Target cell killing by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells represents an important defense barrier against proliferation of tumor cells, virus-infected cells, and other foreign agents. Intimate surface contact between effector and target cells is required to trigger lysis. This project is focused on the dissection of the cytolytic mechanisms/mediators involved in this killing event. A granule exocytosis model for lymphocyte-mediated killing has previously been postulated that involves the release of granule contents from lymphocytes onto the target cell surface. A granule pore-forming (PFP) thought to be released during cell killing has already been identified. The lymphocyte PFP to be structurally/functionally/immunologically related to the 9th component of the complement cascade (C9), which draws paradoxal similarities between the cell-mediated and humoral killing. We plan to purify both murine and human PFP to homogeneity for (i) partial sequencing, (ii) derivation of monospecific polyclonal and monoclonal antibodies, and (iii) characterization of its mechanism of pore assembly though reconstitution of PFP into high impedance voltage-clamped planar lipid bilayers. Monoclonal antibodies directed against PFP will be useful tools in mapping the antigenic epitopes shared by PFP and C9. Specific anti-PFP antibodies will also be used in tissue localization of CTL/NK cells and in assessing the relevance of PFP in the killing mediated by both primary cytotoxic cells and cytolytic cell lines. The partial sequence information will be used for molecular cloning of PFP. cDNA libraries of murine CTL and human NK cells will be screened with oligonucleotide probes and antibodies specific for PFP and C9. Peptides will be constructed from the known sequence information of relevant cDNA clones and tested in their ability to mimic given functions of the native protein (membrane binding, insertion and pore-formation). We will pursue on the identification, isolation and characterization of an """"""""immunity"""""""" polypeptide on the surface of lymphocytes thought to complex with PFP rapidly in the of the membrane, thus preventing its further aggregation to form a functional pore and that would explain how lymphocytes are spared of self-kill. We will also design assays to isolate and characterize a second cytotoxin recently identified in lymphocyte granules that is distinct from PFP. Characterization of this novel cytotoxin and assessment of its lytic role may lead to a much more complex concept of cell-mediated killing, evoking multiple mechanisms/mediators.
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