Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in ginbuna crucian carp, Carassius auratus langsdorfii

T cell-mediated cytotoxicity occurs via pathways based on perforin or Fas mechanisms. Perforin is a protein present in the cytoplasmic granules of CD8⁺ cytotoxic T lymphocytes and is secreted to form pores on target cell membranes. In fish, although the involvement of perforin in cytotoxicity have been suggested for several species, perforin-mediated cytotoxicity of CD8α⁺ lymphocyte in conjunction with expression of the perforin gene has not been reported. In order to investigate the killing mechanism of CD8α⁺ lymphocytes by perforin-mediated pathway in fish, we measured apoptosis of target cells triggered by CD8α⁺ lymphocytes, performed cytotoxic assays in the presence or absence of perforin inhibitor; concanamycin A and EGTA, and analysed the expression of perforin1, perforin2 and perforin3 isotypic genes in ginbuna crucian carp. In the present study, we found that CTLs attached with target cells. CTL should have direct contact with target cells to kill them. Approximately 50% of target cells were positive for annexin V after co-cultured with CD8α⁺ lymphocytes, indicating the induction of apoptotic cell death. Concanamycin A, which induces depolymerization of perforin resulting in lytic function, suppressed the cytotoxicity of CD8α⁺ cells in a dose-dependent manner. In addition, cytotoxicity mediated by CD8α⁺ lymphocytes were significantly suppressed by the addition of the Ca²⁺-chelating agents EGTA or EGTA–Mg²⁺, and the addition of Ca²⁺ restored the killing mechanism of target cells. We further found enhanced expression of perforin1 but not perforin2 or perforin3 in CTLs from allo-sensitized fish. The present study has demonstrated that ginbuna CTLs kill target cells through perforin-mediated pathway, suggesting that perforin-mediated pathway is conserved throughout vertebrate.