A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeated dose toxicity studies

A recent regulatory document for immunotoxicity testing of new pharmaceutical drugs includes cytotoxic natural killer (NK)-cell function as a required parameter in repeated dose toxicity studies. The classical 51Cr-release assay is the conventional test for cytotoxicity testing but several drawbacks with this assay has increased the demand for new reliable test systems.Here, we describe the optimisation of a flow-cytometric cytotoxicity assay especially adapted for regulatory rat studies in drug development. The test principle is based on target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The final format of the assay includes 0.5ml peripheral blood, 1.25x10(5) effector cells per sample, and collection of 500 target events by flow-cytometry. When NKR-P1+ cells were removed from the effector cell population by magnetic depletion the relative proportion decreased from 6 to 0.08%. The corresponding cytotoxic activity decreased from 68 to 8%. Also, the cytotoxic activity showed a significant and positive correlation with the proportion of NK-cells present in the effector cell suspension. Thus, the cytotoxicity measured is almost exclusively exerted by NK-cells. The current flow-cytometric test benefits from using peripheral blood as a source for effector cells since it will not conflict with the use of spleen for histopathological investigations in repeated dose toxicity studies. Additionally, since only a minimal number of effector cells are required per sample repeated testing of the same animal is enabled.