Expression data from granulocytic MDSC cultured with T cells in the absence of IFNgamma
Ontology highlight
ABSTRACT: Myeloid derived Suppressor cells (MDSC) are heterogenous popluation of cells consists of two major subsets namely the monocytic Gr-1dull/int. and granulocytic (Gr-1high). These distinct two subsets use different mechanism to inhibit T cell response. In addition, how the function of these subsets is regulated is not known yet. The Gr-1dull/int. MDSC are suppressing T cells through IFNg dependent nitric oxide dependent manner. However, the exact suppressive mechanism of Gr-1high MDSC is not clear. Here we studied the role of a cytokine IFNg on the suppressive function of Gr-1high MDSC by comparing the gene expression of Gr-1high cells cultured alone versus those cultured with T cells which donot produce IFNgamma. CD11b+Gr-1high cells were purified from the splenocyte of CT-26 colon tumor bearing mice. The purified CD11b+Gr-1high MDSCs were cultured with IFNg-/- antigen specific T cells and re- sorted after 48h and RNA was extracted and gene expression was analyzed using topic-defined PIQORTM Immunology Microarrays.
Project description:Myeloid derived Suppressor cells (MDSC) are heterogenous popluation of cells consists of two major subsets namely the monocytic Gr-1dull/int. and granulocytic (Gr-1high). These distinct two subsets use different mechanism to inhibit T cell response. In addition, how the function of these subsets is regulated is not known yet. The Gr-1dull/int. MDSC are suppressing T cells through IFNg dependent nitric oxide dependent manner. However, the exact suppressive mechanism of Gr-1high MDSC is not clear. Here we studied the role of a cytokine IFNg on the suppressive function of Gr-1high MDSC by comparing the gene expression of Gr-1high cells cultured alone versus those cultured with T cells which donot produce IFNgamma.
Project description:Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC. PBMC were isolated from fresh blood healthy donor by density centrifugation. CD14+ cells were isolated by AutoMACS CD14 microbeads using a AutoMACS (Miltenyi), and then stained with CD14 and HLA-DR antibodies. MDSC and monocytes control cells were sorted as CD14+ HLA-DR-neg/low and CD14+HLA-DR-high cells respectively. The sorted two populations were immediately frozen in liquid nitrogen and shipped to the company on dry ice for RNA isolation and further microarray.
Project description:Myeloid-derived suppressor cells (MDSC) are a major barrier to anticancer responses. Although much is known about how MDSC promote tumor progression, little is known about how they develop. We hypothesized that MDSC develop as a consequence of tumor-induced downregulation of interferon regulatory factor-8 (IRF-8), a key myeloid developmental transcription factor. We showed that: 1) IRF8-deficiency in mice generated myeloid populations highly homologous to tumor-induced MDSC; 2) IRF-8 overexpression in mice reduced MDSC accumulation and retarded tumor growth; 3) MDSC-inducing factors, G-CSF or GM-CSF, facilitated IRF-8 downregulation via STAT3- or STAT5-dependent pathways, respectively; and 4) IRF-8 levels in MDSC-like subsets of breast cancer patients were depressed compared to healthy donors. Altogether, our data implicate IRF-8 as a novel MDSC-dependent transcription factor. Splenic CD11b+Gr-1high cell populations from tumor-bearing mice, IRF8 knockout mice or non-tumor-bearing control mice were purified in two independent experiments by flow cytometry (> 97% purity) and subjected to whole genome expression profiling using Illumina microarrays.
Project description:Global gene expression studies were performed to determine whether the granulocytic-like MDSC populations from G-CSF treated mice resembled those of tumor-bearing (TB) mice more so than those of the non-tumor-bearing control (i.e., WT) at a molecular level. Splenic CD11b+Gr-1high cell populations from WT, G-CSF-treated or 4T1-TB mice were purified in two independent experiments by flow cytometry (> 98% purity) and subjected to whole genome expression profiling using Illumina microarrays.
Project description:Myeloid derived suppressor cells (MDSC) playing the immune suppressive roles in tumor bearing host consists of two major subsets of granulocytic and monocytic cells. Granulocytic MDSC (G-MDSC) express CD11b+ Gr-1high Ly6G+ Ly6Clow and produce high level of reactive oxygen species (ROS). Interestingly, neutrophils are well known ROS producing cells during immune defensive process and share same surface markers with G-MDSC. These similar features always brought the fundamental questions what’s the difference between G-MDSC and neutrophils but it’s not yet proven clearly. In this study, we examined the gene expression of G-MDSC and neutrophils using Affymetrix microarray
Project description:Myeloid derived suppressor cells (MDSC) playing the immune suppressive roles in tumor bearing host consists of two major subsets of granulocytic and monocytic cells. Granulocytic MDSC (G-MDSC) express CD11b+ Gr-1high Ly6G+ Ly6Clow and produce high level of reactive oxygen species (ROS). Interestingly, neutrophils are well known ROS producing cells during immune defensive process and share same surface markers with G-MDSC. These similar features always brought the fundamental questions what’s the difference between G-MDSC and neutrophils but it’s not yet proven clearly. In this study, we examined the gene expression of G-MDSC and neutrophils using Affymetrix microarray G-MDSC (CD11b+Ly6G+Ly6Clow) were purifed from splenocytes in EL4 lymphoma tumor bearing mice by positive selection of Ly6G using microbeads isolation. Neutrophils were purified from ascitic fluids induced after injection of milk protein, casein by negative selection of F4/80 and positive selection of Ly6G using microbeads isolation. Their RNA was extracted and gene expression was analyzed using Affymetrix microarray.
Project description:We employed GeneChip analysis to investigate the global gene expression profiles of neutrophils from BM Gr-1high/CD48– neutrophils were sorted from the BM of C57BL/6 mice by FACS in two independent experiments
Project description:This SuperSeries is composed of the following subset Series: GSE30064: Cultured human amniotic fluid-derived mesenchymal stromal cells [PIQOR data] GSE30065: Cultured human amniotic fluid-derived mesenchymal stromal cells [miRXplore data] Refer to individual Series
Project description:The COP9 signalosome (CSN) is a conserved protein complex occurring in all eukaryotes. The mammalian CSN consists of eight subunits (CSN1 – CSN8) and possesses an intrinsic metalloprotease JAMM motif localised to CSN5. In addition, it is associated with kinases such as protein kinase CK2 and D and the ubiquitin (Ub) specific protease 15. It regulates cullin-RING Ub ligases (CRLs) that label proteins for proteolysis with poly-Ub chains in the Ub proteasome system (UPS). CRLs contain one of the scaffolding cullins 1 – 7, a RING domain protein e. g. Rbx1, and one of hundreds of substrate binding units such as F-box or BTB proteins that specifically target proteins for ubiquitination. The CSN forms functional supercomplexes with CRLs. It removes the Ub-like protein Nedd8 from cullins by its metalloprotease activity and thereby inactivates CRLs. On the other hand, it is essential for CRL function by protecting components and allowing reassembly of the complexes. As a regulator of the UPS the CSN is involved in cell cycle10, DNA repair and development. Overexpression of CSN subunits has been reported in tumour cells. Up to date nothing is known about CSN biogenesis and its regulation. Ten years ago we have observed that overexpression of selected CSN subunits caused a coordinated increase of all CSN subunits and a de novo biosynthesis of the entire complex. Here we show that overexpression of CSN1 mediates an increase of c-Myc which coordinates CSN subunit expression via microRNAs (miRNAs). miRNAs are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides that function as posttranscriptional gene regulators. We found that inhibitors of let-7 family miRNAs or upregulation of c-Myc, which represses let-7 miRNAs, resulted in a coordinated increase of CSN subunits and de novo CSN biogenesis. Keywords: Gene expression analysis of human RNA samples using topic-defined PIQOR™ Ubiquitin-PS Microarrays. Two different platforms were used (these are two different microarray batches which are identical in terms of the spotted cDNAs, but differ in the position of some cDNAs) Gene expression analysis of Hela cells overexpressing either CSN2 or CSN1 was performed to study CSN biogenesis and its regulation. Curcumin, Nr. 8 (Curcumin analogon) and Piceatannol were used for the treatment of Hela cells since they inhibit the CSN associated kinases. The aim was to investigate the effect on the signalosome. For each treatment 50 µM Curcumin, Nr. 8 and Piceatannol were used.
Project description:Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation, do not form teratomas when transplanted into animal models and their stemness seems to be between embryonic cells and adult mesenchymal cells. Prior to their use in cell therapy they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of a hAMSC population kept under in vitro culture conditions for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D. The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the reduction in the proliferative capacity of these cells in a TP53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine. The variations in the expression patterns of the mRNAs were studied using topic-defined PIQOR Stem Cell microarrays. Human amniotic fluid-derived mesenchymal stromal cells pellets of 5 x 10E5 cells were cryopreserved in liquid nitrogen and send to Miltenyi Biotec for their analysis. A pool of non-stimulated lymphocytes (non-mitotic cell cycle) obtained by Ficoll isolation in the Immunology Service of Carlos Haya hospital was used as a control of the expression.