BIOPHYSICAL BASIS OF CELL KILLING BY CTLS

The objective of the proposed research is to elucidate the mechanism of cell killing by cytotoxic T lymphocytes (CTL), an important phenomenon in cellular immunology. Experiments will be performed on single cell basis, by using micropipette- micromanipulation, video recording and computer analysis, to determine the junction avidity of CTL and target cell (TC) conjugates, the biophysical properties of these cells individually and also during their interaction in the killing process, and the changes in cell nuclei. Studies on junction avidity will be performed with two micropipettes to hold separately a CTL and a TC, thus allowing the control of the time of onset for conjugation. These experiments will be performed by using appropriate TC with or without antigen expression induced by gamma-interferon, and non-appropriate target cells resulting from activation (ConA or PHA) of CTL. The energy of interaction will be estimated from the forces required to separate the two cells. The role of cytoskeleton organization in affecting the biophysical properties (viscosity and elasticity) of CTL and TC will be determined by using fluorescent labeled monoclonal antibodies (mAbs). The number and kinds of membrane molecules in the interfacial area of CTL and TC conjugates will be determined by using fluorescent and radioactive labeled mAbs. Together with the determination of the force required to separate the CTL-TC conjugate and using fluorescent labeling and inhibition of cytotoxicity with mAbs, these experiments will allow the estimation of the conjugation force per molecule in the CTL killing process. The membrane molecules involved in the interdigitation, lethal hit programming, or secretion in CTL-TC conjugation will be determined by labeling mAbs with different fluorescent compounds. The effects of biochemical modification (nonspecific) of TC membrane and genetic modification of antigen expression on the TC by DNA- mediated gene transfer (DNA-MGT) or exon shuffling (specific modification) on the juntional avidity of CTL-TC conjugates and the biophysical properties of CTL and TC will be assessed during the killing process. Correlation of the results of these biophysical studies with the immunochemical events studied by other investigators will serve to increase our understanding of the chemico-mechanical transduction process in cell killing by CTL.