Figure 1. Generation of memory-like NK cells with enhanced IFNγ production by in vivo interleukin pre-activation and re-stimulation.

Rag1KO B6 mice were divided into three groups: (a) the IL re-stimulation group, in which mice received via tail vein a combination of IL-12 (4.5 μg/mouse), IL-15 (5.5 μg/mouse), and IL-18 (22 ng/mouse) for pre-activation, after three weeks, further received IL-12 (5 μg/mouse) and IL-15 (8 μg/mouse) for re-stimulation; (b) the negative control group, in which mice only received pre-activation as described above. For these two groups, NK cells were harvested from the spleen of the mice in next day after in vivo IL pre-activation and/or re-stimulation and subjected to further flow cytometric analyses; and (c) the positive control group, NK cells from the spleen of Rag1KO B6 donor mice were in vitro pre-activated with IL-12 (10 ng/mL), IL-15 (10 ng/mL), and IL-18 (50 ng/mL) overnight, after which NK cells were labeled with CFSE and adoptively transferred into Rag1KO B6 recipient mice. Three weeks later, enriched NK cells (2 × 10⁶) from spleen were harvested by negative selection and in vitro re-stimulated with IL-12 (10 ng/mL) and IL-15 (100 ng/mL) for 4 hrs, after which cells were further analyzed by flow cytometry. (A) Experiment schema for three groups. (B) The percentage of NK cell proliferation was analyzed by flow cytometry. (●) the negative control group; (○) the IL re-stimulation group; (▲) the positive control group. (C) The percentage of IFNγ⁺ NK cells in three groups. (D) Equal numbers (2 × 10⁶) of CFSE-labeled NK cells from the spleen of Rag1KO B6 (CD45.2+) mice pre-activated and/or re-stimulated with ILs as above were adoptively transferred to syngeneic B6 (CD45.1+) recipient mice, after which NK cells were harvested from peripheral blood of the recipients every week. Donor NK cells were distinguished from recipient NK cells using anti-A20 antibody. In A20⁻ donor NK cells, the percentage of NK cell proliferation were determined by flow cytometry at the indicated intervals after the adoptive infusion. (■) the negative control group; (□) the IL re-stimulation group. (E) In parallel, the percentage of IFNγ⁺ NK cells were also determined in A20⁻ donor NK cells. (■) the negative control group; (□) the IL re-stimulation group. Values indicate mean ± SD (n = 10 per group, *** p < 0.0001).