John D. Hansen

Lymphocyte development in fish and amphibians

Summary: Recently, molecular markers such as recombination activating genes (RAG), terminal deoxynucleotidyl transferase (TdT), stem cell leukemia hematopoletic transcription factor (SCL), Ikaros and gata‐binding protein (Gata) ‐family members have been isolated and characterized from key lower vertebrates, adding to our growing knowledge of lymphopolesis in ectotherms. In all gnathostomes there appear to be two main embryonic locations derived from the early mesoderm, both intra‐ and extraembryonic, which contribute to primitive and dermitive hematopolesis based upon their differential expression of SCL, Gaia‐1, Gata‐2 and tnyeloblasto‐sis oncogene (c‐myb). In teleosts. a unique intraembryonic location for hematopoletic stem cells termed the intermediate cell mass (ICM) of Oellacter appears to be responsible for primitive or definitive hematopolesis depending upon the species being investigated. In Xenopus, elegant grafting studies in combination with specific molecular markers has led to a better definition of the roles that ventral blood islands and dorsal lateral plate play in amphibian hematopolesis, that of primitive and deffinitive lymphopolesis. After the early embryonic contribution to hematopolesis. specialized tissues must assume the role of providing the proper microenvironment for T and B‐lymphocyte development from progenitor stem cells. In all gnathostomes, the thymus is the major site for T‐cell maturation as evidenced by strong expression of developmental markers such as Ikaros, Rag and TdT plus expression of T‐cell specific markers such as T‐cell receptor β and lck. In this respect, several zebrafish mutants have provided new insights on the development of the thymopoletic environment. On the other band, the sites for B‐cell lymphopolesis are less clear among the lower vertebrates. In elasmobranchs, the spleen, Leydigs organ and the spiral valve may all contribute to B‐cell development, although pre‐B cells have yet to be fully addressed in fish. In teleosts, the kidney is undeniably the major source of B‐cell development based upon functional, cellular and molecular indices. Amphibians appear to use several different sites (spleen, bone marrow and/or kidney) depending upon the species in question.

Description of an Ectothermic TCR Coreceptor, CD8α, in Rainbow Trout

We have cloned the first CD8α gene from an ectothermic source using a degenerate primer for Ig superfamily V domains. Similar to homologues in higher vertebrates, the rainbow trout CD8α gene encodes a 204-aa mature protein composed of two extracellular domains including an Ig superfamily V domain and hinge region. Differing from mammalian CD8α V domains, lower vertebrate (trout and chicken) sequences do not contain the extra cysteine residue (C strand) involved in the abnormal intrachain disulfide bridging within the CD8α V domain of mice and rats. The trout membrane proximal hinge region contains the two essential cysteine residues involved in CD8 dimerization (αα or αβ) and threonine, serine, and proline residues which may be involved in multiple O-linked glycosylation events. Although the transmembrane region is well conserved in all CD8α sequences analyzed to date, the putative trout cytoplasmic region differs and, in fact, lacks the consensus p56lck motif common to other CD8α sequences. We then determined that the trout CD8α genomic structure is similar to that of humans (six exons) but differs from that of mice (five exons). Additionally, Northern blotting and RT-PCR demonstrate that trout CD8α is expressed at high levels within the thymus and at weaker levels in the spleen, kidney, intestine, and peripheral blood leukocytes. Finally, we show that trout CD8α can be expressed on the surface of cells via transfection. Together, our results demonstrate that the basic structure and expression of CD8α has been maintained for more than 400 million years of evolution.

Induction of the rainbow trout MHC class I pathway during acute IHNV infection.

Abstract. Interferons are essential for establishing cytotoxic T-lymphocyte immunity against viral pathogens through different mechanisms including the modulation of antigen presentation to T-cell subsets. At the present time, interferons have yet to be isolated from teleost fish. We have developed a salmonid model to examine whether MHC gene regulation is modulated during acute viral infection in trout, an event attributable to interferons in mammals. During peak infection with infectious hematopoietic necrosis virus, induction of STAT-1, PSMB9A and ABCB2 mRNA was evident in all tissues within infected fish, as compared with controls. In addition, MHC class Ia and β2 microglobulin (β2m) transcript levels were enhanced within the experimental group but surprisingly, splenic and pronephric class IIB mRNA expression was virtually absent. A time-course study looking at 24, 72 and 192xa0h post-infection was then performed to determine the overall kinetics of this response. STAT-1 and PSMB9A message levels increased early during the immune response and remain at relatively high levels until the final time point. MHC class Ia expression is not consistently upregulated until midway in the response. MHC class IIB transcripts are downregulated by 72xa0h in the spleen and pronephros and then partially restored by 192xa0h. Finally, analysis of the putative promoter regions for PSMB9A and ABCB2 identified interferon (IFN) regulatory factory (IRF-1) and INF-γ (GAS) activation sites that may be involved in the regulation of these genes during viral infection.

CHARACTERIZATION OF RAINBOW TROUT TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE STRUCTURE AND EXPRESSION. TDT AND RAG1 CO-EXPRESSION DEFINE THE TROUT PRIMARY LYMPHOID TISSUES

Abstractu2003One component of antigen receptor diversity shared by all gnathostomes characterized to date is mediated by a unique DNA polymerase, terminal deoxynucleotidyl transferase (TdT), which generates significant functional diversity during immunoglobulin and T-cell receptor rearrangement. To gain further insight into the evolutionary origin(s) of this unique enzyme and the immune system, a thymic cDNA clone encoding TdT was isolated from rainbow trout. The 2.3 kilobase (kb) full-length clone contained an open reading frame of 1u200a506 base pairs with a deduced protein product of Mr 57u200a000. Sequence comparisons demonstrate that TdT has been highly conserved in both sequence (>70% aa similarity, >50 aa identity) and overall structure during the course of vertebrate evolution. An amino acid alignment of all known TdT sequences (chicken, Xenopus, mouse, human, cattle, and trout) reveals that some, but not all, structural motifs believed to be critical for TdT activity have been conserved. The TdT alignment, in conjuction with the recently solved crystal structure for rat beta-polymerase, supports the hypothesis that both may have evolved from a common ancestral repair gene. In addition, four PKC phosphorylation sites are conserved, and hence may be involved in TdT regulation. Because TdT contributes to the generation of junctional diversity in antigen receptors of immature lymphocytes, its expression serves as a developmental marker for the sites of teleost lymphopoiesis. Northern blot (2.3 kb message) analysis shows that TdT mRNA is highly expressed within the thymus and to a lesser extent in the pronephros. In addition, reverse transcriptase-polymerase chain reaction analysis detected transcipts of both RAG1 and TdT in the thymus, pronephros, mesonephros, spleen, and intestine, but not within muscle, liver, or brain. Finally, TdT cDNA was amplified from embryos at 20 days post-fertilization (pf), which correlates with the appearence of the thymus and pronephros anlage during trout ontogeny.

Conservation of an alpha 2 domain within the teleostean world, mhc class i from the rainbow trout Oncorhynchus mykiss

A full-length cDNA clone (Onmy-UA-C32) encoding a major histocompatibility complex (MHC) class I heavy chain was isolated from a rainbow trout thymus cDNA library. Onmy-UA-C32 alpha I and III extracellular domains were most similar to other salmonids (92 and 86% at the nucleotide and amino acid level) but interestingly the alpha II domain is closer to that of the carp (74 and 73%) and zebrafish (75 and 70%). In addition, Onmy-UA-C32 displays conservation of residues known to be essential for the function and structure of MHC class Ia molecules. Northern blot hybridization with alpha 2 or 2-3 domain probes of Onmy-UA-C32 detected high expression (2.6 kb) of this gene in the spleen, thymus, kidney, heart and intestine with lower levels being observed in the brain and liver. No tissues were found to be negative indicating a ubiquitous pattern of expression for Onmy-UA-C32. Onmy-UA-C32 may therefore represent a MHC class Ia gene in trout as well as providing new insights regarding the evolution of the MHC within teleost species.

Characterization of a C3a Receptor in Rainbow Trout and Xenopus: The First Identification of C3a Receptors in Nonmammalian Species

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Characterization of the OmyY1 Region on the Rainbow Trout Y Chromosome.

We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800u2009kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed.

Cloning, structure, and function of two rainbow trout Bf molecules: Both classical and alternative pathway activities require the presence of Bf-2☆

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+ EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.

Preface to the special issue “Impact of omics on comparative immunology”

The term “omics” evokes global, high-throughput approaches for producing large biological datasets used by systems biologists. These data describe natural systems, or refer to manipulated ones such as is the case for large-scale screens for gene function (e.g. RNAi) and the bioactivity of various compounds. Omics-based approaches are trans-disciplinary per se, but their target (tissues, cell types) or their output (expression of gene family members or identification of molecular pathways) can have critical impacts on the understanding of a particular field. Assembling a well-integrated special issue covering omics approaches in comparative immunology across the large evolutionary distance between its multiple models may appear unrealistic. In this issue, we did not aim at providing a comprehensive view of omics approaches that are potentially useful for, or already employed by, comparative immunologists. Instead, we collected multiple points of view on the past and future impacts of these technologies for the strategies undertaken by comparative immunologists to develop new avenues of research within their discipline. We also hint at some of the issues that should be taken into account when using high-throughput sequencing (HTS) in comparative studies. A comprehensive understanding of immune systems and responses frommany species is required to define selection pressures that have sorted out mechanisms, molecules and specialized cells or structures during the evolution of immunity. While the importance of comparative approaches is recognized through studies of the innate immune system, research efforts and scientific communities have remained largely unbalanced in favor of human/mouse immunology, and the relative lack of data available from other species has significantly hampered the realization of the full potential of comparative immunology. Omics approaches drastically change this situation, since now we can quickly produce very large data sets from any species in a relatively short period of time. In fact, the limiting factor in building a truly comparative perspective in immunology is no longer the lack of data, but rather the capacity to analyze and integrate the mass of available information. The articles of this Special Issue highlight the enormous potential and challenges associated with this technological breakthrough. The two first articles describe how HTS have contributed to the current state of knowledge about the FREP (Fibrinogen-related proteins), highly diverse mollusk lectins which bind mucin-like proteins on blood flukes. Schultz and Adema first describe the agglutination process, and the history of the genomic description