BIOPHYSICAL BASIS OF CELL KILLING BY CTL

The objectives of this research are to elucidate the mechanism of cell killing by cytotoxic T lymphocytes (CTL), an important phenomenon in cellular immunology. The specific aims are: (1) to observe microscopically the dynamic morphological changes of CTL and target cells following controled onset of conjugation of a pair of single cells; (2) to determine the biophysical properties of CTL and target cells on a single-cell basis and their interaction during killing; and (3) to assess the effects of biochemical and immunochemical modifications on the biophysical properties of CTL and their correlation with the killing process. The experiments will be performed on two types of cell systems: (1) a human cytolytic T-cell line (F1) with JY (HLA-A2[unreadable]+[unreadable], -B7[unreadable]+[unreadable], DR4,6[unreadable]+[unreadable]) B-lymphoblastoid cell lines as target cells, and (2) a mouse cytotoxic T-lymphocyte hybridoma with EL4 cells as target cells. The biophysical properties of the CTLs and their specific target cells will be determined individually and also during their interaction in the killing process. These tests will be made by using the micropipette aspiration technique already established in our laboratory for investigations on various types of blood cells including T lymphocytes. With the aid of a video recording and analysis system and computer assistance, the viscoelsstic properties of these cells will be derived from their deformational behavior in response to an aspiration pressure applied via the micropipette. By using two micropipettes, one to hold a CTL and the other a target cell, these cells will be brought into contact, thus controlling the time of onset for conjugation. Subsequent measurements will provide dynamic information on the biophysical states of both cells throughout the course of the various phases of the killing process. The energy of interaction between the two cells will be estimated from the forces required to separate them. These biophysical measurements will also be made following several types of treatments to modify the properties of the CTL and/or target cells, including the blockade of cytotoxicity by using monoclonal antibodies, modifications of cell surface charge, ionic environment, cytoskeletal apparatus and lipid composition, variations in temperature, and induction of membrane capping. Correlation of the results of these biophysical studies with the biochemical events studied by other investigators will serve to increase our understanding of the chemico-mechanical transduction process in cell killing by cytotoxic T lymphocytes. (LB)