scholarly journals The Tec kinase ITK differentially optimizes NFAT, NF-κB, and MAPK signaling during early T cell activation to regulate graded gene induction

2020 ◽  
Author(s):  
Michael P. Gallagher ◽  
James M. Conley ◽  
Pranitha Vangala ◽  
Andrea Reboldi ◽  
Manuel Garber ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Signal Strength ◽  
Mapk Signaling ◽  
Early Gene ◽  
Cell Nuclei ◽  
Activated T Cells

ABSTRACTThe strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal kinase ITK simultaneously trigger many biochemically separate TCR signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, we examined Erk1/2 activation and NFAT, NF-κB translocation in naive OT-I CD8+ cell nuclei. We observed consistent digital activation of NFAT1 and Erk-MAPK, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength and was tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis also revealed genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

scholarly journals Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK

2021 ◽  
Vol 118 (35) ◽  
pp. e2025825118
Author(s):  
Michael P. Gallagher ◽  
James M. Conley ◽  
Pranitha Vangala ◽  
Manuel Garber ◽  
Andrea Reboldi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Signal Strength ◽  
Cell Receptor ◽  
Early Gene ◽  
Sequencing Analysis ◽  
Nuclear Factor ◽  
Activated T Cells

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2–inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

scholarly journals An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

2013 ◽  
Vol 87 (23) ◽  
pp. 12701-12720 ◽  
Author(s):  
Benjamin Trinité ◽  
Eric C. Ohlson ◽  
Igor Voznesensky ◽  
Shashank P. Rana ◽  
Chi N. Chan ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
De Novo ◽  
Virus Production ◽  
Early Gene ◽  
Viral Dna ◽  
Hiv 1

Integration is a central event in the replication of retroviruses, yet ≥90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4+T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4+T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4+T cells. We found that infection of cytokine-treated resting CD4+T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yieldedde novovirus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression andde novovirus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.

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.

The costimulatory activity of Tim-3 requires Akt and MAPK signaling and its recruitment to the immune synapse

2021 ◽  
Vol 14 (687) ◽  
pp. eaba0717
Author(s):  
Shunsuke Kataoka ◽  
Priyanka Manandhar ◽  
Judong Lee ◽  
Creg J. Workman ◽  
Hridesh Banerjee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Cell Receptor ◽  
Mapk Signaling ◽  
Inhibitory Protein ◽  
Immune Synapse

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3–mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)–dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

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 Nanoconfinement of Microvilli Alters Gene Expression and Boosts T cell Activation

2021 ◽  
Author(s):  
Morteza Aramesh ◽  
Diana Stoycheva ◽  
Ioana Sandu ◽  
Stephan J. Ihle ◽  
Tamara Zund ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Signaling ◽  
T Cell Activation ◽  
Cell Activation ◽  
Local Environment ◽  
T Cell Signaling ◽  
Sense And Respond ◽  
Altered Gene

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanisms by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation, and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200 nm pores, but not in 400 nm pores. Consequently, formation of TCR nanoclustered hotspots within 200 nm pores, allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

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.

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