Mitsuru Ototake

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 PBL were magnetically separated into different subpopulations using monoclonal antibodies (mabs) specific to thrombocytes, IgM, granulocytes and monocytes. Of the isolated subpopulations only the sIgM(-) lymphocytes were capable of lysing allogeneic targets. The separated PBL fractions were characterized by RT-PCR analysis using specific primers for the amplification of trout IgM heavy chain constant region (CH1), T cell receptor alpha chain (TCRalpha), CD8alpha and major histocompatibility complex (MHC) class I gene fragments. Most importantly, CD8alpha was expressed only by the sIgM(-) population. Combined with the requirement for sensitization to detect CMC, this strongly suggests T cell involvement in fish as in higher vertebrates. The involvement of CD8alpha-positive cytotoxic T cells in allograft rejection was supported by additional in vivo and in vitro observations. CD8alpha expression was barely detectable in the blood of unsensitized trout or trout that received xenografts, but was easily detected in the blood of allogeneically stimulated trout. Furthermore, CD8alpha expression in sIgM(-) lymphocytes from immunized trout was secondarily enhanced by addition of allogeneic targets in vitro. Collectively, these functional and genetic data suggest that fish possess specific cytotoxic cells with phenotype and gene expression pattern similar to those of cytotoxic T cells in higher vertebrates.

The Most Primitive Vertebrates with Jaws Possess Highly Polymorphic MHC Class I Genes Comparable to Those of Humans

We report the isolation and extensive analysis of highly polymorphic MHC class I genes from sharks (Triakis scyllia), which belong to the most primitive vertebrate group with jaws, the cartilaginous fish. Predicted complete peptide-binding domains showed retention of the critical amino acid residues that would interact with antigenic peptide termini and revealed extensive allelic polymorphisms comparable to those of classic human MHC class I molecules. Mosaic structures were apparent in these domains, suggesting recombinational mechanisms to create allelic diversity. The present study demonstrates the establishment of the basic strategy for antigen-presentation employed by MHC class I molecules and documents complete divergence of two polymorphic MHC classes at a phylogenetically primitive stage of vertebrate evolution.

Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits

AbstractWe have isolated and characterized cDNAs and genes for pufferfish, Fugu rubripes, (Fugu) orthologues of mammalian interleukin (IL)-12 subunits (IL-12 p35 and IL-12 p40). The deduced amino acid sequences of the Fugu IL-12 subunits showed homology with mammalian IL-12 subunits (p35: 50.4–58.0% similarity; p40: 51.2–55.4% similarity). Phylogenetic analysis confirmed that Fugu IL-12 p35 and p40 genes cluster with their mammalian counterpart lineages. The genomic organization of each of the Fugu IL-12 subunit genes is similar to that of the corresponding mouse IL-12 subunit genes, although the Fugu genes are very compact due to small intron size. Comparative genomic analysis showed conserved syntenies within the IL-12 p35 and p40 regions between Fugu and human, indicating that the Fugu IL-12 p35 and p40 genes are orthologues for mammalian IL-12 p35 and p40 encoding genes, respectively. Expression of IL-12 p35 mRNA was observed in lymphoid tissues and several non-lymphoid tissues, while expression of IL-12 p40 mRNA was constitutive and nearly ubiquitous. In the spleen and head kidney, expression of IL-12 p35 was induced by polyriboinosinic polyribocytidylic acid [poly(I:C)] and not by lipopolysaccharide (LPS), while expression of IL-12 p40 was constitutive and unresponsive to both poly(I:C) and LPS. These results indicate that IL-12 levels are regulated by production of IL-12 p35 mRNA and suggest that IL-12 in fish may be involved in antiviral defense. This is the first report of the identification and characterization of IL-12 subunit cDNAs and genes in a non-mammalian vertebrate.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

Classical MHC Class I Genes Composed of Highly Divergent Sequence Lineages Share a Single Locus in Rainbow Trout (Oncorhynchus mykiss)

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the α3 and transmembrane domains is unprecedented.

Alloantigen-specific killing is mediated by CD8-positive T cells in fish

CD8-positive (CD8(+)) cytotoxic T lymphocytes (CTL) have antigen-specific cytotoxic activity. In fish, however, CTL expressing CD8 on their cell surface have not been identified. In order to characterize the cells involved in specific cell-mediated cytotoxicity in teleosts, we separated and sorted ginbuna kidney leucocytes into CD8alpha(+), CD4(+) and surface IgM (sIgM)(+) cells by magnetic activated cell sorting using monoclonal antibodies and examined their cytotoxic activities. Effector donor ginbuna (OB1 clone) were sensitized by allografting scales from S3N clone fish followed by injection of an allogeneic cell line (CFS) derived from S3N fish. In cytotoxic assays, target cells were labeled with CFSE and cytotoxicity was calculated based on the number of viable target cells using flow cytometry. CD8alpha(+) cells from sensitized OB1 fish showed relatively high cytotoxicity against CFS cells (immunogen) but not against allogeneic CFK cells (third party) nor isogeneic CFO cells. Pre-sensitized sIgM(+) cells exhibited cytotoxicity against not only CFS cells but also CFK cells. However, CD4(+) or CD8alpha(-) CD4(-)sIgM(-) cells as well as cells from non-sensitized fish did not show any significant cytotoxic activity. These results suggest that CD8alpha(+) cells in fish have characteristics similar to those of CTL in mammals, and that the sIgM(+) cells include NK-like cells which non-specifically killed the target cells.

IN VITRO CELL-MEDIATED CYTOTOXICITY AGAINST ALLOGENEIC ERYTHROCYTES IN GINBUNA CRUCIAN CARP AND GOLDFISH USING A NON-RADIOACTIVE ASSAY

Cell-mediated cytotoxicity of clonal ginbuna crucian carp leukocytes against allogeneic erythrocytes is described using a sensitive non-radioactive in vitro assay. Hemoglobin released from target erythrocytes after cell-mediated erythrolysis was detected by tetramethylbenzidine (TMB). TMB assay showed clear correlation with a 51Cr-release assay and even exhibited higher cytotoxicity. The use of erythrocytes as target cells has several advantages over a conventional 51Cr-release assay. Erythrocytes do not have cytotoxic activity, are relatively homogeneous, are available in large numbers and erythrocyte donors need not be killed. Leukocytes from fish sensitized by erythrocyte injection or scale grafting efficiently lysed allogeneic erythrocytes, but did not kill isogeneic or autologous erythrocytes. In contrast, leukocytes from unsensitized fish did not lyse allogeneic erythrocytes and repeated sensitizations by allogeneic grafts were necessary to induce cytotoxic cells. Effector cells isolated from peripheral blood showed a higher cytotoxic effect toward allogeneic target cells than effector cells isolated from kidney. These studies support the hypothesis that fish are capable of a genetically restricted specific cell-mediated cytotoxicity.

Conservation and diversification of MHC class I and its related molecules in vertebrates

Summary: The elucidation of the complete peptide‐binding domains of the highly polymorphic shark MHC class I genes offered us an opportunity to examine the characteristics of their predicted protein products in the light of the latest advance m the structural studies of the MHC class I molecules. The results suggest that the fundamental characteristics in the T‐cell recognition of the MHC class I molecule/peptide complex are expected to have been established at the early stage of the vertebrate evolution. The elucidation of the typical classical class I molecules from fishes and also of some MHC class I‐related molecules may help us Co explore the common denominator of the ancient class I molecules.

The graft-versus-host reaction (GVHR) in the ginbuna crucian carp, Carassius auratus langsdorfii

A model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids was employed to demonstrate the presence of graft-versus-host reaction (GVHR) in a teleost fish. Tetraploid scale grafts on triploid clone members evoked an acute rejection, whereas the reverse transplants were accepted. When sensitized triploid cells were injected into tetraploid recipients, a typical GVHR was induced, leading to death of the recipients within one month. The onset of illness appeared about one week after cell injection as a loss of appetite and constipation, followed by a scale protrusion, severe haemorrhage, local destruction of the ventral skin and prominent splenomegaly. GVHR was most effectively induced by head-kidney cells and peripheral blood leukocytes (PBL), followed by spleen and thymus cells. Donors had to be sensitized at least twice by scale grafting to induce the reaction. A considerable number of recipients injected with cells from donors which had been sensitized by allogenetically different tetraploids died, suggesting a limited polymorphism or heavy cross-reactions between the alleles of the histocompatibility antigens. Ploidy analyses revealed that donor cells greatly increased in the host liver and spleen, constituting approximately 30% of total cells after 2 3 weeks. Most of these features of acute GVHR observed in this fish system are quite similar to those found in mammals and birds. thereby suggesting the presence of allo-reactive cytotoxic T lymphocytes in teleosts.

Development of a new vaccine delivery method for fish: percutaneous administration by immersion with application of a multiple puncture instrument

A new administration method was developed for vaccination of juvenile rainbow trout against beta-haemolytic Streptococcus. This simple and convenient method was equal in effectiveness to intra-peritoneal injection. Small skin lesions were produced using a multiple puncture instrument while fish were immersed in a vaccine suspension containing formalin-killed Streptococcus iniae. Upon challenge 2 weeks after vaccination, mortality of fish vaccinated by this method was 40%, equal to that by intra-peritoneal injection, while non-vaccinated control fish and fish vaccinated by immersion (without multiple puncture) each experienced 80% mortality. High efficacy was obtained with the multiple puncture/immersion method even when vaccine was diluted 10-fold. Quantitative analysis using fluorescent microspheres revealed that both antigen uptake by skin and delivery to the kidney and spleen were more effective with this method than with immersion alone. Microspheres were found in the skin within the pinpoint lesions and pressure mark caused by multiple puncture instrument. The greater protection gained by the present method can be attributed to higher numbers of particulate antigens taken up by fish and delivered to the lymphoid tissues.