Fig. 5
The sustained existence of the memory-like NK cells induced by the combination of STING-LNPs and CpG-ODNs resulted in prolonged protection against the development of melanoma lung metastasis. (A) The prophylactic effect of monotherapies compared with that of combination therapy against B16-F10 lung metastasis. Mice were intravenously injected with either PBS, the STING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy for two cycles. After 10 days, mice were intravenously injected with 2 × 105 B16-F10-Luc2 cells. Representative photos show the lungs of the different treatment groups, and quantitative analysis of the lung metastasis was based on the values of luciferase activities. The values represent the mean ± SEM (n = 3–5, *P < 0.05). (B) The sustained persistence of the memory-like NK cells induced by STING-LNPs and CpG-ODNs vaccination. Mice were intravenously injected with either PBS or a combination of STING-LNPs (4 µg/mouse of c-di-GMP) and CpG-ODNs (250 µg) for two cycles. After 20 days one group of mice was intravenously injected with (2 × 105) B16-F10-Luc2 cells. (C) The percentage of CD11b+ SP (CD11b+ CD27−) NK cells on the indicated days after vaccination. Upper graph: cell percentage among days for the SP CD11b+ NK vaccinated group. Bottom graph: comparison of the SP CD11b+ NK cell percentage of the vaccinated group with that of the PBS group on each termination day. The values represent the mean ± SEM (n = 3–4, **p < 0.01). (D) The FI (Median) of KLRG1 expression on CD11b+ SP (CD11b+ CD27−) NK cells on the indicated days after vaccination. Upper graph: comparison of the KLRG1 expression level of SP CD11b+ NK cells for the vaccinated group among days. Bottom graph: KLRG1 expression levels of SP CD11b+ NK cells comparing the PBS and vaccinated groups on each termination day. The values represent the mean ± SEM (n = 3, ****p < 0.0001; **p < 0.01; *p < 0.05). (E) Prolonged vaccination effect following two cycles of the STING-LNPs and CpG-ODNs. Mice were intravenously injected with either PBS, the STING-LNPs (4 µg/mouse of c-di-GMP), CpG-ODNs (250 µg), or the combination therapy for two cycles. After 20 days, mice were intravenously injected with B16-F10-Luc2 cells (2 × 105). Representative photos show the lungs of different treatment groups, and quantitative analysis of the lung metastasis was based on the values of luciferase activities. The values represent the mean ± SEM (n = 4–5, *P < 0.05)