Ig Gene Segments Research Articles

Differential Usage of VH Gene Segments Is Mediated bycisElements

AbstractIg diversity is generated in large part by the combinatorial joining of the Ig gene segments, VH, D, and JH, that together encode the variable domain of Ig. The final Ig repertoire, however, not only reflects the diversity generated through V(D)J recombinatorial joining, but it is also the product of a number of developmental restraints and selections. To avoid such restrictions and assess the recombination potential of individual Ig gene segments, we constructed Ig heavy (H) chain microlocus plasmids, each of which contain germline coding, recombination signal, and flanking sequences of a VH, D, and JH gene segment. These plasmids allow us to assess the recombination potential of the segments in the context of their natural flanking DNA sequences, but in the absence of any higher order chromatin structure or cellular selection. We found that the frequency and extent of deletions and additions at the recombination breakpoints are similar to those observed at rearranged Ig H chain loci in intact animals. The relative frequencies of the types of rearrangements—VD-J, V-DJ, VinvD-J (invD = inverted D), and VDJ—however, differ strongly. Moreover, V81x, the most used VH gene segment in intact mice, also is overused in this plasmid assay, 15 to 30 times that of another VH segment. This result indicates that the overuse of V81x in the early B cell repertoire can be a consequence of its DNA sequence and not of cellular activities.

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells.

V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed.

Cell Type–Specific Chromatin Structure Determines the Targeting of V(D)J Recombinase Activity In Vitro

A common V(D)J recombinase that recognizes a conserved recombination signal sequence (RSS) mediates the assembly of immunoglobulin (Ig) and T cell receptor (TCR) genes in B and T cell precursors. The rearrangement of particular Ig and TCR gene segments, however, is tightly regulated with respect to cell lineage and developmental stage. Using an in vitro system, we analyzed recombinase cleavage of RSSs flanking Ig and TCR gene segments in nuclei. We found that both the lineage-specificity and temporal ordering of gene rearrangement is reflected in the accessibility of RSSs within chromatin to in vitro cleavage.

The perinatal presence of antigen (p-azophenylarsonate) or anti-mu antibodies lead to the loss of the recurrent idiotype (CRIA) in A/J mice.

The immune response of A/J mice against p-azophenylarsonate (Ars)-keyhole limpet hemocyanin (KLH) is characterized by the dominance, late in primary and during the secondary, of a recurrent idiotype called CRIA, encoded by a canonical combination of Ig gene segments. In this study, A/J mice were given Ars coupled to deaggregated human gamma globulins (dHGG) within 24 h after delivery. The offsprings from these mice were then exposed as adults to Ars-KLH. These animals developed an unusual immune response. The level of anti-Ars antibodies was nearly normal but a dramatic shift in repertoire was observed: the cross-reactive idiotype which is the hallmark of the anti-Ars response in A/J mice was completely absent. The idiotype could be recovered by injection of anti-idiotypic antibodies alone, with no need of lipopolysaccharide coupling. Therefore the presence of antigen at birth can lead to a strong perturbation of idiotype selection. Similar results were obtained with neonatal treatment using anti-IgM antibodies. After recovery of suppression, A/J mice can mount an anti-arsonate response of normal level but devoid of the dominant idiotype.

Isolation of peritoneal precursors of B-1 cells in the adult mouse.

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220+IgM-lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of lambda 5, VpreB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220+IgM- cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220+IgM- cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.

Expression and Characterization of Recombinant Anti-Rh(D) Antibodies on Filamentous Phage: A Model System for Isolating Human Red Blood Cell Antibodies by Repertoire Cloning

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)-expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)-positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)- expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)- positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

Measuring CDR3 length variability in individuals during ontogeny

The portion of the antibody sequence that contributes most to the confornation of the antigen-combining site is the third complementarity-determining region (CDR3). The CDR3 in antibody heavy chains are somatically produced by the rearrangement of three different immunoglobulin (Ig) gene segments, V H, D and J H, and are therefore highly variable in sequence and length. We have devised a technique for rapid analysis of CDR3 length variability from a large number of cDNA samples. This technique allows comparative evaluation of IgH heterogeneity in individuals without recourse to DNA cloning and sequencing. We also present data on Ig sequences from different stages of development in the amphibian, Xenopus laevis. Most of the larval and post-metamorphic Ig samples contained CDR3 of 3–10 codons, two codons shorter than those of the adult CDR3, which were 5–12 codons. Antibodies made by immunized tadpoles have been shown to be much less diverse than those produced by adults to the same antigen. The shorter tadpole CDR3 perhaps generates a more limited spectrum of antigen-combing site structures and, in this way, makes for a more restricted antibody repertoire.

Gene rearrangement and B-cell development

The differentiation of B lymphocytes from their progenitors progresses through a series of successive stages that are defined by sequential rearrangement of Ig loci and surface expression of various stage-specific markers, including Ig heavy and light chain proteins. Considerable evidence suggests that the appearance of cells with an orderly progression of Ig gene rearrangements is linked to the expression of the rearranged Ig gene products. Recent experiments have clarified our understanding of mechanisms by which rearrangement of Ig gene segments is controlled and how Ig gene products participate in the regulation of the B-cell differentiation program.

Map position and usage of 3' VH family members: usage is not position dependent.

The primary repertoire in mice, in large part, is determined by the Ig gene segments joined to form the variable region genes-VDJ for the heavy chain genes and VJ for the light chain genes. However, the mechanisms that determine which VH gene, of several hundred available, is joined to a DJH structure remain unexplained. One theory proposes that the VH gene segments closest to the 3' end of the VH locus are chosen because of their location, i.e. that proximity to a DJH structure is a prime determinant for VH selection. Alternatively, the content of the DNA may be the determining factor regardless of chromosomal position. According to this hypothesis 3' preference is due to the coincidence of preferred content and proximity to DJH structures. Since the 3' VH families span more than a centimorgan of DNA we were able to distinguish between these concepts by determining VH usage of gene segments within this region. The BALB/c strain is ideal for this study since it uses 3' VH families at a very high frequency. We therefore mapped the positions of the 3' VH gene families and used this map to investigate the usage of these gene segments in BALB/c fetal liver derived A-MuLV transformed cell lines. We found that while VH gene segments throughout the Ig locus, both 3' and 5' were used, there was a clear preference for 3' VH families. However, within the 3' VH segments the most proximal were not preferred, indicating that proximity to DJH structure was not a strict determinant of VH utilization.

CLL in three sisters: Preferred usage of specific IG gene segments

CLL in three sisters: Preferred usage of specific IG gene segments

Avian B-Cell Development: Generation of an Immunoglobulin Repertoire by Gene Conversion

The vertebrate B-cell repertoire is capable of generating up to 10(9) different antibody molecules using relatively few germline immunoglobulin (Ig) gene segments. To generate diversity, humans and mice depend on combinatorial and junctional variations that occur during the gene rearrangement events that produce complete heavy and light chain Ig genes. This gene rearrangement process goes on continuously in the bone marrow, where each developing B cell assembles a unique heavy and light chain Ig gene from families of functional V, D, and J gene segments. In contrast, chickens have only single functional V and J segments for the heavy and light chain loci, and chicken Ig gene rearrangement occurs only during a brief period of embryonic development. A specialized organ involved in avian B-cell development, the bursa of Fabricius, provides the microenvironment necessary for the amplification of B cells that have undergone productive Ig gene rearrangements. Within the bursa, B cells also acquire somatic diversity within the rearranged V gene segments of the heavy and light chain Ig loci. Somatic diversification of chicken V gene segments occurs by intrachromosomal gene conversion, a DNA recombination process which involves unidirectional transfer of nucleotide sequence blocks from families of V region pseudogenes into the functional rearranged VH and VL genes.

Adoptive transfer of neonatal T lymphocytes rescues immunoglobulin production in mice with severe combined immune deficiency.

Mice with the autosomal recessive severe combined immune deficiency (scid) mutation lack mature lymphocytes because of defective joining of T cell receptor and immunoglobulin (Ig) gene segments. Penetrance of this mutation is incomplete since 10-25% of SCID mice produce some T or B lymphocytes. This "leaky" phenotype could be due to a reversion of the mutation in some mice or to a constant, low frequency of functional lymphocytes generated in all SCID mice with variable survival of such cells. We report here that all SCID mice can be stimulated to produce functional B cells by the transfer of normal neonatal, but not adult, T cells. T cell-induced rescue of C.B-17scid B cells results in high levels of Ig expressing the Ighb allotype of the SCID recipient. These results show that all SCID mice generate some functional B cells, the majority of which do not survive in the absence of a subset of T cells present in high frequency in the neonate.

Generation of a recombinant mouse-human chimaeric monoclonal antibody directed against human carcinoembryonic antigen.

A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high-affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally-rearranged murine Ig H-chain and L-chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse-human antibody molecule with human (gamma 4, kappa) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electroporation and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen-binding affinity. This anti-CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non-human immunoglobulins in human patients.

T cell receptor DJ but not VDJ rearrangement within a recombination substrate introduced into a pre-B cell line

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.

Molecular Genetics of Murine Lupus Models

Publisher Summary The chapter that addresses the Ig germ line gene organization in lupus-prone strains of mice suggested that the disease can develop in different Ig heavy and light chain haplotypes, and that the Ig germ line genes in lupus mice are probably normal. Analyses of the Ig gene segments expressed in monoclonal autoantibodies from autoimmune mice revealed that similar, moreover, in some instances even identical, gene segments are expressed in autoantibodies and in antibodies to exogenous antigens, and that antiself and antiforeign responses are encoded by the same, or at least an overlapping, germ line gene repertoire. Evidence has been obtained that, in an individual lupus mouse, the number of autoantibody-secreting clonotypes decreases after class switch, while that of productive mutations increases, suggesting that antiself responses might be (auto) antigen-driven responses, but this conclusion should be considered tentative. Finally, based on the investigations of I-E and Mls tolerance-related Vβ clonal deletions, the chapter proposes that the abnormally proliferating, autoimmunity-inducing/enhancing double-negative TcR α:β + lpr and gld cells are not related to immature CD4 - 8 - thymocytes, but instead are derived from CD4 + 8 + precursors through a process resulting in down-regulation of both accessory molecules.

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.

Rearrangement of exogenous immunoglobulin VH and DJH gene segments after retroviral transduction into immature lymphoid cell lines.

A model substrate for the joining of Ig VH and DJH elements has been constructed in a retroviral vector carrying a selectable marker whose expression is independent of the arrangement of the resident Ig gene segments. The substrate was introduced into lymphoid and nonlymphoid cells, and site-specific recombination between the VH and DJH elements was monitored by a direct hybridization assay. Joining of the exogenous gene segments was observed in cell lines representative of three distinct stages in early B cell differentiation. Rearrangement was not observed in three cell lines derived from mature B cells, or in a fibroblastoid cell line. The VH and DJH elements were initially arranged so that the VH-DJH junction and the recombined flanking sequences could be recovered after rearrangement. By molecular cloning and nucleotide sequence determination, VH-DJH junctions formed upon rearrangement of the substrate were found to resemble closely similar junctions in functional H chain genes. The joining of VH and DJH elements was observed to be asymmetric; loss of nucleotides occurred at the coding joints, but not at the junctions between flanking sequences. Our results suggest that Ig H and L chain gene segments are joined by a common mechanism that is more active in B cell precursors than in mature B cells. These observations provide further evidence that the rearrangement of Ig gene segments occurs by a nonreciprocal recombinational mechanism. The model substrate described here is likely to be of use in defining the nucleotide sequences that mediate rearrangement and in examining the developmental specificity of this process.

The genetic origin of murine lupus-associated autoantibodies.

Systemic lupus erythematosus and rheumatoid arthritis in human and murine systems are characterized by circulating autoantibodies and immune complex deposition in various organs causing tissue damage and disease. To define the molecular and clonotypic origin of these anti-self responses, and to determine whether abnormalities in Ig genes or somatic mechanisms generating autoantibody diversity may contribute to lupus etiology, we performed molecular analyses of the Ig germline gene organization and the Ig gene segments expressed in monoclonal autoantibodies from autoimmune mice. Comparative restriction fragment length polymorphism analysis of a large number of Ig gene loci from autoimmune and normal mice indicated that (a) lupus can develop in different Ig heavy and kappa light chain variable region gene haplotypes, and (b) the Ig germline genes in lupus mice might be normal. To determine whether autoantibodies are encoded by unique Ig gene segments present in the normal germline repertoire, but not expressed in exogenous responses, we compared nucleic acid sequences encoding lupus autoantibodies and antibodies against foreign antigens. Similar, and in some instances even identical, gene segments were expressed in both types of antibodies, indicating that anti-self and anti-foreign responses use the same, or at least an overlapping, germline gene repertoire. A large variety of Ig variable, diversity, and joining gene segments encoded these autoantibodies with different specificities. Hence, the overall murine lupus-associated anti-self response may be essentially unrestricted. Furthermore, limited evidence has been obtained that both germline genes and somatically mutated genes encode autospecificity, making gross abnormalities in mechanisms for somatic mutation of Ig variable genes unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduced T cell receptor variable region gene segments recombine in pre-B cells: Evidence that B and T cells use a common recombinase

We have recently proposed that a common recombinase performs all of the many variable region gene assembly events in B and T cells, and that the specificity of these joining events is mediated by regulating the “accessibility” of the involved gene segments. To test this possibility, we have introduced “accessible” T cell receptor (TCR) variable region gene segments into a pre-B cell line capable of recombining endogenous and transfected immunoglobulin (Ig) variable region gene segments. Although the corresponding “inaccessible” endogenous TCR gene segments do not rearrange in this line or in B cells in general, the introduced TCR gene segments join very frequently and, in fact, closely resemble introduced Ig gene segments in their recombination characteristics. These observations suggest a new role for conventional Ig transcriptional enhancers — recombinational enhancement. Our studies provide insight into additional aspects of the joining mechanism such as N region insertion, aberrant joining, and recombination-recognition sequence requirements for joining.