V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.     

Relationship of human variable region heavy chain germ-line genes to genes encoding anti-DNA autoantibodies

In order to identify the V region genes encoding systemic lupus erythematosus (SLE)-derived anti-DNA autoantibodies, we have determined the nucleotide sequence of heavy chain mRNA from several DNA-binding immunoglobulins secreted by human hybridomas. We used the technique of cDNA primer extension for determining sequences of the VH, D, and JH gene segments of anti-DNA autoantibodies from three different primary hybridoma growths from an SLE patient and one hybridoma from a leprosy patient. Immunoglobulins from two of the SLE hybridomas expressed the same idiotype, Id-16/6, which is also expressed on immunoglobulins in sera of patients with active SLE. Their mRNA sequences showed complete homology to each other in the V, D, and J genes and more than 99% homology to the VH26 germ-line gene sequence, a member of the human VHIII gene family. The VH mRNA sequence of the third SLE hybridoma, 21/28, which was idiotypically unrelated to the other two, was 93% homologous to a different VH germ-line gene sequence, HA2, a member of the human VHI gene family. The fourth anti-DNA-producing hybridoma, 8E10, was derived from a leprosy patient of different ethnic origin than the SLE patient. It was idiotypically related to 21/28 and expressed a VH segment gene identical to that of 21/28. Hybridomas 21/28 and 8E10 shared sequence homology with the VH26 anti-DNA antibodies in the first complementarity-determining region. In addition, 21/28 shared sequence homology with the Id-16/6+ group in the region encoded by the D and J gene segments. Our findings indicate that some SLE autoantibodies are encoded by unmodified or scarcely modified VH germ-line genes that are conserved in the human population and identify two distinct VH germ-line genes that can encode segments of anti-DNA immunoglobulins.     

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.     

Novel V genes encode virtually identical variable regions of six murine monoclonal anti-bromelain-treated red blood cell autoantibodies

The variable (V) region sequences of six immunoglobulin M (IgM, kappa) monoclonal autoantibodies that recognize bromelinized isologous red blood cells, obtained by fusions of peritoneal cells from NZB or CBA/J nonimmunized mice with BALB/c myeloma cells, were determined by direct mRNA sequencing. The V regions of the light chains (VL) are almost identical with one another, as are the V regions of the heavy chains (VH), which, however, differ by six linked-base substitutions, depending on the strain of mice producing the autoantibodies. Such variations may reflect allelic differences. The VH segments determined have no obvious correspondence to any VH genes identified so far. They may belong to the small VH group 4, where 73% homology, at the most, can be calculated at the protein level for codons 1 to 94. Alternatively, the VH regions may be members of a new group of VH sequences not previously found. The V kappa regions appear closely homologous to members of the V kappa-9 subgroup of myeloma proteins of unknown antigen-binding specificity. The joining segments, J kappa and JH, used by the autoantibodies investigated, originate from the J kappa 2 and JH1 germ-line gene segments, respectively. The nine base-long diversity segments, D, derive from one member of the germ-line D gene SP2 family.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Origin of the autoreactive anti-type II collagen response. II. Specificities, antibody isotypes and usage of V gene families of anti-type II collagen B cells

Autoantibodies play an important role in the pathogenesis of type II collagen-induced arthritis in mice. We have earlier reported a high frequency of cells producing anti-CII autoantibodies and a low frequency of cells producing multispecific antibodies, in regional lymph nodes 9 to 11 days after primary immunization with CII. It is shown here that anti-CII antibodies produced during primary immune response are IgG-antibodies mainly of IgG2a, IgG1 and IgG2b subclasses while IgM antibodies dominate primary responses elicited by OVA and denatured CII as analyzed with a large panel of hybridomas. Anti-CII antibodies generated during the primary response recognize at least five different epitopes on the CII molecule. The specificities of these antibodies for various epitopes result from combinational association of products encoded by genes derived from various VH and VK families and/or by the occurrence of somatic mutations. It is suggested that the primary anti-CII autoantibody response involves activation of memory B cells and is in this aspect different from the origin of "natural" autoantibodies.     

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.     

Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites

By examining a large database of amino acid sequences of antibodies of various specificities, we have found that many antibodies of distinctly different specificities assemble identical VL domains with different VH domains. In contrast, rarely is the same VH domain found in sets of antibodies of different specificities. We identified additional sets of antibodies of different specificities and identical sequences covering amino acid residues VH 1 to 94 and VL 1 to 95. In addition, there were segments of additional antibodies for which complete sequences were not available, but identities were seen in VL CDR1, VL CDR2, and VL CDR3 up to the VL-JL junction. The finding that there are many identical VL 1 to 95 segments with different VH 1 to 94 sequences, and vice versa, raises important questions as to the role of VH in influencing the conformation of VL and, conversely, the role of VL in influencing the conformation of VH. Evidence is also cited indicating that a single amino acid change may seriously disrupt site structure and in some instances abolish binding. Our findings suggest that it will be important in the future to investigate further conformational effects on antibody structure by using X-ray crystallography, nuclear magnetic resonance spectroscopy, or other methods, to obtain a better understanding of the functions and topography of antibody-combining sites.     

Preferential autoantibody reactivity of the preimmune B cell repertoire in normal mice

Naturally occurring autoantibodies are frequently found in the sera of healthy individuals. They usually exhibit low binding affinities for autoantigens and often react with multiple antigenic determinants. To determine whether their frequencies have been overestimated by sensitive testing procedures, the germ-line B cell repertoire of strain A mice was examined for reactivity with a panel of auto- and foreign Ag. If the high frequencies of autoantibodies result from testing procedures, equally high frequencies would be expected for foreign Ag specificities detected in the same manner. The presence of specific autoantibodies was confirmed in this study by the disparate frequencies observed for antibodies reactive with individual Ag. The frequencies were highest for autoantigens associated with SLE, indicating a bias toward autoreactivity in the preimmune repertoire. Analysis of VH gene usage did not indicate any selection in V gene expression with autoreactivity.     

V region sequences of anti-DNA and anti-RNA autoantibodies from NZB/NZW F1 mice

The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.     

The structural basis of rabbit VH allotypes: serologic studies on a1 H chains with defined amino acid sequence.

The amino acid sequences for the VH regions of three homogeneous antibodies elicited by type III pneumococcal vaccine were determined. All three antibodies had the group a allotype a1. Two of the antibody H chains (3372, 3381) had identical amino acid sequences in all framework positions that are considered correlates of the VH allotype, whereas the third H chain (3T72) differed from these at positions 15 and 16. The a1 allotypic specificities of the three homogeneous antibodies were compared by quantitative radiobinding and inhibition assays by using both insolubilized anti-a1 antisera and allotypic antiserum fractions rendered specific for the homogeneous antibody 3374. It was found that antibodies 3374 and 3381 are allotypically indistinguishable and have in common an a1 allotypic specificity that predominates in pooled a1 IgG. The allotypic specificity of the 3T72 antibody, on the other hand, was markedly deficient to those of 3374, 3381, and the a1 IgG pool. This correlation of allotypic difference with amino acid sequence variation at position 15 and 16 of the H chain indicates the involvement of these two residues in a major a1 allotypic determinant.     

Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation

In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves' disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vlambda1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves' disease patients tested were able to strongly inhibit (60-100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.     

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Two families of monoclonal antibodies to alpha(1----6)dextran, VH19.1.2 and VH9.14.7, show distinct patterns of J kappa and JH minigene usage and amino acid substitutions in CDR3.

Nine groove-type mAb to alpha(1----6)dextran were cloned and sequenced. Together with previous reports from this laboratory, the VH and VL of 34 mAb have been sequenced, in which 10 VH19.1.2 and 11 VH9.14.7 combined with the V kappa-Ox1 gene to form two major families of anti-alpha(1----6)dextrans. The same D minigene (DFL16) was used by all VH19.1.2 and VH9.14.7 mAb; however, the patterns of JH and J kappa usage are quite different. VH19.1.2 mAb used only JH3 and J kappa 2, whereas VH9.14.7 mAb used three JH (JH1, JH2, and JH3) and all four active J kappa (J kappa 1, J kappa 2, J kappa 4, and J kappa 5). Relative uniformity in the lengths of VH CDR3 and the junctional sequences is seen in both families. Some mAb from different mouse strains share common structural features. The differences in idiotypic specificities and in the amino acid sequences suggest that VH19.1.2 and VH9.14.7 may differ in the conformation of CDR1 and CDR2. Combining with V kappa-Ox1 gene to generate groove-type combining sites to the single site-filling epitope of alpha(1----6)dextran, the two VH chains may require certain conformations of CDR3. Whether such conformational requirements influence the choice of J minigenes, the selection of the length of VH CDR3 and the sequences at junctions, are discussed.     

Partial molecular genetic map of the rabbit VH chromosomal region

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.     

VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions

In this study we investigate the molecular genetic basis for VHa- Ig. Knowing that the expression of VHa allotype Ig is suppressed by neonatal injection of rabbits with anti-VHa allotype antibody, and that the decreased level of VHa allotype Ig, VHa+, in the suppressed rabbits is compensated for by an increase in VHa- Ig, we determined the nucleotide sequences of 41 VDJ genes from a2/a2 rabbits neonatally suppressed for the expression of a2 Ig. We compared these nucleotide sequences to each other and identified two groups of VH sequences. We predict that the molecules of each group are encoded by one germline VH gene. Inasmuch as VHa+ Ig is encoded predominantly by one germline VH gene, VH1, it appears that more than 95% of the VDJ repertoire of rabbits may be encoded by as few as three germline VH genes. A genomic VDJ gene whose VH sequence was similar to those of group I molecules was expressed in vitro and was shown by ELISA to encode molecules of the VHa- allotype, y33. Analysis of the D regions in the VDJ gene indicated that germline D2b and D3 gene segments were preferentially used in the VDJ gene rearrangement. A comparison of sequences of D regions of the 41 VDJ gene rearrangements in 3-, 6-, and 9-wk-old rabbits to sequences of germline D gene segments showed an accumulation of mutations in the D region. Inasmuch as we have previously shown that V regions of rabbit VDJ genes are diversified, in part, by somatic gene conversion, it appears now that rabbit VDJ genes diversify by a combination of somatic mutation and somatic gene conversion.     

Structure, map position, and evolution of two newly diverged mouse Ig VH gene families.

We have characterized two novel mouse VH gene families, VH3609N and VHSM7. These VH families have recently diverged from previously defined VH families. The VH3609N family, which may contain only one member in most inbred strains of mice, shares sequence similarity with the VHJ606 family and is located to the 3' side of VHJ606. VHSM7, with at least three members, is related to the VHJ558 family but maps 3' of VHJ558. These findings suggest that physical displacement of VH sequences may facilitate their subsequent divergence. During the early stages of VH gene family evolution that are exemplified by these new families, amino acid replacements have been selected against in frame-work regions and selected for in complementarity-determining regions. This pattern of nucleotide substitution appears to reflect evolutionary pressures to maintain germ-line VH diversity and, possibly, to select for new antibody specificities, as well as to select against mutations resulting in aberrant Ig. The classification of VH sequences with borderline similarity to previously defined VH families is discussed.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.