Analysis of the Molecular Basis of Synovial Rheumatoid Factors in Rheumatoid Arthritis

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 84, No. 3, September, pp. 307–317, 1997 Article No. II974399

Analysis of the Molecular Basis of Synovial Rheumatoid Factors in Rheumatoid Arthritis Richard W. Ermel,* Thomas P. Kenny,† Alice Wong,† Pojen P. Chen,‡ Wasyl Malyj,§ and Dick L. Robbins†,Ø *Department of Medicine and Epidemiology, School of Veterinary Medicine, †Division of Rheumatology, Allergy, and Clinical Immunology, School of Medicine, and §Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, California; ‡Department of Medicine, School of Medicine, University of California, San Diego, California; and Ø Department of Veterans Affairs, Northern California Health Care Systems, Pleasant Hill, California 94523

The objective of this study was to better understand the molecular basis of IgM rheumatoid factor in rheumatoid arthritis (RA). We recently generated 10 different monoclonal IgM RF (mRF) molecules isolated from the synovium of a single patient with RA. The heavy (H) and light chain (L) variable region (V) genes of these 10 mRFs were cloned and sequenced. Six mRFs used kappa light chains and 4 mRFs used lambda light chains. Of particular interest, 8 of 10 heavy chains used the JH4 joining region gene, and all five VH4 heavy chains used the DK4 diversity region gene with the JH4. Four of the VH4 clones used the same germline gene, likely representing a novel but closely related germline gene to VH4.18, and may be clonally related because of the extensive homology in their heavy chain sequence. Two VH4 clones shared the same light chain gene, VkIIIb kv325 (99% homology) and the same JK4 joining region gene, while three VH4 clones used two different light chain genes, an uncommon Vk4 and a Vl4 gene, respectively. In this RA patient, there was recurrent utilization of VH4-DK421/10-JH4 genes and a recurring association with gene elements Vk3 and Vl4. Recurring usage of Vk3 (kv325) and Vl4 (lv418) gene elements may result from a light chain editing process whereby immature autoreactive B cells encountering self-antigen attempt, and often succeed, in altering their specificities through secondary Ig light chain gene rearrangement. Moreover, the oligoclonality of these RFs suggest clonal relatedness secondary to an antigen-driven response. q 1997 Academic Press

clonal RF (mRF) (4). Our studies of nonstimulated lines and those from other laboratories of stimulated lines have provided evidence that the RSC RFs utilize a relatively large variable region (V) gene repertoire (5–22). Many of the mRFs characterized illustrate unmutated germline gene expression for autoantibody activity, suggesting that specific germline genes are capable of directly encoding autoantibodies (9, 16, 17). However, patterns of mutation do exist in many of the characterized RFs and are suggestive of an antigen-driven response in the rheumatoid synovium; presumably mutated RFs that have a higher avidity for antigen are selected and expanded preferentially (10–12). In this study we examine the molecular genetic structures of the heavy (H) and light (L) chain V genes of IgM mRFs derived from the synovium of a single patient with RA and correlate them with antigenic specificity. This study documents, in this RA patient, recurrent utilization of VH4-DK4-21/10-JH4 genes in over 50% of the mRF VH chain genes sequenced and a recurring usage of gene elements Vk3 and Vl4 in the VL chain genes. This is the first report of an apparent preferential association of VH4 to DK4-21/10 genes in RF associated with RA. Furthermore, we demonstrate a bias in gene usage toward the VH4.18 heavy chain germline gene (or closely related VH4 germline genes), the Vk3 light chain germline gene kv325, and the Vl4 light chain germline gene lv418. Of particular interest is the apparent preferential association of lv418 with VH4 in the formation of RF from this patient.

INTRODUCTION

MATERIALS AND METHODS

The major autoantibody present in rheumatoid arthritis (RA) is rheumatoid factor (RF), a polyclonal autoantibody directed against the Fc portion of human IgG (1). RF synthesis is a major characteristic of rheumatoid synovial cells (RSC) and intrasynovial immune complex formation is a prominent feature of RA synovitis (2, 3). Recent advances in hybridoma technology have permitted the production and characterization of stimulated and nonstimulated RSC expressing mono-

Generation and Characterization of IgM RF-Secreting Hybridomas from RSC The methods were described previously (7). Briefly, F3B6 human/mouse heterohybridoma cells were fused with cells from rheumatoid synovial tissue, using the plate fusion technique (7, 22). The cells were mixed and seeded in six-well plates and fused with 40% PEG. Growing hybrids were screened for RF production and sub-

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0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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cloned repeatedly by limiting dilution. In this case, the patient was a 48-year-old female with an 8-year history of seropositive erosive RA who underwent metatarsal head resection. HLA typing was not available. Direct-Binding Enzyme Linked Immunoabsorbant Assay (ELISA) for IgM RF Direct binding was performed as previously described (23). Briefly, various antigen IgGs were coated onto microtiter plates and blocked with 1% bovine serum albumin–phosphate buffered saline. The IgM RF supernatants were added and subsequently detected with enzyme-conjugated anti-human IgM, and developed as previously described (23). Amplification and Cloning of VH and VL Complementary DNA (cDNA) Total RNA was prepared from 106 hybridoma cells using a modified protocol from the Genehunter kit (Genehunter, Brookline, MA). The first strand of cDNA was synthesized using Superscript II (Gibco-BRL, Grand Island, NY) following the manufacturers instructions. Oligonucleotide polymerase chain reaction (PCR) primers were designed as described previously (9, 11, 22). One microgram of cDNA and 2.5 ml of each primer (1 mM final concentration) were added to 40 ml of the PCR mixture. The mixture was heated to 947C for 5 min before the addition of Taq DNA polymerase (Perkin Elmer, Foster City, CA). The mixture was amplified in a thermal cycler for 30 cycles, with each cycle consisting of melting at 947C for 1 min, annealing at 50–557C for 1 min, and extending at 727C for 2 min. The PCR products were ligated into TA vector (Invitrogen, San Diego, CA) following the manufacturers instructions. Appropriate clones were isolated and sequenced. Sequencing and Analysis of IgM mRFs VH and VL Genes The Taq Dyedeoxy terminator cycle sequencing kit was used to prepare the DNA for sequencing. This modification of the dideoxynucleotide chain-termination method uses fluorescent dye-labeled dideoxynucleotides (Applied Biosystems, Foster City, CA). The clones were sequenced in both orientations on the Applied Biosystems 373A automated DNA sequencer. The data were analyzed using the 373A software program. The computer programs of the Genetics Computer Group (24) were also used to analyze the sequence data.

VH Gene Usage Table 1 shows the gene elements used by the 10 mRFs derived from the RSC of this one RA patient. In each

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D Gene Usage The DK4-21/10 diversity segments were utilized in four cases with all four mRFs using VH4 (VH4.18) and JH4 gene segments. This is likely because of the 13/14 bp homology with DK4 and the 8/9 or 7/9 bp homology with D21/10. Alternatively, rather than representing a second D gene segment, the 9 additional bases could represent N region additions. In addition, the DK4FL16 diversity segment (26) was used with VH4 (V71-2) and J4 genes in one mRF. Two D21/10 D genes were used, associating with VH1 (1-58) and VH3 genes (VH26), respectively. FL6r-21/10-FL16, DXP4-D1, and DXP1 genes were used one time each. JH Gene Usage The JH4 gene element was heavily represented in the 10 RSC mRFs. In particular, 7 of the mRFs utilized JH4 gene elements. Five of them associated with VH4 genes (V71-2 and VH4.18) and two with VH1 genes (hv1051 and 1-18). Moreover, a JH4 or JH5 (JH4/5) gene element was used with another of the VH1 genes (1-58). The other two JH elements utilized were JH5, each associating with a VH2 (2-5) and VH3 gene (VH26), respectively. Vk Gene Usage

RESULTS

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case the closest matched germline gene is listed with percentage of homology indicated. Of interest, three mRFs (1AB1, 1AD8, and A1D12) used VH1 genes (Fig. 1). 1AC10 mRF used a VH2 gene, which is, to the best of our knowledge, the first report of a VH2 germline gene being utilized in the formation of RF in an RA patient (Fig. 1), although it was found in an RF from the peripheral blood lymphocytes (PBL) of a normal individual (25). 2BF7 mRF used a VH3 gene with the closest match to VH26 with only 93% homology (Fig. 1). Of particular interest, 5 of the 10 RSC mRFs from this patient used VH4 genes and 4 of those were derived from the VH4.18 germline gene or a novel but closely related gene, showing 94% homology in each case. Furthermore, the similarity of the 4 VH sequences of these mRFs (1AF1, 3DG5, E4C3, and 1BD10) indicates that they may have derived from the same VH4 germline gene. Moreover, the finding that these 4 sequences were identical in their CDR3s (except for the one nucleotide substitution not shared by 1AF1) indicates that at least 3 of them are probably clonally related, and thus represent the same rearrangement (Fig. 2).

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In all, 6 of the 10 mRFs utilized kappa genes. Of interest, Vk3 genes were used in 4 of the cases with the VkIIIb subgroup gene kv325 used in 3 of the 4 mRFs (1AB1, 1AF1, 3DG5). The kv325 gene was used

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V71-2 DP-66 VH4.18 DP-79 VH4.18 DP-79 VH4.18 DP-79 VH4.18 DP-79

VH26 DP-47

2-5 DP-76

94

94

94

94

99

93

99

99 96

99

%H

Dk4 FL16r Dk4 D21/10 Dk4 D21/10 Dk4 D21/10 Dk4 D21/10

D21/10

Dxp1

FL16r D21/10 FL16 Dxp4 d1 D21/10

D

13/14 7/9

13/14 7/9

13/14 7/9

13/14 8/9

18/20 6/6

20/27

26/26

12/13 8/9 6/6 24/24 5/5 17/20

H bp

4

4

4

4

4

5

5

4 4/5

4

JH

40/42

40/40

39/42

40/42

41/42

40/43

46/48

41/42 32/33

42/43

H bp

kv4 kv325 kv325 1v418 1v418

V k3 V k3 V l4 V l4

A20

Vg L6

kv325 A27 1042 1v801

Germline

V k4

V k1

V k3

Vl1 V l8

Vk3

VL

99

98

99

99

99

96

100

99 96

99

%H

Jl1

Jl2/3

J k4

J k2

J k4

J k3

J k3

Jl2 Jl2/3

J k1

JL

41/41

41/41

41/41

41/41

39/39

35/36

39/39

37/37 31/35

38/39

H bp

Note. VH, variable heavy chain; VL, variable light chain; %H, percentage homology; D, diversity gene segment; H bp, homology in base pairs; JH, heavy chain joining gene segment; JL, light chain joining gene segment.

1BD10

E4C3

3DG5

1AF1

VH4 E4D6

VH3 2BF7

VH2 1AC1O

1-18 DP14 1-58 DP-2

1051 DP-10

VH1 1AB1

1AD8 A1D12

Germline

VH

Gene Elements Used by 10 Monoclonal IgM Rheumatoid Factors Derived from the Rheumatoid Synovial Cells of One Rheumatoid Arthritis Patient

TABLE 1

SYNOVIAL RHEUMATOID FACTOR GENES

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FIG. 1. Heavy chain variable region nucleotide sequences of six monoclonal rheumatoid factor genes derived from the rheumatoid synovium from one patient with rheumatoid arthritis. The framework regions (FR) and joining regions (J) are designated. The complementarity determining regions (CDR) are underlined.

in one mRF with the VH1 gene hv1051 (1AB1) (Fig. 3) and in two other mRFs with the VH4.18 gene or its closely related counterpart (1AF1 and 3DG5) (Fig.

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4). In all cases of Vk3 gene usage there was very little suggestion of somatic mutational activity based on the percent of homology with the native germline

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FIG. 2. Heavy chain variable region nucleotide sequences of four clonally related monoclonal rheumatoid factor genes (derived from the rheumatoid synovium from one patient with rheumatoid arthritis) and the most homologous human Ig heavy chain variable germline gene (VH4.18). The framework regions (FR) and joining regions (J) are designated. The complementarity determining regions (CDR) are underlined. Homology is indicated by dashes. N additions are designated by the letter N.

gene. For example, the Vk1 gene found in mRF 2BF7 was 96% homologous to the Vk1 germline gene A20 (Fig. 3) while the Vk1 gene associated with the VH3 gene was only 93% homologous to VH26 (Fig. 3). In the case of 2BF7, it may be that either or both the Vk1 and VH3 genes are somatically mutated or represent closely related genes to the closest germline match as indicated. A Vk4 gene was found in mRF E4D6 which

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was 99.3% homologous with the germline gene kv4 (Fig. 3). This light chain was associated with the VH4 germline gene V71-2. Vl Gene Usage Four of the 10 RSC mRFs utilized Vl light chain genes. Of particular interest, Vl4 genes were used in

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FIG. 3. Kappa and lambda light chain variable region nucleotide sequences of six monoclonal rheumatoid factor genes derived from the rheumatoid synovium from one patient with rheumatoid arthritis. The framework regions (FR) and joining regions (J) are designated. The complementarity determining regions (CDR) are underlined. 312

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SYNOVIAL RHEUMATOID FACTOR GENES

2 of the 4 mRFs utilizing Vl genes; mRF E4C3 and 1BD10 both utilized lv418 in association with VH4.18 or its closely related germline counterpart (Fig. 4). In addition, the Vl8 germline gene lv801 was found in association with the VH1 germline gene 1–58 in mRF A1D12, while the Vl1 germline gene lv1042 was found in association with the VH1 germline 1-18 in mRF 1AD8 (Fig. 3). JL Gene Usage Two RSC mRFs utilized Jk4 genes, two utilized Jk3 genes, one utilized the Jk2 gene, and one utilized a Jk1 gene element. The Jl2/3 sequence was used twice, the Jl2 once and the Jl1 once. Antigenic mRF Specificities Figure 5 shows the antigenic specifities for four of the VH4 clones (mRFs 1AF1, DG5, E4C3, and 1BD10). Of particular interest is the absence of IgG3 binding and the presence of rabbit reactivity in 1AF1 and E4C3. Conversely, mRF DG5 exhibited a classical Ga specificity (i.e., binding to human IgG1, -2, and, -4 without binding to rabbit IgG), while mRF 1BD10 was reactive with all four IgG subclasses (pan specific). DISCUSSION

Immunogenetic studies of the RF response in RA have focused on whether there is a bias in the RF repertoire toward the usage of particular V genes, i.e., whether antigen-driven clonal selection and somatic mutation and/or stochastic forces lead to the generation of immunoglobulin genes that encode high affinity, RA associated autoantibodies. B cell lines producing mRFs have been derived from both RA PBL and from RSC and their RF genes have been characterized (5 – 22). These studies have identified a variety of VH and VL genes that appear to be associated with RF activity in RA patients. Furthermore, our previous studies of nonstimulated RSC mRF lines and those from other laboratories of stimulated mRF lines have provided abundant evidence that the RSC RFs utilize a relatively large V gene repertoire (11). Recent germline sequence comparisons and functional affinity studies indicate that in RA many B cells produce high-affinity autoantibodies that reflect an antigen-driven response (11, 27 – 30). This antigen-driven clonal selection of mutated RFs presumably leads to the development of autoantibodies with higher avidity for antigen. However, many of the RSC mRFs characterized illustrate unmutated germline gene expression for autoantibody activity, suggesting that specific germline genes are capable of directly encoding autoantibodies (9, 16, 17, 31).

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Many RFs from RA patients are monospecific and display a mutation pattern suggestive of an antigendriven response. For example, when eight IgG RFsecreting hybridomas (i.e., C1 – C5, D1 and L1 – L2) from the synovial fluid of three RA patients (i.e., JC, AD, and ML) were analyzed, 7/8 IgG RFs had substantial numbers of somatic mutations, which occurred mainly in the CDRs and often led to amino acid substitutions (6, 8, 15). Moreover, the two IgG RFs from patient ML (i.e., L1 and L2) shared identical VH and VL region sequences, while the five IgG RFs from patient JC consisted of a group of three identical clones and another group of two identical clones (32). Together, these data argue that IgG RFs in rheumatoid synovia are induced and driven by the Fc fragment of human IgG molecules and that they are oligoclonal in individual RA patients. To further understand the mechanisms involved in generating autoantibodies, we amplified the genes encoding the VH and VL chain regions from mRFs derived from RSC of this patient that are directed against the Fc portion of IgG. Surprisingly, in this RA patient, there was recurrent utilization of VH4DK4-JH4 in 50% of the mRFs sequenced and a recurring association of VH4 with gene elements V k3 (kv325) and V l4 (lv418). The overrepresentation of VH4 genes in this patient indicated a bias toward the utilization of VH4.18 or a closely related germline gene. Specifically, four of our RSC mRFs were 94% identical to the same heavy chain germline, VH4.18. Two mRFs, 1AF1 and 3DG5, used a kappa light chain (kv325), and the other two mRFs, E4C3 and 1BD10, used a lambda light chain (lv418). Interestingly, 1AF1 and 3DG5 utilized an identical V germline, being 99 and 99.7% homologous to humkv325. However, there are three differences between the two mRFs on both the VH and VL chains. In addition, 1AF1 binds with rabbit IgG and 3DG5 does not. The other two mRFs (1BD10 and E4C3) that use the VH4 germline, utilized a Vl gene, humlv418, and were 99.7 and 98% homologous, respectively. These two genes differ in five amino acid positions in the heavy chain and three amino acid positions in the light chain. Although it has been reported that IgM/ B cells can express mutations, VH genes of IgM/ B cells are usually 99 to 100% homologous to the germline gene, whereas most of the IgG/ B cells express VH genes with homology less than 95% to germline sequences because of somatic mutation (32). Thus, because these four genes utilize the same VH gene and the closest germline gene match was to VH4.18 with only 94% homology, these mRF most likely were derived from a novel autoreactive closely related VH4 germline gene. Moreover, 1AF1, 1BD10, E4C3, and 3DG5 are likely to be related clones because of the extensive

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FIG. 4. Kappa and lambda light chain variable region nucleotide sequences of four monoclonal rheumatoid factor genes (derived from the rheumatoid synovium from one patient with rheumatoid arthritis) and the most homologous human Ig kappa and lambda light chain variable germline genes (kv325 and lv418). The framework regions (FR) and joining regions (J) are designated. The complementarity determining regions (CDR) are underlined. Homology is indicated by dashes.

homology in their H chain CDR3, which is composed of a 7-bp N region, a 14-bp Dk4 segment, another 7bp N region, and a 9-bp 21/10 D gene segment. Since

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the VH locus is rearranged first in a B cell, the data argue strongly that these four clones are related to each other. Their occurrence also suggests that these

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FIG. 5. Antigenic specificities for four clonally related synovial monoclonal rheumatoid factor hybridomas measured by a direct binding ELISA. Each clone was tested against human IgG (HUM), human IgG3 (IgG3), monkey IgG (Mon), and rabbit IgG (RAB).

four RFs probably have been driven and selected by antigens. Because these four clones pair with different light chains (1AF1 and 3DG5 use kv325; 1BD10 and E4C3 use lv418), these data suggest that their RF activity resides mainly in the VH chain, contrib-

315

uted by both the VH4 and the composite CDR3 amino acid residues. In addition, we believe that the four RSC mRFs (1AF1, 3DG5, 1BD10, and E4C3) are the result of receptor editing via rearrangements in the VL genes (33 – 41). Because the VH gene may encode for most of the autoreactivity, light chain editing may be occurring at the level of VH/VL pairing in an attempt to decrease or eliminate the autoreactivity of the antibody. For example, an autoreactive Vk/VH4 gene pairing may be modified by rearrangement and expression of a less autoreactive Vk/VH4 gene pairing. This process involves secondary VL chain rearrangements that continue to substitute new VL genes for autoreactive VL genes and may ultimately lead to the expression of Vl genes. The VL genes utilized are a result of editing to downgrade the degree of autoreactivity expressed by the autoantibodies or RFs (Fig. 6). The process of light chain editing, however, may not completely eliminate the autoreactivity of the expressed antibodies because of the dependence upon the availability of nonreactive VL genes to replace the reactive VL genes and the potential autoreactive influence of the associated VH genes.

FIG. 6. Schematic representation of light chain receptor editing. B cell maturation occurs in the bone marrow where the heavy chain receptor undergoes rearrangement. If successful, rearrangement of the kappa light chain receptor occurs. Cell death can occur if the immunoglobulin rearrangements result in the production of an autoreactive B cell. To save the cell from death and to produce a nonautoreactive B cell, the light chain receptor may undergo editing, where further rearrangement may occur on the same locus or on the other kappa locus. Anytime during these rearrangements, cell death may occur. Furthermore, rearrangements of both lambda loci may occur to produce a nonautoreactive B cell, resulting in cell death or proliferation of the cell in the presence of antigen.

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Thus, since the k locus is generally rearranged before the l locus, 1BD10 and E4C3 may be derived from 1AF1 and 3DG5 through receptor editing. Another phenomenon which maybe related to the development of these four closely related mRFs is that of epitope spreading. For example, the data suggest that, in this RA synovium, stimulation and selection of the initial RF by IgG and/or IgG antibody – antigen complexes lead to the appearance of a second RF which reacts with the original human IgG specific epitope plus additional epitopes which are shared between human IgG and rabbit IgG. This phenomenon has been reported in other autoantibody systems (42, 43) and is termed epitope spreading. Recurrent usage of the Vk3 and Vl4 genes is seen in our mRFs isolated from the RSC of this patient. Four of the five mRFs that use kappa light chain genes utilize Vk3, while three of the five mRF that use lambda light chain genes use Vl4. The Vk3 genes are nearly identical to the germline genes (kv325 and Vg), 98 – 100% homologous. This is in contrast to the report by Bridges et al. (44), which indicated the presence of somatic mutations in the kappa light chain genes isolated from patients with RA. However, in their study k chains of unselected B cells, not those from a particular isotype or antigen specificity were reported, which may explain the observed differences. In addition, their Vk genes had predominantly longer CDR3 regions and more frequent N additions. The VJ rearrangements from our RA patient tended to have shorter CDR3 segments. This could be related to the increased probability of introducing out of frame rearrangements and/or stop codons with more complex rearrangements or degrees of N-segment addition. Thus, recurrent usage of light chain genes can exist in mRFs isolated from patients with RA, but the mechanisms involved in the expression of these RA autoantibodies are unknown. In summary, the recurrent utilization of four VH4 genes with identical D gene and J gene rearrangements and the recurring association with VL genes Vk3 (kv325) and Vl4 (lv418) in this patient suggest the existence of genetic mechanisms that can bias the functional rearrangement, recombination, and selection of gene elements in the formation of autoreactive RFs in RA. Further elucidation of the specific mechanisms involved should lead to better understanding of disease pathogenesis. ACKNOWLEDGMENTS The authors thank Nikki Phipps for her excellent secretarial assistance and our orthopedic colleagues for providing us with synovial tissue. Grant support was provided by NIH Grant AR-39831, AMAERF, and a UCD-HEW award.

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REFERENCES 1. Carson, D., Rheumatoid factor. In ‘‘Textbook of Rheumatology,’’ Edited by (W. M. Kelley, E. D. Harris, Jr., S. Ruddy, C. B. Sledge, Eds.), 3rd ed., pp. 198–207, Saunders, Philadelphia, 1989. 2. Zvalifler, N., The immunopathology of joint inflammation in rheumatoid arthritis. Adv. Immunol. 16, 265–336, 1973. 3. Fehr, K., Zelvart, M., Rauber, M., Knopfel, M., Baici, A., Salgam, P., and Boni, A., Production of agglutinators and rheumatoid factors in plasma cells of rheumatoid and non-rheumatoid synovial tissues. Arthritis Rheum. 24, 510–519, 1981. 4. Robbins, D. L., Kenny, T. P., Larrick, J. W., and Wistar, R., Production of human monoclonal rheumatoid factor secreting hybridomas derived from rheumatoid synovial cells. Med. Sci. Res. 17, 157–159, 1989. 5. Randen, I., Pascual, V., Victor, K., Thompson, K. M., Forre, O., Capra, J. D., and Natvig, J. B., Synovial IgG rheumatoid factors show evidence of an antigen-driven immune response and a shift in the V gene repertoire compared to IgM rheumatoid factors. Eur. J. Immunol. 23, 1220–1225, 1993. 6. Lu, E. W., Deftos, M., Olee, T., Huang, D.-F., Soto-Gil, R. W., Carson, D. A., and Chen, P. P., Generation and molecular analyses of two rheumatoid synovial fluid-derived IgG rheumatoid factors. Arthritis Rheum. 36, 927–937, 1993. 7. Fang, W., Kannapell, C. C., Gaskin, F., Solomon, A., Koopman, W. J., and Fu, S. M., Human rheumatoid factors with restrictive specificity for rabbit immunoglobulin G: Auto- and multi-reactivity, diverse VH gene segment usage and preferential usage of VlambdaIIIb. J. Exp. Med. 179, 1445–1456, 1994. 8. Deftos, M., Olee, T., Carson, D. A., and Chen, P. P., Defining the genetic origins of three rheumatoid synovium-derived IgG rheumatoid factors. J. Clin. Invest. 93, 2545–2553, 1994. 9. Ermel, R. W., Kenny, T. P., Wong, A., Solomon, A., Chen, P. P., and Robbins, D. L., Preferential utilization of a novel Vl3 gene in monoclonal rheumatoid factors derived from the synovial cells of rheumatoid arthritis patients. Arthritis Rheum. 37, 860–868, 1994. 10. Youngblood, K., Fruchter, L., Ding, G., Lopez, J., Bonagura, V., and Davidson, A., Rheumatoid factors from the peripheral blood of two patients with rheumatoid arthritis are genetically heterogeneous and somatically mutated. J. Clin. Invest. 93, 852–861, 1994. 11. Ermel, R. W., Kenny, T. P., Chen, P. P., and Robbins, D. L., Molecular analysis of rheumatoid factors derived from rheumatoid synovium suggests an antigen-driven response in inflamed joints. Arthritis Rheum. 36, 380–388, 1993. 12. Mantovani, L., Wilder, R. L., and Casali, P., Human rheumatoid B-1a (CD5/ B) cells make somatically hypermutated high affinity IgM rheumatoid factors. J. Immunol. 151, 473–488, 1993. 13. Pascual, V., Victor, K., Randen, I., Thompson, K., Steinitz, M., Forre, O., Fu, S. M., Natvig, J. B., and Capra, J. D., Nucleotide sequence analysis of rheumatoid factors and polyreactive antibodies derived from patients with rheumatoid arthritis reveals diverse use of VH and VL gene segments and extensive variability in CDR-3. Scand. J. Immunol. 36, 349–362, 1992. 14. Pascual, V., and Capra, J. D., VH4-21, a human VH gene segment overrepresented in the autoimmune repertoire. Arthritis Rheum. 35, 11–18, 1992. 15. Olee, T., Lu, E., Huang, D., Soto-Gil, R., Deftos, M., Kozin, F., Carson, D., and Chen, P., Genetic analysis of self-associating immunoglobulin G rheumatoid factors from two rheumatoid synovia implicates an antigen-driven response. J. Exp. Med. 175, 831–842, 1992.

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Received January 29, 1997; accepted with revision May 17, 1997

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