Expression data from developing thymocytes of N15TCR transgenic Rag2 deficient mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE13493 | GEO | 2009/03/31
SECONDARY ACCESSION(S): PRJNA110173
REPOSITORIES: GEO
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Project description:T cell development relies on the precise developmental control of various cellular functions for appropriate positive and negative selection. Previously, gene expression profiling of peptide-driven negative selection events in the N15 TCR class I MHC-restricted mouse and D011.10 TCR class II MHC-restricted mouse has offered insights into the coordinate engagement of biological processes affecting thymocyte development. However, there has been little comparable detailed in vivo global genome expression analysis reported for positive selection. We used microarrays to identify the genes differentially expressed during CD8 single positive T cell development in N15 TCR transgenic Rag2 deficient mice. We have analyzed gene expression in the individual populations as development proceeds from DP, intermediate (Int), to SP subsets. To this end, we sorted the individual populations from six age and sex matched 5-6 week old N15 TCR tg+/+ RAG-2-/- H-2b mice for two independent experiments. For sorting subpopulations, cells were stained with anti-CD4 (L3T4) and anti-CD8 (Ly-2) antibodies (Pharmingen, San Diego, CA USA) and DP, Int and SP thymocytes were sorted on a MoFlo (Cytomation, Fort Collins, CO USA).
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Project description:Pluripotent stem cells (PSCs) present promising engineering opportunities as sources of allogeneic T cells for off-the-shelf immunotherapies. However, the complex in vitro process of differentiation required to generate mature T cells from genetically engineered PSCs introduces unique challenges, as positive selection is dependent on the same molecular machinery that must be removed to prevent alloreactivity. We report an organoid-based method in which engineering of the stromal microenvironment induced generation of antigen-restricted, mature, conventional T cells from PSCs which lacked all endogenous TCR and Class I MHC expression. Transgenic signals required to rescue positive selection were supplied by separate cellular sources; T cell precursors from edited PSC expressed a single TCR, and a murine stromal line provided cognate human MHC and other critical T cell development signals. Edited T cells exhibited superior tumour control in vivo likely due to the absence of TCR mispairing. This in vitro method overcomes key biological impediments inherent to developing T cell immunotherapies from allogeneic PSCs.
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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.
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Project description:T-cell development is predicated on the successful rearrangement of the T-cell receptor (TCR) gene loci, which encode for antigen-specific receptors that enable an immune response. Recombination Activating Gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T-cell development. Here, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCRb chain in mediating b-selection during human T-cell development in vitro. In stark contrast to what is seen in mice, human RAG2-KO T-lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRb rearrangements. Nevertheless, RAG2-KO DPs retrovirally-transduced to ectopically express a rearranged TCRb chain showed increased survival and proliferation as compared to control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRb-transduced and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide new insights as to the distinct requirement for the TCRb chain during human T-cell development.
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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.
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Project description:During T cell development in the thymus, negative selection eliminates autoreactive and potentially harmful T cells, leading to central tolerance. This process, involves specific apoptosis programs thought to be governed by the transcription factor Nur77. To analyze the genetic pathways involved in thymic negative selection and to point to new genes involved in thymic selection, we performed a high throughput temporal expression profiling of thymocytes from HNT-TCR transgenic mice undergoing specific peptide-mediated thymic negative selection, using oligonucleotide microarrays. We used HNT-TCR transgenic mice that are on a B10D2 background and express a transgenic TCR (V beta 8), specific for the HA peptide (HNTNGVTAACSHE) presented by MHC class II I-A^d. In this model of thymic negative selection, a single injection of the specific HA peptide induces massive and synchronized deletion of thymocytes. In this study, we analyzed the dynamically regulated genes over a 24 hours period, with a focus on the early time points of 3 and 6 hours after peptide injection.
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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
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