Recent technical advances in tissue culture methodology allow for the analysis of T-cell mediated antitumor immunity on a clonal level and have enhanced the feasibility of using culture-expanded or cloned cytotoxic T-lymphocytes (CTL) for immunotherapy. As a result, there is significant need for methods to precisely quantitate cellular cytotoxicity in order to compare various heterogeneous and cloned CTL populations. Lytic efficiency of CTL can be quantitated using methods developed for the study of enzyme catalyzed reactions. For this type of kinetic analysis, the effector and target cells are analogous to the enzyme and substrate, respectively. The objective of this research is to apply enzyme-like kinetic analysis to the study of effector/target cell interactions with the goal of using this analysis to identify CTL with high-target affinity. This analysis may be of predictive value in determining which CTL population would be useful for adoptive immunotherapy of leukemia. Preliminary data have demonstrated the applicability of enzyme-like kinetic analysis to the study of cloned CTL lysis of normal and leukemia cells. Using these methods, the affininty of CTL effector populations for leukemic and normal target cells will be quantitated. Data generated by kinetic analysis of lysis will be used to quantitate the maximum rate of lysis, the lytic efficiency of effector population, and the susceptibility of target cells to lysis (i.e., to define the extent of target cell heterogeneity). Heterogeneous and cloned CTL populations with different functional characteristics (e.g., T-helper dependent versus T-helper independent) and/or antigen specificity (e.g., class I or class II antigen-specific, H-2-restricted or -unrestricted, etc.) will be tested. The effect of variation in the density of target antigen or restricting element on lytic parameters also will be evaluated. Inhibition or enhancement of lysis by the presence of drugs, lymphokines, antibodies, or by-stander cells that lack the appropriate target antigen will be studied and quantitated for comparison purposes. The type of inhibition (competitive versus noncompetitive) will be defined using lytic parameters. Finally, CTL with high affinity for leukemic and/or normal cells will be tested using bioassay techniques to determine if kinetic parameters of lysis can be used to predict antileukemic or antihost reactivity in vivo. (LB)
