Biomedical subjects
M S Neuberger
Publications and source records attributed to M S Neuberger.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Membrane immunoglobulin without sheath or anchor.
The canonical form of the B cell antigen receptor is composed of membrane immunoglobulin sheathed by the alpha/beta heterodimer. Whereas membrane IgM cannot be transported to the cell surface in the absence of alpha/beta, both IgD and IgG2b can be expressed naked (i.e. without alpha/beta) on the surface of myeloma transfectants. In the case of one cell-line, such naked IgD has been shown to be inserted into the membrane by a glycosyl-phosphatidylinositol anchor. Here, however, we show that both IgD and IgG2b (but not IgM) can be expressed on the surface of myeloma transfectants without either sheath or anchor. This distinction between the isotypes is attributable to differences in the region of the transmembrane segment.
Creation of mice expressing human antibody light chains by introduction of a yeast artificial chromosome containing the core region of the human immunoglobulin kappa locus.
We have previously described a strategy for integrating selectable marker genes into yeast artificial chromosomes (YACs) to facilitate their transfer into embryonic stem (ES) cells. Here we apply this technology to create mice carrying the core region of the human immunoglobulin (Ig) kappa light chain locus. A YAC was isolated which contains a 300 kb insert spanning three V kappa segments, the J kappa cluster, the C kappa region and extending downstream of the Kde element. After modification of this YAC to integrate the selectable neo marker gene, the YAC was introduced into ES cells by protoplast fusion. Several ES cell clones were obtained which appeared to harbor one complete copy of the YAC while retaining little or no other yeast DNA. The ES cells were injected into blastocysts and the chimaeric mice were shown to rearrange the introduced human light chain genes with the resultant production of antibodies containing human kappa light chains in the serum.
The B-cell antigen receptor of the five immunoglobulin classes.
Several proteins associate with surface IgM to form the antigen receptor. We show that just two, the alpha and beta associated chains, are sufficient to reconstitute an IgM surface receptor in fibroblasts. Contrary to expectation, a common alpha chain associates with all five immunoglobulin classes. We propose that B-cell antigen receptors consist of a common alpha/beta heterodimer associated with each immunoglobulin class. But the classes differ both in the glycosylation of their associated alpha chain and in their dependence on alpha/beta for surface transport.
The mouse IgH 3'-enhancer.
A lymphoid-specific transcription enhancer element has recently been identified at the far 3' end of the rat immunoglobulin heavy chain (IgH) locus. Sequence analysis presented here reveals that this enhancer is flanked by a 350-bp invert repeat, giving a structure reminiscent of a transposable element. We therefore screened for the equivalent enhancer in the mouse to determine whether its presence was conserved during evolution. A mouse homologue was indeed identified and is located 16 kb downstream of the C alpha 1 exon. It is also flanked by invert repeats and these are not repeated throughout the genome. The mouse and rat enhancers retain high sequence homology. As regard activity, the IgH 3'-enhancer is lymphoid specific. However, this activity was detected in two plasmacytoma lines tested but not in two B cell lymphomas nor in HeLa cells suggesting that the enhancer may only play a stage-specific role during lymphocyte differentiation. As regards function within the IgH locus, we found that inclusion of the mouse IgH 3'-enhancer (in addition to the intron-enhancer) on mu gene expression plasmids effected a small increase in mu mRNA levels in stable plasmacytoma transfectants.
Somatic hypermutation of immunoglobulin kappa may depend on sequences 3' of C kappa and occurs on passenger transgenes.
We have compared the pattern of somatic mutation in different immunoglobulin kappa transgenes and suggest that an element(s) located between 1 kb and 9 kb 3' of C kappa is necessary for somatic hypermutation of the antibody V gene. The sequences of transgenic and endogenous Ig V regions were determined in antigen-specific B cell hybridomas specific for 2-phenyloxazolone from independent lines of hyperimmunized transgenic mice. We analysed somatic mutation of the transgene both in hybridomas in which the transgenic kappa chain contributes to the antigen combining site as well as in hybridomas in which the transgene is a passenger with the expressed antibody being composed of endogenously-encoded heavy and light chains. In both cases, nucleotide changes in the transgene are correctly targeted to the V region and are absent from the C region. They accumulate at a similar rate to that in the endogenous Ig genes within the same cell and we find that, irrespective of whether or not the transgene kappa is directly selected by antigen, somatic mutation occurs at a similar rate and involves only single base substitutions. Furthermore, the pattern of mutations in passenger transgenes gives information about the intrinsic sequence specificities of the somatic hypermutation mechanism.
The importance of the 3'-enhancer region in immunoglobulin kappa gene expression.
The first enhancers to be identified in the immunoglobulin gene loci are located in the J-C intron. However, deletion of the immunoglobulin kappa intron-enhancer has little effect on the transcription of kappa transgenes. Here we ask whether the second kappa enhancer which we recently identified at the 3'-end of the locus plays a role in kappa gene expression. We show that its omission leads to 20-40 fold lower expression of kappa transgenes and to poor allelic exclusion. Transfection experiments show that activity of the 3'-enhancer, like that of the kappa-intron enhancer, can be induced in a pre-B cell line by incubation with bacterial lipopolysaccharide. Whereas induction of the kappa-intron enhancer is due to induction of NF-kappa B activity, deletion mapping of the 3'-enhancer localises its activity to a 50 nucleotide region that lacks an NF-kappa B site; indeed the 3'-enhancer allows kappa expression in a cell line which lacks NF-kappa B. Thus, both the 3'- and intron-enhancers can be induced at the same stage of differentiation but by distinct pathways. Furthermore, unlike the intron-enhancer, the 3'-enhancer plays a critical role in the transcription of rearranged immunoglobulin kappa genes.
Lymphoid-specific transcriptional activation by components of the IgH enhancer: studies on the E2/E3 and octanucleotide elements.
The IgH enhancer is a strong lymphoid-specific activator and is composed of multiple factor-binding motifs. One of these, the octamer, is common to enhancer and promoter, binds ubiquitous and lymphoid-specific factors and is able to act as a lymphoid-specific transcriptional activator. However, it is also found as an essential component of promoters active in non-lymphoid cells. From analysis of the activities of synthetic promoters, we suggest that recruitment of the lymphoid-specific octamer-binding protein next to the TATA is sufficient to create a functional lymphoid-specific promoter whereas the ubiquitous octamer binding protein is not active in single copy but can act in concert with other promoter binding factors. However, the activity of the IgH enhancer is not dependent on the octamer and we identify the E2/E3 elements as also being sufficient to confer lymphoid-specificity on a linked gene. Activity of the E2/E3 region results from the synergistic activity of the two motifs, E2 alone being able to confer a low level of activity which is dramatically increased by the adjacent E3. Thus, in the case of both the E2/E3 and the octamer motifs, interactions between adjacent elements can play a critical role in determining the tissue specificity of activity.
A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus.
The expression of immunoglobulin heavy-chain (IgH) genes is generally thought to be regulated by the combination of the VH promoter with the enhancer element which is located in the JH-CH intron. This is probably an oversimplification: there are cell lines that transcribe IgH genes despite the deletion of the intron-enhancer. These findings could imply that other enhancer element(s) exist in the IgH locus. Here we show that a strong B-cell-specific enhancer is indeed located at the 3'-end of the rat IgH locus, 25 kilobases downstream of C alpha. This enhancer should be retained downstream of all rearranged IgH genes, regardless of the VH or CH segment used. Taken together with analogous findings for the mouse kappa locus, the results prompt a re-evaluation of the mechanism of regulation of immunoglobulin gene transcription. Furthermore, unlike the intron-enhancer, the IgH 3' enhancer would become linked to a c-myc that rearranges into an IgH switch region. The IgH 3' enhancer could therefore play a part in the activation of the translocated c-myc genes in rat immunocytomas, mouse plasmacytomas and Burkitt lymphomas.