Project description:Functionally distinct CD4+ helper T (Th) cell subsets, such as Th1, Th2, Th17, and regulatory T cells (Treg), play a pivotal role in the host-defense against pathogen invasion and the pathogenesis of inflammatory disorders. In this project, DIA-MS-based proteome analysis was performed on naïve CD4+ T, Th0, Th1, Th2, Th17 and iTreg cells using Q Exactive HF-X (Thermo Fisher Scientific) to search for proteins that differ among the cell subsets.
Project description:The genetic changes underlying metastatic melanoma need to be deciphered to develop new and effective therapeutics. Previously, genome-wide microarray analyses of human melanoma identified two reciprocal gene expression programs, that included expression of mRNAs regulated by either transforming growth factor, beta 1 (TGFB1) pathways or microphthalmia-associated transcription factor (MITF)/SRY-box containing gene 10 (SOX10) pathways. We extend this knowledge to include gene expression analyses of 5 additional human melanoma lines, and show that these lines also fall into either TGFB1 or MITF/SOX10 gene expression groups. These mRNA expression studies were followed up by miRNA expression analyses. Microarray analyses were performed on 5 metastatic melanoma lines in 3 replicates. Standard Affymetrix protocols were used.
Project description:Type 1 regulatory T (Tr1) cells are one of the regulatory T cell subsets that are characterized by the production of high amount of IL-10 and lack of FOXP3 expression. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue molecule and we have previously reported that LAG3 is expressed on IL-10 producing regulatory T cells. However, naturally occurring Tr1 cells in human secondary lymphoid tissue have not been detected. We identified CD4+CD25-LAG3+ T cells in human tonsil. We compared mRNA expression of five CD4+ T cell subsets in tonsil using microarray analysis (CD4+CD25-LAG3+ T cells, CD4+CD25-CXCR5+PD-1+ follicular helper T cells (TFH), CD4+CD25+ T cells, CD4+CD25-LAG3-CD45RO+ cells and CD4+CD25-LAG3-CD45RO- cells). A human tonsil was obtained from a patient undergoing routine tonsillectomy, and five tonsillar CD4+ T cell subsets were sorted (each 1 x 10^5 cells). There is no biological replication.
Project description:Cerebral metastases occur in a majority of metastatic melanoma patients and are a major cause of mortality. Despite this, there is a poor understanding of the molecules/pathways that lead to the brain-metastatic phenotype. Studies designed to address this deficiency and test novel therapeutic approaches have until recently been slowed by an absence of preclinical models of spontaneous CNS metastatic melanoma disease. To address this, we isolated two variants of the human melanoma cell line WM239 (named 131/4-5B1 and 131/4-5B2) which can metastasize spontaneously to brain parenchyma from an orthotopic primary transplant. We have performed gene expression profiling on both brain metastatic cell lines (131/4-5B1 and 131/4-5B2) and compared to the poorly metastatic parental cell line WM239A and a derived highly metastatic variant 113/6-4L in order to examine the mechanisms that influence the progression of malignant melanoma to a brain-metastatic phenotype. Two-condition experiment, brain metastatic cell lines (131/4-5B1 and 131/4-5B2) and compared to the poorly metastatic parental cell line WM239A and a derived highly metastatic variant 113/6-4L. Biological replicates: 4 independently grown and harvested cell line passages. Two technical replicate per condition (including dye swap).
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:Differentiation of CD4+T-cells into effector subsets is a critical component of the adaptive immune system and an incorrect response can lead to autoimmunity or immune deficiency. Cellular differentiation including T-cell differentiation is accompanied by large-scale epigenetic remodeling, including changes in DNA methylation at key regulators of T-cell differentiation. The TET family of enzymes were recently shown to be able to catalyse methylated cytosine (5mC) into 5-hydroxymethylcytosine (5hmC) enabling a pathway of active removal of DNA methylation. Here, we characterize 5hmC, 5mC and transcriptional dynamics during human CD4+T-cell polarisation in a time series approach and relate these changes to profiles in ex-vivo CD4+memory subsets. We observed large-scale remodelling during early CD4+T-cell differentiation which was predictive of subsequent changes during late time points, these changes were also related to disease associated regions which we show can act as functional regulatory elements. This dataset was designed to assess how DNA methylation differs between in-vivo derived CD4+memory T-cell subsets. DNA methylation was assessed in relationship to gene expression levels and changes (see data series), we observed anticorrelation between promoter DNA methylation levels and gene expression. This submission contains data from DNA methylation profiling of primary human CD4+T-cell memory subsets. This is part of a series, containing transcription and DNA methylation profiling of the same samples. See related experiments E-MTAB-4685, E-MTAB-4686, E-MTAB-4687, E-MTAB-4688
Project description:Type 1 regulatory T (Tr1) cells are one of the regulatory T cell subsets that are characterized by the production of high amount of IL-10 and lack of FOXP3 expression. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue molecule and we have previously reported that LAG3 is expressed on IL-10 producing regulatory T cells. However, naturally occurring Tr1 cells in human secondary lymphoid tissue have not been detected. We identified CD4+CD25-LAG3+ T cells in human tonsil. We compared mRNA expression of five CD4+ T cell subsets in tonsil using microarray analysis (CD4+CD25-LAG3+ T cells, CD4+CD25-CXCR5+PD-1+ follicular helper T cells (TFH), CD4+CD25+ T cells, CD4+CD25-LAG3-CD45RO+ cells and CD4+CD25-LAG3-CD45RO- cells).
Project description:Cutaneous melanoma is an increasingly common form of skin cancer. The molecular mechanisms regulating melanoma progression are not completely understood. We speculated that specific miRNAs may be involved in melanoma development. We compared the miRNA expression profiles of benign nevi and metastatic melanomas. Unsupervised hierarchical clustering demonstrated a distinct miRNA expression pattern in metastatic melanomas compared to nevi. We identified miRNAs that were differentially expressed in melanoma. Notably, miR-193b was significantly down-regulated in the melanoma tissue examined. Using functional studies we demonstrated that over-expression of miR-193b significantly reduced melanoma cell proliferation, and arrested cell at G1 phase. Further gene expression analysis revealed that miR-193b regulated targets involved in cell cycle. Cyclin D1 was down-regulated by miR-193b at both the mRNA and protein level. This is the first study to show that the miR-193b may reduce cell proliferation by directly repressing cyclin D1. Overall, our study suggests that miRNAs are dysregulated in metastatic melanoma, and that miR-193b may play an important role in melanoma. 8 benign nevi and 8 metastatic melanoma tissue samples were profiled by Agilent MicroRNA Microarray (V1.5).