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. Deduced Eomes-a and -b amino acid sequences in both fish species contain a highly conserved T-box DNA binding domain. In RT-PCR, both Eomes transcripts were readily detectable in a variety of tissues in rainbow trout and ginbuna. The high expression of Eomes-a and -b in brain and ovary suggests involvement in neurogenesis and oogenesis, respectively, while their expression in lymphoid tissues presumably is associated with immune functions. Investigation of separated lymphocyte populations from pronephros indicated that both Eomes-a and -b transcripts were few or absent in IgM(+) lymphocytes, while relatively abundant in IgM(-)/CD8α(+) and IgM(-)/CD8α(-) populations. Moreover, we sorted trout CD8α(+) lymphocytes from mucosal and non-mucosal lymphoid tissues and compared the expression profiles of Eomes-a and -b with those of other T cell-related transcription factor genes (GATA-3, T-bet and Runx3), a Th1 cytokine gene (IFN-γ) and a Th2 cytokine gene (IL-4/13A). Interestingly, the tissue distribution of Eomes-a/b, T-bet, and Runx3 versus IFN-γ transcripts did not reveal simple correlations, suggesting tissue-specific properties of CD8α(+) lymphocytes and/or multiple modes that drive IFN-γ expressions.

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(+) αßT 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. The majority of the clones displayed characteristics consistent with CD4(+) αßT cell identity. These clones expressed both TCRα and TCRß, but could not produce a TCRγδ heterodimer since they typically only expressed either TCRγ or TCRδ. These clones also expressed the TCR co-receptor genes CD4-1 or CD4-2, whereas they did not express CD8α or CD8ß. In addition, GATA-3 was expressed whereas T-bet was not. Among these clones, one clone (KoThL5) continued to proliferate on the supporting cells and was successively transferred for more than 10 months and 90-100 passages. To characterize the KoThL5 cells by their cytokine production profile, they were stimulated with PHA and investigated by real-time RT-PCR. mRNA expression of Th2-related cytokine (IL-4/13B) was only enhanced in KoThL5 cells whereas both Th1-related cytokine (IFNγ) and Th2-related cytokines (IL-4/13A and IL-4/13B) were significantly enhanced in bulk-cultured T cells. Taken together, KoThL5 cells share some features with mammalian Th2 cells. This is the first study to describe in vitro cultures of teleost cell with Th2-like features. The KoThL5 cell line has considerable potential for addressing questions concerning the properties of teleost Th2 cells.

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. In mammals, combined high expression of IL-4/13 and GATA-3 in tissues other than thymus is mostly indicative of Th2 responses. Th2-skewage may protect fish skin and gill from parasites and from damage by inflammatory Th1 and Th17 responses. The immune milieus of fish gill and skin are relevant to aquaculture, because these tissues are preferred sites for vaccine administration. The similarities between the immune milieus of fish gill and thymus may reflect an evolutionary relationship, since these tissues map close together lining the gill cavity. Expression patterns of IL-4/13A and interferon gamma (IFN-γ) in isolated trout gill cells and pronephrocytes were consistent with Th2 identity of IL-4/13A.

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. Accordingly, tissue sections revealed many CD8α+ cells in thymus, numerous intra- and subepithelial CD8α+ cells in intestine and gill and few scattered CD8α+ cells in spleen and pronephros. In secondary lymphoid tissues, CD8α+ lymphocytes, which did not react with anti-thrombocyte or anti-IgM mAbs, expressed CD8α, CD8ß and TCRα, while Ig and CD4 transcripts were found in CD8α⁻ lymphocytes. In contrast, considerable CD4 expression in CD8α+ thymocytes suggests the presence of double-positive early T cells. Highly expressed TCRγ, LAG3 and CTLA4 in CD8α+ lymphocytes imply that they constitute a heterogeneous population different from found in non-mucosal tissues. PHA stimulation resulted in an up-regulation of CTL effector genes (perforin, granulysin and IFN-γ) in CD8α+ pronephrocytes, while both Th1 (IFN-γ) and Th2 (IL-4/13A) cytokines were up-regulated in CD8α⁻ pronephrocytes. Although the basic characteristics of CD8α+ lymphocytes seem similar in teleost and mammals, features such as the low proportion of teleost CD8α+ lymphocytes in blood and their high abundance in respiratory tissue reveal a unique dynamics and distribution.

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. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.

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. Surface plasmon resonance (SPR) analysis indicated that also in fish these molecules bind to each other in a zinc-dependent manner.

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. (Mol Immunol, 44:2078-2086, 2007), but this annotation was not well substantiated. However, the present study concludes that the reported pufferfish gene belongs to the IL-4/13 lineage indeed, while also describing an additional IL-4/13 copy in a paralogous genomic region. Our analyses of IL-4/13 loci in fish describe (1) genomic region history, (2) characteristic intron-exon organization, (3) deduced IL-4/13 molecules for several teleost fish species, (4) IL-4/13 lineage-specific protein motifs including a cysteine pair (pair 1), and (5) computer software predictions of a type I cytokine fold. Teleost IL-4/13 molecules have an additional cysteine pair (pair 2) or remnants thereof, which is absent in mammalian IL-4 and IL-13. We were unable to determine if the teleost IL-4/13 genes are orthologous to either IL-4 or IL-13, or if these mammalian genes separated later in evolution.

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. O-linked glycosylation sites were found in the stalk of both CD8 chains, suggesting possible stalk formation. Phylogenetic analysis showed that fugu CD8alpha and CD8beta chains cluster with other vertebrate CD8alpha and CD8beta chains, respectively. The fugu CD8 genes comprise six exons separated by five introns. The genes are tandemly aligned 3.6 kb apart and are in the same transcription orientation. Quantitative RT-PCR analysis demonstrated that fugu CD8 is expressed predominantly in lymphoid tissues. In situ hybridization showed that fugu CD8 genes are expressed in thymocytes and lymphocytes within lymphoid organs. Molecular characterization of CD8 in fish provides the basis for development of specific antibodies to identify T-cell subsets, as well as potentially important insights into the evolution of CD8 and the adaptive immunity.

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. Namely, one zebrafish L locus is closely linked with MHC class II loci, despite the extensively documented nonlinkage of teleost class I with class II. The L lineage consists of highly variable, nonclassical MHC class I genes, and has no apparent orthologues outside teleost fishes. Characteristics that distinguish the L lineage from most other MHC class I are (1) absence of two otherwise highly conserved tryptophan residues W51 and W60 in the alpha1 domain, (2) a low GC content of the alpha1 and alpha2 exons, and (3) an HINLTL motif including a possible glycosylation site in the alpha3 domain. In rainbow trout (Oncorhynchus mykiss) we analyzed several intact L genes in detail, including their genomic organization and transcription pattern. The gene Onmy-LAA is quite different from the genes Onmy-LBA, Onmy-LCA, Onmy-LDA, and Onmy-LEA, while the latter four are similar and categorized as "Onmy-LBA-like." Whereas the Onmy-LAA gene is organized like a canonical MHC class I gene, the Onmy-LBA-like genes are processed and lack all introns except intron 1. Onmy-LAA is predominantly expressed in the intestine, while the Onmy-LBA-like transcripts display a rather homogeneous tissue distribution. To our knowledge, this is the first description of an MHC class I lineage with multiple copies of processed genes, which are intact and transcribed. The present study significantly improves the knowledge of MHC class I variation in teleosts.

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. The deduced amino acid sequences suggest that the UCA and UDA molecules are transported to endosomal compartments and may bind peptides in their binding groove. Our survey revealed seven UCA and eight UDA alleles. Similarity indices overlap when comparing within and between UCA and UDA alleles and some cross-locus motif variation is observed. In most trout both UCA and UDA transcripts were found. However, there probably is functional redundancy, because some trout lacked transcription of one of the two loci. Furthermore, for some UCA and UDA alleles, splicing deficiencies, early stop codons, and upstream start codons were found, which may interfere with efficient protein expression. The present study is the first extensive report on MHC class Ib polymorphism assigned to locus in ectotherm species.

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. The 2a/2b duplication may be related to tetraploid ancestry of salmonid fishes. In the Fugu genome CD4L-2 lies head to tail with an earlier reported, very different CD4-like gene [Suetake, H., Araki, K., Suzuki, Y., 2004. Cloning, expression, and characterization of fugu CD4, the first ectothermic animal CD4. Immunogenetics 56, 368-374], which was designated CD4L-1 in the present article. The flanking genes of the Fugu CD4L-1 and CD4L-2 are reminiscent of the genes surrounding CD4 and LAG-3 in mammals. However, neither synteny nor phylogenetic analysis could decide between CD4 and LAG-3 identity for the fish CD4L genes. CD4L-1 and CD4L-2 share a tyrosine protein kinase p56(lck) binding motif in the cytoplasmic tail with CD4 but not with LAG-3. Trout CD4L-2 expression is highest in the thymus, similar to mammalian and chicken CD4, whereas Fugu CD4L-1 expression was highest in the spleen. However, CD4L-2 encodes only two IG-like domains, whereas CD4L-1, CD4 and LAG-3 encode four. The CD4-like genes 1 and 2 in fish apparently went through an evolution different from that of LAG-3 and CD4 in higher vertebrates.

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. The first intron of the trout IFN-gamma gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-gamma is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-gamma biological activities is also present in the C-terminal region of the trout IFN-gamma. The IFN-gamma expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-gamma produced in Escherichia coli significantly stimulated gene expression of IFN-gamma-inducible protein 10 (gammaIP-10), MHC class II beta-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of gammaIP-10 in RTS-11 cells. Moreover, IFN-gamma-induced gammaIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-gamma homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

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. These behavioral traits were investigated in fish having Onmy-UBA*401/*401 or *4901/*4901 homozygous, or Onmy-UBA*401/*4901 heterozygous genotypes (referred to as BB, FF and BF, respectively). The BB fish exhibited boldness, aggression, faster growth and crepuscular activity, while the FF fish showed little boldness, smaller body size, and diurnal activity with no aggressive behavior. The BF fish displayed traits intermediary to those of the BB and FF fish. These results are consistent with polymorphism in a single MHC class Ia locus driving variation in neural circuits, thereby creating behavioral variation in the trout. This is the first study in any animal to show a potential correlation between polymorphism in MHC class Ia genes with polymorphism of behavioral traits such as 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. The MHC class I duplicated regions, mapped by fluorescence in situ hybridization (FISH), were shown to be located on different metaphase chromosomes, Chr 14 and 18. Gene organization in both duplications is similar to that in other fishes, in that the class I loci are tightly linked with the PSMB8, PSMB9, PSMB10 and ABCB3 genes. Whereas one region, Onmy-IA, has a classical MHC class I locus (UBA), Onmy-IB encodes only non-classical class Ib proteins. The nucleotide diversity between the Onmy-IA and Onmy-IB noncoding regions is about 14%. This suggests that the MHC class I duplication event has occurred about 60 mya close to the time of an hypothesized ancestral tetraploid event. The present article is the first convincing report on the co-existence of two closely related MHC class I core regions on two different chromosomes. The interchromosomal duplication and the homology levels are supportive of the tetraploid model.

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. It shows low amino acid identities (24-34%) with other known IL-2 sequences. The predicted 155-amino acid IL-21 homologue has a predicted secondary structure of four alpha helixes and has the four cysteines important in disulphide-bond formation. It shows low amino acid identities (29-31%) with other known IL-21 sequences. The gene organisation of Fugu IL-2 and IL-21 and the level of synteny between the human and Fugu genomes has been well conserved during evolution, with the order and orientation of the genes matching exactly to human Chromosome 4. Phytohaemagglutinin stimulation of Fugu kidney cells resulted in a large increase in the Fugu IL-2 and IL-21 transcripts. In vivo stimulation of Fugu with LPS and poly I:C showed IL-21 expression to be localised within mucosal tissues. The discovery of IL-2 and IL-21 in fish will now allow more detailed investigations into T-helper cell responses.

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. This survey has practical value for researchers investigating salmonid MHC class Ia variation. In the present study it was used to find new domain lineages. Applied for 24 hatchery strains in Japan, the survey identified two new rainbow trout alpha1 lineages and one new rainbow trout alpha2 lineage. The alpha2 lineage and one of the alpha1 lineages had been described in Atlantic salmon, but the other alpha1 lineage is novel. The newly identified trout alpha1 lineages are evolutionary very old. The present study should be the most extensive description of very deep MHC class Ia lineages to date: six trout alpha1 lineages cluster with non-salmonid sequences whereas previous studies mentioned this for only two salmonid alpha1 lineages. Although exon-shuffling events significantly contributed to salmonid MHC class Ia variation, analysis of 800 trout siblings did not detect such events within a single generation.

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.) on, TCR expression was detectable from 2 weeks p.f. Immunohistochemistry indicated an early and distinct expression of MHC class I protein in the thymus. Positive lymphoid, epithelial and endothelial cells were found in the pronephros, in the spleen and in the inner and outer epithelia at later stages. Whereas in older rainbow trout the intestine is counted among the organs of the highest class I expression, during ontogeny it was the last site (39 days after hatching) where such expression was detectable. Knowledge on the appearance of the assayed key molecules during fish development is relevant for the pathogenesis of infections as well as for early vaccine delivery. Besides such information regarding the development of the adaptive immune system, immunohistochemistry revealed that in early larvae MHC class I was expressed in neurons whereas in older rainbow trout this was not observed.

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. 2001). The present study investigated whether the teleost MHC class I linkage group can be involved in allograft rejection. Erythrocytes were chosen as grafts since they express MHC class I, but do not express class II. Rainbow trout erythrocytes expressing different MHC class I alleles were differentially stained, mixed and injected into recipients that were of the same sibling group as the donors. The MHC class I linkage group was the major determinant for in vivo graft rejection.

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. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.