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T Manser

Publications and source records attributed to T Manser.

At least 55 records · Page 3Linked to original sources
PubMed1991

Position of the rearranged V kappa and its 5' flanking sequences determines the location of somatic mutations in the J kappa locus.

Somatic hypermutation is known to occur in the VJ kappa exon and its flanking sequences, yet little is known about the hypermutation mechanism or its exact target within the rearranged locus. Mutations may occur at the same frequency, spanning a region from the leader intron to 3' of J kappa 5, regardless of which J is chosen for VJ rearrangement. Another possibility is that mutations may be limited to the rearranged VJ kappa and its immediate flanking sequences. To distinguish between these possibilities, the JC introns of 21 alleles with V kappa rearranged to J kappa 1 were sequenced, and mutations were located. The frequency of mutations was determined for different sections of the intron and compared with the frequencies of mutations found in the JC intron of a set of VJ kappa 5 alleles. The results showed that mutations were concentrated in and around the rearranged VJ, regardless of whether J kappa 1 or J kappa 5 was used. These data imply that the hypermutational mechanism focuses on rearranged V genes.

Base Sequence↗
PubMed1991

Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.

Amino Acid Sequence↗
PubMed1990

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.

Alleles↗
PubMed1990

Limits on heavy chain junctional diversity contribute to the recurrence of an antibody variable region.

Antibody V region structural diversity in the mouse is generated, in part, by the combinatorial joining of different gene segments, as well as by the "imprecision" of these joining events. The same two gene segments can be joined at different locations, and nucleotides can be deleted or added de novo to the segment junction. While it is clear that such junctional processes are a major contributor to V region diversity, the mechanisms that generate this diversity are poorly understood. Here I present sequences in the VH-D-JH region of 34 VH genes that are composed of the same three VH gene segments. In combination with a single V kappa-J kappa pair, these VH genes encode a family of V regions that are recurrently expressed in the immune response of A/J mice to p-azophenylarsonate (Ars). The germline sequences of the three constituent gene segments for these VH genes are known, making it possible to determine the origin of the nucleotides in junctional regions. An examination of the frequency and type of nucleotides present in these regions provides insight into the properties of the segment joining mechanism. In addition, the data suggest that recurrent expression of the anti-Ars V regions which these VH genes partially encode is due not only to antigenic selection, but to the high probability with which these VH genes are formed during B cell differentiation.

Animals↗
PubMed1990

The efficiency of antibody affinity maturation: can the rate of B-cell division be limiting?

It has been known for many years that the affinity of antibodies for antigen increases with time during an immune response. It is now clear that two processes play fundamental roles in this affinity 'maturation' in the mouse - V gene somatic mutation and antigen affinity-based selection. Exactly how these two processes work in concert is not fully understood. In this article Tim Manser argues that models of affinity maturation based on the assumption that somatic mutation, antigen selection and B-cell division are interdependent may not explain the high efficiency of the process, and he suggests an alternative model.

Animals↗
PubMed1990

The intraclonal diversity and control of antibody isotype switch recombination.

The configuration of the expressed and unexpressed IgH alleles within two groups of hybridomas, both derived from single clones of B cells at an intermediate stage of the immune response of one mouse, were characterized. The data obtained confirm that individual 'naive' B cells are totipotent in their capacity to generate, via deletions in the IgH locus, offspring that express different IgG isotypes. Further, the number of distinct isotype switch deletions that took place during the proliferation and differentiation of these clones was enormous. Nevertheless, switching within one clone occurred exclusively to the IgG1 isotype at expressed and unexpressed IgH alleles. The data support the conclusion that, despite the fact that isotype switch recombination is occurring on a global scale within responding clones during the immune response, exclusive intraclonal expression of only one isotype can result from the direct control of the switching process.

Alleles↗
PubMed1989

Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response.

To determine how the memory B cell population elicited to one epitope might be used in immune responses to other, structurally related epitopes, we explored the phenomenon of original antigenic sin. Strain A/J mice reproducibly respond to immunization with p-azophenylarsonate (Ars) by production of anti-Ars antibodies encoded predominantly by a single VH gene segment (VHIdCR). The structural analogue of Ars p-azophenylsulfonate (Sulf) fails alone to elicit such V regions, but can do so in A/J mice previously immunized with Ars, providing a means to specifically examine B cells capable of responding secondarily to a crossreactive antigen (i.e., memory cells). VHIdCR-expressing hybridomas were derived from the Ars-primed, Sulf-boosted original antigenic sin response of A/J mice at various times after Ars priming, and the properties of the antibodies they express and the structure of the genes encoding these antibodies were characterized. The data obtained support the following conclusions: (a) The Ars-induced memory B cell population capable of being crossreactively stimulated by Sulf is largely formed from a small fraction of all B cells participating in the anti-Ars primary response that express somatically mutated V regions; (b) the antibody repertoire and clonal composition of this population are stable over long periods of time; (c) memory B cells are capable of clonal expansion in the absence of a high rate of V gene somatic mutation; (d) the activation requirements for clonal selection of memory, versus naive B cells appear to differ; and (e) a major fraction of Ars-induced memory B cells express either IgM or IgG3 prior to and during the initial stages of the sin response.

Animals↗
PubMed1989

Evolution of antibody structure during the immune response. The differentiative potential of a single B lymphocyte.

Changes in the structure and function of antibodies occur during the course of an immune response due to variable (V) region gene somatic mutation and isotype switch recombination. While the end products of both these processes are now well documented, their mechanisms, timing, and regulation during clonal expansion remain unclear. Here I describe the characterization of antibodies expressed by a large number of hybridomas derived from single B cell clones at an intermediate stage of an immune response. These data provide new insights into the mechanism, relative timing, and potential of V gene mutation and isotype switching. The data suggest that somatic mutation and isotype switching are completely independent processes that may, but need not, occur simultaneously during clonal expansion. In addition, the results of this analysis demonstrate that individual B cell clones are far more efficient than previously imagined at generating and fixing particular V region somatic mutations that result in increased affinity for the eliciting epitope. Models to account for this high efficiency are discussed. Taken together with previous data, the results of this analysis also suggest that the "somatic evolution" of V region structure to a single epitope takes place in two stages; the first in which particular mutations are sustained and fixed by antigen selection in the CDR regions of the V region genes expressed in a clone over a short period of clonal expansion, and the second in which these selected CDR mutations are maintained in the growing clone, deleterious mutations are lost, and selectively neutral mutations accumulate throughout the length of V genes over long periods of clonal expansion.

Amino Acid Sequence↗
PubMed1987

Somatically mutated forms of a major anti-p-azophenylarsonate antibody variable region with drastically reduced affinity for p-azophenylarsonate. By-products of an antigen-driven immune response?

The pivotal role played by antigen in the clonal selection of B cells for initial participation in an immune response is well established. Antigen selective mechanisms ensure that antigen-binding antibodies are produced during all stages of the immune response. However, antibodies that lack specificity for the immunogen might also be produced during the course of an antigen-driven immune response . It has been suggested that, through idiotype-antiidiotype network interactions within the immune system, production of antibodies that lack specificity for the immunogen but that share idiotopes with antigen-binding antibodies could result (1). In addition, data obtained by a number of investigators suggest that somatic mutation of antibody V region genes occurs at a rate of 10(-3)/basepair/cell division in B cells participating in an immune response (2, 3). One outcome of such V region structural alteration could be antibodies that lack, or have drastically reduced affinity for the immunogen . We sought to identify and characterize some of the antibody by-products of the antigen-driven immune response that are expected to be created by the mechanisms described above.

Amino Acid Sequence↗
PubMed1987

Influence of the macromolecular form of a B cell epitope on the expression of antibody variable and constant region structure.

We investigated the influence of the macromolecular form of an epitope on the structure of antibody variable and constant regions expressed by the B cell population participating in an immune response to that epitope. Hybridomas were constructed from strain A/J mice undergoing either primary or secondary immune responses to p-azophenylarsonate conjugated to Brucella abortus (Ars-Bruc). We determined the sequences of the V genes expressed by hybridomas selected on the basis of expression of a single VH gene segment known to encode a large family of anti-Ars antibodies. These sequences were compared with the sequences of V genes expressed by a previously characterized panel of hybridomas isolated in the same way during the primary and secondary responses of A/J mice to Ars-KLH. The repertoire of Ars-specific V domains expressed among primary and secondary hybridomas elicited with these two forms of Ars were similar, as were the differences between primary and secondary V region somatic mutational alteration and affinity for Ars. In contrast, predominant expression of IgG2 anti-Ars antibodies was elicited in the secondary Ars-Bruc response, whereas secondary anti-Ars antibodies elicited with Ars-KLH are predominantly IgG1. Thus, differences in the macromolecular form of Ars clearly influence the isotypic profile of the anti-Ars response, but the expression, diversification, and selection of V domains elicited with this hapten are not greatly affected by such differences. Our results suggest that while isotype regulation is highly perceptive of the macromolecular form of a B cell epitope, V region regulation is primarily influenced by the molecular structure of that epitope.

Animals↗
PubMed1987

Mitogen-driven B cell proliferation and differentiation are not accompanied by hypermutation of immunoglobulin variable region genes.

Hybridomas were constructed from splenic B cells after mitogen stimulation in vitro with lipopolysaccharide and dextran sulfate for 9 to 11 days. Extensive proliferation and differentiation (secretion of IgG isotypes) was evident in these cultures before fusion. Hybridomas that express a VH gene segment whose germ-line sequence is known were isolated, and the nucleotide sequences of these expressed VH genes were determined. A total of 3775 VH nucleotides was analyzed in this way, and only one difference from the germ-line VH sequence was observed. The rate of V gene somatic mutation that has been estimated to occur during antigen-driven immune responses in vivo is 10(-3)/base pair/cell division. Given an estimated value for the number of cell divisions that occurred before hybridoma formation, at least 15 changes from the germ-line VH sequence should have been observed if mutation had been occurring at the in vivo rate during the culture period. Therefore, the data suggest that mitogen-driven B cell proliferation and differentiation are not sufficient to induce the hypermutation of Ig V region genes.

Animals↗
PubMed1987

Evolution of antibody variable region structure during the immune response.

The results reviewed above reveal that during the anti-Ars immune response of strain A mice a somatic process that results in the evolution of V region structure occurs. This process involves both the selection of V regions encoded by particular gene segment combinations as well as the selection of structural variants of these V regions produced by somatic mutation as the immune response progresses. As a result, both quantitative and qualitative changes in the V region population initially elicited by immunization take place. The structural and functional character of the immune V region repertoire appears to be largely determined by this process of "somatic evolution" occurring in the primary response.

Animals↗
PubMed1986

A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus.

These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes.

Animals↗
PubMed1986

The molecular evolution of the immune response: idiotope-specific suppression indicates that B cells express germ-line-encoded V genes prior to antigenic stimulation.

Antibodies expressed by the immune B cell population are characterized by variable region amino acid substitutions resulting from somatic nucleotide replacement (somatic mutation). This is not true of antibodies expressed by the "naive" B cell population. It is at present unclear whether this discrepancy is due to the preferential clonal selection of a pre-existing subpopulation of naive B cells that express variable regions altered via nucleotide replacement, or whether the process of nucleotide replacement occurs only during the antigen-dependent stages of B cell differentiation. To address this question we have used anti-idiotypic suppression to functionally delete B cells that express particular variable-region structures from the antigen-responsive repertoire. Suppression of the major cross-reactive idiotype (IdCR) expressed in strain A mice in response to p-azophenylarsonate (Ars) was induced using the monoclonal anti-IdCR antibody AD8. The idiotope recognized by AD8 is easily destroyed by alteration of IdCR variable-region structure via nucleotide replacement. The IdCR anti-Ars immune repertoire is characterized by antibodies that lack the AD8-cognate idiotope due to nucleotide replacement. However, complete suppression of the IdCR could reproducibly be achieved by administration of AD8 prior to Ars immunization. This result indicates that all IdCR-expressing B cells also express the AD8-cognate idiotope prior to immunization. Thus, somatic nucleotide replacement must occur exclusively during the antigen-dependent stages of B cell differentiation in this system.

Animals↗
PubMed1986

Somatic evolution of variable region structures during an immune response.

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.

Animals↗
PubMed1986

Site-directed mutagenesis of an invariant amino acid residue at the variable-diversity segments junction of an antibody.

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.

Amino Acid Sequence↗