Reshaping the murine immunoglobulin heavy chain repertoire with bovine DH genes.

Having a limited number of VH segments, cattle rely on uniquely long DH gene segments to generate CDRH3 length variation (3-70 aa) far greater than that in humans or mice. Bovine antibodies with ultralong CDRH3s (>50 aa) possess unusual structures and abilities to bind to special antigens. In this study, we replaced most murine endogenous DH segments with bovine DH genes, generating a mouse line termed B-DH. The use of bovine DH genes significantly increased the length variation of CDRH3 and consequently the Ig heavy chain repertoire in B-DH mice. However, no ultralong CDRH3 was observed in B-DH mice, suggesting that other factors, in addition to long DH genes, are also involved in the formation of ultralong CDRH3. The B-DH mice mounted a normal humoral immune response to various antigens, although the B-cell developmental paradigm was obviously altered compared with wild-type mice. Additionally, B-DH mice are not predisposed to the generation of autoantibodies despite the interspecies DH gene replacement. The B-DH mice reported in this study provide a unique model to answer basic questions regarding the synergistic evolution of DH and VH genes, VDJ recombination and BCR selection in B-cell development.

FcRn is not the receptor mediating the transfer of serum IgG to colostrum in pigs.

In contrast to humans or rabbits, in which maternal IgG is transmitted to offspring prenatally via the placenta or the yolk sac, large domestic animals such as pigs, cows and sheep transmit IgG exclusively through colostrum feeding after delivery. The extremely high IgG content in colostrum is absorbed by newborns via the small intestine. Although it is widely accepted that the neonatal Fc receptor, FcRn, is the receptor mediating IgG transfer across both the placenta and small intestine, it remains unclear whether FcRn also mediates serum IgG transfer across the mammary barrier to colostrum/milk, especially in large domestic animals. In this study, using a FcRn knockout pig model generated with a CRISPR-Cas9-based approach, we clearly demonstrate that FcRn is not responsible for the IgG transfer from serum to colostrum in pigs, although like in other mammals, it is involved in IgG homeostasis and mediates IgG absorption in the small intestine of newborns.

Revisiting the Pig IGHC Gene Locus in Different Breeds Uncovers Nine Distinct IGHG Genes.

IgG subclass diversification is common in placental mammals. It has been well documented in humans and mice that different IgG subclasses, with diversified functions, synergistically regulate humoral immunity. However, our knowledge on the genomic and functional diversification of IgG subclasses in the pig, a mammalian species with high agricultural and biomedical importance, is incomplete. Using bacterial artificial chromosome sequencing and newly assembled genomes generated by the PacBio sequencing approach, we characterized and mapped the IgH C region gene locus in three indigenous Chinese breeds (Erhualian, Xiang, and Luchuan) and compared them to that of Duroc. Our data revealed that IGHG genes in Chinese pigs differ from the Duroc, whereas the IGHM, IGHD, IGHA, and IGHE genes were all single copy and highly conserved in the pig breeds examined. Most striking were differences in numbers of IGHG genes: there are seven genes in Erhualian pigs, six in the Duroc, but only five in Xiang pigs. Phylogenetic analysis suggested that all reported porcine IGHG genes could be classified into nine subclasses: IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c. Using sequence information, we developed a mouse mAb specific for IgG3. This study offers a starting point to investigate the structure-function relationship of IgG subclasses in pigs.

Analysis of the Chinese Alligator TCRα/δ Loci Reveals the Evolutionary Pattern of Atypical TCRδ/TCRµ in Tetrapods.

Atypical TCRδ found in sharks, amphibians, birds, and monotremes and TCRµ found in monotremes and marsupials are TCR chains that use Ig or BCR-like variable domains (VHδ/Vµ) rather than conventional TCR V domains. These unconventional TCR are consistent with a scenario in which TCR and BCR, although having diverged from each other more than 400 million years ago, continue to exchange variable gene segments in generating diversity for Ag recognition. However, the process underlying this exchange and leading to the evolution of these atypical TCR receptor genes remains elusive. In this study, we identified two TCRα/δ gene loci in the Chinese alligator (Alligator sinensis). In total, there were 144 V, 154 Jα, nine Jδ, eight Dδ, two Cα, and five Cδ gene segments in the TCRα/δ loci of the Chinese alligator, representing the most complicated TCRα/δ gene system in both genomic structure and gene content in any tetrapod examined so far. A pool of 32 VHδ genes divided into 18 subfamilies was found to be scattered over the two loci. Phylogenetic analyses revealed that these VHδ genes could be related to bird VHδ genes, VHδ/Vµ genes in platypus or opossum, or alligator VH genes. Based on these findings, a model explaining the evolutionary pattern of atypical TCRδ/TCRµ genes in tetrapods is proposed. This study sheds new light on the evolution of TCR and BCR genes, two of the most essential components of adaptive immunity.

Genetic removal of the CH1 exon leads to the production of hypofunctional heavy chain-only IgG2a in rats.

Despite great values in many applications, heavy chain-only antibodies (HcAbs) are naturally only produced in camelids and sharks, which are not easy to access and handle. Production of the type of antibodies in small laboratory animals would remarkably facilitate their applications. We previously reported a mouse line in which the CH1 exon of mouse γ1 was deleted that could express heavy chain-only IgG1 antibodies. However, these mice showed an extremely weak IgG1 response to specific antigens when immunized, and we could only achieve single VH domains with low affinity to antigens using these mice. One possibility is that the mouse germline VH repertoire was not sufficient to support the expression of functional heavy chain-only antibodies. In this study, we report the generation of a rat line in which the CH1 exon of the γ2a gene was removed and the γ1 and γ2b genes were silenced. Although the genetically modified rats expressed heavy chain-only IgG2a, they also exhibited a very weak IgG2a response to antigen immunization. Panning of a phage library constructed using IgG2a VH segments amplified from immunized rats identified antigen-specific single VH antibodies, which also exhibited much lower affinity than that of commercial mAbs. Together with our previous report, this study suggests that the simple genetic removal of the CH1 exon does not guarantee the successful expression of functional heavy chain-only antibodies.

Generation of porcine monoclonal antibodies based on single cell technologies.

The development of a rapid and efficient system to generate porcine monoclonal antibodies (mAbs) is an important step toward the discovery of critical neutralizing targets for designing rational vaccines against porcine viruses. In this study, we established a platform for producing porcine mAbs based on single cell technologies. First, we singled out an optimal donor from 507 pigs based on serum antibody neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). After identifying the contribution of IgG to the neutralizing activity, single CD45R+IgG+Ag+ B cells were sorted from peripheral blood mononuclear cells (PBMCs). Single B cell RT-PCR was performed using primers designed to cover the germline repertoire of the porcine VH/VL gene segments. Paired VH/VLs were cloned into a eukaryotic expression vector and transfected into 293T cells. We demonstrate that full-length porcine mAbs were produced, and antigen-specific mAbs were obtained after further validation. The approach reported in this study can be applied to generate porcine mAbs against any given antigen and may help with the screening of neutralizing antibodies against porcine pathogens.

A high-throughput screen for genes essential for PRRSV infection using a piggyBac-based system.

In this study, using a dual-functional, piggyBac transposon-based system, we developed a method to systematically decipher the host genes that may be associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection. A Marc145 cell library, which was randomly mutated by transfecting piggyBac plasmids, was challenged with PRRSV. The surviving cell clones were subjected to inverse PCR and high-throughput sequencing to map the integration sites of the transposon. Detailed annotation of the genes flanking the integration sites allowed us to generate a ranked list of candidate genes. Among the predicted genes with a high priority, four genes, CDK17, RNF168, BCL2L15, and TRIM33, were strongly correlated with PRRSV infection in both Marc145 cells and porcine primary alveolar macrophages. This study not only assists in identifying the genes essential for PRRSV infection but also confirms the possibility of using the piggyBac system to study other virus-host genetic interactions in a high-throughput manner.

Corrigendum: Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

[This corrects the article DOI: 10.3389/fimmu.2018.02202.].

Identification of Two Nonrearranging IgSF Genes in Chicken Reveals a Novel Family of Putative Remnants of an Antigen Receptor Precursor.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Identification of a Transcriptionally Forward α Gene and Two υ Genes within the Pigeon (Columba livia) IgH Gene Locus.

Compared with mammals, the bird Ig genetic system relies on gene conversion to create an Ab repertoire, with inversion of the IgA-encoding gene and very few cases of Ig subclass diversification. Although gene conversion has been studied intensively, class-switch recombination, a mechanism by which the IgH C region is exchanged, has rarely been investigated in birds. In this study, based on the published genome of pigeon (Columba livia) and high-throughput transcriptome sequencing of immune-related tissues, we identified a transcriptionally forward α gene and found that the pigeon IgH gene locus is arranged as µ-α-υ1-υ2. In this article, we show that both DNA deletion and inversion may result from IgA and IgY class switching, and similar junction patterns were observed for both types of class-switch recombination. We also identified two subclasses of υ genes in pigeon, which share low sequence identity. Phylogenetic analysis suggests that divergence of the two pigeon υ genes occurred during the early stage of bird evolution. The data obtained in this study provide new insight into class-switch recombination and Ig gene evolution in birds.

Analysis of TCRß and TCRγ genes in Chinese alligator provides insights into the evolution of TCR genes in jawed vertebrates.

All jawed vertebrates have four T cell receptor (TCR) chains that are expressed by thymus-derived lymphocytes and play a major role in animal immune defence. However, few studies have investigated the TCR chains of crocodilians compared with those of birds and mammals, despite their key evolutionary position linking amphibians, reptiles, birds and mammals. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization, evolution and expression of TRB and TRG loci in Alligator sinensis. According to the sequencing data, the Alligator sinensis TRB locus spans approximately 500 Kb of genomic DNA containing two D-J-C clusters and 43 V gene segments and is organized as Vß(39)-pJß1-pCß1-pDß1-Dß2- Jß2(12)-Cß2-Vß(4), whereas the TRG locus spans 115 Kb of DNA genomic sequence consisting of 18 V gene segments, nine J gene segments and one C gene segment and is organized in a classical translocon pattern as Vγ(18)-Jγ(9)-Cγ. Moreover, syntenic analysis of TRB and TRG chain loci suggested a high degree of conserved synteny in the genomic regions across mammals, birds and Alligator sinensis. By analysing the cloned TRB/TRG cDNA, we identified the usage pattern of V families in the expressed TRB and TRG. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed TRB and TRG sequences. Phylogenetic analysis revealed that TRB and TRG loci possess distinct evolutionary patterns. Most Alligator sinensis V subgroups have closely related orthologues in chicken and duck, and a small number of Alligator sinensis V subgroups have orthologues in mammals, which supports the hypothesis that crocodiles are the closest relatives of birds and mammals. Collectively, these data provide insights into TCR gene evolution in vertebrates and improve our understanding of the Alligator sinensis immune system.

Truncation of the Murine Neonatal Fc Receptor Cytoplasmic Tail Does Not Alter IgG Metabolism or Transport In Vivo.

The neonatal Fc receptor (FcRn) is involved in IgG metabolism and transport in placental mammals. However, whether FcRn is responsible for IgG transfer from maternal serum to colostrum/milk is controversial. Interestingly, large domestic animals, such as cows, pigs, sheep, and horses, in which passive IgG transfer is exclusively completed via colostrum/milk, all express an FcRn α-chain that is shorter in the cytoplasmic tail (CYT) than its counterparts in humans and rodents. To address whether the length variation has any functional significance, we performed in vitro experiments using the Transwell system with the MDCK cell line stably transfected with various FcRn constructs; these clearly suggested that truncation of the CYT tail caused a polar change in IgG transfer. However, we observed no evidence supporting functional changes in IgG in vivo using mice in which the FcRn CYT was precisely truncated. These data suggest that the length variation in FcRn is not functionally associated with passive IgG transfer routes in mammals.

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch.

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

A comprehensive analysis of the germline and expressed TCR repertoire in White Peking duck.

Recently, many immune-related genes have been extensively studied in ducks, but relatively little is known about their TCR genes. Here, we determined the germline and expressed repertoire of TCR genes in White Peking duck. The genomic organization of the duck TCRα/δ, TCRγ and unconventional TCRδ2 loci are highly conserved with their counterparts in mammals or chickens. By contrast, the duck TCRß locus is organized in an unusual pattern, (Vß)n-Dß-(Jß)2-Cß1-(Jß)4-Cß2, which differs from the tandem-aligned clusters in mammals or the translocon organization in some teleosts. Excluding the first exon encoding the immunoglobulin domain, the subsequent exons of the two Cß show significant diversity in nucleotide sequence and exon structure. Based on the nucleotide sequence identity, 49 Vα, 30 Vδ, 13 Vß and 15 Vγ unique gene segments are classified into 3 Vα, 5 Vδ, 4 Vß and 6 Vγ subgroups, respectively. Phylogenetic analyses revealed that most duck V subgroups, excluding Vß1, Vγ5 and Vγ6, have closely related orthologues in chicken. The coding joints of all cDNA clones demonstrate conserved mechanisms that are used to increase junctional diversity. Collectively, these data provide insight into the evolution of TCRs in vertebrates and improve our understanding of the avian immune system.

Internal Duplications of DH, JH, and C Region Genes Create an Unusual IgH Gene Locus in Cattle.

It has been suspected for many years that cattle possess two functional IgH gene loci, located on Bos taurus autosome (BTA) 21 and BTA11, respectively. In this study, based on fluorescence in situ hybridization and additional experiments, we showed that all functional bovine IgH genes were located on BTA21, and only a truncated µCH2 exon was present on BTA11. By sequencing of seven bacterial artificial chromosome clones screened from a Hostein cow bacterial artificial chromosome library, we generated a 678-kb continuous genomic sequence covering the bovine IGHV, IGHD, IGHJ, and IGHC genes, which are organized as IGHVn-IGHDn-IGHJn-IGHM1-(IGHDP-IGHV3-IGHDn)3-IGHJn-IGHM2-IGHD-IGHG3-IGHG1-IGHG2-IGHE-IGHA. Although both of two functional IGHM genes, IGHM1 and IGHM2, can be expressed via independent VDJ recombinations, the IGHM2 can also be expressed through class switch recombination. Likely because more IGHD segments can be involved in the expression of IGHM2, the IGHM2 gene was shown to be dominantly expressed in most tissues throughout different developmental stages. Based on the length and identity of the coding sequence, the 23 IGHD segments identified in the locus could be divided into nine subgroups (termed IGHD1 to IGHD9). Except two members of IGHD9 (14 nt in size), all other functional IGHD segments are longer than 30 nt, with the IGHD8 gene (149 bp) to be the longest. These remarkably long germline IGHD segments play a pivotal role in generating the exceptionally great H chain CDR 3 length variability in cattle.

Multiple germline functional VL genes contribute to the IgL repertoire in ducks.

In the immunoglobulin light chain gene loci of nearly all bird species examined to date, there is only a single functional variable gene segment that can recombine with joining gene segments. Thus, Ig light chain diversity relies on gene conversion using pseudogenes as sequence donors to modify the single rearranged variable gene. In the present study, we have sequenced a bacterial artificial chromosome (BAC) clone containing the entire duck Igλ light chain gene locus. Although only a single pair of Jλ and Cλ was found, 88 Vλ gene segments were identified upstream of the Jλ and Cλ segments. Among the identified Vλ gene segments, 79 appear to be pseudogenes, the remaining 9 are structurally intact and all are able to functionally rearrange with the Jλ. Phylogenetic analyses suggest that the 9 functional variable genes may have been derived from a single gene through duplication events. Although these multiple functional variable gene segments can be subject to VJ recombination, both gene conversion and somatic hypermutation are also actively involved in the generation of diversity in duck Igλ light chains. These data provide significant insight into understanding the duck Ig system.

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.

The effect of albumen removal before incubation (embryonic protein under-nutrition) on the post-hatch performance, regulators of protein translation activation and proteolysis in neonatal broilers.

Albumen was removed from broiler eggs before the start of incubation to induce prenatal protein under-nutrition in chicken embryos. With this method, the direct effect of protein deficiency was investigated, differing from mammalian models manipulating the maternal diet where indirect, hormonal effects can interfere. Based on the estimated albumen/egg weight ratio, 10 % of albumen was removed with an 18G needle, after making a hole at the sharp end of the egg with another 18G needle. Eggs were taped thereafter. The sham group underwent the same procedure, except that no albumen was removed. Control eggs did not receive any treatment. The removal of albumen decreased both embryonic and post-hatch body weight up to day 7 compared with the control group. On embryonic day 18, embryos from the albumen-deprived group had higher plasma uric acid levels compared with the sham (P= 0·016) and control (P= 0·009) groups. Moreover, a lower plasma amino acid concentration was observed at hatch compared with the sham (P= 0·038) and control (P= 0·152) groups. These findings indicate an altered protein metabolism. At hatch, a higher mRNA expression of muscle ring finger-1 (MuRF1), a gene related to proteolysis, was observed in albumen-deprived chicks compared with the control and sham chicks, together with an up-regulated expression of atrogin-1 (another atrogene) at this time point in the male protein-deficient chicks. These findings suggest that muscle proteolysis is transiently increased by the removal of albumen before the start of incubation. No evidence was found for altered protein synthesis capacity and translational efficiency in albumen-deprived chicks.

Contribution of blastoderm cells to Japanese quail (Coturnix coturnix japonica)-Peking duck (Anas platyrhynchos) chimeras.

The purpose of this study was to produce quail-duck chimeras by transferring stage X blastoderm cells and to detect the distribution of donor cells in heterogeneous embryos using PCR. Four experimental groups were made by transferring different amounts of quail blastoderm cells into duck recipients. In early embryonic stages, donor cells labeled with PKH26 fluorescent dye were observed in the head, neural tube and gonads by fluorescent microscopy. A total of 194 duck recipient embryos were injected and 93 survived to hatch. The average hatching rate was 48% (93/194); the hatching rate showed a significant difference among all the groups (P < 0.05). Sixteen somatic chimeras were obtained, 10 of which had black feathers derived from the donor quail. The PCR results showed that donor cells were distributed in various tissues and organs of the phenotypic chimeras. This is the first report on producing Japanese quail-Peking duck chimeras by transferring quail blastoderm cells into the subgerminal cavity of the duck. This technique will provide a basis for the investigation of fertilization barriers in interspecies germline chimeras and will aid conservation of endangered wild birds.