982263ae-2ce6-4f92-90a7-db209d895cea - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001009754 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:To investigate the mechanism by which Cul5 regulates CD8+ T cell cytokine-dependent differentiation and TCR-dependent activation, we performed quantitative mass spectrometry (MS) by data-independent acquisition (DIA)-MS of total proteins in the Cul5 KO and NC primary CD8+ T cells in the following conditions: 1) Cytokine dependent expansion and differentiation (T0); 2) 8 hours cytokine withdraw prior to TCR stimulation (T8); and 3) 16 hours TCR stimulation post 8-hour cytokine withdraw (T16). Principal component analysis (PCA) and correlation analysis revealed that replicates in each condition clustered together while different conditions separated from each other, suggesting significant proteomic changes among different conditions of the same T cells as well as between Cul5 KO and NC T cells in each condition. Together with similar total protein quantities among all detected samples, DIA-MS analyses were of high quality. Additionally, the strong reductions of Cul5 abundances in the Cul5 KO cells from all three conditions compared to the NC cells confirms high KO efficiency. Of note, the proteomic analysis showed that the Cul5 protein level was significantly increased upon TCR stimulation post cytokine starvation in the NC cells, suggesting a potential negative feedback regulatory role of Cul5 in CD8+ T cell activation. Consistent with this idea, we observed more markedly upregulation of Cul5 expression upon of TCR stimulation of na•ve primary CD8+ cells. To identify Cul5 interacting proteins in CD8+ T cells, we overexpressed Cul5 with a C terminal HA-tag (Cul5-HA) in mouse primary CD8+ T cells by retroviral transduction. The cells were subjected to TCR stimulation for 12 hr, and Cul5-HA was immunoprecipitated by anti-HA, followed by DIA-MS analysis (co-IP-MS). Compared to the negative control samples (cells transduced with the empty vector), 65 proteins were enriched (p value <0.05 and fold change >1.5) in the anti-HA IP samples. Altogether, we report that Cul5 KO alters CD8+ T cell proteome and the Cul5 interactome.
2024-01-03 | PXD036793 | Pride
Project description:<p>The etiologies of primary immunodeficiencies often yield novel insights about the immune system. Although a genetic etiology has been suspected for patients with abnormally low CD4+ T cells in the absence of HIV infection or any known causes of lymphopenia, no genetic mutation has been described to date for any case of primary CD4 lymphopenia. In this study, we characterized a non-consanguineous family with two non-HIV infected boys exhibiting an inverted CD4 to CD8 T cell ratio and a history of recurrent chronic viral infections since birth. Consistent with a decreased thymic output of CD4+ T cells, the percentage of CD31+ cells in the CD4+ naive population of these patients was decreased. In addition, the activation of T cells was significantly impaired in the patient upon TCR stimulation. Given the mother's T cells show completely skewed X chromosome inactivation, we suspected that the nature of this disease is X-linked. We performed X-chromosome exon-capture targeted single-end Solexa sequencing on two brothers and the mother and found a 10 base pair deletion at an intron-exon junction of Magnesium Transporter 1 (MAGT1), a Mg<sup>2+</sup> specific transporter. We confirmed that this deletion leads to altered splicing, frameshift, early termination of the mRNA, and deficient protein expression in the lymphocytes of the two patients. Moreover, knockdown of this transporter in T cells isolated from healthy donors can recapitulate the observed T cell activation defect while its ectopic expression in the patients' lymphocytes can restore T cell stimulation. Our discovery highlights the significance of this transporter to T cell function.</p>
| phs000365 | dbGaP
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
Project description:Here we report bulk TCR sequencing data associated with open repertoire murine CD4+, CD4+CD8+, and CD8+ T cells isolated from B16 melanoma tumor resections
2022-05-20 | GSE203187 | GEO
Project description:Gene expression analysis of tumor-specific CD8 T cells encountering a tumor-specific antigen in pre-malignant lesions in the liver at different time points post tumor initiation. The overall goal of this mouse study was to elucidate the molecular program in tumor-specific T cells encountering a tumor-specific antigen in pre-malignant lesions. The study identifies the genes and pathways that are dysregulated in tumor-specific T cells associated with T cell unresponsiveness in tumors. To identify the genes and pathways that are dysregulated in tumor-specific T cells. Gene signatures of the following sample groups were compared: 1. Naïve CD8 T cells; 2. Effector CD8 T cells; 3. Dysfunctional tumor-specific CD8 T cells isolated from pre-malignant lesions 8-12 days after tumor induction. 4. Dysfunctional tumor-specific CD8 T cells isolated from pre-malignant lesions 32-34 days after tumor initiation. The mouse tumor model that was used is an autochthonous, tamoxifen-inducible liver cancer model (ASTxCre-ERT2; AST=Albumin-floxStop-SV40 large T antigen; Cre-ERT2 = TAM-dependent Cre-recombinase) in which the SV40 large T antigen serves as the oncogenic driver and tumor-specific antigen; SV40-I TCR transgenic mice, whose CD8 T cells express a Db-restricted TCR specific for the Tag epitope I were used as source of naïve tumor-specific CD8 T cells (TCRSV40-I) Total RNA was isolated from flow-sorted transgenic CD8 TCRSV40-I T cells from the following sample groups: Naïve, effector, D8-12-pre-malignant lesions, and D32-34 pre-malignant lesions.
2016-08-11 | E-GEOD-60501 | biostudies-arrayexpress
Project description:Naive T cells experience accumulation of TCR signaling in response to self-antigens in the steady state. However, how these signals influence the responsiveness of naive CD8+ T cells to subsequent agonist TCR stimulation remains incompletely understood. We investigated how naive CD8+ T cells that experienced relatively low or high levels of TCR signaling in response to self-antigens respond to stimulation with foreign antigens. A transcriptional reporter of Nr4a1 (Nur77-GFP) revealed substantial heterogeneity of the amount of TCR signaling naive CD8+ T cells accumulate in the steady state. To further understand the impact of differential TCR signaling on CD8 T cells we performed RNA-seq on naive Nur77-GFPhi and lo populations.
2023-03-02 | GSE223457 | GEO
Project description:T cells have the remarkable ability to sense and discriminate between a wide range of antigenic peptides bound to major histocompatibility complex molecules (pMHC). Here, we use interactomics to monitor in a time dependent manner the early molecular events taking place upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR. We used the mouse OT-I model in which T cells specifically recognize the ovalbumin OVA 257-264 peptide (SIINFEKL, also named N4) bound to the MHC-I molecule H-2Kb (pMHC). The OT-I TCR also recognizes altered peptide ligands amongst which SIITFEKL (T4) and SIIGFEKL (G4) with sub-optimal binding capacities (ligand affinity N4>T4>G4). For affinity purification (AP) of signalling complexes, we crossed OT-I TCR transgenic mice onto mice expressing an endogenous tagged (One-Strep-Tag: OST) version of the CD3z (also called CD247), ZAP70 or SLP76 molecules. CD8+ T cells were purified from pooled lymph nodes and spleens, shortly expanded, and were left unstimulated or stimulated with N4, T4 and G4 soluble pMHC tetramers for 30, 120 or 300 s. Complexes formed around either CD247, ZAP70 or SLP76 were then purified and analysed by mass spectrometry. Each AP-MS purification is associated with a corresponding control (purification from OT-I CD8+ T cells expressing only the endogenous version of each bait, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS. In addition, the whole proteome of OT-I cell was analyzed by label-free shotgun proteomics in order to calculate cellular protein abundances for each interactor.
2022-07-05 | PXD029974 | Pride
Project description:During persistent antigen stimulation, CD8+ cytolytic T cells (CTL) show a gradual decrease in effector function, or “exhaustion”, which impairs the immune response to tumors and infections. Here we show that NFAT, a transcription factor with an established role in T cell activation, in parallel controls a second transcriptional program conferring the characteristic features of CD8+ T cell exhaustion, including upregulation of genes encoding inhibitory cell surface receptors and diminished TCR signaling. Expression of an engineered NFAT1, which induces this negative regulatory program in the absence of the effector program, interferes with the ability of CD8+ T cells to protect against Listeria infection or attenuate tumor growth in vivo. NFAT elicits this second program of gene expression in large part by binding to a subset of the sites occupied by NFAT during a typical effector response, suggesting that a balance between the two pathways determines the outcome of TCR signaling. Determination of NFAT1 binding sites in CD8 T cells in vitro
2015-02-13 | E-GEOD-64407 | biostudies-arrayexpress
Project description:CD8 T cells are the main effectors of the adaptive immune response against intracellular pathogens and cancer. Chronic antigen stimulation and inflammation lead to CD8 T cell differentiation and function that differ from those generated during acute infections. A comprehensive definition of the heterogeneity existing within both tumor-specific and total CD8 tumor-infiltrating T cells remains to be clearly established. To investigate this heterogeneity at the transcriptomic level, we performed paired single-cell RNA and TCR sequencing of >3500 CD8 T cells infiltrating B16 murine melanoma tumors, including cells of known tumor-specificity.
2019-10-10 | GSE116390 | GEO
Project description:Durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8 T cells (Tpex). Tpex serve as a source for the pool of effector T cells, and in the absence of their cognate antigen, they are able to preserve their quantity through a process of self-renewal. However, it remains unknown how TCR engagement impacts the self-renewal capacity of Tpex in settings of continued antigen exposure. Here, we used a Lewis lung carcinoma model that elicits an optimal or attenuated TCR signal in CD8 T cells to investigate its effect on Tpex persistence during tumor development. Longitudinal phenotyping and single-cell transcriptomics of tumor-specific T cells revealed that formation of the Tpex reservoir in tumor-draining lymph nodes, and its subsequent replenishment of intratumoral Tpex, is dependent on optimal TCR engagement. Notably, adoptive transfer of optimally primed Tpex into a tumor setting with attenuated TCR stimulation significantly accelerates their terminal differentiation, contrasting to the sustained self-renewal occuring with optimal TCR stimulation. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cell niches and epigenetic imprinting that involves increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, our study highlights the critical role of TCR engagement in sustaining Tpex during tumor progression
2024-04-05 | GSE262842 | GEO