V genes of the primary antibody response of C57BL/10 mice to the hapten phenyloxazolone

The mRNA of ten monoclonal phenyloxazolone (phOx) antibodies originating from the primary (day 7) response of C57BL/10 mice were partially sequenced. The sequences were analyzed together with those of two previously published antibodies. The C57BL response does not have a predominant subset of antibodies like the BALB/c response has (VH-Ox1/V kappa-Ox1 JK5). Probably, C57BL mice lack the VH-Ox1 gene and, as a consequence, their V kappa-Ox1 gene does not have a main role in the anti-phOx response. Five V kappa and six VH genes were found to participate. All five V kappa genes or their "alleles" had previously been found from the BALB/c response to 2-phenyloxazolone (phOx). On the other hand, the two strains use different VH genes for the anti-phOx response. Most C57BL antibodies were coded by VH genes of group 1 which has only minor role in the BALB/c response. The remaining VH genes were from group 7. Our data show that one V kappa segment (e.g. V kappa-Ox1) can code for anti-phOx antibodies with several, even widely different, VH genes. On the other hand, they emphasize the role of certain VH/VL gene combinations for the anti-phOx specificity. Thus, VH genes of group 7 were found to code for anti-phOx antibodies only together with the V kappa 45.1 gene.     

Quantitative representation of two germ-line V genes in the early antibody response to 2-phenyloxazolone

Early anti-phenyloxazolone antibodies of BALB/c mice include a subset that bears the nearly or totally unmutated VH-Ox1 sequence. A fraction of this subset also bears the nearly or totally unmutated V kappa-Ox1 sequence. The whole subset is recognized by an anti-idiotype serum 495 and the VH-Ox1/V kappa-Ox1 fraction also by another anti-idiotype serum 260. Frequencies of the VH-Ox1 subset and its V kappa-Ox1 fraction in early IgM and IgG antibodies were determined by typing hybridomas with anti-idiotype antisera. The whole subset appears to make up approximately one half of IgG antibodies, two thirds of this being 495+/260+ in both isotypes. IgM data are less certain but the same frequencies may be valid. Affinities of 495+/260+ antibodies ranged from 1.3 X 10(6) to 11 X 10(6), affinities of 495+/260- antibodies from 0.47 X 10(6) to 1.1 X 10(6), and affinities of doubly negative antibodies were less than 0.41 X 10(6). High affinity is probably an explanation for the high proportion (one third) of the 495+/260+ antibodies in the early response. Doubly negative (and low-affinity) hybridomas may not have been classified as producers of phenyloxazolone antibodies in earlier studies, and this could explain the still higher reported frequency (73%) of VH-Ox1/V kappa-Ox1 antibodies.     

Heavy and light chain sequences of four monoclonal antibodies that bind galactosylgloboside (GalGb4)

We recently described IgM monoclonal antibodies directed against the glycospingolipid galactosylgloboside (GalGb4; Marcus, M. D. et al., Arch. Biochem. Biophys, 1988.262: 620). We now present the nucleotide and deduced amino acid sequences of the heavy and light chains of these antibodies. The antibodies were generated in a single fusion, their heavy as well as their light chains are almost identical, and they appear to be clonally related. The light chains were encoded by J kappa 5 and a V kappa gene belonging to the Ox1 family, but they are only 93% homologous to the most closely related germ-line gene, and they are probably encoded by a germ-line gene that has not yet been identified. The heavy chains were all encoded by VH441 and JH2, and have identical N segments. The VH441 germ-line gene encodes a potential glycosylation site at Asn58 in the complementarity-determining region 2. This site, which has been retained in all VH441-encoded monoclonal antibodies sequenced previously, was mutated out by a single base change in all four anti-GalGb4 antibodies.     

Molecular and serological diversity of anti-DNA autoantibodies from NZB and (NZB X NZW) F1 mice

We have carried out an analysis of the serological and molecular diversity of a panel of monoclonal anti-DNA autoantibodies and serum autoantibodies from NZB and (NZB X NZW) F1 mice, in an attempt to obtain insights into the mechanisms responsible for the development of systemic autoimmune disease. Our data show that the autoantibodies are quite diverse. A dominant, binding-site idiotope on one of our monoclonal autoantibodies is expressed at variable levels in anti-DNA binding antibodies in the sera of both NZB and (NZB X NZW) F1 mice, but on none of the other monoclonal autoantibodies in our panel. We have cloned and sequenced the heavy chain variable region (VH) gene of one anti-DNA hybridoma and by hybridization have determined the VH and V kappa gene segments expressed by 14 others. All of the autoantibodies express members of known V gene subfamilies. A total of four different VH and at least six V kappa subfamilies are expressed by the hybridomas. Thus, a broad spectrum of the total murine Ig repertoire is represented in the anti-DNA autoantibodies present in these strains.     

Molecular heterogeneity of auto-anti-idiotypic antibodies in MLR-lpr/lpr mice

The VH and V kappa gene families expressed by 20 monoclonal auto-anti-idiotypes (Ab2) derived from unmanipulated MLR-lpr/lpr mice were determined by Northern blotting. Complete variable region sequences of six Ab2, along with three additional V kappa-JH Ab2 sequences, were obtained. These auto-anti-idiotypes arose spontaneously in the animals, and they bound specifically to an idiotypic determinant (Id/r) on mAb 28/12, a monoclonal IgG2b MLR-lpr/lpr anti-small nuclear ribonucleoprotein antibody. The 16 Ab2 heavy chains belonged to 7 different VH gene families, and the 10 Ab2 light chains were derived from 8 V kappa families. The light chains of two Ab2 were approximately 99% identical; the remaining variable region sequences were highly heterogeneous. There was no correlation between primary amino acid sequence of either heavy or light chain and idiotypic properties of the auto-anti-idiotypes. Six Ab2 used VH or V kappa genes that are identical to known germ-line genes. A high proportion of the spontaneous auto-anti-idiotypes was shown to have autoantibody activity (anti-DNA, anti-ribonucleoprotein), or specific binding reactions with lipopolysaccharide of Salmonella RE, or both properties. The structural diversity of spontaneous MLR-lpr/lpr auto-anti-idiotypes differs sharply from the structural homogeneity reported for Ab2 induced in normal animals against syngeneic Ab1. Our results suggest that auto-anti-idiotypes might arise independently of an immunogenic stimulus from an Ab1.     

Combinatorial association of V genes: one VH gene codes for three non-cross-reactive monoclonal antibodies each specific for a different antigen (phoxazolone, NP or gat)

Two anti-phenyloxazolone (phOx3) and one anti-GAT MAbs from C57BL mice are shown to be coded by VH gene 186.2. This gene has been found earlier to code for several anti-NP (NNP) antibodies (Bothwell et al., 1981) and anti-GT antibodies (Rocca-Serra et al., 1983; Carmack and Pincus, 1986). The L chain partner of the VH 186.2 gene is different in anti-NP and anti-GAT antibodies (Bothwell et al., 1981; Rocca-Serra et al., 1983; Carmack and Pincus, 1986); in anti-phOx antibodies two new unrelated kappa chain V regions were found. Both of the new VK genes involved code frequently for anti-phOx antibodies in BALB/c mice but then with different VH genes. We tested five 186.2-coded antibodies for cross-reactions. Four antibodies were specific, one bound only to NNP, one only to phOx and two only to GT (GAT). The fifth antibody (anti-phOx) bound also to NNP, GAT and ABA-HOP though probably with a low affinity. This is the first demonstration that one V gene can code for three different antibody specificities. It emphasizes the role of the combinatorial element in antibody diversity.     

The V kappa gene repertoire in the human germ line

The question of how many V kappa gene segments exist in the human germ line was addressed. Seventy-five V kappa genes of the kappa locus and twenty-five V kappa genes localized outside of the locus ("orphons") had been cloned previously; 67 of the genes and 19 of the orphons had already been sequenced yielding 36 and 1 potentially functional V kappa genes, respectively, the remaining ones being pseudogenes. We now (a) determined the relative hybridization intensities of the cloned V kappa genes and orphons, (b) identified the bands in blot hybridizations of genomic DNA digests with the cloned genes and orphons, (c) determined the band intensities in the genomic DNA digests from two individuals and one cell line, (d) normalized the results with the help of the C kappa gene segment which is present in the haploid genome in one copy, (e) compared the genomic blot hybridization patterns with patterns of equimolar mixtures of the cloned V kappa genes and orphons, and (f) defined the bands and fractional intensities in bands that could not be assigned to cloned genes or orphons. From the resulting data we conclude that there are 5-7 still uncloned V kappa genes in germ-line DNA in addition to the 75 known V kappa genes and in addition to the 25 orphons 12-15 orphon candidates. It appears that the rheumatoid factor light chains of the Wa and 6B6.6 idiotypes are coded for by one V kappa III gene each. It is concluded that the kappa locus comprises no more than 50 potentially functional genes and no more than 85 V kappa genes altogether.     

The same few V genes account for a majority of oxazolone antibodies in most mouse strains.

The early primary anti-phenyloxazolone antibodies of 12 mouse strains were studied by determining proportions of two defined subsets id495 (the classical phOx idiotype) and id350. Id495-positive antibodies bear an H chain encoded by VHOx1 gene (family Q52) and an L chain usually coded for by VKOx1 but occasionally by other VK genes. Id350-positive antibodies are encoded by a VK gene VK45.1, and usually by a VH gene of the S107 family. All 12 strains (representing nine H-chain and four kappa-chain haplotypes) produced id350-positive anti-phOx antibodies. While id495 is the predominant major subset in the BALB/c response (originally studied), id350 seems to be the predominant subset of early anti-phOx antibodies in the mouse species. The combined proportion of the two subsets varied from ca. 50 to almost 100% of the total in all strains except C57BL.     

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.     

V genes of oxazolone antibodies in 10 strains of mice

One pair of V genes (V kappa 45.1 and V11) code for a great portion of phenyloxazolone (anti-phOx) antibodies in 10 strains of mice. This combination replaces the first-known major combination VHOx1-V kappa Ox1 in some strains, and is important in most strains. C57BL/10 and SJL mice have an additional subset of antibodies encoded by genes V kappa 45.1 and V13 (a relative of V11). All three genes involved (V kappa 45.1, V11 and V13) have "allelic" variation. Four alleles of V11 were found, one in Igh haplotypes a, c and g, the second in haplotypes d, j and n, the third in b, and the fourth in f. The most distant alleles d, j, n and f had 10 nucleotide differences out of 429 determined (97.7% homology). Only one allele of the V13 gene was found from anti-phOx hybridomas but two others have been published. Three alleles of the V kappa 45.1 gene were found; one in NZB mice (Ig kappa haplotype b) another in CE (haplotype f), and the third in eight strains including representatives of three Ig kappa haplotypes (a, c and e). The three alleles had greater than 99.0% homology. The V11 and V13 genes that code for anti-phOx antibodies in C57BL/10 and SJL mice were different from the related genes found from the C57BL/10 germ line. C57BL/10 mice must have a chromosome bearing two V11 and two V13 genes. RF mice were found to have two V11 genes, and both code for anti-phOx antibodies. Our data show that the majority of antibodies in the anti-phOx response are encoded by the same restricted collection of V genes in most mouse strains. Antibody responses appear to be no less heritable than other functions of the body.     

Repertoire of human antibodies against the polysaccharide capsule of Streptococcus pneumoniae serotype 6B

We examined the repertoire of antibodies to Streptococcus pneumoniae 6B capsular polysaccharide induced with the conventional polysaccharide vaccine in adults at the molecular level two ways. In the first, we purified from the sera of seven vaccinees antipneumococcal antibodies and determined their amino acid sequences. Their VH regions are mainly the products of VH3 family genes (candidate genes, 3-23, 3-07, 3-66, and 3-74), but the product of a VH1 family gene (candidate gene, 1-03) is occasionally used. All seven individuals have small amounts of polyclonal kappa+ antibodies (Vkappa1 to Vkappa4 families), although kappa+ antibodies are occasionally dominated by antibodies formed with the product of the A27 Vkappa gene. In contrast, lambda+ anti-6B antibodies are dominated by the antibodies derived from one of 3 very similar Vlambda2 family genes (candidate genes, 2c, 2e, and 2a2) and Clambda1 gene product. The Vlambda2(+) antibodies express the 8.12 idiotype, which is expressed on anti-double-stranded-DNA antibodies. In one case, Vlambda is derived from a rarely expressed Vlambda gene, 10a. In the second approach, we studied a human hybridoma (Dob1) producing anti-6B antibody. Its VH region sequence is closely related to those of the 3-15 VH gene (88% nucleotide homology) and JH4 (92% homology). Its VL region is homologous to the 2a2 Vlambda2 gene (91%) and Jlambda1/Clambda1. Taken together, the V region of human anti-6B antibodies is commonly formed by a VH3 and a Vlambda2 family gene product.     

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.     

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.     

V gene analysis of anti-cardiolipin antibodies from (NZW x BXSB) F1 mice.

In (NZW x BXSB) F1 (W/B F1) male mice, systemic lupus-like disease, thrombocytopenia and coronary vascular disease with myocardial infarction occur, due to the presence of platelet-associated antibodies, anti-platelet antibodies and anti-cardiolipin antibodies (aCL). We developed monoclonal aCL and analysed the specificity of aCL. In the W/B F1 mice, there are aCL with pathogenic properties, which have an IgG isotype and reveal a cofactor-dependent binding to CL, binding activity to platelets, and lupus anti-coagulant (LA) activity. Here, we analysed the usage of VH and V kappa genes of six aCL, including two pathogenic aCL, from W/B F1 mice, in an attempt to address the question of whether or not aCL with pathogenic properties use restricted Ig V genes. Sequence analysis of VH and V kappa genes of aCL showed that the pathogenic aCL had VHJ558 and V kappa 21 or V kappa 23 genes, whereas the other aCL without pathogenic features used mainly the 7183 VH family and the random V kappa gene group. However, two pathogenic aCL showed a 86.6% homology with the IgV region, each other, indicating that they were not closely related clones. Thus, these findings suggest the possibility that usage of Ig VH genes in pathogenic aCL is not random, but that there may exist a few epitopes of antigen recognized by the pathogenic aCL.     

Differences in Vϰ gene utilization and VH CDR3 sequence among anti-DNA from C3H-lpr mice and lupus mice with nephritis

To investigate the molecular properties of anti-DNA from lpr mice that express high levels of anti-DNA without immune-mediated nephritis, the sequences of VH and Vϰ genes encoding 11 monoclonal anti-DNA antibodies derived from C3H-lpr/lpr (C3H-lpr) mice were studied. All of the C3H-lpr monoclonal anti-DNA bound single-stranded DNA while five also bound double-stranded DNA. Two of the hybridomas were clonally related as determined by Southern analysis and sequencing. Sequence analysis of C3H-lpr anti-DNA revealed the use of VH genes that encode anti-DNA from the MRL-lpr/lpr and (NZB X NZW)F1 mouse models of lupus, although differences occurred in the VH CDR3 amino acid content. In contrast, the Vϰ genes from C3H-lpr mice lacked significant identity with previously reported Vϰ genes for anti-DNA from lupus models. These results indicate that anti-DNA from C3H-lpr mice differ from anti-DNA from lupus mice with nephritis in patterns of V gene expression and suggest a molecular basis for the lack of pathogenicity of anti-DNA in these mice.     

Analysis of variable region genes encoding anti-Sm and anti-cardiolipin antibodies from a systemic lupus erythematosus patient.

We have analysed the heavy and light chain variable region genes of two monoclonal antibodies, specific for the Sm antigen (RSP1; IgG kappa) and for cardiolipin (RSP4; IgM lambda), derived from a patient with active systemic lupus erythematosus (SLE). We have established that the variable region genes of the RSP1 autoantibody are somatic mutants of two germ line genes from the VH4 and V kappa 1 gene families. RSP4 antibody uses gene segments closely related to a VH3 gene member and to a V lambda 1 gene. The presence and distribution of the somatic mutations on both monoclonal autoantibodies are compatible with an antigen-driven immune process. These data suggest that in SLE a common antigenic stimulus may govern the autoantibody response against a wide spectrum of unrelated antigens, including native DNA, cardiolipin or Sm antigens, and provide further evidence that disease-associated autoantibodies are generated through antigen-selected somatic mutations.     

Isolation and molecular characterization of the B cells producing the paraprotein in a case of benign monoclonal gammapathy in C57BL mice

Benign monoclonal gammapathy (BMG) is defined as a benign monoclonal B cell proliferative disorder characterized by the presence of a persisting component of homogenous immunoglobulins (H-Ig) in the serum. A possible role of antigenic stimulation in the development of BMG has been suggested. From a C57BL mouse, a murine model for BMG, we have isolated clonally related B cells in order to investigate the occurrence of somatic mutations in the variable heavy chain (VH) region of the genes of H-Ig-producing B cell clones. Therefore, B cells were immortalized by hybridoma technology. The hybridomas were screened for resemblance of the serum H-Ig component by Wieme agar electrophoresis, followed by immunoblotting and isoelectrofocusing. Clonal relationship was investigated by Southern blot analysis using a JH probe. In this way we isolated five hybridomas producing an IgG2a, kappa that was identical to the original serum H-Ig component according to testing with anti-idiotypic antisera. mRNA sequencing of four hybridomas showed only one base pair difference in the VH genes. This particular gene belonged to the J558 VH gene family. When compared to the most closely related known VH sequence, three base pair differences were found. The almost complete absence of base pair differences in the VH genes of the four sequenced hybridomas, compared with an independently derived hybridoma, suggests that the same germ-line VH gene has been used and that somatic mutations were infrequent in our BMG clone.     

Variable region cDNA sequences of three mouse monoclonal anti-idiotypic antibodies specific for anti-alpha(1----6)dextrans with groove- or cavity-type combining sites.

The variables regions of three syngeneic anti-idiotypic antibodies (Ab2s) were cloned and sequenced. They are encoded by different VL genes, two are from different members of V kappa-Ox1 superfamily. The H chains are encoded by VH genes belonging to three different VH families, J558, Q52 and 7183. Together with a previous report from this laboratory, the nucleotide sequences of four Ab2s to anti-alpha(1----6)dextrans have been presented. They are derived from a number of unrelated germline genes, and differ from similar studies in anti-NP, anti-GAT and anti-Ars systems. Three of four Ab2s in the anti-alpha(1----6)dextran system appear to have D-D fusions, which has also been reported in several other Ab2s.     

The VH gene sequences of anti-DNA antibodies in two different strains of lupus-prone mice are highly related

The heavy and light chain V region sequences of an IgG anti-DNA autoantibody (PME77), derived from a lupus-prone (NZB x NZW)F1 mouse have been determined by mRNA sequencing. The V kappa gene segment belongs to the V kappa 1A gene sub-group and is found in several (NZB x NZW)F1 and MRL lpr/lpr anti-DNA antibodies, as well as in other antibodies of unrelated specificities. The VH gene segment appears to represent a unique gene or a subfamily of the large J558 VH gene family of the mouse, and is highly related to a germ-line sequence of a major anti-DNA idiotype (H130, IgM) of MRL mice. This anti-DNA-related VH segment has not been found, so far, to be expressed in antibodies with specificities for external or synthetic antigens; therefore, expression of such specificities may be regulated by powerful mechanisms of self tolerance in the healthy animal. In addition, both the heavy and light chain of the PME77 IgG antibody were found to contain somatic point mutations with a high ratio of replacement to silent mutations in complementarity determining regions. This IgM to IgG sequence relationship suggests an affinity maturation process, which is driven by the autoantigen.