Natural Killer Cell Functions and Subsets After In Vitro Stimulation with IL‐2 and IL‐12, with Special Emphasis on Intracellular IFN‐γ and NK‐Cell Cytotoxicity

Nyboe, R.; Rix, T.; Krog, J.; Tønnesen, E.; Hokland, M.

Materials and methods: Isolated cryopreserved human peripheral blood mononuclear cells (PBMCs) were stimulated with IL‐2 and IL‐12. This stimulation has previously been shown to activate NK cells. Cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. This method was compared to the current standard 51Cr release assay. Cells were treated with BFA to accumulate IFN‐γ, stained for surface markers, permeabilized and stained for intracellular IFN‐γ. Flow cytometry was then performed to measure intracellular IFN‐γ production in PBMC, especially in NK cells. Results: We have demonstrated that stimulation with IL‐2 and IL‐12 is effective in increasing the number of IFN‐γ‐positive cells. There is a distinct difference between the CD3‐CD56dim and the CD3‐CD56bright subsets, with a much greater proportion of IFN‐γ‐positive cells in the CD3‐CD56bright subset. The effects of stimulation with IL‐2 and IL‐12 on cytotoxicity will be presented, as will the relation between IFN‐γ production and cytotoxicity. In addition, we will present results of these assays applied to porcine cells. Discussion: In combination, these tests will address NK cell function by combining cytotoxicity with IFN‐γ production in NK cell subsets. The results will demonstrate whether this could serve as a useful tool in describing NK‐cell function, which could be of value in clinical and experimental settings.