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 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 IL-15 Serum Concentrations and CD19 CAR T-Cell Therapy: Impact on Clinical Outcomes and In Vivo CAR T Cell Kinetics

2021 ◽  
Vol 27 (3) ◽  
pp. S196-S197
Author(s):  
Jordan Gauthier ◽  
Cassie Chou ◽  
Alexandre V. Hirayama ◽  
Salvatore Fiorenza ◽  
Erik Kimble ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Clinical Outcomes ◽  
Cell Kinetics ◽  
Serum Concentrations ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

scholarly journals 735 Identification of a small molecule that prevents T cell exhaustion using machine learning algorithms paired with high-resolution single cell RNAseq

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A766-A766
Author(s):  
Isabelle Le Mercier ◽  
Sunny Sun ◽  
Dongmei Xiao ◽  
Laura Isacco ◽  
Daniel Treacy ◽  
...  
Keyword(s):  
T Cell ◽  
Low Dose ◽  
Machine Learning Algorithms ◽  
High Dose ◽  
T Cell Exhaustion ◽  
Tcr Signaling ◽  
Compound A ◽  
Cell Exhaustion

BackgroundT cell responses are tightly regulated and require a constant balance of signals during the different stages of their activation, expansion, and differentiation. As a result of chronic antigen exposure, T cells become exhausted in solid tumors, preventing them from controlling tumor growth.MethodsWe identified a transcriptional signature associated with T cell exhaustion in patients with melanoma and used our proprietary machine learning algorithms to predict molecules that would prevent T cell exhaustion and improve T cell function. Among the predictions, an orally available small molecule, Compound A, was highly predicted.ResultsCompound A was tested in an in vitro T cell Exhaustion assay and shown to prevent loss of proliferation and expression of immune checkpoint receptors. Transcriptionally, Compound A-treated cells looked indistinguishable from conventionally expanded, non-exhausted T cells. However, when assessed in a classical T cell activation assay, Compound A demonstrated dose dependent activity. At low dose, Compound A was immuno-stimulatory, allowing cells to divide further by preventing activation induced cell death. At higher doses, Compound A demonstrated immuno-suppressive activity preventing early CD69 upregulation and T cell proliferation. All together, these observations suggest that Compound A prevented exhaustion with a mechanism of action involving TCR signaling inhibition. While cessation of TCR signaling or rest has been recently associated with improved CAR-T efficacy by preventing or reversing exhaustion during the in vitro manufacturing phase, it is unclear if that mechanism would translate in vivo.Compound A was evaluated in the CT26 and MC38 syngeneic mouse models alongside anti-PD1. At low dose Compound A closely recapitulated anti-PD1 mediated cell behavior changes by scRNA-seq and flow cytometry in CT26 mice. At high dose, Compound A led to the accumulation of naive cells in the tumor microenvironment (TME) confirming the proposed mechanism of action. Low dose treatment was ineffective in MC38 mouse model but a pulsed treatment at high dose also recapitulated anti-PD1 activity in most animals. Importantly, we identified a new T cell population responding to anti-PD1 that was particularly increased in the MC38 mouse model; Compound A treatment also impacted this population.ConclusionsThese data confirm that mild TCR inhibition either suboptimal or fractionated can prevent exhaustion in vivo. However, this approach has a very limited window of activity between immuno-modulatory and immuno-suppressive effects, thereby limiting potential clinical benefit. Finally, these results demonstrate that our approach and platform was able to predict molecules that would prevent T cell exhaustion 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.

MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2181-2181
Author(s):  
Tao Zou ◽  
Atsushi Satake ◽  
Jonathan Maltzman ◽  
Taku Kambayashi
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Adaptor Protein ◽  
Class Ii ◽  
Sh2 Domain ◽  
Tcr Signaling ◽  
Mhc Ii

Abstract Abstract 2181 Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s. Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency. We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway. In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures: No relevant conflicts of interest to declare.

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Arnab Ghosh ◽  
Marco L. Davila ◽  
Lauren F. Young ◽  
Christopher Kloss ◽  
Gertrude Gunset ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Acute Gvhd ◽  
Tcr Signaling ◽  
Car T Cells ◽  
Donor T Cells ◽  
Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

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.

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