Fig 2: IL-12/15/18 preactivated CD3+CD56+ NKT-like cells enhance the intrinsic potential and show an increase in IFN-γ release when stimulated by K562 tumor cells. (a) Percentages of IFN-γ+ TNF-α+ and Ki67+ cell populations by flow cytometry plots. Expression of IFN-γ, TNF-α, and Ki67 in cells restimulated on day 7 by cell activation cocktail. n = 8, ∗P > 0.05,∗∗P < 0.01,∗∗∗P < 0.001 (error bars, mean ± SEM).(b) The gating strategy for percentages of IFN-γ+ cell populations cocultured with K652 on Day 7; an overlaid histogram shows increased expression of Ki67 and enhanced level of GzmB. n = 8. (c) Overlaid histograms show expression of IFN-γ, Ki67, and GzmB cocultured with K562 on day 7 by different concentrations of cytokines preactivating.

Fig 3: Enhanced inhibitory function of Tregs from DLBCL patients. A Cytotoxic activity of activated CD8+CTLs cocultured with or without Tregs from DLBCL patients (D-Treg) and healthy donors (N-Treg) toward CFSE-labelled SU-DHL4 cells at the E/T ratios of 10:1. The percentage of PI+ cells were used to calculate the % lysis of target cells by CD8+ CTLs. n = 3. B PI staining of SU-DHL4 cells from the co-culture system of CD8+CTLs with Tregs from DLBCL patients (D-Treg) and healthy donors (N-Treg), or without Tregs at the E/T ratios of 5:1,10:1 and 20:1. n = 3. C, D Proliferation of CD8+ CTLs was determined by coculturing CD8+ CTLs with or without Tregs from DLBCL patients (D-Treg) and healthy donors (N-Treg) for 3 days. The percentage of Ki67+ cells was used to calculate proliferation of CD8+ CTLs. n = 3. E, F Intracellular expression of GzmB in CD8+ CTLs cocultured either alone or with Tregs from DLBCL patients (D-Treg) and healthy donors (N-Treg) for 3 days. n = 3. G, H CD107a surface expression on CD8+ CTLs exposed to SU-DHL4 cells alone or in the presence of Treg cells from DLBCL patients (D-Treg) and healthy donors (N-Treg) for 4-5 h. n = 3. *P < 0.05; **P < 0.01, ***P < 0.001. Asterisk indicates significant difference from control group as assessed. Pound sign indicates significant difference from different Treg group. p values were determined by one-way ANOVA; data are presented as mean values ± SEM

Fig 4: Trajectory analysis reveals the developmental pathway of cytokine-trained NK cells. (A) Analysis of day 7 NK cells with Monocle2 pseudotime trajectory analysis to simulate the ontology (left) and positions of cells in all 4 clusters (right). (B) Individual clusters of day 7 NK cells are demonstrated in the trajectory. (C) Heatmap of selected gene expression profiles along the pseudotime trajectory. Group 1 genes, including B3GAT1, ITGA5 (CD49e), and KLRK1 (NKG2D), were more highly expressed at the late developmental stage. Group 2 genes, including PRF1, FCGR3A (CD16), and KLRF1 (NKp80), were more highly expressed at the late-middle stage. Group 3 genes, including SELL (CD62 L) and IL7R, were more highly expressed at the early-middle stage of development. Group 4 genes were expressed at an early stage of the developmental pathway, which was cluster #6. The group 5 gene GZMB showed higher expression at the initial and late developmental stages, and the group 6 gene ITGAM (CD11b) showed higher expression at the middle and late stages of development.

Fig 5: NK cells with trained immunity exhibit enhanced effector responses. (A) Overview of the experimental design. PBMCs were preactivated with IL-12 (10 ng/mL), IL-15 (1 ng/mL), and IL-18 (50 ng/mL), or control (1 ng/mL of IL-15 alone) for 16 h, washed, and then cultured under low concentrations of IL-15 (1 ng/mL) for differentiation. Flow cytometry analysis was performed at the indicated time points after preactivation. (B) Left panel: flow cytometry plot showing the gating strategy for CD3−CD56+ NK cells; right panel: histogram showing enhanced IFN-γ production by cytokine-trained NK cells restimulated with IL-12/18 on day 7. (C) Increased killing of K562 leukemia target cells by cytokine-trained NK cells on day 7. (D) Upper panel: representative flow cytometry data showing increased basal levels of granzyme B (GzmB) and expression of TNF-α, IFN-γ and Ki67 in cytokine-trained NK cells restimulated on day 7 by PMA+ionomycin; lower panel: summary data showing the median fluorescence intensity (MFI) of GzmB, TNF-α, IFN-γ, and Ki67. For all experiments, n = 7, *P < 0.05, **P < 0.01, and ***P < 0.001 (error bars, mean ± SEM). Data are representative of at least 3 independent experiments.