Functional Analysis of Human and Mouse Splenic B cells

Transcription

1 University of Miami Scholarly Repository Open Access Dissertations Electronic Theses and Dissertations Functional Analysis of Human and Mouse Splenic B cells Justin C. Boucher University of Miami, justboucher@gmail.com Follow this and additional works at: Recommended Citation Boucher, Justin C., "Functional Analysis of Human and Mouse Splenic B cells" (2016). Open Access Dissertations This Embargoed is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarly Repository. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of Scholarly Repository. For more information, please contact repository.library@miami.edu.

2 UNIVERSITY OF MIAMI FUNCTIONAL ANALYSIS OF HUMAN AND MOUSE SPLENIC B CELLS By Justin Charles Boucher A DISSERTATION Submitted to the Faculty of the University of Miami in partial fulfillment of the requirements for the degree of Doctor of Philosophy Coral Gables, Florida December 2016

3 2016 Justin Charles Boucher All Rights Reserved

4 UNIVERSITY OF MIAMI A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy FUNCTIONAL ANALYSIS OF HUMAN AND MOUSE SPLENIC B CELLS Justin Charles Boucher Approved: Wasif N. Khan, Ph.D. Professor of Microbiology and Immunology Kurt R. Schesser, Ph.D. Associate Professor of Microbiology and Immunology Robert B. Levy, Ph.D. Professor of Microbiology and Immunology Maria T. Abreu, M.D. Professor of Medicine Robert W. Keane, Ph.D. Professor of Physiology Guillermo Prado, Ph.D. Dean of the Graduate School Laurence M. Morel, Ph.D Professor of Pathology University of Florida

5 BOUCHER, JUSTIN CHARLES (Ph.D., Microbiology and Immunology) Functional Analysis of Human and Mouse (December 2016) Splenic B Cells Abstract of a dissertation at the University of Miami. Dissertation supervised by Professor Wasif N. Khan. No. of pages in text. (113) T-cell independent (TI) antibodies may be critical in closing the gap between early innate and late adaptive immune responses against bacterial pathogens. In the mouse, innate marginal zone (MZ) B cells rapidly produce these antibodies. In humans, the characterization of a MZ B cell equivalent to that of mice is controversial because of phenotypic differences between the two species. Studies suggest that TLR activation upregulate BAFF receptors, TACI and BR3 in mouse splenic B. Upregulation of BAFF receptors is important because the ligands for these receptors, BAFF and APRIL, are critical in the regulation of humoral immune responses and immunoglobulin (Ig) isotype class switching in the context of TI antigens. By comparison of human splenic MZ B cells (MZ) with their well characterized mouse counterparts we investigated a role for innate TLRs and BAFF receptors (BR3 and TACI) in TI antibody responses. I show in this dissertation that among splenic B cells, the bulk of TI antibodies are produced by MZ B cells relative to follicular I and II B cells (FOBI and FOBII) both in human and mouse. Although MZ B cells were most responsive to TLR stimulation in both species, they differed qualitatively in the regulation of BAFF receptors. Human MZ B cells increased

6 TACI, but not BR3. Once the TACI was increased, human MZ B cells produced more IgM and switched to IgA and to a lesser extent to IgG1 in the presence of BAFF or APRIL. Bruton s tyrosine kinase (Btk) was found to be required to mediate TLR signaling that enhanced expression of antibody regulators, TACI and an atypical I B, NF- Bid. Together these results suggest that like mouse, human splenic MZ B cells are the major responders to TLR ligand containing TI antigens and require Btk signaling for these TLR driven TI antibody responses.

7 Acknowledgements I would like to acknowledge the help I received from my mentor Dr. Wasif Khan and my fellow labmates Dr. Emily Clark and Dr. Eden Kleiman. This thesis was only possible because of all the help and guidance I received from my lab. I would also like to thank Dr. Schesser and the Schesser lab, the Malek lab, the Blomberg lab, and the Chen lab for their help and use of reagents. I also wish to thank all of the Microbiology and Immunology staff and administrators for their support and help in preparing this thesis I would also like to acknowledge Dr. Oliver Umland (Diabetes Research Institute, University of Miami) and Jeffrey Carrell (MedImmune) for their expertise and help with flow cytometry and sorting experiments. I would also like to recognize the help I received at MedImmune from Dr. Gianluca Carlesso, Jeff Riggs, Ellen Kutae, Nannette Mittereder, Geetha Bhat, and Madhu Ramaswamy. This work would not have been possible without their help during my time at MedImmune Finally I would like to acknowledge the support and help I have received from my wife Dr. Jennifer Pernas Boucher, my parents Mickey and Julie Boucher, my sister Janine Boucher, and all of my family and friends. iii

8 Table of Contents Page LIST OF FIGURES... vii LIST OF TABLES ix CHAPTER 1 Introduction B cell Development and Function Splenic Structure and Function The Role of Marginal Zone B cells in Immune Responses Differences Between Human and Mouse Marginal Zone B cells T-cell Independent Immune Response Role of BAFF/APRIL Ligand Receptor System in Humoral Immunity System Primary and Secondary Signals for T-cell Independent Responses Toll-like Receptors and their Ligands Toll-like Receptor Signaling Bruton s Tyrosine Kinase Cytokine Secretion by B cells Human and Mouse Marginal Zone B cells Apply Distinct TLR and BAFF Receptors During T-cell Independent Antibody Responses Characterization of Human MZ/MZP B cells Human MZ/MZP B cells Innately Express Increased TACI Compared to FOB cells Mouse and Human MZ B cells Upregulate Distinct BAFF Receptors In Response to TLR Ligands BAFF and APRIL Deliver Helper Signals to TLR-primed MZ B cells Summary of Work in Chapter iv

9 3 Role of Bruton s tyrosine kinase Signaling in MZ B cell T-cell Independent Immune Responses Btk is Required for Steady State and TLR Induced TACI Expression and TI-1 Antibody Response Steady State and TLR Induced Expression of TACI and BR3 is Reduced in btk -/- B cells MZ B cells Require Btk Function to Produce TLR Induced IgM and IgG Btk/c-Rel Signaling Axis Transcriptionally Activates Antibody Response Regulator NF- 3.5 MZ B cell Subsets are Responsible for Cytokine Secretion in Response to TLR Stimulation in Mouse and Human Btk is Required for MZ B cell Cytokine Production in Mouse and Human Summary of Work in Chapter Discussion and Future Directions Improved Resolution of Human Splenic MZ B cells Few But Significant Differences Exist between Human and Mouse MZ B cells TI Antibody Secretion is the Domain of MZ Lineage B cells TLRs and TACI Coordinate TI Antibody Secretion Btk Function is required for TI-1 Antibody Response NF- 4.7 Importance of Btk and TACI Signaling in Primary Immune Deficiency Diseases Antibody Independent Functions of MZ Lineage B cells A Model of TI Immune Responses by MZ Lineage B cells Methods Animals. 91 v

10 5.2 Flow Cytometric Analysis and Cell Sorting Isolation and Sorting of Mouse Primary B cells Isolation and Sorting of Primary Human B cells in vitro cell culture ELISA and ELISpot in vivo LPS Treatment Real-time PCR VPLEX Cytokine Assay Statistical Analysis. 95 REFERENCES.. 96 vi

11 List of Figures FIGURE Page 1 Maturation of splenic B cells The splenic marginal zone and B cell follicle T cell independent type-1 and type-2 antibody production by B cells Bruton s tyrosine kinase is critical for adaptor protein signaling In BCR and TLR pathways Characterization of human MZ/MZP B cells Human MZ/MZP B cells innately express distinct BAFF recptors Mouse MZ B cells innately express distinct BAFF receptors Mouse MZ B cells upregulate BAFF and TACI in response TLR ligands Human MZ B cells upregulate only TACI in response to TLR ligands BAFF and APRIL deliver helper signals to enhance antibody production and switching in TLR-primed mouse MZ B cells BAFF and APRIL deliver helper signals to enhance antibody production and switching in TLR-primed MZ and MZP B cells LPS-induced splenic B cell expansion and priming for BAFF/APRIL sensitivity, but not BAFF secretion by innate cells, requires Btk in vivo Steady state and in vivo LPS-induced expression of TACI and BR3 is reduced in btk -/- B cells In vitro TLR-induced expression of TACI and BR3 is reduced in btk -/- B cells TLR-priming for BAFF and APRIL sensitivity requires Btk in mouse MZ B cells TLR-priming for BAFF and APRIL sensitivity requires Btk vii

12 In human splenic MZ/MZP B cells Btk/c-Rel signaling axis regulates NF- 18 Mouse MZ B cells are responsible for cytokine secretion in response to TLR stimulation Human MZ lineage B cells are responsible for cytokine secretion in response to TLR stimulation Btk is required for MZ B cell cytokine production in mice Btk is required for MZ/MZP B cell cytokine production in humans A model of TI immune responses in MZ lineage B cells. 88 viii

13 List of Tables TABLE Page 1 Spenic B cell populations can be defined by distinct proteins on their cell surface. 5 2 Percentages and cell surface phenotype of Non-switched human splenic B cells 36 ix

14 Chapter 1: Introduction The immune system provides protection against viral, bacterial, and fungal pathogens. This immune defense is provided for in layers for maximum effectiveness. The first layer of defense is epithelial barriers at mucosal surfaces. The skin serves as a mechanical barrier against infection. Where the skin is not present, mucus secreted by the respiratory and gastrointestinal tracts can trap pathogens that have entered the body. Innate immune system cells such as macrophage, dendritic cells, neutrophils, innate lymphoid cells, and natural killer cells serve as a second line of defense. These cells can identify pathogens using pattern recognition receptors which recognize pathogen associated molecular patterns [1]. After recognition, innate immune cells are able to eliminate pathogens using the complement system or phagocytosis [2]. All of this is backed up and reinforced by the adaptive immune system. B lymphocytes (B cells) and T lymphocytes (T cells) are the two cell types found in the adaptive immune system. Upon activation T cells expressing CD8 can directly kill infected cells while CD4 T cells can help activate B cells [3, 4]. B cells are predominately found in the bone marrow, spleen, and lymph nodes and are made up of many different subsets. B cells secrete antibodies as part of the humoral arm of adaptive immune system. We are beginning to learn other functions of B cells such as their ability to secrete cytokines that influence homeostasis and functionality of other immune cells [5, 6]. B cell functional is integral to the prevention and successful defense against pathogens. 1

15 2 1.1 B cell Development and Function B cells originate in the bone marrow. The earliest B cell progenitor is the hematopoietic stem cells (HSCs) [7]. HSCs differentiate into multipotent progenitor cells which then differentiate into common lymphoid progenitor cells (CLPs) [7]. Pro-B cells are generated from CLPs by rearrangement of the D and J segments of the heavy chain [8]. However, pro-b cells do not express surface immunoglobulins [8]. For a pro-b cell to become a pre-b cell, rearrangement of the -heavy chain gene segments is required [9]. This results in surface expression of the pre-b cell receptor (pre-bcr). Pre-B cells are then positively selected through pre-bcr signaling. If the pre-bcr fails to bind its ligand the pre-b cell will cease to develop and die [8, 10]. After successful positive selection, a pre-b cell will become an immature B cell. Immature B cells express fully functional B cell receptors (BCRs). Negative selection of immature B cells then occurs in the bone marrow through BCR/self-antigen interactions [8]. If BCR signaling from self-antigens is too strong an immature B cell can undergo deletion, receptor editing, or anergy [11]. After passing negative selection immature B cells then exit the bone marrow. B cell development continues after exit from the bone marrow in the spleen. Upon arrival to the spleen immature B cells are called early transitional (T1) B cells and make up 2-4% of the overall splenic B cell population (Fig 1). Many of these cells have autoreactive B cell receptors (BCRs) and undergo negative selection in the spleen to ensure peripheral tolerance [12]. T1 B cells are distinguished by their surface expression of AA4.1, IgM, and CD24 (Table 1). The expression of AA4.1 and CD24 were key in

16 3 Figure 1. Maturation of splenic B cells. Immature B cells from the bone marrow are called T1 B cells after arrival to the spleen. Many of these cells have autoreactive BCRs and undergo negative selection in the spleen to ensure peripheral tolerance. T1 B cells develop into T2 B cells upon successfully passing this checkpoint and then go through positive selection to mature into a follicular or MZ B cell. The majority of the splenic B cell compartment is made up of FOBI and FOBII cells. FOBI and FOBII cells are the primary subset which produce TD antibodies. PreMZ B cells are the direct precursor to MZ B cells and share characteristics similar both to T2 B cells and MZ B cells. MZ B cells are fully mature cells which reside in the splenic marginal zone and make up only 5-10% of total splenic B cells. As discussed later in this chapter, patients with X-linked agammaglobulinemia (XLA) have a blockage in splenic B cell development between the T1 and T2 stages. This is caused by mutations in Bruton s tyrosine kinase. Mice with X-linked immunodeficiency (Xid) also have mutations in Bruton s tyrosine kinase. Xid mice have a blockage in B cell development between the T2 and FOBI/FOBII stages. This blockage in B cell development is also found in some XLA patients.

17 4 distinguishing T1 B cells from mature follicular B cells [13]. T1 B cells develop into late transitional (T2) B cells upon successfully negative selection. T2 B cells also have surface expression of AA4.1, IgM, and CD24, but in contrast to T1 B cells, they also express CD23 and intermediate levels of CD21 (Table 1). CD21 expression is the critical cell surface marker allowing for T2 to be distinguished from precursor MZ B cells [14]. T2 B cells compose 5-10% of splenic B cells and are a pivotal stage of B cell maturation. T2 B cells acquire signature gene expression profiles from the strength of BCR signaling they receive during positive selection at this stage [15]. Their gene expression profile determines what type of mature B cell a T2 B cell will become. Immature splenic B cells mature into follicular or marginal zone (MZ) B cells. The majority of the splenic B cell compartment is made up of follicular I and II B cells (FOBI, FOBII). Cell surface expression of IgM and IgD is used to differentiate between FOBI and FOBII cells. FOBI cells have high levels of IgD but low levels of IgM, while FOBII cells have high level of both IgM and IgD [16] (Table 1). FOBI cells comprise about 50% of all splenic B cells, are fully mature, and are the primary subset which produces T-cell dependent (TD) antibodies. FOBII cells make up about 15-20% of splenic B cells and can secrete TD antibodies similar to FOBI cells. MZ B cells are fully mature B cells that make up 5-10% of splenic B cells in mice [17] and reside in the marginal zone of the spleen. Surface expression of CD21 and CD23 are critical to discriminate between MZ B cells and FOB cells. MZ B cells have high expression of CD21 compared to intermediate CD21 expression on FOBI/II cells [18, 19]. Also, CD23 expression is low on MZ B cells while on FOBI/II cells CD23

18 5 T1 T2 premz MZ FOBI FOBII AA IgM IgD CD CD CD Table 1. Splenic B cell populations can be defined by distinct proteins on their cell surface.

19 6 levels are high [19]. Proper development of these cells requires Notch2 signaling [20]. Inactivation of Notch2 signaling in B cells results in a specific reduction of MZ B cells without affecting other subsets. In mice, MZ B cells do not circulate in the blood and are long lived. Premarginal zone (premz) B cells are the direct precursor to the MZ B cell [21]. PreMZ B cells have a distinct transcriptome profile compared to either T2 B cells or MZ B cells [22]. High expression of CD21 on premz B cells is used to differentiate them from T2 B cells [14]. PreMZ B cells also express high surface levels of CD24 allowing their separation from MZ B cells (Table 1). Recent evidence suggests FOBII cells may also be an MZ B cell precursor. This has been hypothesized because FOBII cells have the most similar transcriptome profile to MZ B cells compared to all other splenic B cell types which suggests they may be able to differentiate to MZ B cells in certain conditions [22]. MZ B cells are innate-like B cells which participate in both the innate and adaptive response [23]. Another type of innate-like B cells are B1 B cells which are found in the peritoneum. Unlike typical innate cells, innate-like MZ and B1 B cells express antigenspecific BCRs as well as Toll-like receptors (TLRs), which recognize microbial products [24]. In response to TLR stimulation, MZ and B1 B cells can become activated, present antigen, and secrete cytokines like macrophage, dendritic, and other innate cell types [18, 25]. MZ B cells can also participate in the adaptive response via T-cell dependent (TD) or T-cell independent (TI) antibody production while B1 B cells can only produce TI antibody response [26].

20 Splenic Structure and Function The spleen is a secondary lymphatic organ present in all vertebrate species with the primary function of immune surveillance of the blood and an important role in the defense of the body against pathogens. While the spleen is non-essential in adults it is important for immune development in children. For example, infants younger than 2 years of age have a suboptimal antibody response to polysaccharides because the splenic marginal zone has not been fully developed yet [27]. Also, patients with splenic dysfunctions or splenectomy are highly at risk of infections by encapsulated bacteria and lack antibody responses to them [28, 29]. These examples demonstrate the importance of splenic B cell antibody production. The spleen is an important site for the clearance of particulates, antigens, microorganisms, and aged red blood cells flowing through the spleen [30]. This is facilitated by splenic capillaries which feed an open microcirculation unique to the spleen. This allows blood to freely percolate through the connective tissue and recollect in the venous sinuses for drainage [31]. As a consequence of this entering blood cells do not pass through an endothelial barrier which allows antigens to be easily sampled by innate cells. The spleen is typically divided into two main compartments, the red pulp and white pulp. The red pulp is important for blood filtering and iron recycling by red pulp macrophages. The red pulp is made up of arteries, arterioles, sinuses, veins, and intersinusoidal space which is composed of splenic cords [31]. Red pulp macrophages, which are found beneath the venous sinus endothelium, can easily move into the sinus lumen and out again. The spleen s open circulation allows macrophage easy access to any material that needs to be removed making it the body s largest phagocytic

21 8 compartment. Splenic cords contain large amounts of erythrocytes and platelets which can be used as a reserve pool for the body. In the perifollicular zone of the red pulp, erythrocytes accumulate with granulocytes and monocytes and can also serve as a reservoir in case of tissue injury or inflammation [32, 33]. The white pulp is the lymphoid region of the spleen and contains the T cell zone (PALS) and the B cell zone (B cell follicles) (Fig 2). In both mice and rats it is composed of central arteries surrounded by periarterial lymphatic sheaths (PALS) consisting of dendritic cells, macrophage, and T cells. In rodents the white pulp is predominant and the B cell follicles tend to fuse around the T cell zone [31]. The number of B cell follicles in a given area of the spleen is higher in mice then it is in humans but it is not known if the number of follicles per spleen is different in the two species [31]. The marginal zone of the spleen surrounds the follicle and PALS and separates the red and white pulps. It is an important transit area for cells leaving the bloodstream through the marginal sinus and erythrocyte-rich red pulp entering the lymphocyte-rich white pulp. Part of the specialized organization of the marginal zone allows for the filtration of blood-borne pathogens and the initiation of both innate and adaptive immune responses. In addition to marginal zone (MZ) B cells, there are two unique macrophage populations within the marginal zone that are potent stimulators of B cell responses. The first are marginal zone metophilic macrophage which are found between the sinus and the PALS or follicles. These form an inner ring of macrophage and are characterized by their expression of Siglec1. The second population are marginal zone macrophage which form an outer ring of macrophage and are characterized by SIGNR1 and MARCO receptors which belong to the scavenger receptor family [34-36]. It is believed these marginal zone

22 Figure 2. The splenic marginal zone and B cell follicle. The splenic B cell follicle, T cell zone, and marginal zone, which make up the white pulp, are surrounded by the red pulp. The red pulp is important for blood filtering and iron recycling and consists of arteries, arterioles, sinuses, and veins. The B cell follicle is where follicular B cells reside. During a T cell dependent immune response follicular B cells receive help from T cells in the form of antigen presentation and CD40-CD40L interactions. This results in follicular B cell antibody production. The marginal zone surround the B cell follicle and T cell zone and separates these areas from the red pulp. The marginal zone is an important transit area for cells leaving the bloodstream through the splenic capillaries and allowing for the filtration of blood-borne pathogens and the initiation of both innate and adaptive immune response. Marginal zone (MZ) B cells are able to quickly respond to blood-borne pathogens based on their location and preactived state. This allows MZ B cells to produce T cell independent antibodies. MZ B cells are also able to present antigens found in the marginal zone to follicular B cells by shuttling between the marginal zone and B cell follicle. 9

23 10 macrophage capture antigens and present them to MZ B cells [37]. Between the two macrophage populations reside the MZ B cells and dendritic cells [38]. These macrophage and dendritic cells sample the blood draining into the marginal zone and can influence B cell responses through secretion of cytokines BAFF and APRIL. The human spleen has some distinct differences compared to rodents. In humans the white pulp occupies less space than the red pulp and follicles are more prevalent compared to PALS which are limited in number and size [31]. There has been debate as to whether humans possess a true marginal zone because there is only a very thin line of B cells surrounding the PALS which express different markers then mouse MZ B cells. In addition unlike rodents, humans do not have a marginal sinus or phenotypically distinct MZ macrophage [32] The Role of Marginal Zone B cells in Immune Responses MZ B cells are unique among splenic B cell subsets because of their many innate-like characteristics. MZ B cells phenotypically exhibit a preactivated state where they are physically larger, express higher basal levels of activation markers, such as CD69 and CD86, and have a lower threshold for activation compared to FOBI/II cells [26, 39]. MZ B cells can mount rapid and efficient responses to soluble antigens and also can promote T cell proliferation and cytokine production after uptake of antigens. Recently our lab has shown MZ B cells have higher transcription levels of TLR1, TLR3, TLR4, TLR7, and TLR9 compared to other splenic B cells further indicating their innate-like phenotype [22]. This enables MZ B cells to mount rapid immune responses once challenged.

24 11 As discussed above, MZ B cell s location in the spleen allows them to provide rapid responses to blood-borne antigens (Fig 2). This results in MZ B cell s ability to be a bridge between the innate and adaptive immune systems. T-cell independent (TI) antibody secretion is a type of adaptive response MZ B cells can have to these bloodborne challenges. Recent work shows mice which lack MZ B cells are unable to mount a TI Ab response suggesting MZ B cells are the primary subset which responses to TI antigens [40]. Another example of MZ B cells serving as a bridge between innate and adaptive immune systems is their role in activating T cell responses. It has been shown MZ B cell depleted mice infected with B. burgdorferi had reduced levels of TD IgG and diminished CD4 + T cell response [41]. MZ B cells can also transport and present antigens to T cells and other B cells by shuttling between the marginal zone and B cell follicle [6, 42] (Fig 2). Antigen (Ag) activated B cells are also very effective antigen presenting cells (APCs) at later times during infection when Ag becomes limited because their specific BCR allows high affinity binding in low concentration [43]. Ag presenting B cells also regulate the initial expansion and maintenance of follicular helper T cell responses and their contraction later. In addition, recent evidence has shown MZ B cell cytokine secretion can led to T and B cell activation and responses [44] Differences Between Human and Mouse Marginal Zone B cells In humans, existence of an equivalent MZ B cell to that of mice is controversial. As discussed above, there are many differences between the mouse and human splenic marginal zone which makes identification of human MZ B cells that are analogous to mouse MZ B cells challenging. Human B cells with a similar phenotype to mouse MZ B cells (IgM hi IgD lo ) also display the memory B cell marker CD27 and have somatically

25 12 mutated immunolglobulin (Ig) genes [45-47]. This suggests those cells are not naïve, antigen inexperienced MZ B cells like their mouse counterparts. Humans also lack a distinct histological compartment in the spleen where the marginal zone would be, making identification by microscopy difficult. Clinical evidence has support the existence of a human MZ B cell equivalent. Patients who have mutations in the CD40 or CD40L gene which prevent TD formation of GCs and switched memory B cells still have IgD + IgM + CD27 + B cells suggesting these cells are involved in TI responses [48]. More recent studies have also shown human splenic and blood IgD + IgM + CD27 + B cells are the predominat cell type in response to TI antigens and represent 15-20% of total B cells [49-51]. In addition these cells do not expand or have antigen dependent selection in children younger than 2 years of age despite several vaccinations which matches mouse MZ B cells [52]. However, other studies have contradicted the hypothesis that IgD + IgM + CD27 + B cells are the human equivalent of rodent MZ B cells. Transcripitional and phenotypic analyses has shown this subset and switched B cells are closely related suggesting they are not antigen inexperienced like in the mouse [53, 54]. Also, it has been shown VDJ junctions are frequently shared between switched and IgD + IgM + CD27 + B cells indicating they are not naïve like mouse MZ B cells [55]. It is also thought that human IgD + IgM + CD27 + B cells, unlike mouse MZ B cells, are migratory and can be found in the blood and other lymphoid organs [56-58]. These results together suggest most if not all IgD + IgM + CD27 + B cells found in the blood are actually memory B cells which have left the GC before Ig switching after TD immune response.

26 13 Within the last 2 years there have now been studies which have positively identified human MZ and MZ precursor (MZP) B cells as a distinct population similar to their mouse counterparts. A recent study by Descatoire et al. shows human MZ B cells are different from hypermutated IgM + memory B cells by their surface expression MEM55 and CD300a but not IgD [59]. They also used expression of transcription factors SOX7, TOX, COCH, and HOPX to define them as naïve MZ B cells [59]. In addition, this study found Alagille syndrome patients with a NOTCH2 haploinsufficiency had a significant reduction in MZ B cells but their switched memory B cells were not affected [59]. This is important because Notch2 signaling in mice is known to be important for MZ B cell development. Another recent study found coexpression of CD21 and CD24 on human splenic CD19 + IgM + B cells allowed for the identification of MZ B cells similar to mouse MZ B cells [60]. Recently, a third study used expression of CD19, IgD, and CD27 to identify human MZ B cells [61]. Taken together these results argue for the existence of MZ B cell lineages in humans similar to their mouse counterparts however, they do not have one consistent identification scheme. These studies also have not compare the functional aspects of these human MZ B cells such as antibody or cytokine secretion to their mouse counterparts. Using the studies mentioned above as a guide I developed my own scheme to identify and sort MZ B cells from human spleens utilizing CD19, IgM, IgD, CD300a, CD21, CD24, and CD27. My studies found the bulk of TI antibodies are produced by MZ B cells relative to FOBI and FOBII in both human and mouse. As shown in Chapter 2 of this thesis my results suggest that MZ B cell function in TI antibody responses is conserved in mouse and human, however, they differ in the choice of TLR and BAFF

27 14 receptor in this process. In response to TLR agonists mouse MZ B cells robustly induced both TACI and BR3. In contrast, human MZ B cells selectively increased TACI in response to TLR agonists (CpG and CL097). My work shows while human and mouse MZ B cells have many similar properties there are key differences between the species which are important for antibody responses T-cell Independent Immune Response B cell antibody response is critical for effective host defense against pathogens. A TD antibody response requires CD4 + T cells to present antigen to B cells and occurs in follicular germinal centers of the spleen. This process generates memory B cells and plasma cells expressing high affinity antibodies (Abs) [62]. However, this requires 5-7 days which is too much time to neutralize quickly replicating pathogens. TI antibody responses are much quicker, 2-3 days, providing a first line of defense for the host. TI immune responses by B cells results in the rapid production of low affinity and polyreactive Abs encoded by unmutated V(D)J genes that are able to recognize highly conserved epitopes on pathogens [63]. TI antigens are typically large structures that express multiple repeating antigenic epitopes such as bacterial and fungal polysaccharides, DNA and RNA, and viral envelope proteins. Immune responses against such pathogens require antibodies that recognize conserved bacterial components typically polysaccharides, glycoproteins, and glycolipids [64]. A specific example of a TI antigen is polysaccharide-encapsulated extracellular bacteria. A TI antibody response is particularly important during an infection with this type of bacteria, which remains a major clinical challenge, partly due to developing resistance to antibiotics.

28 15 Relative to TD antibody production, the mechanisms of TI antibody secretion remain poorly understood, particularly in humans [65]. TI antigens are divided into type-1 (TI- 1) and type-2 (TI-2) based on responsiveness in Bruton s tyrosine kinase (Btk) mutant X- linked immunodeficient mice (xid) and on their antigens. TI-1 antigens such as LPS induce a polyclonal non-antigen specific B cell response. TI-2 antigens have repetitive biochemical structures such as polysaccharides and glycoproteins that can cross-link BCRs resulting in B cell activation in the absence of T cells [66]. Xid mice are an animal model which has a naturally occurring mutation in the gene btk resulting in defective TI responses. This model has been used to demonstrate the differences between TI-1 and TI-2 antigens [67-69]. For example, TI-2 antigens including haptenated Ficoll [68], pneumococcal polysaccharides [70], and phosphocholine [71] all fail to produce antibody responses in xid mice [67, 72]. TI antigens that can elicit some response in xid mice were therefore designated TI-1 antigens. These include trinitrophenyl (TNP)-lipopolysaccharide (LPS), TNP-Brucell abortus and TNP-Streptococcus pneumonia [67, 69, 73]. The reason btk mutation affects TI antibody response for xid mice is poorly understood. While, these mice have significantly reduced splenic FOBI and peritoneal B1 B cells, the splenic transitional, FOBII and MZ B cell cellularity is relatively comparable to wild type controls [17, 74]. It is known Btk is critical for BCR signaling which likely contributes to the impaired immune responses in Btk-deficient xid mice particularly to TI-2 antigens and will be discussed later in this Chapter. The role of Btk in TI-1 antibody responses in the mouse remains unclear and little is known about human splenic MZ B cell function and their role in TI-1 antibody responses. Work in Chapter 3

29 Figure 3. T cell independent type-1 and type-2 antibody production by B cells. Antigens that produce a TI-1 response activate B cells using a different mechanism than antigens that result in a TI-2 response. TI-1 antigens such as LPS induce a polyclonal non-antigen specific B cell response. TI-2 antigens have repetitive biochemical structures such as polysaccharides and glycoproteins that can cross-link BCRs resulting in B cell activation in the absence of T cells. TI-1 antigens weakly bind BCRs but are predominately regulated by TLR signaling. TLR binding by TI-I antigens serve as a strong second signal required for this response. In contrast, TI-2 antigens activate B cells by strongly cross-linking cell surface BCR molecules using repetitive epitopes and require only a weak second signal. The TI-1 second signal is from the cytokines BAFF and APRIL which are secreted by macrophage and dendritic cells. Both TI responses result in antibody production and isotype switching. 16

30 17 of my thesis will demonstrate Btk is necessary for optimal antibody production to TI-1 and not just TI-2 antigens in mouse and humans. Data in Chapter 3 will also examine the mechanism with which Btk affects TI antibody production. TI-1 and TI-2 antigens also use different mechanisms to activate B cells (Fig 3). TI-2 antigens activate B cells by strongly cross-linking cell surface IgM molecules using repetitive epitopes and a weak second signal, whereas TI-1 immune responses involve BCR but it is predominantly regulated by TLR signaling serving as a strong second signal [66]. This allows TI-1 responses to be polyclonal by virtue of mitogenic activity of TLRs, independent of BCR specificity. It is thought that TI antibody responses are critical for filling the temporal gap between early innate immune response and the delayed adaptive humoral response [75-77]. Studies with mouse models have revealed that in addition to BCR, B cells require costimulatory signals in response to TI-1 antigens. Many TI-1 antigens that are bacterial and viral DNA, RNA, and polysaccharide in nature, are recognized by pattern recognition receptors such as TLRs as well as by BCRs [65, 78]. These TI-1 antigens also stimulate innate cells via TLRs, leading to their activation and cytokine production including BAFF and APRIL [79]. However, whether this knowledge is applicable to human TI-1 antibody responses remains unclear Role of the BAFF/APRIL Ligand Receptor System in Humoral Immunity The cytokines B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are both type II transmembrane proteins. APRIL is mainly produced in a soluble form while BAFF can be processed into a soluble form after cleavage at a furin

31 18 protease site [80, 81]. BAFF s receptor binding domain is trimeric but can also be assembled into a BAFF 60-mer [82, 83]. It is a member of the TNF superfamily and an important regulator of normal B cell development and function. APRIL is also a member of the TNF superfamily and shares homology with BAFF. APRIL has been shown to stimulate the growth of B cells and tumor cells. These cytokines which are expressed by activated neutrophils, macrophages and dendritic cells both promote Ig secretion and isotype class switching in B cells [84]. BAFF has been shown to be critical for B cell maturation from T1 to T2 stage by mice which either lack BAFF or have transgenically increased BAFF expression. The latter having increased circulating mature B cells while the former has fewer [85, 86]. Several studies also suggest that survival signals provided by APRIL and BAFF play a crucial role during TI responses [87-89]. There are three known TNF receptors which bind BAFF or APRIL: B cell maturation Ag (BCMA), a transmembrane activator and calcium-modulating/cyclophilin ligand-interacting protein (TACI), and BAFF receptor (BR3). BR3 and TACI are expressed on all B cells while BCMA is primarily expressed on plasma cells and is therefore dispensable for B cell development [90]. BAFF receptors differ from other TNF receptor family members because they have a single cysteine-rich domain for each ligand subunit while other family members have several [91, 92] while only TACI has a specific hydrophobic residue in the hairpin which is required for binding APRIL [91, 93]. BR3 ligation leads to activation of the alternative NF- pathway which results in production and migration of p52 to the nucleus [94, 95]. Tonic BR3 and BCR signaling allows for the development and maintenance of the mature B

32 19 cell subsets while remaining self-tolerant [96]. For example the deletion of BR3 results in profoundly reduced numbers of transitional and mature splenic B cells [97, 98]. The magnitude of BAFF-mediated survival effects on B cells may increase during a TI response as both BCR- and TLR-mediated signaling have been shown to induce expression of BAFF receptors BR3 and TACI. BCR stimulation predominantly increases BR3 levels, whereas TLR signaling induces both BR3 and TACI expression [99]. A role for BAFF receptors in TLR-mediated immune responses is also suggested by a study showing peritoneal B1 B cells elevate BR3 expression upon interaction with CpG oligonucleotides [100]. Furthermore, mice deficient for BAFF receptors including BR3 and TACI, display defects in the production of antibodies in response to TI-2 antigens and in IgG1 and IgE class switching [98, 101]. APRIL s role in TI responses is less clear but it may play a part in Ig isotype switching [88, 102]. This is evident from studies which have shown TACI deficient mice have decreased antibody secretion after TI-2 antigens and IgA class switching [103]. It has also been shown TACI is required for CD40L, LPS, and CpG mediated Ig secretion [104]. TACI is also important for the down regulation of proapoptotic molecule BIM in plasma cells [105]. Conversely, TACI has also been shown to upregulate both Fas and FasL on MZ B cells as a way to regulate TI B cell responses [106]. Results in Chapters 2 and 3 of this thesis will demonstrate TLR stimulation causing induction of both TACI and BR3 primarily on MZ B cells. Data will also show that signaling from these receptors synergizes with TLRs to induce a greater TI antibody response compared to TLR signaling alone and that this is dependent on Btk.

33 Primary and Secondary Signals for T-cell Independent Responses The B cell receptor (BCR) is a protein complex found on the surface of B cells containing immobilized Ig and is responsible for B cell antigen recognition. Each naïve B cell expresses a unique BCR that interacts with a specific antigen [107]. After the antigen is bound a signal transduction cascade takes place resulting in a highly specific antibody response [108]. Activation also results in BCR internalization allowing for MHCII antigen presentation to T cells [109, 110]. This ensures further assistance in pathogen clearance by T-cell dependent (TD) means. BCR signaling is also important for the maturation as well as activation of B cells. Once an antigen binds to the BCR, a complex signaling cascade is induced that, depending on the antigen, may or may not require T cell help to result in antibody production. To initiate this, BCRs begin to cluster in lipid rafts on the B cell membrane. These rafts contain the protein tyrosine kinase lyn and exclude the phosphotase CD45R [111] [112, 113]. The BCR itself does not have intrinsic kinase activity but the molecules including adaptor protein BLNK [108]. After phosphorylation, syk can then -K. After activation, PI3- -localize to the BCR complex [114] (Fig 4). cleaves PIP2 into DAG and IP3 [108]. Finally, generation of IP3 allows for calcium flux from the endoplasmic reticulum. Calcium flux through BCR signaling is important for induction

34 21 differentiation, and proliferation [115]. This ensures full activation of B cells and their maturation and differentiation resulting in a functional B cell antibody response [116]. While BCR signaling can allow B cells to proliferate without additional help, in order to secrete antibodies a second signal is required. A second signal to induce antibody secretion is important to prevent multivalent antigens like DNA, collagen, actin, or tubulin from causing autoimmune B cell responses [117]. These second signals can be divided into two groups, the first which directly target B cells, and the second which indirectly activate B cells by inducing cytokines or costimulatory molecules on other immune cells [65]. TLR activation is one example of secondary signaling which results in TI B cell Ab secretion [118, 119]. MZ B cell s high expression of TLRs relative to other B cell types indicates their importance for MZ B cell responses such as antibody production. The complement pathway can also be a secondary signal [120]. MZ B cells are particularly able to utilize the complement system as a secondary signal because of their high expression of CD21 which binds C3d [27]. Cytokines from other immune cells can also act as important second signals for B cell Ab secretion. BAFF and APRIL, which are critical for B cell survival and TI Ab production, are produced predominantly by macrophage, neutrophils, and dendritic cells [97]. BAFF production is also found in the peri-marginal zone of the spleen further indicating the marginal zone s importance during TI responses [121]. In addition, recent work has shown activated TFH cells are a major source of BAFF and are necessary for high affinity GC B cells [122]. Macrophage can also secrete IL1, IL6, and IL12 in response to CpG stimulation that, with BCR cross-

35 22 linking, results in TI B cell responses [123]. This suggests cytokines secreted from myleod cells can stimulate B cells and are important for TI Ab secretion Toll-like Receptors and their Ligands Host cells are equipped with cellular sensors which can detect specific microbial components. Triggering these sensors, such as Toll-like receptors (TLRs), RIG-I receptors, Nod like receptors, and cytoplasmic DNA sensors, results in antimicrobial responses. TLRs were the first family of Pattern Recognition Receptor to be identified and have been studied most extensively. They were originally discovered in mammals by their homology to the Toll receptor found in Drosophila and are evolutionarly conserved in lower organisms such as sponges [124, 125]. TLRs are type I transmembrane proteins which mediate the recognition of pathogen-associated molecular patterns (PAMPs). They are mainly expressed in macrophages, dendritic cells (DCs), epithelial cells, neutrophils, and B cells. Currently, 13 TLRs have been found, 10 functional TLRs have been identified in mice and 12 in humans. TLRs can be divided into 2 major groups based on whether their ligand is found extracellularly or endosomally. TLR1, TLR2, TLR5, TLR6, and TLR10 are expressed on the surface of cells and mainly recognize components of bacterial membranes such as lipids, lipoproteins, and proteins. TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 are expressed in endosomes and recognize microbial nucleic acids [126]. TLR4 is found on both the plasma membrane and the endosomal membrane. All TLR members have an ectodomain containing leucine-rich-repeats, a transmembrane domain, and a short cytoplasmic region containing a Toll/interleukin-1 receptor (TIR) domain [126, 127].

36 23 TLRs can recognize a variety of PAMPS such as lipids, sugars, lipopolysaccharides, lipoproteins, proteins, and nucleic acids. Bacterial cell wall components like lipoproteins and peptidoglycans can be sensed by TLR1, TLR2, TLR6, or TLR10. LPS is recognized by TLR4 which is present on the surface of Gram negative bacteria while TLR5 can detect flagellin. TLRs which sense nucleic acids of from bacteria, viruses, or other cells are found in the endosome. These include TLR3 which recognizes viral double-stranded RNA, TLR7 and 8 which sense viral single stranded RNA, and TLR9 which detects CpG DNA. Profilin which is a parasitic protein form Toxoplasma gondii is recognized by TLR12 and TLR 13 detects 23S ribosomal RNA from bacteria [128]. When a ligand binds to a TLR it induces conformational changes allowing homo- or hetro-dimerization of the TLRs. This enables their cytoplasmic domains to become accessible to binding by signaling proteins. In order for TLRs to trigger biological responses they require TIR domain containing adaptors such as MyD88, TRIF, Mal, or TRAM [129]. MyD88 was the first member of this family to be identified and is used by all TLRs except TLR3. TLR3 is the only family member which signals exclusively through TRIF while TLR4 can signal through both MyD88 and TRIF Toll-like Receptor Signaling Signaling through MyD88 recruits the kinases IRAK1 and IRAK4 which interact with TRAF6. Activation of TRAF6 and IRAK1 by K63-linked ubiquitination recruits which then phosporylates is an inhibitor of NF- subunits p65 and results in its degradation allowing p65 and p50 to translocate to the nucleus and bind to

37 24 [1]. This alters gene expression and induces cytokine production, cell maturation, and upregulation of costimulatory molecules such as CD80 and CD86 [127]. MyD88 signaling can also activate the MAP kinase pathway by activating Erk1, Erk2, p38, and JNK. An alternative TLR adaptor to MyD88 is TRIF. The TRIF dependent pathway can be used by TLR4 and is exclusively used by TLR3. TLR4 signaling requires an additional adaptor protein TRAM in order to signal through TRIF, whereas TRIF can directly bind to TLR3 s TIR domain. TRAF6 and RIP1 along with TRADD and Pellino- 1 are then recruited by TRIF to activate TAK1 [130]. TAK1 is then able to activate NF- -dependent pathway [131]. which phosphorylates IRF-3. TRIF signaling also This results in a conformational change of IRF-3 allowing it to dimerize. Dimeric IRF-3 then translocates to the nucleus where it binds to the ISRE sequence near the promoters of target genes. [132]. Cellular localization of TLRs is very important for their function. TLR7 and 9 are exclusively found in the ER of unstimulated cells and rapidly traffic to endosomes after ligand stimulation [133]. It has been shown that inhibition of endosomal acidification prevents TLR7 and 9 induced responses suggesting the delivery of internalized nucleic acids to the endosome is pivotal to these TLRs. TLR4 is unique because it can signal through either MyD88 or TRIF depending on where it is localized. When signaling through MyD88 TLR4 requires Mal as an additional adaptor protein. After binding LPS at the cell surface TLR4 recruits Mal which facilitates its interaction with MyD88 resulting in a signaling cascade which activates NF- B and MAP kinases.

38 25 Tyrosine phosphorylation of TLR cytoplasmic domains is required for recruitment of adaptor proteins and their activation. Using chemical inhibitors it has been shown tyrosine kinase activity is critical for both MyD88 and TRIF dependent TLR4 signaling [134, 135]. It has been shown PI3 kinase is essential for the activation of downstream transcription factors during TLR signaling and tyrosine phosphorylation is needed to recruit PI3K. Therefore, tyrosine phosphorylation is a critical part of the TLR signaling pathway. One of the most well studied examples of how protein tyrosine kinases (PTKs) affect TLR functions is the TLR3/Src interaction. Phosphorylation of Tyr759 of TLR3 by Src is essential for TLR3 signaling [136]. Src binds to the TLR3- EGFR complex which allows for Src to change conformation resulting in its activation by autophosphorylation at Tyr416. Btk is also a relevant PTK for TLR signaling because it can phosphorylate tyrosine amino acids in the Mal TIR domain after LPS stimulation (Fig 4). Recent work demonstrates Btk inhibitors can block tyrosine phosphorylation of Mal further showing Btk s importance as a PTK [137]. Dual BCR/TLR engagement induces antibody secretion and class switch recombination (CSR) to all isotypes. Integration of these two signals results in activation of the NF- -induced cytidine deaminase (AID). An example of this is TLR9 ability to initiate CSR through NF- [138]. It has also been shown that stimulation with BAFF or APRIL after preincubation with CpG results in IgM and class switched Ig secretion [104]. One of the promoter regions targeted by NF- clear translocation is for the constant heavy chain genes. After this early CSR step, TLR9 signaling also upregulates expression of AID allowing for AID to

39 26 Figure 4. Bruton s tyrosine kinase is critical for adaptor protein signaling in BCR and TLR pathways. The BCR itself does not have intrinsic kinase activity signaling molecules including adaptor protein BLNK. After phosphorylation, syk and lyn and in turn allowing b -localize to the BCR complex. Btk then phosphorylates allows for calcium resulting NF- signaling requires adaptor proteins MyD88 or TRIF. During TLR4 signaling MyD88 requires Mal as an additional adaptor protein. Btk then is needed to phosphorylate tyrosine amino acids in the Mal TIR domain. Btk phosphorylation of Mal results in activation NF-

40 27 be recruited to switch regions on the gene. This demonstrates the importance of TLR signaling in TI CSR. Work in Chapter 2 will demonstrate TLR signaling is important for splenic TI antibody production and class switching in both mouse and humans. My data on human splenic MZ B cells supports previous work on human circulating MZ B cells demonstrating TLR s importance in TI antibody secretion. Data in Chapter 3 will illustrate the TLR s relevance in splenic MZ B cell cytokine production and is the first work to show TLR mediated cytokine secretion in human splenic MZ B cells Bruton s Tyrosine Kinase Bruton s tyrosine kinase (Btk) is a nonreceptor tyrosine kinase that is a member of the Tec family of kinases and essential for B cell development. Other members of this family include Tec and Itk. Btk is present in all B cell lineages except plasma cells [139]. It is also expressed in myeloid cells, mast cells, and erythroid precursors but is not present in T cells. Btk contains an N-terminal pleckstrin homology (PH) domain, a Techomology domain, a Src-homology 2 (SH2) domain and a C-terminal SH3 domain. Btk also has multiple tyrosine residues important for its activation and signaling function. Human X-linked agammaglobulinemia (XLA) is caused by mutations in the BTK gene [ ]. XLA was the first identified primary immunodeficiency disease however, there is still no mechanistic explanation on how mutations in BTK results in hypogammaglobulinaemia. Human males affected with primary immunodeficiency XLA have a severe reduction in circulating mature B-lymphocytes and hypogammaglobulinaemia, and suffer severe and recurrent bacterial infections [142, 143]

41 28 (Fig 1). This suggests a defect TI antibody responses however, it is unknown whether B cells in XLA patients can respond to TI antigens due to their paucity or near absence in XLA patients [144]. Similar susceptibility to encapsulated bacterial infection and lack of Ab responses has been seen in mouse and human newborns which lack TACI expression suggesting a link between XLA and TACI expression [101]. It is well established that Btk is critical for BCR signaling and defects in this signaling pathway contribute to the impaired immune responses in Btk-deficient mice [ ]. Loss of Btk function in mice either by a naturally occurring point mutation (R28C) or gene targeted deletion results in a similar but less severe immunodeficiency, compared to humans, termed x-linked immunodeficiency (xid) (Fig 1). Btk mutation preferentially affects splenic follicular and peritoneal B1 B cells, whereas the splenic MZ B cell compartment appears unaffected [ ]. This immune defect primarily manifests in failed antibody responses to TI-2 antigens including haptenated Ficoll [68], pneumococcal polysaccharides [70] and phosphocholin [71] and reduced antibody responses to TI-1 antigens including trinitrophenyl (TNP)-lipopolysaccharide (LPS), TNP-Brucell abortus and TNP-Streptococcus pneumoniae [69]. These findings suggest a role for Btk in the regulation of mouse TI immune responses to bacterial pathogens. While recent studies suggest a direct role for Btk in TLR4 signaling in macrophages [153, 154], it remains unclear whether Btk function in TLR and BR3 signaling plays a role in the regulation of TI responses. A recent study demonstrated Btk and MyD88 were critical for TI IgM secretion in B cells infected with Borrelia hermsii [155]. Btk has also recently been shown to be needed for TLR9 signaling in human

42 29 dendritic cells [156]. These studies indicate that Btk may play an important role in TLR driven B cell TI response in both mice and humans. A small molecule inhibitor specifically against Btk named ibrutinib, formerly called PCI-32765, is a potent, specific, irreversible, and relatively safe has recently been described [157]. This inhibitor binds the Cys-481 of Btk by forming a high affinity covalent bond. Cys-481 is in the active site of Btk and plays a critical role in Btk s function. Several recent studies have found that use of ibrutinib can be an effective treatment for several B cell lymphomas including CLL [ ], DLBCL [161], MCL [162, 163], and WM [164]. I will demonstrate in Chapter 3 Btk s importance in human and mouse splenic MZ B cell antibody secretion and class switching, BR3 and TACI induction, and cytokine production. The mouse work supports previous findings and extends them by showing Btk is critical for TACI induction in response to TI stimulation. Human results in Chapter 3 using Btk inhibitor (Btki) ibrutinib illustrate the importance of BTK in splenic MZ B cells. My dissertation research is the first to show BTK is required for TACI induction and antibody secretion and class switching in response to TI stimulation of human splenic MZ B cells Cytokine Secretion by B cells It is becoming clear that B cells have functions to enhance or suppress immune responses other than antibody secretion. Recent experiments have shown B cells can produce both inflammatory and suppressive cytokines after TLR stimulation. These cytokines are important for lymphoid cell development, regulation of T cell responses,

43 30 and suppression of the immune system. New work has also shown TLR1/2 stimulation of mouse B cells results in production of proinflammatory cytokines such as IL6 and [165]. An important factor for determining which cytokine is produced in B cells is which TLRs are stimulated. A recent study demonstrated activation of TLR4 or TLR9 [166]. During infection, the type of pathogen can also dramatically affect B cell cytokine production. Mice infected with T. gondii secrete cytokines which activate a Th1 response while H. polygyrus infected mice secrete Th2 cytokines [167]. In addition to generating a proinflammatory response, B cell cytokines can also suppress the immune response. B cells which generate these suppressive affects are called regulatory B cells (Bregs) or B10 cells, based on their ability to produce IL10. Currently there are no unique phenotypic, transcription factor, or lineage markers to identify Bregs for they share surface markers with many B cell subsets including transitional, MZ, premz, memory, and B1 B cells [5]. Instead Bregs are typically defined by their ability to produce IL10 after 5hrs of ex vivo stimulation with PMA and ionomycin [ ]. Recent studies suggest Bregs originate from progenitor cells that cannot produce IL10 ex vivo but mature into functional Bregs after 48hrs of CD40 costimulation [5]. BCR signaling has been shown to play a key role in Breg development. In mice which have a transgenically fixed BCR or decreased BCR signaling as a result of CD19 deficiency there are dramatically fewer Bregs [168, 171]. In contrast, overexpression of CD19 results in an expansion of Bregs. This suggests BCR signal strength is important for Breg development. Strong BCR signaling in Bregs may

44 31 allow these cells to respond rapidly to antigens and form a first line of defense. Innate signals such as TLRs have also been found to cause maturation of Bregs. LPS has been shown to mature progenitor Bregs and both LPS and CpG are able to induce IL10 secretion [166, 171]. These previous studies show both innate (TLR) and adaptive (BCR) signals are important for Breg maturation and IL10 production. Bregs ability to downregulate immune responses has been demonstrated to dampen many inflammatory conditions and autoimmune diseases. Loss or absence of Breg cells increase the severity of symptoms in mouse inflammatory bowel disease (IBD) [172], experiment autoimmune encephalomyelitis (EAE) [173, 174], type 1 diabetes [175], lupus [176, 177], and allergy [178] models. In mouse IBD models antibody blockage of CD40 or B7-2 eliminates B cell suppressive effects [179]. EAE recovery has been shown to require B cells with their absence resulting in delayed IL10 and Treg detection [173, 180]. It has also been shown IL10 producing B cells can regulate T cell responses by shifting the balance between type 1 T helper (Th1) and type 2 T helper (Th2) responses which is important for autoimmune diseases [181]. Together this evidence shows regulatory B cells serve as a link between innate and adaptive immunity and are important for disease recovery and suppression of autoimmunity. Human B cells have also been shown to produce many different cytokines. Naïve B cells from the blood, when stimulated with CD40, are able to secrete IL10 but not proinflammatory cytokines similar to Bregs. However, when these cells were stimulated with anti-ig and CD40 they had elevated level IL10 [182]. Similar to mouse Bregs discussed above, in human Bregs the type of cytokine secreted is also influenced by which TLR is activated. After TLR1/2

45 32 stimulation alone IL13 is produced but when TLR1/2 is combined with TLR7 or TLR9 IL10 is secreted [165] in human B cells by increasing the TLR7/8 induced activation of ERK and STAT3 [183]. This is consistent with previous work which shows IL10 is important for the effectiveness of IFN treatment in patients with multiple sclerosis [184, 185] sensitizes human monocytes to IL10 resulting in increased IL10-mediated suppression [186]. The studies described above show there are similarities between mouse and human Breg responses. However, there are still many questions unanswered in how these processes occur and what B cell subsets are the important players in these responses. In Chapter 3 of my thesis will show that MZ B cells in both mouse and humans are the main mature splenic pop Further, the data shows Btk is critical for the production of these cytokines. This data is the first to show human splenic MZ B cells can secrete cytokines similar to mouse and that Btk is required for their production.

46 Chapter 2: Human and Mouse Marginal Zone B cells Apply Distinct TLR and BAFF Receptors During T-cell Independent Antibody Responses T-cell independent (TI) antibodies may be critical in closing the gap between early innate and late adaptive immune responses against bacterial pathogens. In the mouse, innate marginal zone (MZ) and B1 B cells rapidly produce these antibodies. MZ B cells can also participate in the adaptive response via TI and T-cell dependent (TD) antibody production while B1s can only produce TI antibody response. In humans, existence of an equivalent MZ B cell to that of mice is controversial because of their expression of CD27, which is typically found on memory B cells [54, 55, 187]. Studies suggest that survival signals provided by BAFF, a member of the TNF superfamily, also play a crucial role during TI responses [87-89]. The magnitude of BAFF-mediated survival effects on B cells may increase during a TI response as both BCR- and TLRmediated signaling has been shown to induce expression of BAFF receptors; BCR predominantly increase BR3 levels, whereas TLRs induce both BR3 and TACI expression [99]. Furthermore, mice deficient for BAFF receptors including BR3 and TACI, display defects in the production of antibodies in response to TI-type 2 antigens [98]. By comparing poorly characterized human splenic MZ B cells with the wellcharacterized mouse counterparts, I investigated a role for innate mechanisms under TLRs and BAFF receptors (BR3 and TACI) that control TI antibody responses. I found the bulk of TI antibodies are produced by MZ B cells relative to follicular I and II B cells in both human and mouse. In response to TLR agonists mouse MZ B cells robustly 33

47 34 induced both TACI and BR3. In contrast, human MZ B cells selectively increased TACI in response to TLR agonists (CpG and CL097). Provision of BAFF or APRIL to TACI hi human MZ B cells further increased IgM and switched IgG1 and IgA production. Together these results suggest that MZ B cell function in TI-1 antibody responses is conserved in mouse and human, however, they differ in the choice of TLR and BAFF receptor in this process Characterization of Human MZ/MZP B cells Recent identification of human splenic MZ B cells using IgM, IgD, CD27, CD300a, CD24, and CD21 markers allowed me to functionally characterize and assess the relevance of the knowledge gained about mouse MZ B cells for human splenic MZ B cells. For my analysis of human splenic B cells I used identification schemes developed in previous studies by Benitez et al. and Descatoire et al.[59, 60]. To do this I first took all non-switched CD19 + B cells and examined their CD21 and CD24 expression (Fig. 5A). Cells which were CD21 hi and CD24 hi were then further separated by CD300a and IgD expression. Cells that were found to be CD300a + IgD lo were labeled marginal zone (MZ) B cells while cells which were CD300a + IgD hi were labeled marginal zone precursor (MZP) B cells. Follicular B cells were separated by their expression of CD21 lo and CD24 lo. Follicular I (FOBI) B cells were then identified by their IgD hi IgM lo expression while follicular II (FOBII) B cells were identified by their IgD hi IgM hi expression. To confirm that the MZ population I described was the same as the MZ population described by Descatoire et al. we compared gene expression to their gene

48 Figure 5. Characterization of human splenic MZ/MZP B cells. (A-B) Splenic human B cells were purified by negative selection and then analyzed by flow cytometry (FCM). Data is of all non-switch CD19+ cells. (A) MZ and MZP B cells were identified by their expression of CD21, CD24, CD300a, and IgD. FOBI and FOBII B cells were identified by surface expression of CD21, CD24, IgM, and IgD. (B) MZ, MZP, FOBI, and FOBII B cells populations were then analyzed for their expression of common B cell markers CD21, IgM, IgD, CD27, CD23, CD300a, and CD24. This allowed for phenotypic comparison between the populations. Results shown are one out of three total donor spleens. 35

49 36 Percentage of non-switched splenic B cells MZ MZP FOBI FOBII 27.9 ± ± ± ±2.2 CD CD CD CD CD300a IgM IgD Table 2. Percentages and characterization of non-switched human splenic B cells.

50 37 expression data. I found my MZ B cells increased expression of many genes found to have increased expression by Descatoire et al [59]. Importantly I found increased expression of SOX7 in MZ B cells which is known to be important for the identification of non-memory MZ B cells [59] (data not shown). After identifying these four population we examined their expression of other surface phenotype markers by FACS (Fig. 5B). I found human MZ B cells expressed high levels of CD21, IgM, CD27, CD300a, and CD24 while they had low expression of CD23 and IgD (Table 2). MZP B cells were found to have high expression of CD21, IgM, IgD, CD300a, and CD24 while having intermediate expression of CD23 and CD27. I then examined the relative amount of non-switched splenic B cells and found the proportion of human splenic MZ B cells was around 28% and MZP B cells constituted approximately 2.5% (Table 2). Approximately 37% of non-switched splenic B cells were FOBI cells while about 8% were FOBII cells. Overall, the proportions of human splenic B cell populations were different from what have been described for the mouse splenic B cell populations [17]. The increased percent of splenic MZ B cells compared to mouse suggests they may have a more important role for host defense in humans compared to mice 2.2. Human MZ/MZP B cells Innately Express Increased TACI Compared to FOB cells Recent studies with mice suggest a critical role for TLR ligands and cytokines BAFF and APRIL in the regulation of humoral immune responses and Ig isotype class switching, in the context of TI antigens [90, 98, ]. Further, the Khan laboratory

51 Figure 6. Human MZ/MZP B cells innately express distinct BAFF receptors. (A) Splenic B cells were enriched by magnetic beads and MZ, MZP, FOBI, and FOBII B cell populations identified using recently described human MZ B cell markers CD300a, CD27, and CD21 and used for FACS sorting. (B) RNA isolated from FACS sorted non-stimulated B cell populations from (A) were subjected to RNA extraction and qrt-pcr analysis for TACI mrna. MZ and MZP B cells expressed more TACI mrna relative to FOB cells. (C) BR3 expression analyzed as in (B). In contrast to TACI, BR3 expression was greater in FOBI cells relative to MZ and MZP B cells. (D) Cell surface TACI expression was increased on MZ and MZP B cells compared to FOB cells. (E) BR3 is higher on human MZP B cells relative to FOB cells. Surface expression of TACI and BR3 was determined by FCM and shown as median fluorescence intensity (MFI). Histograms show a representative example for each B cell population. Statistical analyses of TACI and BR3 expression in MZ or MZP B cells compared to all other B cell populations was done using student s T test (N=3). Error bars indicate SD around means of samples. Results shown from one experiment representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

52 Figure 7. Mouse MZ B cells innately express distinct BAFF receptors. In the mouse, cell surface expression of both TACI (A) and BR3 was greater in MZ and premz B cells compared to all other B cell subsets. (B) Surface expression of TACI and BR3 was determined by FCM and shown as median fluorescence intensity (MFI). Histograms show a representative example for each B cell population. Statistical analyses of TACI and BR3 expression in MZ or MZP B cells compared to all other B cell populations was done using student s T test (N=3). Error bars indicate SD around means of samples. Results shown from one experiment representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

53 40 had demonstrated that TLRs upregulate TACI and BR3 expression in mice [99]. To determine human B cell intrinsic TLR signaling and its effects on BAFF and APRIL mediated TI immune responses, FACS-sorted splenic FOBI, FOBII, MZP and MZ B cell populations (Fig. 6A) were used to isolate RNA and the relative mrna expression of TACI and BR3 determined by RT-PCR (Fig 6B and 6C). I found MZ and MZP B cells expressed 2-3 fold higher TACI mrna and corresponding TACI protein on the cell surface (Fig 6D). BR3 mrna expression was relatively higher in FOBI than MZ and MZP cells (Fig 6C) but MZP displayed modestly higher TACI protein (Fig 6E). To compare mouse B cell intrinsic TACI and BR3 surface expression splenic FOBI, FOBII, premz, and MZ B cell populations were analyzed by FACS. Similar to human, mouse splenic MZ and premz B cells displayed the most surface TACI (Fig. 7A). However, cell surface BR3 was elevated in MZ and premz relative to the FOBI, FOBII, and T1 B cell populations (Fig. 7B). These results show human splenic MZ and MZP B cells express similarly high levels of TACI which are much greater than FOBI/II cells. However, BR3 expression was similar in all of the human B cell subsets. In contrast, mouse splenic TACI and BR3 expression was highest exclusively in the MZ B cells, although premz B cells did display higher expression of TACI relative to FOB cells. Together, this comparative analysis shows TACI is more important in antibody responses in human B cells compared to BR Mouse and Human MZ B cells Upregulate Distinct BAFF Receptors in Response to TLR Ligands The Khan laboratory previously demonstrated that TACI expression can be induced in mouse splenic B cells upon BCR or TLR stimulation in vitro [99]. To extend

54 41 these in vitro findings, LPS was intraperitoneal (i.p.) injected into mice. After 48hrs LPS-induced expression of TACI and BR3 in splenic transitional and mature B cell populations was compared by FCM. In vivo LPS stimulation MZ B cells showed the highest increase of TACI in agreement with previous in vitro results [99] (Fig 8A). To ascertain if the in vivo effects resulted from direct activation of B cells by LPS and determine whether these findings with TLR4 could be extended to additional TLRs we compared MZ, premz, FOBI, FOBII, and T1 B cell populations for expression of TACI after in vitro TLR4, 7 (CL097) and 9 (CpG) stimulation. FCM analysis of these B cell populations not only confirmed preferential expression of TACI on MZ B cells but also clarified the sharp contrast between MZ/preMZ relative to the other splenic B cell populations (Fig. 8C). I also examined BR3 induction after in vivo and in vitro TLR stimulation. Although LPS-induced BR3 expression was highest in the MZ B-lineage, it was also induced in FoBI and FoBII cells to a lesser extent (Fig. 8B). Likewise BR3 was also increased in MZ B cells compared to other B cell populations after in vitro LPS and CL097 stimulation (Fig 8D). CpG induced BR3 expression in all B cell populations but it was most significantly increased in premz B cells. Together, these results show that TLR4 stimulation dramatically increases expression of TACI in both MZ and premz B cells, while TLR7 and TLR9 stimulation appear to increase BR3 in MZ/preMZ B cells but also in other B cell types, albeit modestly. These results are the first to demonstrate the highest induction of TACI predominantly in the MZ B cell lineage after stimulation with the three TLR agonists

55 Figure 8. Mouse MZ B cells upregulate BR3 and TACI in response to TLR ligands. (A-B) Injection of mice with LPS increased TACI and BR3 expression preferentially in MZ and premz B cells compared to FOBI. WT C57BL/6 mice were injected with either PBS (white bars) or 50μg of LPS (black bars). After 48hrs the spleens were removed and single cell suspensions of splenocytes stained for TACI (A) and BR3 (B) along with cell surface markers IgM, IgD, CD21, CD23, CD9, and CD19 to identify B cell subsets by flow cytometry. (C-D) In vitro stimulation of mouse B cells induced higher TACI (C) and BR3 (D) in MZ and premz B cells compared to other B cell populations. Freshly isolated splenocytes were cultured with agonists for either TLR4 (LPS), TLR7 (CL097), Cells were then stained as described in (A-B). Surface TACI and BR3 expression for B cell populations is expressed as median fluorescence intensity (MFI). Histograms show a representative example for each B cell population. Statistical analyses of MZ B cell TACI and BR3 compared to all other B cell populations done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

56 43 Figure 9. Human MZ B cells upregulate only TACI in response to TLR ligands. TLR activation of human splenic B cells induced higher TACI expression in MZ and MZP compared to FOB cells. Splenocytes were stimulated with agonists for TLR7 and TLR9 (CL097 and CpG, 1μg/mL) (white bars) or left unstimulated (black bars) for 24hrs and stained for TACI and BR3 along with cell surface markers CD19, IgM, IgD, CD300a, CD27, CD24, and CD21 to distinguish human splenic B cell populations by flow cytometry. Surface TACI and BR3 expression for B cell populations is expressed as median fluorescence intensity (MFI). Histograms show a representative example for each B cell population. Statistical analyses of MZ B cell TACI and BR3 compared to all other B cell populations done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

57 44 used and suggest that MZ B cells and possibly their immediate precursors (premz) are the primary B cell populations dedicated to respond to TLR-ligand containing antigens. Having found that the mouse splenic MZ/preMZ B cells are the key TLRresponsive cell populations for enhanced expression of TACI and BR3, I investigated whether the human splenic MZ B cell population is also the main responder to TLR agonists in BAFF receptor upregulation. Results in figure 9A show that TLR7 and TLR9 increase TACI expression equally well in MZ and MZP B cells, whereas TLR3 had no effect on TACI expression (data not shown). In contrast to mouse, TLR agonists did not increase BR3 in any of the human splenic B cell populations (Fig. 9B). Thus, in human TI antibody response may be regulated exclusively by TACI BAFF and APRIL Deliver Helper Signals to TLR-primed MZ B cells Because MZ B cells possess most BR3 and TACI, I reasoned that this contrast would reflect in enhanced BAFF and APRIL stimulatory effects on antibody production relative to FOBI/II cells. Murine splenic FOBI, FOBII and MZ B cells were FACS sorted (Fig. 10A) and stimulated with LPS, CpG or CL097 with and without BAFF or APRIL for 6 days. Analysis of culture supernatants showed that MZ B cells secreted massive amounts of IgM, which was particularly high in cells stimulated with LPS or 4 cells relative to MZ B cells stimulated with CpG alone (Fig. 10B). Addition of BAFF or APRIL to MZ B cells significantly increased IgM secretion compared to TLR agonist alone. In contrast, FOBI and FOBII cells produced very little or no IgM when cultured in the presence of TLR agonists and addition of cytokines had little to no effect on FOB cells (Fig 10B). These

58 Figure 10. BAFF and APRIL deliver helper signals to enhance antibody production and switching in TLR-primed mouse MZ B cells. MZ B cells have higher IgM and IgG3 antibody production compared to FOBI or FOBII cells. Addition of APRIL or BAFF resulted in increased antibody secretion compared to TLR agonist alone in MZ B cell samples. WT mouse MZ (black bars), FOBI (gray bars), and FOBII (white bars) B cells were purified from total splenocytes by a combination of magnetic beads (BD) and FACS sorting (A). 2.2x10 4 cells per well were stimulated in triplicate with LPS (200ng/mL), CpG (150ng/mL), or CL097 (200ng/mL) in the presence or absence of BAFF or APRIL (500ng/mL) in 200μL of RPMI for 6 days. Supernatants were analyzed for IgM (B) and IgG3 (C) secretion by ELISA in duplicate. Statistical analyses of MZ B cell compared to all other B cell populations done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments (B) and two of three independent experiments (C). *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

59 46 data suggest TLR stimulation sensitizes MZ B cells to BAFF and APRIL by increasing TACI and BR3 expression resulting in enhanced IgM production. However, TLR7- activated MZ B cells secreted high levels of IgM even without BAFF and APRIL, which had no discernable effect on IgM levels. Thus, it is possible that TLR4 and TLR9 deliver signals that cooperate with BAFF/APRIL signals for IgM production whereas TLR7 induced signals do not. Together these data suggest that among splenic B cell populations MZ B cells are the primary source of IgM in response to TI antigens. LPS is known to stimulate isotype class switching from IgM to IgG3 [191]. Further, BAFF and APRIL can cooperate with TLR agonists to induce isotype class switching from IgM to IgG and IgA [192]. Therefore, I reasoned TLR primed MZ B cells would be highly sensitive to BAFF and APRIL and may produce isotype switched antibodies in the presence of these cytokines. Results show that TLR4 and BAFF/APRIL signals synergized for the production of switched IgG3 isotype in MZ B cells (Fig 10C). In contrast to LPS, CpG induced minimal IgG3, which was increased by BAFF but not APRIL. Consistent with their high responsiveness to TLRs and BAFF/APRIL, mouse splenic MZ B cells are the main splenic cell type producing isotype class switched antibodies in response to some TI antigens. Having found that the mouse splenic MZ B cells are the key cell population for TLR and BAFF/APRIL mediated TI antibody production, I sought to identify the human B cell population that produced antibodies after TI stimulation. Highly purified MZ, MZP, FOBI and FOBII B cell populations were obtained by FACS sorting as in Fig. 6A and cultured with CpG or CL097 in combination with or without BAFF or APRIL for 6

60 47 Figure 11. BAFF and APRIL deliver helper signals to enhance antibody production and switching in TLR-primed MZ and MZP B cells. (A-C) Human MZ and MZP B cells have increased IgM secretion compared to FOBI and FOBII cells after TLR stimulation. Addition of APRIL or BAFF further increases IgM secretion compared to TLR agonist alone. Human MZ (black bars), MZP (light gray bars), FOBI (dark gray bars), and FOBII (white bars) B cells were purified from splenocytes by magnetic beads and FACS sorting (Fig 2). 1.2x10 4 cells per well were stimulated with and FOBI) or duplicate (MZP and FOBII) based on available cell numbers. After 6 days supernatants were collected and IgM (A), IgG1 (B), and IgA (C) secretion determined by ELISA. Statistical analyses of MZ B cell compared to FOBI and FOBII (A-B) or MZP compared to MZ, FOBI, and FOBII (C) B cell populations done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from

61 48 days. TLR7 or TLR9 treated human MZ B cells produced significantly higher level of IgM which was further increased in the presence of BAFF and APRIL (Fig 11A). Unlike mouse however human MZP B cells also robustly produced IgM in response to CpG and increased IgM similarly to human MZ B cells in the presence of BAFF or APRIL (Fig 11A). In contrast, FOBI and FOBII cells did not produce detectable amounts of IgM, which remained unchanged even in the presence of both CpG and BAFF or APRIL (Fig 11A). Likewise, isotype class switched IgG1 and IgA antibody secretion was also limited to MZ/MZP cells following CpG stimulation and BAFF and APRIL further increased this response (Fig 11B and C). Interestingly, human MZP B cells produced higher level of IgA after CpG stimulation than MZ B cells (Fig 11C). This suggests MZP cells differ from MZ B cells in their response to TLR and BAFF cytokines in terms switching to specific Ig isotype. Taken together these findings suggest that the two species differ considerably in the ways they produce TI antibody response. For example mouse MZ B cells respond most robustly to TLR4 and to a lesser extent TLR7 and both TACI and BR3 appear to contribute to this process. In contrast, human MZP B cells also robustly produce antibodies to TI antigens and human MZ/MZP B cells respond most robustly to CpG and exclusively depend on TACI for at least isotype switched antibody production. Despite these differences, the MZ B cell-lineage is the dedicated splenic B cell population that produces antibodies in response to TI stimulation Summary of Work in Chapter 2 My studies in this chapter have shown that among the human splenic B cell populations, MZ and MZP B cells are the primary source of antibody production in response to TI antigen, and this response is orchestrated by the TLR and BAFF

62 49 ligand/receptor system. Human MZ and MZP B cells upregulate TACI following TLR7 and TLR9 engagement, making them hypersensitive to APRIL and BAFF. In turn, TACI and TLR signals synergize to enhance production of IgM as well as IgG1 and IgA antibodies. Comparative analyses of mouse and human splenic B cell populations revealed some differences in the mechanisms of TI antibody responses. This data suggests human MZ/MZP B cells exclusively rely on TACI for TI antibody responses, whereas in the mouse, both TACI and BR3 appear to contribute to TI antibody response. While TLR7 and TLR9 stimulation potently induced TACI in human MZ/MZP B cells, the mouse MZ B cells responded most robustly to TLR4. I found that MZ B-lineage cells produced the bulk of TI antibodies in both mouse and human, whereas antibody production by FOB cells was negligible under similar stimulatory conditions. In this context, my dissertation research and others have shown that mouse MZ B cells express higher levels of TACI relative to FOBI and FOBII cells, and TLR engagement increases TACI expression [99, 193]. This primes MZ B cells to receive signals from BAFF and APRIL ligands that bind to TACI and activate downstream signals. The Cerutti group demonstrated that TACI, unlike BR3 or CD40, activates intracellular signaling via MyD88 [192]. Signaling through MyD88 is also required for most TLRs suggesting that TLR and TACI signals may converge on MyD88 and enhance NF- switched antibodies. Here we demonstrated that human splenic MZ B cells employ this TLR and TACI mechanism to regulate TI IgM, IgA, and IgG1 antibody responses.

63 50 This dissertation is the first work that has identified FOBII-like cells in human splenic B cells. It is also the first to demonstrate TACI is exclusively induced on TLR stimulated MZ B cells and the first to show TI antibody secretion and class switching in human splenic MZ B cells.

64 Chapter 3: Role of Bruton s tyrosine kinase Signaling in MZ B cell T-cell Independent Immune Responses B cell activation by BCR and TLR requires Bruton s tyrosine kinase (Btk) [67]. X-linked agammaglobulinemia (XLA) in humans and X-liked immunodeficiency (xid) in mice results from Btk mutations, which severely reduce circulating B cells and immunoglobulins [139]. Patients with XLA lack a TI response and suffer recurrent bacterial infections. Btk has been found to be necessary for B cell maturation, signaling, and development [194]. Btk-deficient B cells fail to reach a mature follicular or B1 state, but do mature into MZ B cells, yet fail to produce TI antibody responses. An outstanding question about the mechanisms of humoral immunity is the relative contribution of TLR signaling in innate cells and B cells that drive antibody production. The data presented in Chapter 2 demonstrates that TLR signaling is critical for TI immune responses by B cells, primarily of MZ type. The Khan laboratory has previously shown that mice with targeted deletion of Btk fail to produce antibody response to TNP-Ficol (TI-2) and exhibit reduced antibody responses to TNP-LPS (TI-1). Further, purified B cells lacking Btk function do not proliferate to the same extent as WT B cells [67, 73]. In contrast to some prior reports, work in this chapter shows Btk is required to mediate TLR4, TLR7 and, TLR9 dependent antibody production and cytokine secretion. Together with prior studies, my results suggest that Btk is necessary for optimal antibody production to both TI-2 and TI-1 antigens. Further, upon TLR stimulation Btk mediated expression of an atypical NF-kB member NF- 51

65 52 regulation of antibody responses may involve NF- Btk was also required to mediate TLR signaling that enhanced expression of antibody regulators, TACI and NF- Bid. Together our results suggest that like mouse, human splenic MZ B cells are the major responders to TLR ligands containing TI antigens and require Btk signaling pathway for these TLR driven antibody and cytokine responses. Studies in this chapter are the first to explain the basis for the defects in xid mice and XLA patients Btk is Required for Steady State and TLR Induced TACI Expression and TI-1 Antibody Response The Khan laboratory has previously shown that btk -/- B cells proliferate poorly in response to LPS compared to WT controls independent of LPS concentration [67, 73]. Thus, Btk plays a role in TLR signaling in B cells, however, its significance in innate cell TLR signaling remains controversial [195]. Whether the defective Ab response in btk -/- mice is the result of defective signaling in innate cells or B cells or both, remains unclear. To address this, I injected WT and btk -/- mice with LPS and after 48 hours the effects on splenic B cell numbers were examined. Because B cell clonal expansion is a hallmark of humoral immune responses, splenic B cells were enumerated and it was found that WT mice B cell numbers almost doubled whereas they remained unchanged in Btk -/- mice following LPS injection (Fig 7A). Together with previous observation this data suggests that LPS-induced B cell proliferation relies more heavily on Btk in vivo than in vitro [67, 73]. To test whether btk -/- B cells can be primed by LPS in vivo to secrete IgM, we injected mice with LPS and 48 hours later splenic B cells were purified. Following

66 53 Figure 12. LPS-induced splenic B cell expansion and priming for BAFF/APRIL sensitivity, but not BAFF secretion by innate cells, requires Btk in vivo. (A) TLR4 stimulation in vivo increased B cell numbers in WT but not in btk -/- mice after LPS injection. WT C57/B6 (black bars) and btk -/- (white bars) mice were injected with PBS or 50μg of LPS. After 48hrs spleens were removed and single cell suspensions were analyzed for total splenic B cells numbers using flow cytometry. (B) WT B cells can be primed in vivo within 48 hours to secrete antibodies in vitro and become more sensitive to APRIL and BAFF while btk -/- cannot. Mice were injected with 50μg of LPS. After 48hrs spleens were removed and B cells were purified using magnetic beads (BD). Purified total B cells were then cultured with and without BAFF (250ng/mL) or APRIL (500ng/mL). After 3 days in culture, B cells were transferred to ELISpot plates and analyzed for spots after 24hrs. (C-D) Btk -/- mice have less cell-bound BAFF and increased circulating BAFF compared to WT after LPS injection. Bound BAFF was measured 48hrs after PBS or 50μg LPS injection. To do this the spleens were removed and biotinylated BAFF was added to single cell suspensions. After 20 minutes the excess BAFF was washed out and a secondary streptavidin was added to determine bound BAFF by FCM (C). To determine serum BAFF levels 48hrs after PBS or LPS injection as described above mice were bleed. The whole blood was then spun to collect the serum which was then analyzed for BAFF using ELISA (D). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

67 54 overnight culture to strip any bound LPS, BAFF or other stimulatory components, B cells were plated into ELISPOT plates and cultured with or without BAFF or APRIL for 2 days. Significant numbers WT B cells primed in vivo produced IgM antibody secreting cells (ASCs) without addition of any agonist or cytokine and addition of APRIL led to a 2.5-fold increase in the numbers of IgM ASCs compared to media alone (Fig 12B). In contrast, LPS could not prime B cells in btk -/- mice to secrete IgM ex vivo with or without provision of cytokines in vitro (Fig 12B, media). These results suggest that btk -/- B cells are defective in responding to TLR4 stimulation and remain refractory to priming and secreting IgM even in the presence of BAFF or APRIL. In response to TLR stimulation innate cells including macrophages, dendritic cells, and possibly neutrophils produce BAFF and APRIL, while MZ B cells enhance expression of their receptors BR3 and TACI thereby sensitizing these cells to BAFF and APRIL action. Because the TI-1 response is decreased in btk -/- mice, I examined their innate cells ability to produce BAFF in vivo following LPS injection. Unfortunately, suitable reagents to measure APRIL are not available. Although serum BAFF levels were similar in PBS injected btk -/- and WT control mice, significantly more BAFF was detected in btk -/- mice than in WT controls (Fig. 12D). I also found LPS injection increased the amount of B cell bound BAFF in the WT but not btk -/-. Because B cell numbers and free BAFF are inversely proportional, it stands to reason that disparity in bound BAFF would reflect levels of BR3 and TACI receptors on the surface or numbers of B cells that can bind BAFF. The results suggest that more BAFF was bound to WT than to btk -/- B cells indicating that WT B cells increased the expression of BR3 and TACI on their cell surface, whereas btk -/- B cells fail to do so (Fig 12C). Conversely,

68 55 there was more free BAFF in LPS injected btk -/- mice because B cell numbers did not increase and cell surface TACI and BR3 did not increase in btk -/- B cells (Fig 12D). These results suggest that the innate cells ability to produce BAFF upon TLR4 stimulation is not dependent on Btk. However, the ability of B cells to proliferate and upregulate BR3 and TACI in response to increased BAFF following TLR4 stimulation in vivo is dependent on Btk. This supports the idea that like in innate cells, Btk is important in TLR4 signaling in B cells as well Steady State and TLR Induced Expression of TACI and BR3 is Reduced in btk -/- B cells To gain further insight into how the TLR and BAFF/APRIL signaling axis is regulated and my data (Fig 12) implicating Btk in this signaling axis, I compared the levels of TACI and BR3 in btk -/- with WT B cell populations by FCM. Relative to WT, all btk -/- B cell subsets express reduced TACI (Fig 13A left panel) and BR3 (Fig 13A, right panel). Next, I determined if Btk is required for TLR4-induced increase in TACI and BR3 expression in vivo. Mice were treated similarly as in Figure 8 and analyzed for cell surface TACI and BR3. I found that all btk -/- B cell subsets analyzed failed to increase TACI and BR3 (Fig 13B). Thus, Btk is required for the TLR signaling that upregulates TACI and BR3 in B cells. To verify and extend my in vivo findings, I compared TACI and BR3 expression in response to LPS and agonists for TLR7 (CL097) and TLR9 (CpG) in vitro. LPS treated B cells recapitulated in vivo outcome (Fig 14A). In response to TLR7 and TLR9 agonists WT cells showed large increases in TACI expression while btk -/- B cells failed to (Fig 14B and C). BR3 was also decreased in btk -/- B cells compared to WT after TLR7

69 Figure 13. Steady state and in vivo LPS-induced expression of TACI and BR3 is reduced in btk -/- B cells (A-B) TACI and BR3 levels are reduced in btk -/- splenic B cell populations in vivo before (A), and after LPS injection (B) compared to WT. WT (black bars and lines) and btk -/- (white bars and gray lines) mice were injected with 50μg LPS. After 48hrs spleens were removed and single cell suspensions were stained to determine TACI and BR3 expression in B cell subsets by FCM. TACI and BR3 expression for B cell subsets was expressed as median fluorescence intensity (MFI). Statistical analyses of B cell populations was done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment are representative of three independent experiments *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

70 57 Figure 14. In vitro TLR-induced expression of TACI and BR3 is reduced in btk -/- B cells (A-C) WT and btk -/- splenocytes were stimulated with either LPS (A), CL097 (B), or CpG (B) (all 1μg/mL) for 24hrs then stained to identify B cell subsets and TACI and BAFF expression by flow cytometry. TACI and BR3 expression for B cell subsets was expressed as median fluorescence intensity (MFI). Statistical analyses of B cell populations was done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment are representative of three independent experiments *p< 0.05, **p< 0.01, ***p< 0.001, ****p<0.0001

71 58 stimulation but equal to WT after TLR9 stimulation. These results suggest that mouse MZ B cells respond robustly to TLR4, TLR7 and TLR9 agonists and loss of Btk reduces this increase in TLR4 and 7 stimulated B cells. Thus, Btk is important for TACI and BR3 regulation and therefore for TI-1 Ab responses MZ B cells Require Btk Function to Produce TLR-induced IgM and IgG Next, I examined Btk s function in antibody production by MZ B cells. Relative to controls, btk -/- MZ B cells secreted significantly less IgM in response to all TLR agonists including TLR9 and addition of BAFF or APRIL could not rescue this response (Fig 15A). Further, compared to WT, btk -/- MZ B cells produced significantly less switched IgG3 (Fig 15B). Together these data show mouse IgM and IgG3 production in response to the three TLR agonists tested and by inference for TI-1 antibody response depends on Btk. To determine the relevance of my findings in a mouse model with human TI-1 immune responses, purified human splenic MZ and MZP B cells were treated in vitro with TLR agonists in the presence of the Btk inhibitor (Btki) Ibrutinib. This inhibitor was recently approved for clinical use to treat diffuse large B cell lymphoma (Wilson RH, 2015), mantle cell lymphoma [160] and CLL [158, 159]. Ibrutinib is an irreversible inhibitor blocks Btk tyrosine phosphorylation [157] and is relatively specific at the nm concentration used in this study. I found inhibition of Btk kinase activity resulted in a significant reduction of IgM secretion in response to CpG and CL097 in MZ and MZP B cells and that addition of BAFF or APRIL had no effect, consistent with our findings in

72 59 Figure 15. TLR-priming for BAFF and APRIL sensitivity requires Btk in mouse MZ B cells. (A-B) Mouse MZ B cells that lack Btk secrete significantly less IgM and switched antibodies compared to WT cells. WT (black bars) and btk -/- (white bars) mouse MZ B cells were purified from total splenocytes by magnetic bead (BD) and FACS sorting as described in Fig x10 4 purified MZ B cells per well were stimulated in triplicate with LPS (200ng/mL), CpG (150ng/mL), or CL097 (200ng/mL) and BAFF or APRIL (500ng/mL) in 200μL of RPMI for 6 days. Supernatants were analyzed for IgM (A) and IgG3 (B) secretion by ELISA in duplicate. Statistical analyses done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

73 60 Figure 16. TLR-priming for BAFF and APRIL sensitivity requires Btk in human splenic MZ/MZP B cells. (A-C) Human MZ/MZP B cells that lack Btk secrete significantly less IgM and switched antibodies compared to WT cells. Human MZ and MZP B cells were purified from splenocytes by magnetic bead selection and FACS sorting as described in Fig x10 4 cells per well were pretreated 1 hr. prior to stimulation with or without Btk inhibitor ibrutinib (20ng/mL). Cells were then in triplicate. After 6 days supernatants were collected and IgM(A), IgG1(B), and IgA(C) secretion determined by ELISA. Statistical analyses done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of three independent experiments *p< 0.05, **p< 0.01, ***p< 0.001, ****p<

74 61 the mouse (Fig 16A). I also found that inhibition of Btk prevented Ig class switching in these cells. IgG1 produced by MZ B cells was reduced with the addition of Btk inhibitor (Fig 16B). While addition of BAFF or APRIL enhanced this response, inhibition of Btk by Ibrutinib prevented class switching to IgG1 and IgA (Fig 16B and 16C). CpG plus APRIL showed some IgG1 and IgA secretion even after Btk inhibition by MZ and MZP B cells, however this marginal effect was not investigated further. These data demonstrate that, like in the mouse, TLR and BAFF or APRIL combination induces IgM secretion and switching to IgG1 and IgA and this is dependent on Btk expression and kinase activity Btk/c-Rel Signaling Axis Transcriptionally Activates Antibody Response Regulator NF- Bid The Khan laboratory has previously shown that Btk is required for BCR induced c-rel activation by sustained de novo protein synthesis [15]. Therefore, I tested whether c-rel plays a role in TLR-induced TACI. I found that like Btk, crel is required for TLRinduced TACI expression (Fig 17A). This shows Btk and c-rel signaling is directly needed for TI-1 immune responses. I next wanted to further investigate the Btk/c-Rel signaling axis. It is known that B protein of the BCl3 subfamily, NF- Bid has been identified as a critical regulator of both TD and TI antibody responses and for B cell proliferation in response to LPS [196]. NF- transduction to occur. Knockout mice for NF-

75 62 Figure 17. Btk/c-Rel signaling axis regulates NF- -B) B cells which lack Btk or c-rel have less induction of TACI, BR3, or NF- btk -/- (white bars) and crel -/- (gray bars) B cells express less TACI and BR3 mrna after TLR or BCR stimulation compared to WT (black bars)(a). NF- WTB cells (black bars) compared to btk -/- (white bars) (B). Total splenocytes were purified by B220 + selection and cultured overnight (A) or for 2hrs (B) with LPS extracted and expression TACI, BR3, or NF- qrt-pcr. Statistical analyses done using student s T test (N=3). Error bars indicate SD around the means of samples. Results shown from one experiment representative of two independent experiments *p< 0.05, **p< 0.01, ***p< 0.001, ****p<