Project description:Little is known about the global transcriptional program underlying CD8+ T-cell activation. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of human CD8+ T-cell activation. The goal of this study was to identify genes involved in the various facets of human CD8+ T-cell activation. Keywords: time course
Project description:Little is known about the global transcriptional program underlying CD8+ T-cell activation. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of human CD8+ T-cell activation. The goal of this study was to identify genes involved in the various facets of human CD8+ T-cell activation. Experiment Overall Design: CD8+ T cells isolated from peripheral blood were cultured with CD3, CD28, with or without IL-2 to induce T-cell activation. At each timepoint, cells were harvested and frozen for RNA isolation. Three biological replicate experiments were analyzed and approximately one-half of the samples from each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference thymus total RNA labeled with Cy5.
Project description:Little is known about the global transcriptional program underlying CD4+ T-cell activation. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of human CD4+ T-cell activation. The goal of this study was to identify genes involved in the various facets of human CD4+ T-cell activation. Keywords: time course
Project description:T-cells are a critical component of the adaptive immune system and play a key role in immunological surveillance. Upon engagement of T-cell receptor (TCR), CD4+ and CD8+ T-cells acquire effector functions through a complex interplay between mRNA and proteins yet to be fully understood. In this study we explored the temporal transcriptomic and proteomic changes mediated by TCR engagement in both CD4+ and CD8+ T-cells. T-cells isolated from peripheral blood mononuclear cells of three healthy volunteers over 90% purity as assessed by fluorescence-labeled flow cytometry (FACS) and monoclonal antibodies were in vitro activated using anti-CD3/CD28 Dynabeads. Samples obtained before the activation, and 6h, 12h, 24h, 3 days (d), and 7d following activation were analyzed using label-free data-dependent acquisition mass spectrometry-based proteomics (DDA-proteomics), to identify the temporal dynamics in CD4+ and CD8+ T-cell proteomes during activation. A parallel analysis was performed to explore the transcriptomic dynamics during T-cell activation. Our data revealed a time-dependent dissociation between the T-cell transcriptome and proteome: the onset of activation was driven by rapid changes of the mRNA content with sluggish increase in protein synthesis, ultimately leading to rewired transcriptome and proteome. We surprisingly found that CD4+ and CD8+ T-cells became transcriptionally more divergent while their proteome became more similar over the time course of activation. Several changes in the content of mRNAs and proteins associated with metabolic pathways were detected through KEGG pathway analysis, revealing a transient disconnection between the aerobic glycolysis and glutaminolysis pathways in activated T-cells. This dataset provides a comprehensive framework for understanding the main temporal changes that regulate metabolic pathways governing the acquisition of effector functions by CD4+ and CD8+ T-cells.
Project description:CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.
Project description:CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective; proximal TCR signaling, cytokine secretion, and cytolysis; and enhanced AICD. pcdh18 contains a motif (centered at Y505) shared with src kinases (QGQYQP) which is required for the inhibitory phenotype. Thus, pcdh18 is a novel marker of CD8+ effector memory T cells expressed upon cell activation that can function as a negative regulator by restricting the effector phase. We used microarrays to detail the global programme of gene expression underlying CD8+ 'Central memory' T cells activation and identified distinct transcriptional pattern clusters. Ten spleens were pooled from 62 week old C57BL/6 male mice and enriched for CD8+ T cells by negative selection using a cocktail of biotinylated antibodies to deplete CD4, CD11c, CD11b, MHC-II, B220, and NK cells. Cells were then stained for CD8, CD44, CD62L, and CD127 and sorted (using a iCyt Reflection parallel cell sorter). Cells were collected and cultured (0.5 x106 cells/well) in 10% complete RPMI media supplemented with 0.005 ugr Con A and RNA prepared using TRIZOL and the 'RNeasy clean-up kit' (Invitrogen) after activation for different times. We established single transcriptional profiles for 6 time points after T cell activation (4, 6, 8, 16, 20, 24 h) including a control zero h time point (prior to activation). RNA was isolated by standard procedures and its quality was assessed by the NYU Center for Health Informatics and Bioinformatics. cDNAs were hybridized to GeneSpring arrays using the mouse genome MOE430 2.0 array (Affymetrix) which interrogates ~45,000 transcripts.
Project description:Gene expression of Tfap4â/â and WT CD8+ T cells were compared after activation with anti-CD3 and anti-CD28 antibodies in vitro or with Listeria monocytogenes infection in vivo For in vitro activation, naive CD8+ T cells were purified from WT and Tfap4â/â mice and activated with anti-CD3 and anti-CD28 antibodies for 72 hours. For in vivo activation, naive CD8+ T cells from Tfap4â/â OT-I or control WT OT-I TCR transgenice mice were adoptively transferred to congenic host mice that were subsequently infected with Listeria mnocytogenes expression ovalbumin. Activated OT-I cells were harvested 48 hours after infection.
Project description:This SuperSeries is composed of the following subset Series: GSE34216: miRNA signatures of antigen specific CD8+ T cells at different stages of immune response to LCMV infection GSE34217: Expression profile of miR-17-92a-MSCV-IRES-Thy1.1 transduced P14 CD8+ T cells Refer to individual Series