Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation

The first checkpoint during T cell development, known as beta selection, requires the successful rearrangement of the TCR-beta gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during beta selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4(-)CD8(-) thymocytes to the CD4(+)CD8(+) stage. Furthermore, we address the minimal signaling requirements underlying beta selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Notch signalling in B cells

Notch signalling is likely to regulate multiple aspects of lymphoid development and function. During T cell development, Notch signalling is required for commitment of the earliest progenitor, and may also function during other developmental stages. T cell commitment from a common lymphoid progenitor occurs at the expense of B cell development, suggesting that Notch signalling inhibits the earliest stage of B lymphopoiesis. In contrast, recent evidence suggests that Notch promotes the development of marginal zone lymphocytes. Not only is Notch required for later stages of B cell development, but several viral proteins appear to utilize Notch signalling in B cells to mediate their functions. In this review, we will focus on potential roles of Notch signalling in B lymphopoiesis and also consider how viral proteins may utilize Notch signalling in B cells.     

Active form of Notch members can enforce T lymphopoiesis on lymphoid progenitors in the monolayer culture specific for B cell development

The in vitro induction of T lymphopoiesis needs the precise stereoscopic structure of thymus tissues as seen in fetal thymus organ culture. In this study, we demonstrated for the first time that the introduction of the intracellular region of Notch1 can induce T cells expressing TCR without any thymic environment. In the coculture on the monolayer of OP-9, which was originally known to support B cell specific development, hemopoietic progenitors developed into Thy-1(+)CD25(+) T lineage cells if the progenitor cells were infected with the retrovirus containing Notch1 intracellular domains. The Thy-1(+) cells progressed to a further developmental stage, CD4 and CD8 double-positive cells expressing TCR on the cell surface, if they were further cultured on OP-9 or in the thymus. However, T cell induction by intracellular Notch1 failed unless both OP-9 and IL-7 were present. It is notable that Notch2 and Notch3 showed an effect on T lymphopoiesis similar to that of Notch1. These results indicate that in vitro T lymphopoiesis is inducible by signaling via Notch family members in a lineage-specific manner but shares other stroma-derived factors including IL-7 with B lymphopoiesis.     

Phosphorylation of Rictor at Thr1135 impairs the Rictor/Cullin-1 complex to ubiquitinate SGK1

The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism, cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase. However, its upstream regulatory pathways as well as whether it has additional function(s) remain largely unknown. We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1, but not with other Cullin family members. Furthermore, we identified SGK1 as its downstream target. Interestingly, Rictor, but not Raptor or mTOR, promotes SGK1 ubiquitination. As a result, SGK1 expression is elevated in Rictor^--/-- MEFs. We further defined that as a feedback mechanism, Rictor can be phosphorylated by multiple AGC family kinases including Akt, S6K and SGK1. Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1. On the other hand, it disrupted the interaction between Rictor and Cullin-1. Consequently, T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction. This finding further expands our knowledge of Rictor’s function. Furthermore, our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades, and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.     

A reassessment of the effect of activated Notch1 on CD4 and CD8 T cell development

The Notch signaling pathway plays an important role in the early steps of T cell development and in the generation of T cell tumors, but its role in the CD4 vs CD8 lineage decision is controversial. Notch1 is not essential for CD4 or CD8 T cell development; however, there are suggestions that multiple Notch family members may act in a redundant fashion during thymic development. In theory, expressing a constitutively activated form of Notch in CD4(+)CD8(+) thymocytes could provide clues about the normal role of Notch in developing CD4 and CD8 T cells. Unfortunately, two different studies of transgenic mice expressing activated forms of Notch1 (Notch1IC) led to conflicting conclusions. In this study, we re-examine the effect of the two Notch1IC transgenes on thymocyte development. We find that both Notch1IC transgenic lines display a decrease in CD4 single positive (SP) thymocytes and a corresponding increase in CD8 SP thymocytes. The enhanced development of CD8 SP thymocytes is dependent on either class I or II MHC. Thus, data from two different Notch1IC transgenic lines indicate that Notch activity promotes CD8 and inhibits CD4 SP development. We suggest that the discrepancies in previous reports of Notch1IC transgenic mice are due to differences in the propensity of the two different transgenic lines to develop tumors.     

Pontin is required for pre-TCR signaling at the β-selection checkpoint in T cell development

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Based on high expression in lymphoid tissues, we examined whether Pontin has a T cell-specific function. We generated Pontin^f/f;Lck-Cre mice, in which Pontin can be conditionally deleted in T cells and then explored T cell-specific function of Pontin in vivo. Here, we show that specific abrogation of Pontin expression in T cells almost completely blocked development of αβ T cells at the β-selection checkpoint by inducing cell apoptosis indicating that Pontin is essential for early T cell development. Pontin-deficient thymocytes show a comparable expression level of T cell receptor (TCR)β chain, but have enhanced activation of p53 and Notch signaling compared to wild-type thymocytes. Intriguingly, the developmental block of αβ T cells can be partially rescued by loss of p53. Together, our data demonstrate a novel role of Pontin as a crucial regulator in pre-TCR signaling during T cell development.     

Impairment of thymocyte development by dominant-negative Kuzbanian (ADAM-10) is rescued by the Notch ligand, delta-1

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.     

mTOR Complex 1 Plays Critical Roles in Hematopoiesis and Pten-Loss-Evoked Leukemogenesis

The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state?and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.     

Lunatic fringe controls T cell differentiation through modulating notch signaling

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4-CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4-CD8- (double-negative (DN)) and mature CD4+CD8- (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25- stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.     

Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development

The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.     

Surface expression of Notch1 on thymocytes: correlation with the double-negative to double-positive transition

Notch1 plays a critical role in regulating T lineage commitment during the differentiation of lymphoid precursors. The physiological relevance of Notch1 signaling during subsequent stages of T cell differentiation has been more controversial. This is due in part to conflicting data from studies examining the overexpression or targeted deletion of Notch1 and to difficulties in distinguishing between the activities of multiple Notch family members and their ligands, which are expressed in the thymus. We employed a polyclonal antiserum against the extracellular domain of Notch1 to study surface expression during thymopoiesis. We found high levels of Notch1 on the cell surface only on double negative (DN) stage 2 through the immature single-positive stage of thymocyte development, before the double-positive (DP) stage. The Notch signaling pathway, as read out by Deltex1 expression levels, is highly active in DN thymocytes. When an active Notch1 transgene, Notch1IC, is exogenously introduced into thymocytes of recombinase-activating gene 2-deficient mice, it promotes proliferation and development to the DP stage following anti-CD3 treatment without apparently affecting the intensity of pre-TCR signaling. In addition, a stromal cell line expressing the Notch ligand, Delta-like-1, promotes the in vitro expansion of wild-type DN3 thymocytes in vitro. Consistent with other recent reports, these data suggest a role for Notch1 during the DN to DP stage of thymocyte maturation and suggest a cellular mechanism by which Notch1IC oncogenes could contribute to thymoma development and maintenance.     

Conditional Fas-associated death domain protein (FADD): GFP knockout mice reveal FADD is dispensable in thymic development but essential in peripheral T cell homeostasis

Fas-associated death domain protein (FADD)/mediator of receptor-induced toxicity-1 is required for signaling induced by death receptors such as Fas. In earlier studies, FADD-deficient mice died in utero, and a FADD deficiency in embryonic stem cells inhibited T cell production in viable FADD-/- -->RAG-1-/- chimeras. To analyze the temporal requirement of FADD in the development and function in the T lineage, it is necessary to establish viable mutant mice producing detectable FADD-deficient T cells. We generated mice that express a functional FADD:GFP fusion gene reconstituting normal embryogenesis and lymphopoiesis in the absence of the endogenous FADD. Efficient T cell-specific deletion of FADD:GFP was achieved, as indicated by the presence of a high percentage of GFP-negative thymocytes and peripheral T cells in mice expressing Lck-Cre or CD4-Cre. Sorted GFP-negative thymocytes and peripheral T cells contained undetectable levels of FADD and were resistant to apoptosis induced by Fas, TNF, and TCR restimulation. These T cell-specific FADD-deficient mice contain normal thymocyte numbers, but fewer peripheral T cells. Purified peripheral FADD-deficient T cells failed to undergo extensive homeostatic expansion after adoptive transfer into lymphocyte-deficient hosts, and responded poorly to proliferation induced by ex vivo TCR stimulation. Furthermore, deletion of FADD in preactivated mature T cells using retrovirus-Cre resulted in no proliferation. These results demonstrate that FADD plays a dispensable role during thymocyte development, but is essential in maintaining peripheral T cell homeostasis and regulating both apoptotic and proliferation signals.     

Increased Levels of Rictor Prevent Mutant Huntingtin-Induced Neuronal Degeneration

Rictor associates with mTOR to form the mTORC2 complex, which activity regulates neuronal function and survival. Neurodegenerative diseases are characterized by the presence of neuronal dysfunction and cell death in specific brain regions such as for example Huntington’s disease (HD), which is characterized by the loss of striatal projection neurons leading to motor dysfunction. Although HD is caused by the expression of mutant huntingtin, cell death occurs gradually suggesting that neurons have the capability to activate compensatory mechanisms to deal with neuronal dysfunction and later cell death. Here, we analyzed whether mTORC2 activity could be altered by the presence of mutant huntingtin. We observed that Rictor levels are specifically increased in the striatum of HD mouse models and in the putamen of HD patients. Rictor-mTOR interaction and the phosphorylation levels of Akt, one of the targets of the mTORC2 complex, were increased in the striatum of the R6/1 mouse model of HD suggesting increased mTORC2 signaling. Interestingly, acute downregulation of Rictor in striatal cells in vitro reduced mTORC2 activity, as shown by reduced levels of phospho-Akt, and increased mutant huntingtin-induced cell death. Accordingly, overexpression of Rictor increased mTORC2 activity counteracting cell death. Furthermore, normalization of endogenous Rictor levels in the striatum of R6/1 mouse worsened motor symptoms suggesting an induction of neuronal dysfunction. In conclusion, our results suggest that increased Rictor striatal levels could counteract neuronal dysfunction induced by mutant huntingtin.     

The Notch regulator Numb links the Notch and TCR signaling pathways

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.     

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.