Organization Of Immunoglobulin Genes Research Articles

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

BackgroundThe Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod’s immune response on a molecular level.ResultsA comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5’RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.ConclusionsThis study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Primer Design and Amplification of IgM Antibody Repertoire of Philippine Carabao (Bubalus kerabau Fitzinger, 1860) for Construction of Phage Display Library

Antibody display library is a robust platform for the production of recombinant or monoclonal antibodies that are of great value in diagnostics and therapeutics. A naïve library constructed based on immunoglobulin M (IgM) can reflect the natural immunity of the animal and can offer broad antigen targets. As a natural host of various zoonotic pathogens, the Philippine carabao (Bubalus kerabau Fitzinger, 1860) offers great potential as a surrogate model for molecular biology and biomedical applications. However, studies on its antibody repertoire are very scarce, if not available. Hence, this study was undertaken to design and evaluate degenerate primers targeting the antigen-binding paratope of B. kerabau Fitzinger, 1860 IgM – specifically, lambda variable light chain (VLL), kappa variable light chain (VLK), and variable heavy chain (VH). These primers were mainly based on carabao’s close relative in the Bovidae family, the cattle (Bos taurus). A total of 32 degenerate primers, 14 designed and 18 adapted from literature, were used to amplify the target antibody fragments using a high-fidelity DNA polymerase with a modified touchdown–polymerase chain reaction method. Results demonstrate the amplification of VLL, VLK, and VH genes derived from B-cells of carabao splenic tissues using the degenerate primers. The amplicons will be used in the construction of an antibody phage library to elucidate the IgM repertoire of the Philippine carabao. In addition, DNA sequencing surprisingly revealed longer VLL and VLK genes than VH genes in contrast to what is found in other bovines, suggesting a novel organization of immunoglobulin genes of B. kerabau Fitzinger, 1860 that merits further investigation.

Origin of Immunoglobulin Isotype Switching

From humans to frogs, immunoglobulin class switching introduces different effector functions to antibodies through an intrachromosomal DNA recombination process at the heavy-chain locus. Although there are two conventional antibody classes (IgM, IgW) in sharks, their heavy chains are encoded by 20 to >100 miniloci. These representatives of the earliest jawed vertebrates possess a primordial immunoglobulin gene organization where each gene cluster is autonomous and contains a few rearranging gene segments (VH-D1-D2-JH) with one constant region, μ or ω. V(D)J rearrangement always takes place within the μ cluster, but here we show that the VDJ can be expressed with constant regions from different clusters, although IgH genes are spatially distant, at >120 kb. Moreover, reciprocal exchanges take place between Igω and Igμ genes. Switching is augmented with deliberate immunization and is concomitant with somatic hypermutation activity. Because switching occurs independently of the partners' linkage position, some events involve transchromosomal recombination. The switch sites consist of direct joins between two genes in the 3' intron flanking JH. Our data are consistent with a mechanism of cutting or joining of distal DNA lesions initiated by activation-induced cytidine deaminase (AID), in the absence of mammalian-type switch regions. We suggest that, in shark, with its many autonomous IgH targeted by programmed DNA breakage, factors predisposing broken DNA ends to translocate configured the earliest version of class switch recombination.

Haplotype exclusion: the unique case presented by multiple immunoglobulin gene loci in cartilaginous fish

Cartilaginous fish represent the most phylogenetically distant species from man in which immunoglobulin and T cell antigen receptor genes have been identified. Immunoglobulin genes in cartilaginous fish are organized in hundreds of clusters, located on different chromosomes and presumably are under independent regulation; large numbers of immunoglobulin gene clusters are germline-joined and thus their expression is not directly dependent on somatic rearrangement. Despite the unusual nature of immunoglobulin gene genetics in these species, preliminary characterization of the transcription products of immunoglobulin loci in single cell isolates is consistent with haplotype exclusion. Certain features of immunoglobulin gene organization and expression in cartilaginous fish are remarkably similar to that of odorant receptors and suggest that at the level of transcriptional regulation, at least two different mechanisms could exist that relate to haplotype exclusion.

Receptor Editing in B Cells

Publisher Summary This chapter reviews the essential facts related to the organization of immunoglobulin genes, V(D)J recombination, and B-cell development. The chapter also presents how signals from the antigen receptor influence gene rearrangements during development, tolerance-induction, and immunity. The classical view of the immune system is that lymphocytes, like individuals in a natural animal population, have preexisting genetic diversity upon which selection can act. According to this Darwinian view, the selected growth or death of clones regulates immune specificity. The chapter additionally reviews the evidence, derived primarily from studies of mouse B cells, that a second mechanism of immune regulation, receptor selection, can modify the lymphocyte repertoire by regulating the receptor gene diversification mechanism itself. However, if antigen-specific positive selection is associated with the cessation of V(D)J recombination in B cells, then allelic inclusion would be predicted to occur frequently among normal B cells in vivo (as is the case for TCRa exclusion in T cells). On the contrary, there is substantial evidence from the study of autoantibody transgenic mice that negative selection (i.e., tolerance mediated by encounter with self-antigens) blocks developmental progression and prevents recombinase downregulation.

Immunoglobulin Gene Organization and the Mechanism of Repertoire Development

Recombinant DNA technology has made it possible to comparatively examine the immunoglobulin (Ig) gene constitution of numerous species. These studies reveal that many species diverge from the pattern seen in rodents and primates while revealing a number of interesting correlations involving Ig gene organization, B‐cell lymphogenesis and the mechanism of repertoire development; many of these fail to correlate with traditional phylogenetic relationships. These correlates pose new questions regarding repertoire developments, which suggest that considerable diversity exists in the process. These findings also allow new questions to emerge regarding the role of IgD, the size of the B‐cell repertoire, and the role that antigen may or may not play in repertoire development. These issues are discussed with the intention of stimulating experimentation to test a number of speculative hypotheses regarding B‐cell and repertoire development.

Immunoglobulin gene organization and the mechanism of repertoire development.

Recombinant DNA technology has made it possible to comparatively examine the immunoglobulin (Ig) gene constitution of numerous species. These studies reveal that many species diverge from the pattern seen in rodents and primates while revealing a number of interesting correlations involving Ig gene organization, B-cell lymphogenesis and the mechanism of repertoire development; many of these fail to correlate with traditional phylogenetic relationships. These correlates pose new questions regarding repertoire developments, which suggest that considerable diversity exists in the process. These findings also allow new questions to emerge regarding the role of IgD, the size of the B-cell repertoire, and the role that antigen may or may not play in repertoire development. These issues are discussed with the intention of stimulating experimentation to test a number of speculative hypotheses regarding B-cell and repertoire development.

Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.

Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving.

Control of recombination events during lymphocyte differentiation. Heavy chain variable region gene assembly and heavy chain class switching.

Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.

The use of chromosomal translocations to study human immunoglobulin gene organization: mapping DH segments within 35 kb of the C mu gene and identification of a new DH locus.

We have studied the Burkitt's lymphoma cell line Daudi which carries the translocation t(8;14). The breakpoint of this translocation on the 14q+ chromosome occurs near to a rearranged DH-JH join, and the actual chromosome junction is a few hundred base pairs upstream of the joined DH element. The nucleotide sequence of the rearranged DH segment shows that it does not come from the previously described D cluster. Using this DH sequence as a probe we have identified two separate DH clusters. One of these is the major DH cluster and is located only 20 kb upstream of the JH segments. A pseudo-VH (probably the first VH segment) is also found approximately 98 kb from JH. A second, minor DH locus has been found which seems to be located on the distal side of the VH locus on chromosome 14, since there is little evidence for rearrangement or deletion of this locus in any B cell DNA analysed. A single VHIII subgroup gene is located within 25 kb of the newly identified DH element: it is possible that this minor locus occurs near the limit of the Igh locus.

Kappa-chain gene rearrangement in an apparent T-lineage lymphoma.

We describe a 10-yr-old boy with T-lineage non-Hodgkin's lymphoma. He had a mediastinal mass, swollen supraclavicular lymph nodes, and pleural effusion. A supraclavicular lymph node biopsy under light microscopy showed a malignant lymphoma of diffuse lymphoblastic type. Most of the cells taken from the malignant pleural effusion expressed T cell-associated antigens such as Leu-1 and OKT 8. To confirm these antigens as T-lineage lymphoma, we examined genomic DNA from malignant cells obtained from the pleural effusion. As was expected, T cell receptor beta-chain gene rearrangements were demonstrated. However, when the immunoglobulin gene organization was analyzed, we detected rearrangements in both the heavy- and kappa-chain genes. To our knowledge, this is the first case in which kappa-chain gene rearrangement was detected in apparent T-lineage cells. These findings provide important information relating to determination of the cellular lineage of lymphoid malignancy.

Mitogen-induced human IgG subclass expression.

To investigate human isotype expression among lymphocyte populations, we have studied IgG subclass production by splenocytes, tonsil cells, and peripheral blood lymphocytes (PBL) after stimulation with a panel of nine mitogens. Response magnitudes varied with tissues: all mitogens produced the strongest responses with splenocytes and the smallest with PBL. In addition, the IgG subclass maximally stimulated by a particular mitogen also depended on the tissue studied. For example, LPS mainly stimulated IgG2 in PBL and IgG1 in spleen. Interestingly, the response patterns seen in splenocytes suggest a large and coordinate expression of IgG1 and IgG3 subclasses. Implications of these findings are discussed with respect to immunoglobulin gene organization and human disease states.

9 - The Immunoglobulin Genes: Genetics, Biological and Clinical Significance

The chromosomal organization of immunoglobulin genes, and the synthesis of various heavy and light chains to molecules with an infinitive variety of antibody specificities, is discussed. These molecules carry antigenic determinants (epitopes) that may be individual (idiotypes), shared (isotypes and isoallotypes) or polymorphic (allotypes). Since biological effector functions correlate with epitopes, its determination can be relevant to clinical disease. In particular, in cases of an excessive or a decreased production of immunoglobulins or of incomplete proteins, the determination of isotypes and allotypes will supply information on the abnormality. Allotypes are genetic markers that are useful for family and population studies, for investigation of paternity, blood stains, zygosity of twins and other identification problems. Testing for antibodies to immunoglobulins is indicated when severe transfusion reactions occur that cannot be explained by blood cell incompatibility. Research on the association of immunoglobulin alleles with immune response, and their interactive effect with HLA in diseases, is gradually developing. The study of the Ig and HLA systems may contribute to the elucidation of the aetiology of immunological disorders and malignancies.

Functional V region formation during in vitro culture of a murine immature B precursor cell line.

B lymphocytes originate from pluripotential haematopoietic stem cells and differentiate into immunoglobulin (Ig)-producing cells. B-cell lineage differentiation is accompanied by two types of immunoglobulin gene rearrangements--rearrangement of V, D and J gene segments to create a functional V gene and rearrangement of CH genes for heavy-chain switching. These results, however, have been obtained mainly by analysis of immunoglobulin gene organization of myeloma cells. Baltimore and his colleagues have established Abelson murine leukaemia virus (A-MuLV)-transformed cell lines and found a few lines capable of carrying out kappa-gene rearrangement or undergoing isotype switching during in vitro culture. To study early B-cell lineage differentiation events, we have now also established A-MuLV-transformed cell lines which are capable of differentiating from mu- to mu+ and of undergoing continuing rearrangement of heavy-chain genes in culture. Analysis of immunoglobulin gene organization of these transformed cells revealed that mu- cells have already undergone DNA rearrangements involving JH segments but an additional rearrangement of JH segments is required for initiation of mu-chain synthesis. Southern blot analysis of the DNA and two-dimensional gel electrophoresis of intracytoplasmic mu-chain show that mu-chain diversity with respect to antigen specificity may be generated during this second rearrangement process. As no rearrangement of light-chain genes takes place in these cells, this implies that light-chain gene rearrangement requires some further change, or a different enzyme.

Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B cell development.

The first part of this article discusses the isolation and characterization of several monoclonal antibodies to the B lineage-specific surface molecule, B220. B220 was shown to be expressed on precursors of B cells by the demonstration that removal of B220+ cells from B cell-depleted bone marrow removes the ability of bone marrow to regenerate B cells. Although these antibodies recognize a broad range of differentiation stages within the B lineage, they can be used to isolate highly enriched populations of pre-B cells from mouse bone marrow. We also describe the use of antibodies to the surface markers B220 and ThB to define two sequential stages of pre-B cell differentiation. A simplified diagram of our current view of the B lineage differentiation sequence is shown in Figure 4. No attempt has been made to include the various functionally defined B cell subsets on this diagram since we know almost nothing about the expression of these two surface markers on them. This model reflects an assumption that the early part of B cell differentiation is a linear rather than a branching pathway. At present, there is no evidence for a branching pathway, but little evidence against it either. B220 is the first B cell-specific molecule known to be expressed during differentiation and it continues to be expressed on most subsequent B lineage cells. In this regard, it resembles the Thy-1 molecule on thymus-derived lymphocytes and, like Thy-1, B220 should be quite useful for identifying and classifying B lineage cells. One example of this is the use of B220 expression to clearly assign germinal center cells to the B lineage. The example of Thy-1+, RA3-2C2+ cells from mice with the lpr/lpr genotype, however, suggests that some caution should be used when interpreting data, especially with pathological samples. The availability of substantially purified pre-B cell populations has made it possible to follow changes in immunoglobulin gene organization and expression during differentiation. Our current understanding of these events is also shown in Figure 4, correlated with cell surface phenotype. The large pre-B cell population has extensive heavy chain rearrangements and synthesizes significant quantities of mu heavy chain, but does not yet have detectable light chain gene rearrangement. The small pre-B population consists of two cell types, some with kappa gene rearrangement and some without. This suggests that kappa rearrangement occurs within this cell population, which is homogeneous with respect to morphology and surface phenotype. The asynchrony of heavy and light chain gene rearrangement results in an asynchrony at the level of expression of these genes as well, but the purpose of this remains one of many unanswered questions about pre-B cell differentiation. Now that it is possible to identify, isolate, and manipulate pre-B cells as readily as B or T cells, many of these questions may now be addressed.

B Cell Ontogeny: Immunoglobulin Genes and Their Expression

B cell development occurs in a series of discrete stages which can be distinguished on the basis of morphologic and functional criteria. Genesis of B cells begins within the fetal liver as early as eight weeks gestation, and is later maintained in bone marrow. The first recognizable developmental stage is the rapidly-dividing pre-B cell which synthesizes cytoplasmic IgM but lacks surface receptors for antigen. Immature B lymphocytes emerging from pre-B cells express surface IgM, but are uniquely susceptible to inactivation as a consequence of specific antigen binding. Progeny of these cells express different classes of cell surface immunoglobulin and become committed to differentiate into plasma cells synthesizing IgM, IgD, IgG, IgA, or IgE immunoglobulins, respectively. By the beginning of the second trimester, the human fetus has an adult proportion of B lymphocytes expressing different immunoglobulin classes and capable of recognizing an extensive variety of antigens. The qualitative and quantitative deficiencies in antibody responses of newborns, as compared to adults, appear to reflect regulatory interactions with T cells and macrophages rather than absence of B lymphocytes capable of generating a response. Recent research with recombinant DNA technology has provided remarkable new knowledge of the structure and organization of immunoglobulin genes. This developing information, in concert with more detailed studies of the cellular expression of these genes during ontogeny, holds the promise of generating insights into the molecular mechanisms regulating gene expression during differentation.

Correlation of protein structure and immunoglobulin gene organization in the light of two new deleted heavy chain disease proteins

Two new γ Heavy Chain Disease proteins are described. After a normal amino terminal sequence PCA-Val they have a 214 residue deletion which ends at the beginning of the hinge with the sequence Glu-Pro-Lys. The remainder of the Fc fragment appears to be intact. These findings support the existence of separate genetic units coding for the hinge and the various H chain domains. This type of complex genetic organization had been previously postulated on the basis of H chain mutants and has been recently documented by cloning and sequencing of DNA.

Cloned pairs of variable region genes for immunoglobulin heavy chains isolated from a clone library of the entire mouse genome.

To investigate the organization of immunoglobulin genes, we have constructed a clone library containing 10(6) randomly generated fragments of mouse embryo DNA, corresponding to eight equivalents of the genome. The cloning method involved methylation of embryo DNA at EcoRI recognition sites, partial digestion by EcoRI* endonclease activity, and direct ligation of the resulting large fragments to the lambda phage vector Charon 4A. The library was searched for sequences homologous to a cloned complementary DNA copy of a mu heavy chain mRNA. Nine clones bearing variable heavy chain (VH) sequences were isolated, representing at least eight distinct VH genes. Thus, multiple related VH genes are available in the genome to contribute to immunoglobulin diversity. Each of the two clones carries a pair of VH genes, one pair separated by 15 +/- 1 kilobase pairs of mouse DNA and the other by 14 +/- 2 kilobase pairs. This indicates that related VH genes are clustered and may occur in a tandem array having a repeating unit of 14--16 kilobase pairs. The large spacer sequences between VH genes cannot, however, be highly conserved.

Human heavy chain disease protein WIS: implications for the organization of immunoglobulin genes.

Protein WIS is a human gamma3 heavy (H) chain disease immunoglobulin variant whose amino acid sequence is most readily interpreted by postulating that three residues of the amino terminus are followed by a deletion of most of the variable (VH) domain, which ends at the variable-constant (VC) joining region. Then there is a stretch of eight residues, three of which are unusual, while the other five have striking homology to the VC junction sequence. This is followed by a second deletion, which ends at the beginning of the quadruplicated hinge region. These findings are consistent with mutations resulting in deletions of most of the gene coding for the V region and CH1 domain followed by splicing at the VC joining region and at the hinge. These structural features fit well the notion of genetic discontinuity between V and C genes and also suggest similar mechanisms of excision and splicing in the interdomain regions of the C gene of the heavy chain.