notch signalling during peripheral t-cell activation and differentiation

Notch proteins are cell-surface receptors, the expres-sion of which has been widely conserved across species. Their main function is to regulate the mutually exclusive decisions that cells make during ontogeny. The epony-mous protein notch was first described in Drosophila melanogaster, when a loss-of-function mutation pro-duced a notched-wing phenotype1. Subsequently, four Notch proteins (Notch1–Notch4) have been described in mammals, as have many signal-inducing ligands of these Notch proteins2. Abrogating the signalling capacity of Notch1 or Notch2, but not Notch3 or Notch4, results in death during embryonic development, and this high-lights the significance and non-redundant functions of these proteins during embryogenesis3–5. Conversely, mice engineered to express activated Notch1 or Notch3 proteins in immature thymocytes develop lymphomas or T-cell leukaemia early in life, which indicates that the controlled expression of Notch proteins is essential for normal T-cell development6–8.

Notch proteins exert their pleiotropic effects through the regulation of expression of various downstream genes, many of which require the interaction of Notch proteins with the nuclear binding partner CSL (CBF1-suppressor of hairless–Lag1), the mouse homologue of which is RBP-J (recombination-signal-binding protein-J)9 and which is, itself, required for embryonic viability10,11. The formation of a complex of activated intracellular Notch protein and CSL converts CSL from a transcriptional repressor to a transcriptional transactivator12. The genes encoding the HES (hairy and enhancer of split) family of basic helix–loop–helix proteins are Notch targets that are known to be essen-tial for T-cell development and signalling13–15. The genes encoding the closely related HEY (HES with YRPW motif) family of transcription factors are also regulated by Notch signalling, although their activity in peripheral T cells awaits further characterization16,17.

Additional genes that function downstream of Notch receptors include NRARP (Notch-regulated ankyrin-repeat protein), PTCRA (pre-T-cell receptor-α) and the genes encoding the Deltex family of E3 ligases18–22. More recently, Notch receptors have been shown to regulate the expression of proteins that are crucial for peripheral T-cell activation and differentiation, including the trans-cription factors nuclear factor-κB (NF-κB) and T-bet, the pro-inflammatory cytokine interferon-γ (IFNγ) and the interleukin-4 (IL-4) enhancer CNS2 (REFS 23–26).

Notch signalling during lymphoid development has been extensively studied, and its essential role in specifying cell fate at many stages during T-cell devel-opment is well characterized. In the absence of Notch1, lymphoid progenitors entering the thymus fail to initi-ate a T-cell developmental program, and adopt a B-cell fate by default27–29. Similarly, RBP-J-deficient mice have increased numbers of B cells in the thymus, even when Notch receptors are expressed, which indicates that the function of RBP-J downstream of Notch1 is cru-cial for normal lymphoid development30. In reciprocal experiments, forced expression of activated Notch1 in bone-marrow precursor cells results in the develop-ment of T cells in the bone marrow at the expense of B-cell development31. Overexpression of HES-family genes in mouse bone-marrow progenitors also inhib-ited B-cell development, indicating that Notch proteins might repress the expression of B-lineage-specific genes through HES-family genes32. At later stages of thymocyte development, T cells rearrange the genes that encode a functional T-cell receptor (TCR) and produce either αβ or γδ TCRs. At this developmental checkpoint, Notch1 has a role in regulating stage-specific commitment to the αβ or γδ T-cell lineage33–35, and recent data indicate that Notch1 might mediate this event by synergizing with signals generated through the TCR36,37. The requirement for Notch signalling in determining CD4+ versus CD8+

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, USA.Correspondence to B.A.O. e-mail: [email protected]:10.1038/nri1998Published online15 December 2006

Notch signalling during peripheral T-cell activation and differentiationBarbara A. Osborne and Lisa M. Minter

Abstract | For many years, researchers have focused on the contribution of Notch signalling to lymphoid development. Only recently have investigators begun to ask what role, if any, Notch has during the activation and differentiation of naive CD4+ T cells in the periphery. As interest in this issue grows, it is becoming increasingly clear that the main role of Notch signalling, to regulate cell-fate decisions, might also be influential in peripheral T cells.

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© 2007 Nature Publishing Group

T helper 1/T helper 2 cells(TH1/TH2 cells). Functional subsets of CD4+ T cells expressing an αβ T-cell receptor that produce either type 1 cytokines (interleukin-2 (IL-2), interferon-γ and other cytokines that support macrophage activation, the generation of cytotoxic T cells and the production of opsonizing antibodies) or type 2 cytokines (IL-4, IL-5, IL-13 and other cytokines that support B-cell activation, the production of non-opsonizing antibodies, allergic reactions and the expulsion of extracellular parasites).

FucosylationAddition of the sugar fucose to the oligosaccharide chains (glycans) of membrane proteins through the enzymatic actions of a particular fucosyltransferase.

S1-cleavage siteA specific site in the Notch pre-protein that is targeted by a furin-like protease in the trans-Golgi during the maturation of Notch proteins.

GlycosylationEnzymatic addition of carbohydrate groups to the side chains of asparagine, serine or threonine residues. This process is important in the synthesis of many secreted and cell-surface proteins.

T-cell-fate decisions remains the most controversial con-tribution of Notch proteins to thymocyte development. Gain-of-function studies using mice expressing activated Notch1 in thymocytes indicate that the overexpression of Notch1 skews T-cell development in favour of CD8+ T cells38–40. However, this conclusion was challenged by studies using mice that were conditionally deficient in Notch1 expression by immature thymocytes, in which no significant differences in CD4+ to CD8+ T-cell ratios were found41.

After entering the periphery, T cells become the adap-tive immune system’s decisive recipients of messages from the innate immune system. As such, their effective and effi-cient mobilization is crucial to the host’s ability to respond to antigenic challenge. A CD4+ T-cell response emanates from the interaction of a TCR with its cognate antigenic peptide bound to an MHC class II molecule on the surface of an antigen-presenting cell (APC). Stimulation through the TCR alone is insufficient to induce full activation of T cells. To prevent inappropriate activation that might be deleterious to the host, T cells require a second ‘co-stimulatory’ signal to link proximal and distal signalling cascades effectively, which results in the transcription of a wide variety of downstream genes42.

In peripheral T cells, many diverse intracellular events comprise the activation process. The actions of protein kinases — such as LCK, protein kinase C-θ (PKCθ) and mitogen-activated protein kinases (MAPKs) — and phosphatases, such as calcineurin, as well as the genera-tion of second messengers, such as intracellular calcium ions, diacylglycerol and inositol-1,4,5-trisphosphate42, all have essential roles in initiating and amplifying the signalling cascades that culminate in the activation of crucial transcription factors, including activator pro-tein 1, nuclear factor of activated T cells, Janus kinase and NF-κB (BOX 1).

Once activated, CD4+ T cells are primed to differenti-ate further into effector T cells that can mediate pathogen-specific immune responses. By convention, these cells can be divided into one of the two main categories of T helper (TH) cells, TH1 or TH2 cells, on the basis of their character-istic cytokine profiles43. As TH cells transit through the cell cycle, their individual commitment to becoming a TH1

or TH2 cell becomes increasingly irreversible, indicating that this process invokes heritable changes in gene expres-sion44,45. The transcription factor T-bet is both necessary and sufficient to drive differentiation towards the TH1-cell lineage46, whereas GATA3 (GATA-binding protein 3), an important TH2-cell regulator, is responsible both for acces-sibility to the IL4 gene regulatory locus47,48 and for direct transcription of the IL5 gene49.

As if the journey from naive T cell to fully differenti-ated effector T cell was not complicated enough, recent studies have introduced a new and somewhat controver-sial travelling companion in the form of Notch proteins. How Notch signalling contributes to cell-fate decisions at crucial junctions in T-cell activation and differentiation has become an area of active and fertile investigation. In this Review, we discuss how Notch receptors might function as molecular switches during peripheral T-cell signalling.

Inside-out and outside-in signallingThe regulation of Notch signalling is exquisitely complex, and has recently been reviewed at length elsewhere50. There are four mammalian Notch receptors, Notch1, Notch2, Notch3 and Notch4, and five canonical Notch ligands, Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3 and DLL4 (REF. 2) (BOX 2). Notch receptors undergo several processing steps before the signalling-competent intra-cellular domain of Notch is cleaved from the membrane, as a result of ligand binding to the extracellular domain, and translocates to the nucleus (FIG. 1). Notch receptors are synthesized as large pre-proteins comprised of extracel-lular and intracellular domains. To exit the endoplasmic reticulum and transit to the Golgi, they must be fucosylated by the O-fucosyltransferase FUT1 (REFS 51,52). Once in the Golgi, the full-length Notch receptor is cleaved by a furin-like protease at the S1-cleavage site, facilitating the formation of a non-covalently linked heterodimer that is comprised of the extracellular domain and the transmembrane domain of the receptor53,54. Before its transport to the cell membrane, the extracellular domain of the receptor undergoes glycosylation by Manic fringe, Radical fringe and Lunatic fringe, which are members of the Fringe glycosyltransferase family55,56. The actions of specific Fringe proteins can influence the signalling competence of unique ligand–receptor interactions without necessarily affecting the ligand-binding affinity of the Notch receptor. Although all Fringe proteins facili-tate Notch signalling by increasing Notch–DLL binding and Notch activation, Manic fringe and Radical fringe both inhibit Notch–Jagged signalling without affecting the binding affinity of Jagged57,58. Therefore, before even encountering ligand, Notch receptors can be ‘primed’ to relay signals from some ligands but not others.

The exact mechanisms by which Notch ligands acti-vate their target receptors have not been fully elucidated. One model proposes that after binding a canonical ligand on a neighbouring cell, the extracellular domain of the Notch receptor is removed by mechanical forces, as the ligand-bearing cell trans-endocytoses the ligand-bound extracellular Notch complex59. Removal of the Notch extracellular domain is postulated to induce a

Box 1 | Nuclear factor-κB proteins and signalling pathway

The nuclear factor-κB (NF-κB) family of transcription factors is comprised of five proteins: NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB and Rel. NF-κB1 and NF-κB2 are synthesized as large pre-proteins (p105 and p100, respectively) that are further modified post-translationally to generate the NF-κB1 and NF-κB2 subunits that are capable of binding DNA129. Unlike the three Rel subunits, NF-κB1 and NF-κB2 lack transactivational domains and are thought to act as transcription repressors when found as homodimers130,131. The remaining NF-κB subunits are characterized by a Rel-homology motif, and they positively regulate gene transcription when paired either as heterodimers or homodimers. The exception is RelB, which is unable to homodimerize. NF-κB proteins are tightly regulated, and are held in an inactive state in the cytoplasm by inhibitor of NF-κB (IκB) proteins. Following activation through the T-cell receptor, IκB proteins are phosphorylated by IκB kinase, and subsequently degraded. NF-κB is released and translocates to the nucleus, where it acts to regulate the expression of genes that are essential for T-cell activation and proliferation, such as those encoding interleukin-2 (IL-2), the IL-2 receptor α-chain (CD25) and interferon-γ132,133.

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Trans-endocytosisThe process by which a tightly associated receptor–ligand complex induces invagination of the plasma membrane and internalization of the complex into the ligand-bearing cell to form a membrane-limited transport vesicle.

ADAM proteases(A disintegrin and metalloproteinase family of proteases). Members of this family contain disintegrin-like and metalloproteinase-like domains and are involved in the regulation of developmental processes, cell–cell interactions and protein processing, including ectodomain shedding.

S2-cleavage siteA specific site in the extracellular domain (close to the membrane) of the Notch heterodimer that becomes accessible to ADAM proteases, which cleave the Notch protein, after ligand binding.

S3-cleavage siteA cleavage site in the transmembrane region (cytoplasmic side) of Notch proteins. Cleavage is thought to occur in endosomes, after cleavage at the S2 site, by a complex of proteins that have γ-secretase activity. In mouse Notch1, this site lies between amino-acid residues 1743 and 1744.

conformational change of the remaining Notch receptor that allows cleavage of the Notch protein by an ADAM (a disintegrin and metalloproteinase) protease at the S2-cleavage site60. Ligand-expressing cells that are defective in their ability to trans-endocytose the extracellular domain of Notch also lose their ability to activate Notch proteins in nearby receptor-bearing cells, which lends support to this model61. However, in vitro studies using synthetic ligand fusion proteins, such as DLL1–Fc or DLL1–IgG, indicate that Notch signalling can proceed in the absence of trans-endocytosis62–65. In these studies, crosslinking or immobilizing the fusion construct was required to activate Notch receptors, as measured by the induction of HES-family gene expression, but high concentrations of either crosslinking antibody or DLL1–Fc resulted in repressed Notch signalling62,64. Whether the signalling events gener-ated using these fusion constructs result in agonistic or antagonistic Notch responses remains highly controver-sial. The final processing event that releases intracellular Notch is γ-secretase-mediated cleavage at the S3-cleavage site of the Notch receptor, which is proximal to the trans-membrane domain66. It is this signalling-competent form of Notch that translocates to the nucleus to modulate the transcription of target genes.

The Notch ligands Jagged1, Jagged2 and DLL1 have been detected on the surface of APCs. Experimental sys-tems involving expression of Jagged1 on mouse fibroblast NIH 3T3 cells resulted in prolonged Notch signalling and p63-dependent upregulation of Jagged1 expression in signal-receiving fibroblasts, which argues against an inherent negative-feedback loop67. However, in vivo,

splenic dendritic cells (DCs) express high levels of mRNA encoding Jagged1 and Jagged2 but have few transcripts encoding DLL1. Conversely, splenic macrophages have high-level expression of DLL1, and significantly lower levels of expression of Jagged1 and Jagged2 (REF. 68). The differential expression of Notch ligands implies that they have different functions, and studies using plate-bound DLL4–IgG, DLL1–IgG and Jagged1–IgG fusion proteins have shown a hierarchy of binding affinity and activa-tion potential when incubated with naive CD4+ T cells65. Finally, stimulation of purified CD4+ T cells with CD3- and CD28-specific antibodies also results in Notch-receptor cleavage and upregulation, by mechanisms that remain obscure25,69. So, further studies are required to determine whether specific Notch ligands relay unique and non-redundant T-cell activation signals under physiological conditions.

Interestingly, non-canonical ligands that can induce Notch signalling have also been described. These include DNER (Delta/Notch-like epidermal-growth-factor-related receptor), contactin, contactin 6 (also known as NB3), NOV (nephroblastoma overexpressed gene; also known as CCN3) and, most recently, MAGP1 (microfibril-associated glycoprotein 1) and MAGP2 (REFS 70–74). Notch signalling through interactions with these non-canonical ligands has so far been described in systems other than the immune system. However, it is reasonable to presume that there might be similar, as yet undefined, ligands that can initiate Notch signalling in peripheral T cells, perhaps by destabilizing the het-erodimerization domain of Notch75, as is the case with

CR EGF-like repeats DSLJagged1 and Jagged2

Notch ligands

DLL1 and DLL4

DLL3

LIN RAM ANK TAD PESTNotch1

Notch2

Notch3

Notch4

HD

Extracellular portion Intracellular portion

NLS

EGF-like repeats

Notch receptors

Box 2 | Mammalian Notch receptors and ligands

There are four mammalian Notch receptors (Notch1–Notch4; see figure). Mature Notch proteins reside on the cell surface as non-covalently linked heterodimers that are comprised of the extracellular and transmembrane (intracellular) Notch polypeptides. Extracellular Notch proteins are characterized by numerous epidermal growth factor (EGF)-like repeats. Transmembrane Notch1 and Notch2 are almost identical in size and share many structural features, including the membrane-proximal RBP-J-associated molecule (RAM) domain, which mediates interaction with several cytosolic and nuclear proteins; the Ankyrin (ANK) domain, which is also important for protein–protein interactions; two nuclear-localization sequences (NLSs); a carboxy-terminal transactivation domain (TAD), which is important for activating transcription; and a PEST (proline-, glutamate-, serine- and threonine-rich) domain, which is important for regulating Notch degradation. Transmembrane Notch3 and Notch4 are shorter and lack the TAD. The heterodimerization domain (HD) spans the region of interaction between the extracellular and transmembrane portions. Mutations in this region result in ligand-independent activation of Notch receptors. LIN, lineage.

There are five mammalian Notch ligands, Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3 and DLL4. Each ligand contains an EGF-like repeat region and a conserved sequence that is also found in Drosophila melanogaster, Caenorhabditis elegans and vertebrates and is known as Delta/Serrate/Lag (DSL). Jagged1 and Jagged2 each have a conserved cysteine-rich (CR) domain.

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Ligand-expressing cell Notchligand

c

d

e

S2 cleavageby ADAMprotease

Ubiquitylation

Endocytosis

TransmembraneNotch

Intracellular Notch

Ub

Ub

bS1 cleavage byfurin-like protease

Trans-GolgiGlycosylation

f S3 cleavageby γ-secretase

CoR

CSLCSL

g

Target genes repressed Target genes active

a

Notch

ERFucosylation

CoR

Nucleus

CytosolCoA

gain-of-function mutations that are frequently seen in the heterodimerization domain of Notch in T cells from patients with T-cell acute lymphoblastic leukaemia (T-ALL)76.

Canonical signalling through CSLOne well-described mechanism by which Notch tar-get genes are regulated involves the nuclear binding protein CSL (known as RBP-J in mice), and transcrip-tion of its downstream target genes of the HES family is considered the gold standard for Notch activity77

(FIG. 2). In the absence of Notch signalling, CSL can

repress transcription by occupying unique bind-ing sites in a gene promoter, in association with a co-repressor complex comprised of SMRT (silencing mediator of retinoic acid and thyroid hormone recep-tor), histone deacetylases (HDACs), SHARP (SMRT/HDAC1-associated repressor protein) and CIR1 (CBF1-interacting co-repressor)78–80. SHARP, in turn, recruits CTBP1 (carboxy-terminal binding protein 1) and CTIP (CTBP-interacting protein) as additional co-repressors of the transcription of Notch target genes. Accordingly, CTBP1-deficient mouse embry-onic fibroblasts show significant de-repression, and

Figure 1 | Notch expression and activation. Notch proteins are synthesized as a single polypeptide of ~300 kDa. a | In the endoplasmic reticulum (ER), the Notch polypeptide is fucosylated by the fucosyltransferase FUT1. b | This facilitates shuttling of the Notch protein to the trans-Golgi, where it is cleaved by a furin-like protease at the S1-cleavage site to generate the non-covalently linked Notch heterodimer, which is comprised of the extracellular portion and the intracellular portion. The heterodimer then undergoes glycosylation by several specific glycosylases, including Manic fringe, Radical fringe and Lunatic fringe, which are all members of the Fringe glycosyltransferase family. c | The Notch heterodimer then associates with the plasma membrane, where it becomes available to interact with Notch ligand on a ligand-expressing cell. d | The interaction with Notch ligand induces proteolytic cleavage of the Notch receptor by the ADAM (a disintegrin and metalloproteinase) protease TACE (tumour-necrosis-factor-α-converting enzyme). e | This is followed by mono-ubiquitylation of the intracellular portion of the transmembrane Notch fragment. f | This results in endocytosis of the transmembrane fragment of the Notch protein, presumably facilitating cleavage by γ-secretase in an early endosome, resulting in the release of the intracellular Notch fragment. g | Intracellular Notch then travels to the nucleus, where it associates with the transcriptional repressor CSL (CBF1-suppressor of hairless–Lag1), resulting in the expression of genes that are regulated by CSL. CoA, co-activator; CoR, co-repressor.

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HDACs

CSLCSL

CIR1

Intracellular Notch

Nucleus

Cytosol

CoR

CSL

CoA

Transcriptionalrepression

Co-repressorsdisplaced by Notch

Transcriptionalactivation

SMRT

SHARP

HDACs CIR1

SMRT

SHARP

p300SKIPMAML1

GCN5HES-, HEY-and Deltex-family genes

Histone acetyl transferasesProteins that mediate the enzymatic addition of an acetyl group to the amino group of an amino-terminal residue of the histone complex to facilitate transcription.

thereby increased transcription, of the Notch-target gene Hey1 (REF. 81). In RBP-J-deficient mouse T cells, there is increased IFNγ production but, by contrast, there does not seem to be any increase in Hes1 or Hes5 gene expression35. These observations raise the ques-tion of whether all Notch target genes are equivalently regulated through CSL (RBP-J). To regulate some genes, does CSL act merely as a repressor, and does its physical association with Notch proteins de-repress transcription, thereby allowing subsequent positive regulation by other transcription factors? This model is consistent with the observed increase in transcrip-tion of some genes, such as Hey1, in mice deficient for CTBP, and the gene encoding IFNγ, in RBP-J-deficient mice. However, during the regulation of other genes, such as the HES gene family or the IL4-gene regulatory locus, does CSL repress transcription in the absence of Notch signalling, and is this complex then converted to a strong transcriptional activator when bound by intracellular Notch? This canonical model of Notch signalling through CSL would explain why such genes are not upregulated in mice deficient for RBP-J.

Following the binding of intracellular Notch proteins to CSL, SKIP (Ski-interacting protein) is recruited to the repressor complex to facilitate its conversion to an activating complex82. Additional engagement of the co-activator Mastermind-like 1 (MAML1) and of the histone acetyl transferases p300, PCAF (p300/CBP-associated factor) and GCN5 (gen-eral control of amino-acid synthesis 5) might assist in reconfiguring the CSL-associated repressor complex into a Notch–CSL-associated activating complex83–87.

Recent elegant studies have revealed the struc-tural interaction of MAML1 with intracellular Notch and CSL bound to DNA, and indicate a function of

MAML1 binding in the context of this complex. The RBP-J-associated molecule (RAM) domain of intracel-lular Notch provides the first contact with DNA-bound CSL after translocation of the Notch protein to the nucleus. The Notch–CSL interaction is further stabilized when the Ankyrin (ANK) domain of intracellular Notch is coupled to the Rel-homology domain of CSL. This interaction creates a composite binding site to which MAML1 binds with high affinity, although its asso-ciation with either intracellular Notch or CSL alone is much weaker. MAML1 recruits p300 and PCAF, which cooperate to facilitate transcription in vitro88. Expression of MAML1 together with CSL can increase the phospho-rylation and subsequent degradation of Notch receptors, indicating that MAML1 might function as a temporal regulator of Notch activity89. Furthermore, retroviral transfection of haematopoietic stem cells with a con-struct encoding a dominant-negative form of MAML1 mimics a Notch loss-of-function phenotype, which is characterized by a block in early T-cell development. This indicates that MAML1 might have an important role in mediating Notch signalling in immune cells90.

Notch signalling through CSL-independent pathways has been previously described in D. melanogaster, mainly involving the E3 ligase deltex91, and the overexpression of Deltex proteins in haematopoietic stem cells mim-ics Notch1 inactivation92. However, as transgenic mice expressing a construct encoding a RING-finger-domain-deficient form of Deltex1 (REF. 93), or mice deficient in both Deltex1 and Deltex2 (REF. 94), have normal devel-opment of the immune system and normal immune responses, it remains unclear whether Deltex proteins mediate CSL-independent Notch signalling pathways in the mammalian immune system. These data also highlight the fact that although many of the founda-tions for our understanding of Notch function stem from observations made originally in D. melanogaster or Caenorhabditis elegans, extrapolating these finding to mice or humans does not always give identical results.

NF-κκB: a new nuclear binding partner for Notch? The precise interaction between Notch receptors and NF-κB has not been clearly defined, but emerging data indicate that these two signalling molecules are, indeed, interconnected. Notch1 can transactivate the gene encoding NF-κB2 (p52) by de-repressing CSL95, and it also regulates basal levels of IκBα (inhibitor of NF-κB-α) through a CSL-dependent mechanism96.

NF-κB signalling might reciprocally affect Notch activity, as expression of Rel (a component of the active NF-κB complex) promotes the upregulation of Jagged1 expression and its functional interaction with Notch receptors on co-cultured T cells97. Consistent with these observations, transgenic mice engineered to overexpress Notch3 have constitutive activation of NF-κB in thymo-cytes, which results in T-cell leukaemias8. Conversely, mice with decreased levels of Notch1 (through expres-sion of a Notch1 antisense transgene) have attenuated NF-κB activity, both in haematopoietic progenitor cells98 and in peripheral T cells, which also have defects in IFNγ production25. Overexpression of intracellular Notch1

Figure 2 | Canonical pathway of Notch signalling through CSL. The ligation of a Notch receptor induces the cleavage of transmembrane Notch by γ-secretase and the release and translocation of intracellular Notch protein to the nucleus. In the nucleus, Notch proteins can bind to CSL (CBF1-suppressor of hairless–Lag1), thereby displacing co-repressor (CoR) proteins such as SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), SHARP (SMRT/HDAC1-associated repressor protein), CIR1 (CBF1-interacting co-repressor 1) and various histone deacetylases (HDACs) from CSL. This results in the recruitment of SKIP (Ski-interacting protein), followed by co-activator (CoA) proteins such as p300, GCN5 (general control of amino-acid synthesis 5) and MAML1 (Mastermind-like 1), to the Notch–CSL complex, which aid in the transcription of target genes such as those encoding members of the HES, HEY and Deltex families.

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HES- andDeltex-familygenes

Competition for bindingintracellular Notch

CSL-binding site

NF-κB-bindingsite

NF-κB

Ifng

HDACs

CSL

CIR1CoR

CSL

CoA

?

SMRT

SHARP

p300SKIPMAML1

GCN5

in the mouse thymoma cell line DO11.10 increased the phosphorylation of IκBα, the activity of NF-κB and the secretion of IFNγ, after stimulation through the TCR. Treating peripheral T cells with an inhibitor of γ-secretase not only abrogated the upregulation of Notch1 expression induced by stimulation with CD3- and CD28-specific antibodies, but also prevented IκBα phosphorylation and subsequent NF-κB activation and IFNγ secretion (B.A.O., unpublished observations). It is well established that NF-κB is activated after TCR stimu-lation. We and others have shown that signalling through the TCR activates Notch receptors in both thymic and peripheral T cells25,69; it is therefore reasonable to sug-gest that Notch and NF-κB signalling pathways could intersect in any TCR-stimulated T cell.

Recent studies have also identified NF-κB subunits as potential nuclear binding partners of intracellular Notch proteins. Complexes of Notch and NF-κB1 (p50), as well as complexes of intracellular Notch and Rel, could be immunoprecipitated bound to DNA from the promoter region of the gene encoding IFNγ23, indicating that there might be nuclear partners in addition to CSL through which intracellular Notch proteins regulate target genes. Further studies are required to determine the precise role of CSL in Notch–NF-κB-mediated gene regulation in T cells.

A close examination of consensus NF-κB- and CSL-binding sites in DNA reveals an interesting find-ing. Although not all CSL-binding motifs are sub-sets of a larger NF-κB-response element, consensus NF-κB-binding sites incorporate a nested CSL-binding

site99–101 (FIG. 3). This raises two important outstanding questions: do NF-κB and CSL compete for binding to these overlapping sites as a means of regulating gene expression; and how does the interaction of intra cellular Notch proteins with one or both of these binding partners affect DNA-binding affinity, recruitment of co-activators and, ultimately, gene expression? Answers to these and other questions will undoubtedly aid in our understanding of Notch-mediated regulation of target genes.

In contrast to the studies described so far, some reports indicate that Notch signalling might inhibit NF-κB activation102,103. In these studies, Notch proteins were shown to interact physically with NF-κB and block its activation. One explanation for these disparate results might be due to differences in the constructs encoding intracellular Notch1 that were used in the different studies. The NF-κB-activating intracellular Notch1 construct more closely resembles the full-length intra-cellular Notch1 that is released when Notch1 is cleaved from the membrane after ligand binding, compared with the intracellular Notch1 construct that fails to activate NF-κB, which is a smaller construct that lacks several amino-terminal amino acids. When we compared the NF-κB-activating potential of two intracellular Notch1 constructs, we found that a loss of as few as 11 amino acids at the N-terminus of intracellular Notch1 could abrogate NF-κB binding to DNA, and subsequent IFNγ production (B.A.O., unpublished observations). In light of these findings, the length of intracellular Notch1 might be important for NF-κB activation and/or for the physical interaction between Notch and NF-κB. How this putative interaction between intracellular Notch1 and NF-κB affects the activity of each remains to be clarified.

Notch in peripheral T-cell signallingIn mature T cells, the activation cascades that generate sustained signals originating from TCR engagement and co-stimulatory molecules are complicated at best. Adding to this complexity, Notch proteins are now emerging as potentiators of TCR signalling.

Early reports indicated that Notch proteins could transduce signals in peripheral T cells and assigned a cell-protective function to Notch proteins, which could bind and inactivate the pro-apoptotic orphan nuclear receptor Nur77, and therefore protect T-cell lines from TCR-induced cell death104. Since then, additional anti-apoptotic mechanisms have been described whereby Notch signalling upregulates the expression of inhibitor of apoptosis proteins (IAPs), BCL-2 (B-cell lymphoma 2) and FLIP (FLICE-like inhibitor protein)105. This study also showed that the interaction of Notch proteins with LCK and with phosphatidylinositol 3-kinase (PI3K) led to the activation of AKT (also known as PKB), and that these interactions were important for mediating the anti-apoptotic effects of AKT, as blocking Notch signalling with pharmacological inhibitors abrogated the protection from apoptosis. Further insight into this signalling cascade has been provided both upstream and downstream of PI3K and AKT. Overexpression of PTEN

Figure 3 | Possible consequences of Notch signalling. The activation of a Notch receptor and release of intracellular Notch protein might have several consequences, including the well-described association of intracellular Notch proteins with CSL (CBF1-suppressor of hairless–Lag1) and the activation of CSL-regulated genes such as those encoding members of the HES and Deltex families. Recent data indicate that intracellular Notch proteins can also associate with nuclear factor-κB (NF-κB) and activate known NF-κB targets including the gene encoding interferon-γ (Ifng). The NF-κB- and CSL-binding sites in Notch proteins overlap, and this indicates that the association of a Notch protein with NF-κB could compete with the Notch–CSL interaction. Possible outcomes of Notch signalling include the interaction of Notch proteins with CSL and the activation of CSL-regulated genes and/or the interaction of Notch proteins with NF-κB and the activation of NF-κB-regulated genes. It is possible that there is competition between NF-κB and CSL for interaction with Notch. CoA, co-activator; CoR, co-repressor; CIR1, CBF1-interacting co-repressor 1; GCN5, general control of amino-acid synthesis 5; HDAC, histone deacetylase; MAML1, Mastermind-like 1; SHARP, SMRT/HDAC1-associated repressor protein; SKIP, Ski-interacting protein; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor.

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TH1-celldevelopment

TH2-celldevelopment

CD4+CD25+

TReg-cell or CD8+

regulatory T-celldevelopment

T-bet

IFNγ

CSL

IL-4

Intracellular Notch

NF-κBMAML1?

?

?

?

?

Notch3 in CD4+

or CD8+ T cellsNotch1 orNotch3 inCD4+ T cells

Notch1 orNotch3 inNKT cells ormemoryCD4+ T cells

(phosphatase and tensin homologue), which inhibits signalling downstream of PI3K, also blocked Notch sig-nalling in Jurkat T cells. In addition, PI3K–AKT signal-ling phosphorylated and inactivated GSK3β (glycogen synthase kinase 3β), a kinase that is known to negatively regulate Notch signalling106–108. So, one function of Notch proteins in activated T cells seems to be the ability to protect stimulated T cells from apoptosis by signalling through the PI3K–AKT pathway.

In independent studies, two groups confirmed roles for Notch receptors in T-cell activation, proliferation and cytokine production, although how T-cell activa-tion specifically results in the upregulation of Notch expression has not been clearly defined. In one report,

pharmacological inhibitors that blocked the upregula-tion of Notch expression in peripheral CD4+ and CD8+ T cells also significantly inhibited T-cell proliferation and IFNγ production after stimulation through the TCR25. The second study showed that Notch signal-ling is involved in a positive-feedback loop that regu-lates IL-2 production by T cells and expression of the high-affinity IL-2 receptor α-subunit, CD25 (REF. 69). Overexpression of intracellular Notch proteins resulted in sustained CD25 expression by T cells, irrespective of the number of cell divisions. A more recent report confirmed increased Notch activity after TCR liga-tion and showed that Notch proteins co-localize with CD4 in human CD4+ T cells109. In contrast to previous data, no differences in IFNγ production were seen in CD4+ T cells stimulated with CD3- and CD28-specific antibodies and cultured in the presence or absence of a Notch inhibitor, although it is possible that the use of different pharmacological inhibitors in these stud-ies contributed to the disparate findings. Consistent with an emerging role for Notch proteins in regulating cytokine production, blocking Notch signalling abro-gated IL-10 secretion by antibody-stimulated CD4+ T cells109.

The observations that Notch proteins co-localize with CD4 (REF. 109) and/or the E3 ligase Numb110 at the T-cell–APC interface place Notch proteins in the cor-rect cellular location to modulate early signalling events in activated T cells, and the documented interactions of Notch proteins with proximal signalling molecules further support this view105.

One well-described outcome of T-cell activation is induced NF-κB activity, but the contribution of Notch signalling to this process, albeit at later time points, has only recently come to light23,25. Although an initial TCR-dependent wave of NF-κB activa-tion does not require Notch signalling, intracellu-lar Notch protein seems to be necessary to sustain NF-κB activation23. After stimulation with CD3- and CD28-specific antibodies, the Notch–NF-κB1 interac-tion might facilitate nuclear retention of the NF-κB1 subunit, thereby preserving its ability to bind DNA and potentiate transcription. The Notch ligand Jagged1 is a downstream target of NF-κB activation, providing a potential signalling loop between Notch proteins and NF-κB97. Tight regulation of NF-κB activity is crucial to maintain normal immune-cell responses. In mice transgenic for expression of intracellular Notch3, cooperative signalling between the pre-TCR and PKCθ results in dysregulated NF-κB activation and T-cell leukaemia111. Additional mechanisms of Notch-mediated T-cell transformation have also been described and recently reviewed elsewhere112.

Notch as a regulator of T-cell effector functionOne of the most intriguing, and perhaps most contro-versial, functions recently assigned to Notch proteins is that of a regulator of TH-cell differentiation. To this end, Notch proteins have been described as having a role in directing the differentiation of activated T cells to TH1-, TH2- or regulatory T-cell lineages (FIG. 4).

Figure 4 | The role of Notch proteins in peripheral T cells. The potential involvement of Notch proteins in peripheral T cells is shown, on the basis of results from several laboratories. Confirmed pathways are depicted with solid arrows, possible pathways are depicted with dashed arrows. The activation of a Notch protein results in the activation of nuclear factor-κB (NF-κB), which drives the expression of T helper 1 (TH1)-cell-associated genes such as those encoding T-bet and interferon-γ (IFNγ). This might indicate a T-cell-intrinsic role for Notch proteins in the differentiation of CD4+ T cells to a TH1-cell phenotype. By contrast, the role of Notch proteins in regulating TH2-cell responses is probably mediated through the canonical CSL (CBF1-suppressor of hairless–Lag1)–MAML1 (Mastermind-like 1) pathway. In this regard, Notch proteins and CSL are both required for the production of interleukin-4 (IL-4) by natural killer T (NKT) cells and memory CD4+ T cells, which indicates that Notch proteins might have a T-cell-extrinsic role in TH2-cell development. It is possible that Notch proteins also have a role in the development of regulatory T cells. Ligation of Notch receptors on T cells with Jagged1 and/or Delta-like 1 on antigen-presenting cells can drive the generation of both CD4+CD25+ regulatory T (TReg) cells and CD8+ regulatory T cells. As shown in animal models, the ligation of Notch3 might be important in promoting regulatory T-cell development and in decreasing susceptibility to autoimmune diabetes.

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Cre–loxP approachA site-specific recombination system. Two short DNA sequences (loxP sites) are engineered to flank the target DNA. Expression of the recombinase Cre leads to excision of the intervening sequence. Depending on the type of promoter that controls Cre expression, Cre can be expressed at specific times during development or by specific subsets of cells.

Regulation of TH1-cell differentiation. In ground-breaking work, interactions between DLL1 and Notch3 were described to influence the differentiation of activated CD4+ T cells, by promoting a TH1-cell phenotype64. In this study, co-culture of purified CD4+ T cells with a DLL1–Fc fusion protein resulted in increased IFNγ production or decreased IL-4 production when cells were stimulated under TH1- or TH2-cell-promoting conditions, respectively. In addition, when CD4+ T cells were retrovirally transfected with a construct encoding either intracellular Notch1 or intracellular Notch3 and then cultured with DLL1–Fc, only those cells expressing intracellular Notch3 secreted IFNγ at levels greater than non-transfected, stimulated control cells. This indicated that the DLL1–Notch3 interaction could direct CD4+ T cells towards a TH1-cell fate. This conclusion was sup-ported by the observation that expression of the canoni-cal TH1-cell-associated transcription factor T-bet46 was induced in CD4+ T cells after incubation with DLL1–Fc. In vivo administration of DLL1–Fc promoted a robust TH1-cell-mediated response to Leishmania major infec-tion in normally susceptible BALB/c mice and, in a reciprocal manner, administration of mutant DLL1–Fc to normally resistant C57BL/6 mice resulted in an exac-erbated disease state when the mice were challenged with L. major64. So, susceptibility to pathogens that require a strong TH1-cell-mediated response for clearance could be altered by manipulating Notch receptor–ligand inter-actions. As noted previously, although Notch signalling induced by plate-bound DLL1–Fc induced Hes1 gene transcription as measured by real-time PCR, in this study, the ability of synthetic ligand fusion constructs to act as Notch agonists remains controversial.

A subsequent report from our laboratory concurs with the above findings, indicating that Notch signalling in CD4+ T cells facilitates a TH1-cell response24. By an in vitro approach using purified CD4+ T cells, we showed that decreased Notch signalling resulted in decreased IFNγ production by cells cultured in TH1-cell-polarizing conditions. We also showed that intact Notch signalling was required within the first 48 hours of the TH-cell lineage decision-making process, as cells that were pre-treated with a single dose of an inhibitor of γ-secretase lost expression of Tbx21 (the gene that encodes T-bet) by 48 hours of culture under TH1-cell-polarizing condi-tions. This was comparable to the loss of Tbx21 gene expression at the same time point in cells cultured under conditions that favoured TH2-cell differentiation (but not pre-treated with a γ-secretase inhibitor). When the γ-secretase inhibitor is used to block Notch activity, one cannot fully rule out its effects on off-target proteins that are also cleaved by γ-secretase113. However, the fact that expression of Tbx21 was restored in cells pre-treated with γ-secretase inhibitor after retroviral transfection with a construct encoding intracellular Notch1, even in those cells cultured under TH2-cell-promoting condi-tions, indicates that active Notch1 can direct TH-cell dif-ferentiation towards a TH1-cell phenotype. Furthermore, both intracellular Notch1 and CSL could be found in complexes bound to DNA on the Tbx21 promoter of a stimulated, intracellular-Notch1-expressing T-cell line.

Together, these data point to a T-cell-intrinsic require-ment for Notch signalling early during TH1-cell polariza-tion. In vivo support for this hypothesis was provided by studying the effects of administration of the γ-secretase inhibitor before induction of experimental autoimmune encephalomyelitis (EAE), a TH1-cell-mediated mouse model of multiple sclerosis. Mice that were pre-treated with this pharmacological inhibitor had attenuated symptoms of EAE, with reduced delayed-type hypersen-sitivity responses on antigen rechallenge, and decreased infiltration of demyelinating lymphocytes into the spinal cord, compared with control mice24.

Regulation of TH2-cell differentiation. Data from fur-ther studies114,115 indicate that Notch proteins might also function in directing TH2-cell differentiation, with one report indicating that Notch ligand specificity might influence T-cell effector fates114. Consistent with the previous two reports24,64, incubating CD4+ T cells with APCs that express DLL1 induced IFNγ secretion and TH1-cell differentiation, whereas those T cells co-cultured with Jagged1-expressing APCs adopted a TH2-cell fate, producing IL-4 and IL-5 (REF. 114). The lat-ter process was further explored and shown to, at least partially, require CSL (RBP-J), as IL-4 production was decreased in mouse CD4+ T cells deficient for RBP-J. Partial rescue of IL-4 production was seen after adding exogenous IL-4 to cultures, which indicates that RBP-J might function upstream of the actions of IL-4, and that RBP-J-independent mechanisms might also control IL-4 production. Finally, various IL-4-reporter trans-genes containing RBP-J-binding regulatory sites were activated when intracellular Notch1 constructs were retrovirally expressed in CD4+ T cells from mice express-ing the IL-4-reporter transgenes. These data indicate that intracellular Notch proteins provide an instructive signal that regulates IL-4 production. A further report outlining a role for Notch proteins in regulating IL-4 production also examines the 3′ enhancer region of IL4 and its regulation by RBP-J26. This study used RBP-J-deficient mice expressing various IL4 enhancer elements to drive expression of green fluorescent protein, and the authors concluded that IL-4 expression by naive natural killer T (NKT) cells and by a subset of memory CD4+ T cells is positively regulated by Notch signalling and requires RBP-J. These data were further supported by an in vivo study involving transgenic mice expressing a dominant-negative form of MAML1 (REF. 115). These animals, which have normal thymocyte development and normal peripheral T-cell function, mounted a TH1-cell-mediated response that was sufficient to clear infection with L. major. However, they were unable to eliminate fully the worm burden after infection with the intestinal parasite Trichuris muris, which requires a robust TH2-cell-mediated response.

Yet another study used mice in which Notch1 was conditionally deleted from CD4+ T cells using a Cre–loxP approach. Data from these experiments indicated that Notch1 was not required for either TH1- or TH2-cell differentiation of polarized CD4+ T cells in vitro, or for TH1-cell-mediated responses in vivo, but this study did

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TolerizationAcquired or induced tolerance describes a lack of responsiveness by the immune system to exogenous antigens, a state that is normally created through experimental manipulation. It is important in organ transplantation, when the body is ‘forced’ to accept an organ from another individual.

Linked suppressionThe phenomenon of suppressing immune responses to a specific antigen by co-presenting it simultaneously with another antigen, against which tolerance has previously been established.

Graft-versus-host responseA deleterious process during which immunocompetent donor cells contained in transplanted tissue recognize immunosuppressed host tissues as ‘foreign’ and mount a destructive immune response against them.

Graft-versus-malignancy responseSimilar to a graft-versus-host response, but the cells targeted for destruction in the host are tumorigenic or leukaemic.

not address the possibility of redundancy of signalling between other Notch receptors116.

Given the markedly disparate conclusions of the stud-ies that have examined the role of Notch signalling in TH-cell differentiation, as well as the varied approaches used to answer this question, it is clear that much work remains to be done before a consensus can be reached.

Induction of a regulatory T-cell phenotype. Further complicating the role of Notch proteins in fine-tuning peripheral T-cell responses are data indicating that Notch signalling can impose a regulatory phenotype on responding T cells (FIG. 4). But here, too, data seem to be conflicting with regard to the mechanism by which Notch proteins might mediate this process.

An initial report showed that the expression of DLL1 was transiently upregulated by CD4+ T cells in mice treated with high-dose intranasal administration of a pep-tide allergen117. Signalling through the DLL1–Notch axis seemed to be responsible for the ensuing T-cell tolerization, as transfer of peptide-specific CD4+ T cells transfected with the gene encoding DLL1 could prevent the proliferation of antigen-primed T cells and attenuate an inflammatory response through a process known as cell–cell-mediated linked suppression. Subsequently, increased numbers of transcripts encoding DLL1 were found in a population of naturally occurring human CD4+CD25+ regulatory T cells, after stimulation with CD3- and CD28-specific antibodies118. Here, too, functional suppression required cell–cell contact, and it seemed to be independent of any inhibitory action by APCs. Follow-up studies showed that APCs can indeed influence T-cell fate. APCs engineered to overexpress Jagged1 and co-cultured with naive CD4+ T cells induced a regulatory T-cell phenotype, which could inhibit both primary and secondary T-cell responses119. Furthermore, these regulatory T cells provided antigen-specific tolerance when transferred to naive mice before antigen challenge.

These data were confirmed and expanded using human cells in a study that explored the regulatory effects of a human B-lymphoblastoid cell line infected with Epstein–Barr virus (EBV) and transfected with a construct encoding Jagged1 (REF. 120). In concordance with the mouse studies, co-culture of Jagged1-expressing B cells with autologous T cells induced a regulatory T-cell phenotype when the T cells were exposed to EBV-derived antigens. Surprisingly, the suppression was not limited to the actions of CD4+CD25+ regulatory T cells, but included contributions from a CD8+CD25– T-cell population also. Further investigation by this group showed that this suppressive phenomenon was antigen specific and did not prevent responses to antigens pre-sented by third-party stimulator cells, which indicates an intriguing means of manipulating T-cell responses to avoid deleterious graft-versus-host responses while maintaining desirable graft-versus-malignancy responses in the context of allogeneic haematopoietic-stem-cell transplantation121.

Using a mouse model of cardiac allograft rejection, the induction of regulatory T cells by manipulation of Notch-ligand expression has been further examined122.

In these experiments, constitutive expression of DLL1 by APCs that also expressed alloantigen prevented an immunogenic response and significantly prolonged allograft survival. This response was characterized by decreased IFNγ production and increased IL-10 secre-tion, and it induced a regulatory CD8+ T-cell population, as depletion of CD8+ T cells abrogated the suppression.

Finally, a role for Notch3 in mediating suppression by regulatory T cells has been indicated in transgenic mice that express activated Notch3. These mice have an increased percentage of CD4+CD25+ regulatory T cells and are refractory to the induction of experi-mental autoimmune diabetes when treated with strep-tozotocin123. Although the contribution of a specific Notch ligand was not addressed in this investigation, Notch3 expression seemed to modulate cytokine secre-tion, as both IL-4 and IL-10 levels were increased in Notch3-transgenic mice.

Consensus, clarification and consistencyWhat consensus, if any, can be reached from the results of the studies carried out so far? Are Notch proteins required for the polarization of CD4+ T cells to TH-cell lineages? What role do Notch proteins have in the de novo generation of regulatory T cells? How does the timing of Notch expression affect these pathways? Do Notch proteins have a crucial function in all of these effector pathways; in some, but not others; or in none at all?

Emerging data in this fast-moving field lend support to an expanded model of how Notch proteins might regulate target genes, one that builds on the canonical model of CSL-mediated co-repression to incorporate co-activation mediated by intracellular-Notch–CSL, and which also serves to unify the disparate findings of recent studies.

Why do RBP-J-deficient T cells spontaneously dif-ferentiate towards a TH1-cell, IFNγ-producing pheno-type but have no upregulation of Hes1 or Hes5 gene transcription35? HES-family genes are well described as being regulated by Notch proteins77, and they are now considered to be targets of canonical Notch–CSL-mediated regulation. That is, in the absence of Notch proteins, there is only basal transcription of HES-family genes but, once recruited to the CSL complex, intracel-lular Notch proteins and other co-activators, including MAML1, can act not only to de-repress the promoter, but also to actively transcribe it. The IL4-gene regula-tory locus might also be regulated in this manner and, without the Notch–CSL interaction, active transcrip-tion of these sites might not occur. This mechanism would explain both why no increase in HES-family gene expression is seen in RBP-J-deficient mice, and why decreased IL-4 production is noted in studies using these animals, as well as in mice transgenic for a dominant-negative form of MAML1. In the studies that examined the intrinsic role of Notch proteins in TH-cell differentiation, adding exogenous IL-4 to polarizing cultures might have circumvented this mechanism by providing differentiation signals that are downstream of and, perhaps, in addition to CSL24,64.

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In regulating other gene promoters, however, does RBP-J act simply to repress transcription? If so, is it conceivable that loss of RBP-J de-represses these sites and allows positive regulation by other transcription factors, such as NF-κB? Is this the mechanism respon-sible for the regulation of Ifng, Tbx21 and/or Hey1 gene expression? NF-κB signalling has been implicated in driving TH1-cell responses by regulating IFNγ produc-tion and T-bet expression124,125. Intracellular Notch1 can regulate IFNγ production through NF-κB23. So, it is pos-sible that this Notch1 signalling pathway remains intact in mice expressing dominant-negative MAML1, at least at a threshold level. Although the construct encod-ing dominant-negative MAML1 lacks the carboxy-terminal domain of MAML1 that is responsible for recruiting p300 and PCAF, there are data indicating that, at some promoters, NF-κB might recruit p300 for opti-mal transcription126. In addition, Notch-independent MAML1 signalling has been described, although not in the immune system127. Adding further support to the notion of differential regulation of target genes through a Notch-mediated NF-κB-dependent mechanism are data indicating that different combinations of NF-κB subunits bind to Ccnd1 (the gene encoding cyclin D1), Bcl2a1 (the gene encoding BCL-2A1) and the Il7a promoter in transgenic animals expressing activated intracellular Notch3 in the presence or absence of the pre-TCR128. These observations indicate that input

signals at the level of the (pre-)TCR might subtly but significantly influence biological responses that are ultimately regulated by Notch signalling.

How, too, do Notch proteins induce a regulatory phenotype in some instances? In many of the studies exploring this aspect of Notch signalling, constitutive expression of a specific Notch ligand by APCs was a unifying component. Does the inability to down-regulate Notch ligands interrupt the bidirectional communication between a T cell and an APC, which ultimately ‘desensitizes’ the nearby T cells and pro-motes a tolerogenic response?

Before these seemingly disparate findings can be fully reconciled, a close examination of the experimental details must be carried out. Constructs, inhibitors and experimental approaches all differ between the studies discussed, and this presumably has contributed to the diversity of the results. Once the experimental systems are more clearly defined and consistently implemented, working models can be tested and emerging hypotheses will hopefully follow.

Notch signalling in peripheral T cells is an area of investigation that is still in its infancy. As such, it is bound to experience growing pains as it matures. As researchers in the field move towards standardized approaches and the consistent use of models, they will undoubtedly move closer to a consensus opinion as to how Notch proteins mediate their pleiotropic effects.

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AcknowledgementsWe wish to thank members of the Osborne laboratory for comments and conversations, and R. Goldsby for critical review and support. We also thank L. Miele and T. Golde for continued collaborations and discussions over the past sev-eral years. We apologize to all of those investigators whose data could not be cited owing to space considerations. B.A.O. and L.M.M. are supported by grants from the United States National Institute of Allergy and Infectious Diseases and National Institute of Aging.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCSL | DLL1 | DLL3 | DLL4 | Jagged1 | Jagged2 | MAML1 | Notch1 | Notch2 | Notch3 | Notch4 | RBP-J

FURTHER INFORMATIONBarbara Osborne’s homepage: http://www.umass.edu/vasci/faculty/osborne/osborne.htmlAccess to this links box is available online.

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notch signalling during peripheral t-cell activation and differentiation

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