The Role of Complement Receptors and Fc Gamma Receptor Iib in Collagen-Induced Arthritis

The Role of Complement Receptors and Fc gamma Receptor IIb in Collagen-induced Arthritis

Anja Mezger

Degree project in biology, Master of science (2 years), 2010 Examensarbete i biologi 45 hp till masterexamen, 2010 Biology Education Centre and Department of Cell and Molecular Biology, Uppsala University Supervisor: Professor Sandra Kleinau Table of Contents Abbreviations ...... 1 Summary ...... 2 1.0 Introduction ...... 3 1.1 Rheumatoid arthritis ...... 3 1.2 Collagen-induced arthritis ...... 3 1.3 The spleen...... 4 1.4 The complement system ...... 4 1.5 Complement receptor 1 and 2 ...... 5 1.6 Fc gamma receptors ...... 6 1.7 Aim of the project...... 7 2.0 Material and Methods ...... 7 2.1 Animals ...... 7 2.2 Immunization of mice ...... 8 2.3 Single cell suspension ...... 8 2.4 Heat-inactivated fetal calf serum ...... 8 2.5 Enzyme-linked immunosorbent spot assay ...... 8 2.6 B cell proliferation assay ...... 9 2.7 Enzyme-linked immunosorbent assay ...... 9 2.8 Magnetic activated cell sorting ...... 9 2.9 Statistical analysis ...... 9 3.0 Results ...... 9 3.1 Optimization of ELISPOT protocol ...... 9 3.2 B cell reactivity to BCII in naïve and immunized mice ...... 10 3.3 B cell reactivity against mouse CII in naïve and immunized mice ...... 11 3.4 The proliferative response of stimulated B cells in naïve and immunized mice ...... 12 3.5 The proliferative response in different cell populations ...... 14 3.6 No BCII-specific antibody response in splenocytes from BCII-immunized WT mice after BCR and TLR stimulation...... 15 4.0 Discussion ...... 15 5.0 Acknowledgement ...... 18 6.0 References ...... 19

Abbreviations

ACK Ammonium chloride-potassium AFC Antibody forming cells APC Antigen-presenting cells BSA Bovine serum albumin BCII Bovine collagen type II CIA Collagen-induced arthritis CII Collagen type II CpG Cytosine-phosphate-guanine CR Complement receptor DC Dendritic cell ELISA Enzyme-linked immunosorbent assay FCA Freund’s complete adjuvant FcR Fc receptor FCS Fetal calf serum FDC Follicular dendritic cells FO B Follicular B cells HLA Human leukocyte antigen IFNγ Interferon-γ Ig Immunoglobulin IL Interleukin ITIM Immunoreceptor tyrosine-based inhibition motif LPS Lipopolysaccharide MACS Magnetic activated cell sorting MCII Mouse collagen type II MHC Major histocompatibility complex MOG Myelin oligodendrocyte glycoprotein MZ Marginal zone OD Optical density PBS Phosphate buffered saline RA Rheumatoid arthritis SI Stimulation index SLE Systemic lupus erythematosus TLR Toll like receptor TNF-α Tumor necrosis factor-α WT Wild-type

Summary

Rheumatoid arthritis is a B cell dependent disease, which causes symmetric inflammation of the small joints. Immune cells infiltrate the synovium and B cells become autoreactive. Nowadays, it is possible to treat the symptoms, but there is still no complete cure for the disease. A lot of research to understand the basic mechanisms underlying this autoimmune disease is done in the collagen-induced arthritis model. Mice are injected intradermally with collagen type II and develop collagen type II-specific antibodies. Their symptoms resemble the ones observed in rheumatoid arthritis. Complement receptor 1 and 2, and the inhibitory receptor FcγRIIb are involved in the regulation of B cells. To gain a more detailed insight into the function of these receptors, I have studied if these receptors regulate proliferation and collagen type II-reactivity in B cells in vitro, using splenocytes from FcγRIIb and complement receptor knock-out mice in proliferation and enzyme-linked immunosorbent spot assays. B cell reactivity to bovine collagen type II in immunized mice is significantly increased in complement receptor 1 and 2 deficient mice. Furthermore, B cell reactivity is not only seen towards bovine, but also to mouse collagen type II. Hence, immunization with bovine collagen type II of DBA/1 mice leads to an autoimmune response against mouse collagen type II. Surprisingly, tolerance is first broken in the lymph nodes and then in the spleen. The lacking of FcγRIIb and complement receptor 1 and 2 leads to a significant increased proliferative response to CpG and B cell specific stimuli. Additionally, stimulation with LPS results in a higher response in naïve FcγRIIb or complement receptor 1 and 2 deficient mice. Overall, my data supports the hypothesis that mice lacking FcγRIIb or complement receptor 1 and 2 have a higher susceptibility and develop a more severe arthritis, respectively.

1.0 Introduction

1.1 Rheumatoid arthritis In this study an animal model for rheumatoid arthritis (RA) has been used to investigate the involvement of certain receptors in the initiation of autoimmune arthritis. RA is a B cell dependent disease, which mechanisms responsible for the development and persistence of this autoimmune disease are still not completely understood. About 0.5 - 1% of the world’s population is suffering from RA1. As in many other autoimmune diseases RA is two to three times more frequent in women than in men. Susceptibility of rheumatoid arthritis is linked to the haplotype of the human leukocyte antigen (HLA)-DR genes. DR4, DR14 and DR1 are associated with RA2. RA is a symmetric inflammation of small joints. The synovium is inflamed and articular structures are destroyed. About 80 % of patients have antibodies against the Fc fragment of immunoglobulin (Ig) G3. These antibodies are also known as the rheumatoid factor. Patients positive for rheumatoid factor usually have a more severe course of disease. Once the rheumatoid factor is bound as immune complexes, the complement system is activated and inflammatory cells are recruited to the site of inflammation4. These immune complexes are commonly found in the cartilage5. It is still unknown if autoantibodies initiate the disease or are responsible for the chronicity. Interleukin (IL) 2 and interferon-γ (IFNγ) producing T cells infiltrate the synovium and activate macrophages and synovial fibroblasts. These cells produce inflammatory cytokines (tumor necrosis factor-α (TNF-α), IL-1 and IL-6) in large amounts. Various pro-inflammatory genes are activated and enhance the local inflammation in the joints and tissue degradation. Besides the named cells, B cells, mast cells and dendritic cells (DCs) reside in the synovial tissue. The joint cartilage is mainly degraded by metalloproteases6. Osteoclasts are responsible for bone destruction7.

1.2 Collagen-induced arthritis Collagen-induced arthritis (CIA) is a very common animal model for RA and is used to gain knowledge about disease mechanisms of autoimmune arthritis. CIA is induced by a single intradermal injection of collagen type II (CII) emulsified in Freund’s complete adjuvant (FCA). Mice develop a polyarthritis in the peripheral articular joints. Symptoms are similar to the ones seen in RA and can include amongst others inflammation of the synovium, pannus formation, bone and cartilage destruction and ankylosis8. Heterologous CII gives usually a much stronger immune response than autologous CII9. The susceptibility of mouse strains is dependent on their major histocompatibility complex (MHC). The most susceptible strains carry the haplotypes H-2q or H-2r10. Female mice are protected through estrogen and have thus a lower susceptibility to CIA than male mice11. Another factor that correlates with disease susceptibility is the age of the mice. Mice younger than 6-7 weeks are usually more resistant to arthritis9. The molecular mechanism is yet not fully understood, but progress to an understanding of CIA is made. Svensson et al. demonstrated that CIA is a B cell-dependent disease, since mice deficient in B cells are resistant12. Once mice or rats are injected with a collagen dose, B cells produce arthritogenic anti-CII antibodies, which attack the cartilage of joints9. T cells are believed to activate additional immune cells, which are responsible for inflammation and destruction of joints. IL-12 and IFNγ are the main cytokines produced during inflammation13, 14. Additionally, TNF-α and IL-1 are secreted14, 15. This reflects a typical Th type 1 response that is initiated through the used adjuvant. Further, the classical complement pathway plays an important role in disease development. Mice inhibited or deficient in C5 are protected against CIA16, 17. It is believed that CII specific

3 antibodies activate the complement system and the formed anaphylatoxins cause the infiltration of granulocytes and monocytes18, 19.

1.3 The spleen The spleen was the organ mainly examined in this project, since naturally polyreactive splenic B cells show reactivity against bovine CII (BCII) in naïve mice20. It might also be the immunological compartment where tolerance is broken and the decision is made whether autoimmunity will develop or not. The spleen is a secondary lymphoid organ and the largest blood filter of the body21. Its main function lays in the filtering of blood and in bacterial and fungal defense. The spleen is structured into two major compartments, the white-pulp (lymphoid region) and the red pulp. The white pulp harbors T cells, arterioles and B cell follicles. The red pulp contains the venous sinuses, endothelial cells and stress fibres22. Plasmablasts migrate to the red pulp after antigen encounter23. The produced antibodies can rapidly enter the blood stream and be distributed throughout the body24. The white pulp is structured into T- and B-cell compartments surrounding the arterial vessels. Chemokines are responsible for the attraction of T and B cells to their respective domains. Clonal expansion of B cells takes place in the B-cell follicles. A distinct subset of B cells, the follicular B cells (FO B) are recirculating and mainly located in the white pulp of the spleen and the lymph nodes25. They account for the biggest part of splenic B cells and are able to differentiate into plasmablasts or memory B cells. The marginal zone (MZ) surrounds the T-cell zone and functions as a transit area for incoming cells, which are about to enter the white pulp. It harvests two different subsets of macrophages – MZ macrophages and MZ metallophilic macrophages24. Additionally, it contains MZ B cells and a subset of dendritic cells (DCs). B cells are needed to maintain the macrophages in the MZ26. The MZ is an important compartment for pathogen removal. Once MZ B cells detect pathogens, they differentiate into IgM-producing plasma cells or antigen-presenting cells (APCs)27. APCs are able to activate CD4+ T cells28. We believe that the MZ B cells play a substantial role in the initiation of arthritis, since they are naturally reactive to CII (unpublished data)20.

Figure 1: The structure of the spleen. The spleen consists of different compartments, such as the white pulp which surrounds the central arteriole. The central arteriole branches into cords which are located in the red pulp. (Figure from Mebius and Kraal. Structure and function of the spleen. Nat Rev Immunol. 5, 606-616 (2005).)

1.4 The complement system The complement system is a phylogenetically ancient system and is even found in the horseshoe crab (Carcinoscorpius rotundicauda)29. It contributes to the defense mechanisms against bacterial, viral and parasitic pathogens. It consists of three different pathways: the classical, lectin and alternative pathway. The main function, which is the same for all pathways, is to generate proinflammatory mediators, coat pathogens’ surfaces with 4 complement proteins and induce cell lysis by introducing pores into the pathogens membrane. In the classical pathway, complement activation is initiated by the binding of Fc parts of antibody immune complexes to C1r and C1s (serine proteases). Different complement proteins are cleaved until C3 is cleaved into C3a and C3b. This is the intersecting point where all pathways meet. The lectin pathway is initiated by the recognition of pathogen-associated molecular patterns. The alternative pathway is initiated through spontaneous hydrolyses of C3 into C3b. Just like the other two pathways, it results in the formation of the C3 and C5 convertases. The resulting C5b is able to recruit additional complement proteins to a pathogen’s surface, form the membrane attack complex and insert a pore into the membrane. The cleavage products C4a, C3a and C5a function as proinflammatory mediators. The complement system seems to be linked to autoimmunity, since mice deficient in C4 or complement receptor (CR) 1 and 2 have an increased susceptibility to autoimmune diseases30. Furthermore, C5 deficient DBA/1 mice are not susceptible to CIA31.

Figure 2: The complement system. The complement system can be initiated by the classical, lectin and alternative pathway. Each pathway ends in the formation of C3 and C5 convertases. C5b is generated through cleavage of C5 and is able to recruit the complement proteins C5b-9 to a pathogen’s membrane to form the membrane attack complex. C4a, C3a and C5a function as anaphylatoxins. (Figure from Dunkelberger and Song. Complement and its role in innate and adaptive immune responses. Cell research 20, 34-50 (2010).)

1.5 Complement receptor 1 and 2 CR1 and CR2 are believed to play a role in the regulation of B cells. In mice, the Cr2 gene encodes both CR1 and CR2 through differential splicing32. CR2, also known as CD21, binds C3d and iC3b33. CR1 (CD35) has short consensus repeats at the amino-terminal end and binds in addition to C3d and iC3b, also C4b and C3b32-34. Both receptors are expressed on B cells and on follicular dendritic cells (FDCs) in mice35. B-1a cells express the lowest and marginal zone (MZ) B cells express the highest level of CR1 and CR233, 34.

CR1 and 2 are linked amongst others to autoimmunity. A possible explanation is the role of CR1/2 in the peripheral tolerance of B cells, since CR2 is involved in regulating the elimination of autoreactive B cells30. Data derived from Nilsson et. al supports this hypothesis where a low dose of BCII resulted in an enhanced CIA in female CR1/2-/- mice36. Just like in the CIA model, lupus-prone mice develop a more severe disease if they are lacking CR1/230. As in humans, levels of CR1/2 are decreased in mice suffering from systemic lupus erythematosus (SLE)37. Despite of data showing an increased autoantibody production in CR1/2-/- mice when crossed with the autoimmune lpr/lpr strain, other groups have shown the opposite: an impairment in antibody production in CR1/2-deficient mice 30, 38-40. Cross-linking of the B cell receptor with CR2 bound to an antigen-C3d complex lowers the threshold for B cell activation, and the blocking of CR1/2 leads to an impaired humoral immunity to protein antigens41, 42. Proliferation and antibody production assays in mice might lead to further evidence of CR1/2 as regulators for the induction of autoimmunity.

Figure 3: Complement receptors CD35 and CD21. CD35 and CD21 are splice variants of the Cr2 gene product in the mouse. CD35 can binds C3b, C4b, iC3b and C3d while CD21 has six short-consensus repeats less and binds only iC3b and C3d. (Figure adapted from Carroll. Complement and humoral immunity. Vaccine 265, 128-133 (2008).)

1.6 Fc gamma receptors Fc receptors (FcR) are an important link between the innate and the adaptive immune system. The group of receptors that binds the Fc part of IgG is called FcγR. In mice, there are four FcγRs. FcγRI is the only high-affinity receptor. FcγRIIb, FcγRIII and FcγRIV are low-affinity receptors with FcγRIIb as the only inhibitory receptor. FcγRIIb signals through an immunoreceptor tyrosine-based inhibition motif (ITIM)43. All low-affinity FcγRs show very high sequence homology and might be the result of duplications44. FcγRs are glycoproteins with two (low affinity receptors) or three (high affinity receptor) Ig domains45, 46. The common γ-chain is responsible for signal transduction in the stimulatory FcγRI, FcγRIII and FcγRIV47. Phagocytosis, release of inflammatory mediators, degranulation and cellular cytotoxicity are the result of binding of the Fc parts to activating receptors48, 49. FcγRIIb is expressed on a variety of cells, such as DCs, monocytes, eosinophils, macrophages and B cells50. It is an important checkpoint of humoral tolerance. FcγRIIb, together with activatory receptors, such as CR2, establishes the threshold for B cell activation. Once mice are lacking FcγRII, the threshold is significantly lowered51. Just like CR1 and 2, FcγRIIb is 6 involved in the peripheral tolerance of B cells and its signaling can lead to the elimination of B cells52. Deficiency of FcγRIIb renders mice with an H-2b background that are otherwise resistant susceptible to CIA53. Furthermore, FcγRIIb-/- mice are more susceptible and develop a more severe CIA than WT mice54. Hence, FcγRIIb plays a suppressive role in arthritis. Kagari et. al showed that older mice, deficient in FcγRIIb, are more susceptible to CIA than younger mice. In addition, they are also more susceptible than WT mice to CIA55. In general, FcγRIIb-/- mice show an earlier onset of CIA, higher severity, higher levels of IgG anti-CII, with all subclasses increased54, 56. Thus, FcγRIIb might play a significant role in the level of antibody production. Taken together, these data indicate an important role for FcγRIIb in the regulation of autoimmunity.

Figure 4: FcγR in mice. The only high-affinity receptor is FcγRI. FcγRIIb is the only inhibitory receptor. It contains an ITIM motif in its intracellular part. All other receptors signal through a common γ-chain. The low- affinity receptors have two Ig domains, while the high-affinity receptor has three Ig domains. (Figure adapted from Willcocks et al. Low-affinity Fcγ receptors, autoimmunity and infection. Expert Rev. Mol. Med. 11, e24, (2009).)

1.7 Aim of the project This study aims at elucidating the role of CR1/2 and FcγRIIb in CIA. The function of these receptors will be investigated in vitro, mainly with two different methods. The ELISPOT assay enables to test for CII-specific B cells, whereas the proliferation assay allows us to examine activation of B cells upon stimulation. Splenocytes from CR1/2-/- and FcγRIIb-/- mice are compared to DBA/1 mice to gain knowledge to which extent these receptors contributed to the observed effects.

2.0 Material and Methods

2.1 Animals Female DBA/1 mice (5-10 weeks old), CR1/2-/- DBA/1 mice (8-14 weeks old) and FcγRIIb-/- DBA/1 mice (10 to 17 weeks old) were used. The CR1/2 and FcγRIIb -/- mice (generated on a C57/Bl6 background) had been back crossed to the DBA/1 strain for 12 generations. Male mice were only used where indicated. The DBA/1 mice were originally derived from Taconic, Denmark. All animals were housed and bred at the animal facility of BMC, Uppsala

University. Rodent chow and water were fed ad libitum. All experiments were approved by the ethical committee at Uppsala Tingsrätt.

2.2 Immunization of mice BCII was dissolved in 0.01 M acetic acid to a concentration of 0.8 mg/ml and mixed with FCA (Difco, Michigan) in a 1:1 ratio. Fifty µl of the emulsion were administered intradermally at the base of the tail under anesthetics with isofluran (Baxter medical AB, Sweden). With this immunization protocol each mouse received 20 µg BCII.

2.3 Single cell suspension The mice were euthanized at day 0, 5-6, and 11-12 post immunization. Spleens were extracted and a single cell suspension was prepared. Spleens were grinded through a metal net into 5 ml phosphate buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 ∙ 2 H2O, 1.5 mM KH2PO4) in a tissue culture plate. The net and the plate were rinsed with 2 and 5 ml PBS, respectively. After one washing step with PBS (centrifuged at 300 g) red blood cells were lysed in 3 min with 5 ml ammonium chloride-potassium (ACK) buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA, pH 7.2 – 7.4). The lysis was stopped with 5 ml PBS. The splenocytes were taken up in 1.8 ml DMEM (National Veterinary Institute, Sweden) complete medium (DMEM, 1 % polyethylene glycol, 0.1 M β-mercaptoethanol, 1M Hepes, 200 mM L- glutamine 100x, 10 % heat-inactivated fetal calf serum (FCS)). The cell number was determined by staining the cells in 0.04 % trypan blue (Sigma, Missouri) using a Bürker chamber.

2.4 Heat-inactivated fetal calf serum FCS was inactivated in a water bath at 56°C for 30 min.

2.5 Enzyme-linked immunosorbent spot assay An enzyme-linked immunosorbent spot (ELISPOT) assay was used to enumerate antigen specific antibody producing B cells. Two different protocols were used, where protocol 1 was replaced by protocol 2, to increase number of specific spots and reduce background spots. For both protocols plates were coated over night at 4 °C with 10 µg BCII/well and 10 µg bovine serum albumin (BSA) (Sigma) per well as control. In indicated experiments plates were coated in addition to BCII and BSA with 10 µg mouse CII (MCII) (Elastin, Missouri) and myelin oligodendrocyte glycoprotein (MOG). Splenocytes (1 × 106 cells in 200 µl DMEM) were plated in 96-well flat-bottom plates (Nunc, Denmark). The plates were read manually (counting of spots) using an inverted microscope (Leitz, Germany). Protocol 1: Cells were incubated for 24 h at 37°C, 5 % CO2. After washing with PBS 50 µl alkaline phosphatase conjugated-IgM (Sigma) (diluted 1:500 in PBS) or alkaline phosphatase conjugated-IgG (Sigma) (diluted 1:500 in PBS) was added per well. The secondary antibody was incubated for 2 h at room temperature. Plates were washed with 200 mM Tris-HCl (pH 9.1) and 50 µl Bcip/NBT liquid substrate system (Sigma) was applied per well for 1 h at room temperature. Plates were rinsed with distilled H2O and air-dried. Protocol 2: Cells were incubated for 3 h at 37°C, 5 % CO2. Plates were washed with PBS- Tween (0.05% Tween20 in PBS) and 50 µl alkaline phosphatase conjugated-IgM (Sigma) (diluted 1:100 in PBS) or 50 µl alkaline phophatase conjugated-IgG (Sigma) (diluted 1:500 in PBS) was added per well. After incubating the plates at 4 °C over night, plates were washed with PBS-Tween and subsequently with PBS. Fifty µl Bcip/NBT liquid substrate (Sigma) were added per well and incubated in dark for 1 h at room temperature. Plates were rinsed with distilled H2O and air-dried.

2.6 B cell proliferation assay Triplicates of 1 × 105 splenocytes per well (200 µl final volume) were plated onto a 96-well round-bottom plate (BD, New Jersey). Goat anti-µ F(ab’)2 anti-mouse (Jackson ImmunoResearch, Pennsylvania) was added to a final concentration of 0.5, 2 and 5 µg/ml. Furthermore, lipopolysaccharide (LPS) (5 µg/ml) and cytosine-phosphate-guanine type B (CpG) (3 µg/ml) (Hycult biotechnology, Netherlands) were applied to the plates. Cells were 3 cultured for 3 days at 37°C, 5% CO2. One µl [ H] thymidine (Perkin Elmer, Massachusetts) was added 22 h prior cell harvesting. Cells were harvested using a cell harvester (LKB, Australia) and proliferation was determined with a scintillation counter (LKB).

2.7 Enzyme-linked immunosorbent assay Secreted antibodies were analyzed in splenocyte cultures stimulated with the above mentioned mitogens by an enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Nunc) were coated over night with 10 µg BCII. Unspecific binding was blocked during 1 h with 1% BSA in PBS. Plates were washed with PBS-Tween 20. Splenocytes from male DBA/1 mice were cultured as described in the B cell proliferation assay (no thymidine was added). Supernatant was transferred to coated plates and incubated over night at room temperature. After washes with PBS-Tween 20, 50 µl/well of alkaline phosphatase sheep anti-mouse IgM (µ-chain specific; diluted 1:500 in 1 % BSA in PBS) (Sigma) was added and incubated for 2 h at room temperature. The plates were thereafter washed with PBS-Tween 20 and dH2O. Fifty µl/well of p-nitrophenylphosphate (Sigma) dissolved in substrate buffer (1 M diethanolamine, 1.05 mM MgCl2, pH 9.8) were applied to the plates and absorption was measured in a spectrophotometer (Molecular Devices, California) at 405 nm after 60 and 90 min of incubation.

2.8 Magnetic activated cell sorting B cells were isolated from splenocytes by magnetic activated cell sorting (MACS). Single cell suspensions of spleens were gained as described above, except that the cells were resuspended in MACS buffer (PBS, 0.5 % BSA, 2 mM EDTA, pH 7.2) instead of DMEM. The cells were resuspended to 1×107 cells/90 µl MACS buffer. Anti-CD43-biotin antibody (eBioscience, California) was added (1 µl/2×106 cells) and incubated for 20-30 min on ice. After centrifugation (7 min, 300 g) MACS buffer (90 µl/1×107 cells) and magnetic beads coupled to streptavidin (Miltenyi Biotec, Germany) (1 µl/2×106 cells) were added and incubated for 25 min on ice. Cells were thereafter washed (3 x 3 ml MACS buffer) and resuspended in 500 µl MACS buffer and applied to a pre-cooled and pre-washed column (Miltenyi Biotec) that was placed in a magnetic field. The negative fraction (mainly B cells) was collected, centrifuged and the pellet resuspended in 2 ml DMEM. The positive fraction (containing CD43 positive cells, non-B cells) was retrieved by releasing the column from the magnetic field and pushing 5 ml MACS buffer through the column. Cells were centrifuged and resuspended in 2 ml DMEM.

2.9 Statistical analysis Statistical significance was determined by Student’s t test assuming unequal variances.

3.0 Results

3.1 Optimization of ELISPOT protocol To optimize the ELISPOT assay two different protocols were tested on splenocytes obtained from male DBA/1 mice 5 days post BCII immunization. The incubation time of the cells, the

9 washing agents and the concentration and incubation time of the secondary antibody differed between the protocols. Protocol 2 led clearly to an optimization of the ELISPOT assay (figure 5). The background was reduced and the number of antigen specific spots was significantly higher in protocol 2.

Figure 5: Optimization of ELISPOT. Two different protocols for an ELISPOT assay were used for the analysis of splenic BCII-specific B cells in a male DBA/1 WT mouse (n=1) immunized i.d. with 20 µg BCII/FCA 5 days previously. Protocol 1 had a longer incubation time of cells, used PBS as washing agent and had a lower concentration of secondary antibody compared to protocol 2. Protocol 2 used PBS-Tween20 as a washing agent. Wells treated according to protocol 2 showed an increase of 88.5 % in the mean number of BCII-specific antibody forming cells (AFC) per well. Values are displayed as mean+SEM. ** p < 0.01.

3.2 B cell reactivity to BCII in naïve and immunized mice B cell reactivity to BCII was investigated in the spleens of WT, CR1/2-/- and FcγRIIb-/- mice in order to elucidate the role of CR1/2 and FcγRIIb. Naïve mice were included in the study to be able to predict whether differences in the amount of antibody forming cells (AFCs) are based on the effect of the immunization or are already present in unimmunized mice. Since no significant differences in naïve mice were observed (figure 6), all differences in immunized mice must be accredited to the involvement of CR1/2 and FcγRIIb, respectively.

Figure 6: B cell reactivity to BCII in naïve mice. B cell reactivity to BCII does not differ significantly in CR1/2-/-, FcγRIIb-/- and WT mice. Results are displayed as the mean amount of AFC/106 cells (+SEM). Data was obtained using 5-6 mice per genotype.

Different time points after BCII immunization were investigated since the exact time point of self tolerance breakage in CIA is not known and might differ between individual mice. WT, CR1/2-/- and FcγRIIb-/- mice were immunized with 20 µg BCII and spleens were extracted on day 5 and day 11 post immunization for analyses of BCII-specific B cells by ELISPOT. No significance in the amount of IgM anti-BCII AFCs was seen at 5 days post immunization between WT and FcγRIIb-/- mice (figure 7A). However, CR1/2-/- mice produced a significantly higher amount of AFCs than FcγRIIb-/- and WT mice. At eleven days after BCII- immunization similar results were obtained as on day 5 (figure 7B). Significantly higher 10 numbers of BCII-specific IgM AFC was seen in CR1/2-deficient mice when compared to FcγRIIb-/- and WT mice

Figure 7: B cell reactivity to BCII in immunized mice. Data is displayed as the mean+SEM amount of IgM anti-BCII antibody forming cells (AFCs) in spleens of BCII-immunized CR1/2-/- (n=4-6), FcgRIIb-/- (n=5-6), WT (n=6) mice. CR1/2-/- mice have a significantly higher percentage of AFCs than WT and FcγRIIb-/- mice. A, Amount of AFCs on day 5. B, Amount of AFCs on day 11. * p < 0.05, ** p < 0.01.

3.3 B cell reactivity against mouse CII in naïve and immunized mice To investigate if immunization with BCII in DBA/1 mice leads to the generation of autoreactive B cells an ELISPOT was performed in which MCII was used as coating antigen in comparison with BCII. Cells were prepared from spleens and lymph nodes of DBA/1 mice on day 0, 5 and 12 after BCII-immunization. Single IgM positive spots in MCII coated wells were seen with splenocytes in immunized mice (table 1 B-C), while naïve mice did not show any B cell reactivity against MCII (table 1 A). All animals that displayed B cell reactivity against MCII produced also BCII-specific B cells. On day 12 after immunization, 50 % of the animals showed B cells reactivity against both BCII and MCII in the lymph nodes (table 1C). The ratio of the number of BCII:MCII spots was ca. 3:2 on day 12 in lymph nodes.

Table 1: B cell reactivity against BCII and MCII in naïve and BCII-immunized mice. One million cells in 200 µl DMEM were plated in 96-well flat-bottom plates. Data is shown as the mean number of spots (IgM) per animal against BCII and MCII. A, Naïve mice. B, Day 5. C, Day 12

Thus, no high reactivity of B cells against MCII was observed in the spleen. To exclude the possibility that this was due to a too early time point, spleens and lymph nodes from mice were investigated 21 days after immunization. The results demonstrated that the number of AFCs secreting IgM and IgG specific for MCII were still low in the spleen 21 days after immunization (table 2). In contrast, the number of MCII specific spots in the lymph nodes was clearly higher. The highest value was 6.5 AFCs/1 × 106 cells for IgG and 2.5 AFCs/1 × 106 cells for IgM. In the spleen, 2 out of 3 animals had MCII-specific AFCs for IgM. 11

Table 2: B cell reactivity against BCII and MCII 21 days post BCII-immunization. One million cells in 200 µl DMEM were plated in 96-well flat-bottom plates. Data is shown as the mean number of spots per animal against BCII and MCII. A, Amount of IgM-producing AFCs. B, Amount of IgG-producing AFCs.

3.4 The proliferative response of stimulated B cells in naïve and immunized mice Proliferation assays were performed on splenocytes obtained from naïve and BCII-immunized mice to elucidate the impact of FcγRIIb and CR1/2 regarding the proliferative potential of B cells. CR2 functions as an activatory receptor, while FcγRIIb is inhibitory. The role of CR1 is still not fully known, but there is evidence supporting an inhibitory role. Loss of one of these receptors on B cells might result in a different response to B cell receptor and toll like receptor (TLR) stimuli compared to WT mice. In addition, we investigated if immunization influences the function of these receptors.

The splenocytes were cultured with the B cell receptor specific stimulus F(ab’)2 anti-µ and TLR ligands LPS and CpG. The proliferation of the cells was analyzed. LPS and CpG bind to TLR4 and TLR9, respectively. TLRs are expressed on various kinds of cells, such as B cells, mast cells, NK cells, dendritic cells, regulatory T cells, macrophages, monocytes, neutrophils, basophils and endothelial cells57.

The proliferative response to the different stimuli was greater in WT mice after BCII immunization compared to naïve WT mice (figure 8A). BCII-immunized WT mice showed the highest stimulation index (SI) with the different doses of anti-µ F(ab’)2 at day 5. The SI decreased again at day 11-12 (figure 8A). No significant changes in SI were detected with the CpG stimuli between naïve and BCII-immunized WT mice. The enhanced proliferation capacity seen following BCII immunization was, however, not as evident in the CR1/2-/- and -/- -/- FcgRIIb mice. Thus, in the CR1/2 mice, only the dose of 0.5 µg/ml anti-µ F(ab’)2 gave a higher SI in immunized mice than in naïve mice (figure 8B). Surprisingly, the immunized CR1/2-/- mice responded significantly less to CpG and LPS than the naïve CR1/2-/-mice. This result differs from what was observed in the WT mice (inducing a higher or similar response in immunized mice). The proliferative response of splenocytes from FcγRIIb-/- mice was, in almost all used stimuli, the same in naïve and BCII-immunized mice (figure 8C). However, splenocytes stimulated with 0.5 µg/ml anti-µ F(ab’)2 on day 11 post immunization showed a higher SI compared to naïve mice.

Figure 8: The proliferative response of splenocytes following BCII immunization in different mouse strains. Splenocytes were cultured and stimulated with different doses of a B cell specific stimulus (anti-µ F(ab’)2), LPS and CpG. Proliferation was measured with a β-counter. The mean+SEM stimulation index (SI) of WT (n=6), CR1/2 -/- (n=4-6) and FcγRIIb-/- (n=5-6) is shown at 0, 5-6 and 11-12 days after i.d. immunization. A, WT mice. B, CR1/2-/- mice. C, FcγRIIb -/- mice. * p < 0.05, ** p < 0.01, *** p < 0.001.

The results in the different strains were then compared with each other (Figure 9). The clearest differences were seen between naïve mice (figure 9A). Thus, in naïve mice, FcγRIIb-/- -/- cells responded significantly higher to the lower dose of anti-µ F(ab’)2 than CR1/2 and WT cells, with WT cells showing the lowest response. The response to LPS was the highest in CR1/2-/- cells and lowest in WT cells. Using CpG as a stimuli resulted in a significantly higher response in CR1/2-/- and FcγRIIb-/- than in WT cells. At day 5 post immunization significant differences between the strains were found when lower doses of anti-µ F(ab’)2 were used. Thus, mice deficient in CR1/2 responded less than -/- FcγRIIb and WT mice. At day 11 post immunization doses of 0.5 and 2 µg/ml anti-µ F(ab’)2 induced the highest response in mice lacking the FcγRIIb compared to WT and CR1/2-/- mice. In addition, CR1/2-/- splenocytes proliferated more than the WT splenocytes when 2 µg/ml of anti-µ 13

F(ab’)2 were used. No significant differences were seen with a dose of 5 µg/ml anti-µ F(ab’)2. In immunized mice, FcγRIIb-/- splenocytes gave the lowest SI when LPS was used as a stimulus, but the highest response when CpG was used.

Figure 9: The proliferative response of splenocytes in different mouse strains following BCII- immunization. Splenocytes were cultured and stimulated with different doses of a B cell specific stimulus (anti- µ F(ab’)2) , LPS and CpG. Proliferation was measured with a β-counter. The mean+SEM stimulation index (SI) of WT (n=6), CR1/2-/- (n=4-6) and FcγRIIb-/- (n=5-6) is shown at 0 (A), 5-6 (B) and 11-12 (C) days after i.d. immunization. * p < 0.05, ** p < 0.01, *** p < 0.001.

3.5 The proliferative response in different cell populations All above described data on proliferative responses were gained from unsorted splenocytes. This led to the question whether the observed effects were derived from B cells only or also from other cell types present in the spleen. To solve this question B cells were sorted using MACS and anti-CD43-biotin antibodies. Most B cells, except B-1 cells, do not express CD43. Therefore, the CD43 negative fraction will mainly contain B cells and the positive fraction will mainly contain T lymphocytes, monocytes and granulocytes. 14

Splenocytes of two naïve FcγRIIb-/- mice were labeled with anti-CD43-biotin and sorted using MACS. A proliferation assay was performed on the i) unsorted cells ii) the negative and iii) the positive fraction. The higher doses of anti-µ F(ab’)2 showed a highly specific effect on unsorted cells and cells in the negative fraction (B cells) (figure 10A). No difference was observed between the cell populations with the lowest dose of 0.5 µg/ml. All cell fractions responded well to CpG (figure 10B), while unsorted cells and cells in the negative fraction appeared to respond better to LPS than the positive fraction. The response of B cells to LPS and CpG was also investigated in two naïve CR1/2-/- (figure 10C) and four WT mice (figure 10D). There was no great difference between the different cell fractions in CR1/2-/- and WT mice when stimulated with LPS. However, CpG induced the lowest response in the B cell fraction compared to the unsorted and positive fractions in both CR1/2-/- and WT mice. Thus, CD43 negative cells lacking FcγRIIb were the only CD43 negative cells responding well to CpG.

Figure 10: Effect of BCR and TLR stimuli on different cell populations. Splenocytes from naïve mice were unsorted or MACS sorted using anti-CD43-biotin antibody, giving a CD43 negative fraction (mostly B cells) and a CD43 positive fraction (mainly non–B cells). The cells were stimulated with anti-µ F(ab’)2 and/or LPS and CpG and cultured for 3 days. The proliferation capacity of the cells is shown as stimulation index (SI). A, B FcγRIIb-/- mice (n=2). C, CR1/2-/- mice (n=2). D, WT mice (n=4).

3.6 No BCII-specific antibody response in splenocytes from BCII-immunized WT mice after BCR and TLR stimulation. Splenocytes from BCII-immunized male DBA/1 mice were harvested on day 5 post immunization. The cells were incubated with the above described stimuli for 3 days. The supernatant was transferred to a BCII-coated microtiter plate and incubated over night. IgM coupled to alkaline phosphatase was added and the optical density (OD) was measured after 60 and 90 min. No significant increase of the OD value to any stimuli was detected.

4.0 Discussion In this study we tried to investigate the role of CR1 and 2 and of FcγRIIb in the initiation phase of CIA. We chose ELISPOT and proliferation assays as an approach to solve these

15 open questions. The ELISPOT assay allows looking at the percentage of antibody-forming colonies that are specific for BCII and MCII, respectively. The protocol of the ELISPOT assay was modified to get a more sensitive assay. Using a detergent, PBS-Tween20, as a washing solution could noticeably reduce the background. The higher concentration of the secondary antibody led to higher sensitivity. Taken together, PBS-Tween and a higher concentration of secondary antibody resulted in a detection of a 85 % higher number of antibody-forming cells.

Regarding the immune response after BCII immunization I found that the CR1/2-/- splenocytes produced significantly higher number of BCII specific IgM spots on both day 5 and 11-12 after immunization than splenocytes from WT and FcγRIIb-/- mice. Thus, CR1/2 functions as an inhibitory receptor in early phases of immune responses. This corresponds to earlier observations made by Nilsson et. al. demonstrating that CR1/2-/- mice have a higher incidence of CIA36. It seems that CR1/2 have an important role in the regulation of autoreactive B cells30. If this receptor is not expressed, tolerance is easier broken and a higher percentage of B cells become specific for CII. About two weeks after BCII immunization very few MCII specific AFCs were observed in the spleen, while 50 % of the animals were positive for MCII specific B cells in the lymph nodes. However, at three weeks after immunization IgM and IgG MCII-specific AFCs were seen both in the spleen and lymph nodes, with the highest frequency in the lymph nodes. This suggests that tolerance is first broken in the lymph nodes, later in the spleen.

Proliferation assays can give information about how cells respond to external stimuli. By using different knock-out strains, it can be determined if single receptors function inhibitory or activating. Naïve CR1/2-/- splenocytes proliferate more than naïve WT splenocytes. Seemingly, the expression of CR1/2 lowers the threshold for activation and consequently the potential to proliferate. MZ B cells express higher amounts of CR1/2 compared to FO B cells. Therefore, the effect of being deficient in these receptors must be mostly credited to MZ B -/- cells. This picture changes on day 5. CR1/2 splenocytes responded less to anti-µ F(ab’)2 than WT splenocytes. There is no evident explanation available for the reduced B cell proliferation in CR1/2-/- on day 5. More experiments are needed to validate and explain these results further. Looking at BCII-specific AFCs, CR1/2-/- mice had the highest number both on day 5 and 11 post immunization compared to WT and FcγRIIb-/- mice. This and the data derived from the proliferation assay at day 0 and day 11 support the inhibitory function of CR1/2. Surprisingly, FcγRIIb-/- mice responded to a higher extent to CpG than WT or CD21 -/- mice. To examine whether this effect was exclusively due to B cells or due to all or other cell populations, a proliferation assay with MACS-sorted splenocytes was carried out. We noted that B cells would only proliferate to a high extent to CpG when lacking FcγRIIb. The high response might be linked to the inability to eliminate B cells reactive against self-DNA. Bolland et. al. showed that C57BL/6 mice deficient in FcγRIIb develop spontaneous antinuclear antibodies58. CpG can bind to these self-reactive BCRs and is delivered into the endosome where it binds to TLR 9. This in turn stimulates proliferation59. Additionally, this experiment demonstrated the BCR specificity of anti-µ F(ab’)2. Only CD43 negative cells (B cells) were stimulated using higher concentrations of the F(ab’)2 fragment. The low, but detectable degree of proliferation in the CD43 positive cells might be accountable to B-1 cells, since they express CD43 as well as the BCR. In addition, FcγRIIb-/-, compared to CR1/2-/- and WT mice, showed the highest proliferation on day 11 with the lower doses of anti-µ F(ab’)2. This is in accordance to Takai et. al. who showed that mice lacking FcγRII have a lowered threshold for activation51. Furthermore, naïve FcγRIIb-/- splenocytes responded higher to LPS stimulation than WT mice, since FcγRIIb is shown to inhibit TLR-4 mediated stimulation60. 16

Our aim to elucidate the role of FcγRIIb and CR1/2 in CIA is only partly achieved. My data shows that FcγRIIb and CR1/2 are important receptors which have a suppressive role in autoimmune diseases. There is still a lot more work to do until one can be certain about their extent of involvement. A future experiment could be proliferation and antigen production assays on sorted cells. It must be known if these receptors function only on certain cell populations or if they even function different on different cell populations. It would also be interesting to continue the experiments with MCII. It might give further clues how and when tolerance is broken.

5.0 Acknowledgement I would like to thank my supervisor Professor Sandra Kleinau for giving me the opportunity to do my degree project in her group. Thank you for your guidance and time to answer all my questions. I would like to thank Kajsa Prokopec for introducing me to the lab work and teaching me all relevant techniques. David Plaza, Alex Beyer, Mike Thorpe, Marius Linkevičius and Marlen Adler, thank you for your company inside and outside of the lab, for our lunch conversations and of course for all the kladdkaka. Finally, I want to thank my family and Michael Bondegård for their endless support and putting up with all my moaning. Thanks!

6.0 References 1. Gabriel, S.E. The epidemiology of rheumatoid arthritis. Rheum Dis Clin North Am 27, 269-81 (2001). 2. Nepom, G.T. et al. HLA genes associated with rheumatoid arthritis. Identification of susceptibility alleles using specific oligonucleotide probes. Arthritis Rheum 32, 15-21 (1989). 3. Franklin, E.C., Holman, H.R., Muller-Eberhard, H.J. & Kunkel, H.G. An unusual protein component of high molecular weight in the serum of certain patients with rheumatoid arthritis. J Exp Med 105, 425-38 (1957). 4. Zvaifler, N.J. The immunopathology of joint inflammation in rheumatoid arthritis. Adv Immunol 16, 265-336 (1973). 5. Ishikawa, H., Smiley, J.D. & Ziff, M. Electron microscopic demonstration of immunoglobulin deposition in rheumatoid cartilage. Arthritis Rheum 18, 563-76 (1975). 6. Teitelbaum, S.L. Bone resorption by osteoclasts. Science 289, 1504-8 (2000). 7. Gravallese, E.M. et al. Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis. Am J Pathol 152, 943-51 (1998). 8. Courtenay, J.S., Dallman, M.J., Dayan, A.D., Martin, A. & Mosedale, B. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283, 666-8 (1980). 9. Holmdahl, R. et al. Incidence of arthritis and autoreactivity of anti-collagen antibodies after immunization of DBA/1 mice with heterologous and autologous collagen II. Clin Exp Immunol 62, 639-46 (1985). 10. Holmdahl, R., Jansson, L., Andersson, M. & Larsson, E. Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis. Immunology 65, 305-10 (1988). 11. Jansson, L. & Holmdahl, R. Enhancement of collagen-induced arthritis in female mice by estrogen receptor blockage. Arthritis Rheum 44, 2168-75 (2001). 12. Svensson, L., Jirholt, J., Holmdahl, R. & Jansson, L. B cell-deficient mice do not develop type II collagen-induced arthritis (CIA). Clin Exp Immunol 111, 521-6 (1998). 13. Okamoto, Y. et al. Characterization of the cytokine network at a single cell level in mice with collagen-induced arthritis using a dual color ELISPOT assay. J Interferon Cytokine Res 20, 55-61 (2000). 14. Stasiuk, L.M., Abehsira-Amar, O. & Fournier, C. Collagen-induced arthritis in DBA/1 mice: cytokine gene activation following immunization with type II collagen. Cell Immunol 173, 269-75 (1996). 15. Mussener, A., Litton, M.J., Lindroos, E. & Klareskog, L. Cytokine production in synovial tissue of mice with collagen-induced arthritis (CIA). Clin Exp Immunol 107, 485-93 (1997). 16. Wang, Y., Rollins, S.A., Madri, J.A. & Matis, L.A. Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease. Proc Natl Acad Sci U S A 92, 8955-9 (1995). 17. Watson, W.C., Brown, P.S., Pitcock, J.A. & Townes, A.S. Passive transfer studies with type II collagen antibody in B10.D2/old and new line and C57Bl/6 normal and beige (Chediak-Higashi) strains: evidence of important roles for C5 and multiple inflammatory cell types in the development of erosive arthritis. Arthritis Rheum 30, 460-5 (1987). 18. Holmdahl, R., Bockermann, R., Backlund, J. & Yamada, H. The molecular pathogenesis of collagen-induced arthritis in mice--a model for rheumatoid arthritis. Ageing Res Rev 1, 135-47 (2002). 19

19. Klos, A. et al. The role of the anaphylatoxins in health and disease. Mol Immunol 46, 2753-66 (2009). 20. Prokopec, K.E., Carnrot, C., Råsbo, K., Karlsson, M.C.I. & Kleinau, S. Marginal zone B cells are naturally reactive to collagen type II and initiate the immune response in collagen-induced arthritis. Manuscript. 21. Steiniger, B. & Barth, P. Microanatomy and function of the spleen. Adv Anat Embryol Cell Biol 151, III-IX, 1-101 (2000). 22. Drenckhahn, D. & Wagner, J. Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility. J Cell Biol 102, 1738-47 (1986). 23. Sze, D.M., Toellner, K.M., Garcia de Vinuesa, C., Taylor, D.R. & MacLennan, I.C. Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J Exp Med 192, 813-21 (2000). 24. Mebius, R.E. & Kraal, G. Structure and function of the spleen. Nat Rev Immunol 5, 606-16 (2005). 25. Pillai, S. & Cariappa, A. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol 9, 767-77 (2009). 26. Nolte, M.A. et al. B cells are crucial for both development and maintenance of the splenic marginal zone. J Immunol 172, 3620-7 (2004). 27. Lopes-Carvalho, T. & Kearney, J.F. Development and selection of marginal zone B cells. Immunol Rev 197, 192-205 (2004). 28. Attanavanich, K. & Kearney, J.F. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J Immunol 172, 803-11 (2004). 29. Dunkelberger, J.R. & Song, W.C. Complement and its role in innate and adaptive immune responses. Cell Res 20, 34-50 (2010). 30. Prodeus, A.P. et al. A critical role for complement in maintenance of self-tolerance. Immunity 9, 721-31 (1998). 31. Wang, Y. et al. A role for complement in antibody-mediated inflammation: C5- deficient DBA/1 mice are resistant to collagen-induced arthritis. J Immunol 164, 4340- 7 (2000). 32. Kurtz, C.B., O'Toole, E., Christensen, S.M. & Weis, J.H. The murine complement receptor gene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct gene products that share homologous domains with both human CR2 and CR1. J Immunol 144, 3581-91 (1990). 33. Haas, K.M. & Tedder, T.F. Role of the CD19 and CD21/35 receptor complex in innate immunity, host defense and autommunity. Mechanisms of Lymphocyte Activation and Immune Regulation X: Innate Immunity 560, 125-139 (2005). 34. Molina, H., Kinoshita, T., Inoue, K., Carel, J.C. & Holers, V.M. A Molecular and Immunochemical Characterization of Mouse Cr-2 - Evidence for a Single Gene Model of Mouse Complement Receptor-1 and Receptor-2. Journal of Immunology 145, 2974- 2983 (1990). 35. Oliver, A.M., Martin, F., Gartland, G.L., Carter, R.H. & Kearney, J.F. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur J Immunol 27, 2366-74 (1997). 36. Nilsson, K.E., Andren, M., Diaz de Stahl, T. & Kleinau, S. Enhanced susceptibility to low-dose collagen-induced arthritis in CR1/2-deficient female mice--possible role of estrogen on CR1 expression. FASEB J 23, 2450-8 (2009). 37. Takahashi, K. et al. Mouse complement receptors type 1 (CR1;CD35) and type 2 (CR2;CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice. J Immunol 159, 1557-69 (1997).

38. Kuhn, K.A., Cozine, C.L., Tomooka, B., Robinson, W.H. & Holers, V.M. Complement receptor CR2/CR1 deficiency protects mice from collagen-induced arthritis and associates with reduced autoantibodies to type II collagen and citrullinated antigens. Mol Immunol 45, 2808-19 (2008). 39. Haas, K.M., Poe, J.C. & Tedder, T.F. CD21/35 Promotes Protective Immunity to Streptococcus pneumoniae through a Complement-Independent but CD19-Dependent Pathway That Regulates PD-1 Expression. Journal of Immunology 183, 3661-3671 (2009). 40. Molina, H. et al. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proceedings of the National Academy of Sciences of the United States of America 93, 3357-3361 (1996). 41. Mongini, P.K., Vilensky, M.A., Highet, P.F. & Inman, J.K. The affinity threshold for human B cell activation via the antigen receptor complex is reduced upon co-ligation of the antigen receptor with CD21 (CR2). J Immunol 159, 3782-91 (1997). 42. Thyphronitis, G. et al. Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo. J Immunol 147, 224-30 (1991). 43. Willcocks, L.C., Smith, K.G. & Clatworthy, M.R. Low-affinity Fcgamma receptors, autoimmunity and infection. Expert Rev Mol Med 11, e24 (2009). 44. Qiu, W.Q., de Bruin, D., Brownstein, B.H., Pearse, R. & Ravetch, J.V. Organization of the human and mouse low-affinity Fc gamma R genes: duplication and recombination. Science 248, 732-5 (1990). 45. Ravetch, J.V. et al. Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors. Science 234, 718-25 (1986). 46. Sears, D.W., Osman, N., Tate, B., McKenzie, I.F. & Hogarth, P.M. Molecular cloning and expression of the mouse high affinity Fc receptor for IgG. J Immunol 144, 371-8 (1990). 47. Reth, M. Antigen receptor tail clue. Nature 338, 383-4 (1989). 48. Titus, J.A., Perez, P., Kaubisch, A., Garrido, M.A. & Segal, D.M. Human K/natural killer cells targeted with hetero-cross-linked antibodies specifically lyse tumor cells in vitro and prevent tumor growth in vivo. J Immunol 139, 3153-8 (1987). 49. Young, J.D., Ko, S.S. & Cohn, Z.A. The increase in intracellular free calcium associated with IgG gamma 2b/gamma 1 Fc receptor-ligand interactions: role in phagocytosis. Proc Natl Acad Sci U S A 81, 5430-4 (1984). 50. Powell, M.S. & Hogarth, P.M. Fc receptors. Adv Exp Med Biol 640, 22-34 (2008). 51. Takai, T., Ono, M., Hikida, M., Ohmori, H. & Ravetch, J.V. Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice. Nature 379, 346-9 (1996). 52. Brauweiler, A.M. & Cambier, J.C. Autonomous SHIP-dependent FcgammaR signaling in pre-B cells leads to inhibition of cell migration and induction of cell death. Immunol Lett 92, 75-81 (2004). 53. Yuasa, T. et al. Deletion of fcgamma receptor IIB renders H-2(b) mice susceptible to collagen-induced arthritis. J Exp Med 189, 187-94 (1999). 54. Kleinau, S., Martinsson, P. & Heyman, B. Induction and suppression of collagen- induced arthritis is dependent on distinct fcgamma receptors. J Exp Med 191, 1611-6 (2000). 55. Kagari, T., Tanaka, D., Doi, H. & Shimozato, T. Essential role of Fc gamma receptors in anti-type II collagen antibody-induced arthritis. J Immunol 170, 4318-24 (2003). 56. Brownlie, R.J. et al. Distinct cell-specific control of autoimmunity and infection by FcgammaRIIb. J Exp Med 205, 883-95 (2008). 57. Pandey, S. & Agrawal, D.K. Immunobiology of Toll-like receptors: emerging trends. Immunol Cell Biol 84, 333-41 (2006).

58. Bolland, S., Yim, Y.S., Tus, K., Wakeland, E.K. & Ravetch, J.V. Genetic modifiers of systemic lupus erythematosus in FcgammaRIIB(-/-) mice. J Exp Med 195, 1167-74 (2002). 59. Goodnow, C.C. Immunology. Discriminating microbe from self suffers a double toll. Science 312, 1606-8 (2006). 60. Wenink, M.H. et al. The inhibitory Fc gamma IIb receptor dampens TLR4-mediated immune responses and is selectively up-regulated on dendritic cells from rheumatoid arthritis patients with quiescent disease. J Immunol 183, 4509-20 (2009).