Project description:Define,(a) intrinsic differences and (b) changes occuring upon TCR activation in genetic profiles of naive CD4+ and CD8+ T cells from young and old animals, to identify candidate genes altered in old T cells involved in impairement of immune response in order to restore immune function in the old. Activation dependant time series on sorted naive (CD62Lhi/CD44lo) CD4 and CD8 T cells from young and old animals-Each time point contains RNA pooled from 4 independent sortings
Project description:To increase the dataset of our proteomic naive CD4+ T cell surface atlas, we applied whole genome microarray expression analysis. The NCBI RefSeq accession number of all transcripts which were detected in naive and stimulated CD4+ T cells (aCD3/aCD28 for 3h) of four different donors, was mapped to the corresponding UniProtKB accession number. For these UniProtKB ACs, we extracted the subcellular localization (UniProt_SL) annotation from the UniProtKB/Swiss-Prot database or if not available the subcellular localization was predicted unsing LocTree3 and PolyPhobius. This led to the identification of 908 genes coding for proteins located on the surface of human naive and/or activated CD4+ T cells. Gene expression in human naive T cells and naive T cells which were stimulated with aCD3/aCD28 for 3h was measured. Naive T cells from four different human blood donors were analyzed.
Project description:Naive CD4+ T cells are the common precursors of multiple effector and memory T cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy.<br>Periodate oxidation and aniline-catalyzed oxime ligation (PAL) technology was applied with subsequent quantitative LC-MS/MS (PAL-qLC-MS/MS) to generate a dataset describing the surface proteome of human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins, of which 24 were previously not known to be expressed on human naive CD4+ T cells or have no defined role within T cell activation. To independently confirm the proteomic dataset and to analyse the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous dataset, resulting in 229 surface proteins which are expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation and predicted subcellular localization, and correlated the proteomics result with this transcriptional dataset.<br>This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments.
Project description:Title: Gene Expression analysis of naive CD8+ T-cell differentiation during a primary immune response Description: C57BL/6 wild type mice are administered GK1.5 (depleting anti CD4 antibody) or isotope control by intraperitoneal injection at 3 and 1 day pre-infection, then every 36-48 hour post infection. 24 hours pre-infection, splenocytes from F5 RAG -/- mice are stained with CFSE and 10^7 splenocytes are transfered to recipient C57BL/6 mice by intravenous injection. 24 hours laters, recipient mice are infected with 10^7 PFU of a recombinant Vaccinia virus expressing NP366-74. At timepoints 0, 12, 24, 48, 72 and 96 splenocytes are obtained and FACS sorted to obtain CD8+ T-cells.
Project description:CD4+CD25-CD62L+ naive T-cells were FACS isolated from wild type mouse lymph node. These were compared to regulatory T cells (Treg; CD4+C25+) and activated T-cells induced to express Foxp3 by treatment with rapamycin and LY294002 (Treg-like cells). RNA was extracted from duplicate cultures using RNA-Bee, labelled with the Affymetrix GeneChip Small Sample protocol and hybridised to Affymetrix Mouse 430 2.0 Arrays.
Project description:Th17 cells are believed to be a critical cell population for driving autoimmune diseases. However, environmental factors that are directly related to the development of Th17 cells are largely unknown. High-salt (NaCl) concentrations enhance Th17 differentiation of human naive CD4+ T cells in vitro. The aim of the study was to analyse the changes in gene expression induced by high-salt conditions during Th17 differentiation. Naive human CD4+ T cells were in vitro differentiated into Th17 cells in the presence or absence of high-salt. We arrayed 2 different donors for each condition (control & high-salt).
Project description:HnRNPLL was identified as a critical regulator of CD45 alternative splicing in a lentiviral shRNA screen. RNAi-mediated depletion of hnRNPLL eliminated the activation-induced induced transition from the CD45RA to the CD45RO isoform. HnRNPLL is induced during the process of T cell activation, raising the possibility that it regulates a broad program of alternative splicing in activated T cells. To test this possibility and to identify additional potential targets of hnRNPLL, we performed exon array analysis on RNA isolated from five cellular conditions: 1) activated peripheral CD4+ T cells, 2) peripheral CD4+ T cells infected with a control shRNA directed against GFP, 3) peripheral CD4+ T infected with an shRNA directed against hnRNPLL, 4) naïve cord blood CD4+ T cells, and 5) cord blood CD4+ T cells that had been activated with anti-CD3 and anti-CD28 for 24 hours. The RNA was hybridized to Affymetrix human exon arrays and the hybridization signals were analyzed with XRAYTM software (Biotique). Using stringent filters for non-expressed probesets, we identified 132 genes that showed significant alternative exon usage (p<0.01) in response to hnRNPLL knockdown, but not in response to shGFP infection. Of these 132 genes, 36 also showed significant alternative exon usage in response to activation of cord blood cells, which results in an approximate 5-fold increase in hnRNPLL expression. We thus conclude that induction of hnRNPLL represents a mechanism by which cells can rapidly shift their transcriptomes during the process of T cell activation. This SuperSeries is composed of the following subset Series: GSE11832: naive/activated ANOVA group GSE11833: LL-sh4/uninfected/shGFP ANOVA group
Project description:The naive CD4 T cell compartment is heterogeneous. Ly-6C- and Ly-6C+ Naive CD4 T cells were compare by microarrays. Ly-6C- and Ly-6C+ Naive CD4 T cells were purified for RNA extraction and hybridization on Affymetrix microarrays.
Project description:The methylation profile of human cytotoxic CD4 T cells was compared to their naive counterpart, to central memory Th1 cells, and to naive and cytotoxic CD8 T cells in order to elucidate the epigenetic basis of cytotoxic differentiation in CD4 T cells. Overall design: Total genomic DNA obtained from ex-vivo isolated blood lymphocytes of 5 CMV-seropositive healthy adults. For each subject, five populations were sorted: naive CD4, naive CD8, central memory CD4 Th1 cells, cytotoxic CD28- CD4 T cells and cytotoxic CD27- CD8 T cells.