Lambda Light Chain Genes Research Articles

Assessment of immunoglobulin heavy chain, immunoglobulin light chain, and T-cell receptor clonality testing in the diagnosis of feline lymphoid neoplasia.

Differentiation between neoplastic and reactive lymphocytic proliferations can be challenging in cats. PCR for antigen receptor rearrangements (PARR) testing is a useful diagnostic tool to assess clonality of a lymphoid population. Previous feline PARR studies evaluated clonality of complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma (TRG) gene rearrangements. We aimed to evaluate the sensitivity and specificity of feline PARR primers targeting complete IGH-VDJ and TRG rearrangements, as well as incomplete IGH-DJ, kappa deleting element (Kde), and immunoglobulin lambda light chain (IGL) gene rearrangements in defined feline neoplasms and nonneoplastic controls. Fluorescently labeled PCR primers were designed to amplify complete IGH-VDJ, incomplete IGH-DJ, Kde, IGL, and TRG gene rearrangements in two multiplexed PCR reactions, and PCR products were analyzed by fragment analysis. Fresh tissue samples from 12 flow cytometrically confirmed B-cell lymphomas, 26 cytologically confirmed gastric and renal lymphomas of presumed B-cell origin, 30 flow cytometrically confirmed T-cell leukemias, and 11 negative control cats were tested. Using four immunoglobulin primer sets (IGH-VDJ, IGH-DJ, Kde, and IGL), clonal immunoglobulin rearrangements were detected in 87% (33/38) of the presumed B-cell neoplasms. The IGH-VDJ reaction alone only detected clonality in 50% (19/38) of these cases. TRG rearrangements were clonal in 97% (29/30) of the T-cell leukemia cases. All negative control samples had polyclonal immunoglobulin and TRG rearrangements. The PARR assay developed in this study is useful for assessing clonality in feline lymphoid neoplasms. Clonality assessment of incomplete IGH-DJ, Kde, and IGL rearrangements helped identify clonal B-cell neoplasms not detected with complete IGH-VDJ PARR alone.

Seeking light-chain amyloidosis very early: The SAVE trial—identifying clonal lambda light chain genes in patients with MGUS or smoldering multiple myeloma.

8010 Background: In systemic AL amyloidosis (AL), caused by clonal Ig free light chains (LC) produced in 75% of cases by λ clones, patients often present with advanced organ damage, making earlier diagnosis a critical unmet need. Five λ IGLV germline genes account for 75% of AL λ-type ( IGLV 6-57, 1-44, 2-14, 1-51, 3-1) (Blood 2017;129:299), representing 56% of all AL patients. Relative risk of AL versus MM with these clonal genes can be high (7.3, 6-57; 2.5, 1-44), intermediate (1.7, 2-14; 1.2, 1-51) or low (0.8, 3-1) (Amyloid 2009;16:1). Progression to AL from smoldering multiple myeloma (SMM) or monoclonal gammopathy of undetermined significance (MGUS) occurs but is not well appreciated (JCO 2014; 32:2679). SAVE is a trial for λ SMM or λ MGUS patients with a κ:λ FLC ratio < 0.26 and difference between involved and uninvolved FLC > 23mg/L (JCO 2014;32:2699). Methods: Eligible patients ship peripheral blood (PB) or bone marrow (BM) samples to us for RT-PCR with cDNA from CD138+ cells, using primers for the Vλ families (Blood 2001;98:714). Amplicons are sequenced and the IGLV germline genes identified in IMGT (ImMunoGene-Tics, www.imgt.org). If the germline gene is AL-related, further evaluation is pursued. Results: Twenty asymptomatic λ patients from 18 states have been enrolled (3M, 17F) and 23 PB and 4 BM specimens obtained. Medians of months from diagnosis, involved FLC, κ:λ ratios, MNC and CD138-selected cells were 20.5 months, 113mg/L, κ:λ 0.06, 8.1x106 (0.8-24) and 3x105 (0-30), respectively. Seventeen patients have had IGLV genes identified, 12 with the first and 5 with additional specimens including 4 BM. Increased risk of AL was identified in 7 patients, 2 of whom had undiagnosed AL (both with IGLV2-14 germline genes). One patient with SMM diagnosed in 2016 had AL λ-type with GI involvement and is 9 months status post MEL 200 stem cell transplant (SCT), and the second with SMM diagnosed in 2009 had cardiac AL λ-type by heart biopsy with an NT-proBNP 171 (but with a suggestive MRI) and is en route to SCT. Conclusions: The SAVE trial enables early diagnosis of AL λ-type based on the λ IGLV gene used by the clonal plasma cells. By RT-PCR in this pilot study we identified the clonal λ gene 85% of the time. Earlier diagnosis will enable treatment with effective therapy such as MEL 200 SCT. Clinical trial information: NCT02741999.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B‐cell neoplasms

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Characterization and comparative analysis of immunoglobulin lambda chain diversity in a neonatal porcine model

To elucidate how antigen exposure and selection shape the porcine antibody repertoires, we investigated the immunoglobulin lambda light chain (IGL) gene repertoires of the binary cross-bred (Yorkshire×Landrace) pig at different developmental stages, pre-suckle neonate (0days), wean piglet (35days) and growing pig (75days) under normal farming conditions. Immunoglobulin lambda light transcript (IGLV-J-C) clones of the peripheral blood mononuclear cells (PBMCs) from these different developmental stages were assessed for IGL combination, junction and sequence diversity. Previous research has revealed that IGLV8 plays a major role in immunity during the early fetus stage and that IGLV3 accounts for 30% of the neonatal IGLV repertoires. Here, we found that the antibody profile exhibited salient features at different stages. The usage of the IGLV3-3 subclass gradually decreased during development, in contrast, the utilization of IGLV8 (IGLV8-10, IGLV8-13 and IGLV8-18), which started in the fetal stage, has increased in the growing stage. Moreover, the junction diversity, as measured by the IGLV hypervariable complementarity determining region 3 (CDR3L) lengths, was constant during the different stages. The complete junction mutation ratio clearly increased in the growing pig compared to that in the younger pig. Our data provide new insights into the postnatal porcine IGLV repertoires maturation which can lay the foundation for porcine antibody gene research.

The Importance of an In-depth Study of Immunoglobulin Gene Rearrangements When Ascertaining the Clonal Relationship between Concomitant Chronic Lymphocytic Leukemia and Multiple Myeloma.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers [particularly solid tumors (3–5)] can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare [for a review of the few reported cases, see Ref. (6)]. The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing, and somatic mutations—events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements [clonality assessment, V(D)J sequencing, and somatic hypermutation analysis] in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same VH and JH genes but different DH genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.

Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire.

Rituximab Therapy Leads to Reduced Imprints of Receptor Revision in Immunoglobulin κ and λ Light Chains

Transient B cell depletion by rituximab (RTX) has become a specific treatment of rheumatoid arthritis (RA). Although phenotypic repopulation kinetics of B cell subsets are well documented, precise molecular analyses of the reconstituting immunoglobulin (Ig) genes encoding the B cell receptor in RA are sparse. A total of 708 individual CD19+CD27+ (memory) and CD19+CD27- (naive) B cells from 2 patients with RA were analyzed at baseline and 7 months after RTX at B cell repopulation. Ig light chain variable kappa (Vκ) and lambda (Vλ) light chain gene rearrangements were amplified, sequenced, and analyzed with a focus on receptor revision. The naive as well as the memory repertoire repopulated polyclonally with diverse use of variable light chain gene families and minigenes. During the reconstitution phase, B cells used significantly fewer Jκ distal Vκ genes (p = 0.0006), with a higher frequency of somatic hypermutation of rearrangements employing Jκ5 compared to baseline in memory B cells. The use of Vλ rearrangements in regenerating B cells was also biased toward use of Vλ genes of the proximal cassette. In general, reemerging CD27+ Ig light chain genes were substantially more highly mutated than before RTX therapy (p < 0.0001, baseline vs during reconstitution). Our data indicate that RTX therapy leads to generation of distinct Vκ/Jκ and Vλ/Jλ gene repertoires consistent with replenishment of antigen-experienced B cells by germinal centers. At baseline, the imprints of receptor revision appeared to be more striking, which indicates that receptor revision is active in patients with RA and can be reduced by RTX.

Frequent Chromosome 1q Aberrations in HIV Associated B-Cell Lymphoma Unclassifiable with Intermediate Features Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma,

Abstract 3572Human Immunodeficiency Virus (HIV) infection is known to be strongly associated with an increased risk of high grade B-Cell Lymphoma (such as Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma). As South Africa has more than 5 million people living with HIV infection, this region sees a sharp increase in the diagnosis of high-grade lymphomas. HIV infection may associate with a number of atypical morphological features making it difficult to differentiate between classically described DLBCL and BL. As these two diseases have different prognosis and treatment, it is essential to accurately differentiate the type of disease and/or to diagnose the intermediate category of B-Cell Lymphoma, unclassifiable, with features intermediate between DLBCL and BL newly introduced in the 2008 WHO classification.In this study, we report on twelve patients (seven males and five females) who presented with high grade B cell lymphoma and a cell morphology comprising a mixture of medium to large cells that expressed B Cell markers. Patients' age ranged from 29 to 43 years old with a mean age of 34 years. Eight patients had a documented HIV positive status. Complex cytogenetic features associated with atypical morphologic features best fitted the diagnosis of B cell lymphoma unclassifiable, with features intermediate between DLBCL and BL.Conventional cytogenetics analysis revealed the presence of a complex karyotype in all cases. The chromosomes number varied from 46 to 49 with the exception of one case that had near-tetraploid cells (69 to 84 chromosomes). Ten cases had a t(8;14) classical Burkitt translocation involving the Immunoglobulin heavy chain gene as a partner, and one case had a variant Burkitt translocation t(8;22) involving the lambda light chain gene. Double hits were seen in two cases. In both cases, a translocation t(3;22)(q27;q11) rearranged the BCL6 gene in addition to a t(8;14). MYC and BCL6 genes rearrangements were confirmed using fluorescence in situ hybridization with the MYC and BCL6 break-apart probes (Abbott Molecular) and with the IGH/MYC/CEP probes. The most common secondary chromosomal aberration associated with a MYC rearrangement, was a chromosome 1 long arm structural abnormality present in nine cases, (75%) and resulting in a full trisomy 1q (n=3), or in partial duplication/ triplication of chromosome 1q (n=3), with both types of aberrations associated in 3 cases.Genome-Wide Affymetrix SNP6.0 array analysis was used to delineate chromosome 1q regions of gains. In brief, high molecular weight tumor DNA was extracted from tumor cells together with normal controls. DNA was processed and hybridized to the arrays according to the manufacturer's instructions. Data analysis was performed using the dChip software and Circulary Binary Segmentation. Copy number analysis using SNP arrays delineated three minimum regions of gains on the chromosome 1 long arm, 1q21.1q23.3, 1q24.1q24.2 and 1q32.1q32.2. The smallest region of amplification 1q24.11q24.2 included 27 genes of which the RCSD1 gene normally expressed in B-cells and previously found to be involved in acute B cell lymphoid leukemia (De Braekeleer et al 2007). These regions were smaller than 1q gains previously mapped by array comparative genomic hybridization in acute lymphoblastic leukemia and BL (Davidsson, et al 2007). Abnormalities of chromosome 7q were also non-randomly detected in four cases, in the form of trisomy 7q, or partial 7q duplication. Gains of 1q and 7q are known to occur in 20% and 10% of Burkitt lymphomas respectively (Boerma et al 2009; Scholtysik et al 2010).On the basis of these molecular cytogenetic features, we argue that these aggressive immunodeficiency associated, mostly t(8;14) positive lymphomas, represent advanced, clonally evolved forms of BL. These findings support the notion that there are distinguishable subgroups in DLBCL/BL intermediate lymphomas. Disclosures:No relevant conflicts of interest to declare.

Allelic exclusion of immunoglobulin genes: models and mechanisms.

The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Ig kappa and Ig lambda light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.

Translocation (18;22)(q21;q11) in B-cell lymphomas: a report of 4 cases and review of the literature

Follicular lymphomas characteristically carry t(14;18)(q32;q21) which results in IGH-BCL-2 fusion. Variant translocations that juxtapose the BCL-2 gene with the immunoglobulin kappa (2p11) and lambda (22q11) light chain genes are rare. We report 4 cases of B-cell lymphoma/leukemia associated with t(18;22)(q21;q11). The t(18;22)(q21;q11) was the sole aberration identified by conventional cytogenetics in 2 cases. Three cases were classified as chronic lymphocytic leukemia, and one as follicular lymphoma based on morphology and immunophenotype. Fluorescence in situ hybridization analysis was performed on all 4 cases using a BCL-2 breakapart probe. The BCL-2 gene was rearranged in all cases. Immunoglobulin lambda light chain gene rearrangement was shown in 3 cases using bacterial artificial chromosome probes spanning the variable and constant clusters of the IGlambda gene. Each case was negative for MALT-1 rearrangement using a MALT-1 breakapart probe. These cases illustrate that t(18;22)(q21;q11) is more commonly observed in chronic lymphocytic leukemia and may represent either an initial or secondary genetic event.

Immunoglobulin light chain gene translocations in non‐Hodgkin’s lymphoma as assessed by fluorescence in situ hybridisation

In non-Hodgkin's lymphoma (NHL), the majority of translocations involve the immunoglobulin heavy chain gene (IGH) locus, while a few involve the immunoglobulin light chain gene (IGL) locus, consisting of the kappa light chain gene (IGkappa) and the lambda light chain gene (IGlambda). Although many reports have dealt with the translocation and/or amplification of IGH in NHL, only a few have identified IGL translocations. To identify cytogenetic abnormalities and the partner chromosomes of IGL translocations in NHL, we performed dual-colour fluorescence in situ hybridisation (DC-FISH) and spectral karyotyping (SKY) in seven NHL cell lines and 40 patients with NHL. We detected IGL translocations in two cell lines and nine patients: four patients with diffuse large B-cell lymphoma, three with follicular lymphoma, one with extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue and one with mantle cell lymphoma. Five distinct partners of IGlambda translocation were identified by SKY analysis: 3q27 in three patients, and 1p13, 6p25, 17p11.2 and 17q21 in one patient each. Three cases featured double translocations of IGH and IGL. These findings warrant the identification of novel genes 1p13, 6p25, 17p11.2 and 17q21.

Nonoverlapping Functions of DNA Polymerases Mu, Lambda, and Terminal Deoxynucleotidyltransferase during Immunoglobulin V(D)J Recombination In Vivo

DNA polymerases mu (pol mu), lambda (pol lambda), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol mu participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol lambda-deficient animals have shorter length with normal N-additions, thus indicating that pol lambda is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol mu, pol lambda, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol lambda maintaining a large heavy chain junctional heterogeneity and pol mu ensuring a restricted light chain junctional variability.

PCR-based clonality analysis of B-cell lymphomas in paraffin-embedded tissues: Diagnostic value of immunoglobulin κ and λ light chain gene rearrangement investigation

Polymerase chain reaction (PCR)-based analysis, employed for detecting immunoglobulin heavy chain (IgH) gene rearrangements, has become a diagnostic tool widely used in the investigation of B-cell lymphomas, but the overall sensitivity of these methods does not exceed 80%, notably in germinal center (GC) and post-GC B-cell origin lymphomas. Many PCR strategies devised for detecting immunoglobulin light chain (IgL) gene rearrangements have been developed to enhance the clonality detection rates. However, the feasibility of these methods in routine clinical diagnosis using paraffin-embedded tissues has not yet been investigated sufficiently. We studied a large series of 108 cases of B-cell lymphomas, as well as 20 reactive lymphoid tissues using degenerate primers to amplify immunoglobulin kappa (Ig κ) and lambda (Ig λ) light chain genes. B-cell clonality was further investigated using semi-nested PCR for IgH gene rearrangements. B-cell clonality was detected in 74%, 56.5%, and 43.5% of cases using IgH, Ig κ, and Ig λ PCR, respectively. By combining these methods, the clonality detection rate increased to 93.5%. Only polyclonal patterns were noted in reactive lymphoid samples. We concluded that in addition to the established methods for IgH analysis, a PCR-based approach for IgL gene rearrangements analysis improves the clonality detection rate in over 90% of B-cell lymphoma cases using routine histological specimens with poor preservation of the genomic DNA.

Immunoglobulins and immunoglobulin genes of the horse

Antibodies of the horse were studied intensively by many notable immunologists throughout the past century until the early 1970's. After a large gap of interest in horse immunology, additional basic studies on horse immunoglobulin genes performed during the past 10 years have resulted in new insights into the equine humoral immune system. These include the characterization of the immunoglobulin lambda and kappa light chain genes, the immunoglobulin heavy chain constant (IGHC) gene regions, and initial studies regarding the heavy chain variable genes. Horses express predominately lambda light chains and seem to have a relatively restricted germline repertoire of both lambda and kappa chain variable genes. The IGHC region contains eleven constant heavy chain genes, seven of which are gamma heavy chain genes. It is suggested that all seven genes encoding IgG isotypes are expressed and have distinct functions in equine immune responses.

Report of two novel chromosomal translocations in chronic lymphocytic leukemia

Two novel chromosomal translocations were identified in 2 patients with chronic lymphocytic leukemia (CLL). Case 1: 60 year-old male, stage Rai 0/Binet A, with mutated immunoglobulin heavy (IgH) and lambda (Igλ) light chain genes; karyotype: 46, XY, t(9;12)(q12;p11) [3]/ 46, XY [22]. Case 2: 56 year-old male, stage Rai 2/Binet B, with mutated IgH and unmutated Igλ genes; karyotype: 46, XY, add(10)(q26), t(13;18)(q14;q21) [8]/ 46, XY [27]. Although both translocations are novel, the involved breakpoints (especially 13q14 and 18q21) have been reported to participate in various aberrations in CLL patients. Aberrations affecting bands 9q12 and 12p11, as in case 1, are generally rare.

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)–hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP–hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.

Establishment and characterization of new B-cell precursor leukemia cell line NALM-35.

A novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) cell line, NALM-35, was established from the peripheral blood of a 40-year-old woman at diagnosis of ALL. Imunophenotyping showed BCP type III characteristics including expression of TdT, CD10, CD19, CD22, CD79a and HLA class II. T-cell and myeloid-associated antigens tested were negative except CD5 and CD28. The surrogate light chains CD179a and CD179b were positive. NALM-35 cells have the morphological appearance of lymphoblasts. Cytogenetic analysis of NALM-35 revealed an abnormal karyotype with 46, XX, add(9)(p11). Southern blot analysis of the immunoglobulin genes status of NALM-35 at 10 months after establishment showed germ line configuration of the kappa and lambda light chain genes, and rearrangement of the mu heavy chain gene. DNA fingerprinting, chromosomal analysis and immunophenotyping proved that NALM-35 was clonally derived from the primary leukemia cells. The established cell line may provide a useful model system and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.

Physical map of the mouse λ light chain and related loci

The lambda light chain genes and the related loci lambda 5, V(preB)1 and V(preB)2 are selectively expressed in the development of B lymphocytes. The sequences and the order of the gene segments are known, but a precise physical map of the lambda locus has not yet been published. Using the Celera mouse genome database we constructed such a map. In this paper, we confirm the order of the gene segments as being V lambda 2 - V lambda X - J lambda 2 - C lambda 2 - J lambda 4 - C lambda 4 - V lambda 1 - J lambda 3 - C lambda 3 - J lambda 1 - C lambda 1 and give precise germ line distances. We located the V(preB)2 locus 3' to the lambda locus, 1,077,001 bp downstream; the V(preB)1 locus, 2,180,618 bp downstream of the lambda locus; and the lambda 5 gene, 4,667 bp downstream of the V(preB)1 locus. Except for the V(preB)2 locus all other loci are in the same transcriptional orientation.

Early Vlambda diversification in sheep.

This study examined a number of tissues during early gestation in foetal sheep to determine the earliest site of Vlambda expression and time of generation of the Vlambda repertoire. Tissues, including spleen, liver, gut, blood and bone marrow, were obtained from 48, 55, 60 and 63 gestational day (g.d.) ovine foetuses and cDNA libraries were prepared from them by reverse transcription-polymerase chain reaction. Clones were randomly selected from cDNA libraries and subjected to sequencing. Analysis of these sequences and comparison with a pool of germline genes led to the following conclusions. The expression of Vlambda occurs earlier in spleen (48 g.d.) than in all of the other tissues examined. Also, diversity is seen earlier and at higher levels in early foetal spleen than in all of the other tissues examined. In this regard, it is notable that splenic Vlambda expression is readily apparent even before such gut-associated lymphoid tissue as the ileal Peyer's patch (IPP) has developed. Two germline Vlambda genes, 5.1 and 5.3 predominate in early immunoglobulin lambda light-chain gene rearrangement. Examination of Jlambda usage revealed the existence of a new Jlambda gene and its utilization during the early phases of the development of the ovine antibody repertoire. This study indicates that sites other than the IPP contribute to the diversification of the Vlambda repertoire in sheep. We suggest that it is likely that foetal spleen may provide a partially diversified B-cell repertoire before the IPP becomes active as a major site for massive clonal expansion and extensive diversification of B cells.

Immunoglobulin V region gene use and structure suggest antigen selection in AIDS-related primary effusion lymphomas.

Primary effusion lymphoma (PEL) is a lymphoproliferation of B cells infected by Kaposi's sarcoma-associated herpes-virus/human herpesvirus-8 and reflecting a late stage of B cell differentiation close to plasma cell. Apart from viral infection, the pathogenesis of PEL is currently unclear. The aim of the present study was to investigate the role of antigen stimulation and selection in the evolution of PEL. In order to assess the specific variable heavy (VH) and light (VL) genes used by PEL and to define the heavy and light chain isotypes expressed by these lymphomas, immunoglobulin (Ig) genes from seven AIDS-related PEL were sequenced (three cell lines and four primary samples). Most of the samples (five out of seven) used lambda light chain genes; the majority of these (n = 4) belonged to the V lambda 3 family. Two cases expressed mu chains, whereas gamma chains were found in two cases. In all cases, significant deviations from the presumed germline counterpart were found in both the expressed VH and VL genes. Statistical evidence for antigen selection was evident in four out of seven samples studied. Evidence for selection was more frequent in the light chain genes than in the heavy chain genes. Collectively, these data indicate that PEL originate from mature, antigen-experienced B cells and bear implications for the pathogenesis and histogenesis of this lymphoma.