Chapter

Chapter 7 The generation and utilization of antibody variable region diversity

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Abstract

It is now well established that the vertebrate immune system is capable of synthesizing an enormous number of distinct antibody variable region structures. In recent years the genetic basis for this ability in the mouse has been elucidated. Diversity of the V region arises from four sources: (a) diversity encoded directly in the germ-line genome in the form of V region heterogeneous multigene families; (b) diversity created by the somatic rearrangement of different combinations of gene segments to form functional V region genes, and the association of different V, and V, polypeptides to form the heterodimeric V domain (combinatorial diversity); (c) diversity created at the junctions of V gene segments due to apparent addition and deletion of nucleotides during segment joining (junctional diversity); and, finally, (d) diversity created by somatic replacement of nucleotides in expressed V, and V, genes (somatic mutation). What now remains to be determined is the manner in which this potential for generating diversity is utilized toward the formation of antibody specificity and immunity. This chapter reviews the genetic mechanisms that create diversity and discusses recent experiments that provide insights into the question of how this diversity is utilized during an immune response.

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Article
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Article
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Structural analysis of 21 murine A/J antibodies specific for the hapten p-azobenzenearsonate (Ars), and bearing the major cross reactive idiotype (IdCRI), has revealed an invariant amino acid residue, serine, encoded by the variable-diversity gene segments junction of the heavy chain. To test whether this serine residue is essential for Ars binding, we changed it either to alanine or to threonine by oligonucleotide-directed mutagenesis of a heavy chain gene. Genes containing the mutations were separately introduced into mouse hybridoma cells producing the homologous light chain, and the resulting proteins were tested for antigen binding and idiotypic expression. Whereas the serine to threonine mutant retains full antigen binding activity, the serine to alanine mutant does not bind either to Ars-bovine serum albumin-Sepharose or to the Ars-tyrosine hapten. Both mutants show the same reactivity as wild type towards a series of anti-idiotypic antibodies. These results suggest that a hydroxyl group at the variable-diversity gene segments junction of A/J anti-Ars antibodies is essential for antigen binding.
Article
The B-cell repertoire in neonatal mice contains predominant clonotypes that are reproducibly expressed at particular times after birth. We have isolated and sequenced heavy and light chain cDNA clones from three 2,4-dinitrophenyl-specific neonatal hybridomas. Two of these hybridomas (TF2-36 and TF5-139) express idiotypes (Ids) that predominate during the first days after birth, and the third hybridoma (TF2-76) expresses an Id that predominates during the second week after birth. The heavy (H) chain variable (V) region of the TF2-76 hybridoma protein is encoded by a member of the 7183 VH family, one of eight families of murine VH genes that have been defined by Brodeur and Riblet [Brodeur, P. H. & Riblet, R. (1984) Eur. J. Immunol. 14, 922-930]. Members of this family have been found to undergo a disproportionately high frequency of rearrangement in fetal and neonatal liver pre-B-cells. Because the 7183 VH family is located close to the H chain joining (J) region gene segments, JH, other workers have proposed that VH rearrangement frequency is related to distance from the JH segments. However, the two earlier-appearing predominant clonotypes expressed by TF2-36 and TF5-139 hybridoma proteins utilize a member of the 36-60 VH family, probably VH 1210.7, which is located distal to the JH gene segments on chromosome 12. Since 20-30% of day 3 dinitrophenyl-specific B cells express either the Id(TF2-36) or the Id(TF5-139), the VH 1210.7 gene must be utilized at high frequency early in development. These results indicate that the utilization of rearranged VH segments is strongly influenced by factors other than distance from JH.
Article
To assess the significance of somatic point mutation in the hyperimmune response to the hapten NP, an in vivo enrichment procedure was followed. Mice that expressed high titers of B1-8 idiotopic determinants were selected as donors for serial transfer of small numbers of immune spleen cells into syngeneic irradiated recipient mice. Cells expressing B1-8 idiotopic determinants were chosen for enrichment because B1-8 cross-reactive determinants constitute a significant portion of the primary response. Furthermore, B1-8 is a monoclonal antibody derived from a primary response to NP, and its heavy and light chains are unmutated products of the germ-line genes VH186.2 and VL lambda 1, respectively. The germ-line sequence is thus available for comparison with the somatic mutants that arise during enrichment and hyperimmunization. The data show that serial transfer of spleen cells from mice with a high titer of idiotypic determinants results in a dramatic decrease in the titers of antibodies that bind antigen. Three lines of evidence indicate that progeny cells from the initial lambda-positive, idiotype-bearing, antigen-binding cells are successfully transferred and expanded during successive adoptive transfers. First, the proportion of lambda-bearing antibodies relative to NP-specific lambda-bearing antibodies increases with transfer, which is consistent with mutation away from antigen binding. Second, analysis of serum antibodies and hybridoma proteins derived from transfer-recipient mice confirm the presence of idiotype-positive antibodies that do not bind antigen. Third, RNA dot blot analysis of hybridomas constructed from a recipient mouse in the fourth transfer indicates a high frequency of expression of the VH gene predominantly used in the NP response. Many of the antibodies expressed by these hybridomas not only do not bind antigen, but have also lost the determinants recognized by the anti-idiotypic reagents. Most of these VH-positive hybridomas express lambda L chain. The most likely interpretation of the data is that somatic mutation is occurring during the hyperimmune response. Because we selected donor mice that expressed a high titer of idiotype-positive, antigen-specific antibody and immunized the recipient mice, we expected to observe a selective expansion of somatic variants that bound antigen. This was not the case. The observed loss of antigen binding suggests that the majority of mutations arising result in antibodies with lower affinity for the immunizing antigen.
Article
The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.
Article
The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.
Article
During B-cell development, the VH genes of immunoglobulin heavy (H) chains are assembled from three different germline components: the variable (VH) segment, the diversity (D) segment and the joining (JH) segment. The joining between two segments involves the recognition of conserved nonamer-heptamer sequences bordering each segment, double-stranded cuts at the heptamer-segment border, and the re-ligation of the two segment ends which have frequently been modified by the deletion and addition of nucleotides. The flexibility of the joint increases VHDJH variability. However, it also results in many pre-B cells which do not produce immunoglobulin H chains and have non-functional VHDJH complexes carrying the VH and JH coding sequences in different reading frames. We show here that such 'null cells' are not dead-end products of the B-cell developmental pathway but can perform a novel VH to VHDJH joining using a 5' VH segment to replace the VH sequence of the VHDJ-H complex. This process can result in the generation of a VHDJ+H complex and the subsequent expression of an immunoglobulin heavy chain.
Article
The chicken immunoglobulin light chain repertoire has been shown to be entirely derived from a single V lambda 1-J rearranged combination. The complete coding information of the lambda locus was determined: it comprises 25 V-hybridizing elements, all of which are pseudogenes, clustered in both orientations within 19 kb of DNA, starting 2.4 kb upstream of the V lambda 1 gene. Sequences of somatically rearranged V lambda 1 genes from embryonic and posthatching bursal cells show that diversification of light chain sequences occurs during ontogeny by a segmental gene conversion mechanism which takes place at a frequency of 0.05-0.1 per cell generation between the pseudogene pool and the unique rearranged functional V gene.
Article
Previous studies have shown that the chicken lambda immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V lambda 1) and joining (J lambda) coding regions. The immunologic repertoire of lambda genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged V lambda 1 segment and not within the 5' leader sequence, the J lambda segment, or the unrearranged V lambda 1 segment. The selective diversification of the rearranged V lambda 1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for V lambda 1 sequences detect multiple homologous V lambda segments, probes for both the 5' leader and J lambda segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the lambda gene.
Article
Initial studies of somatically acquired mutations in immunoglobulin V regions from hybridomas and myelomas that are not derived from joining aberrations, suggested a controlled and specific hypermutation process, because spontaneous mutation rates observed for other genes are extremely low. Some evidence for the idea that mutations are introduced during V-gene rearrangement came from the clustering of mutations at the joining sites, from the absence of mutations in unrearranged V genes and from the low level of mutations in only partially (D-J) rearranged nonproductive heavy-chain alleles. Another model in which mutations accumulate with each cell division, rather than being introduced all at once, was supported by the finding that immunoglobulin genes of hybridomas derived from a single mouse frequently had several mutations in common, and so might be derived from the same precursor cell whose daughters then accumulated additional mutations. But the common mutations in some cases could be due to as yet unidentified related germline genes, or could represent the effect of antigen selection for certain amino acids. To try to detect hypermutation in the absence of V-gene rearrangement, we isolated B lymphocytes with endogenous heavy-chain gene mutations from transgenic mice carrying pre-rearranged kappa-transgenes. We found that these kappa-transgenes were also somatically mutated. This and other observations indicated that: ongoing rearrangement is not required for mutation; there are signals for hypermutation in the transgenes; the mutations are found only in the variable region, so the constant region may not be a target; different transgene insertion sites are compatible with hypermutations and more than one transgene is expressed in the same cell.
Article
Immunoglobulin K genes are constructed during lymphocyte differentiation by the joining of two DNA elements, VK and JK, to form both a VKJK coding unit and a reciprocal recombination product. The two products formed in single VK-to-JK joining events can be directly isolated through the use of a retrovirally introduced recombination substrate. The structural analysis of a number of recombinants and the derivation of secondary recombination products define some of the basic features of the mechanism of immunoglobulin gene assembly.
Article
Immunization of strain A mice with p-azophenylarsonate-conjugated protein stimulates B cells that synthesize anti-p-azophenylarsonate antibodies. A large fraction of these cells produce antibodies with variable (V) regions encoded by a single heavy chain V gene segment together with multiple combinations of diversity, heavy chain joining, light chain variable, and light chain joining gene segments. Early in the immune response, these V regions are not somatically mutated. One of these V regions is initially expressed by only a minority of the responding B cells but binds p-azophenylarsonate with the highest affinity. After a secondary immunization, B cells synthesizing mutated derivatives of this single V region dominate the response and bind p-azophenylarsonate with even higher affinity than does the unmutated V region. These results suggest that antigen directs both the expression of the immune repertoire and the amplification of V region diversity by a sequential process of clonal selection of B cells expressing receptor antibodies encoded by unmutated V genes, induction of mutation in the V genes expressed by the selected cells, and reselection of B cells expressing antibodies with mutated V regions of higher affinity.
Article
Most cells in the well-known pre-B-lymphocyte line 18-81 have correctly assembled genes for both alleles at the immunoglobulin heavy chain locus. Only one allele is "active"; the other, "silent" allele contains an amber termination codon. The rate of reversion of this amber codon was determined to be 0.3-1 X 10(-4) per cell generation. This high rate is termed hypermutation.
Article
An average of 50% of anti-p-azophenylarsonate (Ars) antibodies bear a cross-reactive idiotype, IdCR, and an average of 15% bear a relatively minor idiotype, Id, in A/J mice. To begin to investigate the processes that influence the expressed levels of these idiotype-bearing antibodies in serum, we have examined the frequency among preimmune B cells of cells that utilize the heavy chain variable region gene segment (VH) needed for IdCR and that which is needed for Id anti-Ars antibody expression. Our results indicate these VH gene segments are functionally rearranged at frequencies one would expect for random usage. The frequency of VH gene segment utilization is similar to, if not higher than, that of VHCR, arguing that the predominance of IdCR-over Id-bearing antibodies is not due to preferential usage of the VHCR gene segment. In addition to the analysis of Ars-immune sera pooled from several mice, we have examined 20 individual A/J mice to determine whether the relative serum levels of IdCR- and Id-bearing antibodies are strictly regulated relative to each other. Among individuals, we find that IdCR and Id antibody levels fluctuate over a 28-fold and a 120-fold range, respectively. The ratio of IdCR to Id antibody levels was found not to be strictly regulated, varying over a 300-fold range. Linear regression analysis of IdCR relative to Id concentrations shows a correlation coefficient of only 0.093. Indeed, rare mice can be found that generate greater levels of Id-bearing antibodies than those bearing IdCR. These results are indicative of a stochastic process involved during the generation of these IdCR-and Id-bearing antibody families. Models accounting for the generation of this highly variable serologic response derived from a preimmune repertoire in which VH gene segments are equivalently utilized are discussed.
Article
The chicken immunoglobulin lambda locus contains a single C lambda gene with a unique J lambda element, 1.9 kb upstream. The same V lambda gene (V lambda 1) is rearranged in most cells of the Bursa of Fabricius. This V lambda 1 gene is located, in germ-line configuration, 1.7 kb upstream from J lambda and in the same transcriptional orientation. Eight to twelve variable genes of the same set are found adjacent to the V lambda 1 gene, indicating that V-gene amplification did occur. Three of these genes were sequenced and proved to be pseudogenes, one of them having an inverted polarity. Data suggesting extensive somatic diversification of the V lambda 1 sequence are reported, including the possible use of nonfunctional V elements in a somatic gene-conversion-like process.
Article
Anti-idiotypic immunization triggers the production of antibodies that are structurally related to the idiotype. We have shown that the heavy chain variable regions of antibodies produced after anti-ABPC48 (A48) anti-idiotypic immunization of BALB/c mice are homologous to that of A48, except for the third hypervariable region. We present here partial light chain sequences of A48 and of antibodies induced by anti-idiotypic immunization. Nearly perfect homology is found, suggesting that these chains are the products of genes derived from a unique VK germ-line gene. These observations indicate that the H and L hypervariable regions contribute to define the structure of A48 idiotopes. Remarkably, the VK sequence we identify is the same as that described for anti-arsonate and anti-oxazolone antibodies. We discuss the relative importance of particular amino acids for idiotype expression and antigen-binding activity.
Article
Sequence analysis of the heavy- and light-chain messenger RNA of hybridomas immunized with a specific hapten yields important clues about the interplay between genetic and selective events during the onset and maturation of the immune response. The maturation of the primary response to the hapten 2-phenyl-5-oxazolone is characterized by a drift to higher-affinity somatic variants of a germline-encoded basic sequence, whereas hybridomas from the secondary response demonstrate a further maturation dominated by a shift to alternative germline combinations.