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Abstract
Cytotoxic lymphocytes induce target cell death using common effector pathways that involve either the components of their cytotoxic granules or members of TNF superfamily ligands. We present the first evidence for the existence of both granule exocytosis as well as TNF family ligand mediated killing in cytotoxic cells from ectothermic vertebrates. Nonspecific cytotoxic cells (NCC) are the first identified killer cell population in teleosts. This study presents the molecular evidence for expression of multiple granzymes with different substrate specificities as well as other components of cytotoxic granules in NCC, providing the sequence information for such molecules for the first time in a non-mammalian species. The main substrate specificities for granzymes found in these cells were chymase and tryptase. Teleost granzymes conserve the genomic organization described for mammalian granzymes. Teleost granzymes with chymase activity have novel S1 specificity triplet residues. This study also demonstrates the role of TNF-alpha in NCC functions as an effector molecule of cytotoxicity and in regulation of NCC functions. Teleost cytotoxic cells express both membrane-bound and secreted forms of TNF-alpha, which can induce cell death in susceptible target cells. Membrane-bound TNF-alpha on NCC can mediate activation-induced cell death in other NCCs in a paracrine manner, while the soluble TNF-alpha can inhibit this and protect the killer cells from activation-induced cell death. Soluble TNF-alpha upregulates the expression of other cytotoxic effector molecules like granzymes in teleost killer cells, indicating a major role played by this molecule in NCC functions. TNF-alpha-induced protection of NCC was shown to be mediated though different regulators of apoptosis. One of the important mediators of such function was identified as cellular apoptosis susceptibility protein, which is upregulated upon treatment of NCC with recombinant TNF. These findings suggest a parallel evolution of cell-mediated cytotoxicity in teleosts, leading to effector functions comparable to mammalian counterparts.