The smaller human VH gene families display remarkably little polymorphism.

We report the nucleotide sequence of 30 distinct human VH gene segments from the VHIV, VHV and VHVI gene families. When these sequences were compared to previously published sequences from these smaller human VH families a surprisingly low level of polymorphism was noted. Two VHIV gene segments from unrelated individuals were identical to two previously published VHIV sequences. Five VHV sequences were identical and seven VHVI gene segments were identical. Where differences were found between the sequences, allele specific oligonucleotide probes were used to verify the germline nature of the change and to test for segregation in several large kindreds. These data provide evidence that at least some human VH gene segments are remarkably stable.     

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.     

Nucleotide sequence analysis of the V regions of two IgM cold agglutinins. Evidence that the VH4-21 gene segment is responsible for the major cross-reactive idiotype.

Cold agglutinins are human autoantibodies, usually of the IgM class, which agglutinate RBC at low temperature. The major subset recognizes the I/i carbohydrate Ag, and many of these antibodies bear cross-reacting idiotypic determinants. An anti-idiotypic mAb that is specific for one of the idiotopes largely confined to cold agglutinins has been used to identify and monitor tumor cells that secrete these molecules in two patients. The tumor cells were immortalized with EBV and the idiotope-positive lines used to investigate the utilization of the VH and VL genes by these antibodies. Nucleotide sequence analysis of the two cold agglutinins (FS-1 and FS-2) revealed the utilization of a single common gene segment, VH4-21. Serologic analysis documented that only human antibodies utilizing the VH4-21 gene segment were reactive in the idiotope assay, other VHIV antibodies as well as a panel of antibodies derived from other VH families being negative. The DH, JH, VK, and JK gene segments of FS-1 and FS-2 were structurally distinct. These data suggest that the structural basis for the cross-reactive idiotope as well as cold agglutinin activity is the VH4-21 gene segment. A nucleotide change in H chain CDR1 of both cold agglutinins results in the substitution of an aspartic acid residue for glycine at position 31, suggesting that this amino acid might be critical to recognition of the red cell Ag.     

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.     

Sequence analysis of members of the human Ig VH4 gene family derived from a single VH locus. Identification of novel germ-line members.

We have generated a mouse x human heterohybridoma that contains a single copy of chromosome 14 and, thus, a haploid set of Ig VH genes. This cell line was used to investigate the germ-line content and nucleotide sequences of members of the VH4 gene family in a polymerase chain reaction-based approach. The analysis of 58 full-length sequences revealed the presence of 12 different germ-line VH4 genes, each of which is potentially functional. These germ-line VH4 genes were compared with the nucleotide sequences of published VH4 genes. Three VH4 genes were 100% identical to previously published sequences and belong to a group of VH4 genes that are strongly conserved and highly prevalent in the human population. Three VH4 genes in our collection displayed greater than 99.3% sequence identity with reported germ-line VH4 sequences and likely represent allelic counterparts of these genes. Six genes displayed less than 97.2% sequence identity with published VH4 genes and were identified as novel members of the human VH4 gene family or more distantly related alleles of known VH4 genes. Collectively, these data suggest that, overall, the human VH4 gene family may be more diverse than hitherto assumed, whereas a number of individual members are nonpolymorphic and extremely well conserved.     

VH repertoire in progeny of long term lymphoid-cultured cells used to reconstitute immunodeficient mice

VH gene utilization in the progeny of long term lymphoid-cultured cells used for reconstitution of severe combined immunodeficient mice under varying conditions was determined. Hybridomas made from the spleens of these animals were evaluated for clonality and donor origin and a panel of 146 independent hybridomas were subsequently examined for VH expression. Hybridomas derived from the spleens of SCID mice reconstituted with fresh cells, used as a control, utilized VH families in proportion to their numerical representation in the genome. However, hybridomas from the spleens of mice reconstituted with long term cultured cells utilized a predominance of the two VH gene families most proximal to JH, characteristic of cells early in B lymphocyte development. Coinjection of thymocytes with cultured fetal liver cells, to provide good levels of T lymphocytes, did not alter this pattern of VH utilization. Irradiation (3 Gy) of the mice before cultured cell injection, which leads to more complete reconstitution of the B cell compartment, was effective in removing this bias in the VH repertoire. Hybridomas derived from these mice expressed their VH genes more in proportion to family size, characteristic of cells later in B lymphocyte development. In this manner, long term lymphoid-cultured cells can be used to study the transitions that occur in VH repertoire expression which appear to be mediated by either B lymphocyte developmental microenvironment or population size.     

VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules

The immune response to beta-(1,6)-galactan in the BALB/c mouse has been well characterized and includes the amino acid sequence determination of 13 monoclonal antibodies. The genetic potential encoding the VH regions of these antibodies has been determined by isolation and sequencing of homologous germline genes. The germline repertoire encoding these proteins was found to consist of two closely related genes. One of these directly encodes the VH segments of seven Gal-binding proteins, and the second directly encodes one additional protein sequence. Sequence variations found in the VH regions of five other Gal-binding proteins can be explained by somatic mutations leading to single base substitutions in the more frequently used gene. Since four of the hybridoma proteins exhibiting somatic mutations are of the IgM class, these results indicate that somatic mutation, in this system, is not associated with class switching and can apparently be initiated early in B-cell development. The two Gal genes are the only members of a very restricted multigene family and probably result from a gene duplication estimated to occur 1.4-2.8 million years ago. Three other genes hybridizing at moderate stringency to a VHGal probe were also sequenced and were found to be members of two additional VHIII families. Studies of the silent to replacement substitution ratios of these and other VH genes indicate that the number of silent substitutions found in immunoglobulin VH genes is lower than expected when compared with proteins such as preproinsulin and globin. Analysis of base composition reflected in these sequences indicates a marked increase of A-T% in the first and second codon positions of complementarity determining regions (CDR) which may be important in facilitating point mutations.     

Structural relationships among mouse and human immunoglobulin VH genes in the subgroup III

The mouse VHIII subgroup is composed of four families which share sequence homology. We isolated a VH germ-line genomic clone, which cross hybridizes with a cDNA probe from one of these families, derived from a myeloma secreting an antigalactan antibody. We report here the nucleotide sequence of the cross hybridizing gene and show that very likely it has an anti-sheep red blood cell specificity. Comparison of its nucleotide sequence with those of the three other VHIII families shows that these genes share segmental homologies of variable lengths. This suggests that interchanges of sequence blocks between VH genes could be an important evolutionary mechanism for diversifying the germ-line repertoire. The strong homology (82%) with human VHIII genes suggests that efficient antibody sequences are strongly conserved. This conservation of homology is particularly striking when compared to the more limited homology (63%) between mouse and human C kappa genes.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

Patterns of Ig V region expression among neonatal hemagglutinin-responsive B cells. Evidence for non-random VH gene representation during later stages of development

Hybridoma libraries were established whose specificities reflect those within the BALB/c hemagglutinin-responsive B cell repertoire at 1 or 2 wk of age. These libraries were generated through chronic immunization regimes that induce responses dominated by clonotypes available at the age of initial immunization. Dot blot analyses of cytoplasmic RNA from these hybridomas were performed to determine the Ig H chain V region (VH) families associated with the repertoire at each age. Although genes from most known VH families can generate hemagglutinin-specific antibodies, clonotypes prevalent during the first week of life disproportionately use VH7183 gene segments. In contrast, hybridomas representative of the repertoire in 2-wk-old individuals preferentially use VHS107, VH36-60, and VHX24 gene segments. These results demonstrate changes in VH gene family predominance that correlate with the age-related patterns of clonal emergence and turnover previously shown in the hemagglutinin-reactive B cell pool. Taken together, these findings suggest that the very early neonatal Ag-responsive B cell pool closely reflects preferential VH gene rearrangements within the pre-B cell compartment. Further, they suggest that either non-random strategies of VH gene expression, or selective clonal expansion strategies based on VH, operate even at later stages of development.     

Neonatal and adult primary B cells use the same germ-line VH and V kappa genes in their (T,G)-A-L-specific repertoire

Although there is a nonrandom usage of VH gene families by primary B cells early in ontogeny, at issue is whether the preferential rearrangement of 3' germ-line VH genes, e.g., VH7183 and VHQ52 family genes, influences the neonatal B cell repertoire that can be expressed in response to Ag. In order to address this issue, and to determine whether neonatal B cells can use the same germ-line VH and V kappa genes as adult B cells in their primary response, we have analyzed at the molecular level the neonatal antibody response to (T,G)-A-L and compared it with the adult primary response. Among the TGB5 Id+, GT+ antibodies, which dominate the neonatal response to (T,G)-A-L, two VH gene families were used: J558 (high frequency) and 36-60 (low frequency). The majority of Id+ neonatal hybridomas used the same germ-line VH gene (H10, from the VHJ558 family), but with enormous diversity in the D region, and one of two germ-line V kappa 1 genes (V kappa 1A, V kappa 1C). These are the same germ-line V-genes used by most primary adult Id+ hybridomas, and the frequency of expression of this germ-line V-gene combination appears equivalent in the neonatal and adult primary repertoires. Therefore, it is clear from this study that as early as day 5, neonatal B cells can use the same germ-line V-genes as adult primary B cells in their Ag-specific repertoire.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.     

VH gene segments in the mouse and human genomes

We have examined the mouse genome sequence to determine its VH gene segment repertoire. In all, 141 segments are mapped to a 3 Mb region of chromosome 12. There is evidence that 92 of these are functional in the mouse strain used for the genome sequence, C57BL/6J; 12 are functional in other mouse strains, and 37 are pseudogenes. The mouse VH gene segment repertoire is therefore twice the size of that in humans. The mouse and human loci bear no large-scale similarity to each other. The 104 functional segments belong to one of the 15 known sequence subgroups, which have been further clustered into eight sets here. Seven of these sets, comprising 101 sequences, are related to five of the human VH families and have the same canonical structures in their hypervariable regions. Duplication of members of one set in the distal half of the locus is mainly responsible for the larger size of the mouse repertoire. Phylogenetic analysis of the VH segments indicates that most of the sequences in the human and mouse VH loci have arisen subsequent to the divergence of the two organisms from their common ancestor.     

Strain-dependent expression of VH gene families

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.     

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.     

The Ig VH repertoire of fetal liver-derived pre-B cells is influenced by the expression of a gene linked to X-linked immune deficiency.

Ig VH repertoire differences between normal and x-linked immune deficiency- (xid) expressing mice are well established. To test the hypothesis that such differences might exist as early as the pre-B stage of ontogeny we generated panels of xid fetal liver derived Abelson murine leukemia virus transformants with H chain Ig VDJ rearrangements. Cells from CBA/Tufts.xid mice used VH genes from many families, with no demonstrable preference for 3' genes. Analysis of cells derived from (CBA/Tufts.xid X CBA/Tufts)F1 mice showed preferential usage of 3' family genes in the phenotypically normal females, even though V to DJ joins were made in vivo. The defective male mice did not show this marked preferential usage. A similar, but less marked, effect on VH gene usage was seen in mice with X-linked immune deficiency and a BALB/c background. Taken together, these results show that either X-linked immune deficiency, or a closely linked gene, affects fetal pre-B cells such that the usual pattern of predominant usage of 3' family genes is altered.     

Antibody repertoire development in fetal and neonatal piglets. XIII. Hybrid VH genes and the preimmune repertoire revisited

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise approximately 80% of the preimmune repertoire. However, so-called "hybrid" VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied > 3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for > 95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.     

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.     

Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus.

We present a detailed analysis of the content and organization of the human immunoglobulin VH locus. Human VH genes representing five distinct families were isolated, including novel members belonging to two out of three of the known VH gene families (VH1 and VH3) as well as members of three new families (VH4, VH5, and VH6). We report the nucleotide sequence of 21 novel human VH genes, many of which belong to the three new VH gene families. In addition, we provide a preliminary analysis of the organization of these gene segments over the full extent of the locus. We find that the five multi-segment families (VH1-5) have members interspersed over nearly the full 1500-2000 kb of the VH locus, and estimate that the entire heavy chain locus covers 2500 kb or less. Finally, we provide the first report of the physical linkage of the variable and constant loci of a human Ig gene family by demonstrating that the most proximal known human VH segments lie within 100 kb of the constant region locus.