Immunoglobulin VDJ repertoires reveal hallmarks of germinal centers in unique cell clusters isolated from zebrafish (Danio rerio) lymphoid tissues.

DNA mutagenesis during antibody affinity maturation has potentially oncogenic or autoimmune outcomes if not tightly controlled as it is in mammalian germinal centers. Cold blooded vertebrates lack germinal centers, yet have a functional Ig gene mutator enzyme, Aicda. In fish there are clusters of Aicda+ cells encircled by pigmented 'melano-macrophages' and we test the hypothesis that these clusters are functionally analogous to germinal centers. Sequenced IgH VDJ repertoire libraries from individual isolated clusters showed evidence of B-cell clonal expansion and VDJ somatic hypermutation. Construction of Ig clonal lineage trees revealed that unlike surrounding lymphoid tissue, each cluster is dominated by a few B-cell VDJ clonotypes having hundreds of mutated variants. Recruitment of B-cells to the clusters appears to be ongoing, as there are additional Ig clones having smaller lineages. Finally, we show evidence for positive selection for replacement mutations in regions encoding the antigen contact loops, but not in the framework regions, consistent with functional antibody modification. Melano-macrophages appear to trap the Ag used for post-mutation B-cell selection, performing a role analogous to the follicular dendritic cells of mammalian germinal centers. These findings provide insights into the evolution of the affinity maturation process, the improvement of fish vaccines and possibly also the workings of atypical ectopic germinal centers generated in several human diseases.

The Minor MHC Class I Gene UDA of Ducks Is Regulated by Let-7 MicroRNA.

In many nonmammalian vertebrates, the genomic organization of the MHC class I region leads to biased expression of a single classical MHC class I gene coevolving with TAP transporters, whereas class I genes are poorly expressed. This contrasts to the three codominantly expressed classical MHC class I genes in humans and mice. In a sequenced haplotype from White Pekin duck, Anas platyrhynchos, there is one predominantly expressed MHC class I, UAA, although they have five MHC class I genes in the complex, arranged TAP1-TAP2-UAA-UBA-UCA-UDA-UEA The UAA gene, situated proximal to the TAP2 gene, is expressed at levels 10-fold greater than that of another expressed gene, UDA. Three duck MHC class I genes (UBA, UCA, and UEA) are predicted to be partially or completely inactivated by promoter defects, introduction of in-frame stop codon, or the lack of a polyadenylation signal. In this study, we confirm that UBA, UCA, and UEA are indeed inactivated through genetic defects at the promoter, whereas UAA and UDA have functionally equivalent promoters. To examine promoter accessibility, we performed bisulfite sequencing and show that none of the MHC class I promoters are inactivated by methylation. We determine that UDA is differentially regulated through its 3' untranslated region. Namely, expression of UDA is downregulated by let-7 microRNA, whereas the predominantly expressed MHC class I UAA is not. Regulation of UDA by let-7 microRNA suggests that the lower expression level is maintained for its function in immunity.

Antibody Affinity Maturation in Fishes-Our Current Understanding.

It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes.

Consecutive interactions with HSP90 and eEF1A underlie a functional maturation and storage pathway of AID in the cytoplasm.

Activation-induced deaminase (AID) initiates mutagenic pathways to diversify the antibody genes during immune responses. The access of AID to the nucleus is limited by CRM1-mediated nuclear export and by an uncharacterized mechanism of cytoplasmic retention. Here, we define a conformational motif in AID that dictates its cytoplasmic retention and demonstrate that the translation elongation factor eukaryotic elongation factor 1 α (eEF1A) is necessary for AID cytoplasmic sequestering. The mechanism is independent of protein synthesis but dependent on a tRNA-free form of eEF1A. Inhibiting eEF1A prevents the interaction with AID, which accumulates in the nucleus and increases class switch recombination as well as chromosomal translocation byproducts. Most AID is associated to unspecified cytoplasmic complexes. We find that the interactions of AID with eEF1A and heat-shock protein 90 kD (HSP90) are inversely correlated. Despite both interactions stabilizing AID, the nature of the AID fractions associated with HSP90 or eEF1A are different, defining two complexes that sequentially produce and store functional AID in the cytoplasm. In addition, nuclear export and cytoplasmic retention cooperate to exclude AID from the nucleus but might not be functionally equivalent. Our results elucidate the molecular basis of AID cytoplasmic retention, define its functional relevance and distinguish it from other mechanisms regulating AID.

Isolation and cytochemical characterization of melanomacrophages and melanomacrophage clusters from goldfish (Carassius auratus, L.).

Pigmented or "melano-" macrophages are prominent in lymphoid and non-lymphoid tissues of poikilotherms. Though they have been extensively studied in situ only recently has a means to isolate them from other cell types been established. We provide the first in vitro characterization of isolated melanomacrophage cytochemistry and survival in culture. Unlike non-pigmented tissue macrophages melanomacrophages do not adhere to polystyrene surfaces making them easy to separate from tissue macrophages. In vitro goldfish melanomacrophages are distinguishable from tissue macrophages and neutrophils by being Sudan Black B positive (unlike tissue macrophages) and non-specific esterase positive (unlike neutrophils). Like tissue macrophages they also express acid phosphatase and CSF-1R. As sorted cells melanomacrophages only survive a few days in culture. However in coarsely disaggregated spleen and kidney tissues melanomacrophages survive for at least 3 weeks. Furthermore after 5 days culture disaggregating tissue clumps revealed encapsulated melanomacrophage clusters that remained intact for at least another week. The encapsulated clusters were resilient enough to allow for their isolation for further imaging and isolation of RNA. In some cases the clusters had either melanomacrophages or non-fluorescent cells protruding and in the latter case these could initiate outgrowths onto the plates with subsequent collapse of the cluster. These approaches for the isolation of melanomacrophages and melanomacrophage clusters should allow further study into specific cell and cluster functions.

Transcriptional regulation of teleost Aicda genes. Part 1 - suppressors of promiscuous promoters.

In order to better understand antibody affinity maturation in fishes we sought to identify gene regulatory elements that could drive expression of activated B-cell specific fluorescent reporter transgenes in zebrafish. Specifically the promoter and several non-coding regions of the channel catfish (Ictalurus punctatus) and zebrafish (Danio rerio) were tested for transcriptional activity using a dual luciferase reporter system in transfected fish leukocytes and two mammalian cell lines that constitutively express Aicda (activation-induced cytidine deaminase). The promoters of both fish Aicda genes were as transcriptionally active as an SV40 promoter control in all cell lines tested, regardless of the cells ability to express Aicda. Coupling of a putative intron 1 enhancer or a region 10 kb upstream of the zebrafish promoter effectively silenced transcription from the fish Aicda promoter. Paradoxically these suppressor elements enhanced transcription when they were coupled to the mouse Aicda intron 1 enhancer. The results are considered in context of similar observations for Aicda transcriptional regulation in mice and in light of recent evidence that Aicda is utilized for epigenetic reprogramming of several non-lymphoid cell types.

An evolutionarily conserved program of B-cell development and activation in zebrafish.

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

Activation-induced cytidine deaminase structure and functions: a species comparative view.

In the ten years since the discovery of activation-induced cytidine deaminase (AID) there has been considerable effort to understand the mechanisms behind this enzyme's ability to target and modify immunoglobulin genes leading to somatic hypermutation and class switch recombination. While the majority of research has focused on mouse and human models of AID function, work on other species, from lamprey to rabbit and sheep, has taught us much about the scope of functions of the AID mutator. This review takes a species-comparative approach to what has been learned about the AID mutator enzyme and its role in humoral immunity.

The cellular context of AID expressing cells in fish lymphoid tissues.

It has long been held that the cold-blooded vertebrates lack mammalian-like germinal centers, though they do have affinity maturation and the immunoglobulin mutator activation-induced cytidine deaminase or AID. Using AID as a marker of sites of somatic hypermutation, we have identified discrete cell clusters of up to several thousand cells, in the spleen and kidney of channel catfish (Ictalurus punctatus), which may be primordial germinal centers. In situ hybridization revealed that AID expressing cells are interspersed or surrounded by a population of pigmented CSF1-R expressing cells called melano-macrophages. Significantly, melano-macrophages or associated reticular cells have been previously noted for their ability to retain soluble antigen on or near their surface for several weeks following vaccination. Laser capture microdissection and RT-PCR were used to establish that these cell clusters also contained cells expressing Ig heavy chain transcripts as well as transcripts of TcRbeta and the putative CD4 homologue of fish. These observations, coupled with past work showing that mutations develop in B-cell lineages in fishes, allow us to develop a model for how affinity maturation may have evolved in early gnathostome vertebrates.

Evolution of class switch recombination function in fish activation-induced cytidine deaminase, AID.

Following activation of mammalian B cells, class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig heavy chain (IgH) gene can improve the functions of the expressed antibodies. Activation-induced cytidine deaminase (AID) is the only known B cell-specific protein required for inducing CSR and SHM in mammals. Lower vertebrates have an AID homologue, and there is some evidence of SHM in vivo. However there is no evidence of CSR in the cartilaginous or bony fishes, and this may be due in part to a lack of cis-elements in the IgH gene that are the normal targets of AID-mediated recombination. We have tested whether bony fish (zebrafish and catfish) AID can mediate CSR and SHM in mammalian cells. As expected, ectopic expression of fish AID in mouse fibroblasts resulted in mutations in an introduced SHM reporter gene, indicating that fish AID can mediate SHM. Unexpectedly, expression of fish AID in mouse AID-/- B cells induced surface IgG expression as well as switched transcripts from Ig gene loci, clearly indicating that the fish AID protein can mediate CSR, at least in mouse cells. These results suggest that the AID protein acquired the ability to mediate CSR before the IgH locus evolved the additional exon clusters and switch regions that are the targets of recombination. We discuss how pleiotropic functions of specific domains within the AID protein may have facilitated the early evolution of CSR in lower vertebrates.

Evolution of transcriptional enhancers in the immunoglobulin heavy-chain gene: functional characteristics of the zebrafish Emu3' enhancer.

Past studies of the channel catfish immunoglobulin heavy-chain (IgH) locus indicates that it lacks an Emu enhancer in the J(H)-Cmu1 intron but does have an enhancer, termed Emu3', in the mu-delta intergenic region. The positioning of the catfish enhancer downstream of the mu-chain exons is predicted to be unfavorable for antibody-affinity maturation in catfish, and would also have been an impediment to the evolution of class switch recombination, had it existed in early tetrapods. To determine if this downstream enhancer is a general feature of teleost fish, we have identified the location of the transcriptional enhancer in the zebrafish IgH locus. We find that zebrafish, like catfish, only have an Emu3'-like enhancer that has cross-species activity, but which is B-cell-specific in its activity. A 300-bp region of the zebrafish enhancer shares sequence homology with the core of the catfish Emu3', although there has been loss and gain of specific octamer enhancer motifs. Mutagenesis studies demonstrate that the zebrafish IgH enhancer depends on a pair of E-box motifs that are found in the enhancer core. Similarly spaced E-box motifs appear to exist in the Emu3' enhancer regions of other teleost fish, suggesting this is a common feature among fish IgH enhancers. We discuss how this distal positioning of the enhancer may influence affinity maturation in extant teleosts as well as the evolution of this process in the early tetrapods.

Cloning and expression of the AID gene in the channel catfish.

A full-length activation-induced cytidine deaminase (AID or Aicda) cDNA has been obtained from the channel catfish (Ictalurus punctatus). A single open reading frame predicts a 209 amino acid protein that has 57% identity and 73% similarity with the AID proteins of mouse and human. All residues that have previously been found to be critical for deamination, as well as for somatic hypermutation, are conserved in the catfish AID. These residues are also conserved in AID proteins predicted, from genome database sequences, to be expressed in Fugu and zebrafish. The catfish AID is expressed at low levels in spleen, kidney, intestine and fin margins, but not in muscle, liver or brain. Immunoglobulin heavy chain (IgH) is also expressed in the tissues where AID is expressed. The 'ectopic' expression of AID in non-lymphoid tissue was unexpected and not readily explained. However, the identification of a fish AID gene will allow us to determine the tissue architecture and locations for affinity maturation in fish.

A toll-like receptor (TLR) gene that is up-regulated in activated goldfish macrophages.

An expressed sequence tag screen of a macrophage activation factor and lipopolysaccharide (LPS) stimulated goldfish macrophage subtractive library generated several transcripts of a putative teleost homologue of the toll-like receptor (TLR) family. The full-length TLR cDNA was sequenced and is predicted to encode a type I transmembrane protein with an extracellular domain containing leucine rich repeats and a cytoplasmic tail encoding a toll/interleukin-1 receptor domain. These findings indicate that the gene identified is the first teleost homologue of the TLR family reported. Constitutive expression of TLR was observed in unstimulated macrophages and was also observed in goldfish spleen and kidney but not in heart and liver tissues. A significant up-regulation of the TLR mRNA in cultured macrophages following treatments with each of bacterial LPS, heat-killed Aeromonas salmonicida, and live Mycobacterium chelonei was observed after 3 and 6 h post-stimulation, though with different kinetics from each other. A relative decline in TLR expression was observed after 24 h, but expression levels were still higher than that of unstimulated cells. Thus pathogen-derived factors appear to differentially modulate the expression of TLR in goldfish macrophages, which undoubtedly contributes to the orchestration and/or induction of functional immune responses in fish.