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Home , Complement system, RHD (gene)

Trassla inte till saken genom att komma dragande med fakta

Groucho Marx

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

Ia Rutemark C, Alicot E, Bergman A, Ma M, Getahun A, Ellmerich S, Carroll M, Heyman B. Requirement for complement in responses is not explained by the classic pathway activator IgM. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):E934-42. Epub 2011 Oct 10.

Ib Rutemark C, Alicot E, Bergman A, Ma M, Getahun A, Ellmerich S, Carroll M, Heyman B. Requirement for complement in antibody responses is not explained by the classic pathway activator IgM. Author summary. Proc Natl Acad Sci U S A 2011 108(43): 17589-90

II Carlsson F, Getahun A, Rutemark C, Heyman B. Impaired antibody responses but normal proliferation of specific CD4+ T cells in mice lacking complement receptors 1 and 2. Scand J Immunol. 2009 Aug;70(2):77-84.

III Rutemark C, Bergman A, Getahun A, Henningson-Johnson F, Hallgren J, Heyman B. B cells lacking complement receptors 1 and 2 are equally efficient producers of IgG in vivo as wildtype B cells. Manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Background ...... 12 B cells ...... 12 ...... 12 T cells ...... 13 presentation and ...... 13 Antibody feedback regulation ...... 14 IgG-mediated suppression of antibody responses ...... 14 Antibody-mediated enhancement of antibody responses ...... 15 IgM-mediated enhancement of antibody responses ...... 15 The complement system in antibody responses ...... 18 Component 3 ...... 18 pathway ...... 20 Alternative pathway ...... 20 Classical pathway ...... 21 Activators of C1q ...... 23 The C1q paradox in primary antibody responses ...... 24 Complement Receptors 1 and 2 ...... 25 Possible mechanisms for the importance of complement in antibody responses ...... 27 Present investigation ...... 29 Aims ...... 29 Paper Ia ...... 29 Paper II ...... 29 Paper III ...... 29 Experimental setup ...... 30 Mice ...... 30 Immunizations and ...... 31 Adoptive transfer of T cells ...... 31 Assays ...... 31 Bone marrow chimeras ...... 31 Statistical analyses ...... 32

Results and discussion ...... 33 Requirement for complement in antibody responses is not explained by the classic pathway activator IgM (Paper Ia) ...... 33 Impaired antibody responses but normal proliferation of specific CD4+ T cells in mice lacking complement receptors 1 and 2 (Paper II) ...... 38 B cells lacking complement receptors 1 and 2 are equally efficient producers of IgG in vivo as wildtype B cells (Paper III) ...... 40 Highlights of this thesis ...... 44 Acknowledgements ...... 45 References ...... 47

Abbreviations

APC antigen presenting cells BCIP/NBT 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitro blue tetrazololium HRBC horse red blood cells BCR receptor Bf BM bone marrow BSA bovine albumin CD cluster of differentiation CGG chicken gamma CR1, 2 1, 2 CVF cobra venom factor CRP c-reactive DC dendritic cells DNP 2, 4-dinitrophenol ELISA enzyme-linked immunosorbent assay ELISPOT enzyme-linked immunospot assay Fc fragment crystallizable FDC follicular dendritic cells Fo B follicular B GC germinal center IC IgG, M… , M… IgM-IC antigen-specific IgM in complex with that antigen KLH keyhole limpet hemocyanin mAb MASP mannose-associated serine MAC membrane attack complex MBL mannose-binding lectin NP 4-hydroxy-3-nitrophenylacetyl MZ marginal zone OVA ovalbumin PBS phosphate buffered saline PFC plaque forming cell assay SAP serum amyloid P component

SIGN-R1 specific intracellular adhesion molecule-grabbing noninteg- rin related 1 SCR short consensus repeats SRBC sheep red blood cells TCR T cell receptor TH TD thymus-dependent TI thymus-independent TNP 2, 4, 6-trinitrophenol WT wildtype

Introduction

The is our protection against foreign material such as viruses and , but also in clearing the body from dead cells and cancer cells. Assigned to the immune system are two global functions, recognition and response. In the recognition phase the ability to discriminate between self and non-self-structures, as well as the distinction between different foreign structures is paramount. Following optimal recognition comes then an ap- propriate and specific response. The immune system is often divided into innate and adaptive immunity. The innate cells and molecules are encoded before an has occurred. Thus, this part is our very first line of defense, acting fast and with a broad spectrum of recognition. In contrast, the adaptive part is fairly slow to initiate and requires an intact innate defense to mount a full re- sponse. However, once this is fulfilled the adaptive system utilizes the torpe- does of the immune system, the antibodies, produced by B cells, but also cell-mediated defense caused by cytotoxic T cells. The antibodies are highly specific with the ability to discriminate targets that differ down to only one amino acid. However, antibodies recognizing self-antigens are one of the major causes for . Furthermore, the adaptive system also pos- sesses immunological memory, i e the same infection the second time around is responded to faster and with even higher affinity and specificity. Antibodies themselves have the ability to regulate the production of anti- bodies, a process called antibody feedback regulation. The outcome of this regulation can be either enhancement or suppression of the antibody re- sponse, depending on the antibody type or antigen involved. One of the connections between innate and adaptive immunity is the complement system, which is activated via different pathways upon recogni- tion of foreign surfaces. It consists of a group of soluble and recep- tors that can, in very low concentrations, identify and incapacitate many pathogens. The complement system has shown various characteristics in engaging and enhancing the adaptive response, including antibody respons- es. In addition, complement deficiencies often lead to diseases such as auto- immunity. This thesis focuses on the factors that are activating complement and how complement further is affecting the antibody response. More specifically, the classical activation pathway and its most common activator, IgM, have been studied, along with the effect of the complement receptors 1 and 2.

11 Background

B cells B (B cells) originate from the bone marrow (BM) and when they are let out into the circulation, each B cell carries a unique antigen- binding receptor on its surface. This receptor is an IgM antibody molecule bound to the B- and is referred to as the B cell receptor (BCR). Mature B cells that meet the antigen recognized by the BCR, will start to proliferate and differentiate into antibody producing cells called plasma cells and memory B cells. This is what happens in response to thy- mus-independent (TI) antigens. These antigens are repetitive structures, found on many microbes that can either ligate several BCRs and thereby activate the B cell, or activate the B cell via other, antigen-unspecific recep- tors. TI-antigens, however, do not induce affinity maturation, switch or memory. In contrast, these characteristics are fulfilled in response to thy- mus-dependent (TD) antigens and here, the B cell needs help in this reaction from T helper (TH) cells. B cells can be divided into B1- and B2 B cells, among which the follicu- lar B cell (Fo B) of type B2 make up for the majority of the B cells in the body and these cells are the ones involved in the classic adaptive immune responses (1). The marginal zone B cells (MZ B) of the B2 type together with B1 B cells are resident cells that mostly take part in innate-like func- tions. MZ B cells are located in the marginal zone (MZ), a part of the highly specialized structure of the spleen. Here, they have an excellent position in the vicinity of the blood stream to meet antigens. B1 B cells are self- renewing cells mainly found in the peritoneum. They have a restricted diver- sity meaning that their antigen recognition is limited and antibodies pro- duced are of low affinity.

Antibodies As mentioned above antibodies may appear as cell-bound receptors but they are mostly known as elicited pathogen neutralizers found in serum and tissue fluids. Antibodies, or immunoglobulins (Ig), are that consist of two identical antigen-recognizing parts (F(ab’)2) along with an Fc domain that can bind to cellular receptors. This domain dictates the isotype of the

12 antibodies which can be: IgA, IgD, IgE, IgG, and IgM. Murine IgG can fur- ther be divided into subclasses IgG1, IgG2a, IgG2b and IgG3. For humans the corresponding subclasses are IgG1, IgG2, IgG3 and IgG4. Following antigen-binding, the Fc-part of the antibody interacts with Fc receptors (FcR) on cell surfaces mediating an effector function for the cells, e g , or antibody-dependent cell-mediated cyto- toxicity. Antibodies may also activate complement and regulate further anti- body production which will be described in more detail in this thesis.

T cells Like the B cell, the T cell comes from the BM but it matures in the thymus where it starts to express an antigen-binding receptor on its surface. Howev- er, this T cell receptor (TCR) recognizes only antigen that is bound to major complex (MHC) molecules. There are two types of MHC; class I MHC is expressed on all cells with a nucleus whereas class II MHC expression is restricted to antigen presenting cells (APC). Expression of the glycoproteins CD4 or CD8 on the cell surface divides + + the T cells into two major subtypes, where CD4 cells are TH cells and CD8 cells are T cytotoxic cells. Whereas the TH cells are involved in the priming and activation of the adaptive response, T cytotoxic cells are educated to perform cell-cytotoxicity on target cells, e g virus infected cells.

Antigen presentation and T cell activation Even though B cells are poor APC compared to dendritic cells (DC) and , they can have a role in priming of naïve TH cells. This role benefits from the antigen recognition properties of the B cell (2). between APC and TH cells is needed for a full-blown adaptive against TD-antigens. Antigen-specific B cells encounter- ing that specific antigen in lymphoid organs will be stimulated, start to pro- duce a first wave of IgM and IgG antibodies, and form primary follicles, the foundation for germinal centers (GC), together with a few TH cells. In the GC, B cells are further developed to generate antibodies with higher affinity to the antigen, aided by follicular dendritic cells (FDC). Also, antibody class switch is performed in GC. The TH cells in the GC have been activated by APC that in turn have rec- ognized the antigen with various cell surface receptors, engulfed and digest- ed it. The antigenic fragments are subsequently associated to class II MHC for presentation. Naïve TH cells having a T cell receptor (TCR) recognizing the presented antigen will initiate an interaction between the TCR and class II MHC on the APC. This interaction activates the TH cell via pro-

13 duced by the APC. In the GC, the B cell clone with foremost antigen-affinity is selected to interact with these activated TH cells, which starts to secrete cytokines that contribute to further proliferation and maturation of the B cells into plasma cells and memory cells.

Antibody feedback regulation Antibodies can feedback regulate the production of specific antibodies re- sulting in over a 1000-fold enhancement or >99% suppression, depending on which antibody class and which antigens that are involved (3, 4). The regula- tion is dependent on factors such as antibody affinity, administration routes, and doses of antigen and antibody.

IgG-mediated suppression of antibody responses One of the stronger suppression known is that of IgG together with large, particulate antigens e g erythrocytes. This has been used since the 1960’s in clinical practice for protecting the unborn RhD+ child from antibodies pro- duced by the RhD- mother. As treatment, the mother is injected with low doses of IgG anti-RhD and this has been shown to decrease the occurrence of hemolytic disease in the fetus (5). Proposed are three different mechanisms for how IgG suppresses anti- body responses: 1) by masking, where the IgG molecules sterically hinder specific B cells from recognition and binding to the antigen, 2) im- mune complexes (IC) containing IgG and erythrocytes may be more easily taken up via Fc R+ thereby clearing the IC faster compared with antigen alone, 3) the negatively regulating Fc RIIB, found on B cells, may be co-crosslinked with the BCR by IgG/erythrocyte complexes inhibiting B- cell activation, which has been shown to take place in vitro (6, 7). The first of the above mechanisms would require that the suppression functions independently of FcRs. Indeed, IgG suppression occurs in mice lacking all known Fc Rs including Fc RIIB (8, 9). The two other hypotheses are supported by data that IgG suppression is not epitope-specific (10-16). However, a recent study shows that IgG suppression is epitope-specific in antigens with low-density , but non-epitope-specific when the epitope density is high; referred to as epitope masking (17). Using F(ab’)2 fragments, there are diverging data whether suppression is dependent on the Fc part of the IgG antibody or not (11, 15, 18-20).

14 Antibody-mediated enhancement of antibody responses Enhancement of antibody responses can be caused by IgM, IgE or IgG. Herein, the IgM-mediated enhancement will be dealt with separately. IgE together with small soluble proteins as antigens enhance the production of all antibody isotypes recognizing that antigen (21, 22). No enhancement is seen using larger antigens such as keyhole limpet hemocyanin (KLH) or erythro- cytes (21, 22). The mechanism works through CD23 (21-25), more precisely CD23a (26) which in mice is expressed on B cells and FDC. The B cell is the likely effector cell (23, 25). Recent studies indicate that CD23+ B cells capture IgE-IC in peripheral blood, transport it to the follicles of the spleen where it is delivered to DC which present it to CD4+ T cells (27, 28). All murine IgG subclasses suppress antibody responses to large particu- late antigens such as erythrocytes and malaria (8, 10, 11). Interestingly, the same monoclonal antibody (mAb) acting as a suppressor with such antigen can enhance antibody responses against soluble antigens (12, 15). Enhance- ment with IgG1, IgG2a and IgG2b is dependent on activation of Fc Rs (29). Probably, these complexes are captured by Fc R+ APC capable of increasing antigen presentation to specific CD4+ T cells as shown both in vitro and in vivo (30-35). Fc RIIB is known to down-regulate immune responses by in- hibiting activation of immunoreceptor tyrosine-based activation motifs. This receptor inhibits e g antigen presentation by DC, maturation and release of mediators by DC, B-cell activation and BCR-mediated antigen presentation (reviewed in (36)). IgG-mediated enhancement is not mediated by Fc RIIB but this inhibitory receptor has a negative effect on the antibody response to IgG-complexed soluble antigens (29). The IgG-mediated enhancement is sometimes more than a 100-fold higher in Fc RIIB-deficient mice compared to wildtype (WT) (29, 35). Thus, the profile of enhancement mediated by IgG1, IgG2a and IgG2b is an intricate interplay between the different Fc R, the ratio of expression between activating and inhibitory Fc R as well as the different antibody subclasses involved. In contrast, the enhancement of antibody responses by IgG3 is not dependent on Fc R, but rather on the complement system and does not induce proliferation of specific T cells in vivo (37, 38).

IgM-mediated enhancement of antibody responses IgM is a potent complement activating antibody and since complement is needed for the generation of a normal antibody response, IgM may play a role in mediating this effect. IgM administered prior to, or together with, suboptimal doses of its specific antigen will enhance the specific antibody response against that antigen. This has been shown with large antigens such as sheep red blood cells (SRBC) (39-41), KLH (42, 43) and malaria parasites (44). In contrast, IgM administered up to two days after immunization will

15 significantly suppress the antibody response (45). IgM can also enhance the induction of memory B cells (46). IgM-mediated enhancement is not seen with small antigens and this is probably because the IgM itself is very large, and it needs an even larger antigen to be able to bind with all five arms of the molecule and thereby complete the conformational changes required for C1q binding. In fact, recent studies using atomic force microscopy revealed not only that the pentameric structure is mushroom-shaped rather than planar but also, by free energy calculations, that the binding of large antigens would be favored over the binding of smaller antigens (47). Further, IgM cannot sub- stitute for T cells since no enhancement was seen in mice lacking T cells (48-50). Heyman et al. reported in 1988 that IgM-mediated enhancement was de- pendent on the activation of complement (51). This was shown in two ways. First, a loss of IgM-mediated enhancement was seen when depleting mice of C3 using cobra venom factor (CVF). Second, an IgM molecule unable to activate complement also lost the ability to enhance antibody responses. To show this, a mutated Sp6 B-cell hybridoma line (mutant #13) generated by Shulman et al. (52), described as having normal pentameric secretory IgM but with complement activation defects, was used. After cloning and se- quencing, the mutation was narrowed down to a single point mutation in the third constant domain of the heavy chain, where a nucleotide substitution C T resulted in the amino acid change from proline to serine at position 436 in the IgM molecule. This made the IgM molecule unable to bind C1q and resulted in a 50-fold reduction in cytolytic activity whereas the antigen- binding capacity was intact (53). This IgM molecule, which is specific for the 2, 4, 6-trinitrophenol (TNP), was unable to enhance the antibody response, suggesting that IgM-mediated enhancement was dependent on the activation of complement (51). In analogy, monomeric IgM, without com- plement activation capacity, does not enhance antibody responses (43) and the IgM-mediated enhancement is lost in Cr2-/- mice (41). As early as 1971 it was reported that the strong immune response caused by IgM-containing IC compared to antigen alone was accompanied by en- hanced antigen concentrations in the spleen (54). In more recent studies, pentameric IgM-IC given to C3-depleted or Cr2-/- mice, showed that the complexes are trapped in the MZ of the spleen, associated with MZ macro- phages. In contrast, in WT mice, the complexes were found associated with FDC (43). Similar results were obtained using monomeric IgM. This implies a role for the pentameric IgM in this first step of immune response initiation and that the IC are excluded from the splenic follicles in the absence of complement. Indeed, the same group later showed that IgM-containing IC are bound to MZ B cells, expressing high amounts of CR1/2, in WT mice demonstrating an IgM and complement-dependent role for MZ B cells in efficient binding and transport of IgM containing IC onto FDC (55). More recent findings support these data showing that MZ B cells continuously

16 shuttle between the MZ and the follicle and can transport complement- coated antigen into the follicle via CR1/2 receptors (56). The role of secretory IgM has been studied by several groups. In one re- -/- port mice lacking secretory IgM ( s ), upon immunization with 4-hydroxy- 3-nitrophenylacetyl (NP) conjugated to KLH, had a markedly impaired anti- body response. This response could be restored when IgM from naïve mice -/- was given prior to immunization of the s mice (57). Similar results were also found by others using different antigens such as influenza virus and KLH (58, 59), suggesting an important role for the naturally occurring secre- tory IgM. In contrast, transgenic mice with B cells specific for NP, unable to produce secretory IgM, had normal numbers of antigen-specific B cells upon immunization with NP-CGG. They also exhibited normal GC formation but without detectable IC on FDC (60). This prompted the authors investigate the importance of membrane-bound IgM (i e the BCR). They found that when the BCR in these mice lacks the ability to bind to C1q, the antibody production is impaired (61). This suggests that the BCR itself can activate complement and thereby mediate an enhanced antibody response. In line with this Manderson et al. suggested the possibility of IC formation at the B- cell surface instead of in the circulation (62). It was proposed that an array of antigen covering the B-cell surface first bind to BCRs creating a multimeric surface. This is followed by binding of soluble IgM to the antigen matrix and thereby an activation of complement, generating C3 fragments which bind to CR2 on the cell surface and an activation signal into the B cell.

17 The complement system in antibody responses The complement system is a powerful component in our defense against harmful antigens. It consists of several components, both cell-surface bound proteins and circulating serum proteins. Once activated by foreign motifs or by our own antibodies, a cascade of protein cleavages takes place converting inactive pro-enzymes into active ones. This activation enables complement to promote lysis of recognized cells (e g bacteria), opsonize antigens which facilitate antigen recognition, clear the blood circulation from IC to prevent detrimental deposition, or bind to certain complement receptors triggering various cellular mechanisms. The complement is activated in three different ways: the classical pathway, the mannose-binding lectin (MBL) pathway and the alternative pathway (Figure 1). The generation of a normal antibody response is dependent on a function- al complement system (reviewed in (4)). This is true for both TD- and TI- antigens. The effect of the complement system is usually seen with low anti- gen doses as the complement in those situations can serve as an adjuvant (63). In this investigation, a several-fold increase in B-cell activation in vitro was observed with low doses of antigen tagged with C3d fragments com- pared to antigen alone. Often the effect of complement is seen on the prima- ry antibody response, but in some cases also on the secondary response. In the following pages the different parts of the complement system and its impact on antibody responses, will be described.

Component 3 C3 is the common component for the three activation pathways and is cleaved by either of two C3 convertases, generated in all activation path- ways. One is generated by the classical together with the MBL pathway and is composed of a C4b2a complex, formed when C1s cleaves C2 and C4. The other one is C3bBb and is a complex formed when membrane-bound binds to factor B which in turn is cleaved by into Ba and Bb. Here, has been shown to stabilize this formation (64). The product is an important and excess C3b is joined with either C4b2a from the classical pathway or C3bBb from the alternative pathway to assem- ble into C5 convertases. The primary function of these protein complexes is to activate C5 into C5a and C5b. The activation of C5 into C5b is necessary for the generation of the membrane attack complex (MAC) that consists of components C5b6789. The complex functions as a promoter of lysis of path- ogens and foreign cells by punching holes in the cell membranes. Humans with deficiencies in any of the components C5-C8 show increased infection rate of Neisseria meningitis (65). However, C9 deficiency is usually asymptomatic (66).

18 Classical pathway Alternative pathway

Ab-Ag complexes, Mannose and carbohydrates Spontaneous in the presence SIGN-R1, CRP, and SAP on microbial surfaces of microbial surfaces Non-self patterns via properdin

C1q Mannose-binding lectin (MBL) C3 and C4 C3a C1r C1s MASP1/2 C3b iC3b Factor D

C4b Factor B Bb C2 Ba C2b Properdin C3

C4bC2a C3bBbP (C3 convertase) (C3 convertase)

C3a C3d, g

CR1/2 C3b

C5

C4bC2a3b C3bBb3b (C5 convertase) (C5 convertase) C5a

C5b

C6-9

Membrane Attack Complex (MAC)

Figure 1. The complement system and its importance in antibody responses. Depict- ed here are the cascades of activation for each of the three pathways as well as how different components affect the antibody response. In blue are factors known to activate the complement system. In red are factors that, when depleted, abrogates the antibody response and in green are non-affecting factors.

19 The requirement of C3 for normal antibody responses has been found in several studies. In 1974 Pepys showed that depletion of component C3 in mice with CVF resulted in a severe reduction of the antibody response against SRBC (67). Furthermore, C3-deficient dogs had lower antibody responses against the bacteriophage X174, SRBC and 2, 4-dinitrophenyl (DNP) hapten coupled to Ficoll. This effect was seen foremost in primary and IgG responses (68). Knock-out mice lacking C3, when immunized with a moderate dose of bacteriophage X174, had severely diminished IgG anti- phage titers (69).

Lectin pathway The MBL pathway and the alternative pathway are activated independently of antibodies. The former pathway is activated by the binding to mannose residues on glycoproteins or carbohydrates on the surface of foreign patho- gens. This in turn activates the mannose-associated serine (MASP- 1 and MASP-2), responsible for cleavage of C4. Another lectin family of proteins, the ficolins, has recently been described as complement activators, and like MBL, utilizes the MASP enzymes for this activation (70, 71). Mice lacking the MBL subunit A (MBL A-/-) had a lower IgM, but normal IgG response against Trichuris muris parasite (72) and to OVA in adjuvant (73). Furthermore, MBL double knock-out mice on C57BL/6 background, lacking MBL subunits A and C (MBL A/C-/-), showed a lower antibody re- sponse to hepatitis B surface antigen, whereas there was no difference in MBL A/C-/- mice on a SV129SvEv background (74). Hence, the MBL path- way could have some impact on the antibody response, but inhibition of this pathway alone cannot explain the severe impairment of the humoral re- sponse seen in C3-/- and C4-/- mice (69).

Alternative pathway The alternative pathway is characterized by a spontaneous cleavage of C3 into C3b. This product is normally inactivated by hydrolysis but in the pres- ence of microbial surfaces, C3b may be deposited and stabilized by binding of factor B. Properdin stabilizes the alternative pathway C3 convertase (75). Also, properdin can recognize dangerous non-self (e g (LPS)) and altered-self structures such as breast tumor cell lines and apoptot- ic cells (reviewed in (76)). Mice lacking factor B (Bf-/-) can mount a normal or even somewhat higher antibody response against SRBC and to the hapten NP coupled to bovine serum albumin (BSA) (77). Normal or higher antibody levels are seen when Bf-/- mice are exposed to replicating viruses (78). Thus, the alternative pathway seems to be of minor importance for the antibody response.

20 In summary, data observed in hereditary complement deficiences in humans and other animals as well as in experimental models with genetic mutations, point to the classical pathway as the effector pathway for primary antibody responses.

Classical pathway Classical pathway activation is foremost mediated by antibodies bound to an antigen, forming an IC recognized by complement component C1q, which is the binding unit in the protein complex C1qrs. The binding between C1q and the IC activates the enzymes C1r and C1s, and activated C1s is then capable of cleaving C4 into C4a and C4b, necessary for further activation. Thus, via C4 there is an overlap between the classical and MBL pathway (Figure 1). Further, the structure of MBL and C1q is similar, and, owing to gene dupli- cation, C1r and C1s are homologous to MASP-1 and MASP-2 (79).

Components C4 and C2 Guinea pigs with hereditary deficiencies in components C4 or C2, and hu- mans with deficiency in C4 have severely impaired IgG antibody responses, but normal IgM responses when immunized with bacteriophage X174 (80- 82). This suggested an incapability to perform class switch from IgM to IgG. In C4-/- mice immunized with above mentioned bacteriophage, the total Ig anti-phage titers as well as anti-phage IgG were severely diminished (69). Moreover, when challenged with West Nile Virus, C4-/- mice displayed a lower IgM and IgG response the first 10 days compared to WT (78).

Component C1q The structure of C1q was proposed in the 1970’s as a consequence of bio- chemical studies. The protein consists of 18 polypeptide chains, each chain having one collagenous and one non-collagenous part (83). The chains are divided into three types, A, B and C, and one of each type forms a trimer. This results in 6 trimers that come together via disulphide bonds at the N- terminals (collagenous part) forming a structure analogous to “a bundle-of- tulips” (83, 84) (Figure 2). Besides binding to antibodies, C1q can bind directly to antigen, self or non-self-antigens, such as C-reactive protein (CRP) (85), serum amyloid P component (SAP) (86-88), specific intracellular adhesion molecule-grabbing nonintegrin related gene 1 (SIGN-R1) (89), different bacterial and viral mol- ecules (reviewed in (90)) and apoptotic cells (91, 92). Also, a C1q receptor found on human epithelial cells plays a role in the activation of the classical pathway, independently of antibodies (93, 94). C1q is mainly synthesized by monocytic DC and macrophages (95), unlike other complement proteins, including C1r and C1s, that are mainly produced by hepatocytes (96, 97).

21 It seems that C1q plays a dual role in immune responses; C1q-deficient mice are more susceptible to (98-100) whereas the loss of C1q promotes autoimmunity, probably via impaired clearance of apoptotic cells (101). C1q can act as an , aiding the phagocytosis by e g macrophag- es and this seems to work without activation of the complement cascade (91, 102, 103). C1q has also been implicated in numerous immune regulatory functions. For example, apoptotic cells opsonized with C1q was shown to influence proinflammatory production by DC (104). In C1q-deficient mice the gene for subunit A is disrupted leading to a failure of trimerization. These C1qA-/- was reported having a normal primary antibody response but a lower secondary response upon immunization with a 10% SRBC suspension intraperitoneally (105). When immunized with 6 × 106 SRBC coated with DNP conjugated to KLH (DNP-KLH), the IgG2a anti-DNP was 2-fold lower in the primary response, and IgG2a and IgG3 anti-DNP were lower in the secondary response (105). When challenged with the malaria parasite Plasmodium chabaudi chabaudi, C1qA-/- mice dis- played normal anti-malarial response days 7 and 40 after primary infection. However, on day 100 these mice exhibited reduced levels of primary IgG2a antibodies. In contrast, after secondary infection the mice were more suscep- tible than WT mice, but had higher anti-malarial IgM and IgG2a levels (99). In summary, C1qA-/- mice exhibit a normal or lower antibody response against different antigens. Together with the data described above regarding deficiencies in the different activation pathways of complement, this argues for a dominant role of the classical pathway in antibody responses.

C1r C1s

Target A BC TrimerHexamer

Figure 2. Structure of the C1q molecule and binding of the C1(qrs)-complex to a target. The polypeptide chains A, B and C trimerizes and six trimers are joined into a hexamer, or “a bundle-of-tulips”.

22 Activators of C1q

Antibodies IgM binding to large antigens undergoes a conformational change, revealing a site for C1q-binding, thus allowing one single pentamer to activate com- plement. Besides IgM, also IgG subclasses IgG2a, IgG2b and IgG3 are able to activate complement upon antigen binding (106). IgG3 molecules have the capacity to associate into multivalent complexes via their Fc-parts which in turn increases the probability of C1q-binding (107, 108). In contrast, acti- vation of complement by IgG2a or IgG2b occurs less frequent than with IgG3 since it depends on the mere chance that several IgG2a or IgG2b mole- cules, required to activate complement, bind in the vicinity of each other. Monoclonal IgG1 antibodies on the other hand, directed against NP or DNP, was demonstrated to not activate complement (109, 110). Interestingly, the murine antibodies mentioned herein have human coun- terparts as follows: human IgG1 and IgG3 correspond to murine IgG2a and IgG2b where IgG1 is superior IgG3 in C1q binding but in further comple- ment activation via fixation of C4, IgG3 is more efficient (111, 112). One of these studies showed that human IgG2 (murine IgG3) and IgG4 (murine IgG1) are poor C1q-activators.

Specific intracellular adhesion molecule-grabbing nonintegrin related gene 1 (SIGN-R1) SIGN-R1 is the mouse homologue to the human DC-SIGN (113), which is expressed on DC and known to bind antigens expressed on pathogens, e g HIV surface protein gpl20 (114). The DC-SIGN family in mice, including SIGN-R1, consists of C-type that share common homologous carbo- hydrate recognition domains that enable multivalent interaction with glycan ligands (113). SIGN-R1 is highly expressed by MZ macrophages in the spleen and lymph nodes (115, 116). Because of its localization in the MZ of the spleen, the receptor comes in close contact with blood-borne antigens filtering through the lymphoid organs. Indeed, SIGN-R1 was reported aiding the uptake of capsular polysaccharide of Streptococcus pneumoniae (117). These results were achieved using the antibody 22D1 that transiently down- regulates the expression of SIGN-R1 on MZ macrophages and the same group also showed a loss of dextran uptake once SIGN-R1 was blocked (116). Furthermore, SIGN-R1 is able to aggregate C1q in a similar manner as IC, and thereby activate the classical pathway (89). Notably, mice pre- treated with 22D1 and challenged with Streptococcus displayed a normal antibody response (118).

23 C-reactive protein (CRP) and serum amyloid P component (SAP) Another group of proteins that C1q binds to and can be activated by are the pentraxins, represented by CRP (85, 87, 88) and SAP (86). CRP and SAP are produced in the as a response to inflammatory signals, mostly IL-6 (119). Ligands are both extrinsic, such as glycans and phospholipids, and self-structures, e g apoptotic cells (120). In humans, CRP is a major acute phase protein with dramatically increased levels from less than 50 µg/L in healthy conditions to about 500 mg/L in the initial phase of infections (121). SAP is the murine counterpart to CRP and increases in levels during infec- tion (122). Like IgM, CRP and SAP undergo a conformational change when bound to apoptotic cells and foreign structures, revealing a site for C1q to bind (88, 123). CRP-coated Streptococcus pneumoniae injected into mice induced a higher antibody response to the bacteria compared to the response to uncoated bacteria, suggesting a role for CRP in the adaptive immune re- sponse (124). SAP on the other hand seems to have a more suppressive role in antibody responses; mice lacking SAP had an increased susceptibility to experimental autoimmune encephalomyelitis (125). Pentraxin 3 is another C1q-binding pentraxin and can actually associate with C1q in the fluid phase. However, this association does not promote complement activation (126), nor C1q-binding to apoptotic cells (127).

The C1q paradox in primary antibody responses In summary, studies in knock-out mice have shown that complement com- ponents C3, C2, and C4 are of great importance for the generation of a nor- mal primary antibody response. Together with the fact that deficiencies in the MBL and alternative pathway only had minor impacts, this suggested that the classical pathway is the important effector pathway in antibody re- sponses. Indeed, C1qA-/- mice have collectively a lower antibody response to particulate antigens as described above. It is however a paradox as to how the classical pathway, initiated by anti- bodies in complex with their antigen, can play a crucial role for the genera- tion of a primary antibody response since the levels of specific antibodies are very low in naïve mice. As mentioned above, naturally occurring IgM, circulating in low amounts and low affinity, has been shown to be important in primary antibody re- sponses (57-59, 128). However, these studies did not reveal whether the low antibody response was due to the complement activation ability of IgM or merely to the lack of secretory IgM. Thus, a possible explanation for the role of the classical pathway in primary antibody responses would be that natural IgM in naïve mice binds antigen, activates C1q and initiates an antibody response via the mechanism of IgM-mediated feedback enhancement.

24 Complement Receptors 1 and 2 Activation of components in the complement cascade can also create a link to the adaptive immune response via the complement receptors. The genera- tion of C3 fragments is important for the initial immune response since they are serving as ligands for complement receptors 1 (CR1 [CD35]) and 2 (CR2 [CD21]) (Fig. 3). In humans there are two distinct encoding CR1 and CR2 whereas in mice, the receptors are two different splice products from the same gene, Cr2 (129). In mice, the receptors co-localize on B cells and FDC as shown by several studies (130-134). Interestingly, in one study CR1 and CR2 were found on a subset of T cells (135). Both CR1 and CR2 lack an intracellular signaling part but at least CR2 associate with the CD19/CD81 (CD19/Tapa-1) complex on B cells (136). Murine CR1, a type I transmembrane , is the larger protein (190 kDa) and is assembled from 21 short consensus repeats (SCR), i e conserved units of 60-70 amino acids, a transmembrane region and 35 amino acids that compose the cytoplasmic region (132, 137-139). The receptor is capable of binding C3b, C4b, iC3b, C3dg, and C3d and functions as co- factor for C3 cleavage into iC3b and C3d (138). Furthermore, the binding of C3 split products inhibits continous complement activation by diminishing the formation, and stimulating the decay, of C3 and C5 convertase (reviewed in (140)). Murine CR2 (150 kDa) binds the C3 split products iC3b, C3dg and C3d. As the shorter of the two receptors, CR2 is assembled from 15 SCR and binds fewer ligands than CR1. In antigen uptake and presentation the complement fragments are important since CR2 can bind virtually any com- plement-coated antigen without the antigen being able to bind to the BCR (141, 142). Association between CR2 and CD19/Tapa-1 facilitates the signal into the cell and this has been shown to lower the threshold for B-cell activa- tion (143, 144).

25 C3b C4b

iC3b C3dg C3d

CR1 CR2 (CD35) (CD21) Figure 3. Schematic picture of the murine complement receptors 1 and 2 with their respective ligands and ligand binding sites. The receptors are different splice prod- ucts of the same gene, therefore their structure is similar.

Cr2-/- mice lack both receptors and there are several reports showing that these mice have impaired antibody responses (145-149). The mice have normal numbers of GC but these are smaller than in WT animals (145, 148, 150). Moreover, it has been suggested that there is a heightened state of in- flammation in the splenic environment in these mice (151). Since both CR1 and CR2 are absent in the Cr2-/- mouse, it has been difficult to elucidate which of the receptors is required for antibody responses, although some reports imply a major role for CR2. Heyman and colleagues injected differ- ent mAbs specific either for CR1 alone or for both CR1 and CR2 into BALB/c mice followed by immunization with horse red blood cells (HRBC) or KLH. The anti-CR1/2 antibody suppressed over 99% of the response against HRBC and KLH, whereas, the mAb mono-specific for CR1 only had a minor effect on the antibody response (152). One year later a study was conducted with soluble CR2 injected into mice, competing with cellular CR2 for the binding of C3 fragments. This resulted in a suppressed antibody re- sponse against KLH (153), suggesting a primary role for CR2. Cr2-/- mice expressing human CR2 as a transgene had partially restored antibody re- sponse against SRBC (154). In vitro studies showed that CR2 on human B cells mediates a prolonged BCR signaling that could account for enhance- ment of B cell responses (155), and CR2 needs to form a complex with CD19/Tapa-1 for further signaling into the B cell (144, 156). Taken together, both in vivo and in vitro studies indicate a more pronounced role for CR2 than CR1 in antibody responses.

26 There have been different reports regarding which of these cells needs to express CR1/2 for a normal antibody response to occur. Some reports claim that FDC are the key players, where CR1/2 on these cells are thought to in- crease antigen retention in GC (150, 157). Fang et al. generated BM chime- ras expressing CR1/2 on B cells but not on FDC, or on FDC but not on B cells. When given SRBC, mice with CR1/2- FDC were unable to mount an antibody response regardless of whether the B cells expressed CR1/2 or not. However, others have found B cells to be the dominant effector cells (145, 146, 154). By using RAG-2-/- embryos (containing ES cells that give rise to mice without B and T lymphocytes) and injecting the embryos with either normal or Cr2-deficient ES cells, Croix et al. could generate mice bearing B cells with or without CR1/2 expression in an environment where FDC ex- pressed CR1/2 (146). When they were challenged with NP-KLH in alum, mice with CR1/2- B cells had a severely impaired immune response whereas mice with CR1/2+ B cells had a normal immune response. The same group created Cr2-/- mice and generated BM chimeras with CR1/2+ B cells and CR1/2- FDC. When immunized with bacteriophage X174, the antibody response in the chimeras was restored (145). However, B cells from these mice responded normally when incubated with LPS, anti-IgM or soluble CD40L in vitro, suggesting an intact signaling pathway in the absence of CR1/2 (145). Thus, it is still unclear on which effector cell CR1/2 must be expressed in order to allow a normal antibody response.

Possible mechanisms for the importance of complement in antibody responses Based on these data several, not mutually exclusive, hypotheses for why the complement is crucial for the generation antibody responses, have been dis- cussed.

1. Increased antigen retention by CR1/2+ FDC in GC would increase the effective antigen concentration, giving more effective B-cell stimulation (150, 157).

2. MZ B cells may transport complement-coated antigen or IC via their CR1/2 receptors into the follicle (55, 56).

3. Mechanisms depending on increased signaling via BCR co-crosslinked with CR2/CD19/Tapa-1:

a. The BCR itself (i e membrane-bound IgM) may be able to activate complement. In mice lacking secretory IgM and having a BCR una- ble to bind C1q, no complement was activated upon BCR ligation.

27 Moreover, these mice had diminished responses to chicken gamma globulin as well as to human serum albumin (61).

b. Circulating IC, formed in the periphery, may co-crosslink the BCR and CR2/CD19, thus increasing signaling into the B cell and thereby reducing the amount of antigen needed for B-cell activation (143, 144, 155, 158), as shown by in vitro studies.

c. Antigen may bind directly to BCR creating an array of antigen on the surface of the specific B cell. This would facilitate antigen bind- ing of soluble, natural IgM and co-crosslink several BCRs. The binding also enables complement to be activated, inducing binding of C3 fragments and engagement of CR1/2 (62).

4. B cells can take up complement-coated antigen via CR1/2 and subse- quently present it to TH cells leading to an enhanced antibody produc- tion. This has been shown in several in vitro and ex vivo studies (142, 159-164). In contrast, an in vivo study by Gustavsson et al. showed im- paired antibody responses but normal CD4+ T-cell proliferation in mice treated with antibodies blocking CR1/2 (165).

28 Present investigation

Aims In this thesis, the classical complement activation pathway and its most common activator, IgM, have been studied, along with the effect of the com- plement receptors 1 and 2 in antibody responses. I have tried to answer the following issues:

Paper Ia

1. Is the classical pathway component C1q crucial for normal antibody responses to SRBC?

2. Is the normal antibody response dependent on the ability of natural IgM to activate complement?

Paper II

1. Is antigen presentation, measured as T-cell proliferation, impaired in mice lacking CR1/2?

Paper III

1. Which of the two cell types, B cells or FDC, need to express CR1/2 in order for a normal antibody response against SRBC?

2. Which of the two cell types, B cells or FDC, need to express CR1/2 for the IgM-mediated enhancement of antibody responses against SRBC to occur?

29 Experimental setup

Mice Studies were performed using the murine system as a model. Table 1 de- scribes the different strains used. C 13 mice were created as described in the results section. Transgenic mice were either on BALB/c or C57BL/6 genetic background, but always compared to WT mice with the same genetic back- ground. One exception to this was when Cr2-/- (BALB/c background) mice were used as negative controls in experiments where the transgenic mouse of interest and its corresponding WT were on C57BL/6 background. For all experiments mice were age and sex matched.

Table 1. The different mouse strains used in present investigation. Mouse strain Annotation Appears in C 13 Producing IgM unable to activate complement Paper Ia (BALB/c background)

Cr2-/- Lacking complement receptors 1 and 2, CR1/2 (147) Paper Ia, II, III (BALB/c background)

BALB/c WT Paper Ia, II, III (producing Iga antibodies)

C1qA-/- Lacking subunit A of complement component 1, C1qA, Paper Ia (166) (C57BL/6 background)

CRP-/- Lacking C-reactive protein (unpublished) Paper Ia (C57BL/6 background)

SAP-/- Lacking serum amyloid p component (167) Paper Ia (C57BL/6 background)

C57BL/6 WT Paper Ia (producing Igb allotype antibodies)

DO11.10 Expressing an OVA-specific TCR on most T cells (168) Paper II (BALB/c background)

C.BKa- BALB/c congenic Paper III b b Igh /IcrSMnJ (producing Ig allotype antibodies) (CB17)

30 Immunizations and antigens For evoking and monitoring the antibody response, mice were immunized intravenously with specified doses of antigen diluted in 0.2 ml physiological salt solution (PBS) unless otherwise stated. In studies of antibody feedback enhancement, mice were injected with purified antigen-specific IgM, one hour before immunization. Antigens used were: SRBC and KLH (Paper Ia, II and III), ovalbumin (OVA) covalently coupled to SRBC and the TI-antigens Ficoll and LPS, both conjugated to 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) (Paper III). SRBC and KLH have been used in numerous studies regarding com- plement deficiencies. For blocking SIGN-R1, mice were intravenously injected with the SIGN- R1-specific mAb 22D1 24 hours before immunization with FITC-conjugated dextran (Paper I).

Adoptive transfer of T cells T cells from DO11.10 spleens were isolated using anti-CD4 magnetic beads. CD4+ cells were purified and injected intravenously into recipient mice 24 hours before immunization. The T cells were monitored using a clonotypic mAb, KJ1-26, recognizing the transgenic T-cell receptor specific for OVA.

Assays For measuring antibody responses, the enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay (ELISPOT) and hemagglutina- tion test were used. Here, antigen-specific levels of different immunoglobu- lin isotypes and subclasses were quantified. In order to control for comple- ment activation ability of the IgM molecule, the plaque forming cell assay (PFC), test and flow cytometry monitoring C3 deposition on SRBC were used. In addition, flow cytometry was used to analyze the B-cell compartment in C 13 mice, tracking OVA-specific T cells, and also to veri- fy the reconstitution in chimeras.

Bone marrow chimeras Murine complement receptors 1 and 2 are expressed on B cells and FDC, and consequently, in the Cr2-/- mouse neither of the cell types express the receptors. Therefore, in order to study which of the two cell types that need to express the receptors for a normal antibody response we created bone marrow chimeras, where recipient mice (BALB/c, CB17, or Cr2-/-) were sub- lethally irradiated and reconstituted with appropriate bone marrow, rendering mice either expressing CR1/2 on B cells, FDC or on both cell types.

31 Statistical analyses Statistical differences between groups were determined with Students t-test, either unpaired or paired, with significance levels at: p 0.05 (not signifi- cant, ns); p < 0.05 (*); p <0.01 (**); p < 0.001 (***).

32 Results and discussion Requirement for complement in antibody responses is not explained by the classic pathway activator IgM (Paper Ia)

Antibody responses are impaired in C1qA-/- and Cr2-/- mice Earlier studies with other antigens show that C1q-deficient mice have im- paired clearance of infections (98-100) and a slower onset of IgG antibody response to West Nile Virus. However, this defect was restored by day 10 after immunization (78). Others found reduced primary anti-malarial titers but normal secondary antibody responses (99). In contrast, the primary as well as the secondary antibody response to different conjugates of SRBC were impaired (105). Therefore, we wanted to clarify the importance of C1q in antibody re- sponses to SRBC in our system. C1qA-/- mice together with WT and Cr2-/- mice were immunized with four different doses of SRBC whereupon anti- body responses were analyzed. We found that C1qA-/- mice had impaired IgG anti-SRBC responses, similar to that in Cr2-/- mice, at over 90% of the time points assayed (Fig. 1E-H, Paper Ia). This was true also for the second- ary response after a boost at day 21. Moreover, the IgM anti-SRBC response was lower in C1qA-/- and Cr2-/- mice than in WT, most pronounced with the lowest and highest dose (Fig. 1A-D, Paper Ia). These data confirmed the importance of the classical pathway in antibody responses to SRBC and agrees with findings made by Cutler et al. (105). Using the Cr2-/- mice as negative controls, we confirmed a sustained low response in the C1qA-/- mice.

Creation and characterization of C 13 knock-in mice The phenotype found in C1qA-/- mice led us to pursue the hypothesis that the C1q activator important for antibody responses was natural IgM. Using ho- mologous recombination in embryonic stem cells, we generated a knock-in mouse, C 13. A point-mutation was introduced, rendering a codon change (Pro436 Ser436) within the coding region of the IgM constant region of the Igh . This is the same mutation described earlier to make the IgM mol- ecule unable to bind C1q (169), promote lysis via complement activation (53) and to enhance antibody responses (51). All IgM antibodies produced in C 13, both membrane-bound as well as secreted and regardless of specifici- ty, carry this mutation. B-6 embryonic stem cells derived from C57BL/6 (Ighb) were transfected with a modified version the original expression plas- mid (pCµ13) (53) that contains a region of homology to the Igha allele (Fig. 2A-D, Paper Ia). Southern analysis was used to identify positively transfect- ed clones (Fig. 2E, Paper Ia), germline mice were expanded and peripheral blood analyzed for the IgMa product indicative of a transfected gene. C 13

33 mice were then backcrossed for 12 generations to the BALB/c genetic back- ground.

The B-cell compartment was analyzed as a first characterization of the C 13 mice. No differences between C 13 and BALB/c could be seen in the num- ber of CD19+, B-1a, B-1b, or B-2 B cells (Suppl. Table 1, Paper Ia). Moreo- ver, the quantity of Fo B cells and the expression of surface IgM (BCR) were similar between the two strains. A minor increase of MZ B cells as well as a decrease in B220+ cells was found in C 13 mice (Suppl. Table 2, Paper Ia). To further characterize the mice, complement activation properties of the endogenous C 13 IgM was assayed. Mice were immunized with SRBC and spleen cells were analyzed after five days when IgM antibodies are at peak levels. B cells producing IgM specific for SRBC were detected in plaque forming cell assay (PFC) and ELISPOT. PFC is a complement-dependent test where serum is used as the source of complement and pro- motes lysis of SRBC if complement-activating IgM is present. Here, C 13 IgM did not activate complement since only background levels of PFC were detected (Table 1, Paper Ia). A caveat could be that there are no or very few SRBC-specific B cells in the C 13 mice. However, the ELISPOT results, quantifying the number of single B cells producing IgM anti-SRBC in a non- complement dependent assay, show similar numbers of antigen-specific B cell between C 13 and BALB/c (Table 1, Paper Ia). In summary, C 13 mice produce normal amounts of IgM, but the IgM cannot activate guinea pig complement. Mouse complement, more relevant in this study, is difficult to isolate since it is easily activated. To circumvent this we used the anti-coagulant lepirudin, which does not activate complement. SRBC-specific IgM derived from BALB/c or C 13 was tested for initiation of deposition of C3 on the surface of SRBC with mouse plasma as source of complement (Fig. 2, Paper Ia). Incubation of SRBC with IgM anti-SRBC isolated from BALB/c togeth- er with plasma from C57BL/6, BALB/c, or C 13 mice, led to deposition of mouse C3 while incubation with C1qA-/- plasma resulted in very little C3 deposition. IgM anti-SRBC from C 13 was unable to initiate C3 deposition in all situations. No C3-deposition was seen after incubation with plasma alone, indicating that SRBC by itself does not activate complement. Furthermore, we used the fact that IgM-mediated enhancement of anti- body responses is dependent on the ability of antigen-specific IgM to acti- vate complement. IgM specific for SRBC was purified from WT and C 13 mice and injected into BALB/c mice along with SRBC. As expected, IgM from WT was able to enhance antibody responses whereas IgM from C 13 had no enhancing effect (data not shown). Together with the data from depo- sition of C3, this shows that IgM from C 13 is unable to activate mouse complement. Our findings agree well with earlier data on the IgM mutant

34 #13 having a decrease in affinity for human C1q (169) and lack of ability to lyse erythrocytes (53, 169). To ensure that there were no major structural defects of the mutated IgM, the molecules were compared with WT IgM regarding size and half-life in vivo. Separation of IgM and IgG from immune sera on a size fraction col- umn (Sepharose CL-6B) showed that C 13 IgM and WT IgM had similar profiles, suggesting that there are no discrepancies concerning size or mo- lecular assembly (Suppl. Fig. 1A, Paper Ia). This also confirmed earlier stud- ies where the original IgM mutant generated from B cell hybridomas was tested for hemolysis, titers and pentameric assembly (52). The half-life of immune serum from C 13 or WT IgM injected intravenously into BALB/c mice was similar (Suppl. Fig. 1B, Paper Ia). In summary, C 13 IgM lacks the ability to activate complement whereas assembly of the mole- cule, agglutination ability as well as in vivo half-life is normal.

Absence of complement-activating IgM does not affect the antibody response against SRBC or KLH Next, we wanted to study whether the lack of complement activation by endogenous IgM produced in the C 13 mice led to an impaired antibody response as hypothesized. Interestingly, when C 13, BALB/c and Cr2-/- mice were immunized with four different doses of SRBC there were no dif- ferences between C 13 and BALB/c, whereas Cr2-/- mice responded poorly (Fig. 4E-H, Paper Ia). This was true for both primary and secondary IgG anti-SRBC responses. Occasionally, there was a lower response in C 13 mice although it was far from as low as the response seen in C1qA-/- or Cr2-/- mice. In addition, data shown here are from experiments with the most pro- nounced differences between Cµ13 and BALB/c. In analogy to IgG respons- es, the antigen-specific IgM response was similar in BALB/c and C 13 (Fig. 4A-D, Paper Ia). The antibody response in Cµ13 to three different doses of another large particulate antigen, KLH, was also tested. Similar results as with SRBC were obtained and at some time points the IgG anti-KLH levels in C 13 were slightly higher compared to BALB/c (Fig. 5, Paper Ia). Again, Cr2-/- mice had a severely impaired antibody response. Thus, we have ascer- tained that C1q is crucial for a normal antibody response (Fig. 1, Paper Ia), but that IgM is not the major C1q-activator in this case. The hypothesis partly relied on observations that two different transgenic mouse strains lacking secretory IgM also lacked a normal antibody response (57-59). The antigens used in these studies were different hapten-conjugated antigens such as NP-KLH, and this could be a plausible ground for diverging results. Therefore, C 13 mice were immunized with 1 µg NP-KLH but also using this antigen, Cµ13 mice had similar antibody response as WT controls (Suppl. Fig. 1C, Paper Ia). Thus, the use of different antigens is not a likely explanation for the discrepant results. Both strains lacking secretory IgM

35 were reported to have increased natural B cell repertoire (i e B1 B cells), and that might be a reason for the altered antibody response.

Normal antibody response to SRBC in mice lacking SIGN-R1, SAP, or CRP Surprised by the finding that IgM was not the factor explaining C1q- dependence of antibody responses, we considered three other endogenous activators: SAP (86), CRP (85, 86), and SIGN-R1 (89). When injected into mice, dextran labeled with FITC, bound to SIGN-R1 on MZ macrophages. A mAb called 22D1, that is specific for SIGN-R1, transiently blocked FITC-dextran uptake on SIGN-R1 (89, 116, 117). Using the same antibody, we confirmed the blockade of SIGN-R1 in BALB/c mice (Suppl. Fig. 1D, Paper Ia). However, mice that were transiently blocked of SIGN-R1 had an equally good antibody response against SRBC as mock- treated animals, whereas Cr2-/- again had a low response (Fig. 6B, Paper Ia). SIGN-R1 was also blocked in C 13 mice, in order to elucidate whether there could be a requirement of both SIGN-R1 and activation of complement via IgM. This was not the case, since the antibody response to SRBC was simi- lar with or without SIGN-R1 in C 13 mice (Fig. 6A, Paper Ia). CRP-/- and SAP-/- mice, immunized with SRBC also produced the same amount of specific IgG antibodies as their WT controls (Fig. 6C, Paper Ia). Hicks et al. showed that both human CRP and SAP were able to activate C1q following binding of histone structures (86) but as shown here this activation does not seem to be crucial for the generation of antibody responses to SRBC.

In summary, C1q is indeed required for the generation of a normal antibody response but the activator of C1q remains unknown. Herein, IgM, SIGN-R1, CRP and SAP, four known and potent C1q-activators, were tested but found insignificant in their role of affecting the antibody response. Additionally, the notion that C1q by itself would be the candidate factor seems unlikely since deposition of C3 fragments, ligands for CR1/2, was absent when mouse complement was incubated with SRBC alone (Fig. 2, Paper Ia). IgG antibodies are also able to activate complement the classical pathway and in some cases even to enhance antibody responses. However, all IgG subclasses suppress antibody responses to SRBC (8, 10, 11). Furthermore, in order for IgG to activate complement, a high density of IgG molecules, bound in the vicinity of each other, is required and since our study focuses on primary antibody responses where pre-formed levels of IgG are very low, it seems highly unlikely that IgG-mediated activation of complement would contribute to the normal antibody response observed in C 13. It cannot be excluded that some redundancy might exist in the activation of complement, i e that all, or some, of the activators work together. Thus, if one of them is depleted the others may compensate for this. Moreover, the

36 paradigm is that the activation of complement via C1q, leading to the gen- eration of the C3 fragments, which are the ligands for CR1/2, would be one intact chain of events. An alternative possibility is that C1q-activation ren- ders a function that is independent of CR1/2. In that case, the C3 fragments needed for CR1/2 engagement are generated elsewhere in the complement system, e g in the alternative pathway. Even though we and others have shown that the antibody response is not dependent of a functional MBL pathway or alternative pathway, also here a possible alternative is redundan- cy in the system. Mice lacking factors in two or more activation pathways at the same time would be a way to study this.

Our hypothesis was based on the assumption that the importance of natural IgM for antibody responses would depend on its complement-activating properties. However, results emanating from this work suggest that IgM and complement may act separately. The role of IgM could simply be to aggre- gate the antigen sufficiently to make this complex more antigenic than non- aggregating antigen. Also, two kinds of FcR for IgM have been observed, the Fc /µR and FcµR. The Fc /µR is expressed in various tissues e g thy- mus and spleen, the receptor binds both IgA and IgM, and has been impli- cated as mediator in endocytosis of IgM coated (170). The FcµR receptor has been assigned to B and T cells in humans and binds only IgM (171-173). Thus, a possible mechanism could be that IgM is bound by any of these receptors facilitating either antigen uptake by B cells or trans- portation into lymphoid follicles.

37 Impaired antibody responses but normal proliferation of specific CD4+ T cells in mice lacking complement receptors 1 and 2 (Paper II)

Severely impaired antibody response in Cr2-/- mice to the TD-antigens SRBC and KLH Genetically modified mice on CBA/J (41) or C57BL/6 (145-147) genetic background, lacking CR1/2, display an impaired antibody response to differ- ent antigens. We wanted to test whether this was due to impaired antigen presentation to CD4+ T cells. In vitro, CR1/2 on B cells enhance uptake of complement-coated antigens and subsequently present it to TH cells (142, 159-164). To study this in vivo, we chose to use the DO11.10 adoptive transfer sys- tem, developed by Jenkins et al. Here, the majority of the T cells in the DO11.10 mouse carry a transgenic TCR that recognizes an OVA peptide associated with class II I-Ad MHC. Moreover, the antibody KJ1-26 (174) directed against this transgenic TCR was used to track the T cells in flow cytometry. To be able to use this system, the Cr2-/- mice had to be back- crossed to BALB/c (H-2d). Since genotypic background may have an impact on the phenotype of knock-out mice, the antibody response in the new Cr2-/- mice was monitored. They were immunized with five doses of SRBC or three doses of KLH and compared to the relevant WT BALB/c. The results show that the severe impairment in both SRBC- and KLH-specific IgG re- sponses seen in Cr2-/- mice on other genetic backgrounds was retained (Figs. 1 and 2, Paper II). Also, a slight reduction in the antigen-specific IgM re- sponse was seen five days after immunization (Table 1, Paper II) agreeing with what others have observed (147). Hence, we could now use the DO11.10 adoptive transfer system to monitor T cell proliferation in the Cr2-/- mice.

Normal proliferation by OVA-specific CD4+ T cells in spite of impaired antibody response to OVA-SRBC in Cr2-/- mice BALB/c and Cr2-/- mice were injected with purified OVA-specific CD4+ T cells from DO11.10. The cells could be followed in flow cytometry with the antibody KJ1-26, specific for the transgenic TCR. Following the adoptive transfer, both mouse strains were immunized with OVA conjugated to SRBC or unconjugated SRBC as negative controls. Spleens from the mice were taken on days 2-4 and proliferation of OVA-specific T cells was assayed. The group immunized with OVA-SRBC responded well compared to the group immunized with unconjugated SRBC (Fig. 3A, D, Paper II). Im- portantly, the T-cell proliferation was similar in Cr2-/- mice and BALB/c. To make sure that the low T-cell proliferation seen with 5 × 107 OVA-SRBC was not because of poor T-cell transfer, a number of mice from that group

38 were injected with IgE anti-TNP/OVA-TNP. This is a potent inducer of both T-cell proliferation and antibody responses in DO11.10 mice (25, 175), and indeed the T cells had a high proliferation, proving that the transfer of T cells was successful (Fig. 3A, D, Paper II). Adoptively transferred mice immunized with OVA-SRBC or unconjugat- ed SRBC were also screened for antigen-specific IgG. Here, the response to both OVA and SRBC was severely reduced in Cr2-/- mice (Fig. 3B, C, E, F, Paper II). Thus, undetectable antibody responses in mice lacking CR1/2 are not mirrored by an abrogated T-cell proliferation. These results agree with previous findings in vivo (165) but not with in vitro findings (142, 159-164). Furthermore, our data supports a study where priming of T cells were found to be normal in mice lacking C3 or C4 (69). Although B cells have the ca- pacity to take up antigen via CR1/2, process and present it to TH cells in vitro, this cannot be the major mechanism explaining the abrogated antibody response in Cr2-/- mice.

Impaired antibody responses to the TI-antigens LPS-NIP and Ficoll- NIP in Cr2-/- mice

TI-antigens induce an antibody response that is independent of TH cells and therefore do not require antigen presentation via MHC. Mice were immun- ized with different doses of LPS-NIP or Ficoll-NIP and analyzed for the antigen-specific IgG response. Mice lacking CR1/2 had an impaired anti- body response to all doses of Ficoll-NIP, as well as to an intermediate dose of 5 µg LPS-NIP (Fig. 4B, D-F, Paper II). In our setting the lowest dose of LPS-NIP gave a poor overall response that might explain that no difference between WT and Cr2-/- mice could be detected (Fig. 4A, Paper II). The high- est dose may have been high enough to circumvent the need for CR1/2 in antibody responses (176) (Fig. 4C, Paper II). In summary, Cr2-/- mice have impaired antibody responses to TI-antigens in spite of the fact that these antigens do not require presentation to TH cells. This strongly suggests that antigen presentation to TH cells is not the mecha- nism by which CR1/2 exert their effect on antibody responses in vivo.

39 B cells lacking complement receptors 1 and 2 are equally efficient producers of IgG in vivo as wildtype B cells (Paper III)

Expression of CR1/2 on FDC is required for normal antibody responses against SRBC and B cells from Cr2-/- mice are equally efficient producers of IgG anti-SRBC as are wildtype B cells To elucidate whether B cells or FDC must express CR1/2 in order for a nor- mal in antibody response to take place, several different experiments involv- ing chimeric mice were done. By sub-lethal irradiation of mice, BM-derived cells (including B cells) are depleted while stromal cells (including FDC) survive. When mice are reconstituted with BM, the chimeras will have B cells of donor origin and FDC of recipient origin. In the first experiment, chimeras were generated resulting in four different groups with respect to CR1/2 expression: receptor expression on both or none of the cell types, or expression on either B cells or FDC. The mice were immunized with SRBC after a six week long recovery, establishing the new BM. Two major observations were made from this experiment. First, only mice expressing CR1/2 on their FDC elicited an antibody response to SRBC (Fig. 1, Paper III). This was repeated several times and with different doses yielding similar results. This observation is in line with findings by others and with the hypothesis that FDC expressing CR1/2 can capture comple- ment-coated antigen, prolong antigen time span in the follicle and make the antigen more accessible for Fo B cells (150, 157). Second, provided that CR1/2 were expressed on FDC, both B cells with or without CR1/2 expres- sion were able to produce antibodies against SRBC. Furthermore, they ap- peared to be equally efficient, arguing that CR1/2 expression on B cells is not crucial for the generation of a normal antibody response against SRBC. This was a surprising finding because it diminishes the role of CR1/2 on B cells and it indicates that B cell signaling or transportation via CR1/2 on MZ B cells is not required.

The antibody response in chimeric mice is generated by donor BM B cells In experiments using chimeric mice it is of great importance to carefully monitor the differences between donor and any recipient antibody produc- tion, which might arise because of incomplete irradiation. To control for this, the CB17 mouse, a BALB/c mouse congenic for the Ig locus, was used as recipient. CB17 mice produce Igb allotype antibodies whereas BALB/c and Cr2-/- mice (BALB/c background) are Iga antibody producers and the differ- ent allotypes can be distinguished in an allotype-specific ELISA. Here, CB17 mice were irradiated and reconstituted with BM either from BALB/c or Cr2-/- mice and immunized with two doses of SRBC. Since CB17 were the recipients, CR1/2 were expressed on FDC in all groups. As expected, all

40 chimeras produced normal levels of IgG anti-SRBC, as compared to Cr2-/- mice used as negative control (Fig. 2A, B, Paper III). Hence, we could repeat the results from Figure 1 also in experiments using CB17 as recipient. Im- portantly, we could not detect any IgG1 and IgG2a of allotype Igb indicating that there were no antibody-forming cells derived from CB17 in these chi- meras (Fig. 2C, D, Paper III). In contrast, a significant production of IgG1 and IgG2a of allotype Iga antibodies was observed and again, B cells from BALB/c and Cr2-/- mice produced similar antibody levels against SRBC. To summarize, the sub-lethal irradiation used is sufficient to selectively deplete host BM and normal SRBC-specific antibody responses are not de- pendent on CR1/2 expression on B cells.

B cells from wildtype and Cr2-/- mice produce similar amounts of IgG anti-SRBC although competing for the same antigens in the same environment In the studies described above, B cells with or without expression of CR1/2 were acting in different animals. It was however important to monitor their behavior when acting in the same milieu to minimize influences of environ- mental factors. Thus, we created BM chimeras consisting of two groups, either FDC expressing CR1/2 (CB17 recipient) or FDC without CR1/2 ex- pression (Cr2-/- recipient). Both groups were reconstituted with a mixture of BM cells from CB17 and Cr2-/- mice. Hence, antibody production by B cells expressing CR1/2 could be tracked by Igb allotype screening and Iga anti- body production should be from non-CR1/2-expressing B cells. Mice were immunized with different doses of SRBC. Again, the im- portance of FDC expressing CR1/2 is demonstrated. Cr2-/- recipients failed to produce antibodies against a wide range of SRBC doses whereas CB17 recipients produced high amounts (Fig. 3A-C, Paper III). Only when an ex- tremely high dose was given did Cr2-/- recipients respond to SRBC, support- ing the notion that complement deficiencies can be overcome with high anti- gen doses (176) (Fig. 3D, Paper III). When sera from the responding animals (CB17 recipients) were measured for allotype responses, we found that, in the same animal, B cells expressing and B cells not expressing CR1/2 were able to produce equal amounts of SRBC-specific antibodies. Similar results were achieved when spleen cells from CB17 recipients were assayed day 9 and 11 post immunization. Here, using ELISPOT, the number of antigen- specific antibody-producing cells was similar regardless of CR1/2 expres- sion (Table 1, Paper III). These experiments show that, when competing for the same antigen, CR1/2 expression on B cells has no advantages in the pro- duction of antigen-specific IgG antibodies.

Thus, according to our data, none of the hypotheses for how CR1/2 is in- volved in antibody responses which postulate a role for CR1/2 on B cells is correct. Neither B-cell activation by co-ligation of CR2/CD19/Tapa-1 with

41 the BCR (155, 158) nor transportation on CR1/2 on MZ B cells into the fol- licle (55, 56) seem to be applicable. The data thereby rules out any major beneficial effect of having the CR1/2 receptors expressed on B cells, at least when it comes to the generation of normal antibody responses against SRBC. This is puzzling since SRBC are too big to enter the follicle by sim- ple diffusion. Most likely they need to be transported in some way, and this was thought to be done by CR1/2+ B cells (55, 56). This might occur in sev- eral ways. First, MZ B cells have been shown to continuously shuttle be- tween the MZ and the follicle (56). Moreover, this shuttling is independent of expression of CR1/2 and perhaps another receptor expressed on the B cell can bind the antigen for transportation into the follicle. Second, there are early data describing an FDC-like cell that transported IC between the sub- capsular sinus of the lymph node to the follicles (177). Finally, a more con- troversial but nonetheless straight forward theory, which would explain the requirement for CR1/2 on FDC, could be that the “arms” of the FDC might surpass the border between the follicle and the MZ and directly capture the antigen-complement complexes.

Expression of CR1/2 on both FDC and B cells is required for IgM- mediated enhancement of antibody responses to SRBC Recent studies have shown that MZ B cells are continuously shuttling be- tween the MZ and the follicle, presumably transporting complement-coated antigens for deposition onto CR1/2 on FDC (56). Also, earlier data demon- strated a similar function for transportation of IgM-IC (55). Since we show that CR1/2-expressing B cells are dispensable for responses to antigen alone, we hypothesized that the expression of CR1/2 on MZ B cells could play a role in the responses to IgM-IC. Chimeric mice, generated as in Figure 1, were injected with IgM anti-SRBC followed by two different doses of SRBC one hour later. This allows the IgM to form complexes with the antigen in vivo and the effect of the IC could be analyzed as an IgM-mediated en- hancement of the antibody response to SRBC. As expected, mice with CR1/2 expressed on both B cells and FDC had a strong antibody response when injected with IgM together with antigen compared to antigen alone (Fig. 4A, E, Paper III). In mice lacking CR1/2 on both cell-types, the antibody response was abrogated regardless of whether antigen alone or IC was administrated (Fig. 4D, H, Paper III). When CR1/2 were expressed on FDC but not on B cells, an enhancement of the antibody response occurred (Fig. 4B, F, Paper III) and it was seen foremost with the higher dose of SRBC together with IgM. However, this enhancement was slower and less pronounced than in mice expressing CR1/2 on both cell- types suggesting a role for CR1/2 on B cells. In mice having B cells but not FDC expressing CR1/2, we could not detect any enhancement with the lower antigen dose (Fig. 4C, Paper III). Interestingly, the higher dose yielded a small yet significant enhancement (Fig. 4G, Paper III) even though it de-

42 clined rapidly. Nevertheless, this indicated that extra-follicular responses take place and agrees with findings where GC formation, somatic hypermu- tation and memory-cell formation is normal in mice lacking secretory IgM (60).

Thus, we conclude that CR1/2 on FDC are required for a fast and sustained IgG anti-SRBC response to IgM-IC. The enhancement was stronger in chi- meras where both B cells and FDC expressed CR1/2 compared to chimeras where only FDC expressed the receptors. Thus, B cells played a role and IgM-IC could either facilitate co-ligation between the BCR and CR2/CD19/Tapa-1, described to lower the threshold for B-cell activation (155, 158), or transport IgM-IC into the follicles (55). Transportation via an unknown receptor on B cells could explain the remaining response against IgM-IC in chimeras with CR1/2 expressed on FDC but not on B cells. Here, the described Fc-receptor for IgM (Fc ) found on B cells is an appealing alternative (170-173). It has been observed that the strong immune response caused by IgM- containing IC compared to antigen alone is accompanied by enhanced anti- gen concentrations in the spleen (54). Also, this could account for the adju- vant-like effect the IgM-mediated enhancement exerts. It has been shown that the response to IgM together with one dose of antigen is similar to the response when using ten times that amount of antigen alone (39, 178). To- gether, this indicates that the antigen-transportation into lymphoid follicles is facilitated when the antigen is in complex with IgM compared to antigen alone. Thus, in the presence of CR1/2 on FDC, a basal, robust IgG response to SRBC is formed whereas IgM together with SRBC can further increase this response. This IgM-dependent “icing on the cake” requires that IgM activates complement and that B cells express CR1/2. We believe that this is likely caused by antigen transport by MZ B cells.

43 Highlights of this thesis

The activation of the first component of the classical complement path- way, C1q, is crucial for the generation of a normal antibody response as shown in C1qA-/- mice.

The antibody response in Cµ13, mice producing IgM that lacks the abil- ity to activate complement via C1q, had normal antibody responses against both SRBC and KLH.

Neither SIGN-R1, CRP, nor SAP, three other C1q-activating factors, contributed to the antibody response to SRBC. This was showed using mice knocked-out for CRP or SAP as well as mice that transiently lacked expression of SIGN-R1.

Although mice lacking CR1/2 have severely impaired antibody respons- es they have normal proliferation of specific CD4+ T cells. This implies that the impaired antibody response is not due to lack of CR1/2-mediated antigen presentation to TH cells.

For a normal antibody response to occur against SRBC, CR1/2- expression on FDC is crucial. B cells without CR1/2 are equally good antibody producers as CR1/2-expressing B cells. This was elucidated us- ing chimeric mice that either expressed CR1/2 on B cells or FDC.

In similar chimeric mice, IgM-mediated enhancement was dependent foremost on CR1/2 expression on FDC but also a role for the receptors was also found on B cells. The enhancement of the antibody response was slower and smaller in magnitude when B cells lacked CR1/2.

44 Acknowledgements

This work was performed at the Department of Medical biochemistry and Microbiology, Section for Immunology, Uppsala University. C 13 mice were constructed in collaboration with Prof. Michael Carroll at Harvard University. Collaborations have also been made with Prof. Jens Jensenius and Dr. Steffen Thiel at Aarhus University for elucidating the role of MBL pathway, and with Prof. John Cambier and Dr. Andrew Getahun for the in- fluence of the alternative pathway, in antibody responses.

Especially, I would like to acknowledge the following persons:

Professor Birgitta Heyman, supervisor, for your endless enthusiasm and never-ending immunological knowledge. For always having the time to dis- cuss data and being encouraging in the time of need. Thank you also for your hard work in proof-reading this thesis. Last but not least, for the fun and informative talks about the parent – adult-child relationship.

Research Assistant Jenny Hallgren-Martinsson, co-supervisor, for always asking the tricky questions and coming up with great, laborious new experi- mental ideas. Finally, for the cheerleading during the writing of this thesis!

Doctor Andrew Getahun, for introducing me into the immunological lab. You always had the time for the “newbie”-questions and you have great educational skills.

Research Assistant Frida Henningson-Johnson, for your help in preparing chimeras and for sharing Västerås-anecdotes.

Professor Kjell-Olov Grönvik, for giving me new interesting scientific in- put, “this is how it was 30 years ago”-stories and for always trying to con- vince us about the importance of T cells.

My examinator Bengt Westermark.

Malin Winerdal for the fantastic paintings of mice, cells and antibodies used on the cover.

45 The rest of the Heyman/Hallgren Immunology Group: Anna Bergman for being the best co-worker one could have, and for keeping an eye on the clin- ical relevance. Also, thank you for letting me use your nice illustrations in this thesis. Joakim Dahlin for always having time to help out in the lab when I’ve screwed up some experiments, for fun practical jokes on co- workers, for nice parties, and for fun vocabulary and “Lidingö”-accent! Yue Cui for being always happy. Good luck in Paris and remember: Yue R! Zhoujie Ding for fun parties including wine and tequila mix, and for letting me learn some Chinese! Take care of Flora! Annika Hermansson for a good collaboration and help in the lab the time you worked with us.

Sandra Kleinau for asking the hard questions and creating discussions. Cecilia “Mjölby” Carnrot for fun and lengthy stories and for always mak- ing me happy. Anna-Karin “AlKalinPhosfatas” Palm for funny discus- sions about nothing and everything around the fika-sofa. Lisbeth “Bettan” Fuxler for representing the adult population, for fixing everything in and around the lab and always having that “glimten i ögat”-approach. Viktor “Kattpojken” Ahlberg – you turned out to be a decent guy despite the SLU origin. Let’s stay in touch! Lotta Wik for nice discussions and that sporadic wine rendezvous! Sara Bolin for always bringing a smile on my face and for the laughs and great fun shared the short time you were here. Michael Thorpe for excellent knowledge of the English language!

The rest of the Immunology corridor: Lars Hellman, Kajsa Prokopec, So- fia Magnusson, Caroline Fossum, Bernt Hjertner, Tommy Linné, Mag- nus Åbrink, and Kersti Larsson.

The staff at SVA animal facility, especially Ingela Stake for having patience with us concerning the animal quantity and for dealing with animal care situations with common sense.

All IMBIMers, especially administrators, dish wash staff, Olav Nordli for eminent equipment fixing and Lars-Erik “Nalle” Björnberg for excellent package and liquid nitrogen delivery.

Thanks to all my friends inside and outside of Uppsala, whether you can sing or not, for adding quality to my life.

My family for believing in me and for endless support.

This work was financially supported by Uppsala University, the Swedish Research Council, Ellen, Walter and Lennart Hesselman Foundation, Hans von Kantzow Foundation, Agnes & Mac Rudberg Foundation; King Gustaf V:s 80 Years Foundation, Ollie and Elof Ericsson Foundation.

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