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M S Neuberger

Publications and source records attributed to M S Neuberger.

At least 55 records · Page 3Linked to original sources
PubMed1996

The targeting of somatic hypermutation.

Somatic hypermutation does not occur randomly within immunoglobulin V genes but, rather, is preferentially targeted to certain nucleotide positions (hot spots) and away from others (cold spots). Cold spots often coincide with residues essential for V gene folding. Hotspots, which appear to be strategically located to favour affinity maturation, are most frequently located in the CDRs (particularly CDR1) though conserved hotspots are also found at the base of FR3. Hotspots are in part created by local DNA sequence and the strong biases of codon usage in V genes indicate that the genes have evolved such that somatic hypermutation is targeted to those parts of the V where it is likely to prove most useful. These features of mutational hotspots and biased codon usage are also evident in V genes of lower animals suggesting that diversification by strategic targeting of non-templated mutation may have evolved early in antigen receptor evolution.

Animals↗
PubMed1996

Strategies for expressing human antibody repertoires in transgenic mice.

Repertoires of human antibodies can be created in transgenic mice carrying human immunoglobulin-gene loci in germline configuration. These 'transloci', introduced either as miniloci or as almost locus-sized regions, undergo rearrangement and hypermutation in mouse lymphoid tissue. Here, Marianne Brüggemann and Michael Neuberger review the use of such mice for raising antigen-specific human monoclonal antibodies, as well as their exploitation for studying regulatory aspects of antibody repertoire formation.

Animals↗
PubMed1996

The Ig kappa 3'-enhancer triggers gene expression in early B lymphocytes but its activity is enhanced on B cell activation.

Ig kappa gene expression is controlled by two enhancers, one located within the major intron (Ei) and the other located downstream of C kappa (E3'). Whereas loss of E3' has previously been shown to diminish kappa expression, we show here that a rearranged kappa transgene lacking Ei is well expressed, even at the pre-B cell stage. This suggests that E3' alone might be sufficient to give properly regulated transcription throughout B cell development. Indeed, we show that a transgene composed of a beta-globin reporter linked to E3' is expressed in a B cell-specific manner, becoming activated at the late pro-B to pre-B cell stage but with dramatically enhanced activity on B cell activation. Thus, E3' becomes active as a transcription enhancer at the stage when V kappa-J kappa rearrangement is being initiated and is sufficient to yield an expression pattern in a linked reporter gene similar to that of fully rearranged kappa genes.

Animals↗
PubMed1996

Somatic hypermutation of Ig genes in patients with xeroderma pigmentosum (XP-D).

Antibody diversification by somatic hypermutation occurs by the introduction of nucleotide substitutions in and around the rearranged Ig V gene segments. Several characteristics of the process suggest that the introduction of mutations is linked to Ig gene transcription. Since there is a connection between mutation and repair with indications that both processes might show linkage to transcription, we asked whether defects in a component of the transcription factor TFIIH which lead to an inability to carry out nucleotide excision repair also affect somatic hypermutation. A PCR strategy was devised that required small samples of peripheral blood and enabled us to monitor hypermutation of a single, abundantly used VH gene. However, the results showed that in xeroderma pigmentosum patients (complementation group D), somatic hypermutaton appears to take place unaffected as regard both extent and distribution.

Genes, Immunoglobulin↗
PubMed1996

Somatic hypermutation of immunoglobulin genes.

The relationship between somatic hypermutation and affinity maturation in the mouse is delineated. Recent work on the anatomical and cellular site of this process is surveyed. The molecular characteristics of somatic hypermutation are described in terms of the region mutated and the distinctive patterns of nucleotide changes that are observed. The results of experiments utilizing transgenic mice to find out the minimum cis-acting sequences required to recruit hypermutation are summarized. The hypothesis that V gene sequences have evolved in order to target mutation to certain sites but not others is discussed. The use that different species make of somatic hypermutation to generate either the primary or secondary B cell repertoire is considered. Possible molecular mechanisms for the hypermutation process and future goals of research are outlined.

Animals↗
PubMed1995

Targeting of non-Ig sequences in place of the V segment by somatic hypermutation.

Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.

Animals↗
PubMed1995

Somatic hypermutation.

For the generation of secondary response antibodies, immunoglobulin genes are subjected to hypermutation. Cells expressing antibodies with higher affinity are then selected by antigen. Recent clues to the mechanism of hypermutation come from experiments using transgenic mice enabling analysis of the controlling cis-acting elements and the intrinsic features of the hypermutation, dissociated from the effects of antigenic selection.

Animals↗
PubMed1995

Regulated activity of the IgH intron enhancer (E mu) in the T lymphocyte lineage.

The activity of the IgH (E mu) enhancer in the T lymphocyte lineage has been investigated using both transgenic mice and transfection studies. Thymocyte fractionation experiments indicate that a transgene consisting of the bacterial chloramphenicol acetyl transferase (CAT) gene, linked to E mu and the SV40 early promoter (E mu-CAT), is expressed only in thymocytes with a mature medullary phenotype and not in immature cells. Transfection of this same construct into two thymoma cell lines representing different stages of thymocyte development mimics the pattern of activity observed in vivo. Further transfection experiments suggest that this pattern of expression might be attributed to the differential activity of the E2E3 and octanucleotide motifs of E mu during development. In contrast, an Ig lambda transgene (linked to E mu and an Ig V lambda promoter) is expressed in the majority of thymocytes. We envisage that the different patterns of expression of the two transgenes reflect interactions between their respective promoters and the factors which are bound to E mu at different stages of thymocyte development. Although differing in their pattern of expression within the thymus, the two transgenes share the property of extinction in peripheral T lymphocytes. These results indicate that the expression of E mu-linked transgenes in the thymus cannot simply be explained by activation of the enhancer in a lymphoid progenitor cell prior to B/T lineage divergence. Rather, the enhancer (or components of it) must be independently activated (and inactivated) during T lymphocyte development. Furthermore, this activity is consistent with the developmental timing of Ig DH-JH rearrangements in these cells.

Animals↗
PubMed1994

Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.

The rate and nature of hypermutation of immunoglobulin genes are of prime importance in the affinity maturation of antibodies. Although a considerable body of information has been gathered for kappa light chains, there is much less data for lambda chains. We have derived a large data base of somatic mutants of mouse lambda 1 light chains from Peyer's patches germinal center B cells. The endogenous lambda 1 genes mutate at a rate comparable to that previously found for a kappa transgene (V kappa ox1). There are intrinsic hot spots of mutation common to both in-frame and out-of-frame rearrangements; these hot spots cluster in hypermutating domains. In contrast to the pattern seen for V kappa Ox1, the hot spot clusters are found not only in complementarity-determining region (CDR)1 but also in CDR2 and CDR3; mutations also cluster in the joining/constant region intron. The differences between the pattern of mutations in V kappa Ox1 and lambda 1 light chains are discussed.

Amino Acid Sequence↗
PubMed1994

Antibodies generated from human immunoglobulin miniloci in transgenic mice.

One approach to the production of human monoclonal antibodies focusses on the creation of transgenic mice bearing human immunoglobulin gene miniloci. Whilst such loci undergo lymphoid-specific gene rearrangement, only a small proportion of mouse B cells express the human immunoglobulin chains; the miniloci thus contribute poorly to serum immunoglobulin. Attributing this poor performance to competition between the transgenic and endogenous immunoglobulin loci, we crossed mice bearing a human immunoglobulin heavy-chain (HulgH) minilocus with animals that had been rendered B cell-deficient by disruption of their endogenous heavy-chain locus. The results were dramatic: the human minilocus rescued B cell differentiation such that effectively all B cells now expressed human mu chains. The concentration of antibody in the mouse serum recognised by anti-human mu increased to a concentration about one sixth that in human serum. The HulgH antibodies are heterogenous with diversity being generated by both combinatorial and junctional processes. Following antigen challenge, specific antibody is elicited but at low titre.

Amino Acid Sequence↗
PubMed1994

Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region.

Following encounter with antigen, the immunoglobulin genes in B lymphocytes undergo somatic hypermutation. Most nucleotide substitutions are introduced into a region flanked by the V gene promoter and intron enhancer. Experiments described here using transgenic mice revealed that the V kappa promoter does not contain specific signals since hypermutation was retained on substituting it by a beta-globin promoter. However, both the kappa intron and kappa 3' enhancer regions were found to be essential for full hypermutation. This dependence of hypermutation on both enhancers contrasts with transgene expression in hybridomas in which only the 3' enhancer (and not the intron enhancer) is necessary to achieve high mRNA levels. The results show that full hypermutation depends on multiple elements, removal of some of which may drastically impair but not totally abolish the process.

Amino Acid Sequence↗
PubMed1994

The alpha/beta sheath and its cytoplasmic tyrosines are required for signaling by the B-cell antigen receptor but not for capping or for serine/threonine-kinase recruitment.

The B-cell antigen receptor is composed of membrane immunoglobulin sheathed by an alpha/beta heterodimer. The complex is noncovalently associated with protein kinase activity, and crosslinking of the receptor leads to capping and transmembrane signaling. Here we show that the sheath is not necessary either for this capping or for the association of membrane immunoglobulin with the detergent-insoluble cytoskeletal fraction that occurs following crosslinking. It is also not required for association of membrane immunoglobulin with a casein-kinase-like serine/threonine kinase. The sheath is essential, however, for transmembrane signaling. Provision of just the cytoplasmic domain of the beta sheath polypeptide to a mutant, unsheathed IgM molecule was sufficient to restore full signaling capability as judged by the phosphorylation of a variety of cellular proteins, including the B-cell-specific transmembrane protein CD22. This signaling was destroyed by mutating one of the tyrosines in the beta cytoplasmic domain. These results not only suggest that receptor signaling is mediated through phosphorylation of the tyrosines in the sheath's cytoplasmic domains but, together with previous work, indicate that different motifs within the sheath mediate presentation and signaling.

Amino Acid Sequence↗
PubMed1994

The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci.

An approach to the preparation of antigen-specific human monoclonal antibodies focuses on mice transgenic for human immunoglobulin gene miniloci; the V gene segments in these miniloci undergo productive rearrangement to yield mouse B cells expressing human immunoglobulin (Ig) chains. The general usefulness of this strategy hinges on whether it is feasible to obtain specific, high-affinity antibodies following immunization of such animals with a variety of antigens. To test this, we have investigated the antigen-specific responses in mice which carry human IgH miniloci (constaining just one or two VH segments) instead of a functional mouse IgH locus. Although serum responses were relatively weak, monoclonal antibodies were readily obtained to all immunogens tested (a hapten, foreign proteins and human lymphoma cells). The affinities of two of the hapten-specific (anti-2-phenyl-oxazol-5-one) antibodies were 60 and 160 nM, values intermediate between what is typically obtained in the primary and secondary response of normal mice. Sequence analysis of the rearranged V genes revealed that junctional events made a major contribution to diversity with a considerable amount of apparently non-templated sequence at the V-D and D-J borders. Somatic hypermutation was also evident within the expressed V gene segments of many of the antigen-specific hybridomas. These findings augur well for the general usefulness of the transgenic approach for the isolation of high-affinity human antibodies to a wide range of antigens and suggests that the miniloci need not be particularly large.

Amino Acid Sequence↗
PubMed1993

Antigen presentation by the B cell antigen receptor is driven by the alpha/beta sheath and occurs independently of its cytoplasmic tyrosines.

Membrane immunoglobulin functions to internalize bound antigen for its subsequent processing and presentation to T cells. Although the five immunoglobulin isotypes exhibit considerable differences in their cytoplasmic domains, we show by use of matched B lymphoma transfectants that all isotypes manifest a similar high efficacy in antigen presentation. Experiments using mutant receptors reveal that this efficacy can be ascribed to the alpha/beta sheath of the receptor, where presentation correlates with internalization of polyvalent antigen. Efficient presentation is restored to a sheathless antigen receptor by providing it with only the cytoplasmic domain of the beta sheath polypeptide. This restoration of activity does not depend on the tyrosine residues in the beta cytoplasmic tail, implying that antigen receptor-mediated presentation can occur by a pathway distinct from that used by the Fc receptor Fc gamma RIII.

Amino Acid Sequence↗
PubMed1993

Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots.

We have analyzed somatic hypermutation in mice carrying an immunoglobulin kappa transgene in order to discriminate mutations that reflect the intrinsic specificity of the hypermutation mechanism from those highlighted by antigenic selection. We have immunized animals with three different immunogens. With one immunogen, the antigen-specific B cells express a transgenic kappa chain, which does not form part of the antibody; the transgene is a passenger free to accumulate unselected mutations. With the other two immunogens, the transgenic kappa chain constitutes the light chain of the expressed antibody. A comparison of the transgene mutations obtained under these different circumstances allows us to identify common features that we attribute to the intrinsic specificity of the hypermutation process. In particular, it yields only base substitutions and leads to hot spots occurring in individual positions (e.g., the second base of the Ser-31 codon). The mutations preferentially accumulate around the first complementarity-determining region. The process exhibits specific base substitution preferences with transitions being favored over transversions. We propose that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots. We also note that hypermutation distinguishes between the coding and noncoding strands since pyrimidines (particularly thymidines) mutate less frequently than purines.

Amino Acid Sequence↗
PubMed1993

Association of CD22 with the B cell antigen receptor.

The antigen receptor on B lymphocytes is composed of membrane immunoglobulin sheathed by an alpha/beta heterodimer. This structure is in several respects analogous to the antigen receptor on T cells except that, in the case of the T cell but not the B cell receptor, several receptor-associated proteins have been described which may modulate the effects of antigen interaction (e.g. CD4, CD8, CD2 and CD5). To screen for specific associations with the B cell antigen receptor that might be of only low stoichiometry, we have exploited the sensitivity of in vitro kinase assays. We show that the B cell antigen receptor associates with CD22. The association is specific and stable, but Western blotting reveals it to be of low stoichiometry (0.2 to 2% of membrane immunoglobulin is CD22 associated). The CD22/antigen receptor association was demonstrated with multiple isotypes (IgM, IgD and IgG) and was evident both in Burkitt lymphoma lines and in tonsil cells. Whilst the significance of the association is unknown, it is notable that CD22 is a B cell-specific adhesion molecule which we find contains within its cytoplasmic domain a sequence bearing high homology to the "Reth motif" implicated in signal transduction. Indeed, CD22 becomes tyrosine phosphorylated less than one minute after antigen-receptor cross-linking. Thus, it is tempting to speculate that interactions involving CD22 assist in the antigen-mediated triggering of B cell activation.

Amino Acid Sequence↗