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

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of V_H and V_L gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain 'restrictions' do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of V_H3 genes beyond that one would expect based on random utilization.     

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies. In addition, in contrast to the preponderant kappa L chain usage by the RF in these patients, a lambda L chain was utilized by all RF mAb from our rheumatoid arthritis patient. Two RF mAbs utilized V lambda I, two V lambda IV, and one V lambda III L chains. The VH genes of the two low-affinity polyreactive IgM RF mAb were in germline configuration. When compared with the deduced amino acid sequence of the putatively corresponding genomic segment, the VH gene of the high-affinity monoreactive IgM RF mAb displayed five amino acid differences, all of which are in the complementarity determining regions (CDR), possibly the result of a process of somatic point mutation and clonal selection driven by Ag. The unavailability of the corresponding genomic VH segment sequences made it impossible to infer whether the VH genes utilized by the two IgA1 RF were in a germline or somatically mutated configuration. Sequencing of the genes encoding the H chain CDR3 (D segments) revealed that all three low-affinity polyreactive RF mAb displayed a much longer D segment (36-45 bases) than their high-affinity monoreactive counterparts (15-24 bases), raising the possibility that a long D segment may be one of the factors involved in antibody polyreactivity.     

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.     

Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.     

Sequence analysis of immunoglobulin heavy-chain variable region genes from the synovium of a rheumatoid arthritis patient shows little evidence of mutation but diverse CDR3.

To gain insight into the nature of B-lymphocyte responses in the synovium of rheumatoid arthritis (RA) patients, we amplified and sequenced immunoglobulin heavy-chain variable region genes expressed in seven IgM and three IgG-secreting synovial-derived hybridomas established from one patient. Each hybridoma V-region was encoded by unique VH-D-JH combination demonstrating that none of these hybridomas derived from clonally related B-lymphocytes in vivo. The expressed VH genes closely resembled (95.6%-100% homology) known germline VH genes in most hybridomas, including VH genes frequently used to encode autoantibodies. The antibodies produced by these hybridomas, with the exception of one IgM rheumatoid factor, did not bind to any of a large panel of autoantigens in enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluoresence, suggesting that frequent expression of 'autoantibody-associated' VH genes does not correlate with detectable autoreactivity in this patient. Hybridoma CDR3 DNA was diverse in length and gene composition. Conserved heavy-chain cross-reactive idiotypes were expressed on 4/7 IgM- and 2/3 IgG-secreting hybridomas. The close similarity of expressed VH genes to germline counterparts of these hybridomas suggests that polyclonal activation is a prominent mechanism in B-lymphocyte activation in the synovium of this rheumatoid arthritis patient.     

Molecular analysis of carbohydrate antigen-induced monoclonal IgM anti-IgG antibodies (rheumatoid factors).

Light and heavy chain variable regions of 11 monoclonal rheumatoid factors (MRF) produced after carbohydrate antigen immunization, and one MRF produced after protein immunization have been sequenced. Most carbohydrate antigen induced MRF utilized light chains that were homologous to light chains of MRF obtained from protein immune or LPS stimulated mice, and MRF derived from the autoimmune MRL/lpr mouse strain. VH gene usage was diverse for carbohydrate antigen induced MRF that bound all four isotypes of IgG, or that bound only the IgG3 isotype. In contrast VH gene use among our panel of MRF that bound the IgG1 isotype appeared restricted. Four of the five IgG1 binders used VH genes that were highly homologous to the VH nucleotide sequence of a gene encoding an NP binding monoclonal antibody. Our study confirms the use of a particular group of light chain genes among murine MRF, confirms that there is diversity in the heavy chain genes utilized among MRF, and suggests that a gene(s) homologous to the VH NP 23 J558 gene may be preferentially associated with murine MRF specificity for the IgG1 isotype.     

Organ reactive autoantibodies from non-immunized adult BALB/c mice are polyreactive and express non-biased VH gene usage

To examine the naturally activated autoreactive B cell repertoire, we analyzed a panel of hybridomas from unmanipulated adult BALB/c spleen cells for reactivity patterns and VH gene usage. We found a pattern of VH usage that was diverse and appeared to reflect the germline repertoire. Although all but one natural antibody hybridoma (NAb) were initially selected for organ rather than antigen binding, the majority of organ reactive IgM NAbs were polyreactive, expressing a broad range of reactivity patterns for both self and foreign antigens, that were unique for each NAb and were not indiscriminate. Our results are consistent with the hypothesis that many naturally activated adult B cells are highly polyreactive and that autoreactivity is a consequence of polyreactivity. We suggest that the population of NAbs exhibiting organ reactivity overlaps the populations of other IgM autoantibodies that have been described previously, and that these all derive from a pool of polyreactive IgM antibodies which are polyclonally activated in the early immune response. These polyreactive natural antibodies may represent a first line of defense and offer protection for the host against a variety of foreign agents.     

Molecular structure of eight human autoreactive monoclonal antibodies

The heavy (H) and light (L) chain V-region sequences of eight human autoreactive immunoglobulin M (IgM) monoclonal antibodies (mAbs: BY-4, BY-7, BY-12, IRM-3, IRM-7, IRM-8, IRM-10 and CDC-1) were determined at the cDNA level. All VH and VL families were identified. Four different VH families were represented, VH3 being the most common as five of the mAbs (BY-7, BY-12, IRM-3, IRM-8 and CDC-1) used genetic elements of this family, whereas VH1, VH2 and VH4 were only present in IRM-7, BY-4 and IRM-10, respectively. BY-4, BY-7, BY-12, IRM-7 and IRM-10 reacted with a variety of self as well as non-self antigens, thus exhibiting polyreactive behaviour. Comparison of the gene segments utilized by these mAbs with their germline counterparts revealed that the gene segments were close to germline configuration. The length of H-CDR3 was found to be relatively long (27-60 nucleotides) among the polyreactive mAbs and the presence of Tyr and Trp residues in this region seems to be of vital importance for polyreactivity. We have analysed the utilization of gene elements and the presence of amino acid residues in regions particularly important for antigen binding, such as CDR. Common molecular features relating to the function of the mAbs are discussed.     

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.     

Human monoclonal rheumatoid factors: incidence of cross-reactions with tissue components and correlation with VH gene usage.

Human monoclonal antibodies with rheumatoid factor (RF) activity, derived from lymphocytes from the synovial tissue of rheumatoid arthritis (RA) patients and the peripheral blood of healthy individuals were examined for cross-reactivity with tissue and cellular antigens. The majority of IgM RF from RA patients (68%) showed reactivity with at least one component, and were frequently multispecific. A very significantly smaller proportion (28%) of the RF derived from healthy individuals demonstrated reactivities against tissue/cellular antigens (P = 0.004). RF from RA patients most commonly reacted with gastric glands (61%), nuclei (50%) and smooth muscle (50%), whereas RF from healthy donors most commonly reacted with gastric glands (20%), smooth muscle (16%), endothelium (16%) and glomeruli (16%). The most striking difference between the two groups was the reactivity with nuclear components, demonstrated by 50% of the RA RF, but by none of the healthy donor RF. As the two groups of antibodies share the same specificity for IgG Fc, but show differences in variable region segment usage, we investigated the relationship between VH gene usage and tissue/cell cross-reactivity using these antibodies and anti-blood group antibodies. Antibodies using VH3 or VH4 gene segments showed a very significantly greater frequency of tissue/cell reactions than those using VH1 (P = 0.0095 and 0.0004 respectively).     

Variable region genes of anti-histone autoantibodies from a MRL/Mp-lpr/lpr mouse

The variable region nucleotide sequences of seven (five IgM and two IgG) anti-histone monoclonal antibodies from a single MRL/Mp-lpr/lpr mouse have been determined. These antibodies are not clonally related and used diverse V, D and J genes. However, six of the seven antibodies have VH segments encoded by genes from the J558 family, two of these (an IgM and an IgG) share an identical VH gene. The isoelectric points of MRA3 and MRA12, the two IgG antibodies of the panel, range from 6.3 to 7.0 and from 6.0 to 6.3, respectively. The second conplementarity-determining region (CDR) of the VH gene of MRA12 (the most acidic and the most strongly histone-reactive antibody) includes only two positively charged but five negatively charged amino acid residues. This feature is unusual since the equivalent CDR in most VHJ558 genes are not comprised predominantly of acidic residues and suggests that such negatively charged residues are important for antibody binding to histones.     

Repertoire of CD5+ and CD5- cord blood B cells: specificity and expression of VH I and VH III associated idiotopes.

Epstein-Barr (EBV)-immortalized B cell clones were established from CD5+ and CD5- cord blood B cells separated by flow cytometry. We have previously shown that IgM from many of the clones was polyreactive, exhibiting reactivity with a number of autoantigens. In this study, IgM produced by the clones was analysed by MoAb for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor paraproteins and from defined VH and V kappa subgroups of immunoglobulin heavy and light chains. IgM produced by clones established from CD5+ and CD5- B cells expressed the VH I associated idiotope G8. Furthermore, IgM produced by both sets of clones exhibited a similar frequency of VH III heavy chain subgroup expression, as determined by reactivity with staphylococcal protein A (SpA) and VH III-associated CRI expression (B6 and/or D12). In contrast, expression of the V kappa III-associated 17.109 CRI was significantly higher in IgM antibodies produced by clones established from CD5+ compared with the CD5- clones (32 versus 5%: P less than 0.05). Analysis of the VH and VL subgroup expression by IgM produced by the CD5+ and CD5- cord blood clones, and their autoantigen reactivity profile did not reveal restriction or selection within CD5+ and CD5- populations. However, our data suggest that differences may exist in the expression of certain germ-line genes between CD5+ and CD5- cord blood B cells and might indicate an expansion of CD5+ B cells within the fetal environment.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Properties of polyreactive natural antibodies to self and foreign antigens

Conclusions Natural polyreactive antibodies with the capacity to bind to both self and foreign antigens are present in the sear of healthy individuals. The B lymphocytes that secrete these antibodies belong to the subset of CD5+ B cells and may utilize a restricted set of VH and VL genes, strongly conserved during evolution. The role of these antibodies in the immune system is still a matter of debate. Further studies on their behavior in normal and autoimmune responses are required to elucidate this important issue.     

Autoantibody production by severe combined immunodeficient mice reconstituted with synovial cells from rheumatoid arthritis patients

In an attempt to characterize the heterogeneity of the human autoantibody response, mice with severe combined immunodeficiency were reconstituted with synovial or blood lymphocytes from patients with rheumatoid arthritis (RA). Mononuclear cells extracted from synovial fluid or tissue (SMC) were a greatly enriched source of IgM rheumatoid factor (RF)-producing cells compared to the peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis patients or normal donors. Six to nine weeks after reconstitution of mice with synovial mononuclear cells, 0%-39.3% (mean = 11.4%) of total IgM consisted of IgM RF compared to 0%-0.15% (mean = 0.02%) in mice given RA PBMC and 0%-1.2% (mean = 0.34%) in mice given normal PBMC. Detectable levels of IgM RF were maintained in some mice for as long as 20 weeks after transfer. Mice reconstituted with synovial membrane or synovial fluid lymphocytes produced a heterogeneous mixture of immunoglobulins. These included other autoantibodies, such as anti-nuclear and anti-cytoplasmic antibodies, and antibodies to exogenous antigens such as the Epstein-Barr virus nuclear antigen-1 (EBNA-1). This heterogeneity is further illustrated by the demonstration that the sera from mice given synovial cells also contained IgG antibodies possessing all three major VH families (VH1, VH3 and VH4) and the four major V kappa families (V kappa 1 to V kappa 4). Autoantibody production gradually decreased with time even under circumstances where total immunoglobulin levels increased, and elevated production could not be induced by antigenic stimulation. These findings describe a new model for the analysis of human autoantibody production.     

Immunoglobulin VH genes of the goldfish, Carassius auratus: a re-examination

Five VH-related genomic sequences from the goldfish, Carassius auratus, have been characterized. One of these sequences appeared to be a functional gene, and four to be pseudogenes. The main conclusions drawn from this study were that: (1) With minor exceptions, goldfish VH genes conform to the typical pattern of vertebrate VH genes in terms of their structure (they encode an intron-split hydrophobic leader, 3 framework and 2 complementarity-determining regions, and possess a typical 3' recombination signal sequence for VH to D joining) and regulatory sequences (possession of a typical upstream octameric promoter). (2) The sequences indicate that goldfish possess multiple families of VH sequences (at least three). Two of these families contain approximately 6 and 10 members, as judged from Southern blot hybridization experiments. (3) Goldfish VH gene families are distributed throughout the members of the species in the manner typical of that of VH families in other vertebrate species. Thus, this observation corrects the previous conclusion (Wilson et al., Proc. natn. Acad. Sci. U.S.A. 85, 1566-1570, 1988) that VH genes are discontinuously distributed in the goldfish population.     

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.     

The B lymphocyte in rheumatoid arthritis: analysis of rearranged Vx genes from B cells infiltrating the synovial membrane

The participation of the humoral immune system in rheumatoid arthritis (RA) is characterized by the production of rheumatoid factors (RF). RF are autoantibodies against the Fc part of IgG which are encoded by diverse germ-line genes. Most of the RF-encoding genes are unmutated, but in RA, a substantial quantity is encoded by somatically mutated genes. In addition, the synovial membranes (SM) of the diseased joints of RA patients are infiltrated by B lymphocytes which form germinal center-like aggregates. To analyze the local immune response, B cell foci from two RA SM were isolated by micromanipulation. From DNA of these foci, the rearranged kappa light chain variable region (Vx) genes were amplified by polymerase chain reaction (PCR), cloned and sequenced. The amplification of different Vx-Jx combinations of different foci suggested oligoclonal expansion of B lymphocytes, which was confirmed by sequence analysis: each PCR product contained members of a single B cell clone. The sequence analysis of 29 different clones revealed rearrangements of diverse Vx genes. Both frequent representatives of the Vx3 and the Vx1 family, as well as rarely used genes such as the L10 and B2 genes of the Vx2 and Vx5 families were found. Of the eleven potentially functional gene rearrangements, eight were significantly mutated, indicating their derivation from antigen-selected B cells. Intraclonal diversity in one of these clones may suggest ongoing mutation in the diseased synovial membrane of patients with RA.