A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 VH gene.

We report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The VH gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural autoantibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted VH genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations.     

Mutation pattern of paired immunoglobulin heavy and light variable domains in chronic lymphocytic leukemia B cells

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.     

Ig lambda and heavy chain gene usage in early untreated systemic lupus erythematosus suggests intensive B cell stimulation.

To determine the distribution of Vlambda and Jlambda as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vlambda and Jlambda or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vlambda and VH was noted among the productive rearrangements. The distribution of productive Vlambda rearrangements was skewed, with significantly greater representation of the Jlambda distal cluster C Vlambda genes and the Vlambda distal Jlambda7 element, consistent with the possibility that there was receptor editing of the Vlambda locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vlambda1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.     

The human anti-thyroid peroxidase autoantibody repertoire in Graves' and Hashimoto's autoimmune thyroid diseases

Human anti-thyroid peroxidase (TPO) autoantibodies (aAb) are generated during autoimmune thyroid diseases (AITD). Within recent years, increasing knowledge of the TPO-specific aAb repertoire, gained mainly by the use of combinatorial library methodology, has led to the cloning and sequencing of around 180 human anti-TPO aAb. Analysis of the immunoglobulin (Ig) variable (V) genes encoding the TPO aAb in the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr) reveals major features of the TPO-directed aAb repertoire during AITD. Heavy chain VH domains of TPO-specific aAb from Graves' disease patients preferentially use D proximal IGHV1 genes, whereas those from Hashimoto's thyroiditis are characterized more frequently by IGHV3 genes, mainly located in the middle of the IGH locus. A large proportion of the anti-TPO heavy chain VH domains is obtained following a VDJ recombination process that uses inverted D genes. J distal IGKV1 and IGLV1 genes are predominantly used in TPO aAb. In contrast to the numerous somatic hypermutations in the TPO-specific heavy chains, there is only limited amino acid replacement in most of the TPO-specific light chains, particularly in those encoded by J proximal IGLV or IGKV genes, suggesting that a defect in receptor editing can occur during aAb generation in AITD. Among the predominant IGHV1 or IGKV1 TPO aAb, conserved somatic mutations are the hallmark of the TPO aAb repertoire. The aim of this review is to provide new insights into aAb generation against TPO, a major autoantigen involved in AITD.     

Pathogenic autoantibodies in systemic lupus erythematosus are derived from both self-reactive and non-self-reactive B cells

Previous studies have shown that both murine and human anti-double-stranded DNA (anti-dsDNA) antibodies can develop from non-DNA-reactive B cells and suggest a crucial role for somatic mutation in dsDNA binding. However, since only a limited number of human anti-dsDNA antibodies have been analyzed previously, we could not exclude other mechanisms for the generation of anti-dsDNA antibodies in patients with systemic lupus erythematosus (SLE). Therefore, we isolated IgM anti-dsDNA antibodies from peripheral blood B cells of a patient with SLE. Three somatically mutated IgM anti-DNA antibodies with pathogenic potential (glomerular binding) were reverted to their germline configuration. Although all three IgM anti-dsDNA antibodies came from the same lupus patient, they displayed different profiles. Reversion to the germline sequence of autoantibodies A9 and B5 resulted in decreased dsDNA binding. In contrast, the germline form of G3-recognized dsDNA as well as the mutated counterpart. These results suggest that mutated IgM anti-dsDNA antibodies may develop from both DNA- and non-DNA-reactive B cells. The implications are that B cell activation occurs in response to self and non-self antigens, while selection after activation may be mediated by self antigen in SLE. Moreover, ineffective tolerance checkpoints may exist before and after antigen activation in SLE.     

Organization, complexity and allelic diversity of the porcine (Sus scrofa domestica) immunoglobulin lambda locus

We have characterized the organization, complexity, and expression of the porcine (Sus scrofa domestica) immunoglobulin lambda (IGL) light chain locus, which accounts for about half of antibody light chain usage in swine, yet is nearly totally unknown. Twenty-two IGL variable (IGLV) genes were identified that belong to seven subgroups. Nine genes appear to be functional. Eight possess stop codons, frameshifts, or both, and one is missing the V-EXON. Two additional genes are missing an essential cysteine residue and are classified as ORF (open reading frame). The IGLV genes are organized in two distinct clusters, a constant (C)-proximal cluster dominated by genes similar to the human IGLV3 subgroup, and a C-distal cluster dominated by genes most similar to the human IGLV8 and IGLV5 subgroups. Phylogenetic analysis reveals that the porcine IGLV8 subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens. Moreover, expression of IGLV genes is nearly exclusively restricted to the IGLV3 and IGLV8 genes. The constant locus comprises three tandem cassettes comprised of a joining (IGLJ) gene and a constant (IGLC) gene, whereas a fourth downstream IGLJ gene has no corresponding associated IGLC gene. Comparison of individual BACs generated from the same individual revealed polymorphisms in IGLC2 and several IGLV genes, indicating that allelic variation in IGLV further expands the porcine antibody light chain repertoire.     

Perturbations in the impact of mutational activity on Vλ genes in systemic lupus erythematosus

To assess the impact of somatic hypermutation and selective influences on the Vλ light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of mutations were analyzed in individual CD19⁺ B cells from a patient with previously undiagnosed SLE. The mutational frequency of nonproductive and productive rearrangements in the SLE patient was greater (3.1 × 10^-2 vs 3.4 × 10^-2, respectively) than that in normal B cells (1.2 × 10^-2 vs 2.0 × 10^-2, both P < 0.001). The frequencies of mutated rearrangements in both the nonproductive and productive repertoires were significantly higher in the patient with SLE than in normal subjects. Notably, there were no differences in the ratio of replacement to silent (R/S) mutations in the productive and nonproductive repertoires of the SLE patient, whereas the R/S ratio in the framework regions of productive rearrangements of normal subjects was reduced, consistent with active elimination of replacement mutations in this region. The pattern of mutations was abnormal in the SLE patient, with a significant increase in the frequency of G mutations in both the productive and nonproductive repertoires. As in normal subjects, however, mutations were found frequently in specific nucleotide motifs, the RGYW/WRCY sequences, accounting for 34% (nonproductive) and 46% (productive) of all mutations. These data are most consistent with the conclusion that in this SLE patient, the mutational activity was markedly greater than in normal subjects and exhibited some abnormal features. In addition, there was decreased subsequent positive or negative selection of mutations. The enhanced and abnormal mutational activity along with disturbances in selection may play a role in the emergence of autoreactivity in this patient with SLE.     

Dominance of intrinsic genetic factors in shaping the human immunoglobulin Vlambda repertoire

The expressed human immunoglobulin Vlambda repertoire demonstrates a strong bias in the use of individual Vlambda segments. Mechanisms that underlie such biases can be divided into two categories: intrinsic genetic processes that lead to the preferential rearrangement and/or expression of certain segments; and selection following light chain expression. Here, we have used two approaches to investigate the factors that shape the human Vlambda repertoire. Firstly, we characterised 136 Vlambda rearrangements (59 productive and 77 non-productive) amplified from the human genomic DNA of peripheral blood cells. Secondly, we analysed Vlambda segment use in a library of 2000 cDNA clones from a transgenic mouse containing a 380 kb region (including 15 functional Vlambda segments) from the human immunoglobulin lambda locus. By hybridisation and sequencing we found that the patterns of use of human Vlambda segments in the transgenic mouse were similar to those found in the expressed human peripheral blood repertoire and in productive and non-productive genomic DNA rearrangements. These data indicate the importance of intrinsic genetic factors in shaping the human Vlambda repertoire and highlight the remarkable conservation of the molecular mechanisms involved in the production of the antibody repertoire in mouse and man. Therefore, transgenic mice represent a good model for analysis of the human antibody repertoire and for the production of human antibodies.     

The V<Subscript>H</Subscript> gene repertoire of splenic B cells and somatic hypermutation in systemic lupus erythematosus

In systemic lupus erythematosus (SLE) it has been hypothesized that self-reactive B cells arise from virgin B cells that express low-affinity, nonpathogenic germline V genes that are cross-reactive for self and microbial antigens, which convert to high-affinity autoantibodies via somatic hypermutation. The aim of the present study was to determine whether the V_H family repertoire and pattern of somatic hypermutation in germinal centre (GC) B cells deviates from normal in SLE. Rearranged immunoglobulin V_H genes were cloned and sequenced from GCs of a SLE patient's spleen. From these data the GC V gene repertoire and the pattern of somatic mutation during the proliferation of B-cell clones were determined. The results highlighted a bias in V_H5 gene family usage, previously unreported in SLE, and under-representation of the V_H1 family, which is expressed in 20–30% of IgM⁺ B cells of healthy adults and confirmed a defect in negative selection. This is the first study of the splenic GC response in human SLE.     

The importance of the light chain for the epitope specificity of human anti-U1 small nuclear RNA autoantibodies present in systemic lupus erythematosus patients.

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd approximately 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3-11) and the same lambda (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r lambda light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.     

A new model of sheep Ig diversification: shifting the emphasis toward combinatorial mechanisms and away from hypermutation

The current model of Ig repertoire development in sheep focuses on the rearrangement of a small number (approximately 20) of Vlambda gene segments. It is believed that this limited combinatorial repertoire is then further diversified through postrearrangement somatic hypermutation. This process has been reported to introduce as many as 110 mutations/1000 nucleotides. In contrast, our data have that indicated somatic hypermutation may diversify the preimmune repertoire to a much lesser extent. We have identified 64 new Vlambda gene segments within the rearranged Ig repertoire. As a result, many of the unique nucleotide patterns thought to be the product of somatic hypermutation are actually hard-coded within the germline. We suggest that combinatorial rearrangement makes a much larger contribution, and somatic hypermutation makes a much smaller contribution to the generation of diversity within the sheep Ig repertoire than is currently acknowledged.     

Role of conformational dynamics in sequence-specific autoantibody*ssDNA recognition

11F8 is a sequence-specific monoclonal anti-ssDNA autoantibody isolated from a lupus prone mouse that forms pathogenic complexes with ssDNA, resulting in kidney damage. Prior studies show that specificity is mediated by a somatic mutation from serine at (31)V(H) to arginine. Reversion back to serine in 11F8 resulted in >30-fold decrease in affinity and altered thermodynamic and kinetic parameters for sequence-specific recognition of its cognate ssDNA ligand. Mutagenesis and structural studies suggest that (R31)V(H) contacts ssDNA via a salt bridge and a bidentate hydrogen bond and may further contribute to specificity by altering binding-site conformation. Fluorescence resonance energy transfer experiments were conducted to assess the kinetics of conformational change during 11F8*ssDNA association. The extent of rearrangement between the six complementary determining regions in the 11F8*ssDNA complex with germline serine or somatically mutated arginine at residue 31 of the heavy chain was examined. Our studies show that greater conformational change occurs in five of six complementarity determining regions after the heavy chain germline J558 sequence undergoes mutation to arginine at (31)V(H).     

Use of anti-idiotypic antibodies to explore genetic mechanisms of production of anti-DNA antibodies

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies with a broad range of antigenic specificities, including specificity for double-stranded DNA. Analysis of the idiotypic profile of anti-DNA antibodies both in humans and mice has demonstrated presence of cross-reactive idiotypes, suggesting that they arise from a restricted number of germline genes. Our laboratory has previously reported the generation of 3I, a monoclonal anti-idiotypic antibody which recognizes a cross-reactive idiotype on anti-DNA antibodies in a majority of unrelated humans with SLE. We have recently studied the expression of 3I in sera of three human kindreds with familial SLE. We found 6 of 8 SLE patients and 15 of 19 unaffected family members had elevated 3I reactivity. Eleven of these family members had no anti-DNA activity despite elevated 3I reactivity, suggesting that expression of this idiotype in certain individuals is part of the normal immune response. In another set of experiments using an in vitro culture system we examined somatic mutants of the S107 mouse myeloma cell line. This line makes an antibody which bears the T15 idiotype, a common idiotype on antibodies to the bacterial antigen phosphoryl choline (PC). U4, a mutant, makes an immunoglobulin which varies by one amino acid from the parent protein, retains the T15 idiotype, but loses reactivity with PC and acquires reactivity with DNA. We have found that some anti-DNA antibodies in mice with spontaneous lupus and in mice immunologically induced to make anti-DNA antibodies bear the T15 idiotype and may represent somatic mutants arising in vivo.     

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.     

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.     

Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus

The organization of the human immunoglobulin lambda light chain locus (IGL) was recently described. This locus has been entirely sequenced. To evaluate the extent of the genomic variability existing inside that locus, we compiled all the available sequences of germline IGLV genes to find variants of Vλ sequences. We also looked for RFLP polymorphisms in a reputedly highly polymorphic human population from eastern Senegal, and compiled all RFLP data previously published. Analysis of these data indicates that IGLV alleles are frequent and increase the diversity of the lambda light chain repertoire in the human population. In contrast, RFLP and polymorphism by insertion and/or deletion are limited in that locus. This observation reinforces our hypothesis that the human IGL locus has undergone less evolutionary shuffling than the human kappa or heavy-chain loci. Received: 23 December 1998 / Accepted: 1 March 1999     

Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements.

We have recently characterized the heavy chain variable region (VH) genes expressed by a panel of human anti-DNA antibodies derived from four patients with systemic lupus erythematosus and expressing an idiotypic marker representative of a subset of pathogenic autoantibodies. Here, we have cloned and sequenced the kappa chain variable region genes (V kappa) of the clones whose VH genes had been previously analysed. All the V kappa genes utilized map to the 280 kb portion of the 3' end of the locus, suggesting that they represent essentially the products of primary rearrangements. This proximal clustering of the V kappa genes used contrasts with the broad distribution of immunization-induced human antibody V kappa genes over 1400 kb of the locus. In addition, lupus autoantibodies show no tendency to express the downstream junctional (J kappa) exons--another indication of infrequent secondary variable gene assembly. Since successive rearrangements may extinguish high-affinity recognition of self antigens, we propose that this bias in V kappa and J kappa expression reflects a low rate of secondary light chain rearrangements among lupus autoantibodies. We also postulate that the corrective mechanism capable of editing potentially aggressive, self-reactive antibodies in these patients may be deficient--a deficit that could be genetically determined and/or somatically acquired.     

Lupus IgG VH4.34 antibodies bind to a 220-kDa glycoform of CD45/B220 on the surface of human B lymphocytes

Anti-lymphocyte autoantibodies are a well-recognized component of the autoimmune repertoire in human systemic lupus erythematosus (SLE) and have been postulated to have pathogenic consequences. Early studies indicated that IgM anti-lymphocyte autoantibodies mainly recognized T cells and identified CD45, a protein tyrosine phosphatase of central significance in the modulation of lymphocyte function, as the main antigenic target on T cells. However, more recent work indicates that lupus autoantibodies can also recognize B cells and that CD45 may also represent their antigenic target. In particular, IgM Abs encoded by V(H)4.34 appear to have special tropism for B cells, and strong, but indirect evidence suggests that they may recognize a B cell-specific CD45 isoform. Because V(H)4.34 Abs are greatly expanded in SLE, in the present study we investigated the antigenic reactivity of lupus sera V(H)4.34 IgG Abs and addressed their contribution to the anti-lymphocyte autoantibody repertoire in this disease. Our biochemical studies conclusively demonstrate that lupus IgG V(H)4.34 Abs target a developmentally regulated B220-specific glycoform of CD45, and more specifically, an N-linked N-acetyllactosamine determinant preferentially expressed on naive B cells that is sterically masked by sialic acid on B220-positive memory B cells. Strikingly, our data also indicate that this reactivity in SLE sera is restricted to V(H)4.34 Abs and can be eliminated by depleting these Abs. Overall, our data indicate that V(H)4.34 Abs represent a major component of the lupus IgG autoantibody repertoire and suggest that the carbohydrate moiety they recognize may act as a selecting Ag in SLE.