This research involves the investigation of the mechanism of killing by natural killer (NK) and lymphokine activated killer (LAK) cells. Recently, LAK cells have been used in adoptive immunotherapy against cancer. Cytotoxic cells are an important factor in defense against parasitic and viral infections. In order to study the way in which these cells kill, the main protein responsible for the cytotoxicity C9RP, will be isolated and purified. Membranes of target cells killed with 125I-C9RP can be isolated to determine the final destination of the lytic molecule and to isolate the presumed C9RP lytic complex. Anti-C9RP linked to colloidal gold will be used to locate the protein by electron microscopy in LAK target cell conjugates. The search for additional factors will be undertaken by biosynthetically labelling LAK effector cells with 35S-methionine and incubating them with target cells for 4 hrs. The killed cells will then be isolated, solubilized, and run on SDS gels with subsequent autoradiography in order to identify LAK-cell derived, and therefore radioactive, cytotoxic factors. The mechanism of homologous species restriction of complement cytolysis will also be investigated. Complement is more efficient at lysing heterologous cells than it is at lysing autologous red cells. This phenomenon appears to be mediated by an erythrocyte membrane protein that we have designated homologous restriction factor (HRF). Preliminary evidence indicates that HRF is present on blood cells such as erythrocytes and PMN, but absent on M21 melanoma cells. Antibodies to HRF will be used to identify other cells, including endothelial cells that carry the protein. The quantitative interaction of membrane bound HRF and C9 will be examined using 125I-HRF and 131I-C9. Various domains in HRF will be identified by cleaving HRF, separating the fragments on SDS polyacrylamide gels and noting specific binding of radiolabelled C5, C6, C7, C8 or C9. HRF has been shown to protect erythrocytes from NK attack using the antibody-dependent cellular cytotoxicity reaction. We have shown that HRF is present on the surface of killing lymphocytes and may function to protect them from their own cytotoxic factors. This phenomenon will be studied further by inserting HRF into the membranes of target cells to see its effect on LAK-mediated cytotoxicity. A soluble form of HRF has been found in killing lymphocyte granules which is active in inhibiting LAK cell killing. We will study the interaction of the cytolytic molecule C9RP with the protective molecule HRF in order to understand HRFs role in lymphocyte self-protection.
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| Zalman, L S; Muller-Eberhard, H J (1990) Comparison of channels formed by poly C9, C5b-8 and the membrane attack complex of complement. Mol Immunol 27:533-7 |
