Gene expression profiles of human CD8+ T cells: IL-12 and IFN-a polarized in vitro stimulated and ex vivo sorted CCR7hi and CXCR3hi populations
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE27337 | GEO | 2011/09/30
SECONDARY ACCESSION(S): PRJNA137415
REPOSITORIES: GEO
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Project description:CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. While effector CTLs eliminate the infection, a small population of memory cells are retained that yields more rapid and robust response upon re-infection. Antigen presenting cells secrete an array of innate cytokines including IL-12 and IFN-α after recognition of pathogens. Both IL-12 and IFN-α have been shown to act as the third signal regulating the development of CTLs. We have shown that these two cytokines have a non-redundant effect in generation of human effector CTL. IL-12 alone is sufficient for effector CTL genesis marked by IFN-γ and TNF-α production, as well as increased cytolytic activity. Even in the presence of IFN-α, IL-12 programs CTLs that express the chemokine receptor CXCR3 and effector cytokines. Using microarray analysis we have investigated how IL-12 and IFN-α differentially regulate the genetic programming pathways that give rise to effector CTLs among multiple human donors. We have also analyzed the gene expression patterns of cells sorted from healthy human peripheral blood that display surface markers of effector memory CTL (designated as ex vivo) samples. 5 healthy human donor samples were used for the in vitro cultures. For each donor the CFSE labeled cells (CD8+CD45RA+) were cultured in the presence of neutralized, IL-12, IFN-a, and IL-12+IFN-a conditions and plate-bound anti-CD3+anti-CD28 for 3.5 days. Total RNA from CFSEhi (Undiv) and CFSElo (Div) sorted cells were used for Illumina Bead Array. 4 healthy human donor samples were used for the ex vivo samples. Total RNA was collected from FACS sorted CD8+CCR7hiCXCR3lo and CD8+CCR7loCXCR3hi cells without any stimulation.
2011-09-29 | E-GEOD-27337 | biostudies-arrayexpress
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Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation.
2013-12-02 | GSE51393 | GEO
Project description:Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. Recognition of an antigenic ligand by the CTL’s T cell receptor (TCR) triggers a signalling cascade that ultimately results in the targeted release of cytolytic granules and/or secretion of cytokines and chemokines to alert and recruit additional immune cells. These signalling cascades are capable of initiating transcription, translation, and cytoskeletal rearrangements. While previous work has demonstrated how translation and intracellular reorganisation contribute to CTL effector responses, the role of transcription is less well studied. To address this, we examined the impact of blocking transcription on the CTL proteome during TCR stimulation. These data demonstrated a strong transcriptional requirement for expression of cytokines and chemokines but not cytolytic molecules. Together with functional studies, these data reveal differential molecular control of the cell-cell communication and cytolytic functions of effector CTLs. CTLs exhibit complete and persistent priming for cytolytic activity prior to target cell encounter, but they require de novo transcription to recruit additional immune cells that amplify the response.
2023-10-16 | PXD034920 | Pride
Project description:Recent work in immunometabolism has emphasised the role of mitochondria in both the innate and adaptive immune system. Mitochondria are important in T cell development and differentiation, but less is known about their role in CD8+ effector T cells (CTLs). We found that CTLs that lack the mitochondrial deubiquitinase USP30 undergo promiscuous mitophagy, destroying most of the cellular mitochondria. Surprisingly, this results in a markedly diminished killing capacity, while motility, signalling and secretion remain intact. This study uses quantitative DIA proteomics to measure the impact of USP30 deficiency on the global proteome of IL-2 maintained, 4.5 h TCR retriggered and 4.5 h TCR retriggered + cycloheximide CTL. Unexpectedly, inhibition of mitochondrial translation, through genetic or pharmacologic methods, was the mechanism by which CTL killing was impaired. Reduced mitochondrial translation triggered attenuated cytosolic translation which precluded replenishment of secreted effector molecules thereby limiting the capacity of CTLs to serially kill multiple targets. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.
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Project description:Pathogen-associated molecular patterns decisively influence antiviral immune responses, whereas the contribution of endogenous signals of tissue damage, also known as “damage-associated molecular patterns” or “alarmins”, remains ill-defined. We show that interleukin-33 (IL-33), an alarmin released from necrotic cells, is necessary for potent CD8+ T cell (CTL) responses to replicating, prototypic RNA and DNA viruses in mice. IL-33 signaled through its receptor on activated CTLs, enhanced clonal expansion in a MyD88-dependent, CTL-intrinsic fashion, determined polyfunctional effector cell differentiation and was necessary for virus control. Moreover, recombinant IL-33 augmented vaccine-induced CTL responses. Radio-resistant cells of the splenic T cell zone produced IL-33, and efficient CTL responses required IL-33 from radio-resistant cells but not from hematopoietic cells. Thus, alarmin release by radio-resistant cells orchestrates protective antiviral CTL responses. 2 groups (wt vs. ST-/- P14 cells), 3 replicates per group.
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Project description:CD4+ T-cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. We here reveal how “help” signals delivered during priming impact memory differentiation of CTLs, as informed by genome-wide analyses. “Help” signals promoted the IL-15-dependent maintenance of central memory (TCM) cells. However, they had a much larger impact on the generation of effector memory (TEM) cells and their gene expression program . CD4+ T-cell help created TEM cells that produced Granzyme B and IFNg after antigen-independent recall with IL-12 and IL-18. Furthermore, “helped” memory CTLs expressed the effector program characteristic of “helped” primary CTLs upon recall with MHC class I-restricted antigen only, due to both epigenetic imprinting and sustained mRNA expression of relevant genes . Thus, CD4+ T-cell help delivered during priming creates CD8+ TEM cells with innate and help-independent recall capacities.
2019-10-23 | GSE135141 | GEO
Project description:Pattern recognition receptors (PRR) detect microbial products and induce cytokines which shape the immunological response. Interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α) and IL-1β are proinflammatory cytokines which can be essential for resistance against infection, but if produced at high levels, may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine which dampens proinflammatory responses, but can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. Here, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α and IL-1β, but high levels of IL-10 in response to TLR4 and TLR2 ligands LPS and PamCSK4, and Burkholderia pseudomallei a Gram-negative bacterium which activates TLR 2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN dependent, but IL-27 independent mechanism. Further, type I IFN contributed to differential IL-1β and IL-12 production in B. pseudomallei and LPS stimulated C57BL/6 and BALB/c macrophages, via both IL-10-dependent and independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.
2016-09-07 | GSE79809 | GEO
Project description:Recent work in immunometabolism has emphasised the role of mitochondria in both the innate and adaptive immune system. Mitochondria are important in T cell development and differentiation, but less is known about their role in CD8+ effector T cells (CTLs). We found that CTLs that lack the mitochondrial deubiquitinase USP30 undergo promiscuous mitophagy, destroying most of the cellular mitochondria. Surprisingly, this results in a markedly diminished killing capacity, while motility, signalling and secretion remain intact. This study uses quantitative DIA proteomics to measure the impact of USP30 deficiency on the global proteome of IL-2 maintained, 4.5 h TCR retriggered and 4.5 h TCR retriggered + cycloheximide CTL. We also measured the impact of pharmacologically inhibiting mitochondrial translation by performing Quantitative DIA proteomics on IL2 maintained or TCR retriggered CTL treated with the mitochondrial translation inhibitor doxycycline for 4.5 hrs. Unexpectedly, inhibition of mitochondrial translation, through genetic or pharmacologic methods, was the mechanism by which CTL killing was impaired. Reduced mitochondrial translation triggered attenuated cytosolic translation which precluded replenishment of secreted effector molecules thereby limiting the capacity of CTLs to serially kill multiple targets. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.
2022-06-09 | PXD026948 | Pride
Project description:Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how hypoxia configures the proteome of CD8+ cytotoxic T lymphocytes (CTLs). We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8+ T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts. For this project, P14 cytotoxic T cells (CTLs) were generated from T cells from the spleens of mice. CD8+ T cells were activated for 2 days with the P14-T cell receptor (TCR) cognate ligand (peptide gp33-41 from Lymphocytic Choriomeningitis Virus, LCMV) in the presence of 20 ng/ml IL 2 (Proleukin) and 2 ng/ml IL-12 (Peprotech). Cells were grown in a humidified incubator with a gas atmosphere of 18% oxygen and 5% CO2 and a temperature of 37�C. After 48 hours, the activated CD8+ T cells were removed from TCR stimulation and were cultured in RPMI 1640, with 10% foetal bovine serum (FBS), supplemented with 100 units/ml penicillin-G, 100 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2 (Proleukin). Each day, the T cells were counted and split to 500,000 cells per ml with fresh IL-2 being added to a final concentration of 20 ng/ml. CTLs were differentiated in IL-2 for 4 days, before being counted and re-suspended at a concentration of 300,000 cells per ml in RPMI with 10% foetal bovine serum, 50 units/ml penicillin-G, 50 �g/ml streptomycin, 50 �M-mercaptoethanol and 20 ng/ml IL-2. A volume of 8 mls of the cell suspension was plated per well of a 6 well plate, and the CTLs were either maintained in a gas atmosphere of 18% oxygen and 5% CO2 or transferred to an atmosphere of 1% O2 and 5% CO2 in a Galaxy 48 R incubator (Eppendorf) for 24 hours. Three biological replicates (CTLs generated from three separate spleens) were analysed in this experiment."
2021-10-20 | PXD026223 | Pride