Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation

Abstract We show that Indian Hedgehog (Ihh) regulates T-cell development and homeostasis in both fetal and adult thymus, controlling thymocyte number. Fetal Ihh−/− thymi had reduced differentiation to double-positive (DP) cell and reduced cell numbers compared with wild-type littermates. Surprisingly, fetal Ihh+/− thymi had increased thymocyte numbers and proportion of DP cells relative to wild type, indicating that Ihh also negatively regulates thymocyte development. In vitro treatment of thymus explants with exogenous recombinant Hedgehog protein promoted thymocyte development in Ihh−/− thymi but inhibited thymocyte development in Ihh+/−, confirming both positive and negative regulatory functions of Ihh. Analysis of Rag−/−Ihh+/− thymi showed that Ihh promotes T-cell development before pre–T-cell receptor (pre-TCR) signaling, but negatively regulates T-cell development only after pre-TCR signaling has taken place. We show that Ihh is most highly expressed by the DP population and that Ihh produced by DP cells feeds back to negatively regulate the differentiation and proliferation of their double-negative progenitors. Thus, differentiation from double-negative to DP cell, and hence the size of the DP population, is dependent on the concentration of Ihh in the thymus. Analysis of Ihh conditional knockout and heterozygote adult mice showed that Ihh also influences thymocyte number in the adult.

Download Full-text

Overlapping functions of human CD3δ and mouse CD3γ in αβ T-cell development revealed in a humanized CD3γ-deficient mouse

Blood ◽

10.1182/blood-2006-03-010850 ◽

2006 ◽

Vol 108 (10) ◽

pp. 3420-3427 ◽

Cited By ~ 14

Author(s):

Edgar Fernández-Malavé ◽

Ninghai Wang ◽

Manuel Pulgar ◽

Wolfgang W. A. Schamel ◽

Balbino Alarcón ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Development ◽

Cell Development ◽

Double Negative ◽

Wild Type ◽

Thymocyte Development ◽

Double Positive ◽

Deficient Mice ◽

Αβ T Cells

Abstract Humans lacking the CD3γ subunit of the pre-TCR and TCR complexes exhibit a mild αβ T lymphopenia, but have normal T cells. By contrast, CD3γ-deficient mice are almost devoid of mature αβ T cells due to an early block of intrathymic development at the CD4–CD8– double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3δ chain replaces CD3γ during αβ T-cell development. To determine whether human CD3δ (hCD3δ) functions in a similar manner in the mouse in the absence of CD3γ, we introduced an hCD3δ transgene in mice that were deficient for both CD3δ and CD3γ, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3δ efficiently supported pre-TCR–mediated progression from the DN to the CD4+CD8+ double-positive (DP) stage. However, αβTCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3γ-deficient TCR complexes that had incorporated hCD3δ displayed abnormalities in structural stability resembling those of T cells from CD3γ-deficient humans. Taken together, these data demonstrate that CD3δ and CD3γ play a different role in humans and mice in pre-TCR and TCR function during αβ T-cell development.

Download Full-text

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.

Download Full-text

BMP4 acts upstream of FGF in modulating thymic stroma and regulating thymopoiesis

Blood ◽

10.1182/blood-2003-05-1657 ◽

2003 ◽

Vol 102 (12) ◽

pp. 3947-3953 ◽

Cited By ~ 81

Author(s):

Peter T. Tsai ◽

Robert A. Lee ◽

Hong Wu

Keyword(s):

T Cell ◽

Bone Morphogenetic Proteins ◽

T Cell Development ◽

Cell Development ◽

Dependent Manner ◽

Double Negative ◽

Thymocyte Development ◽

Double Positive ◽

Stromal Compartment ◽

Thymic Stroma

Abstract Thymocyte development is a non–cell-autonomous process that requires signals provided by the thymic stroma. Bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) derived from thymic stroma have been implicated as possible regulators of T-cell development. Using thymic organ culture, this study demonstrates that both BMP4 and FGF7/FGF10 arrest early T-cell development at the CD4-CD8-CD44+CD25- (double-negative 1 [DN1]) population and at the CD4-CD8- double-negative (DN) to CD4+CD8+ double-positive (DP) transition in a stromal compartment–dependent manner. Furthermore, BMP4 functions upstream of FGF7/FGF10, as the effects of BMP can be suppressed by cotreatment with an FGF receptor antagonist. BMP4 also acts directly on the thymic stroma to up-regulate the stroma-specific transcription factor Foxn1 and stroma-expressed chemokines. Taken together, the data in this report demonstrate that BMP acts upstream of FGF in the regulation of early T-cell development and that BMP4 acts primarily through the thymic stroma, thereby altering the thymic microenvironment and affecting thymopoiesis.

Download Full-text

Histone Deacetylase 3 Is Required for Efficient T Cell Development

Molecular and Cellular Biology ◽

10.1128/mcb.00706-15 ◽

2015 ◽

Vol 35 (22) ◽

pp. 3854-3865 ◽

Cited By ~ 27

Author(s):

Kristy R. Stengel ◽

Yue Zhao ◽

Nicholas J. Klus ◽

Jonathan F. Kaiser ◽

Laura E. Gordy ◽

...

Keyword(s):

T Cell ◽

T Cell Development ◽

Hdac Inhibitors ◽

Cell Receptor ◽

Cell Development ◽

Open Chromatin ◽

Double Negative ◽

Thymocyte Development ◽

Double Positive ◽

Single Positive

Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deletedHdac3using theLck-Cretransgene. This strategy inactivatedHdac3in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4+or CD8+single-positive cells being produced. WhenHdac3−/−mice were crossed withBcl-xL-,Bcl2-, orTCRβ-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αβ transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection.

Download Full-text

CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1707 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1707-1718 ◽

Cited By ~ 269

Author(s):

K F Byth ◽

L A Conroy ◽

S Howlett ◽

A J Smith ◽

J May ◽

...

Keyword(s):

Signal Transduction ◽

T Cell ◽

B Cells ◽

Transgenic Mice ◽

B Cell ◽

T Cell Development ◽

Cell Development ◽

Cross Linking ◽

Thymocyte Development ◽

Double Positive

The CD45 transmembrane glycoprotein has been shown to be a protein phosphotyrosine phosphatase and to be important in signal transduction in T and B lymphocytes. We have employed gene targeting to create a strain of transgenic mice that completely lacks expression of all isoforms of CD45. The spleens from CD45-null mice contain approximately twice the number of B cells and one fifth the number of T cells found in normal controls. The increase in B cell numbers is due to the specific expansion of two B cell subpopulations that express high levels of immunoglobulin (IgM) staining. T cell development is significantly inhibited in CD45-null animals at two distinct stages. The efficiency of the development of CD4-CD8- thymocytes into CD4+ CD8+ thymocytes is reduced by twofold, subsequently the frequency of successful maturation of the double positive population into mature, single positive thymocytes is reduced by a further four- to fivefold. In addition, we demonstrate that CD45-null thymocytes are severely impaired in their apoptotic response to cross-linking signals via T cell receptor (TCR) in fetal thymic organ culture. In contrast, apoptosis can be induced normally in CD45-null thymocytes by non-TCR-mediated signals. Since both positive and negative selection require signals through the TCR complex, these findings suggest that CD45 is an important regulator of signal transduction via the TCR complex at multiple stages of T cell development. CD45 is absolutely required for the transmission of mitogenic signals via IgM and IgD. By contrast, CD45-null B cells proliferate as well as wild-type cells to CD40-mediated signals. The proliferation of B cells in response to CD38 cross-linking is significantly reduced but not abolished by the CD45-null mutation. We conclude that CD45 is not required at any stage during the generation of mature peripheral B cells, however its loss reveals a previously unrecognized role for CD45 in the regulation of certain subpopulations of B cells.

Download Full-text

Altered thymocyte and T cell development in neonatal mice with hyperoxia-induced lung injury

Journal of Perinatal Medicine ◽

10.1515/jpm-2016-0234 ◽

2018 ◽

Vol 46 (4) ◽

pp. 441-449

Author(s):

Sowmya Angusamy ◽

Tamer Mansour ◽

Mohammed Abdulmageed ◽

Rachel Han ◽

Brian C. Schutte ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lung Injury ◽

T Cell Development ◽

Adaptive Immune System ◽

Cell Development ◽

Thymocyte Development ◽

Double Positive ◽

Neonatal Mice ◽

Single Positive

Abstract Background: The adaptive immune system of neonates is relatively underdeveloped. The thymus is an essential organ for adaptive T cell development and might be affected during the natural course of oxygen induced lung injury. The effect of prolonged hyperoxia on the thymus, thymocyte and T cell development, and its proliferation has not been studied extensively. Methods: Neonatal mice were exposed to 85% oxygen (hyperoxia) or room air (normoxia) up to 28 days. Flow cytometry using surface markers were used to assay for thymocyte development and proliferation. Results: Mice exposed to prolonged hyperoxia had evidence of lung injury associated alveolar simplification, a significantly lower mean weight, smaller thymic size, lower mean thymocyte count and higher percentage of apoptotic thymocytes. T cells subpopulation in the thymus showed a significant reduction in the count and proliferation of double positive and double negative T cells. There was a significant reduction in the count and proliferation of single positive CD4+ and CD8+ T cells. Conclusions: Prolonged hyperoxia in neonatal mice adversely affected thymic size, thymocyte count and altered the distribution of T cells sub-populations. These results are consistent with the hypothesis that prolonged hyperoxia causes defective development of T cells in the thymus.

Download Full-text

Conditional Knockout Mice Reveal Distinct Functions for the Global Transcriptional Coactivators CBP and p300 in T-Cell Development

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.789-809.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 789-809 ◽

Cited By ~ 136

Author(s):

Lawryn H. Kasper ◽

Tomofusa Fukuyama ◽

Michelle A. Biesen ◽

Fayçal Boussouar ◽

Caili Tong ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Cell Development ◽

Cell Development ◽

Conditional Knockout ◽

Specific Cell ◽

Creb Binding Protein ◽

Thymocyte Development ◽

Transcriptional Coactivators

ABSTRACT The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300 flox ) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300 flox and a CBP conditional knockout allele (CBP flox ) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4− CD8− double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.

Download Full-text

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.

Download Full-text

The Role of CBFβ in T Cell Development.

Blood ◽

10.1182/blood.v104.11.3234.3234 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3234-3234

Author(s):

Ling Zhao ◽

Jennifer L. Cannons ◽

Lucio H. Castilla ◽

Pamela L. Schwartzberg ◽

Pu Paul Liu

Keyword(s):

T Cell ◽

Fusion Gene ◽

Ganglion Cells ◽

T Cell Development ◽

Cell Development ◽

Dominant Negative ◽

Mutant Mice ◽

Lower Percentage ◽

Double Negative ◽

Double Positive

Abstract Core binding factor β (Cbfβ) is a transcription factor that heterodimerizes with Runx (Cbfα) family members, thereby stabilizing the interaction between the Runx proteins and DNA. Genetically manipulated mouse models of Runx and Cbfb genes have demonstrated their critical functions in hematopoiesis (Runx1, Runx3 and Cbfb), bone formation (Runx2, Cbfb), proliferation of gastrointestinal epithelia (Runx3) and differentiation of dorsal root ganglion cells (Runx3). Studies on T cell development showed that Runx1 and Runx3 repress CD4 expression at different stages of development. In addition, Runx 1 and Runx 3 are required for CD8 T cell development during thymopoiesis. No defects were found when Runx2 was inactivated, even though it is expressed throughout T cell development. We have previously generated a knock-in mouse model expressing the Cbfb-MYH11 fusion gene (which is created by inv(16)(p13; q22) in human AML M4Eo). Heterozygous knock-in mice had a phenotype identical to that of the Cbfb and Runx1 null mice (embryonic lethality), suggesting that the fusion gene Cbfb-MYH11 functions in a dominant-negative manner. In order to study the function of Cbfb gene in T cell development, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5′ to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. The LckCre-loxKI mice were viable. However, their thymic development was severely impaired: The size of the thymuses in the mutant mice was about half the normal size, and the total number of thymocytes in the mutant mice was 10–20-fold reduced. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockade at the CD4/CD8-double negative (DN) stage, which was characterized by lower percentage of double positive cells and higher percentage of double negative cells. In addition, the CD4: CD8 ratio was altered. Furthermore, the mature T cell population size in the spleen of the mutant mice was lower than that of the control mice. Our preliminary data suggested that Cbfb plays an important role in T cell development. The mechanism through which Cbfb affects the T cell development is currently under investigation. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone.

Download Full-text