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 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 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 Conditional Knockout Mice Reveal an Essential Role of Protein Phosphatase 4 in Thymocyte Development and Pre-T-Cell Receptor Signaling

2006 ◽  
Vol 27 (1) ◽  
pp. 79-91 ◽  
Author(s):  
Jr-Wen Shui ◽  
Mickey C.-T. Hu ◽  
Tse-Hua Tan
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cell Receptor ◽  
Conditional Knockout ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Deficient Mice

ABSTRACT Okadaic acid-sensitive serine/threonine phosphatases have been shown to regulate interleukin-2 transcription and T-cell activation. Okadaic acid inhibits protein phosphatase 4 (PP4), a novel PP2A-related serine/threonine phosphatase, at a 50% inhibitory concentration (IC50) comparable to that for PP2A. This raises the possibility that some cellular functions of PP2A, determined in T cells by using okadaic acid, may in fact be those of PP4. To investigate the in vivo roles of PP4 in T cells, we generated conventional and T-cell-specific PP4 conditional knockout mice. We found that the ablation of PP4 led to the embryonic lethality of mice. PP4 gene deletion in the T-cell lineage resulted in aberrant thymocyte development, including T-cell arrest at the double-negative 3 stage (CD4− CD8− CD25+ CD44−), abnormal thymocyte maturation, and lower efficacy of positive selection. PP4-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo. Analysis of pre-T-cell receptor (pre-TCR) signaling further revealed impaired calcium flux and phospholipase C-γ1-extracellular signal-regulated kinase activation in the absence of PP4. Anti-CD3 injection in PP4-deficient mice led to enhanced thymocyte apoptosis, accompanied by increased proapoptotic Bim but decreased antiapoptotic Bcl-xL protein levels. In the periphery, antigen-specific T-cell proliferation and T-cell-mediated immune responses in PP4-deficient mice were dramatically compromised. Thus, our results indicate that PP4 is essential for thymocyte development and pre-TCR signaling.

scholarly journals T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

1995 ◽  
Vol 181 (3) ◽  
pp. 927-941 ◽  
Author(s):  
C R Wang ◽  
K Hashimoto ◽  
S Kubo ◽  
T Yokochi ◽  
M Kubo ◽  
...  
Keyword(s):  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Intracellular Signaling ◽  
Negative Selection ◽  
Cell Receptor ◽  
Clonal Deletion ◽  
Signaling Events ◽  
Selection Of

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

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 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 Kinetic Threshold between Negative and Positive Selection Based on the Longevity of the T Cell Receptor–Ligand Complex

1999 ◽  
Vol 189 (10) ◽  
pp. 1531-1544 ◽  
Author(s):  
Calvin B. Williams ◽  
Deborah L. Engle ◽  
Gilbert J. Kersh ◽  
J. Michael White ◽  
Paul M. Allen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Positive Selection ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Ligand Complex ◽  
Altered Peptide Ligands ◽  
Self Peptides ◽  
Selection Of

We have developed a unique in vivo system to determine the relationship between endogenous altered peptide ligands and the development of major histocompatibility complex class II– restricted T cells. Our studies use the 3.L2 T cell receptor (TCR) transgenic mouse, in which T cells are specific for Hb(64–76)/I-Ek and positively selected on I-Ek plus self-peptides. To this endogenous peptide repertoire, we have individually added one of six well-characterized 3.L2 ligands. This transgenic approach expands rather than constrains the repertoire of self-peptides. We find that a broad range of ligands produce negative selection of thymocytes in vivo. When compared with the in vitro TCR–ligand binding kinetics, we find that these negatively selecting ligands all have a half-life of 2 s or greater. Additionally, one of two ligands examined with no detectable binding to the 3.L2 TCR and no activity on mature 3.L2 T cells (Q72) enhances the positive selection of transgenic thymocytes in vivo. Together, these data establish a kinetic threshold between negative and positive selection based on the longevity of TCR–ligand complexes.

scholarly journals The transcription factor Gli3 regulates differentiation of fetal CD4–CD8– double-negative thymocytes

Blood ◽  
2005 ◽  
Vol 106 (4) ◽  
pp. 1296-1304 ◽  
Author(s):  
Ariadne L. Hager-Theodorides ◽  
Johannes T. Dessens ◽  
Susan V. Outram ◽  
Tessa Crompton
Keyword(s):  
Transcription Factor ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Differentiation ◽  
Double Negative ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Double Positive ◽  
Dose Dependent

AbstractGlioblastoma 3 (Gli3) is a transcription factor involved in patterning and oncogenesis. Here, we demonstrate a role for Gli3 in thymocyte development. Gli3 is differentially expressed in fetal CD4–CD8– double-negative (DN) thymocytes and is most highly expressed at the CD44+ CD25– DN (DN1) and CD44–CD25– (DN4) stages of development but was not detected in adult thymocytes. Analysis of null mutants showed that Gli3 is involved at the transitions from DN1 to CD44+ CD25+ DN (DN2) cell and from DN to CD4+CD8+ double-positive (DP) cell. Gli3 is required for differentiation from DN to DP thymocyte, after pre–T-cell receptor (TCR) signaling but is not necessary for pre-TCR–induced proliferation or survival. The effect of Gli3 was dose dependent, suggesting its direct involvement in the transcriptional regulation of genes controlling T-cell differentiation during fetal development.

scholarly journals Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas

2015 ◽  
Vol 112 (25) ◽  
pp. 7773-7778 ◽  
Author(s):  
Hyung-Ok Lee ◽  
Xiao He ◽  
Jayati Mookerjee-Basu ◽  
Dai Zhongping ◽  
Xiang Hua ◽  
...  
Keyword(s):  
Transcription Factor ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Thymocyte Development ◽  
Tcr Signaling ◽  
Double Positive ◽  
Human T Cell

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell–specific ThPOK transgene (ThPOKconst mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOKconst RAG-deficient mice, which promotes development to the CD4+8+ (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

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.

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