Project description:Gene expression changes when naïve T-cells are stimulated with antigen. We have stimulated these cells with surrogate antigens (aCD3-aCD28 coated beads) for 48 hours, and then did microarray experiment to identify differentially exprssed genes in the two conditions CD4 naïve T-cells from C57/Bl6 mouse were stimulated (activated) with aCD3-aCD28 coated beads for 48 hours and used for microarray experiment
Project description:Expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated co-inhibitory receptor expression on CD4+ T cells promotes autoimmunity while sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and cancer. Here, we used RNA and protein expression profiling at single-cell resolution to identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity.
Project description:To investigate the kinetics of chromatin accessibility dynamics in activated CD8+ T cells, we stimulated naïve P14 CD8+ T cells in vitro for 0h, 1h, or 24h We then performed chromatin accessibility profiling analysis by ATAC-seq
Project description:CD4 and CD8 T cells display functional defects during chronic infection such as loss of certain cytokines. Recent studies have suggested that CD4 T cells may actually gain other functions, however. Here, we analyzed gene expression profiles from LCMV-specific CD4 and CD8 T cells throughout the response to either acute LCMV or chronic LCMV infection. This alllowed us to identify CD4-specific changes during chronic infection compared to acute infection but also revealed shared core regulators between CD4 and CD8 T cells. LCMV-specific CD4 and CD8 T cells were isolated 6, 8, 15 and 30 days post infection with LCMV Armstrong or LCMV clone 13. Naïve CD4 and CD8 T cells were also isolated from naïve mice as comparisons. Four replicates of each sample were hybridized. The only exception is LCMV-specific CD4 T cells isolated 6 days post infection with LCMV-Arm where only three replicates were hybridized.
Project description:To examine the broad impact of IL-27 on human T lymphocytes, we performed a microarray analysis assessing >20,000 well annotated genes on purified naïve (CD45RA+CD45RO-CCR7+) and central memory (CD45RA-CD45RO+CCR7+) CD4+ and CD8+ T cells from three healthy donors that were activated in vitro (plate bound anti-CD3 and soluble anti-CD28) in the presence or absence of human recombinant IL-27 (100 ng/mL). Our goal was to investigate the impact of interleukin-27 on the gene expression profil of human CD4 and CD8 T lymphocytes.
Project description:Using a CRISPR/Cas9-based approach, we engineered human primary CD4+ and CD8+ T cells in which a bait protein (LAT, SLP76, VAV1 or ZAP70) was tagged with an affinity Twin-Strep-tag (OST), with the purpose of determining by quantitative mass spectrometry the composition and dynamics of the signalosome assembling around each of these signaling proteins prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells left non-stimulated, or stimulated for 30s, 60s, 120s, or 300s with anti-CD3 and anti-CD28 antibodies. Each AP-MS purification is associated with a corresponding control (purification from non-edited WT CD4+ or CD8+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS.
Project description:We compared differences in fetal and adult T cells by performing whole genome profiling on sort-purified T cells (naïve CD4+ and Treg cells) from human fetal specimens (18-22 gestational weeks) and adult specimens (age 25-40 years old). Fetal and Adult Naïve CD4+ T cells phenotype: CD3+CD4+CD45RA+CCR7+CD27+, Fetal and Adult CD4+CD25+ Treg phenotype: CD3+CD4+CD25bright Four different groups were analyzed: Fetal Naïve CD4+ T cells, Adult Naïve CD4+ T cells, Fetal Treg cells, Adult Treg cells. For each group three independent donors were analyzed.
Project description:Subpopulations of human fetal thymocyte and circulating naïve T cells were obtained through FACS sorting, including CD3-CD4+CD8- intrathymic T progenitor cells (ITTP), CD3intCD4+CD8+ "double positive" thymocytes (DP), CD3highCD4+CD8- "single positive" thymocytes (SP4), CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from cord blood (CB4+), and CD3+CD4+CD8-CD45RA+CD62L+ naïve T cells from adult blood (AB4+). Keywords = Microarray Keywords = gene expression Keywords = thymocytes Keywords = naïve CD4+ T cell Keywords = recent thymic emigrants Keywords: other
Project description:A microarray was performed on T-Rheb -/- (CD4 cre positive Rheb fl/fl) and Wt C57B6J CD4+ and CD8+ cells. T-Rheb -/- mice had reduced fasting blood glucose and we were interested to see if there was a difference in the T cells that could mediate our phenotype.