Mechanism of Cell-Mediated Cytotoxicity at the Single Cell Level

Abstract

Abstract A single cell method has been developed for directly measuring the frequency of cytotoxic T lymphocytes (CTL). The method is based on the ability of CTL to bind corresponding target cells and to form CTL-target conjugates visible by microscopy. Conjugates are incubated at 37°C in agarose to prevent CTL recycling. Single target lysis is assessed by trypan blue uptake. Target cell lysis was evident as early as 15 min and reached a plateau at 2 hr with an average of 60 to 80% of the conjugated target cells lysed. In a population of nylonwool nonadherent BALB/c peritoneal exudate T lymphocytes sensitized to C57BL/6 alloantigens, the frequency of CTL was estimated to range from 12 to 24% of the total T lymphocytes. The frequency of CTL and the kinetics of lysis were a function of the state of immunity, the method of sensitization, and the target cell employed. The single cell method described here has several features, namely, its independence of CTL recycling, the elimination of the variability in 51Cr release by target cells, and its prevision for a direct estimate of CTL frequency. Furthermore, the method is simple, reproducible, highly quantitative with very low numbers of CTL, and adaptable to non-T cell-mediated cytotoxic systems.