The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE164895 | GEO | 2021/02/01
REPOSITORIES: GEO
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Project description:Purpose : The goal of this study is to compare the repertoire of T cell receptor (TCR) alpha and beta chain between WT and CIC-deficient thymic CD4+ single positive (SP) T cells, in both non-Treg and Treg populations, to confirm the effect of CIC deficiency on T cell development in the aspect of TCR repertoire. Methods : Treg (CD4+CD8-CD25+GFP+) and non-Treg (CD4+CD8-GFP-) cells from Foxp3-GFP;Cicf/f and Foxp3-GFP;Cicf/f;Vav1-Cre mice were sorted by MoFlo-XDP (Beckman Coulter). Total RNA was extracted using RiboEX (GeneAll) according to the manufacturer's instructions. RNA was then amplified using a commercially available multiplex primer mix covering the TCR alpha and beta chains in two separate PCR reactions. Reverse transcription and subsequent PCR amplification (RT-PCR1) were performed using the Qiagen OneStep RT PCR mix (Qiagen). The cDNA was selected and unused primers were removed by SPRIselect bead selection (Beckman Coulter) followed by a second round of amplification performed with a pair of primers specific for communal sites engineered onto the 5ʹ end of the C- and V- primers used during RT-PCR1. After library preparation, paired-end sequencing was performed using the Illumina Miseq v3 600-cycle Reagent Kit (Illumina). Results : We observed increased frequency of TCRs with long CDR3 length in CIC-deficient non-Treg and Treg cells. We also found significant alterations in the TCR repertoire of TCR beta chain in CIC-deficient Treg cells compared to WT cells, including altered frequency of V and J chain usage. Conclusions : Our study represents the first comparative analysis of TCR repertoire of thymic CD4 SP cells from WT and hematopoietic lineage cell-specific Cic deficient mice. We concluded that CIC deficiency leads to alterations in TCR repertoire of thymic Treg cells.
2021-05-07 | GSE173996 | GEO
Project description:Conventional CD4 and CD8 single positive T cell lineages constitute the main differentiation pathway in the thymus. In human thymus, a minor TCRαβ differentiation pathway diverges from the early double positive stage, consisting of CD10+ PD-1+ cells. These cells are phenotypically and functionally similar to murine agonist-selected intraepithelial T lymphocyte precursors (IELps) which home to the small intestine. Here, the progeny of the human agonist-selected IEL lineage was identified in antigen-inexperienced cord blood (CB) with a polyclonal T cell receptor (TCR) repertoire exhibiting a bias towards early TCR alpha chain rearrangements and elevated autoreactive indices. Single-cell RNA sequencing allowed further delineation of this unconventional lineage in CB. Trajectory analysis, along with TCR repertoire analysis, transcriptomics and proteomics, suggests a precursor-progeny relationship with the thymic IELps. The distinct, heterogeneous CB population can now be defined as CD3+ TCRαβ+ CD4- CCR7- CD26-. Besides recent thymic emigrants, this population also consists of newly identified effector clusters and previously described populations: the suppressive NK receptor expressing CD8+ Treg population, the KIR/NKG2A+ EOMES+ virtual memory population and the CD8αα+ T cell populations. The population shows a discriminating stable HELIOS expression and is exclusively able to downregulate CD8β expression, resulting in double negative T cells. The functional properties of this population suggest that the cells expand on inflammatory cues and exert cytotoxic and proinflammatory activity.
2023-02-10 | PXD033392 | Pride
Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRα/β-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRα/β-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.
2016-08-20 | E-GEOD-65496 | biostudies-arrayexpress
Project description:Title of Publication: Histone Deacetylase 7 Regulates Cell Survival and TCR Signaling in CD4/CD8 Double-Positive Thymocytes Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR J segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes. Three biological replicate samples were prepared for each non-wild type mouse strain (Samples 7-18). Six biological replicates were prepared for the wild type strain (Samples 1-6). Total RNA was prepared from isolated CD4/CD8 Double-positive thymocytes and labeled with the Affymetrix Whole-Transcript labeling protocol. Labeled probes were hybridized to one Affymetrix Mouse Gene 1.0ST array each. Data from .cel files were normalized using RMA, in normalization groups representing each of the binary comparisons made. These binary comparisons between sample groups represent gene expression changes due to loss of HDAC7 (samples 1-3 vs. 7-9), transgenic expression of an HDAC7-VP16 fusion protein (samples 4-6 vs. 10-12), positive thymic selection (samples 1-6 vs. samples 11-15), and negative thymic selection (Samples 11-15 vs. samples 16-18).
2011-04-06 | E-GEOD-26488 | biostudies-arrayexpress
Project description:The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection
| PRJNA692418 | ENA
Project description:To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter (RIP). Aire-deficiency reduced the number of mature single positive (SP) OVA-specific CD4+ or CD8+ T cells in the thymus, independent of OVA expression. Importantly, it also contributed in two way to OVA-dependent negative selection depending on the T cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to mTEC-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTEC can mediate tolerance by direct presentation of Aireregulated antigens to both CD4 and CD8 T cells. Total RNA was extracted from medullary thymic epithelial cells isolated from wildtype and Aire-deficient C57BL/6 mice for comparison of gene expression profiles.
2011-04-06 | E-GEOD-28393 | biostudies-arrayexpress
Project description:Abstract of publicaton: CD4/CD8 double-positive (DP) thymocytes express the transcriptional repressor Histone Deacetylase 7 (HDAC7), a class IIa HDAC that is exported from the cell nucleus after T cell receptor (TCR) engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in thymocytes, and regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR Jalpha segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAP kinase activity that contributes to the observed loss of viability. Remarkably, the activity of Protein Kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining auto-excitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions. Title of manuscript: Nuclear Export of Histone Deacetylase 7 During Thymic Selection Mediates Immune Self-tolerance. abstract of manuscript: Histone Deacetylase 7 (HDAC7) is a TCR signal-dependent regulator of differentiation that is highly expressed in CD4/CD8 double-positive (DP) thymocytes. Here we examine the effect of blocking TCR-dependent nuclear export of HDAC7 during thymic selection, through expression of a signal-resistant mutant of HDAC7 (HDAC7-?P) in thymocytes. We find that HDAC7-?P Transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection-associated gene expression program in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self-tolerance. Goal of Microarray experiment: We did these experiments to determine how alteration of the function of HDAC7, a site-specific and signal-dependent repressor of transcription, changes gene expression in CD4/CD8 DP thymocytes.
2011-04-06 | GSE26488 | GEO
Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells.
2016-08-20 | GSE65496 | GEO
Project description:To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter (RIP). Aire-deficiency reduced the number of mature single positive (SP) OVA-specific CD4+ or CD8+ T cells in the thymus, independent of OVA expression. Importantly, it also contributed in two way to OVA-dependent negative selection depending on the T cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to mTEC-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTEC can mediate tolerance by direct presentation of Aireregulated antigens to both CD4 and CD8 T cells.
2011-04-06 | GSE28393 | GEO
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.
2011-06-20 | E-TABM-550 | biostudies-arrayexpress