scholarly journals Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation

2019 ◽  
Author(s):  
Julia M. Marchingo ◽  
Linda V. Sinclair ◽  
Andrew J. M. Howden ◽  
Doreen A. Cantrell
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Cell Fate ◽  
T Cell Activation ◽  
Cell Activation ◽  
Amino Acid Transporters ◽  
Activated T Cells ◽  
Factor C ◽  
The Impact

SummaryT cell expansion and differentiation are critically dependent on the transcription factor c-Myc (Myc). Herein we use quantitative mass-spectrometry to reveal how Myc controls antigen receptor driven cell growth and proteome restructuring in T cells. Analysis of copy numbers per cell of >7000 proteins provides new understanding of the selective role of Myc in controlling the protein machinery that govern T cell fate. The data identify both Myc dependent and independent metabolic processes in immune activated T cells. We uncover that a primary function of Myc is to control expression of multiple amino acid transporters and that loss of a single Myc-controlled amino acid transporter effectively phenocopies the impact of Myc deletion. This study provides a comprehensive map of how Myc selectively shapes T cell phenotypes, revealing that Myc induction of amino acid transport is pivotal for subsequent bioenergetic and biosynthetic programs and licences T cell receptor driven proteome reprogramming.

scholarly journals Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia M Marchingo ◽  
Linda V Sinclair ◽  
Andrew JM Howden ◽  
Doreen A Cantrell
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Cell Fate ◽  
T Cell Activation ◽  
Cell Activation ◽  
Amino Acid Transporters ◽  
Activated T Cells ◽  
Factor C ◽  
The Impact

T cell expansion and differentiation are critically dependent on the transcription factor c-Myc (Myc). Herein we use quantitative mass-spectrometry to reveal how Myc controls antigen receptor driven cell growth and proteome restructuring in murine T cells. Analysis of copy numbers per cell of >7000 proteins provides new understanding of the selective role of Myc in controlling the protein machinery that govern T cell fate. The data identify both Myc dependent and independent metabolic processes in immune activated T cells. We uncover that a primary function of Myc is to control expression of multiple amino acid transporters and that loss of a single Myc-controlled amino acid transporter effectively phenocopies the impact of Myc deletion. This study provides a comprehensive map of how Myc selectively shapes T cell phenotypes, revealing that Myc induction of amino acid transport is pivotal for subsequent bioenergetic and biosynthetic programs and licences T cell receptor driven proteome reprogramming.

T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4692-4692
Author(s):  
Mauro Di Ianni ◽  
Lorenzo Moretti ◽  
Beatrice Del Papa ◽  
Maria De Ioanni ◽  
Adelmo Terenzi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activated T Cells ◽  
Mutational Status ◽  
The Impact

Abstract As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

scholarly journals DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

2021 ◽  
Author(s):  
Zachary J Waldrip ◽  
Lyle Burdine ◽  
David K Harrison ◽  
Ana Clara Azevedo-Pouly ◽  
Aaron J Storey ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cell Fate ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Interleukin 2 ◽  
T Cell Response ◽  
Activated T Cells

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and non-homologous end joining (NHEJ). However, like other DNA damage repair kinases (DDR), DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify first order phosphorylation targets of DNA-PKcs. Results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at S301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules. Mutation of serine 301 to alanine via CRISPR-Cas9 resulted in increased proteasomal degradation of Egr1 and a decrease in Egr1-dependent transcription of IL2 (interleukin-2) in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

scholarly journals Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease

2021 ◽  
pp. annrheumdis-2020-219335
Author(s):  
Emma Garcia-Melchor ◽  
Giacomo Cafaro ◽  
Lucy MacDonald ◽  
Lindsay A N Crowe ◽  
Shatakshi Sood ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conditioned Media ◽  
Tissue Remodelling ◽  
Activated T Cells ◽  
T Cell Migration

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Katia Urso ◽  
Arantzazu Alfranca ◽  
Sara Martínez-Martínez ◽  
Amelia Escolano ◽  
Inmaculada Ortega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Gene Knockout ◽  
Activated T Cells ◽  
Knock Down ◽  
And Function

Abstract The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

T cells: a dedicated effector kinase pathways for every trait?

2021 ◽  
Vol 478 (6) ◽  
pp. 1303-1307
Author(s):  
Kriti Bahl ◽  
Jeroen P. Roose
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Spectrometric Analysis ◽  
Activated T Cells ◽  
Biological Responses ◽  
Extracellular Signal ◽  
Differentiation And Proliferation

Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation. RNA sequencing-based methods have provided insights into the gene expression programs that support the above-mentioned cell biological responses. The proteome is often overlooked. A recent study by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] focuses on characterizing the effect of extracellular signal-regulated kinase (ERK) on the remodeling of the proteome of activated CD8+ T cells using Mass spectrometric analysis. Surprisingly, the Effector kinase ERK pathway is responsible for only a select proportion of the proteome that restructures during T cell activation. The primary targets of ERK signaling are transcription factors, cytokines, and cytokine receptors. In this commentary, we discuss the recent findings by Damasio et al. [Biochem. J. (2021) 478, 79–98. doi:10.1042/BCJ20200661] in the context of different Effector kinase pathways in activated T cells.

scholarly journals SLFN2 protection of tRNAs from stress-induced cleavage is essential for T cell–mediated immunity

Science ◽  
2021 ◽  
Vol 372 (6543) ◽  
pp. eaba4220 ◽  
Author(s):  
Tao Yue ◽  
Xiaoming Zhan ◽  
Duanwu Zhang ◽  
Ruchi Jain ◽  
Kuan-wen Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Cell Mediated Immunity ◽  
Activated T Cells ◽  
Granule Formation

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell–specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor β (IL-2Rβ) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2’s mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.

scholarly journals Extracellular signal-regulated kinase (ERK) pathway control of CD8+ T cell differentiation

2020 ◽  
Author(s):  
Marcos P. Damasio ◽  
Julia M. Marchingo ◽  
Laura Spinelli ◽  
Doreen A. Cantrell ◽  
Andrew J.M. Howden
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Antigen Receptor ◽  
T Cell Differentiation ◽  
Signalling Pathways ◽  
Activated T Cells ◽  
Transcriptional Reprogramming ◽  
Extracellular Signal

SummaryThe integration of multiple signalling pathways that co-ordinate T cell metabolism and transcriptional reprogramming is required to drive T cell differentiation and proliferation. One key T cell signalling module is mediated by extracellular signal-regulated kinases (ERKs) which are activated in response to antigen receptor engagement. The activity of ERKs is often used to report antigen receptor occupancy but the full details of how ERKs control T cell activation is not understood. Accordingly, we have used mass spectrometry to explore how ERK signalling pathways control antigen receptor driven proteome restructuring in CD8 + T cells to gain insights about the biological processes controlled by ERKs in primary lymphocytes. Quantitative analysis of >8000 proteins identified only 900 ERK regulated proteins in activated CD8+ T cells. The data identify both positive and negative regulatory roles for ERKs during T cell activation and reveal that ERK signalling primarily controls the repertoire of transcription factors, cytokines and cytokine receptors expressed by activated T cells. The ERKs thus drive the transcriptional reprogramming of activated T cells and the ability of T cells to communicate with external immune cues.

scholarly journals Distinct cellular immune profiles in the airways and blood of critically ill patients with COVID-19

2020 ◽  
Author(s):  
Anno Saris ◽  
Tom D.Y. Reijnders ◽  
Esther J. Nossent ◽  
Alex R. Schuurman ◽  
Jan Verhoeff ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Effector Memory ◽  
Activated T Cells ◽  
Immune Profile ◽  
Prolonged Icu Stay

AbstractOur understanding of the coronavirus disease-19 (COVID-19) immune response is almost exclusively derived from studies that examined blood. To gain insight in the pulmonary immune response we analysed BALF samples and paired blood samples from 17 severe COVID-19 patients. Macrophages and T cells were the most abundant cells in BALF. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells and expressed higher levels of the exhaustion marker PD-1 than in peripheral blood. Prolonged ICU stay associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. In conclusion, the bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood.SummaryThe bronchoalveolar immune response in severe COVID-19 strongly differs from the peripheral blood immune profile. Fatal COVID-19 associated with T cell activation blood, but not in BALF.

scholarly journals Diacylglycerol kinase ζ limits IL-2-dependent control of PD-1 expression in tumor-infiltrating T lymphocytes

2020 ◽  
Vol 8 (2) ◽  
pp. e001521
Author(s):  
Javier Arranz-Nicolás ◽  
Miguel Martin-Salgado ◽  
Cristina Rodríguez-Rodríguez ◽  
Rosa Liébana ◽  
Maria C Moreno-Ortiz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Therapeutic Potential ◽  
Interleukin 2 ◽  
Tumor Rejection ◽  
Cytotoxic T Cell ◽  
Adenocarcinoma Cells ◽  
The Impact

BackgroundThe inhibitory functions triggered by the programmed cell death-1 (PD-1) receptor following binding to its ligand (PD-L1) protect healthy organs from cytotoxic T cells, and neutralize antitumor T cell attack. Antibody-based therapies to block PD-1/PD-L1 interaction have yielded notable results, but most patients eventually develop resistance. This failure is attributed to CD8+ T cells achieving hyporesponsive states from which recovery is hardly feasible. Dysfunctional T cell phenotypes are favored by a sustained imbalance in the diacylglycerol (DAG)- and Ca2+-regulated transcriptional programs. In mice, DAG kinase ζ (DGKζ) facilitates DAG consumption, limiting T cell activation and cytotoxic T cell responses. DGKζ deficiency facilitates tumor rejection in mice without apparent adverse autoimmune effects. Despite its therapeutic potential, little is known about DGKζ function in human T cells, and no known inhibitors target this isoform.MethodsWe used a human triple parameter reporter cell line to examine the consequences of DGKζ depletion on the transcriptional restriction imposed by PD-1 ligation. We studied the effect of DGKζ deficiency on PD-1 expression dynamics, as well as the impact of DGKζ absence on the in vivo growth of MC38 adenocarcinoma cells.ResultsWe demonstrate that DGKζ depletion enhances DAG-regulated transcriptional programs, promoting interleukin-2 production and partially counteracting PD-1 inhibitory functions. DGKζ loss results in limited PD-1 expression and enhanced expansion of cytotoxic CD8+ T cell populations. This is observed even in immunosuppressive milieus, and correlates with the reduced ability of MC38 adenocarcinoma cells to form tumors in DGKζ-deficient mice.ConclusionsOur results, which define a role for DGKζ in the control of PD-1 expression, confirm DGKζ potential as a therapeutic target as well as a biomarker of CD8+ T cell dysfunctional states.

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