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.
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: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.
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:IL-27, a member of the IL-12- family of cytokines, has shown anti-tumor activity in several pre-clinical models due to anti-proliferative, anti-angiogenic and immune-enhancing effects. On the other hand, IL-27 demonstrated immune-regulatory activities and inhibition of auto-immunity in mouse models. Also, we reported that IL-27, similar to IFN-, induces the expression of IL-18BP, IDO and PD-L1 immune-regulatory molecules in human cancer cells. Here, a proteomic analysis reveals that IL-27 and IFN- display a broad overlap of functions on human ovarian cancer cells. Indeed, among 990 proteins modulated by either cytokine treatment in SKOV3 cells, 814 showed a concordant modulation by both cytokines, while a smaller number (176) were differentially modulated. The most up-regulated proteins were common to both IFN-γ and IL-27. In addition, functional analysis of IL-27- regulated protein networks highlighted pathways of interferon signaling and regulation, antigen presentation, protection from natural killer cell-mediated cytotoxicity, regulation of protein polyubiquitination and proteasome, aminoacid catabolism and regulation of viral protein levels.
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).
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).
Project description:Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer, represent promising therapeutic target. However, reduced iNKT-cell numbers and function have been observed in many patients with cancer. To overcome this obstacle, we reprogramed human iNKT cells to pluripotency and then redifferentiated into regenerated iNKT cells in vitro through IL-7/IL-15-based optimized cytokine combination. They showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of cancer antigen-specific cytotoxic T lymphocytes in vitro, and exhibited NKG2D- and DNAM-1-mediated natural killer celllike cytotoxicity against cancer cell lines. Their immunological features and availability in an unlimited supply from induced pluripotent stem cells offer the potential to develop effective immunotherapies against cancer.