Doctorial Thesis from the Department of The Wenner-Gren Institute, Stockholm University Stockholm, Sweden

MATURATION OF HUMORAL IMMUNE RESPONSES STUDIES ON THE EFFECTS OF TYPE, APOPTOSIS AND AGE

KARIN LINDROTH

STOCKHOLM 2004 Summary The humoral immune response is dependent on the formation of . Antibodies are produced by terminally differentiated B cells, plasma cells. Plasma cells are generated either directly from antigen challenged B cells, memory cells or from cells that have undergone the (GC) reaction. The GC is the main site for class switch, and generation of memory cells. Different factors, both internal and external, shape the outcome of the immune response. In this thesis, we have studied a few factors that influence the maturation of the humoral response. We have studied how age affects the response, and we show that responses against thymus dependent (TD) are more affected than responses to thymus independent (TI) antigens, in concordance with the view that the compartment is more affected by age than the compartment. Furthermore, we demonstrate that priming early in life have a big influence on the immune response in the aged individual. Priming with a TI form of the carbohydrate dextran B512 (Dx) induces a reduction of IgG levels in later TD responses against Dx. We have evaluated possible mechanisms for this reduction. The reduction does not seem to be caused by clonal exhaustion or mediated mechanisms. We also showed that the reduced TD response after TI priming can be induced against another molecule than Dx. With the hypothesis that TI antigens induce a biased maturation of the responding B cells, we examined the presence of Blimp-1, a master regulator of plasma cell differentiation, in GCs induced by TD and TI antigen. Blimp-1 was found earlier in GCs induced by TI antigen and the staining intensity in these GCs was stronger than in TD antigen induced GCs, indicating that plasma cells might be continuously recruited from these GCs. B cells undergoing the GC reaction are thought to be under a strict selection pressure that removes cells with low affinity for the antigen and also cells that have acquired self-reactivity. We investigated the effect of apoptotic deficiencies on the accumulation of somatic mutations in GC B cells. In mice lacking the death receptor Fas, lpr mice, the frequency of mutations was increased but the pattern of the mutations did not differ from wild type mice. In contrast, mice over-expressing the anti-apoptotic protein Bcl-2, had a lowered frequency of mutations and the mutations introduced had other characteristics.

© Karin Lindroth Stockholm 2004 ISBN 91-7265-835-5 Akademitryck AB, Edsbruk 2004

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Hávamál TABLE OF CONTENTS

ORIGINAL PAPERS 5 Abbrevations 6 INTRODUCTION TO THE 7 Innate and adaptive 7 Lymphoid organs and tissues 8 Hematopoiesis 8 T cells 9 B CELL IMMUNOLOGY 10 Development to mature B cell 10 Immunoglobulin - the multifunctional receptor of the B cells 12 IgM 13 IgD 13 IgG 13 IgA 14 IgE 14 Fc receptors 15 Activation signals 15 Co-receptors 16 Complement receptor 2 16 FcγRIIB 17 B cell responses against TD antigens 17 B cell responses against TI antigens 17 Affinity maturation 19 Class switch 21 The germinal center reaction 21 Plasma cell differentiation 22 Death and survival of mature B cells 24 26 Humoral responses and aging 27 PRESENT STUDY 28 AIM 28 METHODOLOGY 29 Mice 29 Immunizations, Antigens and Adjuvants 29 ELISA 29 Estimating antibody affinity 30 Immunohistochemistry 30 Generation of hybridoma cell lines 30 Extraction of GC B cells from Peyer’s patches 30 DNA and RNA isolation, RT-PCR amplification and sequencing 31 RESULTS 32 Paper I 32 Paper II 32 Paper III 33 Paper IV 34 CONCLUDING DISCUSSION 35 ACKNOWLEDGEMENTS 38 APPENDIX: PAPER I-IV

Original papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Marta Sánchez, Karin Lindroth, Eva Sverremark, África González Fernández, Carmen Fernández. The response in old mice: positive and negative immune memory after priming in early age. Int. Immunol. 2001.13:1213-1221

II Karin Lindroth, Elena Fernández Mastache, Izaura Roos, África González Fernández and Carmen Fernández. Understanding thymus independent antigen induced reduction of thymus dependent immune responses. Submitted

III Karin Lindroth and Carmen Fernández. The role of Blimp-1 in the GC reaction. Differential expression of Blimp-1 upon immunization with TD and TI antigens. Submitted

IV Elena Fernández Mastache*, Karin Lindroth*, Africa González-Fernández¤ and Carmen Fernández¤. Somatic hypermutation of Ig genes is affected differently by failures in apoptosis caused by disruption of Fas (lpr mutation) or by over- expression of Bcl-2. Submitted * E.F.M. and K.L share first authorship. ¤ A.G.F. and C.F share senior authorship

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Abbreviations AID activation-induced deaminase APRIL a proliferation inducing ligand BAFF B cell activating factor BAFF-R BAFF receptor Bcl B cell lymphoma BCMA B cell maturation antigen BCR B cell receptor Blimp-1 B lymphocyte-induced maturation protein I BLNK B cell linker protein BM bone marrow BSA bovine serum albumin BSAP B cell lineage-specific activator C constant CDR complementarity determining region CR complement receptor CT cholera toxin D diversity DNP dinitrophenyl Dx dextran B512 EBF early B cell factor Fas FS-7 associated surface antigen FDC follicular dendritic cell FR framework region GC germinal center HSC hematopoetic stem cell Ig immunoglobulin IL interleukin IRF4 interferon-regulatory factor 4 ITAM immunoreceptor tyrosin-based activation motif ITIM immunoreceptor tyrosin-based inhibitory motif J joining lpr lymphoproliferation LPS lipopolysaccharide MHC major histocompatibility NK natural killer Ox oxazolone PALS periarteriolar lymphoid sheath PI3K phophatidylinositol 3-kinase PLCγ2 phospholipase Cγ2 PNA peanut agglutinin SHM somatic hypermutation TACI transmembrane activator and CAML-interactor TCR T cell receptor TD thymus-dependent TH TI thymus-independent TI-1/TI-2 thymus-independent type 1/ thymus-independent type 2 TLR Toll-like receptor V variable XBP-1 X box-binding protein 1 XID X-linked

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Introduction to the immune system An organism is constantly threatened by external and internal challenges. These threats have acted as evolutionary pressures leading to the emergence of various protective mechanisms. In vertebrates, mechanical barriers like skin and mucosa act as a first way to stop the entry of a wide variety of harmful organisms, pathogens, e.g. bacteria, viruses and parasites. The body also possesses physiological barriers to further complicate for the invading organism, e.g. the body temperature and the pH of the skin and body fluids. Furthermore, if a pathogen passes these barriers, it will encounter a diverse set of cells and molecules that have the capacity to recognize and eliminate the invading organism. Some of these defense mechanisms also have the ability to act against internal challenges such as transformed cells, which could otherwise lead to cancer growth. All these elements are functionally specialized parts that together make up what we call the immune system.

Innate and adaptive immunity The immune system is often described as consisting of two branches, one innate and one adaptive. The innate (or natural) immune system consists of the body’s mechanical and physiological barriers as well as effector cells and molecules. Distinctive for all these mechanisms is that they are not specific for a particular pathogen, either working unspecifically, i.e. the anatomical barriers, or recognizing elements that are conserved and used in a large group of pathogens, e.g. recognition of molecules in the cell wall of bacteria by pattern-recognition receptors. Although the innate immune system can produce a very rapid and strong response it lacks the ability to adapt to a specific invading pathogen. The innate immune system also lacks the ability to form an immunological memory, which is the capability to elicit an improved response after previous antigen exposure, a feature of the .

While the recognition by the innate immune system is conserved, one hallmark of the other branch of the immune system, the adaptive (or acquired), is its capacity to react specifically against a pathogen. This is due to the vast ability of the adaptive immune system to genetically create receptors with different specificities. These receptors are produced by specialized cells called B and T lymphocytes. Fundamental for the function of the adaptive immune response and also for the formation of memory is that one lymphocyte only carries one type of antigen-specific receptor, and that this one lymphocyte can proliferate and generate a number of identical cells, a clone, carrying the same receptor. When activated by an invading pathogen, the responding lymphocytes can exert a variety of effector functions. B lymphocytes produce a soluble variant of their receptor, an antibody, which when secreted helps to neutralize and eliminate the pathogen and also triggers some of the innate immune responses. T lymphocytes can mature into cytotoxic T cells, that can actively kill infected cells, or T helper (TH) cells which help the activation and maturation of B and T cells. After the

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infection is cleared, some of the lymphocytes with receptors specific for the antigen will remain in the body as so called memory cells. When encountering the same antigen again, these memory cells will make the immune response faster and of higher specificity than the first response. Both adaptive and innate immune responses are needed for an optimal response against pathogens, and the two systems interact with each other.

Lymphoid organs and tissues The immune system operates in several specialized environments. Their function is both to provide a microenvironment that can support the maturation of clonally diverse lymphocytes from pluripotent precursors, as well as providing the right context for bringing lymphocytes in contact with their antigen. The produced effector and memory cells must also be able to migrate to the appropriate sites. The lymphoid tissues are often divided into primary, secondary and tertiary. Examples of primary lymphoid tissues are bone marrow (BM) and thymus, which support the differentiation of progenitors to functionally mature B and T cells respectively. Secondary lymphoid tissues constitute the environment where naïve lymphocytes first meet exogenous antigen, and support the activation of naïve cells and the emergence of expanded effector and memory cell populations. This takes place in lymph nodes, Peyer’s patches and the spleen. Tertiary lymphoid tissues are the tissues with immune effector functions, the “battlefield”. These could be any tissue in the organism, but most often are tissues frequently exposed to the surroundings, such as skin and mucosa.

Hematopoiesis The effector cells of both the innate and the adaptive immune system have a common origin. They arise from hematopoetic stem cells in the BM that have the capacity to produce cells of all blood-cell lineages (figure 1). This is possible since the hematopoetic stem cells have the capacity to self-regenerate as well as allowing further differentiation of daughter cells. An early option of the differentiating cell is to develop into a myeloid or lymphoid progenitor cell. The myeloid progenitor cell can in its turn diverge to forming red blood cells, platelets or phagocytic cells (monocytes and granulocytes). Phagocytic cells have the ability to engulf and digest microorganisms. The lymphoid progenitor on the other hand can further differentiate to and form natural killer (NK) cells, B and T lymphocytes.

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Red blood cells

Platelets

Monocytes

Myeloid Granulocytes progenitor HSC Lymphoid B cells progenitor

T cells

NK cells

Figure 1. Hematopoiesis. The hematopoetic stem cell (HSC) give rise to myeloid and lymphoid progenitors which in turn can differentiate further to form specialized cell types.

T cells T cells share a common progenitor in the BM with B cells; however their development into functionally competent cells takes place in a different organ, the thymus. In the thymic environment the T cell progenitors rearrange the genes coding for their antigen specific receptor. The process in which the cell through rearrangement forms its antigen receptor is closely linked to two selection processes. One process, the positive selection, ensures that the becoming T cell only recognizes antigen in the form of processed peptide bound to the surface of major histocompatiblility complex (MHC) class I or class II molecules. The other process, the negative selection, eliminates those cells that show reactivity against self molecules, thereby removing cells that could potentially cause autoimmune reactions [reviewed in Palmer 2003]. When leaving the thymus the majority of T cells will carry a receptor that consists of the T cell receptor (TCR) α and β chain associated to the CD3 complex, a few cells will express the TCR γ and δ chains. In this complex, the TCR chains are responsible for recognition of antigen (peptide+ MHC class I/II) while the intracellular signaling is conveyed by the CD3 subunits [reviewed in Alarcon et al 2003].

The emerging T cells can further be divided into subsets based on the expression of the CD4 or CD8 co-receptors. CD8+ T cells recognize antigen when it is processed and presented on a cell surface by MHC class I molecules. Almost all nucleated cells express these molecules, in contrast to MHC class II molecules that are only

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constitutively expressed by a group of cells called professional antigen presenting cells, including macrophages, B cells, dendritic cells and thymic epithelial cells. Peptides presented by MHC class II are the recognition element for the other T cell subset, the CD4+ T cells. Due to differences in the process of peptide presentation, MHC class I molecules most often present peptides that have been produced by the presenting cell, while the MHC class II molecule presents peptides that have an extra-cellular origin. This is also reflected in the effector functions of the different T cell subsets. CD8+ T cells have cytotoxic functions and can thus eliminate cells that present internally produced foreign peptides on their surface, e.g. a virus infected cell. The CD4+ T cells on the other hand are potent activators and modulators of other immune cells through the use of different co-stimulatory molecules and soluble factors e.g. interleukins (IL).

These cells are often referred to as TH cells. Interactions between T and B cells are important for several different phases in the humoral immune response. They will be discussed in more detail in their biological context.

B cell immunology

Development to mature B cell Unlike T cells, the development of B cells from progenitor stem cells to functionally mature B cells takes place in the BM. The B cell progenitor assembles the Ig genes during its development in the BM, and different stages of B cell development have been characterized in part based on which stage in the rearrangement process the cell is in [Hardy et al 1991; reviewed in Karasuyama et al 1996; reviewed in Osmond et al 1998]. The development of B cells is characterized by successive rearrangements, first of the gene for the Ig heavy chain and then the gene for the light chain. Originally, the loci for the variable (V) region of the Ig chain contain different elements that will be part of the assembled gene. The heavy chain region has V, diversity (D) and joining (J) elements, and the light chain region V and J elements. The assembly of the heavy chain

V region occurs in two steps, first is one of the DH elements joined with a JH element, and then a VH element is joined to the combined DJ region to complete the gene (figure 2).

When B cell precursors rearrange the VH to the DHJH-rearranged alleles, the rearrangement can be either in or out of frame. An in-frame rearrangement leads to expression of an Ig heavy chain with the potential to form a pair with a surrogate light chain forming a pre-B cell receptor (BCR) on the cell surface. Cells that have been able to express a pre-BCR undergo two important events. The VH to DHJH rearrangement at the second heavy chain allele is stopped, a process leading to allelic exclusion. This

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VH DH JH Cµ germline

VH DH JH Cµ DJ rearranged

VH DH JH Cµ VDJ rearranged Figure 2. Rearrengement of the Ig heavy chain genes. In the first step a D and a J element are joined. To complete the gene the DJ region is joined with a V element.

prevents the B cell from expressing two different heavy chains. Furthermore, a proliferative expansion is induced in the cells carrying the pre-BCR, a kind of positive selection for pre-B cells with a productive VHDHJH rearrangement. After this proliferative burst the cells will lose the expression of the surrogate light chain and start to rearrange the light chain genes. In this case a V region is joined directly to a J element. This assembly of the V genes contributes immensely to the diversity of antigen-binding specificities. The presence of multiple germline V, D, and J elements creates a large number of different possible combinations. Furthermore, the elements can be joined at different positions and extra nucleotides can be inserted in the junctions, thus creating a huge potential for constructing unique receptors. Cells that have managed to produce a light chain rearrangement, resulting in an expressed polypeptide chain that can pair with the heavy chain, will become immature B cells, displaying an IgM receptor on the surface. These immature B cells will undergo a negative selection aiming to remove B cells that recognize self antigens. If an immature B cell encounters its antigen it will result in down regulation of the expression of IgM and B220, and an arrest of further differentiation [Hartley et al 1993]. Recognition of antigen can also result in receptor editing, when the immature B cell further rearranges the light chain with the chance of creating a non-auto reactive receptor and thus avoid negative selection [Gay et al 1993; Radic et al 1993; Tiegs et al 1993].

To develop into a mature B cell, the immature B cell leaves the BM and head out to lymphoid organs in the periphery, where it has the possibility of encountering antigen and differentiating into either a memory or a plasma cell. If not receiving the signals necessary for maturation, the half-life for an immature B cells is measured in days. In fact only 5-10% of the newly generated immature B cells get selected into the pool of mature B cells [reviewed in Osmond 1993].

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Immunoglobulin - the multifunctional receptor of the B cells The characteristic molecule of the B cells is the antigen receptor, the immunoglobulin (Ig). The Ig exists both as a membrane bound receptor, the BCR, as well as in a secreted form that is produced by activated B cells, plasma cells. As surface receptor the Ig is involved in such fundamental cellular processes as differentiation, activation and apoptosis, while the secreted form is capable of both directly neutralizing foreign antigen as well as recruiting other effector systems.

The Ig consists of four polypeptide chains paired together, two larger chains, called heavy chains, and two shorter chains, the light chains (figure 3). Each chain is built up of several smaller units called immunoglobulin homology domains. The Ig light chain contains two such Ig domains, the heavy chain has either four or five depending on the of the antibody.

Antigen binding

VL VH Figure 3. The immunoglobulin (Ig). The Ig is formed by two light and two heavy chains paired together. The variable

CL CH1 (V) parts of the Ig are the part that s s bind to antigen while the constant s s s s (C) regions carry the other functions of the Ig molecule.

CH2 Fc part

CH3

Due to great sequence variability the amino-terminal part of each chain is termed a V region, VH and VL for the heavy and light chains respectively, and is the region that carries the antigen specificity. The antigen binding part of the antibody can be further divided into areas with high or low sequence variability. A V region has four more conserved motives called framework regions (FR1-4), which are separated by the highly variable complementarity determining regions (CDR1-3). When the protein folds the CDR regions come together and form the antigen-binding surface. The interactions between the antibody and the antigenic surface are formed through hydrogen bonds, electrostatic and Van der Waals interactions as well as hydrophobic forces [reviewed in Mariuzza et al 1994]. The sequences of the other Ig domains are not so variable and are consequently called constant (C) regions. The light chain only have one C domain, while the heavy chain can consist of three or four C domains depending on isotype.

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The C region of the heavy chain is responsible for the functions of the antibody apart from antigen binding, for example interacting with receptors and complement and the ability to multimerize. There are five different classes of heavy chain C regions, each corresponding to an Ig isotype with characteristic effector functions. The Ig isotypes, IgM, IgD, IgG, IgA and IgE, can appear both as receptors on the cell surface and in a secreted form, with each C region class giving different specific functions to the Ig molecule. During an antibody response there is often class changes of the antibodies produced. However, although an individual B cell can be induced to produce different Ig classes during its life time, each B cell will only produce Igs with identical binding sites for the antigen, Igs with identical antigen specificity.

IgM IgM is the first isotype expressed during B cell development, and is the major isotype in primary antibody responses. In sera 5-10% of the Igs are of this isotype, and IgM is the second most common isotype in the mucosa after IgA. In responses against thymus independent (TI) antigens like polysaccharides, there is a strong induction of antigen specific IgM. It is also the major isotype of so called “natural antibodies” which are produced without being part of a humoral response against a specific antigen. Generally the strength of the antigen-antibody association, the affinity, of IgM antibodies is low. However, since IgM is secreted as a pentamer it can compensate for the low affinity by carrying a total of ten possible binding sites. The increase in the possible number of antigen-antibody interactions enhances the total affinity, the avidity. The pentamer form enables the IgM molecule able to bind with high avidity to antigens that have the same epitope in multiple copies. IgM is also the most efficient antibody class in binding and activating complement, enabling it to opsonize or destroy targets by lysis.

IgD IgD is only secreted to a minor extent; instead it is expressed together with IgM on the cell surface, for example on mature naïve peripheral B cells and on follicular B cells. IgD and IgM are the only Ig classes that can be co-expressed. Studies of mice lacking IgD have not been able to reveal its exact immunological role. However, the fact that IgD is present in all mammals and is well conserved among species suggests that it has a distinct purpose.

IgG High titers of IgG are characteristic of a secondary immune response, and it is also the most common Ig class in blood and lymph. The membrane IgG, and IgE, have a longer cytoplasmatic tail than the other immunoglobulin classes, which seems to enhance the efficiency of memory responses [Achnatz et al 1997; Kaisho et al 1997; Martin and Goodnow 2002]. The IgG class consists of four subclasses, IgG1, IgG2a, IgG2b and IgG3, in mice, and IgG1, IgG2, IgG3 and IgG4, in humans. Responses against different

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types of antigens tend to preferentially use different IgG subclasses, anti-carbohydrate specificities tend to be IgG2b and IgG3 in mice [Ivars et al 1983; Perlmutter et al 1978; Slack et al 1980] and IgG2 in human [Barrett and Ayoub 1986; Riesen et al 1976; Yount et al 1968]. Responses against proteins induce IgG1 [Riesen et al 1976; Scott and Fleischman 1982] and anti-viral responses IgG2a [Coutelier et al 1987] in mouse, while in man anti-protein and anti-viral antibodies often are IgG1 and IgG3 [reviewed in Papadea and Check 1989; Skvaril and Schilt 1984]. The effector functions of the IgG subclasses also differ, complement is most efficiently fixed by IgG2a and IgG2b [Ey et al 1980; Neuberger and Rajewsky 1981] in mouse and IgG1 and IgG3 in human [Bruggemann et al 1987; Dangl et al 1988].

IgA The daily production of IgA is greater than the production of all other Ig classes combined. IgA is the major Ig class in secretions such as saliva, mucus, gastric fluids and tears. Secretory IgA coats all body surfaces except skin and is thus an important first line of defense. IgA is also the major Ig in colostrum and breast milk, thereby providing the neonate with a defense against intestinal pathogens. Secretory IgA is polymeric, while serum IgA is predominantly monomeric. Serum and secreted IgA originate from separate pools of B cells. Whereas secreted IgA is produced by plasma cells in specialized sites of the respiratory, urogenital, gastrointestinal and mammary tissue, the serum IgA is produced in BM, lymph nodes and the spleen [Craig et al 1971; Fagarasan et al 2001; Kutteh et al 1982]. The protection mediated by secreted IgA consists of different functions. IgA binding and cross-linking pathogens prevent the uptake of the pathogens across the epithelia and thereby facilitate the pathogen removal in the mucus excretions. Furthermore, IgA-antigen complexes can be transported across the epithelia into the lumen and at the stromal side IgA-antigen complexes can be eliminated by expressing receptors for IgA, FcαR [reviewed in van Egmond et al 2001]. It has been shown that mucosal IgA responses can be induced in a T cell independent manner and it was suggested to be a part of an evolutionary old form of specific immune defense [Macperson et al 2000].

IgE IgE is mainly produced by plasma cells in the lung and skin. IgE antibodies can be bound to FcεRI on mast cells and basophils. Multivalent antigens can then cross-link the bound IgE and thereby indirectly cross-link the FcεRI molecules. This will induce degranulation and production by mast cells and basophils. While this type of response can be important for anti-parasite responses, it also causes allergic reactions in predisposed individuals.

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Fc receptors One type of receptors that is involved in antibody recognition is the Fc receptors [reviewed in Daëron 1997; reviewed in Ravetch and Kinet 1991]. They recognize and bind the Fc part of antibodies. Fc receptors exist for all antibody classes. They can be divided into receptors that can promote or inhibit cell activation as well as receptors that are involved in the transport of Ig through epithelia. Receptors that mediate antibody transportation are the neonatal Fc receptor for IgG, which facilitates transport of maternal IgG into the fetal circulation, and the polymeric IgA and IgM receptor. Signaling through Fc receptors induce a great variety of actions depending on the cell type carrying the receptor. Examples are the release of inflammatory mediators, cytolysis and mediation of phagocytosis as well as a negative feedback regulation of B cells.

Activation signals B cell antigen receptors exist as a complex between membrane-bound Ig and at least two other transmembrane polypeptides, Ig-α and Ig-β. Ig-α and Ig-β both have a single external Ig-like domain, a transmembrane region and a cytoplasmatic tail which interacts with signaling components [reviewed in Reth and Wienands 1997]. The cytoplasmatic tail carries a sequence with homology to other leukocyte receptors, the immunoreceptor tyrosin-based activation motif (ITAM). Cross-linking of receptor molecules by oligomeric or multimeric antigens will trigger signaling through the BCR (figure 4). Cross-linking will increase phosphorylation of specific proteins, and this phosphorylation leads to altered activity of these proteins or to assembly of signaling complexes. The initial event is phosphorylation of the ITAMs of Ig-α and Ig-β by Scr family tyrosine kinases (Blk, Lyn, Fyn), which leads to the recruitment of Syk to the receptor complex. When bound to a phosphorylated ITAM, Syk itself becomes phosphorylated, which upregulates its activity. The activated tyrosine kinases will trigger complex series of signaling events. Important signaling pathways are mediated by phospholipase Cγ2 (PLCγ2), phosphatidylinositol 3-kinase (PI3K) and activation of Ras.

Figure 4. antigen BCR mediated signaling pathways. BCR Phosphorylation of the Ig-α/β ITAMs by Scr family kinases such as Lyn recruits Ig-α/β Syk. After phosphorylation BLNK attracts Btk and PLCγ2, which leads to the activation of PLCγ2. Two other important Lyn pathways is also initiated, the Ras pathway γ Btk PLC 2 and the PI3K pathway. P Syk The figure is based upon figures in Gold P BLNK 2002; Niiro and Clark 2002; Wang and Clark 2003.

Ras PI3K

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The activation of PLCγ2 seems to be dependent on Syk as well as Btk. Phosphorylation of B cell linker protein (BLNK), probably by Syk, provides docking sites for Btk as well as PLCγ2. When brought together, PLCγ2 is activated by Btk [reviewed in Kurosaki et al 2000]. The PLCγ2 pathway will eventually lead to activation of the transcription factor CREB (cyclic adenosine monophosphate response element binding protein) [Xie and Rothstein 1995; Xie et al 1996] as well as the NFAT family [Venkataraman et al 1994], Ets-1 [Fisher et al 1991], NF-κB and ATF-2 [Dolmetsch et al 1997].

Co-receptors An important aspect of BCR signaling is the modulation of the signal by so called co- receptors. These are cell surface receptors able to detect different elements of the immune system and, when activated, to adjust the BCR signaling and regulate the cellular response. Two important co-receptors for BCR signaling are the promoting complement receptor CR2, and the inhibiting FcγRIIB [reviewed in Buhl and Cambier 1997]. Thus, if an antigen has been recognized by the complement system it will enhance the B cell response, while high levels of antigen specific IgG will be an indicator that no further antibody response is needed and will lead to the antibody production being “turned off”.

Complement receptor 2 The complement system is a complex system of proteolytic enzymes, regulatory proteins and surface receptors. It can on its own, as well as through interaction with antibodies, mediate lysis of microbes. B cells express two receptors for complement components, the complement receptors 1 and 2 (CR1 and CR2). CR2 enhances B cell activation, and is primarily expressed on B and follicular dendritic cells (FDCs). On the B cell, the CR2 can be found either in complex with CR1 or with CD19 and TAPA-1 [Matsumoto et al 1991]. CD19 can also be found associated with the BCR but at low stoichiometry [Carter et al 1997]. Compared to cross-linking of BCR alone, cross- linking of CR2 or CD19 together with the BCR leads to synergistic PIP2 hydrolysis and enhanced B cell proliferation [Carter and Fearon 1992; Carter et al 1988]. The importance of CD19 and CR2 for the humoral response is apparent in mice deficient for these receptors. Mice lacking CR2 or CD19 have a decreased response against thymus- dependent (TD) antigens [Ahearn et al 1996; Croix et al 1996; Engel et al 1995; Molina et al 1996; Rickert et al 1995]. Interestingly, the response against TI-2 antigens in these mice is unaffected or increased. So although the CR2/CD19/TAPA-1 complex is an important enhancer of the humoral response it does not appear to be essential for B cell activation. The enhancement of the response is thought to be mediated through the cytoplasmatic tail of CD19. When phosphorylated, the tail is recognized by PI3K [Tuveson et al 1993] and Vav [Weng et al 1994]. CD19 can also associate to some

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extent with Lyn and Fyn [Uckun et al 1993; van Noesel et al 1993], which could promote the initial event of ITAM phophorylation.

FcγRIIB Another co-receptor for B cells is the FcγRIIB, which is co-ligated with the BCR when the antigen has the form of a soluble immune complex. FcγRIIB has low affinity for IgG. Murine FcγRIIB binds IgG1, IgG2a and IgG2b subclasses but not IgG3 [Weinshank et al 1988]. The cytoplasmatic tail of FcγRIIB carries an immunoreceptor tyrosine-based inhibitory motif (ITIM) which mediates a negative signal to the B cell, possibly by inducing dephosphorylation of CD19 [Hippen et al 1997]. Examples of the inhibition are decreased B cell proliferation [Phillips and Parker 1984] and antibody secretion [Phillips and Parker 1984; Phillips and Parker 1985].

B cell responses against TD antigens Different types of antigens induce humoral responses through different mechanisms. When recognizing antigens such as proteins, B cells need co-stimulatory signals provided by TH cells to be able to elicit a response. This type of antigen is called TD antigens. Naïve B cells circulate through blood, lymph nodes and spleen until they encounter antigen. This often happens in the T cell area in the spleen or lymph nodes, to which antigen is brought by macrophages and dendritic cells. After encountering the specific antigen, the B cell will stop migrating and will linger in the T cell zone [reviewed in Kelsoe and Zheng 1993; Liu 1997]. B cells specific for the antigen will be able to internalize the antigen via the BCR complex. The antigen is then processed and presented on MHC class II molecules on the cell surface. In this area, naïve antigen- specific T cells can be activated, probably by antigen presenting interdigitating dendritic cells. Upon antigen recognition activated T cells can form stable conjugates with the antigen-presenting B cells, and deliver both receptor mediated and soluble stimulatory signals to the B cells. This induces proliferation and terminal differentiation of the B cells to plasma cells. These plasma cells produce the early wave of antibodies generated after antigen challenge [reviewed in MacLennan et al 2003]. Some activated B cells do not undergo terminal differentiation, but will instead migrate into the follicular region and initiate the germinal center (GC) reaction. Though the complete requirement of the signals needed for differentiation either into plasma cells or GC B cells is unknown, the signaling via CD40 on the B cell induced CD40L on the T cell seems to be required for GC formation [Foy et al 1994; Kawabe et al 1994].

B cell responses against TI antigens Not all antigens need T cell help to activate B cells. This group of antigen is called TI, and is further divided in to TI-1 and TI-2 type of antigens. The subdivision of TI-1 and TI-2 antigens originates in their ability or inability to give rise to a humoral response in XID (X-linked immunodeficiency) mice. These mice have lost the function of the

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tyrosine kinase Btk, which is involved in BCR signal transduction. XID mice can respond against TD and TI-1 antigen but not to polysaccharides, the classical group of TI-2 antigens [Amsbaugh et al 1972; reviewed in Scher 1982]. The most striking feature of the TI-1 antigens is their mitogenic properties. They are polyclonal B cell activators that can induce differentiation of B cells into plasma cells regardless of their antigen specificity. Many TI-1 are molecules found in bacterial cell walls, e.g. lipopolysaccharide (LPS), peptidoglycan, and lipoprotein. The responses against LPS have been studied extensively and it has been shown that B cells in mice carry a specific receptor capable of recognizing LPS, the Toll like receptor (TLR) 4 [Hoshino et al 1999; Poltorak et al 1998].

TI-2 antigens are characterized by their repetitive structure, e.g. polysaccharides, and some are capable of polyclonally activate B cells, e.g. dextran and levan [reviewed in Coutinho et al 1974; reviewed in Coutinho and Möller 1975]. This type of antigens can be found both as capsular polysaccharides in bacterial cell walls and on viral particles. The importance of the repetitive structure for the TI-2 response has been shown by using haptenated polyacrylamide as antigen. To elicit a strong in vivo TI antibody response the molecule had to have a minimum of 12-16 with proper spacing [Dintzis et al 1976], suggesting that TI-2 antigens need to be able to efficiently cross- link BCRs. Although not dependent on T cell help, the TI-2 response is supported by T cells [Endres et al 1983; Letvin et al 1981; Mond et al 1980; Wood et al 1982]. CD40 ligation [Dullforce et al 1998; Garcia de Vinuesa et al 1999; Snapper et al 1995] as well as different [reviewed in Mond et al 1995, reviewed in Snapper and Mond 1996; reviewed in Vos et al 2000] improve the response. The source for these cytokines during the TI response is unknown, both bystander responding T cells as well as NK cells could be producers, and possibly also B cells.

The cross-linking of BCRs by TI-2 antigen is considered to be a first step of B cell activation needing additional stimuli to generate a humoral response [Coutinho and Möller 1974; reviewed in Möller et al 1991; reviewed in Vos et al 2000]. The BCR cross-linking is thought to give a first signal as a result of the formation of small clusters with cross-linked BCRs that induce signaling cascades [review in Vos et al 2000]. A more passive role of the BCR has also been suggested, where it would serve to focus the antigen on the cell surface, thereby enabling pathogen-associated molecular patterns to be recognized by pattern-recognition receptors which activates the B cell [Alarcon-Riquelme and Möller 1990; Möller 1999; reviewed in Möller et al 1991]. TLR receptors, capable of recognizing a wide variety of pathogen-associated molecules [reviewed in Takeda et al 2003], are one type of receptors that could mediate a second activating signal. LPS which is recognized by TLR-4 has proven to enhance TI-2 responses in vivo [Zhang et al 1988] as well as in vitro [Hosokawa et al 1989]. Furthermore, many TI-2 antigens can fix complement and thus induce enhancement of

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the BCR signal by recruiting and activating the CR2/CD19/TAPA-1 complex [reviewed in Carroll 1998].

Affinity maturation As the immune response matures there is a shift in antibody repertoire [Berek et al 1991; Foote and Milstein 1991], the antigen-specific antibodies are of higher affinity than early in the response and other Ig classes are used. This phenomenon is called affinity maturation, and it mainly takes place in the environment of the GC. Somatic hypermutation (SHM) is a process that introduces changes in the Ig genes and thus in the ability of the Ig to bind antigen. During the hypermutation process, mutations will be incorporated with a rate around 10-3/base pair/cell generation [reviewed in Allen et al 1987; Berek et al 1991; McKean et al 1984]. It has been calculated that with an approximate length of the V region of 300 nucleotides, one nucleotide exchange will take place in the VH and in the VL chain every third division.

The hypermutation target sequence, the mutation domain, spans over approximately 1500 nucleotides [reviewed in Wagner and Neuberger 1996]. Nucleotide substitutions are seen only in rearranged V regions [Gorski et al 1983; Roes et al 1989; Weber et al 1991] and their flanking sequences [Lebecque and Gearhart 1990; Motoyama et al 1994; Rothenfluh et al 1993; Weber et al 1991]. The sequence of the V region is not important for marking it a target for hypermutation. It has been shown that replacing the gene with other sequences does not affect the introduction of mutations in the sequence [Azuma et al 1993; reviewed in Storb et al 1996; Tumas-Brundage and Manser 1997; Yelamos et al 1995]. To assess the probability of a base to be replaced, mutation patterns have been studied in sequences not under selection pressure [Betz et al 1993; Dörner et al 1997; Hackett et al 1990]. Transitions (A↔G and C↔T) are more frequent than transversions (A→C/T, G→C/T, T→A/G and C→A/G), and there is a strand bias for the coding strand. Some triplets are also more frequent targets, hot spots, than other, mutational cold spots [Smith et al 1996; reviewed in Wagner and Neuberger 1996]. These hot spots often coincide with the CDRs [Betz et al 1993; Reynaud et al 1995].

The exact mechanism by which the mutations are introduced is not known. Some observations suggest that it is a form of error-prone DNA repair mechanism: 1) the dependence on an active promotor [Peters and Storb 1996; Tumas-Brundage and Manser 1997], 2) the hypermutation domain starts downstream of the promotor region [Lebecque and Gearhart 1990; Rothenfluh et al 1993; Weber et al 1991], 3) the transcription enhancer sequences are required for hypermutation [Betz et al 1994], and 4) there is a strand bias [reviewed in Wagner and Neuberger 1996].

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Recently it has been shown that the hypermutation process requires the expression of activation-induced deaminase (AID), and it has been suggested that AID initiates SHM by cytidine deamination of DNA [Petersen-Mahrt et al 2002]. Interestingly, mutations of the AID gene lead to deficiencies in class switch recombination as well as in SHM and gene conversion indicating that there is a common mechanistic link in these processes [Arakawa et al 2002; Harris et al 2002; Muramatsu et al 2000; Revy et al 2000] (figure 5).

Class switch SHM

VDJ E Sµ Cµ Cδ Sx Cx V region 5' 3' Introduction of Introduction of DNA breaks DNA breaks Cµ Cδ 5' 3' AID Sµ Sx Mutations by dependent error-prone VDJ E Cx DNA synthesis

DNA repair DNA repair Cµ Cδ VDJ E Sµ/x Cx 5' 3'

Sµ/x

Figure 5. Ig class switch and SHM. A schematic model of the two mechanism. Nicking of the switch (S) region and the V region is thought to be dependant on AID. The repair during class switch will result in nonhomologous recombination and excision of the intervening DNA. During SHM the DNA breaks will be filled by an error prone DNA synthesis and the mutations will become fixed after DNA repair. The figure is based upon figures in following papers, Diaz and Casali 2002; Kenter 2003; Kinoshita and Honjo 2001.

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Class switch In order to elicit an optimal antibody response, the B cells also have the ability to alter the antibody class used in the response by exchanging the CH regions. Necessary for class switching to take place is DNA-synthesis and transcription of the germline form of the CH gene to which switching will occur, in addition to undefined processes that affect the activity of a switch recombinase. The major mechanism for class switching includes looping out and deletion of CH genes by nonhomologus recombination [reviewed in Honjo et al 2002]. Switching typically occurs in sequences with limited or no homology within so called “switch regions”. Switch regions act as binding or assembly sites and are located 5´ of each CH gene except Cδ. Different stimuli are important for the induction of class switch. Examples are the cytokines interferon-γ, and IL-10, often described as “switch factors” [reviewed in Snapper et al 1997; reviewed in Stavnezer 1996]. They have the ability to induce transcription of the germline form for the isotype to which switching will occur. Another group of stimuli, including LPS, cross-linking of membrane Ig or CD40, acts in a more unspecific way to induce DNA-synthesis, Ig secretion and class switch. IL-4 has the ability to induce both types of effects.

The germinal center reaction When challenged with a TD antigen the major site for B cell activation is the periarteriolar lymphoid sheath (PALS). The activated B cells will form foci of proliferating cells near the arteriole [Jacob et al 1991a]. These B cells can have two different fates, either they differentiate into plasma cells, or they will migrate into the primary follicle and start the GC formation. The classical GC is divided into a dark and a light zone. The dark zone consists of B cells called centroblasts that are proliferating vigoursly. The light zone B cells, the centrocytes, are nondividing and in close contact with a network of FDCs. The FDCs can present antigen to the B cells in the form of immune complex bound to complement or Fc receptors on the cell surface [reviewed in van Nierop and de Groot 2002]. Competition among the B cells for the bound antigen is thought to create the selection of high affinity B cells. Those B cells that are not selected will die by apoptosis, and the debris engulfed by tingible body macrophages at the border between the dark and light zone. Some T cells can bee seen in the GCs, these are antigen specific [Fuller et al 1993] mainly CD4+ and are found in the light zone [reviewed in Kelsoe 1995].

The first clusters of proliferating GC B cells can be seen in the primary follicles three to four days after antigen challenge. In the follicles the B cells will proliferate rapidly and form a GC [reviewed in MacLennan and Gray 1986; Nieuwenhuis and Opstelten 1984]. The cell cycle of a centroblast can be as short as 12 to 6 hours [Fliedner et al 1964; reviewed in MacLennan et al 1991; Zaitoun 1980]. By day 8 the centroblasts will fill the FDC network. The naïve IgD+IgM+ B cells that occupied the primary follicles have been pushed out and form what is called the mantel zone. The classical dark and light

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zones of the GC will form over the next days. GC B cells will proliferate and accumulate mutations in the dark zone, undergo selection in the light zone, leading either to apoptosis, differentiation into memory or plasma cells or re-entry into the dark zone (figure 6). With time, the number of centroblasts will decrease and around day 15 the GC is mainly occupied by centrocytes, and approximately three weeks after antigenic challenge the GC reaction has faded [reviewed in Kosco-Vilbois et al 1997; Liu et al 1991].

The proliferating B cells in the GC will include mutations in their Ig genes and will thus give rise to daughter cells which carry variants of the original receptor [Jacob et al 1991b; Ziegner et al 1994], variants that have a possible different binding capacity. These cells are selected for the highest affinity to the antigen and can after exiting the GC differentiate into memory or plasma cells [Berek et al 1991; Liu et al 1996; McHeyzer-Williams et al 1993; Smith et al 1997; Ziegner et al 1994;].

Figure 6. Dark zone Light zone The GC reaction. Antigen activated B cells can be induced to form a GC. In the dark zone centroblast undergo rapid proliferation and SHM. When entering the light zone these cells • proliferation will compete for antigen on FDCs • SHM and receive stimulatory or apoptotic signals from GC T cells. The † • selection centrocytes can either be promoted • apoptosis leave the GC and further • re-entry to the dark zone memory differentiate into memory and plasma cells, re-enter the dark zone plasma cell or undergo apoptosis.

Plasma cell differentiation During a humoral immune response two highly differentiated cell types develop, plasma cells which produce and secrete antibodies and memory cells that will facilitate a more rapid secondary response of higher affinity. It is not clear which signals decide if a B cell should leave the GC. Though not firmly supported by experimental evidence, it has been proposed that signaling via high-affinity Ig would end the process of affinity maturation [reviewed in Calame 2001]. The signals governing the decision between plasma cell and memory cell fate is also unclear. The results from one study suggest that plasma cell fate would be favored by expression of high-affinity antibodies [Smith et al 1997]. In vitro studies have also shown that co-stimulation via CD40 favors a memory phenotype, whereas in the absence of CD40L, IL-2 and IL-10 induce plasma cell

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differentiation [Arpin et al 1995]. IL-10 can also interrupt memory cell expansion [Choe and Choi 1998], while IL-4 promotes formation of memory B cells [Zhang et al 2001]. Furthermore, the formation of plasma cells is suggested to be promoted by signaling via OX40L [Stuber and Strober 1996].

Plasma cells are the terminally differentiated B cells. They do not divide and during their life time, which can span between days up to months, they produce and secrete Ig. The phenotype of the cell changes to meet the new demands. The cytoplasmic compartment increases in size, and it contains large amounts of rough endoplasmic reticulum and secretory vacuoles. Various surface molecules are down-regulated when the B cell differentiates into a plasma cell, for example MCH class II, B220, CD19, CD21 and CD22. Accordingly, the levels of different transcription factors will change in the cell. Some transcription factors will decrease such as B cell lineage-specific activator (BSAP), CIITA, early B cell factor (EBF) and Bcl-6, while the expression of others, like B lymphocyte-induced maturation protein I (Blimp-1), interferon-regulatory factor 4 (IRF4) and X box-binding protein 1 (XBP-1), are increased in plasma cells [reviewed in Calame et al 2003]. XBP-1 is needed for plasma cell differentiation, but not in earlier steps of B cell development [Reimold et al 2001]. It is expressed ubiquitously; however, XBP-1 is induced in activated B cells and is strongly expressed in plasma cells [Reimold et al 2001]. It is negatively regulated by BSAP [Reimold et al 1996], a transcription factor that decreases during differentiation to plasma cells [Barberis et al 1990; Rinkenberger et al 1996], which could in part explain the increase in XBP-1 during this stage. IRF4 is expressed mainly in lymphoid cells, and is increased in plasma cells and a plasma cell-like subset of GC cells [Falini et al 2000]. Mice lacking IRF4 have severely reduced serum Ig levels, at least a 99% reduction, and fail to produce antibodies in response to antigen challenge [Mittrücker et al 1997].

Blimp-1 was found as a gene being induced in IL-2 and IL-5 driven differentiation of the cell line BCL1 into a plasma cell state [Turner et al 1994], and was later also observed in splenocytes [Schliephake and Schimpl 1996]. Blimp-1 has been shown in vivo to be expressed in all plasma cells, both in TI and TD responses and in secondary responses [Angelin-Duclos et al 2000], and recently it was shown that Blimp-1 is required for plasma cell differentiation [Shapiro-Shelef et al 2003]. Blimp-1 acts as a transcriptional repressor of the c-myc [Lin et al 1997], CIITA genes [Piskurich et al 2000] and Pax5 [Lin et al 2002]. c-Myc is needed for proliferation and cell growth, and the repression of its transcription can explain the non-dividing state of terminally differentiated plasma cells. Likewise, the repression of CIITA provides an explanation for the down-regulation of MHC class II expression in plasma cells. The repression of Pax5, coding for BSAP, is probably important for triggering the plasma cell development. BSAP is important in early stages of B cell development as well as for isotype switching in GCs [reviewed in Michaelson et al 1996; reviewed in Morrison et

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al 1998]. The formation of antibody secreting cells is dependent on down-regulation of BSAP [Lin et al 2002; Usui et al 1997], probably due to BSAPs ability to repress XBP- 1 [Reimold et al 1996], J chain [Rinkenberger et al 1996] and Ig H chain [Singh and Birshtein 1993] gene transcription. Blimp-1 has also the ability to down-regulate Bcl-6, a protein necessary for GC formation [Dent et al 1997; Ye et al 1997]. Bcl-6 can on the other hand repress the gene encoding Blimp-1, Prdm1, [Shaffer et al 2000] creating a feedback loop controlling the B cell fate. While Bcl-6 is expressed in a GC B cell, Blimp-1 expression is repressed and the differentiation into plasma cell is blocked. However, when Prdm1 is induced, Blimp-1 will be able to inhibit the actions of Bcl-6 and the B cell will terminally differentiate into a plasma cell (figure 7).

Pax5 Bcl-6

Figure 7. XBP-1 Blimp-1 IRF4 Transcriptional regulation of plasma cell differentiation. XBP-1, IRF4 and Blimp-1are important for plasma cell CIITA c-myc differentiation. Blimp-1 negative regulates Bcl-6 and Pax5, both important for controlling the GC B cell stage, as well as CIITA and c-myc. Terminal differentiation • Ig production • stop in cell cycle • change in surface proteins

Death and survival of mature B cells Differentiation into long-lived mature B cells requires signaling by B cell activating factor (BAFF) through BAFF receptor (BAFF-R) [reviewed in Laâbi et al 2001; Schneider and Tschopp 2003]. BAFF deficient mice have normal numbers of early B cell stages but lack mature B cells [Gross et al 2001; Schiemann et al 2001], and mice expressing a BAFF transgene accumulated immature and mature B cells, had increased Ig levels and showed signs of autoimmune disease [Gross et al 2000; Khare et al 2000; Mackay et al 1999]. BAFF seams to support B cell differentiation and survival through regulating expression of Bcl-2 family members [Do et al 2000; Hsu et al 2002]. Furthermore, BCR signaling is crucial for B cell survival, mature B cells that loose their expression of the BCR dies rapidly [Lam et al 1997].

The negative selection in BM and spleen will result in cells either being removed by apoptosis, rendered anergic or induced to undergo receptor editing [reviewed in

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Nemazee 2000]. Whether removal of auto-reactive cells is induced via the death receptor Fas is under debate [Rathmell and Goodnow 1994; Rubio et al 1996; reviewed in van Eijk et al 2001a], but is reported to be blocked by Bcl-2 and Bcl-XL [Fang et al 1998; Hartley et al 1993; Lang et al 1997; Nisitani et al 1993]. Loss of the Bcl-2 family member Bim has been shown to block apoptosis of autoreactive B cells, normally released Bim interacts with Bcl-2 and inhibits its anti-apoptotic function [Enders et al 2003].

Activation of B cells leading to proliferation and differentiation requires signals through the BCR and co-stimulatory receptors. CD40L [reviewed in van Kooten and Banchereau 1997], BAFF [Moore et al 1999; Schneider et al 1999] and APRIL (a proliferating inducing ligand) [Stein et al 2002] mediate important signals for the B cell. Their receptors, CD40, TACI (transmembrane activator and CAML-interactor), BCMA (B cell maturation antigen) and BAFF-R signal through the same group of transcription factors, the Rel/NF-κB family [Berberich et al 1994; Kayagaki et al 2002; reviewed in Laâbi et al 2001; Marsters et al 2000; Yan et al 2000]. This results in signaling induced by CD40, BAFF and APRIL, apart from upregulating genes important for B cell activation, also upregulates genes that promote survival, such as bcl-2, bcl-XL [Do et al 2000] and A1 [Kuss et al 1999]. The signals via BCR and costimulatory receptors work in balance, solely CD40 ligation induces an upregulation of Fas which makes the cell sensitive to apoptosis induced by Fas ligand, but BCR stimulation will protect the cell [Rothstein et al 1995], probably through upregulation of the anti-apoptotic protein FLIP (FLICE-inhibitory protein) [Wang et al 2000].

Antigen activated B cells can terminally differentiate into antibody secreting cells in the spleen or form GCs. The cells in the spleenic foci will eventually undergo apoptosis plausibly due to a limited access to essential cytokines, a death that can be blocked by Bcl-2 over-expression [Smith et al 1994]. When the centroblasts enter the light zone in the GC they compete for antigen in form of immune complexes presented by FDCs. This leads to a selection of high affinity B cells, which are able to interact with the FDC. Interaction with FDC has been shown to protect from Fas mediated apoptosis [Koopman et al 1997; Schwarz et al 1999; van Eijk et al 2001b]. Cells with low affinity receptors are presumed to undergo apoptosis through neglect, and this apoptosis can be blocked by Bcl-2 [Smith et al 2000] or Bcl-XL [Takahashi et al 1999].

Another selection step that B cells must pass in the GC light zone is selection on antigen specific TH cells. Antigen specific T cells present in the light zone can give signals to B cells that both promote apoptosis and protect from it. Interaction between CD40 on the

B cell and CD40L on the T cell leads to up-regulation of Bcl-XL [Choi et al 1995; Wang et al 1995] and of Fas [Garrone et al 1995; Schattner et al 1995]. However, signaling through BCR will mediate rescue signals from Fas mediated apoptosis [Rothstein et al

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1995]. Thus, B cells that do not bind antigen will be eliminated by T cells, which carry the Fas ligand, through Fas induced apoptosis. Mice with a mutation in Fas, the lpr mutation, develop a lymphoproliferative disease with autoreactive B cells that have undergone SHM [Shlomchik et al 1987]. The exact role of Fas in GC selection is not clear, although GC, memory and antibody forming cell populations have been reported to be normal in lpr mice [Smith et al 1995], a more recent study have shown defects in and an accumulation of maturations in these mice [Takahashi et al 2001]. When an immune response ends, the expanded clones of effector cells must be reduced in size. This process is dependent on Bim through an apoptotic pathway that is blocked by Bcl-2. Both bcl-2 transgenic and Bim-deficient mice show a disturbed immunological homeostasis with an accumulation of antibody forming cells [Bouillet et al 1999; Smith et al 1994; Strasser et al 1991].

Humoral immunity Humoral immunity is upheld by three different mechanisms. An immune response will induce the formation of short lived plasma cells that are able to create a fast increase in antibody levels. In a more long term perspective, specific antibodies are being produced by long-lived plasma cells, mainly residing in the BM. In contrast to short lived plasma cells with life spans measured in days these cells live for several months [Manz et al 1997; Slifka et al 1998]. However, these cell populations are terminally differentiated, however, and are not able to maintain the antibody response for prolonged periods. True long term memory is sustained by a heterogeneous group of cells called memory B cells. The phenotypes of memory B cells are rather poorly defined. Most memory cells express switched Ig isotypes, but also IgM memory cells can be found [Klein et al 1997]. Furthermore, memory B cells can be divided into two populations based on the expression of B220 [Driver et al 2001; McHeyzer-Williams et al 2000]. Memory B cells expressing B220 (B220+) have the highest potential of cellular differentiation and proliferation capacity upon antigen re-challenge, while memory B cells lacking B220 (B220-) have the ability to self-replenish and are predisposed to form antibody secreting cells. A model of linear differentiation has been proposed, where B220+ memory cells give rise to B220- cells from which antibody secreting cells are generated [McHeyzer- Williams et al 2000].

The GC has since long been considered the main site for directing B cells to the memory pathway, however, it does not seem to be absolutely required as knock-out mice lacking GCs has been shown to have memory B cells [Toyama et al 2002]. After leaving the GC, memory B cells can be divided into recirculating memory B cells and cells localized in the marginal zone in the spleen and lymph nodes [Liu et al 1988; Liu et al 1991; Liu et al 1996; Nieuwenhuis and Ford 1976; Oldfield et al 1988]. Since marginal zone B cells are in regions where blood-borne antigens drain, they are fast responding during secondary challenges. Rechallenge with an antigen leads to rapid and

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massive differentiation of memory cells into blasts and plasma cells [reviewed in MacLennan et al 1992; reviewed in van Rooijen 1990]. Repeated antigen challenges can further increase the antibody affinity, which suggests that either the reactivated cells are capable of undergoing further affinity maturation or that the newly recruited naïve B cells must show higher affinity than the memory response to survive the GC process. The need for restimulation with antigen for maintaining memory B cells is under debate. Antigen can be stored for a long time as immune complexes trapped at the surface of FDC [reviewed in Szakal et al 1989; Tew and Mandel 1979] and viruses can stay in host tissues for years. In agreement with the notion that maintenance of memory need antigen, it has been shown that transfer of memory B cells, in the absence of antigen, leads to the loss of the population [Gray and Skarvall 1988]. In contrast, an experiment where the use of genetically modified mice enabled the specificity of the memory B cells to be changed indicated that long-lived memory B cells could survive without antigen [Maruyama et al 2000]. It has also been suggested that serological memory is maintained through polyclonal activation of memory B cells [Bernasconi et al 2002].

Humoral responses and aging Aging affects the body and its functions, so also the immune system. Thymic involution, decreased T cell responses, changes in cytokine expression of T cells and accumulation of memory T cells have been considered to contribute to the decline of the aging immune system [Knight 1995; reviewed in Miller 1996]. Humoral responses also decline with age, both in quantity and quality. The titers of antibodies produced in immune responses in aged individuals are often lower than in young adults [Burns et al 1990; reviewed in Miller 1991], and the binding capacity of these antibodies is also lower [Doria et al 1978; Goidl et al 1976; Kishimoto et al 1976; Weksler et al 1978;

Zharhary et al 1977]. Possibly due to a changed function of aged TH cells, aging also induces changes in the V gene usage [Nicoletti et al 1991, reviewed in Song et al 1997; Yang et al 1996], leading to a change in B cell repertoire. The effect of aging is more pronounced in TD immune responses than in TI response [Goidl et al 1976; Smith 1976; Weksler et al 1978]. It has been suggested that it is mainly due to deficiencies in the TH cells [reviewed in Miller 1996; reviewed in Song et al 1997]. The formation of

GC, dependent on TH cells, is impaired in old mice [Miller and Kelsoe 1995; Szakal et al 1990]. However, it has been suggested that the defective GC formation is not caused by an aged T cell compartment, but instead to be due by age related changes of FDC [Aydar et al 2003; Szakal et al 2002]. It is also likely that the efficiency of the GC reaction is reduced by age, since a decrease in VH gene mutation in GC B cells has been reported [Miller and Kelsoe 1995; Yang et al 1996].

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Present study

Aim The work presented in this thesis has dealt with how different factors such as age, antigen type and defective apoptosis influence the humoral immune response. The specific aims for each paper are as follows:

Paper I: Aging affects both the B and T cell compartment. By studying the response against TI and TD antigens we evaluated the effect of aging on humoral responses. In particular, we examined what effect antigenic priming early in life had on humoral immune responses in old individuals.

Paper II: Mice immunized with the TI antigen dextran B512 (Dx) have a reduced IgG1 response to later challenges with a TD form of Dx. We investigated if this phenomenon could be induced by other TI antigens and also examined possible mechanisms contributing to the low IgG1 response.

Paper III: Certain TI antigens can induce the formation of GC, but in general the GC reaction is unproductive. We wanted to see if this could be due to an early commitment to the plasma cell fate by studying the expression of Blimp-1, a master regulator for plasma cell differentiation, in GC induced by a TI antigen.

Paper IV: For a productive GC reaction, apoptosis is though to have an important role. In this paper we studied the effect on the accumulation of SHM in GC B cells from Peyer’s patches in mice with disturbed apoptosis, namely lpr mice which carry a mutated variant of the death receptor Fas, and in mice over-expressing Bcl-2, an anti-apoptotic protein.

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Methodology This section describes briefly the methods used in the different experiments; they are described in detail in each paper.

Mice Several different mouse strains were used in these studies and they all shared a C57BL/6 background. C57BL/6 is a strain that is a high responder against Dx [Fernández and Möller 1977; Fernández et al 1979]. Except from wild type C57BL/6, we also used BLκ6 mice (Paper II). These mice are the progeny from Lk6 transgenic mice, which carry five copies of a gene construct of a mouse VκOx1-Jκ5 rearrangement coupled to a rat constant κ chain [Meyer et al 1990], breed onto a C57BL/6 background. The mice used in Paper IV with a defective apoptosis originated from B6.MRL- Tnfrsf6lpr (The Jackson Laboratory, Maine, USA), which carries a mutated gene coding for the Fas protein, and the transgenic C57BL/6-Eµ-bcl-2-36 strain, which over- expresses human Bcl-2 on both B and T cell compartments [Strasser et al 1990]. These two strains were crossed with the BLκ6 mice generating apoptosis deficient mice carrying the Lk6 transgene called lpr/ox and bcl-2/ox respectively. In Paper II a FcγRIIB knockout mice [Takai et al 1996] were used to study the importance of FcγR. Nude mice (MB, Ry, Denmark), which are athymic due to a developmental failure of the thymic anlage, and consequently lack mature T cells, were used in Paper III to assess the contribution of the T cell compartment.

Immunizations, Antigens and Adjuvants The animals were challenged with antigen by intraperitoneal immunization. Since Dx B512 was used as a model antigen, most of the other antigens used were Dx or Dx protein conjugates. Chicken serum albumin was conjugated with Dx with a MW of 103 to obtain a TD form of Dx. TI forms of Dx carrying the haptens 2-phenyl oxazolone (Ox), and dinitrophenyl (DNP) were generated by conjugating 4-ethoxymethylene-2- phenyl-2-oxazolin-5-one, (Sigma-Aldrich Corp., St. Louis, MO, USA) and Nε-2,4- DNP-L-Lysine, (Sigma-Aldrich Corp.) respectively to Dx with a MW of 2 x 106. Other antigen used were a TD form of Ox, which was obtained by conjugating Ox to bovine serum albumin (BSA), and BSA-DNP, which was obtained from Biosearch Technologies (Novato, CA, USA). When antigen was administered with adjuvant the adjuvant used was cholera toxin (CT) (List Biological Laboratories Inc., Campbell, CA, USA). In paper II Dx specific antibodies originated from hybridoma cell lines were administered.

ELISA The levels of antigen specific antibodies were measured by ELISA. Briefly the plates were coated with the desired antigen. Sera obtained from the immunized animals were

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added in serial dilutions. Bound antibodies were detected using alkaline phosphatase labeled antibodies specific for different mouse Ig isotypes, and p-nitrophenyl phosphate substrate. The optical density was measured at 405 nm. In addition, ELISA was also used to screen the BLκ6 offspring in the different breedings for the presence of rat Cκ. More details on the different ELISAs can be found in the respective paper.

Estimating antibody affinity In paper I the affinity of the antibodies was assessed by measuring the apparent association constant aKa. The aKa value is defined as the reciprocal value of the concentration of free ligand that gives a 50% inhibition of antibody binding to the antigen coated plate. Bound antibodies were detected using antibodies specific for mouse Igs labeled with alkaline phosphatase. The plates were developed using the substrate p-nitrophenyl phosphate and the absorbance was measured at 405 nm.

Immunohistochemistry Spleens taken from treated mice were frozen and stored in -70 to -85°C. Pieces of the frozen spleens were embedded in Tissue Tek OCT compound (Miles, Elkhart, IN, USA), cryosectioned (6µm), mounted on slides and stored. For staining, the slides were allowed to dry before they were fixed in acetone. After the slides had dried and been washed they were blocked using horse serum before adding the primary antibodies. The slides were then washed and incubation with secondary reagents before a last wash and mounting. The slides were then examined using a UV-microscope.

Generation of hybridoma cell lines In paper II, mice were hyper-immunized with either Dx or CSA-Dx together with CT. Spleens were taken day 3 after the third immunization and fused with the non-secreting hybridoma cell line SP2/0 using polyethylene glycol solution Hybri-Max (Sigma- Aldrich Corp.) as fusing agent. The cultures were grown under the selection pressure of HAT-media, Hybri-Max (Sigma-Aldrich Corp.). The antigen specificity of the clones was determined by ELISA as described above.

Extraction of GC B cells from Peyer’s patches A single cell suspension was prepared from Peyer’s patches dissected from the small intestine of the experimental mice. After washing in cold PBS, the cell suspension was filtered before a final wash in cold PBS. The cells were stained using FITC-conjugated peanut agglutinin (PNA) (Sigma-Aldrich Sweden AB, Stockholm, Sweden), PNA being a marker for GC B cells [Rose et al 1980], and phycoerythrin-conjugated anti-mouse CD45R (B220) (Pharmingen, San Diego, CA, USA), a B cell marker. The lymphocyte population was gated using the parameters of size and complexity in a Becton + high Dickinson FACSVantage SE, and the B220 PNA cell population was purified by

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sorting. This population corresponds to GC B cells. The sorted cells were centrifugated and the pellets were stored at -70°C.

DNA and RNA isolation, RT-PCR amplification and sequencing This part of the work was done by our collaborators in Spain, Elena Fernández Mastache and África González-Fernández. Briefly, for analysis of the accumulation of mutations in the VκOx1 gene (paper IV) DNA was extracted from the frozen cell pellets obtained by cell sorting. The sequence of interest was amplified using PCR. The PCR product was purified and re-amplified. The re-amplified product was cloned into plasmid vector pBluescript+. Positive clones were expanded and the plasmid was purified using QIAprep Spin Miniprep Kit (QIAGEN GmbH). The sequence reaction was performed using the Dye terminator Cycle Sequencing kit (Beckman Coulter, Fullerton, CA), and the products sequenced in an automated DNA sequencer (CEQTM 2000, Beckman Coulter). The sequences obtained were analysed using the Clustal X (version 1.8) and the Genedoc (version 2.6.002) software.

For analysis of the VH gene usage (paper II) RNA was isolated from hybridoma cell lines using the SV Total RNA Isolation System (Promega, Madison, WI, USA). cDNA was generated using a primer specific for the constant µ region, and the reverse transcriptase M-MLV (Promega). cDNA reaction mixture was amplified in a PCR reaction using primers complementary to the JH and VH genes. The PCR product purified and the sequence reaction and sequencing was carried out as mentioned above. The program IgBlast (NCBI) (http://www.ncbi.nlm.nih.gov/igblast) was used to determine the identity of the VH genes.

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Results Here follows a summary of the results obtained in the different papers. References to figures and tables indicate figures and tables in each individual paper.

Paper I Age is known to affect the function of the immune system. Both the B and T cell compartment is affected, often leading to an impaired immune responsiveness in old individuals. In this study we investigated the effect of aging on responses to TI and TD antigens, and in particular we examined the effect of early life priming for responses in old animals. When analyzing the response against TI Dx, we could see that the levels of specific IgM after primary immunization were similar in young and old mice (fig 1), indicating, in consistency with other data [Weksler et al 1978], that the B cell compartment is not so affected by aging. Furthermore, though old mice immunized with TI Dx generated approximately as many GC as young mice, the Dx binding capacity was reduced in the GC of the old mice (fig 2). This suggests that the number of Dx specific B cells is lower in these GC, since the IgM antibody affinity is similar in young and old mice (table 2). When looking at TD Dx responses and the levels of Dx specific IgG, the differences between young and old animals are more pronounced (fig 3), as could be expected as a result of an impaired T cell function [reviewed in Song et al 1997; reviewed in Zheng et al 1997]. We were particularly interested in the effects antigen priming in young mice had on the immune response in aged mice. In TD responses we show that early life priming with CSA-Dx, together with CT, greatly improves the IgG response of the aged mice against Dx (fig 3). It is known that priming with TI Dx causes a reduced IgG response to later TD Dx responses [Sverremark and Fernández 1998a]. We show in this paper that the reduction is long lasting, it is distinct still one year after TI priming (fig 5) and that it also decrease the affinity of the Dx specific IgG that is produced (table 3).

Conclusion: In agreement of earlier presented data, we show that the T cell compartment is more affected by aging than the B cell compartment. Furthermore, we highlight the importance of early life priming for later responses. We show that priming can result in both an improvement of the response in the old mice and cause a reduced response which is the case after TI priming.

Paper II Knowing that TI Dx priming can have a major effect on later TD Dx responses we investigated possible mechanisms behind this effect and if this reduction could be induced by other TI antigen. Clonal exhaustion caused by activation in the absence of memory formation, could possibly be one explanation for the effects of TI Dx priming. The use of CT strongly improves the TI Dx response, indicating that either the responding clones are not exhausted or that, in the presence of CT, other clones are

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activated in the secondary response. We compared the VH chain usage in Dx specific hybridomas generated from animals hyperimmunized with either TI or TD Dx together with CT. The VHB512 gene was used in both TI and TD Dx specific responses (Table 1). It is the same gene that codes for a majority of antibodies in primary responses against Dx [Fernández 1992]. Thus, we concluded that neither the use of CT, activation by TI or TD forms of Dx, nor repeated immunizations seem to influence the gene usage in the Dx specific response. Consequently, since there is no shift in gene usage, clonal exhaustion does not seem to be contributing to the reduced TD responses induced by TI Dx priming. To examine the effects of Ig dependent mechanisms on TD Dx responses after TI Dx priming, we administered Dx specific antibodies to mice and challenged the mice with CSA-Dx after a resting period. Treatment with Dx specific antibodies did not lead to a reduction of Dx specific IgG1 in the TD response (fig 1). Furthermore, mice lacking FcγRIIB were as affected by TI Dx priming as wild type mice (fig 2). These results suggest that Ig dependent mechanisms, such as generation of anti-idiotypic antibodies or negative signaling through Fc receptors, do not seem to be the cause to the reduction of Dx specific IgG. We could also demonstrate that the negative effect of TI priming is not exclusive to responses against Dx. Using the restricted hapten Ox, we show that TI priming induced a reduction of IgG1 levels in later TD Ox responses. This led us to propose that the reduced TD response after TI priming is due to a property of the TI activation itself, presumably to directing the responding cells to a non- switch/non-memory pathway.

Conclusion: In this paper we rule out clonal exhaustion and Ig dependent mechanisms as main factors behind the reduced TD IgG1 responses induced by TI priming. We also show that this effect can be directed to another molecule than Dx.

Paper III We continued to examine differences between TI and TD responses, and in particular, if the different types of antigens induce the responding cells to mature differently. The GC is a specialized environment where affinity maturation, class switch and induction of memory formation occur. While the formation of GC is typical of responses against TD antigen some TI antigens such as Dx can also induce GC formation [de Vinuesa et al 2000; Sverremark and Fernández 1998b; Wang et al 1994]. However, the response against TI Dx does not show signs of substantial affinity maturation or memory formation. In this paper we investigated if this could be due to differences in expression of the transcriptional repressor Blimp-1 in the GC. Blimp-1 expression drives the cell to plasma cell maturation and regulates the expression of Bcl-6, needed for the formation of GC, in a negative feedback loop. By staining spleen sections from immunized mice with PNA and Blimp-1 specific antibodies, we show that GCs induced by TI antigen express Blimp-1 earlier in the response (fig 1 and 2) and that the staining is stronger than in TD induced GC (fig 3). Furthermore, in TD responses after TI priming the

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Blimp-1 expression early in the formation of the GC resembles that seen in TI responses, while the expression in more mature GC is similar to that in TD GC (fig 2 and 3).

Conclusions: GCs induced by TI antigen express Blimp-1 at earlier time-points and have a higher staining intensity than TD induced GCs. TD GCs in mice that earlier have been primed with TI antigen show a mixed pattern of Blimp-1 expression, early it resembles more the TI induced GC but as the GC matures the expression becomes similar to that in TD GCs. This suggests that the response is mixed and not simply a suppressed TD response as thought before.

Paper IV By using mice with deficiencies in the apoptotic pathways, mutated Fas or over- expression of Bcl-2, as well as carrying a mouse/rat Vκ gene construct, we studied the effect of these deficiencies on the accumulation of SHM. GC B cells (PNAhigh B220+) were isolated from Peyer´s patches and the mutations in the inserted Vκ genes were studied. We found that the mice carrying the lpr mutation displayed a normal mutation pattern, with regard to mutation of intrinsic hotspots (fig 3A). However, the lpr/ox mice showed an increased frequency of T to A transversions (fig 2A). In addition, there was an increase in the frequency of mutations (table 1) as well as in the incorporation of deleterious mutations (table 3). In contrast, the over-expression of Bcl-2 showed a decreased mutation frequency (table 1). Furthermore, these mice showed alteration in the nucleotide substitutions, normally the A/T ration is close to 2, but these mice showed no mutational bias for the mutation of A, A/T ratio 1.1 (table 2) and the G/C ratio was of 0.6 instead of the expected 1. The mice over-expressing Bcl-2 also showed a reduced number of mutations in the intrinsic hotspots (fig 3B, table 3).

Conclusion: We show that a mutated Fas does not lead to any major changes in the characteristics of the SHM introduced, but contributes to an increase in the frequency of mutations. In contrast, the over-expression of Bcl-2, leads to a different pattern of mutations as well as a reduction of the overall number of mutations. This could be due either to disturbed apoptosis or perhaps more likely to other effects of the over- expression. Over-expression of Bcl-2 is for example known to retard the cell cycle progression [Mazel et al 1996; O’Reilly et al 1996], which could possibly allow for repair of the mutated sequences.

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Concluding discussion Like all biological processes the induction of an immune response is shaped by a combination of both internal and external factors. In this thesis I have investigated a couple of different factors that have an impact on how the responses is induced and developed, e.g. age, type of antigen and apoptosis.

In the GC where B cell clones are greatly expanded in the presence of a mutation- machinery, apoptosis plays an important role to weed out cells that potentially could be dangerous or that have a low affinity for the antigen. Though it is not doubted that apoptosis is important for selection in the GC the debate is ongoing with regard to which apoptotic pathway is the most important. In our studies we could se that mice deficient in Fas mediated apoptosis accumulated mutations in the GC B cells, an accumulation that is mirrored in the phenotype of the lpr mice where there is an accumulation of mutated B lymphocytes. Over-expression of Bcl-2 gave another phenotype, a lower number of mutations and differences in nucleotide preferences compared to wild type mice. This led us to conclude that impaired apoptosis caused by the lpr mutation or over-expression of Bcl-2 affects the GC reaction. Furthermore, these effects can be seen at different stages of the GC reaction.

The fact that age affects the efficiency of the immune system is known. In both very young and very old immune responses are often weakened [reviewed in Castle 2000; reviewed in Siegrist 2001]. A better understanding of why we can se these effects and how to counteract them would of course give big humanitarian and economical benefits. We show that antigen encountered early in life can greatly influence the response in an aged individual, both in a positive and negative manner. The long lasting effects of priming in young individuals, suggests a possible future development where vaccines are constructed not only to give a desired protection, but also to maintain a strong immune response in an aged individual.

One group of antigens that both very young and old individuals have difficulties to respond against is polysaccharides. As these molecules can be found on bacterial cell walls as well as viral capsules they constitute antigens that we are constantly challenged with. The model antigen we have used is the polysaccharide Dx B512 from the bacteria Leuconostoc mesenteroides. In common with other TI-2 antigens, the response against Dx shows no sign of affinity maturation or formation of memory. In fact, secondary responses are suppressed compared to primary ones. However, this suppression can be abrogated by the use of CT as adjuvant. A peculiar effect that is seen after TI Dx priming is that later responses against TD Dx have markedly reduced levels of IgG1. As we found in our studies (paper I), this effect can be very long lasting. After ruling out clonal exhaustion and Ig dependent mechanisms as possible mechanisms for inducing this kind of unresponsiveness, we became increasingly convinced that the lack of switch

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and memory in these responses is caused by properties of the activation induced by the TI antigen.

In the past, differences between TI and TD responses have often been explained by the lack of T cell help in the former. However, although certainly not without importance, lack of T cell help might not be the only thing shaping responses against TI antigen. Activation by TI antigen is thought to occur by cross-linking of the BCR coupled to a second signal for example via pattern recognition receptors or complement receptors. TI activation has been shown to induce a different phenotype of the responding B cells than TD like activation [Cong et al 1991; Haas and Estes 2000; Wortis et al 1995], suggesting that the signals induced by the respective activation lead to differences in the maturation of the responding cells. When a B cell is activated during a TD response, it is pictured that the maturation of the cell can proceed in two different directions. Either it differentiates terminally and become a plasma cell or it can enter the germinal center reaction characterized by the incorporation of SHM, class switch and memory formation. As the responses against Dx largely lack class-switched antibodies and a proper memory formation, we wanted to know if this was due to an early commitment of the responding B cells to the plasma cell fate.

We investigated if GC induced by TI Dx differed from TD antigen induced GC with regard to the expression of Blimp-1. Blimp-1 is the master regulator of terminal differentiation of B cells into plasma cells. It also regulates the expression of Bcl-6, a protein needed for GC formation, through a negative feedback loop. We found that TI GCs express Blimp-1 earlier in the response. Our interpretation is that the responding B cells are constantly recruited to a plasma cell fate, and leave the GC before SHM, switch and induction of the memory pathway has occurred. It has been shown that BCR signaling leads to degradation of Bcl-6 [Niu et al 1998]. It is possible that Dx can induce a strong enough signal, either through the BCR or some other type of receptors, such as pattern recognition receptors, to induce degradation of Bcl-6. Another possibility is that the responding B cells fail to get co-stimulatory signals by TH cells that would normally maintain or increase the levels of Bcl-6.

So, if activation with TI antigen drives the responding cells towards differentiation into plasma cells, is that a good or a bad thing? With a TD antigen perspective the answer is bad. Humoral responses should undergo affinity maturation to improve the affinity, class switch to gain other functions for the antibody and memory to be able to respond quicker the second time the antigen is encountered. However, one can take another viewpoint. First the apparent lack of memory could be a lack of class switched memory cells, there indications that TI antigen can induce an IgM memory [Hosokawa 1979; Kolb et al 1993]. And considering that polysaccharides often are found in bacterial cell walls, IgM might be a very important Ig class for the response. Due to the secretion of

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IgM in form of a pentamer, it can bind with high avidity, and it is the most efficient activator of complement of the Ig isotypes, making it able to mediate lysis of the invading pathogen. So maybe it is time to look at TI responses as a different kind of humoral responses instead of defective TD responses?

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Acknowledgements I would like to express my sincere gratitude to family, friends and colleges who have helped and supported me in so many ways during the years. Especially I would like to thank the following people:

Carmen Fernández, my supervisor, for your inspiration, caring and enthusiasm, and for your ability to always find something positive even in the worst results.

Göran Möller, for accepting me as a student at the department.

The seniors, former and present, Manuchehr Abedi-Valugerdi, Klavs Berzins, Alf Grandien, Eva Sverremark-Ekström, Marita Troye-Blomberg, for all the help and for sharing your passion for and knowledge of different parts of immunology.

Peter and Hedvig Perlmann, you are an inspiration for us all.

Margareta Hagstedt, Lena Israelsson, Ann Sjölund, Gunilla Tillinger, Gelana Yadeta for always helping me when I needed it.

Elena Fernández Mastache, África Gonzalez Fernández and Marta Sánchez, my Spanish collaborators, for your hard work and nice discussions.

My roommates, Monika Hansson, for always being ready to help me and to discuss immunology, dogs and all other things over a cup of coffee! Anna Tjärnlund for all the laughs and being such a nice person, Masahi Hayano, for your humor and for putting up with all the chatty girls.

My former roommate, Eva Nordström, without you I would never have tested green milk!

To the other students that have come and gone during my time at the department, Ariane Rodríguez-Muñoz, Anna-Karin Larsson, Mounira Djerbi, Valentina Screpanti, Cecilia Rietz, Nina-Maria Vasconcelos, Elisabeth Hugosson, Caroline Ekberg, Anki Häglind, Gun Jönsson, Qasi Kaleda Rahman, Salah Eldin Farouk, Ahmed Bolad, Manijeh Vafa, Shiva S. Esfahani, Jacob Minang, Halima Balugon, Ben Adu Gyan, Susanne Gabrielsson for being good friends both in and out-side the lab.

Izaura Roos, the one that got away…

Pia Lundell, Eva Nygren and Solveig Sundberg for your work in the animal house taking care of all my mice.

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Mamma och pappa för all den kärlek och det stöd som gjort mig till är den jag är!

Ulf, Björn och Leif, världens bästa bröder som har finslipat min argumentationsteknik till det yttersta.

Mormor och morfar som oroligt sett mig fara kors och tvärs över världen. Men jag vet, det är bäst hemma i Norrland!

Ronny och Bruno, för goda middagar och gott sällskap.

Sixtenssons, för att ni tagit emot mig med öppna armar och hjälpt till när det behövts.

Alla mina vänner, som haft tålamod med mig när jag bara haft jobb i huvudet, och som jag tillbringat så många roliga timmar med i den verkliga världen (och nog minst lika många i någon overklig…).

Tillsist, Magnus, mitt hjärtas kär, för att du alltid finns där när jag behöver dig. Och för att du ibland vet bättre än jag själv vad jag behöver!

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