Genetic analysis of the variable region genes encoding a monospecific human natural anti-DNA antibody.

Recent evidence suggests that natural autoantibodies may play an integral role in the development of the normal immune repertoire. To explore the genetic origins of these antibodies, we have isolated and sequenced the variable (V) region genes encoding both the heavy (H) and light (L) chains of a natural anti-DNA antibody, Kim11.4. The genes appear to be derived from the VH4.18 (subgroup VHIV), JH5, Hum1L1 (subgroup V lambda I) and J lambda 3 germline genes. The origin of the H chain diversity gene is more obscure, being potentially derived from one or more of several germline genes, arranged in either the forward or reverse orientations. Both the Kim11.4 VH and VL genes share significant degrees of similarity with those utilized in other autoantibodies, indicating that at least some degree of V restriction may exist in human autoreactive B cells. The pattern of nucleotide differences between the Kim11.4 VH and VL genes and their putative germline counterparts suggests that the Kim11.4 genes may have undergone somatic mutation and arisen as a result of antigen selection.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

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.     

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.     

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.     

Diversity and somatic hypermutation of the Ig VHDJH,V kappa J kappa,and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone

Burkitt's lymphoma (BL) is a malignancy of B cells characterized by chromosomal translocations involving the immunoglobulin (Ig) and c-MYC gene loci. To address the putative role of antigen in the clonal expansion of these neoplastic B cells, we analyzed the VHDJH and VLJL gene segments expressed by the established cell lines derived from six endemic BL and six sporadic BL. Eight BL cell lines used genes of the VH3 family, two of the VH4, and two of the VH1. Eight VL chains were kappa (four members of the V kappa3, two of the V kappa1, and two of the V kappa2 subgroups) and four lambda (three members of the V lambda1 and one of the Vl ambda3 subgroup). The VH gene utilization was stochastic (i.e., it reflected the relative representation of the different VH gene family members in the human haploid genome). In contrast, the VL gene utilization was skewed toward the overutilization of the V kappa3 and V lambda1 gene subgroups. When compared with those of the respective germline genes, the sequences of the expressed Ig VDJ genes displayed nucleotide differences that resulted from somatic hypermutation. In three endemic and three sporadic BL cells, nucleotide changes yielding amino acid substitutions segregated within the complementarity determining region, indicating the application of a positive pressure for replacement mutations and suggesting that these neoplastic lymphocytes underwent a process of clonal selection driven by antigen, perhaps emerging from or transitioning through germinal centers.     

Comparison between hybridoma and Fab/phage anti-RhD: their V gene usage and pairings

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 V kappa families (V kappa 1 and V kappa 3). However, the 13 lambda chains were not as restricted, using 10 V lambda germline genes from 4 families (V lambda 1, V lambda 2, V lambda 3 and V lambda 8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from V kappa 1 (97%) and the 24 lambda chains used 10 V lambda germline genes from 5 families (V lambda 1, V lambda 2, V lambda 3, V lambda 4 and V lambda 7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 V kappa germline genes used by the IgG (DPK9). However, the repertoire of the V lambda germline genes used in these two systems is different, with analysis showing greater diversity in V lambda gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% of the IgG hybridoma anti-RhD.     

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.     

Immunoglobulin gene rearrangement analysis in composite hodgkin disease and large B-cell lymphoma: evidence for receptor revision of immunoglobulin heavy chain variable region genes in Hodgkin-Reed-Sternberg cells?

Immunoglobulin heavy chain gene (IgH) rearrangement was studied in a patient showing the occurrence of classical Hodgkin disease and large B-cell lymphoma (LBCL) in the same lymph node. The VHDHJH region was amplified by polymerase chain reaction, the template being the DNA extracted from single Hodgkin and Reed-Sternberg and LBCL cells, microdissected on hematoxylin-eosin-stained sections by laser capture. A repeated VH4DH3JH4 segment was found in Reed-Sternberg cells, whereas a repeated VH3DH3JH4 segment was observed in LBCL cells. Rearranged VH genes carried somatic mutations in both populations, indicating a common germinal center cell origin. The IgH rearrangement found in clonally related Reed-Sternberg cells differed from the one of LBCL cells in the VH region but showed the same JH and DH segments with no variation from the respective germline sequence. The DH-JH junction is the first immunoglobulin gene segment rearranged in precursor B cells. Because the possibility of secondary Ig gene rearrangement in peripheral lymphoid organs has recently been reported, in the patient described here Reed-Sternberg and LBCL cells might originate from a common precursor in which secondary VH replacement took place during the germinal center reaction, giving rise to two different clonally related lymphomas.     

Characterization of immunoglobulin variable regions of two human pathogenic monoclonal cryocrystalglobulins

Cold-precipitating monoclonal immunoglobulins can rarely aggregate in form of crystals (cryocrystalglobulins) and cause serious clinical manifestations. The structural basis underlying this phenomenon remains to be defined. This study was undertaken to provide the first characterization of the heavy (VH) and light chain (VL) variable regions of two human pathogenic cryocrystalglobulins. The immunoglobulins used different heavy and light chain constant regions and germline gene fragments, underwent high degrees of somatic hypermutation, and showed distributions of replacement and silent nucleotide changes suggestive of antigenic selection. Primary sequences analyses and computer-generated modeling identified a positive charge and the introduction of unusual hydrophobic residues in exposed areas of VH and VL. In particular, a rare replacement of a polar residue with proline is shared at the beginning of the VH complementarity-determining region 2, and this residue might be involved in intermolecular contacts.     

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.     

Systematic design and testing of nested (RT-)PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells

The aim of this study was to develop a highly specific and sensitive (RT-)PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells. A database of germline immunoglobulin sequences was used to design 112 primers for a nested (RT-)PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. Most but not all V gene family specific primers designed allow amplification of full-length V genes. The nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vkappa families, and all Vlambda genes. The broad V gene family coverage of our primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner. We successfully amplified the expressed VH and Vkappa gene from a single hybridoma cell by RT-PCR and from 10-15 microdissected B cells by genomic PCR. This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.     

Human monoclonal striational autoantibodies isolated from thymic B lymphocytes of patients with myasthenia gravis use VH and VL gene segments associated with the autoimmune repertoire.

The production of autoantibodies reactive with components of skeletal muscle is characteristic of myasthenia gravis (MG) and is strongly associated with the presence of thymic epithelial tumors in patients with MG. The nucleotide sequences of the heavy and light chain variable regions (VH and VL) of three human monoclonal striated muscle antibodies (StrAb) isolated from thymic B lymphocyte lines from two patients with MG and thymoma were analyzed. The VH and VL gene segments used by these anti-striated muscle antibodies appear to be derived from the same repertoire of gene segments as have been found in other autoantibodies isolated from patients with a number of different autoimmune diseases. While the IgM StrAb SA-1A is a direct copy of germ-line VH and VL gene segments, analysis of the IgG StrAb SA-4A and SA-4B, which may be more representative of antibodies found in the serum of MG patients, suggests that the processes of antigenic selection and somatic mutation may contribute to the generation of autoantibodies in MG.     

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.     

Immunoglobulin gene sequence analysis to further assess B-cell origin of multiple myeloma.

To further characterize the B-cell origin of multiple myeloma, our laboratory performed immunoglobulin gene sequence analyses of four cases of myeloma (three immunoglobulin A and one immunoglobulin G). Three tumors expressed VH3 genes and one expressed a VH1 gene, while the light chains included two V lambda and one V kappa III; one light chain was not isolated. The closest homology to published germ line genes ranged from 91 to 97%. In two cases, the expressed VH genes were compared with the putative germ line precursor VH genes isolated from autologous granulocyte DNA and appeared to have mutated randomly from the germ line gene. By sequencing multiple clonal isolates from each tumor sample, we found no evidence for ongoing mutation in three cases; in one case, however, clonotypic heterogeneity was evident. The analysis of DH- and JH-region genes revealed (i) limited or absent N nucleotide insertions (two of four cases), (ii) the presence of a DH-JH junction resulting from sequence overlap between the DH and JH genes (one of four cases), (iii) the absence of somatic mutations (two of four cases), and (iv) restricted JH gene usage of a JH6 polymorphism (three of four cases). These analyses of DH and JH genes suggest that multiple myeloma, similar to what has been proposed for chronic lymphocytic leukemia, may derive from B cells which have rearranged during fetal development rather than during adult life.     

Analysis of the immunoglobulin heavy chain gene variable region of 101 cases with peripheral B cell neoplasms and B cell chronic lymphocytic leukemia in the Japanese population

We have analyzed the immunoglobulin heavy chain (VH) gene variable regions (CDR2 and FW3) of 101 Japanese cases with peripheral B cell neoplasms. When all except one case with a deletion were graphed by frequency of replacement mutation, the 100 cases could be separated into two groups: 24 cases with zero, one and two mutations (germline or low frequency of somatic mutation); and 76 cases with three or more mutations (medium to high frequency of somatic mutation). While most mantle cell lymphoma cases (11/13) showed germline or low frequency of somatic mutation, all cases of mucosa-associated lymphoid tissue (MALT) lymphoma (11/11), follicular lymphoma (three of three cases), plasma cell myeloma (seven of seven cases) and most cases of diffuse large B cell lymphoma (DLBCL; 42/47) belonged to the latter group. These 76 cases, therefore, may be considered to show somatic hypermutation. More than half of chronic lymphocytic leukemia/small lymphocytic lymphoma cases (CLL/SLL; eight of 13) showed a hypermutated VH gene and the ratio of replacement mutation: silent mutation in CDR2 of CLL/SLL was considerably higher compared with DLBCL and MALT lymphoma, showing somatic hypermutation. When comparing VH gene type of B cell-CLL (B-CLL) among our series and those in the literature, more cases of CD5+ B-CLL in the Western literature have the VH5 and VH6 family types, while more cases in Japan are reported to have VH4 family. The occurrence of VH families in B-CLL between Japanese and Western people seems to be comparable.     

Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.     

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 VH and VL 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 VH3 genes beyond that one would expect based on random utilization.