Differential gene expression between TCR-activated CD4+ T cells from control and Gch1;Lck mice
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PROVIDER: PRJNA422527 | ENA |
REPOSITORIES: ENA
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Project description:To activate primary T cells, lymph nodes were removed and disaggregated. Cells were cultured in RPMI 1640 containing L-glutamine, 10% FBS, 50 μM β-mercaptoethanol and penicillin/streptomycin. Cells were stimulated with 1 μg/ml of the CD3 monoclonal antibody (2C11) and 2 μg/ml anti-CD28 (37.51) in the presence of cytokines IL12 (10 ng/ml) and 20 ng/ml IL2 (20 ng/ml). Cells were stimulated for 24 hours. Live TCR activated CD4+ cells were sorted for CD4+ and CD45.1+ expression and DAP1 exclusion prior to collection for proteomics processing. Mice were “wild-type” (ie non-TCR transgenic) from OT-2 CD45.1 expressing C57bl6 background maintained in-house line.
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2009-05-16 | E-GEOD-16130 | biostudies-arrayexpress
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2020-02-25 | PXD016105 | Pride
Project description:Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis translates TCR signal strength into proportional activation of enhancers and genes essential for T cell function. CD4+ T cells from transgenic AND mice were sequenced under the conditions indicated. Replicates are included for each type of data (RNA-Seq, ChIP-Seq), and are numbered accordingly. The No Peptide condition serves as the untreated control for the peptide-treated samples, and inputs are provided for ChIP-Sequencing samples.
2016-06-01 | E-GEOD-69545 | biostudies-arrayexpress
Project description:Purpose: Despite the well-established association between T cell-mediated inflammation and non-ischemic heart failure (HF), the specific mechanisms triggering T cell activation during the progression of HF and the antigens involved are poorly understood. We hypothesized that clonal expansion of CD4+ T cell in response to cardiac neoantigens promote the progression of HF. Methods: We used transverse aortic constriction (TAC) in mice to trigger HF and profiled the TCR repertoire by mRNA sequencing of CD4+ T cells from 3 pooled Nur77GFP reporter mice, which transiently express GFP upon TCR engagement. We performed TCR clonal analysis of 5000 TCR-activated GFP+CD4+ T cells sorted from the LV, and 5000 CD4+ T cells sorted from the thymus, the inguinal lymph nodes (iLNs) and the heart-draining mediastinal lymph nodes (mLNs) by bulk RNA sequencing of the TCR beta chain after 8wks of TAC surgery. Paired‐end 300 base pair reads were obtained by Illumina sequencing and aligned and assembled using MiXCR software. Results: As expected, the greatest number of unique TCR beta clonotypes (4412 clones) – and therefore the highest TCR diversity – was identified in the thymus. The heart draining mLNs showed a much lower degree of TCR clonal diversity relative to the peripheral iLNs, suggesting that cardiac antigen-driven clonal expansion occurs at the mLNs that directly drain the heart. TCR-activated GFP+CD4+ T cells in the LV represented even lower TCR diversity relative to other sites. Conclusion: Collectively, our study demonstrates that in response to cardiac pressure overload, CD4+ T cells clonally expand in the heart-draining mLN; whilst a limited repertoire of CD4+ T cell clones engage endogenous antigens in the heart.
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Project description:Here we analyze the small RNA species in the following: 1. DN3 thymocytes following inactivation of LoxP flanked Drosha and Dicer alleles with Lck-cre 2. Tregs following inactivation of LoxP flanked Drosha and Dicer alleles with CD4-cre 3. activated CD4+ T cells following inactivation of LoxP flanked Drosha and Dicer alleles with CD4-cre 4. MEFs following inactivation of LoxP flanked Drosha and Dicer alleles with Rosa26-CreER and 4-OH tamoxifen treatment Fractionation of small RNAs (19-30nt) and cloning of only RNA with 5' phosphates and 3' hydroxyls
2010-08-18 | E-GEOD-22760 | biostudies-arrayexpress
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2005-03-31 | E-MEXP-283 | biostudies-arrayexpress
Project description:CD4+ T cells have a central role in controlling Mtb infection (1). Despite immune control, Mtb persists by interfering with macrophage and T cell function, allowing for pathogen survival. Our group has demonstrated direct and indirect inhibition of CD4+ T cell activation by different Mtb molecules including lipoproteins LpqH, LprA and LprG, and ManLAM (2-5). ManLAM is an abundant glycolipid in the Mtb cell wall and interferes with TCR signaling by down-regulating phosphorylation of Lck, CD3, ZAP70 and LAT (6). Activation through the TCR-CD3 complex leads to marked changes in the proteome of T cells. Entry into cell cycle, production of cytokines and differentiation from naïve to effector and memory T cell are all reflected in the production and modification of multiple proteins. Mass spectrometry (MS) has been used to characterize TCR complex formation (7-11) and the effect of a range of stressors on the T cell proteome (12-14). Recent advances have overcome technical issues in quantitative MS and allow analysis of the complexity and dynamic range of the cellular proteome (15). To extract biological meaning different bioinformatic tools have been developed for MS based cellular studies (16, 17). In this study we used label-free quantitative MS to characterize the effect of ManLAM on the proteome of activated CD4+ T cells. Approximately 5000 peptides were identified and quantitated from three biological experimental datasets in primary murine CD4+ T cells treated with or without ManLAM and activated with anti-CD3 and anti-CD28 monoclonal antibodies (mAb). Peptides with varying abundance were selected by likelihood ratio based statistical significance by comparing the intensities under different experimental conditions. ManLAM treatment resulted in abundance changes for 149 peptides representing 131 proteins in the activated CD4+ T cell proteome that affected a variety of enriched cluster modules and pathways. Validation experiments confirmed Man LAM induced inhibition of Proliferating Cell Nuclear Antigen (PCNA) that regulates cell cycle progression. Protein-protein interaction (PPI) network analysis revealed a central role for the Akt/mTOR signaling pathway. WB studies revealed decreased phosphorylation of both Akt and mTOR in ManLAM treated CD4+ T cells as well as decreased expression of Otub1, a key molecule regulated by mTOR. Thus disruption of the activated CD4+ T cell proteome by ManLAM-mediated inhibition of proximal TCR signaling is primarily mediated through inhibition of Akt-mTOR signaling.
2018-07-05 | PXD004164 | Pride
Project description:T-cell receptor (TCR) signaling is essential for the function of T cells. Here we combine mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the composition and dynamics of the signaling complexes that assemble around 15 nodes of the TCR signaling cascade of primary CD4+ T cells. This dataset contains the experiments performed with 14 different bait proteins • Cbl • Cblb • Fyb • Inpp5d • Itk • Lck • Lcp2 • Nck1 • Nfatc2 • Plcg1 • Ptpn22 • Ptpn6 • Themis • Vav1 Each of them contains mass spectrometry results from the analysis of AP-MS experiments, based on the endogenous expression of One-Strep-tagged (OST) bait proteins in engineered mice models, and affinity purification of these proteins from primary CD4+ T cells, using Streptactin beads. Purification of OST proteins was performed at 5 different time points of stimulation of CD4+ T cells with anti-CD3 and anti-CD4 antibodies (0s; 30s; 120s; 300s; 600s). Each AP-MS purification of an OST- protein is associated with a corresponding control (purification from WT CD4+ T cells) at the same time point of stimulation. Several biological replicate experiments (time course OST series + associated WT controls) were performed for each bait. Several MS replicates were acquired for each sample. In addition, we analyzed the total proteome of CD4+ T cells isolated from each engineered mice model (14 different mice expressing an OST bait) and from WT mice, in order to calculate copy numbers of the baits and their associated proteins.
2019-09-26 | PXD007660 | Pride
Project description:Though T cell expansion and effector differentiation are triggered and, perhaps, maintained by antigen, the proliferative behaviors of CD4+ and CD8+ T cells responding to timed antigen presentation have rarely been compared side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal T cells were analyzed following transient and continuous TCR signals. We found CD4+ T cell proliferation to be dependent on prolonged antigen presence, whereas CD8+ T cells were able to divide and differentiate into effector cells in the absence of it. We excluded CD4+ T cell proliferation to be abrogated by coinhibitory signals or the lack of inflammatory stimuli and found that autonomous proliferation of CD8* T cells was independent of any MHC class I signals. Gene expression analyses illustrated differences in global gene transcription between the two subsets following stimulation periods of different lengths. These T cell data reflect the MHC class difference in that class II but not class I molecules were stabilized on activated DCs in vivo, suggesting a coevolution of MHC molecules and their respective T cell subsets. Samples 1-12: Analysis on day 2. Purified CD4+ AND-TCR transgenic cells and CD8+ OT1-TCR transgenic cells were separately stimulated with anti-CD3 and anti-CD28 antibodies. 48 hours later, the cells were sorted again to a purity of >99 %. Extracted total RNA was amplified twice and hybridized on Affymetrix Mouse 430A2 microarrays. First, we analysed the changes of the CD4+ and CD8+ T cells after stimulation. Second, we compared the differences of the changes between the two cell types after stimulation. For each of the four groups (CD4+ and CD8+, stimulated and unstimulated), we analysed three independent biological replicates. Samples 13-28: Analysis on day 5. AND and OT1 TCR-transgenic T cells were prepared as described before, but transferred into mice that do not or do present their respective antigens. 72 hours later, the cells were FACS-sorted twice to >99 % purity, directly into Trizol. For each of the six groups (CD4+ and CD8+, unstimulated, transient (2 days) and continuous (5 days) stimulation), three independent biological replicates were analyzed, except for CD4+ unstimulated and CD4+ transient, with two replicates each.
2014-02-28 | E-GEOD-49063 | biostudies-arrayexpress