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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling analysis of pre-selected DP thymocytes in response to positive and negative selection signals

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Wan-Lin Lo

PROVIDER: E-GEOD-38909 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptional profiling analysis of pre-selected DP thymocytes in response to positive and negative selection signals

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.

2012-07-01 | GSE38909 | GEO

Transcription profiling of mouse thymic deletion in B10 430B strains

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. Experiment 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.

2008-11-13 | E-GEOD-9700 | biostudies-arrayexpress

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during positive selection in the thymus

Project description:Here we show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection at the transition from CD4+CD8+ double positive (DP) to single positive (SP thymocyte). Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes led to a partial arrest at the transition from DP to SP, with a reduction in the number of cells undergoing positive selection, and reduced the number and maturation of both CD4SP and CD8SP populations, with a reduction in peripheral naïve CD4+ T-cells. Foxa1 and Foxa2 were required for physiological levels of expression of several genes which encoding splicing factors (Mbnl1, Sf3b1, Hnrnpa1) in cells undergoing positive selection. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated leading to more than 859 significantly differentially expressed exons compared to control, with many genes important for T cell development and for RNA splicing showing differentially used exons.

2021-07-13 | GSE169602 | GEO

Glycosylation change in mouse CD8 T cells during development

Project description:Dr. Jameson's research focus is on development and regulation of lymphocytes, especially T cells. Recent work has suggested that differential glycosylation effects the sensitivity of the T cell receptors and its coreceptors, suggesting that regulation of glycosylation may be a critical element in controlling T cell development, survival and functional activity. Determination of how glycosylation enzymes/substrates change in gene expression during development of mouse CD8 T cells. Highly purified pre-selection CD4+8+ thymocytes (using transgenic/knockout mice in our colony) are compared to mature CD8 T cells from the lymph node. The goal is to build on data suggesting that this developmental step involves regulated expression of sialyltransferases (and/or neuraminidases). Highly purified pre-selection CD4+8+ thymocytes (using transgenic/knockout mice in our colony) are compared to mature CD8 T cells from the lymph node. Pre-selection CD4+8+ (DP) thymocytes were sorted from TCRa-/- thymi. Post-selection (post-positive selection) DP thymocytes came from an OT-I TCR transgenic mouse. Naïve (CD44lo) CD8 T cells from lymph node of OT-I mice were used as naïve CD8 T cells. For activated cells, naïve OT-I T cells were activated for 48 hours in vitro with cognate antigen (SIINFEKL peptide/Kb) (displayed on cell sized latex beads) in the presence of IL-2 and IL-12.

2010-12-11 | E-GEOD-26012 | biostudies-arrayexpress

Thymic deletion in B10 430B

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

2008-11-01 | GSE9700 | GEO

Expression data from CD4 single positive thymocyte subsets from C57BL/6 mice of 6-8 wks of age

Project description:After positive selection in the thymus, the newly generated single positive (SP) thymocytes are phenotypically and functionally immature and undergo apoptosis upon antigen stimulation. In the thymic medullary microenvironment, SP cells progressively acquire immunocompetence. Negative selection to remove autoreactive T cells also occur at this stage. We have defined four subsets of CD4 SP, namely, SP1, SP2, SP3, and SP4 that follow a functional maturation program and a sequential emergence during mouse ontogeny. We used microarray to detail the global programm of gene expression during the maturation of murine CD4 single positive thymocytes

2012-02-13 | GSE30083 | GEO

Expression data from CD4 single positive thymocyte subsets from C57BL/6 mice of 6-8 wks of age

2012-02-13 | E-GEOD-30083 | biostudies-arrayexpress

Gene expression analysis of thymocyte subsets

2011-08-10 | GSE31082 | GEO

Gene expression analysis of thymocyte subsets

Project description:Mouse thymocytes can be classified into four major subsets based on expression of CD4 and CD8 co-receptors. CD4-CD8- (double negative, DN) cells become CD4+CD8+ (double positive, DP) cells following productive T cell receptor (TCR) beta chain rearrangement. A small proportion of DP cells are selected through interaction of clonal TCRalpha/beta and MHC self peptide complex expressed on thymic stromal cells. DP cell expressing MHC class I-restricted TCR become CD4-CD8+ cells, which will finally differentiate into cytotoxic T cells, while MHC class II restricted selection generates CD4+CD8- helper lineage T cells. We used microarrays to identify genes important for thymocyte differentiation and lineage determination by profiling gene expression in different thymocyte subsets. Mouse thymocytes were divided into four subsets based on CD4, CD8a, and TCRb expression and purified by flw cytometry. FACS purified DN (CD4-CD8a-TCRb-), DP (CD4+CD8a+), CD4SP (CD4+CD8a-TCRbhi) and CD8SP (CD4-CD8a+TCRbhi) populations were lysed in Trizol, and provided to the Genomics Core Facility of the Memorial Sloan-Kettering Cancer Center (MSKCC) for quality control, quantification, reverse transcription, labeling and hybridization to MOE430A 2.0 microarray chips (Affymetrix). Arrays were scanned per the manufacturer’s specifications for the Affymetrix MOE430v2 chip.

2011-08-09 | E-GEOD-31082 | biostudies-arrayexpress

Expression data from BDC2.5 TCR Tg, preselected Rag-/-.B6 and Rag-/-.NOD.H2b thymocytes upon antigenic stimulation

Project description:The aim of this study was to quantify the impact of NOD genetic vatiation on thymic negative selection transcriptional programs. Pre-selected BDC2.5 TCR Tg DP thymocytes from non-selecting B6 and NOD.H2b backgrounds were purified (Dynal CD8 FlowComp), mixed in a 1:1 ratio and stimulated with BDC mimotope-loaded TCRa-/-/NOD splenocytes for indicated periods of time and double sorted by FACS as Thy1.2+Dump-CD4+CD8+; Dump includes CD19, Gr1, CD11b, CD11c, CD49b. Following cell sorting into trizol, RNA was purified, labeled and hybridized to Affymetrix arrays. experiment type: unstimulated versus stimulated BDC/B6.Rag-/- and BDC/NOD.H2b.Rag-/- DP thymocytes

2012-01-10 | E-GEOD-34934 | biostudies-arrayexpress

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