Transcription analysis of two Eomesodermin genes in lymphocyte subsets of two teleost species.

Eomesodermin (Eomes), a T-box transcription factor, is a key molecule associated with function and differentiation of CD8(+) T cells and NK cells. Previously, two teleost Eomes genes (Eomes-a and -b), which are located on different chromosomes, were identified and shown to be expressed in zebrafish lymphocytes. For the present study, we identified these genes in rainbow trout and ginbuna crucian carp.

Clonal growth of carp (Cyprinus carpio) T cells in vitro: long-term proliferation of Th2-like cells.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in proliferation of polyclonal CD4(+) αβT cells as described previously. These bulk-cultured T cells expressed transcripts for both T helper 1 cells (Th1) master regulator (T-bet) and T helper 2 cells (Th2) master regulator (GATA-3). To identify the Th subsets in bulk-cultured T cells, single cells were picked up from the bulk culture, proliferated, and characterized.

Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes suggests that these tissues form Th2-skewed immune environments.

Rainbow trout and Atlantic salmon interleukin-4/13A (IL-4/13A) genes were identified. They were found expressed at high level in thymus, gill, and skin, in concert with the transcription factor gene GATA-3. High expression levels of IL-4, IL-13, and GATA-3 were also detected in murine thymus, suggesting similar importance of the fish and mammalian homologues for early T cell development.

The expression of CD8α discriminates distinct T cell subsets in teleost fish.

CD8, belonging to the TCR complex, is the main marker molecule of CTLs. Although CD8 genes have been detected in many fish species, the analysis of teleost CD8+ cells has been limited because of the lack of antibodies. Using newly established mAbs against rainbow trout CD8α, we found high ratios of CD8α+ cells in trout thymus, gill and intestine, but relatively low abundance in pronephros, spleen and blood.

A molecule in teleost fish, related with human MHC-encoded G6F, has a cytoplasmic tail with ITAM and marks the surface of thrombocytes and in some fishes also of erythrocytes.

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor.

Zinc-dependent binding between peptides derived from rainbow trout CD8alpha and LCK.

The cytoplasmic tail of mammalian CD8alpha binds the kinase LCK in a zinc-dependent manner. In analogy with a previous study for humans (Kim et al., 2003) peptides were synthesized from rainbow trout CD8alpha and LCK.

Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish.

Interleukins 4 and 13 (IL-4 and IL-13) are related cytokines important for Th2 immune responses and encoded by adjacent genes on human chromosome 5. Efforts were made previously to detect these genes in fish, but research was hampered by a lack of sequence conservation. A Tetraodon nigrovirides (green spotted pufferfish) gene was annotated as IL-4 by Li et al.

Genomic organization and expression of CD8alpha and CD8beta genes in fugu Takifugu rubripes.

We have cloned cDNAs encoding the alpha and beta chains of CD8 from the tiger pufferfish (fugu), Takifugu rubripes. The cDNA sequences encode a putative leader peptide, extracellular immunoglobulin variable region-like domain, stalk region, transmembrane region, and cytoplasmic tail. A protein tyrosine kinase p56lck binding motif was not found in the putative fugu CD8alpha cytoplasmic tail.

A third broad lineage of major histocompatibility complex (MHC) class I in teleost fish; MHC class II linkage and processed genes.

Most of the previously studied teleost MHC class I molecules can be classified into two broad lineages: "U" and "Z/ZE." However, database reports on genes in cyprinid and salmonid fishes show that there is a third major lineage, which lacks detailed analysis so far. We designated this lineage "L" because of an intriguing linkage characteristic.

Polymorphism of two very similar MHC class Ib loci in rainbow trout (Oncorhynchus mykiss).

As part of an ongoing elucidation of rainbow trout major histocompatibility complex (MHC) class I, the polymorphism of two MHC class Ib loci was analyzed. These loci, Onmy-UCA and Onmy-UDA, are situated head-to-tail and share more than 89% nucleotide identity in their open reading frames. They share 80% identity with some trout Ia alleles.

Identification and characterization of a second CD4-like gene in teleost fish.

In fish, T cell subdivision is not well studied, although CD8 and CD4 homologues have been reported. This study describes a second teleost CD4-like gene, CD4-like 2 (CD4L-2). Two rainbow trout copies of this gene were found, -2a and -2b, encoding molecules sharing 81% aa identity.

Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish.

IFN-gamma is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-gamma homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-gamma cDNA is 1034 bp in length and translates into a 180-aa protein.

Growth and behavioral traits in Donaldson rainbow trout (Oncorhynchus mykiss) cosegregate with classical major histocompatibility complex (MHC) class I genotype.

Although polymorphism in major histocompatibility complex (MHC) genes has been thought to confer populations with protection against widespread decimation by pathogens, this hypothesis cannot explain the type of large allelic diversity in classical MHC class I (Ia) in rainbow trout. Based on expression of Onmy-UBA (MHC class Ia) in trout neurons, we hypothesized that polymorphism in trout class Ia may contribute to polymorphism in behavioral traits. The present study examined whether polymorphism in Onmy-UBA was associated with behavioral variation in Donaldson rainbow trout (Oncorhynchus mykiss) using experiments on food competition, lure-catch, fright recovery, diel locomotor activity and activity characterized as dominance or aggression.

Interchromosomal duplication of major histocompatibility complex class I regions in rainbow trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid ancestry.

Salmonid fishes are among the few animal taxa with a probable recent tetraploid ancestor. The present study is the first to compare large (>100 kb) duplicated genomic sequence fragments in such species. Two contiguous stretches with major histocompatibility complex (MHC) class I genes were detected in a rainbow trout BAC library, mapped and sequenced.

Characterisation and expression analysis of interleukin 2 (IL-2) and IL-21 homologues in the Japanese pufferfish, Fugu rubripes, following their discovery by synteny.

This investigation provides the first conclusive evidence for the existence of the interleukin 2 (IL-2) and IL-21 genes in bony fish. The IL-2 and IL-21 sequences have been determined in Fugu rubripes by exploiting the conservation of synteny that is found between regions of the human and Fugu genomes. The predicted 149-amino acid IL-2 homologue contains the IL-2 family signature, has a predicted secondary structure of three alpha helixes and has the two cysteines important in disulphide-bond formation.

New MHC class Ia domain lineages in rainbow trout (Oncorhynchus mykiss) which are shared with other fish species.

Major histocompatibility complex (MHC) class Ia genes in salmonid fishes are encoded by a single locus with probably the highest allelic diversity ever described. Various combinations of very different domain lineages contribute to the diversity of alleles. An extensive PCR survey distinguishing most domain lineages and their combinations was established.

The ontogeny of MHC class I expression in rainbow trout (Oncorhynchus mykiss).

In the present study, clonal rainbow trout (Oncorhynchus mykiss) embryos and larvae were assayed for the expression of key molecules involved in specific cell-mediated cytotoxicity using an anti-MHC class I monoclonal Ab and by RT-PCR using specific primers derived from classical MHC class I (class Ia), TCR and CD8. Whereas RT-PCR revealed that MHC class Ia and CD8 were expressed from at least 1 week after fertilisation (p.f.

The MHC class I linkage group is a major determinant in the in vivo rejection of allogeneic erythrocytes in rainbow trout (Oncorhynchus mykiss).

Despite accumulating sequence data, information on the function of major histocompatibility complex (MHC) genes in fish is scarce. In contrast to the genome organization in higher vertebrates, the polymorphic MHC class I and II genes are not linked in the teleost genome. A previous study found an MHC class II linkage group to be a major determinant in the rejection of allogeneic scales by a teleost species (Cardwell et al.

A new putative G-protein coupled receptor gene associated with the immune system of rainbow trout (Oncorhynchus mykiss).

A putative G-protein coupled receptor (GPCR) gene belonging to the rhodopsin-like family was detected in rainbow trout and designated LPSenhR-1. Only moderate homology (<35%) was present with known GPCRs. Semi-quantitative RT-PCR indicated that the gene was expressed predominantly in lymphoid tissues, with highest expression associated with cells of the monocyte/macrophage lineage.

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss).

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function.

Adaptive cell-mediated cytotoxicity against allogeneic targets by CD8-positive lymphocytes of rainbow trout (Oncorhynchus mykiss).

Rainbow trout surface-(s)IgM(-) leukocytes exhibited cell-mediated cytotoxicity (CMC) against allogeneic cells. This is described in concordance with a characterization of gene expression in the effector cells. Peripheral blood leukocytes (PBL) isolated from trout grafted with allogeneic tissue lysed allogeneic target cells (erythrocytes or cells of the RTG-2 cell line) in in vitro assays.

The promoter of the classical MHC class I locus in rainbow trout (Oncorhynchus mykiss).

In rainbow trout only a single classical major histocompatibility complex (MHC) class I locus has been identified. In previous studies it was shown that this locus, Onmy-UBA, is characterised by extensive polymorphism and ubiquitous expression. In the present study the sequence of the promoter region of Onmy-UBA was analysed.

Chromosome mapping of MHC class I in rainbow trout (Oncorhynchus mykiss).

The major histocompatibility complex (MHC) is well-studied in mammals. Much research has addressed the genomic organisation of MHC genes and it is well established that human MHC class I genes are located on chromosome 6. However, information on the organisation of the MHC complex in rainbow trout is only beginning to become available.

Differences in MHC class I genes between strains of rainbow trout (Oncorhynchus mykiss).

In rainbow trout there is only one dominant classical MHC class I locus, Onmy-UBA, for which four very different allelic lineages have been described. The purpose of the present study was to determine if Onmy-UBA polymorphism could be used for strain characterisation. This was performed by lineage-specific PCR investigation of 30 fish, each of the Nikko and Donaldson strains, and by sequence analysis of 25 of the amplified DNA fragments.