Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response. 3,4-dichloroisocoumarin (DCI), which inhibit the activity of serine protease including all members of the granzyme family, markedly suppressed the cytotoxic activity of CTL. However, CTL-mediated cytotoxicity was significantly but not completely suppressed by the addition of carbobenzyloxy-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK) that specifically blocks granzyme B activity. These results suggest that additional serine proteases as well as granzyme B-like are involved in cytotoxicity of CTL in fish. We further compared cytotoxicity with the granzyme B-like hydrolytic activity against fluorogenic substrate acetyl-Ile-Glu-Thr-Asp-4-methylcoumaryl-7-amide (Ac-IETD-MCA) and found that granzyme B-like activity correlated well with the cytotoxicity of CTL in ginbuna crucian carp. Present results suggest that the granzyme activity assays is useful to assess cytotoxic activity of CTL in fish in which genetic information on granzymes and specific tools for cytotoxicity assay are not available because of well conserved catalytic triad residues and substrate binding sites in granzyme B throughout vertebrates.