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.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

Latent a1 VH germline genes in an a2a2 rabbit. Evidence for gene conversion at both the germline and somatic levels

We have previously reported the sequences of putative latent a1 cDNA derived from an alpha 2 alpha 2 rabbit. Significant similarity to nominal a1 cDNA sequences was noted, but none of the latent sequences were completely a1-like. We have now probed a genomic library, produced from the same alpha 2 alpha 2 rabbit, for evidence of germline latent a1 VH genes. Four hundred ninety-four VH+ clones were screened with oligonucleotides specific for a1 diagnostic regions of framework region 1 (FR1) and FR3. Twenty-two percent of the VH+ clones hybridized with an a1FR3 oligonucleotide probe. Two a1 FR1 probes yielded weak signals with 6% to 13% of the VH+ clones. Twenty VH genes from clones positive for one or more of the a1-specific oligonucleotide probes were sequenced, revealing 14 unique germline VH genes. All but one of these genes were 85% to 92% identical to the VH1-a1 nominal gene prototype, with sequence identity extending into the leader intron. Most genes displayed extended regions of similarity to a1 in FR1, FR3, or both and expressed 13 to 17 of the 21 allotype-associated residues, consistent with the nominal a1 sequence. The a1-like sequences were variously interspersed with short non-a1 segments, suggestive of germline gene conversion. Although none of the germline a1-like VH genes we have isolated from the alpha 2 alpha 2 rabbits are identical to the known a1 genes or protein sequences from alpha 1 alpha 1 rabbits and 8 of 14 are pseudogenes, most could make significant contributions to the synthesis of a complete nominal a1 sequence by serving as a pool of sequence donors during somatic gene conversion.     

Antigen-induced somatic diversification of rabbit IgH genes: gene conversion and point mutation.

During T cell-dependent immune responses in mouse and human, Ig genes diversify by somatic hypermutation within germinal centers. Rabbits, in addition to using somatic hypermutation to diversify their IgH genes, use a somatic gene conversion-like mechanism, which involves homologous recombination between upstream VH gene segments and the rearranged VDJ genes. Somatic gene conversion and somatic hypermutation occur in young rabbit gut-associated lymphoid tissue and are thought to diversify a primary Ab repertoire that is otherwise limited by preferential VH gene segment utilization. Because somatic gene conversion is rarely found within Ig genes during immune responses in mouse and human, we investigated whether gene conversion in rabbit also occurs during specific immune responses, in a location other than gut-associated lymphoid tissue. We analyzed clonally related VDJ genes from popliteal lymph node B cells responding to primary, secondary, and tertiary immunization with the hapten FITC coupled to a protein carrier. Clonally related VDJ gene sequences were derived from FITC-specific hybridomas, as well as from Ag-induced germinal centers of the popliteal lymph node. By analyzing the nature of mutations within these clonally related VDJ gene sequences, we found evidence not only of ongoing somatic hypermutation, but also of ongoing somatic gene conversion. Thus in rabbit, both somatic gene conversion and somatic hypermutation occur during the course of an immune response.     

Coevolution of immunoglobulin heavy- and light-chain variable-region gene families

The gene families encoding the immunoglobulin variable regions of heavy (VH) and light (VL) chains in vertebrates are composed of many genes. However, the gene number and the extent of diversity among VH and VL gene copies vary with species. To examine the causes of this variation and the evolutionary forces for these multigene families, we conducted a phylogenetic analysis of VH and VL genes from the species of amniotes. The results of our analysis showed that for each species, VH and VL genes have the same pattern of clustering in the trees, and, according to this clustering pattern, the species can be divided into two groups. In the first group of species (humans and mice), VH and VL genes were extensively intermingled with genes from other organisms; in the second group of species (chickens, rabbits, cattle, sheep, swine, and horses), the genes tended to form clusters within the same group of organisms. These results suggest that the VH and VL multigene families have evolved in the same fashion: they have undergone coordinated contraction and expansion of gene repertoires such that each group of organisms is characterized by a certain level of diversity of VH and VL genes. The extent of diversity among copies of VH and VL genes in each species is related to the mechanism of generation of antibody variety. In humans and mice, DNA rearrangement of immunoglobulin variable, diversity, and joining-segment genes is a main source of antibody diversity, whereas in chickens, rabbits, cattle, sheep, swine, and horses, somatic hypermutation and somatic gene conversion play important roles. The evolutionary pattern of VH and VL multigene families is consistent with the birth-and-death model of evolution, yet different levels of diversifying selection seem to operate in the VH and VL genes of these two groups of species.      

B lymphocyte selection and age-related changes in VH gene usage in mutant Alicia rabbits.

Young Alicia rabbits use VHa-negative genes, VHx and VHy, in most VDJ genes, and their serum Ig is VHa negative. However, as Alicia rabbits age, VHa2 allotype Ig is produced at high levels. We investigated which VH gene segments are used in the VDJ genes of a2 Ig-secreting hybridomas and of a2 Ig+ B cells from adult Alicia rabbits. We found that 21 of the 25 VDJ genes used the a2-encoding genes, VH4 or VH7; the other four VDJ genes used four unknown VH gene segments. Because VH4 and VH7 are rarely found in VDJ genes of normal or young Alicia rabbits, we investigated the timing of rearrangement of these genes in Alicia rabbits. During fetal development, VH4 was used in 60-80% of nonproductively rearranged VDJ genes, and VHx and VHy together were used in 10-26%. These data indicate that during B lymphopoiesis VH4 is preferentially rearranged. However, the percentage of productive VHx- and VHy-utilizing VDJ genes increased from 38% at day 21 of gestation to 89% at birth (gestation day 31), whereas the percentage of VH4-utilizing VDJ genes remained at 15%. These data suggest that during fetal development, either VH4-utilizing B-lineage cells are selectively eliminated, or B cells with VHx- and VHy-utilizing VDJ genes are selectively expanded, or both. The accumulation of peripheral VH4-utilizing a2 B cells with age indicates that these B cells might be selectively expanded in the periphery. We discuss the possible selection mechanisms that regulate VH gene segment usage in rabbit B cells during lymphopoiesis and in the periphery.     

Organization of human immunoglobulin heavy chain diversity gene loci

The variable region of immunoglobulin heavy chain is encoded by three separate genes on the germline genome: variable (VH), diversity (DH) and joining (JH) genes. Most human DH genes are encoded in 9-kb repeating sequences. We determined the nucleotide sequence of a 15-kb DNA fragment containing more than one and a half of these repeating units, and identified 12 different DH genes. Based on the sequence similarities of DH coding and the surrounding regions, they can be classified into six different DH gene families (DXP, DA, DK, DN, DM and DLR). Nucleotide sequences of DH genes belonging to different families diverge greatly, while those belonging to the same families are well conserved. Since the 9-kb DNA containing the six DH genes are multiplied at least five times, the total number of DH genes must be approximately 30. These DH genes are sandwiched by 12-nucleotide spacer signals. Most of the somatic DH sequences found in the published VH-DH-JH structures (the somatic DH segment being defined as the region which is not encoded either by germline VH or JH gene) were assigned to one of the germline DH genes. Other than these typical DH genes, however, we found a new kind of DH gene (which we termed DIR) the spacer lengths of whose neighbouring signals were irregular. The DIR gene appears to be involved in DIR-DH or DH-DIR joining by inversion or deletion. Two of the somatic DH sequences were assigned to the DIR genes. Long N segments might, therefore, originate from DIR genes.     

The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops.

We have used the polymerase chain reaction and VH family-based primers to clone and sequence 74 human germline VH segments from a single individual and built a directory to include all known germline sequences. The directory contains 122 VH segments with different nucleotide sequences, 83 of which have open reading frames. The directory indicates that the structural diversity of the germline repertoire for antigen binding is fixed by about 50 groups of VH segments: each group encodes identical hypervariable loops. The directory should help in mapping the VH locus, in estimating somatic mutation and VH segment usage and in designing and constructing synthetic antibody libraries.     

Intestinal microflora and diversification of the rabbit antibody repertoire

The rabbit establishes its primary Ab repertoire by somatically diversifying an initial repertoire that is limited by restricted VH gene segment usage during VDJ gene rearrangement. Somatic diversification occurs in gut-associated lymphoid tissue (GALT), and by about 1-2 mo of age nearly all Ig VDJ genes are somatically diversified. In other species that are known to establish their primary Ab repertoire by somatic diversification, such as chicken, sheep, and cattle, diversification appears to be developmentally regulated: it begins before birth and occurs independent of exogenous factors. Because somatic diversification in rabbit occurs well after birth in GALT, the diversification process may not be developmentally regulated, but may require interaction with exogenous factors derived from the gut. To test this hypothesis, we examined Ab repertoire diversification in rabbits in which the appendix was ligated shortly after birth to prevent microbial colonization and all other organized GALT was surgically removed. We found that by 12 wk of age nearly 90% of the Ig VDJ genes in PBL were undiversified, indicating that intestinal microflora are required for somatically diversifying the Ab repertoire. We also examined repertoire diversification in sterilely derived remote colony rabbits that were hand raised away from contact with conventional rabbits and thereby acquired a different gut microflora. In these remote colony rabbits, GALT was underdeveloped, and 70% of the Ig VDJ genes in PBL were undiversified. We conclude that specific, currently unidentified intestinal microflora are required for Ab repertoire diversification.     

Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes.

Four potentially productive and two non-productive VDJ gene segments were isolated from the DNA of mouse B-lymphocytes which had been polyclonally activated by bacterial lipopolysaccharide (LPS). Three VDJ regions exhibit VH genes which stem from two novel VH gene families. The complexity of these families is 5-9 genes. One of the non-productive VDJ regions exhibits a D segment which may have been generated by joining of two DSP2 segments. Both non-productive VDJ regions appear to contain rearranged pseudo VH genes. Three potential somatic mutations distributed over two productive VDJ regions are observed.     

iHMMune-align: hidden Markov model-based alignment and identification of germline genes in rearranged immunoglobulin gene sequences

MOTIVATION: Immunoglobulin heavy chain (IGH) genes in mature B lymphocytes are the result of recombination of IGHV, IGHD and IGHJ germline genes, followed by somatic mutation. The correct identification of the germline genes that make up a variable VH domain is essential to our understanding of the process of antibody diversity generation as well as to clinical investigations of some leukaemias and lymphomas. RESULTS: We have developed iHMMune-align, an alignment program that uses a hidden Markov model (HMM) to model the processes involved in human IGH gene rearrangement and maturation. The performance of iHMMune-align was compared to that of other immunoglobulin gene alignment utilities using both clonally related and randomly selected IGH sequences. This evaluation suggests that iHMMune-align provides a more accurate identification of component germline genes than other currently available IGH gene characterization programs. AVAILABILITY: iHMMune-align cross-platform Java executable and web interface are freely available to academic users and can be accessed at http://www.emi.unsw.edu.au/~ihmmune/.     

Relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity in the mouse

The relative contributions of germline gene variation and somatic mutation to immunoglobulin diversity were studied by comparing germline gene sequences with their rearranged counterparts for the mouse VH, V kappa, and V lambda genes. The mutation rate at the amino acid level was estimated to be 7.0% in the first and second complementarity- determining regions (CDRs) and 2.0% in the framework regions (FRs). The difference in the mutation rate at the nucleotide level between the CDRs and FRs was of the same order of magnitude as that for the amino acid level. Analysis of amino acid diversity or nucleotide diversity indicated that the contribution of somatic mutation to immunoglobulin diversity is approximately 5%. However, the contribution of somatic mutation to the number of different amino acid sequences of immunoglobulins is much larger than that estimated by the analysis of amino acid diversity, and more than 90% of the different immunoglobulins seem to be generated by somatic mutation. Examination of the pattern of nucleotide substitution has suggested that clonal selection after somatic mutation may not be as strong as generally believed.      

Evolution of immunoglobulin VH pseudogenes in chickens

In chickens, there is a single functional gene (VH1) coding for the heavy chain variable region of immunoglobulins, and immunoglobulin diversity is generated by gene conversion of the VH1 gene by many variable region pseudogenes (psi VH's) that exist on the 5' side of the VH1 gene. To understand the evolution of this unique genetic system, we conducted statistical analyses of VH1 and psi VH genes together with functional VH genes from other higher vertebrate species. The results indicate, first, that chicken VH genes are all closely related to one another and were derived relatively recently from an ancestral gene belonging to one of the three major groups of VH genes in higher vertebrates. Second, the rate of nonsynonymous substitution is slightly higher than that of synonymous substitution in the complementarity- determining regions (CDRs), which suggests that diversity-enhancing selection has operated in the CDRs even for pseudogenes. However, both the rates of synonymous and nonsynonymous substitution are higher in the CDRs than in the framework regions (FRs), apparently because of an interaction between positive selection and meiotic gene conversion in the CDRs. Third, a dot matrix analysis of the psi VH genes and genomic diversity (D) genes has indicated that the 3' end of psi VH genes is attached by D-gene-like sequences, and this region of psi VH genes has high similarity with D gene sequences. This suggests that V and D genes were fused at some point of evolutionary time and this fused element multiplied by gene duplication. Finally, two alternative hypotheses of explaining the evolution of the chicken VH gene system are presented.      

Detection of a unique human V kappa IV germline gene by a cloned cDNA probe.

We have cloned the cDNA encoding the KIV chain of a human antibody with specificity against the major carbohydrate antigen of Streptococcus A. The cDNA has been used as a genetic probe to estimate the number of germline VKIV genes in human DNA. The presence of unique hybridizing bands on digestion of human DNA with several restriction endonucleases and the equivalence of the DNA in a band to a single gene per haploid genome point to the conclusion that there is a unique human VKIV germline gene. The corollary of this conclusion is that the diversity of human VKIV chains must be exclusively due to somatic mutation. This is supported by examination of the sequences of human KIV chain genes and their KIV chain products. Fusion of the unique germline VKIV gene (1) with one of several JK segments, followed by somatic mutations in the V region of the rearranged KIV gene, can account for the known sequences. The restricted germline gene repertoire may account for the small proportion of human KIV chains in the human K chain sequence library (2).     

Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.     

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.     

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.     

Expressed antibody repertoires in human cord blood cells: 454 sequencing and IMGT/HighV-QUEST analysis of germline gene usage, junctional diversity, and somatic mutations

Human cord blood cell-derived IgM antibodies are important for the neonate immune responses and construction of germline-based immunoglobulin libraries. Several previous studies of a relatively small number of sequences found that they exhibit restrictions in the usage of germline genes and in the diversity of the variable heavy chain complementarity determining region 3 compared to adults. To further characterize such restrictions on a larger scale and to compare the early B-cell diversity to adult IgM repertoires, we performed 454 sequencing and IMGT/HighV-QUEST analysis of cord blood IG libraries from two babies and determined germline gene usage, V-D-J rearrangement, VHCDR3 diversity, and somatic mutations to characterize human neonate repertoire. Most of the germline subgroups were identified with frequencies comparable to those present in the adult IgM repertoire except for the IGHV1-2 gene that was preferentially expressed in the cord blood cells. The gene usage diversity contributed to 1,430 unique IGH V-D-J rearrangement patterns while the exonuclease trimming and N region addition at the V-D-J junctions along with gene diversity created a wide range of VHCDR3 with different lengths and sequence variability. We observed a lower degree of somatic mutations in the CDR and framework regions of antibodies from cord blood cells compared to adults. These results provide insights into the characteristics of human cord blood antibody repertoires, which have gene usage diversity and VHCDR3 lengths similar to that of the adult IgM repertoire but differ significantly in some of the gene usages, V-D-J rearrangements, junctional diversity, and somatic mutations.     

Restricted utilization of germ-line VH genes and diversity of D regions in rabbit splenic Ig mRNA

Previous studies have suggested that the majority of rabbit germ-line VH genes encode molecules that are rarely found in serum or secretory Ig. To examine the repertoire of expressed VH genes, we prepared a cDNA library from splenic mRNA of an alpha 1/alpha 1 rabbit and isolated 10 complete VH-encoding cDNA clones. None of the cDNA clones hybridized to an oligomer that had hybridized to more than 50% of cloned germ-line VH genes. These data indicate that only a subset of germ-line VH genes are used in functional VDJ rearrangements. DNA sequence analysis demonstrated that the 10 cDNA clones contained highly similar VH regions, further suggesting that the repertoire of utilized VH genes is limited. In contrast, the D regions of each of the 10 clones exhibited little similarity to one another, suggesting that the rabbit has a large D region repertoire. We propose that the apparent lack of diversity within the VH segment of VDJ rearrangements is offset by extensive D region diversity.