Identification of conformation-dependent epitopes and V gene selection in the B cell response to type II collagen in the DA rat

Patrik Wernhoff, Christine Unger1,, Estelle Bajtner, Harald Burkhardt1, and Rikard Holmdahl Section for Medical Inflammation Research, I 11 BMC, Lund University, 22184 Lund, Sweden
1 Department of Internal Medicine III and Institute of Clinical Immunology, University of Erlangen-Nürnberg, Kranken hausstrasse 12, 91057 Erlangen, Germany

Correspondence to: P. Wernhoff


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Collagen-induced arthritis (CIA) is a widely used model for rheumatoid arthritis. Induction of CIA in rats using rat type II collagen (CII) results in a chronic arthritis in which anti-CII antibodies are believed to play a pathogenic role. In this study, we analyzed the epitope selection and V gene usage in the anti-CII response in the DA rat. A panel of CII-reactive B cell hybridomas was established from the draining lymph nodes 11 days after immunization. All of the CII-specific antibodies bound cartilage in vivo, showing that these are true autoantibodies. These antibodies were all IgG and specific for several distinct triple helical epitopes on CII. Interestingly, the major epitope, recognized by four different antibodies, was identical with the major B cell epitope in the mouse CII located at position 359–369 (denoted as C1III). The Q52 and PC7183 VH gene families encoded 12 out of 14 sequenced heavy chains. There was a relatively more heterogeneous usage of VL genes as the antibodies were encoded by four different V{kappa} families (V{kappa}1, V{kappa}2, V{kappa}12/13 and V{kappa}RF). As in the mouse, some of the V genes used showed germline characteristics. We conclude that the immune response in the rat shares epitope specificity and a constrained V gene repertoire with the mouse. However, the V genes used for recognition of the closely related collagen structures differed considerably between mouse and rat, indicating an influence of the species-specific variation in the V gene repertoire.

Keywords: autoimmunity, B cells, collagen-induced arthritis, epitope, V gene selection


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic disease in humans affecting peripheral joints, and is characterized by a chronic progressive or relapsing inflammatory destruction of cartilage and subchondral bone. The etiology and pathology of RA is unknown, but several observations indicate an involvement of the immune system. The susceptibility to RA is associated with certain HLA alleles, specifically the HLA-DR4 and -DR1 subtypes, and an infiltrate of a large number of mononuclear cells (T and B cells) can be found in the arthritic joints. The autoantigen is not known for RA, but type II collagen (CII), a major component of the joint cartilage, has been postulated to be a target antigen. T cells specific for CII have been isolated from the synovium membrane of the joint (1). Autoantibodies directed against CII are often detected in sera and synovial fluids (2) as well as in immune complexes found in arthritic joint cartilage (3). The distribution of CII is restricted to cartilage and vitreous humor of the eye, but has a wider distribution during fetal life (4). The humoral anti-CII response includes production of antibodies directed against both native and denatured CII. However, complement-fixing IgG antibodies recognizing native CII seems to dominate in arthritogenic immune responses (5).

Collagen-induced arthritis (CIA) is an experimental autoimmune disease that can be induced in rats, mice and primates by an intradermal immunization with native CII emulsified in mineral oil (68). The CIA model reassembles RA both histologically and clinically, and is characterized by synovial hyperplasia, infiltration of joints by inflammatory cells, marginal erosion and cartilage destruction. CIA is under polygenetic control where the MHC class II region is the dominating contributor (9,10). The importance of T cells for CIA development has been demonstrated (11). Arthritis will not occur in congenitally athymic rats nor in neonatelly thymectomized rats (12) and treatment with antibodies directed against the TCR {alpha}ß framework can prevent CIA development in mice (13). The need of B cells in CIA has also been investigated and found to be of crucial importance. B cell-deficient mice do not develop arthritis (14) and anti-IgM antibody treatment suppresses arthritis (15). Activation of B cells, recognizing native triple helical CII, is needed for the induction of CIA (16). DA rats immunized with denatured rat CII (RCII) only develop an acute arthritis with a weak B cell response against CII, whereas immunization with native RCII induces a strong anti-CII response and a classical chronic CIA. The evident lack of negative selection of CII autoreactive B cells is demonstrated by the strong and specific IgG response against autologous CII upon RCII immunization (17).

The CIA model, which is dependent on B cell interaction, is therefore well suited to study the importance of pathogenic autoantibodies (14). Injected antibodies bind to cartilage in vivo (18), and can induce synovial tissue inflammation and arthritis (19), demonstrating a potential role of autoantibodies recognizing CII in the initial phase of the disease course. The mechanism for this passive transferred arthritis is not known, but an involvement of the complement system is likely (20,21). The requirement of a B cell response raises several questions of how pathogenic B cells are selected and regulated. One way to investigate how B cells are involved is to determine the target structures on CII and the binding properties of the B cell receptors (BCR) to specific epitopes. In mouse strains, several B cell epitopes have been characterized (22) using mAb derived from immunization with heterologous CII. Interestingly, the antibody response tended to be confined to certain dominant epitopes, such as the C1 epitope (23). A further peculiarity of the anti-CII response was the rather restricted usage of germline-encoded V genes in the IgG-switched autoantibodies (24), indicating an epitope-dependent V gene selection (25). The epitope specificity of the anti-CII response in the rat has not previously been identified, although investigations have been undertaken using linear peptides and cyanogen bromide (CB)-cleaved fragments of CII for binding studies of arthritic sera (26).

In the present study we have investigated the B cell response in the initial phase of CIA in DA rats. Thirty hybridomas producing anti-CII mAb were generated. The epitope specificity was analyzed by an approach of recombinant homotrimeric collagen chimeras (23), consisting of selected parts of human CII (HCII) inserted into the human type X collagen (CX). This enabled us to determine epitopes in a triple helical conformation. The binding properties of the anti-CII mAb were analyzed by variable region gene sequencing. The V gene and J segment usage and CDR3 diversity are described.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
DA rats were kept and bred at the animal unit of Medical Inflammation Research. All animals used in the study were 8–12 weeks old at the time of study. They were not infected by commonly occurring pathogens and their environment was standardized as described at http://net.inflam.lu.se/.

Collagen preparations
Native CII from rat, mouse, human, bovine and chick was prepared as earlier described (27). Denatured RCII was prepared from native CII by heat inactivation at 50°C for 30 min. Rat type I collagen (RCI) was purchased (Sigma, St Louis, MO). Rat type IX collagen (RCIX) and rat type XI collagen (RCXI) were prepared as earlier described (28).

Hybridoma production and ELISA
Hybridoma cell clones were produced essentially as previously described (27). DA rats were immunized in the right footpad with 200 µg native RCII emulsified in incomplete Freund's adjuvant (IFA; Difco, Detroit, MI). The draining popliteal lymph nodes at the site of injection were taken day 11 after immunization and fused to the non-secreting mouse myeloma cell line SP2/0 (29). After fusion, cells were seeded in 96-well plates, NUNCLON Surface (Nunc, Roskilde, Denmark) together with thymocyte suspension (5x106 cells/ml) in complete DMEM Glutamax-1 (Life Technologies, Paisley, UK). Selection of hybridoma cells was started 12 h later by adding hypoxanthine, thymidine and aminopterine (Sigma). Cells were incubated in 37°C in 7.5% CO2. The anti-CII mAb production was tested 2–3 weeks later by ELISA in Immulon2 HB (Dynex/Technologies, Chantilly, VA) plates. After coating native RCII (10 µg/ml), plates were pre-blocked to avoid background disturbance with 1% BSA (Sigma) in PBS, pH 7.4. Positive controls, rat anti-CII sera (10 µg/ml) were included in all experiments. The ELISA was developed with the ABTS system (Boehringer, Mannheim, Germany) and the absorbance level determined at 405 nm (ELISA reader; Titertek). Positive clones were recloned twice with limiting dilution. Cells were later expanded in standard tissue flasks. The mAb were purified on Protein G, GammaBind Plus (Pharmacia, Uppsala, Sweden), dialyzed against PBS and sterile filtrated. Purified mAb were kept in –70°C. The titers and concentrations were calculated using limiting dilution (ELISA software: MacELISA 3.0). The isotypes were determined in ELISA with mouse anti-rat reagents specific for IgG1, IgG2a, IgG2b and IgG2c (New England Biolabs, Beverly, MA).

Immunohistochemistry
For immunohistochemical analysis for mAb binding to cartilage, 100 µg purified mAb was injected i.p. into neonatal mice (1-day-old) QD mice. The mice were sacrificed after 48 h, and the hind limbs were removed and immediately frozen in –70°C in OTC compound (Tissue-Tek; Sakura, The Netherlands) and stored in –70°C until usage. Cryosections of the joints (6 µm thick) were cut from the front and fixated on glass slides at 4°C in acetone (Kebo, Lund, Sweden) and air-dried. All staining were made in Tech Mate 500 (Dakopatts, Copenhagen, Denmark). Sections were first incubated with biotinylated Rabbit anti-Rat IgG mAb (10µg/ml) (Dakopatts) then with ExtrAvidin–peroxidase (Dakopatts) and developed with DAB-Safe (Saveen, Lund, Sweden) in accordance with the protocol from the manufacturer. The sections were counter stained with hematoxylin. The binding affinity of the antibodies to cartilage CII was defined from a range of no binding (–), weak binding (+), medium binding (++) and strong binding (+++).

Affinity measurements using Biacore
The affinity was determined using the Biacore 2000 system (Sensor Chip CM5, BIA evaluation software and amine coupling kit; Pharmacia Biosensor, Uppsala, Sweden). HBS buffer (10 mM HEPES with 0.15 M NaCl, 3.4 mM EDTA and 0.005% surfactant P20, pH 7.4) was used as running buffer. Immobilization of RCII was preformed according to previously described principles (Löfås and Johnsson, 1990, personal communication). A continuous flow of HBS buffer was maintained over the sensor surface. The carboxylated dextran matrix on the sensor surface was activated with a 7-min injection of a solution containing 0.2 M N-ethyl-N'-(3-diethylaminopropyl) carbodimide and 0.05 M N-hydroxysuccuinimide. Specific surfaces were obtained by injecting RCII (100 µg/ml) in 10 mM acetate buffer, pH 4.5. The amount of RCII immobilized in each flow cell was controlled by adjusting the injection time for RCII. The injection times used were 3, 7, 9 and 12 min. The immobilization procedure was completed by a 7-min injection of 1 M ethanolamine hydrochloride, pH 8.0, to block remaining ester groups. Antibodies (1 and 10 µg/ml) in HBS buffer were injected at a flow rate of 40 µl/min. The collagen surface was regenerated with 0.2 M NaOH. All of the binding studies were performed at 25°C.

Vector construction, transfection and culturing of HEK 293 cells
The chimeric collagen constructs, transfection and culturing of the HEK 293 (30) cells were done as previously described (23). The chimeric molecules consisted of HCX with inserted parts of HCII generating a full-length triple helical fusion protein. Selected parts of CII were PCR amplified (Perkin-Elmer GeneAmp 2400) from a human CII cDNA template and cloned in frame into CX cDNA (31) (pBluescript SK– vector; New England Biolabs, Beverly, MA), by specific restriction sites (NcoI, PstI and BamHI) introduced to the PCR primers (MWG-Biotech, Ebersberg, Germany). The Taq polymerase was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). The full-length CX sequence had five unique restriction sites: HindIII sites flanking the CX sequence, NcoI and PstI sites internally, and a BamH1 at the 3' junction of the CII/CX sequence. The cloning was done either with NcoI–BamHI or with PstI–BamHI respectively depending on internal restriction sites in the CII sequence. For the expression in eukaryotic cells, the DNA was cut with HindIII and the CX–CII–CX region was subcloned into the expression vector pRCMV (Promega, Madison, WI). Prior to the transfection procedure, the chimeric constructs were controlled by in vitro translation and DNA sequencing. HEK 293 cells were transfected with plasmid DNA constructs using the calcium phosphate precipitation method as described previously (24). The transfected cells were grown in DMEM/F12 and 5% FCS. After 48 h, the selection was started by supplementation with 800 µg/ml G-418 (Gibco/BRL, Paisley, UK). Medium was renewed every 2 days. The collection of supernatants was started when the G-418 resistant cells reached confluence. During harvesting, the transfected HEK 293 cells were kept in FCS-free DMEM/F12 supplemented with 50 µg/ml L-ascorbic acid. The expression of the constructs was controlled in SDS–PAGE using 10% gels. The proper folding of the recombinant collagen chimeras was tested in ELISA using mouse mAb that bind in a strict conformation dependent manner to the respective CII inserts.

Mapping of epitopes
Regenerated chick (CCI) CB fragments (CB8, CB10, CB11 and CB12) (32) were directly coated onto ELISA plates, Immulon2 HB (Dynex/Technologies), and the mAb reactivity tested in duplicates at a concentration of 10 µg/ml. Serum-free supernatants from transfected HEK-293 cells, containing chimeric constructs, were directly coated onto Nunc Immunoplates (Nunc) for ELISA measurements. The mouse anti-CX specific antibody X53 and media from non-transfected HEK 293 cells were used for controlling the coating efficiency and the specificity in the ELISA. The ELISA was performed as described above. Negative controls, recombinant wild- type CX, were included in all assays. The epitopes were mapped in a series of ELISA using a variety of different constructs with overlapping CII sequences. For a rough localization of the epitopes in the beginning, chimeric molecules that harbor large parts of the entire CII sequence were used, while the fine mapping subsequently was performed with smaller constructs (23). mAb not showing binding reactivity to any construct or multiple binding to recombinant chimeric collagens with non-overlapping CII sequences were disclosed from further investigations. Whenever possible, epitopes were narrowed down by the construction of chimeric collagens that contained CII sequences at their minimal length for mAb recognition.

V gene sequencing
Twenty million hybridoma cells were used for mRNA purification in each preparation (QuickPrep Micro mRNA purification kit; Pharmacia Biotech, Uppsala, Sweden). The cDNA was directly PCR amplified (Taq polymerase; Amersham Pharmacia Biotech) from the cDNA reaction according to the of the manufacturer supplied protocol (First-Strand synthesis kit; Amersham Pharmacia Biotech) by using V gene and constant region-specific primers for the VH and VL chains. Primers used for the VH ({gamma}) chain were: framework (FR)-1-specific primer VH1.2/EcoRI: 5'-CGG AAT TCA GGT (GC) (AC) A (AG) CTG CAG (GC) AGT-3' and the constant region-specific primer: C{gamma}1.2/BamH1: 5'- GCG GAT CCG GAC AGG GCT CCA GAG TTC CA-3'. Primers used for the VL ({kappa}) chain were: FR-1-specific primer PWRkV3 {kappa}/EcoR1: 5-GCG AAT TCG ACA (CT) (CT) (AGC) (AT) G (AC) TGA C (ACT) C AGT CTC-3' and the constant region ({kappa})-specific primer PWR594 {kappa}/Hind III: 5'- CGC GAA GCT TTC AGT AAC ACT GT (CT) CAG GAC A-3'. Negative controls (no template) were included for all amplification reactions. Amplified V genes were purified on a 2% agarose gel and cleaved with restriction enzymes (sites introduced in the primers). PCR products were cloned into pBluescript KS+ vector (Stratagene, La Jolla, CA) and electroporated using Gene Pulser II (BioRad, Hercules, CA) into Top-10 cells (Electrocompetant Transformation kit; Invitrogen, Täby, Sweden). Transformed cells were selected by ampicillin (Sigma-Aldrich, Stockholm, Sweden) resistance and positive clones were identified by restriction enzyme cleavage of miniculture preparations (QIAprep spin Miniprep kit 250; Qiagen, Kebo, Lund, Sweden). DNA from the miniculture preparations were used as template DNA in the sequencing reactions (Thermo-Sequence fluorescent labeled primer cycle sequencing kit, RPN 2536; Amersham Pharmacia Biotech). The clones were sequenced automatically (ALF; Amersham Pharmacia Biotech). To avoid and correct misincorporations of nucleotides due to mistakes of the Taq polymerase, three different clones were sequenced for each VH and VL chain. Sequencing primers: fluorescent-labeled Reversed and Universal primer (included in sequencing kit). The sequences were analyzed in the DNA Star program (Madison, WI) and aligned to the GeneBank/EMBL and IMGT databanks.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-CII reactive antibodies
B cells producing high-affinity anti-CII autoantibodies can be found in DA rats after immunization of autologous CII. To isolate and characterize these B cells, we obtained hybridomas from draining lymph nodes day 11 after immunization of autologous CII. Thirty CII-reactive hybridomas were randomly chosen from different fusions and investigated further for different antigens (Table 1Go). In this panel, seven mAb had a wide cross-reactivity as suggested by their ability to bind to RCI or ovalbumin. These multi-reactive mAb also tended to bind to denatured RCII (dRCII) equally well as native RCII. The remaining more specific mAb were in most cases highly specific for CII without cross-reactivities to RCI or to various cartilage collagens such as RCIX and RCXI. Most of these mAb had higher affinity to native than denatured RCII and in most cases; they bound to species conserved epitopes in mouse, bovine, human (data not shown) and chicken CII. However, two of the CII-specific antibodies (122.41 and 125.4) did not cross-react to CCII. Affinity measurements by biosensor-based technique (Biacore) displayed apparent affinity for RCII (ranging from 105 to 107 KA–M). All of the anti-CII reactive antibodies were of IgG isotypes, and the subclasses included IgG1, IgG2a and IgG2b, but not IgG2c. All antibodies used {kappa} as their light chains.


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Table 1. Binding characteristics of anti–CII antibodies and in vivo binding to cartilage
 
The true autoreactivities of these antibodies were tested in vivo by measuring the binding capacity to cartilage (Fig. 1A and BGo). Purified mAb, 100 µg, were injected i.p. into neonatal, 1-day-old, QD mice and the binding to cartilage was detected by immunohistochemical staining for rat mAb. The results showed a non-equivalent binding to cartilage for the different mAb with a strong and clearly detectable binding on cartilage surfaces for 14 mAb (Table 1Go), indicating that the epitopes were naturally exposed on the cartilage. For all the mAb that bound to cartilage in vivo, the biosensor measurements revealed specificity and high or medium affinity for RCII in vitro.




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Fig. 1. In vivo binding of type-CII specific rat mAbs to neonatal mouse cartilage. Immunohistochemical staining of the wrists of hind paws from 3-day old QD mice after i.p. injection of 100 µg rat mAbs. (A) Binding of the ClIII epitope specific mAb 126.35 to the surface of the joint cartilage. Localization of the mAbs is indicated by the dark staining on the surface of the cartilage. (B) A joint from an animal that received the multi-reactive mAb 122.13 (binding to dRC11, RCI). No binding detected.

 
Mapping the B cell epitopes on CII
The collection of hybridoma cells revealed a possibility for a broad selection of different epitopes on RCII. The distribution of the epitopes was determined by screening for binding reactivity against different CB fragments of CCII. All CCII-specific antibodies bound to the CB fragments and the majority bound specifically to the major CB fragments (CB11, CB10 and CB8) indicating a diverse pattern of recognized epitopes along the CII molecule (Table 1Go). The precise locations of the conformational epitopes on the CII were characterized by the use of a total of 130 different chimeric constructs, which harbor selected triple helical regions of HCII. Eleven of the CII-specific mAb bound to specific constructs in accordance with their previously determined reactivity to the distinct CB fragments (Table 1Go). Nine, out of the 11 mAb, could be more closely mapped (Fig. 2Go). The majority of the epitopes were localized to CII regions that corresponded to the CB11 fragment. Shadowed boxes (Fig. 2Go) indicate the five distinct binding sites of the anti-CII mAb on CB11.



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Fig. 2. Schematic overview of the cyanogen bromide (CB) cleaved type II collagen molecule and the locations of the recognized B cell epitopes. The mABs binding to the different CB fragments are shown. The epitopes are marked with shadowed boxes and name and locations are shown above and under the CB fragments (mAbs binding to multiple CB fragments or non-binding mAbs are not shown).

 
The binding site of four CB11 reactive mAb (125.5, 126.35, 145.322 and 146.50) was mapped to the 11 amino acid long C1III epitope (ARGLTGRPGDA), which corresponds to position 359–369 in the human {alpha}1(II) sequence. The `C1' epitope, which has originally been identified in the mouse model, consists of three overlapping, but clearly distinct epitopes, C1I, C1II and C1III, which share a common N-terminus but differ in length (5–11 amino acid residues). The specificity was tested against different chimeric constructs that covered the three epitopes at their minimal length. The binding of all four rat mAb to the C1 region was dependent on the presence of the entire 11 amino acid long sequence in the recombinant constructs, thereby proving their specificity for the C1III epitope.

The binding sites of the other CB11-specific mAb were characterized as distinct epitopes. However, each of these epitopes was only recognized by a single mAb. The mAb 122.9 defined the epitope, T2 (GIAGFKGDQGPKG) at position 256–271 (Table 1Go). In immune responses to heterologous CII in vivo response, the B cell epitope, T2, is identical to a dominant T cell determinant that is well recognized in the rat (33) but of major importance in the mouse model of CIA (34). The mapping strategy for the mAb 122.41 revealed binding to the region `E41' (GPRGLPGERGRTGPAGAAG), at position 124–142. Interestingly, the mAb 122.41 could neither bind to native CCII nor to CB fragments (derived from CCII) as shown in Table 1Go. The inability of mAb 122.41 to bind to CCII indicates the crucial importance of amino acid residues 135 and 138 for mAb recognition since the sequence of CCII differs from RCII and other type II collagens at these two positions in the critical region. The sequence epitope `E47' of the mAb 122.47 could be mapped to the region 181–220 (sequence not shown) that partially overlaps with the binding site for the mAb 122.17, at position 208–220 (GNPGTDGIPGAKG) (`E17').

Of the 12 antibodies binding to the CB8 fragment, three mAb reacted stringently with the chimeric constructs. The mAb 126.30 recognized the epitope `U1' (LVGPRGERGFP), at position 494–504, representing the minimal sequence requirements for mAb binding. The epitopes of mAb 126.26 and 126.40 were mapped to the regions of 400–436 and 400–462 (sequence not shown).

V gene family usage in the anti-CII response
From the collection of 30 hybridoma clones, 14 VH and 17 VL genes were sequenced. The sequences included both V genes from epitope and non-epitope characterized mAb. The majority of the VH segments belonged to the Q52 family, which was represented in 57% of the rearranged genes (see Fig. 3Go). The PC7183 family was used in four VH genes. The J558 and VH13 family was used in one heavy chain respectively. The usage of light chains was more evenly distributed. The V{kappa}RF family was used in 35% of the mAb, followed by V{kappa}12/13 (29%), V{kappa}1 (18%) and V{kappa}2 (18%). The classification of the rat V{kappa} families was based on sequence similarity to mouse V{kappa} families.



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Fig. 3. (A) Phylogenetic tree of the heavy chain nucleotide sequences of the anti-CII reactive hybridomas. The clustal method with `balanced branches' was used to construct the tree. The tree shows the belonging to the different V heavy families, Q52, PC7183, VH 13 and J558. (B) Phylogenetic tree for the light chain nucleotide sequences. The numbering at the bottom of the trees indicates the genetic distances. V gene family identify, epitope specificity and cyanogen bromide (CB) reactivity are shown.

 
V gene selection
The expression of restricted VH and VL genes for epitope-specific mAb was determined by comparing the sequences of the four C1III binding mAb: 125.5, 145.322, 146.50 and 126.35. All used variable segments belonged to the Q52 VH family (Fig. 4Go). Three of the four hybridomas (125.5, 145.322 and 146.50) displayed high nucleotide sequence homology (nearly 100%) and were probably derived from the same Q52 gene. The hybridoma 145.322 had a single replacement mutation in framework region 2 (FR2) (35) generating the amino acid substitution threonine (T) to alanine (A) at residue 41. In comparison to the other mAb, the fourth C1III-specific mAb 126.35 displayed 13 amino acid differences overall indicating an utilization of another Q52 gene. The corresponding VL genes of the C1III mAb (Fig. 5Go) belonged to two different V{kappa} families: V{kappa}12/13 and V{kappa}RF. A comparison of the V{kappa}12/13 chains with known rat sequences revealed a high similarity (97.5% homology) to a rat V{kappa}21/61 sequence (36). Interestingly, the three mAb showing highest homology for the VH genes all used the V{kappa}12/13 family. Minor sequence differences remained confined to a base pair difference in the CDR1 at residue 31 (silent mutation) in the V{kappa}12/13 genes.



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Fig. 4. Nucleotide sequences of the {gamma} chain V regions of the ClIII epitope specific mAbs, 125.5, 145.322, 146.50 and 126.35. The deduced amino acid sequence for mAb 125.5 is illustrated as a prototype. The CDR and FR regions are defined and numbered according to Kabat et al. (35). Dashes indicate nucleotide identify and dots non-corresponding nucleotides. These sequence data are available from EMBL/Genebank under accession number AF217570-AF217583.

 


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Fig. 5. Nucleotide sequences of kappa ({kappa}) chain V regions of the ClIII specific mAbs. The deduced amino acid sequence for mAb 125.5 is shown. The sequence data are available from EMBL/Genebank under accession number AF217584–AF217600.

 
Comparing the VH and VL sequences of the other mAb confirmed a restricted V gene family usage, as highly homologous V genes were used in mAb with different specificity. The VH genes of the CB10 binding 126.26 and 146.42 shared high sequence homology with the two Q52 family-encoded, C1III-specific, VH elements of 126.35 and 125.5 (98.6 and 100% respectively) (Fig. 3Go). Notable, mAb 125.5 and 126.35 used different Q52 genes. The associated light chains of 126.26 and 126.42 (V{kappa}2 family) displayed 100% homology. In addition, the two unmapped mAb, 122.3 and 122.7, shared 99% sequence homology at the nucleotide level in their Q52-related VH sequences. However, these VH genes represent a separate subgroup clearly distinguishable from the other Q52 family members used in the C1III-specific mAb. The VL gene elements used in the T2 binding mAb, 122.9 (unknown VH segment), and mAb 126.30 (U1 epitope) displayed similarity with the V{kappa}12/13 family, but were clearly more distantly related to the VL sequences determined in the C1III-specific mAb. The VH segment of 126.30 differed from the others and was the only heavy chain encoded by the VH J558 family. The VH gene family PC7183 was represented in mAb: 121.4, 122.4, 122.33 and 122.41. In two mAb, the VH PC7183 genes were associated with the V{kappa}1 family, exemplified by the E41 binding 122.41. The mAb 122.33 used a V{kappa}RF family gene. The V{kappa}RF family was used in six light chains (122.13, 122.17, 122.33, 122.77, 125.17 and 126.35).

CDR3 region and J segment use
The heavy chain CDR3 regions (HCDR3) were highly heterogeneous and revealed no common motif. The length varied from 4 to 12 amino acids. None of the D segments could be identified (few rat D segments characterized). There was a biased usage of JH segments, with the majority (11 of 14) using the JH2 segment. The mAb 122.3 and 122.33 used the JH3 segment and 126.35 (C1III reactive) JH4. In the light chains, the CDR3 regions (LCDR3) displayed higher homology (within the V{kappa} families). This is in line with data from mouse anti-CII response (33). The LCDR3 varied in length from 8 to 9 amino acids. The two most used J{kappa} segments, J{kappa}1 and J{kappa}2, were used in 6 and 8 light chains respectively.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell recognition of CII in rats developing chronic arthritis after immunization with homologous rat CII has been investigated. The isolation of B cell clones from primary immune lymph nodes enabled us to show the production of autoantibodies with specificity for a number of distinct epitopes on native CII that are exposed on the cartilage surface in vivo. Since CII is a protein with an evolutionary highly conserved structure it is not too surprising that most of the rat autoantibodies bound to an epitope (`C1') that has already been identified as an immunodominant epitope of the arthritogenic antibody response in mice. Furthermore, as in the mouse, the majority of the antibodies were encoded by selected V genes, most likely in germline configuration and often shared between B cells recognizing different epitopes. However, a comparison of V genes from mAb that recognize the dominant `C1' epitope in mouse and rat reveal considerable interspecies differences, despite pronounced intraspecies similarities. The data suggest that the constrained use of certain germline V genes in the anti-CII antibody response is not only selected by the antigen, but is also influenced by the pre-existing V gene repertoire in the B cell compartment.

Autoreactive B cells specific for cartilage CII are most likely playing a crucial role in the pathogenesis of CIA (15). CIA cannot be initiated without B cells and, in addition, B cells are of importance to maintain the chronic development of arthritis (16). The apparent escape of CII-reactive B cells from negative selection, together with the fact that cartilage is accessible for antibody binding in vivo, is a risk factor in the development of arthritis. To understand how B cells are selected and which role they play in the pathogenesis, we need to identify their specificity and V gene usage. Clearly, the antibody response is mainly directed to conformational epitopes on the triple helical part of the CII molecule (37). This has introduced severe problems in mapping the epitopes and only the expression of CII sequences in a native triple helical conformation allowed us to identify the immunodominant epitopes relevant to the pathogenesis of CIA in DBA/1 mice (23). The present study shows striking similarities between the B cell response in the rat, induced with homologous RCII, and that of the mouse, induced with heterologous RCII. Firstly, the epitopes are widely distributed on the triple helical molecules, although certain epitopes are more frequently recognized. Furthermore, the mAb binds in a type-specific manner and rarely cross-react to other collagens or denatured collagen. This specificity for the collagen type is most likely explained by the deletion of B cells, which have receptors reacting with more abundantly exposed collagens such as type I. The finding that the antibodies did not cross-react to other cartilage collagens, not even with CXI that has one {alpha} chain in common with CII, emphasizes the fine specificity and the strict conformation dependence. Thus, the recognized epitopes are unique structures on the triple helical part of CII. Secondly, the recognition of the major epitope C1III by a significant number of antibodies in both species shows that the specificity of the response is partly shared. The structure of CII, and its tissue location and function, is highly conserved. Due to this, it is likely that the immune systems in both rats and mice, and possibly also in humans, recognize and are selected for the same set of epitopes. Thirdly, the primary response to CII in both mice and rats is highly dominated by B cells that have already switched to IgG, and which also may use germline-encoded V genes. These germline-encoded IgG antibodies bind cartilage in vivo with a significant affinity. Fourthly, in both mice and rats there is a restricted usage of V genes for recognition of CII, irrespective of epitope specificity. This could be due to the triple helical structure of CII allowing the usage of only a limited number of V genes or that the V gene repertoire is pre-selected or constrained. Thus, our results indicate a lack of negative selection and argue for a physiological positive selection of at least a subset of CII-reactive B cells. Interestingly, in the present collection of B cell clones, there is a possible example of BCR editing. The mAb 122.33 and 122.41 completely shared the heavy chain but differed in the usage of the light chains. The VH PC7183 gene sequences were identical, including the JH2 segments and the CDR3 regions, indicating a usage of the same rearrangement of VH–DH–JH genes (unknown DH segment). The light chains belonged to the V{kappa}RF (122.33) and V{kappa}1 (122.41) families. Both hybridomas were derived from the same rat. The apparent shifts of light chain usage and the following of change of antibody specificity indicate a mechanism of receptor editing in anti-CII-reactive B cells. However, the editing process resulted in mAb with approximately the same affinity for the autoantigen. The B cells remained anti-CII specific and are capable of binding to intact cartilage in vivo. The epitope of the mAb 122.41 was mapped to the CB11 fragment (E41, amino acids 124–142), whereas mAb 122.33 recognizes an unknown structure on the CB10 fragment. Accordingly, the observed constrained V gene repertoire used in both mice and rats for CII recognition could have been antigen selected. However, the lack of obvious similarity in the interspecies comparison of V genes used for recognition of the major C1III epitope indicates that the V gene germline repertoire or possibly regulatory events also influence the B cell response to CII.

A striking finding when comparing the sequences of the VH and VL chains is that two VH families are highly represented in the total response. The two VH families, Q52 and PC7183, represent 85.7% of all the VH genes sequenced. This restricted usage of VH genes clearly differs from studies in mice (38), where a random usage of VH genes could be seen in contrast to a heavily biased usage of light chains of V{kappa}19 and V{kappa}21 families. The usage of light chains in the rat is more diverse and no single family dominates. Interestingly, the Q52 and PC7183 V gene families are known to be highly expressed during fetal and neonatal life. The origin of anti-CII antibodies in the rat could be due to positive selection of autoreactive B cells, with rearranged V genes in the fetal life (39) that have escaped negative selection (40). The Q52 and PC7183 V gene families are also known to be expressed in CD5+ B cells, which have been proposed to be associated with autoreactivity (41). Importantly, the selected primers did not amplify all V genes leading to the fact that not all of the VH and VL genes in the antibody panel were sequenced. These non-sequenced V genes mainly belong to antibodies unmapped for their epitopes.

The results concerning the similarities in the specificity of the B cell recognition of CII in the rat when compared to the mouse may have important implications for RA. The human CII-reactive B cells in RA are also specific for the triple helical part of CII, and these conformation-dependent antibodies are highly type specific and do not cross-react with other collagens (42). They are highly dominated by IgG producers and, interestingly, mainly directed towards the parts of the CII triple helix in which the major epitopes in mice and rats are found (43). The pathogenic role of CII-specific B cells, in acute CIA in mice, chronic CIA in rats and RA in humans, needs to be further investigated. It is likely that in all three species CII is exposed to the immune system and that the selection of the autoreactive B cells to this cartilage specific antigen may represent a clue to the pathogenesis of CIA and RA.


    Acknowledgments
 
We wish to thank Lennart Lindström and Carlos Palestro for taking good care of the animals, Margareta Svejme and Eva Bauer for excellent technical assistance, and Alexandra Treschow for critical reading of the manuscript. This work was supported by grants from the Anna Greta Crafoord Foundation for Rheumatological Research, King Gustaf V's 80-year Foundation, the Kock and Österlund Foundations, the Swedish Association Against Rheumatism, the Swedish Medical Research Council, Deutsche Forschungsgemeinschaft (SFB 263, project C3), Bundesministerium für Bildung und Forschung [Kompetenznetz Rheuma] (project C 2.1) and the European Commission (Bio4-98-0479).


    Abbreviations
 
C chicken
CI type I collagen
CII type II collagen
CIX type IX collagen
CX type X collagen
CXI type XI collagen
CB cyanogen bromide
CIA collagen-induced arthritis
FR framework region
H human
R rat
RA rheumatoid arthritis

    Notes
 
Transmitting editor: A. Radbruch

Received 16 October 2000, accepted 4 April 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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