Scope of action of the immunoglobulin mutator system

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.     

Nucleotide sequence of an unequal sister chromatid exchange site in a mouse myeloma cell line.

The mouse myeloma cell line MPC 11 carries two C gamma 2a immunoglobulin heavy-chain genes on the expressed chromosome, a duplication shown to have occurred through unequal sister chromatid exchange (USCE). In the present report, we present the nucleotide sequence of the USCE joint and show that both breaks occurred within tracts of repeated TC dinucleotides. Additional TC dinucleotide tracts and two oligonucleotide segments (N sequences) were inserted at the USCE site.     

Compartment analysis of T cell receptor gamma--and immunoglobulin V(H) rearrangements by microdissection in patients with malt lymphoma and chronic gastritis with lymphatic hyperplasia.

The interpretation of clonality within H. pylori-associated gastritis and low-grade MALT lymphoma remains controversial. Due to the observation of MALT lymphoma regression after H. pylori eradication, new definitions concerning the border between benign reactive lesions and malignant gastric lymphoma are needed. Gene rearrangements for immunoglobulin heavy-chain in low-grade MALT lymphoma (N= 12) and H. pylori-associated chronic gastritis with lymphatic hyperplasia (N= 13) were analyzed by microdissection and polymerase chain reaction. Furthermore, T cell receptor-gamma chain rearrangements were analyzed by gene scan analysis. In 11 of 12 cases with initial low-grade MALT lymphoma, intraepithelial and subepithelial B cell rearrangements showed a restricted usage of the immunoglobulin heavy-chain 3. In H. pylori-associated chronic gastritis, the intraepithelial B cell compartment showed an oligoclonal the immunoglobulin heavy-chain rearrangement pattern with a predominance of VH3. The subepithelial compartment did not show any restrictive immunoglobulin heavy-chain gene usage. Additionally a mono- to oligoclonal rearrangement pattern of the T cell receptor-y chain was observed in low-grade MALT lymphoma, whereas an oligoclonal pattem was observed in chronic gastritis. Our data provide evidence that low-grade MALT lymphoma may start within the epithelium and subsequently infiltrate the subepithelial compartment. The observation of a mono- to oligoclonal TCR-gamma rearrangement suggests that an antigen selecting process also takes place within reactive T cells. Combining TCR-gamma gene scan analysis with IgH chain rearrangement analysis might help in discriminating between chronic gastritis and initial MALT lymphoma in questionable cases.     

Genes activated in the presence of an immunoglobulin enhancer or promoter are negatively regulated by a T-lymphoma cell line.

The tissue-specific expression of immunoglobulin genes can be partially explained by a requirement for activating factors found only in B lymphocytes and their derivatives. However, loss of immunoglobulin expression upon fusion of an immunoglobulin-producing myeloma cell with a T lymphoma cell (BW5147) or fibroblast (L cell) suggests that negatively acting factors also play a role in the tissue specificity of immunoglobulin genes. Expression of a cloned immunoglobulin heavy-chain gene introduced into myeloma cells was suppressed after fusion of the myeloma transformants with BW5147. The presence of either the immunoglobulin heavy-chain enhancer or promoter conferred suppression, under similar conditions, upon a heterologous gene that is normally expressed in both B and T lymphocytes. These immunoglobulin heavy-chain gene control regions, or gene modifications induced by them, are subject to negative control by T-lymphocyte-derived factors.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

B-lymphocyte targeting of gene expression in transgenic mice with the immunoglobulin heavy-chain enhancer.

A hybrid gene containing rabbit beta-globin structural sequences (-9 to +1650), and a chicken conalbumin gene promoter (+62 to -102) in the place of the beta-globin promoter (upstream from -9), was inactive in 5 different transgenic mouse line. Adding the mouse immunoglobulin heavy-chain (IgH) enhancer to this construction specifically stimulated expression in B-cells. These results show that IgH enhancer is specifically active in B-cells. Expression of the hybrid gene was low compared to the endogenous immunoglobulin heavy and light-chain genes. Substituting the mouse immunoglobulin kappa light-chain gene (Ig kappa) promoter (+4 to -800) for the heterologous conalbumin promoter was not sufficient to restore gene expression to level of the endogenous genes. In addition to the reproducible B cell expression, we also found inheritable unexpected expression in certain tissues, which varied from line to line.     

The biology and cytogenetics of multiple myeloma

Despite the advances in our knowledge of myeloma cell biology, our understanding of myeloma pathogenesis is still incomplete. In this review, we present a summary of the cellular and molecular aspects of B-cell development and immunoglobulin (lg) gene rearrangement which have been important in defining the characteristics of the myeloma plasma cell (MPC). The PMC has undergone variable gene recombination, somatic hypermutation and isotype switching, and is therefore at a postgerminal center stage of development. The finding of preswitch clonal cells and isotype variants have raised interesting questions about the cell of origin of myeloma, for which no conclusive data is as yet available. However much information has been obtained about the chromosomal and genetic aberrations in myeloma, including monosomy 13, Ig heavy chain (IgH) switch region translocations, numerical abnormalities and a multitude of heterogeneous changes. A variety of techniques have been developed to overcome the insensitivity of conventional karyotyping, utilizing molecular cytogenetic strategies ranging from the delineation of precise loci by fluorescent in situ hybridization, a more "global" assessment of the genome by multicolor spectral karyotyping, to the quantitation of chromosomal material of specific origin by comparative genomic hybridization. Whether the abnormalities detected represent oncogenic insults, are involved in disease progression or are simply "by-products" of genetic instability is still unclear. For IgH translocations, the role of candidate genes such as Cyclin D1 and FGFR3 has been studied extensively by quantitating their expression and assessment of their oncogenicity (e.g. for FGFR3) in animal models. The significance of other aberrations such as c-myc, ras and p53 has also been investigated. With the advent of oligonucleotide microarrays, the expression of thousands of genes can be efficiently examined. So far, this approach seems promising in defining subgroups of different disease behavior, and may highlight specific genes and molecular mechanisms which are important in myeloma pathogenesis.     

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.     

Isolation and characterization of a variant of mouse plasmacytoma J558 synthesizing a 110,000-dalton immunoglobulin heavy chain and of secondary variants synthesizing either a 55,000-dalton or an 80,000-dalton immunoglobulin heavy chain: possible implications.

A mutant has been isolated from the J558 (immunoglobulin A, lambda, anti-alpha 1 leads to 3 dextran) cell line which synthesizes a heavy-chain immunoglobulin twice the size of normal heavy chain. Secondary variants that synthesized heavy chains either 1.5 times as large as wild type or the same size as wild type were identified. All mutants were serologically immunoglobulin continued to bind antigen, and retained the individual idiotype of the parent. Northern blot analysis and in vitro synthesis studies showed that the large heavy chains were primary synthetic products and not the consequence of abnormal covalent bonds. Cleavage of genomic DNA with restriction endonucleases and molecular hybridization studies showed new fragments in the 2 X and 1.5 X mutants which disappeared in the 1 X revertant. These data cannot easily be reconciled with the mutants arising either by unequal recombination or gene conversion. Further molecular characterization of these mutants should give additional insight into immunoglobulin gene evolution.     

Distinguishing primary and secondary translocations in multiple myeloma

Multiple myeloma (MM) is a malignant post-germinal center tumor of somatically-mutated, isotype-switched plasma cells that accumulate in the bone marrow. It often is preceded by a stable pre-malignant tumor called monoclonal gammopathy of undetermined significance (MGUS), which can sporadically progress to MM. Five recurrent primary translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 have been identified in MGUS and MM tumors. The five partner loci include 11q13, 6p21, 4p16, 16q23, and 20q12, with corresponding dysregulation of CYCLIN D1, CYCLIN D3, FGFR3/MMSET, c-MAF, and MAFB, respectively, by strong enhancers in the IgH locus. The five recurrent translocations, which are present in 40% of MM tumors, typically are simple reciprocal translocations, mostly having breakpoints within or near IgH switch regions but sometimes within or near VDJ or JH sequences. It is thought that these translocations are caused by aberrant IgH switch recombination, and possibly by aberrant somatic hypermutation in germinal center B cells, thus providing an early and perhaps initiating event in transformation. A MYC gene is dysregulated by complex translocations and insertions as a very late event during the progression of MM tumors. Since the IgH switch recombination and somatic hypermutation mechanism are turned off in plasma cells and plasma cell tumors, the MYC rearrangements are thought to be mediated by unknown mechanisms that contribute to structural genomic instability in all kinds of tumors. These rearrangements, which often but not always juxtapose MYC near one of the strong immunoglobulin enhancers, provide a paradigm for secondary translocations. It is hypothesized that secondary translocations not involving a MYC gene can occur at any stage of tumorigenesis, including in pre-malignant MGUS tumor cells.     

Sequence of the full-length immunoglobulin kappa-chain of mouse myeloma MPC 11.

MPC 11 mouse myeloma cells synthesize two immunoglobulin kappa light chains, coded by two separate genes. One of these Kappa-chains has no variable region and is degraded intracellularly. The other is a full-length kappa-chain contaning both variable and constant regions: this chain is secreted, both by itself and combined with heavy chains in molecules of immunoglobulin G. This paper reports the amino acid sequence of the myeloma MPC 11 full-length kappa-chain. The chain is unusual in having 12 extra residues at its N-terminus when its sequence is aligned with those of other mouse kappa-chains; no other anomalies were found in its sequence.     

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.     

Minimal peripheral blood cells carrying clonal markers of B cell disorders: evidence for monoclonality of circulating lymphocytes in patients with multiple myeloma

Peripheral blood lymphocytes (PBL) of 20 patients with multiple myeloma (MM) were assayed for clonality by Southern blot and cell surface marker analysis. Eight samples showed monoclonal origin of circulating lymphocytes by demonstrating rearrangements of the heavy chain immunoglobulin gene (IgH). In selected experiments, comparison of IgH rearrangements of bone marrow plasma cells and peripheral blood-derived mononuclear cells, highly enriched for B lymphocytes, proved to be identical. However, monoclonal circulating cells could not be detected in samples with rearranged IgH genes by surface marker phenotyping using one-color immunofluorescence analysis and a panel of monoclonal and polyclonal antibodies to various B lineage-associated antigens. These results indicate that in a substantial proportion of MM, monoclonal growth involves circulating B lymphocytes and underscores the clinical usefulness of Southern analysis of IgH gene rearrangements for monitoring this disease.