FLOW CYTOMETRIC ANALYSIS ON PERFORIN INDUCTION IN PERIPHERAL-BLOOD MONONUCLEAR-CELLS WITH INTERLEUKIN-2 OR OK-432

Perforin is a protein present in the cytoplasmic granules of killer cells and is considered to be an important effector molecule. We assessed the perforin appearance via flow cytometry in human peripheral blood mononuclear cells stimulated in vitro for 3 days by recombinant interleukin-2 (rIL-2) or OK-432, a biological response modifier. The relationship between the lymphocyte subsets and perforin was investigated via two-color assay. CD4-positive cells had almost no perforin, and most of the CD16-positive cells did. Regarding the relationship with CD8, some of the bright positive cells (which were likely T cells) and most of the dull positive cells (likely NK cells) had perforin. Mean fluorescence was greatest in perforin-positive cells incubated with rIL-2, less in cells incubated with OK-432, and minimal in cells incubated in a medium without additives. Immunohistochemical staining with antiperforin antibody revealed that blast-transformed and enlarged cells were stained positively and that the intensity of staining of each cell alone was enhanced in cells incubated with OK-432 or rIL-2. If the fluorescence intensity of perforin-positive cells correlates with the amount of perforin in those cells, then the appearance of perforin was enhanced with OK-432, more enhanced with rIL-2, and consistent for cytotoxicity against K562 and Daudi cells. IL-2 was induced by OK-432, suggesting that the indirect effect of this IL-2 may play a role in OK-432-perforin induction. The results suggest that perforin may be an effector molecule in killer cells induced by rIL-2 or OK-432.