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:Comparison of gene expression profile of CD4+ CD25+, CD4+ CD25- CD45RBlow LAG3+ and CD4+ CD25- CD45RBlow LAG3- T cells. Naive CD4+CD25-CD45RBhigh T cells were used as a reference for pair comparison with values from the three other subsets.
Project description:Type 1 regulatory T (Tr1) cells are one of the regulatory T cell subsets that are characterized by the production of high amount of IL-10 and lack of FOXP3 expression. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue molecule and we have previously reported that LAG3 is expressed on IL-10 producing regulatory T cells. However, naturally occurring Tr1 cells in human secondary lymphoid tissue have not been detected. We identified CD4+CD25-LAG3+ T cells in human tonsil. We compared mRNA expression of five CD4+ T cell subsets in tonsil using microarray analysis (CD4+CD25-LAG3+ T cells, CD4+CD25-CXCR5+PD-1+ follicular helper T cells (TFH), CD4+CD25+ T cells, CD4+CD25-LAG3-CD45RO+ cells and CD4+CD25-LAG3-CD45RO- cells). A human tonsil was obtained from a patient undergoing routine tonsillectomy, and five tonsillar CD4+ T cell subsets were sorted (each 1 x 10^5 cells). There is no biological replication.
Project description:A comparison of CD4+CD25+ and CD4+CD25- splenic T cells. Although both subsets have regulatory T cell activity in an induced transplantation tolerance model, the CD4+CD25+ subsets are apparently 10 fold enriched in regulatory T cells. SAGE analysis is performed and compared both on resting and CD3 activated populations. Keywords: other
Project description:Type 1 regulatory T (Tr1) cells are one of the regulatory T cell subsets that are characterized by the production of high amount of IL-10 and lack of FOXP3 expression. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue molecule and we have previously reported that LAG3 is expressed on IL-10 producing regulatory T cells. However, naturally occurring Tr1 cells in human secondary lymphoid tissue have not been detected. We identified CD4+CD25-LAG3+ T cells in human tonsil. We compared mRNA expression of five CD4+ T cell subsets in tonsil using microarray analysis (CD4+CD25-LAG3+ T cells, CD4+CD25-CXCR5+PD-1+ follicular helper T cells (TFH), CD4+CD25+ T cells, CD4+CD25-LAG3-CD45RO+ cells and CD4+CD25-LAG3-CD45RO- cells).
Project description:Naïve T cells were obtained by StemCell CD4+ T cell isolation kit of spleen from Mettl3 KO and WT mice followed by FACS sorting (CD4+CD25-CD45RB-Hi). Raw sequencing reads were aligned to the mouse genome (mm10) with Tophat, and gene expression levels were measured by Cufflinks.
Project description:Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a buffy coat (Australian Blood Bank) using Ficoll methodology. CD4+ T cells were isolated using Dynal Beads kit. Pure CD4+ T cells were then stained using a cocktail of monoclonal antobodies (mAbs), including: anti-CD4PE, CD45RO ECD, CD62L APC-Cy7, CD25 APC, CD127 Pacific Blue. After incubation, cells were washed twice in PBS/FCS (0.2%), and sorted into five different cell subsets: CD4+CD25+CD127low CD62L+CD45RO- (naive regulatory T cells), CD4+CD25+CD127low CD62L+/- CD45RO+ (activated regulatory T cells), CD4+CD25+CD127hi CD62L+/- CD45RO+ (memory T cells), CD4+CD25-CD127low CD62L+/- CD45RO+ (effector T cells) and CD4+CD25-CD127hi CD62L+ CD45RO- (naive T cells).
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other