IgL Isotypes Research Articles

Diversity of Immunoglobulin Light Chain Genes in Non-Teleost Ray-Finned Fish Uncovers IgL Subdivision into Five Ancient Isotypes.

The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.

Immunoglobulin light chain (IGL) genes in torafugu: Genomic organization and identification of a third teleost IGL isotype

Here, we report a genome-wide survey of immunoglobulin light chain (IGL) genes of torafugu (Takifugu rubripes) revealing multi-clusters spanning three separate chromosomes (v5 assembly) and 45 scaffolds (v4 assembly). Conventional sequence similarity searches and motif scanning approaches based on recombination signal sequence (RSS) motifs were used. We found that three IGL isotypes (L1, L2, and L3) exist in torafugu and that several loci for each isotype are present. The transcriptional orientations of the variable IGL (VL) segments were found to be either the same (in the L2 isotype) or opposite (in the L1 and L3 isotypes) to the IGL joining (JL) and constant (CL) segments, suggesting they can undergo rearrangement by deletion or inversion when expressed. Alignments of expressed sequence tags (ESTs) to corresponding germline gene segments revealed expression of the three IGL isotypes in torafugu. Taken together, our findings provide a genomic framework for torafugu IGL genes and show that the IG diversity of this species could be attributed to at least three distinct chromosomal regions.

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins

Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied.

A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

A new hypersensitive site, HS10, and the enhancers, E3' and Ed, differentially regulate Igκ gene expression.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E3' interact with a novel DNA sequence near the 3' end of the Igκ locus, specifically in B cells. In the present investigation, we examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E3' in transient transfection assays. Although HS10(-/-) mice exhibited normal patterns of B cell development, they were tested further along with E3'(-/-) and Ed(-/-) mice for their Igκ expression levels in plasma cells, as well as for both allelic and isotype exclusion in splenic B cells. HS10(-/-) and Ed(-/-), but not E3'(-/-), mice exhibited 2.5-fold lower levels of Igκ expression in antigenically challenged plasma cells. E3'(-/-) mice, but not HS10(-/-) mice, exhibited impaired IgL isotype and allelic exclusion in splenic B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS10(-/-) mice no longer exhibited regional chromosome interactions with E3', and they exhibited modestly reduced somatic hypermutation in the Jκ-Cκ intronic region in germinal center B cells from Peyer's patches. We conclude that the HS10, E3', and Ed differentially regulate Igκ gene dynamics.

A Multifunctional Element in the Mouse Igκ Locus That Specifies Repertoire and Ig Loci Subnuclear Location

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.

Three isotypes of immunoglobulin light chains in large yellow croaker, Pseudosciaena crocea: Molecular cloning, characterization, and expression analysis

Both cDNA library mining and transcriptome analysis were used to obtain 21 immunoglobulin light chain (IgL) sequences for the large yellow croaker, Pseudosciaena crocea. Full-length cDNA sequences are available for 10 of these, and they were identified as belonging to the three IgL isotypes of LycIgL1, LycIgL2, and LycIgL3. The LycIgL1 isotype is most abundant in the large yellow croaker IgL repertoire, as in the other teleosts. Tissue expression profile analysis revealed that the three LycIgL isotypes were constitutively expressed at different abundances in the kidney, spleen, liver, gill, heart, intestine, and muscle, although the heart did not express LycIgL3. Real-time polymerase chain reaction revealed that expression of the three LycIgL isotypes in the kidney and spleen tissues was up-regulated during 72 h of inductions with poly(I:C) or bacterial vaccine at different intensities and in different manners. The LycIgL1 isotype responded to stimulations most intensely in the spleen, while the LycIgL3 isotype responded most quickly in the kidney. Compared to the LycIgL1 and LycIgL3 isotypes, the LycIgL2 isotype responded more slowly and weakly in both tissues. These results indicate different isotypes of LycIgL respond to immune stimuli in the spleen and kidney in an isotypic-specific manner.

Identification of Two IgD+ B Cell Populations in Channel Catfish,Ictalurus punctatus

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

Peripheral B Cell Tolerance and Function in Transgenic Mice Expressing an IgD Superantigen

Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM(+) plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.

IgD-bearing B cell populations in the channel catfish, Ictalurus punctatus (43.17)

Abstract Channel catfish, Ictalurus punctatus, express two different IgD+ B cell populations. Catfish IgM+/IgD+ B cells are small and agranular, while IgM-/IgD+ B cells are larger and exhibit plasmablast morphology. The use of cell sorting, flow cytometry and RT-PCR demonstrated that IgD+ B cell expression varies between individual catfish. For example, some catfish have little to no IgD+ B cells in their peripheral blood leukocytes (PBL), while in others the IgM-/IgD+ B cell population can represent as much as 72%. This contrasts to the situation in mammals where IgM-/IgD+ B cells are rare. Also, transfection studies show IgD functions as a typical BCR since Igδ chains associate with accessory molecules CD79a and CD79b. Furthermore, IgM-/IgD+ B cells preferentially express IgL σ chains, while IgM+/IgD+ B cells can express any of the four catfish IgL isotypes. As assessed by 5'-RACE, all membrane IgD transcripts from sorted IgM-/IgD+ B cells contained viable VH rearrangements with no bias in family member usage. However, all secreted IgD transcripts were V-less and began with a leader sequence spliced to the Igδ CH1 domain and it is likely that these transcripts encode for the secreted form of IgD previously found in catfish serum. Importantly, the Igδ leader mediated IgD protein secretion when transfected into catfish B cells. This characterization of distinct IgM+/IgD+ and IgM-/IgD+ B cells in teleosts provides an important first step in understanding the evolution of IgD function.

Analysis of the Immunoglobulin Light Chain Genes in Zebra Finch: Evolutionary Implications

All jawed vertebrates produce immunoglobulins (IGs) as a defense mechanism against pathogens. Typically, IGs are composed of two identical heavy chains (IGH) and two identical light chains (IGL). Most tetrapod species encode more than one isotype of light chains. Chicken is the only representative of birds for which genomic information is currently available and is an exception to the above rule because it encodes only a single IGL isotype (i.e., lambda). Here, we show that the genome of zebra finch, another bird species, encodes a single IGL isotype, that is, lambda, like the chicken. These results strongly suggest that the second isotype (i.e., kappa) present in both reptiles and mammals was lost in a very early stage of bird evolution. Furthermore, we show that both chicken and zebra finch contain a single set of functional variable, joining, and constant region genes and multiple variable region pseudogenes. The latter finding suggests that this type of genomic organization was already present in the common ancestor of these bird species and remained unchanged over a long evolutionary time. This conservation is in contrast with the high levels of variation observed in the mammalian IGL loci. The presence of a single functional variable region gene followed by multiple variable pseudogenes in zebra finch suggest that this species may be generating antibody diversity by a gene conversion-like mechanism like the chicken.

IgD+ cell populations in channel catfish, Ictalurus punctatus (81.8)

Abstract Only two immunoglobulin (Ig) molecules, IgM and IgD have been identified in the channel catfish. While IgM has been extensively studied and in many aspects resembles mammalian IgM, catfish IgD is quite different. It is a chimeric molecule consisting of a rearranged VDJ region spliced to the Cμ1 domain, followed by seven Cδ domains and either a transmembrane or secreted tail. Through the use of anti-IgM and anti-IgD monoclonal antibodies three different B cell populations were identified that differentially express IgM and IgD; IgM+/IgD-, IgM+/IgD+ and IgM-/IgD+. PCR and RACE protocols amplified appropriate Ig rearranged message from these sorted populations indicating that they contained functional B cells. Furthermore, co-immunoprecipitation, Western blot analyses, and RT-PCR showed that IgD in the IgM-/IgD+ sorted cell population preferentially associates with the catfish Ig light (L) chain isotype sigma (IgL σ?. In contrast, the IgM+/IgD-, IgM+/IgD+ B cell populations express all four of the catfish IgL isotypes, IgLσ, IgLλ??and the IgL¿? which includes F and G IgL chains. In addition a third IgD+ cell population was identified consisting of IgM-/IgD+ granular-like cells of unknown origin, which do not express IgD message, and thus appear to be armed with exogenous IgD via a putative IgD binding receptor. Supported by NIH grant R01 AI19530 and UMMC IRSP 59908.

Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers

The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups (kappa, lambda, and sigma isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: kappa, lambda, and sigma. Of these, two (kappa and lambda) are also found in reptiles and some mammals. The lambda isotype is found in all tetrapods tested to date, whereas the kappa isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed.

Characterisation of a fourth immunoglobulin light chain isotype in the common carp

Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp ( Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription–polymerase chain reaction (RT–PCR) method including 5′- and 3′-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5′- and 3′-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences.

The generation of monoclonal antibodies by genetic immunisation: antibodies against trout TCRα and IgL isotypes

Production of monoclonal antibodies (mAb) using genetic immunisation is a potential alternative when purified antigen is difficult to obtain, or when induction of an antibody response to a limited part of an antigen is wanted. DNA immunisation using only the constant parts of trout immunoglobulin light chains coding regions was attempted here, because mAbs against the variable (V) part of immunoglobulins do not recognise the whole repertoire of the isotype. After positive results with the light chains and establishing of a proper screening system (ELISA), generation of monoclonal antibodies against trout T cell receptor was also performed.The DNA constructs were used both for immunisation of mice and for protein expression in EBNA 293 cells. Mice were immunised with the constructs 3–5 times by intramuscular injection, with or without adjuvants during 1–3 months. Spleens of positive mice were fused with myeloma Sp2/0 cells and clones were screened by ELISA using double-screening (recombinant protein/trout cells).MAbs 46E5 (anti-IgL2C), 4F2 (anti-TCRα), 18B3 (anti-TCRαC) and 4E5 (anti-TCRαC) show specific binding to its antigen in Western blot, mAb 18B3 and 7H7(anti-TCRα) shows specific staining of trout splenocytes in flow cytometry and mAb 7H7 induces proliferation of trout peripheral blood leucocytes (PBL) in vitro.

Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes

By screening a cDNA library and analysis of DNA produced by a combined 3′RACE/5′-anchored PCR, we have isolated three isotypes of IgL in the Atlantic salmon. Two of the isotypes were homologous to rainbow trout IgL1 and L2 sequences, while the third represents a previously uncharacterised salmonid IgL. The novel type 3 CL region is homologous to spotted wolffish c1 and yellowtail sequences, while the VL region is more similar to channel catfish F class than to any other fish VL sequences. Southern analysis indicates that the gene segments of all three isotypes are organised in multiple clusters. In addition, the VL gene segments of type 3 are arranged in opposite orientation relative to the JL and CL segments, while gene segments in type 2 clusters are all in the same orientation. Although transcripts of type 1 and 3 were readily found in the spleen and head kidney, only minute amounts of type 2 transcripts were seen. The majority of type 3 messages were truncated, suggesting that spliced and full-length transcripts of this isotype probably are present at a low level compared to type 1 transcripts. The uniqueness of the type 3 VLJL sequences suggests that this isotype offers additional diversity to the antigen-binding site of Atlantic salmon immunoglobulins.

A second immunoglobulin light chain isotype in the rainbow trout.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity.

The amphibian Xenopus laevis expresses several types of immunoglobulin light chain (IgL). cDNA clones for two IgL isotypes, C sigma 1 and C sigma 2, were analysed. C sigma is expressed in spleen and mitogen-stimulated B cells, like another Xenopus IgL type, termed C rho. C sigma shares less than 33% residues with C rho or with CL regions of shark, chicken and mammals. This suggests that C sigma diverged from a common ancestor of CL regions before or at the emergence of amphibians. Two families of VL elements, V sigma 1 and V sigma 2 are associated with C sigma (but not with C rho). They rearrange to their own set of JL elements, J sigma 1 and J sigma 2, which are poorly related to other J elements of the Ig gene family. The Xenopus genome contains a few V sigma 2 and multiple V sigma 1 elements (comparable with mammalian V kappa), but only two C sigma genes. Thus, the organization and expression of Xenopus IgL loci are apparently similar to mammalian IgL loci but different from shark and chicken IgL loci. Only a few VL elements are expressed, since cDNA clones show extensive sharing of CDR1 and CDR2 sequences; some clones differ only in CDR3. Rearranging VL and JL elements increases CDR3 diversity in both V sigma families, but abortive rearrangements are frequent in V sigma 1 regions. The very poor heterogeneity of expressed VL elements therefore appears to limit antibody diversity in Xenopus.