The lambda gene immunoglobulin repertoire of human neonatal B cells

The dynamics of immunoglobulin rearrangements and selection, which depend on age, antigen exposure and tolerance functions, are only partly understood. Thus, we analyzed and compared the lambda chain immunoglobulin repertoire of individual IgD+ human neonatal B cells with the adult peripheral B cell VlambdaJlambda repertoire. Some Vlambda genes, 4C, 2A2, 2B2, 5A, 1G and 4B, were overexpressed in the non-productive neonatal repertoire, whereas other Vlambda genes (2E, 2A2, 3H, 2B2, 1C and 1G) were overexpressed in the productive repertoire. The adult B cell repertoire revealed nearly the same predominance of genes in the non-productive and productive repertoire. A comparison of the non-productive and productive repertoire indicated that the genes 3H and 1C were positively selected, whereas the genes 4C, 2A1, 3I, 5A, 9A, 4A and 4B were negatively selected. All four functional Jlambda genes were used in both repertoires. Jlambda2/3 was used mainly. Insertions of non-templated nucleotides at the VlambdaJlambda-junction by the enzyme TdT were less frequent as compared to the adult, but the CDR3 length was the same. Comparison of CD5+IgD+ and CD5-IgD+ B cells revealed no differences between neonatal productive rearrangements. However, the genes 1C and 1G were used more often in the non-productive repertoire of CD5+ B cells, whereas gene 4B was used significantly more frequent in CD5- B cells. These data provide evidence that the primary usage and subsequent selection of Vlambda genes in the neonate are surprisingly comparable with the adult. This suggests that selection into the productive Vlambda repertoire in principal might be driven mainly by autoantigens in the newborn, as well as in adulthood, since newborns have not been exposed to exogenous antigens.     

A simple technique for the determination of kappa and lambda immunoglobulin light chain expression by B cells in whole blood.

This article describes the development of a simple technique by which the numbers of surface immunoglobulin expressing cells can be analysed by dual fluorescence flow cytometry in samples of whole blood. We have compared the results obtained using this procedure with those obtained using samples prepared by traditional density gradient centrifugation, and demonstrate an excellent correlation between the two techniques. The method is applicable both to blood samples from normal individuals and from patients with B cell malignancies such as B-CLL and B-NHL. We have also confirmed previous findings that density gradient centrifugation may preferentially deplete certain lymphocyte subsets. This technique offers the following advantages: (i) it is rapid, (ii) it is accurate, (iii) it is reliable, (iv) it is useful in cases of lymphopenia, and (v) it requires only a small volume of blood. It is likely to be applicable to other situations in which the presence of serum factors interfere with antibody staining in whole blood.     

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.     

Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire.

To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.     

Diversity and somatic hypermutation of the Ig VHDJH,V kappa J kappa,and V lambda J lambda gene segments in lymphoma B cells: relevance to the origin of the neoplastic B cell clone

Burkitt's lymphoma (BL) is a malignancy of B cells characterized by chromosomal translocations involving the immunoglobulin (Ig) and c-MYC gene loci. To address the putative role of antigen in the clonal expansion of these neoplastic B cells, we analyzed the VHDJH and VLJL gene segments expressed by the established cell lines derived from six endemic BL and six sporadic BL. Eight BL cell lines used genes of the VH3 family, two of the VH4, and two of the VH1. Eight VL chains were kappa (four members of the V kappa3, two of the V kappa1, and two of the V kappa2 subgroups) and four lambda (three members of the V lambda1 and one of the Vl ambda3 subgroup). The VH gene utilization was stochastic (i.e., it reflected the relative representation of the different VH gene family members in the human haploid genome). In contrast, the VL gene utilization was skewed toward the overutilization of the V kappa3 and V lambda1 gene subgroups. When compared with those of the respective germline genes, the sequences of the expressed Ig VDJ genes displayed nucleotide differences that resulted from somatic hypermutation. In three endemic and three sporadic BL cells, nucleotide changes yielding amino acid substitutions segregated within the complementarity determining region, indicating the application of a positive pressure for replacement mutations and suggesting that these neoplastic lymphocytes underwent a process of clonal selection driven by antigen, perhaps emerging from or transitioning through germinal centers.     

Protein displays of the human immunoglobulin heavy, kappa and lambda variable and joining regions

'Protein displays of the Human Immunoglobulin Heavy, Kappa and Lambda Variable and Joining Regions', the 6th report of the 'IMGT Locus on Focus' section, comprises 4 figures: (1) 'Protein display of human IGH V-REGIONs'; (2) 'Protein display of human IGK V-REGIONs'; (3) 'Protein display of human IGL V-REGIONs and V-PREB REGION'; (4) 'Protein display of human IGH, IGK and IGL J-REGIONs', and 1 table entitled: 'FR-IMGT and CDR-IMGT length of the human IGHV, IGKV, IGLV and V-PREB genes'. These figures and table are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cnusc.fr: 8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France. Copyright Copyright 1999 S. Karger AG, Basel     

Bacteriophage lambda P gene shows host killing which is not dependent on lambda DNA replication.

Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation. In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria. Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322. Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present. When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation. We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host. We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination. This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage.     

Definition of the RFLP alleles in the human immunoglobulin IGHG gene locus

In order to define more precisely the polymorphism of the human immunoglobulin IGHG (C gamma) genes and, consequently, to understand the structure and evolution of this multigene family, we have investigated the Restriction Fragment Length Polymorphisms (RFLP) of the IGHG genes in 113 unrelated individuals and 18 families. Using the restriction enzymes Bam HI, Sac I and Eco RI, and hybridization to a IGHG probe and to a specific IGHG3 (C gamma 3) probe, we describe 47 different restriction fragments (RF) in the IGHG locus, allowing us to define at least 15 RFLP alleles for the different IGHG genes. Our data demonstrate that the restriction fragment length polymorphism is occasionally due to the presence or absence of a given restriction site as a consequence of a point mutation, such as the creation of an Eco RI site, at the codon 3 of exon 2 in one IGHG4 allele (IGHG4*D2). However, most of the RFLP we observed seem to result from the insertion or deletion of a few hundred base pairs as illustrated by the IGHG3 polymorphism.     

Polymorphisms and haplotypes in the human immunoglobulin kappa locus.

By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the kappa locus (p for the C kappa proximal, d for the distal copy). Some of these duplication-differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B-J kappa-C kappa region were included into the study. Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11. The latter haplotype in which the whole distal copy of the kappa locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11 are consistent with Mendelian inheritance. Haplotypes N and G are distinguished from each other by eight RFLP markers. Six additional haplotypes, which were found in one or several individuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.     

Characterization of a new subgroup of human Ig V lambda cDNA clone and its expression.

From a human bone marrow cDNA library, we have cloned and sequenced a gene which cross-hybridized with murine pre-B cell-specific gene 8HS-20 cDNA under the low-stringent condition. Sequence analysis predicted that this gene (YM-1) encoded 240 amino acids which had the basic structure of immunoglobulin lambda light chain. The 3' half of the YM-1 sequence was identical to the J lambda 2 C lambda 2 region except for four nucleotides. The 5' part of the gene had 87.6% sequence homology with the reported V lambda gene called T1. Comparison of the deduced amino acid sequences with representative members of the seven other V lambda subgroups showed considerable structural homology, but the maximum homology with these chains was 44%. Therefore, we conclude that YM-1 belongs to a new V lambda subgroup. Interestingly YM-1 showed higher homology with VpreB1 (56%) than with any of the other V lambda subgroups. By Southern blot analysis four to six cross-hybridizing V lambda bands were detected at high stringency. Expression of the V lambda gene was observed in immature as well as mature B cell lines without accompanying V-JC gene joining, suggesting that V lambda of the YM-1 locus is activated at the early stage of maturation.     

Definition of the human immunoglobulin variable lambda (IGLV) gene subgroups

Comparison of 60 human immunoglobulin variable lambda (IGLV) sequences allowed us to define seven subgroups designated V lambda I to V lambda VII. We demonstrate that all lambda proteins sequenced so far fall into the subgroups I, II, III and VI, and that the lambda regions previously assigned to subgroups IV and V belong, in fact, to subgroups III and II, respectively. Four sequences not belonging to any of the subgroups I, II, III and VI define the new subgroups IV, V and VII. Interestingly, these subgroups show a higher homology to rabbit or mouse V lambda genes than to the other human V lambda subgroups. By comparison of the proteins either with the sequences deduced from the germ-line genes or with the consensus sequences, the rate of amino acid changes due to somatic mutations or allelic variations was evaluated in several lambda proteins. Framework and complementarity-determining regions of the human IGLV genes and proteins were delineated. Alignment of the lambda sequences shows that functional V-J rearrangement occurs, with or without deletion of nucleotides encoding one or two amino acids at the 3' end of the V gene. Diversity of the third complementarity-determining region is due to somatic mutations and to flexible V-J junction, but there is no evidence of N-diversity in the human lambda locus.     

Analysis of Ig kappa light chain gene variable regions expressed in the rheumatoid synovial B cells

Sequence analysis of antibody variable (V) regions can provide an insight regarding whether B cells have gone through an antigen-driven process of affinity maturation. In this study, we analyzed 16 V-regions of immunoglobulin (Ig) kappa light chain genes obtained from a cDNA library of a rheumatoid arthritis (RA) synovial tissue. A salient feature of our results is the high frequency utilization of germline V kappa I family genes, especially the O2/O12 gene (38%). All kappa V-regions showed extensive somatic hypermutation with 5.4% of an average mutation rate. Replacement to silent mutation (R/S) ratio in the complementarity determining region (CDR) was > 2.9 in 12 out of 16 clones, indicating that the majority of the RA synovial B cells had undergone affinity maturation. However, the four other clones showed R/S ratios of < 2.9 in the CDR despite a high mutation rate. In contrast to the previous reports, long CDR3 was not a characteristic feature of these clones. In summary, these data show the high frequency utilization of the germline O2/O12 gene and a high rate of mutation with an evidence of antigen selection in most of the Ig kappa genes expressed in the RA synovium.     

The immunoglobulin lambda-like gene cluster (14.1, 16.1 and F lambda 1) contains gene(s) selectively expressed in pre-B cells and is the human counterpart of the mouse lambda 5 gene

A human immunoglobulin (Ig)-related gene, covering approximately 8 kb, was isolated from a cosmid genomic library, by hybridization with a C lambda probe and with a lambda-like probe. This gene was identified as 14.1 It belongs to the human lambda-like cluster which is composed of three genes (14.1, 16.1 and F lambda 1) that do not rearrange. Sequence data indicate that 14.1 is organized similarly to the mouse lambda 5 gene. It contains three exons with lengths of 69, 38, and 106 codons as compared with 65, 38, and 106 for exons 1, 2, and 3 of mouse lambda 5, respectively. The corresponding homology values were 61, 66 and 75.5%. Using a 14.1 specific probe containing exon 1, we showed that this gene was selectively expressed in human pre-B cell lines. It is likely to encode a 213-amino acid lambda-like light chain that would associate with mu chains and play an important role in the early steps of B cell differentiation.     

Insights into the regulation of immunoglobulin light chain gene rearrangements via analysis of the kappa light chain locus in lambda myeloma

Accumulating evidence indicates that B cells may undergo sequential rearrangements at the light chain loci, despite already expressing light chain receptors. This phenomenon may occur in the bone marrow and, perhaps, in germinal centers. As immunoglobulin (Ig)kappa light chains usually rearrange before Iglambda light chains, we analysed, by polymerase chain reaction, the Igkappa locus of bone marrow mononuclear cells from 29 patients with Iglambda myeloma to identify earlier recombinations in marrow plasma cells. The results demonstrated that Igkappa alleles were inactivated via the kappa-deleting element, presumably prior to V(kappa)-J(kappa) rearrangement, in many cases. Eighteen alleles (16 myeloma clones, 55%) showed V(kappa)-J(kappa) rearrangements, with increased utilization of 5' distant V(kappa) and 3' distant Jkappa gene segments (Jkappa4, 56%), an indication of multiple sequential rearrangements. In-frame, potentially functional V(kappa)-J(kappa) rearrangements were found in approximately one-third of available rearrangements (as expected by chance), each one in different myeloma clones: three were germline encoded, while one had several nucleotide substitutions, suggesting inactivation after the onset of somatic hypermutation. Three of four potentially functional V(kappa)-J(kappa)rearrangements involved V(kappa)4-1, a segment considered to be associated with autoimmunity. These findings provide insights into the regulation of light chain rearrangements and support the view that B cells may occasionally undergo sequential light chain rearrangements after the onset of somatic hypermutation.