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.

Specific Patterns of Blood ILCs in Metastatic Melanoma Patients and Their Modulations in Response to Immunotherapy

“…Furthermore, NK-derived extracellular vesicles can compromise NK cell function [ 37 ]. The higher expression of CD69 and lower CD62L in patients indicate that these ILCs may correspond to recirculating tumor-exposed ILCs [ 38 ]. The low CD62L can also indicate their altered cytotoxic function [ 39 , 40 ] and/or capacity to spill out of the tumor and migrate to lymph node.…”

An approach for normalization and quality control for NanoString RNA expression data

“…) on DE analysis 3 across natural killer (NK) cells primed for tumor-specific cells and cytokines from Sabry et al [20]. RLE plots before and after normalization showed minor differences between the two normalization methods…”

“…Given these advantages, nCounter is increasingly used for longitudinal studies involving FFPE samples carried out over several years [3] and diagnostic assays in clinical settings [4,5]. We downloaded raw RCC files from Sabry et al [20] from the NCBI Gene Expression Omnibus (GEO) 1 with accession number GSE130286 and imported them using functions in NanoStringQCPro [21]. This The full quality control and normalization process using nSolver and RUVSeq is summarized in Figure 1, 1 starting with familiarization of the raw data (Figure 1.1), technical quality control (Figure 1.2), pre-1 in at least one of these four QC assessments as technical quality control.…”

“…cancer subtype), with no assumptions on co-expression of all housekeeping 1 genes. To assess the potential for housekeeping correction to introduce bias, housekeeping genes were 1 assessed for differential expression across a primary biological covariate of interest (estrogen receptor 1 status in CBCS, tumor stage in the kidney and bladder cancer data, and treatment groups in Sabry et al 1 [20]) using negative binomial regression on the raw counts from the MASS package [22]. NanoString guidelines suggest background correction [9,12] by either subtraction or thresholding for an 1 estimated background noise level for experiments in which low expressing targets are common, or when 1 the presence of a transcript has an important research implication [7,12].…”

“…Raw RCC files for nCounter expression from Sabry et al[20] are available NCBI Gene Expression 4 Omnibus (GEO) with the accession numbers GSE130286. Raw and normalized expression data from 4 CBCS will be available on GEO upon publication.…”