Immunoglobulin Diversity Research Articles

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.

Immunoglobulin diversity, B-cell and antibody repertoire development inlarge farm animals

The B-cell lineage, the antibodies produced by these cells and the diversification of the antibody repertoire are essential for the health and survival of all mammals. Cattle, sheep, swine and horses, unlike rodents and primates, develop their antibody repertoire from a relatively small number of VH (variable heavy) genes of one or several families and cattle, sheep and horses use almost exclusively lambda-light chains. These large farm animals appear to use templated mutation (gene conversion) in addition to untemplated (point) mutation in repertoire development; this may occur predominantly in the ileal Peyer's patches. Whether B-cell lymphogenesis is continuous throughout life--as in rodents and primates--or whether B cells are largely of the B-1 lineage and develop only in foetal and neonatal life, is uncertain. The fact that immunoglobulin D (IgD) is totally absent from swine and ruminants may be significant, as IgD is expressed weakly on rodent B-1 cells. Information on IgG subclass diversity in large farm animals is incomplete, except for sheep and cattle, and no information is available for any large farm animal to show whether T helper 1 (Th1) and Th2 responses correlate with the expression of any subclass antibody response, as is the case in rodents. All large farm animals exclusively use the mammary gland to transfer immunity to offspring, although the receptor involved in the transport of IgG into colostrum and milk has not been characterised. Efforts to standardise the nomenclature and measurements of antibodies and immunoglobulins in large farm animals are discussed, and a proposal currently under review concerning the standardisation of the nomenclature for bovine immunoglobulins is presented as a model.

Incidence of rat-soluble placental factors on IgE and IgG2a synthesis.

The in vivo effect of soluble factors present in placental culture supernatants (PCSs) on the synthesis of rat immunoglobulin E (IgE) and IgG2a isotypes was investigated. Batches of Wistar SPF rats immunized with a 10-microgram dose of ovalbumin and Al(OH)3 were used: group I, consisted of virgin rats; group II, virgin females injected simultaneously with PCSs; and group III, pregnant females. As controls, nonimmunized batches were included. Serum samples were collected at days 0 (basal) and 10 after antigen challenge, determining levels of total and specific antiovalbumin of both IgE and IgG2a by enzyme-linked immunoadsorbent assay (ELISA). In vivo and at least at the doses administered, PCSs exert an inhibitory effect on the synthesis of specific and total anti-ovalbumin IgE during the course of immune response to such challenge. However, PCSs did not modify serum values of total and specific IgG2a. These results suggest that PCSs exert selective influence on the synthesis of diverse immunoglobulin isotypes during immune response, through the balance of cytokines synthesized by placental cells.

Evaluation of different lymphoid tissue sources for the construction of human immunoglobulin gene libraries

Background: Phage display technology allows the isolation of novel human monoclonal antibodies. The technology relies on the construction of a recombinant antibody library and its display on phage particles. The quality of an antibody library is affected by several factors including the size, diversity and source of immunoglobulin genes.Objective: The aim of the project was to determine the best tissue source for the construction of antibody libraries.Study design: Three tissue sources were used in this study: peripheral blood mononuclear cells from a healthy donor, Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells and lymph node tissue from individuals with breast cancer. The quality of each tissue source was assessed using two criteria: (1) the number of mature and activated B cells in each source; (2) the amount of immunoglobulin heavy and light chain genes amplifiable by polymerase chain reaction (PCR). Results: EBV-transformed peripheral blood mononuclear cells and lymph node tissue were shown to contain more B cells than peripheral blood mononuclear cells. A relatively larger amount of immunoglobulin gene products could be amplified from EBV-transformed peripheral blood mononuclear cells and the lymph node. However, immunoglobulin containing γ1 chains could not be amplified from EBV-transformed mononuclear cells, and the resultant pattern of gene amplification suggests a possible selection bias. Conclusion: This study indicates that among the three tissue sources examined, lymph node tissue is the most suitable source for the construction of antibody libraries.

Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D-D recombination

We have determined the complete nucleotide sequence of the human immunoglobulin D segment locus on chromosome 14q32.3 and identified a total of 27 D segments, of which nine are new. Comparison with a datbase of rearranged heavy chain sequences indicates that the human antibody repertoire is created by VDJ recombination involving 25 of these 27 D segments, extensive processing at the V-D and D-J junctions and use of multiple reading frames. We could find no evidence for the proposed use of DIR segments, inverted D segments, “minor” D segments or D-D recombination. Conventional VDJ recombination, which obeys the 12/23 rule, is therefore sufficient to explain the wealth of lengths and sequences for the third hypervariable loop of human heavy chains.

Immunoglobulin Gene Diversification in Cattle

Research in several species has revealed that different types of mammals have evolved divergent molecular and cellular strategies for generating immunoglobulin diversity. Other chapters in this text have highlighted the specific characteristics unique to chicken, rabbit, mouse, human and sheep B lymphocyte development; namely indicating differences in the mechanisms of diversity and the site of primary B cell development. Studies of the bovine system have indicated that like the sheep system, the ileal Peyer's patch (IPP) is a likely chicken bursal equivalent, and is a site of primary B lymphocyte development. Substantial investigation in sheep has indicated that Ig diversity is created by untemplated somatic mutation and intense selective pressure (Reynaud et al., 1991). The frequency of alteration in the sheep Ig light chain gene locus also is characteristic of the bovine system, however, recent evidence from sequencing of bovine lambda light chain genes indicates that one mechanism that contributes to diversity is gene conversion, utilizing several pseudogenes located in the Ig locus (Parng et al., 1996). The mechanism by which this hyperalteration of Ig genes occurs in both sheep and cattle is poorly understood and is thus the focus of considerable investigation. The study of events in the IPP may also have informative ramifications for secondary diversification of the Ig repertoire by somatic hyperalteration in germinal centers.

The V H and C H immunoglobulin genes of swine: implications for repertoire development

Swine have the largest number of IgG subclass genes of all species so far studied but have a single gene for IgA which occurs in two allelic forms that differ in hinge length. Swine also have constant region genes for Cμ and Cϵ, but lack a gene homologous to that which encodes IgD in rodents and primates, despite the otherwise high degree of sequence similarity of all other swine C H genes with those of humans. Swine have < 20 V H genes, a single J H and perhaps a limited number of D H segments. Newborn piglets show preferential V H and D H usage and may use gene conversion as a mechanism for expanding their antibody repertoire. Despite the close similarity of their Ig gene sequences to humans, swine belong to the group of animals that includes rabbits, chickens and cattle when classified on the basis of B cell development. This group, unlike rodents and humans, have a single V H family, use hindgut follicles early in life (rather than bone marrow throughout life) to diversify their antibody repertoire and probably all use gene conversion. It is proposed that IgD may serve a function in repertoire development in rodents and humans which is unnecessary in the chicken-lagomorph-artiodactyl group. The diversity of immunoglobulins and immunoglobulin genes among species justifies the quest of veterinary immunologists to define the system for their species of interest rather than making extrapolations from mouse and human immune systems.

Immunoglobulin diversity: Rearranging by cutting and repairing

The recombination process that assembles antigen-receptor genes is now understood in some biochemical detail. The initial steps reflect a common theme seen in retroviral integration and prokaryotic transposition, and the later steps involve the enzymatic machinery for double-strand break DNA repair.

Evidence of recombination and an antigenically diverse immunoglobulin A1 protease among strains of Streptococcus pneumoniae

The genetic relationships among 114 isolates of Streptococcus pneumoniae representing mainly nine serotypes that frequently cause severe childhood disease in Northern Europe were examined by use of multilocus enzyme electrophoresis. A comparison was made of the corresponding antigenic variations of excreted immunoglobulin A1 (IgA1) proteases detected by enzyme neutralization assays. Allelic variation at 13 gene loci among 70 electrophoretic types disclosed a comparatively low mean genetic diversity per locus (H = 0.319). In contrast, IgA1 proteases showed extensive antigenic diversity as 17 different inhibition types were distinguished. A lack of overall clonality was apparent from the linkage equilibrium of alleles harbored by 28 isolates chosen to represent the genetic diversity of the study population. However, certain clones, such as those marked by identical electrophoretic type, serotype, and IgA1 protease type, persisted for a sufficiently long time to enable clonal spread between distant geographic areas. Among clonally related isolates, examples illustrating a shift of capsular serotype or IgA1 protease type supported the view that recombination occurs in vivo in corresponding genes. In conclusion, over time, horizontal genetic exchange appears to be sufficiently frequent to disrupt the clonal structure otherwise generated by binary fission in natural populations of S. pneumoniae. The clonal instability combined with considerable antigenic heterogeneity renders the pneumococcal IgA1 protease less attractive as a potential component of future vaccines.

Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes

In chickens, single functional immunoglobulin variable and joining gene segments at each of the heavy and light chain loci undergo V(D)J rearrangement. Diversity is subsequently introduced by conversions templated by upstream pseudo V region genes in such a way that practically all V regions in mature B cells have identical ends. This greatly simplifies the representative amplification of V region genes. Furthermore, the entire naive repertoire of the adult chicken is produced in the bursa of Fabricius of the young bird. These special properties of the generation of immunoglobulin diversity in chickens have been exploited in the development of procedures to produce large libraries of diverse antibody combining sites derived from chicken Ig genes and expressed on filamentous bacteriophage. The utility of this library was assessed by selection of specifically binding phage using three solid phase-bound protein antigens, hen egg white lysozyme, bovine thyroglobulin and bovine serum albumin. The sequences of the V region genes thus isolated demonstrated that selection was specific and that the library contained useful diversity of binding sites. This library provides access to a repertoire whose diversity is based on a mechanism different from that underlying previously available libraries. The demonstrated feasibility of generating chicken phage antibodies may lead to the production of monoclonal reagents from immunised chickens, and the derivation of reagents for studying immunoglobulin mediated selection in avian B cell development.

Divergent evolution and evolution by the birth-and-death process in the immunoglobulin VH gene family.

Immunoglobulin diversity is generated primarily by the heavy- and light-chain variable-region gene families. To understand the pattern of long-term evolution of the heavy-chain variable-region (VH) gene family, which is composed of a large number of member genes, the evolutionary relationships of representative VH genes from diverse organisms of vertebrates were studied by constructing a phylogenetic tree. This tree indicates that the vertebrate VH genes can be classified into group A, B, C, D, and E genes. All VH genes from cartilaginous fishes such as sharks and skates from a monophyletic group and belong to group E, whereas group D consists of bony-fish VH genes. By contrast, group C includes not only some fish genes but also amphibian, reptile, bird, and mammalian genes. Group A and B genes were composed of the genes from mammals and amphibians. The phylogenetic analysis also suggests that mammalian VH genes are classified into three clusters--i.e., mammalian clans I, II, and III-and that these clans have coexisted in the genome for >400 Myr. To study the short-term evolution of VH genes, the phylogenetic analysis of human group A (clan I) and C (clan III) genes was also conducted. The results obtained show that VH pseudogenes have evolved much faster than functional genes and that they have branched off from various functional VH genes. There is little indication that the VH gene family has been subject to concerted evolution that homogenizes member genes. These observations indicate that the VH genes are subject to divergent evolution due to diversifying selection and evolution by the birth-and-death process caused by gene duplication and dysfunctioning mutation. Thus, the evolutionary pattern of this monofunctional multigene family is quite different from that of such gene families as the ribosomal RNA and histone gene families.

Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.

To study the influence of immunoglobulin heavy-chain (HC) and light-chain (LC) expression in promoting B-cell differentiation, we have introduced functional immunoglobulin HC and/or LC transgenes into the recombinase activating gene-2-deficient background (RAG-2-/-). RAG-2-/- mice do not undergo endogenous V(D)J rearrangement events and, therefore, are blocked in B- and T-cell development at the early pro-B- and pro-T-cell stages. Introduction of immunoglobulin HC transgenes into the RAG-2-/- background promotes the development of a B-lineage cell population that phenotypically has the characteristics of pre-B cells. We have shown further that this population has altered growth characteristics as measured by interleukin-7 responsiveness in culture. Bone marrow cells from immunoglobulin HC transgenic RAG-2-/- mice have up-regulated expression of germ-line kappa LC gene transcripts and down-regulated expression of lambda 5 surrogate LCs (SLCs). Although mu HC/SLC complexes are detectable intracellularly in HC/RAG-2-/- pre-B-cell populations, HC expression is not readily detectable on the surface of these cells. lambda LC RAG-2-/- mice had a bone marrow B-lineage cell phenotype indistinguishable from that of RAG-2-/- littermates, indicating that LC expression by itself has no influence on pro-B cell differentiation. Strikingly, simultaneous introduction of mu HC and lambda LC transgenes into RAG-2-/- mice led to the generation of a substantial population of "monoclonal" peripheral B-cells that were functional with regard to immunoglobulin secretion, indicating that T cells or diverse immunoglobulin repertoires are not necessary for peripheral B-cell development.

Recombinators, recombinases and recombination genes of yeasts

Genetic recombination in the nuclear, organellar, and plasmid genomes of eukaryotic organisms occurs during mitosis and meiosis. Mitotic recombination is an important mechanism for the repair of DNA damaged by ultraviolet light, ionizing radiation, and chemical agents (Friedberg et al. 1991). Moreover, nuclear chromosomal mitotic recombination is involved in a number of basic cellular processes seen in a wide variety of eukaryotic systems including: cell-type differentiation of homothallic budding yeast (Saccharomyces cerevisiae) (Strathern et al. 1982) and fission yeast (Schizosaccharomyces pombe) (Egel et al. 1984); transpositions of P-elements in Drosophila melanogaster (Gloor et al. 1991); mammalian and human site-specific V-D-J gene rearrangements that give rise to immunoglobulin and T-cell receptor diversity (Oettinger et al. 1990; Kallenbach and Rougeon 1992); and loss of heterozygosity implicated in carcinogenesis (Solomon et al. 1991). Meiotic recombination plays a key role in ensuring accurate chromosome segregation at the first meiotic division (the reductional division). Additionally, meiotic interchromosomal and intrachromosomal recombination of homologous DNA sequences between members of multigene families, such as the mammalian major histocompatibility complex, MHC, (Kuhner et al. 1991) and the isoaccepting multiple tRNA genes of fission yeast (Amstutz et al. 1985), contributes to allelic diversity and concerted evolution of members of multigene families. These recombinational events and conventional allelic meiotic gene conversion, crossing over, and independent assortment, produce an abundance of gametic genotypes.

Germ Line Maintenance of the Pseudogene Donor Pool for Somatic Immunoglobulin Gene Conversion in Chickens

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.

Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens.

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.

Phenotype and function of stromal cells cloned from the ileal Peyer's patch of sheep.

The ileal Peyer's patch (PP) is the major site of B cell production and immunoglobulin diversification in lambs, but the factors which regulate these processes are poorly understood. As a first step toward identifying possible regulatory mechanisms, stable long-term cultures of ileal PP stromal cells were established at the clonal level. Four distinct cell types were identified by their phenotype and growth requirements. Immunohistochemical staining confirmed that all clones were mesenchymal (vimentin+; cytokeratin-) in origin and were negative for T cell, B cell, and macrophage markers. Three cell lines were negative for major histocompatibility complex (MHC) I and II molecules, but one cell line, SCN, expressed MHC I, MHC II and CD44 molecules, and a subpopulation of SCN cells expressed BAQ44A, a B cell differentiation molecule. The four cell lines produced different types and amounts of extracellular matrix proteins, and their growth was not influenced by exogenous human interleukin 1 (IL-1), IL-2, transforming growth factor beta 1 (TGF beta 1), or bovine fibroblast growth factor (FGF) but was influenced by serum. When tested for their capacity to support lymphocyte growth, all clones produced a soluble factor(s) that was mitogenic for ileal and jejunal PP cells and thymocytes. Similar growth promoting activity was observed with culture supernatants of murine, human and bovine fibroblasts but could not be reproduced using recombinant human cytokines. Furthermore, coculture of stromal cells with ileal PP follicular B cells elicited a proliferative response unique to each stromal cell line. Coculture with increasing numbers of SCN cells inhibited B cell proliferative responses, whereas coculture with SCG2 and SCF32 cells enhanced B cell proliferative response at both low and high stromal cell densities. Ileal PP follicular B cells rapidly bound to the surface of all stromal cell clones, and this interaction was specific when compared with thymocytes or peripheral blood lymphocytes. These results suggest that ileal PP stromal cells are a phenotypically and functionally heterogeneous population that may enhance or inhibit B lymphopoiesis in the ileal PP.

Mechanisms that generate human immunoglobulin diversity operate from the 8th week of gestation in fetal liver

The repertoire of immunoglobulin expressed very early in human development was approached by cloning and sequencing 55 rearranged and 11 germ-line VH transcripts, after amplification by polymerase chain reaction of cDNA libraries derived from two fetal livers at 8 and 13 weeks of gestation. All families with the exception of VH2, were expressed as soon as 8 weeks, with preferential usage of certain germ-line genes. Very few somatic mutations, randomly localized, were identified. By contrast, in a series of clones derived from the same VDJ rearrangement using the VH6 family, extensive mutations had taken place, mostly accumulated in the third complementarity-determining region (CDR3) suggesting that the specialized enzymatic machinery was at hand very early during human development. Some other characteristics of the fetal repertoire also emerged, namely increased usage of JH3 and JH2, as compared to the adult pattern, where JH4 is dominant and reduced length of the D/CDR3 regions. All D gene families were identified, and their usage frequently involved D-D fusions. N diversity was present very early, and increased with age. Identification of germ-line transcripts pertaining to all six VH families including pseudogenes, in the E55 library, revealed a population very different as compared to rearranged gene transcripts. This suggests that a large portion of VH locus is accessible for transcription, bringing no evidence of correlation between preferential rearrangement of a given VH gene and its localization in the locus.

Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes.

The somatic diversity immunoglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Rag1 and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Rag1 and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types of junctional diversity observed during lymphoid development.

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.

Creation of immunoglobulin diversity by intrachromosomal gene conversion

Creation of immunoglobulin diversity by intrachromosomal gene conversion