Expression of immunoglobulin lambda chains in the laboratory rat

We immunized a BALB/c mouse with the lambda-bearing rat IgG1 myeloma IR31, fused its spleen cells with the hybridoma parent line P3.X63.Ag8.653, and isolated a monoclonal antibody (G33/11) directed against rat immunoglobulin lambda chains. We used this antibody to classify two existing rat hybridomas as lambda-bearing proteins (D4.37HL.252 and PC61.5), and isolated one new lambda-bearing rat IgM hybridoma, G36/1. All the normal inbred rat sera that were tested contained lambda-bearing Ig as detected by G33/11, at levels ranging from 1.5% to 13% of the total serum Ig, the mean value being 7.9%. This antibody will be valuable for broadening our understanding of the immunogenetics of the rat, and for the characterization of monoclonal antibodies made in this species.     

Expression of immunoglobulin kappa and lambda chains in mink

The ratio of ? and λ chains of immunoglobulins varies significantly from one species to another. It has previously been thought that λ was only type expressed in mink. We tested mink immunoglobulin light chains using two monoclonal antibodies G80 and G88. It has been shown that G80 and G88 specifically recognize two antigenically different subpopulations of the light chains. Immunochemical analysis of these subpopulations separated by affinity chromatography suggested that they represent λ and ? types of light chains, respectively. Screening of a mink cDNA library with monoclonal antibody G88 resulted in the isolation of clone pIGK-1 containing ? chain-encoding sequence. The cDNA insert of pIGK-1 included most of the V segment, as well as the J, C and 3′ untranslated sequences. Mink V? sequence shown the highest homology with the human V?II subgroup genes (76-79%). Mink C? sequence was 53-63% homologous to C? of other species. The striking feature of mink C? chain is the presence of glutamine in the C-terminal position. Southern blot analysis suggested that mink haploid genome has one C? gene and multiple V? genes. The ?: λ chain ratio in the 12 minks studied was, on the average, 46:54. The same ratio was observed for the ?- and X-producing cells in the mesenteric lymph nodes. The five previously identified mink light chain allotypes were assigned to the λ chains, thereby confirming that λ chains in this species are additionally subdivided into several subtypes.     

Expression of two mRNAs for immunoglobulin lambda chains in the rat

We have analyzed the expression of immunoglobulin lambda chains in the rat by hybridizing RNA from various sources with C lambda 1-like and C lambda 2-like sequences recovered from a rat genomic library. A 1.0 kb lambda 2-like sequence is readily detected in lambda-producing hybridomas and in normal rat spleen RNAs; a 1.0 kb lambda 1-like message is also present, although at much lower levels. An additional 700 b.p. C lambda 2-like fragment is found in all normal rat spleens, and presumably represents a defective message. The nucleotide sequence of one cDNA clone isolated from the lambda-producing hybridoma G36/1 shows a lambda 2-like sequence, and six lambda-secreting hybridomas produced from the spleen of a kappa-suppressed rat all express a C lambda 2-like message. The great majority of rat lambda chains therefore appear to be lambda 2-like. Northern blot analysis of RNA from the spleen of this kappa-suppressed rat shows a considerable increase in the expression of both lambda 2-like and (at lower levels) lambda 1-like message. The coordinate rise of lambda 1 and lambda 2 RNA in this rat suggests that there may be at least two functional lambda chain genes in the rat, although there is as yet no evidence for the existence of rat lambda 1-like proteins.     

Junctional diversity in Xenopus immunoglobulin light chains

Xenopus cDNA sequences encoding the homolog of mammalian kappa (kappa) light (L) chains were isolated from isogenic tadpole and adult individuals to investigate whether there existed stage-specific immunoglobulin L chain expression and somatic diversification. In the course of these studies rearrangements to a sixth J(L) gene segment and a pseudogene (J(L)psi) were found, and it is suggested that the order of these gene segments with respect to the L chain constant (C) region exon is: J(L)6-J(L)1-J(L)2-J(L)3-J(L)4-J(L)5-J(L)psi-C(L). The cDNA junctional diversity was analyzed; few N and P regions were found and almost all the CDR3 were 9 codons in length. There were restricted patterns of recombination site resolution, and this is attributed to some constraint in JL coding end processing.     

Properties of monoclonal antibodies to human immunoglobulin kappa and lambda chains

Hybridomas have been produced from mice immunized with human IgG. Culture supernates were assayed for the presence of antibody-producing cells by passive haemagglutination. Hybridomas producing antibodies to human kappa (kappa) and lambda (lambda) light chains have been cloned and grown as ascitic tumours in BALB/c mice. The antigen-binding characteristics of the monoclonal antibodies, contained in the ascitic fluid, were assessed by haemagglutination inhibition, ELISA and radioimmunoassay systems and by the binding of radiolabelled antigen in analytical flat-bed iso-electric focussing gels. One monoclonal anti-kappa reacted better with free than with combined kappa chains; for another the reverse was true. Antibody fractions separated by DEAE chromatography of ascitic fluids were coupled to ox red cells with chromic chloride and compared with polyclonal antibodies for the detection of cell-surface immunoglobulins.     

Genetic polymorphism of IgG in the mink. V. Two new genetic markers of the lambda light chains of mink immunoglobulins, L4 and L5

Two new allotypes of the light (L) chains IgG, L4 and L5, were identified in the mink with dispecific antiserum produced by immunization with allogenic IgG. By means of hybrid IgG molecules and proteolytic fragments, L4 and L5 were localized on the C region of the L chain. L4 and L5 occurred frequently in the three mink populations studied and L4 and L5 are inherited independently of the known mink C gamma allotypes. L4 and L5 are encoded by closely linked genes. The antigenic specificities of L4 and L5 were not identified in the closely related Mustelidae and in the other mammalian representatives. Consequently, L4 and L5 are species specific to mink. Determination of the phenotype combinations of the five allotypes on the L chains (including the new L4 and L5) demonstrated the existence of seven combinations only with a predominance of L1,2,3; L4,5, and L1,2,3,4,5 phenotypes. Based on the results obtained, it is concluded that the mink C lambda locus has a complex organization. A model for the mink C lambda locus with at least three or possibly five linked genes is suggested.     

Murine anti-TNP antibodies positive for either lambda 1 or lambda 2 light chains express common idiotypic determinants

Although the lambda-bearing antibodies represent only 5% of the total mouse serum immunoglobulins, some antigens such as B1355 dextran (alpha (1-3)Dex), the 4-hydroxy-3-nitrophenyl acetyl (NP) and 2,4-dinitro or 2,4,6-trinitrophenyl (DNP/TNP) antigens can induce lambda-positive immune responses. In contrast to the lambda antibody response against alpha (1-3)Dex and NP antigens which is restricted to the lambda 1 isotype it was shown that the response to the DNP (or TNP) antigen uses lambda 1 and lambda 2 and lambda 3 isotypes. The idiotypy of the alpha (1-3)Dex and NP systems has been well characterized contrary to that of the lambda-positive anti-TNP/DNP response which has been poorly studied. In this paper, we describe two idiotopes (Id C19-3 and Id D11-2) shared by two BALB/c monoclonal anti-TNP antibodies (TNP5 and TNP9) which, respectively, use the lambda 1 and lambda 2 light chains. These idiotopes were independently expressed on other monoclonal anti-TNP/DNP antibodies and appear to require the use of a unique VH gene associated with a particular V lambda region. After TNP-Ficoll immunization, BALB/c mice recurrently express both idiotopes on lambda 1 and (lambda 2 + lambda 3) anti-TNP antibodies. In addition, all the mouse strains immunized against TNP-Ficoll give a lambda 1- and (lambda 2 + lambda 3)-positive immune response with the exception of SJL and SJA strains which present a deficit for the expression of lambda 1 light chain. The expression of Id C19-3 was restricted to the strains with the Igh-Va allotypic haplotype (including SJA) whereas the Id D11-2 was extensively expressed in the various strains.     

Genetic polymorphism of IgG in the mink. IV. Identification and genetic control of L3 allotype of the light chains

A new allotype of the mink light chains, designated L3, was identified. This allotype is inherited as a Mendelian character at a frequency of 0.46 in the mink population. Data were obtained indicating that L3 is independent of the C gamma-heavy chain allotypes of mink immunoglobulins. The gene L3 is closely linked to a gene encoding L1, another light chain allotype. Alloantigens L1 and L3 are presumably markers of the light chains of two different subtypes. In contrast to L1, which occurs in many mammalian species, L3 is species-specific, i.e., it is a case of light chain polymorphism representative of the whole mink species.     

Guinea pig immunoglobulin light chain isotypes. I. Separation of kappa and lambda chains and the identification of three isotypes of the lambda chain constant homology region.

The separation of intact kappa chain and two fragments comprising lambda (lambda) chain from immunoglobulin light chain pools isolated from strain 13 guinea pigs is achieved by cyanogen bromide digestion and gel filtration before and after reductive clevage of disulfide bonds. The smaller lambda chain fragment derives from the original carboxyl-terminus of lambda chain. The partial sequence of component tryptic and thermolytic peptides of this thirty-nine residue fragment allowed its complete sequence to be deduced. Three isotypic forms of this lambda chain constant region fragment, distinguished by four residue positions showing alternative amino acids, are found expressed by guinea pigs. Each of these three isotypes is more homologous to the other two than to lambda chains of any other species. The distribution of amino acid substitutions differentiating these isotypes further supports the view that lambda chain isotypes arose in guinea pigs, and in several other species, independently by gene duplication.     

Characterization of an amyloid fibril protein from localized amyloidosis of the skin as lambda immunoglobulin light chains of variable subgroup I (A lambda I)

Amyloid fibrils obtained from a case (Hud) of localized, tumorous amyloidosis of the skin were investigated by immunological and biochemical methods including amino acid sequence studies. Evidence was obtained that a lambda immunoglobulin light chain protein, A lambda I, and differently sized N terminal fragments (mol. wt 14,000 and 10,000) of the same protein comprised a major component of the fibrils. The chemical nature of this protein suggested that this case of cutaneous amyloidosis belonged to the idiopathic form of amyloidosis, probably as a special type of systemic, idiopathic amyloid disease.     

Serum kappa and lambda light immunoglobulin chains in cynomolgus macaques (Macaca fascicularis) during the first twenty months of age

Growth is coupled to physiological modifications of the immune system which reaches the functional capabilities according to age-related milestones. Few data are available on the circulating immunoglobulin levels and no data exist on total immunoglobulin light chains in infant macaques. Therefore we studied by a nephelometric assay, the age-dependent variations of κ and λ serum light chains in the experimental animal model Macaca fascicularis during the first 20 months of age. Both κ and λ showed a marked increase in their concentrations during the first 7–8 months of life. Infants' light chain levels were anyhow significantly lower than those of the nursing dams and of the control group, never attaining, even at the 20th month, the same concentration as the adult, although the value of the κ/λ ratio was apparently the same.     

cDNA clones encoding immunoglobulin lambda chains from rabbit expressing the phenotype c7

A cDNA library derived from spleen cells of an unimmunized rabbit expressing the c7 phenotype of Ig lambda chains (c7+, c21-) was screened with V lambda or C lambda probes of a lambda light chain bearing c21 epitopes. The nucleotide sequences of three hybridizing clones were found to be identical within the V lambda, J lambda and C lambda regions. The V lambda region was 97% similar to that of the functional germ-line gene V lambda 2, and the C lambda region was identical to that of gene C lambda 6, recently identified. Gene C lambda 6 exhibited four codon differences when compared with gene C lambda 5, the latter encoding c21 epitopes. The data presented here and in the accompanying report (Jaton, J.-C. et al., Eur. J. Immunol. 1990, 20:2713) support the view that gene C lambda 6 encodes the C region of c7 lambda chains and that c7 and c21 markers designate two distinct isotypic forms of lambda chains. On the basis of comparative Southern blotting analyses and restriction maps of cloned genomic regions containing V lambda and C lambda genes, a scheme is proposed to account for the c7- and c21- phenotypes.     

High resolution two-dimensional electrophoretic mapping of immunoglobulin light chains

Staphylococcus protein A immunoadsorbents were used to purify immunoglobulins from the sera of adult inbred (BALB/c) or outbred (Peromyscus leucopus) mice which had been raised under similar conditions. High resolution two-dimensional gel electrophoretic patterns of light-chains from individual BABL/c mice showed that 70–80% of the 1̃00 spots observed occur in patterns from all animals. The light-chain patterns from outbred P. leucopus showed only 3̃0% commonality with many more total spots. Possible implications of these results and the advantages of the 2-D technique for monitoring antibody repertoires are discussed.     

Monoclonal hybridoma screening by analysis of immunoglobulin light chains

A technique is described for the characterization of immunoglobulin light chains of hybridomas in culture. Immunoglobulins biosynthetically labelled with 14C are obtained from culture supernatants. Following complete reduction and alkylation light chains are separated from heavy chains and most other labelled contaminants by urea-formate gel electrophoresis. They are subsequently analyzed by isoelectric focusing using a simple transfer procedure. The method can be used to analyze up to 30 samples at a time and has a potential for the distinction of 750 light chains. The technique is especially useful (1) to determine the monoclonality of antibodies at an early stage in production, (2) to identify and classify antibodies having different structures but similar specificities, (3) to identify any alterations which may occur in quantity or quality of antibodies in long term culture, (4) to identify different hybridomas which produce antibodies of identical light chain subgroup.     

Recognition of lambda 1 and lambda 2 murine light chains by carrier-specific isologous T helper cells; effect of L-H chain assembly

Previous studies from this laboratory have revealed an antigenic site located on the variable domain of the lambda 2 light chain of BALB/c myeloma protein 315 (the V lambda 2(315) site). This site is recognized by conventional carrier-specific T helper cells (Th) of BALB/c mice and is expressed on both the free and assembled V lambda 2(315) domain. The present work defines two new antigenic sites associated with murine lambda chains. The first site was associated with free lambda 1-chain of myeloma protein J558. It was recognized by splenic Th from animals that had been primed with free lambda 1J558 in complete Freund's adjuvant; when transferred to irradiated animals the primed Th responded to a boost with (4-hydroxy-5-iodo-3-nitro-phenyl)acetate (NIP)-free lambda 1J558 in saline, but did not respond to NIP-complete J558 or NIP-free lambda 2(315). Priming with complete J558 failed to elicit Th that responded to NIP-free lambda 1J558. This determinant was therefore only expressed on the free (as opposed to the assembled) form of lambda 1J558, and it was not shared with free lambda 2(315). The second antigenic site was shared between free lambda 1J558 and free lambda 2(315). It was defined by free lambda 2(315)-primed Th which responded to a boost with NIP-free lambda 1J558. Since priming with free lambda 1J558 did not elicit Th that recognized NIP-free lambda 2(315), the cross-reaction was undirectional. The free lambda 2(315)-primed Th failed to respond to the complete J558, and M315-primed Th failed to respond to NIP-free lambda 1J558, indicating that the second (cross-reactive) antigenic site, like the first, was only expressed on free lambda chains. Completely reduced and alkylated (unfolded) free lambda 1J558 and free lambda 2(315) chains elicited Th that recognized native (folded) free chains. Thus, free lambda 1J558 bears two antigenic determinants recognized by Th, one private and a second shared with free lambda 2(315). Lambda 2(315) also bears two determinants, a cross-reactive one on free lambda 2(315) shared with free lambda 1J558, and a private one located on the V lambda 2(315) domain of the complete M315. The discussion is focused on possible explanations for the quenching of the two new lambda chain determinants upon light-heavy chain assembly and why, by contrast, the private V lambda 2(315) site is maintained in the complete M315.     

Mapping the binding domain of immunoglobulin light chains for Tamm-Horsfall protein

Cast nephropathy, or myeloma kidney, is a potentially reversible cause of chronic renal failure. In this condition, filtered light chains bind to a common site on Tamm-Horsfall protein (THP), which is produced by cells of the thick ascending limb of the loop of HENLE: Subsequent aggregation of these proteins produces casts that obstruct tubule fluid flow and results in renal failure. In the present study, we used the yeast two-hybrid system to determine the site of interaction of light chains with THP. The third complementarity-determining region (CDR3) of both kappa and lambda light chains interacted with THP. These findings were confirmed in a series of competition studies using a synthetic peptide that corresponded to the CDR3 region and purified THP and light chains. Variations in the CDR3 sequence of the light chain affected binding. Thus, the current studies increase our understanding of the process of cast formation and provide an opportunity to develop strategies that may inhibit this interaction and prevent the clinical manifestations of myeloma kidney.     

Immunogold quantitation of immunoglobulin light chains in renal amyloidosis and kappa light chain nephropathy.

By quantitative immunoelectron microscopy using protein A-gold, the authors compared the content and distribution of immunoglobulin light chain (LC) antigens in glomeruli from 11 cases of renal amyloidosis with that in two cases of kappa LC glomerulopathy and two cases of diabetic glomerulosclerosis. In a supplementary study and using a similar immunogold technique, the authors identified amyloid A in deparaffinized renal tissue from three of the 11 cases of renal amyloidosis. Each patient had similar clinical manifestations (chronic renal failure with proteinuria) and similar glomerular morphology (thickened glomerular basement membranes and nodular expansion of the mesangium). In 12 cases (10 amyloid, 2 kappa LC), immunoelectron microscopy localized LC antigens over the glomerular deposits and allowed indirect tissue quantitation of each LC antigen to the various cellular and interstitial compartments. In 6 of the 11 cases of renal amyloidosis, the amyloid labeled only for lambda, and in one, only for kappa. In one patient with Waldenström''s macroglobulinemia, who had a biclonal gammopathy, both LC were identified in the amyloid. In two cases, both of whom had a history of chronic suppurative lung disease, both LC antigens as well as amyloid A were localized to the amyloid fibrils. In only one case, in which glomerular amyloid labeled for amyloid A, the amyloid did not label for either LC. Whereas lambda LC-derived fibrils often appeared as spicules in the glomerular subepithelial space, other amyloid deposits usually accumulated in the subendothelial zone and did not form spicules. The epimembranous location of spicules suggested that the amyloid precursor protein transformed into amyloid fibrils after filtration into the urinary space. Presence of epimembranous spicules may explain the more severe proteinuric renal failure and the more rapid progression to glomerulosclerosis described in primary amyloidosis.