Transcription profiling of mouse IL-7Rhi/lo effector CD8 T cells on day 6/7 after LCMV armstrong infection
ABSTRACT: At the peak of the CD8 T cell response to acture viral and bacterial infections, expression of the Interleukin-7 Receptor (IL-7R) marks Memory Precursor Effector CD8 T Cells (MPECs) from other Short-Lived Effector CD8 T cells (SLECs), which are IL-7Rlo. This study was designed to determine the gene expression differences between these two subsets of effector CD8 T cells. Experiment Overall Design: This study compared IL-7Rhi and IL-7Rlo LCMV-specific P14 Transgenic CD8 T cells, sorted from LCMV armstrong infected recipient mice 6/7 days after infection. Data includes 3 independent replicates for the IL-7Rhi and IL-7Rlo groups.
Project description:At the peak of the CD8 T cell response to acture viral and bacterial infections, expression of the Interleukin-7 Receptor (IL-7R) marks Memory Precursor Effector CD8 T Cells (MPECs) from other Short-Lived Effector CD8 T cells (SLECs), which are IL-7Rlo. This study was designed to determine the gene expression differences between these two subsets of effector CD8 T cells. Keywords: expression comparison Overall design: This study compared IL-7Rhi and IL-7Rlo LCMV-specific P14 Transgenic CD8 T cells, sorted from LCMV armstrong infected recipient mice 6/7 days after infection. Data includes 3 independent replicates for the IL-7Rhi and IL-7Rlo groups.
Project description:In response to acute infection CD8 T cells differentiate into effector cells capable of clearing the antigen. While the transcriptional and functional changes have previously been studied little is known of the epigenetic modifications that accompany this differentiation process. To gain insights into CD8 T cell effector differentiation and the role of epigenetics, we mapped DNA methylation by MeDIP-seq in naive CD8 T cells and day 8 effector CD8 T cells that are induced following an acute infection. We identified hundreds of thousands of differentially methylated regions (DMRs). Promoter DNA methylation inversely correlated with gene expression and DMRs were enriched for functional transcription factor binding sites. These data indicated that DNA methylation is dynamic during CD8 T cell differentiation and provide a map of possible regulatory regions important in this process. Examination of DNA methylation during CD8 T cell differentiation from naïve to day 8 effectors following acute infection
Project description:During acute viral infections, effector CD8+ T cells differentiate into memory precursors or short-lived terminal effectors. miR-17-92a over-expression skews CD8+ effector cells to the terminal differentiation. We used microarray to identify the genes that are differentially expressed caused by miR-17-92a over-expression. CD8+ T cells from P14 TCR transgenic mice were infected with miR-17-92a-MSCV-IRES-Thy1.1 vector and transfer to C57BL6 recipients. Chimeras were infected with LCMV Armstrong. Thy1.1+ miR-17-92a-MSCV-IRES-Thy1.1 transduced P14 cells and Thy1.1- non-transduced P14 cells were sorted by FACS. RNA was extracted from samples, labeled, and hybridized to Affymetrix microarrays.
Project description:miRNAs play an important role in regulating CD8+ T cell response. We used the microarray approach to profile the miRNA signatures of naïve, day 5 effector, day 8 effector, and memory CD8+ T cells. We identified a miRNA signature associated with rapidly proliferating effector CD8+ T cells. CD8+ T cells from P14 TCR transgenic mice were transferred to C57BL6 recipients which were subsequently infected with LCMV Armstrong. Donor P14 CD8+ T cells were sorted on day 5, day 8, or >day 60 post-infection. Naïve P14 CD8+ T cells were sorted directly from naive P14 splenocytes. The total RNA including miRNAs was extracted from sorted samples, labeled, and hybridized to Agilent Mouse miRNA microarray.
Project description:The PI3K/Akt signaling pathway impacts various aspects of CD8 T cell homeostasis, such as effect versus memory cell differentiation, during viral infection. We used microarrays to determine which downstream molecules were affected and what other signaling pathways were interconnected with the Akt pathway by constitutive activation of Akt in LCMV-infected CD8 T cells. Splenocytes from naive P14/WT or P14/Akt mice were stained with anti-CD8 and anti-Ly5.1, and CD8 T cells were sorted using a FACSAria II instrument. Purified Ly5.1+ CD8 T cells from P14/WT or P14/Akt mice were transferred into B6 mice, which were subsequently infected with LCMV Armstrong. At day 8 post infection, splenocytes were stained with anti-CD8, anti-Ly5.1, anti-KLRG1, and anti-CD127. Following staining, short-lived effector cells (SLECs) and memory precursor effector cells (MPECs) were sorted using the FACSAria II instrument; the purity of the sorted cells was >95%. A total of 5 samples were analyzed, including WT naive, WT SLEC, WT MPEC, Akt naive and Akt SLEC.
Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong
Project description:Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection. However, despite heightened readiness to respond, memory cells exist in a functionally quiescent state. The paradigm is that memory cells remain inactive due to lack of TCR stimuli. Here we report a unique role of Tregs in orchestrating memory quiescence by inhibiting effector and proliferation programs through CTLA-4. Loss of Tregs resulted in activation of genome-wide transcriptional programs characteristic of potent effectors, and both developing and established memory quickly reverted to a terminally differentiated (KLRG-1hi/IL-7R±lo/GzmBhi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality and protective efficacy. CTLA-4, an inhibitory receptor overexpressed on Tregs, functionally replaced Tregs in trans to rescue Treg-less memory defects and restore homeostasis of secondary mediators as well. These studies present CD28-CTLA-4-CD80/CD86 axis as a novel target to potentially accelerate vaccine-induced immunity and improve T-cell memory quality in current cancer immunotherapies proposing transient Treg-depletion. We used microarray analysis to detail the global programming of gene expression in LCMV GP33-specific CD8 T cells differentiated in the presence or absence of regulatory T cells Differentiation of memory CD8 T cells entails a progressive transition of highly activated effector program to a quiescent memory program. A key question in the field is to understand the factors that aid in the differentiation of memory cells from effector cells. It is a generally accepted paradigm that effector cells transition to a memory state by default after antigen clearance, since TCR stimuli is the key driver of effector programs in CD8 T cells. We hypothesized that the effector to memory transition of CD8 T cells involves active immunological brakes through regulatory T cells (Tregs) that allow the highly activated effector cells to convert into quiescent memory cells. To address this hypothesis, we used FoxP3-DTR mice to deplete Tregs during the window following antigen clearance, during which the effector CD8 T cells convert to long-lived memory cells. To get a deeper understanding of the global transcriptome of CD8 T cells as they transition from an effector to a memory state, we isolated and arrayed the antigen-specific CD8 T cells at day 16 post-infection that have experienced the transitional environment with and without the presence of Tregs.
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
Project description:The forkhead O transcription factors (FOXO) integrate a range of extracellular signals including growth factor signaling, inflammation, oxidative stress and nutrient availability, to substantially alter the program of gene expression and modulate cell survival, cell cycle progression, and many cell-type specific responses yet to be unraveled. Naive antigen-specific CD8+ T cells undergo a rapid expansion and arming of effector function within days of pathogen exposure, but in addition, by the peak of expansion, they form precursors to memory T cells capable of self-renewal and indefinite survival. We used microarrays to determine whether FOXO1 broadly affects effector and memory differentiation, and to what extent FOXO1 determines the program of memory T cell gene expression. To obtain an unbiased analysis of genes differentially expressed in antigen-specific Foxo1-/- CD8+ T cells responding to infection, we obtained RNA and performed Affymetrix microarray analysis from KLRG1low and KLRG1high FACS-sorted congenically-marked WT and Foxo1-/- P14 cells obtained from mixed transfers, eight days post-infection with LCMV-Armstrong. We carried out gene deletion in Rosa26Cre-ERT2 Foxo1f/f (Foxo1-/-) P14 mice just prior to adoptive transfer (Kerdiles et al., 2009), and transfer equal numbers of P14 cells from the spleens of KO (Foxo1-/- P14) and WT P14 mice. Day8 post infection
Project description:During acute viral infections, naïve CD8+ T cells differentiate into effector CD8+ T cells and, after viral control, into memory CD8+ T cells. Memory CD8+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD8+ T cells become “exhausted” and have poor effector function, express multiple inhibitory receptors, possess low proliferative capacity, and cannot persist without antigen. To compare the development of functional memory T cells with poorly functional exhausted T cells, we generated longitudinal transcriptional profiles for each. Naive CD44Lo CD8+ T cells were isolated and sorted from uninfected C57BL/6 mice and H2-Db GP33-specific CD8+ T cells were sorted using MHC-I tetramers at d6, 8, 15, and 30 p.i. with either LCMV Arm or LCMV clone 13. RNA from these CD8+ T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays