Project description:ab and gd T cells originate from a common, multi-potential precursor population in the thymus, but the molecular mechanisms regulating this lineage fate decision process are unknown. We have identified Sox13 as a gd-specific gene in the immune system. Using Sox13 transgenic mice, we show that SOX13 promotes gd T cell development while opposing ab T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gd T cells, but not ab T cells. One mechanism of SOX13 function is the inhibition of WNT/TCF signaling, suggesting that differential WNT/TCF activity is an essential parameter for this binary cell fate choice. Experiment Overall Design: CD4+CD8+ double positive thymocyte subsets were sorted by FACS using total pooled thymocytes from minimum of two mice and immediately lysed in Trizol. Comparison groups in each experiment were DP thymocytes from Sox13 transgenic mice and wild type B6 littermate controls. Gene expression profiling was performed according to the manufacturerâ??s protocol (Affymetrix). Labeled cRNA (from total RNA) was generated and applied to Affymetrix Mu11K(A and B) (expt 1) or muU74Av2 (expt 2) microarrays. Results were analyzed using Microarray Analysis Software v4 and v5 (Affymetrix).
Project description:Sustained Ca2+ entry into CD4+CD8+ double-positive thymocytes is required for positive selection. We identified a voltage-gated Na+ channel (VGSC), essential for positive selection of CD4+ T cells. Pharmacological inhibition of VGSC activity inhibited sustained Ca2+ influx induced by positive-selecting ligands and in vitro positive selection of CD4+ but not CD8+ T cells. In vivo shRNA knockdown of Scn5a specifically inhibited positive selection of CD4+ T cells. Ectopic expression of VGSC in peripheral AND CD4+ T cells bestowed the ability to respond to a positively selecting ligand, directly demonstrating VGSC expression was responsible for increased sensitivity. Thus active VGSCs in thymocytes provides a mechanism by which a weak positive selecting signal can induce sustained Ca2+ signals required for CD4+ T cell development. Pre-selected AND DP thymocytes were stimulated with plate bound peptide-loaded I-Ek Ig dimers. Genes were differentially regulated by positive and negative selection signals. The goal of this study is to identify ion-channel related genes critical for thymic positive selection, given the sustained Ca2+ entry into double positive thymocytes is required for positive selection. Total RNA of pre-selected AND thymocytes stimulated with I-Ek Ig dimers loaded with the positively-selecting peptide gp250, the negatively-selecting agonist peptide MCC, or the non-selecting control peptide Hb were used for the transcriptional profiling analysis. We found 28 genes were differentially down-regulated by MCC stimulation but upregulated by gp250 stimulation. One of them, scn4b, was ion-channel related.
Project description:Cellular binary fate decisions require the progeny to silence genes associated with the alternative fate. The major subsets of alpha:beta T cells have been extensively studied as a model system for fate decisions. While the transcription factor RUNX3 is required for the initiation of Cd4 silencing in CD8 T cell progenitors, it is not required to maintain the silencing of Cd4 and other helper T lineage genes. The other runt domain containing protein, RUNX1, silences Cd4 in an earlier T cell progenitor, but this silencing is reversed whereas the gene silencing after RUNX3 expression is not reverse. Therefore, we hypothesized that RUNX3 and not RUNX1 recruits other factors that maintains the silencing of helper T lineage genes in CD8 T cells. To this end, we performed a proteomics screen of RUNX1 and RUNX3 to determine candidate silencing factors.
Project description:Single cell suspensions of total thymocytes were obtained from Pten enhancer (PE) wild-type or knockout mice. This single-cell suspension was enriched in CD4-CD3- immature thymocyte progenitor cells. CD4-CD3- enriched thymocytes were then mixed 1:1 with single-cell suspensions from total unenriched thymocytes and subsequntly loaded in a 10x Chromium instrument for single-cell RNAseq analyses. Our results revealed Pten levels are signifcantly decreased in CD4-CD8- double negative (DN) thymocytes, CD8+ intermediate single positive (ISP) thymocytes and CD4+CD8+ double positive (DP) thymocytes in PE knockout mice, compared to PE wild-type mice.
Project description:Comparative gene expression profiling of thymocytes at the DP, CD4 SP and CD8 SP stage derived from FoxN1-Gpr177 mice (FoxN1-Cre mediated deletion of (Exon3 of) Gpr177/Wtls) or C57Bl/6N mice as comparison. Objective was to test the influence of TEC-secreted Wnt ligands on the transcriptome of thymocytes at the respective developmental stages. Total RNA extracted from FACS-sorted primary mouse thymocytes. CD4/8 double positive (DP) thymocytes, CD4 single positive (CD4 SP) thymocytes and CD8 single positive (CD8 SP) thymocytes were FACS-sorted from conditional knock-out mice (FoxN1-Gpr177) and C57Bl/6N mice as comparison.
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+).
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:To determine the effect of mutation of the KIX domain of CBP and p300 on gene expression, thymuses dissected from four to seven week old wild type and CBP KIX/KIX;p300 +/KIX adult mice were dissociated into single cell suspesions, antibody stained for CD4 and CD8, sorted for CD4+ CD8+ double positive thymocytes, RNA was made from the sorted cells and transcriptional profiling by array was carried out. CD4+CD8+ double positive thymocytes were chosen because these cells express c-Myb.