Gene expression anaylsis of tumor stimulated human NK cells
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ABSTRACT: Genome wide expression profiling of human NK cells stimulated with K562 erythroleukemic tumor cells after four hours of NK-tumor co-culture. Responding NK cells were compared to non-responding NK cells, delineated by display of CD107 on the NK cell surface following cytotoxic granule release. We hypothesized that tumor responses would initiate rapid changes in gene expression in the NK cell that would identify new features of the anti-tumor response of NK cells. Results identify NK cell activation responses and induction of TNF superfamily molecules with immunoregulatory activity. Human peripheral blood NK cells were co-cultured with tumor target cell line K562 for 4 hours with GolgiStop (brefeldin) then stained for granule exocytosis marker CD107a / CD107b, and NK cell markers then FACS sorted for responding NK cells (CD107+) and non-responding NK cells (CD107-). Pooled donor sample comprised NK cells from 3 individuals.
Project description:Genome wide expression profiling of human NK cells stimulated with K562 erythroleukemic tumor cells after four hours of NK-tumor co-culture. Responding NK cells were compared to non-responding NK cells, delineated by display of CD107 on the NK cell surface following cytotoxic granule release. We hypothesized that tumor responses would initiate rapid changes in gene expression in the NK cell that would identify new features of the anti-tumor response of NK cells. Results identify NK cell activation responses and induction of TNF superfamily molecules with immunoregulatory activity.
Project description:Natural Killer (NK) cells present natural cytotoxicity against tumor cells, although their activity is increased after activation. NK cell activation depends on a complex intracellular signaling process mediated by activating and inhibitory receptors and the functional outcome depends on the integration of the activating and inhibitory signals received. Soluble cytokines and/or ligands on target cells bind the NK cell receptors, and hence, influence the final NK cell response: attack versus ignorance. We used microarrays to detail the global programme of gene expression underlying NK cell activation by IL-2, a MHC-I-deficient target cell (K562)+IL-2 and an EBV-target cell (R69). PBLs from 4 different donors were activated by 100 U/ml IL-2; K562+IL-2 or R69 cells. After 5 days we obtained RNA and miRNA from naïve NK cells or from NK cells activated with the above mentioned stimuli, with more than 90% of purity. The 16 RNA samples were used to generate cDNA libraries that were hybridized on Human Gene 1.1ST arrays (Affymetrix) and analyzed with the Affymetrix Gene Chip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
Project description:Natural Killer (NK) cells present natural cytotoxicity against tumor cells, although their activity is increased after activation.NK cell activation depends on a complex intracellular signaling process mediated by activating and inhibitory receptors and the functional outcome depends on the integration of the activating and inhibitory signals received. Soluble cytokines and/or ligands on target cells bind the NK cell receptors, and hence, influence the final NK cell response: attack versus ignorance. We used microarrays to detail the global programme of gene expression underlying NK cell activation by IL-2, a MHC-I-deficient target cell (K562)+IL-2 and an EBV-target cell (R69). PBLs from 4 different donors were activated by 100 U/ml IL-2; K562+IL-2 or R69 cells. After 5 days we obtained RNA and miRNA from naïve NK cells or from NK cells activated with the above mentioned stimuli, with more than 90% of purity. The 16 RNA samples were used to generate cDNA libraries that were hybridized on Human Gene 1.1ST arrays (Affymetrix) and analyzed with the Affymetrix Gene Chip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
Project description:Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NKEVs are constitutively secreted, are biologically active, reflect the protein and genetic repertoire of their originating cells and exert anti-tumor activity in vitro and in vivo. NKEVs from tumor-conditioned NK cells interact with naïve NK cells promoting their cytotoxic activity. In cancer NK cells exhibit profound defects in degranulation ability, a status probably reflected by their NKEVs. Hence, NKEVs could contribute to improve cancer therapy by interacting with tumor and/or immune cells at the same time sensing the actual NK cell status in cancer patients. Here we investigated the role of NKEVs in stimulating the immune system and developed an immune enzymatic test (NKExoELISA) to sense the systemic NK cell status by measuring plasma NK-derived exosomes through combined capture of exosomes, expressing typical EV (tsg101) and NK cell (CD56) markers. We analyzed by LC-MS/MS the protein content from NKEVs evaluating proteins differentially expressed in exosomes (NKExo), vescicles (NKMV) and total cell extract (Tot extr) from parental NK cells. Proteomic data confirmed the presence of many EV markers and detected several proteins involved in immune response, cell adhesion and complement biological processes.
Project description:Natural Killer cells (NK), a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients Eight healthy donor and eight myeloma patients were used in the study. Non-expanded natural killer (NK) cells were isolated from PBMCs of healthy donors and myeloma patients. Expanded natural killer (ENK) cells were generated from same set of samples as mentioned in expansion protocol. All ENK and NK cells were used for gene expression profiling.
Project description:NK cells may acquire under certain conditions features of adaptive immune cells. As the functional role of memory NK cells in cancer has so far remained elusive, we reasoned whether tumor-priming itself might promote memory NK cell generation. We provide substantial evidence that independent from pro-inflammatory stimulation, tumor-induced memory-like (TIML) NK cells exhibit a heightened, tumor-restricted cytotoxicity which is dependent on a higher/faster perforin but not IFN-γ release. Comparative transcriptome analysis reveals that gene expression patterns differ between TIML- and Cytokine-induced memory-like (CIML)-NK cells.
Project description:Natural killer (NK) cells are a type of innate lymphocytes that play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell-cell interaction with cell surface proteins and then attack target cells via multiple mechanisms involving TRAIL, Fas Ligand, cytokine secretion, perforin, and granzymes. In addition, extracellular vesicles (EVs), including exosomes derived from NK cells (NK-EVs), possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells in a granzyme B independent manner. In addition, small RNA-seq and mass spectrometry analyses indicate that miRNA and protein profiles in EVs are altered by cytokine stimulation. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings reveal novel characteristics of NK-EVs and the mechanism of their incorporation into target cells.
Project description:Natural killer (NK) cells are lymphocytes that participate in immune responses through their cytotoxic activity and secretion of cytokines and chemokines. They can be activated by interaction with ligands on target cells or by soluble mediators such as cytokines. In addition, soluble HLA-G, a major histocompatibility complex molecule secreted by fetal trophoblast cells during early pregnancy, stimulates resting NK cells to secrete proinflammatory and proangiogenic factors. Human NK cells are abundant in uterus, where they remain after implantation. Soluble HLA-G is endocytosed into early endosomes of NK cells where its receptor, CD158d, initiates a signaling cascade through DNA-PKcs, Akt and NF-kB3. The physiological relevance of this endosomal signaling pathway, and how the fate and function of NK cells during early pregnancy is regulated, is unknown. Here we show that soluble agonists of CD158d trigger DNA damage response signaling and p21 (CIP1/WAF1) expression to promote senescence in primary NK cells. CD158d engagement resulted in morphological alterations in cell size and shape, chromatin remodeling, and survival in the absence of proliferation, all hallmarks of senescence. Microarray analysis revealed a senescence signature of upregulated genes upon sustained activation through CD158d. The proinflammatory and proangiogenic factors secreted by these metabolically active NK cells are part of a senescence associated secretory phenotype (SASP) that promoted tissue remodeling and angiogenesis as assessed by functional readouts of vascular permeability and endothelial cell tube formation. We propose that ligand-induced senescence is a molecular switch for the sustained activation of NK cells in response to soluble HLA-G for the purpose of remodeling the maternal vasculature in early pregnancy. Time series (4 hr, 16 hr, 64 hr) of NK cells treated with agonist (anti-CD158d mAb) or control. NK cells were from 4 donors.
Project description:NK cells are part of the innate immune system and therefore directly involved in the response to a Candida albicans infection. Our aim is to better understand the interaction of NK cells and C.albicans cells. Natural Killer cells (NK cells) were isolated from blood of 3 different donors and coincubated with Candida albicans. Transcriptome analysis was performed after 3 and 6 hours using an Illumina HumanHT-12 v4 Expression BeadChip.