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.