Heterogeneity of memory marginal zone B cells in the rat

University of Groningen Heterogeneity of memory marginal zone B cells in the rat Hendricks, Jacobus IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hendricks, J. (2015). Heterogeneity of memory marginal zone B cells in the rat. University of Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverneamendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 05-12-2021 Heterogeneity of memory marginal zone B cells in the rat Jacobus Hendricks Heterogeneity of memory marginal zone B cells in the rat Author: Jacobus Hendricks Lay out: Mariska Offerman (Mikka Art) and Gildeprint Drukkerijen, Enschede Cover image: Peter M. Dammers, Groningen Cover lay out: Siyabonga Nikwe Thesis printing: Gildeprint, Enschede ISBN: 978-94-6233-152-5 Copyright © 2015 by J. Hendricks Heterogeneity of memory marginal zone B cells in the rat PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. E. Sterken and in accordance with the decision by the College of Deans. This thesis will be defended in public on Wednesday 18 November 2015 at 11:00 Jacobus Hendricks born on 5 December 1973 in Wynberg, Western Cape, South Africa Supervisors: Prof. dr. F.G.M. Kroese Prof. dr. N.A. Bos Assessment committee: Prof. dr. J.L. Hillebrands Prof. dr. G.T. Rijkers Prof. dr. ir. H. Savelkoul Paranymphs: P.M. Dammers W.H. Abdulahad This book is lovingly dedicated to: My wife and child (Natasha and Eva Hendricks) My father and late mother (Pieter and Eva Hendricks) My late wife (Theresa Lucinda Hendricks) My brothers and sisters; Derick, Janet (late), Ebrahim, Willem, Magdelene, Stephanus (late), Erno Pasquallie, extended Hendricks, Hesselman, Adams, Smith, Galant and Ockhuis families. CHAPTER 1 Introduction and aims CHAPTER 2 Organization of the variable region of the immunoglobulin 9 37 heavy-chain gene locus of the rat CHAPTER 3 Organization of the immunoglobulin heavy-and light-chain loci in 53 the rat CHAPTER 4 The proportion of mutated IgM positive marginal zone B cells 75 varies between IGHV gene families CHAPTER 5 Marginal zone B cells in neonatal rats express unmutated IgM 101 molecules CHAPTER 6 Class switched marginal zone B cells in spleen have relatively 115 low numbers of somatic mutations CHAPTER 7 Summary and general discussion 139 Nederlandse samenvatting en discussie 153 Opsomming en algemene bespreking in het Afrikaans 163 Acknowledgements 173 Chapter 1 Introduction and aims R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 1. General: the marginal zone as an anatomical compartment The spleen is the largest secondary lymphoid organ of the body that filters the blood, to remove bacteria, virusses, dead cells, cellular debris, immune complexes etc, and where adaptive immune responses towards blood-borne antigens take place (Cesta, 2006; Hofmann et al., 2010; Steiniger et al., 2006). Anatomically, the spleen is divided into two main compartments, the red pulp (RP) and the white pulp (WP) (Figure 1). Figure 1. Histological structural organization of the rat spleen. The spleen is divided into red pulp (RP) and white pulp (WP) regions. The WP are further divided into B and T lymphocyte regions including the follicles, marginal zone and periarteriole lymphatic sheath (PALS). Visible in the WP are a secondary follicle i.e. a follicle containing a germinal center. The marginal zone (MZ) forms an interface between the RP and the WP. The MZ is further separated from the follicle and PALS by the marginal sinus. The RP is primarily responsible for filtering of the blood. The RP consists of a sinusoidal venous system and RP cords. Blood vessels that carry blood into the spleen deliver their contents largely “open” into the spleen. The venous sinuses of the RP collect the blood again. The RP cords are characterised by loose lymphatic tissue that harbour mainly macrophages, plasma cells and lymphoid cells. Macrophages in the RP not only function in removal of antigens, dead cells and immune complexes from the circulation but also to recycle heme iron from senescent red blood cells. The WP is arranged around blood vessels (arterioles) and is mainly composed of lymphocytes, in addition to a variety of non-lymphoid cells. T and B cells are predominantly, but not exclusively, located in their own distinct compartments, the pariarteriolar lymphocyte she- Introduction and aims | 11 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 aths (PALS) and follicles, respectively. The follicles contain recirculating B cells, which are in search for their appropriate antigen. Most B cells in these follicles are naive IgM and IgD expressing B cells which are called follicular B (FO-B) cells. Upon antigenic stimulation with protein antigens, areas of proliferating B cells can be found within these follicles, the so-called germinal centers (GC). GC’s are sites where memory B cells are generated, a process that is associated with class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) genes (Victora and Mesin, 2014). Although the classical mechanism of CSR involves direct switching from IgM to any other class (isotype) of Ig molecule, CSR can also occur sequentially from IgM via IgG to IgA (van Zelm, 2014) or e.g. from IgG to IgA (Takahashi et al., 1986). SHM is the process whereby point mutations are introduced in the variable (V) region of Ig genes of B cells. In addition to follicles, a second large, anatomically distinct, B cell compartment is found in the spleen: the marginal zone (MZ). The MZ is located around the PALS and follicles and forms the interface between the splenic WP and RP (Pillai and Cariappa, 2009). The MZ is highly vascularized, similar to the RP, but can be distinguised from the RP by the presence of densely arranged lymphocytes. In rats and mice (Figure 2), but not in humans, the MZ is separated from the WP by the marginal sinus. This sinus is very porous, by gaps in the endothelium that lines the sinusoids, allowing the transit of lymphocytes and dendritic cells (DC) from the circulation to enter the WP. The MZ is largely composed of a unique subpopulation of B cells, the so-called MZ-B cells, in addition to non-lymphoid cells i.e. MZ macrophages (MZM), marginal metallophilic macrophages (MMM) and DC. In mice, MZM can be distinctively recognised by the expression of MARCO (macrophage receptor with collagenous structure) and C type lectin SIGN-R1, whereas MMM express SIGLEC 1 (Sialic acid-binding lectins 1) (Munday et al., 1999). In humans, marginal sinus and MMM are absent (Steiniger et al., 2006). Another structural difference between MZ in human, mice and rats is that humans contain a fibroblast-like layer that express mucosal vascular addressin cell-adhesion molecule 1 (MAdCAM-1), which divides the MZ into an inner and outer layer. These fibroblasts are often associated with an accumulation of CD4+ T cells, which are also absent in rodent spleens (Steiniger et al. 2001). MAdCAM-1 is also expressed on endothelial cells of the marginal sinus in mice, but not in the spleens of rats (Iizuka et al., 2000). In rodents, the response of MZ-B cells to infectious agents present in the blood is facilitated by the leaky MS endothelial layer that is adjacent to the MZ. Part of the blood that exits the circulation enters the spleen through the MS and therefore brings MZ-B cells in close contact with blood borne pathogens. In humans a so-called perifollicular zone (PFZ) is located between the MZ and the RP. This zone is not present in rodents. Because of the terminal capillaries that directly open in the PFZ, Steiniger et al. proposed that the PFZ serves as exit for recirculating CD4+ T cells and B cells from the general circulation into the WP (Steiniger et 12 | Chapter 1 al., 2001). In addition, the PFZ contains other blood cells such as erythrocytes, macrophages and monocytes. Together, the anatomical location of the MZ in close proximity with the RP and the rich supply with blood makes it the ideal environment to make initial contact with blood borne pathogens. MZ macrophage MARGINAL ZONE MZ B cell RED PULP FOLLICLE Bob Crimi T ZONE DC Terminal arteriole Marginal sinus Marginal metallophillic macrophage Central arteriole Figure 2. Diagram of the rat spleen highlighting the anatomical location and cellular composition of the MZ. The MZ contains marginal zone B (MZ-B) cells, MZ macrophages, marginal metallophilic macrophages and dendritic cells (DC). Blood that exit the circulation via central arterioles enter the spleen through the marginal sinus and therefore bring the MZ-B cells in first contact with blood to remove and detect blood borne pathogens (reproduced with permission from (Cyster, 2000)). 2. Marginal zone B cells 2.1. Phenotype MZ-B cells have been described as a distinct cell type with an unique cell surface phenotype and physiological function (Garraud et al., 2012; Martin and Kearney, 2002; Mebius and Kraal, 2005; Pillai and Cariappa, 2009; Zouali and Richard, 2011). In rats and mice, MZ-B cells are IgMhighIgDlowCD21highCD23low cells that distinguish them from the majority population of naïve, recirculating, FO-B cells in these animals, which are IgMlowIgDhighCD21intermediateCD23high (Figure 3). In mice MZ-B cells also characteristically express high levels of CD1d, a MHC class-I like molecule that is likely involved in presenting lipid molecules to NK cells (Gumperz et al., Introduction and aims | 13 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 2000). In rats, MZ-B cells can be further discriminated from FO-B cells by their low levels of CD45R, recognized by the HIS24 monoclonal antibody (Kroese et al., 1987b) and high levels of binding to the monoclonal antibody HIS57, directed to an undefined molecule (Dammers et al., 1999). In rats, CD90 (Thy-1) distinguishes immature B cells from mature B cells (Kroese et al., 1995); mature MZ-B cells lack therefore CD90. Also human MZ-B cells are IgMhigh and IgDlow (Weill et al., 2009). Importantly, human MZ-B cells also express CD27 (Klein et al., 1998; Tangye et al., 1998; Weller et al., 2004b; Zandvoort et al., 2001). CD27 is a member of the TNF-receptor family, and is in humans expressed by memory B cells (Klein et al., 1998). Klein and colleagues observed that not only class switched B cells express CD27, but that in human peripheral blood also IgM+IgD+CD27+ B cells are present in large numbers (Klein et al., 1998). Studies by Weller et al. (Weller et al, 2004) revealed that these IgM+IgD+CD27+ B cells in blood have a similar gene expression profile as splenic MZ-B cells, suggesting that peripheral blood IgM+IgD+CD27+ B cells may represent circulating MZ-B cells. In humans, these IgM+IgD+CD27+ B cells in the blood are also called natural effector cells (Tangye and Good, 2007). In line with the notion that the IgM+IgD+CD27+ B cells are circulating MZ-B cells is the observation that numbers of IgM+IgD+CD27+ B cells in blood are reduced after splenectomy and are almost completely undetectable in children younger than 2 years of age, when also mature MZ-B cells are absent in spleen (Kruetzmann et al., 2003). This absence of MZ-B cells in very young children is associated with a reduction in antibody responses to encapsulated bacteria (Timens et al., 1989b). In humans MZ-B cells (or at least MZ-B like cells) are not only found in spleen and blood but are also present in other human lymphoid sites such as the inner wall of the subcapsular sinus in lymph nodes (Spencer et al., 1998), in the crypt epithelium of tonsils (Dono et al., 2000), and under the dome epithelium of Peyer’s patches in gut-associated lymphoid tissues (GALT) (Spencer et al., 1985). 14 | Chapter 1 Figure 3. Immunohistology structure of the rat spleen stained for IgM and IgD. B cells located in marginal zone and follicles of the spleen have a distinct phenotype and express reciprocal levels of cell surface molecules recognized by mouse anti-rat IgM and mouse anti-rat IgD mAb’s. MZ-B cells are IgMhighIgDlow and FO-B cells are IgDhighIgMlow. Visible is also the PALS which is mainly a T cell zone. 2.2. Activation The activation status of MZ-B cells also distinguishes these cells from FO-B cells. Even “resting” MZ-B cells seem to be in a kind of “pre-activated” state as e.g. reflected by their larger size with nuclei that are more pale and irregular, as well as their higher levels of B7.1/CD80 and B7.2/CD86 on their cell membrane, compared to FO-B cells (Oliver et al., 1999). Furthermore, in vitro studies have shown that MZ-B cells have less stringent requirements for their activation, causing MZ-B cells to react more rapidly in comparison to FO-B cells (Martin et al., 2001). MZ-B cells require lower levels of lipopolysaccharide (LPS) antigens for their activation compared to FO-B cells (Martin et al., 2001; Oliver et al., 1999). MZ-B cells also express high levels of complement receptor (CR) CD21 (Timens et al., 1989a) and toll like receptors (TLR’s) (Gunn and Brewer, 2006). These high levels of CR and TLR’s underline the fact that MZ-B cells are equipped for rapid and easy activation in (primary) immune responses (Ferguson et al., 2004; Snapper et al., 1993). MZ-B cells can respond to both T-cell independent (TI) antigens and T-cell dependent (TD) antigens (Liu et al., 1991). Upon the engagement of antigens through their B cell receptor (BCR) and/or in cooperation with either the CR CD21, (Molina et al., 1996) or TLR’s (Pone et al., 2012) MZ-B cells can become activated. The activation of MZ-B cells can be enhanced by macrophages and dendritic cells in the MZ through the secretion of cytokines such as BAFF (B cell activating factor) and APRIL (a proliferation inducing ligand) (Litinskiy et al., 2002). Human splenic MZ also contain IgG+ cells (Ettinger et al., 2007). Uniquely these IgG+ MZ-B cells can be synergistically stimulated by IL-21 and BAFF, a member of the TNF family, to induce B-lymphocyte-induced maturation protein-1 Introduction and aims | 15 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 (BLIMP-1) in the absence of any further co-stimulation (Ettinger et al., 2007). BLIMP-1 is an essential transcription factor for the differentiation of B cells to plasma cells. In vitro assays have shown that IL-21 and BAFF are secreted respectively by CD4+ T cells (Coquet et al., 2007; Kobayashi et al., 2009) and DC (Fayette et al., 1998). In view of this, Ettinger et al. (Ettinger et al., 2007) speculated that IgG+ MZ-B cells contribute to serological memory in an antigen-independent fashion. 2.3. Function of marginal zone B cells Although MZ-B cells can respond to both TD-antigens (Gatto et al., 2007) and TI-antigens (Martin et al., 2001), they were initially thought to play an indispensable role in the protection against polysaccharide antigens (Guinamard et al., 2000). MacLennan and co-workers were the first to propose that MZ-B cells in rats are involved in immune responses directed against Type 2, T cell independent (TI-2) antigens (MacLennan et al., 1985). These antigens are polysaccharide antigens, expressed by the surface of encapsulated bacteria. Much of the currently available evidence of this function of MZ-B cells is, however, largely circumstantial, i.e. absence of MZ-B cells results in reduced antibody responses towards these kinds of antigens. For example, tyrosine kinase (Pyk-2) deficient mice lack MZ-B cells and this result in a marked suppression of IgM, IgG3 and IgG2c antibody production when immunized with TI-2 antigens (Guinamard et al., 2000). In addition to their involvement of anti-polysaccharide responses MZ-B cells also have the ability to launch polyreactive antibody responses (Chen et al., 1997). In humans polyreactive antibody responses i.e. the binding of an antibody to several different structural antigen elements, are associated with shorter sequence lengths of heavy chain complementary determining region 3 (H-CDR3) regions (Schroeder et al., 1995). In accordance with this finding our group also showed that, on average, MZ-B cells have a shorter H-CDR3 length compared to FO-B cells in rats (Dammers et al., 2000b). Available evidence thus suggests that MZ-B cells are polyreactive and directed against TI-2 antigens. Polysaccharide antigens are present on the surface of bacteria such as Hemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitides (Guinamard et al., 2000; Martin et al., 2001). The strategic location of MZ-B cells and their “pre-activated” state make it possible for MZ-B cells to make a rapid response to foreign particles in the blood, in particular caused by encapsulated bacteria. This response was found to occur as rapid as 3 days after immunization with TI-2 antigens, producing massive plasmablast in the bridging channels the areas where the PALS borders the RP directly, and also in the RP itself (Martin et al., 2001). Studies by Balazs et al. (Balazs et al., 2002) have shown that blood-derived neutrophils and DC carrying bacterial cargo can interact with splenic MZ-B cells. Puga et al. (Puga et al., 2012) have implicated the involvement of neutrophils to assist B cells in the clearance of TI-2 antigens. These authors observed that neutrophils exclusively present in the spleen stimulate 16 | Chapter 1 IgM production to TI-2 antigens such as LPS and even do so better then MZM, DC and as effective as CD4+ helper T cells. Furthermore, they showed that neutrophils stimulate MZ-B cells to upregulate the expression of activation induced deaminase (AID), the different class (isotype) switched transcripts and also show that in the presence of neutrophils MZ-B cells accumulate SHM. In conclusion, neutrophils activate MZ-B cells via BAFF, APRIL and IL-21 to make antibody responses to LPS (Puga et al., 2012). A newly defined subset of innate lymphoid cells (ILC) ILC’s has been identified in the splenic MZ by Magri et al. (Magri et al., 2014). Several subsets of ILC can be discriminated based on their cytokine secretion profiles (Walker et al., 2013). Magri and co-workers showed that these MZ-related ICL’s activate MZ-B cells through BAFF, the ligand of the costimulatory factor CD40 (CD40L) and notch2 ligand Delta-Like 1 (DLL1) molecule. They further showed that these ICL’s amplify the response of MZ-B cells by activating neutrophils through granulocyte macrophage-colony stimulating factor (GM-CSF). Importantly, the depletion of ICL’s results in the impairment of TI antibody responses and reflect the involvement of ILC’s in MZ-B cell responses against TI blood borne particulate antigens. IL-7 is required for the development of ILC’s (Satoh-Takayama et al., 2010). Importantly, Willems et al. (Willems et al., 2011) using IL-7 deficient mice, have demonstrated that IL-7 signaling is required in the development of the intrinsic MZ-B cell function to rapidly induce IgM production against polysaccharide antigens, providing additional evidence that ILC’s are involved in MZ-B cell responses. 2.4. Exit of activated marginal B cells from the marginal zone Activation of MZ-B cells induces their migration from the MZ. Either, they shuttle between the MZ and follicular areas (Cinamon et al., 2008) or they proliferate and differentiate to plasmablasts leading to the generation of extrafollicular foci (Liu, 1997; MacLennan et al., 2003). It is possible that the type of antigens i.e. TI-antigens or TD-antigens might be responsible for diverting the development of activated MZ-B cells into either the follicular or the extrafollicular pathway (MacLennan et al., 2003). Antigens can stimulate the exit of MZ-B cells from MZ by inducing the downregulation of SIP1 and SIP3 (Cinamon et al., 2004) and by the upregulation of chemokine receptor CXCR5 (Cinamon et al., 2008; Lu and Cyster, 2002). The expression of CXCR5 allows MZ-B cells to be attracted along a gradient induced by chemokine CXCL13 produced by i.e. follicular dendritic cells (FDC’s) in the follicles. When MZ-B cells bind either to TD-antigens (Liu et al., 1988) or TI-antigens (Vinuesa et al., 2001) with their BCR in combination with crosslinking to the complement receptor CD21 (as part of the BCR co-receptor) they become permissive to make a cognate interaction with CD4+ T cells at the T-B cells border (outer PALS) in the spleen where they can proliferate and produce an antibody response (Liu et al., 1988) forming extracellular foci or further proliferate inside the follicles to form GC. Possibly, TI-antigens stimulate MZ-B cells to proliferate and differentiate to become Introduction and aims | 17 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 plasmablasts at extracellular foci, whereas TD-antigens will most likely cause the migration of MZ-B cells into the follicles to generate GC. Although a role of MZ-B cells in the generation of plasmablasts/-cells is well known, their capacity to generate GC is less well understood. The work of Song and Cerny (Song and Cerny, 2003) have shed some light on this aspect. They provide experimental evidence showing that MZ-B cells are capable to form GC albeit with a delay in comparison to FO-B cells. However it is still unclear what signals determine the divergence into either the GC independent (i.e. extrafollicular foci) or GC dependent pathway. 3. Heterogeneity of marginal zone B cells and their developmental origin 3.1. Naive marginal zone B cells Naive MZ-B cells are antigen-inexperienced cells which express germline (unmutated) encoded immunoglobulin variable (IGV) region genes, whereas memory B cells are antigenexperienced cells that carry somatically mutated IGV region genes which are selected during the humoral immune responses by the immunizing antigen. Both naïve and memory B cells are present in the MZ, but their relative frequencies appear to be different dependent upon the species. In rodents the vast majority (>80%) of the B cells located in the MZ express IgM encoded by unmutated IGHV region genes, whereas in humans only a small percentage of B cells present in the MZ express unmutated IGHV genes (Dammers et al., 2000b; Makowska et al., 1999). These cells thus likely represent naïve, IgM+ MZ-B cells. In rats, MZ-B cells appear to be derived from (mature) FO-B cells (Dammers et al., 1999; Kumararatne and MacLennan, 1981). In the mouse, newly generated immature B cells are called early transitional B cells. Three populations, designated as T1, T2 and T3 cells, have been identified in this species (Allman et al., 2001), of which T2 cells can subsequently either become FO-B cells or MZ-B cells (Srivastava et al., 2005). Not all FO-B cells (rats) or T2 B cells (mice) develop into MZ-B cells. Naïve MZ-B cells have a somewhat shorter H-CDR3 region compared to FO-B cells in rats (Dammers et al., 2000b) and mice (Carey et al., 2008). Apparently there is some form of selection of B cells that may enter the MZ-B cell pool. Dammers and colleagues have speculated that in rats a more tonic signaling of the BCR of MZ-B cell precursor cells would result in positive selection of these B cells into the MZ-B cell compartment (Dammers et al., 2000). They also reasoned that this enhanced tonic signaling might be the consequence of shorter H-CDR3 region, which leads to multireactivity and hence also more (low-affinity) binding of autoantigens to the BCR (Dammers and Kroese, 2005). As mentioned above, in mice there is a checkpoint for cells to become either FO-B cells or MZ-B cells at the T2 cell stage. Also Pillai and colleagues (Cariappa et al., 2001) argue that signaling strength determines to which B cell subset the cells develop, but in contrast to rats they reasoned that reduced tonic signa- 18 | Chapter 1 ling results in MZ-B cells. Different signaling proteins such as Btk (Bruton’s tyrosine kinase), Aiolos (a zinc finger protein of the Ikaros family), MINT (MSH-homeoboxhomologue 2-interacting nuclear target), neurogenic locus notch homolog protein 2 (Notch2) and protein tyrosine kinase (Pyk-2) contribute to BCR signaling in the development of either FO-B cells or naïve MZ-B cells in mice (Mebius and Kraal, 2005; Pillai and Cariappa, 2009). Sufficient stimulation by (self) antigens will allow BCR signaling via activation of the Btk pathway, to induce FO-B cell development, whereas lower stimulation induces MZ-B cell development (Cariappa et al., 2001). Aiolos is a negative regulator of Btk and absence of Aiolos will therefore enhance BCR signaling. As a consequence, precursor B cells preferentially develop into FO-B cells, at the expense of MZ-B cells, which are significantly reduced in Aiolos-/- mice (Wang et al., 1998). Notch-2 signaling has been shown to be indispensable for development of MZ-B cells. In mice with a conditionally targeted deletion of Notch-2 mice their T2 precursor cells and MZ-B cells do not develop (Saito et al., 2003). The work of Saito et al. (Saito et al., 2003) have also shown that Notch-2 are preferentially expressed on mature B cells whereas Descatoire et al. (Descatoire et al., 2014) have shown that Notch-2 ligand Delta-Like1 expression on precursor MZ-B cells results in the development of MZ-B cells. MINT acts as a negative regulator of Notch-2. MINT deficient B cells differentiate more effectively into MZ-B cells (Kuroda et al., 2003). Studies by Astier et al. (Astier et al., 1997) revealed in both transformed and normal human B cells that Pyk-2 phosphorylation is induced by integrins and BCR ligation. Pyk-2 deficient mice fail to develop into MZ-B cells and therefore also Pyk-2 is considered to be important in the development of MZ-B cells (Guinamard et al., 2000). Together, these data support the notion that variable levels of BCR-mediated activation are required for either FO-B or MZ-B cell development. Since also MZ-B cells from neonatal and germfree animals already express shorter H-CDR3 regions, we speculated that endogenous (auto) antigens are involved as ligand in this (positive) selection of naive MZ-B cells (Dammers and Kroese, 2005). 3.2. Memory B cells Memory B cells provide the host with long term protection against pathogens. These B cells launch a rapid humoral immune response upon secondary stimulation. The Ig’s produced by memory B cells have a high affinity for antigens. Memory B cells are generally thought to be generated within a GC (see below) (Figure 4). GC’s are specialized areas arising in primary lymphoid follicles in response to foreign antigenic stimulation or immunization. At these sites memory B cells are generated. These cells are generally characterized by the expression of high-affinity, class switched Ig classes (MacLennan, 1994; Thorbecke et al., 1994; Victora and Mesin, 2014). In these structures high-affinity antigen-specific B cells are generated by SHM of IGV genes followed by positive selection. GC B cells can be distinguished by the expression of distinctive markers. Peanut agglutinin (PNA) was originally reported to probe for GC by the group of Thorbecke et al. (Coico et al., 1983). Additional surface markers have been Introduction and aims | 19 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 discovered to distinguish GC cells from other mature B cells e.g. in humans CD77 (Hogerkorp and Borrebaeck, 2006), CD38 (Pascual et al., 1994), CD55 and CNA.42 (Kasajima-Akatsuka and Maeda, 2006), and in mice GL7 (Balogh et al., 2010). At a molecular level, memory B cells are characterized by the presence of somatically mutated IGV genes. Memory B cells can either be class switched, or non-class switched B cells. In rats and mice a minor fraction (10%-20%) of the MZ-B cells carries SHM IGV genes and are therefore considered to represent memory B cells (Dammers et al., 2000b; Makowska et al., 1999). Early experiments by Liu et al. (Liu et al., 1988) in rodents have provided evidence that GC derived memory antigen-specific B cells can colonize the MZ. This was further supported by the work of Gatto et al. (Gatto et al., 2007; Gatto et al., 2004) who observed QB-specific IgG expressing B cells after immunization with viral QB capsid that remain associated with the MZ in mice. In contrast to rodents, in humans the proportion of mutated B cells in the MZ is much larger: more than 90% of the B cells carry mutated Ig’s (Dunn-Walters et al., 1995). Furthermore in adults, as mentioned before, nearly all splenic MZ-B cells express CD27 (Tangye et al., 1998; Zandvoort et al., 2001), which is a marker for mutated (memory) B cells (Klein et al., 1998). The origin of these CD27+ MZ-B cells in humans is hotly debated in the literature. On one hand some authors (Seifert and Kuppers, 2009; Tangye and Good, 2007) consider these cells as bona fide GC derived memory B cells, whereas others (Weill et al., 2009; Weller et al., 2004a; Weller et al., 2008) proposed that these cells mutate their Ig receptor outside the GC during their generation. This last conclusion was largely based upon the observation that the IGHV genes of MZ-B cells are also mutated in patients with hyper IgM type I syndrome (Weller et al., 2001). Hyper IgM type I patients are characterised by a mutation in the CD40L gene and therefore do not express functional CD40L protein. These patients cannot generate GC or produce class switched Ig molecules. Because these patients lack GC, but still have MZ-B cells, the authors proposed that CD27+ MZ-B cells are apparently not generated inside GC and therefore are distinct from the classical GC derived memory B cells. The authors postulate that mutations are introduced during their generation, outside the GC’s in a T cell and antigen independent fashion, and that the reason for these mutations is to diversify their primary Ig repertoire. 3.3. Germinal centers: A source of memory B cells Although other pathways are proposed for the development of memory B cells, GC’s are still considered as the primary site for memory B cell development (Takemori et al., 2014). In brief, upon infection or immunization in a TD immune response a few (1-3) precursor B cells (Jacob and Kelsoe, 1992; Kroese et al., 1987c) are recruited to the follicles of secondary lymphoid organs, were they rapidly expand. The initiation step for GC formation involves the cognate interaction of B cells and follicular helper CD4+ T (Tfh) cells at the border between 20 | Chapter 1 the T cell zone and the B cell follicle (Garside et al., 1998; Kerfoot et al., 2011; Okada et al., 2005). B cells receive help of Tfh cells via CD40L expressed on T cells (Kawabe et al., 1994a) and Interleukin 21 (IL-21) (Linterman et al., 2010) to proliferate and seed a primary follicle to become a GC. A fully developed GC develops into two zones i.e. dark zone (DZ) and a light zone (LZ) (Victora et al., 2012). In the DZ B cells proliferate. These proliferative B cells are known as centroblasts and express Ki-67 and CD77. Centroblasts upregulate AID (Kolar et al., 2007; Muramatsu et al., 2000) to undergo SHM and CSR. AID is an enzyme that belongs to the cytosine deaminase family and it has been shown to be indispensable for both SHM and CSR (Arakawa et al., 2002; Muramatsu et al., 2000; Petersen-Mahrt et al., 2002; Revy et al., 2000). AID expression is induced upon antigen stimulation and is predominantly expressed in GC B cells (Muramatsu et al., 1999). The ultimate aim of SHM is to generate high affinity antibodies, whereas CSR will change the functionality of the secreted antibodies. Centroblasts that exit the DZ become centrocytes and form the so-called LZ, which are CD77-. In the LZ these cells are positively selected for their affinity to the antigen that induces the GC reaction. FDC and Tfh cells both located in the follicles play a critical role in this process. In the LZ centrocytes test their newly develop hypermutated Ig receptors by binding and processing antigens presented on FDC. These centrocytes present antigen peptides to Tfh cells and can return to the DZ to further increase their affinity against the specific antigen. Positive selection occurs when centrocytes with a higher affinity for the antigen present on FDC receive more help from Tfh cells compared to B cells with lower affinity (Cerutti et al., 2012; Victora and Nussenzweig, 2012). Tfh deliver survival signals to B cells via CD40L, ICOS and cytokines (Cerutti et al., 2012). Most GC B cells also undergo CSR. In vitro studies have shown that CSR by B cells require both ligation of CD40 by CD40L on T cells and T cell derived cytokines (Tangye et al., 2002). Memory B cell development is driven by IL-4 that induces Bcl-6 through STAT-6 (STAT: signal transducers and activators of transcription), a transcription factor that is essential for IL-4 induction of CSR to IgG1 (Linehan et al., 1998). A unique profile of cytokines will induce a particular isotype. In mouse for example CD40 in the presence of IL-4 will induce CSR to IgG1 and IgE (Bergstedt-Lindqvist et al., 1988). Conversely, when IL-4 is substituted with interferon-gamma (IFN-γ) the CSR is more towards IgG2a in mice. Interestingly Pihlgren et al. (Pihlgren et al., 2013) have provided experimental evidence that in mice lacking T cells, CD40L and CD28 B cells can still undergo CSR. This TI IgG class switching was strongly driven by ligation of TLR-4 and mediated via TIR-domain-containing adapter inducing interferon-β (TRIF). However TI IgG CSR was still associated with GC formation as indicated by the expression of PNA, GL-7 and AID on GC B cells. High affinity selected centrocytes can either become a plasma cell or a memory B cell, which depends on certain transcription factors (Calame et al., 2003; Crotty et al., 2010). Transcription factors, such as paired box 5 (Pax5), B lymphocyte-induced maturation protein-1 (Blimp-1) and Bcl-6 have been shown to play a key role in directing either plasma cell or memory B cell development. Transcription Introduction and aims | 21 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 factors Pax-5 and Bcl-6 are required for GC B cells to develop into memory B cells and these transcription factors are inhibitory to plasma cell development (Angelin-Duclos et al., 2000; Calame et al., 2003). Bcl-6 is a transcriptional repressor that regulates lymphocyte differentiation during immune responses and is particularly highly expressed in GC B cells (Shaffer et al., 2000). Although GC’s are certainly the main site for generation of memory B cells (Shlomchik and Weisel, 2012), there is also evidence from studies in mice that memory B cells can develop outside GC’s (Good-Jacobson and Shlomchik, 2010; Takemori et al., 2014). Toyama et al. (Toyama et al., 2002) have shown that memory B cells can be generated in Bcl-6 deficient mice. Although these mice completely lack GC’s, their B cells were able to differentiate into IgM and IgG1 memory B cells. Also, CD40 deficient mice that lack GC formation revealed normal IgG and IgM responses to TI-antigens (Kawabe et al., 1994b). These have led to the proposal that some memory B cells might be formed independent of GC. Also memory towards TD antigens can be GC independent as shown by (Kaji et al., 2012). However these IgG memory B cells having hallmarks of memory cells, i.e. antigenic experienced B cells that express class switched isotype, are unmutated, most likely indicate that mutated memory B cells are primarily generated in a GC (Kaji et al., 2012). Figure 4. Germinal center: site for SHM, selection and CSR. Illustrated in the above diagram is the GC where high affinity antibody-producing B cells are selected. B cells that are activated by an antigen and have made contact with a CD4+ Tfh cell initiate the formation of the GC. A fully formed GC contains a DZ and LZ. The environment of the DZ and LZ is well suited for the expansion and selection of high affinity memory B cells. The DZ contains tightly pack centroblast cells that proliferate and undergo somatic hypermutation mutations caused by the AID enzyme either results in antibodies with diminished affinity or improved affinity of antibody-antigen binding. These B cells will undergo selection after each round of mutations in the LZ. B cells in the LZ that encounter antigen with high affinity from FDCs will internalize 22 | Chapter 1 the antigen for presentation to antigen activated CD4+ Tfh cells. Activated CD4+ Tfh cells express CD40L which binds to CD40 on the B cells, resulting selection of high affinity antibody producing B cells. High affinity B cells receive more “help” signals than low affinity B cells. Selected B cells will either differentiate into a plasma or memory cell. By cytokine signals CD4+ Tfh cells also instruct B cell to undergo CSR. AID is also required for CSR (switching from IgM to IgG, IgA). B cells that binds antigen with low affinity will be negatively selected and undergo apoptosis. 4.Introduction to the thesis In this thesis we aimed at studying the memory compartment of MZ-B cells and compare it to recirculating memory B cells present in follicles in the rat. The earliest studies in the rat by the group of Liu et al. (Liu et al., 1988) have documented the presence of hapten specific MZ-B cells after immunization in the MZ. This work has received later experimental support by the group of Kroese et al. (Dammers et al., 2000a) demonstrating that the IGHV-Cµ transcripts expressed by phenotypically defined splenic MZ-B cells (defined as CD90negIgMhighIgDlow B cells) can carry SHM. However, only a minor fraction (less than 10%-20%) of these MZ-B cells is mutated and is considered to represent memory B cells. The presence of mutations in Ig genes is an important hallmark of memory B cells and determination of nucleotide mismatches with their counterpart germline IGHV gene sequence are required to enumerate mutations. In order to analyse the presence of these mutations in Ig genes it is therefore necessary to have a complete insight into the germline IGHV gene repertoire and understanding of the rat IGHV locus. Germline genes refer to the unrearranged DNA (nucleotide) sequences of the different IGHV, immunoglobulin heavy diversity (IGHD) and immunoglobulin heavy joining (IGHJ) gene segments present at the IGH locus (Figure 5). The chromosomal maps of the IGH locus was already published for humans (Matsuda et al., 1998) and the mouse (Johnston et al., 2006), but not for the rat. In 2004 the (entire) rat genome was unraveled (Gibbs et al., 2004), although a detailed analysis of the IGH loci was lacking. For this analysis genomic sequences of the Brown Norway (BN) rat strain were taken. For the analysis of the presence of mutations in IGHV genes the availability of genomic sequences of IGHV genes are indispensable. In Chapter 2 we therefore aimed at the analysis of these genomic sequences and to give a comprehensive description of the organization of the IGH locus in the BN rat. In Chapter 3, not only the organization of the IGH locus, but also the organization of the Ig light (IGL) chain locus of the rat is reviewed. We used the information of the IGH locus to analyse the presence of mutations in rat MZ-B cells. As mentioned before in rats and mice only a minority of the MZ B-cells are mutated, in contrast to humans. The reason for this discrepancy is not clear. So far the information on SHM in rat and mouse MZ-B cells was limited to only one IGHV family. The IGHV locus consist of multiple IGHV genes, which can be subdivided into V gene families based on homology of 80% between individual IGHV genes (Brodeur and Riblet, 1984). The conclusion by Dammers et al. that in rats only a small fraction of the MZ-B cells are mutated, Introduction and aims | 23 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 was based only on the analysis of one IGHV gene family, the IGHV5 (PC7183) gene. This study was carried out in the PVG rat strain. As mentioned above, however, the rat IGH locus, including germline IGHV gene sequences were from another rat strain, the BN rat (Chapter 2). For these reasons we analysed in Chapter 4 the presence of mutations in IGHV genes expressed by MZ-B cells isolated from BN rats. Furthermore we extended our analysis also to other IGHV gene families that contained fewer IGHV gene family members. Analysis was done on rearranged IGHV-Cµ (IgM) transcripts derived from FACS defined MZ-B cells (CD90IgMhighIgDlow) and these transcripts were compared to such transcripts of FO-B cells (CD90IgMlowIgDhigh) to address whether the proportion of 10-20% mutated MZ-B cells are a property of the IGHV5 gene family or whether it is uniformly distributed over the other gene families as well. Although the GC still prevails as the classical site where memory B cells are generated, the origin of mutated IgM expressing MZ-B cells is an issue of much debate. The findings of Weller et al. (Weller et al., 2004b) have stirred much attention and discussion on the development of IgM memory B cells. These authors proposed that human IgM memory MZ-B cells provide the MZ early in life with a diversified and protective preimmune repertoire (formed in the absence of antigen) involved in responses against encapsulated bacteria. The lack of phenotypically defined MZ-B cells in the blood in infants under 2 years of age and immaturity in neonatal rats results in poor immune responses to TI-antigens (Kruetzmann et al., 2003; Lane et al., 1986; Timens et al., 1989b). According to Weller et al. (Weller et al., 2004b) IgM memory MZ-B cells are distinct from the classical GC-derived memory B cells, as they are found in patients that cannot make GC nor class-switched memory cells. These authors postulated that these cells are generated in a T cell and GC independent fashion, and that these mutations are introduced to provide the MZ-B population with a pre-diversified Ig repertoire. In Chapter 5 we investigate if a similar pre-diversified (mutated) memory B cell population also exists in rats. Since GC are absent in the first few weeks of neonatal rats (Kroese et al., 1987a; van Rees et al., 1986) analysis of the mutation profiles of IGHV genes from neonatal rats might reveal whether mutated IgM MZ-B cells can be generated in an antigen independent and GC-independent process in rats as well as proposed by Weller et al. for humans (Weller et al., 2004a). In chapter 6 we studied whether in addition to IgM memory MZ-B cells also IgG memory MZ-B cells exist in the rat, and if present what the origin is of these isotype switched MZ-B cells. They might be derived from GC, as is the case for typical memory B cells. In Chapter 6 we analysed the expression of rearranged IGHVDJ-Cγ transcripts in MZ-B cells that were defined in an Ig isotype independent fashion. To this end, HIS57highHIS24low MZ-B cells were analysed and compared to classical switched isotype IgMnegIgDneg B cells and IgG expressing HIS24highHIS57neg FO-B cells. Overall analysis is done on rearranged IGHVDJ-Cγ transcripts derived from the respective B cell subsets in terms of the patterns of mutations in the IG genes, the usage of the different IGHV genes, H-CDR3 lengths and the distribution pattern of IgG subclasses. This analysis may help us to elucidate whether IgG 24 | Chapter 1 HIS57highHIS24low MZ-B cells are a distinct subset of memory B cells in comparison to other classical memory B cells and may give us insights into the origin of these memory MZ-B cells. Finally, in Chapter 7 we discuss the findings and implications of this thesis. Introduction and aims | 25 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 26 | Chapter 1 IGHD gene segment n=20 IGHV gene segment n=252 Germ Line DNA V H1 V H2 IGHJ gene segment n=5 V Hn CH RSS RSS IGHV RSS Organization of IGHV, IGHD, IGHJ RSS IGHV CH gene 5’IGHD RSS CACTGTG GGTTTTTGG GGTTTTTGG CACTGTG V-HEPTAMER n=9 V-NONAMER n=40 D-NONAMER n=5 D-HEPTAMER n=3 23 bp RSS Spacer CACAGTG TCAAAAACC D-HEPTAMER n=5 D-NONAMER n=7 GGTTTTTGT FR1 FR2 CDR1 RAG1/2 IGHJ gene FR4 FR2 CDR2 CH Potential Functional Recombinations 119 IGHV x 13 IGHD x 4 IGHJ Rearranged IGHVDJ gene IGHV gene IGHJ J-HEPTAMER n=4 23 bp RSS Spacer Recombination and deletion RAG1/2 RAG1/2 CACAGTG J-NONAMER n=4 12 bp RSS Spacer 12 bp RSS Spacer Organization of rearranged IGHVDJ gene IGHJ RSS 3’IGHD RSS IGHD CH CDR3region (V-D-J junction) Immunoglobulin structure Antigen Binding site Variable region IGLV gene Light chain Constant region V L1 VL2 Kappa n= 163 Lambda n= 7 IGHV Heavy chain IGLJ gene CL n= 7 n=4 IGH J CL Figure 5. Structural organization of IGH and IGL in the rat. The above diagram demonstrates the organization of IGH gene segments position on chromosome 6q32-33 on the IGH locus of the Brown Norway (BN) rat. The IGH locus consists of an IGH variable region and heavy constant (CH) region. IGH variable region is grouped into three germline elements: IGHV, IGHD and IGHJ gene segments respectively. Also depicted in the above figure are the recognition signal sequenced (RSS) flanking each of IGHV, IGHJ on one side and IGHD on both sides. RSS consist of conserved heptamer and nonamer sequences, separated from each other by either a 12 or a 23 base pair (bp) RSS spacer. Recombination of germline IGHV, IGHD, IGHJ (VDJ rearrangement) gene segments follow a 12/23 rule i.e. VDJ rearrangement occurs only between a 12 bp RSS spacer and a 23 bp RSS spacer. Recombination activating Genes (RAG1/2) bind to RSS’s initiating VDJ rearrangement that results in the sequential random joining of a single gene segment of IGHD to IGHJ and IGHV to IGHDJ forming a rearranged IGHVDJ gene that encodes for the variable part of the immunoglobulin molecule. The IGHV gene is further arranged into CDR1 and CDR2 that are separated from each other by conserved frame work regions (FR1, FR2, FR3). FR4 is completely encoded by the IGHJ gene. The CDR3 (junctional) region is the product of VDJ rearrangement. CH chain genes are separately joined by splicing to the rearranged IGHVDJ gene. The total number of gene segments, heptamer and nonamer sequences are designated by “n”. Introduction and aims | 27 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Allman D., Lindsley R. C., DeMuth W., Rudd K., Shinton S. A. and Hardy R. R., 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. Journal of immunology 167, 6834-40. Angelin-Duclos C., Cattoretti G., Lin K. I. and Calame K., 2000. Commitment of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in vivo. Journal of immunology 165, 5462-71. Arakawa H., Hauschild J. and Buerstedde J. M., 2002. Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science (New York, N.Y.) 295, 1301-6. Astier A., Avraham H., Manie S. N., Groopman J., Canty T., Avraham S. and Freedman A. S., 1997. The related adhesion focal tyrosine kinase is tyrosine-phosphorylated after beta1-integrin stimulation in B cells and binds to p130cas. The Journal of biological chemistry 272, 228-32. Balazs M., Martin F., Zhou T. and Kearney J., 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17, 341-52. Balogh A., Adori M., Torok K., Matko J. and Laszlo G., 2010. A closer look into the GL7 antigen: its spatio-temporally selective differential expression and localization in lymphoid cells and organs in human. Immunology letters 130, 89-96. Bergstedt-Lindqvist S., Moon H. B., Persson U., Moller G., Heusser C. and Severinson E., 1988. Interleukin 4 instructs uncommitted B lymphocytes to switch to IgG1 and IgE. European journal of immunology 18, 1073-7. Brodeur P. H. and Riblet R., 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. European journal of immunology 14, 922-30. Calame K. L., Lin K. I. and Tunyaplin C., 2003. Regulatory mechanisms that determine the development and function of plasma cells. Annual review of immunology 21, 205-30. Carey J. B., Moffatt-Blue C. S., Watson L. C., Gavin A. L. and Feeney A. J., 2008. Repertoire-based selection into the marginal zone compartment during B cell development. The Journal of experimental medicine 205, 2043-52. Cariappa A., Tang M., Parng C., Nebelitskiy E., Carroll M., Georgopoulos K. and Pillai S., 2001. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14, 603-15. Cerutti A., Puga I. and Cols M., 2012. New helping friends for B cells. European journal of immunology 42, 1956-68. Cesta M. F., 2006. Normal structure, function, and histology of the spleen. Toxicologic pathology 34, 455-65. Chen X., Martin F., Forbush K. A., Perlmutter R. M. and Kearney J. F., 1997. Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone. International immunology 9, 27-41. Cinamon G., Matloubian M., Lesneski M. J., Xu Y., Low C., Lu T., Proia R. L. and Cyster J. G., 2004. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nature immunology 5, 713-20. Cinamon G., Zachariah M. A., Lam O. M., Foss F. W., Jr. and Cyster J. G., 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nature immunology 9, 54-62. Coico R. F., Bhogal B. S. and Thorbecke G. J., 1983. Relationship of germinal centers in lymphoid tissue to immunologic memory. VI. Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin. Journal of immunology 131, 2254-7. Coquet J. M., Kyparissoudis K., Pellicci D. G., Besra G., Berzins S. P., Smyth M. J. and Godfrey D. I., 2007. IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production. Journal of immunology 178, 2827-34. Crotty S., Johnston R. J. and Schoenberger S. P., 2010. Effectors and memories: Bcl-6 and Blimp-1 in T and B lymphocyte differentiation. Nature immunology 11, 114-20. 28 | Chapter 1 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Cyster J. G., 2000. B cells on the front line. Nature immunology 1, 9-10. Dammers P. M., de Boer N. K., Deenen G. J., Nieuwenhuis P. and Kroese F. G., 1999. The origin of marginal zone B cells in the rat. European journal of immunology 29, 1522-31. Dammers P. M. and Kroese F. G., 2005. Recruitment and selection of marginal zone B cells is independent of exogenous antigens. European journal of immunology 35, 2089-99. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000a. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of immunology 165, 6156-6169. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000b. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of immunology 165, 6156-69. Descatoire M., Weller S., Irtan S., Sarnacki S., Feuillard J., Storck S., Guiochon-Mantel A., Bouligand J., Morali A., Cohen J., Jacquemin E., Iascone M., Bole-Feysot C., Cagnard N., Weill J. C. and Reynaud C. A., 2014. Identification of a human splenic marginal zone B cell precursor with NOTCH2-dependent differentiation properties. The Journal of experimental medicine 211, 9871000. Dono M., Zupo S., Leanza N., Melioli G., Fogli M., Melagrana A., Chiorazzi N. and Ferrarini M., 2000. Heterogeneity of tonsillar subepithelial B lymphocytes, the splenic marginal zone equivalents. Journal of immunology 164, 5596-604. Dunn-Walters D. K., Isaacson P. G. and Spencer J., 1995. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. The Journal of experimental medicine 182, 559-66. Ettinger R., Sims G. P., Robbins R., Withers D., Fischer R. T., Grammer A. C., Kuchen S. and Lipsky P. E., 2007. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. Journal of immunology 178, 2872-82. Fayette J., Durand I., Bridon J. M., Arpin C., Dubois B., Caux C., Liu Y. J., Banchereau J. and Briere F., 1998. Dendritic cells enhance the differentiation of naive B cells into plasma cells in vitro. Scandinavian journal of immunology 48, 563-70. Ferguson A. R., Youd M. E. and Corley R. B., 2004. Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells. International immunology 16, 1411-22. Garraud O., Borhis G., Badr G., Degrelle S., Pozzetto B., Cognasse F. and Richard Y., 2012. Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond. BMC immunology 13, 63. Garside P., Ingulli E., Merica R. R., Johnson J. G., Noelle R. J. and Jenkins M. K., 1998. Visualization of specific B and T lymphocyte interactions in the lymph node. Science (New York, N.Y.) 281, 96-9. Gatto D., Bauer M., Martin S. W. and Bachmann M. F., 2007. Heterogeneous antibody repertoire of marginal zone B cells specific for virus-like particles. Microbes.Infect. 9, 391-399. Gatto D., Ruedl C., Odermatt B. and Bachmann M. F., 2004. Rapid response of marginal zone B cells to viral particles. Journal of immunology 173, 4308-4316. Good-Jacobson K. L. and Shlomchik M. J., 2010. Plasticity and heterogeneity in the generation of memory B cells and long-lived plasma cells: the influence of germinal center interactions and dynamics. Journal of immunology 185, 3117-25. Guinamard R., Okigaki M., Schlessinger J. and Ravetch J. V., 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nature immunology 1, 31-6. Gumperz J. E., Roy C., Makowska A., Lum D., Sugita M., Podrebarac T., Koezuka Y., Porcelli S. A., Cardell S., Brenner M. B. and Behar S. M., 2000. Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12, 211-21. Gunn K. E. and Brewer J. W., 2006. Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity. Journal of immunology 177, 3791-8. Hofmann J., Greter M., Du Pasquier L. and Becher B., 2010. B-cells need a proper house, whereas Introduction and aims | 29 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. T-cells are happy in a cave: the dependence of lymphocytes on secondary lymphoid tissues during evolution. Trends in immunology 31, 144-53. Hogerkorp C. M. and Borrebaeck C. A., 2006. The human CD77- B cell population represents a heterogeneous subset of cells comprising centroblasts, centrocytes, and plasmablasts, prompting phenotypical revision. Journal of immunology 177, 4341-9. Iizuka T., Tanaka T., Suematsu M., Miura S., Watanabe T., Koike R., Ishimura Y., Ishii H., Miyasaka N. and Miyasaka M., 2000. Stage-specific expression of mucosal addressin cell adhesion molecule-1 during embryogenesis in rats. Journal of immunology 164, 2463-71. Jacob J. and Kelsoe G., 1992. In situ studies of the primary immune response to (4-hydroxy3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. The Journal of experimental medicine 176, 679-87. Johnston C. M., Wood A. L., Bolland D. J. and Corcoran A. E., 2006. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. Journal of immunology 176, 4221-34. Kaji T., Ishige A., Hikida M., Taka J., Hijikata A., Kubo M., Nagashima T., Takahashi Y., Kurosaki T., Okada M., Ohara O., Rajewsky K. and Takemori T., 2012. Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory. The Journal of experimental medicine 209, 2079-97. Kasajima-Akatsuka N. and Maeda K., 2006. Development, maturation and subsequent activation of follicular dendritic cells (FDC): immunohistochemical observation of human fetal and adult lymph nodes. Histochemistry and cell biology 126, 261-73. Kawabe T., Naka T., Yoshida K., Tanaka T., Fujiwara H., Suematsu S., Yoshida N., Kishimoto T. and Kikutani H., 1994a. The immune responses in CD40-deficient mice: Impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167-178. Kawabe T., Naka T., Yoshida K., Tanaka T., Fujiwara H., Suematsu S., Yoshida N., Kishimoto T. and Kikutani H., 1994b. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167-78. Kerfoot Steven M., Yaari G., Patel Jaymin R., Johnson Kody L., Gonzalez David G., Kleinstein Steven H. and Haberman Ann M., 2011. Germinal Center B Cell and T Follicular Helper Cell Development Initiates in the Interfollicular Zone. Immunity 34, 947-960. Klein U., Rajewsky K. and Kuppers R., 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. The Journal of experimental medicine 188, 1679-89. Kobayashi S., Haruo N., Sugane K., Ochs H. D. and Agematsu K., 2009. Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings concomitantly with activation-induced cytidine deaminase expression and differentiation into plasma cells. Human immunology 70, 35-40. Kolar G. R., Mehta D., Pelayo R. and Capra J. D., 2007. A novel human B cell subpopulation representing the initial germinal center population to express AID. Blood 109, 2545-52. Kroese F. G., de Boer N. K., de Boer T., Nieuwenhuis P., Kantor A. B. and Deenen G. J., 1995. Identification and kinetics of two recently bone marrow-derived B cell populations in peripheral lymphoid tissues. Cellular immunology 162, 185-93. Kroese F. G., Wubbena A. S., Kuijpers K. C. and Nieuwenhuis P., 1987a. The ontogeny of germinal centre forming capacity of neonatal rat spleen. Immunology 60, 597-602. Kroese F. G., Wubbena A. S., Opstelten D., Deenen G. J., Schwander E. H., De L. L., Vos H., Poppema S., Volberda J. and Nieuwenhuis P., 1987b. B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens. Eur.J.Immunol. 17, 921-928. Kroese F. G. M., Wubbena A. S., Seijen H. G. and Nieuwenhuis P., 1987c. Germinal centers develop oligoclonally. European journal of immunology 17, 1069-1072. Kruetzmann S., Rosado M. M., Weber H., Germing U., Tournilhac O., Peter H. H., Berner R., Peters A., Boehm T., Plebani A., Quinti I. and Carsetti R., 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. The Journal of 30 | Chapter 1 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. experimental medicine 197, 939-45. Kumararatne D. S. and MacLennan I. C., 1981. Cells of the marginal zone of the spleen are lymphocytes derived from recirculating precursors. European journal of immunology 11, 865-9. Kuroda K., Han H., Tani S., Tanigaki K., Tun T., Furukawa T., Taniguchi Y., Kurooka H., Hamada Y., Toyokuni S. and Honjo T., 2003. Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18, 301-12. Lane P. J., Gray D., Oldfield S. and MacLennan I. C., 1986. Differences in the recruitment of virgin B cells into antibody responses to thymus-dependent and thymus-independent type-2 antigens. European journal of immunology 16, 1569-75. Linehan L. A., Warren W. D., Thompson P. A., Grusby M. J. and Berton M. T., 1998. STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination. Journal of immunology 161, 302-10. Linterman M. A., Beaton L., Yu D., Ramiscal R. R., Srivastava M., Hogan J. J., Verma N. K., Smyth M. J., Rigby R. J. and Vinuesa C. G., 2010. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. The Journal of experimental medicine 207, 353-63. Litinskiy M. B., Nardelli B., Hilbert D. M., He B., Schaffer A., Casali P. and Cerutti A., 2002. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nature immunology 3, 822-9. Liu Y. J., 1997. Sites of B lymphocyte selection, activation, and tolerance in spleen. The Journal of experimental medicine 186, 625-9. Liu Y. J., Oldfield S. and MacLennan I. C., 1988. Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. European journal of immunology 18, 355-62. Liu Y. J., Zhang J., Lane P. J., Chan E. Y. and MacLennan I. C., 1991. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. European journal of immunology 21, 2951-62. Lu T. T. and Cyster J. G., 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science (New York, N.Y.) 297, 409-12. MacLennan I. C., 1994. Germinal centers. Annu.Rev.Immunol. 12, 117-139. MacLennan I. C., Bazin H., Chassoux D., Gray D. and Lortan J., 1985. Comparative analysis of the development of B cells in marginal zones and follicles. Advances in experimental medicine and biology 186, 139-44. MacLennan I. C., Toellner K. M., Cunningham A. F., Serre K., Sze D. M., Zuniga E., Cook M. C. and Vinuesa C. G., 2003. Extrafollicular antibody responses. Immunological reviews 194, 8-18. Magri G., Miyajima M., Bascones S., Mortha A., Puga I., Cassis L., Barra C. M., Comerma L., Chudnovskiy A., Gentile M., Llige D., Cols M., Serrano S., Arostegui J. I., Juan M., Yague J., Merad M., Fagarasan S. and Cerutti A., 2014. Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells. Nature immunology 15, 354-64. Makowska A., Faizunnessa N. N., Anderson P., Midtvedt T. and Cardell S., 1999. CD1high B cells: a population of mixed origin. European journal of immunology 29, 3285-94. Martin F. and Kearney J. F., 2002. Marginal-zone B cells. Nature reviews. Immunology 2, 323-35. Martin F., Oliver A. M. and Kearney J. F., 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14, 617-29. Matsuda F., Ishii K., Bourvagnet P., Kuma K., Hayashida H., Miyata T. and Honjo T., 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. The Journal of experimental medicine 188, 2151-62. Mebius R. E. and Kraal G., 2005. Structure and function of the spleen. Nature reviews. Immunology 5, 606-16. Molina H., Holers V. M., Li B., Fung Y., Mariathasan S., Goellner J., Strauss-Schoenberger J., Karr R. W. and Chaplin D. D., 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proceedings of the National Academy of Sciences of the United States of America 93, 3357-61. Munday J., Floyd H. and Crocker P. R., 1999. Sialic acid binding receptors (siglecs) expressed by macrophages. Journal of leukocyte biology 66, 705-11. Introduction and aims | 31 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. Muramatsu M., Kinoshita K., Fagarasan S., Yamada S., Shinkai Y. and Honjo T., 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553-63. Muramatsu M., Sankaranand V. S., Anant S., Sugai M., Kinoshita K., Davidson N. O. and Honjo T., 1999. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. The Journal of biological chemistry 274, 18470-6. Okada T., Miller M. J., Parker I., Krummel M. F., Neighbors M., Hartley S. B., O’Garra A., Cahalan M. D. and Cyster J. G., 2005. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS biology 3, e150. Oliver A. M., Martin F. and Kearney J. F., 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. Journal of immunology 162, 7198-207. Pascual V., Liu Y. J., Magalski A., de Bouteiller O., Banchereau J. and Capra J. D., 1994. Analysis of somatic mutation in five B cell subsets of human tonsil. The Journal of experimental medicine 180, 329-39. Petersen-Mahrt S. K., Harris R. S. and Neuberger M. S., 2002. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99-103. Pihlgren M., Silva A. B., Madani R., Giriens V., Waeckerle-Men Y., Fettelschoss A., Hickman D. T., Lopez-Deber M. P., Ndao D. M., Vukicevic M., Buccarello A. L., Gafner V., Chuard N., Reis P., Piorkowska K., Pfeifer A., Kundig T. M., Muhs A. and Johansen P., 2013. TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice. Blood 121, 85-94. Pillai S. and Cariappa A., 2009. The follicular versus marginal zone B lymphocyte cell fate decision. Nature reviews. Immunology 9, 767-77. Pone E. J., Zhang J., Mai T., White C. A., Li G., Sakakura J. K., Patel P. J., Al-Qahtani A., Zan H., Xu Z. and Casali P., 2012. BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-kappaB pathway. Nature communications 3, 767. Puga I., Cols M., Barra C. M., He B., Cassis L., Gentile M., Comerma L., Chorny A., Shan M., Xu W., Magri G., Knowles D. M., Tam W., Chiu A., Bussel J. B., Serrano S., Lorente J. A., Bellosillo B., Lloreta J., Juanpere N., Alameda F., Baro T., de Heredia C. D., Toran N., Catala A., Torrebadell M., Fortuny C., Cusi V., Carreras C., Diaz G. A., Blander J. M., Farber C. M., Silvestri G., CunninghamRundles C., Calvillo M., Dufour C., Notarangelo L. D., Lougaris V., Plebani A., Casanova J. L., Ganal S. C., Diefenbach A., Arostegui J. I., Juan M., Yague J., Mahlaoui N., Donadieu J., Chen K. and Cerutti A., 2012. B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nature immunology 13, 170-80. Revy P., Muto T., Levy Y., Geissmann F., Plebani A., Sanal O., Catalan N., Forveille M., DufourcqLabelouse R., Gennery A., Tezcan I., Ersoy F., Kayserili H., Ugazio A. G., Brousse N., Muramatsu M., Notarangelo L. D., Kinoshita K., Honjo T., Fischer A. and Durandy A., 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565-75. Saito T., Chiba S., Ichikawa M., Kunisato A., Asai T., Shimizu K., Yamaguchi T., Yamamoto G., Seo S., Kumano K., Nakagami-Yamaguchi E., Hamada Y., Aizawa S. and Hirai H., 2003. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity 18, 675-85. Satoh-Takayama N., Lesjean-Pottier S., Vieira P., Sawa S., Eberl G., Vosshenrich C. A. and Di Santo J. P., 2010. IL-7 and IL-15 independently program the differentiation of intestinal CD3-NKp46+ cell subsets from Id2-dependent precursors. The Journal of experimental medicine 207, 273-80. Schroeder H. W., Jr., Mortari F., Shiokawa S., Kirkham P. M., Elgavish R. A. and Bertrand F. E., 3rd, 1995. Developmental regulation of the human antibody repertoire. Annals of the New York Academy of Sciences 764, 242-60. Seifert M. and Kuppers R., 2009. Molecular footprints of a germinal center derivation of human 32 | Chapter 1 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation. The Journal of experimental medicine 206, 2659-69. Shaffer A. L., Yu X., He Y., Boldrick J., Chan E. P. and Staudt L. M., 2000. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity 13, 199212. Shlomchik M. J. and Weisel F., 2012. Germinal center selection and the development of memory B and plasma cells. Immunological reviews 247, 52-63. Snapper C. M., Waegell W., Beernink H. and Dasch J. R., 1993. Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro. Journal of immunology 151, 4625-36. Song H. and Cerny J., 2003. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. The Journal of experimental medicine 198, 192335. Spencer J., Finn T., Pulford K. A., Mason D. Y. and Isaacson P. G., 1985. The human gut contains a novel population of B lymphocytes which resemble marginal zone cells. Clinical and experimental immunology 62, 607-12. Spencer J., Perry M. E. and Dunn-Walters D. K., 1998. Human marginal-zone B cells. Immunol Today 19, 421-6. Srivastava B., Quinn W. J., 3rd, Hazard K., Erikson J. and Allman D., 2005. Characterization of marginal zone B cell precursors. The Journal of experimental medicine 202, 1225-34. Steiniger B., Barth P. and Hellinger A., 2001. The perifollicular and marginal zones of the human splenic white pulp : do fibroblasts guide lymphocyte immigration? The American journal of pathology 159, 501-12. Steiniger B., Timphus E. M. and Barth P. J., 2006. The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants. Histochemistry and cell biology 126, 641-8. Takahashi M., Tsukada T., Kojima M., Koide T., Koike T., Takahashi H., Sakai C., Kashimura M. and Shibata A., 1986. Immunoglobulin class switch from IgG to IgA in a patient with smoldering multiple myeloma. Blood 67, 1710-3. Takemori T., Kaji T., Takahashi Y., Shimoda M. and Rajewsky K., 2014. Generation of memory B cells inside and outside germinal centers. European journal of immunology 44, 1258-64. Tangye S. G., Ferguson A., Avery D. T., Ma C. S. and Hodgkin P. D., 2002. Isotype switching by human B cells is division-associated and regulated by cytokines. Journal of immunology 169, 4298-306. Tangye S. G. and Good K. L., 2007. Human IgM+CD27+ B cells: memory B cells or “memory” B cells? Journal of immunology 179, 13-9. Tangye S. G., Liu Y. J., Aversa G., Phillips J. H. and de Vries J. E., 1998. Identification of functional human splenic memory B cells by expression of CD148 and CD27. The Journal of experimental medicine 188, 1691-703. Thorbecke G. J., Amin A. R. and Tsiagbe V. K., 1994. Biology of germinal centers in lymphoid tissue. FASEB J. 8, 832-840. Timens W., Boes A. and Poppema S., 1989a. Human marginal zone B cells are not an activated B cell subset: strong expression of CD21 as a putative mediator for rapid B cell activation. European journal of immunology 19, 2163-6. Timens W., Boes A., Rozeboom-Uiterwijk T. and Poppema S., 1989b. Immaturity of the human splenic marginal zone in infancy. Possible contribution to the deficient infant immune response. Journal of immunology 143, 3200-6. Toyama H., Okada S., Hatano M., Takahashi Y., Takeda N., Ichii H., Takemori T., Kuroda Y. and Tokuhisa T., 2002. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 17, 329-39. van Rees E. P., Dijkstra C. D. and van Rooijen N., 1986. The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat. Cell and tissue research 246, 673-7. van Zelm M. C., 2014. B cells take their time: sequential IgG class switching over the course of an Introduction and aims | 33 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 immune response[quest]. Immunol Cell Biol 92, 645-646. 113. Victora G. D., Dominguez-Sola D., Holmes A. B., Deroubaix S., Dalla-Favera R. and Nussenzweig M. C., 2012. Identification of human germinal center light and dark zone cells and their relationship to human B-cell lymphomas. Blood 120, 2240-8. 114. Victora G. D. and Mesin L., 2014. Clonal and cellular dynamics in germinal centers. Current opinion in immunology 28, 90-6. 115. Victora G. D. and Nussenzweig M. C., 2012. Germinal centers. Annual review of immunology 30, 429-57. 116. Vinuesa C. G., Sunners Y., Pongracz J., Ball J., Toellner K. M., Taylor D., MacLennan I. C. and Cook M. C., 2001. Tracking the response of Xid B cells in vivo: TI-2 antigen induces migration and proliferation but Btk is essential for terminal differentiation. European journal of immunology 31, 1340-50. 117. Walker J. A., Barlow J. L. and McKenzie A. N., 2013. Innate lymphoid cells--how did we miss them? Nature reviews. Immunology 13, 75-87. 118. Wang J. H., Avitahl N., Cariappa A., Friedrich C., Ikeda T., Renold A., Andrikopoulos K., Liang L., Pillai S., Morgan B. A. and Georgopoulos K., 1998. Aiolos regulates B cell activation and maturation to effector state. Immunity 9, 543-53. 119. Weill J. C., Weller S. and Reynaud C. A., 2009. Human marginal zone B cells. Annual review of immunology 27, 267-85. 120. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004a. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-54. 121. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004b. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-3654. 122. Weller S., Faili A., Garcia C., Braun M. C., Le Deist F. F., de Saint Basile G. G., Hermine O., Fischer A., Reynaud C. A. and Weill J. C., 2001. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proceedings of the National Academy of Sciences of the United States of America 98, 1166-70. 123. Weller S., Mamani-Matsuda M., Picard C., Cordier C., Lecoeuche D., Gauthier F., Weill J. C. and Reynaud C. A., 2008. Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants. The Journal of experimental medicine 205, 1331-42. 124. Willems L., Li S., Rutgeerts O., Lenaerts C., Waer M. and Billiau A. D., 2011. IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment. Journal of immunology 187, 3587-94. 125. Zandvoort A., Lodewijk M. E., de Boer N. K., Dammers P. M., Kroese F. G. and Timens W., 2001. CD27 expression in the human splenic marginal zone: the infant marginal zone is populated by naive B cells. Tissue antigens 58, 234-42. 126. Zouali M. and Richard Y., 2011. Marginal zone B-cells, a gatekeeper of innate immunity. Frontiers in immunology 2, 63 34 | Chapter 1 Chapter 2 Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat. Jacobus Hendricks Peter Terpstra Peter M. Dammers Rajesh Somasundaram Annie Visser Maaike Stoel Nicolaas A. Bos Frans G. M. Kroese Immunogenetics (2010) 62:479–486 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Abstract We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species. 38 | Chapter 2 1. Introduction The antigen recognition site of an antibody is the product of a pairing set of immunoglobulin heavy (IGH) and immunoglobulin light (IGL) chain variable domains. Both the IGH-chain variable domain and the IGL-chain variable domain are composed of conserved framework sequences that alternate with three hypervariable regions, the complementary-determining regions (CDRs), which are responsible for actual antigen recognition. The variable domains of the IGH chain are encoded by three different genes (or gene segments): variable (IGHV), diversity (IGHD), and joining (IGHJ) genes. The IGH gene locus of most mammalian species contains a large number of IGHV genes, fewer IGHD genes, and some IGHJ genes (Marchalonis et al., 1998). In many species such as humans, mice, and rats, these gene segments recombine by DNA rearrangements during B-cell genesis in the bone marrow. These recombinations result in a so-called combinatorial diversity of the variable domain of the heavy chain (Yancopoulos and Alt, 1986). Similarly, recombination of IGLV and IGLJ gene segments leads to the combinatorial diversity of IGL-chains. Imprecision of the rearrangements by addition or removal of nucleotides between the segments during the recombination process results in further enlargement of the primary repertoire of the variable domain of both IGH and IGL chains (junctional diversity). Another form of combinatorial diversity is created by the combination of IGH and IGL chains that are required to form the actual antigen recognition site. All mammals use combinatorial diversity (and junctional diversity) of IGHV, IGHD, and IGHJ genes to form a diverse primary preimmune H-chain repertoire (Marchalonis et al., 1998). The extent of this combinatorial diversity varies, however, significantly between different species (Flajnik, 2002; Marchalonis et al., 1998). Mammalian cross-species comparisons have demonstrated considerable divergence in the number and/or expression of IGHV, IGHD, and IGHJ genes (Das et al., 2008; Flajnik, 2002; Marchalonis et al., 1998). For example, the number of potentially functional germline IGHV genes may vary from only 20 in pigs (Butler et al., 2006) to >100 in rats and mice (Das et al., 2008; Johnston et al., 2006). Some mammalian species such as rabbit, sheep, and cow use only a very limited number of possible IGHV, IGHD, and IGHJ genes (Dufour et al., 1996; Gontier et al., 2005; Mage et al., 2006; Saini et al., 1997). In chickens, even only one unique functional IGHV gene is present in the heavy-chain locus (Reynaud et al., 1995). Chickens and mammals that use only very few IGHV genes must therefore rely on additional mechanisms to compensate for the presence of a relatively limited combinatorial preimmune repertoire of their IGH chains. The strategies used to form a diverse primary IGH chain repertoire in these species include gene conversion in chickens and rabbits (Mage et al., 2006; Reynaud et al., 1995); hypermutation in chickens, sheep, and rabbits (Dufour et al., 1996; Gontier et al., 2005; Kothapalli et al., 2008; Mage et al., 2006; Reynaud et al., 1995); and extra-long H-CDR3 regions in cows (Saini et al., 1999). For a better understanding of the generation of the primary antibody repertoire during B-cell Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 39 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 development and the changes (somatic hypermutations) that occur in this repertoire during humoral immune responses, detailed knowledge of the germline IGHV, IGHD, and IGHJ genes and organization of the IGHVDJ locus are of critical importance. This information is also essential in giving insight into how various species have evolved different mechanisms to create a diverse preimmune specificity repertoire of their antibodies. With the unraveling of the mouse and human genomes, a detailed complete physical annotated map of the IGHVDJ locus has become available for these species (Johnston et al., 2006; Matsuda et al., 1998). One of the remarkable findings of these studies was that the number of functional IGHV genes in both species appeared to be much lower than previously estimated, whereas the number of nonfunctional IGHV genes [pseudogenes and open reading frame (ORF) genes lacking appropriate signal sequences] was relatively high. The genome of the rat has been unraveled, and there is a nearly complete sequence of the IGH locus (Gibbs et al., 2004). In rats, the exact number and location of IGHV genes are not known. Preliminary data suggest, however, that they are among the species with the highest number of (functional) IGHV genes in the genome (Das et al., 2008). Our previous studies (Dammers et al., 2000a) have indicated that, similar to the mouse, rat IGHV genes can be subdivided into IGHV gene families, on the basis of nucleotide sequence identity. IGHV genes belong to the same family when the IGHV genes share more than 80% of their nucleotides (Brodeur and Riblet, 1984). We have detected previously the existence of at least 28 functional IGHV (germline) genes that belong to the IGHV5 family (PC7183) in the PVG rat strain (Dammers et al., 2001; Stoel et al., 2008). Here we present an annotated map of the variable region of the IGH locus of the Brown Norway (BN) rat, including not only functional and nonfunctional IGHV genes but also IGHD and IGHJ genes. 2. Materials and Methods 2.1. Genomic sequence of the rat IGH locus: The genomic sequence of the BN/SsNHsdMCW rat (Rattus norvegicus) was generated by the Rat Genomic Sequence Consortium (RGSC) (Gibbs et al., 2004; Havlak et al., 2004). This sequence is available through the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). Analysis of the variable region of the rat IGH gene locus, located on chromosome 6q32–33, was based on the Human Genome Sequencing Center assembly version RGSC V3.4 (November 2004 release; Baylor College of Medicine, Houston, TX, USA). Assembly RGSC V3.4 has been established in a hybrid approach combining the cloneby-clone method and the whole genome shotgun method. 40 | Chapter 2 2.2. Mapping of the variable region of the BN rat: IGHV and IGHD gene sequences of the BN rat were obtained from the International Immunogenetics (IMGT) database (http://imgt.cines.fr) (Lefranc et al., 1999). IGHJ genes were taken from (Lang and Mocikat, 1991) (accession number X56791). Additional previously unreported IGHV genes were searched for in mapped and unmapped sequences of the rat genome. Unmapped sequences were taken from contigs in the “unplaced section” of the NCBI database or from newly established bactigs of the BN rat genome (Baylor College of Medicine; http://www.hgsc.bcm.tmc.edu/projects/rat) not yet present in assembly RGSC V3.4. Nucleotide alignments were carried out using the NCBI BLASTN program (Altschul et al., 1990). New IGHV gene sequences were manually analysed for the presence of an ORF of the coding region and for the presence of functional recombination signal sequences (RSS) and leader sequences using V-QUEST alignment software (http://www.imgt.cines.fr) (Giudicelli et al., 2004). Previously unreported IGHD genes were identified by searching manually in the genomic assembly RGSC V3.4 with sets of rat nonamer and heptamer sequences. The relative positions of the IGHV, IGHD, and IGHJ genes on the chromosomal map were determined by aligning the encoding parts of the IGHV, IGHD, and IGHJ genes (functional and nonfunctional) against the BN rat genome using the Genome Browser and BLAT programs (http:// www.genome.ucsc.edu) (Karolchik et al., 2008; Kent, 2002). Because of the relatively small size of IGHD genes, we included the IGHD 5′ and 3′ flanking RSS regions in the alignment. IGHD genes and flanking RSS were obtained from the IMGT database (accession numbers AABR03049813, AABR03051895, and M13798). The location of other predicted genes in the IGH locus was taken from NCBI annotation. The complete physical map and annotation of the genomic IGHVDJ region were drawn using the software package Genvision (Dnastar, Madison, WI, USA). 2.3. Nomenclature of rat IGHV, IGHD, and IGHJ genes: IGHV, IGHD, and IGHJ gene nomenclature and classification (functional, nonfunctional, ORF gene, and pseudogene) were adopted from the IMGT (Lefranc et al., 1999). Briefly, nonfunctional IGHV, IGHD, and IGHJ genes are either genes with an intact ORF but erroneous regulatory sequences (“ORF genes”) or genes lacking a correct ORF (pseudogenes). 3. Results and Discussion 3.1. Organization of the variable region of the IGH locus of the BN rat: Recently, DNA sequencing resulted in elucidation of the vast majority of the genomic nucleotide sequence of the BN rat, including the IGH locus located on chromosome 6q32–33 (Gibbs et al., 2004). The current IMGT database (Lefranc et al., 1999) contains 342 IGHV genes (120 Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 41 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 functional and 222 nonfunctional), 20 IGHD genes (13 functional and 7 nonfunctional), and 5 IGHJ genes (4 functional and 1 nonfunctional). In order to establish a detailed chromosomal map of this part of the IGH locus containing the exact chromosomal location and orientation of the individual genes, we aligned the coding sequences of all known IGHV, IGHD, and IGHJ genes (functional and nonfunctional) from the IMGT database to the rat genome assembly RGSC V3.4. As depicted in Figure 1, the variable region of the IGH locus of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization (many IGHV genes, a dozen IGHD genes, and a few IGHJ genes) similar to mice and humans. The locus has a telomeric to centromeric orientation and runs from the distally located IGHV7S16 gene towards the proximally located IGHJ4 gene. The upstream boundary of the IGHV region is marked by the non-IGH zinc-finger-protein type 386 gene, similar to the situation in mice (Johnston et al., 2006). The IGHV genes of the BN rat can be classified into 13 IGHV gene families based on nucleotide sequence identity (IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, IGHV7, IGHV8, IGHV9, IGHV10, IGHV11, IGHV12, and IGHV15) (Table 1). In comparison to rats, the IGHV genes in humans and mice can be grouped together into 7 and 16 IGHV gene families, respectively. Most BN rat IGHV gene families are composed of various members, except for the IGHV15 gene family, which is composed of only one gene. In rats and mice, the various members (both functional and non-functional genes) of these IGHV gene families are more or less clustered together on the genome. The order of various IGHV gene families on the genome appears to be well preserved between rats and mice. Similar to mice (Johnston et al., 2006), the IGHV genes that belong to the IGHV1 and IGHV8 gene families are the most telomeric IGHV genes, whereas the members of the IGHV2 and IGHV5 gene families are located centromeric and closest to the IGHD genes. IGHV gene family members of the rat (and also of the mouse) are not completely spatially separated, and members of various IGHV gene families are frequently found intermingled with each other (e.g., IGHV2/IGHV5 and IGHV1/ IGHV8 genes). The members of the IGHV1 gene family are more widely distributed over the locus and are mixed with members of various other IGHV gene families (such as IGHV7, IGHV8, IGHV11, etc.). In comparison to rat and mouse, the human IGHV gene family gene members are more extensively interspersed and less clustered on the IGH locus (Matsuda et al., 1998). The almost identical distribution pattern of IGHV gene families between mouse and rat strongly suggests a close evolutionary relationship shared between these species. The vast majority (>90%) of rat IGHV genes are orientated in the direction of the IGHD cluster, whereas a small number of IGHV genes have an inverted orientation (in the direction of the telomere). These inverted IGHV genes are grouped together on four inverted regions of the chromosomal map. Three of these regions are inverted repeats (positions 3.74, 3.79, and 4.76 Mb on the map of Figure 1) containing eight pairs of 100% identical IGHV genes. The majority of the inverted IGHV genes are, however, located on a large inverted region of the 42 | Chapter 2 Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 43 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Figure 1. Chromosomal map of the variable region of the IGH locus of the BN rat. Shown is the IGHV region from chromosome 6 ranging from gene Znf386 to IGHJ4 (RGSC V3.4: 138,451,833–143,326,393 bp).The map was established on the basis of the IMGT database (http://imgt.cines.fr), as indicated in “Materials and Methods,” and does not contain the 12 newly identified IGHV genes. Genes and their orientation are indicated by an arrow point. The IGHV, IGHD, and IGHJ genes are numbered according to the IMGT nomenclature (Lefranc et al., 1999). Members of the same IGHV family share identical colors (non-IGHV genes are shown in gray). Nonfunctional genes are indicated with a “p” for pseudogene or with an “r” for ORF gene after the family number. The last number in the gene name is the rank number of the gene in the locus, starting at the centromeric end. Black dotted lines indicate near-perfect inverted repeats. Gaps in assembly V3.4 are marked by solid gray bars. This map is also available as an MS Excel file (Online Resource 1) and as a “bed”-type file (Online Resource 2) that can be projected on the current rat genome version V3.4 at the UCSC genome website (www.genome.ucsc.edu). IGHV locus (between the genes IGHV2S28p, 39 and IGHV2S12, 16) close to the centromeric end of the IGHV region of the locus. This area of the locus also contains a gene, IGHV5S47, which is identical to the non-inverted gene IGHV5S8. These two genes are approximately 1.1 Mb separated from each other. We further noted the presence of another pair of identical IGHV genes (IGHV5S51p) located only 1,183 bp apart (Figure 1; 3.41 Mb). In contrast to the other repeated pairs of genes, the latter pair of IGHV genes has the same (normal) orientation on the chromosome. If the rat VDJ locus indeed contains inverted regions, then this would imply that the VDJ recombination mechanism for these genes would use inversion instead of deletion. The presence of inverted repeats and completely 100% identical IGHV genes has not been identified in humans (Matsuda et al., 1998) and mice (Johnston et al., 2006). It might well be that the presence of inverted repeats in rats in the IGH locus may reflect inconsistencies in the current genome assembly (Worley et al., 2008). Our map must therefore be taken as tentative, and it will be interesting to see whether an “upgraded” version of the rat genome sequences confirms the current assembly in regions with inverted repeats. In addition to IGHV genes, there are also six non-IGHV genes mapped on the IGHV gene locus (Figure 1). A metallopeptidase domain 6 gene (Adam6) is found at the proximal end of this locus. Of the remaining five non-IGHV genes, two have reported annotations: nuclear-case independent kinase substrate 1 (Nucks1) and the olfactory receptor pseudogene 874. These genes are located between IGHV1 and IGHV8 gene family members at the distal end of the IGHV gene locus. The other three genes are NCBI predicted genes: homolog of the Brix domain gene BXDC1 (RGD1560842) and two prematurely terminated fragments of potential rat homologs (RGD1559843 and LOC691867). It is unknown whether these non-IGHV genes are functionally expressed. If these genes are functional, their expression might well be influenced by immunoglobulin enhancers as a consequence of the VDJ recombination process, or they may even be lost during this process. For these reasons, we assume that these non-IGHV genes in this locus are nonfunctional genes. 44 | Chapter 2 3.2. Identification of novel IGHV genes: The current assembly (RGSC V3.4) has a number of gaps in the IGHV region of the IGH locus (~300 kb) of which the nucleotide sequence still has to be determined (Twigger et al., 2008). Approximately 7% of the variable region of the IGH locus has not been mapped yet. These regions are indicated in Figure 1 as gap regions. The largest gap is found in the IGHV2–IGHV5 region (Figure 1; between 3.6 and 3.7 Mb). These gaps may potentially contain novel IGHV genes. There are available genomic sequences of the BN rat that have not yet been incorporated into the current assembly (RGSC V3.4). These sequences are present as contigs and are grouped together as “unplaced sequences” in the NCBI database. To explore the presence of unidentified IGHV genes, we used BLASTN to align all IMGT-listed rat IGHV genes to the unplaced contigs NW_047922.1 and NW_047772.1. Contig NW_047922.1 contains genomic sequences that are not yet assigned to any specific chromosome, whereas contig NW_047772.1 contains genomic sequences that are specific for chromosome 6. This search resulted in the identification of 18 IGHV genes (17 genes in contig NW_ 047922.1 and 1 gene in contig NW_047772.1). All these 18 IGHV genes share 100% identity to an IGHV gene already present in the IMGT database. All other potential IGHV homologs (i.e. sequences with ≥80% identity with a known IGHV sequence in the IMGT database) found in unplaced sequences did not comply with the IMGT criteria for functional IGHV genes (Lefranc et al., 1999). Thus, the unplaced genomic sequences did not reveal any new previously unidentified rat IGHV genes. In addition to the unplaced sequences mentioned in the previous paragraph, there are also available bactig sequences (Baylor College of Medicine) that are not included in assembly RGSC V3.4. These bactigs are composed of overlapping bacterial artificial chromosome (BAC) sequences that may contain sequences that could map on the gap regions of this assembly. We analysed two nonoverlapping bactigs that span the entire variable region of the IGH locus, including the gap regions in assembly V3.4 (Figure 1), for the presence of additional unreported IGHV genes. One of these bactigs (gpwy_grzy) (7.5 Mb) includes 71 BACs and extends into the major gap region. The other bactig (kdyb_kdzq; 1.65 Mb) is located centromeric to the largest gap region and consists of 18 overlapping BACs and probably also extends into the major gap region. Bactig gpwy_grzy did not reveal any previously unknown IGHV genes, although this bactig partially overlaps the major gap region. Because the nucleotide sequences of these BACs are currently not complete and also lack sufficient accuracy, this does not, however, imply that the major gap region does not contain any IGHV gene. On the other hand, 11 novel IGHV sequences that meet the IMGT criteria for functional germline IGHV genes (preliminary third-party annotation accession numbers BN001223–BN001233) were found in bactig kdyb_kdzq. These criteria include the appropriate sequence length, at least one ORF, a proper leader sequence, and a functional RSS (Lefranc et al., 1999). Furthermore, the Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 45 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 flanking intron regions of these genes were not identical to the flanking regions of previously established IGHV genes already listed in the IMGT database. Based on sequence identity, five of these novel IGHV genes belong to the IGHV5 gene family (designated IGHV5-1 to IGHV5-5), and six belong to the IGHV2 gene family (designated IGHV2-1 to IGHV2-6). The finding that these novel IGHV genes all belong to either the IGHV2 gene family or the IGHV5 gene family is consistent with the notion that most gap regions are found in the area of the IGHV locus where members of these two IGHV gene families are located. To reveal whether these 11 newly identified IGHV genes are also functionally expressed in rearranged IGHVDJ transcripts, we aligned the newly identified IGHV sequences to the R. norvegicus nucleotide collection database of the NCBI. Two of these IGHV genes (IGHV2-3 and IGHV5-1) share a 100% identity with rearranged IGHVDJ BN rat complementary DNA sequences (accession numbers L07402 and X78897, respectively). In a recent study, we looked at the expression of IGHV5 genes in rat B cell subsets (Hendricks et al., manuscript in preparation). We found the expression of 100% identical IGHV5-1 and IGHV5-2 genes in mature B cells. In addition, we detected another previously unidentified IGHV5 gene (named IGHV5-6). This IGHV gene is also 100% identical to the IGHV gene expressed in BN hybridoma Hg16 (Dammers et al., 2001) (accession number Z75899). These findings indicate that at least some of these novel germline genes are also functionally used. 3.3. Identification of an additional IGHD gene: So far, 20 (13 functional and 7 nonfunctional) IGHD genes have been described by the IMGT. We manually searched genomic assembly RGSC V3.4 for the presence of additional IGHD genes by using available RSS from functional rat IGHD genes. With this approach, we found a previously unidentified member of the IGHD1 subgroup (Figure 1). Remarkably, this gene, named IGHD1-9 (third-party annotation accession number pending), is located among IGHV2/IGHV5 genes ~200 kb upstream of the IGHD gene cluster (Figure 1). This IGHD gene contains an ORF and has functional RSS (12-bp spacer) flanking the gene on both sides (chromosomal coordinates can be found in supplementary files). Most probably, IGHD1-9 is also functionally expressed, since it is used in a rearranged IGHVDJ sequence (accession number AJ286179), albeit this IGHVDJ sequence is derived from another rat strain (PVG). The total number of functional IGHD genes in the BN rat is therefore most likely 14. 4. Concluding remarks Together, our data imply that the total number of unique IGHV genes in the BN rat is at least 353 (see Table 1), including the 12 newly identified IGHV genes. In this estimate, the pairs of identical genes (ten pairs in total) are counted as one. Of these 353 IGHV genes, 131 46 | Chapter 2 (37%) meet all criteria for functional germline IGHV genes and can therefore be potentially expressed. The remaining genes are nonfunctional because they either do not have at least one ORF (pseudogenes) or lack an appropriate RSS (ORF genes). Nearly all nonfunctional IGHV genes are pseudogenes. It should be noted here that there may be more nonfunctional IGHV genes because the bactigs (see the previous discussion) were only analysed for the presence of functional IGHV genes. Also in humans, the number of nonfunctional IGHV genes exceeds the number of functional IGHV genes (approximately one third) (Matsuda et al., 1998); however, in the mouse, there seems to be a higher number of functional (55%) IGHV genes than non-functional IGHV genes (Johnston et al., 2006). In general, however, there is a positive correlation between the number of functional IGHV genes and the number of nonfunctional IGHV genes (Das et al., 2008). The presence of large numbers of nonfunctional genes reflects the diversification of IGHV genes in evolution, as proposed before (Ota and Nei, 1994). This process involves gene duplications and functional elimination after deleterious mutations (pseudogenes and ORF genes). The rat genome harbors the highest number of functional IGHV genes (at least 131) of all mammalian species studied so far (Table 1). The (antigen-independent) recombination of this large number of IGHV genes with one of the 14 IGHD genes and with one of the 4 IGHJ genes accounts for a more diverse combinatorial IGH repertoire in rats compared to other species. In addition, this repertoire is enlarged by junctional diversity, as illustrated by the presence of TdT in rat B cell precursor cells (Opstelten et al., 1986) and variable numbers of N insertions in sequenced rat IGHV genes (Dammers et al., 2000b). Our main conclusion is that the overall organization of the variable region of the IGH locus and the distribution of IGHV gene family members in the rat are strikingly similar to the corresponding region in the mouse (Johnston et al., 2006), despite the fact that these two species diverged from each other 41 million years ago (Kumar and Hedges, 1998). Also in the mouse, the IGHV1 (J558), IGHV2 (Q52), and IGHV5 (PC7183) gene families have the highest number of IGHV members. Both in rat and in mice, members of these IGHV gene families represent approximately two thirds of all functional IGHV genes. These genes therefore contribute the most to the available germline repertoire. In the mouse, the IGHV1 family (J588) is, by far, the largest IGHV gene family (almost half of all functional IGHV genes), whereas in the rat, the largest IGHV gene family is the IGHV2 (Q52) family, with 40 unique members (31% of all functional IGHV genes). Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 47 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Table 1. numbers of IGHV gene family members in the IGHV locus of the BN rat IGHV gene family Number of IGHV genes IMGT subgroupsa Previous mouse nomenclatureb Potentially functional genesc Nonfunctional genesc Total number of IGHV genes IGHV1 J558 25 42 67 d IGHV2 Q52 39 (+4 ) 60 (+ 2) 99 (+6) IGHV3 36–60 4 4 8 IGHV4 X-24 2 2 4 IGHV5 7183 26 (+ 1) 53 (+ 3) 79 (+4) IGHV6 J606 8 11 19 IGHV7 S107(T15) 6 9 15 IGHV8 3609 9 10 19 IGHV9 VGAM3-8 4 4 8 IGHV10 VH10 2 17 19 IGHV11 CP3 5 7 12 IGHV12 CH27 1 2 3 IGHV15 VH15A 0 1 1 131 (+ 5) 222 (+ 5) Total The table also contains the 12 newly identified IGHV genes (see the text for further explanation). a IGHV nomenclature according to IMGT. b IGHV family nomenclature according to (Brodeur and Riblet, 1984). c Functional and nonfunctional (pseudogenes or ORF genes), according to IMGT standards. d The number of 100% identical genes is presented in parentheses. 48 | Chapter 2 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Altschul S. F., Gish W., Miller W., Myers E. W. and Lipman D. J., 1990. Basic local alignment search tool. Journal of molecular biology 215, 403-10. Brodeur P. H. and Riblet R., 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. European journal of immunology 14, 922-30. Butler J. E., Sun J., Wertz N. and Sinkora M., 2006. Antibody repertoire development in swine. Developmental and comparative immunology 30, 199-221. Dammers P. M., Bun J. C., Bellon B., Kroese F. G., Aten J. and Bos N. A., 2001. Immunoglobulin VH-gene usage of autoantibodies in mercuric chloride-induced membranous glomerulopathy in the rat. Immunology 103, 199-209. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P., Bos N. A. and Kroese F. G., 2000a. Immunoglobulin VH gene analysis in rat: most marginal zone B cells express germline encoded VH genes and are ligand selected. Current topics in microbiology and immunology 252, 107-17. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000b. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. J.Immunol. 165, 6156-6169. Das S., Nozawa M., Klein J. and Nei M., 2008. Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates. Immunogenetics 60, 47-55. Dufour V., Malinge S. and Nau F., 1996. The sheep Ig variable region repertoire consists of a single VH family. Journal of immunology (Baltimore, Md. : 1950) 156, 2163-70. Flajnik M. F., 2002. Comparative analyses of immunoglobulin genes: surprises and portents. Nature reviews. Immunology 2, 688-98. Gibbs R. A., Weinstock G. M., Metzker M. L., Muzny D. M., Sodergren E. J., Scherer S., Scott G., Steffen D., Worley K. C., Burch P. E., Okwuonu G., Hines S., Lewis L., DeRamo C., Delgado O., Dugan-Rocha S., Miner G., Morgan M., Hawes A., Gill R., Celera, Holt R. A., Adams M. D., Amanatides P. G., Baden-Tillson H., Barnstead M., Chin S., Evans C. A., Ferriera S., Fosler C., Glodek A., Gu Z., Jennings D., Kraft C. L., Nguyen T., Pfannkoch C. M., Sitter C., Sutton G. G., Venter J. C., Woodage T., Smith D., Lee H. M., Gustafson E., Cahill P., Kana A., Doucette-Stamm L., Weinstock K., Fechtel K., Weiss R. B., Dunn D. M., Green E. D., Blakesley R. W., Bouffard G. G., De Jong P. J., Osoegawa K., Zhu B., Marra M., Schein J., Bosdet I., Fjell C., Jones S., Krzywinski M., Mathewson C., Siddiqui A., Wye N., McPherson J., Zhao S., Fraser C. M., Shetty J., Shatsman S., Geer K., Chen Y., Abramzon S., Nierman W. C., Havlak P. H., Chen R., Durbin K. J., Egan A., Ren Y., Song X. Z., Li B., Liu Y., Qin X., Cawley S., Worley K. C., Cooney A. J., D’Souza L. M., Martin K., Wu J. Q., Gonzalez-Garay M. L., Jackson A. R., Kalafus K. J., McLeod M. P., Milosavljevic A., Virk D., Volkov A., Wheeler D. A., Zhang Z., Bailey J. A., Eichler E. E., et al., 2004. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493-521. Giudicelli V., Chaume D. and Lefranc M. P., 2004. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic acids research 32, W435-40. Gontier E., Ayrault O., Godet I., Nau F. and Ladeveze V., 2005. Developmental progression of immunoglobulin heavy chain diversity in sheep. Veterinary immunology and immunopathology 103, 31-51. Havlak P., Chen R., Durbin K. J., Egan A., Ren Y., Song X. Z., Weinstock G. M. and Gibbs R. A., 2004. The Atlas genome assembly system. Genome research 14, 721-32. Johnston C. M., Wood A. L., Bolland D. J. and Corcoran A. E., 2006. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. Journal of immunology (Baltimore, Md. : 1950) 176, 4221-34. Karolchik D., Kuhn R. M., Baertsch R., Barber G. P., Clawson H., Diekhans M., Giardine B., Harte R. A., Hinrichs A. S., Hsu F., Kober K. M., Miller W., Pedersen J. S., Pohl A., Raney B. J., Rhead B., Rosenbloom K. R., Smith K. E., Stanke M., Thakkapallayil A., Trumbower H., Wang T., Zweig A. S., Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat | 49 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Haussler D. and Kent W. J., 2008. The UCSC Genome Browser Database: 2008 update. Nucleic acids research 36, D773-9. Kent W. J., 2002. BLAT--the BLAST-like alignment tool. Genome research 12, 656-64. Kothapalli N., Norton D. D. and Fugmann S. D., 2008. Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus. Journal of immunology (Baltimore, Md. : 1950) 180, 2019-23. Kumar S. and Hedges S. B., 1998. A molecular timescale for vertebrate evolution. Nature 392, 917-20. Lang P. and Mocikat R., 1991. Immunoglobulin heavy-chain joining genes in the rat: comparison with mouse and human. Gene 102, 261-4. Lefranc M. P., Giudicelli V., Ginestoux C., Bodmer J., Muller W., Bontrop R., Lemaitre M., Malik A., Barbie V. and Chaume D., 1999. IMGT, the international ImMunoGeneTics database. Nucleic acids research 27, 209-12. Mage R. G., Lanning D. and Knight K. L., 2006. B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues. Developmental and comparative immunology 30, 137-53. Marchalonis J. J., Schluter S. F., Bernstein R. M., Shen S. and Edmundson A. B., 1998. Phylogenetic emergence and molecular evolution of the immunoglobulin family. Advances in immunology 70, 417-506. Matsuda F., Ishii K., Bourvagnet P., Kuma K., Hayashida H., Miyata T. and Honjo T., 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. The Journal of experimental medicine 188, 2151-62. Opstelten D., Deenen G. J., Rozing J. and Hunt S. V., 1986. B lymphocyte-associated antigens on terminal deoxynucleotidyl transferase-positive cells and pre-B cells in bone marrow of the rat. Journal of immunology (Baltimore, Md. : 1950) 137, 76-84. Ota T. and Nei M., 1994. Divergent evolution and evolution by the birth-and-death process in the immunoglobulin VH gene family. Molecular biology and evolution 11, 469-82. Reynaud C. A., Garcia C., Hein W. R. and Weill J. C., 1995. Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process. Cell 80, 115-25. Saini S. S., Allore B., Jacobs R. M. and Kaushik A., 1999. Exceptionally long CDR3H region with multiple cysteine residues in functional bovine IgM antibodies. European journal of immunology 29, 2420-6. Saini S. S., Hein W. R. and Kaushik A., 1997. A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle. Molecular immunology 34, 641-51. Stoel M., Evenhuis W. N., Kroese F. G. and Bos N. A., 2008. Rat salivary gland reveals a more restricted IgA repertoire than ileum. Molecular immunology 45, 719-27. Twigger S. N., Pruitt K. D., Fernandez-Suarez X. M., Karolchik D., Worley K. C., Maglott D. R., Brown G., Weinstock G., Gibbs R. A., Kent J., Birney E. and Jacob H. J., 2008. What everybody should know about the rat genome and its online resources. Nature genetics 40, 523-7. Worley K. C., Weinstock G. M. and Gibbs R. A., 2008. Rats in the genomic era. Physiological genomics 32, 273-82. Yancopoulos G. D. and Alt F. W., 1986. Regulation of the assembly and expression of variableregion genes. Annual review of immunology 4, 339-68. 50 | Chapter 2 Chapter 3 Organization of the immunoglobulin heavy-and light-chain loci in the rat Peter M. Dammers Jacobus Hendricks Peter Terpstra Nicolaas A. Bos Frans G. M. Kroese In Azad K. Kaushik and Yfke Pasman (Eds.) 2014, Comparative Immunoglobulin Genetics (pp108-130). New Jersey, US: Apple Academic Press. Inc. R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Abstract Rats of the genus Rattus norvegicus are undoubtedly one of the most popular animals used for experimental biomedical research. Despite the fact that the initial publications on immunoglobulin (IG) genes and their organization in the rat were shortly behind those of mouse and human, our knowledge on the complexity and organization of the IG loci in this rodent species has been hampered for years, due to lack of (genomic) nucleotide sequence information. The tide turned as of the establishment of most of the genomic nucleotide sequence from the Brown Norway (BN) rat strain. This genomic sequence information allows researchers to more accurately map the IG genes in this so commonly used species. To date, the organization of the rat immunoglobulin heavy (IGH) locus has been described in detail, whereas effort is undertaken to resolve the organization of the IG light (IGL) chain loci including IG kappa (IGK) and IG lambda (IGL) loci. This chapter summarizes the current knowledge on the complexity and organization of the IGH, IGK, and IGL loci in the rat and reveals that the IG loci in this animal bear much resemblance to mouse and human. 54 | Chapter 3 1. Introduction Rats belong to the largest order of mammals, named Rodentia (rodents; family Muridae). This order comprises more than 2,000 species, including mice (family Muridae), guinea pigs (family Caviidae), squirrels (family Sciuridae), beavers (family Castoridae) and hamsters (family Cricetidae) (Carleton and Musser, 2005). The most well-known rat species belong to the genus Rattus (“true rats” or “Old World” rats) of which the roof rat (Rattus rattus) and the brown rat (Rattus norvegicus) are the most common members. The evolutionary history of genus Rattus goes back to Asia, where the oldest fossil specimens of Rattus sp. were excavated from sediments that extend to the late Pliocene (2.5 MYA) (Carleton and Musser, 2005). Nowadays, both Rattus rattus and Rattus norvegicus are established worldwide in the temperate climate areas and parts of tropical and subantarctic zones. Most laboratory rats used today trace their heritage to Wistar ancestors (R. norvegicus), originally held at the Wistar Institute of Anatomy and Biology, Philadelphia PA, in the beginning of the twentieth century (Clause, 1993). At present, a large number of genetically well-defined inbred rat strains are established that serve as models for important human disease traits, including susceptibility to cancer, hypertension, ischemia, obesity, diabetes, and autoimmune diseases. So, the rat became “from evil harbinger of pestilence to hero of modern medicine”, as described by Clause (Clause, 1993), and has undoubtedly become one of the most frequently used animals in experimental biology and medical research. As in other mammalian species, diversity in the immunoglobulin (IG) antigen-recognition site in the rat is established during early B cell development by rearrangement of variable region genes (or gene segments) located at the IG heavy (H)-chain and light (L)-chain loci. The Variable domain of the IGH chain is encoded by three different genes: Variable (IGHV), diversity (IGHD), and joining (IGHJ), whereas the IGK and IGL chain Variable domains are encoded by a combination of IGKV–IGKJ and IGLV–IGLJ, respectively (Roth, 1996). Recombination at the IGH, IGK and IGL loci is mediated (among other enzymes) by two “recombinase activation genes”―RAG-1 and RAG-2―that recognize a recombination signal sequence (RSS), flanking each of the IG Variable region genes (Fugmann et al., 2000; Roth, 1996). During recombination, RAG-1 and RAG-2 produce double-strand breaks between the IG Variable region genes to be joined and the flanking RSS. The joining-ends are subsequently subjected to hairpin opening nicks (generating small palindrome sequences called P-nucleotides), exonuclease activity, and nucleotide additions (N-nucleotides) by terminal deoxy-nucleotidyl transferase (TdT) (Gilfillan et al., 1993; Komori et al., 1993). The end result is the generation of highly variable nucleotide sequences at the junctions of the rearranged gene segments. Rats are able to produce the following eight Ab isotypes: IgM, IgD, IgG1, IgG2a, IgG2b, IgG2c, IgA, and IgE (Bazin et al., 1974). The constant region exons (IGHC) necessary to encode these different isotypes are located at the 3′-end of the IGH locus. Organization of the immunoglobulin heavy-and light-chain loci in the rat | 55 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 2. Genomic organization of the rat immunoglobulin heavy chain locus 2.1. Immunoglobulin heavy variable chain locus The Vast majority of the genomic nucleotide sequence of the Brown Norway (BN) rat has been elucidated and this confirmed the location of the IGH locus on chromosome 6q32–33 (Gibbs et al., 2004), as determined before by Pear et al. (Pear et al., 1986). The IGH locus in rats bears much resemblance to that of mice, both with respect to its genomic organization as well as the nucleotide sequence of the coding regions (Bruggemann et al., 1986; Dammers and Kroese, 2001; Dammers et al., 2000; Hendricks et al., 2010; Lang and Mocikat, 1991). The rat IGH locus consists of a typical translocon organization (Marchalonis et al., 1998), comprised of 353 individual IGHV genes (of which 131 are functional), 21 IGHD genes (14 functional), and five IGHJ genes (four functional) (Hendricks et al., 2010). A detailed map of the organization of the rat IGH variable region locus has been published by Hendricks et al. (Hendricks et al., 2010). The IGH locus in rat is orientated from telomere (distal) towards centromere (proximal). Similar to mouse and human, the Vast majority of rat IGHV genes are orientated towards the IGHD and IGHJ genes, and deletional joining, instead of inversional joining, is therefore most preferably used as major recombination mechanism at the IGH locus. The size of the rat IGHV locus spans about 4.8 Mb and is therefore two times larger than the IGHV region in mouse (2.5 Mb) (Johnston et al., 2006) and five times larger than in human (0.9 Mb) (Matsuda et al., 1998; Pallares et al., 1999). The rat IGHV region comprises two and three times as many IGHV genes in comparison to mouse (195 IGHV genes) and human (123–129 IGHV genes), respectively. This indicates that the overall density of IGHV genes in the IGHV region is very similar between rat and mouse (74 en 78 genes per Mb, respectively), but lower in comparison to humans (137–143 genes per Mb). The intergenic distance between the IGHV genes is rather large in comparison to the size of the IGHV genes (~500 bp). For mouse and rat, the density of IGHV genes is somewhat higher at 3′ end of the IGHV region than at the 5′ end. The total number of functional IGHV genes (i.e., germline IGHV genes with an open reading frame in the coding regions and no defects in splice sites, recombination signals and/or regulatory elements) is more or less the same between mouse and rat (110 Vs. 131), and significantly more in comparison to human (39–46). Furthermore, the mouse IGHV locus contains relatively more functional IGHV genes (56%), compared to rat and human (37% and 36%, respectively). In Table 1, the total numbers of IGHV genes in the IGH locus of rat, mouse, and human are depicted, according to current IGH chromosomal maps (Hendricks et al., 2010; Johnston et al., 2006; Matsuda et al., 1998). For this table, numbers of human IGHV genes were obtained from the study of Matsuda et al. (Matsuda et al., 1998). However, due to allelic variation and structural Variants (i.e., deletions, insertions, and duplications that result in changes in gene copy number), the numbers of IGHV genes in the human IGH locus differs among individuals (Lefranc, 2001; Pallares et al., 1999; Watson 56 | Chapter 3 et al., 2013). The human IGH locus is estimated to contain between 123–129 IGHV genes of which 39–46 are considered functional (Pallares et al., 1999). The data of Matsuda et al. (Matsuda et al., 1998) are therefore not applicable for every individual. Nearly all nonfunctional IGVH genes in rat are pseudogenes that lack an open reading frame (ORF). Some nonfunctional genes, however, have an ORF. In ORF genes, alterations in the splice, regulatory, and/or RSS signal sequence(s), and/or amino acid constitution, and/or chromosomal position (i.e., orphan) renders the gene or product nonfunctional (Lefranc, 2001). Das et al. (Das et al., 2008) showed that there is a positive correlation between the number of nonfunctional and functional IGVH genes (in general, the more IGHV genes, the higher the number of nonfunctional genes in the IGH locus). This observation is assumed to result from the evolution of the IGVH genes. As proposed by Ota and Nei (Ota and Nei, 1994), the evolution of IGVH genes is the consequence of two evolutionary processes: the birth-and-death process and diversifying selection. Whereas some duplicated IGVH genes diversify and acquire new functions by mutations and evolutionary selection (“birth”; diversifying selection), others become functionally eliminated after obtaining deleterious mutations (“death”; pseudogenes) (Nei et al., 1997; Ota and Nei, 1994; Tanaka and Nei, 1989). As a result of this evolutionary process, IGHV genes can be grouped into genes with highly homologous sequences, the IGHV gene families or subgroups. As in mice (Brodeur and Riblet, 1984), IGHV subgroup members in rat share at least 80% identity over the coding exon nucleotide sequence, whereas the sequence identity between members of different IGVH subgroups is generally less than 70% (Dammers et al., 2000). Rat, mouse and human IGHV genes can be grouped into 13, 16, and 7 IGHV gene families, respectively. The IGHV genes in the BN rat can be subdivided into 13 IGHV subgroups based on nucleotide sequence identity: IGHV1, IGHV2, IGHV3, IGHV4, IGHV5, IGHV6, IGHV7, IGHV8, IGHV9, IGHV10, IGHV11, IGHV12, and IGHV15 (Hendricks et al., 2010). In rat, most IGHV gene families are composed of various members, ranging from 3 to 99 (total genes, i.e., functional and nonfunctional), except for the IGHV15 gene family, which consists of only one (nonfunctional) member. The largest IGHV gene families in rat are represented by IGHV1, IGHV2, and IGHV5, which contain a total of 67, 99, and 79 IGHV genes, respectively. In the IGHV locus of mouse and rat, the various IGHV gene family members are often found clustered together (Hendricks et al., 2010; Johnston et al., 2006). However, the IGHV gene families are not completely spatially separated from each other and members of different IGHV gene families are frequently found intermingled with one another. Similar to mouse, the IGVH1 and IGHV8 gene family members in rat are located at the 5′end of the IGHV locus, whereas members of the IGHV2 and IGHV5 gene families are located at the 3′ end of the IGHV locus, closely to the IGHD region (Hendricks et al., 2010; Johnston et al., 2006). In comparison to mouse and rat, the IGHV gene family members in human are more extensively intermingled with each other and Organization of the immunoglobulin heavy-and light-chain loci in the rat | 57 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 therefore clustering of IGHV gene family members is hardly observed (Matsuda et al., 1998). The almost identical IGHV locus organization between mouse and rat strongly implies a close evolutionary relationship between the two species. Table 1. Numbers of IGHV genes in the IGH locus of rat, mouse, and human grouped by IMGT subgroup Total number of IGHV genesa IMGT Subgroup Previous mouse nomenclature BN Rat Humanb C57BL/6 Mouse IGHV1 J558 67 (25) 89 (52) 14 (9) IGHV2 Q52 105 (43) 13 (9) 4 (3) IGHV3 36–60 8 (4) 8 (6) 65 (19) IGHV4 X24 4 (2) 2 (1) 32 (6) IGHV5 PC7183 83 (27) 21 (10) 2 (1) IGHV6 J606 19 (8) 5 (5) 1 (1) IGHV7 S107(T15) 15 (6) 4 (3) 5 (0) IGHV8 3609 19 (9) 16 (8) — IGHV9 VGAM3.8 8 (4) 4 (4) — IGHV10 VH10 19 (2) 3 (2) — IGHV11 VH11(CP3) 12 (5) 2 (2) — IGHV12 VH12(CH27) 3 (1) 1 (1) — IGHV13 3609N — 2 (1) — IGHV14 SM7 — 4 (4) — IGHV15 VH15 1 1 (1) — IGHV16 VH16 — 1 (1) — Unclassified — — 19 (0) — 195 (110) 123 Total a 363 (0) (136) (39) Total numbers of IGHV genes grouped by IMGT subgroup (functional + ORF + pseudogenes) (Lefranc, 2001; Lefranc et al., 2009). Numbers of functional IGHV genes are shown in parentheses. A germline IGHV gene is regarded functional if the coding region has an open reading frame without stop codon and if there are no defects in splice sites, RSS and/or regulatory elements (Lefranc, 2001). Numbers of IGHV genes for the different species were obtained from (Hendricks et al., 2010; Johnston et al., 2006; Matsuda et al., 1998). Please note that the human IGHV subgroup nomenclature is not fully compatible between with rat and mouse, for instance human IGHV3 subgroup members share the highest identity with members of the rat/mouse IGHV5 subgroup and human IGHV4 with rat/mouse IGHV3. b Number of IGHV genes in the human IGH locus can Vary among individuals due to copy-number Variation (Pallares et al., 1999; Sudmant et al., 2010; Watson et al., 2013). 58 | Chapter 3 2.2. Immunoglobulin heavy diversity chain locus The rat IGHD gene-cluster spans about 70 kB and is similar in size in comparison to mouse (Hendricks et al., 2010; Kurosawa and Tonegawa, 1982; Lang and Mocikat, 1991; Sakano et al., 1980; Ye, 2004). In addition to the size of the loci, rat IGHD genes (both coding as well as RSS regions) also show high sequence identity to those of the mouse (Figure 1). The IGHD locus in BN rat is comprised of 14 functional IGHD genes (Table 2), which include nine IGHD1 (DFL16), four IGHD4 (DST4) genes, and one IGHD5 (DQ52) gene (previous mouse nomenclature is shown in parentheses). The number of functional IGHD genes in rat is very comparable to the situation in mouse, where the IGH locus contains about 10–13 functional IGHD genes, depending on the mouse strain (Chang et al., 1992; Feeney, 1990; Feeney and Riblet, 1993; Gerondakis et al., 1988; Kurosawa and Tonegawa, 1982; Lawler et al., 1987; Ye, 2004). As indicated in Figure 1, rat IGHD genes are flanked on both sides by a typical RSS, characterized by a 12-bp spacer and the rather conserved heptamer and nonamer sequences (Ramsden et al., 1994). This allows the proper rearrangement of IGHD genes with an IGHV and an IGHJ gene at the 5′ and 3′ side, respectively. In addition to the functional IGHD genes, there are seven nonfunctional ORF genes containing non-canonical/aberrant RSS sequences, which probably render them nonfunctional. All these nonfunctional IGHD genes either belong to the subgroups IGHD2 (3 genes) or IGHD3 (4 genes). Rat IGHD2 genes show high nucleotide identity to mouse IGHD5 genes (Figure 1), which are also considered to be nonfunctional (Ye, 2004). R|IGHD1-7 R|IGHD1-1 R|IGHD1-9 R|IGHD1-8 R|IGHD1-6 M|IGHD1-1 R|IGHD1-4 R|IGHD1-2 R|IGHD1-3 R|IGHD1-5 GC T T T T T GT GA A GGGA T T T A T T A C T GT GT T T A T - - - T A C T A T GA T GGT A GT T A T T A C T A C - - - C A C A GT GGT A C GT C C A A C A GC A A A A A C C . . . . . . . . . . . . . . . . . A C . C . . . . . . . . . . . . GT A . . . . . C . G- A T . - A . . . C . . . - - - - - - . . . . . . . . . . T A . . . . G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . - - - . . . . . . - - - A . C - A - - GC . . T A T . T A C . . . . . . . A . . T A . . . . G. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - . . . . . . . . . . . . T A . . . . C. . - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . C . . . . . . . - . . . - - - - - . . . . C . GA . . GT A . A G. G. G- - - - - - . . . . . . . T . . T A . . . . GT . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . C . . . . . . . . . . . . - - - . . . . . C . G. A . . - A - - GC . . . - - - - - - . . . . . . . C . . T A . . . . T . . . . . . . . . . . . A . . . . . . . . . . . . C . . A C . C . . . . . . . . A C . . - - - - . . . . . . - - . . . - A . A . C . . . - - - - - - . . . . . . . . A . T A . . . . G. . . . . . . . . . . . A . . . . . . . . . . . . C . . A C . C . . . . . . . . . C . . - - - - . . . . . . - - . . . - A . G. C . . . - - - - - - . . . . . . . . A . T A . . . . G. . A . . . . . C . . A T . . . . . . . . . . . . . . . C . . C . . . . . . . . . . T . - - - A . . . . C - - - A . . - A - - GC . . . - - - - - - . . . . . . . . A . T A . T . . G. . A . . . . . . . . . . . . . . . . . . . . . . A . . A . . C . . . . . . . . . . . . - - - A . . - - - - - - - - - - A - - . C . . . - - - - - - . . . . . C . A . . T A . A . T G. . . . . . . . . . . R|IGHD4-1 R|IGHD4-2 R|IGHD4-3 M|IGHD3-1 R|IGHD4-4 GGA T T T T GA A C T A GT T A GT GT C A C A GT GGGT A T A A T T C GGGGT A C C A C T GT A GGA A A A GC T GC A A C GA A A AC T A . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . G. . . . . . . . . . . T A . T A . . . . A . . . . . . . . . . . . . C . . . . . A . . . . . . . . . . . A . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . C T C . . . CT G . . . . . . . . . . . A . . . . . C . . . . . . . . . . . . C . C . GC . . . . . C . . . . . . . . . . A . . . . . . . . C A . . . C . . . . . . . . . . . . . . . . . . . . G. . C . . . . . T G. . . . . . . . . - . . T . . . C . T . . . T . . . . . . . . . . T . . . . T . . T . . . . . . R|IGHD5-1 GGT T T T GA C T A A GC A A A GC A T C A C A GT GC T A A C T GGGA GC A C A GT GA C T T GT GGC T C A A C A A A A A C C M|IGHD4-1 . . . . . . . . . . . . . . GG. . . . C . . . . . . . . . . . . . . . . . C . . . G. . . . . A C . . . . . . . . . . . . . . . . . IGHD ORF+pseudogenes R|IGHD2-1 R|IGHD2-3 M|IGHD5-3 R|IGHD2-2 GGA T T T T GT A T GGGC C T C T C T C A C T GT GGGA T A C T T A C C A T A GT A GT A T A GT T C A GT C C C A A A A GC . . . . . C C . . . . . . . T . C . . . . . . . . . . . . . . . . . C . . . . . A . . . GA . . . . . A . . . . . . . . . . . T T . . . . . . C . . . . . . T . . . . . . . . . . . C . . . . A . . . . C . . . . . C . T . G. . . C . . A G. . . . . T . . . . T T . . . . . . C . . A . . . . . . . . T . . . . . . . . . . . . . . . . C . . T . . C . . . GT . . . . . A . T . A . . . T . . . GC A R|IGHD3-1 R|IGHD3-4 R|IGHD3-2 R|IGHD3-3 M|UNDEF A C A A C C C A T GGT GGC T T C A GGC A GC A GGC C T C T GC A GT GC C C C C A A C C C A A C A T GC C A T C T GC T C T GGGA C C A GA A A GT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . GT . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . - . T . . . . . . . . . . . . . T . . . . . . . . T . . . . . . . . . GT . . . . . . . . . . . . . . . . . A . . T . . . . . . . . . . . . . . A T . . . . . . T . . . . . . . . A . . . . . T . . . . . . . . . T . . . . GT . . GT . . . . . . . . . . A A . . . . . . . . . . T A . . . . . . . . . . . . . - . A . . . . . . . C A . . . A . T . G. . A C . . - - T . . A . . . . . . G Figure 1. Alignment of the IGHD sequences of the BN rat. Sequences are described in Ref. (Hendricks et al., 2010) and obtained from the UCSC (http://genome.ucsc.edu) (Meyer et al., 2013) using Baylor 3.4/ Organization of the immunoglobulin heavy-and light-chain loci in the rat | 59 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 rn4 rat genome assembly (Nov. 2004) (Gibbs et al., 2004). All rat IGHD genes have the same orientation in the locus (5′ to 3′ on the minus strand). In each IGHD subgroup, a mouse IGHD gene from the most homologous subgroup is included for comparison (R: rat, M: mouse). Dots and dashes denote nucleotide identities and gaps, respectively. The RSS heptamer and nonamer sequences are underlined. The IGHD region is marked in bold. The coordinates of the IGHD genes in Baylor 3.4/rn4 assembly are as follows: IGHD1–1 chr6:138,521,262–138,521,344; IGHD1–3 chr6:138,505,008–138,505,083; IGHD1–5 chr6:138,496,518–138,496,587; IGHD1–7 chr6:138,483,608–138,483,692; IGHD1–9 chr6:138,743,870–138,743,951; IGHD4–2 chr6:138,501,150–138,501,222; IGHD4–4 chr6:138,486,905–138,486,976; IGHD2–1 chr6:138,518,929–138,518,994; IGHD2–3 chr6:138,495,637–138,495,702; IGHD3–2 chr6:138,509,882–138,509,959; IGHD3–4 chr6:138,495,398–138,495,476. IGHD1–2 chr6:138,511,394–138,511,471 IGHD1–4 chr6:138,503,455–138,503,532; IGHD1–6 chr6:138,490,407–138,490,485; IGHD1–8 chr6:138,471,761–138,471,842; IGHD4–1 chr6:138,509,072–138,509,144; IGHD4–3 chr6:138,494,453–138,494,525; IGHD5–1 chr6:138,453,776–138,453,842; IGHD2–2 chr6:138,510,119–138,510,184; IGHD3–1 chr6:138,518,691–138,518,769; IGHD3–3 chr6:138,488,933–138,489,011; The rat IGHD3–1 sequence matches with a previous undefined (UNDEF) region in the mouse IGH locus at chromosomal position chr12:113,469,160–113,469,235 of Genome Reference Consortium assembly Dec. 2011 (GRCm38/mm10) (Church et al., 2009). Table 2. Numbers of IGHD genes in the IGH locus of rat, mouse, and human grouped by IMGT subgroup. IMGT subgroupa Number of genesb Rat Mouse Human BN Rat C57BL/6 Mouse Human IGHD1 IGHD1 (DFL16) IGHD4 9 (9) 1 (1) 4 (2) — IGHD2 (DSP2) — — 6 (6) — — — IGHD5 — — 4 (3) IGHD2 IGHD5 — 3 (0) 8 (0) — c IGHD3 UNDEF — 4 (0) 6 (0) — IGHD4 IGHD3 (DST4) IGHD1 4 (4) 2 (2) 5 (4) IGHD5 IGHD4 (DQ52) IGHD7 1 (1) 1 (1) 1 (1) — IGHD6 IGHD2 — 2 (0) 4 (4) — — IGHD3 — — 5 (5) — — IGHD6 Total a — — 4 (4) 21 (14) 26 (10) 27 (23) IGHD genes are grouped by IMGT nomenclature and arranged in corresponding subgroups. Grouping of the mouse IGHD6 subgroup with human IGHD2 is tentative. b Numbers of IGHD genes for the different species were obtained from (Hendricks et al., 2010; Lefranc, 2001; Watson et al., 2013; Ye, 2004) and IMGT (mouse IGHD5 genes) (Lefranc et al., 2009). Numbers of functional IGHD genes are shown in parentheses. c Previously undefined murine IGHD subgroup, provisionally consisting of six nonfunctional IGHD genes (pseudo/ORF). 60 | Chapter 3 Rat IGHD3 homologous, however, have not been described for mouse, but alignment of the rat IGHD3–1 sequence to the mouse genome using the BLAT genome search program (Kent, 2002), revealed the presence of a previously undefined IGHD gene in the mouse IGH locus equivalent to the rat IGHD3 subgroup (Figure 1; M|UNDEF). This murine IGHD gene is most probably also nonfunctional, since it contains a 2-bp deletion in the 3′ RSS spacer and has heptamer and nonamer sequences that carry aberrant (non-canonical) bases. A preliminary BLAT search (assembly Dec. 2011, GRCm38/mm10) (Church et al., 2009; Kent, 2002), indicates that this new IGHD gene most likely belongs to a new murine IGHD subgroup consisting of six (presumably nonfunctional) members. The distribution of IGHD genes in the IGH locus is not completely similar between rat and mouse (Figure 2). Except for the single functional DQ52 gene (rat IGHD5–1 and mouse IGHD4–1), which is located in close proximity (approx. 0.6 kB) to the IGHJ locus, the relative position and numbers of IGHD subgroup members are less well preserved between these species. For instance, the IGH locus of C57BL/6 mice harbors one DFL16 (IGHD1) gene, six DSP2 (IGHD2) genes, two DST4 (IGHD3) genes, and Figure 2. Comparison of the IGHD locus organization of BN rat Vs. C57BL/6 mouse. Rat and mouse IGHD sequences are described by Hendricks et al. (Hendricks et al., 2010) and Ye (Ye, 2004), respectively, and retrieved from the IMGT (http://www.imgt.org) (Lefranc et al., 2009). Relative positions were inferred using the BLAT genome search program (http://genome.ucsc.edu) (Kent, 2002; Meyer et al., 2013) on the basis of genome assemblies Baylor 3.4/rn4 (Nov. 2004) and GRCm38/mm10 (Dec. 2011) (Church et al., 2009; Gibbs et al., 2004). IGHD genes are designated in abbreviated IMGT nomenclature. Solid and open triangles represent functional and pseudo(p)/ORF(r) genes, respectively. The IGHJ locus is marked by an open box and is approx. 0.6 kB downstream from gene DQ52 (rat D5–1and mouse D4–1). For rat, the IGHD gene-clusters (see text) are indicated by bidirectional arrows. Rat IGHD1–9 is located 200 kB upstream of the IGHD gene-cluster and is not included in the figure. one DQ52 (IGHD4) gene, revealing that the IGHD locus of the BN rat contains relatively more DFL16 (IGHD1) genes, whereas genes belonging to the DSP2 subgroup, which is the largest in mouse, seem to be absent from the rat IGH locus. Typically, five clusters of tandem IGHD genes can be found in the rat IGH locus, generally consisting of a functional Organization of the immunoglobulin heavy-and light-chain loci in the rat | 61 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 IGHD1 and IGHD4 gene separated from each other by an approx. 6 kB region harboring most frequently two pseudo(p)/ORF(r) genes of the IGHD2 and IGHD3 subgroup, respectively ([IGHD1–IGHD2r–IGHD3p–IGHD4] gene-cluster). A similar phenomenon is observed in mouse, where five out of six IGHD2 genes are flanked at the 3′ side by a pseudogene of the IGHD5 subgroup. Most likely, such clusters of tandem IGHD genes evolved by duplication of a common ancestral IGHD gene-cluster and may well result in a rapid expansion of certain IGHD subgroups during species evolution. The human IGHD gene locus, on the other hand, is about 40 kB and contains 27 IGHD genes (23 functional) subdivided into seven subgroups (Lefranc, 2001; Matsuda et al., 1998). The number of functional IGHD genes in the human IGH locus is therefore about twice the number of functional IGHD genes in rat and mouse. Figure 3 shows the evolutionary relationship between the functional IGHD genes of rat, mouse, and human, as inferred from the nucleotide sequences of the IGHD genes. This phylogenetic analysis shows that rat IGHD1 genes are closely related to mouse IGHD1 (DFL16), mouse IGHD2 (DSP2), and human IGHD4 genes. Also the rat IGHD5–1, mouse IGHD4–1 (DQ52), and human IGHD7–27 genes, seem to be relatively preserved among the three species. On the other hand, the human IGHD2, IGHD3, IGHD5, and IGHD6, form more isolated phylogenetic clusters. It seems likely that the human IGHD5 genes evolved from an ancestor shared with the human IGHD4 (rat/mouse IGHD1) subgroup, but the IGHD5 genes have substantially diverged from this group. 2.3. Immunoglobulin heavy joining chain locus The rat IGHJ locus was initially partly described by Brüggemann et al. (Bruggemann et al., 1986) and further completed by Lang and Mocikat (Lang and Mocikat, 1991). The IGHJ locus of rat is comprised of four functional IGHJ genes (IGHJ1 to IGHJ4), with intact 5′ RSS and 3′ splice signal sequences, and one IGHJ pseudogene (Jψ0; coding region is interrupted by a stop codon). The order of the IGHJ genes in the locus is Jψ0–IGHJ1–IGHJ2–IGHJ3–IGHJ4. Both IGHJ2 and IGHJ3 have a 23–bp RSS, whereas IGHJ1 and IGHJ4 (and the pseudogene Jψ0) carry a 22–bp RSS. Considering the preference of 23–bp over 22–bp RSS during recombination (Suzuki and Shiku, 1992), this may result in a higher frequency of IGHJ2 and IGHJ3 gene usage in IGH transcripts. The rat IGHJ locus is about 2 kB in size and bears much resemblance to that of mouse and human, both in respect to organization as well as nucleotide and amino acid (coding region) sequence. In general, rat IGHJ coding regions share around 80% or more identity at both nucleotide and amino acid sequence level with the IGHJ genes of mouse (Lang and Mocikat, 1991). A similar degree of identity is also observed upon comparison with human IGHJ genes (Lang and Mocikat, 1991), although in human the IGHJ locus is comprised of six functional IGHJ genes and three IGHJ pseudogenes (Ravetch et al., 1981). 62 | Chapter 3 rn4|IGHD1-2|R 86 51 rn4|IGHD1-4|R Rat rn4|IGHD1-1|R 6 X13972|IGHD4-4|H Human 12 22 20 X97051|IGHD4-17|H 87 rn4|IGHD1-5|R Rat rn4|IGHD1-9|R Rat rn4|IGHD1-3|R 23 Rat rn4|IGHD1-6|R 27 53 Rat AC073553|IGHD1-1|M Mouse AC073553|IGHD2-3|M 63 AC073553|IGHD2-5|M 31 AC073553|IGHD2-8|M 31 Mouse AC073553|IGHD2-4|M 20 AC073553|IGHD2-7|M 78 rn4|IGHD1-7|R 54 Rat rn4|IGHD1-8|R 25 Rat X13972|IGHD5-12|H 14 X13972|IGHD5-5|H 100 77 J00256|IGHD7-27|H rn4|IGHD5-1|R 96 Human Rat AC073553|IGHD4-1|M 45 Human X97051|IGHD5-18|H Mouse X93616|IGHD3-22|H X13972|IGHD3-10|H 86 X13972|IGHD3-9|H 63 Human X13972|IGHD3-3|H 41 X93614|IGHD3-16|H 47 X97051|IGHD2-2|H 53 41 91 X13972|IGHD2-8|H Human X97051|IGHD2-21|H J00234|IGHD2-15|H X13972|IGHD6-6|H 48 53 48 X97051|IGHD6-25|H Human 93 X13972|IGHD6-13|H X97051|IGHD6-19|H 90 55 86 X97051|IGHD1-1|H X97051|IGHD1-20|H 35 Human X97051|IGHD1-26|H X13972|IGHD1-7|H AC073553|IGHD3-2|M 68 87 Mouse AC073553|IGHD3-1|M Mouse rn4|IGHD4-1|R 35 rn4|IGHD4-3|R 78 Rat rn4|IGHD4-4|R 58 70 rn4|IGHD4-2|R 0.2 Figure 3. Evolutionary relationships of functional IGHD genes from rat, mouse and human. The evolutionary relationship was inferred using the Neighbor-Joining method (Nei and Kumar, 2000). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed from IGHD 5′ to 3′ RSS nonamer sequences using the Kimura 2-parameter method (number of base substitutions per site), and modeled with a gamma distribution (shape parameter = 1). Bootstrap confidence Values of interior branches (500 replicates) are depicted near the branches. Values of 95% or higher are considered to represent the correct topology (Nei and Kumar, 2000). All ambiguous positions were removed for each sequence pair (partial deletion). There were a total of 95 positions in the final dataset. Evolutionary analyses were conducted with MEGA5 software (Tamura et al., 2011). Sequence identifiers show GenBank accession number, IGHD gene number, and whether the sequence is derived from rat (R), mouse (M), or human (H), respectively. Mouse and human IGHD sequences were retrieved from the IMGT (http://www.imgt.org) (Lefranc et al., 2009). Rat IGHD sequences (rn4) were obtained from the UCSC (http://genome.ucsc.edu) (Meyer et al., 2013) using Baylor 3.4/rn4 rat genome assembly (Nov. 2004) (Gibbs et al., 2004). Organization of the immunoglobulin heavy-and light-chain loci in the rat | 63 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 The close evolutionary relationship among rat, mouse, and human IGHJ genes is illustrated by the phylogenetic tree depicted in Figure 4. This phylogenetic analysis, based on the nucleotide sequences of the functional IGHJ genes, reveals that rat, mouse and, human IGHJ genes are related in the following way: rat/mouse IGHJ1 to human IGHJ2, rat/mouse IGHJ2 to human IGHJ1 and IGHJ4, rat/mouse IGHJ3 to human IGHJ5, and rat/mouse IGHJ4 to human IGHJ6. 2.4. Immunoglobulin heavy constant chain locus The eight serological-defined IGH isotypes in the rat (IgM, IgD, IgG1, IgG2a, IgG2b, IgG2c, IgE, and IgA) (Bazin et al., 1974) are encoded by the IGHC genes: Cμ (IGHM), Cδ (IGHD), Cγ1 (IGHG1), Cγ2a (IGHG2A), Cγ2b (IGHG2B), Cγ2c (IGHG2C), Cε (IGHE), and Cα (IGHA), respectively. The coding and surrounding intron nucleotide sequences are described for many of the rat IGHC genes (Alcaraz et al., 1980; Bruggemann, 1988; Bruggemann et al., 1988; Bruggemann et al., 1986; Bruggemann et al., 1989; Dammers et al., 2001; Hellman et al., 1982; Hellman et al., 1988; Parker et al., 1994; Sire et al., 1982; Steen et al., 1984). The chromosomal order of the IGHC genes in the rat was originally resolved by Brüggemann et al. (Bruggemann et al., 1986) and is: Cμ–Cδ– Cγ2c–Cγ2a–Cγ1–Cγ2b–Cε–Cα. To our knowledge, however, an exact chromosomal map of the rat IGHC locus has never been published. On the basis of the rat IGHC exon nucleotide sequences that were retrieved for the IMGT/GENE-DB (Giudicelli et al., 2005; Lefranc et al., 2009), we were able to establish a genomic map of the rat IGHC region using the Baylor 3.4/rn4 genome assembly (Nov. 2004) at the UCSC (http://genome.ucsc.edu) (Gibbs et al., 2004; Meyer et al., 2013). As shown in Figure 5, the size of rat IGHC locus is about 220 kB and the IGHC genes are arranged in the following order: JH–7.5 kB–Cμ–2 kB–Cδ–35 kB–Cγ2c–42 kB–Cγ2a–32 kB–Cγ1–19 kB–Cγ2b–21 kB– Cε–8 kB–Cα. The number of IGHC genes in the rat and the distance between them is rather similar to the IGHC gene organization in mouse [JH–6.5 kB–Cμ–4.5 kB–Cδ–55 kB–Cγ3–34 kB–Cγ1–21 kB–Cγ2b–15 kB–Cγ2a–14 kB–Cε–12 kB–Cα] (Shimizu et al., 1982), but is different from that observed in human, in which a tandem gene-cluster [Cγ–Cγ–Cε–Cα] is found [JH–8 kB–Cμ–8 kB–Cδ–±60 kB–Cγ3–26 kB–Cγ1–19 kB–Cψε2–13 kB–Cα1–±55 kB (incl. Cψγ)–Cγ2–18 kB–Cγ4–23 kB–Cε1–10 kB–Cα2] (Lefranc, 2001; Lefranc and Lefranc, 1987; Ravetch et al., 1981). In contrast to human, the rat and mouse (Shimizu et al., 1982) IGHC locus does not seem to contain any conserved IGHC pseudogenes. Except for Cα, the IGHC genes in the rat are comprised of four exons encoding the IGH chain constant domains and two exons coding for the membrane anchor (M1 and M2). The relative position of the most 5′ (Cμ and Cδ) and 3′ (Cε and Cα) IGHC genes in the IGHC locus is similar between mouse and rat.The Cμ, Cδ, and Cα loci are, however, not extensively described for the rat. Genomic sequences of the exons Cμ2 and Cα3 were established by Brüggemann et al. (Bruggemann et al., 1986), whereas the nucleotide sequence of the Cμ1 region was recovered from cDNA 64 | Chapter 3 (Dammers et al., 2001). The rat Cμ and Cα sequences bear high similarity to their counterpart in the mouse and generally ≥90% sequence identity is revealed upon comparison (both at nucleotide and amino acid level). 88 X56791 |IGHJ2|R 34 Rat V00770 |IGHJ2|M 64 J00256 |IGHJ4|H Mouse Human J00256 |IGHJ1|H 75 J00256 |IGHJ3|H J00256 |IGHJ5|H 61 Human X56791 |IGHJ3|R 82 99 J00256 |IGHJ2|H 80 V00770 |IGHJ3|M Rat V00770 |IGHJ1|M J00256 |IGHJ6|H Mouse Human X56791 |IGHJ4|R 99 91 Rat Mouse Human M13798 |IGHJ1|R 84 Human Human V00770 |IGHJ4|M Rat Mouse 0.1 Figure 4. Phylogenetic tree of the functional IGHJ genes from rat, mouse and human (Neighbor-Joining method; (Nei and Kumar, 2000)). IGHJ sequences are described by (Bruggemann et al., 1986; Gough and Bernard, 1981; Lang and Mocikat, 1991; Sakano et al., 1980) and retrieved from the IMGT (http:// www.imgt.org) (Lefranc et al., 2009). Evolutionary distances were computed from the IGHJ 5′ RSS nonamer sequence up to the IGHJ 3′ splice donor site (based on a total of 105 positions). See Figure 3 legend for further details. A large proportion of the cDNA sequence of the rat Cδ gene was determined by Sire et al. (Sire et al., 1982). The rat Cδ locus spans nearly 20 kB and is therefore twice the size of the other IGHC genes (Figure 5). Comparison to the mouse reveals that the rat Cδ1 exon differs substantially from mouse Cδ1 (50–55% identity), whereas much more identity is observed in the more C-terminal parts of the Cδ gene encoded by the Cδ3 and CHS exons (85–90% identity) (Sire et al., 1982). The genomic organization of the rat Cε gene was described by Steen et al. (Steen et al., 1984) and is about 5 kB in size. Overall, the rat ε-chain yields ≥60% and ≥80% nucleotide identity with the ε-chain in man and mouse, respectively. Of all rat IGHC genes, Cγ genes have been described most extensively (Bruggemann, 1988; Bruggemann et al., 1988; Bruggemann et al., 1986; Bruggemann et al., 1989). The rat Cγ1 and Cγ2a genes share 94% nucleotide identity with each other and best resemble the single mouse Cγ1 gene (87% and 86%, respectively). The rat Cγ2b gene, on the other hand, bears high nucleotide sequence identity to the mouse Cγ2a and Cγ2b genes (82% and 81%, respectively). Furthermore, the rat Cγ2c gene seems to be the equivalent of mouse Cγ3 gene (87% identity), as revealed by nucleotide sequence identity and effector function (Bruggemann et al., 1988). Organization of the immunoglobulin heavy-and light-chain loci in the rat | 65 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Similar to other IGH genes, the IGHC genes in mammals most probably evolved from series of gene duplication(s) followed by mutation and selection of new genes. Some of these duplications occurred before species divergence, whereas others occurred in recent species evolution. In this regard, rat and mouse Cγ genes are most likely derived from a common set of three ancestral genes that evolved to Cγ2c, [Cγ2a–Cγ1], and Cγ2b in rat, and Cγ3, Cγ1, and [Cγ2b–Cγ2a] in mouse (Bruggemann, 1988). The pairs of closely related Cγ2a/Cγ1 genes in rat and Cγ2b/Cγ2a genes in mouse are expected to result from a duplication event in recent evolution of either species. Also the presence of two Cγ gene-clusters [Cγ–Cγ–Cε–Cα] in the human IGHC locus can be explained by a segmental-duplication in recent human evolution. Figure 5. Genomic organization of the entire IGHC region of the BN rat. The 220 kB map was constructed on the basis of rat Baylor 3.4/rn4 genome assembly (Nov. 2004) at UCSC (http://genome.ucsc.edu) (Gibbs et al., 2004; Meyer et al., 2013) and Genvision software (DNASTAR, Madison, WI; http://www. dnastar.com). Direction is from telomere to centromere on chromosome 6q32 (minus strand in UCSC genome browser). The region corresponds to the assembly coordinates chr6:138450912–138230912. Nucleotide sequences of the IGHC exons were obtained using IMGT/GENE-DB (http://www.imgt.org) (Giudicelli et al., 2005; Lefranc et al., 2009). The JGHJ locus is shown for map orientation. Red tick marks indicate positions of (small) assembly gaps in the Baylor 3.4/rn4 assembly (first 6 gaps approx. 10 bp; 66 | Chapter 3 last gap approx. 130 bp). Long arrows represent the loci of individual IGHC genes, including the introns, but excluding the 5′ and 3′ untranslated regions (UTR). IGHC exons are indicated by short arrows and annotated with standard IMGT labels (Lefranc et al., 2009). 3. Genomic organization of the rat immunoglobulin kappa and lambda loci The IGK and IGL chains in rats are transcribed from two loci of which only one copy of either locus is present in the rat haploid genome (Frank et al., 1987; Sheppard and Gutman, 1981a; Steen et al., 1987). The IGK and IGL loci are located on chromosome 4 and 11, respectively (Perlmann et al., 1985; Wahlstrom et al., 1988). In rodents, the IGK and IGL chains are not equally expressed among peripheral B cells and most (>90%) express IGK chains. In contrast to the rat IGH locus, limited information is available on the genomic organization of the rat IGK and IGL loci. Initial Southern blotting experiments by Breiner et al. (Breiner et al., 1982) using Vκ probes revealed the presence of dozens of Vκ genes in the rat genome and that they presumably could be subdivided into subgroups similar to mouse. The current IMGT/GENE-DB (Giudicelli et al., 2005) lists 163 (135 functional) IGKV genes for the IGK locus of the BN rat that are subdivided over 21 IGKV subgroups. The largest IGVK subgroups in rat are IGKV1 (of which 22 are functional), IGKV4 (19 functional), and IGKV12 (21 functional). Together, members of these subgroups comprise almost half (46%) of the functional IGKV gene repertoire. In comparison, the mouse IGK locus on chromosome 6 consists of 169 IGKV genes (98 functional; current IMGT/GENE-DB) (Giudicelli et al., 2005), which belong to 20 subgroups. The total number of IGKV genes is therefore very similar between rat and mouse, but the rat IGK locus contains relatively more functional IGKV genes (83% Vs. 68%). In addition to the IGKV genes, the IGK locus harbors seven IGKJ genes (IGKJ1, IGKJ2–1, IGKJ2–2, IGKJ2–3, IGKJ3, IGKJ4, and IGKJ5) and only one IGKC (Cκ) gene (Breiner et al., 1982; Burstein et al., 1982; Frank et al., 1987; Sheppard and Gutman, 1981b). As in mouse, IGKJ3 is probably not functional, because of a mutation in the splice-donor site, leaving six functional IGKJ genes available (Burstein et al., 1982; Sheppard and Gutman, 1982). The rat IGKJ genes bear much resemblance to their murine homologs (>90% nucleotide identity for the coding regions). The same degree of nucleotide sequence identity is observed for the IGKC genes of rat and mouse (88.5%) (Sheppard and Gutman, 1981a). An interesting observation was made by Sheppard and Gutman (Sheppard and Gutman, 1981a) and Frank et al. (Frank et al., 1987), who demonstrated that on short-term evolutionary scale the mutation rate in the IGKC coding region is higher in comparison to the surrounding noncoding regions. This results in a rapid divergence of the IGKC region in the rat lineage and the creation of many allelic Variants. A phenomenon which is also observed for the IGLC gene (Frank and Gutman, 1988).The rat IGL and related loci (15 and VpreB) have not been as extensively Organization of the immunoglobulin heavy-and light-chain loci in the rat | 67 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 studied as those of mouse and an exact physical map of these loci has still to be established. In mouse, the IGL locus spans a region of about 180 kB on chromosome 16 and is arranged from a limited number of IGL genes. The IGL genes in this species are organized in two [VnJCJC] gene-clusters, about 94 kB separated from each other, which have the following order: IGLV2–18.5 kB–IGLV3–40.4 kB–IGLJ2–1.3 kB–IGLC2–2.0 kB–IGLJ4–1.2 kB–IGLC4 and IGLV1–14.4 kB–IGLJ3–1.3 kB–IGLC3–2.1 kB–IGLJ1–1.1 kB–IGLC1 (Gerdes and Wabl, 2002). The IGLJ4 and IGLC4 combination is considered nonfunctional due to a defective splice donor-site (Blomberg and Tonegawa, 1982). Only three IGLV genes are described for the mouse IGL locus. The number of combinations that can be generated during IGLV–IGLJ rearrangement is therefore Very restricted. In case of the rat, only three IGLC genes and one IGLV gene are described (Frank and Gutman, 1988; Steen et al., 1987). Two of these IGLC genes (originally named Cλ2 and Cλ1) are located at approximately 3 kB from each other. Each of these IGLC genes seems to be preceded by two IGLJ genes, instead of one in case of the mouse. One of these IGLC genes (Cλ1) does not seem to be functionally expressed, as both Jλ1 genes upstream of Cλ1 have aberrant RSS and splice donor-sites. Also one of the Jλ2 genes is not functional, leaving only the Jλ2–Cλ2 combination apparently able to be functionally expressed. A third IGL gene (Cλ1.2) was also described by Frank and Gutman (Frank and Gutman 1988) and initially thought to represent another IGLC pseudogene. Cλ1.2, however, may well represent the rat homologue of the mouse λ5 gene (unpublished observations; accession number Z68145). Rat Cλ2 bears the highest resemblance to the murine IGLC3 gene (>90% sequence identity in coding and surrounding noncoding region), whereas the rat Cλ1 gene is most closely related to both IGLC1 and IGLC4 (Frank and Gutman, 1988; Steen et al., 1987). The rat Cλ2–Cλ1 cluster described before (Frank and Gutman, 1988; Steen et al., 1987) may therefore well be the homologue of the mouse IGLC3–IGLC1 cluster. In addition to the IGLJ and IGLC genes, Aquilar and Gutman (Aguilar and Gutman, 1992) showed that the IGL locus in rats also comprises about 10–15 IGLV genes (including some pseudogenes), representing at least four distantly related IGLV subgroups. At present, the rat IGL locus seems to have a rather low complexity, apparently consisting of only one [VnJJCJJC] cluster. According to the current IMGT/GENE-DB (Giudicelli et al., 2005), the rat IGL locus on chromosome 11 consists of seven functional IGLV (representing three subgroups: IGLV1, IGLV2, and IGLV3), four IGLJ (two functional), and four functional IGLC genes. Whether the two additional IGLC genes listed in the IMGT database represent the members of a second IGL cluster in the rat, as observed in mouse, is currently not known. 68 | Chapter 3 4. Conclusion The rapid evolution of IG loci suggests that these loci are subjected to high diversifying-selection pressure, to establish a large repertoire of antigen-receptors on B cells. This evolutionary process results in variation of complexity and organization of IG loci among different mammalian species and provides optimal protection against threatening pathogens. Knowledge on the IG complexity and organization is prerequisite for the appropriate evaluation of the functional repertoire expressed by B cells. With the background information on the IG loci in the rat that is presented in this chapter, it will be feasible to maximize the usefulness of the rat as an experimental model for many important human disorders. Organization of the immunoglobulin heavy-and light-chain loci in the rat | 69 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Aguilar B. A. and Gutman G. A., 1992. Transcription and diversity of immunoglobulin lambda chain variable genes in the rat. Immunogenetics 37, 39-48. Alcaraz G., Bourgois A., Moulin A., Bazin H. and Fougereau M., 1980. Partial structure of a rat IgD molecule with a deletion in the heavy chain. Ann Immunol (Paris) 131C, 363-88. Bazin H., Beckers A. and Querinjean P., 1974. Three classes and four (sub)classes of rat immunoglobulins: IgM, IgA, IgE and IgG1, IgG2a, IgG2b, IgG2c. Eur J Immunol 4, 44-8. Blomberg B. and Tonegawa S., 1982. DNA sequences of the joining regions of mouse lambda light chain immunoglobulin genes. Proc Natl Acad Sci U S A 79, 530-3. Breiner A. V., Brandt C. R., Milcarek C., Sweet R. W., Ziv E., Burstein Y. and Schechter I., 1982. Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse. Gene 18, 165-74. Brodeur P. H. and Riblet R., 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. Eur J Immunol 14, 922-30. Bruggemann M., 1988. Evolution of the rat immunoglobulin gamma heavy-chain gene family. Gene 74, 473-82. Bruggemann M., Delmastro-Galfre P., Waldmann H. and Calabi F., 1988. Sequence of a rat immunoglobulin gamma 2c heavy chain constant region cDNA: extensive homology to mouse gamma 3. Eur J Immunol 18, 317-9. Bruggemann M., Free J., Diamond A., Howard J., Cobbold S. and Waldmann H., 1986. Immunoglobulin heavy chain locus of the rat: striking homology to mouse antibody genes. Proc Natl Acad Sci U S A 83, 6075-9. Bruggemann M., Teale C., Clark M., Bindon C. and Waldmann H., 1989. A matched set of rat/ mouse chimeric antibodies. Identification and biological properties of rat H chain constant regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha. Journal of Immunology 142, 3145-50. Burstein Y., Breiner A. V., Brandt C. R., Milcarek C., Sweet R. W., Warszawski D., Ziv E. and Schechter I., 1982. Recent duplication and germ-line diversification of rat immunoglobulin kappa chain gene joining segments. Proc Natl Acad Sci U S A 79, 5993-7. Carleton M. D. and Musser G. G., 2005. Order of Rodentia. In Wilson D. E. and Reeder D. M. (Eds.), Vol. 3, The Johns Hopkins University Press, Baltimore, MD, pp. 745-1600. Chang Y., Paige C. J. and Wu G. E., 1992. Enumeration and characterization of DJH structures in mouse fetal liver. The EMBO journal 11, 1891-9. Church D. M., Goodstadt L., Hillier L. W., Zody M. C., Goldstein S., She X., Bult C. J., Agarwala R., Cherry J. L., DiCuccio M., Hlavina W., Kapustin Y., Meric P., Maglott D., Birtle Z., Marques A. C., Graves T., Zhou S., Teague B., Potamousis K., Churas C., Place M., Herschleb J., Runnheim R., Forrest D., Amos-Landgraf J., Schwartz D. C., Cheng Z., Lindblad-Toh K., Eichler E. E. and Ponting C. P., 2009. Lineage-specific biology revealed by a finished genome assembly of the mouse. PLoS Biol 7, e1000112. Clause B. T., 1993. The Wistar Rat as a right choice: establishing mammalian standards and the ideal of a standardized mammal. J Hist Biol 26, 329-49. Dammers P. M., Bun J. C., Bellon B., Kroese F. G., Aten J. and Bos N. A., 2001. Immunoglobulin VH-gene usage of autoantibodies in mercuric chloride-induced membranous glomerulopathy in the rat. Immunology 103, 199-209. Dammers P. M. and Kroese F. G., 2001. Evolutionary relationship between rat and mouse immunoglobulin IGHV5 subgroup genes (PC7183) and human IGHV3 subgroup genes. Immunogenetics 53, 511-7. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P., Bos N. A. and Kroese F. G., 2000. Immunoglobulin VH gene analysis in rat: most marginal zone B cells express germline encoded VH genes and are ligand selected. Current topics in microbiology and immunology 252, 107-17. Das S., Nozawa M., Klein J. and Nei M., 2008. Evolutionary dynamics of the immunoglobulin heavy 70 | Chapter 3 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. chain variable region genes in vertebrates. Immunogenetics 60, 47-55. Feeney A. J., 1990. Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences. J Exp Med 172, 1377-90. Feeney A. J. and Riblet R., 1993. DST4: a new, and probably the last, functional DH gene in the BALB/c mouse. Immunogenetics 37, 217-21. Frank M. B., Besta R. M., Baverstock P. R. and Gutman G. A., 1987. The structure and evolution of immunoglobulin kappa chain constant region genes in the genus Rattus. Mol Immunol 24, 953-61. Frank M. B. and Gutman G. A., 1988. Two pseudogenes among three rat immunoglobulin lambda chain genes. Mol Immunol 25, 953-60. Fugmann S. D., Lee A. I., Shockett P. E., Villey I. J. and Schatz D. G., 2000. The RAG proteins and V(D)J recombination: complexes, ends, and transposition. Annu.Rev.Immunol. 18, 495-527. Gerdes T. and Wabl M., 2002. Physical map of the mouse lambda light chain and related loci. Immunogenetics 54, 62-5. Gerondakis S., Bernard O., Cory S. and Adams J. M., 1988. Immunoglobulin JH rearrangement in a T-cell line reflects fusion to the DH locus at a sequence lacking the nonamer recognition signal. Immunogenetics 28, 255-9. Gibbs R. A., Weinstock G. M., Metzker M. L., Muzny D. M., Sodergren E. J., Scherer S., Scott G., Steffen D., Worley K. C., Burch P. E., Okwuonu G., Hines S., Lewis L., DeRamo C., Delgado O., Dugan-Rocha S., Miner G., Morgan M., Hawes A., Gill R., Celera, Holt R. A., Adams M. D., Amanatides P. G., Baden-Tillson H., Barnstead M., Chin S., Evans C. A., Ferriera S., Fosler C., Glodek A., Gu Z., Jennings D., Kraft C. L., Nguyen T., Pfannkoch C. M., Sitter C., Sutton G. G., Venter J. C., Woodage T., Smith D., Lee H. M., Gustafson E., Cahill P., Kana A., Doucette-Stamm L., Weinstock K., Fechtel K., Weiss R. B., Dunn D. M., Green E. D., Blakesley R. W., Bouffard G. G., De Jong P. J., Osoegawa K., Zhu B., Marra M., Schein J., Bosdet I., Fjell C., Jones S., Krzywinski M., Mathewson C., Siddiqui A., Wye N., McPherson J., Zhao S., Fraser C. M., Shetty J., Shatsman S., Geer K., Chen Y., Abramzon S., Nierman W. C., Havlak P. H., Chen R., Durbin K. J., Egan A., Ren Y., Song X. Z., Li B., Liu Y., Qin X., Cawley S., Worley K. C., Cooney A. J., D’Souza L. M., Martin K., Wu J. Q., Gonzalez-Garay M. L., Jackson A. R., Kalafus K. J., McLeod M. P., Milosavljevic A., Virk D., Volkov A., Wheeler D. A., Zhang Z., Bailey J. A., Eichler E. E., et al., 2004. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493-521. Gilfillan S., Dierich A., Lemeur M., Benoist C. and Mathis D., 1993. Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 261, 1175-1178. Giudicelli V., Chaume D. and Lefranc M. P., 2005. IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes. Nucleic Acids Res 33, D256-61. Gough N. M. and Bernard O., 1981. Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity. Proc Natl Acad Sci U S A 78, 509-13. Hellman L., Pettersson U. and Bennich H., 1982. Characterization and molecular cloning of the mRNA for the heavy (epsilon) chain of rat immunoglobulin E. Proc Natl Acad Sci U S A 79, 1264-8. Hellman L., Steen M. L., Sundvall M. and Pettersson U., 1988. A rapidly evolving region in the immunoglobulin heavy chain loci of rat and mouse: postulated role of (dC-dA)n.(dG-dT)n sequences. Gene 68, 93-100. Hendricks J., Terpstra P., Dammers P. M., Somasundaram R., Visser A., Stoel M., Bos N. A. and Kroese F. G., 2010. Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat. Immunogenetics 62, 479-86. Johnston C. M., Wood A. L., Bolland D. J. and Corcoran A. E., 2006. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. Journal of Immunology 176, 4221-34. Kent W. J., 2002. BLAT--the BLAST-like alignment tool. Genome Res 12, 656-64. Komori T., Okada A., Stewart V. and Alt F. W., 1993. Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261, 1171-1175. Kurosawa Y. and Tonegawa S., 1982. Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments. J Exp Med 155, 201-18. Lang P. and Mocikat R., 1991. Immunoglobulin heavy-chain joining genes in the rat: comparison Organization of the immunoglobulin heavy-and light-chain loci in the rat | 71 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. with mouse and human. Gene 102, 261-4. Lawler A. M., Lin P. S. and Gearhart P. J., 1987. Adult B-cell repertoire is biased toward two heavychain variable-region genes that rearrange frequently in fetal pre-B cells. Proc Natl Acad Sci U S A 84, 2454-8. Lefranc M. P., 2001. Nomenclature of the human immunoglobulin heavy (IGH) genes. Exp Clin Immunogenet 18, 100-16. Lefranc M. P., Giudicelli V., Ginestoux C., Jabado-Michaloud J., Folch G., Bellahcene F., Wu Y., Gemrot E., Brochet X., Lane J., Regnier L., Ehrenmann F., Lefranc G. and Duroux P., 2009. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 37, D1006-12. Lefranc M. P. and Lefranc G., 1987. Human immunoglobulin heavy-chain multigene deletions in healthy individuals. FEBS Lett 213, 231-7. Marchalonis J. J., Schluter S. F., Bernstein R. M., Shen S. and Edmundson A. B., 1998. Phylogenetic emergence and molecular evolution of the immunoglobulin family. Adv Immunol 70, 417-506. Matsuda F., Ishii K., Bourvagnet P., Kuma K., Hayashida H., Miyata T. and Honjo T., 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J Exp Med 188, 2151-62. Meyer L. R., Zweig A. S., Hinrichs A. S., Karolchik D., Kuhn R. M., Wong M., Sloan C. A., Rosenbloom K. R., Roe G., Rhead B., Raney B. J., Pohl A., Malladi V. S., Li C. H., Lee B. T., Learned K., Kirkup V., Hsu F., Heitner S., Harte R. A., Haeussler M., Guruvadoo L., Goldman M., Giardine B. M., Fujita P. A., Dreszer T. R., Diekhans M., Cline M. S., Clawson H., Barber G. P., Haussler D. and Kent W. J., 2013. The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res 41, D64-9. Nei M., Gu X. and Sitnikova T., 1997. Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc Natl Acad Sci U S A 94, 7799-806. Nei M. and Kumar S., 2000. Molecular evolution and phylogenetics. Oxford University Press, New York. Ota T. and Nei M., 1994. Divergent evolution and evolution by the birth-and-death process in the immunoglobulin VH gene family. Mol Biol Evol 11, 469-82. Pallares N., Lefebvre S., Contet V., Matsuda F. and Lefranc M. P., 1999. The human immunoglobulin heavy variable genes. Exp Clin Immunogenet 16, 36-60. Parker K. E., Bugeon L., Cuturi M. C. and Soulillou J. P., 1994. Cloning of cDNA coding for the rat mu heavy chain constant region: differences between rat allotypes. Immunogenetics 39, 159. Pear W. S., Wahlstrom G., Szpirer J., Levan G., Klein G. and Sumegi J., 1986. Localization of the rat immunoglobulin heavy chain locus to chromosome 6. Immunogenetics 23, 393-5. Perlmann C., Sumegi J., Szpirer C., Levan G. and Klein G., 1985. The rat immunoglobulin kappa light chain locus is on chromosome 4. Immunogenetics 22, 97-100. Ramsden D. A., Baetz K. and Wu G. E., 1994. Conservation of sequence in recombination signal sequence spacers. Nucleic Acids Res 22, 1785-96. Ravetch J. V., Siebenlist U., Korsmeyer S., Waldmann T. and Leder P., 1981. Structure of the human immunoglobulin mu locus: characterization of embryonic and rearranged J and D genes. Cell 27, 583-91. Roth D. B., 1996. V(D)J Recombination. In Herzenberg L. A., Weir D. M. and Blackwell C. (Eds.), Blackwell Science, Oxford, pp. 16.1-16.20. Sakano H., Maki R., Kurosawa Y., Roeder W. and Tonegawa S., 1980. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286, 676-83. Sheppard H. W. and Gutman G. A., 1981a. Allelic forms of rat kappa chain genes: evidence for strong selection at the level of nucleotide sequence. Proc Natl Acad Sci U S A 78, 7064-8. Sheppard H. W. and Gutman G. A., 1981b. Complex allotypes of rat kappa chains are encoded by structural alleles. Nature 293, 669-71. Sheppard H. W. and Gutman G. A., 1982. Rat kappa-chain J-segment genes: two recent gene duplications separate rat and mouse. Cell 29, 121-7. Shimizu A., Takahashi N., Yaoita Y. and Honjo T., 1982. Organization of the constant-region gene family of the mouse immunoglobulin heavy chain. Cell 28, 499-506. 72 | Chapter 3 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. Sire J., Auffray C. and Jordan B. R., 1982. Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA. Gene 20, 377-86. Steen M. L., Hellman L. and Pettersson U., 1984. Rat immunoglobulin E heavy chain locus. J Mol Biol 177, 19-32. Steen M. L., Hellman L. and Pettersson U., 1987. The immunoglobulin lambda locus in rat consists of two C lambda genes and a single V lambda gene. Gene 55, 75-84. Sudmant P. H., Kitzman J. O., Antonacci F., Alkan C., Malig M., Tsalenko A., Sampas N., Bruhn L., Shendure J. and Eichler E. E., 2010. Diversity of human copy number variation and multicopy genes. Science 330, 641-6. Suzuki H. and Shiku H., 1992. Preferential usage of JH2 in D-J joinings with DQ52 is determined by the primary DNA sequence and is largely dependent on recombination signal sequences. Eur J Immunol 22, 2225-30. Tamura K., Peterson D., Peterson N., Stecher G., Nei M. and Kumar S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731-9. Tanaka T. and Nei M., 1989. Positive darwinian selection observed at the variable-region genes of immunoglobulins. Mol Biol Evol 6, 447-59. Wahlstrom G., Pear W. S., Steen M. L., Szpirer J., Levan G., Klein G. and Sumegi J., 1988. Localization of the rat immunoglobulin lambda light chain locus to chromosome 11. Immunogenetics 28, 182-3. Watson C. T., Steinberg K. M., Huddleston J., Warren R. L., Malig M., Schein J., Willsey A. J., Joy J. B., Scott J. K., Graves T. A., Wilson R. K., Holt R. A., Eichler E. E. and Breden F., 2013. Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. Am J Hum Genet 92, 530-46. Ye J., 2004. The immunoglobulin IGHD gene locus in C57BL/6 mice. Immunogenetics 56, 399-404. Organization of the immunoglobulin heavy-and light-chain loci in the rat | 73 Chapter 4 The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families Jacobus Hendricks Annie Visser Peter M. Dammers Johannes G.M. Burgerhof Nicolaas A. Bos Frans G.M. Kroese Manuscript in Preparation R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Abstract The splenic marginal zone is a unique anatomical B cell compartment populated with B lymphocytes with distinctive phenotypical and functional capabilities. Previous studies have shown that in rodents the vast majority of B cells are naïve cells with unmutated immunoglobulin genes, which is in marked contrast to humans, where nearly all splenic marginal zone B (MZ-B) cells are memory type B cells with mutated immunoglobulin genes. Here, we show that the proportion of mutated IgM+ MZ-B cells varies significantly between the various IGHV genes analysed, ranging from 27% mutated IGHV5 transcripts to 66% mutated IGHV4 transcripts. Also mutated IgM+ follicular B (FO-B) cells can be observed, but their proportion is much lower compared to MZ-B cells, Excitingly we observed mutated sequences from clonally related B cells with a MZ-B cell and FO-B cell phenotype indicating that mutated IgM+ MZ-B and FO-B cells have a common origin. Together we conclude that the splenic MZ of rats harbors a significant number of memory type IgM+ MZ-B cells with mutated immunoglobulin genes and that these memory MZ-B cells are probably generated as a result of an antigen driven immune response in germinal centers. 76 | Chapter 4 1. Introduction The marginal zone is a distinct anatomical white pulp compartment of the spleen, at the border with the red pulp (Steiniger et al., 2006). This unique compartment is characterized by a high blood flow and is separated, in rodents, from follicles and T cell zones by a layer containing a small blood sinus, the so-called marginal sinus. The splenic marginal zone harbors B cells, macrophages and dendritic cells, and in humans also CD4+ T cells. In rodents, B cells in the marginal zone (MZ-B cells) are pre-activated, non-recirculating cells, expressing high levels of IgM and low levels of IgD (Oliver et al., 1999). This phenotype distinguishes them from the majority population of naïve B cells, which express low levels of IgM and high levels of IgD. This latter population of B cells is recirculating B cells that home to lymphoid follicles of secondary lymphoid organs, and hence are called follicular B (FO-B) cells. The location of MZ-B cells, together with their functional properties make these cells perfectly suited for mounting rapid antibody responses to blood-borne antigens. In particular, these cells appear to be effectively involved in T cell independent humoral immune responses directed towards carbohydrate (Type 2) antigens (Guinamard et al., 2000). Another important role of MZ-B cells comprises the facilitation of antigen transport towards the follicles (Cinamon et al., 2008). MZ-B cells constitute a heterogeneous population of cells. In rats and mice, the vast majority (80-90%) of the MZ-B cells is naïve cells, with unmutated immunoglobulin heavy variable (IGHV) region genes (Dammers et al., 2000; Makowska et al., 1999). Their IGHV genes differs from FO-B cells and show more negatively charges amino acids (Carey et al., 2008; Schelonka et al., 2007). On average the heavy chain complementarity regions 3 (HCDR3) are 2-3 amino acids smaller, compared to their FO-B cell counterparts (Dammers et al., 2000). Autoantigens, rather than exogenous antigens might likely play a role in the ligand selection of these naïve MZ-B cells (Dammers and Kroese, 2005; Martin and Kearney, 2000). In addition to naïve B cells a small fraction of the MZ-B cells might be either unswitched or class switched memory B cells as shown by their appearance after immunization (Gatto et al., 2007; Gatto et al., 2004; Obukhanych and Nussenzweig, 2006; Pape et al., 2003; Phan et al., 2005; White and Meng, 2012). A hallmark of memory B cells is the presence of somatic mutations in the IGV genes (Tangye and Tarlinton, 2009). Indeed, approximately 10-20% of rodent sIgM+ MZ-B cells carry mutated IgM-encoding IGHV genes (Dammers et al., 2000; Makowska et al., 1999). Recently, we revealed in rats the presence of class switched B cells with a MZ-B cell phenotype, as defined by non-Ig markers expressing somatically mutated IGHV genes encoding for IgG (Hendricks et al., 2011). Interestingly, these class switched memory MZ-B cells exhibit significantly fewer mutations, compared to memory B cells with a FO-B cell phenotype (Hendricks et al., 2011). There is some evidence that these class switched memory MZ-B cells and FO-B cells have a common, germinal center (GC) origin. In marked contrast with rodents, the vast majority MZ-B cells in adult human spleens are unswitched The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 77 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 (IgM+) memory B cells as revealed by their expression of CD27 (Tangye et al., 1998; Zandvoort et al., 2001) and the notion that virtually all MZ-B cells expressed mutated IGHV genes (Dunn-Walters et al., 1995; Spencer et al., 1998). The discrepancy between the frequency of mutated MZ-B cells in rodents and humans is not clear. There might be developmental differences between the species. Weller and co-workers (Weill et al., 2009; Weller et al., 2004; Weller et al., 2008) speculated that presence of mutations in human MZ-B cells is an intrinsic property of these cells. According to their view human MZ-B cells are not generated during antigen-induced immune responses in GC and thus do not represent memory B cells. Rather, mutations are introduced in the IGHV genes of MZ-B cells in an antigen independent fashion, in order to diversify their naïve Ig repertoire, during the generation of these MZ-B cells (Weller et al., 2008). Another factor that might contribute to this discrepancy between (adult) human and rodent MZ-B cells is the analysis of IGHV genes themselves. Both in humans (DunnWalters et al., 1995; Tierens et al., 1999) and rodents (Dammers et al., 2000; Makowska et al., 1999) mutational analysis on IGHV genes derived from splenic MZ-B cells was carried out on only a very restricted set of IGHV genes of certain IGHV gene families. Whether these IGHV genes are representative for the mutation frequencies of the IGHV genes of the entire MZ-B cell pool is not known. MZ-B cells are ligand selected and for this reason, there might well be significant differences in the frequencies of mutated IGHV genes between the individual IGHV genes or IGHV gene families in rodents and humans. This issue was addressed in the work described here by looking at the mutation frequencies of individual IGHV genes that belong to several IGHV gene families that varied in size (viz. IGHV3, IGHV4, IGHV5) expressed in rat MZ-B or FO-B cells. 2. Materials and Methods 2.1. Animals Adult male BN/SsNOlaHsd rats were obtained from Harlan (Horst, The Netherlands) at 4.5 months of age. Animals were housed until the age of 9 months under clean conventional conditions at the Central Animal Facility of the University Medical Center Groningen. All experiments were approved by the Animal Ethics Committee of the University of Groningen. 2.2. Isolation and purification of B cell subsets by flow-cytometry Rat B-lymphocytes were isolated and purified from splenic tissue as described previously (Dammers et al., 1999). Briefly, spleen cell suspensions from 2 animals were labeled for flow-cytometry with the following mouse monoclonal antibodies: FITC conjugated anti-rat IgM (HIS40; eBioscience, San Diego, CA, USA), biotinylated anti-rat IgD (MaRD3; AbD Serotec, Oxford, UK), APC conjugated anti-rat CD90/Thy1.1 (HIS51; eBioscience) and PE conjugated anti-rat TCRαβ (R73; eBioscience); TCRγδ (V65; eBioscience), CD161a/NKRP1a (10/78; BD 78 | Chapter 4 Pharmingen). Biotinylated mAb were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5 (Ebioscience). The PE channel was used as a “dump” channel: only PE negative (i.e. Dump- and CD90- cells) were sorted. Herewith, we were able to exclude immature B cells (i.e. CD90+ B-cells: (Dammers et al., 1999), T cells and NK cells from our sorts. Cell analysis and cell sortings were performed with a MoFlo flow cytometer (Cytomation, Ft Collins, CO). Dead cell, plasma cell, monocyte/macrophage, and erythrocyte contamination was excluded from sorting by using forward and side scatter profiles. Sorted FO-B cells (CD90-IgDhighIgMlow) and MZ-B cells (CD90-IgMhighIgDlow) cells were collected in sterile FACS tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) containing 500 μl of newborn calf serum (PAA laboratories GmbH, Pasching, Austria). At least one million cells per B cell subset were sorted. B cell subsets were obtained with >95% purity. FlowJo software (Tree Star, San Carlos, CA) was used for flow cytometry data analysis. 2.3. Molecular cloning of IGHV-Cμ transcripts Total RNA was extracted from sorted cells using the Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA, USA) according to instructions of the manufacturer. Briefly, sorted cells were pelleted by 300xg centrifugation for 10 min at 4oC and then resuspended in a total volume of 350 μl lysis buffer containing β-mercaptoethanol (Stratagene). First strand cDNA was synthesized using an oligo-(dT) 12-18 primer (Invitrogen, Breda, The Netherlands) and SuperScriptTMII reverse transcriptase (200U/μl; Invitrogen) as described in the manufacturer’s protocol. Rearranged IGHV3-Cµ, IGHV4-Cµ IGHV5-Cµ transcripts were amplified in a 50 μl reaction mixture, containing 2 μl cDNA of either IGHV3-Cµ, IGHV4-Cµ and IGHV5Cµ family specific primer ,plus 0.6 pmol/µl universal Cµ constant region primer and 2.5U Taq DNA Polymerase (Invitrogen). The IGHV gene family specific primers were: IGHV3:5’TGAAACCCTCACAGTCACTC-3’, IGHV4:5’-GGTGCARCCTGGAAGATCCT-3’ and IGHV5’CTTAGTGCAGCCTGGAAGGT-3’ (Dammers et al., 2000). Individual IGHV gene family specific primers were used in separate RT-PCR reactions in combination with the constant region Cµ primer 5’-CAACACTGAAGTCATCCAGGG-3’. To assess the amount and quality of the cDNA, PCR was also performed for β-actin, using β-actin specific primers as described by Stoel et al. (Stoel et al., 2005). The PCR program for amplification of IGHV-Cμ transcripts and β-actin consisted of 35 cycles of 30 sec at 94°C (2 min in first cycle), 1 min at 58°C and 1 min at 72°C, respectively. This program was followed by an additional incubation period of 25 min at 72°C to allow extension of all IGHV-Cμ products. The quality and size of the PCR products was evaluated by agarose gel electrophoresis. 2.4. Cloning and sequencing PCR products were cloned into the pJET1/blunt vectors using the GeneJETTM PCR Cloning Kit (Fermentas Life Sciences). TOP10F E. coli competent cells (Invitrogen) were transformed The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 79 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 with plasmids containing the PCR product. Plasmid DNA was isolated from randomly picked colonies with the Nucleospin Plasmid QuickPure kit (Bioke, Leiden, The Netherlands). Size of the inserts was determined by digestion of a DNA sample of the plasmids with the appropriate restriction enzymes followed by agarose gel electrophoresis. Plasmids containing an insert of approximately 500 bp were sequenced in both directions by ServiceXS (Leiden, The Netherlands). Sequence processing was performed using EMBL/Genbank Data Libraries and Chromas software (Digital River GmbH, Cologne, Germany). IGHV-Cµ sequences displaying 100% identity and obtained from the same PCR amplification, might be derived from a single B cell and were therefore counted only once in our subsequent analysis. Mutational analysis was performed using IMGT/V QUEST database (www.imgt.org/IMGT_vquest/share/textes/). 2.5. Statistical analysis of IGHV gene analysis Statistical analysis of the data was performed using SPSS 16 software (SPSS Inc. Chicago, Ill. USA). Since Taq DNA polymerase errors might be responsible for 1–2 mutations per sequence, only sequences with more than 2 mutations were considered as truly mutated sequences (Dammers et al., 2000). The number of mutations was determined by counting the number of nucleotide mismatches in comparison with each IGHV gene sequence to its closest germline counterpart (Hendricks et al., 2010). Statistical analysis was performed by Fisher’s exact test to determine possible differences between groups with regard to frequency of mutated sequences. Nonparametric tests (Kruskal-Wallis and Mann-Whitney tests) were used to compare groups with respect to the number of mutations in mutated sequences. In all statistical tests a P-value < 0.05 was considered to be statistically significant different. 3. Results 3.1. Analysis of the IGHV genes in FO-B cells and MZ-B cells. Recently, we have constructed and annotated the complete genomic repertoire of the IGH locus of the BN rat (Hendricks et al., 2010). The completion of the IGH locus has allowed us to analyse individual IGHV genes among different IGHV gene families by FO-B and MZ-B cells. In Figure 1 the strategy for sorting of FO-B and MZ-B cells is illustrated. Viable lymphocytes were gated on the basis of forward-sideward scatter profiles, and non-T, non-NK cells (Dumpcells) were further analysed. FO-B cells and MZ-B cells were subsequently defined as CD90IgMlowIgDhigh and CD90-IgMhighIgDlow. The post sort purity for FO-B cells and MZ-B cells was >95%. Expressed IGHV3, IGHV4 and IGHV5 genes were amplified, cloned and sequenced. These IGHV gene families were chosen, because of their difference in size, with respectively 4, 2 and 26 potentially functional IGHV genes in the BN rat. 80 | Chapter 4 Figure 1. Four colour cytometry of FO-B cells and MZ-B cells. Single cell suspensions of spleen from rats were stained with FITC conjugated anti-rat IgM, biotinylated anti-rat IgD, APC conjugated anti-rat CD90/ Thy1.1 and PE conjugated anti-rat TCRαβ, TCRγδ, CD161a/NKRa. Biotinylated monoclonal antibodies were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5. Viable lympocytes were gated by forward scatter and side scatter profiles. Acquisition gates were set to exclude PE positive cells (T cells and NK cells) and CD90 positive (immature) B cells. Mature FO-B cells, defined as CD90IgDhighIgMlow and MZ-B defined as CD90-IgMhighIgDlow were sorted. Post sort reanalysis showed that the purity of FO-B cells and MZ-B cells was >95%. We obtained 19 and 16 complete IGHV3-Cµ transcripts from FO-B cells and MZ-B cells, respectively. Three of the four IGHV3 germline genes were expressed as productive genes in both B cell subsets i.e. IGHV3S1, IGHV3S3 and IGHV3S5 (Table 1). From the IGHV4 gene family we were able to successfully amplify only one of the two potentially functional genes (viz. IGHV4S2). In total we obtained 12 IGHV4-Cµ transcripts from FO-B cells and 59 IGHV4Cµ transcripts from MZ-B cells (Table 1). From the second largest IGHV gene family in the BN rat, the IGHV5 gene family (also called the PC7183 family), 16 different, out of 26 potentially functional IGHV5 genes were found among 40 and 61 IGHV5-Cµ transcripts (see Table 1) that were amplified from FO-B cells and MZ-B cells, respectively. 3.2. MZ-B cells express more mutated IGHV-Cµ transcripts then FO-B cells We subsequently analysed the obtained IGHV-Cµ transcripts that were amplified from both B cell subpopulations. The number of mutations within each rearranged IGHV gene was assessed on the bases of the nucleotide identity to the closest corresponding germline of the IGHV gene counterparts. Sequences with only 1 or 2 mutations were considered to be germline because we cannot exclude the possibility that these differences compared to germline IGHV genes were due to PCR artifacts (Dammers et al., 2000). We first analysed the proportion of mutated among the combined IGHV3, IGHV4 and IGHV5 sequences. As we show in Figure 2 18% of FO-B cells and 45% of MZ-B cells expressed mutated IGHV-Cµ transcripts. This percentage of mutated sequences is significantly higher within the MZ-B cell subset compared to the percentage of mutated sequences present within the FO-B cell subset (Fisher’s exact test: P < 0,001). The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 81 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 3.3. The difference in frequency of mutated sequences between MZ-B cells and FO-B cells is largely due to the IGHV3 gene family. We subsequently analysed whether this difference in percentage of mutated sequences between FO-B cells and MZ-B cells was present in all three IGHV gene families tested. There was no statistical difference in percentage mutated sequences between the two B cell subsets among sequences of the IGHV4 and IGHV5 gene families, albeit that there was a strong trend that a higher percentage of MZ-B cells expressing mutated IGHV4 genes were present compared to the percentage of mutated IGHV4 genes of FO-B cells (Fisher’s exact test: P = 0,051) (Figure 2). In contrast, within the IGHV3 gene family, 31% of the sequences derived from MZ-B cells were mutated, whereas none of IGHV3 sequences derived from FO-B cells were mutated (Fisher’s exact test: P = 0,016) (Figure 2). Thus, the higher frequency of MZ-B cells expressing mutated IGHV sequences is largely due to the contribution of the IGHV3 gene family. 3.4. The IGHV4 gene family contains the highest percentage of mutated IGHV-Cµ transcripts, both among MZ-B cells and among FO-B cells We analysed whether there was a difference in the percentage of mutated IGHV-Cµ transcripts between the three IGHV gene families (IGHV3, IGHV4, and IGHV5) in the two B cell subsets. As we show in Figure 2 within both MZ-B cells and FO-B cells, the IGHV4 gene family contains a significantly higher proportion of mutated IGHV-Cµ transcripts, compared to the two other IGHV gene families (IGHV3 and IGHV5) (Fisher’s exact test: P = 0,023 and P < 0,001, respectively). Of the IGHV4 sequences two-third of the MZ-B cell derived sequences and one-third of the FO-B cell sequences were mutated. Thus, based upon these findings we conclude that there is a significant difference in the percentage of mutated sequences between the various IGHV gene families, for both MZ-B cells and FO-B cells. 82 | Chapter 4 100 Percentage of mutated sequences 90 80 70 60 FO-B 50 MZ-B 40 30 20 10 0 IGHV3 IGHV4 IGHV5 TOTAL IGHV gene family Figure 2. Percentage of mutated IgM+ FO-B cells and MZ-B cells within different IGHV gene families. Analysis of the proportion of mutated (>2 mutations compared to the closest germline gene) shows that MZ-B cells express more mutated sequences than FO-B cells, when all sequences from the three IGHV gene families are combined (i.e. “total”) (Fisher’s exact test: P < 0.001). This difference between MZ-B cells and FO-B cells was largely due to a significant difference in percentage mutated sequences within the IGHV3 family (Fisher’s exact test: P = 0.016). There are relatively more mutated sequence found in the IGHV4 gene family compared to IGHV3 and IGHV5 both for the MZ-B cells (Fisher’s exact test: P < 0.001) and FO-B cells (Fisher’s exact test: P = 0.023). The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 83 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Table 1. Sequence analysis of IGHV3, 4 and 5-Cµ transcripts expressed by FO-B cells and MZ-B cells from adult BN rat spleen Clonea IGHV IGHD IGHJ member member member Mutationsb H-CDR3c Nf Amino acids d Sequences of IGHV3 gene family from FO-B cells A2RFV3-A4 IGHV3S1 IGHD1-7 IGHJ1 0 12 ARTTRVYWYFDF A2RFV3-B2 IGHV3S1 IGHD4-4 IGHJ2 0 9 ARFVGYFDY A2RFV3-B3 IGHV3S1 IGHD4-1 IGHJ2 1 11 AYSPGGYRFDY A2RFV3-C2 IGHV3S1 IGHD1-6 IGHJ2 0 12 ARYGATEGIVDY A2RFV3-C4 IGHV3S1 IGHD4-1 IGHJ3 0 14 VNMSNNSGYDWFAY A2RFV3-D1 IGHV3S1 IGHD1-7 IGHJ1 0 16 ARFYDGSYYYDWYFDF A2RFV3-D3 IGHV3S1 IGHD1-7 IGHJ3 0 17 ARYYGIYYYSSYDWFAY A2RFV3-E1 IGHV3S1 IGHD1-8 IGHJ3 0 17 ARAGGRDSYAHVGWFAY A2RFV3-E2 IGHV3S1 IGHD1-3 IGHJ2 1 11 ARLYSIAAPYY A2RFV3-E3 IGHV3S1 IGHD4-1 IGHJ3 0 8 ARNPGFAY A2RFV3-G3 IGHV3S1 IGHD3-3 IGHJ2 2 11 ARSGQKSLFDY A2RFV3-H1 IGHV3S1 IGHD1-7 IGHJ2 0 16 ARYGDYYDGSYYAFDY A2RFV3-H3 IGHV3S1 IGHD3-1 IGHJ2 0 10 ASPYPGQRWY A2RFV3-A3 IGHV3S3 IGHD1-3 IGHJ2 1 12 ARSELQWYYFDY A2RFV3-B4 IGHV3S3 IGHD5-1 IGHJ2 0 16 ARRPITGSGGGYYFDY A2RFV3-F2 IGHV3S3 IGHD5-1 IGHJ4 0 12 ASGNWDSYVMDA A2RFV3-G1 IGHV3S3 IGHD1-7 IGHJ2 0 15 ARSSYYYDGSYSLDY A2RFV3-A1 IGHV3S5 IGHD1-5 IGHJ3 0 10 AGNNLDWFAY A2RFV3-F3 IGHV3S5 IGHD1-8 IGHJ4 1 16 ASPLDGYYPYYYVMDA 0 9 ARRTVSFDY e Sequences of IGHV3 gene family from MZ-B cells A2MZV3-A5 IGHV3S1 IGHD1-1 IGHJ2 A2MZV3-A6 IGHV3S1 IGHD1-4 IGHJ2 8 12 ARRDPGITLFDY A2MZV3-B7g IGHV3S1 IGHD1-3 IGHJ2 0 13 ARGQQLSEYYFDYC#1 A2MZV3-F5g IGHV3S1 IGHD1-3 IGHJ2 1 13 ARGQQLSEYYFDYC#1 A2MZV3-C5 IGHV3S1 IGHD1-7 IGHJ1 0 14 ARYDGSYYYWYFDF h IGHV3S1 IGHD1-4 IGHJ2 1 12 ARSGGYNYYFDYC#2 h IGHV3S1 IGHD1-4 IGHJ2 0 12 ARSGGYNYYFDYC#2 A2MZV3-D4 IGHV3S1 IGHD1-6 IGHJ3 5 10 ARYSERGFAY A2MZV3-E4 IGHV3S1 IGHD1-5 IGHJ2 0 12 ARGGIYNTYFDY A2MZV3-E5 IGHV3S1 IGHD4-1 IGHJ2 7 13 ARKGDSNSGLFDY A2MZV3-F6 IGHV3S1 IGHD1-1 IGHJ2 0 15 ARGGVYYGLLSSFDY A2MZV3-G6 IGHV3S1 IGHD1-7 IGHJ2 0 13 ARSTTVVHYYFDY A2MZV3-G7 IGHV3S1 IGHD1-1 IGHJ2 4 12 ARSGYTTDYPDY A2MZV3-H4 IGHV3S1 IGHD3-2 IGHJ2 0 9 ARSTDYFDY A2MZV3-C7 A2MZV3-E7 84 | Chapter 4 Clonea IGHV IGHD IGHJ member member member A2MZV3-E6 IGHV3S3 IGHD3-1 IGHJ2 A2MZV3-B5 IGHV3S5 IGHD1-5 IGHJ4 Mutationsb H-CDR3c Nf Amino acids 0 10 ARSGSGDFDY 4 12 ARRTTSDYVMDA d Sequences of IGHV4 gene family from FO-B cells A2RFV4-10 IGHV4S2 IGHD4-1 IGHJ2 6 10 IRERNSGAEY A2RFV4-2 IGHV4S2 IGHD4-1 IGHJ2 9 9 VREAFGVREC#3 A2RFV4-2.25 IGHV4S2 IGHD1-7 IGHJ4 0 14 ARVYDGSYYYVMDA A2RFV4-2.27 IGHV4S2 IGHD1-2 IGHJ1 0 10 GGSLYWYFDF A2RFV4-5 IGHV4S2 No IGHD IGHJ3 0 5 ASRAY A2RFV4-6 IGHV4S2 IGHD1-6 IGHJ4 4 11 TRAGTVLQMDA i A3RFV4-12 IGHV4S2 IGHD1-8 IGHJ2 0 12 ARASYYDGYGDY A3RFV4-3j IGHV4S2 IGHD4-1 IGHJ2 11 9 VREAFGVDYC#4 A3RFV4-3.2 IGHV4S2 IGHD1-4 IGHJ3 0 13 ARADGYNFNWFAY A3RFV4-3.4 IGHV4S2 IGHD3-3 IGHJ1 0 12 ARLWRRYWYFDF A3RFV4-43 IGHV4S2 IGHD1-5 IGHJ2 1 12 ARWNNYDYYFDY A3RFV4-9 IGHV4S2 IGHD1-5 IGHJ2 0 11 AREDYNNIGDH e Sequences of IGHV4 gene family from MZ-B cells A2MZV4-1i IGHV4S2 IGHD4-1 IGHJ2 9 9 VREAFGVREC#3 A2MZV4-10 IGHV4S2 IGHD1-6 IGHJ2 1 9 AREVGYFDY A2MZV4-11 IGHV4S2 IGHD3-4 IGHJ2 1 9 TRARKSVDY A2MZV4-12 IGHV4S2 IGHD1-1 IGHJ3 2 6 EGGIIG A2MZV4-13 IGHV4S2 IGHD1-6 IGHJ4 11 11 ARASGQRVLDA A2MZV4-14k IGHV4S2 IGHD4-1 IGHJ4 5 14 TRREFGPHYYVMDAC#5 A2MZV4-3k IGHV4S2 IGHD4-1 IGHJ4 7 14 TRREFGPHYYVMDAC#5 A2MZV4-2.1 IGHV4S2 IGHD1-1 IGHJ3 9 12 ARGLYYGFGFAY A2MZV4-2.10 IGHV4S2 IGHD4-1 IGHJ4 0 13 ARARNSDYYVMDA A2MZV4-2.11 IGHV4S2 IGHD4-1 IGHJ2 0 10 ASHERYTSDY l A2MZV4-2.13 IGHV4S2 IGHD4-2 IGHJ2 11 9 VREHFGVDFC#6 l IGHV4S2 IGHD4-2 IGHJ2 13 9 VREHFGVDFC#6 A2MZV4-2.14 IGHV4S2 IGHD4-1 IGHJ2 8 9 AREAFGVRE A2MZV4-2.18 IGHV4S2 IGHD1-6 IGHJ2 10 9 AREEAGIDY A2MZV4-2.20 IGHV4S2 IGHD1-6 IGHJ4 5 9 VREALGVNA A2MZV4-2.21 IGHV4S2 IGHD2-2 IGHJ2 9 9 VREAYGVDY A2MZV4-2.3 IGHV4S2 IGHD1-1 IGHJ2 1 25 AREGVYYYSSYRDVYYGLLPGYFDY A2MZV4-2.4 IGHV4S2 IGHD1-7 IGHJ2 8 15 ARGYYYDGSYYHFDY A2MZV4-2.7 IGHV4S2 IGHD1-5 IGHJ1 2 16 AREALITTTSYWYFDF A2MZV4-2.8 IGHV4S2 IGHD1-6 IGHJ4 16 9 VREALGVDA A2MZV4-2.17 The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 85 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Clonea IGHV IGHD IGHJ member member member IGHV4S2 IGHD1-1 IGHJ2 A2MZV4-7m IGHV4S2 IGHD1-1 A2MZV4-5 IGHV4S2 A2MZV4-8 A2MZV4-9 Mutationsb H-CDR3c Nf Amino acids 5 13 ARARGMSTTDYLYC#7 IGHJ2 4 13 ARARGMSTTDYLYC#7 IGHD4-2 IGHJ2 6 9 VREELGVDY IGHV4S2 IGHD5-1 IGHJ1 10 13 GRLSWELYWYFDF IGHV4S2 IGHD3-2 IGHJ2 3 9 VRAHSSAGD A3MZV4-1 IGHV4S2 IGHD1-4 IGHJ2 2 15 ARGTSYGSSSDYFDY A3MZV4-10 IGHV4S2 IGHD1-3 IGHJ2 0 15 ARALDYYSSYIYLDY A2MZV4-2.9 m A3MZV4-35 IGHV4S2 IGHD1-6 IGHJ2 13 12 ARGDYYRGDFDY A3MZV4-11n IGHV4S2 IGHD1-6 IGHJ2 11 9 VREHLGVDYC#8 A3MZV4-3.1n IGHV4S2 IGHD1-6 IGHJ2 11 9 VREHLGVDYC#8 A3MZV4-9n IGHV4S2 IGHD1-6 IGHJ2 10 9 VREHLGVDYC#8 A3MZV4-12 IGHV4S2 IGHD1-6 IGHJ2 0 10 ARVVTEGIDY A3MZV4-36 IGHV4S2 IGHD1-3 IGHJ2 0 11 AREDYSGDFDY A3MZV4-13 IGHV4S2 IGHD1-6 IGHJ4 0 12 FNGGIAAGVMDA A3MZV4-14 IGHV4S2 IGHD1-8 IGHJ2 5 10 SGGLGWIFDY A3MZV4-15 IGHV4S2 IGHD1-1 IGHJ4 0 17 ARVLFMYTTDYQGVMDA A3MZV4-16 IGHV4S2 IGHD4-1 IGHJ2 19 9 VREDFGVDY A3MZV4-17 IGHV4S2 IGHD5-1 IGHJ2 0 11 ARARETGNFDY A3MZV4-18 IGHV4S2 IGHD1-2 IGHJ2 9 15 TRGPSYGSDSDFFDY A3MZV4-19 IGHV4S2 IGHD1-4 IGHJ2 8 15 ARGTSYGSNSDYFDY A3MZV4-20 IGHV4S2 IGHD4-1 IGHJ2 8 9 AREAFGVDY A3MZV4-20B IGHV4S2 IGHD1-4 IGHJ2 14 10 AKSGPGIIEYC#9 A3MZV4-7o IGHV4S2 IGHD1-4 IGHJ2 13 10 AKSGPGIIEYC#9 A3MZV4-22 IGHV4S2 IGHD4-1 IGHJ2 9 9 IREAFGVDY A3MZV4-37 IGHV4S2 IGHD3-2 IGHJ1 15 14 AGLRSGAPYWYLDF A3MZV4-23 IGHV4S2 IGHD1-6 IGHJ3 9 12 ARELSTGEWFAY A3MZV4-24 IGHV4S2 IGHD1-3 IGHJ2 1 9 GREIPPVDY A3MZV4-29 IGHV4S2 IGHD1-7 IGHJ2 2 14 ARSLMVVISHYFDY A3MZV4-3 IGHV4S2 IGHD1-6 IGHJ4 0 10 ARRRSDVMDA A3MZV4-3.12 IGHV4S2 IGHD1-6 IGHJ4 0 14 ARVGDSSYYYVMDA o A3MZV4-3.14 IGHV4S2 IGHD1-6 IGHJ3 1 11 VRERSTEGFAY A3MZV4-3.2j IGHV4S2 IGHD4-1 IGHJ2 14 9 VREAFGVDYC#4 A3MZV4-3.5 IGHV4S2 IGHD4-1 IGHJ2 12 9 VREDLGVDY A3MZV4-30 IGHV4S2 IGHD2-2 IGHJ2 14 9 AREIPPVDY A3MZV4-31 IGHV4S2 IGHD1-4 IGHJ4 11 11 ARAVISRVLDA A3MZV4-32 IGHV4S2 IGHD4-1 IGHJ2 6 9 VREEFGVDY 86 | Chapter 4 Clonea IGHV IGHD IGHJ member member member A3MZV4-5N IGHV4S2 IGHD1-6 IGHJ2 A3MZV4-5A IGHV4S2 IGHD1-2 IGHJ2 A3MZV4-9 IGHV4S2 IGHD1-5 IGHJ2 Mutationsb H-CDR3c Nf Amino acids 11 9 VREQRGVDYC15 8 15 TRGPSYGSDSDYFDY 0 11 ARADNNSGFDY Sequences of IGHV5 genes from FO-Bd cells A2RFV5-39 IGHV5S16 IGHD1-1 IGHJ3 0 12 ARPNYYSGPLAY A3RFV5-11 IGHV5S13 IGHD1-2 IGHJ2 0 9 ARRAMGFDY A2RFV5-42 IGHV5-1 IGHD1-6 IGHJ2 17 9 TKGVGGPDYC#10 A2RFV5-46 IGHV5S10 IGHD5-1 IGHJ2 0 9 ATHLGYFDY A2RFV5-38 IGHV5S14 IGHD1-1 IGHJ2 1 12 VRLCGERDYFDY A2RFV5-45 IGHV5S14 IGHD1-6 IGHJ1 1 16 ARHVPLHYGGHGYFDF A3RFV5-14N IGHV5S14 IGHD1-2 IGHJ2 0 8 ARRDDFDY A3RFV5-48 IGHV5S14 IGHD1-6 IGHJ1 0 17 ARLPAYYGGYSELPFAY A3RFV5-5 IGHV5S14 IGHD1-1 IGHJ2 0 18 ARHLMYTTDYYYPGAFDY A2RFV5-17 IGHV5S23 IGHD1-4 IGHJ3 4 14 ARGDYPGITGWFAY A2RFV5-19 IGHV5S23 IGHD1-4 IGHJ2 0 6 ARPYSV A3RFV5-12 IGHV5S23 IGHD1-8 IGHJ1 1 20 ARPPRWDYDGYYHVGWYFDF A3RFV5-15 IGHV5S23 IGHD1-1 IGHJ4 6 17 ARSLMYTTDYYYGVMDA A3RFV5-8 IGHV5S23 IGHD1-7 IGHJ2 4 12 ARGDDGSYYFDY A3RFV5-3 IGHV5S27 IGHD1-4 IGHJ2 4 12 ARRPPGYNPFDY A3RFV5-41 IGHV5S27 IGHD1-8 IGHJ2 0 7 ARHYPDY A3RFV5-45 IGHV5S27 IGHD1-7 IGHJ3 0 20 ARHGADGAMMVVITNGWFAY A3RFV5-47 IGHV5S29 IGHD2-3 IGHJ2 2 10 TTDRLSTFDY A2RFV5-20 IGHV5S30 IGHD1-1 IGHJ3 1 16 ATEVYTTDYYEGWFAY A2RFV5-21 IGHV5S30 IGHD1-5 IGHJ2 1 11 ARPHYNNYFDY A2RFV5-22 IGHV5S30 IGHD1-1 IGHJ3 4 17 ARHMYTTDYYHGDWFAY A2RFV5-23 IGHV5S30 IGHD1-4 IGHJ3 2 14 ATRPLPGYNYGFAY A2RFV5-35 IGHV5S30 IGHD1-8 IGHJ3 11 9 ARQDQEFAY A2RFV5-37 IGHV5S30 IGHD1-7 IGHJ2 4 13 ARLDYYDGSYYDY A2RFV5-42 IGHV5S30 IGHD4-1 IGHJ2 0 9 ATVAGYFDY A2RFV5-8 IGHV5S30 IGHD1-3 IGHJ2 0 8 ATLLYSGH A3RFV5-13N IGHV5S30 IGHD1-1 IGHJ3 0 16 ATDSPTTDYYSNWFAY A3RFV5-2 IGHV5S30 IGHD1-6 IGHJ3 0 17 ATDTDYGGYSELGGFAY A3RFV5-4 IGHV5S43 IGHD4-2 IGHJ3 1 13 TRDRGYSSHWFAY A3RFV5-46 IGHV5S43 IGHD1-3 IGHJ4 0 14 TREPGDYSSYVMDA A3RFV5-13 IGHV5S43 IGHD1-7 IGHJ2 2 13 TRVGHYYSSYFDY p The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 87 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Clonea IGHV IGHD IGHJ member member member A2RFV5-18 IGHV5S45 IGHD1-1 IGHJ2 A2RFV5-88 IGHV5S45 IGHD1-7 IGHJ1 A2RFV5-41 IGHV5S45 IGHD1-2 A3RFV5-43 IGHV5S45 A3RFV5-42 A2RFV5-47 Mutationsb H-CDR3c Nf Amino acids 2 12 ARRYTTDYWFDY 0 21 A R G L G L I T M M V V I T TDWYFDF IGHJ2 0 10 ARPPYGAFDY IGHD1-1 IGHJ3 4 24 T T G AY S S YAV M Y T TDYYYAGWFAY IGHV5S45 IGHD2-2 IGHJ1 2 12 ARRDTLYWYFDF IGHV5S57 IGHD3-3 IGHJ1 1 15 TRASSSYVSDWYFDF A3RFV5-44 IGHV5S57 IGHD1-2 IGHJ2 0 11 TRTRVSYYFDY A2RFV5-43 IGHV5S65 IGHD1-5 IGHJ2 1 13 AKDQGNNYGYFDY A3RFV5-2 IGHV5S74 IGHD5-1 IGHJ2 2 11 ARGHGDYYFDY Sequences of IGHV5 genes from MZ-Be cells A2MZV5-37p IGHV5-1 IGHD1-6 IGHJ2 13 9 TKGVGGPDYC#10 A2MZV5-39 IGHV5S10 IGHD4-1 IGHJ2 0 11 ATHPGEYYFDY A3MZV5-4 IGHV5-1 IGHD1-6 IGHJ2 16 9 AKGVGGPDY A2MZV5-2.5 IGHV5S13 IGHD1-1 IGHJ2 0 14 ARFGLITVAVHFDY A2MZV5-20 IGHV5S13 IGHD1-6 IGHJ1 1 18 ARTTGLTTEGIGYWYFDF A2MZV5-38 IGHV5S13 IGHD1-1 IGHJ3 4 7 GYYGFAY A3MZV5-16 IGHV5S13 IGHD1-6 IGHJ3 2 14 ARHETTVVTGWFAY A3MZV5-38 IGHV5S13 IGHD1-1 IGHJ2 1 11 ASIITTGYFDY A3MZV5-66 IGHV5S13 IGHD1-3 IGHJ1 1 12 ASQSSYNWYFDF A2MZV5-11 IGHV5S14 IGHD1-1 IGHJ2 0 9 ARRLLQWDY A2MZV5-33 IGHV5S14 IGHD1-4 IGHJ2 1 17 ARGGINNIGTTRGVMDA A3MZV5-3.8 IGHV5S14 IGHD1-7 IGHJ2 0 12 ARYYYDGPWGDY A3MZV5-5 IGHV5S14 IGHD1-7 IGHJ2 2 15 ARTGFYYYSGDYFDY A3MZV5-8 IGHV5S14 IGHD1-7 IGHJ2 1 13 ARHYYDGSYYFDY A2MZV5-14 IGHV5S16 IGHD5-1 IGHJ2 4 7 TTDLNNY A2MZV5-19 IGHV5S16 IGHD1-8 IGHJ1 0 11 ATCSPYWYFDF A2MZV5-22 IGHV5S16 IGHD1-7 IGHJ4 4 11 ATDEGGGVMDA A3MZV5-13 IGHV5S16 IGHD1-6 IGHJ3 4 12 TTLYGGPPWFAY A3MZV5-8 IGHV5S16 IGHD4-1 IGHJ4 0 7 ARRGMDA A2MZV5-21 IGHV5S23 IGHD1-2 IGHJ1 5 15 ARQSTYYEDGWYFDF A3MZV5-3.3 IGHV5S23 IGHD1-2 IGHJ3 4 14 ATEGTMGMSDWFAY A2MZV5-23 IGHV5S27 IGHD1-4 IGHJ3 0 13 ARPYGYNYRWFAY A2MZV5-24 IGHV5S27 IGHD1-1 IGHJ3 1 19 ARHPYSSYIYGYTTDWFAY A3MZV5-61 IGHV5S29 IGHD1-6 IGHJ3 0 12 TTDRGNYGWFAY A3MZV5-65 IGHV5S29 IGHD1-7 IGHJ2 1 13 TSPLTTVVPYFDY 88 | Chapter 4 Clonea IGHV IGHD IGHJ member member member A2MZV5-16 IGHV5S30 IGHD1-3 IGHJ2 A2MZV5-18 IGHV5S30 IGHD1-5 IGHJ2 A2MZV5-2.2 IGHV5S30 IGHD1-3 A3MZV5.57 IGHV5S30 IGHD1-3 A3MZV5-10 IGHV5S30 A3MZV5-14 IGHV5S30 A3MZV5-17 A3MZV5-2N Mutationsb H-CDR3c Nf Amino acids 6 10 ATDQLYLRAF 1 13 ARHDNNYVAYFDY IGHJ2 2 17 ATDQYYSSYTLAGYFDY IGHJ3 1 15 ATDRAYRSYIPTFAY IGHD1-6 IGHJ1 0 12 ATEIDSDWYFDF IGHD1-8 IGHJ2 0 6 ATLSYY IGHV5S30 IGHD1-7 IGHJ2 5 15 AKMWGGSYYYVPFDY IGHV5S30 IGHD4-1 IGHJ2 0 6 ATDSSG A3MZV5-24 IGHV5S30 IGHD5-1 IGHJ3 0 11 ATDDQLDWFAY A3MZV5-25 IGHV5S30 IGHD4-1 IGHJ3 11 11 AHNAGDVWFPY A3MZV5-26 IGHV5S30 IGHD1-3 IGHJ2 0 13 ATGVHYSSYIFDY A3MZV5-3.11 IGHV5S30 IGHD1-2 IGHJ2 2 10 ATQLGGSFDY A3MZV5-3.13 IGHV5S30 IGHD1-6 IGHJ4 0 12 ATDQTEGPPMDA A3MZV5-3.4 IGHV5S30 IGHD1-8 IGHJ2 1 12 ATGDYYDGYPDY A3MZV5-3.6 IGHV5S30 IGHD1-7 IGHJ2 0 13 ATDRSDDGGFFDY A3MZV5-3.7 IGHV5S30 IGHD1-1 IGHJ2 0 12 ATDHVYYGLLGA A3MZV5-33 IGHV5S30 IGHD1-1 IGHJ3 0 14 ATAGDTTDYSRFAY A3MZV5-39 IGHV5S30 IGHD1-6 IGHJ2 0 11 ARGINYGGYAH A3MZV5-6 IGHV5S30 IGHD1-1 IGHJ3 2 14 ATEVYYGLSDWFAY A3MZV5-63 IGHV5S30 IGHD1-4 IGHJ2 1 11 ATDEAGDTGDY A3MZV5-8 IGHV5S30 IGHD1-5 IGHJ2 0 12 ATAFITTTGFDY A2MZV5-4 IGHV5S30 IGHD1-7 IGHJ3 0 13 ATDGGYAPRWFAY A2MZV5-25 IGHV5S45 IGHD1-4 IGHJ2 2 10 TTGDMGITPY A2MZV5-35 IGHV5S45 IGHD1-2 IGHJ4 2 11 ARQGDYGPMDA A2MZV5-9 IGHV5S45 IGHD4-2 IGHJ1 1 13 ARRGGSAYWYFDF A3MZV5-11 IGHV5S32 IGHD1-1 IGHJ4 4 17 ATDGAFTTNYFYDVMAA A3MZV5-12 IGHV5S32 IGHD1-6 IGHJ2 1 12 ARQGYGGYPFDY A2MZV5-36 IGHV5S36 IGHD1-6 IGHJ2 7 11 TTEVLQWVFDY A2MZV5-40 IGHV5S36 IGHD1-3 IGHJ3 1 12 TTGTIAANWFAY A3MZV5-21 IGHV5S36 IGHD1-2 IGHJ2 6 7 ATGLGDY A2MZV5-12 IGHV5S43 IGHD1-4 IGHJ2 0 13 TREGPYGYNYFDY A3MZV5-3.15 IGHV5S43 IGHD1-1 IGHJ4 14 10 TIYSNYVMDA A3MZV5-6 IGHV5S43 IGHD1-6 IGHJ3 0 9 TRGTTEAAY A3MZV5-10 IGHV5S65 IGHD1-2 IGHJ2 0 9 AKESTMGMG A3MZV5-36 IGHV5S65 IGHD1-1 IGHJ2 19 7 AINKYNY A3MZV5-7 IGHV5S65 IGHD1-2 IGHJ2 6 13 AKDSYGGYRYFDY The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 89 a R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Cµ (IgM) transcripts from FO-B cells and MZ-B cells Mutations, nucleotide differences between IMGT germline gene and rearranged Cµ transcript c H-CDR3, heavy chain complementarity determining region 3 d FO-B cells, recirculating follicular B cells e MZ-B cells, marginal zone B cells f Lenght of H-CDR3 in amino acids g The sequence A2MZV3-B7 and A2MZV3-F5 are from clonally related B cells and designated as clone set C#1 h The sequence A2MZV3-C7 and A2MZV3-E7 are from clonally related B cells and designated as clone set C#2 i The sequences A2RFV4-2 and A2MZV4-1 are from clonally related B cells and designated as clone set C#3 j The sequences A3RFV4-3 and A3MZV4-3.2 are from clonally related B cells and designated as clone set C#4 k The sequences A2MZV4-14 and A2MZV4-3 are from clonally related B cells and designated as clone set C#5 l The sequences A2MZV4-2.13 and A2MZV4-2.17 are from clonally related B cells and designated as clone set C#6 m The sequences A2MZV4-2.9 and A2MZV4-7 are from clonally related B cells and designated as clone set C#7 n The sequences A3MZV4-11, A3MZV4-3.1 and A3MZV4-9 are from clonally related B cells and designated as clone set C#8 o The sequences A3MZV4-20B and A3MZV4-7 are from clonally related B cells and designated as clone set C#9 p The sequences A2RFV5-42 and A2MZV5-37 are from clonally related B cells and designated as clone set C#10 b 3.5. Mutation frequency of MZ-B cells with mutated IGHV-Cµ transcripts is higher compared to FO-B cells. We next compared the number of mutations among the mutated IGHV-Cµ transcripts (i.e. > 2 mutations per transcript) IGHV genes in FO-B cells and MZ-B cells. When taking all mutated sequences from the three families together, MZ-B cells have a significantly (Mann-Whitney, P = 0.046) higher number of mutations then FO-B cells (Figure 3). In MZ-B cells the number of mutations is 8.8±4.0 (median 8) and in FO-B cells 6.8±3.9 (median 4). Further analysis reveals that the mutation frequency of mutated IGHV-Cµ sequences from MZ-B cells is significantly higher among IGHV4 sequences than among IGHV3 or IGHV5 sequences (KruskalWallis, P = 0.011). These results indicate that the number of mutations in mutated MZ-B cells is higher in comparison to FO-B cells, which appears largely to be due to the higher number of mutations among IGHV4 sequences (Figure 3). 3.5. Clones of B cells are found within the MZ-B cell subset and some of these clones have members that are also present within the FO-B cell subset. H-CDR3 regions can be used to assess for clonal relationships between B cells because the H-CDR3 region is virtually unique for each different IGHV rearrangement. A total of 10 independent clone sets (designated as C#1-C#10) were found among the two B cell subsets. 90 | Chapter 4 Seven clone sets (2-3 members per clone) have members exclusively found within the MZ-B cell subset and the remaining three clone sets have shared members between the MZ-B cell subset and the FO-B cell subset (see Table 1). The sequences that belong to clonally related cells with 2 or 3 members only found in the MZ-B cell subset include clone sets using IGHV3: clone set C#1 (A2MZV3-B7, A2MZV3-F5), clone set C#2 (A2MZV3-C6, A2MZV3-C7, A2MZV3-E7), clone set C#8 (A3MZV4-11, A3MZV4-3.1, A3MZV4-9) and clone set C#9 (A3MZV4-20B, A3MZV4-7) or clone sets using IGHV4: clone set C#5 (IGA2MZV4-14, A2MZV4-3), clone set C#6 (A2MZV4-2.13, A2MZV4-2.17) and clone set C#7 (A2MZV4-2.9, A2MZV4-7). IGHV genes used by members of clone sets C#1 and C#2 had none or only one mutation in their IGHV genes. Members from other clone sets (C#5, 6, 8 and 9) exhibit more mutations (more than 6 mutations per IGHV sequence); most of these mutations were shared between the members of a clone. Sequences from three clone sets C#3 (A2RFV4-2, A2MZV4-1), C#10 (A2RFV5-42, A2MZV5-37) and C#4 (A3RFV4-3, A3MZV4) have members found in both B cell subsets. Clone sets C#3 and C#10 exhibits an identical mutation pattern, while clone set C#4 show many shared mutations. Overall, most of these clonally related sequences thus display both shared mutations in combination with unique mutations, indicating that members from one clone set were probably derived from the same naive precursor cell. The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 91 100 Percentage of sequences 90 IGHV3 80 70 60 FO-B 50 40 MZ-B 30 20 10 0 0-2 3-5 6-8 >8 100 Percentage of sequences 90 IGHV4 80 70 60 FO-B 50 40 MZ-B 30 20 10 0 0-2 3-5 6-8 >8 100 Percentage of sequences 90 IGHV5 80 70 60 FO-B 50 40 MZ-B 30 20 10 0 0-2 3-5 6-8 >8 100 90 Percentage of sequences R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 TOTAL 80 70 60 FO-B 50 40 MZ-B 30 20 10 0 0-2 3-5 6-8 >8 Number of mutations Figure 3. Mutation frequency of IGHV-Cµ trancripts of different IGHV gene families within MZ-B cells and FO-B cells. Analysis of the distribution of the number of mutations of all (“total”) IGH-Cµ transcripts shows that MZ-B cells have significantly more mutations per transcript then FO-B cells (Mann-Whitney P = 0.046). Within the MZ-B cell subset, IGHV4 sequences contain more mutations than in IGHV3 or IGHV5 sequences (Kruskal-Wallis, P = 0.011). 92 | Chapter 4 3. Discussion In this study we demonstrate that both FO-B cells and MZ-B cell populations contain mutated IgM sequences, albeit that among MZ-B cells the frequency of mutated sequences is generally higher compared to that in FO-B cells. Previous studies revealed that human FO-B cells (naïve mature cells) contain largely unmutated IgM encoding transcripts for various IGHV genes and families (Berkowska et al., 2011; Dunn-Walters et al., 1995; Klein et al., 1998; Kuppers et al., 1993; Pascual et al., 1994; Tangye et al., 1998). In mice, IGHV1 (J558) sequences derived from splenic FO-B cells are also unmutated (Gu et al., 1991). On the other hand Carey et al. (Carey et al., 2008) observed in mice that 10% of the IgM transcripts encoding for IGHV5 (PC7183) genes in splenic FO-B cells were mutated (i.e. contained more than 2 mutations). As we show here, there is a significant difference in frequency of IGHV3, IGHV4, and IGHV5). For example we did not observe FO-B cells with mutated IGHV3 sequences mutated sequences between the three studied IGHV gene families in rats (whereas up to 20-30% of FO-B cells express mutated IGHV4 or IGHV5 transcripts. Apparently, rodent B cells with a FO-B cell phenotype can be mutated, but the frequency varies with the expressed IGHV gene family. The finding that in human FO-B cells (mature naïve B cells) are virtually unmutated can be explained by species differences or that in most studies usually only a limited set of IGHV genes or gene families was used for analysis. Another, more likely explanation might be how naïve (unmutated) and memory (mutated) B cells are defined in humans. In humans unmutated B cells lack CD27 whereas mutated B cells express CD27 (Klein et al., 1998). CD27 is therefore widely used in human studies to discriminate naïve (unmutated) B cells from memory (mutated) B cells. However, some IgG+ or IgA+ B cells express mutated IgG or IgA genes but lack CD27 expression. (Berkowska et al., 2011; Fecteau et al., 2006) IgM and IgD expressing human B cells, lacking CD27 are all unmutated and are truly naïve B cells. In contrast, IgM and IgD expressing B cells that express CD27 are all mutated and are considered to represent memory B cells (Berkowska et al., 2011; Klein et al., 1998; Weill et al., 2009). In contrast to humans, in rodents CD27 expression cannot be used to distinguish memory B cells from naïve B cells (Xiao et al., 2004). In rats and mice, B cells expressing IgM and IgD (including FO-B cells), might therefore represent a heterogeneous population of cells composed of both naïve and memory B cells. As we show here, in comparison to FO-B cells, a much higher proportion of splenic MZ-B cells in (BN) rats express mutated IgM encoding transcripts. This proportion of mutated IgM+ MZ-B cells varied significantly between the different IGHV gene families and ranged from 28% to 66%. Previous studies in another rat strain (PVG) or in mice revealed a much smaller fraction (up to ~10%) of mutated MZ-B cell derived sequences (Carey et al., 2008; Dammers et al., 2000; Makowska et al., 1999). Differences in rat strain or species differences may explain The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 93 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 (part of) the observed discrepancy in percentage of mutated MZ-B cells. We show here, there is a significant variation in frequency of mutated IgM transcripts encoded by the various IGHV gene families. The highest frequency of mutated sequences (66%) was observed among MZ-B cells expressing IgM transcripts using IGHV4 gene family genes. This IGHV gene family consists of only two potentially functional members (Hendricks et al., 2010), of which only one appeared to be expressed. Based upon our findings, conclusions about frequency of mutated sequences among MZ-B cells should be drawn with care and analysis of a single IGHV gene, or a small IGHV gene family cannot be extrapolated for the entire MZ-B cell population. In contrast to MZ-B cells in rodents, nearly all human splenic MZ-B cells appear to be mutated (Dunn-Walters et al., 1995; Tangye et al., 1998; Tierens et al., 1999; Weller et al., 2004). Human splenic MZ-B cells express CD27 (Tangye et al., 1998), which is, as mentioned before, expressed by B cells with mutated IGHV genes. In these studies, however, only a limited set of IGHV genes expressed by human splenic MZ-B was analysed. However, given the notion that human splenic MZ-B cells express CD27 (Tangye et al., 1998), which is, as mentioned before, expressed by B cells with mutated IGHV genes, it is very likely that nearly all adult human splenic MZ-B cells are mutated. However, definite conclusions about frequencies of mutated versus unmutated MZ-B cells in various species awaits high throughput sequencing of large numbers of cells. Taken together, apparently both FO-B cells and MZ-B cells can express either unmutated or mutated IgM molecules on their cell membrane. The origin of mutated IgM expressing FO-B and MZ-B cells is not clear. Among the mutated sequences we observed groups of mutated sequences that were derived from clonally related B cells, i.e. these sequences had identical H-CDR3 regions, used the same IGHV gene, expressed shared mutations and were from the same rat. Most (70%) of clonally related groups of cells had members that were confined to the MZ-B cell compartment. However, importantly, some clonally related groups of cells had members that were found among MZ-B cells and FO-B cells. This clonal relationship strongly suggests that there is a common origin between mutated IgM+ MZ-B cells and mutated IgM+ FO-B cells. Remarkably, we have previously shown the existence of clonally related class switched, mutated B cells with members in both the MZ-B and FO-B cell compartment (Hendricks et al., 2011). Possibly both unswitched and class switched B cells are generated in the same fashion. Somatic hypermutations (SHM) are usually introduced in B cells proliferating in the GC environment. Mutated B cells are subsequently subjected to some form of positive selection for B cells expressing immunoglobulins that bind with high affinity to antigen, presented by follicular dendritic cells. Genetically engineered mice that cannot form GC (such as Bcl6 deficient or CD40 deficient mice) indeed lack B cells with mutated IGHV genes, including mutated IgM genes (Bergqvist et al., 2010; Toyama et al., 2002). Thus, at least in mice GC appear to be critically involved in SHM of IGHV genes during regular immune responses. This indicates that both mutated IgM+ 94 | Chapter 4 FO-B cells and MZ-B cells are probably GC derived. In contrast to mice, mutated IgM+ B cells can still be found in humans with CD40 or CD40L deficiency (hyper IgM syndrome patients, HIGM), that lack classical CD40L mediated T cell help and lack GC formation (Agematsu et al., 1998; Berkowska et al., 2011; Weller et al., 2004; Weller et al., 2001). These mutated B cells are IgM+IgD+CD27+ cells, also called natural effector cells, and correspond to splenic MZ-B cells (Weller et al., 2004). Other CD27+ B cell populations could not be formed in CD40/ CD40L deficient HIGM patients. Berkowska et al. observed that IgM+IgD+CD27+ cells have a relatively low replicative history (Berkowska et al., 2011). Furthermore they are already present in very young (<2 years) children and in human fetus (Scheeren et al., 2008; Weller et al., 2004; Weller et al., 2008). These findings suggest that at least a significant proportion of these mutated IgM+IgD+CD27+ MZ-B (-like) cells are not derived from GC and are generated in the absence of T cell help. Weill and colleagues (Weill et al., 2004; Weill et al., 2009; Weller et al., 2004) speculate that these MZ-B (-like) cells use SHM in order to diversify their repertoire early during ontogeny outside T-dependent or T-independent humoral immune responses. This hypothesis is, however, challenged in the literature (Seifert and Kuppers, 2009; Tangye and Good, 2007). Our observation that there are clonally related FO-B cells and MZ-B cells, expressing mutated IgM molecules indicates that these cells have a common origin and argues against the notion that such a postulated diversification process would then be unique for MZ-B cells. Furthermore, the variation in percentage of mutated sequences between the various IGHV gene families also argues against the notion, that SHM is used for diversification. In that case is would be more likely that mutations occur more or less at a similar rate in all IGHV gene families. This variation in mutation frequency between different IGHV gene families is more in favor of a diversification as a result of antigenic stimulation. Thus, a high percentage of IgM+ MZ-B cells in rat spleen have mutated IgM transcripts. This percentage is much higher than observed previously in this species (Dammers et al., 2000). These mutated IgM+ FO-B cells and MZ-B cells are likely generated in GC during antigendriven immune responses. We do not know which cells give rise to the GC that generate (simultaneously) IgM+ mutated FO-B cells and MZ-B cells. However, it is known that both naïve FO-B cells and naïve MZ-B cells can give rise to GC, although MZ-B cells appear to be far less effective (Phan et al., 2005; Song and Cerny, 2003). In any case, given our observation that some mutated IgM+ FO-B cells and MZ-B cells are clonally related, the phenotype of at least a proportion of the mutated cells has to change. Where and how they acquire their phenotype is also not known. Interestingly, the average number of mutations in mutated IgM transcripts is slightly higher among MZ-B cells than in FO-B cells. The reason for this is not clear. It could suggest that mutated IgM+ MZ-B cells have a longer history in successive GC reactions, or that they expand more within the GC environment. The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 95 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Agematsu K., Nagumo H., Shinozaki K., Hokibara S., Yasui K., Terada K., Kawamura N., Toba T., Nonoyama S., Ochs H. D. and Komiyama A., 1998. Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome. The Journal of clinical investigation 102, 853-60. Bergqvist P., Stensson A., Lycke N. Y. and Bemark M., 2010. T cell-independent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation. Journal of immunology 184, 3545-53. Berkowska M. A., Driessen G. J., Bikos V., Grosserichter-Wagener C., Stamatopoulos K., Cerutti A., He B., Biermann K., Lange J. F., van der Burg M., van Dongen J. J. and van Zelm M. C., 2011. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways. Blood 118, 2150-8. Carey J. B., Moffatt-Blue C. S., Watson L. C., Gavin A. L. and Feeney A. J., 2008. Repertoire-based selection into the marginal zone compartment during B cell development. The Journal of experimental medicine 205, 2043-52. Cinamon G., Zachariah M. A., Lam O. M., Foss F. W., Jr. and Cyster J. G., 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nature immunology 9, 54-62. Dammers P. M., de Boer N. K., Deenen G. J., Nieuwenhuis P. and Kroese F. G., 1999. The origin of marginal zone B cells in the rat. Eur J Immunol 29, 1522-31. Dammers P. M. and Kroese F. G., 2005. Recruitment and selection of marginal zone B cells is independent of exogenous antigens. Eur J Immunol 35, 2089-99. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of immunology 165, 6156-69. Dunn-Walters D. K., Isaacson P. G. and Spencer J., 1995. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. The Journal of experimental medicine 182, 559-66. Fecteau J. F., Cote G. and Neron S., 2006. A new memory CD27-IgG+ B cell population in peripheral blood expressing VH genes with low frequency of somatic mutation. Journal of immunology 177, 3728-36. Gatto D., Bauer M., Martin S. W. and Bachmann M. F., 2007. Heterogeneous antibody repertoire of marginal zone B cells specific for virus-like particles. Microbes Infect 9, 391-9. Gatto D., Ruedl C., Odermatt B. and Bachmann M. F., 2004. Rapid response of marginal zone B cells to viral particles. Journal of immunology 173, 4308-16. Gu H., Tarlinton D., Muller W., Rajewsky K. and Forster I., 1991. Most peripheral B cells in mice are ligand selected. The Journal of experimental medicine 173, 1357-71. Guinamard R., Okigaki M., Schlessinger J. and Ravetch J. V., 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nature immunology 1, 31-6. Hendricks J., Terpstra P., Dammers P. M., Somasundaram R., Visser A., Stoel M., Bos N. A. and Kroese F. G., 2010. Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat. Immunogenetics 62, 479-86. Hendricks J., Visser A., Dammers P. M., Burgerhof J. G., Bos N. A. and Kroese F. G., 2011. Classswitched marginal zone B cells in spleen have relatively low numbers of somatic mutations. Molecular immunology 48, 874-82. Klein U., Rajewsky K. and Kuppers R., 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. The Journal of experimental medicine 188, 1679-89. Kuppers R., Zhao M., Hansmann M. L. and Rajewsky K., 1993. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J 12, 4955-67. 96 | Chapter 4 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Makowska A., Faizunnessa N. N., Anderson P., Midtvedt T. and Cardell S., 1999. CD1high B cells: a population of mixed origin. Eur J Immunol 29, 3285-94. Martin F. and Kearney J. F., 2000. Selection in the mature B cell repertoire. Current topics in microbiology and immunology 252, 97-105. Obukhanych T. V. and Nussenzweig M. C., 2006. T-independent type II immune responses generate memory B cells. The Journal of experimental medicine 203, 305-10. Oliver A. M., Martin F. and Kearney J. F., 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. Journal of immunology 162, 7198-207. Pape K. A., Kouskoff V., Nemazee D., Tang H. L., Cyster J. G., Tze L. E., Hippen K. L., Behrens T. W. and Jenkins M. K., 2003. Visualization of the genesis and fate of isotype-switched B cells during a primary immune response. The Journal of experimental medicine 197, 1677-87. Pascual V., Liu Y. J., Magalski A., de Bouteiller O., Banchereau J. and Capra J. D., 1994. Analysis of somatic mutation in five B cell subsets of human tonsil. The Journal of experimental medicine 180, 329-39. Phan T. G., Gardam S., Basten A. and Brink R., 2005. Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen. Journal of immunology 174, 4567-78. Scheeren F. A., Nagasawa M., Weijer K., Cupedo T., Kirberg J., Legrand N. and Spits H., 2008. T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells. The Journal of experimental medicine 205, 2033-42. Schelonka R. L., Tanner J., Zhuang Y., Gartland G. L., Zemlin M. and Schroeder H. W., Jr., 2007. Categorical selection of the antibody repertoire in splenic B cells. Eur J Immunol 37, 1010-21. Seifert M. and Kuppers R., 2009. Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation. The Journal of experimental medicine 206, 2659-69. Song H. and Cerny J., 2003. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. The Journal of experimental medicine 198, 192335. Spencer J., Perry M. E. and Dunn-Walters D. K., 1998. Human marginal-zone B cells. Immunol Today 19, 421-6. Steiniger B., Timphus E. M. and Barth P. J., 2006. The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants. Histochemistry and cell biology 126, 641-8. Stoel M., Jiang H. Q., van Diemen C. C., Bun J. C., Dammers P. M., Thurnheer M. C., Kroese F. G., Cebra J. J. and Bos N. A., 2005. Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts. Journal of immunology 174, 1046-54. Tangye S. G. and Good K. L., 2007. Human IgM+CD27+ B cells: memory B cells or “memory” B cells? Journal of immunology 179, 13-9. Tangye S. G., Liu Y. J., Aversa G., Phillips J. H. and de Vries J. E., 1998. Identification of functional human splenic memory B cells by expression of CD148 and CD27. The Journal of experimental medicine 188, 1691-703. Tangye S. G. and Tarlinton D. M., 2009. Memory B cells: effectors of long-lived immune responses. Eur J Immunol 39, 2065-75. Tierens A., Delabie J., Michiels L., Vandenberghe P. and De Wolf-Peeters C., 1999. Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion. Blood 93, 226-34. Toyama H., Okada S., Hatano M., Takahashi Y., Takeda N., Ichii H., Takemori T., Kuroda Y. and Tokuhisa T., 2002. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 17, 329-39. Weill J. C., Weller S. and Reynaud C. A., 2004. A bird’s eye view on human B cells. Semin Immunol 16, 277-81. Weill J. C., Weller S. and Reynaud C. A., 2009. Human marginal zone B cells. Annual review of The proportion of mutated IgM positive marginal zone B cells varies between IGHV gene families | 97 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 40. 41. 42. 43. 44. 45. immunology 27, 267-85. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-54. Weller S., Faili A., Garcia C., Braun M. C., Le Deist F. F., de Saint Basile G. G., Hermine O., Fischer A., Reynaud C. A. and Weill J. C., 2001. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proceedings of the National Academy of Sciences of the United States of America 98, 1166-70. Weller S., Mamani-Matsuda M., Picard C., Cordier C., Lecoeuche D., Gauthier F., Weill J. C. and Reynaud C. A., 2008. Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants. The Journal of experimental medicine 205, 1331-42. White H. N. and Meng Q. H., 2012. Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response. Journal of immunology 188, 287-93. Xiao Y., Hendriks J., Langerak P., Jacobs H. and Borst J., 2004. CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation. Journal of immunology 172, 7432-41. Zandvoort A., Lodewijk M. E., de Boer N. K., Dammers P. M., Kroese F. G. and Timens W., 2001. CD27 expression in the human splenic marginal zone: the infant marginal zone is populated by naive B cells. Tissue antigens 58, 234-42. 98 | Chapter 4 Chapter 5 Marginal zone B cells in neonatal rats express unmutated IgM molecules Jacobus Hendricks Annie Visser Peter M. Dammers Johannes G.M. Burgerhof Nicolaas A. Bos Frans G.M. Kroese Manuscript in Preparation R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Abstract The majority of marginal zone (MZ)-B cells (80%) in the rat are naive (antigen inexperienced) B cells that express unmutated immunoglobulin variable region (IGHV) genes. In this species only a minor fraction of the IGHV-Cµ transcripts carry somatic mutations, and are considered to be memory B cells. This is in marked contrast to humans where nearly all MZ-B cells are mutated. Although memory (mutated) B cells are generally believed to be derived from germinal centers (GC’s) the origin of mutated IgM MZ-B cells remains unclear. In this study, we analysed whether rearranged IGHV-Cµ transcripts using IGHV4 and IGHV5 genes from neonatal MZ-B cells and follicular B (FO-B) cells carry mutations. We were not able to detect mutations in any of the IGHV4 and IGHV5 genes expressed by MZ-B cells or FO-B cells from neonatal rat spleen. Since GC’s are absent from neonatal rat spleen, these data argue against the notion that MZ-B cells mutate their IGHV genes as part of their developmental program, but are consistent with the notion that mutated rat MZ-B cells require GCs for their generation. 102 | Chapter 5 1. Introduction The splenic marginal zone (MZ) is a distinct anatomical compartment dominated by a unique population of B lymphocytes, in addition to macrophages, dendritic cells and other cell types (Martin and Kearney, 2002; Mebius and Kraal, 2005; Pillai et al., 2005). This compartment forms an interface between the splenic red pulp and white pulp. This unique localization in combination with the blood flow through this compartment, allows intimate contact between antigens in the blood and cells present. The B cells in the MZ (MZ-B) cells have a distinctive phenotype, generally characterized by their high levels of IgM and low levels of IgD (IgMhigh IgDlow). This contrasts to the majority population of mature (naïve) B cells located in follicles of peripheral lymphoid organs which express low levels of IgM and high levels of IgD (IgMlow IgDhigh), the follicular B (FO-B) cells. MZ-B cells appear to be in a “pre-activated” state, which is illustrated for example by their high expression levels of CD80/CD86 and complement receptor 2 (CD21) on their membrane surface in comparison with FO-B cells (Oliver et al., 1999). MZ-B cells have important physiological functions and are primarily held responsible for T cell-independent (TI) responses to polysaccharides antigens, present on the surface of encapsulated bacteria (Guinamard et al., 2000; Martin et al., 2001). The majority of MZ-B cells in rats and mice express unmutated transcripts for IgM heavy chain molecules and are considered to represent naïve B cells. A small proportion of the cells, however, express mutated IgM transcripts and is therefore classified as IgM memory B cells (Dammers et al., 2000; Makowska et al., 1999). We have recently observed that the percentage of these mutated IgM memory MZ-B cells varies significantly between the various immunoglobulin heavy variable (IGHV) gene families analysed and ranges from 28% to 66%. Furthermore we also observed a clonal relationship between IgM memory (mutated) MZ-B cells and IgM memory (mutated) FO-B cells, suggesting that these two B cell subsets have a common origin. In addition to IgM memory B cells, a small (but unknown) fraction of the splenic MZ-B cells are classically class switched (IgG expressing) memory B cells (Hendricks et al., 2011). In contrast to rodents, nearly all MZ-B cells in human spleens express mutated IGVH genes (Dunn-Walters et al., 1995; Tierens et al., 1999). Phenotypically, these B cells express CD27, which is an important, but not conclusive, characteristic property of human memory B cells (Klein et al., 1998). In humans MZ-B cells are therefore defined as IgM+IgD+CD27+ B cells (Weller et al., 2004). Whether the (mutated) MZ-B cells in humans represent bona fide memory B cells is a matter of debate. On one hand these B cells are considered to be true memory cells, generated in germinal centers (GC) during antigen-driven humoral immune responses (Seifert and Kuppers, 2009; Tangye and Good, 2007). On the other hand these Marginal zone B cells in neonatal rats express unmutated IgM molecules | 103 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 cells were proposed to be generated during TI immune responses (Kruetzmann et al., 2003). Weller and co-workers argued that the presence of mutations reflects an intrinsic property of MZ-B cells that is exploited by these cells to diversify their primary antibody repertoire (Weller et al., 2004; Weller et al., 2001; Weller et al., 2008). According to Weller and colleagues the IgM+IgD+CD27+ MZ-B cells are not memory cells, but cells that develop in the absence of antigen, in a TI fashion, outside GC, along a pathway that differs from classical memory B cells. This conclusion was initially based on the finding that also patients with CD40L or CD40 deficiency harbour mutated IgM+IgD+CD27+ MZ-B cells in blood (Weller et al., 2004; Weller et al., 2001). These patients lack classical cognate T-B cell collaboration leading to GC. Furthermore, also in very young children under the age of 2 years, the majority of blood and splenic MZ-B cells appear to be mutated, with no signs of antigen-driven clonal expansion, in contrast to classical class switched memory B cells (Weller et al., 2004; Weller et al., 2008). Furthermore Scheeren et al. (Scheeren et al., 2008) observed that a low fraction (~20%) of human fetal splenic IgM+IgD+CD27+ B cells are mutated, and these authors hypothesized that somatic hypermutation (SHM) in this population occurs mainly during fetal development and in very young children. MZ-B cells are already found early during ontogeny in rat spleen (Dammers and Kroese, 2005; Dammers et al., 2002). At that time GCs are absent from spleen (Kroese et al., 1987; van Rees et al., 1986). Whether early in neonatal life IGHV genes expressed by MZ-B cells in rodents are mutated or not is not known and was studied here. We show that in contrast to humans, neonatal MZ-B cells in rats are all unmutated, supporting the view that, at least in rodents, mutated MZ-B cells are memory B cells, generated in GC. 2. Materials and Methods 2.1. Animals Pregnant BN/SsNOlaHsd rats were obtained from (Harlan Horst, The Netherlands). Rats were housed under clean, conventional conditions at the Central Animal Facility of the University Medical Center Groningen. Two day old neonatal rats of both sexes were killed by decapitation. The experiment was approved by the Animal Experimental Committee of the University of Groningen. 2.2. Isolation and purification of B cell subsets by Flow-cytometry Single cell suspensions were prepared from 5 pooled spleens from two day old neonatal rats and labelled with monoclonal antibodies for cell sorting as described previously (Dammers et al., 2000). Briefly, cells were incubated with FITC conjugated anti-rat IgM (HIS40; 104 | Chapter 5 eBioscience, San Diego, CA, USA), biotinylated anti-rat IgD (MaRD3; AbDSerotec, Oxford, UK), APC conjugated anti-rat CD90/Thy1.1 (HIS51; eBioscience) and PE conjugated anti-rat TCRαβ (R73; eBioscience), anti-TCRγδ (V65; eBioscience) and anti-CD161a/NKRa (10/78; BD Pharmingen). BiotinylatedmAb were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5 (Ebioscience). The PE channel was used as a “Dump” channel; only PE negative (Dumpneg) cells were sorted. Herewith, we were able to exclude immature B cells (i.e. CD90+ B cells), T cells and NK cells from our sorts. Analysis and sorting of B cells was carried out on a MoFlo flow cytometer (Cytomation, Ft Collins, CO). Dead cells, plasma cells, monocytes/macrophages, and erythrocytes were excluded from cell sorting by using forward and side scatter profiles. Sorted FO-B cells (CD90-IgDhighIgMlow) and MZ-B cells (CD90-IgMhigh IgDlow) were collected in sterile FACS tubes (Greiner Bio-One,Alphen a/d Rijn, The Nether- lands) containing 500 μl of newborn calf serum (PAA laboratories GmbH, Pasching, Austria). FlowJo software (Tree Star, San Carlos, CA) was used for flow cytometry data analysis. 2.3. Molecular cloning of IGHVDJ-Cμ transcripts Total RNA was extracted from sorted cells using the Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA, USA) according to instructions of the manufacturer. Briefly, sorted cells were pelleted by 300xg centrifugation for 10 min at 4oC and then resuspended in a total volume of 350µl lysis buffer containing β-mercaptoethanol (Stratagene). First strand cDNA was synthesized using an oligo-(dT)12-18 primer (Invitrogen, Breda, The Netherlands) and SuperScriptTMII reverse transcriptase (200U/μl; Invitrogen) as described in the manufacturer’s protocol. Rearranged IGHV4-Cµ and IGHV5-Cµ gene transcripts were amplified in a 50 μl reaction mixture, containing 2 μl cDNA of either IGHV4 or IGHV5 family specific primer, plus 0.6 pmol/µl universal Cµ constant region primer and 2.5U Taq DNA Polymerase (Invitrogen). The following primers were used: IGHV4 (5’-GGTGCA(A/G)CCTGGAAGATCCT-3’), IGHV5 (5’-CTTAGTGCAGCCTGGAAGGT) (Dammers et al., 2000) and the constant region Cµ primer (5’-CAACACTGAAGTCATCCAGGG-3’). To assess the amount and quality of the cDNA, PCR was also performed for β-actin, using β-actin specific primers as described by (Stoel et al., 2005). The PCR program for amplification of IGHVDJ-Cμ transcripts and β-actin consisted of 35 cycles of 30 sec at 94°C (2 min in first cycle), 1 min at 58°C and 1 min at 72°C, respectively. This program was followed by an additional incubation period of 25 min at 72°C to allow extension of all IGHVDJ-Cμ transcripts. The quality and size of the PCR products was evaluated by agarose gel electrophoresis. 2.4. Cloning and sequencing PCR products were subsequently cloned into the pJET1/blunt vectors using the GeneJETTM PCR Cloning Kit, respectively (Fermentas Life Sciences). TOP10F E. coli competent cells (Invitrogen) were transformed with plasmids containing the PCR product. Plasmid DNA was Marginal zone B cells in neonatal rats express unmutated IgM molecules | 105 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 isolated from randomly picked colonies with the Nucleospin Plasmid QuickPure kit (Bioke, Leiden, The Netherlands). Size of the inserts was determined by digestion of a DNA sample of the plasmids with the appropriate restriction enzymes followed by agarose gel electrophoresis. Plasmids containing an insert of approximately 500 bp were sequenced in both directions by Service XS (Leiden, The Netherlands). Sequence processing was performed using EMBL/ Genbank Data Libraries and Chromas software (Digital River GmbH, Cologne, Germany). 2.5. Statistical analysis of IGHVDJ –Cµ transcripts Statistical analysis of the sequence data was performed using SPSS 16 software (SPSS Inc. Chicago, Ill. USA). IGHV-Cµ sequences displaying 100% identity were considered to be derived from a single B cell and counted only once for statistical analysis. Since Taq DNA polymerase errors might be responsible for 1–2 mutations per sequence, we considered only sequences with more than 2 mutations as truly mutated (Dammers et al., 2000). The number of mutations was determined by counting the number of nucleotide mismatches in comparison with each IGHV gene sequence to its closest germline counterpart. We used Fisher’s exact test to determine possible differences between groups with regard to the binary response variable indicating mutation or not. Non parametric tests, Kruskal-Wallis and Mann-Whitney, were used to compare groups with respect to the number of mutations. In all statistical tests a p-value < 0.05 was considered to be significant. 3. Results and Discussion Adult MZ-B cells in rodents and humans are a heterogeneous population of B cells, comprising both unmutated IgM expressing B cells (naïve MZ-B cells) and either class switched or unswitched B cells with mutated IGHV genes. Both subsets of mutated MZ-B cells are generally believed to represent memory type B cells. Weller et al and others, however, postulate that mutations present in MZ-B cells are not the consequence of an antigen driven response, but are an intrinsic property of these B cells, introduced during their development (Weller et al., 2004; Weller et al., 2008). In this study we addressed this issue, by analysing the occurrence of SHM in IGHV genes, expressed by MZ-B cells present in neonatal rats, i.e. at a time point that antigen-driven humoral immune responses have not been taken place yet, as witnessed by the absence of GC in lymphoid organs during the first few weeks of life (Kroese et al., 1987; van Rees et al., 1986). MZ-B cells and FO-B cells were sorted from (pooled) neonatal rat spleen, as is illustrated in Figure 1. A large proportion of MZ-B cells in neonatal rats express CD90 and are therefore considered to represent immature MZ-B cells (Dammers et al., 2000); mature MZ-B cells and FO-B cells are defined as CD90-IgMhighIgDlow cells and CD90-IgMlowIgDhigh respectively. By including CD90 in our staining combination, we excluded 106 | Chapter 5 immature MZ-B and FO-B cells from our analysis. IGHV-Cµ mRNA transcripts from both neonatal FO-B cells and MZ-B cells were amplified by RT-PCR, cloned, sequenced and analysed for the presence of SHM. Lymphoid cells TCRαβ TCRᵞᵟ CD90+ Dump channel FO-B cells MZ-B cells Forward scatter IgM Figure 1. Three colour cytometry was used to analyse FO-B cells and MZ-B cells. Single splenic cell suspension of neonatal rats was stain with FITC conjugated anti-rat IgM (HIS40; eBioscience, San Diego, CA, USA), biotinylated anti-rat IgD (MaRD3; AbD Serotec, Oxford, UK), APC anti-rat CD90/Thy1.1 (HIS51; eBioscience). Biotinylated mAb were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5 (Ebioscience). Lymphocytes were sequentially gated by forward scatter and side scatter. Acquisition gates were set to exclude the unwanted immature B cells (CD90+-APC). Gate settings were set appropriately for FO-B cells (CD90negIgDhighIgMlow) and MZ-B (CD90negIgMhighIgDlow) cells. Table 1. Neonatal IGHV Cµ mRNA transcripts from FO-B cells and MZ-B cells Clonea IGHV IGHD IGHJ member member member Mutationsb H-CDR3c Nf Amino acid d Sequences of IGHV4 gene family from FO-B cells NRFVH4-60 NRFVH4-71 g NRFVH4-16 IGHV4S2 IGHD5-1 IGHJ2 0 7 ATGSFDY IGHV4S2 IGHD1-6 IGHJ2 1 9 ARAPGGYDY C#1 IGHV4S2 IGHD1-1 IGHJ2 0 14 ARESYYYYSGDFDY g IGHV4S2 IGHD1-6 IGHJ2 1 9 ARAPGGYDYC#1 NRFVH4-2.1 IGHV4S2 IGHD1-6 IGHJ2 0 11 ARAGGYYYFDY NRFVH4-2.4 IGHV4S2 IGHD1-2 IGHJ2 0 10 ARVLWVYFDY NRFVH4-2.5 IGHV4S2 IGHD1-4 IGHJ2 0 12 ARAYYGYNYFDY NRFVH4-2.6 IGHV4S2 IGHD1-4 IGHJ2 0 11 ARYYGYNYFDY NRFVH4-2.7 IGHV4S2 IGHD1-4 IGHJ2 0 12 ATYYGYNYYFDY NRFVH4-37 Sequences of IGHV4 gene family from MZ-Be cells NMZVH4-1 IGHV4S2 IGHD1-1 IGHJ1 0 15 AIMYTTDYXYWYFDF NMZVH4-3 IGHV4S2 IGHD1-7 IGHJ2 0 11 ARAYYDGSYYY Marginal zone B cells in neonatal rats express unmutated IgM molecules | 107 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Clonea IGHV IGHD IGHJ member member member NMZVH4-4 IGHV4S2 IGHD1-1 IGHJ4 NMZVH4-5 IGHV4S2 IGHD1-5 IGHJ1 NMZVH4-6 IGHV4S2 IGHD1-4 NMZVH4-9 IGHV4S2 IGHD2-2 NMZVH4-13 IGHV4S2 NMZVH4-17 IGHV4S2 NMZVH4-20 NMZVH4-3.1 Mutationsb H-CDR3c Nf Amino acid 0 16 ARAHMYTTDYYYVMDA 0 10 AIYNNWYFDF IGHJ2 0 10 ARLPGYNFDY IGHJ2 0 9 ARDTYYFDY IGHD1-3 IGHJ1 0 12 ARARSSYWYFDF IGHD1-7 IGHJ2 0 12 ARNYPGMYYFDY IGHV4S2 IGHD1-6 IGHJ2 0 8 ARTEGIDY IGHV4S2 IGHD3-2 IGHJ2 0 10 ARARYNYFDY NMZVH4-3.2h IGHV4S2 IGHD1-7 IGHJ1 1 16 ARFYYDGSYYYWYFDFC#2 NMZVH4-3.3 IGHV4S2 IGHD1-3 IGHJ2 0 10 ARYSSYYFDY NMZVH4-3.4 IGHV4S2 IGHD5-1 IGHJ2 0 8 ATGSYFDY IGHV4S2 IGHD1-7 IGHJ1 0 16 ARDYYDGSYYYWYFDFC#2 NMZVH4-3.7 IGHV4S2 IGHD1-3 IGHJ2 0 6 ARGSYY NMZVH4-3.8 IGHV4S2 IGHD4-1 IGHJ2 0 9 ARAQFGVDY NMZVH4-3.9 IGHV4S2 IGHD1-5 IGHJ2 0 9 ARIYNNFDY NMZVH4-3.10 IGHV4S2 IGHD5-1 IGHJ2 1 11 ARTGYYWSFDF NMZVH4-3.11 IGHV4S2 IGHD5-1 IGHJ2 1 10 ARDWELYFDY NMZVH4-3.12 IGHV4S2 IGHD5-1 IGHJ1 0 12 ARTGSYYWYFDF NMZVH4-3.13 IGHV4S2 IGHD1-4 IGHJ2 0 13 ARRYYGYNYYFDY NMZVH4-3.6 h Sequences of IGHV5 gene family from FO-Bd cells NRFVH5-50 IGHV5S30 IGHD4-1 IGHJ1 1 13 ATDNSGYYWYFDF NRFVH5-72 IGHV5S30 IGHD1-4 IGHJ2 0 13 ATIAAISTYYFDY NRFVH5-8 IGHV5S30 IGHD5-1 IGHJ2 0 8 ATGSYFDY NRFVH5-18 IGHV5S30 IGHD1-2 IGHJ3 1 9 ATGYNWFAY NRFVH5-1 IGHV5S27 IGHD1-7 IGHJ2 0 12 ARHYYSGDYFDY NRFVH5-3 IGHV5S13 IGHD1-8 IGHJ2 0 14 ARHYYDGYYHYFDY NRFVH5-4 IGHV5S11 IGHD4-1 IGHJ3 1 12 ARHNSGYNWFAY NRFVH5-6 IGHV5-6 IGHD1-2 IGHJ2 0 8 TTDHYGDY NRFVH5-7 IGHV5S74 IGHD1-2 IGHJ3 1 12 ARTYYGYNWFAY NRFVH5-8 IGHV5S27 IGHD1-7 IGHJ2 0 14 ARHYYDGSYYYFDY NRFVH5-9 IGHV5S10 IGHD1-5 IGHJ1 2 12 ATHNNYYWYFDF NRFVH5-10 IGHV5-1 IGHD1-3 IGHJ2 0 11 ANYYYSSYIDY NRFVH5-11 IGHV5S74 IGHD1-1 IGHJ3 1 12 ARMYTTDNWFAY NRFVH5-12 IGHV5S10 IGHD1-8 IGHJ2 0 10 ATHYYDGYYY NRFVH5-13 IGHV5-6 IGHD4-1 IGHJ2 0 10 TTNSGYYFDY NRFVH5-14 IGHV5S57 IGHD1-8 IGHJ2 1 12 TNYRDSYAYFDY NRFVH5-15 IGHV5S45 IGHD1-6 IGHJ2 1 10 ARQLRRVFDY 108 | Chapter 5 Clonea IGHV IGHD IGHJ member member member NRFVH5-16 IGHV5S10 IGHD1-6 IGHJ3 NRFVH5-17 IGHV5S57 no results IGHJ3 NRFVH5-18 IGHV5S29 IGHD1-7 NRFVH5-19 IGHV5S16 NRFVH5-2.3 IGHV5S30 Mutationsb H-CDR3c Nf Amino acid 0 10 ATYGGYWFAY 0 8 TRGYWFAY IGHJ2 0 15 TTETYYYDGSYYFDY IGHD5-1 IGHJ1 0 9 ARGSWYFDF IGHD4-1 IGHJ2 0 11 ATDNSGYYFDY IGHJ2 0 4 ATNY e Sequences from IGHV5 gene family from MZ-B cells a b c d e f g NMZVH5-2 IGHV5S30 no results NMZVH5-10 IGHV5S30 IGHD4-1 IGHJ2 0 10 ATDSGYYFDY NMZVH5-2 IGHV5-3 IGHD1-2 IGHJ4 0 10 ARHGYYVMDA NMZVH5-3 IGHV5-2 IGHD1-8 IGHJ2 0 10 ARHDGYYFDY NMZVH5-5 IGHV5S43 IGHD1-5 IGHJ2 0 10 TRDNNYYFDY NMZVH5-6 IGHV5S65 no results IGHJ2 1 8 AKAHYFDY NMZVH5-11 IGHV5S10 IGHD1-4 IGHJ2 1 11 ATHYGYNYFDY NMZVH5-12 IGHV5-6 IGHD1-7 IGHJ2 1 12 ARHYYDGSYYDY NMZVH5-14 IGHV5S32 IGHD1-7 IGHJ2 1 14 ARHYDGSYYYYFDY NMZVH5-15 IGHV5S16 IGHD4-1 IGHJ3 0 9 ARHNSGFAY NMZVH5-16 IGHV5S16 IGHD1-4 IGHJ2 0 6 ATHNDY NMZVH5-17 IGHV5S30 IGHD1-3 IGHJ4 0 10 ATYSSYVMDA NMZVH5-18 IGHV5S43 IGHD1-5 IGHJ2 0 11 TRDHNNYYFDY NMZVH5-20 IGHV5S11 IGHD1-6 IGHJ1 0 16 ARHNYGGYSDYWYFDF NMZVH5-3.15 IGHV5S30 IGHD1-3 IGHJ2 0 8 ATEYWSDY NMZVH5-3.16 IGHV5S30 IGHD5-1 IGHJ2 0 9 ATTGSYFDY Cµ (IgM) transcripts from FO-B cells and MZ-B cells Mutations, nucleotide differences of one or more basis between IMGT germline gene and rearranged Cµ transcript H-CDR3, heavy chain complementarity determining region 3 FO-B cells, recirculating follicular B cells MZ-B cells, marginal zone B cells Lenght of H-CDR3 in amino acids The sequence NRFVH4-71 and NRFVH4-37 are from clonally related B cells and designated as clone set C#1 In a previous study we showed that the proportion of mutated IgM+ MZ-B cells varies significantly when different IGHV gene families were analysed. This proportion mutated MZ-B cell derived sequences ranged up to 28% for IGHV5 transcripts and up to 66% for IGHV4 transcripts in adult rats (chapter 4). For this reason we chose to analyse IGHV-Cµ transcripts Marginal zone B cells in neonatal rats express unmutated IgM molecules | 109 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 encoding for IGHV5 and IGHV4 family genes. The IGHV5 gene family is the second largest IGHV gene family in the rat and consists of 27 potentially functional IGHV genes (Hendricks et al., 2010). In total we amplified 16 unique IGHV5-Cµ transcripts from neonatal MZ-B cells and 22 IGHV5-Cµ transcripts from neonatal FO-B cells (Table 1). These transcripts were encoded by various germline genes: 10 different IGHV5 germline genes were used by MZ-B cells and 12 by FO-B cells. The IGHV4 gene family is composed of only 2 potentially functional IGHV genes (Hendricks et al., 2010), of which only one (IGHV4S2) seems to be expressed (chapter 4). We obtained 21 unique IGHV4-Cµ sequences derived neonatal MZ-B cells and 9 IGHV4Cµ sequences from neonatal FO-B cells (Table 1). All these transcripts were encoded by the IGHV4S2 gene, confirming our previous findings in adult rats, that only this family member is functionally expressed by rat B cells. Importantly, as is shown in Table 1, mutational analysis of the IGHV5-Cµ and IGHV4-Cµ transcripts revealed that none of the transcripts from either MZ-B cells or FO-B cells were mutated (i.e. expressed more than two nucleotide differences compared to their germline counterparts). This is in marked contrast to adult rat MZ-B cells and FO-B cells, of which a significant proportion of the transcripts encoding for IgM heavy chains are mutated (Dammers et al., 2000; chapter 4). SHM of (heavy and light) IGV genes are generally believed to take place during the generation of memory B cells within the GC and is associated with selection of B cells expressing high affinity surface Ig. GC’s are typically antigen- and T-cell dependent structures. GC’s are absent in neonatal rats during the first few weeks of life, and in fact, GC’s can even not be induced by deliberate antigenic stimulation after birth until spleen and lymph nodes have reached a certain stage of maturity (Kroese et al., 1987; van Rees et al., 1986). The appearance of follicular dendritic cell networks might be a critical factor in the maturation process, allowing GC formation (Kroese et al., 1985; Kroese et al., 1987). The absence of B cells with mutated IgM transcripts in neonatal rats thus corresponds with the absence of GC in these animals and favours the hypothesis that mutated MZ-B and FO-B cells in adult animals are both memory B cells which are generated in GC during antigen driven immune responses. A possible GC origin of mutated IgM+IgD+CD27+ B cells is supported by findings of Seifert and Küppers (Seifert and Kuppers, 2009). They showed that, similar to class switched memory B cells, mutated peripheral blood IgM+IgD+CD27+ B cells carry mutated Bcl6 genes, similar to class switched memory B cells. Bcl6 is expressed at high levels by GC B cells and is considered to be the master transcriptional regulator of these B cells (Basso and Dalla-Favera, 2010). When GC B cells acquire SHM of their IGHV genes also the Bcl6 gene can become mutated (Pasqualucci et al., 1998). Presence of mutations in the Bcl6 gene is therefore considered to be a genetic feature of GC experience (Seifert and Kuppers, 2009). Importantly, Seifert and Küppers (Seifert and Kuppers, 2009) also identified mutated IgM+IgD+CD27+ B cells that were clonally related to class switched, classical IgG+ memory B cells, with both shared and unshared mutation, which is strongly indicative for a common, GC origin. Taken together, the absence of mutated Cµ transcripts 110 | Chapter 5 from both IgM+ MZ-B cells and FO-B cells in neonatal rats led us to postulate that both IgM+ memory B cell subsets in adult rats are likely derived from GC, as a consequence of stimulation by exogenous antigens, similar to IgG+ memory B cells. At least in rats it is thus unlikely that MZ-B cells acquire SHM during their antigen-independent generation as proposed for humans (Scheeren et al., 2008; Weller et al., 2004; Weller et al., 2001). Marginal zone B cells in neonatal rats express unmutated IgM molecules | 111 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Basso K. and Dalla-Favera R., 2010. BCL6: master regulator of the germinal center reaction and key oncogene in B cell lymphomagenesis. Advances in immunology 105, 193-210. Dammers P. M. and Kroese F. G., 2005. Recruitment and selection of marginal zone B cells is independent of exogenous antigens. Eur J Immunol 35, 2089-99. Dammers P. M., Lodewijk M. E., Zandvoort A. and Kroese F. G., 2002. Marginal zone B cells in neonatal rats express intermediate levels of CD90 (Thy-1). Developmental immunology 9, 187-95. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of immunology 165, 6156-69. Dunn-Walters D. K., Isaacson P. G. and Spencer J., 1995. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. The Journal of experimental medicine 182, 559-66. Guinamard R., Okigaki M., Schlessinger J. and Ravetch J. V., 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nature immunology 1, 31-6. Hendricks J., Terpstra P., Dammers P. M., Somasundaram R., Visser A., Stoel M., Bos N. A. and Kroese F. G., 2010. Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat. Immunogenetics 62, 479-86. Hendricks J., Visser A., Dammers P. M., Burgerhof J. G., Bos N. A. and Kroese F. G., 2011. Classswitched marginal zone B cells in spleen have relatively low numbers of somatic mutations. Molecular immunology 48, 874-82. Klein U., Rajewsky K. and Kuppers R., 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. The Journal of experimental medicine 188, 1679-89. Kroese F. G., Leceta J., Dopp E. A., Herraez M. P., Nieuwenhuis P. and Zapata A., 1985. Dendritic immune complex trapping cells in the spleen of the snake, Python reticulatus. Developmental and comparative immunology 9, 641-52. Kroese F. G., Wubbena A. S., Kuijpers K. C. and Nieuwenhuis P., 1987. The ontogeny of germinal centre forming capacity of neonatal rat spleen. Immunology 60, 597-602. Kruetzmann S., Rosado M. M., Weber H., Germing U., Tournilhac O., Peter H. H., Berner R., Peters A., Boehm T., Plebani A., Quinti I. and Carsetti R., 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. The Journal of experimental medicine 197, 939-45. Makowska A., Faizunnessa N. N., Anderson P., Midtvedt T. and Cardell S., 1999. CD1high B cells: a population of mixed origin. Eur J Immunol 29, 3285-94. Martin F. and Kearney J. F., 2002. Marginal-zone B cells. Nature reviews. Immunology 2, 323-35. Martin F., Oliver A. M. and Kearney J. F., 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14, 617-29. Mebius R. E. and Kraal G., 2005. Structure and function of the spleen. Nature reviews. Immunology 5, 606-16. Oliver A. M., Martin F. and Kearney J. F., 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. Journal of immunology 162, 7198-207. Pasqualucci L., Migliazza A., Fracchiolla N., William C., Neri A., Baldini L., Chaganti R. S., Klein U., Kuppers R., Rajewsky K. and Dalla-Favera R., 1998. BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci. Proceedings of the National Academy of Sciences of the United States of America 95, 11816-21. Pillai S., Cariappa A. and Moran S. T., 2005. Marginal zone B cells. Annual review of immunology 23, 161-96. 112 | Chapter 5 20. 21. 22. 23. 24. 25. 26. 27. 28. Scheeren F. A., Nagasawa M., Weijer K., Cupedo T., Kirberg J., Legrand N. and Spits H., 2008. T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells. The Journal of experimental medicine 205, 2033-42. Seifert M. and Kuppers R., 2009. Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation. The Journal of experimental medicine 206, 2659-69. Stoel M., Jiang H. Q., van Diemen C. C., Bun J. C., Dammers P. M., Thurnheer M. C., Kroese F. G., Cebra J. J. and Bos N. A., 2005. Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts. Journal of immunology 174, 1046-54. Tangye S. G. and Good K. L., 2007. Human IgM+CD27+ B cells: memory B cells or “memory” B cells? Journal of immunology 179, 13-9. Tierens A., Delabie J., Michiels L., Vandenberghe P. and De Wolf-Peeters C., 1999. Marginal-zone B cells in the human lymph node and spleen show somatic hypermutations and display clonal expansion. Blood 93, 226-34. van Rees E. P., Dijkstra C. D. and van Rooijen N., 1986. The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat. Cell and tissue research 246, 673-7. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-54. Weller S., Faili A., Garcia C., Braun M. C., Le Deist F. F., de Saint Basile G. G., Hermine O., Fischer A., Reynaud C. A. and Weill J. C., 2001. CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans. Proceedings of the National Academy of Sciences of the United States of America 98, 1166-70. Weller S., Mamani-Matsuda M., Picard C., Cordier C., Lecoeuche D., Gauthier F., Weill J. C. and Reynaud C. A., 2008. Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants. The Journal of experimental medicine 205, 1331-42. 29. Marginal zone B cells in neonatal rats express unmutated IgM molecules | 113 Chapter 6 Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations Jacobus Hendricks Annie Visser Peter M. Dammers Johannes G.M. Burgerhof Nicolaas A. Bos Frans G.M. Kroese Molecular Immunology 48 (2011) 874–882 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Abstract The vast majority of rodent splenic marginal zone B (MZ-B) cells are naive IgM+ cells. A small fraction of these MZ-B cells carry mutated V-genes, and represent IgM+ memory MZ-B cells. Here we reveal further heterogeneity of B cells with a MZ-B cell phenotype, by providing evidence for the existence of class switched memory MZ-B cells in the rat. In essence, we observed IGHV5 encoded Cγ transcripts, among FACS-purified MZ-B cells, defined as HIS24lowHIS57bright cells. Furthermore, we found that most IgG encoding transcripts are mutated. There is no significant difference in IGHV5 repertoire and subclass usage of these IgG encoding transcripts collected from B cells with a MZ-B cell phenotype and B cells with a follicular (FO-B) cell phenotype. However, the IGHV5 genes encoding for IgG antibodies of MZ-B cells exhibited significantly fewer mutations, compared to those with a FO-B cell phenotype. In one rat we found a clonally related set of IgG encoding sequences, of which one was derived from the MZ-B cell fraction and the other from the FO-B cell fraction. We speculate that these two subpopulations of class switched B cells are both descendants from naive FO-B cells and are generated in germinal centers. Class switched memory B cells with a MZ-B cell phenotype may provide the animal with a population of IgG memory B cells that can respond rapidly to blood-borne pathogens. 116 | Chapter 6 1. Introduction The marginal zone (MZ) represents a distinct anatomical B cell compartment in the spleen located at the outer areas of the white pulp, at the border of the red pulp (for review see e.g. (Steiniger et al., 2006). The circulatory system of the spleen ensures an intimate contact of blood and cells of the MZ. Most of the cells in this compartment are B cells, but macrophages and dendritic cells (and in humans also CD4+ T cells) are also present. MZ-B cells have unique characteristics (for reviews see e.g. (Martin and Kearney, 2002; Pillai et al., 2005; Weill et al., 2009). In rodents the vast majority of MZ-B cells expresses high levels of IgM and low levels of IgD (IgMhiIgDlo) in combination with high levels of CD21 and low levels of CD23 (CD21hiCD23lo) (Oliver et al., 1997). This unique phenotype distinguishes them from the majority population of mature, naive B cells, i.e. follicular B (FO-B) cells, which are IgMloIgDhi CD21loCD23hi. Rat FO-B cells can also be defined as mature (i.e. CD90− (Kroese et al., 1995) small-sized, HIS24highHIS57neg/low B cells whereas MZ-B cells are slightly larger cells and can be distinguished as CD90−HIS24lowHIS57high cells (Dammers et al., 1999; Kroese et al., 1990; Kroese et al., 1995). Importantly, MZ-B cells also have different functional characteristics, such as their pre-activated status and their proliferative and stimulatory requirements (Oliver et al., 1997; Oliver et al., 1999). Rodent MZ-B cells appear to be biased towards T cell-independent (TI-2) immune responses against micro-organism-derived polysaccharide antigens (Guinamard et al., 2000; Martin et al., 2001; Vinuesa et al., 2003). These properties in combination with their topographical localization in spleen, allow them to respond rapidly to bloodborne pathogens by the generation of massive numbers of antibody secreting cells during the first few days after infection (Martin et al., 2001). MZ-B cells are a heterogeneous population of cells, and comprise both naive and memory cells. In rats and mice, the majority (up to 80%) of MZ-B cells are naive cells which express germline encoded V region of the Ig genes (Dammers et al., 2000; Makowska et al., 1999). Occurrence of memory B cells in the MZ was first demonstrated by (Liu et al., 1988), showing the appearance of hapten-binding, IgM+ memory cells with a MZ-B cell phenotype in the MZ of spleens from immunized rats. Hapten-binding MZ-B cells were also demonstrated by flow-cytometry and/or immunohistology in several subsequent studies in immunized normal and Ig-gene targeted mice (Gatto et al., 2007; Gatto et al., 2004; Obukhanych and Nussenzweig, 2006; Pape et al., 2003; Phan et al., 2005). In rodents, memory cells constitute a minority MZ-B cell population. Up to 20% of the rodent MZ-B cells might be IgM memory B cells as indicated by the presence of mutated IgH chain V gene (IGHV) trancripts encoding for IgM antibodies (IGHV-Cγ transcripts) among purified sIgM+ MZ-B cells (Dammers et al., 2000; Makowska et al., 1999). There are some data that suggest that, in addition to these unswitched IgM-expressing memory MZ-B cells, also some class switched memory B cells are found among the MZ-B cell population in rodent spleens after immunization (Gatto et al., 2004; Liu et al., 1988; Obukhanych and Nussenzweig, 2006; Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 117 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Pape et al., 2003). For example, (Gatto et al., 2004) observed the presence of IgG+ phage (Qβ)-specific B cells with a MZ-B cell phenotype (i.e. CD21hiCD23low B cells), up to 21 days upon immunization of normal mice. Whether these class switched MZ-B cells were indeed “classical” memory B cells with their characteristic mutated high affinity BCR’s was, however, not investigated. The presence of mutated, antigen-specific memory MZ-B cells were subsequently demonstrated in mice 12 days after immunization with viral particles (Gatto et al., 2007). The isotype of these B cells was, however, not known, but the authors speculated that these mutated MZ-B cell sequences were derived from class switched memory B cells. In humans, a relative large proportion (30%) of the MZ-B cells (defined as CD21+CD23−CD27+ cells) appear to express IgG (Ettinger et al., 2007); the mutational status of these class switched IGHV genes is also not known. Thus, although both IgM+ and IgG+ memory cells appear to be present among the pool of MZ-B cells in rodents and humans, direct evidence for presence of MZ-B cells with mutated IgG encoding genes is currently lacking. Furthermore, the origin of the IgG+ memory MZ-B cells is enigmatic. This prompted us to analyse in detail the nucleotide sequences of IgG encoding (IGHV-Cγ) transcripts from purified rat MZ-B cells, defined in a sIg independent fashion. We show that indeed naturally occurring (i.e. without deliberate antigenic stimulation) MZ-B cells express mutated IGHV genes encoding for IgG antibodies. The repertoire of the MZ-B cell derived IgG encoding transcripts does not differ from that obtained from class switched B cells with a FO-B cell phenotype, albeit that MZ-B cell derived IgG encoding transcripts exhibit lower numbers of mutations. 2. Materials and methods 2.1. Animals Male PVG rats were purchased from Harlan (Horst, The Netherlands) at the age of 6–8 weeks. Animals were maintained until use under clean conventional conditions at the central animal facility of the University Medical Center Groningen. Experiments were approved by the Animal Ethics Committee of the University of Groningen. 2.2. Flow-cytometry Spleens were taken from 4.5 to 8 months old animals. Single cell suspensions were prepared from spleen and labeled with mAb as described previously (Dammers et al., 1999). Briefly, spleen cell suspensions from 4 animals were stained for flow-cytometry with the following two sets of mouse monoclonal antibodies: FITC conjugated anti-rat IgM (HIS40; eBioscience, San Diego, CA, USA) and biotinylated anti-rat IgD (MaRD3; AbD Serotec, Oxford, UK) or FITC anti-rat MZ-B cell marker (HIS57; BD Pharmingen, San Diego, CA, USA; (Dammers et al., 1999) and biotinylated anti-rat CD45R (HIS24; Ebioscience). Biotinylated mAb were revealed with 118 | Chapter 6 streptavidin conjugated to the tandem fluorochrome PE-Cy5.5 (Ebioscience). The two sets of antibodies were used in combination with a mixture of PE conjugated anti-rat TCRαβ (R73; eBioscience); TCRγδ (V65; eBioscience), CD90/Thy1.1 (HIS51; eBioscience) and CD161a/ NKR-P1a (10/78; BD Pharmingen). The PE channel was used as a “Dump” channel; only PE negative (Dump−) cells were sorted. Herewith, we were able to exclude immature B cells (i.e. CD90 positive B cells: (Kroese et al., 1995), T cells and NK cells from our sorts. Cell analysis and cell sortings were performed on a MoFlo flow cytometer (Cytomation, Ft Collins, CO). Dead cell, plasma cell, monocyte/macrophage, and erythrocyte contamination was excluded from sorting by using forward and side scatter profiles. Sorted cells were collected in sterile FACS tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) containing 500µl of newborn calf serum (PAA laboratories GmbH, Pasching, Austria). At least one million cells per B cell subset were sorted. B cell subsets were obtained with >95% purity. FlowJo software (Tree Star, San Carlos, CA) was used for flow cytometry data analysis. 2.3. Molecular cloning of rearranged IGHV5-Cγ transcripts Total RNA was extracted from sorted cells using the Absolutely RNA Miniprep kit Stratagene, La Jolla, CA, USA) according to instructions of the manufacturer. Briefly, sorted cells were pelleted by 300×g centrifugation for 10 min at 4°C and then resuspended in a total volume of 350 ml lysis buffer containing β-mercaptoethanol (Stratagene). First strand cDNA was synthesized using an oligo-(dT) 12–18 primer (Invitrogen, Breda, The Netherlands) and SuperScriptTMII reverse transcriptase (200 U/ml; Invitrogen) as described in the manufacturer’s protocol. Rearranged immunoglobulin IGHV5-Cγ transcripts were amplified in a 50ml reaction mixture, containing 2µl cDNA and 0.6 pmol/ml IGHV5 (PC7183) family specific primer (5ˈ-CTTAGTGCAGCCTGGAAGGT-3ˈ; (Dammers et al., 2000), 0.6 pmol/ml universal Cγ constant region primer (5ˈ-GACAGGGATCCAGAGTTCCA-3ˈ) and 2.5U Taq DNA Polymerase (Invitrogen). The universal Cγ region primer was designed on the basis of a conserved sequence found in exon 1 of all rat IgG subclasses. To assess the amount and quality of the cDNA, PCR was also performed for β-actin, using β-actin specific primers as described by (Stoel et al., 2008). The PCR program for amplification of IGHV5-Cγ transcripts and β-actin consisted of 35 cycles of 30s at 94°C (2min in first cycle), 1min at 58°C and 1min at 72°C, respectively. This program was followed by an additional incubation period of 25 min at 72°C to allow extension of all IGHV5-Cγ products. The quality and size of the PCR products was evaluated by agarose gel electrophoresis. PCR products were subsequently cloned into the pCR4-TOPO vector using the TOPO TA cloning kit (Invitrogen). Plasmid DNA was isolated from randomly picked colonies with the Nucleospin Plasmid QuickPure kit (Clontech, Mountain View, CA, USA). Plasmids containing an insert of approximately 600bp were sequenced in both directions at our local sequence facility (Department of Pathology and Laboratory Medicine, Division of Medical Biology, University Medical Center Groningen, Groningen, The Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 119 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Netherlands). Sequence processing was performed using ClustalW from the European Molecular Biology Laboratory and Chromas software (Digital River GmbH, Cologne, Germany). 2.4. Analysis of IGHV5-Cγ transcripts Analysis of IGHV5-Cγ transcripts (accession numbers pending), was carried out as described previously (Dammers et al., 2000). Briefly, IGHV region sequences were compiled according to the format of the International Immunogenetics (IMGT) database (http://imgt.cines.fr) (Lefranc et al., 1999b). Rearranged IGHV5 genes were compared to the 28 previously established PVG germline IGHV5 genes (Dammers and Kroese, 2001) and to two newly identified germline IGHV5 genes from PVG rat (PC-39 and PC-41). PC-39 and PC-41 were established on the basis the following GenBank database accession numbers: AJ286206, AJ286170, and AJ286224 (PC-39); AJ286269, AJ286226, and AJ286210 (PC-41). IGHV5 gene sequences were considered to be germline when two or more independently sampled, rearranged or genomic, IGHV5 gene sequence(s) share 100% identity upon alignment. Germline IGHD and IGHJ gene sequences were taken from the IMGT database. IgG subclasses were determined by aligning the Cγ nucleotide sequence from the IGHV5-Cγ transcripts to the NCBI rat genome database using the BLASTN program (http://www.ncbi.nlm.nih.gov). The accession numbers for the C region genes encoding for rat IgG subclasses are: IgG2a, BC088240; IgG2b, M28671; IgG1, BC095846; and IgG2c, X07189. 2.5. Statistical analysis Statistical analysis of the data was performed using SPSS 16 software (SPSS Inc. Chicago, IL, USA). IGHV5 sequences displaying 100% identity were considered to be derived from a single B cell and counted only once for statistical analysis. We used Fisher’s exact test for comparison of IGHV, IGHD, IGHJ gene usage, the subclass distribution of the expressed Cγ regions and the number of mutations between the different groups. In all statistical tests we considered a P-value < 0.05 to be significant. The number of mutations was determined by counting the number of nucleotide mismatches in comparison with each IGHV5 gene sequence to its closest germline counterpart. Possible differences in H-CDR3 length between different groups were tested with the Mann–Whitney test. The R/S mutation ratio is the quotient of replacement (R) to silent (S) mutations. R/S mutation ratios are calculated separately for HCDR1 and 2 and the H-FR chain. The theoretical expected (inherent) R/S mutation ratio is the quotient of total possible R to total possible S mutations in the germline gene, as described by (Chang and Casali, 1994). The probability that an excess or scarcity of R mutations in the H-CDR or the H-FR results solely from chance is negated by the significantly low probability values (P < 0.05) calculated according to the Binomial distribution. 120 | Chapter 6 3. Results 3.1. Mutated IGHV5-Cγ transcripts are found among B cells with a MZ-B cell phenotype Classical memory B cells are class switched and carry somatically hypermutated IGHV genes (see e.g. (Tangye and Tarlinton, 2009). In order to see whether class switched memory B cells with a MZ-B cell phenotype exist, we first analysed the presence of IgG encoding (IGHVCγ) transcripts among FACS-purified B cell subsets defined in a sIg-independent fashion. As shown in Figure. 1, FO-B cells are mature (i.e. CD90−; (Kroese et al., 1995) small-sized, HIS24highHIS57neg/low B cells whereas MZ-B cells are slightly larger cells and are defined as CD90−HIS24lowHIS57high cells (Dammers et al., 1999; Kroese et al., 1990). Plasma cells are not included in these two B cell fractions, since they lack expression of HIS24-determinant (CD45R) (Kroese et al., 1987). We first looked for the presence of IGHV-Cγ transcripts encoding for IGHV5 (PC7183) family genes using RT-PCR. For comparison, we also analysed IGHV5-Cγ transcripts from B cells with a FO-B cell phenotype (CD90−HIS24highHIS57neg/low) and from a fraction of cells that should include classical, class switched memory B cells, i.e. IgM−IgD− (non-T, non-NK) B cells. This IgM−IgD− fraction of B cells comprises class switched B cells with a FO-B cell and a MZ-B cell phenotype. In all four rats analysed, we found expression of IGHV5-Cγ transcripts in all three B cell fractions (i.e. MZ-B, FO-B, and IgM−IgD− cell phenotypes). These observations indicate that class switched B cells are present both among the sorted MZ-B cells and FO-B cells. Since presence of mutations is another hallmark of (most) classical memory B cells, IGHV5-Cγ transcripts from the three B cell subsets were subsequently cloned and sequenced. As shown in Table 1 (see below), nearly all sequences were uniquely and productively rearranged. Only very few 100% identical IGHV5-Cγ sequences were found (three sets in the FO-B cell fraction and one set in the IgM−IgD− cell fraction). These sequences were counted only once in our further analysis, since we could not rule out the possibility that they originate from the same cell. In total, we obtained 33 unique productive IGHV5-Cγ transcripts from the MZ-B cell fraction (HIS24lowHIS57high), 27 from the FO-B cell fraction (HIS24highHIS57neg/low) and 37 from the class switched B cell fraction (IgM−IgD−). Alignment of the constant region of these transcripts to known constant regions of rat revealed that all sequences were indeed encoded by Cγ genes (see below in Table 1), and that the V-regions of the transcripts were encoded by IGHV5 genes. Nearly all sequences reveal somatic hypermutations (SHM) upon comparison to the nearest germline sequence of PVG rats. Since Taq errors might be responsible for 1–2 mutations per sequence, we considered only sequences with more than 2 mutations as truly mutated (Dammers et al., 2000). Using this criterion, at least 80% of the IGHV5-Cγ sequences of all three fractions display SHM (see below in Table 1). Because of the presence of Cγ transcripts and mutated IGHV5 genes, we provide evidence for the existence of classical, class switched memory B cells in the phenotypically defined MZ-B cell and FO-B cell compartments. Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 121 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Dump- cells FO-B cells a MZ-B cells HIS24 HIS57 Dump- cells SSC b FSC TCRαβ, TCRγδ CD90, CD161a IgD IgM-IgDcells IgM Figure 1. Phenotype of MZ-B cells and FO-B cells in rat spleen. Spleen cell suspensions of rat spleen were stained with mAb directed against CD90, TCRαβ, TCRγδ and CD161a to exclude immature B cells, T cells and NK cells from the analysis (Dump channel). These mAb’s were combined with HIS24 (antiCD45R) HIS57, a mAb directed to an unknown determinant on MZ-B cells (panel a) or combined with mAb directed against IgM and IgD (panel b). MZ-B cells are defined as Dump-HIS24loHis57hi cells, and FO-B cells as Dump-HIS24hiHIS57-/low cells. Classical, class switched memory B cells are found among the Dump-IgM-IgD- fraction. SSC = side scatter, FSC = forward scatter. 3.2. IGHV5-Cγ transcripts from B cells with a MZ-B cell phenotype exhibit fewer somatic mutations compared to B cells with a FO-B cell phenotype The average number of mutations and the mutation frequency of the IGHV5-Cγ transcripts from B cells with a MZ-B cell phenotype appeared to be lower than that obtained from B cells with a FO-B cell phenotype: 7±4.9 (i.e. 2.9±2.07%) (mean±s.d.) vs 10±8.0 mutations (i.e. 4.3±3.36%) per IGHV gene, respectively (Table 1). Dividing the IGHV5-Cγ transcripts into four categories regarding the number of mutations, i.e. unmutated (0–2 mutations), low (3–5 mutations), intermediate (6–10 mutations) and high (>10 mutations), revealed that the number of mutations in IGHV5-Cγ transcripts from sorted MZ-B cells differ statistically significantly from transcripts from sorted FO-B cells (Fisher’s exact test, P = 0.004). As we show in Figure 2, transcripts from B cells with a MZ-B cell phenotype are enriched in the category with an intermediate number of mutations whereas most sequences from B cells with a FO-B cell phenotype and IgM−IgD− B cells are found in the category of a high number of mutations. 122 | Chapter 6 60 50 IgM-IgD-B cells Percentage 40 MZ-B cells 30 FO-B cells 20 10 0 0-2 3-5 6 - 10 > 10 Number of mutations Figure 2. IGHV genes encoding for IgG antibodies from B cells with a MZ-B or FO-B cell phenotype differ in mutation frequencies. PCR products from IGHV5-Cγ transcripts from FACS-purified MZ-B cells, FO-B cells and IgM-IgD- cells were cloned and sequenced. Sequences were compared to known germline IGHV5 genes from PVG rats, and the numbers of mutations compared to these germline genes was calculated. Sequences were divided into four groups based upon the number of mutations. Sequences with 0-2 mutations were considered to be unmutated, because we could not exclude the possibility that 1-2 mutations are the result of Taq errors. Class switched B cells with a FO-B and MZ-B cell phenotype have a statistically different mutation profile (Fisher’s exact test, P = 0.004). 3.3. Class switched memory type MZ-B cells have a similar IgG subclass distribution as their FO-B cell counterpart There are four IgG subclasses in rat: IgG1, IgG2a, IgG2b and IgG2c. Based upon sequence identity, we were able to establish the IgG subclasses of the IGHV5-Cγ trancripts. The constant region primer used to amplify IGHV5-Cγ trancripts was located in the first exon of the Cγ regions. Since this part of the Cγ region is identical for both the Cγ1 and Cγ2a regions (Bruggemann, 1988), it was not possible to discriminate between these two IgG subclasses. Figure 3 shows that the three B cell fractions express IGHV5-Cγ transcripts encoding for all IgG subclasses (IgG1/IgG2a, IgG2b and IgG2c). The usage of the various IgG subclasses appear, however, not to be statistically significant between the various B cell fractions (Fisher’s exact P = 0.127). Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 123 70 60 50 Percentage R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 IgM-IgD-B cells 40 MZ-B cells 30 FO-B cells 20 12 0 IgG1/IgG2a IgG2b IgG2c Isotype Figure 3. Subclasses distribution of IGHV5-Cγ transcripts derived from B cells with a MZ-B or FO-B cell phenotype are comparable. The subclasses of IGHV5-Cγ transcripts obtained from purified MZ-B cells, FO-B cells and IgM-IgD- B cells was determined by comparing the part of the PCR-amplified Cγ region to known rat Cγ genes encoding for the various rat IgG subclasses. The sequence of this part of Cγ1 and Cγ2a regions are identical to each other and cannot be discriminated. The usage of the various IgG subclasses is similar in the three B cell subsets (Fisher’s exact test, P = 0.127) 3.4. The repertoire of class switched IGHV region genes obtained from B cells with a MZ-B cell or FO-B cell phenotype is highly similar There are no substantial differences in the usage of individual IGHV5 family members among the IGHV5-Cγ transcripts derived from the three B cells fractions (see below Table 1). Some IGHV5 family members are, however, more frequently expressed in the different fractions. For example, B cells with a MZ-B cell phenotype appear to use most frequently PC-5 (20%), PC-29 (16%) and PC-4 (13%) to encode for their IgG antibodies, whereas B cells with a FO-B cell phenotype use most frequently PC-5 (15%), PC-1 (15%) and PC-34 (15%). These differences are, however, not statistically significant (Fisher’s exact test, P = 0.60). Of the four IGHJ genes, the IGHJ2 gene was the most abundantly found among the IGHV-Cγ transcripts in all three B cell fractions, compared to the other IGHJ gene members. Approximately 50– 60% of these IgG encoding transcripts appear to utilize the IGHJ2 gene, followed by IGHJ3 (20–30%) and the two other IGHJ genes. This biased usage of IGHJ2 genes is similar to the usage of IGHJ2 genes by mature, naive FO-B and MZ-B cells (Dammers et al., 2000). Also the utilization of IGHD genes by the IGHV5-Cγ transcripts is similar for all three B cell fractions (Fisher’s exact test, P = 0.794). Of the known functional IGHD genes (Hendricks et al., 2010) the IGHD genes 1–6 and 1–7 appear to be preferentially used to encode for IgG antibodies in all three fractions. We have previously shown that naive MZ-B cells and FO-B cells differ with respect to the length of H-CDR3 region (Dammers and Kroese, 2005; Dammers et al., 2000). This H-CDR3 region is the most important part of the H chain for antigen recognition. 124 | Chapter 6 On average, the length of the H-CDR3 region of naive MZ-B cells is 10.9±2.8 codons, which is 1.7 codons shorter than used by FO-B cells (Dammers et al., 2000). Here we show that the average length of the H-CDR3 regions of the sequenced IGHV5-Cγ transcripts derived from B cells with an MZ-B cell phenotype is 12.2±2.8 codons and does not differ from the H-CDR3 codon length of encoding for IgG antibodies expressed by B cells with a FO-B cell phenotype (12.6±3.4 codons) (Mann–Whitney test, P = 0.61). In summary, we conclude that there are no major differences in the primary IGHVDJ repertoire between the IgG expressing B cells with a MZ-B cell phenotype and a FO-B cell phenotype. 3.5. IGHV-Cγ transcripts from B cells with an MZ-B cell phenotype show signs of antigen selection High affinity antibodies which are generated during humoral immune responses generally result in amino acid replacements in the H-CDR regions. Selection of antibodies that can bind with high affinity to a particular antigen takes place within the germinal center (GC) during the formation of memory B cells. The replacement over silent mutation (R/S) ratio of antigenselected antibodies in the H-CDR regions is therefore higher than expected in case these mutations are randomly introduced. In contrast, selected antibodies favor relatively more silent mutations in the framework (FR) regions, resulting in lower R/S values than expected. In accordance to the method developed by (Chang and Casali, 1994) we calculated the binomial change for the R/S ratio of H-CDR and H-FR regions of the IGHV5-Cγ transcripts with more than 4 mutations. As we show in Table 1 (see below), approximately 40% of the IGHV5-Cγ sequences in all B cell fractions show signs of antigen selection, i.e. reveal a significantly higher R/S ratio for the H-CDR regions and/or a significantly lower R/S ratio for the H-FR regions than expected. Thus, there is evidence that both IgG antibodies produced by B cells with a MZ-B cell phenotype and FO-B cell phenotype are likely the result of antigen-selection. Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 125 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 126 | Chapter 6 Table 1. Sequence analysis of IGHV5-Cγ transcripts from MZ-B cells, FO-B cells and IgM-IgD- B cells from adult rat spleen. Clone Rat IGHV5 IGHD IGHJ Subclass H- member member member CDR H-CDR3 Mutationsb Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg 16 TTRTIAAISTSY- 2.53% 6 1/3 3.04 0.03689 1/1 3.63 0.40130 0.42% 1 0/0 3.1 0.39710 0/1 4.75 0.83270 1.27% 3 3/0 3.07 0.21760 0/0 4.44 0.58160 0.42% 1 1/0 3.07 0.60140 0/0 4.44 0.83470 1.69% 4 2/0 2.99 0.34710 2/0 3.21 0.10230 R/S mutationrate FR R/S mutation rate A. sequences from MZ-B cells MZ1 1 PC-15 IGHD1-7 IGHJ4 IgG2b VLDA MZ2 1 PC-3 IGHD5-1 IGHJ1 IgG1/IgG2a 13 ARPKNWEGWCFDF MZ3 1 PC-4 IGHD1-7 IGHJ2 IgG2c 14 ARHDGMMVVSPFGY MZ4 1 PC-39 IGHD1-7 IGHJ3 IgG2c 16 ARPGVTTVVTWNWFAY MZ5 1 PC-29 IGHD1-7 IGHJ4 IgG1/IgG2a 15 SKDYYYDASYYVMDA MZ6 1 PC-34 IGHD1-2 IGHJ3 IgG2c 12 TREDPDITWFSY 4.64% 11 4/1 3.13 0.06680 3/3 3.79 0.16800 MZ7 1 PC-5 IGHD1-2 IGHJ1 IgG2c 10 ARKDSWFFDF 0.00% 0 0/0 2.99 1.00000 0/0 4.48 1.00000 MZ8 1 PC-1 IGHD5-1 IGHJ2 IgG2b 9 ASLNWELDY 1.27% 3 1/1 3.11 0.28480 1/0 3.48 0.33520 MZ9 1 PC-4 IGHD1-7 IGHJ3 IgG2c 14 ARHSGMVVIT- 0.84% 2 2/0 3.07 0.36170 0/0 4.44 0.69670 0.27630 PFAY MZ10 1 PC-5 IGHD1-6 IGHJ2 IgG1/IgG2a 11 ASESYGGLFDY 0.84% 2 0/1 2.99 0.16210 1/0 4.48 MZ11 2 PC-5 IGHD4-1 IGHJ2 IgG2b 10 ARAIRDYFDY 4.64% 11 4/3 2.99 0.07209 2/2 4.48 0.29570 MZ12 2 PC-4 IGHD1-6 IGHJ2 IgG1/IgG2a 13 ARHDYGGY- 3.80% 9 4/1 3.07 0.16580 3/1 4.44 0.12830 MZ13 2 PC-29 IGHD1-3 IGHJ3 IgG2b 15 2.95% 7 2/1 2.99 0.07930 3/1 3.21 0.06591 0.14200 SELGY AKASYYFSSYNWFTY MZ14 2 PC-28 IGHD1-7 IGHJ3 IgG2b 9 ARYDAPLTY 4.22% 10 5/1 2.99 0.20280 3/1 3.55 MZ15 2 PC-6 IGHD1-6 IGHJ2 IgG2b 8 TTGGYGDY 3.38% 8 5/0 3.21 0.28030 3/0 3.79 0.09627 MZ16 2 PC-31 IGHD1-6 IGHJ4 IgG1/IgG2a 12 ARHPNYG- 2.11% 5 0/1 3.01 0.01039 2/2 3.86 0.15290 2 PC-39 IGHD5-1 IGHJ2 IgG2c 8 TAGTGFEY 2.53% 6 4/1 3.07 0.31180 1/0 4.44 0.40190 3 PC-5 IGHD1-6 IGHJ2 IgG1/IgG2a 12 ARHEEGAGYFDY 3.38% 8 3/3 2.99 0.12630 2/0 4.48 0.25910 3 PC-11 IGHD1-7 IGHJ2 IgG1/IgG2a 11 ARQRGSYYPDY 6.33% 15 8/3 3.04 0.17710 2/2 4.56 0.27320 PLMDA MZ17 MZ18 MZ19 j k j Clone Rat IGHV5 IGHD IGHJ Subclass H- member member member CDR H-CDR3 Mutationsb R/S mutationrate FR R/S mutation rate Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 127 Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg MZ20 3 PC-36 IGHD4-4 IGHJ4 IgG1/IgG2a 11 ARLGIAGVMDA 3.38% 8 4/3 3.03 0.23250 1/0 3.96 0.37630 MZ21 3 PC-28 IGHD1-4 IGHJ3 IgG2b 13 STLRYYGYNPFGY 3.38% 8 3/1 2.99 0.12630 4/0 3.55 0.02193 MZ22 3 PC-22 IGHD1-5 IGHJ2 IgG2b 10 ARHFITTFDY 1.27% 3 1/0 2.96 0.29190 2/0 3.86 0.06515 3 PC-4 IGHD1-1 IGHJ2 IgG2b 15 ARHLGATTVVT- 2.53% 6 2/0 3.07 0.13690 3/1 4.44 0.05254 0.00% 0 0/0 3.11 1.00000 0/0 3.48 1.00000 10.55% 25 12/5 2.99 0.07848 7/1 4.48 0.06307 MZ23 l PFDY MZ24 3 PC-1 IGHD1-4 IGHJ1 IgG2c 16 ARTYYGYNPHYWYFDF MZ25 3 PC-5 IGHD1-7 IGHJ4 IgG2b 20 ARPKPVTIFSDGSLGFVLDA MZ26 4 PC-3 IGHD3-3 IGHJ2 IgG2b 11 VRAEYLHYFDY 3.80% 9 4/0 3.1 0.16440 4/1 4.75 0.03952 MZ27 4 PC-26 IGHD3-4 IGHJ3 IgG1/IgG2a 8 AGDVPFTY 3.80% 9 2/3 3.16 0.01951 4/0 4.48 0.03831 MZ28 4 PC-22 No D IGHJ2 IgG2b 8 ARHPYFDY 3.80% 9 5/0 2.96 0.25250 4/0 3.86 0.03511 0.03099 present MZ29 4 PC-29 IGHD1-6 IGHJ2 IgG2b 11 ARQEPLRGFDY 3.80% 9 3/2 2.99 0.07629 4/0 3.21 MZ30 4 PC-36 IGHD1-3 IGHJ1 IgG2b 13 TRFGYSRYWYFDF 4.64% 11 4/4 3.03 0.07036 2/1 3.96 0.29400 MZ31 4 PC-29 IGHD1-6 IGHJ2 IgG1/IgG2a 13 VKDGINNGG- 3.80% 9 6/0 2.99 0.24910 2/1 3.21 0.26540 MZ32 4 PC-5 IGHD5-1 IGHJ2 IgG1/IgG2a 12 ARETTGDYYFDY 3.38% 8 3/1 2.99 0.12630 4/0 4.48 0.02550 MZ33 4 PC-27 IGHD1-4 IGHJ2 IgG2b 12 ARRELGITLFDY 1.69% 4 0/0 3.16 0.02412 4/0 3,93 0,00068 PFDY B. Sequences from FO-B cells FO1 1 PC-22 IGHD1-3 IGHJ2 IgG2b 10 ARRAYSSYPY 0.42% 1 0/0 2.96 0.40410 1/0 3.86 0.16090 FO2 1 PC-3 IGHD4-1 IGHJ2 IgG2b 13 AAGNSGQRG- 0.84% 2 1/0 3.1 0.47880 1/0 4.75 0.27860 FFDY FO3 1 PC-34 IGHD1-4 IGHJ3 IgG1/IgG2a 13 TRLSPGITRPFAY 4.64% 11 4/4 3.13 0.06680 3/0 3.79 0.16800 FO4 1 PC-31 IGHD1-6 IGHJ1 IgG1/IgG2a 13 ARQGEGIT- 2.11% 5 3/0 3.01 0.34560 2/0 3.86 0.15290 FO5 1 PC-41 IGHD1-7 IGHJ3 IgG1/IgG2a 15 ARHQDG- 0.42% 1 0/1 3.2 0.39260 0/0 4.44 0.83470 3.80% 9 3/1 3.11 0.07190 2/3 4.56 0.27850 WYFDF SYYYSWFAY FO6 1 PC-24 IGHD3-1 IGHJ4 IgG1/IgG2a 14 TTDANYPGTYIMDA R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 128 | Chapter 6 Clone Rat IGHV5 IGHD IGHJ Subclass H- member member member CDR H-CDR3 Mutationsb R/S mutationrate FR R/S mutation rate Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg FO7 1 PC-27 IGHD1-7 IGHJ2 IgG2b 10 ARGWSGTLDY 2.53% 6 2/1 3.16 0.13280 3/0 3.93 0.04962 FO8 1 PC-28 IGHD1-7 IGHJ3 IgG2c 10 TRGWNNWFPY 2.53% 6 1/1 2.99 0.03793 3/1 3.55 0.04709 FO9 1 PC-4 IGHD4-4 IGHJ4 IgG1/IgG2a 16 ARHPPNLLLG- 2.11% 5 3/0 3.07 .34560 2/0 4.44 0.15890 FO10 1 PC-5 IGHD1-1 IGHJ3 IgG1/IgG2a 13 2.11% 5 1/2 2.99 0.07850 2/0 4.48 0.15930 GYVMDA ARRDMDPWDWFAY FO11 2 PC-1 IGHD1-6 IGHJ2 IgG2b 7 ATSGGSY 5.49% 13 5/3 3.11 0.06310 5/0 3.48 0.03157 FO12 2 PC-1 IGHD1-6 IGHJ3 IgG1/IgG2a 14 AREGDMAAG- 0.84% 2 0/1 3.11 0.15740 0/1 3.48 0.71000 AWFAY FO13 i FO14 2 PC-34 IGHD3-2 IGHJ2 IgG1/IgG2a 14 SRGGGFIAAIYFDY 4.64% 11 4/0 3.13 0.06680 5/2 3.79 0.01713 2 PC-1 IGHD1-7 IGHJ1 IgG2c 14 ARHRTMVVIT- 5.49% 13 5/1 3.11 0.06310 5/2 3.48 0.03157 PFDF FO15 2 PC-5 IGHD1-4 IGHJ2 IgG1/IgG2a 9 VRHGPGYKF 7.59% 18 6/5 2.99 0.01531 6/1 4.48 0.04358 FO16 2 PC-14 IGHD1-7 IGHJ3 IgG2b 19 AKKGTYFYHG- 8.02% 19 8/5 2.96 0.05618 5/1 4.15 0.11110 SYDVGWFAY i,j FO17 2 PC-5 IGHD3-4 IGHJ3 IgG2b 11 AKASTANWFAY 8.02% 19 10/3 2.99 0.14870 6/0 4.48 0.05315 FO18 2 PC-11 IGHD1-7 IGHJ2 IgG1/IgG2a 18 TRGDPIYYYDGSY- 15.61% 37 17/9 3.04 0.02963 9/2 4.56 0.07403 FO19 3 PC-26 IGHD5-1 IGHJ1 IgG1/IgG2a 13 6.75% 16 7/3 3.16 GYFDY ARLLNWEL- 0.07865 6/0 4.48 0.02700 WYFDF FO20 3 PC-1 IGHD1-6 IGHJ2 IgG1/IgG2a 10 AKDKNYGGFY 6.75% 16 7/5 3.11 0.08098 3/1 3.48 0.23560 FO21 3 PC-2 No D IGHJ2 IgG2b 5 TTRDY 0.42% 1 0/0 3.03 0.40040 1/0 3.48 0.15740 0.06171 present FO22 3 PC-23 IGHD1-6 IGHJ2 IgG2a/IgG1 9 TTTEALFDY 4.64% 11 5/2 3.11 0.14390 4/0 3.55 FO23 3 PC-34 IGHD5-1 IGHJ2 IgG2c 10 TRENWLPGYN 1.27% 3 1/0 3.13 0.28380 2/0 3.79 0.06471 FO24 3 PC-4 IGHD1-6 IGHJ1 IgG1/IgG2a 18 TRHPPGE- 6.75% 16 9/2 3.07 0.18820 3/2 4.44 0.24150 FO25 3 PC-26 IGHD1-7 IGHJ3 IgG1/IgG2a 18 5.06% 12 5/3 3.16 0.09550 3/1 4.48 0.19580 5.06% 12 7/2 2.99 0.22750 2/1 4.48 0.29610 i GFSDHSWYFDF ARLAYYYDGSYYYGRFAY FO26k 3 PC-5 IGHD1-6 IGHJ2 IgG1/IgG2a 12 ARHEEGAGYFDY Clone FO27 Rat 3 IGHV5 IGHD IGHJ Subclass H- member member member CDR PC-24 IGHD1-4 IGHJ2 IgG2b H-CDR3 Mutationsb R/S mutationrate FR R/S mutation rate Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg 11 TRDPGITGFDY 2.11% 5 3/1 3.11 0.34560 1/0 4.56 0.40160 C. Sequences from IgM− IgD−cells h Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 129 CM1 1 PC-29 IGHD1-5 IGHJ2 IgG1/IgG2a CM2 1 PC-35 IGHD5-1 IGHJ2 IgG1/IgG2a 1 PC-24 IGHD4-4 IGHJ3 1 PC-27 IGHD1-7 IGHJ2 1 PC-31 IGHD1-4 IGHJ2 CM3 CM4 i 10 AKDGKQLFDS 4.64% 11 2.99 0.02430 4/0 3.21 0.05803 12 AKRLPGYHYFDY 3.38% 8 4/2 3/4 3.10 0.23000 2/0 3.79 0.25220 IgG1/IgG2a 4 GMGQ 5.06% 12 2/4 3.11 6/0 4.56 IgG1/IgG2a 14 ASPSWHYT- 2.11% 5 4/1 3.16 0.26560 0/0 3.93 0.41460 IgG2b 15 ARHPLFRYNSLGF- 0.84% 2 1/0 3.01 0.48050 1/0 3.86 0.27000 0.00% 0 0/0 2.99 1.00000 0/0 4.48 1.00000 5.91% 14 4/3 3.11 0.01280 7/0 3.48 0.00247 0.42% 1 1/0 3.11 0/0 4.56 0.00232 0.00653 GHAGDY CM5 DY CM6 1 PC-5 IGHD3-4 IGHJ3 IgG1/IgG2a 11 ARHEVGGWFAY CM7 j 1 PC-1 IGHD4-1 IGHJ3 IgG1/IgG2a 16 TTDLIIRGRDPNWFVY CM8 1 PC-24 IGHD1-6 IGHJ1 IgG1/IgG2a 17 TTGGGSSYIYPG- 0.60330 0.83390 WYFDF CM9 1 PC-24 IGHD1-6 IGHJ2 IgG1/IgG2a 12 TTETYGGSYFDY 5.91% 14 3/3 3.11 0.00306 8/0 4.56 CM10 2 PC-34 IGHD1-8 IGHJ2 IgG2b 14 TSGDYDGYY- 2.95% 7 2/3 3.13 0.07435 2/0 3.79 0.22520 0.00059 CM11 2 PC-14 IGHD5-1 IGHJ2 IgG2b 12 ARHGPSNSLFDY 0.84% 2 1/1 3.04 0.48000 0/0 4.15 0.70020 CM12 2 PC-15 IGHD1-8 IGHJ2 IgG2b 12 ARHPIVEDYFDY 0.84% 2 1/1 3.04 0.48000 0/0 3.63 0.70760 CM13 2 PC-4 IGHD4-2 IGHJ3 IgG1/IgG2a 12 TRRGKVGDWFAY 1.27% 3 1/0 3.07 0.28660 0/2 4.44 0.58160 CM14 2 PC-34 IGHD1-3 IGHJ2 IgG1/IgG2a 13 ARQPFYS- 1.69% 4 3/0 3.13 0.34930 1/0 3.79 0.37960 2.53% 6 2/0 2.96 0.14210 2/2 3.86 0.19250 PAGDY GYPFDY CM15 2 PC-22 IGHD1-6 IGHJ4 IgG2b 14 TRSGRLTTKGVMDA CM16 2 PC-14 IGHD1-7 IGHJ2 IgG2b 11 SRHDFYGFPED 4.64% 11 5/2 3.04 0.14720 4/0 4.15 0.06725 CM17 2 PC-34 IGHD3-2 IGHJ1 IgG1/IgG2a 13 SRQGSHQN- 2.95% 7 3/3 3.13 0.18930 1/0 3.79 0.39340 2.11% 5 1/1 3.03 0.07706 3/0 3.48 0.02767 WYFDF CM18 2 PC-2 IGHD1-3 IGHJ2 IgG1/IgG2a 16 TRDRSKFDYSGYYFDY R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 130 | Chapter 6 Clone Rat CM19 3 IGHV5 IGHD IGHJ Subclass H- member member member CDR PC-8 IGHD1-7 IGHJ2 IgG2c H-CDR3 Mutationsb Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg 14 TRQRFTMMP- 1.27% 3 1/0 3.14 0.28340 2/0 4.00 0.06600 7.17% 17 9/4 3.11 0.15790 4/0 3.48 0.15760 2.11% 5 2/0 2.99 0.23290 3/0 4.48 0.03160 5.91% 14 5/1 3.07 0.03998 7/1 4.44 0.00327 R/S mutationrate FR R/S mutation rate VDFDY CM20 3 PC-1 IGHD1-6 IGHJ2 IgG2b 13 ARPPYGGYGLLDY CM21 3 PC-5 IGHD1-7 IGHJ1 IgG1/IgG2a 20 ARQTYFYDGSYYYRYWYFDF CM22 l 3 PC-4 IGHD1-1 IGHJ2 IgG2c 15 ARHLGATTVVTPFDY CM23 3 PC-25 IGHD1-8 IGHJ3 IgG1/IgG2a 14 ARSTITAISNWFAY 4.64% 11 7/2 2.96 0.23480 2/0 3.86 0.29360 CM24 3 PC-27 IGHD1-4 IGHJ2 IgG2b 13 ARHEYRY- 0.84% 2 0/2 3.16 0.15530 0/0 3.93 0.70320 2.53% 6 2/0 3.21 0.13080 3/1 3.79 0.04876 NYYFDY CM25 3 PC-6 IGHD1-6 IGHJ1 IgG2b 15 AAYGGYSELAWYFDL CM26 3 PC-8 IGHD1-2 IGHJ2 IgG1/IgG2a 13 ARRGILWVPYFDY 5.06% 12 7/1 3.14 0.22600 4/0 4.00 0.08294 CM27 3 PC-41 No D IGHJ3 IgG2b 7 ARHWFAY 0.42% 1 0/0 3.20 0.39260 0/1 4.44 0.83470 IGHJ2 IgG1/IgG2a 20 ARQERFYSTF- 5.06% 12 3/3 2.99 0.01304 6/0 4.48 0.00643 4.22% 10 5/3 3.04 0.20070 1/1 3.63 0.33490 present CM28 3 PC-5 IGHD1-6 SYSGLDFFDY CM29 4 PC-15 IGHD1-1 IGHJ2 IgG1/IgG2a 17 SRYPIIYYGLLSRPFDY CM30 4 PC-5 IGHD1-5 IGHJ2 IgG2b 11 AREELDTYYGY 1.27% 3 1/0 2.99 0.29050 2/0 4.48 0.06863 CM31 4 PC-1 IGHD1-2 IGHJ3 IgG2b 12 ARPGHSGFWFAY 3.38% 8 4/0 3.11 0.22970 3/1 3.48 0.09270 CM32 4 PC-5 IGHD3-1 IGHJ2 IgG1/IgG2a 11 ARRPTVSPFDY 4.64% 11 4/1 2.99 0.07209 4/2 4.48 0.06985 CM33 4 PC-29 IGHD3-3 IGHJ3 IgG1/IgG2a 9 TKVSNCFGY 1.69% 4 1/0 2.99 0.15600 3/0 3.21 0.01246 CM34 4 PC-39 IGHD1-3 IGHJ4 IgG2b 15 ARHGQYSSYD- 3.80% 9 4/1 3.07 0.16580 4/0 4.44 0.03812 DVMDV CM35 4 PC-5 IGHD1-4 IGHJ2 IgG1/IgG2a 9 TPVGYGHNY 7.59% 18 9/5 2.99 0.13100 3/1 4.48 0.24520 CM36 4 PC-15 IGHD1-7 IGHJ2 IgG2b 14 TRDPSYYYSNSL- 0.84% 2 2/0 3.04 0.35990 0/0 3.63 0.70760 DY Clone CM37 a Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 131 b c d e f g h i j k l Rat 4 IGHV5 IGHD IGHJ Subclass H- member member member CDR PC-24 IGHD1-3 IGHJ2 IgG1/IgG2a H-CDR3 Mutationsb Na Amino acids Fc Nd Obse Expf Pg Obse Expf Pg 9 TTWDYYSSY 2.11% 5 3/1 3.11 0.34560 1/0 4.56 0.40160 R/S mutationrate FR R/S mutation rate Length of H-CDR3 in amino acids. Revealed from nucleotide position 52 (codon 18) up to and including 312 (codon 104) according to IMGT nomenclature (http://imgt.cines.fr) (Lefranc et al., 1999a). Mutation frequency (percent). Number of mutations. Observed R/S mutation ratio is the quotient of observed R to observed S mutations in H-FR of H-CDR3 regions. The actual number of R and S mutations are given for the observed R/S ratios. The theoretical expected R/S ratio is the quotient of total possible R and total possible S mutations in the germline gene, and is calculated according to (Chang and Casali, 1994). The possibility (P) that an excess or scarcity of replacement mutations in H-FR or H-CDR3 regions result solely by chance is negated by the significantly low probability values (P < 0.05) calculated according to the binomial distribution (Chang and Casali, 1994). Since the used constant region primer was located in the first exon of the C regions, it is not possible to discriminate between IgG1 and IgG2a subclas ses. Sequences found twice. Alternative family member are possible: for CM7: PC-22; for MZ17: PC-41; for MZ19: PC-26 or PC-33; for FO17: PC-14. The alternative family members give rise to same number of mutations and have no effect on significance levels of R/S ratios. The sequences MZ18 and FO26 are from clonally related B cells and form clone set #1 (see text). The sequences CM22 and MZ23 are from clonally related B cells and form clone set #2 (see text). R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 3.6. Clonally related B cells are found in both MZ-B cell fraction and FO-B cell fractions IGHV sequences with identical H-CDR3 regions obtained incompletely separate PCR’s are considered to be derived from clonally related cells. We found two sets of two clonally related sequences in one rat (rat #3). In this animal one of the MZ-B cell derived sequences (MZ18) was clonally related to a FO-B cell derived sequence (FO26) (set #1), and another MZ-B cell derived sequence (MZ23) was clonally related to an IgM−IgD− B cell derived sequence (CM22), whereas the two members of set #2 express different IgG subclasses (IgG2b and IgG2c, respectively). There are no shared mutations between the sequences of both members of set #1 and set #2, indicating that the two members of each clone may have developed independently from each other from one single naive B cell, upon antigenic stimulation. 4. Discussion Most MZ-B cells present in rodent spleen show characteristic features of naive B cells, i.e. they express IgM molecules on their membrane of which the variable domains are encoded by unmutated V-region genes. A small fraction of IgM+ MZ-B cells, however, carry mutated V-genes and are qualified as IgM+ memory MZ-B cells. In this study we provide molecular evidence for the existence of a third type naturally occurring B cell population with a MZ-B cell phenotype in rat spleen, viz. class switched memory MZ-B cells. Previous studies described the appearance of antigen-specific, class switched MZ-B cells (and FO-B cells) after immunization of rats and mice with protein antigens (Gatto et al., 2004; Liu et al., 1988; Obukhanych and Nussenzweig, 2006), and that antigen (virus like particle)-specific MZ-B cells are encoded by somatically mutated IGHV genes (Bergqvist et al., 2010; Gatto et al., 2007). The work presented here, combines these data by directly showing the presence of somatically mutated IGHV5 transcripts encoding for IgG antibodies among the pool of purified rat MZ-B cells. These two characteristics, class switching recombination (CSR) and SHM, are hallmarks of classical, class switched memory B cells (Tangye and Tarlinton, 2009). The developmental origin of class switched memory MZ-B cells is not clear. Also the developmental relationship with their FO-B cell counterpart remains to be established. Memory B cells are generally believed to be generated in germinal centers (GC’s) (for reviews see e.g. (MacLennan, 1994; Manser, 2004). Proliferating GC B cells alter their BCR’s by SHM and CSR on their way to become memory B cells. (Toyama et al., 2002) have shown that Bcl6-deficient mice, which cannot develop GC’s, are still able to generate class switched memory B cells after immunization with a foreign protein antigen, albeit that the V-genes of these memory B cells are not mutated. Similarly, CD40−/− mice, which also cannot form GC, are still able to generate intestinal IgA plasma cells whereas SHM in these cells are absent (Bergqvist et al., 2010). These findings illustrate that SHM, but not CSR, during the humoral immune response to exogenous 132 | Chapter 6 antigens are indispensable of the GC microenvironment. The somatic mutations are, in principle, randomly introduced into the IGHV genes (Winter and Gearhart, 1998). Mutated B cells are subsequently subjected to positive selection for B cells expressing BCR’s that bind with high affinity to the inducing antigen. To this end, follicular dendritic cells uniquely located in the GC’s present immune complexes to the proliferating B cells. Many of the IGHV5-Cγ transcripts from class switched memory B cells with a MZ-B or FO-B cell phenotype show signs of selection. An appreciable proportion of the IGHV5-Cγ genes exhibit significantly more R mutations in H-CDR’s and/or significantly fewer R mutations in the H-FR’s, than expected from a random distribution of mutations. This indicates that after acquiring somatic mutations during proliferation the cells must have undergone some form of selection of their BCR’s. Thus, the observation that the mutation patterns of IgG transcripts obtained from both B cells with a MZ-B or FO-B cell phenotype show signs of antigen-selection therefore favors the notion that both types of memory cells are probably GC derived. It could be that class switched memory B cells with a MZ-B or FO-B cell phenotype are derived from antigen stimulated naive MZ-B cells and naive FO-B cells, respectively. Transfer experiments of purified B cell subsets into SCID mice have indeed revealed that both MZ-B cells and FO-B cells can generate GC’s upon stimulation with T cell-dependent (protein) antigen, albeit that MZ-B cells appear to be far less effective (Song and Cerny, 2003). Furthermore, both anti-hen egg lysozyme (HEL) transgenic MZ-B cells and FO-B cells, transferred into wild type recipients, are capable of generating a robust anti-HEL IgG1 response and forming GC’s after immunization with a protein antigen (Phan et al., 2005). The entry of MZ-B cells into GC’s in these experiments was, however, delayed. Although it thus seems possible that class switched MZ-B cells and FO-B cells are derived from their own naïve counterparts, we have some arguments to assume that naive B cells that give rise to class switched memory B cells with a MZ-B or FO-B cell phenotype belong to the same B cell pool. First, even within the relatively small number of sequences analysed in this study, we detected a set of sequences derived from clonally related cells (i.e. IGHV5 sequences with identical H-CDR3 regions, and same IGHV genes) with members in both the MZ-B and FO-B cell fraction (clone #1). Apparently, descendants of one and the same activated (naive) B cell can become either a class switched B cell with a FO-B cell phenotype or a class switched MZ-B cell. Second, there are no differences in the primary, unmutated, H-chain repertoire between these two memory B cell subsets since usage of IGHV, IGHD and IGHJ gene segments is similar. Third, also the H-CDR3 lengths of the IGHV-Cγ transcripts between B cells with a FO-B or MZ-B cell phenotype are comparable: 12.2±2.8 codons for class switched MZ-B cells and 12.6±3.4 codons for the class switched FO-B cells, respectively. This is in contrast to naive, IgM expressing, MZ-B cells which have significantly shorter H-CDR3 regions compared to naive, IgM expressing, FO-B cells (Dammers et al., 2000; Makowska et al., 1999). Also (Gatto et al., 2007) observed that the length of H-CDR3 regions expressed by virus-specific (possibly class switched) MZ-B cells and FO B cells are Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 133 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 comparable. Notably, the average H-CDR3 length of both class switched B cell subsets is identical to the reported H-CDR3 length of naive FO-B cells (Dammers et al., 2000). Together, these observations may suggest that class switched memory MZ-B cells are probably derived from naive FO-B cells and not from naive MZ-B cells, with their shorter H-CDR3 regions. In this context we like to mention that also naive FO-B cells can develop into naive MZ-B cells (Dammers et al., 1999; Guay et al., 2009; Srivastava et al., 2005; Vinuesa et al., 2003). Although the repertoire of naturally occurring class switched memory MZ-B cells is very similar to the repertoire of naturally occurring class switched B cells with a FO-B cell phenotype, the numbers of mutations between the two subsets vary significantly. Class switched MZ-B cells are enriched in the category of 7–10 mutations per sequence, whereas class-switched B cells with a FO-B cell phenotype have most sequences in the category of >10 mutations per sequence (Fig. 2). A similar mutational difference has been observed by (Gatto et al., 2007) between antigen-specific B cells with a FO-B cell phenotype and a MZ-B cell phenotype after immunization with virus like particles. As shown by (Bende et al., 2007) recirculating class switched IgG memory cells in humans can participate in several successive GC reactions herewith acquiring possibly more mutations. In vivo intravital imaging in mice revealed that GC’s are open structures and that high affinity antigen-specific B cells can participate in preexisting GC’s (Schwickert et al., 2009). The difference in mutation frequency between class switched memory B cells with a MZ-B or FO-B cell phenotype might therefore be explained by the migratory properties of MZ-B cells. It could be that similar to naive MZ-B cells also class switched memory MZ-B cells are sessile cells. In contrast, FOB cells recirculate between the various lymphoid organs, herewith giving them the opportunity to participate in multiple GC reactions, and acquiring more mutations. An alternative explanation for the difference in mutation frequencies between class switched B cells with a MZ and FO-B cell phenotype might be that there are intrinsic differences between the precursor cells for the two different types of memory cells, such as levels in activation induced deaminase. This enzyme plays a critical role in both CSR and SHM (Maul and Gearhart, 2010). As mentioned before, purified murine MZ-B cells and FO-B cells can give rise to IgG expressing memory B cells upon transfer into recipient animals followed by immunization (Phan et al., 2005; Song and Cerny, 2003). It could be that levels of activation induced deaminase in activated MZ-B cells are lower, compared to activated FO-B cells, possibly as a result of differences in the signaling requirements of the two subsets. Lower levels of this enzyme may consequently result in lower mutation frequencies. Preliminary data suggest that there are no significant differences in levels of mRNA encoding for activation induced deaminase in flow cytometry purified rat B cells with a MZ-B or FO-B cell phenotype (data not shown). Since class switched B cells with a FO-B cell phenotype carry more mutations in their IgG encoding transcripts than MZ-B cells, it is very unlikely that class switched B cells with FO-B cell phenotype simply acquire a MZ-B cell phenotype, e.g. during differentiation towards plasma cells. This is supported by the observation 134 | Chapter 6 that class switched B cells with a MZ-B cell phenotype are absent from lymph nodes, whereas memory cells with a FO-B cell phenotype are present (Bergqvist et al., 2010; Gatto et al., 2007). In summary, the present study shows that in addition to naïve MZ-B cells and mutated IgM+ memory MZ-B cells, also class switched somatically mutated cells with a MZ-B cell phenotype are present in rat spleen. It remains, however, to be formerly proven that these class switched B cells also reside in the anatomically defined splenic MZ. We speculate that these class switched memory MZ-B cells are derived from naïve FO-B cells and are generated in GC’s. The function of these classical, memory type MZ-B cells, is not known. Excitingly, (Ettinger et al., 2007) demonstrated that human IgG+ MZ (like) B cells can respond vigorously in an antigen- and T cell-independent fashion to the combination of IL-21 and B cell activating factor belonging to the TNF family (BAFF). Triggering by these cytokines results in the rapid differentiation of IgG+ MZ-B cells into IgG-secreting plasma cells. Whether class switched memory MZ-B cells in rodents respond similarly remains to be seen. These B cells in human splenic MZ’s are in close association with CD4+ TH-cells and dendritic cells that could potentially secrete IL-21 and BAFF, respectively (Ettinger et al., 2007). In rodents, however, T cells are absent from the MZ. Class switched MZ-B cells may provide the immune system with a sessile pool of memory B cells that reflects the antigenic experience of the animal. These cells may respond rapidly to the presence of blood-borne antigens by producing IgG antibodies in addition to naive MZ-B cell-derived IgM antibodies, herewith contributing to humoral immunity in this extremely dangerous situation. Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 135 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Bende R. J., van Maldegem F., Triesscheijn M., Wormhoudt T. A., Guijt R. and van Noesel C. J., 2007. Germinal centers in human lymph nodes contain reactivated memory B cells. The Journal of experimental medicine 204, 2655-65. Bergqvist P., Stensson A., Lycke N. Y. and Bemark M., 2010. T cell-independent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation. Journal of immunology (Baltimore, Md. : 1950) 184, 3545-53. Bruggemann M., 1988. Evolution of the rat immunoglobulin gamma heavy-chain gene family. Gene 74, 473-482. Chang B. and Casali P., 1994. The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement. Immunol.Today 15, 367-373. Dammers P. M., de Boer N. K., Deenen G. J., Nieuwenhuis P. and Kroese F. G., 1999. The origin of marginal zone B cells in the rat. Eur.J.Immunol. 29, 1522-1531. Dammers P. M. and Kroese F. G., 2001. Evolutionary relationship between rat and mouse immunoglobulin IGHV5 subgroup genes (PC7183) and human IGHV3 subgroup genes. Immunogenetics 53, 511-517. Dammers P. M. and Kroese F. G., 2005. Recruitment and selection of marginal zone B cells is independent of exogenous antigens. European journal of immunology 35, 2089-99. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of Immunology. 165, 6156-6169. Ettinger R., Sims G. P., Robbins R., Withers D., Fischer R. T., Grammer A. C., Kuchen S. and Lipsky P. E., 2007. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. J.Immunol. 178, 2872-2882. Gatto D., Bauer M., Martin S. W. and Bachmann M. F., 2007. Heterogeneous antibody repertoire of marginal zone B cells specific for virus-like particles. Microbes.Infect. 9, 391-399. Gatto D., Ruedl C., Odermatt B. and Bachmann M. F., 2004. Rapid response of marginal zone B cells to viral particles. Journal of Immunology. 173, 4308-4316. Guay H. M., Mishra R., Garcea R. L., Welsh R. M. and Szomolanyi-Tsuda E., 2009. Generation of protective T cell-independent antiviral antibody responses in SCID mice reconstituted with follicular or marginal zone B cells. Journal of immunology 183, 518-23. Guinamard R., Okigaki M., Schlessinger J. and Ravetch J. V., 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat.Immunol. 1, 31-36. Hendricks J., Terpstra P., Dammers P. M., Somasundaram R., Visser A., Stoel M., Bos N. A. and Kroese F. G., 2010. Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat. Immunogenetics 62, 479-86. Kroese F. G., Butcher E. C., Lalor P. A., Stall A. M. and Herzenberg L. A., 1990. The rat B cell system: the anatomical localization of flow cytometry-defined B cell subpopulations. European journal of immunology 20, 1527-34. Kroese F. G., de Boer N. K., de Boer T., Nieuwenhuis P., Kantor A. B. and Deenen G. J., 1995. Identification and kinetics of two recently bone marrow-derived B cell populations in peripheral lymphoid tissues. Cellular immunology 162, 185-93. Kroese F. G., Wubbena A. S., Opstelten D., Deenen G. J., Schwander E. H., De L. L., Vos H., Poppema S., Volberda J. and Nieuwenhuis P., 1987. B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens. Eur.J.Immunol. 17, 921-928. Lefranc M. P., Giudicelli V., Ginestoux C., Bodmer J., Muller W., Bontrop R., Lemaitre M., Malik A., Barbie V. and Chaume D., 1999a. IMGT, the international ImMunoGeneTics database. Nucleic acids research 27, 209-12. Lefranc M. P., Giudicelli V., Ginestoux C., Bodmer J., Muller W., Bontrop R., Lemaitre M., Malik A., Barbie V. and Chaume D., 1999b. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 27, 209-212. 136 | Chapter 6 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. Liu Y. J., Oldfield S. and MacLennan I. C., 1988. Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. European journal of immunology 18, 355-62. MacLennan I. C., 1994. Germinal centers. Annu.Rev.Immunol. 12, 117-139. Makowska A., Faizunnessa N. N., Anderson P., Midtvedt T. and Cardell S., 1999. CD1high B cells: a population of mixed origin. European journal of immunology 29, 3285-94. Manser T., 2004. Textbook germinal centers? The Journal of Immunology 172, 3369-3375. Martin F. and Kearney J. F., 2002. Marginal-zone B cells. Nat.Rev.Immunol. 2, 323-335. Martin F., Oliver A. M. and Kearney J. F., 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity. 14, 617-629. Maul R. W. and Gearhart P. J., 2010. AID and somatic hypermutation. Advances in immunology 105, 159-91. Obukhanych T. V. and Nussenzweig M. C., 2006. T-independent type II immune responses generate memory B cells. The Journal of experimental medicine 203, 305-10. Oliver A. M., Martin F., Gartland G. L., Carter R. H. and Kearney J. F., 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. European journal of immunology 27, 2366-74. Oliver A. M., Martin F. and Kearney J. F., 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. Journal of immunology 162, 7198-207. Pape K. A., Kouskoff V., Nemazee D., Tang H. L., Cyster J. G., Tze L. E., Hippen K. L., Behrens T. W. and Jenkins M. K., 2003. Visualization of the genesis and fate of isotype-switched B cells during a primary immune response. The Journal of experimental medicine 197, 1677-87. Phan T. G., Gardam S., Basten A. and Brink R., 2005. Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen. Journal of immunology 174, 4567-78. Pillai S., Cariappa A. and Moran S. T., 2005. Marginal zone B cells. Annu.Rev.Immunol. 23, 161196. Schwickert T. A., Alabyev B., Manser T. and Nussenzweig M. C., 2009. Germinal center reutilization by newly activated B cells. The Journal of experimental medicine 206, 2907-14. Song H. and Cerny J., 2003. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. The Journal of experimental medicine 198, 192335. Srivastava B., Quinn W. J., 3rd, Hazard K., Erikson J. and Allman D., 2005. Characterization of marginal zone B cell precursors. The Journal of experimental medicine 202, 1225-34. Steiniger B., Timphus E. M. and Barth P. J., 2006. The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants. Histochemistry and cell biology 126, 641-8. Stoel M., Evenhuis W. N., Kroese F. G. and Bos N. A., 2008. Rat salivary gland reveals a more restricted IgA repertoire than ileum. Mol.Immunol. 45, 719-727. Tangye S. G. and Tarlinton D. M., 2009. Memory B cells: effectors of long-lived immune responses. European journal of immunology 39, 2065-75. Toyama H., Okada S., Hatano M., Takahashi Y., Takeda N., Ichii H., Takemori T., Kuroda Y. and Tokuhisa T., 2002. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 17, 329-39. Vinuesa C. G., Sze D. M., Cook M. C., Toellner K. M., Klaus G. G., Ball J. and MacLennan I. C., 2003. Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens. European journal of immunology 33, 297-305. Weill J. C., Weller S. and Reynaud C. A., 2009. Human marginal zone B cells. Annual review of immunology 27, 267-85. Winter D. B. and Gearhart P. J., 1998. Dual enigma of somatic hypermutation of immunoglobulin variable genes: targeting and mechanism. Immunological reviews 162, 89-96. Class switched marginal zone B cells in spleen have relatively low numbers of somatic mutations | 137 Chapter 7 Summary and general discussion Nederlandse samenvatting en discussie Opsomming en algemene bespreking in het Afrikaans Acknowledgements R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Summary and general discussion The marginal zone (MZ) is a well-defined anatomical compartment of the spleen that encloses the follicles and periarteriolar lymphocyte sheaths (PALS). Together, these three structures form the so-called “the white pulp”, which contain mostly lymphocytes. The MZ forms an interface with the red pulp, an area which is very rich in venous sinuses. Next to macrophages and dendritic cells, the MZ is primarily populated with a unique population of B cells, the MZ-B cells, with unique phenotypical, developmental and functional properties. Because of their anatomical location at the border of the red pulp, and their rich supply of capillaries, that are widely open (Kusumi et al., 2015). MZ-B cells are thought to play an important role in rapid immune responses against blood-borne pathogens. In particular, they are believed to respond rapidly to polysaccharide antigens (TI-2 antigens) which are present on the surface of encapsulated bacteria, such as pneumococci and meningococci (Guinamard et al., 2000; Kruetzmann et al., 2003; Martin et al., 2001; Vinuesa et al., 2003). Herewith the MZ and MZ-B cells in particular, may play an important role in the prevention of sepsis. Previous experimental data have provided evidence of the presence of both naive B cells and memory B cells in the MZ (Colombo et al., 2013; Dammers et al., 2000a; Dunn-Walters et al., 1995; Gatto et al., 2007; Gatto et al., 2004; Makowska et al., 1999; Tangye et al., 1998; Weller et al., 2004b; Zandvoort et al., 2001). Naïve B cells are cells that have not responded yet to antigen and are characterized by the expression of IgM molecules on their cell surface membrane, which are encoded by unmutated immunoglobulin (Ig) variable region (IGV) genes. Naïve MZ-B cells are likely derived from transitional B cells or follicular (FO) B cells. In contrast to naïve B cells, memory B-cells are antigen experienced cells, which frequently express also non-IgM isotypes on their membrane, which are encoded by mutated IGV genes. The presence of somatic hypermutations (SHM) in the IGV regions of Ig genes are the primary hallmark of memory B cells. Mutations are introduced both in the Ig heavy chain (IGH) and Ig light chain V regions genes during humoral immune responses when antigen-activated B cells expand in a T cell dependent (TD) fashion in the germinal centers (GC’s). Although, GC’s are generally believed to be the sole sites where this SHM of IGV genes takes place, the origin of mutated MZ-B cells is, however, still elusive. This thesis aims to analyse the memory compartment of splenic MZ-B cells in the rat, in particular to get some insights into the origin and function of these memory MZ-B cells. As experimental animal model for the study of the immune system, rats offer a selective advantage over mice, because in comparison to mice, rats have a much larger MZ which can be easily visualised. Rat immunoglobulin variable region genes For establishing the presence of SHM in IGV genes it is of crucial importance to have information about the genomic sequences of their germline V gene counterparts (unrearranged ge- Summary and general discussion | 141 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 nomic sequences) available. In view of this it was necessary to identify germline IGV genes of the rat. Furthermore, understanding the genomic organization of the organization of the IGH locus of the rat, including the localization of germline nucleotide sequences of all IGH chain variable genes, may help to unravel further possible differences in humoral immune responses and Ig repertoires between rats on the one hand and humans and mice on the other hand. These studies in rats became possible when the genome sequence of the Brown Norway (BN/SsNHsdMCW) rat (Rattus norvegicus) became available in 2004 and generated by the Rat Genomic Sequence Consortium (RGSC) (Gibbs et al., 2004; Havlak et al., 2004). This genome sequence includes the IGH locus located on chromosome 6q32–33. Of note, the BN rat strain that was used in this analysis differs from other inbred rats strains, also at the IGH locus. For example, Dammers et al. found by restriction fragment length polymorphism analysis that the BN rat differs significantly from other (e.g. PVG, AO, Lew) rat strains in terms of numbers of IGV genes at the IGH locus that belong to the IGHV5 family (Dammers, 2001). These data indicate that the BN rat strain contains probably a lower number of IGHV5 genes compared to other rat strains. The V part of the IGH chain is encoded by three different gene segments (genes): IGH variable (IGHV), IGH diversity (IGHD), and IGH joining (IGHJ) gene segments, whereas the immunoglobulin kappa variable (IGK) and immunoglobulin lambda (IGL) chain V domains are encoded by a combination of (IGKV–IGKJ) and immunoglobulin kappa variable (IGLV–IGLJ) genes, respectively (Roth, 1996). Although the genomic sequence of the rat was published, the exact number and location of IGHV genes including IGHD and IGHJ genes were not established. Therefore we first analysed several available sources for germline IGHV, IGHD, and IGHJ genes. These sources were: the genomic sequence RGSC genome assembly version 3.4, the unmapped sequences from contigs in the “unplaced section” of the NCBI database and newly established bactigs of the BN rat genome (Baylor College of Medicine; http://www.hgsc.bcm. tmc.edu/projects/rat) not yet present in assembly RGSC V3.4. In chapter 2 we present an annotated map of the V region of the IGH locus of the BN rat, including not only functional and non-functional IGHV genes but also the IGHD and IGHJ genes. In chapter 3 we provide an update on the rat IGH chain locus in the rat in comparison to mouse and humans, and describe what is currently known about the organization of the Ig light chain locus in the rat. In total we were able to identify 353 IGHV genes, of which 131 appear to be functional. This number of 131 functional IGHV genes in rats is the highest among mammalian species of which the whole genome sequences are available, in line with the prediction of Das (2009). This number might even increase since the whole genome map of the variable region of the IGH locus displays a number of small gaps (~300 kb) of which the nucleotide sequence still has to be determined. By analysing the rat genomic sequence of the IGVH locus we have identified 11 new IGHV genes in the BN rat. These novel IGHV genes were unidentified IGHV genes that were not 142 | Chapter 7 placed in the current assembly (RGSC V3.4) available at the NCBI database and also not listed in the International Immunogenetics (IMGT) database (http://imgt.cines.fr) (Lefranc et al., 1999). These genes were found among rat genomic sequences available from the Baylor College of Medicine Human Genome Sequencing Group that were not yet incorporated into the rat assembly of the BN rat at that time. Thus including the 11 newly identified IGHV genes, the BN rat genome comprises at least 353 individual IGHV genes. Similar to the mouse, rat IGHV genes can be subdivided into IGHV gene families, on the basis of nucleotide sequence identity. Individual IGHV genes belong to the same family when the IGHV genes share more than 80% of their nucleotides (Brodeur and Riblet, 1984). From the 11 newly discovered IGHV genes, 6 belong to the IGHV2 gene family and 5 belong to the IGHV5 gene family. Furthermore because the largest gap regions on the IGH locus of which the nucleotide sequence still needs to be determined is also located in the area of the IGHV2 and IGHV5 gene families it is plausible to assume that these 11 newly discovered IGHV genes are located in gap region found in the IGHV2-IGHV5 regions of assembly V3.4. In summary our studies have provided evidence that rats harbours the highest number of (functional) germline IGHV genes among all mammalian species studies so far. Given the notion that BN rats may have a lower number of IGHV5 genes compared to other rat strains (Dammers et al., 2001), and the fact that there are some gaps in the genomic sequence, in particular in the area of the IGHV2 and IGHV5 gene families it is plausible to assume that the number of IGHV genes in the rat might even be higher. Similar to the mouse and human, the vast majority of rat IGHV genes are orientated in the same direction as the IGHD and IGHJ genes and therefore deletional joining, instead of inversional joining, is most preferably used as recombination mechanism at the IGH locus. For both rats and mouse the overall density of IGHV genes (number of IGHV genes per Mb nucleotide sequence) is the highest at the 3’end of the IGHV region in comparison to the 5’end. As expected, rats and mouse share a close evolutionary relationship as shown by the almost identical organization of IGHV gene families and the distribution of IGHV genes at the IGH locus. At present, little is known about the genomic organization of the rat Ig light chain loci including IGK and IGL chain genes. Similar to the IGHV chain genes, rats and mouse share almost the same number of IGKV genes with rats harbouring slightly more functional IGKV genes than the mouse. Overall, the comparison between the two species indicates that they have maintained the same basic structure of their IG loci including a similar order of IGHVDJ arrangement. In these species diversity in the IG antigen-recognition site is established during early B cell development by rearrangement of V region genes (or gene segments) located at the IGH and IGL chain loci. Recombination at the IGH, IGK and IGL loci result in the generation of a highly diversified IG (antibody) primary repertoire with a wide range of antibody specificities. Summary and general discussion | 143 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 IgM expressing marginal zone memory B cells Previous experiments provide evidence for the existence of mutated, IgM expressing, memory MZ-B cells in rat (Dammers et al., 2000b). Dammers et al. demonstrated that less than 20% of the MZ-B cells isolated from spleens of PVG rats carried mutated IGHV genes. These findings were in marked contrast with humans, where >95% of the splenic MZ-B cells are mutated (Colombo et al., 2013; Dunn-Walters et al., 1995; Tangye et al., 1998). One possible explanation for this difference could be that only one particular IGHV gene family (viz. the IGHV5 family, the homologue of PC7183 in the mouse) has been analysed in the (PVG) rat and that this IGHV gene family was not representative for other IGHV genes, or IGHV gene families. By establishing the genomic germline IGHV gene repertoire of the BN rat (chapter 2), it became possible to accurately analyse other IGHV gene families as well. To avoid possible strain differences we used the BN rat strain, instead of the PVG rat strain that was used before (Dammers et al., 2000b). In chapter 4 we report on the frequency of mutated sequences in rearranged IGHV-Cµ transcripts derived from FACS sorted MZ-B cells (IgMhighIgDlow) in comparison with FO-B cells (IgMlowIgDhigh) from adult BN rat spleen. The analysis was confined to three different IGHV gene families, which differ in size: IGHV3, IGHV4 and IGHV5. These three IGHV gene families have 4, 2 and 26 functional IGHV genes, respectively. The IGHV3 and IGHV4 genes families were chosen to determine whether there is a difference in mutation frequencies among members of IGHV gene families that are relatively small and to compare this frequency to the second largest IGHV gene family (IGHV5) in the rat, which had also been analysed before in the PVG rat (Dammers et al., 2000a). The BN rat strain contains 26 functional IGHV5 (germline) genes compared to the 28 germline genes in the PVG rat. As was shown before, we found that splenic MZ-B cells express a significantly higher percentage of mutated sequences than FO-B cells and all three analysed IGHV gene families contributed to this difference. In BN rats a slightly higher proportion (27%) of the MZ-B cells expressed mutated IgM molecules encoded by IGHV5 family genes, compared to this proportion in the PVG rat (10-20%) (Dammers et al., 2000a). This difference in mutation frequency can be due to the strain differences that exist between the PVG and BN rat strain, such as for example the fact that BN rats have fewer IGHV genes, or might also be caused by different environmental conditions (microbial environment; different microbiota) of the two rat strains. Analysis of the IGHV3 gene family showed a similar proportion (approximately 30%) of mutated IgM encoding sequences can be found among BN rat derived MZ-B cells. In marked contrast, to these two IGHV families, a very high proportion (66%) of the IGHV4 sequences from purified MZ-B cells was mutated. This family consists of only two potential functional IGHV genes, albeit that only one of these members appeared to be functionally expressed. Our findings thus show that the proportion of mutated sequences derived from MZ-B cells varies between the different IGHV gene families in the BN rat and that in total a higher proportion (27-66%) of IGHV genes were mutated compared to the (10-20%) of mutated sequences found previously 144 | Chapter 7 for the IGHV5 gene family in PVG rats (Dammers et al., 2000a; Makowska et al., 1999). Our observation that the highest percentage of mutated frequencies occurred in the single functional member IGVH4 gene family, suggests that there could be more antigen selection pressure on this particular IGHV4 gene in expanding its available repertoire by SHM. Although in total a higher average number of mutated sequences among rat MZ-B cells was observed than previously, the frequency of mutated sequences among human MZ-B cells is still much higher. In humans, nearly all MZ-B cells are mutated (Colombo et al., 2013; Dunn-Walters et al., 1995; Tangye et al., 1998). Our observation that there is variation in frequency of mutated IGHV genes between the different IGHV families, may also contribute to the difference in mutated MZ-B cells between humans and rats, since in humans the analysis of mutated IGHV genes was restricted to a restrictive set of IGHV genes. Dunn-Walters et al. analysed only two particular IGHV genes: the IGHV6 gene and IGHV4.21 gene. It is formally possible that these IGHV genes are more mutated than other genes. However the analysis of Tangye et al. (1998) has proven that Ig genes isolated from IgM+ memory B cells among IGHV5 and IGHV6 gene families were all mutated and shows that the high frequency of mutations is not only due to individual IGHV genes. Further, Colombo et al. (2013) investigated and compared the presence of mutations in human IGHV1, IGHV3 and IGHV4 gene families among splenic derived MZ-B cells (IgMhighCD27+), GC B cells and class switched B cells. Also they found that most of the MZ-B cells were mutated, albeit with a lower average number of mutations than both GC and class switched B cells. However, the average number of mutations in human MZ-B cells (11.8) (Colombo et al., 2013) is higher both for rat IgM+ MZ-B cells (8.8) and rat IgG+ MZ-B cells (7) (Hendricks et al., 2011). This might be due to the fact that humans have fewer functional IGHV genes than rats. We postulate that the higher number of germline IGHV genes in rodents have as a consequence that rats require fewer mutations to diversify their antibody repertoire after immunization than humans, because rats can encode for a larger pool of different antibodies for their primary repertoire. In addition, it is possible that differences in life span and environmental conditions also contribute to differences in average mutation frequency per IGHV gene. During their (long) lives humans may encounter much more different antigens than laboratory rats that live in well-controlled laboratorial conditions. Memory cells are generally believed to be generated in GC’s. It is still controversial, however, whether mutated (memory) IgM+ MZ-B cells are derived from GC’s or whether they represent a GC-independent B cell population. In humans, Colombo et al. (Colombo et al., 2013) observed a small number of clonally related sequences that were shared between MZ-B cells and GC B cells, indicative that mutated IgM+ MZ-B cells can be derived from GC’s. In contrast, Weill et al. (Weill et al., 2009; Weller et al., 2004a; Weller et al., 2008) suggested that the (mutated) IgM+ MZ-B cells are not GC-derived memory B cells. Instead, these authors postulated that the mutations in human MZ-B cells are acquired during their development in order to diversity their primary repertoire, in a GC (and T-cell) independent fashion. To test this Summary and general discussion | 145 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 hypothesis in rats we investigated in chapter 5 the possible presence of mutated IgM+ MZ-B cells in neonatal rats. Neonatal rats do not develop GC in the first weeks of their life (Kroese et al., 1987; van Rees et al., 1986). Thus, when MZ-B cells are unmutated in neonatal rats this would strongly argue against the hypothesis of Weill et al. (Weill et al., 2009; Weller et al., 2004a; Weller et al., 2008), that SHM is part of the developmental program of MZ-B cells. To this end, we analysed IGHV-Cµ transcripts using IGHV4 and IGHV5 gene families from both MZ-B cells and FO-B cells (chapter 4). However, no mutations at all were found in any of the neonatal sequences, even not in IGHV4 gene family genes with the highest number of mutated sequences (66%) in the adult rat. These results support the notion that at least in rats, mutated IgM+ MZ-B cells seen in adult animals are bona fide memory cells, which are most probably generated under the influence of external antigenic stimuli in GC. IgG expressing marginal zone memory B cells In addition to unswitched (IgM+) MZ-B cells, also class switched B cells can be found within the human (Ettinger et al., 2007b) and rodent MZ (Gatto et al., 2004). The phenotype of these cells is not clear, nor whether their IGHV genes exhibit SHM, as hallmark of memory B cells. This issue was analysed in chapter 6, where we analysed class switched (IgG+) MZ-B cells. To analyse IgG+ expressing B cells with a MZ-B cell phenotype we obviously could not use IgM and IgD to define MZ-B cells. Therefore, we made use of the fact that rat MZ-B cells express low levels of CD45R, defined by monoclonal antibody HIS24, and high levels of a surface molecule defined by the monoclonal antibody HIS57. Mature (CD90-) MZ-B cells can thus defined as CD90−HIS24lowHIS57high cells, and FO-B cells CD90−HIS24highHIS57neg/low cells. From purified (FACS-sorted) MZ-B cells (CD90−HIS24lowHIS57high) and FO-B cells (CD90−HIS24highHIS57neg/lo) we amplified rearranged IGHV-Cγ transcripts, specific for the IGHV5 gene family. For comparison, we also amplified IGHV-Cγ transcripts from a fraction of cells that should include classical class switched memory B cells, i.e. IgM−IgD− B cells. We were able to detect the presence of IGHV5-Cγ transcripts in all B cell subsets analysed, implying that IgG expressing cells can apparently exhibit at least two different phenotypes: cells with a MZ-B cell phenotype and cells with a FO-B cell phenotype. Analysis of the individual IGHV5 genes used by these IgG transcripts revealed that almost all IGHV5 genes were mutated, as is typical for memory B cells. We did observe that IgG expressing MZ-B cells had a lower number of mutations compared to IgG expressing FO-B cells. There were no differences in the usage of IGHV, IGHD and IGHJ genes between the IgG+ MZ-B cells and IgG+ FO-B cell subsets and the H-CDR3 lengths were also comparable between these two subsets. An important observation was that we found sets of clonally related IgG-encoding sequences (sequences with identical H-CDR3 regions, and usage of the same IGHV genes) derived from cells with members both in MZ-B cell (CD90−HIS24lowHIS57high) and FO-B cell (CD90−HIS24highHIS57neg/ low ) fractions. Such sets of clonally related sequences were also found among the mutated 146 | Chapter 7 IgM+ MZ-B cells and IgM+ FO-B cells (chapter 4). These observations strongly indicate that (mutated) memory B cells with a MZ-B cells phenotype and a FO-B cell phenotype have a common origin. The origin of these cells is not clear. Classical memory B cells (i.e. mutated class switched B cells) are usually generated in the GC. At these sites the mutated B cells undergo some form of selection for the antigen that drives the GC reaction. This antigen selection is crucial to increase the affinity of the Ig molecules that recognize the antigen, and is reflected in the fact that the IGHV genes that encode for the Ig molecules exhibit not random mutations. Some of the mutation patterns of IgG encoded sequences from both the MZ-B cell fraction and FO-B cell fraction show indeed signs of antigen selection (Chapter 6), and thus favours the hypothesis that they are both generated in the GC’s. The finding that mutated IgM+ memory B cells are absent in neonatal animals, in which GC’s cannot be formed yet, supports the hypothesis that also IgM+ memory MZ-B cells are generated in the GC. The common origin of IgM+ and IgG+ memory MZ-B cells and FO-B cells, illustrated by the presence of clonally related mutated sequences between MZ-B and FO-B cells, suggest that at least some GCderived memory cells can either acquire a FO-B cell phenotype or a MZ-B cell phenotype. The factors that drive this differentiation towards these two phenotypes are not known. Concluding remarks B cells in the MZ are a heterogeneous population of cells and both naïve MZ-B cells, class switched and unswitched memory MZ-B cells are present at this unique site in the spleen. Naive MZ-B cells carry unmutated Ig genes, produce low affinity IgM molecules and constitute a first line of defense against invading pathogens. The antibody repertoire expressed by these B cells has been suggested to be selected to bind to carbohydrate, carried by micro-organisms (e.g. Galson et al., 2015). These antigens do not require T cell help for their responses. Furthermore, the heavy chains of the IgM molecules expressed by naïve MZ-B cells contain shorter H-CDR3 regions compared to FO-B cells in rats (Dammers et al., 2000a) and mice (Carey et al., 2008). Shorter H-CDR3 is associated with polyreactive antibody responses i.e. the binding of an antibody to several different structural antigenic elements (Schroeder et al., 1995). Memory MZ-B cells express high affinity Ig molecules, directed to (microbial) antigens that have been encountered during life. Thus the presence of naïve MZ-B cells and memory MZ-B cells allows the MZ to make rapid innate-like and adaptive antibody responses to microbial antigens, to both TI and TD antigens. A novel role for neutrophils in the response of MZ-B cells has been proposed by Puga et al. (Puga et al., 2012). Neutrophils present in the spleen induce IgM+ production by activating MZ-B cells via BAFF, APRIL and IL-21 to make antibody responses to TI-2 antigens such as LPS, after induction of BLIMP-1 in the activated MZ-B cells (Puga et al., 2012). BAFF and IL-21 can also stimulate IgG+ MZ-B cells in a T cell independent fashion, to become antibody secreting plasma cells (Ettinger et al., 2007a). Summary and general discussion | 147 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 MZ-B cells, are in some form of pre-activated state, and they express high levels of complement receptor (CR) CD21 (Timens et al., 1989) and toll like receptors (TLR’s) (Gunn and Brewer, 2006) which underlie the fact that MZ-B cells are equipped for rapid and easy activation in (primary) immune responses. Further Garraud et al. (Garraud et al., 2012) suggest that shuttling of MZ-B cells between the MZ and follicles and transport of antigens (immune complexes) to follicular dendritic cells (FDC’s) decipher the role of MZ-B cells as antigen presenting cells to participate in immune responses that generate high affinity antibodies. They observed that type 1 interferon produced in response to blood-borne pathogens, inactivates the sphingosin-1-phosphate receptor 1 (S1P1) and S1P3, allowing MZ-B cells to migrate from the MZ in response to CXCL13, which is highly expressed in follicles. Down regulation of CXCR5 allows MZ-B cells to exit from the follicles and return to the MZ. Herewith MZ-B cells transport immune complexes towards the follicles where the immune complexes are subsequently be captured by FDC’s in a complement dependent manner. The immune complexes trapped by the FDC’s are involved in the selection of mutated B cells that express Ig with higher affinities with the help of follicular helper T (Tfh) cells. First FDC’s will present antigen to the B cells that undergo SHM, and class switch recombination (CSR). Then these B cells will go on to present antigens to Tfh cells which will deliver survival signals to this high affinity GC B–cells, leading to selection. The selected GC B cells will then either differentiate into memory B cells or plasma cells. MZ-B cells express the inhibitory IgG binding Fc-like receptor FcRL5, (Wilson et al., 2012). After binding IgG, this receptor can inhibit the BCR. As is known for FcRL4, that binds IgA, and also inhibits the BCR, it is possible that occupation of FcRL5 by IgG similarly acts as an adaptive innate molecular switch dampening the BCR signaling and enhancing the TLR signaling (Sohn et al., 2011). Herewith MZ-B cells become more innate-like B cells that do not rely so much anymore on their BCR for their activation, but more on TLRs. Herewith these cells can respond rapidly to microbiological antigens present in the blood, in a BCR independent fashion. Since MZ-B cells have a broad repertoire, contain memory B cells (IgM and IgG) and are in a kind of pre-activated state these cells are ideally suited to respond rapidly to a broad range of blood borne antigens to prevent sepsis to occur. Targeting these B cells by vaccination in the future will be crucial for efficient protection against life-threatening infections. 148 | Chapter 7 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Brodeur P. H. and Riblet R., 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. European journal of immunology 14, 922-30. Carey J. B., Moffatt-Blue C. S., Watson L. C., Gavin A. L. and Feeney A. J., 2008. Repertoire-based selection into the marginal zone compartment during B cell development. The Journal of experimental medicine 205, 2043-52. Colombo M., Cutrona G., Reverberi D., Bruno S., Ghiotto F., Tenca C., Stamatopoulos K., Hadzidimitriou A., Ceccarelli J., Salvi S., Boccardo S., Calevo M. G., De Santanna A., Truini M., Fais F. and Ferrarini M., 2013. Expression of immunoglobulin receptors with distinctive features indicating antigen selection by marginal zone B cells from human spleen. Molecular medicine (Cambridge, Mass.) 19, 294-302. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000a. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. Journal of immunology (Baltimore, Md. : 1950) 165, 6156-69. Dammers P. M., Visser A., Popa E. R., Nieuwenhuis P. and Kroese F. G., 2000b. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. J.Immunol. 165, 6156-6169. Dunn-Walters D. K., Isaacson P. G. and Spencer J., 1995. Analysis of mutations in immunoglobulin heavy chain variable region genes of microdissected marginal zone (MGZ) B cells suggests that the MGZ of human spleen is a reservoir of memory B cells. The Journal of experimental medicine 182, 559-66. Ettinger R., Sims G. P., Robbins R., Withers D., Fischer R. T., Grammer A. C., Kuchen S. and Lipsky P. E., 2007a. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. J.Immunology 178, 2872-82. Ettinger R., Sims G. P., Robbins R., Withers D., Fischer R. T., Grammer A. C., Kuchen S. and Lipsky P. E., 2007b. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. J.Immunol. 178, 2872-2882. Galson J. D., Clutterbuck E. A., Truck J., Ramasamy M. N., Munz M., Fowler A., Cerundolo V., Pollard A. J., Lunter G. and Kelly D. F., 2015. BCR repertoire sequencing: different patterns of B-cell activation after two Meningococcal vaccines. Immunol Cell Biol. Garraud O., Borhis G., Badr G., Degrelle S., Pozzetto B., Cognasse F. and Richard Y., 2012. Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond. BMC immunology 13, 63. Gatto D., Bauer M., Martin S. W. and Bachmann M. F., 2007. Heterogeneous antibody repertoire of marginal zone B cells specific for virus-like particles. Microbes.Infect. 9, 391-399. Gatto D., Ruedl C., Odermatt B. and Bachmann M. F., 2004. Rapid response of marginal zone B cells to viral particles. J.Immunol. 173, 4308-4316. Gibbs R. A., Weinstock G. M., Metzker M. L., Muzny D. M., Sodergren E. J., Scherer S., Scott G., Steffen D., Worley K. C., Burch P. E., Okwuonu G., Hines S., Lewis L., DeRamo C., Delgado O., Dugan-Rocha S., Miner G., Morgan M., Hawes A., Gill R., Celera, Holt R. A., Adams M. D., Amanatides P. G., Baden-Tillson H., Barnstead M., Chin S., Evans C. A., Ferriera S., Fosler C., Glodek A., Gu Z., Jennings D., Kraft C. L., Nguyen T., Pfannkoch C. M., Sitter C., Sutton G. G., Venter J. C., Woodage T., Smith D., Lee H. M., Gustafson E., Cahill P., Kana A., Doucette-Stamm L., Weinstock K., Fechtel K., Weiss R. B., Dunn D. M., Green E. D., Blakesley R. W., Bouffard G. G., De Jong P. J., Osoegawa K., Zhu B., Marra M., Schein J., Bosdet I., Fjell C., Jones S., Krzywinski M., Mathewson C., Siddiqui A., Wye N., McPherson J., Zhao S., Fraser C. M., Shetty J., Shatsman S., Geer K., Chen Y., Abramzon S., Nierman W. C., Havlak P. H., Chen R., Durbin K. J., Egan A., Ren Y., Song X. Z., Li B., Liu Y., Qin X., Cawley S., Worley K. C., Cooney A. J., D’Souza L. M., Martin K., Wu J. Q., Gonzalez-Garay M. L., Jackson A. R., Kalafus K. J., McLeod M. P., Milosavljevic A., Virk D., Volkov A., Wheeler D. A., Zhang Z., Bailey J. A., Eichler E. E., et al., 2004. Genome sequence of Summary and general discussion | 149 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493-521. Guinamard R., Okigaki M., Schlessinger J. and Ravetch J. V., 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat.Immunol. 1, 31-36. Gunn K. E. and Brewer J. W., 2006. Evidence that marginal zone B cells possess an enhanced secretory apparatus and exhibit superior secretory activity. Journal of immunology (Baltimore, Md. : 1950) 177, 3791-8. Havlak P., Chen R., Durbin K. J., Egan A., Ren Y., Song X. Z., Weinstock G. M. and Gibbs R. A., 2004. The Atlas genome assembly system. Genome research 14, 721-32. Hendricks J., Visser A., Dammers P. M., Burgerhof J. G., Bos N. A. and Kroese F. G., 2011. Classswitched marginal zone B cells in spleen have relatively low numbers of somatic mutations. Molecular immunology 48, 874-82. Kroese F. G., Wubbena A. S., Kuijpers K. C. and Nieuwenhuis P., 1987. The ontogeny of germinal centre forming capacity of neonatal rat spleen. Immunology 60, 597-602. Kruetzmann S., Rosado M. M., Weber H., Germing U., Tournilhac O., Peter H. H., Berner R., Peters A., Boehm T., Plebani A., Quinti I. and Carsetti R., 2003. Human immunoglobulin M memory B cells controlling Streptococcus pneumoniae infections are generated in the spleen. J.Exp.Med. 197, 939-945. Kusumi S., Koga D., Kanda T. and Ushiki T., 2015. Three-dimensional reconstruction of serial sections for analysis of the microvasculature of the white pulp and the marginal zone in the human spleen. Biomedical research (Tokyo, Japan) 36, 195-203. Lefranc M. P., Giudicelli V., Ginestoux C., Bodmer J., Muller W., Bontrop R., Lemaitre M., Malik A., Barbie V. and Chaume D., 1999. IMGT, the international ImMunoGeneTics database. Nucleic acids research 27, 209-12. Makowska A., Faizunnessa N. N., Anderson P., Midtvedt T. and Cardell S., 1999. CD1high B cells: a population of mixed origin. European journal of immunology 29, 3285-94. Martin F., Oliver A. M. and Kearney J. F., 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity. 14, 617-629. Puga I., Cols M., Barra C. M., He B., Cassis L., Gentile M., Comerma L., Chorny A., Shan M., Xu W., Magri G., Knowles D. M., Tam W., Chiu A., Bussel J. B., Serrano S., Lorente J. A., Bellosillo B., Lloreta J., Juanpere N., Alameda F., Baro T., de Heredia C. D., Toran N., Catala A., Torrebadell M., Fortuny C., Cusi V., Carreras C., Diaz G. A., Blander J. M., Farber C. M., Silvestri G., CunninghamRundles C., Calvillo M., Dufour C., Notarangelo L. D., Lougaris V., Plebani A., Casanova J. L., Ganal S. C., Diefenbach A., Arostegui J. I., Juan M., Yague J., Mahlaoui N., Donadieu J., Chen K. and Cerutti A., 2012. B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen. Nature immunology 13, 170-80. Roth D. B., 1996. V(D)J Recombination. In Herzenberg L. A., Weir D. M. and Blackwell C. (Eds.), Blackwell Science, Oxford, pp. 16.1-16.20. Schroeder H. W., Jr., Mortari F., Shiokawa S., Kirkham P. M., Elgavish R. A. and Bertrand F. E., 3rd, 1995. Developmental regulation of the human antibody repertoire. Annals of the New York Academy of Sciences 764, 242-60. Sohn H. W., Krueger P. D., Davis R. S. and Pierce S. K., 2011. FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling. Blood 118, 6332-41. Tangye S. G., Liu Y. J., Aversa G., Phillips J. H. and de Vries J. E., 1998. Identification of functional human splenic memory B cells by expression of CD148 and CD27. The Journal of experimental medicine 188, 1691-703. Timens W., Boes A. and Poppema S., 1989. Human marginal zone B cells are not an activated B cell subset: strong expression of CD21 as a putative mediator for rapid B cell activation. European journal of immunology 19, 2163-6. van Rees E. P., Dijkstra C. D. and van Rooijen N., 1986. The early postnatal development of the primary immune response in TNP-KLH-stimulated popliteal lymph node in the rat. Cell and tissue research 246, 673-7. Vinuesa C. G., Sze D. M., Cook M. C., Toellner K. M., Klaus G. G., Ball J. and MacLennan I. C., 2003. Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens. European journal of immunology 33, 297-305. 150 | Chapter 7 32. 33. 34. 35. 36. 37. Weill J. C., Weller S. and Reynaud C. A., 2009. Human marginal zone B cells. Annual review of immunology 27, 267-85. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004a. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-54. Weller S., Braun M. C., Tan B. K., Rosenwald A., Cordier C., Conley M. E., Plebani A., Kumararatne D. S., Bonnet D., Tournilhac O., Tchernia G., Steiniger B., Staudt L. M., Casanova J. L., Reynaud C. A. and Weill J. C., 2004b. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood 104, 3647-3654. Weller S., Mamani-Matsuda M., Picard C., Cordier C., Lecoeuche D., Gauthier F., Weill J. C. and Reynaud C. A., 2008. Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+ IgD+ CD27+ B cell repertoire in infants. The Journal of experimental medicine 205, 1331-42. Wilson T. J., Fuchs A. and Colonna M., 2012. Cutting edge: human FcRL4 and FcRL5 are receptors for IgA and IgG. Journal of immunology (Baltimore, Md. : 1950) 188, 4741-5. Zandvoort A., Lodewijk M. E., de Boer N. K., Dammers P. M., Kroese F. G. and Timens W., 2001. CD27 expression in the human splenic marginal zone: the infant marginal zone is populated by naive B cells. Tissue antigens 58, 234-42. Summary and general discussion | 151 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Nederlandse samenvatting en discussie De marginale zone (MZ) is een anatomisch omschreven compartiment van de milt dat de follikels en de peri-arteriolaire lymfocytenschede (PALS) omsluit. Samen vormen deze drie structuren de zogenaamde “witte pulpa” In de witte pulpa liggen vooral lymfocyten naast kleinere aantallen niet-lymfoïde cellen. De MZ vormt de grens met de “rode pulpa” en bevat veel veneuze sinussen. In de MZ is een unieke populatie van B cellen aanwezig, de zogeheten MZ-B cellen, naast macrofagen en dendritische cellen. Deze MZ-B cellen hebben unieke fenotypische-, ontwikkelings- en functionele eigenschappen. Vanwege hun anatomische positie op de grens met de rode pulpa en de vele bloedvaten, wordt verondersteld dat MZ-B cellen een belangrijke rol spelen bij de immuunrespons tegen infecties in het bloed. Ze worden met name gedacht snel te kunnen reageren tegen de polysaccharide antigenen (TI-2 antigenen) van kapseldragende bacteriën, zoals pneumococcen en meningococcen. Hierdoor speelt de MZ en met name de MZ-B cellen een belangrijke rol bij de preventie van sepsis. Voorgaande experimenten hebben laten zien dat er in de MZ zowel naïeve B cellen als geheugen B cellen aanwezig zijn. Naïeve B cellen zijn cellen die nog niet met een antigeen gereageerd hebben. Deze worden gekarakteriseerd door de expressie van IgM moleculen op hun oppervlak die gecodeerd worden door ongemuteerde immuunglobuline (Ig) variabele regio (IGV) genen. Naïeve MZ B cellen zijn waarschijnlijk ontstaan uit onrijpe B cellen die net gevormd zijn in het beenmerg (transitionele B cellen) of uit rijpe B cellen die recirculeren en in de lymfoïde organen vooral in de follikels liggen. In tegenstelling tot naïeve B cellen zijn geheugen B cellen die al antigeen-gestimuleerd zijn en meegedaan hebben aan een immuunrespons. Zij brengen vaak andere isotypen dan IgM tot expressie op hun celoppervlak, en deze immuunglobulinen worden over hel algemeen gecodeerd door gemuteerde IGV genen. De aanwezigheid van somatische hypermutaties (SHM) in de IGV genen is dan ook het primaire kenmerk van geheugen B cellen. Mutaties treden op in zowel de Ig zware keten (IGH) en de Ig lichte keten V regio genen gedurende humorale immuun responsen. Dit gebeurt tijdens de proliferatie van geactiveerde B cellen in de zogenaamde kiemcentra (germinal centers, GC’s). Deze GC’s worden gevormd in de follikels van de lymfoïde organen. In de MZ zijn ook B cellen aanwezig waarvan de immuunglobulinen gemuteerd zijn, en dus zeer waarschijnlijk geheugen cellen zijn. Over het algemeen wordt gedacht dat het GC de enige locatie is waar SHM van IGV genen plaats vindt. Desondanks is de origine van de MZ aanwezige gemuteerde B cellen nog onbekend. Dit proefschrift heeft tot doel om het geheugencompartiment van de MZ-B cellen in de milt van de rat te bestuderen, met name om meer inzicht te verkrijgen in de oorsprong en functie van deze geheugen MZ-B cellen. De rat heeft als experimenteel proefdier model van het immuunsysteem voordelen ten opzichte van de Nederlandse samenvatting en discussie | 153 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 muis als het gaat om de MZ, omdat in ratten een veel grotere MZ aanwezig is, die anatomisch gezien veel duidelijker te onderscheiden is van de overige compartimenten van de milt. Rat immuunoglobuline variabele regio genen Om vast te kunnen stellen of er mutaties aanwezig zijn in de IGV genen is het essentieel om de nucleotide volgorde van de kiemlijn V regio genen (de niet-geherschikte genomische sequenties) beschikbaar te hebben. Daarom was het noodzakelijk om eerst de kiemlijn IGV genen in het genoom van ratten te identificeren. Inzicht in de genomische organisatie van de IGH locus van de rat kan daarnaast helpen om mogelijke verschillen tussen de humorale immuun respons van ratten ten opzichte van die bij mens en muis beter te begrijpen. De identificatie van de genomische organisatie van de IGH locus van de rat werd mogelijk toen in 2004 de nucleotide sequenties van het genoom van de Brown Norway (BN/SsNHsdMCW) rat (Rattus norvegicus) beschikbaar werd gemaakt door het Rat Genomic Sequence Consortium (RGSC). De beschikbare sequenties omvatte ook de IGH locus op chromosoom 6q32-33. Wat wel van belang is, is dat voor deze analyse de BN rat gebruikt was. Deze rattenstam vertoont in het IGH locus ten opzichte van andere ingeteelde rattenstammen grote verschillen. Dammers et al. hebben bijvoorbeeld gevonden met behulp van “restriction fragment length polymorphismes” dat de BN rat minder IGV genen heeft dan andere ratten stammen (bijvoorbeeld PVG. AO, Lewis ratten). Het V gedeelte van de IGH keten wordt gecodeerd door drie verschillende gen segmenten: IGH variabele (IGHV), IGH diversity (IGHD) en IGH joining (IGHJ) gen segmenten. Door herschikking van deze drie gen segmenten (VDJ recombinatie) worden er één V gen segment gekoppeld aan één D gen segment en één J gen segment (VDJ recombinatie). Slechts na herschikking kunnen deze coderen voor het V gedeelte van de IGH keten. Ook al was het totale genoom van de rat gepubliceerd, het exacte aantal en de lokalisatie van de IGHV genen alsmede van de IGHD en IGHJ genen, was nog niet bekend. Daarom hebben we eerst, gebruik makend van verschillende beschikbare bronnen, de kiemlijn sequenties van de IGHV, IGHD en IGHJ genen geanalyseerd. De bronnen die gebruikt werden waren: de genoom volgorde van het RGSC “genome assembly version 3.4”, de niet-gelokaliseerde sequenties (contigs) in de “unplaced section” van de NCBI database en in de nieuw gevonden zogenaamde bactigs van het BN ratten genoom (Baylor College of Medicine; http://www.hgsc. bcm. tmc.edu/projects/rat). In hoofdstuk 2 hebben we de organisatie van de V regio van de BN rat gedetailleerd in kaart gebracht. Dit is uitgevoerd voor zowel de functionele en niet-functionele IGHV genen als ook voor de IGHD en IGHJ genen. In hoofdstuk 3 presenteren we een update van de IGH locus van de rat in vergelijking met die van de muis en van de mens. Tevens beschrijven we wat er op dit moment bekend is over de organisatie van de lichte keten locus bij de rat. In totaal 154 | Chapter 7 waren wij in staat om 353 IGHV genen te identificeren waarvan er 131 functioneel lijken te zijn. Dit aantal van 131 functionele IGHV genen bij de rat is het grootste aantal wat gevonden is bij zoogdieren waarvan het gehele genoom beschikbaar is. Door de analyse van de genomische sequenties van het IGVH locus hebben we 11 nieuwe IGHV genen geïdentificeerd in de BN rat. Deze nieuwe IGHV genen waren nog niet geïdentificeerd in de huidige samenstelling (RGSC V3.4) van de NCBI database en ook nog niet genoemd in de International Immunogenetics (IMGT) database (http://imgt.cines.fr). Deze genen werden gevonden in de rat genoom sequenties die beschikbaar waren gemaakt door de Baylor College of Medicine Human Genome Sequencing Group die nog niet waren opgenomen in de samengestelde map van de BN rat op dat moment. Net als bij de muis kunnen bij ratten IGHV genen onderverdeeld worden in zogeheten IGHV gen families op basis overeenkomsten in nucleotide volgorden. Individuele IGHV genen behoren tot dezelfde familie als hun IGHV genen voor meer van 80% van hun nucleotiden volgorde met elkaar overeenkomen. Van de 11 nieuw gevonden IGHV genen behoren er 6 tot de IGHV2 gen familie en 5 tot de IGHV5 gen familie. De onbekende regio van het IGH locus waarvan de nucleotide volgorde nog bepaald moet worden is precies gelegen in het gebied van de IGHV2 en IGHV5 gen families. Daarom is het redelijk om te veronderstellen dat deze 11 nieuw gevonden IGHV genen waarschijnlijk gelegen zijn in de IGHV2-IGHV5 regio van assembly V3.4. Net als bij de muis en bij de mens is het overgrote merendeel van de ratten IGHV genen in dezelfde richting georiënteerd als de IGHD en IGHJ genen en daarom zal VDJ recombinatie meestal gebruik maken van deletie en van inversie tijdens. Voor zowel rat als muis is de totale dichtheid van IGHV genen (dat is het aantal IGHV genen per Mb nucleotiden volgorde) het hoogst aan de 3’ kant van de IGHV regio in vergelijking met de 5’ kant. Zoals verwacht tonen de rat en de muis een sterke evolutionaire overeenkomst zoals aangetoond met de bijna identieke organisatie van de IGHV gen families en de verspreiding van de verschillende IGHV genen op het IGH locus. Samenvattend laten onze studies (Hoofdstuk 2) zien dat ratten het hoogste aantal (functionele) kiemlijn IGHV genen hebben van alle bestudeerde zoogdieren soorten. Gezien het feit dat de BN rat misschien wel een lager aantal IGHV5 genen heeft ten opzichte van andere ratten stammen en het feit dat er nog wat gaten zitten in de genoom volgorde in het gebied van de IGHV2 en IGHV5 gen families is het niet onwaarschijnlijk dat het aantal van IGHV genen in de rat mogelijk zelfs nog hoger is. Op dit moment is er nog weinig bekend over de genomische organisatie van het ratten Ig lichte keten locus die gevormd wordt door genen die coderen voor de kappa lichte keten (IGK) en de lambda lichte keten (IGL) genen. De variabele delen van de IGK en IGL worden gecodeerd door respectievelijk een combinatie van (IGKV-IGKJ) of (IGLV-IGLJ) gen segmenten. Nederlandse samenvatting en discussie | 155 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Net als bij de IGHV genen, hebben ratten en muizen ongeveer hetzelfde aantal IGKV genen waarbij de ratten een paar meer functionele IGKV genen hebben dan de muis. IgM positieve marginale zone geheugen B cellen Eerdere experimenten lieten zien dat er in gemuteerde IgM-positieve geheugen MZ-B cellen aanwezig zijn in de rat. Dammers et al. hebben aangetoond dat ongeveer 20% van de MZ-B cellen afkomstig van de milt van PVG ratten gemuteerde IGHV genen hebben. Deze bevindingen stonden in scherp contrast met de situatie bij mensen waarbij meer dan 95% van de MZ-B cellen uit de milt gemuteerd zijn. Een mogelijke verklaring voor dit verschil zou kunnen zijn dat er in de (PVG) rat alleen maar een bepaalde IGHV gen familie (met name de IGHV5 familie, de homoloog van de PC7183 in de muis) was geanalyseerd. Mogelijk was deze IGHV gen familie niet representatief voor andere IGHV gen families. Door het in kaart brengen van de op het genoom aanwezige kiemlijn IGHV genen van de BN rat (hoofdstuk 2), werd het mogelijk om nauwkeurig ook andere IGHV gen families te analyseren. Om bovendien mogelijke verschillen tussen rattenstammen te voorkomen, gebruikten we de BN ratten stam in plaats van de PVG ratten stam die eerder was onderzocht. In hoofdstuk 4 laten we de frequentie zien van het aantal gemuteerde sequenties in herschikte IgM transcripten (IGHVCµ transcripte) afkomstig van MZ-B cellen (IgMhighIgDlow) in vergelijking met die van de FO-B cellen (IgMlowIgDhigh) afkomstig uit de milt van volwassen BN ratten. Deze B cel subpopulaties werden verkregen door gebruik te maken van sortering met behulp van flow-cytometrie (FACS). De analyse van de transcripten was gericht op drie IGHV gen families die verschillend zijn in het aantal leden dat deel uit maakt van deze families: IGHV3, IGHV4 en IGHV5. Deze drie IGHV gen families hebben in de BN rat respectievelijk 4, 2 en 26 functionele IGHV genen. De IGHV3 en IGHV4 gen zijn dus relatief klein vergeleken met de IGHV5 gen familie, welke de op-één-na grootste familie is. De IGHV5 familie was ook eerder al eerder geanalyseerd in de PVG rat.. Zoals eerder was aangetoond, vonden we dat in de milt een significant hoger percentage van de IgM sequenties van de MZ-B cellen gemuteerd is dan de FO-B cellen. Bovendien bleek dat alle drie de geanalyseerde IGHV gen families bijdragen aan dit verschil. In BN ratten waren 27% van de MZ-B cellen die gebruik maken IgM moleculen gecodeerd door de IGHV5 familie gemuteerd, wat iets hoger is dan in de PVG rat (10-20%). Dit verschil in frequentie van gemuteerde IgM sequenties kan veroorzaakt zijn door stam verschillen tussen de PVG en de BN rattenstam, zoals bijvoorbeeld het feit dat BN ratten minder IGHV genen hebben, of kan veroorzaakt worden door verschillen in omgevingsfactoren (andere microbiota) bij de twee ratten stammen. Analyse van de IGHV3 gen familie liet zien dat een vergelijkbaar percentage (30%) van MZ-B cellen gemuteerde IgM sequenties heeft. In tegenstelling tot deze twee IGHV families is een groot gedeelte (66%) van de IGHV4 sequenties afkomstig van MZ-B cellen gemuteerd. Deze familie bestaat uit maar twee potentieel functionele IGHV genen, 156 | Chapter 7 waarvan er waarschijnlijk slechts één van deze leden functioneel tot expressie komt. Onze bevindingen laten dus zien dat het aandeel van gemuteerde sequenties afkomstig van MZ-B cellen varieert tussen de verschillende IGHV gen families en dat een groter deel (2766%) van de IGHV genen in de BN rat gemuteerd is, dan eerder was gevonden voor alleen de IGHV5 gen familie in de PVG rat (10-20%). Onze observatie dat het hoogste percentage van gemuteerde sequenties te vinden was in de IGHV4 gen familie met één enkel functioneel lid, suggereert dat er mogelijk meer antigene selectiedruk is op dit bepaalde IGHV4 gen om het beschikbare repertoire te expanderen door middel van somatische hypermutatie. Ondanks dat er een hoger gemiddeld aantal gemuteerde sequenties werd gevonden in MZ-B cellen van de rat dan eerder werd gevonden, is bij de MZ-B cellen van de mens nog steeds een veel groter percentage gemuteerd. Bij de mens zijn nagenoeg alle MZ-B cellen van de milt gemuteerd. Onze waarneming dat er een variatie bestaat in de percentage gemuteerde IGHV genen tussen verschillende IGHV families kan voor een deel het verschil verklaren tussen het aantal gemuteerde MZ-B cellen van mens en rat. Immers de analyse van humane MZ-B cellen is sterk beperkt tot een smalle set van IGHV genen. Zo analyseerden Dunn-Walters et al. alleen twee individuele IGHV genen: het IGHV6 gen en het IGHV4.21 gen. Colombo et al onderzochten de aanwezigheid van mutaties in humane IGHV1, IGHV3 en IGHV4 gen families in onder andere MZ-B cellen. Ook zij vonden dat de meeste MZ-B cellen gemuteerd waren. Het gemiddeld aantal mutaties per sequentie is in MZ-B cellen bij de mens (gemiddeld 11.8 mutaties per sequentie) is echter hoger dan bij ratten IgM+ geheugen MZ-B cellen (gemiddeld 8.8 mutaties per sequentie). Mogelijk dat dit veroorzaakt wordt door het feit dat bij de mens er minder functionele IGHV genen zijn dan in de rat. We speculeren dat misschien het hogere aantal kiemlijn IGHV genen in ratten en muizen tot gevolg heeft dat ratten minder mutaties nodig hebben om hun antistof repertoire te vergroten na immunisatie dan mensen, omdat ratten een grotere diversiteit van verschillende antistoffen kunnen maken met hun primaire (kiemlijn) repertoire. Bovendien is het mogelijk dat een verschil in levensverwachting tussen mens en rat alsmede omgevingsfactoren ook bijdragen aan het verschil van de gemiddelde mutatie frequentie per IGHV gen. Gedurende hun (lange) leven kunnen mensen meer verschillende antigenen tegenkomen dan laboratorium ratten die leven onder streng gecontroleerde laboratorium omstandigheden. Geheugen B cellen worden over het algemeen gedacht gevormd te worden in de GC’s, tijdens de T cel afhankelijke immuunrespons van antigeen-specifieke B cellen. In deze GC’s worden, zoals eerder gezegd de V genen (van zowel de lichte als de zware ketens) gemuteerd door middel van somatische hypermutatie (SHM). Het is echter nog niet goed bekend of ook gemuteerde (geheugen) IgM+ MZ-B cellen afkomstig zijn van de GC’s of dat ze een GC-onafhankelijke B cel populatie zijn. Bij de mens vond Colombo et al. een klein aantal van klonaal-gerelateerde IGH sequenties, afkomstig van B-cellen afkomstig van MZ-B cellen en Nederlandse samenvatting en discussie | 157 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 GC-B cellen. Deze bevinding suggereert dat gemuteerde IgM+ MZ-B cellen inderdaad mogelijk afkomstig zouden zijn van GC’s. Daartegenover stelt Weil et al. dat bij de mens de (gemuteerde) IgM+ MZ-B cellen juist niet gevoprmd worden in het GC, maar ontstaan gedurende de ontwikkeling van het immuunsysteem. Dit zou niet het gevolg zijn van een immuunrespons maar een eigenschap van MZ-B cellen om het primaire repertoire te verbreden op een GC- (en T-cel) onafhankelijke manier. De hypothese van Weill et al. is vooral gebaseerd op de waarneming dat bij patiënten met een bepaalde immuundeficiëntie waarbij GC’s afwezig zouden zijn, er toch gemuteerde MZ-B cellen gevonden worden. Om de oorsprong van gemuteerde IgM+ MZ-B cellen te onderzoeken bij de rat hebben we in hoofdstuk 5 de mogelijke aanwezigheid van deze cellen onderzocht in neonatale ratten. Neonatale ratten kunnen in de eerste paar weken van hun leven nog geen GC’s vormen in de lymfoïde organen. Dus wanneer de MZ-B cellen in de milt van deze neonatale ratten gemuteerd zijn, zou dit een sterk argument zijn voor de hypothese van Weill et al. dat SHM een onderdeel is van de natuurlijke ontwikkeling van MZ-B cellen. Daarom hebben we in dit hoofdstuk de IGHV-Cµ transcripten van de IGHV4 en IGHV5 gen families van zowel de MZ-B cellen als de FO-B cellen geanalyseerd. In geen van de neonatale sequenties werd een mutatie gevonden, zelfs niet in de IGHV4 gen familie met het hoogste aantal gemuteerde sequenties in MZ-B cellen afkomstig van volwassen ratten. Deze resultaten ondersteunen het feit dat ten minste in de rat, de gemuteerde IgM+ MZ-B cellen zoals te vinden in de volwassen rat het meest waarschijnlijk echte geheugen cellen zijn, die ontstaan onder de invloed van externe antigene stimulatie in GC’s. IgG positieve marginale zone geheugen B cellen Naast IgM+ MZ-B cellen, kunnen er ook in de MZ ook B cellen gevonden worden waarvan de immuunglobulinene genen isotype switching (klasse switch recombinatie) hebben Dergelijke IgG+ B cellen zijn gevonden in de MZ van de milt van zowel mens als rat. B cellen met een geswitcht isotype worden over het algemeen beschouwd als geheugen B cellen, en deze cellen hebben dan ook meestal gemuteerde IGHV genen. Of geschwitchte B cellen in de MZ ook mutaties hebben en of deze B cellen met betrekking tot hun fenotype lijken op niet-geswitchte (IgM+) was niet bekend. Dit werd geanalyseerd in hoofdstuk 6 waar we IgG+ MZ-B cellen van de milt van de rat hebben geanalyseerd. Om de te onderzoeken of er IgG+ B cellen met een MZ-B cel fenotype voorkomen en vervolgens te analyseren hebben we gebruik gemaakt van het feit dat ratten MZ-B cellen een laag niveau van CD45R tot expressie brengen, zoals dat gedefinieerd wordt door de monoclonale antistof HIS24, en hoge niveaus van het oppervlakte molecuul dat gedefinieerd wordt door de monoclonale antistof HIS57. Uitgerijpte B cellen, inclusief MZ-B cellen, brengen daarentegen het eiwit CD90 niet meer op hun oppervlak tot expressie. Op deze manier kunnen MZ-B celle van de rat gedefinieerd worden als CD90−HIS24lowHIS57high cellen en FO-B cellen als CD90−HIS24highHIS57neg/low. We hebben 158 | Chapter 7 eerst onderzocht of er van gezuiverde (FACS-gesorteerde) MZ-B cellen en FO-B cellen geherrangschikte IGHV-Cγ transcripten geamplificeerd konden worden, die specifiek zijn voor de IGHV5 gen familie. Ter vergelijking hebben we ook IGHV-Cγ transcripten geamplificeerd van klassieke “geswichte”” geheugen (IgM-IgD-) B cellen. We waren in staat om de aanwezigheid van IGHV5-Cγ transcripten aan te tonen in alle B cel subsets die we geanalyseerd hebben. Dit betekent dat IgG+ B cellen tenminste twee fenotypes kunnen hebben: cellen met een MZ-B cel fenotype en cellen met een FO-B cel fenotype. Analyse van de individuele IGHV5 genen onder deze IgG transcripten liet zien dat bijna alle IGHV5 genen gemuteerd waren, zoals karakteristiek is voor geheugen B cellen. We zagen wel dat de IgG+ MZ-B cellen een lager aantal mutaties had in vergelijking tot de IgG+ FO-B cellen. Er was geen verschil in het gebruik van IGHV, IGHD en IGHJ genen tussen de IgG+ MZ-B cellen en de IgG+ FO-B cellen en de H-CDR3 lengte was ook hetzelfde tussen beide subpopulaties. Een belangrijke observatie was dat we sets van klonaal- gerelateerde IgG-coderende sequenties (sequenties met identieke H-CDR3 regio en gebruik van dezelfde IGHV genen) vonden afkomstig van cellen met leden in zowel de MZ-B cel (CD90−HIS24lowHIS57high) fractie als in de FO-B cel (CD90−HIS24highHIS57neg/low) fractie. Degelijke sets van klonaal-gerelateerde sequenties werden ook gevonden bij de gemuteerde IgM+ MZ-B cellen en IgM+ FO-B cellen (hoofdstuk 4). Deze waarnemingen suggereren zeer sterk dat (gemuteerde) geheugen B cellen met een MZ-B cel fenotype en met een FO-B cel fenotype een gezamenlijke oorsprong hebben. De oorsprong van die cellen is niet helemaal duidelijk. Zoals eerder gezegd, worden klassieke geheugen B cellen (dat wil zeggen gemuteerde die ook een geswitcht isotype hebben B cellen) meestal geproduceerd in de GC’s. Op die lokaties ondergaan de gemuteerde B cellen een vorm van selectie voor het antigeen dat de GC reactie aanzet. Deze antigene selectie is een cruciale stap in het proces van de affiniteitsrijping van geheugen B cellen. Hierbij worden B cellen die door de somatische hypermutatie een hogere affiniteit voor het antigeen gekregen hebben geselecteerd, ten koste van B cellen met een lagere affiniteit voor het antigeen. Dit is terug te zien in het feit dat de IGHV genen die coderen voor de Ig moleculen in geheugen B cellen geen willekeurige mutaties hebben. Aan de hand van de mutatie patronen van de IgG–coderende sequenties van zowel de MZ-B cel fractie als van de F0-B fractie konden we vaststellen (hoofdstuk 6) dat er in sprake was van antigene selectie. Dit past bij de hypothese dat deze ook IgG+ MZ-B cellen gegenereerd zijn in de GC’s. De bevinding dat gemuteerde IgM+ geheugen B cellen afwezig zijn in neonatale dieren (hoofdstuk 5) waar nog geen GC’s aanweizg zijn, ondersteunt de hypothese dat ook de IgM+ geheugen MZ-B cellen afkomstig zijn van de GC’s. De gezamenlijke oorsprong van IgM+ en IgG+ geheugen MZ-B cellen en FO-B cellen, zoals geïllustreerd door de aanwezigheid van klonaal-gerelateerde sequenties tussen MZ-B en FO-B cellen, suggereert dat tenminste een aantal van de GC-afkomstige geheugen cellen zowel een FO-B cel fenotype als een MZ-B cel fenotype kunnen verkrijgen. De factoren die deze differentiatie Nederlandse samenvatting en discussie | 159 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 tussen deze twee fenotypes aan sturen zijn onbekend. Concluderende opmerkingen B cellen in de MZ vormen een heterogene populatie van cellen en zowel naïeve MZ-B cellen, als geswitchte en niet-geswitchte geheugen MZ-B cellen zijn aanwezig op deze unieke locatie in de milt. Naïeve MZ-B cellen hebben ongemuteerde Ig genen, produceren IgM moleculen met een lage affiniteit en vormen een eerste lijn van afweer tegen binnendringende pathogenen. Het antistof repertoire van deze B cellen wordt verondersteld geselecteerd te zijn te binden aan suiker-antigenen zoals aanwezig op sommige micro-organismen. Voor de immuunrespons tegen deze antigenen is er geen T cel hulp nodig. Daarnaast hebben in rat en muis de IgM moleculen zoals te vinden in naïeve MZ-B cellen kortere H-CDR3 regio’s in vergelijking met die van FO-B cellen. Kortere H-CDR3 regio’s zijn geassocieerd met poly-reactiviteit, dat wil zeggen dat de antistoffen kunnen binden aan meerdere antigenen. Geheugen (MZ-)B cellen hebben daarentegen Ig moleculen met een hoge affiniteit, die gericht zijn tegen de microbiële antigenen waarmee in aanraking is gekomen tijdens het leven. De aanwezigheid van naïeve MZ-B cellen en geheugen MZ-B cellen zorgen ervoor dat in de MZ zowel aangeboren (laag affiene, polyreactieve) als verworven (hoog affiene) antistof responsen tegen T cel onafhankelijke (koolhydraten) als T cel afhankelijke (eiwitten) microbiële antigenen kunnen plaatsvinden. MZ-B cellen zijn in een soort van pre-geactiveerde staat. Hierdoor lijken ze uitermate geschikt om snel te kunnen reageren op antigeen, wat zeker in het geval van een dreigende sepsis van groot belang is. Ze brengen hoge niveaus van de complement receptor CD21 tot expressie alsmede Toll-like receptoren (TLR’s) wat verder bewijs is van het feit dat MZ-B cellen goed zijn uitgerust om snel en gemakkelijk geactiveerd te worden in (primaire) immuunresponsen. Een nieuwe rol van neutrofiele granulocyten in de immuunrespons van MZ-B cellen is voorgesteld door Puga et al. Deze neutrofielen in de milt induceren IgM productie door de MZ-B cellen te activeren via de cytokinen BAFF, APRIL en IL-21 om zo een antistof respons te maken tegen TI-2 antigenen zoals LPS. Deze cytokinen kunnen de transcriptie factor de inductie van BLIMP-1 induceren in de geactiveerde MZ-B cellen. BAFF en IL-21 kunnen samen ook IgG+ MZ-B cellen stimuleren op een T-cel onafhankelijke manier om te differentiëren tot antistofproducerende plasma cellen. Behalve (snelle) antistof productie spelen MZ-B cellen ook mogelijke een andere rol. Garraud et al. hebben gevonden dat MZ-B cellen op en neer gaan tussen de MZ en de follikels en daarmee het transport van antigenen (in de vorm van immuuncomplexen) naar folliculair dendritische cellen (FDC’s) verzorgen. Hierbij is er dus ook een rol weggelegd voor MZ-B cellen als antigeen-presenterende cellen en zo een aandeel hebben aan immuunresponsen waarbij hoog-affiene antistoffen geproduceerd worden. Deze onderzoekers vonden dat type 160 | Chapter 7 1 interferon dat wordt gemaakt in reactie op pathogenen dit transport stuurt. Onder invloed van het interferon wordt de sphingosine-1-phosphate receptor 1 (S1P1) en S1P3 geïnactiveerd waardoor de MZ-B cellen de gelegenheid gegeven wordt om de MZ te verlaten en te migreren naar de follikels toe onder invloed van het chemokine CXCL13. Het verlagen van de expressie van CXCR5, de receptor voor CXCL13, staat de MZ-B cellen dan weer toe om de follikels weer te verlaten en terug te keren naar de MZ. Op die manier transporteren MZ-B cellen immuuncomplexen naar de follikels toe waarna deze immuuncomplexen vervolgens vast gehouden worden voor een lange tijd door de FDC’s op een complement-afhankelijke manier. De immuuncomplexen die worden vastgehouden door de FDC’s zijn betrokken bij de selectie van gemuteerde B cellen in het GC die een hogere affiniteit hebben voor het antigeen dat de reactie induceerde. Dit gebeurt samen met de hulp van folliculaire helper T (Tfh) cellen. Eerst presenteren de FDC’s het antigeen aan GC-B cellen die het proces van SHM en isotype switching ondergaan. Dan presenteren deze B cellen het antigeen aan Tfh cellen die op hun beurt overlevingssignalen voor deze hoog-affiene GC-B cellen afgeven waardoor deze geselecteerd worden. De geselecteerde GC-B cellen zullen dan daarna differentiëren in geheugen B cellen of in plasma cellen. Recent is gevonden dat MZ-B cellen ook de Fc-like receptor FcRL5 tot expressie brengen. Dit is een receptor, die na binding van IgG de signalering via de B cel receptor (BCR) kan remmen. Zoals dat al bekend is voor FcRL4 dat bind aan IgA en dat ook de BCR signalering inhibeert, maar niet de signalering via TLR’s. TLR’s kunnen slechts een beperkt aantal moleculaire patronen van vooral micro-organismen herkennen, en zijn dus niet zo specifiek. Deze receptoren zijn vooral betrokken bij responsen van het aangeboren immuunsysteem. Het is mogelijk dat bezetting van de FcRL5 door IgG op een vergelijkbare manier als FcRL4 werkt. Hierdoor zou het BCR signaal van de MZ-B cel geremd kunnen worden en het signaal van de TLR’s versterkt. Op deze manier krijgen de MZ-B cellen als het ware meer eigenschappen van het aangeboren immuunsysteem en worden de MZ-B cellen meer B cellen die reageren op microbiële antigenen. Op deze manier kunnen deze cellen heel snel reageren op microbiele antigenen in het bloed op een BCR-onafhankelijke manier. Omdat de MZ-B cellen zo’n breed repertoire hebben en ook geheugen B cellen (zowel IgM als IgG) omvatten en zij zich bevinden in een soort van pre-geactiveerd stadium zijn deze cellen uitermate geschikt om snel te kunnen reageren op een breed spectrum van antigenen in het bloed om op die manier bloedvergiftiging (sepsis) te voorkomen. Het aanspreken van deze B cellen door middel van vaccinatie zal in de toekomst cruciaal zijn voor een effectieve bescherming tegen deze levensbedreigende infecties. Nederlandse samenvatting en discussie | 161 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Opsomming en algemene bespreking in het Afrikaans Die marginale zone (MZ) is ‘n goed-gedefinieerde, anatomiese kompartement van die milt wat die follikels en peri-arteriolêre limfosiet skedes (PALS) omsluit. Saam vorm hierdie drie strukture die sogenaamde “wit pulp”, wat meestal limfosiete bevat. Die MZ vorm ‘n koppelvlak met die rooi pulp, ‘n gebied wat baie ryk is in veneuse sinusse. Benewens makrofage en dendritiese selle, bestaan die MZ hoofsaaklik uit ‘n unieke bevolking van B-selle, die MZ-B selle, wat unieke fenotipiese, ontwikkelings- en funksionele eienskappe besit. As gevolg van hul anatomiese posisie op die grens van die rooi pulp, asook die ryk toevoer van kapillêre, wat wyd oop presenteer ( Kusumi et al, 2015), speel die MZ-B selle vermoedelik ‘n belangrike rol in die vinnige immuunreaksies teen bloed-oordraagbare patogene. Daar word besonders geglo dat hulle vinnig reageer tenopsigte van polisakkaried antigene (TI-2 antigene) op die oppervlak van geinkapsuleerde bakterieë, soos pneumokokke en meningokokke (Guinamard et al, 2000; Kruetzmann et al, 2003; Martin et al, 2001; Vinuesa et al, 2003). Dus kan die MZ en MZ-B selle in die besonder, ‘n belangrike rol vervul in die voorkoming van sepse. Vorige eksperimentele data lewer bewyse van die teenswoordigheid van beide naïef B selle en geheue B selle binne in die MZ (Colombo et al, 2013; Dammers et al, 2000a; Dunn-Walters et al, 1995; Gatto et al, 2007; Gatto et al, 2004; Makowska et al., 1999; Tangye et al, 1998; Weller et al, 2004b; Zandvoort et al, 2001). Naïewe B selle is selle wat nog nie teenoor antigene gereageer het nie, en word gekenmerk deur die vorming van immunoglobulien M (IgM) molekules op hul seloppervlakmembraan, wat gekodeer word deur die ongemuteerde gene van die immunoglobulien veranderlike gebied (IGV). Naïewe MZ-B selle word waarskynlik gevorm vanaf oorgangs B selle, óf follikulêre (FO) B selle. Inteenstelling met naïewe B selle, is geheue B selle antigeen ervare selle, wat dikwels ook nie-IgM isotypes uitdruk op hul membrane, wat gekodeer word deur gemuteerde IGV gene. Die teenswoordigheid van somatiese hypermutasies (SHM) binnein die IGV streke van Ig gene is die primêre kenmerk van geheue B selle. Mutasies vorm in beide die Ig swaarketting (IGH) en Ig ligteketting V streekgene tydens die humorale immuunrespons, wanneer antigeengeaktiveerde B selle uitbrei in ‘n T-sel afhanklike (TD) wyse binne in die kiemsentrums (GC). Alhoewel, daar geglo word dat GC’s oor die algemeen die plekke is waar hierdie SHM van IGV gene uitsluitlik plaasvind, is die oorsprong van gemuteerde MZ-B selle egter nog steeds ontseker. Die doel van hierdie tesis is om milt MZ-B selle te analiseer in die geheue kompartement van die rot, omsodoende tot insigte te bekom oor die oorsprong en funksie van hierdie geheue MZ-B-selle. As proefdiermodel vir die bestudering van die immuunstelsel, bied rotte ‘n selektiewe voordeel bo muise, omdat rotte ‘n veel groter MZ het wat maklik gevisualiseer kan word. Opsomming en algemene bespreking in het Afrikaans | 163 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Rot immunoglobulien veranderlike streek gene Om die teenswoordigheid van SHM in die IGV gene te bepaal, is dit van kardinale belang om inligting beskikbaar te hê oor die genomiese volgorde van hul kiemlyn V geen eweknieë (ongeherrangskikte genomiese volgordes). Dit was dus belangrik om die kiemlyn IGV gene in die rot te identifiseer. Daarbenewens sal die kennis van die genomiese rangskiking van die organisasie van die IGH lokus van die rot, insluitend die lokalisering van kiemlyn nukleotied volgordes van alle IGH veranderlike ketting gene, help om moontlike verdere verskille in humorale immuunreaksies, asook die Ig repertoires tussen rotte aan die een kant en mense en muise aan die ander, te ontrafel. Hierdie studies in rotte het moontlik geword toe die genoom van die Brown Norway (BN/ SsNHsdMCW) rot (Rattus norvegicus) beskikbaar gestel word deur die Rat Genomic Sequence Consortium (RGSC) in 2004 (Gibbs et al, 2004; Havlak et al, 2004). Die genomieseinligting bevat ook die IGH lokus geleë te chromosoom 6q32-33. Dit is tog belangrik om te weet dat die BN rot wat gebruik is vir hierdie analise wel verskil van ander ingeteelde rotstamme, en ook by die IGH lokus. Byvoorbeeld, Dammers et al. het deur beperkings fragment lengte polimorfisme analise gevind dat die BN rot aansienlik verskil van ander rotstamme (soos bv PVG, AO, Lew) in terme van die getal IGV gene in die IGH lokus wat aan die IGHV5 familie behoort (Dammers, 2001). Hierdie data dui daarop dat die BN rot waarskynlik ‘n laer aantal IGHV5 gene bevat in vergelyking met ander rotstamme. Die V gedeelte van die IGH ketting word gekodeer deur drie verskillende geensegmente (gene): IGH veranderlike (IGHV), IGH diversiteit (IGHD), en IGH aansluitend (IGHJ) geensegmente, terwyl die immunoglobulien kappa veranderlike (IGK) en immunoglobulien lambda (IGL) ketting V domeine geïnkripteer word deur ‘n kombinasie van hierdie (IGKV-IGKJ) en immunoglobulien kappa veranderlike (IGLV-IGLJ) gene, onderskeidelik (Roth, 1996). Hoewel die genomiese volgorde van die rat gepubliseerd is, is die presiese aantal en ligging van IGHV gene insluitend IGHD en IGHJ gene nie vasgestel nie. Daarom het ons eers verskeie beskikbare bronne ontleed vir kiemlyn IGHV, IGHD en IGHJ gene. Hierdie bronne was: die genomiese volgorde RGSC genoomsamestelling weergawe 3.4, die ongekarteerde volgordes van “contigs” in die “ongeplaasde afdeling” van die NCBI databasis en nuut gestigte “bactigs” van die BN rotgenoom, nog nie in die RGSC V3.4 samevatting nie. In hoofstuk 2 bied ons aan ‘n geannoteerde kaart van die V-streek van die IGH lokus van die BN rot, wat nie net funksionele en nie-funksionele IGHV gene insluit nie, maar ook die IGHD en IGHJ gene. In hoofstuk 3 bied ons ‘n update van die rot IGH kettinglokus en vergelyk dit met die muis en die mens, asook ‘n beskrywing van wat tans bekend is oor die organisasie van die Ig ligtekettinglokus in die rot. In geheel was ons instaat om 353 IGHV gene te identifiseer, waarvan 131 vermoedelik funksioneel is. Hierdie getal van 131 funksionele IGHV gene in rotte is die grootste onder soogdiere waarvan die hele genoomvolgorde beskikbaar is, in lyn met die voorspelling van Das (2009). Hierdie getal kan selfs verhoog word omdat die hele genoomkaart van die veranderlike streek van die IGH 164 | Chapter 7 lokus nog ‘n aantal klein gapings vertoon (~ 300 KB) waarvan die nukleotiedvolgorde nog bepaal moet word. Deur die rot genomiese volgorde van die IGVH lokus te ontleed het ons 11 nuwe IGHV gene in die BN rot geïdentifiseer. Dié NOVEL IGHV gene was ongeïdentifiseerde IGHV gene wat nie geplaas is in die huidige beskikbare NCBI databasis samestelling (RGSC v3.4); en is ook nie gelys in die Internasionale Immunogenetiese (IMGT) databasis (http://imgt.cines.fr) nie (Lefranc et al, 1999). Hierdie gene is gevind tussen rot genomiese volgordes beskikbaar vanaf die Mensgenoom Volgordebepalingsgroep van die Baylor College of Medicine, en is nog nie opgeneem nie in die rot samestelling van die BN rot op die hedige tydstip nie. Dus saam met die 11 nuut geïdentifiseer IGHV gene, bestaan die BN rotgenoom uit ten minste 353 individuele IGHV gene. Soortgelyk aan die muis, kan rot IGHV gene onder verdeel word in IGHV gene families, op die basis van nukleotiedvolgorde identiteit. Individuele IGHV gene behoort aan dieselfde familie indien die IGHV gene meer as 80% van hul nukleotiedes deel (Brodeur en Riblet, 1984). Uit die 11 nuut ontdekte IGHV gene, behoort 6 aan die IGHV2 gene familie en 5 behoort aan die IGHV5 gene familie. Verder, omdat die grootste gapingsgebiede op die IGH lokus waarvan die nukleotiedvolgorde nog bepaal moet word, geleë is in die gebied van die IGHV2 en IGHV5 geen families is dit geloofwaardig om te aanvaar dat hierdie 11 nuut ontdekte IGHV gene geleë is in ‘n gapingstreek van die IGHV2-IGHV5 streke van samestelling v3.4. Ter opsomming het ons studies bewys gelewer dat rotte die hoogste aantal (funksionele) kiemlyn IGHV gene bevat onder alle soogdierspesies wat tot dusver bestudeer is. Gegewe die idee dat BN rotte ‘n laer aantal IGHV5 gene het in vergelyking met ander rotstamme (Dammers et al., 2001), en die feit dat daar ‘n paar gapings is in die genomiesevolgorde, veral in die gebied van die IGHV2 en IGHV5 gene families, is dit geloofwaardig om te aanvaar dat die getal IGHV gene in die rot selfs hoër kan wees. Soortgelyk aan die muis en die mens is die oorgrote meerderheid van die rot IGHV gene georiënteerde in dieselfde rigting as die IGHD en IGHJ gene en dus word wegdoen aansluiting, in plaas van inversionale aansluiting, verkieslik die meeste gebruik as rekombinasie meganisme by die IGH locus. In beide rotte en muise is die algehele digtheidsverspreiding van IGHV gene (aantal IGHV gene per Mb nukleotiedvolgorde) die hoogste by die 3’-kant van die IGHV streek in vergelyking met die 5’-kant. Soos verwag, deel rotte en muise ‘n noue evolusionêre verwantskap soos vertoon deur die byna identies organisasie van IGHV geenfamilies en die verspreiding van IGHV gene by die IGH locus. Op hede, is min bekend oor die genomiese organisasie van die rot Ig ligteketting loci, wat IGK en IGL ketting gene insluit. Soortgelyk aan die IGHV kettinggene, deel rotte en muise byna dieselfde aantal IGKV gene, met rotte wat effens meer funksionele IGKV gene as die muis bevat. Oor die algemeen, toon die vergelyking tussen die twee spesies daarop dat hulle dieselfde basiese struktuur van hul IG loci behou, insluitend ‘n soortgelyke rangskiking van die IGHVDJ orde. In hierdie spesie is diversiteit in die IG antigeen-erkennings setel gestig Opsomming en algemene bespreking in het Afrikaans | 165 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 gedurende die vroeë B sel ontwikkeling deur herrangskikking van V streekgene (of gene segmente) geleë op die IGH en IGL ketting loci. Rekombinasie by die IGH, IGK en IGL loci veroorsaak die ontwikkeling van ‘n hoogs gediversifiseerde IG (teenliggaam) primêre repertoire met ‘n wye verskeidenheid van teenliggaam spesifieke eienskappe. IgM uitdrukkende marginale zone geheue B-selle Vorige eksperimente het bewys gelewer vir die bestaan van gemuteerde, IgM uitdrukkende, geheue MZ-B-selle in die rot (Dammers et al, 2000b ). Dammers et al. het bewys dat minder as 20% van die MZ-B selle wat vanaf die milt van PVG rotte geïsoleer was gemuteerde IGHV gene bevat. Hierdie bevindinge is in sterk kontras met die mens, waar >95% van die miltsiekte MZ-B selle gemuteerde is (Colombo et al, 2013; Dunn-Walters et al, 1995;Tangye et al, 1998). Een moontlike verklaring vir hierdie verskil kan wees dat slegs een spesifieke IGHV geenfamilie (nl. die IGHV5 familie, die homoloog van PC7183 in die muis) in die (PVG) rot ontleed is, en dat hierdie IGHV geenfamilie nie verteenwoordigend was vir ander IGHV gene, of IGHV geenfamilies nie. Met die vorming van die genomiese kiemlyn IGHV geen repertoire van die BN rot (hoofstuk 2), het dit moontlik geword om ander IGHV geenfamilies ook akkuraat te analiseer. Om moontlike stam verskille te vermy het ons die BN rotstam gebruik, in plaas van die PVG rotstam, wat voorheen gebruik is (Dammers et al, 2000b). In hoofstuk 4 lewer ons verslag oor die frekwensie van gemuteerde volgordes in herrangskikte IGHV-Cμ transkripte wat vanaf FACS gesorteer MZ-B selle afgelei is (IgM met FO-B selle (IgM laag IgD hoog) hoog IgD laag) ), in vergelyking ) vanaf die volwasse BN rot milt. Die analise is beperk tot drie verskillende IGHV geenfamilies, wat in grootte verskil: IGHV3, IGHV4 en IGHV5. Hierdie drie IGHV geenfamilies het 4, 2 en 26 funksionele IGHV gene, onderskeidelik. Die IGHV3 en IGHV4 geenfamilies is gekies om te bepaal of daar ‘n verskil in mutasie frekwensies onder lede van die IGHV geenfamilies is, wat relatief klein is, en om hierdie frekwensie te vergelyk met die tweede grootste IGHV geenfamilie (IGHV5) in die rot, wat voorheen ook ontleed is in die PVG rot (Dammers et al, 2000a). Die BN rotstam bevat 26 funksionele IGHV5 (kiemlyn) gene in vergelyking met die 28 kiemlyn gene in die PVG rot. Soos reeds aangetoon, het ons gevind dat milt MZ-B selle ‘n aansienlik hoër persentasie gemuteerde volgordes uitdruk as wat daar FO-B selle is, en al drie van die IGHV geenfamilies wat ontleed is het bygedra tot hierdie verskil. BN rotte het ‘n effens hoër proporsie (27%) van MZ-B selle gemuteerde IgM molekules uitgedruk wat deur IGHV5 familie gene gekodeer is, in vergelyking met hierdie proporsie in die PVG rot (10-20%) (Dammers et al, 2000a). Dié verskil in mutasie frekwensie kan wees as gevolg van die stam verskille wat tussen die PVG en BN rot stamme bestaan, soos byvoorbeeld, die feit dat BN rotte minder IGHV gene het, of mag dalk ook veroorsaak word deur verskillende omgewingstoestande (mikrobiese omgewing mikrobiota) van die twee rotstamme. Ontleding van die IGHV3 geenfamilie het getoon dat ‘n soortgelyke proporsie (ongeveer 30%) van gemuteerde IgM gekodeerde volgordes gevind kan word in MZ-B selle 166 | Chapter 7 wat van die BN rot afkomstig is. In sterk kontras met hierdie twee IGHV families, is ‘n baie hoë persentasie (66%) van die IGHV4 volgordes van gesuiwerde MZ-B selle gemuteer. Hierdie familie bestaan uit slegs twee moontlike funksionele IGHV gene, hoewel slegs een van hierdie lede waarskynlik funksioneel uitgedruk word. Ons bevindinge toon dus dat die verhouding van gemuteerde volgordes afkomstig van MZ-B selle wissellend is tussen verskillende IGHV geenfamilies in die BN rot, en dat in total, ‘n hoër proporsie (27-66%) IGHV gene gemuteer is in vergelyking met dié (10-20%) van gemuteerde volgordes wat voorheen vir die IGHV5 geenfamilie in PVG rotte gevind is (Dammers et al, 2000a; Makowska et al, 1999). Ons waarneming dat die hoogste persentasie van gemuteerde frekwensies plaasgevind het in die enkele funksionele lid IGVH4 geenfamilie, dui daarop dat daar meer antigeen drukseleksie kan wees op hierdie spesifieke IGHV4 geen in die uitbreiding van sy beskikbare repertoire deur SHM. Alhoewel daar in totaal ‘n hoër gemiddelde aantal gemuteerde volgordes onder rot MZ-B selle is as wat voorheen waargeneem is, is die frekwensie van gemuteerde volgordes onder menslike MZ-B selle nog baie hoër. In die mens, is byna al die MZ-B selle gemuteerde (Colombo et al, 2013; Dunn-Walters et al, 1995; Tangye et al, 1998). Ons waarneming dat daar variasie in frekwensie is vir gemuteerde IGHV gene tussen die verskillende IGHV families, kan ook bydra tot die verskil in gemuteerde MZ-B selle tussen mense en rotte, juis omdat in die mens die ontleding van gemuteerde IGHV gene beperk is tot ‘n beperkende stel van IGHV gene. Dunn-Walters et al. het slegs twee spesifieke IGHV gene ontleed: die IGHV6 gene en IGHV4.21 gene. Dit is formeel moontlik dat hierdie IGHV gene meer gemuteerde is as ander gene. Maar die ontleding van Tangye et al (1998) het bewys dat Ig gene geïsoleerd van IgM + geheue B-selle onder IGHV5 en IGHV6 geenfamilies is almal gemuteer en bewys dat die hoë frekwensie van mutasies nie net as gevolg van individuele IGHV gene is nie. Verder, het Colombo et al. (2013) die teenwoordigheid van mutasies in die menslike IGHV1, IGHV3 en IGHV4 geenfamilies ondersoek en vergelyk met onder milt afgelei MZ-B selle (IgMhoë CD27+), GC B selle en klas verwisselde B selle. Ook het hulle gevind dat die meeste van die MZ-B selle gemuteer is, alhoewel teen ‘n laer gemiddelde aantal mutasies as beide GC en klas verwisselde B selle. Tog is die gemiddelde aantal mutasies in die menslike MZ-B selle (11,8) ( Colombo et al, 2013) hoër vir beide rot IgM+ MZ-B selle (8,8) en rot IgG+ MZ-B selle (7) ( Hendricks et al, 2011). Dit kan wees as gevolg van die feit dat die mens minder funksionele IGHV gene het as rotte. Ons postuleer dat die groter aantal kiemlyn IGHV gene in knaagdiere is as gevolg dat rotte minder mutasies benodig om hul teenliggaam repertoire na immunisering te diversifiseer as mense, want rotte kan kodeer vir ‘n groter poel van verskillende teenliggaampies vir hul primêre repertoire. Daarbenewens is dit moontlik dat die verskille in lewensduur en omgewingstoestande ook bydra tot verskille in die gemiddelde mutasie frekwensie per IGHV gene. Tydens hul (lang) lewens mag mense baie meer verskillende antigene teëkom as laboratorium rotte wat in goed beheerde laboratoriumteostande leef. Geheueselle soos oor die algemeen geglo word gegenereer in GCs. Dit is egter nog steeds Opsomming en algemene bespreking in het Afrikaans | 167 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 omstrede of gemuteerde (geheue) IgM+ MZ-B selle afkomstig is van GCs en of hulle ‘n GConafhanklike B sel bevolking verteenwoordig. In die mens, het Colombo et al. (Colombo et al, 2013) ‘n klein aantal klonaal verwante volgordes waargeneem wat gedeel word tussen MZ-B selle en GC B selle, wat ‘n aanduiding gee dat die gemuteerde IgM+ MZ-B selle van GCs afgelei kan word. In teenstelling hiermee het Weill et al (Weill et al, 2009; Weller et al, 2004a; Weller et al, 2008) voorgestel dat die (gemuteerde) IgM+ MZ-B selle nie GC-afgelei geheue B-selle is nie. Instede hiermee het hierdie skrywers gepostuleer dat die mutasies in die menslike MZ-B selle tydens hul ontwikkeling verkry word om hul primêre repertoire te diversifiseer, in ‘n GC (en T-sel) onafhanklike manier. Om hierdie hipotese in rotte te toets het ons in hoofstuk 5 ondersoek onderneem van die moontlike teenwoordigheid van gemuteerde IgM+ MZB-selle in neonatale rotte. Neonatale rotte ontwikkel nie GC in die eerste weke van hul lewens nie (Kroese et al, 1987; van Rees et al, 1986). Dus, wanneer MZ-B selle ongemuteerd in neonatale rotte voorkom, sou dit sterk argumenteer teen die hipotese van Weill et al. (Weill et al, 2009; Weller et al, 2004a; Weller et al, 2008), dat SHM deel is van die ontwikkelingsprogram van MZ-B selle. Vir hierdie doel, het ons IGHV-Cμ transkripte ontleed met behulp van IGHV4 en IGHV5 geenfamilies van beide MZ-B selle en FO-B selle (hoofstuk 4). Daar was egter geen mutasies gevind in enige van die neonatale volgordes, selfs nie in IGHV4 geenfamilie gene met die hoogste aantal gemuteerde volgordes (66%) in die volwasse rot. Hierdie resultate ondersteun die idee dat ten minste in rotte, gemuteerde IgM+ MZ-B selle wat in volwasse diere voorkom bona fide geheue selle is, wat waarskynlik ontwikkel onder die invloed van eksterne antigeniese stimuli in GC. IgG uitdrukkende marginale zone geheue B-selle Bykomend tot onverwisselde (IgM+) MZ-B selle, kan klas verwisselde B selle ook gevind word binne die menslike (Ettinger et al, 2007b) en knaagdier MZ (Gatto et al, 2004). Die fenotipe van hierdie selle is nie duidelik nie, nog of hulle IGHV gene SHM uitstal, as kenmerk van geheue B-selle. Hierdie kwessie is in hoofstuk 6 ontleed, waar ons die klas verwisselde (IgG+) MZ-B selle ontleed. Om IgG+ uitdrukkende B selle met ‘n MZ-B sel fenotipe te ontleed kon ons natuurlik nie IgM en IgD gebruik nie om MZ-B selle te definieer. Daarom het ons gebruik gemaak van die feit dat rot MZ-B selle lae vlakke van CD45R uitdruk, gedefinieer deur monoklonale antiliggaam HIS24, en hoë vlakke van ‘n oppervlak molekuul gedefinieer deur die monoklonale antiliggaam HIS57. Volwasse (CD90-) MZ-B selle kan dus gedefinieer word as CD90- HIS24lae HIS57hoë selle, en FO-B selle CD90- HIS24hoë HIS57neg/lae selle. Van gesuiwerde (FACS-gesorteer) MZ-B selle (CD90- HIS24lae HIS57hoog) en FO-B selle (CD90- HIS24hoë HIS57neg/lo) het ons herrangskikte IGHV-Cγ transkripte geamplifiseer, spesifiek vir die IGHV5 geenfamilie. Ter vergelyking, het ons ook IGHV-Cγ transkripte geamplifiseer van ‘n fraksie van selle wat klassieke klas verwisselde geheue B-selle moet insluit, naamlik IgM- IgD- B selle. Ons was in staat om die teenwoordigheid van IGHV5-Cγ transkripte in alle B sel subklasse 168 | Chapter 7 te ontleed, wat impliseer dat IgG uitdrukkende selle blykbaar ten minste twee verskillende fenotipes kan uitstal: selle met ‘n MZ-B sel fenotipe en selle met ‘n FO-B sel fenotipe. Ontleding van die individuele IGHV5 gene wat deur hierdie IgG transkripte gebring word het aan die lig gebring dat byna alle IGHV5 gene gemuteerde is, soos tipies is vir geheue B-selle. Ons het wel gesien dat die IgG uitdrukkende MZ-B selle het ‘n laer aantal mutasies in vergelyking met IgG uitdrukkende FO-B selle. Daar was geen verskille in die gebruik van IGHV, IGHD en IGHJ gene tussen die IgG+ MZ-B selle en IgG+ FO-B sel subversamelings en die H-CDR3 lengtes was ook vergelykbaar tussen hierdie twee subversamelings. ‘n Belangrike waarneming was dat ons stelle klonaal verwante IgG-koderende volgordes gevind het (volgordes met identiese H-CDR3 streke, en gebruik van dieselfde IGHV gene) wat verkry is uit die selle met lede in beide MZ-B sel (CD90- HIS24lae HIS57hoë) en FO-B sel (CD90- HIS24hoë HIS57neg/laag) fraksies. Sulke stelle klonaal verwante reekse is ook gevind onder die gemuteerde IgM+ MZ-B selle en IgM+ FO-B selle (hoofstuk 4). Hierdie waarnemings dui sterk daarop dat (gemuteerde) geheue B-selle met ‘n MZ-B sel fenotipe en ‘n FO-B sel fenotipe ‘n gemeenskaplike oorsprong het. Die oorsprong van hierdie selle is nie duidelik nie. Klassieke geheue B-selle (dit wil sê gemuteerde klas verwisselde B-selle) word gewoonlik gegenereer in die GC. By hierdie setels ondergaan die gemuteerde B selle ‘n vorm van keuring vir die antigeen wat die GC reaksie dryf. Dié antigeen seleksie is van kardinale belang om die affiniteit van die Ig molekules wat die antigeen kan herken te verhoog, en word weerspieël in die feit dat die IGHV gene wat kodeer vir die Ig molekules nie toevallige mutasies uitstal. Sommige van die mutasie patrone van IgG ingebou volgordes van beide die MZ-B selfraksie en FO-B selfraksie wys inderdaad tekens van antigeen seleksie (Hoofstuk 6), en bevoordeel dus die hipotese dat hulle albei gegenereer word in die GCs. Die bevinding dat gemuteerde IgM+ geheue B-selle afwesig is in neonatale diere, waarin GCs nog nie gevorm kan word nie, ondersteun die hipotese dat ook IgM+ geheue MZ-B selle in die GC gegenereer word. Die gemeenskaplike oorsprong van IgM+ en IgG+ geheue MZ-B selle en FO-B selle, geïllustreer deur die teenswoordigheid van klonaalverwante gemuteerde volgordes tussen MZ-B en FO-B selle, dui daarop dat ten minste sommige GC-afgeleide geheue selle kan óf ‘n FO-B sel fenotipe of ‘n MZ-B sel fenotipe kry. Die faktore wat hierdie differensiasie teenoor hierdie twee fenotipes aandryf is nie bekend nie. Slotopmerkings B-selle in die MZ is ‘n heterogene selbevolking en beide naïef MZ-B selle, klas verwisselend en onverwisselende geheue MZ-B selle is teenwoordig by hierdie unieke plek in die milt. Naïef MZ-B selle besit ongemuteerde Ig gene, produseer lae affiniteit IgM molekules en vorm ‘n eerste linie van verdediging teen indringer patogene. Die teenliggaam repertoire wat deur hierdie B selle uitgedruk word is veronderstel om selekteer te word om koolhidrate te bind, wat op mikro-organismes voorkom (Galson et al, 2015). Hierdie antigene benodig nie die hulp van T selle om hul respons uit te voer nie. Verder bevat die swaar kettings van die IgM Opsomming en algemene bespreking in het Afrikaans | 169 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 molekules wat deur naïef MZ-B selle uitgedruk word korter H-CDR3 streke in vergelyking met FO-B-selle in rotte (Dammers et al, 2000a) en muise (Carey et al, 2008). Korter H-CDR3 word geassosieer met polireaktiewe teenliggaam response, dit wil sê die binding van ‘n teenliggaam met verskillende strukturele antigeniese elemente (Schroeder et al, 1995). Geheue MZ-B selle druk hoë affiniteit Ig molekules uit, wat gerig word op (mikrobiese) antigene wat hul tydens hul leeftyd teekom. Dus laat die teenswoordigheid van naïef MZ-B selle en geheue MZ-B selle toe dat die MZ ‘n vinnige ingebore en verworwe teenliggaam response kan uitvoer teen mikrobiese antigene, teen beide TI en TD antigene. ‘n Nuwe rol vir neutrofiele is deur Puga et al. voorgestel in die reaksie van MZ-B selle (Puga et al, 2012). Neutrofiele teenswoordig in die milt veroorsaak die produksie van IgM+ deur die aktivering van MZ-B selle via BAFF, April en IL-21 om teenliggaam response te maak teen TI-2 antigene soos LPS, na induksie van Blimp-1 binnein die geaktiveerde MZ-B selle (Puga et al, 2012). BAFF en IL-21 kan ook IgG+ MZ-B selle stimuleer, in ‘n T-sel onafhanklike wyse, om teenliggaam afskeidende plasma selle te word (Ettinger et al., 2007a). MZ-B selle, kom voor in ‘n pre-geaktiveerde vorm, en hulle druk hoë vlakke van komplementreseptor (CR) CD21 (Timens et al, 1989) en toll-like reseptore (TLRs) uit (Gunn en Brewer, 2006) wat onderliggend is van die feit dat MZ-B selle toegerus is vir ‘n vinnige en maklike aktivering in (primêre) immuun response. Verdere stel Garraud et al. (Garraud et al, 2012) voor dat die heen en weer oorskakeling van MZ-B selle tussen die MZ en follikels, en die vervoer van antigene (immuun komplekse) na follikulêre dendritiese selle (FDC) ontsluit die rol van MZ-B selle as antigeen-presenterende selle wat deel neem in immuunreaksies om hoë affiniteit teenliggaampies te genereer. Hulle het opgemerk dat die tipe 1 interferon wat geproduseer word in reaksie op bloed-oordraagbare patogene, die sphingosin-1-fosfaat reseptor 1 (S1P1) en S1P3 inaktiveer sodat MZ-B selle uit die MZ kan migreer in reaksie op CXCL13, wat ten hoogste in follikels uitgedruk word. Die afwaartse regulering van CXCR5 kan toelaat dat MZ-B selle uit die follikels migreer en terug keer na die MZ. Hiermee vervoer MZ-B selle immuunkomplekse na die follikels waar die immuunkomplekse derhalwe opgevang word deur FDC in ‘n komplement afhanklike wyse. Die immuunstelsel komplekse vasgevang deur die FDCs is betrokke by die seleksie van gemuteerde B selle wat hoër affiniteit Ig uitdruk met behulp van follikulêre hulper T (TFH) selle. Eers sal FDCs antigeen presenteer aan die B selle wat SHM, en klas wisselendsrekombinasie (CSR) ondergaan. Daarna sal hierdie B selle antigene aan TFH selle presenteer wat oorlewingseine sal oordra aan hierdie hoë affiniteit GC B-selle, wat tot seleksie lei. Die geselekteerde GC B selle sal dan óf in geheue B-selle of plasma selle differensieer. MZ-B selle druk die inhiberende IgG bindende Fc tipe receptor FcRL5 uit (Wilson et al, 2012). Na IgG binding, kan dié reseptor die BCR inhibeer. Soos bekend is vir FcRL4, wat IgA bind, en ook die BCR inhibeer, dit is moontlik dat die besetting van FcRL5 deur IgG ‘n soortgelyke 170 | Chapter 7 rol vervul as ‘n aangepaste ingebore molekulêre skakelaar wat die BCR sein demper en die sein van die TLR verhoog (Sohn et al, 2011). Hiermee word MZ-B selle meer soos ingebore B selle wat nie meer so veel staatmaak op hul BCR vir hul aktivering nie, maar meer op die TLRs. Sodoende kan hierdie selle vinnig reageer op mikrobiologiese antigene wat in die bloed teenswoordig is, in ‘n onafhanklike BCR wyse. Aangesien MZ-B selle ‘n breë repertoire het, geheue B-selle bevat (IgM en IgG) en in ‘n soort van pre-geaktiveer staat is, is hierdie selle ideaal geskik om vinnig te reageer op ‘n wye verskeidenheid van die bloedsgedraagte antigene om sepse te verhoed. Toespitsing op hierdie B selle deur inenting in die toekoms sal van kardinale belang wees vir doeltreffende beskerming teen lewensdreigende infeksies. Opsomming en algemene bespreking in het Afrikaans | 171 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 Acknowledgements Leaving for Groningen, The Netherlands was not easy. My journey did not start off well; just before leaving to Holland I lost my beloved wife Theresa Lucinda Hendricks. During my studies I lost my mother (Eva Hendricks) and beloved brother (Peter Stephanus Hendricks) as well. Thanks to God Almighty who has carried my soul during these tough times when I have lost so much that I have love. God has blessed me with a wonderful and loving wife, Natasha and my baby girl, Eva. I would like to express my deep gratitude to Professor Frans G.M Kroese and Professor Nico Bos, my research supervisors, for their patient guidance, enthusiastic encouragement and useful critiques of this research work. With great gratitude I would also like to thank Anne Visser and Peter Dammers for their valuable contributions that they have made in training me in the laboratory techniques essential for my studies. A special thanks to Dr. Peter Terpstra, who have helped me with the Bioinformatics analysis that have resulted in my first publication for this thesis. I also like to thank all my colleagues who has helped me in this project and was also involved during my stay in the Netherlands including, all (former) PhD students, technicians, staff and students: Andre, Annie, Arjan, Carmen, Davina, Flip, Geanina, Greetje, Gwenny, Dr. Caesar Hulstaert, Jeroen, Jan, Kaushal, Karla, Lisa, Maaike, Maikel, Moniret, Neinke, Nishath, Saravanan, Rajesh, Roberta, Silvia, Sylvia, Wayel. A big thanks to all my Dutch friends for giving me a live time experiencing in Holland: Michel Lageman (my roommate), John Ditu, Mulders and Wubs families (Helperkerk), Tirzah Kock, Johan and Tessa Proost (Destiny Kerk). Also I want to thank Albert and Doreen Wiggins, Dawid and Gloria Samuels, Colin Lafoy, Rodney & Yvonne Alvis, Renuka, Jansen’s and Straus families, Charles Smurdon, Andre du Toit, Swartz, Dennis Makhubela, Le Fleur family, Yvette and Grand Daniels, Harold and Geraldine Daniels, Du Plooys, Patrick Hendricks, ma June and oom Daniel Trompetter, Heidi Opperman, Gerald January, Eben, Freddie, Leon, Dirkie, William Claassen, Mervin Coetzee and all members from the Logos Assembly and Redhill Assembly for they support in my quest. A special thanks to the Department of Human Physiology from the University of KwaZulu Natal for their financial support and particularly the Dean, Prof. Willie Daniels and Dr. Musa Mbandla, for their constant help and advice especially in the times when I thought this PhD degree will never materialised. My sincere thanks go to GUIDE for offering me a PhD fellowship and a life time enriching experience that will stay with me forever. And last but not the least, to all my brothers and sisters, father and in-laws (Pelstons, mummy Moira, Dawn, Charlton, Steffie, Theodore, Auntie Joyce, Nita, Nola and uncle Terence), thank you for all your encouragements, love and support. You will always be with me. Acknowledgements | 173