scholarly journals A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

2018 ◽  
Vol 217 (8) ◽  
pp. 2931-2950 ◽  
Author(s):  
David Bending ◽  
Paz Prieto Martín ◽  
Alina Paduraru ◽  
Catherine Ducker ◽  
Erik Marzaganov ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Dynamic Processes ◽  
Computer Algorithms ◽  
Tcr Signaling ◽  
Sequence Of Events ◽  
Downstream Effects ◽  
Tcr Signals ◽  
Treg Differentiation

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

scholarly journals A Timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

2017 ◽  
Author(s):  
David Bending ◽  
Paz Prieto Martín ◽  
Alina Paduraru ◽  
Catherine Ducker ◽  
Erik Marzaganov ◽  
...  
Keyword(s):  
T Cell ◽  
Cellular Differentiation ◽  
Cell Kinetics ◽  
Temporal Dynamics ◽  
Temporal Sequence ◽  
Dynamic Processes ◽  
Computer Algorithms ◽  
Tcr Signaling ◽  
Sequence Of Events

AbstractUnderstanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyse in vivo. We establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky [toki], time in Japanese). Tocky uses the Fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue-to-red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR)-signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a Tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.SummaryThe authors establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky) revealing the temporal dynamics of cellular activation and differentiation in vivo. The tool analyses the temporal sequence of molecular processes during cellular differentiation and identifies cells that receive persistent signals in vivo.

scholarly journals Loss of tonic T-cell receptor signals alters the generation but not the persistence of CD8+ memory T cells

Blood ◽  
2010 ◽  
Vol 116 (25) ◽  
pp. 5560-5570 ◽  
Author(s):  
Karla R. Wiehagen ◽  
Evann Corbo ◽  
Michelle Schmidt ◽  
Haina Shin ◽  
E. John Wherry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Lymphocytic Choriomeningitis ◽  
Sh2 Domain ◽  
Normal Numbers ◽  
Tcr Signaling ◽  
Tcr Signals

Abstract The requirements for tonic T-cell receptor (TCR) signaling in CD8+ memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain–containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76–dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.

scholarly journals Restricting Zap70 Expression to CD4+CD8+ Thymocytes Reveals a T Cell Receptor–dependent Proofreading Mechanism Controlling the Completion of Positive Selection

2003 ◽  
Vol 197 (3) ◽  
pp. 363-373 ◽  
Author(s):  
Xiaolong Liu ◽  
Anthony Adams ◽  
Kathryn F. Wildt ◽  
Bruce Aronow ◽  
Lionel Feigenbaum ◽  
...  
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Novel Approach ◽  
Single Positive ◽  
Tcr Signals

Although T cell receptor (TCR) signals are essential for intrathymic T cell–positive selection, it remains controversial whether they only serve to initiate this process, or whether they are required throughout to promote thymocyte differentiation and survival. To address this issue, we have devised a novel approach to interfere with thymocyte TCR signaling in a developmental stage-specific manner in vivo. We have reconstituted mice deficient for Zap70, a tyrosine kinase required for TCR signaling and normally expressed throughout T cell development, with a Zap70 transgene driven by the adenosine deaminase (ADA) gene enhancer, which is active in CD4+CD8+ thymocytes but inactive in CD4+ or CD8+ single-positive (SP) thymocytes. In such mice, termination of Zap70 expression impaired TCR signal transduction and arrested thymocyte development after the initiation, but before the completion, of positive selection. Arrested thymocytes had terminated Rag gene expression and up-regulated TCR and Bcl-2 expression, but failed to differentiate into mature CD4 or CD8 SP thymocytes, to be rescued from death by neglect or to sustain interleukin 7Rα expression. These observations identify a TCR-dependent proofreading mechanism that verifies thymocyte TCR specificity and differentiation choices before the completion of positive selection.

scholarly journals Thymic progenitors of TCRαβ+ CD8αα intestinal intraepithelial lymphocytes require RasGRP1 for development

2017 ◽  
Vol 214 (8) ◽  
pp. 2421-2435 ◽  
Author(s):  
Dominic P. Golec ◽  
Romy E. Hoeppli ◽  
Laura M. Henao Caviedes ◽  
Jillian McCann ◽  
Megan K. Levings ◽  
...  
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
Cell Receptor ◽  
Intraepithelial Lymphocytes ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Clonal Deletion ◽  
Selection Processes ◽  
Tcr Signals

Strong T cell receptor (TCR) signaling largely induces cell death during thymocyte development, whereas weak TCR signals induce positive selection. However, some T cell lineages require strong TCR signals for differentiation through a process termed agonist selection. The signaling relationships that underlie these three fates are unknown. RasGRP1 is a Ras activator required to transmit weak TCR signals leading to positive selection. Here, we report that, despite being dispensable for thymocyte clonal deletion, RasGRP1 is critical for agonist selection of TCRαβ+CD8αα intraepithelial lymphocyte (IEL) progenitors (IELps), even though both outcomes require strong TCR signaling. Bim deficiency rescued IELp development in RasGRP1−/− mice, suggesting that RasGRP1 functions to promote survival during IELp generation. Additionally, expression of CD122 and the adhesion molecules α4β7 and CD103 define distinct IELp subsets with differing abilities to generate TCRαβ+CD8αα IEL in vivo. These findings demonstrate that RasGRP1-dependent signaling underpins thymic selection processes induced by both weak and strong TCR signals and is differentially required for fate decisions derived from a strong TCR stimulus.

scholarly journals SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ–dependent manner

2017 ◽  
Vol 115 (3) ◽  
pp. E458-E467 ◽  
Author(s):  
Subha Sen ◽  
Fei Wang ◽  
Jing Zhang ◽  
Zhiheng He ◽  
Jian Ma ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Orphan Nuclear Receptor ◽  
Tcr Signaling ◽  
Th17 Differentiation ◽  
Tcr Signals

Th17 cells are major players in multiple autoimmune diseases and are developmentally contingent on reciprocal functionality between the transcription factor Retineic acid receptor-related orphan nuclear receptor gamma (RORγt) and Forkhead box protein P3 (Foxp3). Here we deciphered a previously unappreciated role of Steroid receptor coactivator 1 (SRC1) in defining the lineage decision for the development of Th17 versus induced T-regulatory (iTreg) cells. We demonstrate that SRC1 functions as a critical coactivator for RORγt in vivo to promote the functional dominance of RORγt over Foxp3 and thus establishing an unopposed Th17 differentiation program. In the absence of SRC1, T cell polarization resulted in decreased IL-17+ and increased Foxp3+ cells during both in vitro differentiation and in vivo development of experimental autoimmune encephalomyelitis. Mechanistically, T cell receptor (TCR) signaling molecule protein kinase C theta (PKC-θ)–mediated phosphorylation of SRC1 is important for inducing enhanced RORγt–SRC1 interaction, stable DNA binding, and resultant IL-17A transcription. Furthermore, phospho-SRC1–mediated recruitment of CARM1 induced prominent asymmetric dimethylation of H3R17 while preventing repressive H3K9 trimethylation and hence further modifying the IL-17 locus for optimal transcription. Moreover, binding of phospho-SRC1 to RORγt displaced bound Foxp3, leading to prompt degradation of the dissociated Foxp3 via a ubiquitin–proteosomal pathway and hence reversing the inhibitory action of Foxp3 on RORγt activity. Thus, SRC1 acts as a crucial molecular mediator to integrate positive PKC-θ–dependent TCR signals to induce peak RORγt activity and establish phenotypic dominance of Th17 over the iTreg pathway.

scholarly journals Preclinical characterization of Sintilimab, a fully human anti-PD-1 therapeutic monoclonal antibody for cancer

2018 ◽  
Vol 1 (2) ◽  
pp. 65-73 ◽  
Author(s):  
Shuang Zhang ◽  
Min Zhang ◽  
Weiwei Wu ◽  
Zhijun Yuan ◽  
Andy Tsun ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Luciferase Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tcr Signaling ◽  
Igg Antibody ◽  
Dissociation Kinetics ◽  
Programmed Death

ABSTRACT Background Programmed cell death 1 (PD-1) is an inhibitory immune checkpoint expressed on activatedT cells. Upon the formation of T cell receptor (TCR)-pMHC complexes, concomitant PD-1 ligation to its ligands programmed death-ligand 1 (PD-L1) or programmed death-ligand 2 (PD-L2) downregulates TCR signaling and effector function. Here we describe the preclinical characterization of Sintilimab, a fully human IgG4 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. Methods The binding affinity and blockade function were detected by using surface plasmon resonance (SPR), Enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biology function properties were measured with luciferase assay and mixed lymphocyte reaction assay. In vivo anti-tumor function and preclinical pharmacokinetic (PK) were identified with human PD-1 transgenic mice and non-human primates separately. Results Sintilimab can specifically and strongly bind to human PD-1 (hPD-1) and cynomolgus PD-1 and the affinity of Sintilimab to human PD-1 was measured at 0.3 nm via surface SPR, and displayed slow dissociation kinetics. Sintilimab can block the interaction of PD-1 to PD-L1 and PD-L2 and induce high secretion levels of interferon (IFN)-γ and interleukin (IL)-2 in primary T cell assays. In humanized hPD-1 knock-in mouse models, Sintilimab showed potent anti-tumor activity and increased tumor-infiltrating CD8/CD4 T cell and CD8/ Treg ratios. Preclinical experimentation in non-human primates following a single intravenous infusion of Sintilimab at 1, 6 and 30 mg/kg presented with no signs of drug-related toxicity, and showed typical PK characteristics of an IgG antibody. Conclusions Sintilimab has desirable preclinical attributes that supports its clinical development for cancer treatment.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

scholarly journals Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor SignalingIn Vivo

2016 ◽  
Vol 36 (18) ◽  
pp. 2396-2402 ◽  
Author(s):  
Peter A. Thill ◽  
Arthur Weiss ◽  
Arup K. Chakraborty
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
New Model ◽  
Downstream Signaling ◽  
Conventional Models

The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase Zap70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines,in vivo, Zap70 is bound to phosphorylated ITAMs in resting T cells. However, Zap70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates thesein vivoobservations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed.

scholarly journals A Stat5b transgene is capable of inducing CD8+ lymphoblastic lymphoma in the absence of normal TCR/MHC signaling

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 344-350 ◽  
Author(s):  
Katherine Bessette ◽  
Mark L. Lang ◽  
Roy A. Fava ◽  
Martin Grundy ◽  
Jennifer Heinen ◽  
...  
Keyword(s):  
T Cell ◽  
Significant Proportion ◽  
Cell Receptor ◽  
Tumor Formation ◽  
Tcr Signaling ◽  
Double Positive ◽  
Oncogenic Potential ◽  
Histocompatibility Complex ◽  
T Cell Lymphomas ◽  
Tcr Signals

Stat5 proteins are critical signaling molecules activated by many cytokines. Within the immune system, Stat5 plays important roles related to the development of thymocytes and proliferation of T cells. Stat5 has been implicated in malignant transformation, and moreover, the activated tyrosine phosphorylated form of Stat5 is frequently observed in human lymphomas. We previously demonstrated the oncogenic potential of Stat5, with thymic lymphoblastic lymphomas developing in a significant proportion of transgenic (TG) mice overexpressing Stat5a or Stat5b in lymphocytes. In addition, immunization or expression of a T-cell receptor (TCR) transgene augmented the rate of tumor formation. Here, we investigate the mechanism of Stat5-mediated lymphomagenesis by exploring the contributions of major histocompatibility complex (MHC)/TCR and pre-TCR signals. We present data demonstrating that Stat5b TG mice unexpectedly develop CD8+ lymphoma even in the absence of either pre-TCR signaling or normal thymic selection. Indeed, acceleration of Stat5b transgene-mediated lymphoma occurred on TCRα−/− and pre-TCRα−/− backgrounds. In light of these data, we propose a model in which alterations in T-cell development at the double-negative/double-positive (DN/DP) stages cooperate with cytokine-mediated pathways in immature thymocytes to give rise to lymphoblastic T-cell lymphomas in Stat5b TG mice.

scholarly journals LRCH1 deficiency enhances LAT signalosome formation and CD8+T cell responses against tumors and pathogens

2020 ◽  
Vol 117 (32) ◽  
pp. 19388-19398 ◽  
Author(s):  
Chang Liu ◽  
Xiaoyan Xu ◽  
Lei Han ◽  
Xiaopeng Wan ◽  
Lingming Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Tcr Signaling ◽  
T Cell Migration ◽  
Car T ◽  
Leucine Rich Repeats

CD8+T cells play pivotal roles in eradicating pathogens and tumor cells. T cell receptor (TCR) signaling is vital for the optimal activation of CD8+T cells. Upon TCR engagement, the transmembrane adapter protein LAT (linker for activation of T cells) recruits other key signaling molecules and forms the “LAT signalosome” for downstream signal transduction. However, little is known about which functional partners could restrain the formation of the LAT signalosome and inhibit CD8+cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Here we have demonstrated that LRCH1 (leucine-rich repeats and calponin homology domain containing 1) directly binds LAT, reduces LAT phosphorylation and interaction with GRB2, and also promotes the endocytosis of LAT.Lrch1−/−mice display better protection against influenza virus andListeriainfection, with enhanced CD8+T cell proliferation and cytotoxicity. Adoptive transfer ofLrch1−/−CD8+CTLs leads to increased B16-MO5 tumor clearance in vivo. Furthermore, knockout ofLRCH1in human chimeric antigen receptor (CAR) T cells that recognize the liver tumor-associated antigen glypican-3 could improve CAR T cell migration and proliferation in vitro. These findings suggest LRCH1 as a potential translational target to improve T cell immunotherapy against infection and tumors.

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