pre-TCR Signaling Research Articles

The roles of cyclin‐dependent kinase 11 (CDK11) during mammalian development

CDK11 gene encodes two proteins, p110 and p58. CDK11p110 proteins are part of large-molecular-weight complexes containing RNA polymerase II, transcriptional elongation and general pre-mRNA splicing factors, suggesting they couple transcription and pre-mRNA splicing. CDK11p58 is expressed only during G2/M from an internal ribosome entry site (IRES) present in the mRNA encoding CDK11p110 and appears to regulate mitosis. To examine the in vivo role of CDK11p110/p58 kinases, we generated CDK11p110/p58-null mice and found CDK11p110/p58deficiency results in early embryonic lethality and apoptosis between E3.5 and E4.0. Cells within CDK11p110/p58−/− embryos exhibit both proliferative defects and a mitotic arrest. These results indicated that the CDK11p110/p58kinases are essential for cellular viability and normal early embryonic development. CDK11p110/p58 T cell-specific knockout mice generated by using Cre/loxP system show defects in T cell development. Deletion of CDK11p110/p58 in thymocytes results in a block at the double negative (DN) stage due to the defective pre-TCR signaling, impaired proliferation and apoptosis. CDK11p110/p58 deficiency in thymocytes also affects alternative splicing of CD45. Taken together, these results indicate that CDK11 protein kinases play important roles in transcription, splicing and mitotic regulation. (Supported by NIH grants GM44088, CA0211765, and ALSAC)

Regulation of T‐cell progenitor survival and cell‐cycle entry by the pre‐T‐cell receptor

Pre-T-cell receptor (pre-TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. Although multiple reports have addressed several aspects of pre-TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR-beta locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T-cell leukemia and lymphomas. In this review, we integrate pre-TCR signaling pathways and study their effects on the regulation of T-cell progenitor cell-cycle entry and cell survival. We also connect aberrant pre-TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.

Critical Function for SIP, a Ubiquitin E3 Ligase Component of the β-Catenin Degradation Pathway, for Thymocyte Development and G1 Checkpoint

Beta-catenin has been implicated in thymocyte development because of its function as a coactivator of Tcf/LEF-family transcription factors. Previously, we discovered a novel pathway for p53-induced beta-catenin degradation through a ubiquitin E3 ligase complex involving Siah1, SIP (CacyBP), Skp1, and Ebi. To gain insights into the physiological relevance of this new degradation pathway in vivo, we generated mutant mice lacking SIP. We demonstrate here that SIP-/- thymocytes have an impaired pre-TCR checkpoint with failure of TCRbeta gene rearrangement and increased apoptosis, resulting in reduced cellularity of the thymus. Moreover, the degradation of beta-catenin in response to DNA damage is significantly impaired in SIP-/- cells. SIP-/- embryonic fibroblasts show a growth-rate increase resulting from defects in G1 arrest. Thus, the beta-catenin degradation pathway mediated by SIP defines an essential checkpoint for thymocyte development and cell-cycle progression.

Mechanistic basis of pre–T cell receptor–mediated autonomous signaling critical for thymocyte development

The pre-T cell receptor (TCR) is crucial for early T cell development and is proposed to function in a ligand-independent way. However, the molecular mechanism underlying the autonomous signals remains elusive. Here we show that the pre-TCR complex spontaneously formed oligomers. Specific charged residues in the extracellular domain of the pre-TCR alpha-chain mediated formation of the oligomers in vitro. Alteration of these residues eliminated the ability of the pre-TCR alpha-chain to support pre-TCR signaling in vivo. Dimerization but not raft localization of CD3epsilon was sufficient to simulate pre-TCR function and promote beta-selection. These results suggest that the pre-TCR complex can deliver its signal autonomously through oligomerization of the pre-TCR alpha-chain mediated by charged residues.

Overexpression of the antiapoptotic protein A1 promotes the survival of double positive thymocytes awaiting positive selection

As it has been shown for Mcl-1, Bcl-xl and Bcl-2, proteins of the Bcl-2 family play a crucial role during T-cell development in the thymus. We here show that the expression of the antiapoptotic gene A1 is specifically enhanced at the DN3/DN4 transition and in DP thymocytes that have been positively selected suggesting that A1 expression might be considered as a transcriptional signature of thymocytes that have received pre-TCR or TCR survival signal. Furthermore, we observed that A1-a overexpression in recombination activation gene 1-deficient mice transgenic for the major histocompatibillity complex class I-restricted F5 TCR enhances cell survival of DP thymocytes and permits accumulation of DP cells awaiting positive selection. However, A1-a overexpression has no effect on negative selection. Therefore, our results suggest that A1 plays a specialized role in allowing survival of DP thymocytes and that its role can be distinguished from that of Mcl-1, Bcl-xl and Bcl-2.

Cooperating pre–T-cell receptor and TCF-1–dependent signals ensure thymocyte survival

AbstractIntrathymic T-cell maturation critically depends on the selective expansion of thymocytes expressing a functionally rearranged T-cell receptor (TCR) β chain. In addition, TCR-independent signals also contribute to normal T-cell development. It is unclear whether and how signals from the 2 types of pathways are integrated. Here, we show that T-cell factor-1 (TCF-1), a nuclear effector of the canonical wingless/int (wnt)/catenin signaling pathway, ensures the survival of proliferating, pre-TCR+ thymocytes. The survival of pre-TCR+ thymocytes requires the presence of the N-terminal catenin-binding domain in TCF-1. This domain can bind the transcriptional coactivator β-catenin and may also bind γ-catenin (plakoglobin). However, in the absence of γ-catenin, T-cell development is normal, supporting a role for β-catenin. Signaling competent β-catenin is present prior to and thus arises independently from pre-TCR signaling and does not substantially increase on pre-TCR signaling. In contrast, pre-TCR signaling significantly induces TCF-1 expression. This coincides with the activation of a wnt/catenin/TCF reporter transgene in vivo. Collectively, these data suggest that efficient TCF-dependent transcription requires that pre-TCR signaling induces TCF-1 expression, whereas wnt signals may provide the coactivator such as β-catenin. The 2 pathways thus have to cooperate to ensure thymocyte survival at the pre-TCR stage. (Blood. 2005;106:1726-1733)

Enforced Expression of Spi-B Reverses T Lineage Commitment and Blocks β-Selection

The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4(-)CD8(-)CD44(-)CD25(+) (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the beta-selection checkpoint to the CD4(-)CD8(-)CD44(-)CD25(-) (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the beta-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the beta-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the beta-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.

PKCθ mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

The Ets-1 transcription factor is required for complete pre-T cell receptor function and allelic exclusion at the T cell receptor β locus

The pre-T cell receptor (TCR) functions as a critical checkpoint during alphabeta T cell development. Signaling through the pre-TCR controls the differentiation of immature CD4(-)CD8(-)CD25(+)CD44(-) [double-negative (DN)3] thymocytes into CD4(+)CD8(+) double-positive (DP) cells through the CD4(-)CD8(-)CD25(-)CD44(-)(DN4) stage. In addition, pre-TCR activity triggers expansion and survival of thymocytes and inhibits TCRbeta gene rearrangement through a process referred to as allelic exclusion. Whereas many proteins involved in the pre-TCR transduction cascade have been identified, little is known about the nuclear factors associated with receptor function. Here, we use gene targeting to inactivate the Ets-1 transcription factor in mice and analyze pre-TCR function in developing Ets-1-deficient (Ets-1(-/-)) thymocytes. We find that inactivation of Ets-1 impairs the development of DN3 into DP thymocytes and induces an elevated rate of cell death in the DN4 subset. This defect appears specific to the alphabeta lineage because gammadelta T cells maturate efficiently. Finally, the percentage of thymocytes coexpressing two different TCRbeta chains is increased in the Ets-1(-/-) background and, in contrast with wild type, forced activation of pre-TCR signaling does not block endogenous TCRbeta gene rearrangement. These data identify Ets-1 as a critical transcription factor for pre-TCR functioning and for allelic exclusion at the TCRbeta locus.

Deranged Early T Cell Development in Immunodeficient Strains of Nonobese Diabetic Mice

NOD mice exhibit defects in T cell functions that have been postulated to contribute to diabetes susceptibility in this strain. However, early T cell development in NOD mice has been largely unexplored. NOD mice with the scid mutation and Rag1 deficiency were analyzed for pre-T cell development in the NOD genetic background. These strains reveal an age-dependent, programmed breakdown in beta selection checkpoint enforcement. At 5-8 wk of age, even in the absence of TCRbeta expression, CD4+ and CD4+CD8+ blasts appear spontaneously. However, these breakthrough cells fail to restore normal thymic cellularity. The breakthrough phenotype is recessive in hybrid (NODxB6)F1-scid and -Rag1null mice. The breakthrough cells show a mosaic phenotype with respect to components of the beta selection program. They mimic normal beta selection by up-regulating germline TCR-Calpha transcripts, CD2, and Bcl-xL and down-regulating Bcl-2. However, they fail to down-regulate transcription factors HEB-alt and Hes1 and initially express aberrantly high levels of Spi-B, c-kit (CD117), and IL-7Ralpha. Other genes examined distinguish this form of breakthrough from previously reported models. Some of the abnormalities appear first in a cohort of postnatal thymocytes as early as the double-negative 2/double-negative 3 transitional stage. Thus, our results reveal an NOD genetic defect in T cell developmental programming and checkpoint control that permits a subset of the normal outcomes of pre-TCR signaling to proceed even in the absence of TCRbeta rearrangement. Furthermore, this breakthrough may initiate thymic lymphomagenesis that occurs with high frequency in both NOD-scid and -Rag1null mice.

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

The function of the Src-family kinases (SFKs) Lck and Fyn in T cells has been intensively studied over the past 15 years. Animal models and cell line studies both indicate a critical role for Lck and Fyn in proximal T-cell antigen receptor (TCR) signal transduction. Recruited SFKs phosphorylate TCR ITAMs (immunoreceptor tyrosine-based activation motifs) in the CD3 and zeta chains, which then serve as docking sites for Syk-family kinases. SFKs then phosphorylate and activate the recruited Syk-family kinase. Lck and Fyn are spatially segregated in cell membranes due to differential lipid raft localization, and may undergo sequential activation. In addition to the CD4 and CD8 coreceptors, a recently described adaptor, Unc119, may link SFKs to the TCR. CD45 and Csk provide positive and negative regulatory control of SFK functions, respectively, and Csk is constitutively bound to the transmembrane adapter protein, PAG/Cbp. TCR-based signaling is required at several stages of T-cell development, including at least pre-TCR signaling, positive selection, peripheral maintenance of naive T cells, and lymphopenia-induced proliferation. SFKs are required for each of these TCR-based signals, and Lck seems to be the major contributor.

Sustained early growth response gene 3 expression inhibits the survival of CD4/CD8 double-positive thymocytes.

In the absence of selection, CD4+, CD8+ double-positive (DP) thymocytes will die after 3-4 days. The mechanism for regulating the life span of DP cells is unknown. Previously, we demonstrated that the zinc finger transcription factor, early growth response gene 3 (Egr3), promotes proliferation during the transition from double negative (DN) to DP. In this study we demonstrate a novel role for Egr3 in controlling DP thymocyte survival in mice. Constitutive transgenic expression of Egr3 in thymocytes increases apoptosis among DP cells and shortens their survival in vitro. In addition, DP cells in Egr3 transgenic mice have poor expression of TCRalpha, and based on the predominant usage of 3' Valpha and 5' Jalpha gene segments, the low level of TCRalpha expression is a result of DP death soon after the initiation of TCRalpha rearrangements. Constitutive transgenic expression of Egr3 results in poor expression of Bcl-x(L) and the thymic isoform of retinoic acid receptor-related orphan receptor gamma (RORgammat) in DP thymocytes, two molecules that are required in DP cells for normal life span. Egr3 expression induced by pre-TCR signals in nontransgenic mice is transient and returns to background levels before RORgammat or Bcl-x(L) is induced. The data support a model in which Egr3 must be transiently induced in response to pre-TCR signals, so that the expression of the prosurvival molecules, RORgammat and Bcl-x(L), can be elevated only after the proliferative signal provided by Egr3 has subsided.

Notch and T cell malignancy

Notch signaling is required for normal T cell development. However, Notch expression must be precisely regulated as constitutive Notch signaling leads to T cell lymphomas. Recent evidence has provided insights into potential mechanisms of Notch-mediated lymphomagenesis and its relationship to T cell development. The evidence suggests that Notch likely interacts with several important cellular pathways and can cooperate with other oncogenes during lymphomagenesis. In particular, Notch appears to modulate pre-TCR signaling, inhibit the E2A pathway, and in murine leukemia models, frequently cooperates with Myc, E2A-PBX and dominant negative Ikaros dysregulation. This review will present current knowledge in these areas and explore theories on Notch-mediated T cell lymphomagenesis.

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.

Early growth response gene 3 regulates thymocyte proliferation during the transition from CD4-CD8- to CD4+CD8+.

In thymocytes developing in the alphabeta lineage, the transition from CD4, CD8 double negative (DN) to CD4, CD8 double positive (DP) is associated with several rounds of cell division and changes in the expression of multiple genes. This transition is induced by the formation of a pre-TCR that includes a rearranged TCR beta-chain and the pre-TCR alpha-chain. The mechanism by which the pre-TCR influences both gene expression and proliferation has not been defined. We have evaluated the role played by early growth response gene 3 (Egr3) in translating pre-TCR signals into differentiation and proliferation. Egr3 is a transcriptional regulator that contains a zinc-finger DNA binding domain. We find that Egr3-deficient mice have a reduced number of thymocytes compared with wild-type mice, and that this is due to poor proliferation during the DN to DP transition. Treatment of both Egr3(+/+) and Egr3(-/-) mice on the Rag1(-/-) background with anti-CD3epsilon Ab in vivo results in similar differentiation events, but reduced cell recovery in the Egr3(-/-) mice. We have also generated transgenic mice that express high levels of Egr3 constitutively, and when these mice are bred onto a Rag1(-/-) background they exhibit increased proliferation in the absence of stimulation and have pre-TCR alpha-chain and CD25 down-regulation, as well as increased Calpha expression. The results show that Egr3 is an important regulator of proliferation in response to pre-TCR signals, and that it also may regulate some specific aspects of differentiation.

E2A proteins enforce a proliferation checkpoint in developing thymocytes.

E2A proteins regulate multiple stages of thymocyte development and suppress T-cell lymphoma. The activity of E2A proteins throughout thymocyte development is modulated by signals emanating from the pre-TCR and TCR. Here we demonstrate that E2A is required for the complete arrest in both differentiation and proliferation observed in thymocytes with defects in proteins that mediate pre-TCR signaling, including LAT, Lck and Fyn. We show that E2A proteins are required to prevent the accumulation of TCRbeta negative cells beyond the pre-TCR checkpoint. E2A-deficient thymocytes also exhibit abnormal cell-cycle progression prior to pre-TCR expression. Furthermore, we demonstrate that E47 can act in concert with Bcl-2 to induce cell-cycle arrest in vitro. These observations indicate that E2A proteins function during early thymocyte development to block cell-cycle progression prior to the expression of TCRbeta. In addition, these data provide further insight into how deficiencies in E2A lead to T lymphoma.

Pre-TCR-triggered ERK signalling-dependent downregulation of E2A activity in Notch3-induced T-cell lymphoma

Notch and basic helix-loop-helix E2A pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3-induced T-cell leukaemia is abrogated by the inactivation of pTalpha/pre-T-cell antigen receptor (pre-TCR). We report here that Notch3-induced transcriptional activation of pTalpha/pre-TCR is responsible for the downregulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor Id1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in Id1 expression is achieved by pre-TCR-induced extracellular-signalling-regulated kinase 1/2. These observations support a model in which the upregulation of pre-TCR signalling seems to be the prerequi-site for Notch3-induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice.

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.

Pre-TCR signaling regulates IL-7 receptor alpha expression promoting thymocyte survival at the transition from the double-negative to double-positive stage.

The pre-T cell receptor (pre-TCR) and IL-7 receptor (IL-7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre-TCR signaling directly maintains IL-7Ralpha expression as developing thymocytes undergo beta-selection. Inhibition of IL-7/IL-7R signaling in (CD44-CD25-) DN4 cells results in decreased generation of double-positive thymocytes due to increased death of rapidly proliferating beta-selected cells. Thus, we identify a mechanism by which pre-TCR signaling controls the selective survival of TCRbeta+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.

A Role for the Cytoplasmic Tail of the Pre-T Cell Receptor (TCR) α Chain in Promoting Constitutive Internalization and Degradation of the Pre-TCR

Engagement of the alpha beta T cell receptor (TCR) by its ligand results in the down-modulation of TCR cell surface expression, which is thought to be a central event in T cell activation. On the other hand, pre-TCR signaling is a key process in alpha beta T cell development, which appears to proceed in a constitutive and ligand-independent manner. Here, comparative analyses on the dynamics of pre-TCR and TCR cell surface expression show that unligated pre-TCR complexes expressed on human pre-T cells behave as engaged TCR complexes, i.e. they are rapidly internalized and degraded in lysosomes and proteasomes but do not recycle back to the cell surface. Thus, pre-TCR down-regulation takes place constitutively without the need for extracellular ligation. By using TCR alpha/p Tau alpha chain chimeras, we demonstrate that prevention of recycling and induction of degradation are unique pre-TCR properties conferred by the cytoplasmic domain of the pT alpha chain. Finally, we show that pre-TCR internalization is a protein kinase C-independent process that involves the combination of src kinase-dependent and -independent pathways. These data suggest that constitutive pre-TCR down-modulation regulates pre-TCR surface expression levels and hence the extent of ligand-independent signaling through the pre-TCR.