Lambda Light Chain Genes Research Articles

B-less: a strain of profoundly B cell-deficient mice expressing a human lambda transgene.

We have created several transgenic mouse strains that bear the human lambda light chain gene driven by its own promoter and a mouse immunoglobulin heavy chain enhancer. The transgene is expressed in many tissues, with particularly high levels of expression in the bone marrow, thymus, spleen, and lymph nodes. One of these transgenic lines, B-less, displays a dramatic phenotype characterized by an acute susceptibility to bacterial and viral infections. Analysis of this strain shows it to be profoundly deficient in both immature (pre-B) and mature B cells, as well as in circulating immunoglobulin. The pre-B and B cell defects are cell autonomous, as judged by cell culture and bone marrow graft chimeras. Despite this B cell deficiency, the T cell lineage appears grossly normal as assessed by flow cytometric analysis and by its response to mitogen stimulation. Since an independently derived transgenic strain bearing the same human lambda construct displays a partial B-less phenotype, it is likely that the B lineage deficiency is due to a dominant effect of transgene expression rather than to the insertional perturbation of an endogenous mouse gene. It is interesting that the deficiency phenotype is fully expressed in the FVB/N genetic background, but is suppressed in F1 hybrids formed between the FVB/N and C57BL/6 inbred strains. Evidently, there are one or more dominant genetic suppressors of B-less in the C57BL/6 genome.

Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo.

Cell lines and clones were established from PB76-positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum-substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL-7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP-1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP-1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ-line configuration. Either one or both of their H-chain-gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg-positive surface, MHC class II negative, LPS-reactive B cells within 2-3 days, in the absence of contacts with stromal cells and/or IL-7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig-, MHC class II-positive LPS-reactive cells for greater than 25 weeks. The long-term in vitro proliferative capacity of these DH-JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.

Endogenous immunoglobulin expression in mu transgenic mice

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.

Immunoglobulin gene rearrangements in acute lymphoblastic leukemia with the 9;II 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.

Genotypic and Immunophenotypic Characterization of Two Human Light Chain– – Only B-Cell Non-Hodgkin's Lymphomas

Two atypical human non-Hodgkin's lymphomas (NHLs) that exhibited unusual genotypic and in situ immunophenotypic abnormalities are described. Immunophenotypically, both NHLs lacked surface Ig heavy chains. With the exception of the MB2 B-cell-associated antigen, no B- and T-cell differentiation antigen was detected in case 1. NHL 2 failed to show evidence of clonality by immunohistochemical analysis but revealed the presence of many B-lymphocytes with an abnormal phenotypic profile: CD19+, CD20+, CD22+, kappa-, lambda-, CD9-, CD10-, CD21-, and CD24-. Genotypic analysis indicated that both lymphomas derived from anomalously matured pre-B-cells that had rearranged the lambda or kappa light chain genes but not the Ig heavy chain gene. The neoplastic cells of the two NHLs resemble the light chain-only B-cells recently discovered, following Epstein-Barr virus immortalization, in the human bone marrow. The authors' data confirm, therefore, the existence of the light chain-only B-cells in the human hematopoietic compartment. Moreover, their results emphasize the conclusive role of the immunogenotypic analysis in defining clonality, lineage, and maturation abnormalities of such atypical NHLs.

Heterogeneity of immunoglobulin gene rearrangements in B‐cell lymphomas

We have examined 69 B-cell non-Hodgkin's lymphomas (NHL) for rearrangements of the immunoglobulin (Ig) or T-cell antigen receptor (TCR) genes. The lymphomas were assigned to the categories of the Kiel classification and their B-cell nature was confirmed by immunostaining. Only 2 cases (with CLL) displayed clonal T beta-chain TCR gene rearrangements together with rearranged heavy- and light-chain Ig genes. The remaining 67 lymphomas had a germline beta-chain TCR-gene configuration. Three different patterns of Ig gene rearrangements were identified; (A) presence of both heavy- and light-chain rearrangements (H+L+); (B) rearrangement of heavy-chain gene only (H+L-); (C) heavy- and light-chain genes in germline configuration (H-L-). All the 45 low-grade NHLs and the 4 immunoblastic lymphomas exhibited pattern A and all had their kappa gene rearranged or deleted. Of 24 low-grade lymphomas tested, 13 (54%) had an addition rearrangement of the lambda light-chain gene. In contrast, the 19 high-grade centroblastic (cb) B-NHLs had distinct patterns of Ig-gene rearrangement: 12 with pattern A, 4 with B and 2 with C. In this group only 2 of 17 (12%) cases analyzed had evidence of lambda light-chain rearrangement whereas 12 of 18 (67%) had a kappa gene rearrangement or deletion. In one case expressing sIgM/lambda and with heavy chain Ig-rearrangement, no DNA was available for Ig light-chain analysis.

Molecular Evidence for a Single Clonal Origin in Biphenotypic Concomitant Chronic Lymphocytic Leukemia and Multiple Myeloma

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.

Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.

Polymemse Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes from Single Hybridoma Cells

We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies. (Less)

Plasma Cell Malignancy in the Acquired Immune Deficiency Syndrome: Association with Epstein-Barr Virus

The development of high-grade, malignant B-cell lymphoma is a well-recognized complication of human immunodeficiency virus (HIV) infection. Plasma cell neoplasms, however, have been rarely encountered in HIV-infected people. This study presents the morphologic and immunologic features of an unusual plasma cell tumor occurring in a 31-year-old HIV-antibody-positive male. The malignancy was characterized by widespread dissemination and hypercalcemia at presentation and a clinically aggressive course. Immunoperoxidase staining of tumor tissue obtained from biopsy and at autopsy had positive results for IgM and lambda. In the patient's serum, only an IgG kappa paraprotein was detected, indicating that the tumor was nonsecretory. DNA analysis of autopsy-derived tumor tissues demonstrated clonal rearrangements of the immunoglobulin (Ig) heavy chain gene locus and rearrangements in both kappa and lambda light chain gene loci. Furthermore, DNA hybridization studies revealed the presence of Epstein-Barr virus (EBV) genomes in tumor tissue but not in nontumor tissue from this patient.

Immunoglobulin lambda light-chain-related genes 14.1 and 16.1 are expressed in pre-B cells and may encode the human immunoglobulin omega light-chain protein.

Human pre-B cells, which produce immunoglobulin heavy chain but do not produce immunoglobulin light chain, are shown to contain a 1-kilobase transcript homologous to immunoglobulin lambda light-chain genes. Detailed analysis of RNA and cDNA clones derived from these transcripts reveals that they originate from the distinct immunoglobulin lambda-like genes 14.1/16.1. Sequence analysis of these clones reveals a long open reading frame, beginning with an ATG, capable of encoding a protein of 214 amino acids with an unprocessed molecular weight of 22,944. The C-terminal half of this predicted protein is highly homologous to immunoglobulin lambda light-chain joining and constant region protein sequence, while the amino-terminal end does not share homology with variable regions. Unlike immunoglobulin genes, these genes do not undergo rearrangement prior to expression. Analysis of a panel of 26 hematopoietic cell lines reveals that expression of 14.1/16.1 is limited to pre-B cells and one B-cell line, which, like the pre-B cells, is surface immunoglobulin negative. Antisera raised against a peptide whose sequence was predicted from the 14.1 cDNA sequence identifies a 22-kDa protein in human pre-B cells. Immunoprecipitation of immunoglobulin mu-chain from these pre-B cells with anti-immunoglobulin mu antibody coprecipitates a 22-kDa protein, which is a candidate for the human immunoglobulin omega light-chain protein and may be the protein product of the 14.1/16.1 genes.

Variant translocation of the bcl-2 gene to immunoglobulin lambda light chain gene in chronic lymphocytic leukemia.

The bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18), which is found in approximately 90% of human follicular lymphoma cases, and is a prime candidate for the oncogene playing a crucial role in follicular lymphomagenesis. In this paper, we describe a case of chronic lymphocytic leukemia showing the juxtaposition of the bcl-2 gene on chromosome 18 to immunoglobulin lambda light chain (Ig lambda) gene on chromosome 22 in a head-to-head configuration. Sequencing analysis of the joining site of the bcl-2 gene and Ig lambda gene has shown that the breakpoint is within the 5' flanking region of the bcl-2 gene and about 2.2 kilobases 5' to the joining segment of Ig lambda locus in a germ-line configuration. The extranucleotide, commonly appearing at the joining site of the t(14;18) translocation involving the IgH locus, is absent from the joining site of bcl-2 and Ig lambda. The lack of extranucleotide suggests that the juxtaposition of the bcl-2 and Ig lambda genes occurred during physiological rearrangement of the Ig lambda gene since it has been shown that the rearrangement of the Ig lambda locus is not accompanied by extranucleotides.

A linkage map of the mouse immunoglobulin lambda light chain locus

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes. The order of the lambda gene segments is V2-J2C2J4C4-V1-J3C3J1C1, as was originally proposed. No evidence for nonmethylated CpG rich areas (HTF islands) within the region was found. Fine mapping using the lambda 1, lambda 3 rearranged cell line J558 mapped the gap between the V and J-C gene segments in the lambda 1 gene cluster (V1-J3C3J1C1) to approximately 70 kb. The similar distance (60-100 kb) found in the lambda 2 gene cluster (V2-J2C2J4C4) is further evidence that duplication of an ancestral locus occurred.

Immunophenotypic and gene probe analysis of a case of Richter's syndrome.

A rare case of diffuse large cell lymphoma (DLL) arising in the stomach of a patient with long-standing chronic lymphocytic leukemia (CLL) has been examined by a combination of immunophenotypic and gene probe methods. Both the resected lymphoma and the CLL (obtained from an infiltrate in perigastric lymph nodes) expressed surface membrane markers consistent with a B-cell origin (HLA-DR+; B1+; B4+), but surface membrane immunoglobulin (SmIg) could be detected only on the CLL cells (SmIgM lambda+). Neither neoplasm expressed cytoplasmic immunoglobulin. DNA probe analysis using the Southern blot method and probes to the joining (J-H) region of the immunoglobulin heavy chain gene and to the constant (C) regions of the immunoglobulin kappa and lambda light chain genes revealed different light and heavy chain gene rearrangements in the two neoplasms. These findings suggest that the large cell lymphoma arose from a different clone than the CLL and the development of large cell lymphoma may have represented a de novo neoplastic event in a predisposed person.

Both immunoglobulin promoter and enhancer sequences are targets for suppression in myeloma-fibroblast hybrid cells.

When immunoglobulin (Ig)-producing B cells are fused with fibroblastic cells, expression of Igs is suppressed by a mechanism that selectively abolishes transcription of Ig genes. The suppression is also maintained in proliferating hybrids. We have used gene transfer followed by cell fusion to study this phenomenon further. Here we report that expression of a rearranged Ig heavy chain gene, stably integrated into a myeloma genome, is completely suppressed upon fusion with fibroblasts by a mechanism that is equally active on the endogenous myeloma lambda light chain gene. To define regulatory sequences within the Ig transcriptional unit that are involved in this down-regulation, we examined the transcriptional contributions of the IgH chain gene enhancer and the kappa light chain gene promoter individually by linking them to a heterologous reporter gene. Mouse myeloma cells were stably transformed with such test constructs and subsequently fused with mouse fibroblasts. To avoid any significant loss of chromosomes, hybrid cells were isolated shortly after fusion by fluorescence-activated cell sorting, and proliferating hybrids were harvested within 2-3 weeks. On the basis of RNase protection mapping of cytoplasmic RNA, and of nuclear run-on assays we showed that both the kappa light chain promoter and the IgH chain enhancer contain regulatory information that is made redundant or is suppressed in the hybrid environment.

Two pseudogenes among three rat immunoglobulin lambda chain genes

In order to examine the number and organization of the immunoglobulin lambda light chain genes of the rat, we have used mouse lambda chain cDNA probes to isolate hybridizing fragments from a partial Eco RI rat liver DNA library. We have determined the partial nucleotide sequence of two such clones. One clone, containing a 5.8 kb EcoRI insert which hybridizes to both mouse Cλ1 and Cλ2 probes, includes an apparently expressible Cλ2-like gene as well as a CλI-like pseudogene (ψCλ1.1), arranged similarly to the mouse Cλ gene complexes. Sequence analysis of a second cloned EcoRI fragment (1.15 kb in length) revealed part of a second CλI-like pseudogene (ψCλ1.2), the coding regions of both pseudogenes being interrupted by multiple frame-shifting size differences. In the case of υCλ 1.2, the degree of sequence identity with mouse Cλ 1 drops abruptly immediately following the termination codon, suggesting that translocation events have played a role in its generation. These two rat pseudogenes, and the mouse Cλ4 pseudogene, clearly have been rendered unexpressible by separate evolutionary events. Comparisons between Cλ coding and non-coding regions of rats and mice indicate that some of the unusual patterns of divergence we have observed in recently diverged C k genes may exist in Cλ genes as well.

Immunoglobulin heavy chain gene rearrangements and mixed lineage characteristics in acute leukemias with the 11;19 translocation.

Although the origin of acute leukemia with the 4;11 translocation has been shown to be an early myeloid progenitor cell or a stem cell with the potential for differentiation into both lymphoid and myeloid lineage, few precise studies on acute leukemia with the 11;19 translocation have thus far been reported. This study focused on the clinical, morphologic, ultrastructural, and immunologic characteristics as well as the DNA in three cases of acute leukemia with the 11;19 translocation. All three patients were infants and showed hyperleukocytosis. The morphologic feature was French-American-British (FAB)-L2 in two patients, in one of which a few monocytoid blasts were also seen by electron microscopy. Cells from the third patient underwent morphologic changes from FAB-L2 at the time of diagnosis to M5b at relapse. Immunologic marker studies revealed that the blast cells from all three patients expressed Ia and B4, but none expressed B1, CALLA(J5), T antigens, or SIg. Cells from one patient simultaneously expressed myeloid antigen (MCS-II) both at diagnosis and relapse. Cells from two patients expressed myeloid antigen after being cultured for a short time in vitro. An analysis of immunoglobulin genes and T-cell receptor genes revealed rearrangements of the heavy chain genes and germ line configurations of the kappa and lambda light chain genes, and of the T-cell receptor beta chain genes. These findings suggest that acute leukemia with the 11;19 translocation has mixed lineage characteristics as a result of leukemogenesis in a stem cell with the potential for both lymphoid and myeloid, especially monocytic, differentiation.

Dual Genotype in Cutaneous T Cell Lymphoma: Immunoglobulin Gene Rearrangement in Clonal T Cell Malignancy

Genomic DNA digests of peripheral blood lymphocytes from 13 patients with the leukemic phase of the T cell neoplasm cutaneous T cell lymphoma were studied by hydridization using probes for the constant region of the beta chain of the T cell receptor, the joining region of the immunoglobulin heavy chain gene, and the kappa and lambda light chain genes. Lymphocytes from all 13 cutaneous T cell lymphoma patients contained DNA with clonal rearrangements of the beta chain gene of the T cell receptor. In addition, DNA from 4 patients contained an immunoglobulin gene rearrangement. T cell enrichment studies of peripheral blood lymphocytes from 2 patients confirmed that the immunoglobulin gene joining region rearrangement was confined to the T cell population. These results demonstrate that cutaneous T cell lymphoma is a clonal T cell malignancy that frequently expresses a dual genotype. A multiparameter approach, including DNA probes for the beta chain of the T cell receptor, as well as the immunoglobulin genes, immunophenotyping, and cytogenetics, is valuable in the diagnosis of cutaneous T cell lymphoma.

Molecular evidence that SJL reticulum cell sarcomas are derived from pre-B cell. Clonal rearrangement of heavy chain but not of light chain immunoglobulin genes.

In order to investigate the clonal origin of SJL reticulum cell sarcoma (RCS), two-color cell membrane-staining and molecular biologic analyses were performed. Flow cytometric analysis revealed that the SJL RCS consists of about 50 to 60% Thy-1-positive and 20 to 30% B220-positive cells and that the majority of the Thy-1-positive cells are L3T4-positive, whereas a few are Lyt-2-positive. In spite of this pleomorphic nature of SJL RCS shown by cell membrane analysis, when the immunoglobulin heavy chain J segment (JH) was used to probe the DNA obtained from the tumor, we observed clonal rearrangements of the gene. Results of the cell-sorting experiment combined with Southern hybridization using the JH gene probe confirmed that those clonally expanding cells are Ia and B220 antigen-positive. Furthermore, all tumors derived from a given mouse showed the same rearrangement pattern. However, no clonal rearrangement was observed when the DNA was probed with the T cell receptor beta-chain gene or with immunoglobulin kappa- or lambda-light chain genes. From these data, we conclude that SJL RCS is a tumor of B cells and that the neoplastic event takes place at an immature B cell stage.

Rearranged antigen receptor genes in Hodgkin's disease [published erratum appears in Blood 1987 Sep;70(3):893

Despite intensive efforts using a wide variety of approaches, the cellular lineage and clonality of the abnormal cells of Hodgkin's disease have remained an enigma. In the present study, cell separation techniques that enriched for Reed-Sternberg cells and their variants were used to generate sufficient percentages of abnormal cells to allow detection of rearrangements in these cell fractions. DNA from the involved tissues of eight Hodgkin's disease patients was subjected to Southern blot analysis to detect rearrangements of T cell antigen receptor genes and immunoglobulin genes. Immunoglobulin gene rearrangements were found in three of five cases in which Reed-Sternberg cells and their variants were enriched by cell separation techniques to cell frequencies greater than 1%. Rearrangements of immunoglobulin heavy chain genes occurred in two cases, and a lambda light chain gene rearrangement occurred in a third case. Rearrangements were not detected in lymphocyte fractions or in unseparated cells prepared from the same tissues. The putative Hodgkin's cell line, L428, also contained rearrangements of immunoglobulin heavy and kappa and lambda light chain genes and, in addition, harbored a single T cell receptor beta gene rearrangement. These findings indicate that Reed-Sternberg cell-enriched fractions contain clonal cell populations and provide a lead, at the molecular genetic level, to a possible lymphoid derivation of the Reed-Sternberg cell.