Tumor- and cytokine-primed human natural killer cells exhibit distinct phenotypic and transcriptional signatures

Abstract

An emerging cellular immunotherapy for cancer is based on the cytolytic activity of natural killer (NK) cells against a wide range of tumors. Although in vitro activation, or "priming," of NK cells by exposure to pro-inflammatory cytokines, such as interleukin (IL)-2, has been extensively studied, the biological consequences of NK cell activation in response to target cell interactions have not been thoroughly characterized. We investigated the consequences of co-incubation with K562, CTV-1, Daudi RPMI-8226, and MCF-7 tumor cell lines on the phenotype, cytokine expression profile, and transcriptome of human NK cells. We observe the downregulation of several activation receptors including CD16, CD62L, C-X-C chemokine receptor (CXCR)-4, natural killer group 2 member D (NKG2D), DNAX accessory molecule (DNAM)-1, and NKp46 following tumor-priming. Although this NK cell phenotype is typically associated with NK cell dysfunction in cancer, we reveal the upregulation of NK cell activation markers, such as CD69 and CD25; secretion of pro-inflammatory cytokines, including macrophage inflammatory proteins (MIP-1) α /β and IL-1β/6/8; and overexpression of numerous genes associated with enhanced NK cell cytotoxicity and immunomodulatory functions, such as FAS, TNFSF10, MAPK11, TNF, and IFNG. Thus, it appears that tumor-mediated ligation of receptors on NK cells may induce a primed state which may or may not lead to full triggering of the lytic or cytokine secreting machinery. Key signaling molecules exclusively affected by tumor-priming include MAP2K3, MARCKSL1, STAT5A, and TNFAIP3, which are specifically associated with NK cell cytotoxicity against tumor targets. Collectively, these findings help define the phenotypic and transcriptional signature of NK cells following their encounters with tumor cells, independent of cytokine stimulation, and provide insight into tumor-specific NK cell responses to inform the transition toward harnessing the therapeutic potential of NK cells in cancer.

Conflict of interest statement

UCL has patented the use of CTV-1 and other tumor cells for the exvivo priming of NK cells for therapy. Some of the research presented in this manuscript was funded by INmuneBio Inc. and by Novamune Ltd., which are companies that develop clinical products involving NK-priming technology with the intention of taking them into clinical trials. MWL is a co-founder and chief scientific officer of INmuneBio Inc. and MS is a consultant to INmuneBio Inc. The following patent held by UCL is pertinent to this work: PCT/GB2010/051135, “Preserved Compositions of Activated NK Cells and Methods of Using the Same.” This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

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

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.