Yuta Matsuura

T Cells in Fish

Cartilaginous and bony fish are the most primitive vertebrates with a thymus, and possess T cells equivalent to those in mammals. There are a number of studies in fish demonstrating that the thymus is the essential organ for development of T lymphocytes from early thymocyte progenitors to functionally competent T cells. A high number of T cells in the intestine and gills has been reported in several fish species. Involvement of CD4+ and CD8α+ T cells in allograft rejection and graft-versus-host reaction (GVHR) has been demonstrated using monoclonal antibodies. Conservation of CD4+ helper T cell functions among teleost fishes has been suggested in a number studies employing mixed leukocyte culture (MLC) and hapten/carrier effect. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ginbuna and rainbow trout. Furthermore, the important role of cell-mediated immunity rather than humoral immunity has been reported in the protection against intracellular bacterial infection. Recently, the direct antibacterial activity of CD8α+, CD4+ T-cells and sIgM+ cells in fish has been reported. In this review, we summarize the recent progress in T cell research focusing on the tissue distribution and function of fish T cells.

Kinetics of lymphocyte subpopulations in allogeneic grafted scales of ginbuna crucian carp

In mammals the rejection of allografts is primarily accomplished by cell-mediated immunity including T cells. Recently, considerable studies reveal the existence of helper and cytotoxic T cell subsets in fish. Here we investigate the kinetics of CD4(+) and CD8α(+) T cells along with sIgM(+) cells and phagocytic cells in an allogeneic scale graft model using ginbuna crucian carp for understanding the mechanisms of cell-mediated immune response. The results showed that CD4(+) T cells first infiltrated into allogeneic scales followed by CD8α(+) and sIgM(+) cells, and finally phagocytic cells appeared in the graft. Furthermore, most of the CD8α(+) T cells appeared on the border of the allografted scales at the time of rejection. These results suggest that T cells play crucial roles and work together with other cell types for completion of allograft rejection.

Identification of a novel fish granzyme involved in cell-mediated immunity

Granzymes (Gzms) are serine proteases released from cytoplasmic granules within cytotoxic T lymphocytes and natural killer (NK) cells. Gzms induce apoptosis within virus-infected and transformed cells. In fish as well as mammals, Gzms appear to play a major role in inducing target cell death. However, information on the function of fish Gzms is limited, although Gzm-like genes have been reported in several species. We identified and characterized a fish Gzm (termed gcGzm) in ginbuna crucian carp, Carassius auratus langsdorfii. The primary structure of gcGzm resembled mammalian GzmB, and gcGzm clustered with mammalian GzmB by phylogenetic tree analysis. gcGzm was secreted from HEK293T cells transfected with gcgzm cDNA and was predominantly expressed in CD8(+) T cells, as in mammals. Expression of gcgzm mRNA was greatly enhanced by allo-sensitization and infection with the intracellular pathogen Edwardsiella tarda, indicating that gcGzm is involved in cell-mediated immunity. However, its enzymatic activity was different from mammalian Gzms because gcGzm did not cleave the known substrates for mammalian Gzms. Thus we conclude that the newly discovered gcGzm is a novel secretory serine protease involved in cell-mediated immunity in fish, with similar structure to human GzmB but different substrate specificity.

Stimulatory effects of heat-killed Enterococcus faecalis on cell-mediated immunity in fish

&NA; Intracellular bacterial and viral diseases are widespread in the aquaculture industry and cause serious economic losses. Development of effective vaccines and adjuvants that can induce cell‐mediated immunity is urgently needed for prevention of these diseases. Here we report the immunostimulatory effects of probiotic bacteria “E. faecalis” in ginbuna crucian carp Carassius auratus langsdorfii. Intraperitoneal injection of heat‐killed E. faecalis induced an increase in CD4‐1+ lymphocytes, CD8&agr;+ lymphocytes and macrophages in vivo. Expression of Th1 cytokine genes was enhanced by exposure to the bacteria in vitro. We identified the leukocyte subsets that expressed specific Th1 cytokine genes: granulocytes and macrophages produced IL12 and IFN&ggr;rel2, respectively, while lymphocytes produced IFN&ggr;s including IFN&ggr;1 and IFN&ggr;2. Finally, expression of Th1 cytokines was also enhanced by intraperitoneal injection of heat‐killed E. faecalis in vivo, while expression of Th2 cytokine was unchanged. Together, these findings suggest that heat‐killed E. faecalis can induce cell‐mediated immunity in fish. HighlightsHeat‐killed Enterococcus faecalis induced an increase in CD4‐1+ lymphocytes, CD8&agr;+ lymphocytes and macrophages in vivo.Heat‐killed E. faecalis enhanced expression of Th1 cytokine genes including IFN&ggr;s, IFN&ggr;rels and IL12 in vitro.Stimulated granulocytes and lymphocytes by heat‐killed E. faecalis in vitro expressed IL12 and IFN&ggr;s mRNA, respectively.Enhanced expression of Th1 cytokines by heat‐killed E. faecalis was also observed in in vivo analysis.

EFFECTS OF IFNγ ADMINISTRATION ON ALLOGRAFT REJECTION IN GINBUNA CRUCIAN CARP

In vertebrates, the rejection of allografts is primarily accomplished by cell-mediated immunity. We recently identified four IFNγ isoforms with antiviral activity in ginbuna crucian carp, Carassius auratus langsdorfii. However, involvement of the IFNγ isoforms in cell-mediated immunity, especially in T cell function remains unknown. Here we investigate expression of the IFNγ isoforms and effects of administration of recombinant IFNγ (rgIFNγ) isoforms in ginbuna scale allograft rejection. All four IFNγ isoforms showed significantly higher expression with the progression of graft rejection. Administration of rgIFNγrel 1 but not rgIFNγrel 2, rgIFNγ1 nor rgIFNγ2 enhanced allograft rejection. The number of CD4(+) and CD8α(+) cells increased in early stages of rejection, while sIgM(+) cells were higher than controls at day 0 and 5 in the rgIFNγrel 1 administrated group. Expression of IFNγ1 and IFNγ2 mRNA was significantly up-regulated by rgIFNγrel 1 administration, while that of IFNγrel 1 and IFNγrel 2 was not. These results suggest different contributions of the four IFNγ isoforms toward the immune responses comprising allograft rejection.

Cross-reactivity of monoclonal antibodies against CD4-1 and CD8α of ginbuna crucian carp with lymphocytes of zebrafish and other cyprinid species

ABSTRACT We have monoclonal antibodies (mAbs) against CD4‐1 (6D1) and CD8&agr; (2C3) in ginbuna crucian carp Carassius auratus langsdorfii. In our previous studies we showed that 2C3 mAb positive cells are the primary cell type showing specific cytotoxicity against allogeneic targets, suggesting that CD8&agr;+ lymphocytes in ginbuna are equivalent to cytotoxic T lymphocytes (CTLs) in mammals. We further demonstrated the helper T cell function of 6D1 mAb positive cells by studying mixed leukocyte culture (MLC) and hapten/carrier effects. Here, we report that our mAbs cross‐react with zebrafish lymphocytes. First, mAbs 6D1 and 2C3 recognized 7–11% of zebrafish lymphocytes that were ZAP‐70 positive and had the typical morphology of lymphocytes. Second, to verify the cell types reacting with the 6D1 and 2C3 mAbs we examined the expression profiles of zebrafish lymphocyte surface markers in FACS‐sorted lymphocytes from kidney. cd4‐1 (cd8a) and tcrac but not iglc transcripts were detected in 6D1(2C3)+ lymphocytes, whereas cd4‐1 (cd8a) transcripts were not found in 6D1 (2C3)‐ lymphocytes. Third, we further confirmed that 6D1 reacted with zebrafish CD4‐1 but not CD4‐2, and 2C3 recognized zebrafish CD8&agr; expressed on HEK293T cells. Collectively, these findings suggest that 6D1+ and 2C3+ lymphocytes in zebrafish are equivalent to CD4+ and CD8&agr;+ T lymphocytes in mammals, respectively. Furthermore, we found the cross‐reactivity of our 6D1 and 2C3 mAbs with other cyprinid species including goldfish, common carp and grass carp. HighlightsMonoclonal antibodies 6D1(anti‐CD4‐1) and 2C3 (anti‐CD8&agr;) of ginbuna carp cross‐react with zebrafish lymphocytes.MAb positive lymphocytes were all ZAP‐70 positive and had the typical morphology of lymphocytes.FACS‐sorted 6D1 and 2C3 mAb positive lymphocytes expressed cd4‐1 and d8a, respectively together with tcrac but not iglc.6D1 mAb reacted with zebrafish CD4‐1 but not CD4‐2, and 2C3 mAb recognized zebrafish CD8&agr; expressed on HEK293T cells.6D1 and 2C3 mAbs cross‐react with other cyprinid species including goldfish, common carp and grass carp.

A simple and non-invasive method for analyzing local immune responses in vivo using fish fin

&NA; Immunocompetence is an important parameter that reflects disease resistance in fish. Very few methods to examine immunocompetence in vivo have been developed, even for mammals. In the present study, we present a unique method for analyzing local immune responses using fish fin. We first demonstrated the migration of granulocytes to the site of zymosan injection in fin in a dose‐dependent manner and that this could be easily observed macroscopically due to the fin membrane transparency. We also demonstrated phagocytic activity of accumulated leukocytes after zymosan administration and that almost all phagocytes were granulocytes. In addition, we succeeded to detect respiratory burst activity of granulocytes in vivo without any in vitro treatment of cells, indicating that our present method is suitable for the analysis of granulocyte phagocytic function in vivo. The method provides a unique tool applicable for fishes that possess transparent fins and may lead to better understanding of the mechanisms of local immune responses in fish. HighlightsA simple and unique method for analyzing local immune responses using fish fin in vivo was developed.Migration of granulocytes to the site of zymosan injection in fin was observed in a dose‐dependent manner.Migration of granulocytes was easily observed macroscopically due to transparency of the fin membrane.In vivo phagocytic activity of granulocytes that migrated to the fin was observed after zymosan administration.Respiratory burst activity of granulocytes at the fin was also detected in vivo.

Lack of a contact requirement for direct antibacterial activity of lymphocyte subpopulations in ginbuna crucian carp.

Cytotoxic T lymphocytes (CTL) recognize and kill cells infected with viruses, intracellular bacteria and tumors with MHC restriction and antigen specificity. In addition to these activities, recent studies in mammals have suggested that CTL can exhibit direct microbicidal activity. In our previous study we documented direct antibacterial activity of CD4(+) T cells and sIgM(+) cells as well as CD8α(+) T cells from immunized fish. However, we also found weak non-specific killing activity of lymphocytes against bacteria. In the present study we further analyzed the weak killing activity of lymphocytes, increasing the effector cell to target bacteria ratio from 10:1 to 10(3):1. Sensitized and non-sensitized effector lymphocytes (CD8α(+), CD4(+) and sIgM(+)) separated by MACS were incubated with target bacteria. CD8α(+) T cells from Edwardsiella tarda-immunized ginbuna crucian carp killed 98%, 100% and 70% of E. tarda, Streptococcus iniae and Escherichia coli, respectively. CD8α(+) T cells from non-immunized fish showed similar but slightly lower killing activity than sensitized cells. CD4(+) and sIgM(+) lymphocytes also showed high killing activity against E. tarda and S. iniae as found for CD8α(+) T cells, although the activity was lower against E. coli. Supernatants from all three types of lymphocytes showed microbicidal activity, although the activity was lower than that evoked by effector lymphocytes. Furthermore, the presence of a membrane between effectors and targets did not affect the killing activity. The present results suggest that both sensitized and non-sensitized lymphocytes non-specifically killed target bacteria without the need of contact. The major difference between the present and previous experiments is the E:T ratio. We suspect that there are two different mechanisms in the direct bacterial killing by lymphocytes in ginbuna.

Peculiar Expression of CD3-Epsilon in Kidney of Ginbuna Crucian Carp

TCR/CD3 complex is composed of the disulfide-linked TCR-αβ heterodimer that recognizes the antigen as a peptide presented by the MHC, and non-covalently paired CD3γε- and δε-chains together with disulfide-linked ζ-chain homodimers. The CD3 chains play key roles in T cell development and T cell activation. In the present study, we found nor or extremely lower expression of CD3ε in head- and trunk-kidney lymphocytes by flow cytometric analysis, while CD3ε was expressed at the normal level in lymphocytes from thymus, spleen, intestine, gill, and peripheral blood. Furthermore, CD4-1+ and CD8α+ T cells from kidney express Zap-70, but not CD3ε, while the T cells from other tissues express both Zap-70 and CD3ε, although expression of CD3ε was low. Quantitative analysis of mRNA expression revealed that the expression level of T cell-related genes including tcrb, cd3ε, zap-70, and lck in CD4-1+ and CD8α+ T cells was not different between kidney and spleen. Western blot analysis showed that CD3ε band was detected in the cell lysates of spleen but not kidney. To be interested, CD3ε-positive cells greatly increased after 24 h in in vitro culture of kidney leukocytes. Furthermore, expression of CD3ε in both transferred kidney and spleen leukocytes was not detected or very low in kidney, while both leukocytes expressed CD3ε at normal level in spleen when kidney and spleen leukocytes were injected into the isogeneic recipient. Lower expression of CD3ε was also found in kidney T lymphocytes of goldfish and carp. These results indicate that kidney lymphocytes express no or lower level of CD3ε protein in the kidney, although the mRNA of the gene was expressed. Here, we discuss this phenomenon from the point of function of kidney as reservoir for T lymphocytes in teleost, which lacks lymph node and bone marrow.