Fig 1.
Loss of NK cell activation receptor expression following tumor-priming.
(A) Freshly isolated NK cells were incubated in medium alone, with K562, CTV-1, Daudi, RPMI-8226, or MCF-7 cells or with IL-2/12/15/18 (individually or in combination) overnight at 37°C. Thereafter, cells were washed. NK cell activation receptor expression was analyzed using flow cytometry. The percentage change of NK cell expression was calculated for each receptor after stimulation and results are presented as the mean of 3–5 different donors. Red indicates an increase, and green indicates a decrease in receptor expression, according to the key on the right, relative to NK cells in medium alone. (B) Freshly isolated NK cells were incubated in medium alone or with CTV-1 cells overnight at 37°C. Cells were washed, the surface expression of 20 different NK cell activation receptors was analyzed. Bars represent the means ± SD of 5 different donors. The percentages of NK cell expression of different receptors after stimulation was compared with those in cells treated with medium alone using the paired t-test. Statistical significance is indicated as: *P <0.05; **P <0.01; ***P <0.001.
Fig 2.
Influence of CTV-1 cell priming on pro-inflammatory cytokine secretion by NK cells.
(A) Freshly isolated NK cells were incubated alone or with CTV-1 cells for 6 hours at 37°C. Supernatants were harvested, and the concentrations of 25 different cytokines and chemokines were determined using a multiplex immunoassay. Bars represent means ± SD of 4 different donors. Post-stimulation cytokine secretion by NK cells was compared with that of cells incubated in medium alone using the paired t-test. Statistical significance is indicated as: *P <0.05; **P <0.01; ***P <0.001. (B) NK cells were incubated with CTV-1 cells at 37°C. Supernatants were harvested at different time points, as indicated, and the concentrations of IFN-γ, TNF-α, MIP-1α, MIP-1β, and RANTES were determined by a multiplex immunoassay. Values represent mean ± SD of 4 different donors.
Fig 3.
Influence of tumor- or cytokine-priming on cytokine secretion by NK cells.
NK cells were incubated in medium alone, with K562, or CTV-1 cells for 6 hours or with IL-2 or IL-12 overnight at 37°C. Supernatants were harvested, and the concentrations of IFN-γ, TNF-α, MIP-1α, MIP-1β, RANTES, IL-R2α, IL-1α, and IL-1β were determined by a multiplex immunoassay. Bars represent means ± SD of 4 different donors. Post-stimulation NK cell cytokine secretion was compared with that in cells incubated in medium alone using the paired t-test. Statistical significance is indicated as: *P <0.05; **P <0.01; ***P <0.001.
Fig 4.
Differential influence of tumor cell or cytokine priming on the transcriptome of NK cells.
(A) Schematic of the workflow in obtaining NK cell gene expression profiles after tumor cell or cytokine priming. Human NK cells were isolated from peripheral blood and incubated in medium alone, mitomycin-C-treated K562, or CTV-1 for 6 h or with IL-2 overnight. NK cells were enriched from co-cultures, and RNA extraction and gene expression profiling were performed using RNA-Seq or the NanoString nCounter FLEX platform. (B) Hierarchical clustering was applied to the gene expression values, and the top differentially expressed genes are shown in a heat map with rows representing the relative expression of each gene across 12 samples. Red corresponds to higher gene expression, and blue corresponds to lower gene expression. Similarity in gene expression patterns between different samples are reflected in the resulting dendrogram. Z-Score is calculated using the following formula: (gene expression value in sample of interest)—(mean expression across all samples) / SD. Venn diagrams generated by the intersection of list of genes upregulated (C) or downregulated (D) by at least 1 Log2fold (P <0.05) vs. NK cells incubated in medium alone are shown.
Fig 5.
Top cell signaling network induced in tumor-primed NK (TpNK) cells as generated by Ingenuity pathway analysis.
Nodes represent genes that are either upregulated (red), downregulated (green), or absent from our original differential expression analysis (white), but play an important role in the tumor-specific signaling network according to Ingenuity Pathway Analysis. The intensity of node color signifies the extent of change in expression of the respective gene relative to NK cells incubated in medium alone. Node shapes and the relationships between different genes are outlined in the figure legend.
Table 1.
NK cell gene expression exclusively affected by CTV1- or IL-2-priming.