Survivin Loss in Thymocytes Triggers p53-mediated Growth Arrest and p53-independent Cell Death

Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4− CD8− double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre–T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.

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Gimap4 accelerates T-cell death

Blood ◽

10.1182/blood-2005-11-4616 ◽

2006 ◽

Vol 108 (2) ◽

pp. 591-599 ◽

Cited By ~ 40

Author(s):

Silke Schnell ◽

Corinne Démollière ◽

Paul van den Berk ◽

Heinz Jacobs

Keyword(s):

Cell Death ◽

T Cell ◽

Critical Role ◽

T Cell Development ◽

Cell Receptor ◽

Cell Physiology ◽

Double Positive ◽

Pkc Activation ◽

Null Mutant Mouse

Gimap4, a member of the newly identified GTPase of the immunity-associated protein family (Gimap), is strongly induced by the pre–T-cell receptor in precursor T lymphocytes, transiently shut off in double-positive thymocytes, and reappears after TCR-mediated positive selection. Here, we show that Gimap4 remains expressed constitutively in the cytosol of mature T cells. A C-terminal IQ domain binds calmodulin in the absence of calcium, and conserved PKC phosphorylation motifs are targets of concanavalin A (ConA)– or PMA/ionomycin-induced PKC activation. To address the role of Gimap4 in T-cell physiology, we completed the genomic organization of the gimap4 locus and generated a Gimap4-null mutant mouse. Studies in these mice revealed no critical role of Gimap4 in T-cell development but in the regulation of apoptosis. We have found that Gimap4 accelerates the execution of programmed cell death induced by intrinsic stimuli downstream of caspase-3 activation and phosphatidylserine exposure. Apoptosis directly correlates with the phosphorylation status of Gimap4.

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Normal Immune Development and Glucocorticoid-Induced Thymocyte Apoptosis in Mice Deficient for the T-Cell Death-Associated Gene 8 Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.26.2.668-677.2006 ◽

2006 ◽

Vol 26 (2) ◽

pp. 668-677 ◽

Cited By ~ 52

Author(s):

Caius G. Radu ◽

Donghui Cheng ◽

Amar Nijagal ◽

Mireille Riedinger ◽

Jami McLaughlin ◽

...

Keyword(s):

Cell Death ◽

T Cell ◽

Fluorescent Protein ◽

Critical Role ◽

Immune Functions ◽

Thymocyte Development ◽

Double Positive ◽

Immune Development ◽

Induced Apoptosis

ABSTRACT T-cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor transcriptionally upregulated by glucocorticoids (GCs) and implicated by overexpression studies in psychosine-mediated inhibition of cytokinesis and in GC-induced apoptosis. To examine the physiological function of TDAG8, we generated knockout (KO) mice by homologous recombination. An enhanced green fluorescent protein reporter was knocked into the disrupted tdag8 locus to allow the analysis of TDAG8 expression in living cells. Interestingly, we found that during thymocyte development, TDAG8 expression resembled the dynamic regulation described for known modulators of GC-induced apoptosis, including Bcl-2, Notch1, and GC receptor. TDAG8 was expressed in double-negative cells, was downregulated at the double-positive transition, and was upregulated in single-positive thymocytes. However, despite this striking expression pattern, maturation and selection of thymocytes, as well as major immune functions, were not affected in TDAG8 KO mice. In contrast to previous overexpression results, TDAG8 was dispensable for psychosine-induced formation of multinucleated cells. Furthermore, TDAG8 KO thymocytes showed normal apoptosis following in vivo and in vitro GC treatment. These results, while establishing a useful reporter strain to study T-lymphocyte maturation, argue against a critical role for TDAG8 in immune development, psychosine-mediated inhibition of cytokinesis, and GC-induced cell death.

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Critical role of WNK1 in MYC-dependent early mouse thymocyte development

eLife ◽

10.7554/elife.56934 ◽

2020 ◽

Vol 9 ◽

Author(s):

Robert Köchl ◽

Lesley Vanes ◽

Miriam Llorian Sopena ◽

Probir Chakravarty ◽

Harald Hartweger ◽

...

Keyword(s):

Critical Role ◽

Ion Homeostasis ◽

Thymocyte Development ◽

Double Positive ◽

Mouse Thymocyte ◽

Proliferation And Differentiation ◽

Tcr Signals ◽

And Migration ◽

Early Mouse

WNK1, a kinase that controls kidney salt homeostasis, also regulates adhesion and migration in CD4+ T cells. Wnk1 is highly expressed in thymocytes, and since migration is important for thymocyte maturation, we investigated a role for WNK1 in mouse thymocyte development. We find that WNK1 is required for the transition of double negative (DN) thymocytes through the β-selection checkpoint and subsequent proliferation and differentiation into double positive (DP) thymocytes. Furthermore, we show that WNK1 negatively regulates LFA1-mediated adhesion and positively regulates CXCL12-induced migration in DN thymocytes. Despite this, migration defects of WNK1-deficient thymocytes do not account for the developmental arrest. Instead, we show that in DN thymocytes WNK1 transduces pre-TCR signals via OXSR1 and STK39 kinases, and the SLC12A2 ion co-transporter that are required for post-transcriptional upregulation of MYC and subsequent proliferation and differentiation into DP thymocytes. Thus, a pathway regulating ion homeostasis is a critical regulator of thymocyte development.

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

Blood ◽

10.1182/blood-2005-03-0998 ◽

2005 ◽

Vol 106 (4) ◽

pp. 1296-1304 ◽

Cited By ~ 32

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.

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Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes.

Journal of Experimental Medicine ◽

10.1084/jem.178.6.1867 ◽

1993 ◽

Vol 178 (6) ◽

pp. 1867-1875 ◽

Cited By ~ 43

Author(s):

C N Levelt ◽

R Carsetti ◽

K Eichmann

Keyword(s):

Signal Transduction ◽

T Cell ◽

T Cell Receptor ◽

Alternative Hypothesis ◽

Cell Receptor ◽

Surface Expression ◽

Beta Chain ◽

Thymocyte Development ◽

Double Positive ◽

Highly Correlated

Recent studies have shown that maturation of CD4-8- double negative (DN) thymocytes to the CD4+8+ double positive (DP) stage is dependent on expression of the T cell receptor (TCR)-beta polypeptide. The exact mechanism by which the TCR-beta chain regulates this maturation step remains unknown. Previous experiments had suggested that in the presence of some TCR+ thymocytes, additional DN thymocytes not expressing a TCR-beta chain may be recruited to mature to the DP stage. The recent demonstration of an immature TCR-beta-CD3 complex on early thymocytes lead to the alternative hypothesis that signal transduction through an immature TCR-CD3 complex may induce maturation to the DP stage. In the latter case, maturation to the DP stage would depend on the expression of TCR-beta-CD3 in the same cell. We examined these two hypotheses by studying the expression of the intra- and extracellular CD3 epsilon, CD3 zeta, and TCR-beta polypeptides in intrathymic subpopulations during embryogenesis. CD3 epsilon and CD3 zeta were expressed intracellularly 2 and 1 d, respectively, before intracellular expression of the TCR-beta chain, potentially allowing immediate surface expression of an immature TCR-beta-CD3 complex as soon as functional rearrangement of a TCR-beta gene locus has been accomplished. Calcium mobilization could be induced by stimulation with anti-CD3 epsilon mAb as soon as intracellular TCR-beta was detectable, suggesting that a functional TCR-beta-CD3 complex is indeed expressed on the surface of early thymocytes. From day 17 on, most cells were in the DP stage, and over 95% of the DP cells expressed on the TCR-beta chain intracellularly. At day 19 of gestation, extremely low concentrations of TCR-beta chain and CD3 epsilon were detectable on the cell surface of nearly all thymocytes previously thought to be TCR-CD3 negative. These findings strongly support the hypothesis that maturation to the DP stage depends on surface expression of and subsequent signal transduction through an immature TCR-beta-CD3 complex and suggest that maturation to the DP stage by recruitment, if it occurs at all, is of minor relevance.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424104112 ◽

2015 ◽

Vol 112 (25) ◽

pp. 7773-7778 ◽

Cited By ~ 9

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.

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BCL11B is required for positive selection and survival of double-positive thymocytes

Journal of Experimental Medicine ◽

10.1084/jem.20070863 ◽

2007 ◽

Vol 204 (12) ◽

pp. 3003-3015 ◽

Cited By ~ 92

Author(s):

Diana I. Albu ◽

Dongyun Feng ◽

Debarati Bhattacharya ◽

Nancy A. Jenkins ◽

Neal G. Copeland ◽

...

Keyword(s):

Positive Selection ◽

Transcriptional Control ◽

Critical Role ◽

Cell Receptor ◽

Calcium Flux ◽

Tcr Signaling ◽

Double Positive ◽

Control Of Gene Expression ◽

Altered Expression ◽

Selection Of

Transcriptional control of gene expression in double-positive (DP) thymocytes remains poorly understood. We show that the transcription factor BCL11B plays a critical role in DP thymocytes by controlling positive selection of both CD4 and CD8 lineages. BCL11B-deficient DP thymocytes rearrange T cell receptor (TCR) α; however, they display impaired proximal TCR signaling and attenuated extracellular signal-regulated kinase phosphorylation and calcium flux, which are all required for initiation of positive selection. Further, provision of transgenic TCRs did not improve positive selection of BCL11B-deficient DP thymocytes. BCL11B-deficient DP thymocytes have altered expression of genes with a role in positive selection, TCR signaling, and other signaling pathways intersecting the TCR, which may account for the defect. BCL11B-deficient DP thymocytes also presented increased susceptibility to spontaneous apoptosis associated with high levels of cleaved caspase-3 and an altered balance of proapoptotic/prosurvival factors. This latter susceptibility was manifested even in the absence of TCR signaling and was only partially rescued by provision of the BCL2 transgene, indicating that control of DP thymocyte survival by BCL11B is nonredundant and, at least in part, independent of BCL2 prosurvival factors.

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Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon

Blood ◽

10.1182/blood-2008-11-189118 ◽

2009 ◽

Vol 114 (7) ◽

pp. 1355-1365 ◽

Cited By ~ 11

Author(s):

Shella Saint Fleur ◽

Akemi Hoshino ◽

Kimie Kondo ◽

Takeshi Egawa ◽

Hodaka Fujii

Keyword(s):

T Cells ◽

Cell Death ◽

Critical Role ◽

Cell Receptor ◽

Surface Proteins ◽

Immune Homeostasis ◽

Activated T Cells ◽

Transgenic Expression ◽

Activation Induced Cell Death ◽

Inducible Protein

Abstract Activation-induced cell death (AICD) plays an essential role in the contraction of activated T cells after eradication of pathogen. Fas (APO-1/CD95) is one of the key cell surface proteins that mediate AICD in CD4+ and CD8+ T cells. Despite its prime importance in cell death, regulation of Fas expression in T cells is poorly understood. Here we show that Cyclon, a newly identified cytokine-inducible protein, is induced in T cells on T-cell receptor ligation and important for immune homeostasis. Transgenic expression of Cyclon ameliorated autoimmune phenotype in mice lacking subunits of IL-2R. Transgenic expression of Cyclon markedly enhanced AICD through increased expression of Fas whose expression is essential for Cyclon action. Finally, we demonstrated that activated but not resting CD4+ T cells with targeted deletion of a Cyclon allele show reduced AICD and expression of Fas, indicating a critical role of Cyclon in Fas expression in activated T cells. We think that our data provide insight into expression regulation of Fas in T cells.

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Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

10.21203/rs.3.rs-1069634/v1 ◽

2021 ◽

Author(s):

Delong Feng ◽

Yanhong Chen ◽

Ranran Dai ◽

Shasha Bian ◽

Wei Xue ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Receptor Gene ◽

Cell Receptor ◽

Regulation Mechanism ◽

Thymocyte Development ◽

Double Positive ◽

Enhancer Activity ◽

Cell Identity ◽

Single Positive

Abstract CD4+ and CD8+ double-positive (DP) thymocytes are at a crucial stage during the T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRβ. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, Ets2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.

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Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism.

Journal of Experimental Medicine ◽

10.1084/jem.179.3.785 ◽

1994 ◽

Vol 179 (3) ◽

pp. 785-796 ◽

Cited By ~ 50

Author(s):

A O Hueber ◽

G Raposo ◽

M Pierres ◽

H T He

Keyword(s):

Cell Death ◽

Protein Synthesis ◽

Structural Domain ◽

Protein Synthesis Inhibitors ◽

Thymocyte Development ◽

Dot Blot ◽

Double Positive ◽

Mouse Thymocyte ◽

Major Interest ◽

Thymocyte Apoptosis

Programmed cell death plays an important role during thymocyte development, since a vast majority (97%) of mouse cortical thymocytes die in thymus, whereas only 3% of these cells are rescued from cell death and positively selected. Although it seems well established that thymocyte fate depends upon appropriate surface-expressed T cell receptor, little is known about the molecular mechanism(s) responsible for the massive thymocyte elimination that occurs in the thymus. We report here that Thy-1 is capable of triggering mouse thymocyte death in vitro through a bcl-2-resistant mechanism. We have previously shown that Thy-1 is involved in mouse thymocyte adhesion to thymic stroma through interaction with an epithelial cell ligand. To examine the Thy-1 signaling function in thymocytes, we have mimicked its interaction with stromal cells by culturing mouse thymocytes onto tissue culture plates coated with monoclonal antibodies (mAb) directed at distinct Thy-1 epitope regions. mAb recognizing determinants in a defined Thy-1 structural domain, but not others, were found to induce marked thymocyte apoptosis as evidenced by morphological and biochemical data. Use of a quantitative DNA dot blot assay indicated that Thy-1-mediated thymocyte apoptosis was not blocked by RNA or protein synthesis inhibitors, EGTA, or by cyclosporin A, and differed, therefore, from "activation-driven cell death". Moreover, Thy-1(+)-transfected, but not wild-type AKR1 (Thy-1-d) thymoma cells underwent apoptosis after ligation with apoptosis-inducing, Thy-1-specific mAb. In contrast to thymocytes, the latter event was inhibitable by RNA and protein synthesis inhibitors, an indication that thymocytes, but not thymoma cells, contain the molecular components necessary for Thy-1-driven apoptosis. We further showed that Thy-1-triggered thymocyte death is a developmentally regulated process operative in fetal thymocytes from day 17 of gestation, but not in peripheral T cells. Indeed, the target of apoptosis by anti-Thy-1 was found to reside mainly within the CD4+8+3- and CD4+8+3lo double positive immature thymocyte subsets. Finally, it is of major interest that Thy-1-mediated apoptosis, which was found to be readily detectable in thymocytes from bcl-2-transgenic mice, represents a thus far unique experimental system for studying bcl-2-resistant thymocyte death mechanism(s).

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