Transcriptomic analysis of Responsive and Non-responsive Human NK cells to Plasmodium falciparum-infected red blood cells
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ABSTRACT: NK cells are important cells of the innate immune system that respond to malaria infection in human. Howver, NK cell responses to malaria infection vary significantly in the human population. Base on their ability to kill infected red blood cells (iRBC), NK cells from malaria-naive individuals can be classified as responsive or non-responsive. NK cells used for microarray was obtained as follows: 10^6 NK cells were cocultured with either 10^5 iRBC at a starting parasitemia of 0.5% or with the same amount of RBC for 96 hours. Thereafter live NK cells were purified by FACS and RNA extracted via Trizol method. RNA quality was assessed by electrophoretic assay on the Agilent 2100 Bioanalyzer using a Nano chip (Agilent RNA 6000 Nano chip). RNA with a RIN value of >7.0 was used for genome-wide transcriptional analysis by microarray using HumanHT-12 v4 BeadChip (Illumina).
Project description:NK cells are innate immune cells that recognize and kill foreign, virally-infected and tumor cells without the need for prior immunization. NK expansion following viral infection is IL-2 or IL-15-dependent. To identify Runx3 responsive genes, NK cells were isolated from spleen of WT and Runx3-/- mice . Six samples (3 WT and 3 Runx3-/-) of freshly isolated NK cells (resting) were separately obtained from individual mice.
Project description:NK cells are innate immune cells that recognize and kill foreign, virally-infected and tumor cells without the need for prior immunization. NK expansion following viral infection is IL-2 or IL-15-dependent. To identify Runx3 responsive genes, NK cells were isolated from spleen of WT and Runx3-/- mice . Ten samples (5 WT and 5 Runx3-/-) of freshly isolated NK cells were separately obtained from individual mice. Cells were cultured for 7 days with IL-2 or IL-15.
Project description:To study the effect of Plasmodium falciparum-infected erythrocytes on gene expression in NK92 cells, microarray analysis after 6, 12 and 24 hours of co-culture with either uRBC or iRBC was performed. The aim was to identify pathways in NK92 cells that are switched on after iRBC encounter in a time-dependent manner that will help to understand the mechanisms in innate immune defenses against Plasmodium falciparum infection. Variation in gene expression of NK92 cells was determined after 6, 12, and 24 hours of co-culture with either infected or uninfected RBC compared to timepoint 0 (start of co-culture, untreated control). All experiments were done in triplicate, that means that samples were collected in 3 different experiments (A, B, and C) each time after 0, 6, 12 and 24 hours of co-culture.
Project description:Cerebral malaria is a multifactorial condition that begins with high numbers of infected erythrocytes binding to human brain endothelium without invasion into the brain. Here we investigated the global transcriptional gene response of primary human brain endothelial cells after incubation with high numbers of infected erythrocytes; High or low parasite binding phenotypes did not alter gene response. Predominate amongst signaling pathways were the NF-kB proinflammatory response. The inflammatory pathways were validated by direct measurement of proteins. This study delineates the strong inflammatory component of human brain endothelium contributing to cerebral malaria. Experiment Overall Design: Total of 8 samples (4 control and 4 treated) were analyzed. 4 control samples included two normal RBC control and two medium controls. 4 treated samples includes 2 exposed to low binding Pf-IRBC and 2 exposed to high binding Pf-IRBC (Pf-IRBC-P). Medium and RBC controls were finally used as four replicates of control and all four Pf-IRBC or Pf-IRBC-P exposed endothelial cells were used as 4 separate treated controls.
Project description:Single-cell transcriptomics was performed to investigate the bone marrow NK cell compartment of myeloma patients at diagnosis (n=19), during treatment (n=21) and at relapse (n=6). The bone marrow of myeloma patients is characterized by a reduction in conventional cytotoxic NK cells that persists throughout treatment. We show in 20% of newly diagnosed myeloma patients that an altered balance between cytotoxic and cytokine-producing NK cells translates into a reduced cytotoxic ability in response to therapeutic antibodies. The relative loss of cytotoxic NK cells persists at relapse and is accompanied by an expansion of IFN-responsive NK cells. These findings reveal previously unappreciated alterations in bone marrow NK cell composition and highlight the importance of understanding the bone marrow immune system in patients receiving immunotherapies.
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 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: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:Somatic STAT5B gain-of-function mutations have been frequently found in patients with T- and NK-cell neoplasms. STAT5BN642H represents the most frequently occuring STAT5B mutation. To investigate the molecular mechanism of STAT5BN642H-driven NK-cell leukemia, we performed RNA-Seq of liver derived FACS-sorted diseased N642HNK/NK and aged non-diseased control (Cre neg, GFPNK/NK), STAT5BNK/NK, N642HNK/NK NK cells.
Project description:STAT1 is an important regulator of NK cell maturation and cytotoxicity. Although the consequences of Stat1-deficiency have been described in detail the underlying molecular functions of STAT1 in NK cells are only partially understood. Here we describe a novel non-canonical role of STAT1 that was unmasked in NK cells expressing Stat1-Y701F. This mutation prevents JAK-dependent phosphorylation, subsequent nuclear translocation and cytokine-induced transcriptional activity. As expected Stat1-Y701F mice displayed impaired NK cell maturation comparable to Stat1-/- animals. In contrast Stat1-Y701F NK cells exerted a significantly enhanced cytotoxicity in vitro and in vivo suggesting a so-far unknown cytoplasmic function. Using immunofluorescence technology we uncovered the recruitment of STAT1 to the immunological synapse during NK cell killing. A Stat1ind mouse expressing FLAG-tagged STAT1α was used to study the STAT1α interactome in NK cells. Mass spectrometry revealed that STAT1 directly binds proteins involved in cell junction formation and proteins associated to membrane or membrane-bound vesicles. We propose a novel function for STAT1 in the immunological synapse of NK cells regulating tumor surveillance and cytotoxicity.