Histology and immunology of the placenta in the Atlantic sharpnose shark, Rhizoprionodon terraenovae.

The Atlantic sharpnose shark, Rhizoprionodon terraenovae, is viviparous species that forms a yolk sac placenta to facilitate exchange between mother and embryo. However, very little is known about the immunological aspects of this organ in sharks. To begin to understand this, we used histology, histochemistry and immunohistochemistry to investigate the sharpnose shark placenta throughout gestation. We report the presence of lymphoid aggregates in the maternal portion of the placenta during all stages of gestation, and their increasing size and vascularity near term. Immunoglobulin is found in the maternal tissues of the placenta, but its presence in embryonic tissue and potential transfer from maternal circulation remains unclear. Placental cells resembling mammalian uterine NK cells and melanomacrophages of lower vertebrates are described for the first time. Similarities with mammalian placentae point to shared aspects in the co-evolution of reproductive and immune systems, even between two phylogenetically diverse groups in which placentation arose by convergent evolution.

Antibody repertoire development in cartilaginous fish.

There are 3 H chain and 3 L chain isotypes in the cartilaginous fish, all encoded by genes in the so-called cluster (VDDJ, VJ) organization. The H chain isotypes IgM and IgNAR, are readily detected at the protein level in most species. The third is readily identified at the protein level in skates (IgR) but only via immunoprecipitation or at the transcript level in sharks (IgW). High levels of diversity in CDR3 and up to 200 germline genes have been detected for IgM depending upon the species examined. IgNAR displays very high levels of CDR3 diversity but almost none in the germline. At least IgNAR and L chain genes have been shown to hypermutate to very high levels, apparently in response to antigen. The mutation footprints are similar to those in mammals except that the shark genes uniquely mutate nucleotide residues in tandem. A conspicuous feature of cartilaginous fish Ig genes is the presence of germline-joined genes, which are a result of RAG activity in germ cells. Such genes are expressed early in ontogeny and then extinguished or expressed at lower levels. 19S IgM and IgW expression precede that of 7S IgM and IgNAR during ontogeny. The 'switch' from 19S to 7S IgM, the regulation of expression of the Ig clusters, and the microenvironments for mutation/selection of cartilaginous fish B cells are all areas of ongoing research.

The development of primary and secondary lymphoid tissues in the nurse shark Ginglymostoma cirratum: B-cell zones precede dendritic cell immigration and T-cell zone formation during ontogeny of the spleen.

Secondary lymphoid tissue and immunoglobulin (Ig) production in mammals is not fully developed at birth, requiring time postnatally to attain all features required for adaptive immune responses. The immune system of newborn sharks - the oldest vertebrate group having adaptive immunity - also displays immature characteristics such as low serum IgM concentration and high levels of IgM1gj, an innate-like Ig. Primary and secondary lymphoid tissues in sharks and other cartilaginous fish were identified previously, but their cellular organization was not examined in detail. In this study of nurse shark lymphoid tissue, we demonstrate that the adult spleen contains well-defined, highly vascularized white pulp (WP) areas, composed of a central T-cell zone containing a major histocompatibility complex (MHC) class II+ dendritic cell (DC) network and a small number of Ig+ secretory cells, surrounded by smaller zones of surface Ig+ (sIg+) B cells. In neonates, splenic WPs are exclusively B-cell zones containing sIgM+-MHC class IIlow B cells; thus compartmentalized areas with T cells and DCs, as well as surface Ig novel antigen receptor (sIgNAR)-expressing B cells are absent at birth. Not until the pups are 5 months old do these WP areas become adult-like; concomitantly, sIgNAR+ B cells are readily detectable, indicating that this Ig class requires a 'mature immune-responsive environment'. The epigonal organ is the major site of neonatal B lymphopoiesis, based on the presence of developing B cells and recombination-activating gene 1 (RAG1)/terminal deoxynucleotidyl transferase (TdT) expression, indicative of antigen receptor rearrangement; such expression persists into adult life, whereas the spleen has negligible lymphopoietic activity. In adults but not neonates, many secretory B cells reside in the epigonal organ, suggesting, like in mammals, that B cells home to this primary lymphoid tissue after activation in other areas of the body.

Comparative genomics of the MHC: glimpses into the evolution of the adaptive immune system.

MHC gene organization (size, complexity, gene order) differs markedly among different species, and yet all nonmammalian vertebrates examined to date have a true "class I region" with tight linkage of genes encoding the class I presenting and processing molecules. Three paralogous regions of the human genome contain sets of linked genes homologous to various loci in the MHC class I, class II, and/or class III regions, providing insight into the organization of the "proto MHC" before the emergence of the adaptive immune system in the jawed vertebrates.

Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase zeta.

To examine a role of DNA polymerase zeta in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the V(H) genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase zeta, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.

The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation.

Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol zeta, which effectively extends DNA past mismatched bases (mispair extender), and pol eta, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4+ T cells, these lymphocytes upregulated pol zeta, downregulated pol eta, and mutated the Ig and bcl-6 genes. Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol zeta plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol eta.

Evolution and the molecular basis of somatic hypermutation of antigen receptor genes.

Somatic hypermutation of immunoglobulin genes occurs in many vertebrates including sharks, frogs, camels, humans and mice. Similarities among species reveal a common mechanism and these include the AGC/T sequence hot spot, preponderance of base substitutions, a bias towards transitions and strand bias. There are some differences among species, however, that may unveil layers of the mechanism. These include a G:C bias in frog and shark IgM but not in nurse shark antigen receptor (NAR), a high frequency of doublets in NAR hypermutation, and the co-occurrence of somatic hypermutation with gene conversion in some species. Here we argue that some of the similarities and differences among species are best explained by error-prone DNA synthesis by the translesion synthesis DNA polymerase zeta (Pol zeta) and, as suggested by others, induction of DNA synthesis by DNA breaks in antigen receptor variable genes. Finally, targeting of the variable genes is probably obtained via transcription-related elements, and it is the targeting phase of somatic hypermutation that is the most likely to reveal molecules unique to adaptive immunity.

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Rearrangement of immunoglobulin genes in shark germ cells.

The variable (V), (diversity [D]), and joining (J) region recombinases (recombination activating genes [RAGs]) can perform like transposases and are thought to have initiated development of the adaptive immune system in early vertebrates by splitting archaic V genes with transposable elements. In cartilaginous fishes, the immunoglobulin (Ig) light chain genes are organized as multiple VJ-constant (C) clusters; some loci are capable of rearrangement while others contain fused VJ. The latter may be key to understanding the evolutionary role of RAG. Are they relics of the archaic genes, or are they results of rearrangement in germ cells? Our data suggest that some fused VJ genes are not only recently rearranged, but also resulted from RAG-like activity involving hairpin intermediates. Expression studies show that these, like some other germline-joined Ig sequences, are expressed at significant levels only early in ontogeny. We suggest that a rejoined Ig gene may not merely be a sequence restricting antibody diversity, but is potentially a novel receptor no longer tied to somatic RAG expression and rearrangement. From the combined data, we arrived at the unexpected conclusion that, in some vertebrates, RAG is still an active force in changing the genome.

Primitive synteny of vertebrate major histocompatibility complex class I and class II genes.

Major histocompatibility complex (MHC) class I and class II molecules bind to and display peptidic antigens acquired from pathogens that are recognized by lymphocytes coordinating and executing adaptive immune responses. The two classes of MHC proteins have nearly identical tertiary structures and were derived from a common ancestor that probably existed not long before the emergence of the cartilaginous fish. Class I and class II genes are genetically linked in tetrapods but are not syntenic in teleost fish, a phylogenetic taxon derived from the oldest vertebrate ancestor examined to date. Cartilaginous fish (sharks, skates, and rays) are in the oldest taxon of extant jawed vertebrates; we have carried out segregation analyses in two families of nurse sharks and one family of the banded houndshark that revealed a close linkage of class IIalpha and beta genes both with each other and with the classical class I (class Ia) gene. These results strongly suggest that the primordial duplication giving rise to classical class I and class II occurred in cis, and the close linkage between these two classes of genes has been maintained for at least 460 million years in representatives of most vertebrate taxa.

Trans-species polymorphism of the major histocompatibility complex-encoded proteasome subunit LMP7 in an amphibian genus, Xenopus.

LMP7 (PSMB8) is a major histocompatibility complex (MHC)-encoded catalytic subunit of 20S immunoproteasome, which is responsible for the production of antigenic peptide to be presented by the MHC class I molecules. Two highly diverged allelic lineages of LMP7, termed LMP7A and LMP7B, have been identified previously in an amphibian, Xenopus laevis. Fourteen Xenopus species were analyzed by genomic Southern hybridization using LMP7A- and LMP7B-specific probes. Ten had both LMP7A and LMP7B, and the other 4 had only LMP7A. Identification of LMP7A and LMP7B was confirmed by reverse transcription-polymerase chain reaction/sequencing analysis of LMP7 mRNA including eight diagnostic amino acid residues that discriminate the two allelic lineages. These data suggest that these two allelic lineages were established more than 80 million years ago, and were transmitted from species to species. Trans-species evolution has so far been reported for MHC class I and II molecules in mammals and teleost fish, and is believed to be a basis for the extraordinary polymorphism of these molecules. A similar mode of evolution of the LMP7 alleles in Xenopus provides a possible explanation for the linkage of the LMP7 gene with the MHC in all vertebrates analyzed to date.

Two ancient allelic lineages at the single classical class I locus in the Xenopus MHC.

Unlike all other vertebrates examined to date, there is only one detectable class I locus in the Xenopus MHC. On the bases of a nearly ubiquitous and high tissue expression, extensive polymorphism, and MHC linkage, this gene is of the classical or class Ia type. Sequencing analysis of class Ia cDNAs encoded by eight defined MHC haplotypes reveals two very old allelic lineages that perhaps emerged when humans and mice diverged from a common ancestor up to 100 million years ago. The unprecedented age of these lineages suggests that different class Ia genes from ancestors of the laboratory model Xenopus laevis are now expressed as alleles in this species. The lineages are best defined by their cytoplasmic and alpha2 peptide-binding domains, and there are highly diverse alleles (defined by the alpha1 peptide-binding domain) in each lineage. Surprisingly, the alpha3 domains are homogenized in both lineages, suggesting that interallelic gene conversion/recombination maintains the high sequence similarity.

Insight into the primordial MHC from studies in ectothermic vertebrates.

MHC classical class I and class II genes have been identified in representative species from all major jawed vertebrate taxa, the oldest group being the cartilaginous fish, whereas no class I/II genes of any type have been detected in animals from older taxa. Among ectothermic vertebrate classes, studies of MHC architecture have been done in cartilaginous fish (sharks), bony fish (several teleost species), and amphibians (the frog Xenopus). The Xenopus MHC contains class I, class II, and class III genes, demonstrating that all of these genes were linked in the ancestor of the tetrapods, but the gene order is not the same as that in mouse/man. Studies of polyploid Xenopus suggest that MHC genes can be differentially silenced when multiple copies are present; i.e. MHC 'subregions' can be silenced. Surprisingly, in all teleosts examined to date class I and class II genes are not linked. Likewise, class III genes like the complement genes factor B (Bf) and C4 are scattered throughout the genome of teleosts. However, the presumed classical class I genes are closely linked to the 'immune' proteasome genes, LMP2 and LMP7, and to the peptide-transporter genes (TAP), implying that a true 'class I region' exists in this group. A similar type of linkage group is found in chickens and perhaps Xenopus, and thus it may reveal the ancestral organization of class I-associated genes. In cartilaginous fish, classical and non-classical class I genes have been isolated from three shark species, and class II A and B chain genes from nurse sharks. Studies of MHC linkage in sharks are being carried out to provide further understanding of the putative primordial organization of MHC Segregation studies in one shark family point to linkage of classical class I and class II genes, suggesting that the non-linkage of these genes in teleosts is a derived characteristic.

Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.

The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations. Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG. Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity. As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot. The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes. In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase zeta (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions. Therefore, we propose a model of somatic hypermutation where DNA polymerase zeta is recruited to the Ig locus. An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair.

Identification and genetic mapping of Xenopus TAP2 genes.

The amphibian Xenopus laevis is one non-mammalian vertebrate in which the major histocompatibility complex (MHC) has been analyzed extensively. Class IIbeta, class Ia, LMP2, LMP7, HSP70, C4, Factor B, and Ring3 genes have been identified and mapped to the MHC. Here, we report the isolation of a transporter associated with antigen processing (TAP) gene, TAP2, and demonstrate its linkage to the MHC. While the ATP-binding region of Xenopus TAP2 is highly conserved in evolution, amino acid identity to other vertebrate TAP proteins was not detected in the N-terminal region. Segregation analysis of 34 individuals from two families showed exact restriction fragment length polymorphism matching between the MHC class Ia gene and the one TAP2 gene demonstrating linkage conservation since the mammalian/amphibian divergence approximately 350 million years ago. In addition, one non-MHC-linked TAP2-hybridizing fragment was detected in approximately half of the individuals tested. Interestingly, TAP2 allelic lineages appear to match those of LMP7 and classical class I, which previously were categorized into two highly divergent groups that emerged at least 60 million years ago. Similar to LMP7 and class Ia,TAP2 is expressed ubiquitously with highest levels in intestine and spleen.

Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers.

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.

Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins.

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (VH) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.