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Cytotoxic T cells are known to have the ability to kill microbe-infected host cells, which makes them essential in the adaptive immunity processes of various vertebrates. In this study, we demonstrated innate cell-mediated cytotoxicity of CD8

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8

Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

Although many recent studies have suggested that CD4 + helper T cell (Th-cell) functions are well conserved among teleost fishes and mammals, there is little evidence that CD4 + Th-cells in fish are actually involved in both humoral and cell-mediated immunity during a secondary immune response. In the present study, adoptive transfer using clonal ginbuna crucian carp and crucian carp hematopoietic necrosis virus (CHNV) was used to investigate the functions of CD4 + cells during humoral and cell-mediated immunity. With regard to humoral immunity, transplanting CHNV-sensitized donor cells, containing CD4 + cells, into naive fish induced more rapid and stronger antibody production than by transplanting non-sensitized donor cells or sensitized donor cells lacking CD4 + cells. During cell-mediated immunity, no significant differences were found in recipients that received sensitized cells regardless of whether the donor cells contained CD4 + cells, although recipients that received both sensitized donor cells (with and without CD4 + cells) exhibited more efficient cell-mediated cytotoxicity than those that received non-sensitized donor cells. These findings suggest that inducing a secondary antibody response requires CD4 + cell help, and secondary cell-mediated immunity can be induced both by CD4 + cells and leukocytes other than CD4 + cells.

Studies on the complement system of bony fish are now finishing a stage of homologue-hunting identification of the components, unveiling existence of almost all the orthologues of mammalian complement components in teleost. Genomic and transcriptomic data for several teleost species have contributed much for the homologue-hunting research progress. Only an exception is identification of orthologues of mammalian complement regulatory proteins and complement receptors. It is of particular interest that teleost complement components often exist as multiple isoforms with possible functional divergence. This review summarizes research progress of teleost complement system following the molecular identification and sequence analysis of the components. The findings of extensive expression analyses of the complement components with special emphasis of their prominent extrahepatic expression, acute-phase response to immunostimulation and various microbial infections, and ontogenic development including maternal transfer are discussed to infer teleost-specific functions of the complement system. Importance of the protein level characterization of the complement components is also emphasized, especially for understanding of the isotypic diversity of the components, a unique feature of teleost complement system.

The presence of antigen-specific cytotoxic T cells has been suggested in a number of in vivo and in vitro studies in fish. Acute allograft rejection with an accelerated response on second-set grafts and the presence of graft-versus-host reaction (GVHR) has been reported in teleost. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ex vivo studies in ginbuna and rainbow trout. In addition, alloantigen-specific cytotoxic T cell clones have been produced in cultures initiated with peripheral blood leukocytes (PBL) from an alloantigen-immunized channel catfish. Over the last decade several fish genomes have been sequenced and genetic information is rapidly accumulating. Thanks to these genome data bases and EST analysis, mRNA expression of T cell surface marker genes in alloantigen- or virus-specific effector cells has been reported in some fish species, e.g. TCR α or β and CD8α in ginbuna and rainbow trout, and TCR α, β or γ in channel catfish. These findings suggest the presence of CD8(+) cytotoxic T lymphocyte (CTL) in fish similar to those of higher vertebrates. Recently, monoclonal antibodies against CD8α and CD4 antigens have been produced in some fish species. Investigation on the characteristics of CTL and cell-mediated immune mechanisms is now possible at defined T cell subsets, although identification of T cell subset is limited in a few fish species at present. In this review, we describe the recent progress in this field focusing on cells involved in antigen specific cytotoxicity.

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.

In the present study, we attempted to determine whether virus-specific cell-mediated cytotoxicity occurs in fish. Experiments were conducted with clonal ginbuna crucian carp (Carassius auratus langsdorfii) (S3n strain), and the syngeneic cell line (CFS). Two infectious viruses were used: infectious pancreatic necrosis virus (IPNV) and eel virus from America (EVA). Peripheral blood leukocytes, which consisted almost entirely (95%) of lymphocytes and thrombocytes, from S3n ginbuna immunized with virus-infected CFS cells lysed the virus-infected CFS cells (immunogen) more completely than CFS cells infected with a different virus (non-immunogen). This specific cell-mediated cytotoxicity of ginbuna against virus-infected CFS cells was efficiently induced as a result of in vivo secondary immunization. A significant specific cytotoxic activity peaked during 7-10 days after the secondary immunization. In addition, effector cells immunized with IPNV-infected syngeneic cells did not lyse IPNV-infected xenogeneic cells. These results support the hypothesis that fish exhibit specific cytotoxicity against virus-infected cells, resembling the specific cytotoxicites of higher vertebrates.