Gene expression profiles of Themis-deficient thymocytes
ABSTRACT: Determine the biochemical cause of impaired development of Themis-deficient thymocytes. Overall design: We isolated three thymocyte populations (three populations: CD4+CD8+CD5loCD69lo, CD4+CD8+CD5+CD69+, and CD4+CD8-CD24hiH2Klo) from Themis-deficient and WT mice. 8 or 9 individual biological replicates were analyzed for each population. An aliquot of cDNA from each sample was pooled and used as a reference.
Project description:Determine the biochemical cause of impaired development of Themis-deficient thymocytes. We isolated three thymocyte populations (three populations: CD4+CD8+CD5loCD69lo, CD4+CD8+CD5+CD69+, and CD4+CD8-CD24hiH2Klo) from Themis-deficient and WT mice. 8 or 9 individual biological replicates were analyzed for each population. An aliquot of cDNA from each sample was pooled and used as a reference.
Project description:Comparison of transcriptome between control and Tcf1/Lef1-deficient mature CD8 thymocytes Control mice or those are deficient for Tcf1 and Lef1 transcription factors (deleted by CD4-Cre) were used to isolate thymocytes. The thymocytes were surface-stained to identify TCRbeta high, CD69–, CD24– CD8+ subsets. These cells were sorted for RNAseq analysis.
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:The biological functions of histone demethylases Jmjd3 and Utx remain poorly understood. We assessed such functions in developing T cells, using conditional (CD4-Cre-mediated) gene disruption, by inactivating Kdm6a and Kdm6b, respectively encoding Utx and Jmjd3, in immature CD4+CD8+ thymocytes. We compared microarray gene expression in mature (Va2hi CD24lo) mutant and wild-type CD4+CD8- thymocytes carrying the OT-II TCR transgene. We show that Jmjd3 and Utx redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Mature (Va2hi CD24lo) CD4 thymocytes were sorted from freshly prepared single-cell suspensions OT-II TCR transgenic thymocytes deficient for Utx and Jmjd3 (dKO, CD4-Cre conditional deletion of floxed Kdm6a and Kdm6b alleles), and from Cre-negative controls (wild-type). Total RNA was extracted from sorted thymocytes using the RNeasy Plus Mini Kit (Qiagen) and processed for microarray analyses (Affymetrix Mouse Exon 1.0 ST array) at the NCI microarray facility, following the manufacturer’s recommendation. Data is generated from 3 replicates from each experiment.
Project description:Here we utilized a conditional knock-out mouse model to investigate the role of Smarca5, an ISWI subfamily chromatin remodeling ATPase, during thymocyte development using hCD2-iCre transgene. We did transcriptional profiling of FACS-sorted CD4/CD8 double-positive thymocytes as this thymic population was persistent yet strongly underrepresented in adult 6-week mutant thymi. Controls included age-matched CD4/CD8 double-positive thymocytes from wild-type and tumor suppressor protein Trp53-null mice. For comparison, the Smarca5/Trp53-double-deficient thymocytes included in the experiment were partially rescued upon loss of Trp53.
Project description:Thymic lymphomas develop spontaneously in LN3 mice. As for T-ALL in general, ex vivo LN3 lymphoma cells require stromal support to remain viable in culture. We found that primary stromal cells from thymic lymphomas, but not from wild-type thymi, support ex vivo lymphoma survival. By FACS sorting stromal populations, we identified dendritic cells in the tumor microenvironment as the cells capable of supporting lymphoma survival. We used microarrays to analyze the gene expression profiles of T-ALL cells and tumor-associated versus wild-type thymic dendritic cell subsets. Overall design: Sirpa+ and Sirpa- conventional thymic dendritic cells, as well as CD45+MHCII- T-ALL tumor cells, were isolated from three male LN3 tumor-bearing mice. After enzymatic digestion of the thymi, the dendritic cells and tumor cells were FACS purified, and RNA was extracted and hybridized to Affymetrix Mouse 430 2.0 arrays. Raw data were uploaded to Gene Expression Commons for RMA normalization. Both raw CEL and normalized datasets from the 9 samples are included. Normalized datasets for Sirpa+ and Sirpa- conventional thymic DC subsets in WT mice at 6 months of age is included here as well. A model within Gene Expression Commons has been created for comparison of these datasets to previously reported WT thymocytes from mice at 6 months of age. The model within Gene Expression Commons contains 22 populations: 6-month WT Sirpa- DC (average of 2 datasets), 6-month WT Sirpa+ DC (average of 2 datasets), 3 individual tumor-associated Sirpa-DC populations , 3 individual tumor-associated Sirpa+ DC populations, and 3 individual CD45+MHCII- T-ALL cell populations. In addition WT thymocyte subsets (DN1, DN2, DN3a, DN3b, DN4, DP CD69-, DP CD69+, CD4 CD69+, CD4 CD69-, CD8 CD69+, and CD8 CD69+) are included, each as averages of 3 datasets. WT thymocyte datasets were published in Seita et al.,PLoS ONE (2012), vol. 7 (7) e40321 (GSE34723). WT thymic dendritic cell subsets from 6 month old mice were published in Ki et al., Cell Rep (2014), vol. 9, pp. 402-15 (GSE56928).
Project description:Comparison of epigenome and Tcf1 occupancy between control and Tcf1/Lef1-deficient CD8 T cells Control mice or those are deficient for Tcf1 and Lef1 transcription factors (deleted by CD4-Cre) were used to isolate thymocytes. The thymocytes were surface-stained to identify TCRbeta high, CD69–, CD24– CD8+ subsets. These cells were sorted for ChIPseq analysis of various histone marks. Control mice or those are deficient for Tcf1 (deleted by CD4-Cre) were used to isolate thymocytes. The splenocytes were surface-stained to identify TCRbeta high, CD8+ subsets. These cells were sorted for ChIPseq analysis of Tcf1 binding locations.
Project description:Mice lacking components of the linear ubiquitin chain assembly complex (LUBAC) exhibit defects in thymocyte differentiation. To determine how two LUBAC components, HOIL and SHARPIN, mediate their effects, we purified thymocytes from CD4Cre Hoil-fl/fl or Sharpin-cpdm mice and compared their transcriptional profile to WT controls. We focused on the CD69+ MHCI low and CD69+ MHCI high subsets that follow positive selection. Overall design: Thymocytes were prepared from 3 wildtype mice, 3 Cd4Cre Hoil-floxed mice and 3 Sharpin-cpdm mutant mice. CD69+ MHC I high and CD69+ MHC I low cells from each mouse were FACS purified on a MoFlo cell sorter.
Project description:Microarray analysis of thymic deletion in the B10 mouse strain The gene expression patterns of thymocytes at the pre-selection stage and undergoing positive selection or negative selection were compared in the C57BL/10-H2k (B10k) strain. In order to sort thymocytes homogenously undergoing positive selection the 3A9 TCRHEL transgenic was used, which directs thymocyte development towards a MHC class II-restricted HEL-reactive lineage. To sort thymocytes homogenously undergoing negative selection, the 3A9 TCRHEL transgenic was crossed to the insHEL transgenic, which expresses HEL under the control of the insulin promoter in the thymic stroma, leading to efficient negative selection at the early single positive (CD4+CD8low) stage. To prepare the samples, thymic cell suspensions from female 6-8 week old B10k mice, either 3A9 TCRHEL or 3A9 TCRHEL x insHEL transgenics, were stained with CD4-FITC, CD69-PE, CD8-PerCP, and 1G12 supernatant (which recognises the 3A9 TCRHEL complex) followed by anti-IgG1-APC, and sorted. Pre-selection cells (from both transgenics) were defined as CD4+CD8+CD69-1G12low. Cells undergoing positive selection (from 3A9 TCRHEL transgenics) or negative selection (from 3A9 TCRHEL x insHEL transgenics) were defined as CD4+CD8lowCD69+1G12high. Keywords: repeat Overall design: Single positive or double positive thymocytes from B10 mice were compared to those from B10 TCRHEL transgenic mice. 3 biological replicaes were used for each of the 4 different groups of thymocytes.
Project description:T-cell-specific deletion of USP8 (USP8ffCD4Cre) revealed that USP8 is required for thymocyte transition to the CD4+ and CD8+ single positive (SP) stages. To evaluate underlying mechanisms, gene expression profilling was performed in CD4+CD8+ double pos thymocytes derived from control (USP8ff and USP8ffCD4Cre) mice. Microarray analysis of two groups of USP8fl/fl and USP8fl/flCD4Cre thymocytes. Material from two mice was combined for each group (eight mice in total).