Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci.

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.

Novartis Medal Lecture. Antibodies: a paradigm for the evolution of molecular recognition.

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination.

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells.

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

B cells acquire antigen from target cells after synapse formation.

Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.

In vivo and in vitro studies of immunoglobulin gene somatic hypermutation.

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.

Somatic hypermutation of immunoglobulin kappa transgenes: association of mutability with demethylation.

Following antigen encounter, immunoglobulin genes are diversified by somatic hypermutation. The mechanism by which this mutational process preferentially targets immunoglobulin genes is not known, but is likely linked to transcription. However, transcription is not sufficient to ensure mutability. Here, by polymerase chain reaction amplification of bisulfite-modified DNA, the pattern of demethylation within the Igkappa mutation domain is analysed and transgenes are used to identify an association between demethylation and mutability. In mice carrying an Igkappa transgene that is well transcribed but only poorly targeted for hypermutation, the mutated transgene copies have been demethylated within the mutation domain, whereas the methylated copies remain unmutated. Thus, the hypermutation mechanism only acts on immunoglobulin gene targets that are demethylated as well as transcribed, although transcription and demethylation do not themselves guarantee mutability.

Somatic hypermutation in the absence of DNA-dependent protein kinase catalytic subunit (DNA-PK(cs)) or recombination-activating gene (RAG)1 activity.

Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.

Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro.

Multiple DNA polymerases exist in eukaryotes. Polymerases alpha, delta and epsilon are mainly responsible for chromosomal DNA replication in the nucleus and are required for proliferation. In contrast, the repair polymerases beta and eta are not essential for cellular proliferation in yeast or mice, but a lack of either polymerase can lead, respectively, to defects in base excision repair or the ability to replicate past lesions induced by ultraviolet (UV) radiation [1-3]. Here, we have focused on polymerase zeta. This was first described as a non-essential product of the yeast REV3/REV7 genes involved in UV-induced mutagenesis, and was later implicated in trans-lesion synthesis [4,5]. Unlike in yeast, the mouse homologue (mRev3) was found to be essential for life. Homozygous mutant mice died in utero. Mutant embryos were considerably reduced in size at day 10.5 of development and usually aborted around day 12.5. It is likely that this block reflects a need for mRev3 in proliferative clonal expansion (rather than in the production of a particular cell type) as mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture. Thus, mRev3 could be required to repair a form of externally induced DNA damage that otherwise accumulates during clonal expansion or, consistent with the high homology shared between its Rev7 partner and the mitotic checkpoint gene product Mad2 [6], mRev3 might play a role in cell proliferation and genomic stability even in the absence of environmentally induced damage.

The contribution of somatic hypermutation to the diversity of serum immunoglobulin: dramatic increase with age.

Although somatic mutation contributes to the diversity of only a minor fraction of B cells in mouse spleen or blood, its contribution to the diversity of serum immunoglobulin is unknown. We have devised an immunoassay to monitor mutated antibodies in serum using a monoclonal antibody that recognizes a VK only when mutated at its major intrinsic hot spot. Mutation makes essentially no contribution to the diversity of endogenous serum IgM, IgG, or IgA in young mice. However, in response to environmental antigens, the titer of mutated immunoglobulin in T cell-proficient mice rises strikingly with age, such that the major proportion of serum immunoglobulin in adults is somatically mutated, with the mutation load in IgG being some 10-fold greater than in IgM.

Dysregulated expression of the Cd22 gene as a result of a short interspersed nucleotide element insertion in Cd22a lupus-prone mice.

The Cd22 gene encodes a B cell-specific adhesion molecule that modulates B cell Ag receptor-mediated signal transduction, and is allelic to a lupus-susceptibility locus in New Zealand White (NZW) mice. In this study, we show that, in addition to the wild-type transcripts, NZW (Cd22a) mice synthesize aberrant CD22 mRNAs that contain approximately 20-120 nucleotide insertions upstream of the coding region between exons 2 and 3, and/or approximately 100-190 nucleotide deletions of exon 4. Sequence analysis revealed that these aberrant mRNA species arose by alternative splicing due to the presence in the NZW strain of a 794-bp sequence insertion in the second intron, containing a cluster of short interspersed nucleotide elements. Both the presence of sequence insertion and aberrantly spliced mRNAs were specific to mice bearing the Cd22a and Cd22c alleles. Up-regulation of CD22 expression after LPS activation appeared impaired in Cd22a spleen cells (twice lower than in Cd22b B cells). Furthermore, we show that partial CD22 deficiency, i.e., heterozygous level of CD22 expression, markedly promotes the production of IgG anti-DNA autoantibodies in C57BL/6 (Cd22b) mice bearing the Y chromosome-linked autoimmune acceleration gene, Yaa. Taken together, these results suggest that a lower up-regulation of CD22 on activated B cells (resulting from Cd22 gene anomaly in Cd22a mice or from CD22 heterozygosity in mutants obtained by gene targeting) is implicated in autoantibody production, providing support for Cd22a as a possible candidate allele contributing to lupus susceptibility.

Immunology. RNA editing AIDs antibody diversification?

How do B cells generate the enormous diversity of antibodies that are able to recognize and bind to whichever antigen a B cell might happen to encounter in the body? Several genetic mechanisms that manipulate different combinations of immunoglobulin genes are known. In their Perspective, Neuberger and Scott, highlight another genetic mechanism called RNA editing now shown to be involved in the production of antibody diversity.

The c-MYC allele that is translocated into the IgH locus undergoes constitutive hypermutation in a Burkitt's lymphoma line.

Burkitt's lymphomas harbour chromosomal translocations bringing c-MYC into the vicinity of one of the immunoglobulin gene loci. Point mutations have been described within c-MYC in several Burkitt's lymphomas and it has been proposed that translocation into the Ig loci might have transformed c-MYC into a substrate for the antibody hypermutation mechanism. Here we test this hypothesis by exploiting a Burkitt's lymphoma line (Ramos) that we have previously shown to hypermutate its immunoglobulin genes constitutively. We find that, during in vitro culture, Ramos mutates the c-MYC allele that is translocated into the IgH locus whilst leaving the untranslocated c-MYC and other control genes essentially unaffected. The mutations are introduced downstream of the c-MYC transcription start with the pattern of substitutions being characteristic of the antibody hypermutation mechanism; the mutation frequency is 2-3-fold lower than for the endogenous functional IgH allele. Thus chromosomal translocations involving the Ig loci may not only contribute to transformation by deregulating oncogene expression but could also act by potentiating subsequent oncogene hypermutation.

Memory in the B-cell compartment: antibody affinity maturation.

In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene loci. Here we consider how the B-cell response to antigen depends on the affinity of the antigen receptor interaction. We also compare and draw parallels between the two processes, which underpin the generation of secondary-response antibodies: V gene somatic hypermutation and immunoglobulin heavy-chain class switching.

Diversification and selection mechanisms for the production of protein repertoires: lessons from the immune system.

The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.

Deficiency in serum immunoglobulin (Ig)M predisposes to development of IgG autoantibodies.

Serum immunoglobulin (Ig)M provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Here we show that mice deficient in serum IgM have an increased propensity to spontaneous autoimmunity as judged by the development with age of serum IgG anti-DNA antibodies and the renal deposition of IgG and complement. They also exhibit augmented anti-DNA IgG production on exposure to lipopolysaccharide. Thus, deficiency in serum IgM leads to diminished responsiveness to foreign antigens but increased responsiveness to self-a paradoxical association reminiscent of that described in humans deficient in complement or IgA. We wondered whether serum IgM might play an analogous role with regard to the response to self-antigens. However, here-in contrast to the sluggish response to foreign antigens-we find that deficiency in serum IgM actually predisposes to the development of IgG antibodies to autoantigens.

B cells extract and present immobilized antigen: implications for affinity discrimination.

Binding of antigen to B-cell antigen receptor (BCR) leads to antigen internalization and presentation to T cells, a critical process in the initiation of the humoral immune response. However, antigen internalization has been demonstrated for soluble antigen, in vivo antigen is often encountered in insoluble form or tethered to a cell surface. Here, we show that not only can B cells internalize and present large particulate antigen (requiring a signalling-competent BCR to drive antigen uptake), but they can also extract antigen that is tethered tightly to a non-internalizable surface. The form in which the antigen is displayed affects the B cell's ability to discriminate antigen-BCR affinity. Thus, arraying an antigen on a particle or surface allows efficient presentation of low affinity antigens. However, the presentation efficiency of antigen arrayed on an internalizable particle plateaus at low affinity values. In contrast, extraction and presentation of antigen from a non-internalizable surface depends on antigen-BCR affinity over a wide affinity range. The results have implications for understanding both the initiation and affinity maturation of the immune response.

Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes.

We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa. Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use.

Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation.

During maturation of the immune response, IgM+ B cells switch to expression of one of the downstream isotypes (IgG, A or E). This class switching occurs by region-specific recombination within the IgH locus through an unknown mechanism. A lack of switch recombination in mice deficient in components of the DNA-dependent protein kinase (DNA-PK)-Ku complex has pointed to a role for non-homologous end joining. Here we characterize a switching defect in mice lacking a protein involved in DNA mismatch recognition. Mice deficient in Msh2 give diminished IgG (but not IgM) responses following challenge with both T cell-dependent and T cell-independent antigens. This appears to reflect a B cell-intrinsic defect since B cells from Msh2-deficient mice also exhibit impaired switching (but not blasting or proliferation) on in vitro culture with lipopolysaccharide. Furthermore, those switches that do occur in Msh2-deficient B cells reveal a shift in the distribution of recombination sites used: the breakpoints are more likely to occur in consensus motifs. These results, which intriguingly parallel the effects of Msh2 deficiency on hypermutation, suggest a role for Msh2 in the mechanics of class-switch recombination.