by redistributing integrins from intracellular and surface pools. This is referred to as “valency regulation,” which is regulated by outside-in signaling (Bazzoni and Hemler, 1998; Kinashi, 2005). Outside-in signaling is induced upon binding of an integrin ligand to an integrin, which stimulate changes to integrin conformation and subsequent recruitment and activation of intracellular signaling proteins (Kinashi, 2005). These changes result in integrin clustering and have been shown to play a role in valency regulation of integrins (Cambi et al., 2006; Kim et al., 2004). Together, integrin avidity for ligands is mediated by both affinity and valency regulation. Perturbations to integrin expression, activation, signaling, or adhesiveness by using blocking antibodies or via gene disruption all have profound impacts on lymphocyte migration and immune responses. One well-defined example is the disease leukocyte adhesion deficiency I (LAD-I) (Anderson and Springer, 1987). Leukocytes in patients with LAD-I cannot extravasate into inflamed tissues to clear infections. 1.3.8  Integrins and slow rolling  Integrins may also support cell rolling (Ley et al., 2007). Early studies on leukocyte rolling demonstrated that "4#7 (LPAM-1) and "4#1 (VLA-4) integrins could support rolling on immobilized MAdCAM-1, and VCAM-1 respectively (Berlin et al., 1995). Consistent with this finding, another group showed that VLA-4 also supports rolling in central nervous system (CNS) venules (Vajkoczy et al., 2001). #2 integrins like LFA-1 can also support  20  rolling. Human umbilical vein endothelial cells (HUVEC) that co-express ICAM-1 and Lselectin ligands support slower rolling of human lymphocytes but HUVEC cells, which only express selectin ligands, cannot (Kadono et al., 2002). Subsequently, in vivo studies showed that in addition to E-selectin, LFA-1 is also required to mediate leukocyte rolling in tumor necrosis factor-alpha (TNF-") -stimulated venules (Dunne et al., 2002). E-selectin engagement by leukocytes causes LFA-1 to transition from the inactive state to the immediate affinity conformation, allowing the cell to bind to ICAM-1 in venules and undergo rolling in vivo (Salas et al., 2004). The integrin-dependent rolling of cells may promote complete integrin activation, since mechanical forces are important for the induction of maximum ligand affinity (Astrof et al., 2006). 1.3.9  Integrins, adhesion, and outside-in signaling  Chemokines that are produced by lymphoid stromal cells and by venule endothelial cells bind surfaces of endothelial cells in the venules of peripheral lymphoid organs and mediate the trafficking of FO B cells to SLOs. P-selectin-mediated rolling will allow the B cell to sample these chemokines presented on the surfaces on the endothelial, triggering GPCR signaling, and subsequent activation of LFA-1, which mediates firm adhesion to ICAM-1. Firm arrest can also be mediated by other #1, #2, and #7 integrins. Of these, the best studied integrins are LFA-1 ("L#2) and VLA-4 ("4#1). Immobilized chemokines are the most potent physiological activators of these integrins and GPCR-mediated signaling can increase both integrin affinity and valency in milliseconds (Constantin et al., 2000; Kinashi, 2005; Laudanna et al., 2002; Shamri et al., 2005). After LFA-1 binds to ICAM-1, this will induce conformation changes in the cytoplasmic tails of LFA-1 and consequently, initiate intracellular signaling events by recruiting signaling proteins. This outside-in signaling is important for stabilizing adhesion and for inducing cytoskeletal rearrangement that promote cell crawling and migration (Billadeau et al., 2007; Vicente-Manzanares and SanchezMadrid, 2004). For example, adhesion to ICAM-1 induces dramatic cell spreading and Factin remodeling in T lymphocytes (Porter et al., 2002). This may be important for inducing firm adhesion under physiological conditions where lymphocytes are under shear forces due to flowing blood.  21  Signaling proteins that mediate outside-in integrin signaling include Src family kinases such as Hck and Fgr, guanine exchange factors such as Vav1 and Vav3, and PI3K. Inhibiting the activity of these proteins causes severe defects in outside-in signaling and the loss of sustained adhesion (Gakidis et al., 2004; Giagulli et al., 2006; Smith et al., 2006). Syk has also been implicated in outside-in integrin signaling (Abram and Lowell, 2007). The use of Src and Syk in integrin outside-in signaling is reminiscent of lymphocyte antigen receptor signaling, and it is not surprising that Src homology 2 (SH2) domain-containing proteins such as Syk and PI3K are important for the early events of outside-in integrin signaling (Abtahian et al., 2006; Mocsai et al., 2006). Many cytoplasmic adaptor molecules such as talin, paxillin, RAPL, and RIAM also participate in outside-in integrin signaling by linking integrins to the actin cytoskeleton and by recruiting kinases such as ILK, FAK, and Pyk2 (Harburger and Calderwood, 2009). ILK, FAK, and Pyk2 are particularly important in outside-in signaling because they mediate cell motility and spreading by activating Rho family GTPases, which in turn regulate actin cytoskeleton remodeling (Harburger and Calderwood, 2009). 1.3.10 Lymphocytes undergo amoeboid migration There are three types of cell migration strategies including mesenchymal, collective, and amoeboid migration (Friedl and Weigelin, 2008; Pals et al., 2007). Mesenchymal migration or fibroblastic migration is the classical depiction of cell crawling, which is characterized by an elongated shape and extracellular matrix (ECM) proteolysis. Collective migration is characterized by a large group of cells migrating together, while bound to each other by cadherins and gap junctions. This is seen during wound healing or when malignant epithelial cells invade tissues (Pals et al., 2007). Amoeboid migration, as the name suggests, mimics the movement of the amoeba Dictyostelium discoideum. Lymphocytes move by using amoeboid migration. The crawling movement of lymphocytes is a cyclic process involving four general steps. The leading edge sends out pseudopods, which are generated by actin flow and membrane protrusion. This allows surface receptors at these protrusions to adhere to the substrate and to allow contraction of the mid-body by actomyosin-dependent forces. Lastly the uropod, the protruding structure at the trailing end of the migrating cell, detaches as the cell is pulled 22  forward (Friedl and Weigelin, 2008). Amoeboid migration is much faster than mesenchymal and collective migration. For example, leukocytes can move up to 30 $m per minute (B cells move at 10 $m per minute), whereas migration of fibroblasts, smooth muscle cells, and cancer cells proceed at less than 1 $m per minute (Friedl and Weigelin, 2008; Pals et al., 2007). Amoeboid migration also does not depend on degradation of the ECM and the cells do not form stress fibers or strong adhesive interactions such as focal adhesions (Friedl and Weigelin, 2008; Pals et al., 2007). Another important characteristic of amoeboid-like migration is that lymphocytes are able to scan the extracellular environment with its pseudopods and respond quickly to changes in the environment, a feature that is important during intranodal migration and during antigen scanning on APCs. 1.3.11 Cell motility and cytoskeletal remodeling After integrins mediate firm adhesion, lymphocytes will quickly polarize in the presence of chemokines (Gerard et al., 2007; Kinashi and Katagiri, 2005; Manes et al., 2005). A polarized lymphocyte can be divided into three main areas: the leading edge, which consists of rapidly advancing and retracting pseudopods, the main cell body or mid-body, and the uropod (Evans et al., 2009; Friedl and Weigelin, 2008). The leading edge is the site of intense membrane ruffling and filamentous actin (F-actin) remodeling. This is important for exploring the environment so that the migrating cell can respond quickly to signaling by antigen receptors, chemokine receptors, or integrins (Stanley et al., 2008; Wei et al., 1999). The leading edge is rich in signaling proteins and signaling pathways that are activated, this includes PI3K, PLC!, and p38 MAPK, which converge on cytoskeletal regulatory proteins including Rac and Cdc42 GTPases (Thelen and Stein, 2008). Hence, the leading edge is a dynamic site of cytoskeletal remodeling that drives the protrusion of both the F-actin network and the plasma membrane outward. The complex sequence of events in cell motility is dependent on cytoskeletal remodeling, which is regulated by Rho family GTPases (Rac, Cdc42, RhoA) and the Rap GTPases (Heasman and Ridley, 2008; Kinashi, 2005). Rac and Cdc42 are essential for the formation of lamellipodia and filopodia, respectively (Etienne-Manneville and Hall, 2002; Heasman and Ridley, 2008). Lamellipodia are broad membrane protrusions that are rich in branched actin filaments that grow outwards towards the membrane and generate protrusive forces that 23  help the cell move forward. Filopodia are longer, finger-like membrane protrusions containing long, unbranched actin filaments. They serve as sensors of the external environment and appear to play a role in transendothelial cell migration by lymphocytes (Section 1.3.12) (Shulman et al., 2009). Nucleation of actin on pre-existing actin filaments is required for the formation of both lamellipodia and filopodia, and is regulated by the actin-related protein 2/3 (Arp2/3) complex (Pollard and Borisy, 2003). Normally the Arp2/3 complex is inactive and must be activated by the Wiskott-Aldrich syndrome protein (WASP) (Notarangelo and Ochs, 2003; Pollard and Borisy, 2003). Rac1 promotes lamellipodia formation at the leading edge of cells by activating the WASP family member WAVE/Scar, which in turns activates the Arp2/3 complex to induce branched actin polymerization. Cdc42 promotes filopodia formation by acting on WASP, which activates Arp2/3 to create actin branch points that acts as sites for nucleation of filopodia actin filaments (Korobova and Svitkina, 2008). Rac1 and Cdc42 can also initiate actin polymerization by activating the formin (formin homology proteins) protein mDia2 (mammalian diaphanous 2) (Pollard, 2007). Formins are involved in the polymerization of actin at barbed ends (fast-growing end), and mDia2 is particularly important for stimulating the growth of unbranched actin filaments during filpodia formation (Peng et al., 2003). RhoA may also play a role in actin polymerization, but this is independent of the Arp2/3 complex and instead depends on formins such as mDia1 (VicenteManzanares and Sanchez-Madrid, 2004; Zigmond, 2004). More importantly, RhoA promotes contraction of the cell body by regulating the formation of contractile bundles of Factin by myosin motor proteins. Thus, the Rho family GTPases are essential regulators of actin cytoskeleton dynamics that control cell morphology during cell migration. Integrins also play a key role in the dynamic nature of the leading edge and elsewhere in the migrating cell. LFA-1 in the leading edge compartment is quite low and increases as one moves to the uropod, where the density of LFA-1 is the highest (Smith et al., 2005). Furthermore, LFA-1 molecules that are located at the leading edge are of intermediate affinity for ligand binding; possibly because intermediate affinity allows the cell to form and break adhesions as it is exploring or prodding the extracellular environment (Evans et al., 2009). The cytoskeletal protein "-actinin-1 is an important link between these intermediate 24  affinity LFA-1 molecules and the F-actin network (Stanley et al., 2008). Disrupting the binding of "-actinin-1 to LFA-1 with a dominant-negative peptide prevents attachment of the leading edge to ICAM-1 and reduces the in vivo migration of lymphocytes into peripheral lymph nodes, presumably by blocking transendothelial migration (Evans et al., 2009). 1.3.12 Lymphocyte transendothelial and interstitial migration Transendothelial migration is the final stage of the “adhesion cascade” in the trafficking of lymphocytes from the blood to SLOs. After firm adhesion induced by chemokines, which is mediated by LFA-1 binding to ICAM-1, lymphocytes crawl on endothelial surfaces seeking a site of least resistance to initiate transendothelial migration (Wang et al., 2006). There are three obstacles to diapedesis: endothelial cells, the endothelial cell basement membrane, and pericytes, a group of cells that form a discontinuous network around venule endothelial cells (Ley et al., 2007). Migration of cells past the endothelial cell layer is well documented but less is known about migration beyond the basement membrane and past the pericytes. There are two routes for transendothelial migration. One route is migration between endothelial cells, called paracellular migration, and the other route is migration through the endothelial cells, which is called transcellular migration (Ley et al., 2007). During paracellular migration, lymphocyte engagement of adhesion molecules reduces inter-endothelial contacts and allows for migration through endothelial call junctions. For example, the binding of ICAM-1 on endothelial cells by LFA-1 on the lymphocyte induces Rho activation in endothelial cells and subsequent opening of endothelial junction contacts mediated by VEcadherin, PECAM1, and JAM-A (Greenwood et al., 2003; Millan and Ridley, 2005). Signaling by bound receptors on the endothelial cell also opens junctions by mobilizing intracellular calcium stores and activating p38 MAPK, which results in activation of myosin light chain kinase (MLCK) and endothelial cell contraction (Ley et al., 2007). The transcellular route for transendothelial migration is an important mechanism for leukocyte migration in the central nervous system, in in vivo inflammation models, and in in vitro assays (Ley et al., 2007). However, the transcellular route is less common as only 5 to 20% of leukocytes employ this route of migration as determined in vitro (Carman and Springer, 2004). After adhesion and crawling to a site of least resistance, all leukocytes can form invasive podosomes. These are filopodial projections that are rich in high-affinity 25  integrins and F-actin, which penetrate into the endothelial cell (Carman et al., 2007). This process appears to be regulated by shear stress and chemokine signaling, which triggers the activation of high-affinity LFA-1 (Shulman et al., 2009). Src kinases and the WASP protein are also required for the formation of these podosomes (Carman et al., 2007). In response to the leukocyte, the endothelial cell forms a cup-like docking structure around the leukocyte that is rich in ICAM-1 and VCAM-1 (Barreiro et al., 2002; Carman and Springer, 2004). Engagement of these ICAM-1 molecules on endothelial cells results in recruitment to the plasma membrane of vesicles that are rich in fusogenic proteins called vesicle-associated membrane proteins 2 and 3 (VAMP2, VAMP3) (Evans et al., 2009). LFA-1 bound ICAM-1 also relocates to regions of the endothelial cell that are rich in F-actin and caveolae (Millan et al., 2006). These caveolae then link together to form vesiculo-vacuolar organelles (VVOs), which form an intracellular channel for the leukocyte to migrate through (Dvorak and Feng, 2001; Ley and Kansas, 2004). In addition to VVOs, SNAREs also participate in forming the “tunnel” for lymphocyte transmigration (Carman et al., 2007). Because these tunnels through endothelial cells are unstable, these gateways are stabilized with actin and vimentin (Millan et al., 2006; Nieminen et al., 2006). After entering the lymphoid organ, B cells migrate along cellular networks of fibroblastic reticular cells (FRCs), dendritic cells and FDCs (Bajenoff et al., 2006; Lindquist et al., 2004; Miller et al., 2002). The FRC network is a scaffold that supports interstitial migration because of its high expression of ICAM-1 and other adhesion molecules, as well as large amounts of bound chemokines such as CXCL13 (Bajenoff et al., 2006; Cahalan and Parker, 2008). These highly organized reticular-fiber networks are also rich in collagen and are surrounded by stromal fibroblastic cells. In addition, Lammermann et al. has shown that lymphocytes can also migrate within lymph nodes in an integrin-independent fashion (Lammermann et al., 2008). This group elegantly shown that lymphocytes devoid of #1, #2, #7, and "V integrins are still able to migrate effectively within lymph nodes. It appears that lymphocytes use actin-dependent flow and myosin II-mediated squeezing to move within peripheral lymphoid tissues, in addition to integrin-dependent crawling on FRCs. After scanning for antigen within the lymphoid tissue, lymphocytes can exit via the cortical sinus vessels in the medullary region. This egress into efferent lymph appears to be regulated 26  by S1P but does not depend on integrin-mediate adhesion (Arnold et al., 2004; Cyster, 2005; Lo et al., 2005). However, it was shown recently that B cells can also exit from lymph nodes by an S1P-independent mechanism that involves squeezing through the lymphatic endothelium in sinusoids that are located next to B cell follicles (Sinha et al., 2009).  1.4 B cells and antigen encounters Antigen encounter by B cells occurs most effectively in SLOs. Lymph nodes are strategically located to receive draining lymph fluids containing antigens from the periphery, as well as migrating antigen-loaded dendritic cells (Castenholz, 1990; Mueller and Ahmed, 2008). The spleen on the other hand, which does not have an afferent lymphatic system, is ideal for collecting blood-borne antigens as it is highly vascularized (Cesta, 2006; Mebius and Kraal, 2005). 1.4.1  Lymph node and spleen architecture  In order to appreciate the complexity of how B cells encounter antigens, the structure of SLOs will be discussed briefly. The lymph node consists of three major regions, encapsulated by a protective sheath (Castenholz, 1990; Mueller and Ahmed, 2008). The outer most regions, also referred to as the subcapsular sinus, is the site of initial draining of lymph from the afferent lymphatic vessel and is rich in macrophages called subcapsular sinus (SCS) macrophages. Underneath the SCS is the cortex where there are follicles, in which B cells are in close contact with FDCs. FDCs are important for B cell activation and affinity maturation because they express complement receptors, Fc receptors, ICAM-1, and VCAM-1 that mediate antigen presentation and facilitate immune synapse formation (Carroll, 1998; Klaus et al., 1980; Mandel et al., 1980; Qin et al., 2000). Activation of B cells in the follicles leads to the formation of a germinal center, where B cells actively proliferate and differentiate into effector cells. Underneath the follicles is the lymph node paracortex, which is the T cell zone where dendritic cells and macrophages are found. The paracortex is also the site of the high endothelial venule, the entry site for all circulating lymphocytes (Miyasaka and Tanaka, 2004). Lastly, the center of the lymph node, or the medulla, contains a mosaic of B and T cells, dendritic cells, and macrophages. After lymph enters the SCS of the lymph node, it drains into the medulla via the trabecular sinus and FRC conduits (Gretz et  27  al., 2000; Sixt et al., 2005). The lymph is filtered and exits via the efferent lymph vessel, along with exiting lymphocytes. The spleen is a highly vascularized organ in the abdomen that contains an extensive network of branching arterial vessels, making it the body’s largest filter of blood (Mebius and Kraal, 2005). It contains areas that are rich in leukocytes, called the white pulp. In many ways, the white pulp is organized in a similar structure to lymph nodes and contains B and T cell compartments (Cesta, 2006; Mebius and Kraal, 2005). Surrounding the white pulp are regions called the red pulp rich in macrophages. These macrophages are important for iron recycling by via the removal of aged red blood cells and for the removal of blood-borne bacteria (Mebius and Kraal, 2005). Other cell types that are present in the red pulp include dendritic cells, plasma cells, and some lymphocytes (Cesta, 2006). Between the red pulp and the white pulp is a specialized region called the marginal zone. The marginal zone consists of MZ B cells, MZ macrophages, and dendritic cells (Pillai et al., 2005). These cells are ideally positioned to respond to blood-borne antigens because the marginal sinus is where blood enters the spleen via the follicular arteriole branches (Bohnsack and Brown, 1986). 1.4.2  Activation of B cells by antigen  Recognition of antigen by the BCR leads to B cell activation and antibody responses. After BCR binding and receptor-mediated endocytosis, the antigen will be processed and presented via MHC II. Concomitant changes in chemokine receptors will allow the B cell to migrate to the border of the T cell zone to elicit help from CD4+ helper T cells such as TFH. T cell help is required for B-2 cells response, allowing these B cell to proliferate and differentiate (Lanzavecchia, 1985). Some of these will become extrafollicular plasmablasts and secrete IgM antibodies for an early response against the infection. Other activated B cells will migrate the lymphoid follicles and form a germinal center, where plasma cells with high affinity IgG antibodies are generated, along with memory B cells (Allen et al., 2007a). How a B cell encounters and responds to antigen is largely determined by the nature, size and location of the antigen (Batista and Harwood, 2009) (see Figure 1.5 for summary). The various ways in which B cells acquire antigens are discussed in detail below.  28  Figure 1.5  Mechanism of antigen encounter for B cells in the lymph node  (A) B cells can encounter soluble antigen (<70 kDa; red dots) that have diffused through the pores in the subcapsular sinus. Antigen non-specific B cells can also transfer these antigens onto resident and follicular dendritic cells. (B) B cells encounter large particulate antigens presented on the surfaces of subcapsular sinus macrophages. (C) B cells can recognize and bind intact antigens (small and large) that are presented on the surfaces of dendritic cells. (D) Lastly, B cells can also acquire antigen by extending pseudopods into fibroblastic reticular cell conduits to sample small antigens (Adapted and modified from Harwood and Batista, 2009).  1.4.3  Soluble antigens  Soluble antigens are delivered to lymph nodes by the afferent lymph vessels, or to the spleen via blood supplied by follicular arterioles. Antigens can be detected in the SCS within minutes following a subcutaneous injection (Nossal et al., 1968). Electron microscopy has shown that pores ranging from 0.1 to 1 $m diameter are present in the SCS and it is believed that soluble antigen can enter the lymph node via these pores and diffuse into the cortex of the lymph node (Farr et al., 1980; van Ewijk et al., 1988). Consistent with this idea, Pape et al. shown that small soluble antigens (less than 70 kDa) can enter lymph nodes and be taken 29  up by B cells in the follicles (Pape et al., 2007). Antigen that was detected following immunization could be seen rapidly diffusing from the SCS into the cortex of the lymph nodes. This presence of antigen in the lymph node was not mediated by dendritic cells, nor did it involve migration of B cells, indicating that passive diffusion is the mechanism that allows soluble antigen to enter the lymph node (Pape et al., 2007). These findings were recapitulated in another report that showed that small, soluble antigens such as small dextrans and turkey egg lysozyme were able to freely diffuse into the SCS and lymph node cortex (Roozendaal et al., 2009) 1.4.4  Particulate antigens  Particulate antigens such as bacteria and virus particles are also delivered to lymph nodes via lymph and to spleen via blood. Bacteria and viral particles have many repetitive antigen epitopes on their surfaces and are very efficient at inducing innate immune responses as well as B cell responses. These three-dimensional repeating antigen epitopes are very effective at crosslinking the BCRs to induce B cell activation and a strong humoral response. The immunogenicity of particulate antigens is reflected in their use for vaccines (Gander, 2005; Jennings and Bachmann, 2008; Riedmann et al., 2007). Unlike soluble antigens, particulate antigens are unable to passively enter the lymph node. Instead, particulate antigens from the periphery are bound in the SCS by macrophages and actively transported into the lymph node for presentation to B cells (Section 1.4.5) (Carrasco and Batista, 2007; Junt et al., 2007; Phan et al., 2007) or presented on mature DCs from the periphery (Section 1.4.6) (Carrasco and Batista, 2006a). 1.4.5  Membrane-bound antigens presented by macrophages  B cells can respond to both soluble and membrane-associated antigens but it is increasingly appreciated that membrane-associated antigens are more relevant in vivo (Carrasco and Batista, 2006a). In contrast to the free diffusion of small soluble antigens that arrive at the SCS via draining lymph, larger antigens that cannot diffuse through the pores can be actively taken up by SCS macrophages. These macrophages are able to extend processes into the SCS to sample afferent lymph and bind antigens (Farr et al., 1980; Fossum, 1980; Szakal et al., 1983). This is similar to how metallophilic macrophages in the splenic marginal zone capture and concentrate antigen, and appears to be a unique characteristic of these 30  macrophages that are in sentinel locations (Gray et al., 1984; Martinez-Pomares et al., 1996). These macrophages have limited phagocytic abilities and can present intact antigen on their surfaces for up to 72 hours after immunization (Delamarre et al., 2005). Presentation of intact antigen is key for B cells because their BCRs recognize intact complementary threedimensional epitopes. The binding of intact antigens to the surfaces of SCS macrophages can be mediated by multiple receptors including MAC1 ("M#2; CD11b/CD18), Fc!RIIb, DCSIGN (c-type lectin DC-specific ICAM3- grabbing non-integrin; CD209), and complement receptor 1 and 2 (CR1/CD35; CR2/CD21). These receptors are able to bind to complementcoated, antibody-coated, and highly glycosylated antigens (Taylor et al., 2005). The SCS macrophages are able to efficiently present antigens such as immune complexes of C3-bound or IgG-bound particulates, bacteria, and viruses to FO B cells (Carrasco and Batista, 2007; Junt et al., 2007; Phan et al., 2007). Following administration of virus to the animal, virus particles could be seen accumulating underneath the SCS (Junt et al., 2007). This suggests that antigen could be translocated by the macrophages from the lumenal side of the SCS into the interstitial spaces of the lymph node. This allows B cells to cluster underneath the SCS where they were initially immobilized to antigens, but after antigen accumulation and processing they regain the ability to migrate to the border of the T-cell zones to elicit T cell help. The SCS macrophages are critical for this response as depletion of all macrophages using clodronate-loaded liposomes eliminates all antigen transfer to B cells following immunization (Junt et al., 2007). Junt et al. suggested that antigen is not phagocytosed because SCS macrophages lack mannose receptors, which are usually associated with phagocytosis of complement-coated antigens. Furthermore, SCS macrophages express high levels of CD169, a sulphated glycoprotein that is thought to impede phagocytosis (Martinez-Pomares and Gordon, 2007). The SCS macrophages can quickly bind immune complexes in afferent lymph and present it to bystander FO B cells. These antigen non-specific bystander FO B cells are then able to transfer the immune complexes via their CR1 and CR2 receptors to FDCs (Phan et al., 2007). This represents a mechanism by which antigen can be transported to FDCs and may explain how FDCs acquire antigen for presentation to other antigen-specific FO B cells (Section 1.4.6). 31  1.4.6  Membrane-bound antigens presented by dendritic cells  Dendritic cells (DCs) are the most effective antigen presenting cells in the body (Ardavin, 2003; Reis e Sousa, 2006). The lymph node contains three main types of dendritic cells: FDCs in the B cell follicles, resident DCs in the paracortex, and mature DCs that have immigrated from the periphery following antigen uptake. Resident DCs in the paracortex are found in close association with the fibroblastic reticular cell network, clustered around the HEV (Batista et al., 1996). These DCs are ideally located to sample and take up antigens that drain into the paracortex via the trabecular sinus and FRC conduits. B cells entering the lymph node at the HEV can sample antigens trapped by these resident DCs localized near HEV (Colino et al., 2002; Qi et al., 2006; Wykes et al., 1998). Dendritic cells can retain and present intact antigen on their surfaces due to their high level of Fc!RIIb and DC-SIGN expression (Ardavin, 2003; Reis e Sousa, 2006). Moreover, Fc!RIIb on DCs mediates internalization of immune complexes into endosomal compartments that do not fuse with lysosomes (Bergtold et al., 2005). Instead these endosomal compartments undergo recycling, and the bound immune complex along with Fc!RIIb is returned to the surface to allow priming of B cells. FDCs in lymphoid follicles efficiently activate B cells (Batista and Harwood, 2009; Tew et al., 2001). Before it had been shown that FDCs retain and present antigens, earlier studies had shown that extracellular antigens bound by membranes are retained in follicles for long periods after immunization (Batista and Harwood, 2009; Mandel et al., 1980; Mitchell and Abbot, 1965; Nossal et al., 1968). FDCs can retain and present antigen by using CR1, CR2, and Fc!RIIb to antibody- or complement-coated antigens. Mice that lack CR1, CR2, or Fc!RIIb in their stromal cell compartment show impaired antigen presentation by FDCs to B cells (Barrington et al., 2002; Qin et al., 2000; Fang et al., 1998; Yoshida et al., 1993). FDCs do not reside near vasculature but are still able to acquire and present antigen to B cells. In the lymph nodes, FDCs acquire antigen via transfer of antigens by bystander FO B cells. These bystander FO B cells do not recognize the antigens presented on SCS macrophages but instead, they bind the antigens via their complement receptors and transport it to the lymphoid follicles (Phan et al., 2007). Though, it was not shown conclusively that  32  FO B cells mediate transfer of intact antigen to FDCs directly, it is likely that FO B cells can transport antigen into follicles where it can be taken up by FDCs. In the spleen, MZ B cells also shuttle antigen from the marginal sinus to follicles for presentation of antigen by FDCs (Cinamon et al., 2008). MZ B cells also express high levels of CR1 and CR2, and in the study by Cinamon et al., CR2 was the main receptor mediating the transport of antigen. Once the MZ B cells reach the FDCs in the follicles, proteolysis of CR2 on the surfaces of MZ B cells may release the immune complexes, allowing the FDCs to bind it (Whipple et al., 2004). Consistent with these findings, CD19 knockout mice, which lack MZ B cells (Martin and Kearney, 2000) are unable to accumulate IgM-immune complexes on FDCs, illustrating the importance of MZ B cells for transporting intact antigen onto FDCs in vivo (Ferguson et al., 2004). It was previously thought that B cells in the germinal centers were rather sessile, as they remain firmly attached to FDCs in order to receive survival signals and to undergo SHM (Allen et al., 2007a). However, multiphoton in vivo imaging of germinal center dynamics revealed that GC B cells were continuosly circulating within the germinal center and did not make prolonged contacts with FDCs (Allen et al., 2007b; Hauser et al., 2007; Schwickert et al., 2007). GC B cells also have an unusual morphology characterize by many “dendritelike” processes. Gene expression analysis revealed that GC B cells express many neural genes that are associated with neurite formation, and may be involved in the formation of dendrite-like processes in GC B cells (Yu et al., 2008). These processes may be important for antigen uptake from FDCs when GC B cells are crawling on FDCs (Batista and Harwood, 2009). 1.4.7  Antigens in fibroblastic reticular cell conduits  An exciting new study revealed an additional mechanism in which B cells can encounter antigen (Roozendaal et al., 2009). Roozendaal et al. showed that low molecular-weight antigens mimicked by small dextrans and turkey egg lysozyme (TEL) fill the FRC conduits after subcutaneous injections of these antigens. The FRC conduits consist of a discontinuous network of FRCs ensheathed around reticular collagen fibers to form a channel that carries lymph from the SCS into the follicles and paracortex of the lymph node (Harwood and  33  Batista, 2009). Since the conduit is not entirely enclosed, gaps are present between FRCs that allow FO B cells to extend pseudopods into the conduit network to directly sample lymph within the core and acquire antigens. This method may be a highly effective mechanism to initiate B cell-antigen encounters because B cells are activate more quickly when encountering antigen in FRC conduits as opposed to encountering subcapsular antigen that entered the lymph node by diffusion. FRC conduits are also important for presenting antigens to T cells (Sixt et al., 2005). Dendritic cells are closely associated with FRC conduits and can internalize antigen for presentation to T cells, further supporting the idea that the FRC conduit is an “antigen highway” (Gretz et al., 2000).  1.5 B cell activation 1.5.1  Overview  The binding of antigen by the BCR initiates a cascade of signaling events that induces changes in gene expression, cell proliferation, and reorganization of the cytoskeleton (Gold, 2002). Recent advances in fluorescence microscopy techniques such as total internal reflectance fluorescence microscopy (TIRFM) have provided new insights into how B cells initiate and respond to antigen stimulation (Harwood and Batista, 2008). Many groups have identified structures such as microclusters (aggregates of antigen receptors) and immune synapses that were once appreciated only in T cell immune responses, as important requirements for BCR-mediated signaling and B cell activation (Harwood and Batista, 2008). Currently, one proposed model (Figure 1.6) for how B cells respond to antigens presented on APCs is as follows: recognition and binding of antigen by the BCR localizes the BCR within a membrane domain that is deficient in phosphatases (Section 1.5.2), allowing BCR signaling to be initiated (Harwood and Batista, 2008). Next, BCRs aggregate together to form microclusters, which are the main signalosomes (complex of active signaling molecules in association with the BCR) that mediate BCR signaling (Section 1.5.3). BCR signaling then induces localized cytoskeletal rearrangement to allow cell spreading and a concomitant increase in contact area with the APC, leading to further microcluster formation (Section 1.5.5). The spreading B cell will subsequently contract and gather BCR-antigen complexes into an immune synapse (Section 1.5.6). Lastly, antigen will be extracted from the APC at  34  the immune synapse and internalized for antigen processing and presentation to T helper cells. The following sections will describe each step in detail.  Figure 1.6  The stages of B cell response to membrane-bound antigen  The four stages of B cell response to intact antigen presented on the surfaces of APC. The top panel represents the contact area (interface) between the B cell and the APC and the bottom panel represents a side view of the interaction between the B cell and the APC. (A) Recognition of antigen by BCR induces segregation of transmembrane phosphatases such as CD45 (not shown) and initiates BCR signaling. (B) Next, BCR and CD19 (not shown) aggregate together to form microclusters (depicted by medium-sized red circles). (C) B cell spreading follows to increase the contact area between the B cell and APC, allowing it to gather more antigens. This is followed by a contraction phase as the antigen is pulled inwards and concentrated. (D) After the B cell contracts it forms an immunological synapse, which is important for extracting and internalizing antigen, and for B cell activation. The immune synapse is characterized by a central supramolecular activation complex (cSMAC) (depicted by the large red circle) and a ring of ligandbound integrins called peripheral SMAC (pSMAC) (depicted by the ring of purple dots).  35  1.5.2  Kinetic segregation  Many aspects of TCR signaling dynamics may also be applicable to the BCR, including “kinetic segregation,” whereby antigen receptors are localized to phosphatase-deficient compartments of the membrane following antigen binding, may also be applicable to the BCR (Harwood and Batista, 2008). TCR binds to MHC-peptide complexes to initiate signaling but there are challenges to overcome before this can occur. The TCR is dwarfed in the sea of large extracellular receptor domains such as the phosphatase CD45. CD45 presents two challenges to effective TCR signaling: CD45 in close proximity to the TCR will prevent initiation of TCR signaling by dephosphorylating Lck with its intracellular phosphatase domain, and the large extracellular domain of CD40 acts as a physical deterrent to engagement between the much shorter TCR and MHC-peptide complexes. In order to overcome these challenges, T cells form an area of intimate contact between APCs and T cells when the TCR binds to MHC-peptide complexes. This close contact zone facilitates the size-dependent exclusion of larger membrane proteins such as CD45, allowing the TCR to maintain prolonged contact with the MHC-peptide complex, and also excluding the CD45 phosphatase (the brake) from the contact area, allowing activation of Src kinases such as Lck (Choudhuri et al., 2005; Davis and van der Merwe, 2006). CD45, as well as other receptor tyrosine phosphatases are also present on the surfaces of B cells. Consistent with this idea, CD45-null B cells do not show any defects in activation by membrane-bound antigen (Depoil et al., 2008), presumably because they also express the CD148 phosphatase (DEP-1/PTPRJ), which may compensate for the loss of CD45. CD148 contains a large extracellular domain and has been shown to regulate immunoreceptor signaling (Lin and Weiss, 2003). Consistent with this idea, only double knockout of CD45 and CD148 causes severe B cell development defects, resulting from hyperphosphorylation of Src family kinases on a negative regulatory site as well as aberrant BCR signaling (Zhu et al., 2008). This suggests that the separation of receptor tyrosine phosphatases and antigen receptors (kinetic segregation) is an important early event for the initiation of BCR signaling (Harwood and Batista, 2008). Indeed, it has been reported that CD45 is excluded from BCR microclusters, the active signaling structure in B cells (Depoil et al., 2008).  36  1.5.3  BCR microclusters  In T cells, the active signalosomes are TCR microclusters. These are small aggregates of TCRs that form at the DC-T cell contact zone within minutes of antigen binding by the TCR, and before the formation of the immune synapse (Section 1.5.6) (Campi et al., 2005; Yokosuka et al., 2005). These microclusters are essential for initiating and sustaining TCR signaling following antigen engagement and continue to signal even after the immune synapse has formed (Yokosuka et al., 2005). Recently, BCRs were also shown by confocal microscopy to form microclusters following binding of antigen captured on phospholipid bilayers (Depoil et al., 2008). If there is sufficient BCR avidity for membrane-bound antigen, i.e. a sufficient amount of antigen and sufficient BCR affinity for the antigen, then this induces formation of microclusters containing molecules of IgM, IgD, and CD19 (Depoil et al., 2008). BCR microclusters recruit key signaling molecules such as Syk, PLC!2, and Vav (Depoil et al., 2008; Weber et al., 2008). Therefore, the formation of these microclusters is an essential early B cell response to antigen as and is required for subsequent processes including cell spreading, immune synapse formation, and B cell activation (Depoil et al., 2008). This importance is demonstrated by the inability of signaling-deficient B cells, which form microclusters but are unable to signal, to undergo cell spreading, immune synapse formation, or undergo activation. Moreover, microclusters lacking CD19, by using CD19-deficient B cells, are also unable to undergo these post antigen-binding processes, demonstrating that CD19 is an important component of the microcluster (Depoil et al., 2008). Surprisingly, Depiol et al. reported that BCRs also aggregate spontaneously in the absence of antigen but that these clusters are not involved in signaling. This suggests that kinetic segregation of phosphatases from the BCR following antigen engagement allows the microclusters to initiate signaling. Conversely, Tolar et al. recently showed that microcluster formation only occurs after stimulation with membrane bound antigen (Tolar et al., 2009). Using both multivalent and monovalent antigen bound in lipid membranes, they were able to show by real-time confocal microscopy that B cells form microclusters within 5 seconds of antigen recognition. Furthermore, the clustering of antigen-BCR complexes resulted in the immobilization of the complexes, contrary to antigen-free BCRs that freely diffused within the B cell membrane.  37  The authors proposed that immobilization of BCRs is the result of tethering to the cytoskeleton, similar to what has been shown for TCR microclusters (Campi et al., 2005). 1.5.4  B cell receptor signaling  Antigen binding by the BCR will exclude CD45 and CD148 phosphatases, facilitating the initiation of BCR signaling by activating and recruiting the Src family kinases (Fyn, Lyn, and Blk) to the BCR (Kurosaki, 2002b). At the plasma membrane, Lyn, Fyn, and Blk phosphorylate the ITAMs on the cytoplasmic tails of the Ig" and Ig# chains (Gold, 2002). These phosphorylated tyrosines act as homing beacons for SH2-domain containing proteins such as Syk and additional Src kinases, amplifying the signal. Once the SH2 domains on these tyrosine kinases bind the phosphorylated tyrosine residues, conformational changes allow Src kinases to undergo autophosphorylation, and Syk to be phosphorylated by Src kinases (Kurosaki, 2002b; Niiro and Clark, 2002). Activated Syk is the key signaling molecule in BCR signaling because it phosphorylates adaptor proteins that initiate the formation of signaling complexes that activate downstream signaling pathway modules such as the PI3K pathway, the PLC!2 pathway, the Ras-Raf-Erk pathway, and the Rac and Rap GTPase proteins (Gold, 2002). The loss of Syk in knockout mice results in the disruption of BCR signaling and the failure of B cells to develop (Kurosaki, 2002b). The PI3K signaling module regulates many cellular functions in B cells and in other cells (Gold et al., 2000; Koyasu, 2003; Marshall et al., 2000; Okkenhaug and Vanhaesebroeck, 2003a). The PI3K pathway regulates cell growth, proliferation, differentiation, survival, apoptosis, polarization, migration, and metabolism. Syk activation following antigen engagement by the BCR results in phosphorylation of the CD19 co-receptor and the B cell adaptor protein (BCAP). This recruits PI3K to the plasma membrane via interactions between the PI3K SH2 domain and the phosphorylated tyrosine motifs, Y(P)XXM, on BCAP and CD19 (Niiro and Clark, 2002). Once bound to CD19 or BCAP at the membrane, PI3K produces the second messenger PIP3 by phosphorylating PIP2. PIP3 is able to recruit signaling proteins to the plasma membrane via their Pleckstrin-homology (PH) domains that bind PIP3. Molecules that are recruited in this fashion include phosphoinositide-dependent kinase (PDK1), protein kinase B (PKB/Akt), Btk, and PLC!2. The Akt kinase is a major effector in the PI3K pathway that transduces signals generated at the membrane to 38  cytoplasmic signaling molecules and to transcription factors. Akt activates many prosurvival factors and inactivates apoptotic molecules. For example, Akt activates the prosurvival nuclear factor %B (NF-%B) pathway (Kane et al., 1999) and inactivates pro-apoptotic pathways by directly phosphorylating BAD (a member of the Bcl-2 family) (Datta et al., 1997; del Peso et al., 1997) and caspase 9 (Cardone et al., 1998). Furthermore, Akt phosphorylates and inactivates FOXO transcription factors, which induce the expression of genes that cause cell cycle arrest (Coffer and Burgering, 2004; Herzog et al., 2009). Hence, activated Akt plays a key role in executing the PI3K-dependent pro-survival program in B cells (Pogue et al., 2000). PI3K and the phosphatase PTEN are important regulators of cell polarity in migrating cells by generating an intracellular gradient of PIP3 (Kolsch et al., 2008). Localized production of PIP3 may also be important for polarized responses when B cells sample and gather membrane-bound antigens on dendritic cells, or when they bind to particulate antigens such as bacteria. PTEN, the phosphatase specific for PIP3 helps generate polarity by restricting PIP3 production to the leading edge. PIP3 also recruit guanine exchange factors, such as Vav1, Sos1, Tiam1, and P-Rex1 that activate the Rac and Cdc42 GTPases (Barber and Welch, 2006). Another important BCR signaling molecule involves PLC!2 (Gold, 2002; Kurosaki, 2002a; Marshall et al., 2000). PLC!2 is recruited to the BCR complex via its SH2 domain interacting with the adaptor protein BLNK. BLNK is recruited to the phosphorylated Ig" chain following antigen engagement where Syk subsequently phosphorylates BLNK. Once PLC!2 has bound BLNK, Syk and Btk activate PLC!2 by phosphorylation. Activation of PLC!2 is facilitated by PIP3, which directs Btk to the BLNK-PLC!2 complex. Following activation, PLC!2 produces the second messengers IP3 and DAG. IP3 causes the release of Ca2+ from intracellular stores, which activates the NF-AT transcription factor. DAG activates Ras guanyl nucleotide-releasing protein (RasGRP), CalDAG-GEFI (RasGRP2), and protein kinase C (PKC) enzymes. These molecules in turn modulate the activity of other targets including the Ras-Raf-MEK-ERK pathway (Gold, 2000), NF-%B (Moscat et al., 2003), the JNK and p38 MAPKs (Hashimoto et al., 1998), and the Rap GTPases (Katagiri et  39  al., 2004b), which regulates B cell differentiation, proliferation, survival, adhesion, and migration. GTPases play an important role in BCR signaling by linking signals at the membrane to downstream signaling cascades (Ehrhardt et al., 2002; McLeod and Gold, 2001). GTPases exist either in an active GTP-bound form or an inactive GDP-bound form. When GTPases are in their active forms, they able to bind effector proteins that transduce signals further downstream. They function as regulatory, molecular switches because of their ability to bind these effector proteins. As their name suggests, GTPases have an intrinsic ability to hydrolyze bound GTP to GDP. GTPase activating proteins (GAP) negatively regulate GTPases by increasing their intrinsic GTP hydrolyzing activity. Conversely, guanine nucleotide exchange factors (GEF) turn GTPases ‘on’ by enhancing the exchange of bound GDP for the more abundant GTP. Ras, Rac1 and Rap are GTPases that are important components of BCR signaling. The Ras GTPase activates the Raf-MEK-ERK kinase cascade after activation by RasGRP by the DAG produced upon BCR engagement. The RasRaf-MEK-ERK cascade is required for proper B cell development, and for the proliferation of mature B cells (Gold, 2002). Rac1 and PKC links BCR signaling to the JNK and p38 MAPK pathways. This results in the activation of several transcription factors that regulate the expression of genes that promote survival and proliferation. Furthermore, Rac1 is important for lamellipodia formation during B cell spreading and migration. Targeted gene knockout of the Rac1 and Rac2 GTPases in B cells of mice has demonstrated the importance of these molecules in B cell development and signaling (Walmsley et al., 2003). The Rap GTPases are another class of important GTPases that regulate B cell adhesion and motility, which is important for the function and activation of B cells (see Section 1.6) (Caron, 2003; McLeod and Gold, 2001; Stork, 2003). 1.5.5  B cell spreading and contraction  Following the formation of BCR microclusters, B cells undergo a dynamic two-phase response in which the cell spreads on antigen-containing lipid membranes or APCs, and then contracts as it gathers up antigen (Fleire et al., 2006). The rapidly spreading cell “aprons” show remarkable resemblance to lamellipodial protrusions that are characteristic of migrating cells, and it is likely that it is regulated by the same cytoskeletal machinery involved in actin 40  remodeling during migration. Consistent with this hypothesis, Vav and actin polymerization are necessary for this B cell spreading (Fleire et al., 2006; Weber et al., 2008). Vav, a RhoGEF that activates Rac1, is recruited to microclusters and is necessary for B cell spreading (Weber et al., 2008). The Rac1 and Rac2 GTPases are key molecules in regulating the formation of lamellipodia in migrating cells and also mediate B cell spreading as well as the formation of immune synapses (Arana et al., 2008b; Brezski and Monroe, 2007). Other molecules that are recruited to microclusters and play a role in antigen-induced B cell spreading and immune synapse formation include Src kinases (e.g. Lyn), PLC!2, and CD19 (Depoil et al., 2008; Fleire et al., 2006; Weber et al., 2008). B cell spreading increases the contact area with the APC so that more BCRs can interact with, and gather antigen. This results in amplification of the initial antigen signal. It is not known what causes the cell to stop spreading. Perhaps there is a physical limitation as the cell only has a finite amount of actin and membrane and thus cannot infinitely spread out. Alternatively, contractile forces generated by myosin II, actin-based motor proteins that bind to F-actin to generate force, may predominate so that the cell starts to contract. During this contraction phase, the B cell gathers BCR-bound antigen via into a central aggregate that will eventually form the cSMAC of the immune synapse (Fleire et al., 2006). This site is also where the B cell will extract antigen from the DC and internalize it for processing, followed by presentation on MHC II to elicit additional signals from T helper cells (Batista et al., 2001). 1.5.6  B cell immune synapses  The immune synapse was first identified in CD4+ T cells in a series of landmark papers illustrating the importance of this supramolecular structure for T cell activation (Grakoui et al., 1999; Krummel et al., 2000; Monks et al., 1998; Wulfing et al., 1998). We now know that the immune synapse is a common feature of immunoreceptor signaling in B cells, CD8+ T cells, and natural killer (NK) cells (Batista et al., 2001; Davis et al., 1999; Potter et al., 2001; Stinchcombe et al., 2001). In the center of the immune synapse is an accumulation of antigen receptors that form the cSMAC. In T cells this is the site of internalization of TCRs, which terminates TCR signaling (Dustin, 2008; Seminario and Bunnell, 2008). In B cells, antigen is extracted from the DC at the cSMAC and internalized (Batista et al., 2001). 41  However, the functional significance of the cSMAC for BCR signaling is unknown. Surrounding the cSMAC is a ring of activated integrins (LFA-1 and VLA-4) that are engaged to their ligands on the surface of the APC. This ring is called the pSMAC and reduces the threshold for B cell activation (Carrasco and Batista, 2006b; Carrasco et al., 2004). One way in which it may do so is by prolonging adhesion of the B cell to the APC, so that the cell can continually sample antigen and sustain signaling necessary for its activation. Consistent with this idea, BCR microclusters continually form and signal at the periphery of the immune synapse (Dustin, 2008; Harwood and Batista, 2008). Moreover, integrin signaling can contribute to immunoreceptor signaling and B cell activation as both integrin signaling and BCR signaling synergize through the activation and recruitment of key molecules such as Syk, PI3K, and PLC!2 (Arana et al., 2008a).  1.6 Rap GTPases 1.6.1  Overview  The Rap GTPases are a group of 23 kDa monomeric GTPases belonging to the Ras superfamily. There are five Rap GTPases in humans and mice, Rap1a, Rap1b, Rap2a, Rap2b, and Rap2c, which are collectively referred to as Rap. Like all GTPases, Rap cycles between a GTP-bound active state and a GDP-bound inactive state, which is regulated by GEFs and GAPs. In its active state, Rap interacts with multiple effector proteins to regulate cell adhesion, cytoskeletal organization, polarity, and migration (Figure 1.7B). Rap is activated by RapGEFs, which contain two important domains: the Cdc25 homology domain that contains the GDP-GTP exchange activity and a Ras exchange motif (REM) (Raaijmakers and Bos, 2008). RapGEFs catalyze the exchange of bound GDP for GTP by inserting a catalytic helix into the guanine nucleotide-binding pocket of Rap in order to disrupt the binding of GDP (see Figure 1.7A). GDP is released as a result and GTP takes its place because the concentration of GTP in the cytosol is ten times higher than GDP. GTP binding will result in the release of the GEF. There are multiple RapGEFs including C3G, Epac1 and 2, RasGRP2 (CalDAG-GEF1), RasGRP3, PDZ-GEF1 and 2, and PLC( (Bos et al., 2007). Although there are multiple RapGEFs, in B cells the BCR and chemokine receptors activate Rap via a PLC!/DAG signaling pathway. The PLC! pathway may target  42  RasGRP2 (CalDAG-GEF1) because it has a DAG-binding domain (Bos et al., 2001; McLeod and Gold, 2001; McLeod et al., 1998; Stone, 2006). GAPs such as SPA-1, SPA-1-like, E6TP1, RapGAPI, and RapGAPII induce the hydrolysis of GTP to GDP, turning Rap off (Raaijmakers and Bos, 2008). Rap possesses an intrinsic GTPase activity, which is normally quite low. GAPs increase the GTP hydrolysis rate by several fold by inserting an asparagine catalytic side chain into the nucleotide-binding pocket (Bos et al., 2007). Rap is usually membrane-bound and is normally located at intracellular membranes in the perinuclear region, endocytic and exocytic vesicles, and sometimes at the plasma membrane. Active Rap1 localizes to the plasma membrane, as well as to membrane protrusions following cell adhesion and cell spreading (Bivona et al., 2004; Schwamborn and Puschel, 2004). Therefore, precisely-regulated localization of RapGEFs and RapGAPs likely control the time course and subcellular localization of Rap activation (Raaijmakers and Bos, 2008).  43  Figure 1.7  Summary of the Rap GTPase structure and signaling pathway  (A) Several protein motifs in Rap have been identified through crystallography studies. Four key structural features of Rap are outlined here: the GTP binding regions (green), the GEF interaction regions (blue), the switch regions (blue), and the effector interaction regions (yellow). (B) Rap GTPases can activate integrins by interacting with and localizing adaptor molecules such as RAPL and talin. Rap can also modulate actin cytoskeleton dynamics by binding to and activating guanine exchange factors for Rac and Cdc42 such as Vav2 and Tiam. Lastly, Rap has been implicated in cell polarity by mediating the localization of Cdc42 and the Par3/Par6 polarity complex (abbreviated as polarity complex in the figure) (Adapted and modified from Bos, 2005).  44  1.6.2  Overview of Rap1 functions  To examine the function of Rap GTPases, several groups have genetically ablated individual Rap isoforms. Most of these studies implicated Rap as a regulator of adhesion, which is not surprising since Rap regulates the activation of integrins (see below). Rap1a-null mice do not exhibit any development defects and are fertile (Li et al., 2007). However, macrophages isolated from these mice show decreased adhesion to fibronectin and vitronectin, and the chemotaxis of both lymphoid and myeloid cells is also decreased (Li et al., 2007). Another group disrupted Rap1a and although the phenotype was less dramatic, there were defects in the adhesion of hematopoietic cells to ICAM and fibronectin, as well as defects in T cell polarization (Duchniewicz et al., 2006). This implicates Rap1 as a master regulator of lymphocyte adhesion and migration by controlling activation of integrins. Rap1b was genetically ablated to examine functional differences between Rap1a and Rap1b. Rap1b-deficient mice demonstrated a bleeding defect due to the inability of their platelets to aggregate (Chrzanowska-Wodnicka et al., 2005). Examination of the platelets revealed that Rap1b deficiency resulted in decreased activation of "II#3 integrins (Chrzanowska-Wodnicka et al., 2005). Using the same Rap1b-null mice, two independent reports show that Rap1b is the main Rap1 isoform in B cells. However, the two reports were conflicting in regards to the function of Rap1b in B cells. One group found that Rap1b did not impact early B cell development but significantly decreased the numbers of MZ B cells (Chen et al., 2008). The second group showed that B cell development in the bone marrow is disrupted because of the inability of B cell progenitors to adhere to stromal cells (Chu et al., 2008a). It is unclear why there are differences in observations between these two groups. Despite these differences, both groups reported that Rap1b is an important regulator of B cell adhesion and migration as they observed impaired in vitro migration and in vivo homing of Rap1b-deficient B cells (Chen et al., 2008; Chu et al., 2008a). 1.6.3  The mechanism of integrin activation by Rap GTPases  Rap is essential for mediating inside-out signals that activate integrins such as LFA-1 and VLA-4 (Caron et al., 2000; Katagiri et al., 2000; McLeod et al., 2002; Reedquist et al., 2000). Integrins are activated by the BCR and TCR (Boussiotis et al., 1997; McLeod et al., 1998; Reedquist and Bos, 1998), by GPCRs (Durand et al., 2006; McLeod et al., 2002; 45  Shimonaka et al., 2003), and by cytokine receptors (Alsayed et al., 2000). Activation of Rap1 is essential for integrin activation, adhesion, and migration in both T and B cells since converting Rap to its inactive GDP-bound state via expression of the Rap-specific GAPs SPA-1, RapGAP1, or RapGAPII inhibits these processes (McLeod et al., 2002; McLeod et al., 2004; Shimonaka et al., 2003). Furthermore, Rap1-GTP is required for localization of CXCR4 to the leading edge of cells, localization of CD44 to the uropod, and the clustering of LFA-1 integrins (Shimonaka et al., 2003). Therefore, it appears that Rap has an essential role in regulating cell adhesion, morphology, and motility in T and B cells. The active form of Rap (Rap-GTP) activates LFA-1 in lymphocytes via RAPL (regulator of adhesion and cell polarity enriched in lymphoid tissues), an adaptor protein that binds to the Rap1-GTP (Katagiri et al., 2003). TCR signaling results in activation of Rap1, leading to the formation of a Rap1-GTP/RAPL complex, which binds to the GFFKR motif in the cytoplasmic tail of the "L chain of LFA-1 (Katagiri et al., 2003). Binding of RAPL to this motif separates the "L and #2 cytoplasmic tails, which induces the unfolding of the extracellular domains of the integrin into the immediate affinity conformation. When RAPL function is disrupted, T and B cells are less adherent to ICAM-1 and do not polarize or cluster integrins after chemokine stimulation (Katagiri et al., 2003). Moreover, in RAPL-/mice, T and B cells do not migrate properly to the peripheral lymphoid organs, demonstrating the importance of the Rap1/RAPL pathway in lymphocyte adhesion and migration (Katagiri et al., 2004a). Rap1 also promotes integrin-mediated adhesion by increasing the clustering of integrins. Following chemokine or antigen receptor stimulation, Rap1-GTP/RAPL complexes bind to the Mst1 kinase (Katagiri et al., 2006). The Rap1/RAPL/Mst1 complex binds to LFA-1 integrins and increases LFA-1 affinity, while simultaneously promoting vesicle-mediated translocation of intracellular LFA-1 to the leading edge of the cell (Katagiri et al., 2006). In this way, Rap1-GTP promotes the formation of large clusters of intermediate affinity LFA-1 at the leading edge of migrating lymphocytes. Another Rap1 effector is the adaptor protein RIAM (Rap1-GTP-interacting adaptor molecule), which also plays a role in integrin activation. RIAM contains multiple protein interaction domains, including a Ras association (RA) domain that binds Rap1-GTP, a PH domain, an N-terminal coiled-coil motif, a proline-rich C-terminal motif, and multiple FPPPP 46  motifs (Lafuente et al., 2004; Menasche et al., 2007). The FPPPP motifs allow RIAM to interact with the EVH1 domain of Ena/Vasp (Ena-vasodilator-stimulated phosphoprotein) and profilin. Ena/Vasp binds to the barbed ends of F-actin to prevent capping, while profilin primes actin monomers for addition to actin filaments by proteins such as WASP (Krause et al., 2003). Rap1/RIAM complexes also translocate to the plasma membrane to stimulate adhesion of Jurkat T cells via #1 and #2 integrins (Lafuente et al., 2004). Later, it was shown that RIAM activates "IIb#3 integrin by recruiting talin (Han et al., 2006). Talin is a cytoskeletal protein that binds to integrin #-subunit cytoplasmic tails (Tadokoro et al., 2003), disrupting the "-# subunit interaction and allowing the integrin to unclasp into its intermediate affinity state (Wegener et al., 2007). In addition to binding talin, RIAM constitutively associates with ADAP (adhesion and degranulation-promoting adapter protein) and SKAP-55 (55kDA Src kinases-associated phosphoprotein) scaffolding proteins (Wang and Rudd, 2008). Following TCR activation, ADAP/SKAP-55/RIAM recruit Rap1-GTP to the plasma membrane to activate integrins via talin and this is required for T cell adhesion to fibronectin and ICAM-1 in vitro (Menasche et al., 2007). Rap1 also regulates T cell adhesion and integrin avidity via protein kinase D1 (PKD1) (Medeiros et al., 2005). Rap1 binds to the PH domain of PKD1, which promotes activation of Rap1 and translocates Rap1 to the plasma membrane. Furthermore, Rap1/PKD1 complexes localize to #1 integrin tails. It is unclear how PKD1 activates Rap1, as its kinase activity did not appear to be necessary for Rap1 activation. PKD1 may act as an adaptor protein to bring Rap1 into proximity of RapGEFs, which might be localized to the leading edge of migrating cells. 1.6.4  Rap GTPases modulate cytoskeletal dynamics and polarity  Aside from regulating integrin activation, Rap is a key player in cytoskeletal dynamics as recent work has shown that Rap1 acts upstream of several key cytoskelsetal regulators (Bos, 2005). Rap1-GTP binds to and interacts with Afadin (AF-6), a profilin binding protein (Boettner et al., 2000; Boettner et al., 2003). Profilin primes monomeric G-actin for addition to actin filaments (Paavilainen et al., 2004). Rap1-GTP also binds the RIAM adaptor protein (Section 1.6.3), which interacts with Ena/Vasp and profilin, two actin regulatory proteins. RNA interference-mediated silencing of RIAM in Jurkat T cells prevents T cell adhesion and 47  spreading. Moreover, Rap1 fails to localize to the actin cytoskeleton at sites of adhesion in these Jurkat cells (Lafuente et al., 2004). Rap1 also acts upstream of several Rho family GTPase members to modulate cytoskeletal organization and polarity. Rap1-GTP binds to the RacGEFs Vav2 and Tiam1. This interaction is required for the translocation of these GEFs to cell protrusions where they can mediate localized activation of Rac1 and promote the formation of membrane protrusions as well as cell adhesion (Arthur et al., 2004). Rap1-GTP binds to the DH-PH domains of Vav2 and Tiam1, but does not affect the catalytic activity of these GEFs. Instead, this interaction is important for relocating Vav2 to sites of cell spreading. Rap1-GTP has also been reported to bind to the TSS (Tiam-STEF-SIF) homology domain of Tiam1, which results in the activation of Rac1 (Zaldua et al., 2007). Rap1 and Tiam1 establish cell polarity by regulating the localization of the Par3/Par6 polarity complex in migrating T cells (Gerard et al., 2007). Gerard et al. reported that chemokine receptor signaling results in localized activation of Rap1, which binds and recruits Tiam1. Rap1-GTP also recruits the Par polarity complex (Par3, Par6, PKC)) that binds to Tiam1 and activates it. Once activated, Tiam1 increase Rac1 activity, resulting in actin polymerization and lamellipodia formation at the leading edge. Disrupting Tiam1 results in loss of Rap1- and chemokine-mediated polarization and migration. Activation and recruitment of Cdc42 to the leading edge is needed for proper activation of the Par polarity complex in lymphocytes (Krummel and Macara, 2006), and this is regulated by Rap1-GTP in migrating T cells (Gerard et al., 2007). Consistent with this idea, Rap1b is necessary for localizing Cdc42 and the polarity complex in neurons (Schwamborn and Puschel, 2004). This is important for establishing cellular asymmetry in growing neurites in neuronal cells (Schwamborn and Puschel, 2004). How Rap1-GTP activates and localizes Cdc42 in lymphocytes is not known. In yeast, the Rap1 ortholog Bud1 establishes cell polarity during budding by interacting with Cdc24, a GEF specific for Cdc42 (Kang et al., 2001; Park et al., 2002). In endothelial cells, Rap1 at cell junctions mediates activation of Cdc42 via the Cdc42 GEF called FRC (Sato et al., 2005). Thus, Rap1 helps establish cell polarity by modulating the activity and localization of Cdc42 and polarity complex.  48  In Dictyostelium discoideum, Rap1-GTP binds to Phg2 at the leading edges of migrating cells. Phg2 is a serine threonine kinase that phosphorylates myosin II, allowing it to disassemble from actin filaments. This facilitates F-actin-mediated pseudopod formation at the leading edge (Jeon et al., 2007). Thus, Rap1 regulates both cytoskeletal dynamics and cell polarity, which are necessary for migration and activation.  1.7 B cell lymphomas 1.7.1  Overview  B cells are predisposed to developing genetic lesions leading to malignancy. They undergo genetic changes during rearrangement of their Ig gene segments during development and during receptor editing, as well as when they undergo SHM. Furthermore, activated B cells proliferate at very high rates and this proliferative capacity can be detrimental when deregulated. Hence, when a B cell does become aberrant because of genetic lesions, this can lead to the development of a lymphoma or leukemia. B cell leukemia is characterized by abnormal proliferation of B cells in the blood or bone marrow, whereas B cell lymphomas originate from abnormally proliferating cells in the SLOs, which then form solid tumors. B lymphomas will be the focus in the following sections because they are metastatic and are able to disseminate to various organs by employing the same mechanisms that are used by regular B cells as discussed previously (Section 1.3).  49  Type  Incidence Most Previous Ongoing Putative (% of Common Somatic Somatic Cell lymphomas) Translocation Hypermutation Hypermutation Origin  Hodgkin’s lymphoma  ~10  No consensus  Yes  No  GC Bcell Post-GC B-cell  Follicular lymphoma  ~22  bcl-2  Yes  Yes  GC Bcell  Mantle-cell lymphoma  ~6  cyclin D1  No  No  Unclear  Burkitt’s lymphoma  ~2.5  c-myc  Yes  No  GC Bcell  Multiple myeloma  ~10  cyclin D1 cyclin D3 c-Maf MMSET  Yes  No  Post-GC B-cell Plasma cell  Diffuse large B-cell lymphoma  ~30-40  bcl-6 bcl-2  Yes  Yes/No  GC Bcell  MALT lymphoma  ~7.6  bcl-2  Yes  Yes  Marginal zone B-cell  pax5  Yes  Yes  Post-GC B-cell  MLT/MALT1  Yes  Yes  Unclear  Lymphoplasmacytic ~1.5 lymphoma Marginal zone ~1.8 lymphoma Table 1.2  B cell lymphomas  List of mature B cell neoplasms, their percent incidence, common genetic defects, state of SHM, and their origin. Abbreviations: multiple myeloma SET domain (MMSET), mucosal associated lymphoid tissue translocation (MLT/MALT1) (adapted from Jaffe et al., 2001; Kuppers, 2005; Shaffer et al., 2002b)  50  1.7.2  Genetic causes of B cell malignancy  Almost all non-Hodgkin’s lymphomas (NHL), which represent the large majority of B cell lymphomas (~90%), are derived from GC B cells or B cells that have passed through the germinal center (see Table 1.2). The germinal center is a site of multiple genetic events such as Ig class-switch recombination (CSR) and SHM, which lead to mutations or chromosomal translocations. GC B cells are therefore highly susceptible to genetic lesions caused by double-stranded breaks that lead to non-homologous end-joining repair and subsequent chromosomal translocations (Fugmann et al., 2000). These translocations can lead to the loss of regulatory sequences of genes that control proliferation, differentiation, and survival. Translocation that replaces the endogenous regulatory sequence of these genes with an inappropriate constitutive regulatory sequence, could lead to deregulated proliferation and oncogenic transformation. A classical translocation event involved in B cell lymphoma development is t(14;18), which places the gene encoding the anti-apoptotic, pro-survival protein Bcl-2 under the control of the Ig heavy locus (Shaffer et al., 2002b). Thus allowing these cells to survive indefinitely. Another common translocation is t(11;14), which places the cyclin D1 gene under the Ig heavy locus, or sometimes the Ig light chain locus. These cells will then proliferate uncontrollably. In addition to CSR, which generates chromosomal breaks, SHM can introduce nucleotide exchanges in the 5’ ends of various genes. This results in the deregulation of gene expression, and can lead to oncogenic transformation when this occurs in the regulatory regions of genes such as c-myc and Pim1, both which are proto-oncogenes that are commonly deregulated in lymphomas (Pasqualucci et al., 2001). Thus a consequence of BCR editing, which increases Ig affinity for antigen and provides additional functionality via class-switch recombination, is the increased possibility of genetic mutations being introduced into the regulatory regions of proto-oncogenes, such that the B cell becomes malignant. 1.7.3  B cell lymphoma development  Gene rearrangement and SHM induce genetic mutations that deregulate three cellular processes important for B cell lymphoma development: cell cycle deregulation (proliferation), increased cell survival, and blocked terminal differentiation into plasma cells (Shaffer et al., 2002b). Enhanced cell growth and proliferation are two characteristics of GC 51  B cells (Klein and Dalla-Favera, 2008). Consistent with this, GC B cells have high levels of cell cycle progression genes such as cell-division cycle 2 (CDC2), polo-like kinase (PLK), and budding uninhibited by benzimidazoles 1 homolog (BUB1) (Rosenwald et al., 2002; Shaffer et al., 2001). One example of the loss of cell cycle control is the overexpression of cMyc. In many B cell lymphomas, such as diffuse large B-cell lymphoma and Burkitt’s lymphoma, genetic mutations upregulates the expression of c-Myc, which is normally low in these cells. Myc is a prototypic oncogene. It is a transcription factor that turns on the expression of multiple proliferative genes, including CDC2, PLK, and BUB1, thereby causing cells to hyper-proliferate (Meyer and Penn, 2008). Moreover, c-Myc can enhance protein translation and increase DNA replication, enhancing the growth and proliferation of the malignant cells (Cole and Cowling, 2008). Another program that is deregulated in malignant B cells is apoptosis. GC B cells that have non-functional BCRs or BCRs that have decreased affinity for antigen following SHM are destined to die by apoptosis. However, many B cell lymphomas have mutations that promote their survival by increasing the expression of pro-survival genes such as Bcl-2, Bcl-XL, A1, and MALT1 (Kuppers, 2005). Overexpression of these genes is often caused by translocation to the Ig heavy locus. Mutations in the NF-%B pathway can also promote the survival of B cell lymphomas by turning on anti-apoptotic genes (Karin and Lin, 2002). Genetic lesions that affect this pathway can include inactivating mutations in I%B, the inhibitory subunit of NF-%B, or overexpression of upstream activators of NF-%B such as MALT1, Bcl-10, or CARMA1. Overexpression of NF-%B subunits such as c-Rel can also deregulate this pathway, resulting in oncogenic transformation (Cabannes et al., 1999; Lucas et al., 2001; Rosenwald et al., 2002). Lastly, mutations that block terminal differentiation of B cells so that they remain in a proliferative state promote the development of B cell lymphomas. Some NHLs exhibit Bcl-6 translocation. Bcl-6 is an important transcriptional regulator of the differentiation and proliferation of GC B cells (Dalla-Favera et al., 1999; Staudt et al., 1999). Bcl-6 represses genes that are involved in the differentiation of B cells into non-dividing plasma cells (Shaffer et al., 2000). In particular Blimp-1 expression is repressed by Bcl-6. Blimp-1 itself is a repressor of genes expressed in mature B cells (e.g. CD79b, CD19) such that it drives the 52  differentiation of GC B cells into plasma cells (Shaffer et al., 2002a). Normally, following BCR and CD40 signaling in the germinal center, B cells downregulate Bcl-6 expression whereas Blimp-1 expression increases so that the cell can differentiate into a plasma cell. Deregulation of Bcl-6 expression will prevent this from happening and the cell will continue to proliferate. Another protein that is repressed by Bcl-6 is an inhibitor of the cyclindepenent kinases (CDK1), p27Kip1. Loss of p27Kip1 function is a common feature of many cancers and promotes proliferation (Chu et al., 2008b). 1.7.4  B cell lymphoma dissemination  B cell lymphomas are able to spread throughout the body and many organs. Their ability to disseminate is regulated by the same mechanisms that control B cell homing and trafficking during homeostasis (Pals et al., 2007). Some lymphomas spread quickly, such as mantle-cell lymphoma, which is able to disseminate throughout the body right at onset. Some lymphomas such as Burkitt’s lymphoma and diffuse large B-cell lymphoma are confined initially but disseminate at later stages of the disease (Pals et al., 2007). Depending on the type of lymphoma, some will preferentially disseminate to sites of inflammation and trauma because they express adhesion molecules and chemokine receptors that preferentially respond to inflammatory cues (Pals et al., 2007). Other lymphomas can only disseminate and colonize similar types of tissues that they originally developed in. For example, lymphomas that arise in mucosal sites (such as the gastrointestinal tract) can only home to other mucosal sites and not to sites such as the skin. Likewise, lymphomas that arise in the skin cannot home to mucosal areas (Drillenburg and Pals, 2000). This likely reflects the adhesion molecules and chemokine receptors that these lymphomas express. For example, mantle cell lymphoma usually express L-selection and "4#7 integrin, which allows them to home to mucosal areas in the digestive track (Geissmann et al., 1998). Similarly, mucosal associated lymphoid tissue (MALT) tumors and follicular lymphoma of the gastrointestinal track express "4#7 integrins and home only to mucosal sites in the gut (Bende et al., 2003). In contrast, a subset of mantle-cell lymphoma that do not express the gut-targeting "4#7 integrin preferentially home to peripheral lymph nodes (Pals et al., 1994). Moreover, these cells have a higher expression of CCR7 and CXCR4, the receptors for the chemokines CCL19/21 and CXCL12 respectively, which are produced by peripheral lymph node stromal  53  cells (Corcione et al., 2004; Lopez-Giral et al., 2004). Similarly, certain MALT lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma have high expression of CXCR4 and CXCR5, and this correlates with their ability to home to peripheral lymph nodes (Burger and Kipps, 2002; Husson et al., 2002; Lopez-Giral et al., 2004; Trentin et al., 2004). Thus, lymphomas specifically home to certain organs depending on the expression of specific chemokine receptors and integrins. Similar to how lymphomas home to peripheral sites, multiple myelomas (MM) derived from malignant plasma cells are able home to the bone marrow due to their selective expression of adhesion molecules and chemokine receptors that target these cells to the bone marrow (Hideshima et al., 2007). Normally, differentiated plasma cells downregulate their expression of peripheral lymph node homing receptors such as CXCR5 and CCR7, while upregulating their expression of CXCR4, which allows them to respond to CXCL12 secreted by bone stromal marrow cells (Hargreaves et al., 2001; Hauser et al., 2002; Nakayama et al., 2003). MM use the same mechanism to invade the bone marrow. CXCL12 increases transendothelial migration of MM and increases adhesion to VCAM-1 (Sanz-Rodriguez et al., 2001). Once within the bone marrow, adhesion of plasma cells to the bone marrow stroma induces the stromal cells to secrete IL-6, which promotes the survival of plasma cells (Kawano et al., 1995; Roldan et al., 1992b; Uchiyama et al., 1993). The integrin VLA-4 is necessary for adhesion of plasma cells to the bone marrow stroma and for their survival (Cassese et al., 2003; Roldan et al., 1992a). In summary, malignant transformation of B cells into lymphomas requires deregulation of the cell cycle, induction of anti-apoptotic programs, and blocking terminal differentiation. The dissemination of lymphomas employs the same integrin- and chemokine-dependent mechanisms used by normal B cells during their course of immunosurveillance. Therefore, much of what is learned about normal B cell trafficking should be applicable to malignant B cell invasion, and vice versa.  1.8 Rationale, objectives, and hypothesis Changes in B cell morphology are important for the function of B cells. B cell trafficking during immunosurveillance is dependent on changes in cell shape and polarization. B cells  54  are activated by chemokines and adhere to endothelial cells of the lymphoid organs via their integrins. This is followed by subsequent changes in the cytoskeleton and formation of membrane processes such as filopodia and lamellipodia, which allow the cell to crawl and extravasate. When encountering antigen bound on the surfaces of APCs, B cells undergo a biphasic response when sampling antigen on antigen presenting cells. The first phase involves B cell polarization and spreading after the initial BCR engagement. This is then followed by B cell contraction of these projections, which gathers membrane bound antigens into a cSMAC and leads to the formation of a B cell immune synapse. Particulate antigens may also induce B cell cytoskeletal rearrangements such that the B cell wraps itself around the antigen (i.e. bacteria) because the epitopes are immobile. Although changes in B cell morphology are clearly important for B cell activation, the signaling pathways that regulate B cell adhesion, spreading, migration, and immune synapse formation are not fully understood. Thesis Aim: To investigate how BCR signaling and integrin signaling regulate B cell morphological changes. Hypothesis: Activation of the Rap GTPases regulates B cell morphology, which is important for cell spreading, immune synapse formation, responses to particulate antigens, and the dissemination of B cell lymphoma. Specific Aims: 1.  Determine whether Rap activation is required for B cell spreading in response to BCR signaling and integrin signaling  2.  Determine whether Rap activation is required for immune synapse formation  3.  Determine whether Rap activation is required for cytoskeletal reorganization and prolonged signaling in response to particulate antigen  4.  Determine whether Rap activation is required for lymphoma dissemination in vivo  55  1.9 References Abram, C.L., and Lowell, C.A. (2007). Convergence of immunoreceptor and integrin signaling. Immunol Rev 218, 29-44. Abram, C.L., and Lowell, C.A. (2009). The ins and outs of leukocyte integrin signaling. Annu Rev Immunol 27, 339-362. 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Curr Opin Cell Biol 16, 99-105.  83  2. The Rap GTPases regulate B cell morphology, immune synapse formation and signaling by particulate B cell receptor ligands1 2.1 Introduction The ability of B cells to reorganize their cytoskeleton helps them scan for antigens (Ag). Within lymphoid organs, B cells move across the surfaces of follicular reticular cells, dendritic cells (DC), and follicular dendritic cells (FDC), spreading and extending membrane processes as they search for Ags present in the interstitial fluids or displayed on the surface of Ag-presenting cells (APCs) (Allen et al., 2007; Bajenoff et al., 2006; Hauser et al., 2007). Integrins mediate the adhesion of B cells to APCs and “outside in” integrin signaling may promote cell spreading and the extension of membrane processes. Cytoskeletal reorganization and integrin activation are particularly critical for APCdependent B cell activation, an important mode of B cell activation (Carrasco and Batista, 2006a). B cells can be activated by APCs that capture Ags or immune complexes and then display those Ags on their surface (Bergtold et al., 2005; Huang et al., 2005; Qi et al., 2006). When B cells contact membrane-bound Ags, B cell receptor (BCR) signaling initiates formation of an immune synapse (IS) in which a central cluster of BCRs is surrounded by a ring of integrins (Carrasco et al., 2004), similar to the T cell IS. Initial BCR signaling leads to F-actin-dependent spreading of the B cell (Fleire et al., 2006), allowing additional BCRs to bind Ag. The Ag-bound BCRs are then concentrated into a central supramolecular activation cluster (cSMAC). BCR signaling also activates the lymphocyte functionassociated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins via “inside-out” signaling that promotes integrin clustering as well as conformational changes that increase their affinity for ligands. Once activated integrins bind ligands on the APC, they are organized into a peripheral pSMAC surrounding the cSMAC. This integrin-mediated attachment prolongs the B cell:APC interaction and increases the B cell:APC contact area such that more Ags can be bound by BCRs and then concentrated into the cSMAC. In this 1  A version of this chapter has been published. Lin, K.B.L., Freeman, S.A., Zabetian, S., Brugger, H., Weber, M., Lei, V., Dang-Lawson, M., Tse, K.W.T., Santamaria, R., Batista, F.D., and Gold, M.R. (2008) The Rap GTPases regulate B cell morphology, immune synapse formation, and signaling by particulate B cell receptor ligands. Immunity 28(1): 75-87.  84  way integrin engagement reduces the amount of Ag required for B cell activation (Carrasco and Batista, 2006b; Carrasco et al., 2004). Cytoskeletal reorganization and cell spreading may also be important for the activation of B cells by particulate Ags with repeating epitopes (e.g. bacteria, fungi). Unlike APC-associated Ags, epitopes on particulate Ags may not be mobile, precluding IS formation. Instead, B cells extend membrane processes around model particulate Ags such as Ag-coated beads (Vidard et al., 1996). This polarized cell spreading increases the contact area and may allow more BCRs to bind epitopes on the particulate Ag. The signaling pathways that regulate B cell morphology and cytoskeletal organization are not fully elucidated. The Rap1 GTPases (Rap1A and Rap1B) regulate actin dynamics, cell polarity, integrin activation, and cell migration by controlling the actions of effector proteins such RapL, Rap1-GTP-interacting adaptor molecule (RIAM), and the Rac GTPase activators Vav2 and TIAM1 (Arthur et al., 2004; Bos, 2005; Han et al., 2006; Katagiri et al., 2003; Kinashi, 2005; Lafuente et al., 2004; Stork and Dillon, 2005). Although less well characterized, Rap2 has also been implicated in cell migration (McLeod et al., 2002; Miertzchke et al., 2007). Consistent with the idea that the active GTP-bound form of Rap promotes actin polymerization, we showed that phorbol ester-induced increases in total Factin content in the A20 B cell line are substantially reduced when Rap activation is blocked (McLeod et al., 2004). Based on these findings we hypothesized that activation of the Rap GTPases plays a critical role in B cell spreading, polarization, and IS formation. We have previously shown that the BCR and chemoattractant receptors activate Rap1 and Rap2 in B cells and that Rap activation is required for these receptors to induce integrinmediated adhesion (Durand et al., 2006; McLeod et al., 2002). Moreover, expressing a Rapspecific GTPase-activating protein (GAP) that converts Rap1 and Rap2 (referred to collectively as Rap) to the inactive GDP-bound form blocks chemoattractant-induced B cell migration (Durand et al., 2006; McLeod et al., 2002). B cells lacking the Rap effector RapL also exhibit decreased adhesion and migration as well as impaired in vivo homing to lymphoid organs (Katagiri et al., 2003; Katagiri et al., 2004).  85  In addition to cell adhesion and migration, Rap1 regulates cell polarity. Activated Rap1 is at the leading edge of motile cells and is important for chemokine-induced T cell polarization (Bivona et al., 2004; Katagiri et al., 2003; Shimonaka et al., 2003). In S. cerevisiae, the Rap1 ortholog Bud1 (Rsr1) determines the site at which budding occurs by initiating F-actin assembly and by directing polarized vesicle trafficking toward the growing bud (Kang et al., 2001). T cell IS formation also involves polarized vesicle trafficking toward the IS and employs evolutionarily-conserved polarity proteins (Krummel and Macara, 2006; LudfordMenting et al., 2005). Activated Rap1 binds the Par3-Par6-aPKC polarity complex in T cells (Gerard et al., 2007) and has been implicated in trafficking of LFA-1-containing vesicles to the T cell IS (Katagiri et al., 2006). The role of Rap GTPases in establishing B cell polarity during IS formation or when B cells bind particulate Ags has not been assessed. In this report we show that Rap activation plays a key role in B cell spreading, BCR-induced IS formation, and the ability of particulate BCR ligands to induce localized F-actin assembly and membrane process extension. We also show that Rap activation is needed for optimal signaling responses to particulate BCR ligands.  2.2 Results 2.2.1  BCR- and LFA-1-induced B cell spreading  Cell spreading may enhance the ability of B cells to scan for Ags. To study signaling involved in B cell spreading, we plated B cells on surfaces coated with antibodies (Abs) to the BCR or the LFA-1 integrin. In this way the cells were passively captured by the immobilized Abs and cell spreading induced by clustering the BCR or LFA-1 could be assessed independently of adhesion. We found that IgG+ A20 B cell lymphoma cells spread dramatically when plated on immobilized anti-IgG or immobilized LFA-1 monoclonal Abs (mAbs) (Figures 2.1A,B and 2.2). Spread cells had an elongated or irregular shape with membrane extensions and a length >1.5 times the width. By these criteria, 45-75% of the cells spread when plated for 1-4 hr on plates coated with anti-IgG or anti-LFA-1 (Figures 2.1C,E and 2.2). Because these LFA-1 mAbs cluster LFA-1, but block the ligand binding site, LFA-1 clustering must be sufficient to induce cytoskeletal reorganization and B cell  86  spreading. A20 cells exhibited similar spread morphologies when plated on ICAM-1, the physiological ligand for LFA-1 (Figure 2.1D). Spreading was a specific response to engagement of the BCR or LFA-1. A20 cells plated on immobilized mAbs to Fc#RIIB or CD40 rarely spread (Figure 2.1A-C), even though they were captured by these Abs. Both BCR- and LFA-1-induced spreading were dependent on the actin cytoskeleton and were blocked when latrunculin A or cytochalasin D were used to depolymerize F-actin (Figure 2.1E). The Src family kinase inhibitor PP2 also inhibited BCR- and LFA-1-induced spreading (Figure 2.1E), consistent with the idea that Src kinases play a key role in regulating the actin cytoskeleton in lymphocytes (Badour et al., 2004). Other B cell lines, as well as activated splenic B cells, also spread on immobilized Abs to the BCR or LFA-1. WEHI-231 cells, which resemble immature B cells, spread when plated on immobilized anti-IgM, with many cells extending multiple membrane processes (Figure 2.3E). Anti-CD40/IL-4-activated murine splenic B cells also spread dramatically when plated on immobilized anti-IgM Abs, anti-LFA-1 mAb, or ICAM-1 (Figure 2.1F).  87  Figure 2.1  B cells undergo F-actin-dependent spreading on immobilized anti-Ig,  anti-LFA-1 or ICAM-1 (A) A20 cells plated on tissue culture wells coated with anti-IgG or mAbs against LFA-1 (TIB213), CD40, or Fc#RIIB (all at 2.63 $g/cm2). Phase-contrast microscopy, scale bar = 20 $m. (B) Scanning EM of A20 cells plated 16 hr on coverslips coated with 17.7 $g/cm2 anti-IgG, anti-LFA-1, anti-CD40, or anti-Fc#RIIB Abs. Scale bar = 10 $m. (C) A20 cells plated on wells coated with the Abs (2.63 $g/cm2) or 2% BSA as a control. The percent of adherent cells with an elongated or irregular shape with membrane processes and a length >1.5 times the width is shown. Each bar in (C)-(E) is the mean + SEM for >100 cells counted in each of 3-5 experiments. A series of images showing cells scored as spread or not spread is shown. (D) A20 cells plated on wells coated with BSA or 2.63 $g/cm2 ICAM-1. Scale bar = 20 $m. (E) A20 cells were treated with DMSO or 10 $M PP2, latrunculin A, or cytochalasin D for 30 min, then plated on wells coated with 2.63 $g/cm2 anti-IgG or anti-LFA-1. Statistical significance versus DMSO control using Student’s two-tailed t-test: *p <0.05; **p <0.01. (F) Murine splenic B cells that had been activated with anti-CD40 plus IL-4 were plated for 3 hr on wells coated with 4.2 $g/cm2 anti-IgM, anti-LFA-1 or ICAM-1. Scale bar = 20 $m. Insets are 3X higher magnification. For (D) - (F), similar results were obtained in 3 independent experiments.  88  89  Figure 2.2  A20 cell spreading can be induced by different anti-LFA-1 mAbs  A20 cells were plated for 1-4 hr on tissue culture wells coated with 2.63 $g/cm2 of the TIB213 or M17/4 anti-LFA-1 mAbs, both of which are blocking Abs that bind the LFA-1 !L chain. (A) The percent of spread cells was determined as in Figure 1. Each bar represents the mean + SD for >100 cells counted in each of two independent experiments. (B) Phase-contrast microscopy of A20 cells plated on anti-LFA-1 mAbs for 4 hr. Scale bar = 20 $m.  90  2.2.2  BCR- and LFA-1-induced B cell spreading depends on Rap activation  To assess the role of Rap activation in Ab-induced cell spreading, we blocked Rap activation by expressing RapGAPII, a Rap-specific GAP that converts Rap1 and Rap2 to the inactive GDP-bound form while having no effect on activation of the Rac1, Ras, or RhoA GTPases (McLeod et al., 2002; Mochizuki et al., 1999; Polakis et al., 1991). We have shown that expressing RapGAPII in the A20 and WEHI-231 B cell lines blocks BCR-induced activation of Rap1 and Rap2 without affecting the activation of JNK or p38 (Christian et al., 2003), kinases that are downstream of Rac in B cells. RapGAP expression has been widely used to block Rap activation (Mochizuki et al., 1999; Reedquist et al., 2000; Suga et al., 2001) and is more effective than single gene disruptions or knockdowns since Rap1A, Rap1B, Rap2A, Rap2B, and Rap2C are encoded by separate genes and appear to be functionally redundant in B cells (Duchniewicz et al., 2006). We also blocked Rap activation by expressing Rap1N17, a dominant-negative form of Rap1 that interferes with both Rap1 and Rap2 (Fu et al., 2007). We found that Rap activation was critical for B cell spreading. When plated on immobilized anti-IgG or anti-LFA-1, the number of RapGAPII-expressing cells exhibiting an elongated or spread morphology with membrane processes was substantially less than that for control cells (Figure 2.3A,B), even though the numbers of cells captured by the immobilized Abs were similar for the control and RapGAPII-expressing cells (data not shown). Rap1N17 expression also inhibited the spreading of A20 cells on anti-Ig and anti-LFA-1 Abs (Figure 2.3C). Although Rap1N17 expression did not reduce the number of A20 cells that spread to the same extent as RapGAPII, which inactivates Rap in an enzymatic manner, the mean area of Rap1N17 cells was ~50% that of control cells after 4 hr (Figure 2.4). Thus these two approaches showed that Rap activation is important for B cell spreading. Scanning electron microscopy (EM) showed that control A20 cells plated on anti-IgG or antiLFA-1 assumed an elongated morphology, with extensive, asymmetric membrane aprons and a somewhat flattened cell body (Figure 2.3D). In contrast, RapGAPII-expressing A20 cells retained a round cell body and the membrane aprons were uniformly distributed around the cell base (Figure 2.3D). Rhodamine-phalloidin staining showed that control cells formed multiple F-actin-rich processes when plated on immobilized anti-IgG Abs but often adopted an elongated morphology when plated on immobilized LFA-1 mAb (Figure 2.5), similar to 91  what was seen by scanning EM (Figure 2.1B). Rap activation was important for both these modes of spreading, as most RapGAPII-expressing A20 cells remained small and round, without large F-actin processes (Figure 2.5). Rap activation was also required for spreading and membrane process formation by WEHI231 B lymphoma cells and primary B cells. Control WEHI-231 cells underwent dramatic cell spreading and membrane process formation when plated on immobilized anti-IgM (Figure 2.3E) whereas RapGAPII-expressing WEHI-231 cells did not spread or form membrane processes. Similarly, Rap1N17 expression substantially reduced the number of activated murine splenic B cells that could spread on immobilized anti-LFA-1 (Figure 2.3F) and reduced the mean cell area by 50% (Figure 2.4). Thus blocking Rap activation with either RapGAPII or Rap1N17 inhibited the spreading of primary B cells and B cell lines.  92  Figure 2.3  BCR- and LFA-1-induced B cell spreading is dependent on Rap  activation (A) Vector control and RapGAPII-expressing A20 cells plated on wells coated with 2.63 $g/cm2 anti-IgG or anti-LFA-1 for 3 hr. Scale bar = 15 $m. (B) Spreading was quantified as in Figure 1. Each bar is the mean + SEM for >100 cells counted in each of 3-5 experiments. **p <0.01 compared to control cells. (C) A20 cells were transiently transfected with 0.5 $g pmaxEGFP plus 2 $g pcDNA3.1 (vector) or pcDNA3.1-Rap1N17, plated on coverslips coated with 5 $g/cm2 anti-IgG or anti-LFA-1 and imaged by fluorescence microscopy. The percent of EGFP+ cells that spread is shown. Each bar is the mean + SD for >100 cells counted in each of 2 experiments. *p < 0.05; **p <0.005 compared to control cells. Scale bar = 15 $m. (D) Scanning EM of control and RapGAPII-expressing A20 cells plated on coverslips coated with 17.7 $g/cm2 anti-IgG or anti-LFA-1 for 4 hr. Scale bar = 15 $m. (E) Control and RapGAPII-expressing WEHI-231 cells plated on wells coated with 0.26 $g/cm2 anti-IgM Abs for 3 hr. Scale bar = 20 $m. Similar results were obtained in 3 experiments. (F) The spreading of splenic B cells that had been activated with LPS plus IL-4 and transiently transfected with pmaxEGFP plus pcDNA3.1 or pcDNA3.1-Rap1N17 was analyzed as in (C). Each bar is the mean + SD for >100 cells counted in each of 2 experiments. *p < 0.05; **p <0.005 compared to control cells. Scale bar = 15 $m.  93  94  Figure 2.4  Rap1N17 inhibits B cell spreading  Cell area analysis was performed on images from the experiments depicted in Figures 2C and 2F. A20 cells or anti-CD40- plus IL-4-activated splenic B cells were transfected with EGFP or EGFP plus Rap1N17 and then plated on anti-IgG- or anti-LFA-1-coated coverslips for 4 hr. Each data point represents the mean + SD for the areas of >50 EGFP+ transfected cells imaged in 2 independent experiments. Cell areas were quantified using ImagePro software (Media Cybernetics).  95  Figure 2.5  BCR- and LFA-1-induced B cell spreading and F-actin reorganization is  dependent on Rap activation Control and RapGAPII-expressing A20 cells were plated on coverslips coated with (2.63 $g/cm2) anti-IgG or anti-LFA-1 (TIB213) for 4 hr. F-actin was visualized with rhodamine-phalloidin (red) and nuclei were stained with DAPI (blue). Scale bar = 20 $m. Similar results were obtained in 3 experiments.  96  Consistent with the finding that BCR- and LFA-1-mediated spreading was dependent on Rap activation, plating control A20 cells on wells coated with LFA-1 mAbs or with anti-IgG resulted in increased amounts of Rap1-GTP compared to cells plated on BSA (Figure 2.6). Expressing RapGAPII blocked both anti-LFA-1- and anti-Ig-induced Rap1 activation (Figure 2.6B). This supports the idea that the BCR and LFA-1 induced cell spreading via Rap activation.  Figure 2.6  Plating cells on anti-Ig or anti-LFA-1 induces Rap1 activation  Control and RapGAPII-expressing A20 cells were plated on BSA, 2.63 $g/cm2 LFA-1 mAb (TIB213 or M17/4) or 2.63 $g/cm2 anti-IgG for 5-30 min. Scanned X-ray film images were saved as TIFFs and relative amounts of Rap1-GTP were quantified using ImageJ. In (A) the Rap1-GTP amounts are normalized to the amount of total Rap1 in the cell lysates. Rap1-GTP levels in cells plated on BSA were defined as 1. Similar results were obtained in 4 experiments.  97  2.2.3  Rap activation is required for immune synapse pSMAC formation  B cell recognition of membrane-bound Ag leads to IS formation. BCR signaling induces rapid cell spreading and subsequent contraction that gathers Ag-bound BCRs into a cSMAC (Fleire et al., 2006). BCR signaling also triggers LFA-1 activation, leading to ICAM-1 binding and organization of LFA-1 into a pSMAC. pSMAC formation increases the contact area between the B cell and the Ag-bearing membrane, allowing more BCRs to bind Ag (Carrasco et al., 2004). The signaling pathways involved in Ag accumulation and pSMAC formation at the IS are poorly understood. Since Rap activation is required for BCR-induced integrin activation (McLeod et al., 2004) and for actin-dependent B cell spreading (Figure 2.3), we asked if Rap activation contributes to IS formation. We compared the ability of control and RapGAPII-expressing A20 cells to form an IS on lipid bilayers containing anti-$ light chain Abs as a surrogate Ag, with or without GPI-linked ICAM-1. In the absence of ICAM-1, the RapGAPII-expressing cells had a somewhat smaller contact area between the cell and the bilayer (Figure 2.7A,C) than the control cells. This was accompanied by a 50% decrease in the amount of Ag accumulated in the cSMAC (Figure 2.7A,D). Much more dramatic differences were seen when ICAM-1 was included in the bilayer. In the presence of ICAM-1, ~40% of the control cells formed pSMACs and this was accompanied by increased contact area with the bilayer (Figure 2.7A,C) as well as substantially greater Ag accumulation (Figure 2.7A,D) than in the absence of ICAM-1. Strikingly, the RapGAPII-expressing cells were virtually unable to form pSMACs (Figure 2.7A,B). This was accompanied by a significant (p < 0.01) reduction in the contact area between the B cell and the bilayer, compared to control cells (Figure 2.7A,C). As a consequence, Ag accumulation was greatly diminished when Rap activation was blocked (Figure 2.7A, D). Thus in addition to contributing to BCR-mediated Ag accumulation, Rap activation was critical for formation of LFA-1-containing pSMACs and for LFA-1 to enhance Ag accumulation at the IS.  98  Figure 2.7  Rap activation is important for IS formation  Vector control and RapGAPII-expressing A20 cells were allowed to settle onto lipid bilayers containing Alexa633-conjugated anti-$ light chain Ab (green) as a surrogate Ag, with or without Alexa532-conjugated ICAM-1 (red). After 30 min the spatial localization of the surrogate Ag and ICAM-1 was imaged by confocal microscopy. (A) Differential interference contrast (DIC), fluorescence, and interference reflection microscopy (IRM) images of representative cells from 2 independent experiments. Scale bar = 5 $m. (B)-(D), The percent of cells forming a pSMAC (B), the area of B cell contacts with the bilayer (determined by IRM) (C), and the relative amounts of Ag accumulated (expressed as the sum of FITC fluorescence) (D) are shown. Each data point is the mean + 95% confidence interval for 40-50 cells analyzed in 2 independent experiments. *p <0.05; **p <0.01; ***p <0.001.  99  2.2.4  Particulate BCR ligands induce the formation of F-actin-rich cups via Rap activation  Multivalent particulate Ags effectively activate B cells. Unlike soluble Ags, particulate Ags initiate BCR signaling at a focused contact site, thereby establishing a cell polarity that is accompanied by localized cytoskeletal reorganization and signaling (Batista et al., 2001). Because Rap1-GTP regulates cell polarity as well as F-actin organization, we asked whether Rap activation is needed for BCR-induced actin reorganization at the site of contact with anti-Ig-coated beads, a model particulate Ag. Binding of anti-Ig-coated beads to A20 cells or resting splenic B cells resulted in rapid (within 3 min) accumulation of F-actin at the contact site, followed by extension of F-actinrich membrane protrusions part way around the bead (Figure 2.8A,B). Both 3D reconstruction of confocal images (Figure 2.8C) and scanning EM (Figure 2.12C) show that the F-actin-rich membrane protrusions form a cup around the bead. Formation of these cups can also be induced by Ag-coated beads (Figure 2.8C,D). A20 cells expressing a dinitrophenyl (DNP)-specific membrane IgM formed F-actin-rich cups at the site of contact with DNP32-BSA-coated beads. The BCR accumulated at the bead:cell contact site (Figure 2.9A) and BCR-induced tyrosine phosphorylation (pTyr) was concentrated at the contact site in both A20 cells and splenic B cells (Figure 2.9B). In addition, there was dramatic relocalization of the Rap effector RapL to the bead contact site in splenic B cells (Figure 2.9C). Since RapL binds activated Rap, these data suggest that Rap-GTP is at the contact site. Formation of F-actin-rich cups was a specific, actin-dependent response to BCR engagement. Beads coated with Abs to CD40 or LFA-1 did not induce cup formation (Figure 2.10, 2.11C). Moreover latrunculin A, which caused almost complete loss of polymerized F-actin, prevented the formation of membrane cups at bead contact sites (data not shown).  100  Figure 2.8  B cells form F-actin-rich cups when they contact anti-Ig- or Ag-coated  beads F-actin was visualized with rhodamine-phalloidin (red) and nuclei were stained with DAPI (blue). DIC images of the same cells are shown on the left. Asterisks indicate position of the bead. In (A), (B), and (D), single confocal slices through the centers of representative cells are shown. (A) A20 cells mixed with anti-IgG-coated beads. (B) Resting splenic B cells mixed with anti-IgM-coated beads for 5 min. (C) 3-dimensional reconstructions of F-actin-rich cups. (D) A20 cells expressing a DNP-specific membrane IgM mixed with DNP32-BSA-coated beads for 20 min. Scale bars = 5 $m. Each panel is from one of 3 experiments with similar results.  101  Figure 2.9  BCR accumulation and BCR signaling at the site of bead:cell contact  (A) Vector control A20 cells expressing a transfected Ig"-YFP fusion protein were mixed with anti-Igcoated beads for 5 min. The localization of Ig"-YFP-containing BCRs (blue) was assessed by confocal microscopy. The position of the bead is indicated by an asterisk. (B) A20 cells and resting splenic B cells were incubated with anti-Ig-coated beads for 5 min before staining for F-actin (red), phosphotyrosine (pTyr, green) and nuclei (blue). Single confocal slices through the center of representative cells are shown. Each panel is from one of 3 experiments with similar results. Asterisks indicate position of the beads. (C) Anti-CD40- plus IL-4-activated splenic B cells expressing a RapL-EGFP fusion protein were imaged continuously by scanning fluorescence microscopy both before and after the addition of anti-Ig beads. Still images captured from the real time movies show cells at ~5 min after addition of the beads. RapL-EGFP was localized at the bead:cell contact site in >85% of the cells imaged (left and center panels; n = 60 bead:cell conjugates). Cells that did not bind beads were highly motile and the RapL-EGFP was localized in the rear portion of the cell. All scale bars = 5 $m.  102  Figure 2.10 Beads coated with anti-CD40 mAb do not induce cup formation Vector control A20 cells were mixed with beads coated with anti-CD40 (1C10 mAb) for 30 min. F-actin was visualized with rhodamine-phalloidin (red) and nuclei were stained with DAPI (blue). Single confocal slices through the center of the cell are shown. DIC images of the same cells are shown on the left. Two representative cells are shown. Similar results were obtained for >50 bead:cell conjugates imaged in 3 independent experiments. Asterisks indicate position of the beads. Scale bars = 5 $m.  103  Figure 2.11 Anti-Ig- and anti-LFA-1-coated beads induce Rap1 activation; antiLFA-1-coated beads do not induce cup formation (A,B) Vector control and RapGAPII-expressing A20 cells were mixed with anti-IgG beads or anti-LFA-1 (TIB213) beads for the indicated times before assaying for Rap1 activation (A) or Rap2 activation (B). Note that RapGAPII expression does not alter the amounts of total Rap1 or Rap2 in the cell lysates. (C) A20 cells were mixed with beads coated with anti-LFA-1 (TIB213) for 30 min. F-actin was visualized with rhodamine-phalloidin (red) and nuclei were stained with DAPI (blue). Confocal and DIC images of the same cells are shown for 2 representative fields. F-actin-rich cups were formed in <3% of >300 bead:cell conjugates imaged. Asterisks indicate position of the beads. Scale bars = 10 $m.  104  105  We found that Rap activation is required for the cytoskeletal reorganization involved in forming F-actin-rich cups. Anti-Ig beads induced robust activation of Rap1 in A20 cells, and this could be blocked by RapGAPII expression (Figure 2.11A). Importantly, confocal microscopy of bead:cell conjugates showed that preventing Rap activation substantially decreased the percent of bead-bound cells that formed F-actin-rich cups, and also slowed the kinetics of cup formation (Figure 2.12A,B). The RapGAPII-expressing A20 cells bound the anti-Ig-coated beads at the same rate as the control cells (Figure 2.13C) but fewer of these cells showed increased F-actin at the bead:cell contact site or formed F-actin-rich cups (Figure 2.12A,B). This difference was most evident at early times (e.g. 3 min), but at all time points up to 2 hr the RapGAPII-expressing cells formed F-actin-rich cups less frequently than control cells (Figure 2.12B). Scanning EM showed that control A20 cells extended membrane processes around the beads whereas many RapGAPII-expressing cells bound anti-Ig beads but did not extend membrane processes (Figure 2.12C). Similarly, realtime imaging showed that control cells extended dynamic membrane processes that contacted the anti-Ig beads (Movie S1) whereas RapGAPII-expressing cells that bound beads did not exhibit this extensive membrane dynamics (Movie S2). Expressing the dominant negative Rap1N17 protein also reduced the ability of both A20 cells and splenic B cells to form Factin-rich cups with anti-Ig-coated beads (Figure 2.12D,E). Thus Rap activation is important for BCR engagement to initiate localized F-actin accumulation and the extension of membrane processes that increase the contact area with a particulate BCR ligand. Interestingly, anti-LFA-1-coated beads activated Rap1 and Rap2 in A20 cells but did not induce cup formation (Figure 2.11C), either because they do not activate Rap to the same extent as anti-Ig beads or because other BCR-specific signals are also required for forming Factin-rich cups.  106  Figure 2.12 Rap activation is important for formation of F-actin-rich cups at contact site with anti-Ig beads (A) Vector control and RapGAPII-expressing A20 cells were mixed with anti-IgG-coated beads, then stained for F-actin (red) and nuclei (blue). Single confocal slices through the centers of representative cells are shown. Asterisks indicate position of the bead. (B) The percent of bead:cell conjugates in which F-actin-rich cups formed at the contact site was determined by confocal microscopy. Each data point is the mean + SEM for >30 bead:cell conjugates imaged in each of 3-4 experiments. *p <0.05, **p <0.01 compared to control cells. Two independent sets of experiments are combined in the line graph. (C) Scanning EM images of control and RapGAPII-expressing A20 cells mixed with anti-Ig beads for 20 min. (D, E) A20 cells or anti-CD40- plus IL-4-activated splenic B cells were transiently transfected with pmaxEGFP plus pcDNA3.1 or pcDNA3.1-Rap1N17 before being mixed with anti-Ig beads. The percent of bead-bound EGFP+ cells forming F-actin-rich cups is shown in the graphs. For A20 cells, each bar is the mean + SEM for >30 bead:cell conjugates imaged in each of 4 experiments. *p <0.05, **p <0.005 compared to control cells. For splenic B cells, several independent experiments were performed and >45 bead:cell conjugates were imaged for each point. Scale bars = 5 $m.  107  108  Figure 2.13 Formation of bead:cell conjugates does not depend on Rap activation (A) Beads coated with anti-IgG-FITC were incubated for 2 h with control A20 cells. FITC fluorescence and forward scatter (FSC) allow bead:cell conjugates (in box) to be distinguished from unbound beads and from cells that have not bound beads. (B) Beads coated with anti-IgG-FITC were incubated for 2 h with vector control or RapGAPII-expressing A20 cells. The percent of cells forming conjugates with the anti-IgG-FITC beads (indicated by box) is shown. (C) Vector control and RapGAPII-expressing A20 cells were incubated with anti-IgG-FITC-coated beads for the indicated times and the percent of cells forming bead:cell conjugates was determined as in (B). Each data point represents the mean + SEM for 3 experiments in which 10,000 events were collected.  109  110  2.2.5  Rap activation is important for BCR signaling initiated by anti-Ig beads  Since IS formation promotes sustained TCR signaling (Gomez et al., 2006), we asked if Rapdependent formation of F-actin-rich cups was important for BCR signaling in response to particulate Ags. We stimulated control and RapGAPII-expressing A20 cells with beads coated with anti-IgG-FITC and then performed intracellular staining to quantify total pTyr levels as well as phosphorylation of ERK and Akt. Gating on forward scatter and FITC fluorescence allowed us to assess BCR signaling in bead:cell conjugates (Figure 2.13A). The percent of cells that formed bead:cell conjugates was similar for control and RapGAPIIexpressing cells (Figure 2.13B,C). Staining for pTyr revealed only small differences between control and RapGAPII-expressing A20 cells stimulated with anti-Ig beads (Figures 2.14A, 2.15A). In both cell populations, an increase in the number of cells with pTyr levels above those in unstimulated cells was seen at 5 min and was sustained for 2 hr. Slightly fewer RapGAPII-expressing cells had increased pTyr levels (Figures 2.14A) but these differences were not highly significant (p values of 0.15 to 0.63). Moreover, the mean fluorescence intensity of the pTyr signal was identical in the control and RapGAPII cells that did respond (Figure 2.15A). Thus the ability of anti-Ig beads to induce tyrosine phosphorylation, a proximal BCR signaling event, was not highly dependent on Rap activation. In contrast, blocking Rap activation impaired the ability of anti-Ig beads to induce sustained phosphorylation of ERK and Akt. Intracellular staining showed that ERK phosphorylation induced by anti-Ig beads was relatively unaffected at 5 min and 30 min, but at 1 hr and 2 hr both the magnitude of ERK phosphorylation and the number of responding cells were reduced when Rap activation was blocked (Figure 2.14C). Immunoblotting also showed that sustained ERK phosphorylation required Rap activation. The ability of anti-Ig beads to induce Akt phosphorylation was also dependent on Rap activation at later time points (Figure 2.14D). At 5 min after the addition of anti-Ig beads, RapGAPII-expressing cells had slightly reduced phospho-Akt (p-Akt) in some experiments, but not others. However at the 30-120 min time points, blocking Rap activation consistently reduced the number of cells with high amounts of p-Akt as judged by intracellular staining. Immunoblotting also showed that sustained Akt phosphorylation required Rap activation. The inability of anti-Ig beads to 111  induce sustained phosphorylation of ERK and Akt in RapGAPII-expressing cells was not due to a decreased ability of these cells to form stable bead:cell conjugates. The percent of cells binding anti-Ig-FITC beads was nearly identical in control and RapGAPII-expressing A20 cells at all time points from 5 min to 2 hr (Figure 2.13C). A striking observation was that Rap activation was important for sustained BCR signaling in response to particulate, but not soluble, surrogate Ag. RapGAPII expression did not reduce the ability of soluble anti-Ig Abs to induce phosphorylation of ERK or Akt (Figures 2.14B, 2.15B-D). The decreased phospho-ERK (p-ERK) and p-Akt responses induced by anti-Ig beads in RapGAPII-expressing A20 cells could be due to the decreased ability of these cells to accumulate F-actin and form cups at the contact site. Indeed, using latrunculin A to disrupt the actin cytoskeleton and prevent cup formation had the same effect as blocking Rap activation. In latrunculin A-treated cells stimulated with anti-Ig beads, sustained ERK phosphorylation at 60 and 120 min was reduced and Akt phosphorylation was reduced at all time points (Figure 2.14E). We showed this by immunoblotting since the amount of DMSO used to deliver 10 $M latruculin A interfered with FACS analysis. However, we could analyze Akt phosphorylation by FACS when 2.5 $M latrunculin A was used and found that this reduced the number of cells in which anti-Ig beads induced Akt phosphorylation (Figure 2.16). These data are consistent with the idea that Rap-dependent formation of F-actin-rich cups is important for signaling by particulate Ags.  112  Figure 2.14 Rap activation and F-actin are important for anti-Ig beads to induce phosphorylation of ERK and Akt (A) Control and RapGAPII-expressing A20 cells were mixed with anti-Ig-FITC-coated beads for 5-120 min and then analyzed by intracellular staining with anti-pTyr. FITC fluorescence and forward scatter were used to gate on bead:cell conjugates (see Figure S7). Cells with a staining intensity higher than that in 95% of unstimulated cells (0 min, no beads added), as indicated by the gates on the FACS plots in Figure S8A, were considered responding cells. The number of responding cells is expressed as percent of bead:cell conjugates. Each bar is the mean + SEM for 2000 conjugates analyzed in each of 3 experiments. (B) Control and RapGAPII-expressing A20 cells were stimulated with 1 $g/ml soluble anti-IgG and analyzed by intracellular staining with anti-p-ERK or anti-p-Akt Abs. Cells with a staining intensity higher than that in 95% of the unstimulated cells, as indicated on the FACS plots in Figures S8B and C, were considered responding cells. The number of responding cells is expressed as percent of all cells. Each bar is the mean + SEM for 10,000 cells analyzed in 3 experiments. Similar results were obtained by immunoblotting (Figure S8D). (C,D) Control and RapGAPII-expressing A20 cells were mixed with anti-Ig-FITC beads and analyzed by intracellular staining or by immunoblotting with anti-p-ERK (C) or p-Akt (D) Abs. Representative experiments are shown. The bar graphs show the number of responding cells expressed as percent of bead:cell conjugates. Each bar is the mean + SEM for 2000 conjugates analyzed in each of 3 experiments. *p <0.05, **p <0.01 compared to control cells. (E) A20 cells were treated with 10 $M latrunculin A or DMSO for 30 min and then mixed with anti-Ig beads. p-ERK and p-Akt levels were assessed by immunoblotting. Similar results were obtained in 3 experiments. For each panel, all lanes are from the same blot. Irrelevant intervening lanes were excised.  113  114  Figure 2.15 Rap activation is not essential for anti-Ig beads to induce protein tyrosine phosphorylation or for soluble anti-Ig to induce phosphorylation of ERK or Akt (A) Vector control and RapGAPII-expressing A20 cells were mixed with anti-Ig-FITC-coated beads as in Figure 7 and analyzed by intracellular staining with anti-pTyr. Bead-bound cells with a staining intensity higher than that of 95% of the unstimulated cells (0 min) were considered responding cells and are indicated by the gates on the histograms. In some experiments, e.g. the one shown here, fewer RapGAPII-expressing cells had increased pTyr amounts at 5 min, but this difference was not statistically significant (p = 0.47) when all experiments were combined (see Figure 7A). (B-D) Control and RapGAPII-expressing A20 cells were stimulated with 1 $g/ml soluble anti-Ig for the indicated times and then analyzed by intracellular staining or immunoblotting with Abs to p-ERK1/2 or pAkt. Data from representative experiments are shown.  115  116  Figure 2.16 Akt phosphorylation induced by anti-Ig beads is dependent on F-actin A20 cells were treated with 2.5 $M latrunculin A or DMSO for 30 min and then mixed with anti-Ig-FITCcoated beads. Intracellular staining was used to assess Akt phosphorylation. The data are presented as percent of bead bound cells with phospho-Akt staining higher than that of 95% of the unstimulated cells (no beads added). Each data point is the mean + SEM for 3 experiments in which 2000 conjugates were analyzed. *p <0.05 compared to DMSO-treated cells.  117  2.3 Discussion We have shown that the Rap GTPases link the BCR and LFA-1 to cell spreading and IS formation, morphological changes that promote Ag acquisition and facilitate the activation of B cells by membrane-bound Ag. We also showed that Rap activation and localized actin polymerization are required for optimal BCR signaling in response to particulate ligands. Thus Rap may play a key role in the activation of B cells by membrane-bound and particulate Ags. BCR- and integrin-induced spreading, which we showed are dependent on Rap activation, would enhance the ability of B cells to scan the surface of APCs for Ags. This may be particularly important for germinal center B cells, which compete for FDC-bound Ags that provide survival and differentiation signals. Indeed, germinal center B cells interacting with FDCs in vivo exhibit a spread morphology with multiple membrane processes (Allen et al., 2007; Hauser et al., 2007). We found that LFA-1 clustering led to Rap activation, a response that is also induced by ICAM-1 binding (Miertzchke et al., 2007). In addition to promoting cell spreading, Rap1GTP initiates inside-out signaling that leads to further integrin activation. This positive feedback loop could enhance the ability of B cells to adhere to ICAM-1-expressing cells and spread on their surface. The initial binding of small numbers of activated LFA-1 integrins to ICAM-1 would lead to increased Rap-GTP, which in turn would activate additional LFA-1 molecules. Eventually, sufficient LFA-1 engagement would be achieved to promote cell spreading. This may account for the ability of unstimulated A20 cells and splenic B cells to spread on ICAM-1. A key finding is that Rap activation is essential for B cells to form an IS. This likely reflects the dual role of Rap-GTP in promoting cytoskeletal reorganization that drives localized cell spreading and contraction and in activating integrins. By using lipid bilayers with mobile Ags and ICAM-1 to model IS formation, Batista and colleagues showed that B cells interacting with such membranes undergo rapid BCR-dependent spreading, followed by contraction and concentration of the Ag into a cSMAC (Fleire et al., 2006). At the same time, BCR-induced activation of LFA-1 and the subsequent binding of LFA-1 to ICAM-1  118  stabilizes the B cell:membrane interaction, increases the contact area, and greatly enhances Ag accumulation at the IS by promoting pSMAC formation (Carrasco and Batista, 2006b; Carrasco et al., 2004). We found that LFA-1-dependent pSMAC formation is strongly dependent on Rap activation. Consequently, blocking Rap activation resulted in a substantial reduction in the ability of the B cell to gather Ag into the cSMAC. A key function of the IS is to reduce the amount of Ag required for B cell activation by generating high local concentrations of Ag (Carrasco and Batista, 2006b; Carrasco et al., 2004). Since pSMAC formation and efficient Ag accumulation at the IS require Rap-GTP, Rap activation likely plays an important role in the activation of B cells by Ags that are present at low concentrations on the surfaces of APCs. The inability of RapGAPII-expressing A20 cells to form a pSMAC may reflect the decreased ability of the LFA-1 on these cells to bind ICAM-1 (McLeod et al., 2004). However, it is also possible that some LFA-1 molecules on RapGAPII-expressing A20 cells can bind ICAM-1 but the cells cannot organize them into a pSMAC. In T cells, the initial formation of the IS as well as maintenance of the pSMAC depends on actin polymerization (Dustin and Cooper, 2000) and this process could be controlled by Rap-GTP. Another striking observation was that Rap activation is required for sustained BCR-induced activation of ERK and Akt in response to anti-Ig-coated beads, a model particulate Ag. Since the duration of BCR signaling may be a critical determinant of B cell activation, the role of Rap-GTP in sustaining BCR-induced phosphorylation of ERK and Akt may be important for the activation of B cells by particulate or APC-bound Ags. Interestingly, Rap activation was not required for activation of ERK and Akt by soluble anti-Ig Abs, a uniformly distributed stimulus. Thus Rap-GTP plays a specific role in the ability of B cells to respond to spatially localized Ags. This may reflect the ability of Rap-GTP to promote localized assembly of cytoskeletal structures that establish cell polarity, for example at the leading edge of a migrating cell. The IS has been likened to the leading edge of a migrating cell (Krummel and Macara, 2006) and this analogy may extend to F-actin-rich cups that form when B cells bind particulate Ags. We found that Rap activation was important for optimal activation of ERK and Akt in response to a particulate Ag but that BCR-induced tyrosine phosphorylation was relatively 119  normal in RapGAPII-expressing cells. Thus Rap activation appears to play a more important role in the activation of downstream signaling pathways by particulate Ags that have immobile epitopes on their surface than in the activation of tyrosine kinases, a proximal BCR signaling event. Using latrunculin A to promote the depolymerization of F-actin also impaired the ability of anti-Ig beads to induce phosphorylation of ERK and Akt. This suggests that Rap-dependent formation of an F-actin-rich structure (e.g. cups) may be important for sustained activation of ERK and Akt by a particulate Ag. Consistent with this idea, RNAi knockdown of dynamin 2 or HS1 prevents F-actin accumulation at the T cell IS, another polarized signaling structure, and this results in a failure to sustain TCR signaling (Gomez et al., 2005; Gomez et al., 2006). Polymerized F-actin could act as a platform that helps assemble, localize, and then maintain signaling complexes such that signaling can be sustained. Actin filaments may also stabilize aggregated lipid rafts that contain signaling proteins (Gupta et al., 2006). The roles of individual Rap effectors in B cell spreading, IS formation, and the formation of F-actin-rich cups remain to be determined. Rap1-GTP can promote actin polymerization by recruiting TIAM1 and Vav2, activators of the Rac and Cdc42 GTPases (Arthur et al., 2004; Gerard et al., 2007). Rac and Cdc42 activate the Arp2/3 complex, which initiates actin polymerization. Rap-GTP also binds AF-6 and RIAM, both of which bind profilin (Boettner et al., 2000; Lafuente et al., 2004). Profilin primes actin monomers for addition to actin filaments (Paavilainen et al., 2004). Myosin II, which regulates actin-dependent contraction, is also a target of Rap signaling (Jeon et al., 2007). In addition, integrin clustering driven by Rap-GTP may allow integrins to nucleate the assembly of signaling complexes that regulate the cytoskeleton. Whether clustered integrins can do this even in the absence of ligand binding is not known. In summary, we have shown that the Rap GTPases are key regulators of cytoskeleton and membrane dynamics in B cells and that this may have important implications for B cell activation. Further work is needed to elucidate the relative roles of Rap1 and Rap2 in these processes. Although Rap1 and Rap2 bind overlapping sets of effector proteins, differences in the relative affinities of Rap1-GTP and Rap2-GTP for individual effector proteins such as RapL (Miertzchke et al., 2007), as well as differences in the spatial or temporal pattern of 120  Rap1 activation versus Rap2 activation (Ohba et al., 2000), could allow Rap1 and Rap2 to control different aspects of cytoskeletal organization and membrane dynamics.  2.4 Experimental procedures 2.4.1  Cells  Several independent populations of A20 and WEHI-231 cells (ATCC) stably transduced with pMSCVpuro (BD Biosciences Clontech) or pMSCVpuro-FLAG-RapGAPII (Durand et al., 2006; McLeod et al., 2004) were used. A20 cells expressing DNP-specific human IgM (Lankar et al., 2002) were a gift from D. Lankar (Institut Curie, Paris, France). Murine splenic B cells (>95% CD19+) were isolated by depleting non-B cells (Durand et al., 2006). The UBC Animal Care Committee approved all protocols. Where indicated, splenic B cells were activated for 48 hr with 5 ng/ml IL-4 (R&D Systems) plus 5 $g/ml LPS or CD40 mAb. Cells were transiently transfected by nucleofection (Amaxa) using Amaxa kit V for A20 cells or a splenic B cell transfection kit and used 24 hr later. 2.4.2  Cell spreading  Tissue culture plates or glass coverslips were coated overnight at 4°C with goat anti-mouse IgG or IgM Abs (Jackson Immunoresearch), ICAM-1-Fc fusion protein (Stemcell Technologies), or rat mAbs against mouse !L integrin (TIB213 [ATCC], M17/4 [eBioscience]), CD40 (1C10 [eBioscience]), or Fc#RIIB (2.4G2 [ATCC]), and then blocked with PBS/2% BSA for 2 hr. Cells (105 in 0.5 ml culture medium) were plated on the coated surfaces and incubated at 37°C. Where indicated, cells were treated with PP2, latrunculin A or cytochalsin D (Calbiochem-EMD). Cells scored as spread had an elongated or irregular shape with membrane processes and a length >1.5 times the width. 2.4.3  Bead:cell conjugates  4 x 107 4.5-$m polystyrene beads (Polyscience Inc.) were mixed with 2 $g anti-IgG, antiIgM, anti-LFA-1, or DNP32-BSA (Biosearch Technologies) for 1 hr at 37°C, blocked with 2% BSA for 30 min, and resuspended in modified HEPES-buffered saline (mHBS; see supplemental data). To form bead:cell conjugates, 2.5 x105 cells in 0.1 ml mHBS/1% FCS were mixed with 25 $l (5 x 105) beads to yield a 2:1 bead:cell ratio and incubated at 37°C. 121  At the end of the incubation, the cells and beads were pipetted onto coverslips coated with 0.1% poly-L-lysine (PLL). Cells were fixed by adding an equal volume of 8% paraformaldehyde (PFA) and imaged by confocal microscopy or scanning EM. 2.4.4  Scanning EM  A20 cells were plated on glass coverslips coated with immobilized Abs or were mixed with anti-Ig beads and then added to PLL-coated coverslips. After fixing the cells with glutaraldehyde, the samples were processed as described in the supplemental data. Images were obtained using a Hitachi S4700 scanning electron microscope (UBC BioImaging Facility). 2.4.5  Confocal microscopy  Cells were plated on glass coverslips coated with immobilized Abs or mixed with anti-Ig beads and then adhered to PLL-coated coverslips before adding an equal volume of 8% PFA. Fixed cells were washed with TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl), permeabilized with TBS/0.5% Triton X-100 for 10 min, and blocked with TBS/0.1% Triton X-100/2% BSA. F-actin was visualized using rhodamine-phalloidin. Coverslips were treated with ProLong Gold anti-fade reagent containing DAPI (Molecular Probes-Invitrogen), mounted onto slides, and imaged using an Olympus IX81/Fluoview FV1000 confocal microscope. Images and 3D reconstructions were processed using Olympus Fluoview 1.6 software. 2.4.6  Rap activation  A20 cells (5 x 106) in 0.8 ml mHBS were added to 6-well plates coated with anti-IgG or antiLFA-1 Abs. Reactions were stopped by adding 0.2 ml of cold 5X Rap lysis buffer (see supplemental data). Alternatively, 5 x 106 cells in 0.4 ml mHBS were mixed with 0.1 ml (107) anti-IgG or anti-LFA-1 beads and reactions were stopped with 0.5 ml 2X lysis buffer. A GST-RalGDS fusion protein was used to precipitate Rap1-GTP and Rap2-GTP, which were detected by immunoblotting (McLeod et al., 2002). 2.4.7  Intracellular staining analysis of signaling  A20 cells (106) in 0.5 ml mHBS were incubated with soluble anti-IgG or with 2 x 106 beads coated with FITC-conjugated goat anti-mouse IgG. Reactions were stopped with 0.5 ml 8% 122  PFA. Fixed cells were pelleted, permeabilized with 90% methanol for 10 min on ice, washed, and then incubated with rabbit Abs to pTyr (BD Pharmingen), p-ERK, or p-Akt (Ser473) (Cell Signaling Technologies). Donkey anti-rabbit IgG-Alexa647 or -Alexa488 was used for detection by a BD Bioscience LSRII cytometer. FlowJo software (Tree Star) was used for analysis. 2.4.8  Immune synapse (IS) formation on lipid bilayers  Lipid bilayers containing Alexa633-conjugated anti-mouse $ chain Abs and GPI-linked Alexa532-conjugated ICAM-1 were prepared as described (Carrasco et al., 2004). A20 cells were allowed to settle onto the lipid bilayers for 30 min. IS were imaged by interferencereflection microscopy (IRM) and fluorescence microscopy using a Zeiss Axiovert LSM 510META inverted microscope with a 63X oil immersion objective. Volocity software (Improvision) was used for image analysis. 2.4.9  Statistics  Student’s two-tailed t-test was used to compare sets of matched samples.  2.5 Supplemental movies Movie S1. Membrane dynamics of control A20 cells bound to anti-IgG-coated beads. Vector control A20 cells were mixed with anti-IgG-coated beads, plated on PLL-treated glass bottom dishes, and analyzed by real-time confocal microscopy for 15 min. Link: http://download.cell.com/immunity/mmcs/journals/10747613/PIIS1074761307005845.mmc2.avi Movie S2. Membrane dynamics of RapGAPII-expressing A20 cells bound to anti-IgGcoated beads. RapGAPII-expressing A20 cells were mixed with anti-IgG-coated beads, plated on PLL-treated glass bottom dishes, and analyzed by real-time confocal microscopy for 20 min. Link: http://download.cell.com/immunity/mmcs/journals/10747613/PIIS1074761307005845.mmc3.avi  123  2.6 Supplemental procedures Solutions. Modified HEPES-buffered saline (mHBS) used for stimulating cells consisted of 25 mM sodium HEPES pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 mg/ml glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 $M 2mercaptoethanol. For Rap activation assays, 0.2 ml 5X Rap lysis buffer (125 mM Tris-HCl pH 7.5, 25% glycerol, 2.5% Igepal (Sigma), 500 mM NaCl, 5 mM MgCl2, 2.5 mM PMSF, 2.5 $g/ml aprotinin, 25 $g/ml leupeptin, 2.5 mM Na3VO4) was added to 0.8 ml cells in mHBS in order to stop the reactions and solubilize the cells. Capture of A20 cells by immobilized Abs. A20 cells were labeled with CMFDA (Molecular Probes-Invitrogen) and adhesion assays were performed as described previously (Durand et al., 2006) except that the wells were coated with 2.63 $g/cm2 anti-IgG or antiLFA-1. The binding of control and RapGAPII-expressing A20 cells to immobilized anti-IgG or anti-LFA-1 was nearly identical (data not shown). Processing of samples for scanning EM. The cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.37 and then processed in a Pelco Biowave microwave processing system (Ted Pella, Redding, CA) at 37ºC under vacuum with microwaving at 100W for four 2 min cycles, each separated by 2 min. After rinsing with 0.1 M cacodylate buffer pH 7.2 and microwaving at 22ºC without vacuum for 40 s at 300W, the samples were treated with 1% osmium tetroxide at 22ºC under vacuum with microwaving at 100W for four 2 min cycles, each separated by 2 min. The samples were then rinsed with water, microwaved at 110W for 40 s and dehydrated by sequential treatments with 50%, 70%, and 95% ethanol, followed by three treatments with 100% ethanol, in each case microwaving the samples under vacuum for 40 s at 110W. The coverslips were dried using a CPD 020 critical point dryer (Bal-Tec, Balzers, Lichtenstein) and covered with a ~25 nm layer of gold/palladium using a SEMPrep II sputter coater (Nanotech, Manchester, UK). Localization of the BCR in bead:cell conjugates. Transient transfection of a pcDNA3 vector encoding an Ig"-yellow fluorescent protein (YFP) fusion protein (a gift from S. Pierce, NIH) into A20 cells was performed using the Amaxa nucleofection system and transfection kit V (Amaxa). After 18 hr, 105 cells in 0.1 ml mHBS/1% FCS were mixed 124  with 25 $l (5 x 105) anti-Ig-coated beads for 5 min. The cells were then imaged by confocal microscopy. Localization of phosphotyrosine (pTyr) in bead:cell conjugates. Vector control A20 cells or naïve splenic B cells were mixed with anti-Ig-coated beads and then fixed, permeabilized and stained with rhodamine-phalloidin as described in the main text. The cells were also stained with rabbit anti-pTyr (BD Pharmingen) followed by Alexa488-conjugated donkey anti-rabbit IgG (Molecular Probes-Invitrogen). The coverslips were treated with anti-fade reagent containing DAPI (Molecular Probes-Invitrogen) before being mounted onto slides. Localization of RapL-EGFP. RapL-EGFP cDNA (a gift from T. Kinashi, Kyoto University, Kyoto, Japan) was transiently transfected into anti-CD40- plus IL-4-activated splenic B cells by nucleofection (Amaxa). The cells were cultured overnight, plated on $slide 8-well tissue culture-treated slides (Integrated BioDiagnostics) and imaged in real time at 37oC with an Olympus FV1000 confocal microscope. Real-time imaging of bead:cell conjugates. A20 cells in mHBS/2% FCS were mixed with anti-IgG-coated beads in an Eppendorf tube. After centrifugation at 800 RCF for 10 sec and inverting the tube several times, the cell:bead mixture was pipetted onto PLL-treated glass bottom dishes (No. 1.5; MatTek Corp., Ashland, MA). The dishes were placed on the stage of an Olympus IX81/Fluoview FV1000 confocal microscope with a 37°C chamber and imaged using a 60X oil objective. Time-lapse video recordings were collected for 20 min and movies were created using Olympus Fluoview 1.6 software. Immunoblotting analysis. Vector control and RapGAPII-expressing A20 cells (5 x 106 in 0.4 ml mHBS) were incubated at 37°C with anti-IgG beads (107 in 0.1 ml) or with 1 $g/ml soluble anti-IgG Abs. The cells were then pelleted and solubilized by addition of RIPA buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Igepal (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 10 $g/ml leupeptin, 1 $g/ml aprotinin, 1 mM Na3VO4, 25 mM "-glycerophosphate, 1 $g/ml microcystin-LR) and 5X SDS-PAGE sample buffer. Proteins were transferred to nitrocellulose and blots were probed with anti-pERK or anti-p-Akt Abs before being re-probed with Abs specific for total ERK or Akt (Cell  125  Signaling Technologies). Bands were visualized by enhanced chemiluminescence (ECL; GE Life Sciences).  126  2.7 References Allen, C.D., Okada, T., Tang, H.L., and Cyster, J.G. (2007). Imaging of germinal center selection events during affinity maturation. Science 315, 528-531. Arthur, W. 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Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion. Nat. Immunol. 7, 919-928. Katagiri, K., Maeda, A., Shimonaka, M., and Kinashi, T. (2003). RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4, 741-748. Katagiri, K., Ohnishi, N., Kabashima, K., Iyoda, T., Takeda, N., Shinkai, Y., Inaba, K., and Kinashi, T. (2004). Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafficking. Nat. Immunol. 5, 1045-1051. Kinashi, T. (2005). Intracellular signalling controlling integrin activation in lymphocytes. Nat. Rev. Immunol. 5, 546-559. Krummel, M.F., and Macara, I. (2006). Maintenance and modulation of T cell polarity. Nat. Immunol. 7, 1143-1149. Lafuente, E.M., van Puijenbroek, A.A., Krause, M., Carman, C.V., Freeman, G.J., Berezovskaya, A., Constantine, E., Springer, T.A., Gertler, F.B., and Boussiotis, V.A. (2004). RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev. Cell 7, 585-595. Lankar, D., Vincent-Schneider, H., Briken, V., Yokozeki, T., Raposo, G., and Bonnerot, C. (2002). Dynamics of major histocompatibility complex class II compartments during B cell receptor-mediated cell activation. J. Exp. Med. 195, 461-472. Ludford-Menting, M.J., Oliaro, J., Sacirbegovic, F., Cheah, E. T., Pedersen, N., Thomas, S.J., Pasam, A., Iazzolino, R., Dow, L.E., Waterhouse, N.J., et al. (2005). A network of PDZcontaining proteins regulates T cell polarity and morphology during migration and immunological synapse formation. Immunity 22, 737-748. McLeod, S.J., Ingham, R.J., Bos, J.L., Kurosaki, T., and Gold, M.R. (1998). Activation of the Rap1 GTPase by the B cell antigen receptor. J. Biol. Chem. 273, 29218-29223. McLeod, S.J., Li, A.H., Lee, R.L., Burgess, A.E., and Gold, M.R. (2002). The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor-1 (CXCL12): potential role for Rap2 in promoting B cell migration. J. Immunol. 169, 1365-1371. 129  McLeod, S.J., Shum, A.J., Lee, R.L., Takei, F., and Gold, M.R. (2004). The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J. Biol. Chem. 279, 12009-12019. Miertzchke, M., Stanley, P., Bunney, T.D., Rodrigues-Lima, F., Hogg, N., and Katan, M. (2007). Characterisation of interactions of adaptor protein RAPL/Nore1 with Rap GTPases and their role in T cell migration. J. Biol. Chem. 282, 30629-30642. Mochizuki, N., Ohba, Y., Kiyokawa, E., Kurata, T., Murakami, T., Ozaki, T., Kitabatake, A., Nagashima, K., and Matsuda, M. (1999). Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i). Nature 400, 891-894. Ohba, Y., Mochizuki, N., Matsuo, K., Yamashita, S., Nakaya, M., Hashimoto, Y., Hamaguchi, M., Kurata, T., Nagashima, K., and Matsuda, M. (2000). Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade. Mol. Cell. Biol. 20, 6074-6083. Paavilainen, V.O., Bertling, E., Falck, S., and Lappalainen, P. (2004). Regulation of cytoskeletal dynamics by actin-monomer-binding proteins. Trends Cell Biol. 14, 386-394. Polakis, P.G., Rubinfeld, B., Evans, T., and McCormick, F. (1991). Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells. Proc. Natl. Acad. Sci. USA 88, 239-243. Qi, H., Egen, J.G., Huang, A.Y., and Germain, R.N. (2006). Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312, 1672-1676. Reedquist, K.A., Ross, E., Koop, E.A., Wolthuis, R.M., Zwartkruis, F.J., van Kooyk, Y., Salmon, M., Buckley, C.D., and Bos, J.L. (2000). The small GTPase, Rap1, mediates CD31induced integrin adhesion. J. Cell Biol. 148, 1151-1158. Shimonaka, M., Katagiri, K., Nakayama, T., Fujita, N., Tsuruo, T., Yoshie, O., and Kinashi, T. (2003). Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J. Cell Biol. 161, 417-427. Stork, P.J., and Dillon, T.J. (2005). Multiple roles of Rap1 in hematopoietic cells: complementary versus antagonistic functions. Blood 106, 2952-2961. Suga, K., Katagiri, K., Kinashi, T., Harazaki, M., Iizuka, T., Hattori, M., and Minato, N. (2001). CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1. FEBS Lett. 489, 249-253. Vidard, L., Kovacsovics-Bankowski, M., Kraeft, S.K., Chen, L.B., Benacerraf, B., and Rock, K.L. (1996). Analysis of MHC class II presentation of particulate antigens of B lymphocytes. J. Immunol. 156, 2809-2818.  130  3. The Rap GTPases regulate the migration, invasiveness, and in vivo dissemination of B-cell lymphoma2 3.1 Introduction The ability of normal and malignant B cells to leave the circulation and enter tissues depends on homeostatic chemokines such as CXCL12, CCL19, CCL21, and CXCL13, which are immobilized on the surface of vascular endothelial cells (Pals et al., 2007; Thelen and Stein, 2008). Chemokine receptor signaling activates the lymphocyte integrins LFA-1 and VLA-4, permitting firm adhesion of the lymphocyte to endothelial cells. This leads to chemokineinduced migration of the lymphocyte along the surface of the endothelial cells, followed by transendothelial migration into the underlying tissue. In the case of lymphoma cells, these cells then migrate along chemokine gradients to sites where they can establish tumors. The Rap GTPases (Rap1a, Rap1b, Rap2a, Rap2b, and Rap2c; collectively referred to as Rap) are signal-transducing switches that cycle between an active GTP-bound form and an inactive GDP-bound form. Activated Rap is a master regulator of cytoskeletal organization and integrin activation (Bos, 2005). In lymphocytes, chemokine receptor signaling leads to Rap activation and this is critical for chemokines to stimulate cell migration and integrinmediated adhesion (Durand et al., 2006; McLeod et al., 2002; McLeod et al., 2004; Shimonaka et al., 2003). However, the role of Rap activation in the in vivo dissemination and establishment of B cell lymphoma is not known. Therefore, we asked whether blocking Rap activation in the well-characterized A20 murine B lymphoma cell line (Kim et al., 1979) would reduce the ability of these cells to form tumors when injected i.v. into Balb/c mice, a well-defined model of B cell lymphoma (Staveley-O'Carroll et al., 1998). The A20 line is a relevant model because they resemble human diffuse large B cell lymphoma, which account for 40% of all non-Hodgkin’s lymphomas.  2  A version of this chapter has been submitted for publication. Lin, K.B.L., Freeman, S.A., Tan, T., Lam, M., McNagny, K., and Gold, M.R. (2009) The Rap GTPases regulate the migration, invasiveness, and in vivo dissemination of B-cell lymphomas.  131  3.2 Results and discussion To block Rap activation, we transduced A20 B lymphoma cells with a bicistronic vector designed to co-express GFP and RapGAPII (Fig. 3.1a), a Rap-specific GTPase-activating protein (GAP) that converts Rap1 and Rap2 to their inactive GDP-bound forms. Expressing RapGAPII in A20 cells completely blocked the activation of Rap1 and Rap2 by the chemokine CXCL12 (Fig. 3.1b). CXCL12 plays a key role in the dissemination of many types of tumor cells and is produced by lymphoid organs, as well as by the bone marrow, liver, lungs, and ovaries (Shirozu et al., 1995). RapGAPII expression selectively blocks CXCL12-induced Rap activation and does not affect CXCL12-induced activation of Akt/protein kinase B, MAP kinases, or the Rac1 GTPase in B cell lines (Durand et al., 2006; McLeod et al., 2002). Moreover, expressing RapGAPII in A20 cells did not alter the levels of Rap1 or Rap2 (Fig. 3.1b), did not decrease the cell surface expression of CXCR4 (the receptor for CXCL12), LFA-1 or VLA-4 (Fig. 3.1c), and did not alter the in vitro proliferation of A20 cells (Fig. 3.1d).  132  Figure 3.1  RapGAPII expression blocks CXCL12-induced activation of Rap1 and  Rap2 in A20 cells. (a) Bulk populations of A20 cells stably expressing either the GFP-encoding pMXPIE retroviral vector, or the pMXPIE/RapGAPII vector, which encodes RapGAPII and GFP on a single transcript separated by an internal ribosome entry site (IRES), were generated using retroviruses, as described (Krebs et al., 1999). (b) A20/vector and A20/RapGAPII cells were stimulated with 100 ng/ml CXCL12 (R&D Systems). Upper panels show the active GTP-bound forms of Rap1 and Rap2, which were precipitated using a GST-RalGDS fusion protein and visualized by immunoblotting, as described (McLeod et al., 1998). Lower panels show total Rap1 and Rap2 in the cell lysates. (c) FACS analysis of A20/vector and A20/RapGAPII cells stained with antibodies to CXCR4 (clone 2B11; eBioscience), LFA-1 (clone M17/4; eBioscience), VLA-4 (clone R1-2; eBioscience) or with isotypematched control antibodies. (d) Cell proliferation was assessed by culturing cells (2 x 104 per well of a 96-well plate) for 48 h before adding 1 $Ci 3H-thymidine per well and measuring 3H-thymidine incorporation (mean + s.d. for triplicate wells) 4 h later.  133  To test whether Rap activation is important for the in vivo dissemination and establishment of B cell lymphomas, GFP-expressing A20/vector cells or A20/RapGAPII cells were injected i.v. into Balb/c mice. Mice were monitored daily for signs of tumor progression (abdominal swelling, scruffy fur, decreased movement, weight loss, hunched posture), at which point they were euthanized and examined for tumors. When injected with A20/vector cells, 50% of the mice exhibited these symptoms after 31 days and by day 43 all of the mice had to be euthanized (Fig. 3.2a). Strikingly, preventing Rap activation delayed and reduced the incidence of tumor formation. When the mice were injected with A20/RapGAPII cells, 50% of the mice did not require euthanization until day 49, 18 days later than mice injected with A20/vector cells. Moreover, 30% of the mice injected with A20/RapGAPII cells did not develop any signs of tumor formation up to 90 days post-injection (Fig. 3.2a). In the mice that developed tumors, lymphoma occurred in multiple organs including the liver, ovaries, lymph nodes, peritoneal cavity, spleen, and bone marrow. In general, A20/vector cells established tumors at more sites than A20/RapGAPII cells. The liver, which produces large amounts of CXCL12 (Goddard et al., 2001), was the preferential site of lymphoma development. All mice that developed tumors first exhibited tumors in the liver, before developing visible tumors at other sites. When we examined the livers of mice at three and four weeks after injection with A20 cells, we found that fewer of the mice injected with A20/RapGAPII cells had detectable liver tumors at these time points than mice injected with A20/vector cells (Fig. 3.2b). These data suggest that blocking Rap activation delays the ability of A20 cells to form tumors in vivo. Intriguingly, closer examination of tumors isolated from mice injected with A20/RapGAPII cells revealed that they had reduced levels of GFP compared to A20/RapGAPII cells that were kept in culture (Fig. 3.2c, right panel). This in vivo loss of GFP expression was not observed for A20/vector cells (Fig. 3.2c, left panel). The bicistronic expression vector used to generate the A20/RapGAPII cells was designed to co-express the RapGAPII and GFP genes (Fig. 3.1a); immunoblotting confirmed that the loss of GFP expression was accompanied by the loss of RapGAPII expression (data not shown). Importantly, cells isolated from tumors arising in mice injected with A20/RapGAPII cells exhibited strong CXCL12-induced Rap activation, consistent with a loss of RapGapII expression (Fig. 3.2d). 134  This is in contrast to A20/RapGAPII cells that were cultured in vitro, which maintained expression of GFP (Fig. 3.2c), as well as RapGAPII, the latter being indicated by continued suppression of Rap activation after 3 months in culture (Fig. 3.2d). Thus, the tumors that formed in mice injected with A20/RapGAPII cells were derived either from a minor population of cells that had very low RapGAPII expression prior to injection, or from cells that silenced the pro-viral vector expression after integration. Because both retroviral and GFP sequences are frequent targets of epigenetic silencing, the latter explanation is likely the case. In support of this idea, A20/RapGAPII cells that were present in the liver 24 h after injection (see below) were GFP+, and presumably expressed RapGAPII, whereas the cells isolated from the tumors that developed in these mice were GFPlow and exhibited robust Rap activation. The delayed kinetics of tumor formation in mice receiving A20/RapGAPII cells (Fig. 3.2b) is also consistent with in vivo selection for cells that had regained the ability to activate Rap. Thus, the in vivo dissemination and development of B cell lymphomas appears to be dependent on Rap activation because cells in which Rap activation remained blocked did not form tumors.  135  Figure 3.2  Blocking Rap activation inhibits lymphoma development in vivo.  (a) Survival curves for groups of ten 6-8 weeks old Balb/c mice that received 5 x 106 GFP-expressing A20/vector or A20/RapGAPII cells via i.v. injection into the tail vein. Mice were euthanized when they exhibited signs of tumor development such as abdominal swelling, scruffy fur, decreased movement, weight loss, or hunched posture, in accord with UBC Animal Care Committee policies. The data are from two independent experiments. (b) Groups of six Balb/c mice were injected with either A20/vector or A20/RapGAPII cells and the presence of visible liver tumors was assessed after 3 or 4 weeks. (c) Representative FACS plots of GFP expression in cells kept in culture for 3 months versus cells isolated from tumors. (d) Cells isolated from tumors, as well as A20/RapGAPII cells that had been kept in culture, were stimulated with 100 ng/ml CXCL12 and assayed for Rap1 activation.  136  The ability of circulating lymphoma cells, to enter organs and to form tumors, depends on their ability to exit the vasculature and migrate to a niche that can sustain tumor growth. Interestingly, we found that both CXCL12-dependent migration and extravastion were dependent on Rap activation. A20/RapGAPII cells exhibited a significantly reduced ability to migrate across fibronectin-coated Transwells in response to CXCL12 (Fig. 3.3a), an in vitro assay that may mimic the movement of lymphoma cells across endothelial cells prior to extravasation. Moreover, we found that blocking Rap activation reduced the transmigration of A20 cells through a confluent monolayer of bEND.3 mouse microvascular endothelial cells by more than 50% (Fig. 3.3b). Confocal imaging revealed that A20/vector cells were able to extend membrane processes between and underneath the endothelial cells (Fig. 3.3c), mimicking the initial steps in transmigration. In contrast, the majority of A20/RapGAPII cells remained on top of the endothelial cells and did not extend processes through these endothelial monolayers (Fig. 3.3c). Thus, in these in vitro assays, Rap activation is required for efficient chemokine-induced migration and transendothelial migration by B lymphoma cells.  137  Figure 3.3  Rap activation is important for A20 cells to undergo chemokine-induced  migration and transendothelial invasion. (a) The migration of A20/vector and A20/RapGAPII cells towards 100 ng/ml CXCL12 was assessed using Transwell assays, as described (McLeod et al., 2002). After 5 h, cells that had migrated across 5-$m Transwell filters (Costar) that had been coated with 2 $g of fibronectin (R&D Systems) were counted. The percent of input cells that had migrated into the lower chamber (mean + s.e.m. for 5 experiments) is shown. P values were determined using Student’s paired t-test. (b) Transendothelial migration was assessed in a similar manner except that 8-$m clear cell culture inserts (BD) were coated with fibronectin and then seeded with bEnd.3 murine endothelial cells (Sikorski et al., 1993), which were grown to confluence and then activated with 10 ng/ml TNF-" (eBioscience) overnight. The percent of cells that had migrated into the lower chamber after 16 h (mean + s.e.m. for 4 experiments) is shown. (c and d) Rap activation regulates transendothelial invasion of A20 cells. bEnd.3 cells were cultured overnight with 10 ng/ml TNF-" in 8-well $-Slides (ibidi) that had been coated with fibronectin. A20/vector or A20/RapGAPII cells were labeled with CellTracker Green (Invitrogen) and added to the confluent monolayer of bEnd.3 cells. After 16 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Tween, and stained with rhodamine-phalloidin to detect F-actin and with DAPI to mark nuclei. Images were captured using an Olympus IX81/Fluoview FV1000 confocal microscope. The top confocal slices (above the endothelial cells) and bottom slices (closest to the substrate, i.e. below the endothelial cells) of Z-axis stacks are shown (c) along with 3D reconstructions of X and Y projections (d). Arrows indicate membrane processes that A20/vector cells have extended under the endothelial cells.  138  139  To determine whether the decreased ability of A20/RapGAPII cells to cross endothelial cell layers and migrate on extracellular matrix components in vitro correlated with a decreased ability to exit the vasculature and invade organs in vivo, equal numbers of A20/vector cells and A20/RapGAPII cells that had been differentially labeled with CellTracker dyes were coinjected into mice. After 24 h, the mice were perfused with PBS to flush circulating cells from the vasculature such that the relative number of A20/vector cells and A20/RapGAPII cells that had lodged in the liver could be determined. Confocal microscopy on liver sections showed that A20/vector cells readily invaded the liver over a 24 h period whereas A20/RapGAPII cells did so at a lower frequency (Fig. 3.4a,b). Although injected in equal numbers, 65-75% of the cells lodging in the liver were vector control cells whereas only 2535% were A20/RapGAPII cells (Fig. 3.4b). The A20/RapGAPII cells that lodged in the liver after 24 h were GFP+ (data not shown), suggesting that they still expressed RapGAPII at this point. The A20/vector cells that lodged in the liver frequently (~60%) assumed a classic amoeboid morphology that is characteristic of extravasating and migrating cells, whereas the A20/RapGAPII cells do not assume this morphology as often (~40%) (Fig. 3.4c,d). Their intimate association with hepatocytes, as determined by DAPI staining and autofluorescence of the hepatocytes, suggested that the A20 cells had lodged in the liver parenchyma. By these criteria, approximately 70% of the A20/vector cells that had accumulated in the liver within 24 h had infiltrated the liver parenchyma. The remainder appeared to be in unstained interstitial spaces and may represent cells that were not flushed out of the vasculature by the perfusion process. Of the A20/RapGAPII cells that lodged in the liver, approximately 45% appeared to have infiltrated the liver parenchyma, a lower percentage than for A20/vector cells. Consistent with this interpretation, approximately 60% of the A20/RapGAPII cells in the liver were in interstitial spaces and were round (Fig. 3.4c,d), as opposed to amoeboid, suggesting that they were not in contact with hepatocytes. Thus, Rap activation contributes to lymphoma dissemination by promoting the ability of these cells to infiltrate organs such as the liver and lodge in sites that may be suitable microenvironments for tumor growth.  140  Figure 3.4  Blocking Rap activation impairs the ability of A20 B lymphoma cells to  invade the liver. A20/vector and A20/RapGAPII cells were differentially labeled with CellTracker Green and CellTracker Orange. Five million cells of each type were mixed together and injected into a Balb/c mouse. After 24 h, the mice were euthanized, perfused with PBS to wash out cells in the vasculature, and liver sections were prepared. Liver samples were embedded in OCT (Fisher Scientific), frozen on dry ice, cut into 7-$m sections using a Cryotome (Thermo Scientific), fixed in 4% paraformaldehyde, and mounted onto slides with DAPI-Prolong Gold (Invitrogen). (a) Confocal images of liver sections. Arrows indicate A20 cells. DAPI-stained hepatocytes appear blue/purple. (b) Quantitative analysis of the relative numbers of A20/vector and A20/RapGAPII cells in the liver after 24 h. For each mouse injected, 40 cells from multiple sections were counted. Combined data for 4 mice is shown. Dye-swap experiments were performed to rule out any effects of the dyes. (c) Higher magnification images showing that A20/vector cells that had entered the liver often exhibited a spread, amoeboid morphology and were localized primarily within the liver parenchyma. A20/RapGAPII cells were usually round and were more frequently in interstitial spaces. (d) Quantitative analysis of in vivo morphology and invasion of A20/vector and A20/RapGAPII cells in the liver after 24 h. For each mouse injected, 40 cells were analyzed (n = 4; mean ± s.e.m).  141  142  In this report we have shown for the first time that activation of the Rap GTPases is critical for the in vivo dissemination of B cell lymphomas. This likely reflects the role of Rap activation in multiple processes that involve cell adhesion and cell migration. We had previously shown that Rap activation is required for A20 cells to undergo CXCL12-induced adhesion to ICAM-1-expressing cells (McLeod et al., 2004) and have now shown that Rap activation is important for A20 cells to undergo transendothelial migration in vitro, as has been shown for T cells and chronic lymphocytic leukemia cells (Shimonaka et al., 2003; Till et al., 2008). Importantly, we found that Rap activation is critical for circulating lymphoma cells to exit the vasculature in vivo and enter the liver parenchyma, a site where these cells readily form tumors. The Rap GTPases are master regulators of actin cytoskeleton organization, and are known to control changes in cell morphology that are required for cell motility (Bos, 2005). Activated Rap regulates the activity and subcellular localization of multiple proteins that control actin dynamics and cell polarity including the Rac GTPase activators Vav2 and TIAM1, profilin, AF-6, myosin II, and the Par3/Par6 polarity complex (Bos, 2005; Gerard et al., 2007; Jeon et al., 2007). We have previously shown that Rap activation is important for A20 cells to reorganize their actin cytoskeleton, undergo cell spreading, and form immune synapses in response to B cell receptor engagement (Lin et al., 2008). A novel finding reported here is that Rap activation is required for A20 cells to form invasive membrane processes that are associated with extravasation. A20/vector control cells were able to extend membrane processes between and underneath vascular endothelial cells in vitro and within liver parenchyma in vivo, whereas A20/RapGAPII cells were unable to do so. The decreased ability to form such invadopodia may account for the significantly reduced ability of A20/RapGAPII cells to undergo transendothelial migration both in vitro and in vivo. In addition to playing an important role in the ability of lymphoma cells to migrate into and within organs, our data suggest that Rap activation is also important for individual A20 cells to establish a suitable microenvironment in the liver and form tumors. When A20/RapGAPII cells entered the liver parenchyma during the first 24 h after i.v. injection, they expressed GFP (data not shown), and presumably RapGAPII. However, the cells present in the tumors that developed 3-8 weeks later in these mice had lost the expression of GFP and RapGAPII 143  and exhibited robust Rap activation in vitro. Thus, there was an in vivo selection for cells that had regained the capacity to activate Rap. This suggests that the ability of a newly arrived A20 cell to colonize the liver microenvironment and form a tumor depends strongly on Rap activation. A20/RapGAPII did form tumors when 5 x 105 cells were injected subcutaneously into the flanks of Balb/c mice. When localized together in large numbers via subcutaneous injection, A20/RapGAPII cells may be more adept at establishing a supportive microenvironment or promoting their own survival via paracrine signals than when they lodge in the liver as single cells. Tumor formation by isolated malignant cells that have arrested in an organ is an inefficient process that is strongly dependent on localized survival and growth factors as well as the interactions between the tumor cells and the surrounding stromal cells (Chambers et al., 2002). For example, isolated A20 cells may require adhesion-dependent survival signals, as is the case for many types of malignant B cells including B-CLL cells, lymphomas, and multiple myelomas; in each of these cases, adhesion-dependent survival signals are required for disease progression (Drillenburg and Pals, 2000; Zhou et al., 2005). In B cells, activated Rap promotes the conversion of integrins to their high affinity/high avidity state that can bind ligands and we have shown previously that Rap activation is required for VLA-4-dependent adhesion of A20 cells to bone marrow stromal cells (McLeod et al., 2004). Thus in A20 cells, survival signals generated by integrin-dependent adhesion would be dependent on Rap activation. Although A20 cells are not dependent on adhesion for their in vitro survival or growth, adhesion-dependent signals may be required for single A20 cells to survive and form tumors in vivo. In summary we have shown that Rap activation plays a key role in the establishment and dissemination of B cell lymphomas in vivo, suggesting that Rap and its downstream effectors may be good therapeutic targets for limiting the dissemination of B cell lymphomas to critical organs.  144  3.3 References Bos JL. (2005). Linking Rap to cell adhesion. Curr Opin Cell Biol 17: 123-128. Chambers AF, Groom AC, MacDonald IC. (2002). Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2: 563-572. Drillenburg P, Pals ST. (2000). Cell adhesion receptors in lymphoma dissemination. Blood 95: 1900-1910. Durand CA, Westendorf J, Tse KW, Gold MR. (2006). The Rap GTPases mediate CXCL13and sphingosine1-phosphate-induced chemotaxis, adhesion, and Pyk2 tyrosine phosphorylation in B lymphocytes. Eur J Immunol. 36: 2235-2249. Gerard A, Mertens AE, van der Kammen RA, Collard JG. (2007). The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization. J Cell Biol 176: 863-875. Goddard S, Williams A, Morland C, Qin S, Gladue R, Hubscher SG et al. (2001). Differential expression of chemokines and chemokine receptors shapes the inflammatory response in rejecting human liver transplants. Transplantation 72: 1957-67. Jeon TJ, Lee DJ, Lee S, Weeks G, Firtel RA. (2007). Regulation of Rap1 activity by RapGAP1 controls cell adhesion at the front of chemotaxing cells. J Cell Biol 179: 833843. Kim KJ, Kanellopoulos-Langevin C, Merwin RM, Sachs DH, Asofsky R. (1979). Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol 122: 549-554. Krebs DL, Yang Y, Dang M, Haussmann J, Gold MR. (1999). Rapid and efficient retrovirusmediated gene transfer into B cell lines. Methods Cell Sci 21: 57-68. Lin KB, Freeman SA, Zabetian S, Brugger H, Weber M, Lei V et al. (2008). The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands. Immunity 28: 75-87. McLeod SJ, Ingham RJ, Bos JL, Kurosaki T, Gold MR. (1998). Activation of the Rap1 GTPase by the B cell antigen receptor. J Biol Chem 273: 29218-29223. McLeod SJ, Li AH, Lee RL, Burgess AE, Gold MR. (2002). The Rap GTPases regulate B cell migration toward the chemokine stromal cell-derived factor-1 (CXCL12): potential role for Rap2 in promoting B cell migration. J Immunol 169: 1365-1371. McLeod SJ, Shum AJ, Lee RL, Takei F, Gold MR. (2004). The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem 279: 12009-12019.  145  Pals ST, de Gorter DJ, Spaargaren M. (2007). Lymphoma dissemination: the other face of lymphocyte homing. Blood 110: 3102-3111. Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O et al. (2003). Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J Cell Biol 161: 417-427. Shirozu M, Nakano T, Inazawa J, Tashiro K, Tada H, Shinohara T et al. (1995). Structure and chromosomal localization of the human stromal cell-derived factor 1 (SDF1) gene. Genomics 28: 495-500. Sikorski EE, Hallmann R, Berg EL, Butcher EC. (1993). The Peyer's patch high endothelial receptor for lymphocytes, the mucosal vascular addressin, is induced on a murine endothelial cell line by tumor necrosis factor-alpha and IL-1. J Immunol 151: 5239-5250. Staveley-O'Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang L, Fein S et al. (1998). Induction of antigen-specific T cell anergy: An early event in the course of tumor progression. Proc Natl Acad Sci USA 95: 1178-1183. Thelen M, Stein JV. (2008). How chemokines invite leukocytes to dance. Nat Immunol 9: 953-959. Till KJ, Harris RJ, Linford A, Spiller DG, Zuzel M, Cawley JC. (2008). Cell motility in chronic lymphocytic leukemia: defective Rap1 and !L"2 activation by chemokine. Cancer Res 68: 8429-8436. Zhou J, Mauerer K, Farina L, Gribben JG. (2005). The role of the tumor microenvironment in hematological malignancies and implication for therapy. Front Biosci 10: 1581-1596.  146  4. Concluding chapter 4.1 Summary of main findings  Chapter 2: The Rap GTPases regulate B cell morphology, immune synapse formation, and signaling by particulate antigens •  B cells spread on immobilized antibodies that cluster the BCR or the LFA-1 integrin  •  Spreading on immobilized anti-Ig and anti-LFA-1 antibodies requires Rap activation  •  Clustering of LFA-1 induces Rap activation  •  B cell immune synapse formation and antigen gathering is regulated by Rap activation  •  Model particulate antigens induces F-actin rich cup formation by B cells  •  F-actin rich cup formation is regulated by Rap activation  •  F-actin rich cup formation is necessary for sustained BCR signaling  Chapter 3: The Rap GTPases regulate the migration, invasiveness, and in vivo dissemination of B-cell lymphomas •  Rap activation is required for effective dissemination of A20 B cell lymphoma cells in vivo  •  In vitro fibronectin-dependent migration and transendothelial migration of A20 cells is regulated by Rap activation  •  In vivo invasiveness of A20 cells is regulated by Rap activation  147  4.2 Discussion and future directions Until recently, there were very few reports describing B cell morphology and its importance for functions such as trafficking, antigen-induced activation, and lymphoma dissemination. Therefore, for my thesis I set out to investigate how signaling through the BCR and integrins might regulate B cell morphology. Specifically, I focused on the Rap GTPases, signaling molecules that transmit information generated at the cell surface to intracellular pathways that regulate integrin activation and cytoskeletal dynamics. In this thesis, I have described B cell morphological changes associated with BCR and integrin signaling that were regulated by activation of Rap GTPases. More importantly, I demonstrated the functional importance of these morphological changes for B cell function, activation, and for the dissemination of B cell lymphomas. Rap-GTP was necessary for B cell spreading, which is integral for motility and transendothelial migration. This was particularly apparent in the studies of lymphoma invasion and motility as blocking Rap activation by expressing RapGAPII in A20 B cell lymphoma cells inhibited adhesion, cell spreading, and subsequent in vivo invasion into liver tissue (Chapter 3). Rap-GTP also regulates integrin activation and I found that blocking Rap activation prevented immune synapse pSMAC formation and as a consequence, B cells were unable to spread and gather as much membrane-bound antigen as their wild type counterparts (Chapter 2). Rap activation was also necessary for B cells to form F-actin rich cups in response to anti-Ig-coated beads, which mimic particulate antigens such as bacteria and viruses that have repeating epitopes on their surface. These cups were also sites of intense localized BCR signaling at the contact site with the particulate antigen. Blocking Rap activation did not prevent the binding of particulate antigens but did decrease the frequency and intensity of the F-actin cup response (Chapter 2). Furthermore, sustained downstream BCR signaling in response to this model particulate antigen was dependent on Rap activation and Rap-mediated actin polymerization (Chapter 2). In summary, the regulation of B cell morphology by Rap GTPases is important for B cell migration, invasion, immune synapse formation, and sustained BCR signaling in response to particulate antigens.  148  4.2.1  Importance of B cell morphology in B cell function and development  A recent report on T cells crawling on, and transmigrating through, endothelial monolayers supported many of the conclusions drawn from this thesis. Shulman et al. reported that T cells form “focal dots” of high affinity LFA-1 when moving across activated endothelial cells and that these focal dots were often localized at the tips of adhesive filopodia (Shulman, Shinder et al. 2009). These invasive filopodia allow T cells to adhere to vessel walls under shear force, and form prior to and during transendothelial migration (TEM). Cells that have more filopodia, such as memory T cells, transmigrate more rapidly and this suggests a positive correlation between filopodia formation and the ability to extravasate. Similarly, I demonstrated that the formation of invasive filopodia, or invadopodia, by A20 lymphoma cells correlated with the ability of these cells to undergo TEM and tissue invasion in vivo (Chapter 3). The observation that memory T cells form more filopodia than naïve T cells is consistent with our data, which showed that primary B cells activated with IL-4 and anti-CD40 spread on immobilized anti-IgG and anti-LFA-1 antibodies whereas naïve B cells did not (Chapter 2). Furthermore, it was reported that T-dependent GC B cells that were antigen-experienced, and hence activated, are able to form long filopodia or neurite-like projections in vitro, whereas resting and T-independent B cells do not (Yu, Cook et al. 2008). Shulman et al. also examined the requirements for crawling and invasive adhesion of T cells. They found that chemokines such as CXCL12, which are bound to the surfaces of endothelial cells, triggered activation of the VLA-4 and LFA-1 integrins, and that shear stress was required for their transition to the high-affinity state (Shulman, Shinder et al. 2009). Cdc42 was also required for the generation of the invasive filopodia by adherent T cells. Rap is a key hub in these signaling pathways that link receptor signaling to integrin activation and to cytoskeletal dynamics such as Cdc42 activation and localization. Therefore, as discussed in this thesis, Rap activation is pivotal for regulating cytoskeletal reorganization and subsequent changes in motility. Consistent with this hypothesis, when Rap activation in T cells was inhibited by overexpression of Spa-1, a Rap1-specific GAP, integrin activation into the high-affinity state was decreased by 50%, as was shear-resistant T cell crawling, and focal dot formation, suggesting a decrease in adhesive filopodia  149  formation (Shulman, Shinder et al. 2009). Even though the authors only used T cells in their study, these observations and conclusions are likely applicable to B cell crawling in venules, as this thesis has strongly suggested (Chapter 2 and 3). Cell crawling and spreading are important processes for sampling and acquiring antigens displayed on the surfaces of APCs such as dendritic cells and subcapsular macrophages (Harwood and Batista 2008). Recent work showed that FO B cells can acquire low molecular weight antigens that are transported in follicular conduits comprised of FRCs, which wrap around collagen fibrils and carry lymph from the SCS to the medulla of lymph nodes (Roozendaal, Mempel et al. 2009). FRC conduits rapidly fill with antigen following subcutaneous injection and FO B cells are able to acquire antigen from these conduits within 1 hour. Electron microscopy revealed that the FRC network contains gaps into which B cells extend pseudopods in order to gain direct access to the lymph and antigens in the conduit. Examination of activation markers such as CD86 showed that when FO B cells acquire antigen in FRC conduits, they are efficiently activated. One question this raises is whether Rap activation is necessary for B cells to acquire antigen from these follicular (FRC) conduits. As demonstrated in this thesis, antigen gathering on lipid bilayers and B cell spreading is severely compromised when Rap activation is blocked (Chapter 2). Therefore, preventing the activation of Rap may inhibit cytoskeletal rearrangements that are necessary for the formation of antigen-sampling pseudopods that extend into the FRC conduits. Furthermore, interstitial migration of B cells might also be impaired as motility within lymph nodes requires actin-based flow and squeezing (Lammermann, Bader et al. 2008). Thus, FO B will likely require actin-based motility regulated by Rap-GTP. Moreover, a gradient of CXCL13 directs FO B cells towards follicular conduits since these conduits are extremely rich in CXCL13 (Roozendaal, Mempel et al. 2009) and it was shown that Rap activation is required for B cells to migrate towards CXCL13 (Durand et al., 2006). From the data presented in this thesis, one can propose that Rap-mediated cell spreading is important during B cell development and mature B cell interactions in the bone marrow with stromal cells. As discussed in Chapter 1, the bone marrow is the site of B cell development as well as long-term survival of differentiated B cells, mainly plasma cells (Tokoyoda, Egawa et al. 2004; Nagasawa 2006). Recently, Pereira et al. used two-photon microscopy 150  to look at developing B cells in the bone marrow of mice (Pereira, An et al. 2009). They observed immature B cells crawling and entering bone marrow sinusoids. Bone marrow sinusoids are thin-walled venous blood vessels that carry blood and newly produced cells from the bone marrow to the large central sinusoid that runs through the middle of the bone and returns these products back into circulation (Tavassoli and Yoffey 1983). Since the Rap GTPases regulate B cell spreading and migration, Rap-GTP may regulate immature B cell crawling in bone marrow sinusoid compartment. The crawling of immature B cells and retention in this bone marrow sinusoids appears to be important for shaping the B cell repertoire (Pereira, An et al. 2009). Crawling and retention in this specialized niche may allow immature B cells to encounter self-antigens that induce receptor editing. Consistent with this idea, a large fraction of B cells that exit bone marrow sinusoids soon after development express Ig%, whereas immature B cells that stay longer in the bone marrow express predominantly Ig&. Ig& expression is usually associated with receptor editing since the & locus is only rearranged after unsuccessful rearrangement or edited rearrangement on the two chromosomes bearing the % loci (Pereira, An et al. 2009). Surprisingly, the retention of immature B cells in this compartment is not regulated by CXCL12/CXCR4 signaling but rather through cannabinoid receptor 2 (CB2), which stimulates VLA-4 on immature B cells to bind VCAM-1 on bone marrow stromal cells and the sinusoidal endothelium. The CB2 receptor is a G"i protein-coupled receptor and it may activate VLA-4 in the same Rap-dependent-manner as CXCR4, which also activate G"i. Whether Rap activation is important for regulating immature B cell crawling on bone marrow stromal cells and endothelium, and for shaping B cell repertoire, remains to be determined. Consistent with the importance of Rap-GTP in regulating the development of B cells in the bone marrow, Hattori et al. reported that expression of the dominant negative Rap1A17 protein results in a block in the transition from the pro-B to pre-B cell stage (Minato and Hattori 2009). The defect in early B cell development was attributed to the inability of progenitor B cells to respond to IL-7, which resulted in progressive cell death. It was not determined whether there were any defects in pro-B cell interactions with bone marrow stromal cells. Adhesion of B cell progenitors to stromal cells is required for their survival, 151  proliferation, and differentiation (Nagasawa 2006). Thus Rap activation may promote the development of early B cell progenitors in several ways. In addition to being the site for B cell development, the bone marrow also functions as a compartment for long-lived plasma cells (Cariappa, Mazo et al. 2005; Moser, Tokoyoda et al. 2006). In addition, mature B cells that respond to TI antigens recirculate through the bone marrow and this process is essential for the survival of mature B cells (Cariappa, Mazo et al. 2005; Sapoznikov, Pewzner-Jung et al. 2008). Sapoznikov et al. identified a new bone marrow niche that contained CD11chigh bone marrow dendritic cells (bmDCs) that are distinct from other subsets of dendritic cells in SLOs (Sapoznikov, Pewzner-Jung et al. 2008). Intravital microscopy revealed perivascular clusters of these bmDCs around blood vessels that were similar in structure to lymphoid follicles, since these clusters contained both T cells and mature B cells that are in intimate association with bmDCs. Ablation of these bmDCs by using CD11c-diphtheria toxin receptor-transgenic mice resulted in a substantial loss of recirculating mature B cells but did not affect T cells numbers. Subsequent work showed that bmDCs produce the cytokine macrophage migration-inhibitor factor (MIF), which maintained the steady-state survival of recirculating B cells by activating the Akt pro-survival pathway. Thus, the interaction of mature B cells with bmDCs may share similarities to the interaction between FO B cells and FDCs. Since Rap GTPases regulate integrin-mediated adhesion, cell spreading, and cell crawling, it would be interesting to test whether Rap mediates the interaction between mature B cells and these bmDCs, thereby promoting the survival of circulating mature B cells. To study whether Rap GTPases regulate in vivo interactions between B cells and cells such as sinusoid endothelial cells or bmDCs would require an inducible loss-of-function model because early B cell development appears to be dependent on Rap activation (Minato and Hattori 2009). An inducible system would allow B cells to develop normally before expression of a RapGAP transgene could be selectively induced in mature B cells. One possible approach would be to selectively express a RapGAP (Spa-1 or RapGAPII) under the control of a tetracycline-regulated promoter (i.e. Tet-Off) in only B cells. Alternatively, one could generate mice with a floxed RapGAP transgene and cross these to mice with the Cre-ERT2 transgene expressed specifically in B cells, then treat the mice with tamoxifen to 152  induce expression of the RapGAP transgene. Another approach would be to transduce purified mature B cells with lentiviruses containing viral genomes encoding RapGAPII or dominant negative Rap1 (Rap1N17 or Rap1A17), label the cells with fluorescent tracker dye, reintroduce them into mice, and monitor their interactions in the bone marrow by intravital two-photon microscopy of the bone marrow. One potential difficulty with the second approach is that transplanted cells in which Rap activation is blocked may not be able to home to the correct compartment as Rap regulates B cell migration and homing. Therefore, intra-femur injection might be necessary to directly reintroduce cells that were transduced ex vivo. 4.2.2  Modulation of B cell activation by the Rap GTPases  B cell spreading is important for immune synapse formation, antigen gathering, and sustained BCR signaling, but it is also important for early B cell activation events. Therefore, understanding how Rap-GTP regulates the cytoskeleton following antigen recognition is essential for understanding B cell activation. For example, BCR microcluster formation occurs even in the absence of BCR signaling (Depoil, Fleire et al. 2008). However, the number of microclusters formed in the absence of signaling is extremely low because the cells are unable to spread due to the lack of BCR signals. Therefore, B cell spreading allows more BCRs to engage antigen and form additional microclusters that will initiate and amplify signaling to promote B cell activation. Consistent with this, microclustering of BCRs occur exclusively in filopodia and in membrane ruffles in the periphery of the spreading cell (Tolar, Hanna et al. 2009). This indicates a role for the actin cytoskeleton in generating microclusters. Similarly, blocking actin polymerization in T cells reduces the number of microclusters formed and inhibits TCR signaling (Campi, Varma et al. 2005; Varma, Campi et al. 2006). As a result, it is likely that Rap-mediated actin polymerization and cell spreading play a key role in early B cell activation by propagating microcluster formation following the initial contact with antigen. Sustained signaling may also be important for B cell activation. In vivo activation of T and B cells involves prolonged adhesion and interaction between lymphocytes and APCs. Therefore, Rap activation may regulate B cell activation by promoting sustained signaling. First, Rap-GTP is important for integrin activation, which contributes to pSMAC formation, 153  antigen gathering, and to prolonged adhesion with antigen presenting cells. Rap activation may also regulate B cell activation by modulating localized actin polymerization at sites of microcluster signaling and at the immune synapse. As demonstrated in T cells, actin polymerization at the site of interaction with APCs is important for sustained TCR signaling and activation (Saito and Yokosuka 2006). Actin polymerization may induce or maintain the localization of actin-interacting adaptor molecules or scaffolding proteins to the sites of signaling. Adaptor molecules and scaffolding proteins are important regulators of signal transduction as they can modulate the location, the threshold, and the intensity of signaling (Shaw and Filbert 2009). Preliminary results examining B cell activation by particulate antigen showed that Rap activation is required for optimal B cell activation as analyzed by expression of the classical activation markers CD80 and CD69 (Section 5; Appendix A). These results are consistent with the idea that Rap activation regulates sustained signaling, leading to the activation of B cells. Furthermore, it has been reported that microcluster signaling at the periphery around the region of cell contact is the main site of active, sustained signaling (Depoil, Fleire et al. 2008; Depoil, Weber et al. 2009). Because wild type B cells are able to form F-actin cups around particulate antigen, this would increase the size of the contact interface, and yield a larger circumference with more microclusters to mediate sustained signaling. In contrast, in B cells in which Rap activation is blocked their contact site is much smaller. This could result in fewer microclusters carrying out sustained signaling. Further work is required to examine how Rap activation promotes sustained signaling and B cell activation. Cantor et al. reported that B cells deficient in CD98hc failed to sustain Erk1/2 activation and produced significantly less antibodies in vivo (Cantor, Browne et al. 2009). CD98hc is a transmembrane molecule whose extracellular domain transports amino acids while its transmembrane and cytoplasmic domains are involved in integrin binding and signaling (Bertran, Magagnin et al. 1992; Fenczik, Sethi et al. 1997). B cells lacking CD98hc are unable to spread on immobilized anti-LFA-1, are unable to maintain sustained activation of Erk1/2, exhibit impaired clonal proliferation, and impaired differentiation into plasma cells, which resulted in lower antibody responses (Cantor, Browne et al. 2009). These data strongly suggest that integrin signaling and cytoskeletal dynamics play an essential role in sustaining BCR signaling and promoting B cell activation and differentiation in vivo. Thus, 154  Rap-mediated adhesion and cytoskeletal dynamics may promote sustained BCR signaling and subsequent B cell activation. Interestingly, Cantor et al. also reported that B cells lacking all leukocyte integrins exhibit decreased proliferation in response to BCR crosslinking compared to wild type cells (Cantor, Browne et al. 2009). This suggests that integrins may enhance BCR signaling, possibly by localizing together within the same membrane compartment, even in the absence of integrin ligands. Integrins have been shown to enhance TCR signaling and this may reflect the common usage of Src kinases and Syk by antigen receptors and integrins (Abram and Lowell 2007). Hikida et al. recently showed that PLC!2 is important for the generation and maintenance of B cell memory (Hikida, Casola et al. 2009). Conditionally ablating PLC!2 expression in GC B cells revealed that PLC!2 signaling is required for the formation of GC and memory B cells, as well as for secondary antibody responses by memory B cells (Hikida, Casola et al. 2009). The authors suggested that PLC!2 conveys survival signals to GC and memory B cells. In B cells, Rap is mainly activated downstream of PLC!2 signaling by DAGdependent GEFs (McLeod, Ingham et al. 1998). Therefore, Rap-GTP may mediate the effects of PLC!2 on B cell survival by promoting integrin-dependent interactions such as cell adhesion and cell spreading of GC B cells on FDCs, which is required for SHM, affinity maturation, and long-term survival (Klein and Dalla-Favera 2008). One obvious question to ask is whether Rap activation is impaired in their conditional PLC!2 knockout mice. If it is, the next questions to address are whether selectively blocking Rap activation in GC and memory B cells prevents their interactions with FDCs and whether sustained activation of pro-survival pathways (e.g. Akt) in RapGAP-expressing GC B cells is less effective than in wild type mice. BCR signaling and activation of B cells are integral components of generating an immune response and memory against pathogens, but they are also necessary first steps in the generation of malignant B cells (Refaeli, Young et al. 2008). BCR signaling leads to activation, proliferation, and differentiation of naïve B cells to activated B cells such as GC B cells. Subsequently, activation leads to genetic remodeling of the Ig loci via class switch recombination and somatic hypermutation, and this may result in oncogenic transforming 155  events (Section 1.7). Therefore, Rap may play a key role in the initiation of lymphomagenesis and lymphoma survival by facilitating adhesion between B cells and APCs and by lowering the activation threshold by promoting formation of the pSMAC. Over-activation of Rap may increase the frequency of productive B cell activations and hence, increase the possibility of a B cell transforming into a malignant cell. Surprisingly, the lost of PLC!2, upstream regulator of Rap activation, is also able to promote the lymphomagenesis of B cell lymphomas (Wen, Chen et al. 2006). Further work will be required to investigate the role of Rap in B cell lymphomagensis, similar to whether Rap regulates activation of B cells, B cell antibody response, and generation of B cell memory. 4.2.3  Novel effectors and pathways regulated by the Rap GTPases  The work in this thesis implicates the Rap GTPases in regulating B cell morphology, B cell activation, and B cell lymphoma dissemination. One key aspect that is being addressed by others in the lab is the mechanism by which Rap regulates B cell morphology. Of the known Rap effectors, only RAPL has been convincingly shown to regulate B cell morphology (Katagiri, Maeda et al. 2003; Katagiri, Ohnishi et al. 2004). Rap can regulate cytoskeletal rearrangement by modulating the activity of GEFs and GAPs for Rho family GTPases in other cell types. However, in B cells blocking Rap-GTP did not induce any global changes in Rac activation (McLeod, Li et al. 2002), and recently it was shown that Rac2-deficient B cells actually have less Rap1-GTP, suggesting that Rap may be downstream of Rac signaling in B cells (Arana, Harwood et al. 2008). Whether Rap-GTP influences the activation of other GTPases such as Cdc42, RhoA, Rabs, and Arfs in B cells, and whether there is crosstalk between Rap and Rac has not been examined. Identifying new effectors and pathways regulated by Rap-GTP will improve our understanding of how Rap regulates B cell morphology. Myosin II appears to be potential target of Rap that may control cell shape and movement. In Dictyostelium discoideum, RapGTP is found at the leading edge with a putative effector Phg2 (Jeon, Lee et al. 2007). Phg2 is a kinase that is responsible for phosphorylating myosin II, leading to its disassembly, and allowing for the formation of actin-dependent pseudopodia formation at the leading edge (Jeon, Lee et al. 2007). Myosins are important modulators of the cytoskeleton because they are actin-based motor proteins that bind to F-actin to generate force and movement along 156  actin filaments (Mermall, Post et al. 1998; Sellers 2000; Houdusse and Sweeney 2001). Myosin IIa promotes T cell amoeboid migration and mesenchymal crawling by modulating adhesion and cell spreading in response to chemokines. Conversely, stimulation through the TCR by antigen induces a stop signal and this correlates with myosin IIa phosphorylation (Jacobelli, Chmura et al. 2004; Jacobelli, Bennett et al. 2009). However, how phosphorylated myosin IIa stops T cell migration is not clear. Nonetheless, myosins are important for anti-CD44-induced B cell spreading in vitro (Sumoza-Toledo, Gillespie et al. 2006). My preliminary data suggests that Rap-GTP may regulate the phosphorylation of myosin IIa (Section 6; Appendix B). When stimulated with anti-Ig beads, phosphorylation of myosin IIa is maintained in wild type A20 cells but not in RapGAPII-expressing A20 cells. In this situation, BCR-stimulation of Rap activation may be important for generating a stop signal in B cells by phosphorylating and disassembling myosin IIa, allowing the antigen-engaged B cell to stop and spread. Further analysis of the role of Rap-GTP in modulating myosin IIa localization, phosphorylation, and activity would reveal whether Rap-GTP acts through myosin IIa to control B cell movement. Cofilin is a protein that promotes actin depolymerization by severing actin filaments, but it can also promote actin filament elongation by generating fresh barbed ends (Huang, DerMardirossian et al. 2006; Van Troys, Huyck et al. 2008). Cofilin is activated by dephosphorylation of serine 3 by phosphatases such as the Slingshot family (SSH1L, SSH2L, SSH3L), and inactivated by being phosphorylated by Lim kinase (LIMK1) (Huang, DerMardirossian et al. 2006; Van Troys, Huyck et al. 2008). In primary murine B cells, cofilin is dephosphorylated, and presumably activated following BCR stimulation by particulate antigen (Section 7; Appendix C). Recent data from our lab showed that RapGTP regulates the phosphorylation of cofilin. Consistent with cofilin acting downstream of Rap, cofilin is important for T cell motility, immune synapse formation, and activation (Burkhardt, Carrizosa et al. 2008). Recently, it was reported that caspase-11 interacts with actin-interacting protein 1 (Aip1), a cofilin activator, and that Aip regulates lymphocyte migration by promoting actin polymerization (Li, Brieher et al. 2007). Furthermore, coronin1b a F-actin binding protein, coordinates leading edge protrusion and migration by binding to and localizing Arp2/3 and SSHL1 at the leading edge to induce actin remodeling (Cai, Marshall et al. 2007). Therefore, the cofilin pathway is an interesting target that may 157  mediate many of Rap’s functions in terms of cytoskeletal remodeling and migration. Whether Rap-GTP modulates cofilin activation by binding proteins such as Aip1, coronin1b, SSHL1, or LIMK1, or indirectly controls their localization or activation is not known and would be interesting to pursue. The actin-interacting proteins dynamin 2 and HS1 are also important regulators of actin polymerization at the immune synapse, and hence of T cell activation (Gomez, Hamann et al. 2005; Gomez, McCarney et al. 2006). Both proteins are recruited to the immune synapse following T cell interaction with APCs and are required for sustained downstream TCR signaling events. These two proteins are interesting because the phenotype caused by genetic ablation of either protein mirrors what is seen in Rap-GTP-deficient B cells. For example, T cells lacking dynamin2 or HS1 exhibit an inability to polymerize F-actin at the immune synapse and a loss of sustained TCR signaling. Although these two proteins do not contain classical Rap-interacting domains, they could be regulated or localized indirectly by Rap-GTP. The clinical relevance of Rap-mediated integrin activation is illustrated by patients that suffer from leukocyte adhesion-deficiency type 1 variant (LAD-I), also known as LAD-III (Alon and Etzioni 2003). A mutation in the Rap GEF (CalDAG-GEF1/RasGRP2) caused these people to suffer symptoms such as spontaneous bleeding and recurrent bacterial infections due to the inability of platelets and leukocytes to activate Rap GTPases and subsequently integrins (Kinashi, Aker et al. 2004; Bergmeier, Goerge et al. 2007). Recently, mutations in the kindlin-3 protein were shown to cause the exact same conditions as those observed in LAD-III patients (Malinin, Zhang et al. 2009; Svensson, Howarth et al. 2009). Kindlin-3 is a cytoplasmic protein that belongs to the kindlin family of focal adhesion proteins (Moser, Bauer et al. 2009). Unlike kindlin-1 and -2, kindlin-3 is specifically expressed in hematopoietic cells and localizes to adhesion sites such as podosomes or focal dots, instead of focal adhesions, which do not exist in leukocytes (Ussar, Wang et al. 2006). Leukocytes from mice that are deficient in kindlin-3 behave very similarly to cells in which Rap activation is blocked. These leukocytes exhibit impaired adhesion and spreading on #2 integrin ligands, and are defective in their ability to adhere to endothelial cells and 158  extravasate in vivo (Moser, Bauer et al. 2009). These defects were attributed to the inability to activate integrins, as kindlin-3 binds directly to #1, #2, and #3 integrin cytoplasmic tails at a NXXY/F motif that is further away from the plasma membrane than the NXXY/F motif that talin binds (Moser, Bauer et al. 2009). Mutations in the integrin-binding domain of kindlin-3 were responsible for the clinical manifestation in patients with kindlin-3 mutations (Malinin, Zhang et al. 2009; Svensson, Howarth et al. 2009). Furthermore, it was shown that CalDAG-GEFI was normal in these patients, suggesting that Rap and its upstream components were intact and that the inability to activate integrins was due to the kindlin-3 mutation. Bone marrow transplant of hematopoietic stem cells reconstituted with wild type kindlin-3 cured these patients and restored the ability of their new leukocytes to activate integrins (Malinin, Zhang et al. 2009; Svensson, Howarth et al. 2009). Thus, these data suggest that kindlin-3 functions downstream of Rap-GTP, like RAPL and RIAM/talin, and could be a third effector protein via which Rap-GTP activates integrins. If Rap-GTP acts upstream of kindlin-3, then blocking Rap activation may prevent kindlin-3 from binding to the cytoplasmic tails of #2 integrins. This can be examined by co-immunoprecipitation or by fluorescence microscopy. Although a number of Rap effectors have been identified, it is possible that additional effectors have yet to be discovered. One approach to identify such proteins would be to use affinity purification and mass spectrometry (MS) (Gingras, Gstaiger et al. 2007). In this approach, the target protein (Rap1) would be fused with a tag such as FLAG, GFP (or CFP), tandem affinity purification tag (TAP), or an improved TAP system called GS-TAP that uses two protein G modules as well as a streptavidin binding peptide (Burckstummer, Bennett et al. 2006; Gavin, Aloy et al. 2006; Krogan, Cagney et al. 2006; Trinkle-Mulcahy, Boulon et al. 2008). For example, CFP-Rap1 would be introduced into mice or into B cell lines. These cells could then be subjected to either BCR or chemokine receptor stimulation. To stabilize protein-protein interactions, cells could be fed photoreactive diazirine analogs of leucine and methionine prior to the start of the experiment, and then proteins complexes could be stabilized by UV crosslinking before cell lysis (Suchanek, Radzikowska et al. 2005). Alternatively, formaldehyde has been used successfully as a cross-linking agent (Gingras, Gstaiger et al. 2007). Protein complexes of interests could then be isolated by affinity purification and the Rap-binding proteins identified (Gingras, Gstaiger et al. 2007). 159  MS is ideal for this because it is sensitive, capable of high-throughput, and can be automated. Potential effectors would require validation after this proteomic approach. This can be accomplished by confocal microscopy or by co-immunoprecipitation. Alternatively, if a CFP-Rap1 expressing B cells are generated, fluorescence resonance energy transfer (FRET) with a YFP-fused effector could be used to valid their interaction. This approach could potentially identify effectors that bind Rap following its activation by the BCR or chemokine receptors.  4.3 Closing remarks The work in this thesis has identified new roles for the Rap GTPases in B cells, and highlighted the importance of changes in cell morphology for B cell activation. Importantly, I demonstrated the importance of Rap activation for immune synapse formation, F-actin-rich cup formation, sustained BCR signaling, B cell activation, and B cell lymphoma invasion and organ colonization. Since B cell lymphomas spread and cause disease, designing inhibitors to block their dissemination by blocking the Rap pathway may have therapeutic potential. In addition, blocking Rap activation could also impair the ability of B cell lymphomas to adhere to stromal cells and receive survival signals. To block the Rap pathway, one could design molecules to target Rap effectors or regulators of Rap that are specifically expressed in hematopoietic cells because Rap itself is ubiquitously expressed in all cell types. Examples of targets that satisfy this criterion include the Rap GAP Spa-1, RAPL, and kindlin-3. Similarly, blocking the Rap pathway could prevent lymphoma metastasis as I have demonstrated in this thesis by genetically overexpressing RapGAPII. Drugs that constitutively activate Spa-1 or molecules that inhibit CalDAG-GEFI (RasGRP2), as well as molecules that inhibit Rap-GTP binding to RAPL could potentially impair lymphoma adhesion and subsequent metastasis. The drawback of designing molecules to block RAPL or kindlin-3 is that these proteins are not enzymes and therefore, require large amounts of inhibitor to sequester these effectors. Moreover, designing an inhibitor from scratch is difficult. Therefore, using a library of small molecules, like those isolated from sea sponges, could be use to screen and identify molecules that may inhibit lymphoma motility and may specifically target the Rap pathway (Roberge, Berlinck et al. 1998). After isolating 160  the compound and identifying its structure, it could be modified to increase its potency and/or decrease its toxicity in vivo. The advantages of using a natural library of small molecules is the biodiversity it possesses; meaning that organisms, such as the sponge, can make many novel compounds via unique biosynthetic pathways that are not easily duplicated with synthetic methods. Conversely, these biosynthetic molecules might be difficult to reproduce synthetically and may present a challenge to the production of large quantities of the inhibitor. B cells also contribute to a variety of autoimmune diseases such as rheumatoid arthritis, systemic sclerosis, multiple sclerosis, and type 1 diabetes by the production of autoantibodies (Martin and Chan 2004). Furthermore, B cells can act as “cellular adjuvants” for CD4+ T cell activation by presenting self-antigens and providing costimulation. Therefore, B cells are very important in initiating autoimmunity (Yanaba, Bouaziz et al. 2008). Depletion of B cells by anti-CD20 antibody (rituximab) treatment is quite effective in controlling autoimmunity and highlights the importance of B cells in autoimmunity (Smith 2003). Therefore, blocking Rap activation or Rap effectors may prevent autoreactive B cell activation and their long-term survival by inhibiting their interactions with FDCs and stromal cells. Further research is necessary to uncover new effectors and regulators of Rap GTPases, and to better understand the mechanism of how Rap regulates B cell adhesion, cytoskeleton organization, migration, immune synapse formation, and activation. When these mechanisms are better characterized, this knowledge can be applied for clinical treatment of lymphomas and autoimmune diseases.  161  4.4 References Abram, C.L., and Lowell, C.A. (2007). Convergence of immunoreceptor and integrin signaling. Immunol Rev 218, 29-44. Alon, R., and Etzioni, A. (2003). LAD-III, a novel group of leukocyte integrin activation deficiencies. Trends Immunol 24, 561-566. Arana, E., Harwood, N.E., and Batista, F.D. (2008). Regulation of integrin activation through the B-cell receptor. J Cell Sci 121, 2279-2286. Bergmeier, W., Goerge, T., Wang, H.W., Crittenden, J.R., Baldwin, A.C., Cifuni, S.M., Housman, D.E., Graybiel, A.M., and Wagner, D.D. (2007). 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Sustained signaling over several hours is required for induction of pathways that regulate proliferation and expression of genes that regulate differentiation. Since Rap-GTP regulates B cell adhesion, antigen gathering, and sustained signaling in response to particulate antigens, Rap-GTP may regulate B cell activation. CD80 and CD69 are classical markers expressed following the activation of B cells and are commonly used to assess B cell activation.  5.2 Experimental procedure A20 cells expressing MSCV (vector control) or RapGAPII were generated as described in Chapter 2. 1x106 anti-IgG-coated beads (prepared as described in Chapter 2) were used to stimulate 1x106 A20 cells resuspended in modified Hepes-buffered saline (1% FBS) overnight. A20 cells (1x106) were also stimulated with 10 $g/ml goat anti-IgG in quine saline (1% FBS) overnight. These conditions were compared to A20 cells resuspended in quine saline (1% FBS) overnight without any stimulation (no stimulation). Cells were stained with anti-CD80-PE and anti-CD69-PE-Cy7 (BD Bioscience) for 20 minutes on ice (1:100) and analyzed by flow cytometry.  167  5.3 Results Figure 5.1  Optimal A20 B cell activation by anti-Ig and anti-Ig-coated beads  require Rap activation B cells were stimulated with goat anti-IgG (10 $g/ml), anti-IgG-coated 4.5 $m beads (1 bead: 1 cell), or left in quine saline (1% FBS) (no stimulation control) for 24 hours. B cell activation was measured by measuring expression of the activation markers CD80 and CD69. (A) Representative FACS blots comparing activation of A20/vector and A20/RapGAPII cells following 24 hours of stimulation with soluble anti-IgG or with anti-IgG-coated beads (B) Percent of A20/vector and A20/RapGAPII cells stimulated with soluble anti-IgG that were CD80+ and CD69+. Data represents the averages from 4 independent experiments ± SD. P-value was determined by two-tailed Student’s t-test. (C) Percent of A20/vector and A20/RapGAPII cells stimulated with anti-IgG-coated beads that were CD80+ and CD69+. Data represents the averages from 4 independent experiments ± SD. P-value was determined by two-tailed Student’s t-test.  168  169  5.4 Conclusions Rap activation is necessary for the optimal activation of A20 lymphoma B cells as determined by their expression of CD80 and CD69. Rap activation is important for sustained BCR signaling in response to particulate antigens (Chapter 2) via regulating actin polymerization by possibly recruiting and maintaining signaling complexes that sustain signaling pathways such as Erk1/2, NF-%B, and the JNK and p38 MAPKs, leading to the activation of B cells. The data also suggests that Rap may be important for B activation in response to soluble anti-Ig stimulation. Actin polymerization is not required for sustained signaling induced by soluble anti-Ig as shown by others in the lab. Therefore, it might be possible that integrins activated by Rap-GTP act as a signaling platform that sustains BCR signaling because both active integrins and BCRs recruit common signaling molecules such as Src kinases and Syk. Consistent with this, B cells lacking all integrins display less proliferation in response to soluble anti-Ig stimulation (Cantor et al., 2009)  170  6. Appendix B: Rap activation is required to sustain phosphorylation of myosin IIa heavy chain 6.1 Rationale Myosin IIa is composed of a heavy-chain dimer and each chain is associated with two myosin light chains. Myosin IIa is important for cell polarization, adhesion, migration, and immune synapse formation (see Discussion 4.2.3). These processes are also regulated by Rap GTPases. Myosin IIa is phosphorylated following TCR stimulation and acts as a stop signal to promote prolonged adhesion to APCs presenting peptide:MHC molecules. Therefore, Rap GTPases may regulate B cell polarization, adhesion, migration and immune synapse formation by regulating the phosphorylation of myosin IIa.  6.2 Experimental procedure A20 MSCV (vector control) and RapGAPII cells were generated as described (Chapter 2). Cells were starved in FBS-free RPMI media for 2 hours prior to stimulation. 5x106 A20 cells were resuspended in quine saline, rested for 30 minutes at 37°C, and then stimulated with anti-Ig-coated beads at a ratio of 2 beads:1 cell. Following stimulation, cells were briefly spun down and lysed with RIPA lysis buffer (see Chapter 2). Immunoblotting was performed to assess phosphorylation of myosin IIa (Thr1939) (antibody gift from Dr. Max Krummel, UCSF).  171  6.3 Results  Figure 6.1  Rap activation is required to sustain phosphorylation of myosin IIa  heavy chain following stimulation with particulate antigen B cells were stimulated with anti-IgG-coated beads (2 bead: 1 cell) for the indicated times, and lysed with RIPA lysis buffer. Western blotting with an anti-phospho-myosin IIa (Thr1939) antibody was used to determine the levels of phosphorylation. Phosphorylation of myosin IIa is a rapid even that persists for up to 20 minutes in A20/vector cells but myosin IIa phosphorylation is not maintained when Rap-GTP is blocked. Blot is representative of three independent experiments.  6.4 Conclusions Anti-IgG beads induced rapid phosphorylation of myosin IIa that is maintained up to 20 minutes in A20 cells. However, in RapGAPII-expressing A20 cells the phosphorylation of myosin IIa was not sustained and this may explain why RapGAPII-expressing cells do not form F-actin rich cups around particulate antigens. Phosphorylation of myosin IIa is an important signal that stops T cell that respond to antigen while crawling on DCs. Therefore, phosphorylation of myosin IIa following antigen recognition in B cells may be important signal that stops their motility and induces B cell spreading and contraction.  172  7. Appendix C: Particulate antigens induce activation of cofilin 7.1 Rationale Cofilin is an actin regulatory protein that severs actin filaments, which is important for generating fresh barbed ends to promote actin cytoskeleton rearrangement. Cofilin is normally phosphorylated in resting lymphocytes by LIMK and is activated by phosphatases such as the Slingshot family. Since actin rearrangement is required for F-actin-rich cup formation following B cell encounters with particulate antigen, cofilin might be activated (dephosphorylated) during these encounters.  7.2 Experimental procedure Primary splenic B cells were isolated from C57/B6 mice (see Chapter 2) and resuspended in quine saline. Conjugates of B cells and anti-Ig-coated beads were prepared (see Chapter 2) and allowed to settle on poly-L-lysine coated coverslips for ten minutes. Following stimulation, cells were fixed with 4% PFA, permeablized with 0.5% TBST, and stained with rabbit anti-phospho-cofilin (Ser3) antibodies (1:200; Cell Signaling). Secondary detection with anti-rabbit-AF488 (1:1000; Invitrogen) was used to visualize the levels of cofilin phosphorylation as imaged on the Olympus FV1000 confocal microscope.  173  7.3 Results  Figure 7.1  Anti-IgG-coated beads induces activation of cofilin  Splenic B cells stimulated with anti-Ig-coated beads showed that cofilin was activated (dephosphorylated) in cells that respond to a model particulate antigen. (A) The top panel shows the dephosphorylation of cofilin (Ser3) following binding of anti-IgG-coated beads. In contrast, cells that did not bind beads still had high levels of phospho-cofilin (inactive). Bottom panel shows the total protein levels of cofilin the cells. (B) Quantification of the relative intensity of phospho-cofilin (Ser3) and total confilin staining as compared to the cells that did not form conjugates (= 1.0). The data represent the mean relative intensity ± SD (n > 150 cells).  174  7.4 Conclusions Cofilin is activated in B cells following stimulation by the BCR with particulate antigens and soluble anti-Ig (data not shown). In particular, B cells that formed F-actin-rich cups demonstrate strong activation of cofilin, in contrast to cells that did not bind the anti-Igcoated beads. Since RapGAPII-expressing cells do not form frequent or extensive F-actinrich cups, this might reflect that activation of cofilin regulated by Rap-GTP is necessary for formation of F-actin-rich cups.  175  8. Appendix D: UBC Research Ethics Board!s Certificates of Approval Listed on following pages (6 total).  176      177      178      179      180      181     182  

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