The complexity of expressed kappa light chains in egg-laying mammals

Complementary DNAs encoding immunoglobulin light chains were isolated from two monotreme species, Ornithorhynchus anatinus (duckbill platypus) and Tachyglossus aculeatus (echidna). The sequences of both the variable and constant regions of these clones had greater similarity to IGK than to other light chain classes and phylogenetic analyses place them squarely within the mammalian IGK group, establishing them as monotreme IGK homologues. The constant region sequences of all clones were essentially identical within each species and, along with Southern blot results, the data are consistent with a single IGKC in each species. The expressed IGKV repertoires from both platypus and echidna were randomly sampled and there appear to be at least four platypus and at least nine echidna IGKV subgroups. The IGKV subgroups are highly divergent within species, in some cases sharing as little as 57% nucleotide identity. Two of the IGKV subgroups are present in both species, so there is some degree of overlap in the germline repertoires of these two monotremes. Overall the complexity seen in platypus and echidna IGK light chains is comparable with that of other mammals considered to have high levels of germline diversity and is in contrast to what has been found so far for monotreme IGL.Electronic Supplementary Material Supplementary material is available for this article at .     

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes,providing for a powerful therapeutic antibody platform

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.     

On the genomics of immunoglobulins in the gray,short-tailed opossum <Emphasis Type="Italic">Monodelphis domestica</Emphasis>

Annotated maps of the IGH, IGK, and IGL loci in the gray, short-tailed opossum Monodelphis domestica were generated from analyses of the available whole genome sequence for this species. Analyses of their content and organization confirmed a number of previous conclusions based on characterization of complementary DNAs encoding opossum immunoglobulin heavy and light chains and limited genomic analysis, including (a) the predominance of a single immunoglobulin heavy chain variable region (IGHV) subgroup and clan, (b) the presence of a single immunoglobulin (Ig)G subclass, (c) the apparent absence of an IgD, and (d) the general organization and V gene complexity of the IGK and IGL light chain loci. In addition, several unexpected discoveries were made including the presence of a partial V to D, germline-joined IGHV segment, the first germline-joined Ig V gene to be found in a mammal. In addition was the presence of a larger number of IGKV subgroups than had been previously identified. With this report, annotated maps of the major histocompatibility complex, T-cell receptor, and immunoglobulin loci have been completed for M. domestica, the only non-eutherian mammalian species for which this has been accomplished, strengthening the utility of this species as a model organism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accuracy and coverage assessment of Oryctolagus cuniculus (rabbit) genes encoding immunoglobulins in the whole genome sequence assembly (OryCun2.0) and localization of the IGH locus to chromosome 20

We report on the analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51× whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG, and IGHE sequences in another. Some IGKV, IGLV, and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3-Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa, and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.     

New alleles of IGKV genes A2 and A18 suggest significant human IGKV locus polymorphism

The human kappa light chain consists of approximately 35 potentially functionalIGKV genes. However, an estimation of the diversity in theIGKV repertoire of an individual will be affected by the extent of polymorphisms for the differentIGKV genes and their patterns of inheritance. To date, little information is available to indicate the extent of allelic variation of theIGKV genes. We examined the extent of allelism for oneIGKV gene pair, the distal regionA2 gene and its closely related proximal region duplicateA18. We found two new alleles forA2 and one new allele forA18, and sequenced ∼ 1 kilobase flanking each gene. The newA18 allele, unlike the originally described allele, appears to be functional. All these alleles were found at relatively high frequencies in the four ethnic populations studied, with the exception of the defectiveA2b allele which was highly represented only in Navajos. The originally describedA2a allele encodes for the predominant protective antibody againstHaemophilus influenzae. Therefore, the patterns of allelic inheritance described for thisIGKV gene pair indicate that allelism in theIGKV locus is likely to have a significant impact on immune responses.     

Dissecting microregulation of a master regulatory network

Background

All jawed-vertebrates have four T cell receptor (TCR) chains: alpha (TRA), beta (TRB), gamma (TRG) and delta (TRD). Marsupials appear unique by having an additional TCR: mu (TRM). The evolutionary origin of TRM and its relationship to other TCR remain obscure, and is confounded by previous results that support TRM being a hybrid between a TCR and immunoglobulin locus. The availability of the first marsupial genome sequence allows investigation of these evolutionary relationships.

Results

The organization of the conventional TCR loci, encoding the TRA, TRB, TRG and TRD chains, in the opossumMonodelphis domesticaare highly conserved with and of similar complexity to that of eutherians (placental mammals). There is a high degree of conserved synteny in the genomic regions encoding the conventional TCR across mammals and birds. In contrast the chromosomal region containing TRM is not well conserved across mammals. None of the conventional TCR loci contain variable region gene segments with homology to those found in TRM; rather TRM variable genes are most similar to that of immunoglobulin heavy chain genes.

Conclusion

Complete genomic analyses of the opossum TCR loci continue to support an origin of TRM as a hybrid between a TCR and immunoglobulin locus. None of the conventional TCR loci contain evidence that such a recombination event occurred, rather they demonstrate a high degree of stability across distantly related mammals. TRM, therefore, appears to be derived from receptor genes no longer extant in placental mammals. These analyses provide the first genomic scale structural detail of marsupial TCR genes, a lineage of mammals used as models of early development and human disease.     

Comparative genomic analysis of mammalian NKG2D ligand family genes provides insights into their origin and evolution

NKG2D is a major activating receptor of natural killer cells. Its ligands are major histocompatibility complex (MHC) class I-like molecules whose expression is induced by cellular stresses such as infections and tumorigenesis. Humans have two families of NKG2D ligands (NKG2DL): MHC class I-related chains (MIC) encoded in the MHC and UL16-binding proteins (ULBP) encoded outside the MHC. By contrast, mice have only the latter family of ligands; instead, they have non-MHC-encoded MILL molecules that are closely related to MIC, but do not function as NKG2DL. To gain insights into the origin and evolution of MIC, ULBP, and MILL gene families, we conducted comparative genomic analysis of NKG2DL family genes in five mammalian species. In the opossum MHC, we identified a ULBP-like gene adjacent to a previously described MIC-like gene, suggesting that ULBP genes were originally encoded in the MHC. The opossum genome also contained a transcribed MILL-like gene in a region syntenic to the rodent regions encoding MILL molecules. These observations indicate that MIC-, ULBP-, and MILL-like genes emerged before the divergence of placental and marsupial mammals. Comparison of the human, cattle, rat, mouse, and opossum genomes indicates that after emigration from the MHC, ULBP genes underwent extensive duplications in each species. In mice, some of the ULBP genes appear to have been translocated telomerically on the same chromosome, forming a major cluster of existent NKG2DL genes.     

Structure and diversity of the T-cell receptor α chain in the Mexican axolotl

 Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) α chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRα-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRα-V segments were all provided by sequences belonging to the human TCRα-V1 family and the mouse TCRα-V3 and TCRα-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 α chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 α chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Cα domain had the typical structure of mammalian and avian Cα domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. Received: 12 June 1996 / Revised: 11 September 1996     

Unusually similar patterns of antibody V segment diversity in distantly related marsupials

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline V(H) diversity tend to have limited germline V(L) diversity, whereas species with high levels of germline V(H) diversity have more diverse V(L) gene segments. Evidence for a limited pool of V(H) but diverse V(L) gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the V(H) and V(L) of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted V(H) pool but a diverse V(L) pool. The V(L) gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of V(H) and V(L) gene segments observed in other mammals. Rather, marsupial V(H) and V(L) complexity appears to be evolving divergently, retaining diversity in V(L) perhaps to compensate for limited V(H) diversity. There is a high degree of similarity between the V(H) and V(L) in M. domestica and T. vulpecula, with the majority of V(L) families being shared between both species. All marsupial V(H) sequences isolated so far form a common clade of closely related sequences, and in contrast to the V(L) genes, the V(H) likely underwent a major loss of diversity early in marsupial evolution.     

Immunoglobulin heavy chain diversity in Pteropid bats: evidence for a diverse and highly specific antigen binding repertoire

Bats are the natural host reservoir for range of emerging and re-emerging viruses, many of which cause significant morbidity and mortality in other mammals, yet appear to result in no clinical consequences for bats. The ability of bats to coexist with a variety of viruses presents an interesting immunological problem that has not been examined in any detail but which could provide significant insights into the evolution of antiviral mechanisms in mammals. Towards a better understanding of the bat immune system, we analysed the expressed heavy chain variable (VH) regions of antibodies from the black flying fox, Pteropus alecto. The germline repertoire of the closely related Pteropid bat, Pteropus vampyrus, whose genome has been sequenced was also examined for comparative purposes. Representative VH genes were found in all three mammalian VH clans (I, II and III) in both the expressed P. alecto VH repertoire and the germline P. vampyrus VH repertoire. Evidence for the use of multiple heavy chain diversity (DH) and joining (JH) segments for the generation of diverse VDJ rearrangements was also present in the expressed antibody repertoire of P. alecto. The long period of co-evolutionary history of bats with viruses may have resulted in a variety of highly specific VH segments being hardwired into the genomes of bats and may have implications for their ability to successfully cope with a diversity of viral antigens.     

Rapid cloning of any rearranged mouse immunoglobulin variable genes

Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologist. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5 and 3 universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36–60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny.The nucloetide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number U32111     

A VpreB3 homologue in a marsupial, the gray short-tailed opossum, Monodelphis domestica

A VpreB surrogate light (SL) chain was identified for the first time in a marsupial, the opossum Monodelphis domestica. Comparing the opossum VpreB to homologues from eutherian (placental mammals) and avian species supported the marsupial gene being VpreB3. VpreB3 is a protein that is not known to traffic to the cell surface as part of the pre-B cell receptor. Rather, VpreB3 associates with nascent immunoglobulin chains in the endoplasmic reticulum. Homologues of other known SL chains VpreB1, VpreB2, and λ5, which are found in eutherian mammals, were not found in the opossum genome, nor have they been identified in the genomes of nonmammals. VpreB3 likely evolved from earlier gene duplication, independent of that which generated VpreB1 and VpreB2 in eutherians. The apparent absence of VpreB1, VpreB2, and λ5 in marsupials suggests that an extracellular pre-B cell receptor containing SL chains, as it has been defined in humans and mice, may be unique to eutherian mammals. In contrast, the conservation of VpreB3 in marsupials and its presence in nonmammals is consistent with previous hypotheses that it is playing a more primordial role in B cell development.     

Diverse endogenous light chains contribute to basement membrane reactivity in nonautoimmune mice transgenic for an anti-laminin Ig heavy chain

 Basement membrane proteins are targeted in a variety of pathologic autoimmune responses, yet little is known regarding the origins and regulation of this subset of pathogenic lymphocytes. To examine the generation and fate of B cells reactive with a matrix autoantigen, nonautoimmune C57BL/6 mice were rendered transgenic for a nephrotropic lupus anti-laminin immunoglobulin (Ig) H chain, termed LamH-Cμ. We previously reported recovery of two distinct phenotypes among LamH-Cμ-transgenic mice: progeny of founders M6 and M29 contained abundant transgene-expressing B cells but little anti-laminin Ig, whereas spontaneous autoreactivity was readily recovered from the M7 lineage that expressed minimal B-cell mIgM. To explore the spectrum of autoreactivity generated in vivo by different LamH-Cμ-endogenous L-chain combinations, we determined in vitro and in vivo antigen reactivity and L-chain V-region sequences of 17 LamH-Cμ-transgenic anti-laminin Igs. The results reveal a heterogeneous population of anti-laminin Igs with different fine specificities encoded by diverse endogenous L chains, encompassing nine different Vk gene families, 11 Vk genes, and three Jk genes. Many of the L chains are identical to known or putative unmutated germline Vk genes used to encode Igs reactive with self and foreign antigens in nonautoimmune and genetically autoimmune-prone mouse strains. These observations confirm that the LamH-Cμ H chain plays a dominant role in determining anti-laminin reactivity, and indicate that nonautoimmune B6 mice are fully capable of generating a diverse pool of basement-membrane-reactive B cells using unmutated Ig genes. When interpreted in the context of the divergent M6/M29 and M7 transgenic mouse phenotypes, our findings further suggest that these matrix-reactive lymphocytes are not spontaneously activated in vivo under normal circumstances. Received: 1 July 1999 / Revised: 28 July 1999     

Immunoglobulin light chains in medaka (Oryzias latipes)

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.