Immunogenic and antigenic epitopes of Ig. XXV. Monoclonal antibodies that differentiate the Mcg+/Mcg- and Oz+/Oz- C region isotypes of human lambda L chains

The C region of human lambda L chains is specified by multiple C lambda genes of which three--C lambda 1, C lambda 2, and C lambda 3--encode for the isotypes designated Mcg+, Kern- Oz-, and Kern- Oz+, respectively. The Mcg, Kern, and Oz factors have been characterized by sequence differences involving specific C lambda amino acid residues. They have also been recognized serologically by polyclonal antisera but, with rare exception, these reagents are no longer available. We have obtained two murine anti-human lambda-chain mAb, 14G1 and 14D1, that recognize antigenic determinants specific for the C lambda isotypes Mcg and Oz, respectively. These antisera have been used to classify as Mcg+/Mcg- or Oz+/Oz- monoclonal lambda-chains (Bence Jones proteins) and intact Ig lambda proteins. There was complete concordance between the chemical and serologic assignment of lambda-chains as Mcg+/Mcg- or as Oz+/Oz-; no single protein expressed both isotypes. There was no evident association between the C region isotype Mcg or Oz and the V region subgroup of the protein tested. However, our finding that four of seven amyloid-associated lambda VI Bence Jones proteins were Oz+ suggests a predominant expression of the C lambda 3 gene product among proteins of this uncommon V lambda subgroup.     

The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7

The human lambda L chain Ig gene complex consists of multiple JC gene segments. A seventh human lambda C region gene segment, C lambda 7, was found 2.7 kb downstream of C lambda 6 in this gene complex. A J lambda gene segment, J lambda 7, was found 1.2 kb upstream of C lambda 7 and contains potentially functional nonamer and heptamer recombination sites, an RNA splice site and J coding region. C lambda 7 maintains an open reading frame and encodes a new lambda isotype. C lambda 7 encodes Kern+ and Oz- determinants, but does not encode any of the Kern+Oz- myeloma proteins published to date. Nevertheless, we present evidence that JC lambda 7 is transcribed in normal lymphocytes and is functional. In contrast, we present new data that the C lambda 6 gene segment, reported by others to encode the Kern+Oz- protein, is non-functional due to a 4-bp insertion in our cosmid clone. The 4-bp insertion was characterized further in 32 genomic DNA samples by producing a distinctive restriction fragment length and verified by the DNA sequences of the polymerase chain reaction products of two different cell lines. We discuss the possibility that the Kern+Oz- myeloma proteins do not define an isotype and are not encoded by JC lambda 7 nor other non-allelic genes, and we discuss the level of expression of JC lamba 7 as compared to that of JC lambda 2 and JC lambda 3.     

Cloning and sequence analysis of an Ig lambda light chain mRNA expressed in the Burkitt''s lymphoma cell line EB4.

A cDNA library was constructed from the mRNA of the Ig lambda producing Burkitt's lymphoma cell line, EB4. Overlapping clones encompassing the coding sequence of the Ig lambda mRNA were isolated and sequenced. The predicted amino acid sequence shows a short hydrophobic leader peptide and a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The V region belongs to subgroup VI and has greatest homology (80%) with the Amyloid-AR protein. The constant region is the Kern- Oz+ isotype. Probing normal human DNA with the subcloned V lambda coding sequence detects one gene at high stringency and a family of 11 members at low stringency. To date, no restriction enzyme site polymorphisms have been detected. The V lambda VI gene is rearranged on both chromosomes of EB4 and is deleted on both chromosomes in the Burkitt's lymphoma cell line BL2.     

Characterization of the human Ig V lambda II gene family and analysis of V lambda II and C lambda polymorphism in systemic lupus erythematosus.

We report the cDNA sequence of an expressed human V lambda II gene and present an RFLP analysis of the Ig gene family defined by this clone. This V lambda II gene was expressed in a monoclonal B cell line generated from a patient with SLE by transformation with EBV. The encoded lambda L chain displays the 8.12 Id, an Id common to anti-DNA antibodies from patients with SLE. Using a coding region probe we estimate from Southern blot analysis that the germline V lambda II gene family contains at least 15 members. Many of the V lambda II restriction fragments are polymorphic both in SLE patients and in nonautoimmune individuals. EcoRI, HindIII, and TaqI RFLP analyses of the V lambda II gene family and EcoRI analysis of the C lambda gene family reveal no polymorphisms specific to SLE. Observed V lambda II and C lambda allele frequencies are the same among SLE patients and nonautoimmune individuals, and show no evidence of linkage disequilibrium between the two loci.     

The Ig light chain restricted B6.kappa(-)lambda(SEG) mouse strain suggests that the IGL locus genomic organization is subject to constant evolution

In laboratory mice, the different Ig lambda light chain subtypes (lambda 1, lambda 2, lambda x and lambda 3) are expressed on 60, 16, 16 and 8%, respectively, of the lambda-positive peripheral B cells. Eighteen years ago, our laboratory characterized a lambda 1(-) wild mouse strain: SPE ( Mus spretus). In this report, we describe the characterization of another wild-derived Mus spretus inbred strain, SEG, that presents the same characteristic, namely the absence of lambda 1 expression. An almost congenic strain, B6.lambda(SEG), was detected in a series of recombinant congenic strains carrying 2% of SEG/Pas genome in a C57BL/6J background. This B6.lambda(SEG) strain was crossed to Igh (a) C kappa (-) mice in order to derive two different additional congenic strains: B6.kappa(-)lambda(SEG) Igh (a) and B6.kappa(-)lambda(SEG) Igh (b). In this paper, we characterize the genomic organization and the expression of the SEG IGL locus. Altogether, our data show that the SEG IGL locus is constituted by a single functional IGLJ2SEG-IGLC2SEG, two pseudo IGLJ4SEG1/2-IGLC4SEG1/2 gene clusters and two V gene segments: IGLV2SEG and IGLVXSEG. In particular, we show the absence of IGLV1 and IGLVSD26 gene segments. IGLVSD26 was reported to be present in some Mus m. musculus mice and absent in BALB/c. Here, we confirm its presence not only in other Mus m. musculus mice but also in Mus spretus mice. Consequently, we propose that IGLVSD26-related gene segments define a new family that we name V lambda 4. The study of the organization of different IGL loci, in addition to the V lambda 4(+) reported here, could elucidate questions concerning the evolution of the lambda locus.     

A rabbit Ig lambda L chain C region gene encoding C21 allotopes

Southern blot analyses of germ-line DNA obtained from rabbits expressing lambda chains of C7 and/or C21 allotypes were performed with a rabbit C lambda region-specific probe; a 12-kbp EcoRI- and a 2-kbp BamHI-hybridizing fragment were detected only in the DNA from rabbits expressing the C21 allotype. The 12-kbp EcoRI fragment was cloned and shown to contain two C lambda region-encoding genes in the same orientation. Each is preceded by a J lambda gene segment. Nonamer-12-bp spacer-heptamer recombination signal sequences were found 5' of each J lambda segment, and splicing signals were identified at the 3' ends of the J lambda segments and the 5' ends of the corresponding C lambda genes. The C lambda 5 gene, which exhibits a sequence identical with that found in several cDNA clones, is carried by the 2-kbp BamHI fragment missing from the genomic DNA of rabbits which do not express the C21 allotype. The second C lambda gene, C lambda 6, lies 3' of C lambda 5, in a 1.6-kbp BamHI fragment which is present in genomic DNAs of all tested rabbits, irrespective of their phenotype. Its sequence is identical with that found in one cDNA clone and differs from that of C lambda 5 in 17 base positions resulting in four amino acid substitutions. A fragment of a cDNA, with a J-C region sequence identical with that encoded by the J lambda 5-C lambda 5 gene pair, was subcloned into a plasmid expression vector. The resulting polypeptide product could be specifically immunoprecipitated by anti-C21 but not anti-C7 alloantisera, showing that some, if not all, C21 allotopes are encoded by the C lambda 5 gene. In contrast, the C lambda 6 gene product was not precipitable, either by anti-C7 or by anti-C21 alloantisera, although it was readily immunoprecipitated by a goat anti-rabbit lambda chain antiserum.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

Association of human lambda light chain V/J/C segments: serologic analysis and primary structure of the lambda VI Bence Jones protein THO

The complete amino acid sequence of the human monoclonal lambda VI light chain Bence Jones protein THO was determined. We have found it to have remarkable similarities to the previously sequenced lambda VI Bence Jones protein SUT. Immunochemical analyses demonstrated that both lambda VI chains belong to a V lambda VI sub-subgroup. The 98-residue V gene-encoded segments of proteins THO and SUT are closely homologous and are distinguished from other lambda VI chains by a one-residue deletion at the V-J recombination site. Proteins THO and SUT have identical 13-residue J segments and therefore are encoded by the same J lambda gene. Further, both proteins have identical 105-residue C regions that by sequence represent products of the C lambda 3 (Kern-, Oz+) gene. The primary structure and serologic properties of proteins THO and SUT imply at the protein level of association between certain types of V lambda, J lambda, and C lambda segments.     

Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus

The organization of the human immunoglobulin lambda light chain locus (IGL) was recently described. This locus has been entirely sequenced. To evaluate the extent of the genomic variability existing inside that locus, we compiled all the available sequences of germline IGLV genes to find variants of Vλ sequences. We also looked for RFLP polymorphisms in a reputedly highly polymorphic human population from eastern Senegal, and compiled all RFLP data previously published. Analysis of these data indicates that IGLV alleles are frequent and increase the diversity of the lambda light chain repertoire in the human population. In contrast, RFLP and polymorphism by insertion and/or deletion are limited in that locus. This observation reinforces our hypothesis that the human IGL locus has undergone less evolutionary shuffling than the human kappa or heavy-chain loci. Received: 23 December 1998 / Accepted: 1 March 1999     

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.     

Mouse V lambda x gene sequence generates no junctional diversity and is conserved in mammalian species

The lambda x, a new mouse Ig lambda L chain, is produced by rearrangement of the V lambda x, J lambda 2, and C lambda 2 gene segments. The V lambda x amino acid sequence is as divergent to other V lambda as to Vk gene sequences. Additionally, its third hypervariable region (CDR3) is four amino acids longer than those of all other variable gene segments of murine L chain. We have cloned and sequenced the germ-line V lambda x gene and found that the unexpected CDR3 length is encoded by the V lambda x gene. Junctional diversity is prevented by a TAA termination codon localized at the V lambda x 3' extremity. Moreover, we show a striking conservation of the V lambda x sequence in various mammalian species. Portions of the V lambda x sequence display more than 70% of nucleotide sequence identity with rabbit and human variable regions. These results suggest that V lambda x predated the divergence of mammalian species.     

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.     

Polymorphism of C lambda genes and units of duplication in the genus Mus

The number of Ig C lambda genes in nine geographically widespread species from the four subgenera in the genus Mus was estimated from the number of Bam HI and Eco RI restriction fragments that hybridize under high stringency conditions to cDNA probes of BALB/c inbred mouse origin (Mus musculus domesticus). Three closely related species in the subgenus Mus, M. musculus, M. spretus, and M. spicelegus, show considerable variation in the number of C lambda genes. Estimates of gene numbers in these animals range from two C lambda genes in M. spretus from Puerto Real, Spain to 12 C lambda genes in M. musculus musculus from Studenec, Czechoslovakia. Strains of mice carrying either six or 10 C lambda genes were derived from a single population of M. musculus domesticus from Centreville, MD. The hybridization patterns of mice exhibiting C lambda gene amplification indicate that duplications are of relatively recent origin and probably occurred by reiteration of a DNA segment closely related to the 6.5 kb [C lambda 3 - C lambda 1] unit found in BALB/c inbred mice. Three more distantly related species in the subgenus Mus, and a species representing the Nannomys subgenus all appear to carry only four C lambda genes. DNA of species representing the Coelomys and Pyromys subgenera hybridized weakly to the C lambda cDNA probes, but these animals also have no more than four C lambda genes. Thus, there may be a base number of four C lambda genes in most species in the genus Mus. All inbred strains of mice so far examined also have only four C lambda genes, but no feral M. musculus examined have fewer than six C lambda genes. One explanation of the discrepancy in the number of genes between inbred and feral M. musculus is that C lambda genes were deleted during the process of inbreeding.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.     

Nucleotide sequence of a chromosomal rearranged lambda 2 immunoglobulin gene of mouse.

The rearranged lambda 2 gene of the mouse plasmacytoma cell line MOPC315 has been cloned and sequenced. A comparison of its sequence with the sequence of the unrearranged (germ-line) V, J and C gene segments shows that the sequences of the V gene segments differ at six positions. The sequence of the J and C gene segments remained unchanged. These results add support to the hypothesis that somatic mutations occur in immunoglobulin in genes and that these mutations do not involve the C gene segment. The degree of homology of the elements of the lambda 2 gene with those of the lambda 1 gene and C lambda 3 and C lambda 4 gene fragments suggest a pathway of evolution by gene duplication of the immunoglobulin lambda light chain locus. According to this scheme the original structure V0-J0C0 gave rise to a structure V0-J1C1-J11C11 by duplication of the J0C0 region. A second duplication encompassing the whole region resulted in the present structure: V1-J3C3-J1C1/V2-J2C2-J4C4.     

Molecular cloning of a human immunoglobulin lambda chain variable sequence

We have cloned a human V lambda cDNA sequence from an Ig lambda-producing human Burkitt lymphoma cell line (BL2) by taking advantage of a cloned constant region gene as a primer for cDNA synthesis instead of an oligo(dT) primer. The amino acid sequence deduced from the nucleotide sequence of V lambda clones is highly related to that of the NEW V lambda protein of subgroup I. Southern blot hybridization of human DNAs with the V lambda I probe showed at least 12 hybridizing V lambda fragments. These fragments are amplified in K562 cells which derive from a case of chronic myelogenous leukemia and contain an amplified c-abl oncogene and amplified C lambda sequences.     

Cloning and nucleotide sequence analysis of the cDNA of the immunoglobulin lambda light chain from the bovine udder

A cDNA library was constructed from the mRNA of bovine mammary gland which contained Ig lambda producing plasmacytes. Overlapping clones encompassing the major portion of the coding sequence of the Ig lambda mRNA were isolated and sequenced. Predicted amino acid sequence shows a mature polypeptide of 217 residues in which V, J and C regions can be distinguished. The constant region has greatest homology with human Ig lambda mRNA constant region. The area of the V region between CDR3 and CDR2 has also great homology with the same area of human lambda chain.     

Variation in V lambda genes in the genus Mus

The complement of Ig V lambda genes in nine species of feral mice representing the four extant subgenera of the genus Mus was examined and compared with that of BALB/c inbred mice. Although all inbred strains examined have two V lambda genes, there is variation in the number of copies of V lambda genes in the wild mice. All feral representatives of M. musculus domesticus, from which inbred strains are derived, have at least three V lambda genes, indicating that a V lambda gene may have been lost during the inbreeding process. At least three V lambda genes are also found in representatives of three other M. musculus subspecies, including the stock of M. musculus musculus "Czech II" shown to have at least 12 C lambda genes. In comparing the complement of V lambda and C lambda genes in these animals, evidence is found that supports a mechanism of lambda gene reiteration involving duplication of a unit containing a V lambda and two C lambda genes. However, the possibility that C lambda gene amplification occurred independent of V lambda gene evolution cannot be ruled out. M. spicelegus and M. spretus, species that are semifertile with M. musculus, have one to three V lambda genes. Species more distantly related to M. musculus, such as M. cookii and M. platythrix, appear to have more (four to six) V lambda genes. Greater V lambda gene heterogeneity is also found in these animals. We propose that the ancestors of the subgenus Mus had more V lambda genes than are seen in modern species and that the paucity of V lambda genes in M. musculus, M. spicelegus, and M. spretus may be the result of V lambda gene deletion events that occurred since the divergence of the ancestor of these three species and those of the distantly related species.