Antibody Variable Region Genes Research Articles

DNA Target Motifs of Somatic Mutagenesis in Antibody Genes

During humoral immunity to T-cell-dependent antigens, responding B lymphocytes selectively mutate their antibody variable region genes at a high rate. This, together with the process of clonal selection, ultimately enhances the affinity and specificity of the antibody molecule and memory B cells that express it as a receptor. Despite several decades of investigation, the mutation mechanism has remained unresolved, largely due to the convoluted nature of experimental systems used to approach it. Somatic mutations preferentially occur within specific oligonudeotide motifs, and this targeting is consistent in all immunoglobulin genes of humans and mice that we have examined, suggesting that a conserved mechanism operates in both species. Our mutation targeting analyses implicate evolution of germline variable gene sequences to direct somatic mutations to specific codon positions in a manner that regulates the frequency of amino acid replacements to the benefit of the antibody product. Finally, our recent strand bias analyses support the idea that somatic mutation occurs preferentially, perhaps exclusively, at two bases on both strands of DNA. These and related observations from other laboratories support a mutation model that invokes at least two error-prone polymerases that have distinct template biases and requirements for elements of postreplicative mismatch repair.

Double break-point in the match for the mutator

Mammalian B cells use heretical genetic mechanisms to generate an astonishingly diverse array of antibody molecules. In fact, the creation of a single antibody molecule involves three separate events that permanently alter genomic DNA. The variable (V) regions of the immunoglobulin gene are created when smaller gene segments are recombined, the constant (C) regions of the immunoglobulin genes can be replaced or ‘switched’ at the gene level and single base pair changes can appear within the recombined antibody variable region genes in a process referred to as somatic hypermutation.

Sequence heterogeneity in Ig kappa transcripts from single B lymphocytes

Individually amplified kappa cDNA molecules from single B lymphocytes revealed sequence heterogeneity and aberrantly spliced products. The nature and frequency of the base changes and their absence from similarly amplified β2 microglobulin transcripts indicate that they were not derived by Taq polymerase misincorporations or by a general infidelity in RNA polymerase. The trinucleotide sequences in which the base changes occurred are disfavored targets of the somatic hypermutation mechanism that modifies antibody variable (V) region genes during immunity. Taken together with the observation that the transcript alterations were absent from the kappa Ig gene, this suggests that somatic mutations were acquired by the kappa gene and rapidly repaired following limited transcription. Preferential repair of mutations located in specific trinucleotide contexts could be the basis for some of the microsequence-specific bias in mutation frequencies observed in antibody V region genes.

Production of Recombinant Antibody Against Potato Virus Y by using Phage Display Technology

ABSTRACTPotato virus Y (PVY) is distributed around the world and causes an important economic loss. We report here, the development of a recombinant antibody in the form of single chain variable fragment (scFv) against PVY by using phage display technology. A library of single chain variable fragments was constructed by using the antibody variable region (V) genes of B cells derived from the spleens of PVY immunized Balb/CJ mice. The selection of scFv with ability to bind PVY was performed by biopanning and tested against PVY by phage-enzyme linked immunosorbent assay (ELISA). ELISA tests showed that scFvs produced by five clones from randomly selected 44 clones reacted specifically with PVY. These five clones didn't show cross reaction with tobacco mosaic virus(TMV). These results suggest that the application of phage display technology can be used for developing immunodiagnostic kits for detection of PVY and trangenic plant against PVY.

Somatic origin of T-cell epitopes within antibody variable regions: significance to monoclonal therapy and genesis of systemic autoimmune disease.

During an immune response, specific antibody variable region genes are diversified by a somatic point mutation process that generates de novo "foreign" V-region sequences. This creates an interesting problem in immune regulation because B cells are highly proficient at self-presenting V-region peptides in the context of class II MHC. Though our studies indicate that the corresponding T-cell repertoire attains a state of tolerance to germline-encoded antibody V-region diversity, it is presently unknown whether the same is true of mutationally generated diversity. On the basis of immunoregulatory considerations, we hypothesize that contact exclusion or tolerance normally precludes T cells from helping B cells via self-presented mutant V-region peptides. The lack of recurrent somatic mutations that create known T-cell epitopes in antibody V regions lends some support to this idea. In contrast, our studies of spontaneously autoreactive B cells in systemic autoimmune disease strongly suggest that precursors of such cells are recruited by T-cell help directed to self-presented mutant idiopeptides. Failures in tolerance or contact exclusion mechanisms may be responsible for this apparently abnormal event. In addition to their importance in immune regulation, somatic mutations or other differences from germline-encoded V-region sequence may be largely responsible for undesirable patient responses to therapeutic monoclonal antibodies. These reactions might be averted or diminished by inducing tolerance in the T-cell repertoire with synthetic peptide correlates of non-germline-encoded V-region sequences in humanized antibodies.

The transcriptional promoter regulates hypermutation of the antibody heavy chain locus.

A somatic process introduces mutations into antibody variable (V) region genes at a high rate in many vertebrates, and is a major source of antibody diversity. The mechanism of this hypermutation process remains enigmatic, although retrospective studies and transgenic experiments have recently suggested a role for transcriptional regulatory elements. Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation. However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not. Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA. These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.

Review: Human Antibody Variable Region Gene Usage in HIV-1 Infection

Human antibody variable region gene usage during human immunodeficiency virus type 1 (HIV-1) infection is examined in the following review, and several hypotheses are presented to account for the distinct patterns of antibody gene expression associated with infection. Evidence supporting qualitatively biased antibody gene expression has been derived from analysis of the human humoral immune response by isoelectric focusing (IEF) and serological and molecular studies of immunoglobulin (Ig) from different lymphoid compartments of HIV-1-infected patients. Preferential usage of heavy-chain variable region (VH) gene families 1 and 4 is supported by serological studies of serum Ig and molecular characterization of anti-HIV-1 human monoclonal antibodies derived from infected patients. Negative biases against VH3 family gene usage are detected by polymerase chain reaction (PCR) studies of peripheral blood lymphocytes from AIDS patients but not by combinatorial phage display library techniques. Biased antibody gene usage and expression during HIV-1 infection may be related to HIV-1 pathogenesis by limiting the available HIV-1 neutralizing repertoire. Further molecular characterization of anti-HIV-1 antibodies and in vivo expression of V-region genes during HIV-1 infection should provide important information regarding antibody gene expression and its relationship to HIV-1 pathogenesis.

Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes

Previously described primers for PCR amplification of variable immunoglobulin (Ig) genes were based on gene sequences. To include the large number of amino acid sequences of antibodies whose DNA has not been sequenced and to ensure a maximal fit to rearranged human Ig variable region genes, we have made a comprehensive comparison of both protein and nucleotide sequences. The resulting set of 15 primers was able to amplify a wide range of rearranged antibody variable region genes. Restriction sites included in the primers facilitate cloning of the PCR products into various expression vectors. Sequence analyses of PCR-amplified cDNA derived from a polyclonal B cell population showed that maximal enrichment is obtained for highly represented variable Ig gene subgroups. Rarely occurring Vκ4 and Vλ5 subgroups were not detected. Rearranged Ig variable region genes from each of 19 human B cell lines were also amplified. Comparisons to germline sequences allowed the allocation of rearranged genes to the original Ig genes. This primer set should be very useful for generating large repertoires of rearranged V genes and for amplifying genes of individual B cell clones.

Antibody recognition of a neutralization epitope on the major outer membrane protein of Chlamydia trachomatis.

Two BALB/c mice were immunized with serovar C Chlamydia trachomatis elementary bodies, and 63 hybridomas producing monoclonal antibodies to C. trachomatis were recovered. Eight hybridomas which were specific for an identical peptide epitope (AGLQND) in serovar C major outer membrane protein variable domain I were identified. Detailed immunochemical study of the antigen-antibody interaction and genetic characterization of the antibody variable-region gene sequences showed that distinct B-cell clonal lineages were elicited by the epitope sequence. Since each antibody had a distinct pattern of fine specificity for recognition of the epitope and displayed different degrees of cross-reactivity with a related serovar (serovar A), we conclude that B-cell recognition of an immunodominant neutralization epitope can be pleiotropic. Differences in B-cell recognition of a neutralization epitope may delay the emergence by mutation of antigenic-drift variants of the C. trachomatis major outer membrane protein.

An improved method for generating single-chain antibodies from hybridomas

Cloning the correct V L kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the V L gene-derived products obtained on PCR amplicication of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V regions genes are then spliced by PCR, in the form 5′-V L-LINKER-V H-3′ (where the linker encodes [GlyGlyGlyGlySer] 3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged V L kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.

Low frequencies of somatic mutation in two expressed V kappa genes: unequal distribution of mutation in 5' and 3' flanking regions.

Somatic mutation of antibody variable region genes is a hallmark of secondary responses. Most often the coexpressed VH and V kappa genes of a B cell mutate at nearly equal rates. We have previously identified hybridomas from two B cell clones that exhibited > 10-fold lower frequency of mutation in their expressed V kappa 12 or V kappa 1A genes relative to their coexpressed VH genes. To gain insight into the mechanism(s) responsible for this low frequency V kappa mutation, we determined the frequency of mutation in non-coding flanking DNA from multiple members of each clone. We find a low frequency of mutation in the 5' (4/700) and 3' (1/670) non-coding regions of the expressed V kappa 1A genes consistent with the low frequency of coding region mutation. In contrast, the distribution and frequency of mutation surrounding the expressed V kappa 12.37 gene are unusual. Among the six members of this clone there are 31 mutations 3' (31/2100 bp) and no mutations 5' (0/2010 bp) of the V kappa exon. This V kappa exon has acquired mutations that are intermediate in number to its flanking regions and are significantly skewed in distribution to the 3' end. None of the 31 3' mutations are shared by two or more members of this clone, indicating that they all occurred late in clonal expansion. These results raise the possibility that some V kappa genes may lack functional cis-regulatory elements which direct V kappa coding region mutation.

Molecular characterization of single memory B cells

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.

Idiotypic cross-reactivity of immunoglobulins expressed in Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia, and mantle zone lymphocytes of secondary B-cell follicles

Monoclonal antibodies (MoAbs) specific for autoantibody-associated cross-reactive idiotypes (CRIs) of Waldenstrom's IgM react frequently with the surface Ig (slg) expressed by leukemia cells of patients with chronic lymphocytic leukemia (CLL). Evaluation of the molecular basis for this cross-reactivity indicates that such CRIs are encoded by conserved antibody variable region genes (V genes) that have undergone little or no somatic hypermutation. We find that such anti-CRI MoAbs stain a subpopulation of cells within the mantle zones surrounding the germinal centers of normal human tonsil. In contrast, MoAbs specific for variable region subgroup determinants react with cells in both the mantle zones and germinal centers of secondary B-cell follicles. To test whether mantle zone B cells not reactive with existing anti-CRI MoAbs may express slg bearing as-yet-unrecognized CRIs present on Igs produced by neoplastic cells of some patients with Waldenstrom's macroglobulinemia or CLL, we immunized mice with purified Waldenstrom's IgM that have been characterized for their variable region subgroups using subgroup-specific antisera raised against synthetic peptides. The supernatants of hybridomas generated from the splenocytes of immunized mice were screened for their ability to stain a subpopulation of mantle zone lymphocytes in human tonsil. With this approach, two new anti-CRI MoAbs were identified, designated OAK1 and VOH3. OAK1 binds to a CRI present on a subset of kappa light chains of the VK1 subgroup. VOH3 recognizes a CRI determinant(s) present on a subset of antibody heavy chains of the VH3 subgroup. Flow cytometric analyses demonstrated that OAK1 specifically binds leukemia cells from 5 to 20 patients (25%) with kappa light chain expressing CLL. In addition, VOH3 reacted with the leukemia cells from 1 of 17 (6%) patients tested. The success of these methods demonstrates that the variable regions of the Igs produced by mantle zone B cells share idiotypic determinants with Igs expressed in B-cell CLL (B-CLL) and Waldenstrom's macroglobulinemia.

Idiotypic Cross-Reactivity of Immunoglobulins Expressed in Waldenström's Macroglobulinemia, Chronic Lymphocytic Leukemia, and Mantle Zone Lymphocytes of Secondary B-Cell Follicles

AbstractMonoclonal antibodies (MoAbs) specific for autoantibody-associated cross-reactive idiotypes (CRIs) of Waldenström's IgM react frequently with the surface Ig (slg) expressed by leukemia cells of patients with chronic lymphocytic leukemia (CLL). Evaluation of the molecular basis for this cross-reactivity indicates that such CRIs are encoded by conserved antibody variable region genes (V genes) that have undergone little or no somatic hypermutation. We find that such anti-CRI MoAbs stain a subpopulation of cells within the mantle zones surrounding the germinal centers of normal human tonsil. In contrast, MoAbs specific for variable region subgroup determinants react with cells in both the mantle zones and germinal centers of secondary B-cell follicles. To test whether mantle zone B cells not reactive with existing anti-CRI MoAbs may express slg bearing as-yet-unrecognized CRIs present on Igs produced by neoplastic cells of some patients with Waldenström's macroglobulinemia or CLL, we immunized mice with purified Waldenström's IgM that have been characterized for their variable region subgroups using subgroup-specific antisera raised against synthetic peptides. The supernatants of hybridomas generated from the splenocytes of immunized mice were screened for their ability to stain a subpopulation of mantle zone lymphocytes in human tonsil. With this approach, two new anti-CRI MoAbs were identified, designated OAK1 and VOH3. OAK1 binds to a CRI present on a subset of K light chains of the VK1 subgroup. VOH3 recognizes a CRI determinant(s) present on a subset of antibody heavy chains of the VH3 subgroup. Flow cytometric analyses demonstrated that OAK1 specifically binds leukemia cells from 5 to 20 patients (25%) with K light chain expressing CLL. In addition, VOH3 reacted with the leukemia cells from 1 of 17 (6%) patients tested. The success of these methods demonstrates that the variable regions of the Igs produced by mantle zone B cells share idiotypic determinants with Igs expressed in B-cell CLL (B-CLL) and Waldenström's macroglobulinemia.

Evolution of a VH gene family in low Vertebrates

Antibody genes represent one of the most complex receptor gene systems which have evolved in the vertebrate lineage. One of the central questions is how antibody variable region genes diversify the complementarity determining region (CDR) in evolution while they are subject to forces for sequence homogenization operating in multigene families. Information on germ line antibody genes in lower vertebrates is still fragmentary and it would be fruitful to gain insight into this question. We have studied the evolutionary stability of a rainbow trout VH family (RTVH 431) by Southern blot hybridization in various fish species. Comparison of our results with paleontological/zoological evidence suggests that this VH family has been preserved in the genomes of many fish species over a time span of 150 million years. We also show that there exist species-specific residues common to rainbow trout VH genes, which strongly supports the presence of genetic processes for homogenization. These results are compatible with the view that antibody V genes evolve at a fairly constant rate and that the homogenization process (e.g. gene duplication) may be slow enough to allow diversification of CDR by mutation, selection and drift.

Distribution of Mutations around Rearranged Heavy-Chain Antibody Variable-Region Genes

The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.

Distribution of mutations around rearranged heavy-chain antibody variable-region genes.

The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.

Polymemse Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes from Single Hybridoma Cells

We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies. (Less)

The cellular basis of T-cell memory.

One of the major characteristics of the immune system is its ability to react more quickly and more intensely on the second exposure to an antigen. This characteristic, usually referred to as immunologic memory, was first observed in studies of antibody production in vivo (reviewed in 1). The secondary, or anamnestic, response to an antigen not only results in the production of increased levels of antibodies, but these antibodies also generally have a higher affinity for antigen compared to those antibodies produced during the primary response (2). Memory at the B-cell level is generally accepted as the result of selection and specific expansion of clones secreting high affinity antibody molecules (3, 4). Molecular studies suggest that somatic hypermutation of antibody variable-region genes during the primary response may be responsible for the occurrence of B cells express­ ing high affinity receptors (reviewed in 5). However, the differentiation pathway involved in the generation of memory B cells is still poorly understood. In particular, it is not clear how, in the primary response, some of the B cells differentiate into terminal plasma cells and others into memory cells. While there is ample evidence that immunologic memory also involves T cells, our understanding of the molecular and cellular basis of T-cell secondary responses is quite limited. Following early studies dem­ onstrating that cytolytic T lymphocytes (CTL) are not necessarily terminal cells but may differentiate, at least in vitro, into cells exhibiting the expected properties of memory cells (6), attempts have been made to define quan­ titative and qualitative differences between memory T cells and T cells that have not yet been stimulated by antigen (which are often designated as

Isotype progression and clonality of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice.

Antibodies to the nuclear ribonucleoprotein Sm are found in 25% of MRL/Mp-lpr/lpr mice, which develop a syndrome similar to human systemic lupus erythematosus. We have previously described that these autoantibodies are relatively restricted to the IgG2a isotype. In the current study, we analyze the isotype distribution of anti-Sm antibodies in these mice over time. The most common pattern observed was an initial response of the IgG2a isotype, which progressed such that this isotype was the major one at the time of peak response. No IgM to IgG class switch was observed. Additional studies directed at the clonality of the anti-Sm response indicated that both kappa- and lambda-light chains could be involved, and that the isoelectric focusing pattern of the anti-Sm antibodies was in general characteristic of multiple spectrotypes. These results also support a special role for the IgG2a isotype in the anti-Sm response in MRL/Mp-lpr/lpr mice. Despite this heavy chain isotype restriction, the response usually evidences substantial diversity, which suggests either multiple B cell clones or somatic mutation of antibody variable region genes.