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.