Immunoglobulin λ Gene Rearrangement Can Precede κ Gene Rearrangement

Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two lightchain loci: κ and λ.It has been reported that κ loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged λ-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the κ locus, and that only upon failure to produce a functional κ chain is there an attempt to rearrange the λ locus; and (b) the stochastic theory, which postulates that rearrangement at the λ locus proceeds at a rate that is intrinsically much slower than that at the κ locus. We show here that λ-chain genes are generated whether or not the κ locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.     

Independent rearrangement of Ig lambda genes in tissue culture-derived murine B cell lines

Rearrangement of the lambda light chain locus is considered a late event in pre-B cell differentiation which occurs after successful heavy chain and unsuccessful kappa light chain rearrangement. However, this view has recently been challenged by the observation of apparently independent lambda rearrangement in certain B cell lines and Ig transgenic populations. In this study we have examined the pattern of Ig rearrangements and expression in several tissue culture-derived murine B cell lines. One pre-B cell line (BDL-1) displayed germline heavy and kappa light chain genes despite the presence of a productive lambda 1 light chain rearrangement. Two other cell lines (DAC-2, BDL-2) had multiple lambda rearrangements despite the presence of productive kappa chain rearrangements. These cell lines provide new precedents for rearrangement of the lambda locus independent of the kappa locus. Their phenotype suggests that accessibility at the different Ig loci may be controlled by a non-sequential mechanism.     

The level of lambda 1 light chain expression in the mouse reflects the probability of rearrangement of the relevant gene

The predominance of kappa over lambda light chain expression in mice can either reflect the probability of rearrangement of the relevant locus or be the result of antigen-driven clonal expansion. To discriminate between these two possibilities we determined, by limiting dilution analysis, the frequencies of kappa- and lambda-producing cells in B lymphocytes generated in vitro from bone marrow pre-B cells. The frequencies obtained in these cultures are not significantly different from those obtained with mature spleen cells. In addition, Southern blot analysis of bone marrow-derived and splenic cell DNA revealed that in both populations the extent of B lymphocytes having undergone lambda 1 gene rearrangement does not exceed 4%. These results, therefore, establish that in the mouse the low level of lambda light chain expression directly reflects the probability of rearrangement of the relevant locus.     

Lambda5 is required for rearrangement of the Ig kappa light chain gene in pro-B cell lines.

Lambda5 associates with V(pre-B) to form the surrogate light (L) chain. The phenotype of lambda5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of lambda5 to lambda5-deficient pro-B cell lines which were established from bone marrow cells of lambda5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in lambda5 knockout mice. Restoration of lambda5 protein by retroviral-mediated gene transfer into established lambda5-deficient pro-B cell lines induced rearrangement of the Ig kappa L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored lambda5 in the cell line is coupled with V(pre-B) to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both lambda5 and V(pre-B), stimulates efficient rearrangement of the kappa L chain genes.     

A mouse with a monoclonal primary immunoglobulin repertoire not further diversified by V-gene replacement

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.     

The scid gene encodes a trans-acting factor that mediates the rejoining event of Ig gene rearrangement

Homozygous mutation at the scid locus in the mouse impairs lymphoid development and results in animals deficient in B and T cells. We found that immunoglobulin heavy-chain gene rearrangement was blocked at the D-JH stage in Abelson-transformed scid pre-B cell lines. Examination of the recombinational junctions indicated that the correct gene elements (D and JH) were assembled, as shown by the presence of D region and JH-region DNA on the breakpoint restriction fragments cloned from the genome of the scid cell lines. All rearrangement events were accompanied by deletions of varying sizes such that none of the rearrangements resulted in the production of functional immunoglobulins. The breakpoints of the rearrangement events did not correspond to the utilization of a novel heptamer-nonamer recombination signal but probably arose by nonspecific deletion from distal JH and D heptamer-nonamer signals in the process of recombination. scid pre-B cell lines were infected with a recombinant retrovirus (DGR) containing Ig joining signals. Aberrant rearrangements were observed in DGR DNA that was integrated randomly throughout the mouse genome, which suggested that the mutation in scid mice encodes a trans-acting factor that is part of the lymphoid gene recombination machinery.     

The lambda5-VpreB1 locus--a model system for studying gene regulation during early B cell development

The lambda5 and VpreB genes encode the components of the surrogate light-chain, which forms part of the pre-B cell receptor. In mouse, the lambda5 and VpreB1 genes of mouse are closely linked and coordinately regulated by a locus control region (LCR). Activation of the genes in pro-B cells depends on the combined effects of early B cell factor (EBF) and the E2A factors E12 and E47. Silencing of lambda5 expression in mature B cells occurs through the action of Ikaros on the gene promoter where it may compete for binding of EBF and initiate the formation of a silent chromatin structure.     

Immunoglobulin gene rearrangements in acute lymphoblastic leukemia with the 9;11 translocation

The recurring chromosomal 9;11 translocation [t(9;11) (p22;q23)] typically is associated with acute monoblastic leukemia, but a number of patients with acute lymphoblastic leukemia also have been reported to have the t(9;11). To investigate the cell lineage in the latter cases, we analyzed DNA from the leukemic cells of an 8-year-old girl with acute lymphoblastic leukemia and a t(9;11) for rearrangements of the immunoglobulin and T-cell receptor genes. Rearrangements of both immunoglobulin heavy-chain loci and of one lambda light-chain gene were detected, as well as deletions affecting both alleles of the kappa light-chain genes; T-cell receptor genes were in germline configuration. These results provide further evidence that the 9;11 translocation is not limited to myeloid lineage leukemia and may be observed in acute lymphoblastic leukemia.     

Uniform detection of immunoglobulin-gene rearrangement in benign lymphoepithelial lesions

The term "benign lymphoepithelial lesion" is used to describe the salivary-gland lymphocytic infiltration and epithelial changes typically found in association with Sj?gren's syndrome. We used Southern blot hybridization techniques to examine the immunoglobulin genes in salivary-gland tissue derived from eight patients with benign lymphoepithelial lesions. Three of these patients had intrasalivary non-Hodgkin's lymphoma complicating the lesions, whereas the lesions in the remaining five were all histologically benign. Ten samples from the eight patients all revealed rearrangement of both the heavy-chain and light-chain immunoglobulin genes. In one of the patients in whom non-Hodgkin's lymphoma involved both the salivary-gland lesion and an ipsilateral lymph node, the rearrangements of the heavy-chain and light-chain immunoglobulin genes detected at the two sites were identical. One other patient had two distinct benign lymphoepithelial lesions removed two years apart. The rearrangements of the heavy-chain as well as the kappa light-chain genes detected in these two lesions were entirely different. These data suggest that B-cell clonal expansion has an integral role in the pathophysiology of the benign lymphoepithelial lesion and may explain the increased incidence of lymphoma noted in association with this disorder.     

Mapping and functional analysis of regulatory sequences in the mouse lambda5-VpreB1 domain

The lambda5 and VpreB genes encode the components of the surrogate light-chain which forms part of the pre-B cell receptor and plays a key role in B cell development. In the mouse, the lambda5 and VpreB1 genes are closely linked and are co-regulated by a multi-component locus control region. To identify the sequences that regulate lambda5 and VpreB1 expression during B cell development, we have comprehensively mapped the DNaseI hypersensitive sites (HS) in the lambda5-VpreB1 functional domain. The active domain contains 12 HS that are distributed at high density across the 18.3 kb region that forms the lambda5 and VpreB1 functional unit. Analysis of a reporter gene driven by the VpreB1 promoter in transgenic mice identified a novel enhancer associated with two HS located upstream of lambda5. The lambda5-VpreB1 locus was also found to be closely linked to the ubiquitously expressed Topoisomerase-3beta (Topo3beta) gene. The VpreB1 and Topo3beta genes have entirely different expression patterns despite the fact that the two promoters are separated by a distance of only 1.5 kb.     

The kappa/lambda ratio in surface immunoglobulin molecules on B lymphocytes differentiating from DHJH-rearranged murine pre-B cell clones in vitro

The expression of kappa and lambda light chains in surface immunoglobulin (sIg) molecules on B lymphocytes differentiating from murine pre-B cell clones in vitro was analyzed. The four pre-B cell clones used represent a very early pre-B cell stage. They have their heavy chain loci DJ rearranged and their light chain loci in germ-line configuration. In order to grow in vitro, these clones require contact with stromal cells and the stimulatory activity of interleukin (IL) 7. Upon removal of IL 7 from the cultures, these clones differentiate within 3 days into sIg+ B cells. Between 7% and 12% of IgM+ B cells could be detected in these cultures. The majority (78%-92%) of the IgM+ B cells co-expressed kappa light chains. The percentage of lambda light chain expressing B cells was below detectable level. Upon lipopolysaccharide (LPS) stimulation, the percentages of IgM+ B cells increased dramatically (from 32%-64%). The majority (91%-97%) of the IgM+ B cells express kappa chains, but a very small percentage (3.1%-5.0%) express lambda. A similarly high kappa/lambda ratio was found in 418 hybridomas prepared from these LPS-stimulated B cells (388 kappa+ and 30 lambda+). Thus, the high kappa/lambda ratio characteristic of the mouse peripheral B cell repertoire is already evident in the antigen-independent transition from pre-B to B cells.     

B lineage-restricted rearrangement of a human Igx transgene

To study the control of immunoglobulin x light chain gene rearrangement, we generated transgenic mice carrying a germ-line human x minilocus (HK) containing the Jx-proximal V gene, VxIV, the V-J intergenic region, the five Jx segments and the Cx gene. This construct includes the intronic, but not the 3′ x enhancer. Rearrangement of the HK transgene was found to be lymphoid specific and restricted to the B cell lineage. Quantification of x gene rearrangement in pre-B cell lines established from HK transgenic mice showed that, like endogenous x genes, rearrangement of the transgene is repressed in μ-negative early B cell precursors. These results indicate that rearrangement of the HK transgene is subjected to the same B/T cell and developmental regulation as Vx-Jx rearrangement at the endogenous locus. Comparison with an unrearranged x transgenic construct lacking the V-J intergenic region, suggests that this region, or elements associated with the proximal V gene, may act to restrict x gene rearrangement to the B cell lineage.     

IgD/λ plasmocytoma with immunoglobulin κ light-chain genes in the germ-line configuration

Human immunoglobulin (Ig) genes are rearranged in an ordered sequence of events during B-cell differentiation: starting at the IgH locus, a productive V_HDJ_H rearrangement leads to the expression of chains. Light-chain gene rearrangements have been found in pre-B cells which express chains. In these cells rearrangements of Ig light-chain genes precede that of genes. In an IgD/-producing plasmocytoma, however, we found an apparent exception to this rule: the genes were not rearranged. Together with the observation that roughly 90% of human IgD plasmocytomas produce light-chain proteins, the finding reported here leads us to suggest that light-chain genes are rearranged preferentially in IgD-producing plasma cells. Ig gene rearrangement, isotype switch, and the phenomenon of isotypic and allelic exclusion are discussed with special reference to our findings.     

Production of heavy-chain class-switch variants of human monoclonal antibody by recombinant DNA technology

We have previously established a human-mouse heterohybridoma (H6-3C4), which produced a human sperm-immobilizing antibody (mu, lambda of human type). The human rearranged immunoglobulin mu-chain and lambda-chain genes were cloned from the hybridoma H6-3C4. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglobulin gamma 1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switch from mu-chain to gamma 1-chain of H6-3C4 antibody. The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation. The stable transformants produced the human IgG monoclonal antibody, which fully retained specificity to human sperm cells and sperm-immobilizing activity.