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Cytotoxic lymphocytes have been shown to be a potent immunological mechanism for the highly specific destruction of virus-infected and foreign cells, and most current immunological approaches to cancer therapy envision stimulating the differentiation of cytotoxic T lymphocytes with the ability to selectively recognize tumor cells. We have been studying the basic mechanism by which cytotoxic lymphocytes kill their target cells in vitro, and have developed the now well accepted granule exocytosis model for the lethal mechanism. The model proposes that cytotoxic lymphocytes rapidly secrete preformed mediators in response to target binding, utilizing a modified form of the regulated secretory process found in many cell types. However, the details of how proteins secreted by the killer lymphocyte cause target cell death remain to be elucidated. While we initially focussed on the pore-forming properties of the lymphocyte cytolysin/perforin molecule, we have recently found that the secreted serine proteases known as granzymes enter the target cell cytoplasm where they activate a resident apoptotic death pathway. We studied the properties of model cytotoxic lymphocytes created by transfecting the rat mast cell tumor RBL to express components of cytotoxic lymphocyte granules. A combination of cytolysin/perforin, granzyme A and granzyme B endowed these originally non-cytotoxic cells with a cytotoxic activity close to cloned cytotoxic T lymphocytes, including apoptotic target cell damage. Cytotoxic activity required cytolysin expression, and showed a synergism between granzymes A and B expression. Using enucleated target cells, we further showed that the nucleus is not required for the apoptotic target cell death due to secreted granzymes.
We have also been defining the molecular events of apoptotic death in T lymphocytes, particularly the role of two different types of cytoplasmic cysteine proteases. In the Fas-mediated death pathway triggered by T cell receptor crosslinking in T cell blasts, the TcR triggers the upregulation of Fas Ligand, which in turn crosslinks Fas. We have shown the calcium-dependent protease calpain is required for upregulation of Fas Ligand mRNA, based on selective blocking by calpain inhibitors including the highly specific protein calpastatin (expressed by transfection). We have also demonstrated a novel Fas-independent route of antigen-triggered T cell death by showing that T blasts are killed by TNF and lymphotoxin, cytokines secreted by activated T cells.
Developmental cell death in the nematode C. elegans requires the death gene ced-3, which codes for an intracellular cysteine protease cleaving proteins at aspartic acid. This protease is structurally and functionally related to the mammalian interleukin-1Beta; converting enzyme (ICE) and several other recently described human proteases. There is increasing evidence for a functional role for such ICE-like proteases in apoptotic death of mammalian cells. We are currently assessing their role in T lymphocyte programmed cell death and their involvement in death triggered by cytotoxic lymphocyes.