Essential role of Rap signal in pre-TCR–mediated β-selection checkpoint in αβ T-cell development

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4565-4573 ◽  
Author(s):  
Kohei Kometani ◽  
Masaki Moriyama ◽  
Yasuhiro Nakashima ◽  
Yoshinori Katayama ◽  
Shu-Fang Wang ◽  
...  
Keyword(s):  
T Cell ◽  
Essential Role ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Double Positive ◽  
Conditional Expression

Abstract We demonstrate that lck promoter–driven conditional expression of transgenic SPA-1, a Rap GTPase-activation protein, causes a profound defect of αβ T-cell development at the CD4/CD8 double-negative (DN) stage due to enhanced cell death without affecting γδ T-cell development. The effect was specific to the DN stage, because CD4 promoter–driven SPA-1 expression hardly affected T-cell development. Rap1A17, a dominant-negative Rap mutant, interfered with the generation of double-positive (DP) cells from Rag2−/− fetal thymocytes in vitro in the presence of anti-CD3ϵ antibody and Notch ligand. Rap GTPases were activated in a DN cell line by the expression of self-oligomerizing CD3 (CD8:CD3ϵ chimera), which substituted autonomous pre–T-cell receptor (TCR) signal, inducing CD69 expression and CD25 down-regulation. Reciprocally, expression of C3G, a Rap guanine nucleotide exchange factor, in both normal and Rag2−/− DN cells markedly enhanced Notch-dependent generation and expansion of DP cells without additional anti-CD3ϵ antibody, thus bypassing pre-TCR. Defective αβ T-cell development in the conditional SPA-1–transgenic mice was restored completely by introducing a p53−/− mutation. These results suggest that endogenous Rap GTPases downstream of pre-TCR play an essential role in rescuing pre-T cells from the p53-mediated checkpoint response, thus allowing Notch-mediated expansion and differentiation.

Loss of Function of the Homeobox Gene Hoxa-9 Perturbs Early T-Cell Development and Induces Apoptosis in Primitive Thymocytes

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 383-393 ◽  
Author(s):  
David J. Izon ◽  
Sofia Rozenfeld ◽  
Stephen T. Fong ◽  
László Kömüves ◽  
Corey Largman ◽  
...  
Keyword(s):  
T Cell ◽  
Hox Genes ◽  
Apoptotic Cell ◽  
T Cell Development ◽  
Interleukin 2 ◽  
Homeobox Gene ◽  
Cell Receptor ◽  
Cell Development ◽  
Loss Of Function ◽  
Double Positive

Abstract Hox homeobox genes play a crucial role in specifying the embryonic body pattern. However, a role for Hox genes in T-cell development has not been explored. The Hoxa-9 gene is expressed in normal adult and fetal thymuses. Fetal thymuses of mice homozygous for an interruption of the Hoxa-9 gene are one eighth normal size and have a 25-fold decrease in the number of primitive thymocytes expressing the interleukin-2 receptor (IL-2R, CD25). Progression to the double positive (CD4+CD8+) stage is dramatically retarded in fetal thymic organ cultures. This aberrant development is associated with decreased amounts of intracellular CD3 and T-cell receptor β (TCRβ) and reduced surface expression of IL-7R and E-cadherin. Mutant thymocytes show a significant increase in apoptotic cell death and premature downregulation of bcl-2 expression. A similar phenotype is seen in primitive thymocytes from adult Hoxa-9−/− mice and from mice transplanted with Hoxa-9−/−marrow. Hoxa-9 appears to play a previously unsuspected role in T-cell ontogeny by modulating cell survival of early thymocytes and by regulating their subsequent differentiation.

scholarly journals Functions of E2A-HEB Heterodimers in T-Cell Development Revealed by a Dominant Negative Mutation of HEB

2000 ◽  
Vol 20 (18) ◽  
pp. 6677-6685 ◽  
Author(s):  
Robert J. Barndt ◽  
Meifang Dai ◽  
Yuan Zhuang
Keyword(s):  
T Cell ◽  
Target Genes ◽  
T Cell Development ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Cell Development ◽  
Dominant Negative ◽  
Helix Loop Helix ◽  
Dominant Negative Mutation ◽  
Bhlh Proteins

ABSTRACT Lymphocyte development and differentiation are regulated by the basic helix-loop-helix (bHLH) transcription factors encoded by theE2A and HEB genes. These bHLH proteins bind to E-box enhancers in the form of homodimers or heterodimers and, consequently, activate transcription of the target genes. E2A homodimers are the predominant bHLH proteins present in B-lineage cells and are shown genetically to play critical roles in B-cell development. E2A-HEB heterodimers, the major bHLH dimers found in thymocyte extracts, are thought to play a similar role in T-cell development. However, disruption of either the E2A or HEBgene led to only partial blocks in T-cell development. The exact role of E2A-HEB heterodimers and possibly the E2A and HEB homodimers in T-cell development cannot be distinguished in simple disruption analysis due to a functional compensation from the residual bHLH homodimers. To further define the function of E2A-HEB heterodimers, we generated and analyzed a dominant negative allele of HEB, which produces a physiological amount of HEB proteins capable of forming nonfunctional heterodimers with E2A proteins. Mice carrying this mutation show a stronger and earlier block in T-cell development than HEB complete knockout mice. The developmental block is specific to the α/β T-cell lineage at a stage before the completion of V(D)J recombination at the TCRβ gene locus. This defect is intrinsic to the T-cell lineage and cannot be rescued by expression of a functional T-cell receptor transgene. These results indicate that E2A-HEB heterodimers play obligatory roles both before and after TCRβ gene rearrangement during the α/β lineage T-cell development.

scholarly journals VßGene Family Usage in Spontaneous Lymphomas of AKR Mice: Evidence for Defective Clonal Deletion

1992 ◽  
Vol 2 (2) ◽  
pp. 95-101 ◽  
Author(s):  
Cees de Heer ◽  
Bernard de Geus ◽  
Henk-Jan Schuurma ◽  
Henk Van Loveren ◽  
Jan Rozing
Keyword(s):  
T Cell ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Mesenteric Lymph Nodes ◽  
Double Positive ◽  
Clonal Deletion ◽  
Lymphoma Cells ◽  
Single Positive ◽  
Akr Mice

T-cell receptor (TCR)ß-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4+8+) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR Vßgene family usage, 2 out of 14 lymphomas expressed Vß6. Normally, Vß6+thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of Vß6+lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.

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 Separation of Notch1 Promoted Lineage Commitment and Expansion/Transformation in Developing T Cells

2001 ◽  
Vol 194 (1) ◽  
pp. 99-106 ◽  
Author(s):  
David Allman ◽  
Fredrick G. Karnell ◽  
Jennifer A. Punt ◽  
Sonia Bakkour ◽  
Lanwei Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Receptor ◽  
Cell Development ◽  
Surface Proteins ◽  
Lineage Commitment ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4+CD8+ double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2−/−) or Src homology 2 domain–containing leukocyte protein of 76 kD (SLP-76)−/− mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2−/− progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3ε and pre-Tα mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2−/− mice with a TCRβ transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell–specific signals associated with development of DP thymocytes.

Loss of Function of the Homeobox Gene Hoxa-9 Perturbs Early T-Cell Development and Induces Apoptosis in Primitive Thymocytes

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 383-393 ◽  
Author(s):  
David J. Izon ◽  
Sofia Rozenfeld ◽  
Stephen T. Fong ◽  
László Kömüves ◽  
Corey Largman ◽  
...  
Keyword(s):  
T Cell ◽  
Hox Genes ◽  
Apoptotic Cell ◽  
T Cell Development ◽  
Interleukin 2 ◽  
Homeobox Gene ◽  
Cell Receptor ◽  
Cell Development ◽  
Loss Of Function ◽  
Double Positive

Hox homeobox genes play a crucial role in specifying the embryonic body pattern. However, a role for Hox genes in T-cell development has not been explored. The Hoxa-9 gene is expressed in normal adult and fetal thymuses. Fetal thymuses of mice homozygous for an interruption of the Hoxa-9 gene are one eighth normal size and have a 25-fold decrease in the number of primitive thymocytes expressing the interleukin-2 receptor (IL-2R, CD25). Progression to the double positive (CD4+CD8+) stage is dramatically retarded in fetal thymic organ cultures. This aberrant development is associated with decreased amounts of intracellular CD3 and T-cell receptor β (TCRβ) and reduced surface expression of IL-7R and E-cadherin. Mutant thymocytes show a significant increase in apoptotic cell death and premature downregulation of bcl-2 expression. A similar phenotype is seen in primitive thymocytes from adult Hoxa-9−/− mice and from mice transplanted with Hoxa-9−/−marrow. Hoxa-9 appears to play a previously unsuspected role in T-cell ontogeny by modulating cell survival of early thymocytes and by regulating their subsequent differentiation.

T-cell developmental blockage by tachykinin antagonists and the role of hemokinin 1 in T lymphopoiesis

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2165-2172 ◽  
Author(s):  
Yu Zhang ◽  
Christopher J. Paige
Keyword(s):  
T Cell ◽  
B Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Double Positive ◽  
Peptide Family ◽  
Tachykinin Antagonists ◽  
B Cell Precursors

Abstract Hemokinin 1 (HK-1) is a new member of the tachykinin peptide family that is expressed in hematopoietic cells. Recent reports studying mouse, rat, and human orthologs of HK-1 demonstrate a broader distribution than originally reported. Our previous studies demonstrated that HK-1, by promoting proliferation, survival, and possibly maturation of B-cell precursors, plays an important role in B lymphopoiesis. Here we present data showing that HK-1 also influences T-cell development at a similar stage of differentiation. This peptide enhanced the proliferation of T-cell precursors and increased the number of thymocytes in fetal thymus organ cultures (FTOCs). Tachykinin antagonists, on the other hand, greatly reduced the cellularity of thymi both in vivo and in vitro. The major reduction occurred in the CD4/CD8 double-positive (DP) cells and the CD44–CD25+ subset of the CD4/CD8 double-negative (DN) cells. Of note, these populations also express HK-1, raising the possibility of autocrine or paracrine pathways influencing T-cell development as we previously reported for B-cell development. Consistent with this, the detrimental effect of tachykinin antagonists could be partially overcome with exogenous HK-1 peptide.

The Role of CBFβ in T Cell Development.

Blood ◽  
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.

CBFB Is Required for Early T Cell Development in the Thymus.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 648-648
Author(s):  
Ling Zhao ◽  
Jennifer L. Cannons ◽  
Stacie Anderson ◽  
Martha R. Kirby ◽  
Liping Xu ◽  
...  
Keyword(s):  
T Cell ◽  
Bone Formation ◽  
Fusion Gene ◽  
T Cell Development ◽  
Cell Lineage ◽  
Cell Development ◽  
Dominant Negative ◽  
Compound Heterozygous ◽  
Thymocyte Development

Abstract Runx1 and Runx3 play important roles in early T cell development. Runx1 is required in the development of double negative cells. Runx1 is also required to repress CD4 expression in DN cells while Runx 3 is essential for epigenetic silencing of CD4 expression in CD8 cells. Both Runx1 and Runx3 are required for CD8 cell development. Because Cbfβ heterodimerizes with both Runx1 and Runx3, we hypothesized that Cbfb is also important in T cell development. To address this issue we analyzed transgenic mouse models with three Cbfb alleles. The first is a null allele for Cbfb and embryos homozygous for this allele die in midgestation due to failure of definitive hematopoiesis and hemorrhage. The second one is a GFP knockin. This Cbfb-GFP allele is a hypomorphic one in that the fusion protein Cbfβ-GFP produced from the allele behaves similarly as the wildtype Cbfβ protein but RNA and protein production from the allele is lower than that of the wildtype allele. Interestingly, AGM hematopoiesis is relatively normal and there is no hemorrhage in the CbfbGFP/GFP embryos, which die at birth due to bone formation defects. The third model is our knock-in mouse model expressing Cbfb-MYH11, the fusion gene found in human AML MeEo with inv (16)(p13; q22). Heterozygous knock-in mice had a phenotype identical to that of the Cbfb and Runx1 null mice, suggesting that the fusion gene Cbfb-MYH11 functions in a dominant-negative manner. We conditionally expressed the Cbfb-MYH11 fusion gene in T cells by using Cre-lox recombination with a floxed Cbfb-MYH11 allele and a Lck-Cre transgene, which starts to express the Cre enzyme at the DN2 stage. By analyzing embryos compound-heterozygous for the null and the hypomorphic GFP knockin alleles (Cbfb−/GFP), we found that CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb−/GFP embryos, which also had much smaller thymi with reduced cellularity compared to their litter mate controls. Further studies on cell proliferation and apoptosis indicated that increased cell death might account for the reduced cellularity. The compound heterozygous Cbfb−/GFP mice died at birth with severe bone formation defects. The Tg(Lck-Cre)/conditional Cbfb-MYH11 mice were viable. In adult thymus, Cbfb-MYH11 expression led to a 10-fold reduction in thymocyte numbers, resulting from both impaired survival of CD4+CD8+ thymocytes (similar as in Cbfb−/GFP embryos) and a differentiation block at DN3 stage. The reduced cellularity could be rescued by over expression of Bcl2 through crossing with Tg(Lck-hBcl2) mice. Cbfb-MYH11 did not derepress CD4 expression in the thymus even though it did so in reporter assays in vitro, which could be due to incomplete Cre-lox reaction, or that Cbfb-MYH11 acts more than just a pure dominant negative. Our data suggest that Cbfβ is critical for several stages of T cell development and may help to explain why CBFB-MYH11+ cells cannot be detected in the T cell lineage in AML patients with this fusion gene.

scholarly journals Delta-like 4 is indispensable in thymic environment specific for T cell development

2008 ◽  
Vol 205 (11) ◽  
pp. 2507-2513 ◽  
Author(s):  
Katsuto Hozumi ◽  
Carolina Mailhos ◽  
Naoko Negishi ◽  
Ken-ichi Hirano ◽  
Takashi Yahata ◽  
...  
Keyword(s):  
T Cell ◽  
Notch Signaling ◽  
Cell Fate ◽  
T Cell Development ◽  
Cell Development ◽  
Thymic Epithelial Cells ◽  
Hematopoietic Progenitors ◽  
Double Positive

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

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