Kinetic analysis of cloned cytotoxic T lymphocyte reactivity against normal and leukaemic target cells.

Abstract

The kinetics of unrestricted killing of normal and leukaemic lymphocyte target cells by a Qa-1b-specific murine cytotoxic T lymphocyte (CTL) clone were evaluated in a manner analogous to enzyme kinetic assays in which the effector and target cells corresponded to the enzyme and substrate, respectively. In order to apply the enzyme-substrate analogy to clonal cytolytic reactions, it was first established that the lytic reactions exhibited initial steady-state velocity of lysis at the effector and target cell concentrations used. The lytic reaction maintained linearity for velocity of lysis during the first 90 min of incubation, then plateaued. Vmax (the maximal rate of target cell lysis achieved by a given effector population) and Km (the target cell number resulting in 1/2Vmax) values were determined over a wide range of target and effector cell concentrations. Both parameters were found to be directly proportional to the number of effector cells. At a given concentration of cloned CTL, the lytic parameters of Vmax and Km were not significantly different for normal or leukaemic target cells that express Qa-1b. Additional kinetic parameters for lysis of normal and leukaemic target cells by a cloned CTL were also compared. The lytic efficiency of the CTL clone (i.e. maximal killing rate with an infinite number of targets) and the intrinsic affinity between effector and target cells were the same with either normal or leukaemic targets. However, the maximal lysis of target cells at an infinite number of effectors was significantly less for normal compared with leukaemic targets. This suggests that the normal target cells were more heterogeneous in their expression of the target (Qa-1b) antigen. Enzyme-like kinetic analysis of cell-mediated lysis reactions can be useful for comparing the relative affinities of effector and target cells obtained from various sources.