Transcription profiling of mouse naive T cells from T-cell specific furin knockouts and wild type littermates
ABSTRACT: Purified naive (CD4+ CD62L+ CD44-) T cells from 10-11 weeks old T cell specific Furin knockout (CD4-cre fur flox/flox) and littermate wild type (fur flox/flox) control mice were profiled for gene expression using Affymetrix MOE 430 2.0 microarray platform.
Project description:Analysis of T-cells lacking the proprotein convertase furin. Proprotein convertases promote the proteolytic maturation of proproteins. Furin is induced in activated T-cells. Results provide insight into the function of furin in T-cells. CD4+CD62L+CD44- naive, CD4+CD62L-CD44+ memory and CD4+CD25+FoxP3+ regulatory T cells were isolated from Fur flox/flox and CD4 cre Fur flox/flox mice. Naive T cells were activated via TCR. Total RNA was extracted from all cells and hybridized to Affymetrix microarrays.
Project description:Furin is a proprotein convertase induced in activated T cells, reported to processes the anti-inflammatory cytokine TGFb-1. Herein, we show that conditional deletion of furin in T cells allowed for normal T cell development but impaired the function of regulatory T cells and effector cells, which produced less TGFb-1. Furin-deficient Treg cells, were less protective in a T cell transfer colitis model and failed to induce Foxp3 in normal T cells. Furin-deficient effector cells were inherently overly active and were resistant to suppressive activity of wild-type Tregs. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its nonredundant, essential function in regulating TGFb-1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. The current work suggests that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance. Experiment Overall Design: Naive CD4+ CD62L+ CD44- T cells were isolated from Fur flox/flox and CD4 cre Fur flox/flox mice. Replicated samples were achieved for wild type and knockout conditions.
Project description:Furin is a proprotein convertase induced in activated T cells, reported to processes the anti-inflammatory cytokine TGFb-1. Herein, we show that conditional deletion of furin in T cells allowed for normal T cell development but impaired the function of regulatory T cells and effector cells, which produced less TGFb-1. Furin-deficient Treg cells, were less protective in a T cell transfer colitis model and failed to induce Foxp3 in normal T cells. Furin-deficient effector cells were inherently overly active and were resistant to suppressive activity of wild-type Tregs. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its nonredundant, essential function in regulating TGFb-1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. The current work suggests that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance. Overall design: Naive CD4+ CD62L+ CD44- T cells were isolated from Fur flox/flox and CD4 cre Fur flox/flox mice. Replicated samples were achieved for wild type and knockout conditions.
Project description:The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo. Overall design: Two biological replicates of FURIN KO and WT peritoneal macrophages were left unstimulated or were stimulated for 1, 4 and 24 hours with LPS.
Project description:Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a buffy coat (Australian Blood Bank) using Ficoll methodology. CD4+ T cells were isolated using Dynal Beads kit. Pure CD4+ T cells were then stained using a cocktail of monoclonal antobodies (mAbs), including: anti-CD4PE, CD45RO ECD, CD62L APC-Cy7, CD25 APC, CD127 Pacific Blue. After incubation, cells were washed twice in PBS/FCS (0.2%), and sorted into five different cell subsets: CD4+CD25+CD127low CD62L+CD45RO- (naive regulatory T cells), CD4+CD25+CD127low CD62L+/- CD45RO+ (activated regulatory T cells), CD4+CD25+CD127hi CD62L+/- CD45RO+ (memory T cells), CD4+CD25-CD127low CD62L+/- CD45RO+ (effector T cells) and CD4+CD25-CD127hi CD62L+ CD45RO- (naive T cells).
Project description:Subpopulations of human fetal thymocyte and circulating naïve T cells were obtained through FACS sorting, including CD3-CD4+CD8- intrathymic T progenitor cells (ITTP), CD3intCD4+CD8+ "double positive" thymocytes (DP), CD3highCD4+CD8- "single positive" thymocytes (SP4), CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+), and CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+).
Project description:Liver cirrhosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes patients to tumorigenesis are not well understood. Transgenic mice expressing platelet-derived growth factor C (Pdgf-c) under the control of the albumin promoter provide a unique animal model that mimics the step-wise disease progression in humans from fibrosis to HCC. The livers of Pdgf-c Tg mice show evidence of liver injury, including inflammation, proliferation, fibrosis and steatosis, and as the mice age, angiogenesis and dysplasia. Eighty-five percent of these mice develop HCC spontaneously, and have reduced survival that is related to their liver pathology. Through measurement of protein, RNA, and histological markers, we provide evidence to support the hypothesis that changes in liver stromal cells play an essential role in tumorigenesis in this model. A paracrine signaling model is proposed where ectopic expression of Pdgf-c in hepatocytes results in activation of hepatic stellate cells, which subsequently activates endothelial and Kupffer cells. Activation of these non-parenchymal cells promotes the release of hepatocyte growth factors that, together with changes in extracellular matrix, lead to the formation of HCC. Pdgf-c Tg mice provide a useful pre-clinical model in which to test novel drugs for chronic liver disease and HCC that focus on blocking the processes that alter the liver's fibrotic microenvironment. Two strains of mice, C57BL/6 and C57/BL6 Pdgf-c transgenic, were analyzed to see if liver stromal cells play an essential role in tumorigenesis.
Project description:The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on Array Comparative Genomic Hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal (GI) carcinoids. In contrast to the relatively stable karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. Recurrent CN alterations of a total of 203 genes, including the RB1 gene, and 59 microRNAs, most of which locate in the DLK1-DIO3 domain, were observed in SCLC tumors, bronchial carcinoids and carcinoids of GI origin; in contrast, CN alterations of the TP53 gene and the MYC family members were observed more frequently in SCLC. These findings suggest the existence of partially shared tumor-specific CN alterations in these tumors. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resemble that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC. Carcinoids, including 19 bronchial carcinoids and 9 carcinoid of gastrointestinal origin, and small cell lung cancer, including 33 patients' tumor samples and 13 cell line samples, were compared.
Project description:Naïve CD4 cells (CD45.2+CD4+,CD62L high,CD44 low) isolated from lymph nodes of mixed bone marrow chimeras 5 days after induced S1pr1 deletion. Overall design: CD45.1+ mice were reconstituted with a mix of BM from WT UBC:GFP mice and BM from S1pr1f/fUBC-CreERT2 CD45.2+ mice or littermate controls. S1pr1 was deleted by tamoxifen injection for 5 days. Naïve CD4+ CD62L high CD44 low CD45.2+ GFP- cells were sorted 5 days after last tamoxifen injection.
Project description:Given their precipitous encounter with the environment, newborn infants might be expected to possess abundant immunoprotective mechanisms. Paradoxically, their T cells display grossly impaired Th1 anti-bacterial and anti-viral responses. This study identifies factors produced by neonatal CD4 T cells when compared with adult naive CD4 T cells and highlights CXCL8 as a pivotal effector molecule in neonatal T cells. Using Affimetrix microarray we compared gene expression in cord blood derived CD4 T cells with naive CD4 T cells from adults after polyclonal stimulation. The Affymetrix GeneChip Human Genome 1.0 ST array were used to define gene expression profiles in each of the CD4 T cell samples.