The effect of HTLV-1 bZIP factor (HBZ) RNA and protein in mouse CD4 T cells
ABSTRACT: Human T-cell leukemia virus type 1 (HTLV-1) bZIP factor (HBZ) which is encoded in the minus strand of HTLV-1 provirus, possesses dual function as protein, and also as RNA. To know the effect of HBZ RNA and protein in primary T-cells, we introduced HBZ, or its mutant TTG (exclude protein activity), or SM (exclude RNA activity) with retrovirus vector into CD4 positive murine T cells, and analysed the transcriptome profiling. We found that HBZ RNA altered cell cycle progression, cell survival related genes, while HBZ protein altered immunology related genes. This microarray results demonstrated that HBZ RNA and protein possess distinct functions in primary cells. CD4 positive cells were enriched from murine splenocyte, and cultured with irradiated antigen presenting cells (APC). HBZ, or its mutant were introduced with retroviral vector. Forty eight hours after introduction, cells were washed and cultured with recombinant human IL-2. Further 48 hours after culture, virus infected cells were sorted and analysed by agilent microarray.
Project description:Human T-cell leukemia virus type 1 (HTLV-1) encodes HTLV-1 bZIP factor (HBZ), which is thought to be crucial for neoplastic and inflammatory diseases caused by HTLV-1. So, we analyzed the transcriptional profile of HBZ expressing cells and how HBZ affect the expression of apoptosis-related genes. We used microarrays to detail the effect of HTLV-1 bZIP factor (HBZ), which is encoded in the minus strand of HTLV-1 genome on gene expression. Especially how HBZ affect the expression of apoptosis-related genes. Jurkat cells stably expressing HBZ were stimulated with or without PMA and ionomycin for 9hours. Then RNA extraction and hybridization on Affymetrix microarrays were performed.
Project description:HTLV-1 preferentially infects CD4+ T cells and these cells play a central role in HTLV-1 infection. In this study, we investigated the global gene expression profile of circulating CD4+ T cells from distinct clinical status of HTLV-1-infected individuals in regard to Tax expression levels. CD4+ T cells were isolated from asymptomatic HTLV-1 carrier (HAC) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, in order to identify genes involved in the HAM/TSP development. Hierarchical clustering analysis showed that healthy controls (CT) and HTLV-1-infected samples clustered separately. We also observed that HAC and HAM/TSP groups clustered separately regardless Tax expression. The gene expression profile of CD4+ T cells was compared among CT, HAC and HAM/TSP groups. The IL-27, PXN, CXCR4, GZMA, PRF1 and Foxp3 genes were differentially expressed between HAC and HAM/TSP groups and the frequency of CD4+Foxp3+ regulatory T cells (Treg) were higher in HTLV-1-infected individuals. These findings suggest that CD4+ T cells activity is distinct between HAC and HAM/TSP groups as expected. In order to study the transcriptional changes in CD4 T cell from HTLV-1-infected individuals, immunomagnetically purified CD4+ T-cells from the peripheral blood of 4 asymptomatic HTLV-1 carrier individuals (HAC) and 4 individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), as well as from 4 healthy controls (CT) were isolated and processed the microarray assay according Agilent's protocol. The differential expressed genes, molecular characterization and networks analysis were evaluated using robust bioinformatic tools, then the real time PCR was done to validate the genes.
Project description:Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the causative agent of adult T cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected T cells have been hypothesized to contribute to the development of these disorders, although the precise mechanisms are not well understood. Recently, we and others have proposed that HTLV-1 preferentially infects CD4+CD25+CCR4+ T-cells. In this study, we used microarrays to examine the gene expression profiles of CD4+CD25+CCR4+ T-cells isolated from a HAM/TSP patient, an ATLL patient and a healthy donor to identify gene signatures that may be characteristic for these particular diseases. Overall design: We performed microarray gene expression profiling of immune cells selected on the marker of HTLV-1 infection, CD4, CD25 and CCR4 from a HAM/TSP patient, an ATLL patient and a healthy donor. Total RNA samples from CD4+CVD25+CCR4+ T-cells were subjected to Cy-3 labeling followed by human whole genome gene expression microarray analyses.
Project description:HTLV-1 infected individuals stay as carriers for their lifetimes, while the rest of 5% are killed by ATL. ATL leukemogenesis is a complex process involving accumulation of multiple genetic abnormalities in HTLV-1 infected cells. To clarify genetic events underlying ATL leukemogenesis, we conducted comprehensive miRNA expression profiling in 40 ATL patients and in 22 healthy volunteers. Total RNA samples from PBMC of ATL patients (mostly HTLV-1 inefcted CD4+ T-cells) and from CD4+ T-cells from healthy donors were subjected to Cy-3 labeling followed by miRNA expression microarray analyses.
Project description:To address whether changes in miRNA expression accompany cell-associated replication of HTLV-1 in vivo, we carried out the miRNA expression profiling of CD4+ and CD8+ T-cells derived from naturally HTLV-1 infected individuals with no clinical sign of malignancy. T-cell clones were obtained by limiting dilution culture of PBMCs of HTLV-1 carriers. miRNA expression was assessed by Agilent V3 miRNA array according to the manufacturer's instructions. miRNA expression profiles of cloned CD4+ and CD8+ T-cells (carrying or not HTLV-1) derived from naturally HTLV-1 infected individuals.
Project description:HTLV-1 infected individuals stay as carriers for their lifetimes, while the rest of 5% are killed by ATL. ATL leukemogenesis is a complex process involving accumulation of multiple genetic abnormalities in HTLV-1 infected cells. To clarify genetic events underlying ATL leukemogenesis, we conducted comprehensive gene expression profiling in 52 ATL patients and in 21 healthy volunteers. Total RNA samples from PBMC of ATL patients (mostly HTLV-1 inefcted CD4+ T-cells) and from CD4+ T-cells from healthy donors were subjected to Cy-3 labeling followed by human whole genome gene expression microarray analyses.
Project description:We used microarrays to compare the global programme of gene expression in HTLV-positive, ATL-derived and HTLV-positive in vitro-transformed cell lines with that of uninfected primary CD4 T cells. Cells were harvested one day after splitting, in case of primary T cells, cells were harvested after reaching a postmitotic state.
Project description:We used microarrays to compare the global programme of gene expression in HTLV-positive, ATL-derived and HTLV-positive in vitro-transformed cell lines with that of uninfected primary CD4 T cells. Overall design: Cells were harvested one day after splitting, in case of primary T cells, cells were harvested after reaching a postmitotic state.
Project description:We performed ChIP-on-chip analysis of primary ATL patient cells, normal CD4+ T cells, and Tax-transduced cells to decipher the ATL-specific ‘epigenetic-code’ that was critical in ATL pathogenesis. Overall design: Comparison of histone H3K27me3 and H3K4me3 marks in normal, Tax-transduced, and ATL cells. Investigation of the biological effects of HTLV-1 Tax on the host epigenome in HTLV-1 infected cells.
Project description:The human T-lymphotropic virus type-1 (HTLV-1) is the cause of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spatic paraparesis (HAM/TSP). Both diseases have a late onset, although ATL has a medium survivor of 7.7 months after the onset despise aggressive treatment. T CD4+ cells are the main target of HTLV-1, but other cells types are known to be infected as T CD8+, CD34+ progenitor, dendritic cells and immature lymphocytes. The thymus gland is a primary lymphoid organ, in which the lymphocytes undergo differentiation, where selected T cell ultimately being exported from the organ and going to peripheral lymphoid organs. This process occurs along with immature lymphocyte migration and interaction with thymic microenvironment. Thymic epithelial cells (TECs) are the main component of the thymic stroma and are responsible for the process of intrathymic T cell maturation. This process are dependent of T cell receptor (TCR)-mediated recognition of antigenic peptide fragments presented by major histocompatibility complex (MHC) molecules in TEC and culminate in TCR repertoire formation. In this study, we show that TECs have the receptors for HTLV-1 entry and can be infected by cell-cell contact and cell-free virus. These cells change gene expression of anti-apoptosis, chemokine and adhesion molecules genes; however, there is no difference in antigen presentation molecules. Furthermore, HTLV-1 infected TECs can transmit the virus to a CD4 T cell lineage and CD4 T cells derived from peripheral blood of healthy donors after in vitro co-cultivation. Conjointly, our data points to the possibility that the human thymic epithelial cells must favor the HTLV-1 infection as a reservoir, transmitting the virus to maturing lymphocytes that cause the disease in the periphery. Overall design: We used non-treated Thymic Epithelial Cell (TEC) and TEC treated with non-infected T cell supernatant (CEM). We analyze three independent samples (biological replicates) for each conditions (total 2 conditions) and also technical replicates using dye-swap. In total we use 1.5 slides with 4 array in each.