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 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:By comparing CD94-CD56dim NK cell with or without primary stimulation treatment after 5 days, cytokine induced NK memory related molecules were identified.
Project description:Natural killer cells are innate lymphocytes that play a pivotal role in the immune surveillance and elimination of transformed or virally infected cells. Using a combined chemico-genetic approach, we have identified that BET bromodomains BRD2 and BRD4 are central regulators of NK cell responses. We show that both BRD2 and BRD4 play a key regulatory function in controlling NK cell specific inflammatory responses. However, knockdown of BRD2 but not BRD4 impairs NK cell cytolytic response, highlighting a redundant role for BRD4 in regulating NK cell killing. We further show that the prototypic monovalent BET inhibitor impairs in vitro NK cell mediated killing of cancer target cells, while the bivalent BET bromodomain AZD5153 does not. We ascribe these differences to the preferential affinity of JQ1(+) to BRD2, while AZD5153 has a higher affinity for BRD4. Our work suggests that inhibiting BET bromodomains may be an effective therapeutic strategy for controlling inflammatory function. Given that BRD2 but not BRD4 inhibition can impair NK cell mediated killing, our findings also have clinical significance in light of the ongoing clinical application of BET bromodomains in oncology.
Project description:MicroRNAs (miRNAs) are 20–24 nt RNAs that can be packaged into exosomes and play important regulatory roles. Here we show that miRNA-containing exosomes from NK cells could alleviate symptoms of chronic mild stress in mice. In vivo experiment, these exosomes decreased the levels of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α in the medium of astrocytes. By microarray analysis of exosome miRNA profiles, miR-207 was found to be an overexpressed miRNA in exosomes derived from unstressed mice. Experiments confirmed that miR-207 directly targets TLR4 interactor with leucine-rich repeats (Tril) and inhibits NF-κB signaling downsream of TLRs in astrocytes. When overexpressed in astrocytes, miR-207 decreased the release of pro-inflammatory cytokines and by inclusion of miR-207 inhibitor in astrocytes, higher release of pro-inflammatory cytokines and higher expression of Tril was found in vitro experiments. When NK cells were transfected with miR-207 inhibitor, miR-207 levels in NK cells derived exosomes were also declined. These exosomes with low miR-207 levels showed a decreased antidepressant activity in the vivo experiment. Collectively, our findings revealed that exosomal miR-207 alleviated the depression symptoms of stress mice by targeting Tril to inhibit NF-κB signaling downstream of TLRs in astrocytes.
Project description:Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor post-infusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK cell functions following cytokine withdrawal to model post-infusion performance. In contrasts to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided functional advantages. Genome-wide analysis of cytosolic and polysome-associated mRNA revealed cytokine dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mTOR signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced functional advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15 stimulated NK cells was also observed using a clinically applicable protocol for NK cell expansion. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK cell cancer therapy. Freshly isolated NK cells from 6 donors were activated with IL-2 or IL-15 for 48 hours, followed by cytokine withdrawal for 24 hours, resulting in four RNA samples per donor. From each sample, both the cytosolic as well as the polysomal fraction were collected. Donor 3 contains activation and post withdrawal data from two different donors due to poor RNA-quality obtained for some samples which did not allow for processing of the complete set of 6 donors (resulting in a total of 40 samples).
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 (NK) cells are important components of the immune system in the defense against tumor transformation. They release exosomes containing proteins and nucleic acids, including microRNAs (miRNAs) that play a role in the anti-tumor NK cell functions since they are able to recognize and kill cancer cells. In this study, we explored the miRNA content of NK exosomes by microarray as compared to their cellular counterpart and we identified a small subset of miRNAs highly expressed in NK exosomes.