Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA127977 | ENA |
REPOSITORIES: ENA
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Project description:We used Affymetrix expression arrays to determine changes in gene expression associated with activation of human NK cells mediated through treatment with cytokines IL-2, IL-12 and IL-18 over a 24 hour period. Human natural killer cells were isolated via negative selection from PBMCs of healthy donors. Cells were found to be > 95% CD3-CD56+. RNA was harvested at time of isolation or after 24 hour stimulation from 8 x 10^6 cells per condition. For stimulations, cells were incubated at 37C (5% CO2) in RPMI, suuplemented with 10% Fetal Bovine Serum at 1.5 x 10^6 cells/ml. Cytokine stimualtions were conducted with IL-2 (100U/mL), IL-12 (10ng/mL) and IL-18 (100ng/mL) from 2 male donors (N = 4). Expression analysis was carried out to determine transcriptional changes associated with 24 hr stimulation relative to freshly isolated cells.
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Project description:Regulated chromatin states control genome accessibility and thus influence gene expression. Here we report an analysis pipeline termed ATAC-mass that capitalizes on isotopic labeling to detect the accessible genome by multiplexed ion beam imaging (MIBI) and mass cytometry. With MIBI the accessible genome can be visualized at approximately 100-nm resolution simultaneously with metabolic labeling to enable multi-parameter three-dimensional imaging of nuclear features. Extension of this approach to non-spatial mass cytometry enabled the simultaneous measurement of multiple parameters and total genome accessibility in millions of individual cells. We used ATAC-mass to analyze natural killer cells after stimulation with interleukin (IL)-12 or IL-18 -- demonstrating that IL-18 treatment leads to increased total genome accessibility. Analysis of the spatial organization of open chromatin suggest that IL-12 and IL-18 both induce an increase in chromatin accessibility in noncompacted DNA regions. Deep sequencing of the genomic distribution of open chromatin revealed that IL-18 increased the accessibility of quiescent enhancers whereas genomic loci that become more accessible by IL-12 stimulation are mainly localized in the active promoter regions. This integration of epigenomics, proteomics and high-resolution imaging at the single-cell level provides a tool that can enhance our appreciation of the molecular mechanisms underlying gene regulation.
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Project description:Adaptive CD4 T helper cells and their innate counterparts, innate lymphoid cells, utilize an identical set of lineage-determining transcription factors (LDTFs) for their differentiation and functions. However, the similarities and differences in the induction of the LDTFs in these lymphocytes are still elusive. Here we show that type 1 T helper (Th1) cells and natural killer (NK) cells displayed distinct epigenomes at the Tbx21 locus, which encodes T-bet, the LDTF for type 1 lymphocytes. The initial induction of T-bet in NK precursors was partially dependent on the NK-specific DNase I hypersensitive site Tbx21-CNS-3 and the expression of IL-18 receptor; IL-18 induced T-bet expression through RUNX3, which binds to Tbx21-CNS-3. By contrast, Tbx21-CNS-12 containing STAT binding motifs was critical for IL-12-induced T-bet expression during Th1 cell differentiation both in vitro and in vivo. Thus, innate and adaptive lymphocytes of same class may utilize distinct enhancer elements for their development and differentiation.
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Project description:Human natural killer T cells (NKTs) are innate-like T lymphocytes that are increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintain a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observed that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates de novo programming of memory NKTs in CD62L negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 showed enhanced antitumor activity in tumor models, persisted long-term in vivo and conserved the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic and unappreciated plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.
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Project description:Analysis of human natural killer cells following stimulation with immobilized IgG1 for 4 and 24 hours. Results identified significantly differentially expressed genes in natural killer cells stimulated with immobilized IgG1 providing insight into how antibodies modulate the transcriptome in way that may contribute to cell activation and immune tolerance
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