Nancy Wertz

Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis.

Only three Ig isotypes, IgM, IgX, and IgY, were previously known in amphibians. Here, we describe a heavy-chain isotype in Xenopus tropicalis, IgF (encoded by Cφ), with only two constant region domains. IgF is similar to amphibian IgY in sequence, but the gene contains a hinge exon, making it the earliest example, in evolution, of an Ig isotype with a separately encoded genetic hinge. We also characterized a gene for the heavy chain of IgD, located immediately 3′ of Cμ, that shares features with the Cδ gene in fish and mammals. The latter gene contains eight constant-region-encoding exons and, unlike the chimeric splicing of μCH1 onto the IgD heavy chain in teleost fish, it is expressed as a unique IgD heavy chain. The IgH locus of X. tropicalis shows a 5′ VH-DH-JH-Cμ-Cδ-Cχ-Cυ-Cφ 3′ organization, suggesting that the mammalian and amphibian Ig heavy-chain loci share a common ancestor.

The piglet as a model for B cell and immune system development.

Abstract The ability to identify factors responsible for disease in all species depends on the ability to separate those factors which are environmental from those that are intrinsic. This is particularly important for studies on the development of the adaptive immune response of neonates. Studies on laboratory rodents or primates have been ambiguous because neither the effect of environmental nor maternal factors on the newborn can be controlled in mammals that: (i) transmit potential maternal immunoregulatory factors in utero and (ii) are altricial and cannot be reared after birth without their mothers. Employing the newborn piglet model can address each of these concerns. However, it comes at the price of having first to characterize the immune system of swine and its development. This review focuses on the porcine B cell system, especially on the methods used for its characterization in fetal studies and neonatal piglets. Understanding these procedures is important in the interpretation of the data obtained. Studies on neonatal piglets have (a) provided valuable information on the development of the adaptive immune system, (b) lead to important advances in evolutionary biology, (c) aided our understanding of passive immunity and (d) provided opportunities to use swine to address specific issues in veterinary and biomedical research and immunotherapy. This review summarizes the history of the development of the piglet as a model for antibody repertoire development, thus providing a framework to guide future investigators.

Porcine IgG: structure, genetics, and evolution

Eleven genomic porcine Cγ gene sequences are described that represent six putative subclasses that appear to have originated by gene duplication and exon shuffle. The genes previously described as encoding porcine IgG1 and IgG3 were shown to be the IgG1a and IgG1b allelic variants of the IGHG1 gene, IgG2a and IgG2b are allelic variants of the IGHG2 gene, while “new” IgG3 is monomorphic, has an extended hinge, is structurally unique, and appears to encode the most evolutionarily conserved porcine IgG. IgG5b differs most from its putative allele, and its CH1 domain shares sequence homology with the CH1 of IgG3. Four animals were identified that lacked either IgG4 or IgG6. Alternative splice variants were also recovered, some lacking the CH1 domain and potentially encoding heavy chain only antibodies. Potentially, swine can transcribe >20 different Cγ chains. A comparison of mammalian Cγ gene sequences revealed that IgG diversified into subclasses after speciation. Thus, the effector functions for the IgG subclasses of each species should not be extrapolated from “same name subclasses” in other species. Sequence analysis identified motifs likely to interact with Fcγ receptors, FcRn, protein A, protein G, and C1q. These revealed IgG3 to be most likely to activate complement and bind FcγRs. All except IgG5a and IgG6a should bind to FcγRs, while all except IgG6a and the putative IgG5 subclass proteins should bind well to porcine FcRn, protein A, and protein G.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XI. The Relationship of Variable Heavy Chain Gene Usage and the Genomic Organization of the Variable Heavy Chain Locus

In this study, we have mapped the 3′ H chain V region (VH) genes and those in the H chain diversity, H chain joining, and 5′ portion of the H chain constant locus. We show that swine possess only two functional H chain diversity segments and only one functional H chain joining segment. These data help to explain more than a decade of observations on the preimmune repertoire of this species and reveal the vulnerability of swine to natural or designed mutational events. The results are consistent with earlier studies on the region containing Enh, Cμ, and Cδ while revealing that the ancestral IgG3 is the most 5′ Cγ gene. We also observed a recent duplication (∼1.6 million years ago) in the VH locus that contains six of the seven VH genes that comprise 75% of the preimmune repertoire. Because there are no known transfers of immune regulators or Ags that cross the placenta as in mice and humans, fetal VH usage must be intrinsically regulated. Therefore, we quantified VH usage in fetal piglets and demonstrated that usage is independent of the position of VH genes in the genome; the most 3′ functional VH gene (IGHV2) is rarely used, whereas certain upstream genes (IGHV14 and IGHV15) are predominately used early in fetal liver but seldom thereafter. Similar to previous studies, three VH genes account for 40% of the repertoire and six for ∼70%. This limited combinatorial diversity of the porcine VH repertoire further emphasizes the dependence on CDR3 diversity for generating the preimmune Ab repertoire of this species.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XVII. IgG Subclass Transcription Revisited with Emphasis on New IgG3

Fetal piglets offer an in vivo model for determining whether Ag-independent IgG subclass transcription proceeds in a manner that differs from subclass transcription in pigs exposed to environmental Ags and TLR ligands. Our data from ≃12,000 Cγ clones from >60 piglets provide the first report on the relative usage of all known porcine Cγ genes in fetal and young pigs. Studies revealed that among the six Cγ genes, allelic variants of IgG1 comprised 50–80% of the repertoire, and IgG2 alleles comprised <10% in nearly all tissues. However, relative transcription of allelic variants of IgG1 randomly deviate from the 1:1 ratio expected in heterozygotes. Most surprising was the finding that IgG3 accounted for half of all Cγ transcripts in the ileal Peyer’s patches (IPPs) and mesenteric lymph nodes but on average only ≃5% of the clones from the thymus, tonsil, spleen, peripheral blood, and bone marrow of newborns. Lymphoid tissues from late term fetuses revealed a similar expression pattern. Except for IgG3 in the IPPs and mesenteric lymph nodes, no stochastic pattern of Cγ expression during development was seen in animals from mid-gestation through 5 mo. The age and tissue dependence of IgG3 transcription paralleled the developmental persistence of the IPP, and its near disappearance corresponds to the diversification of the preimmune VDJ repertoire in young piglets. We hypothesize that long-hinged porcine IgG3 may be important in preadaptive responses to T cell-independent Ags similar to those described for its murine namesake.

Antibody repertoire development in fetal and neonatal piglets. XIII. Hybrid VH genes and the preimmune repertoire revisited.

The expressed porcine VH genes belong to the VH3 family (clan), four of which, VHA, VHB, VHC, and VHE, alone comprise ∼80% of the preimmune repertoire. However, so-called “hybrid” VH genes that use CDR1 of one VH gene and the CDR2 of another are frequently encountered. We studied >3000 cloned VDJs and found that such hybrids can contribute up to 10% of the preimmune repertoire. Based on the 1) recovery of hybrid VH genes from bacterial artificial chromosome clones, 2) frequency of occurrence of certain hybrids in the preimmune repertoire, and 3) failure to recover equal numbers of reciprocal hybrids, we concluded that some chimeric genes are present in the genome and are not PCR artifacts. Two chimeric germline genes (VHZ and VHY), together with VHF and the four genes mentioned above, constitute the major VH genes and these account for >95% of the preimmune repertoire. Diversification of the preimmune IgG and IgM repertoires after environmental exposure was mainly due to somatic hypermutation of major VH genes with no evidence of gene conversion. Somatic hypermutation was 3- to 10-fold higher in CDRs than in framework regions, most were R mutations and transversions and transitions equally contributed. Data were used to 1) develop an index to quantify the degree of VH repertoire diversification and 2) establish a library of 29 putative porcine VH genes. One-third of these genes are chimeric genes and their sequences suggest that the porcine VH genome developed by duplication and splicing from a small number of prototypic genes.

Antibody repertoire development in fetal and neonatal pigs. VII. Characterization of the preimmune κ light chain repertoire

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vκ genes in the porcine preimmune repertoire belong to three subfamilies of Vκ genes that share 87% sequence similarity with human IGKV2. This porcine Vκ family also shares sequence similarity with some, but not all, Vκ genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess ∼60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jκ segments, complete with recombination signal sequences and separated by ∼300 nt, were identified ∼3 kb upstream of a single Cκ. Surprisingly, Jκ2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use ∼10 IGVK2 genes from three of six subfamilies and preferentially one Jκ segment to generate their preimmune κ repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.

The two suborders of chiropterans have the canonical heavy-chain immunoglobulin (Ig) gene repertoire of eutherian mammals

Bats comprise 20% of all mammals, yet little is known about their immune system and virtually nothing about their immunoglobulin genes. We show that four different bat species transcribe genes encoding IgM, IgE, IgA and IgG subclasses, the latter which have diversified after speciation; the canonical pattern for eutherian mammals. IgD transcripts were only recovered from insectivorous bats and were comprised of CH1, CH3 and two hinge exons; the second hinge exon was fused to CH3. IgA in all species resembles human IgA2 with the putative cysteine forming the bridge to the light chain found at position 77. Sequence comparisons yielded no evidence for a diphyletic origin of the suborders. Bats show no close similarity to another mammalian order; the strongest association was with carnivores. Data reveal that CH diversity and VDJ and CDR3 organization are similar to other eutherian mammals, although the expressed VH3 family repertoire was unusually diverse.

Antibody Repertoire Development in Fetal and Neonatal Piglets. XX. B Cell Lymphogenesis Is Absent in the Ileal Peyer’s Patches, Their Repertoire Development Is Antigen Dependent, and They Are Not Required for B Cell Maintenance

The continuous ileal Peyer’s patches (IPP) of sheep are regarded as a type of mammalian bursal equivalent where B cells diversify their repertoire in an Ag-independent fashion. Anatomically and developmentally similar IPP occur in swine. Resection of ∼90% of the IPP in piglets at birth did not alter Ig levels in serum and secretions or retard diversification of the Ab repertoire when animals were maintained in isolators and colonized with a defined gut flora. Resection or sham surgery elevated IgG and IgA in serum and in lavage fluid from the gut, lung, and in saliva. No changes in the frequency of IgG-, IgA-, and IgM-containing cells in the spleen and peripheral lymph node were observed. Using an index that quantifies diversification of the VDJ repertoire, no differences were seen in three secondary lymphoid tissues between piglets lacking IPP and colonized controls, whereas both groups displayed >10-fold greater diversification than did late-term fetal piglets or piglets maintained germ-free. Somatic hypermutation was very low in fetal IPP and the IPP of germ-free piglets but increased 3- to 5-fold after colonization. D–J signal joint circles were not recovered in IPP, and V–DJ signal joint circles were 5-fold lower than in bone marrow and similar to those in thymus and spleen. We conclude that the porcine IPP are not a site of B cell lymphogenesis, do not undergo Ag-independent repertoire diversification, and are not primary lymphoid tissue since they are not required for maintenance of Ig levels in serum and secretions.

Porcine Reproductive and Respiratory Syndrome Virus Subverts Repertoire Development by Proliferation of Germline-Encoded B Cells of All Isotypes Bearing Hydrophobic Heavy Chain CDR3

Isolator piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), which is related to the lactate dehydrogenase-elevating virus of mice, develop severe hypergammaglobulinemia, lymph node adenopathy, and autoimmune disease. Many of the polyclonally activated B cell clones bear hydrophobic H chain CDR3s (HCDR3s) and are disseminated to most lymphoid tissues. We show in this study that B cells with identical hydrophobic HCDR3s are expressed with all major isotypes in PRRSV-infected piglets (PIPs), explaining why PRRSV-induced hypergammaglobulinemia is seen in all major isotypes. Up to one-third of randomly selected VDJ clones from the respiratory tract of PIPs have hydrophobic HCDR3s exclusively bearing VDJ rearrangements with CDR1, CDR2, and nearly intact DH segments in germline configuration. These HCDR3s are long and DHA and DHB are exclusively used in reading frame 3. A minimal tripeptide motif containing three hydrophobic amino acids (Leu, Val, and Ile) or any two plus alanine is common to this hydrophobic patch. We propose that PRRSV infection causes generalized Ag-independent B cell activation and hypergammaglobulinemia with biased expansion of a subpopulation of the preimmune repertoire with hydrophobic binding sites that normally disappears during Ag-driven repertoire diversification. Elevated Ig levels in PIP cannot be explained as antiviral Abs; some Igs can account for autoantibodies to dsDNA and Golgi, whereas those with hydrophobic binding sites may account for the Ig aggregates seen in PIPs and lactate dehydrogenase-elevating virus-infected mice. This diversion from normal repertoire development may explain the delayed immune response to PRRSV.