The Notch and Sonic hedgehog signalling pathways in immunity

Molecular Immunology 41 (2004) 715–725

The Notch and Sonic hedgehog signalling pathways in immunity Robert A. Benson, Jacqueline A. Lowrey, Jonathan R. Lamb, Sarah E.M. Howie∗ Immunobiology Group, MRC Centre for Inflammation Research, College of Medicine and Veterinary Medicine, University of Edinburgh, Medical Buildings, Teviot Place, Edinburgh, EH8 9AG, UK

Abstract There is an increasing body of knowledge demonstrating that genes involved in cell fate decisions during development also play a role in the continuous cell fate decisions made by the mature immune system in response to foreign antigen. This review concentrates on the role of the Notch and Sonic hedgehog (Shh) signalling pathways in the development and function of CD4+ T lymphocytes. © 2004 Elsevier Ltd. All rights reserved. Keywords: Notch; Sonic hedgehog; CD4+ T cells; Cytokines; Thymocytes

1. Introduction Protective immunity depends on appropriate priming of CD4+ T cells by antigen presenting cells (APCs). Under normal circumstances, this is true regardless of how the final effector function is mediated. Activation via the T cell receptor (TCR) provides antigen specificity while the influence of TCR/MHC/peptide avidity, co-stimulatory signals such as ligation of CD28, CD40L, OX40, ICOS, CTLA-4, and cytokines determine whether CD4+ T cells differentiate as Th1/Th2, memory or regulatory/anergic cells (Santana and Rosenstein, 2003; Kaech et al., 2002; Lechler et al., 2001). Flexibility within the immune system allows differentiation between harmful and innocuous antigens, and cell fate decisions are made to produce the appropriate response. The identification of components of the Notch and Sonic hedgehog (Shh) signalling pathways in cells of the immune system has shed new light on how the specification of T effector cell phenotype might be achieved. 2. Notch signalling and immunity The Notch pathway is classically associated with cell fate choices during development (Artavanis-Tsakonas et al., 1999), but the effects of Notch signalling are now known to extend beyond embryonic patterning and into regulation of the mature immune system. Notch signalling regulates differentiation of macrophages, dendritic cells (DCs) and lymphocytes (Masuya et al., 2002; Ohishi et al., 2001; ∗

Corresponding author. Fax: +44-131-650-6528. E-mail address: [email protected] (S.E.M. Howie).

0161-5890/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2004.04.017

Cheng et al., 2003; Tanigaki et al., 2003). A large body of evidence details the critical role of Notch in controlling thymocyte development where it promotes T versus B lymphocyte lineage commitment, determines T cell receptor usage and influences expression of CD4 and CD8 (Guidos, 2002; Doerfler et al., 2001; Hadland et al., 2001) (Fig. 1). However, this review will concentrate on how Notch signalling influences the function of mature CD4+ T cells and how this may contribute to both immunity and tolerance.

3. The Notch signalling pathway The mammalian Notch family consists of four receptors, Notch1, 2, 3 and 4 (Baron, 2003). The mature receptor is generated from a single polypeptide cleaved (S1 cleavage event) by a furin-like convertase (Logeat et al., 1998), producing the functional heterodimeric single-pass transmembrane receptor. The extracellular domain consists of multiple epidermal growth factor (EGF)-like repeats and three Lin12/Notch/Glp-1 (LNG) domains. Binding of the ligand DSL (Delta/Serrate/Lag) domain to Notch EGF repeats induces signal transduction (Fig. 2) by allowing disintegrin–metalloprotease-mediated release of the Notch extracellular domain (S2 cleavage) (Brou et al., 2000). Subsequent presenilin-dependent ␥-secretase cleavage (S3 cleavage) of the remaining Notch receptor releases the intracellular domain (NICD) (De Strooper et al., 1999) which contains two nuclear-localisation sequences. Once inside the nucleus, NICD binds CBF-1/RBP-J␬ via the RAM23 domain and ankyrin repeats. Binding of NICD converts CBF-1 from a transcriptional repressor to an activator by

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Fig. 1. Notch and Shh signalling and thymocyte development. A simplified over-view of thymocyte development is shown, indicating stages where Notch or Shh signal transduction has been shown to promote a particular fate commitment. Thymocyte development is strongly influenced by a combination of TCR and Notch signalling. Arrows indicate where development is promoted by Notch signalling. Common lymphoid progenitors (CLPs) can develop as either B cells or T cells in the bone marrow or thymus, respectively. Development of CD4-, CD8-, TCR- (DN, double negative) cells is promoted by Notch signalling, promoting expansion of the DN1 population. At this time the first TCR gene rearrangements are made. Enhanced Notch signalling at this stage increases the number of cells eventually expressing ␣␤ TCRs at the expense of ␥␦ expression. The role that Notch has in determining development of MHC class I or class II restricted T cells is controversial. However, it appears that Notch1 signalling promotes CD8 development in the absence of MHC class I but not CD4 in the absence of class II. For details of Shh receptor expression, see text.

displacing histone deacetylases and recruiting histone acetylases along with co-activators such as Mastermind-like1 and SKIP (Bray and Furriols, 2001). Typically, this pathway activates transcription of the basic helix-loop-helix (bHLH) proteins hairy-enhancer of split family (HES and HERP) (Iso et al., 2003). Notch can also signal independently of CBF-1 via a less well-defined pathway involving the zinc-finger-containing cytoplasmic protein Deltex. The role of Deltex in augmenting or inhibiting Notch signalling remains to be clarified, but it is able to bind Grb2 and modulate Ras and consequently c-Jun N-terminal kinase (JNK) activity (Ordentlich et al., 1998; Matsuno et al., 1998).

Fig. 2. Notch signal transduction. Binding of ligand by Notch allows receptor cleavage by a metalloprotease (possibly TACE). The intra-membrane region of the receptor is then cleaved by the ␥-secretase complex, releasing the Notch intra-cellular domain (NICD). NICD translocates to the nucleus where it binds to CBF-1 and displaces co-repressors and recruits co-activators inducing transcription of hes1. Signalling by Notch through the Deltex pathway is little understood but does inhibit the Ras/MAPK pathway through an interaction with Grb-2.

rified peripheral CD4+ T cells can be seen using RT-PCR (Benson et al., submitted manuscript). The highly conserved nature of this pathway has made generation of high-affinity antibodies difficult. However, Notch1 has been identified in unstimulated spleen and lymph node cells by western blot (Palaga et al., 2003) and on purified CD4+ T cells by immunofluorescence (Benson et al., submitted manuscript). Activation of these cells with anti-CD3 and anti-CD28 antibodies enhances expression of Notch1 (Palaga et al., 2003; Adler et al., 2003). Real-time RT-PCR confirmed this but also revealed differential expression of the other Notch family receptors by purified CD4+ T cells (Adler et al., 2003). Notch1, 2, 3 and 4 expression was up-regulated after 16 h (Adler et al., 2003). Our own findings show that notch3 and 4 expression is down-regulated 24 h post-stimulation (Benson, unpublished observation).

5. Notch signalling in activated T cells 4. Expression of Notch components by CD4+ T cells Gene expression of notch1 and jagged1 (Hoyne et al., 2000) as well as notch2, 3 and 4, delta1 and jagged2 by pu-

Expression of the Notch target gene hes1 is up-regulated upon CD4+ T cell activation by western blot, RT-PCR and real-time RT-PCR (Palaga et al., 2003; Adler et al., 2003;

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Benson et al., submitted manuscript). The significance of this is unclear but is likely to be related to increased Notch receptor expression and enhanced signal transduction observed as elevated free NICD (Adler et al., 2003).The first important steps in identifying expression of Notch pathway components in T cells have been taken; however, much remains to be done. Current literature relates primarily to expression of Notch and particularly Notch1 by CD4+ T cells. It has been shown that APCs express Notch ligands and as such, it has been assumed that they deliver Notch signals to T cells. Our own data demonstrate presence of transcripts for jagged1, 2 and delta1 in purified CD4+ T cells. Human CD25+ CD4+ T regulatory cells have been shown to express high levels of delta1 (Ng et al., 2001) and preliminary evidence suggests that CD4+ T cell sub-populations have differential expression of other Notch ligands (Benson, unpublished observation). This raises the possibility that some T cells may also be capable of influencing other cell types by delivery of Notch signals. 6. Notch and CD4+ T cell activation Immunological synapse (IS) formation stabilises interactions between an APC and CD4+ T cells (Bromley et al., 2001). This promotes TCR signalling and recruitment of co-stimulatory molecules. We have demonstrated that Notch1 and CD4 co-localise on activated but not resting CD4+ T cells (Benson et al., submitted manuscript), indicating recruitment of Notch to the IS and a potential interaction with TCR/co-stimulatory signals. In support of this, Notch signalling is known to influence signalling pathways also utilised in T cell activation such as Ras, AP-1 and NF␬B (Ordentlich et al., 1998; Chu et al., 2002; Cheng et al., 2001). The first direct evidence for an interaction between TCR and Notch signalling came from the observation that NICD1 over-expression in thymocytes reduced CD25 and CD69 expression, indicating that Notch signalling inhibited activation (Izon et al., 2001). The same NICD1 construct was demonstrated as inhibiting NFAT/AP-1 promoter activity upon TCR stimulation in a Jurkat cell line (Izon et al., 2001). Based on this finding, it might be expected that enhanced Notch signalling in mature CD4+ T cells would hamper activation, block IL-2 gene transcription and eventually limit T cell proliferation. Inhibitors of ␥-secretase activity were initially developed as a therapy for Alzheimer’s disease. They have also been demonstrated to be potent inhibitors of Notch signalling (Adler et al., 2003). Inhibition of the ␥-secretase complex prevents release of NICD upon ligand binding, and hence blocks signal transduction. Blockade of Notch signalling in mature CD4+ T cell activation has produced some exciting results, although somewhat contradictory to those discussed earlier (Izon et al., 2001). Inhibition of Notch signalling does not enhance proliferation and IL-2 secretion; indeed quite the

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opposite was demonstrated upon treatment of whole spleen and lymph node preparations with ␥-secretase inhibitors and anti-CD3 antibody (Palaga et al., 2003; Adler et al., 2003). Reduced CD4+ T cell proliferation was observed in the absence of Notch signalling, accompanied by reduced IL-2 secretion. IL-2 secretion by anti-CD3 antibody treated splenocytes from Notch1 antisense transgenic (Tg) mice was found to be significantly reduced but proliferation was unaffected (Palaga et al., 2003). This may indicate that Notch1 is itself not responsible for regulation of proliferation or may simply reflect a degree of redundancy between the four mammalian Notch receptors. Retroviral mediated over-expression of NICD1 in CD4+ T cells enhanced antigen-specific proliferation; however, no reference to IL-2 secretion was made in this experiment. Anti-CD3 and anti-CD28 antibody induction of IL-2 transcription involves activation of NFAT, AP-1 and NF␬B. Electromobility shift assays (Palaga et al., 2003) and the use of ovalbumin (OVA)-specific DO11.10 TCR Tg hybridomas transfected with an NFAT/GFP reporter construct (Benson, unpublished observation) have revealed that inhibition of Notch does not affect NFAT activity. However, NF␬B activity is dramatically reduced (Palaga et al., 2003), consistent with other findings demonstrating enhanced activity of this transcription factor when T cells over-express NICD3 (intracellular domain of Notch3) (Bellavia et al., 2000) and is decreased in haemopoietic progenitors from Notch1 antisense mice (Cheng et al., 2001). While AP-1 activity has not been assessed in T cells in the absence or presence of enhanced Notch signalling, evidence suggests that active Notch1 inhibits AP-1 activity in several cell lines (Chu et al., 2002). This may relate to inhibition of NFAT/AP-1 activity by NICD1 in Jurkat cells (Izon et al., 2001); however, it does not correlate with the enhanced proliferation seen in T cells retrovirally transduced with NICD1 (Adler et al., 2003), given that diminished AP-1 activity is associated with CD4+ T cell anergy (Macian et al., 2002). Interestingly, our own data do not suggest that Notch signalling is necessary for CD4+ T cell proliferation. Treatment of cells with the ␥-secretase inhibitor MW167 did not reduce proliferation (Benson et al., submitted manuscript), whereas use of IL-CHO or compound E did (Palaga et al., 2003; Adler et al., 2003). This may relate to slightly different activities between the inhibitors, reflecting possible biases in target specificities. Indeed, proliferation of T cells from Notch1 antisense mice was unaffected (Palaga et al., 2003). A more in-depth study of the biological functions of different ␥-secretase inhibitors may facilitate further dissection of the roles played by different Notch receptors in T cell activation. Addition of IL-2 to ␥-secretase inhibitor treated cultures did not restore proliferation to anti-CD3 antibody (Adler et al., 2003). Flow cytometric analysis revealed down-regulation of CD25 expression in the absence of Notch signalling, suggesting that an inability to respond to IL-2 was responsible for reduced proliferation (Adler

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et al., 2003). What may make these findings conflict with those from thymocyte reports is the fact that ␥-secretase inhibitors are believed to block signalling from all four Notch receptors, where over-expression experiments detail the effect of one specific receptor. It may also reflect effects of inhibition of other ␥-secretase substrates that have a role in T cell activation. Contradictory evidence to the idea that increasing Notch signalling enhances proliferative responses comes from the use of a recombinant Delta1–Fc fusion protein. Delivery of Notch signals using this recombinant ligand did not affect T cell proliferation at doses affecting cytokine production (Maekawa et al., 2003). Addition of high concentrations of Delta1–Fc inhibited proliferation (Maekawa et al., 2003), the opposite effect to NICD1 retroviral transduction of CD4+ T cells (Adler et al., 2003). This may be a result of NICD1 over-expression representing particularly artificial concentrations of Notch signalling. Alternatively, differential signals may be derived from different Notch receptors since Delta1–Fc can potentially activate all four Notch receptors. The binding of ligand may also induce a qualitatively different signal in comparison to simply over-expressing truncated Notch receptors. However, this does not detract from the fact that Notch signalling can influence T cell proliferation in response to TCR stimulation. Apparent contradictions as to how Notch may affect T cell activation may stem from the differences in TCR signalling strength and consequent masking of any effect Notch may exert. This was demonstrated using ␥-secretase inhibitor treated DO11.10 TCR Tg mononuclear cells (Adler et al., 2003). At low dose OVA, cells proliferated less in the absence of Notch. Increasing the concentration of OVA restored proliferation in the absence of Notch. This suggests that Notch functions as a co-stimulatory molecule increasing the sensitivity of TCR signalling. Our own data also support the idea of Notch as a co-stimulator, being required for CD4+ T cell secretion of IFN␥, TNF␣, IL-4 and IL-5 in the absence of CD28 co-stimulation (Benson et al., submitted manuscript), and is consistent with Notch activation of NF␬B activity (Bellavia et al., 2000; Cheng et al., 2001; Palaga et al., 2003). Although no effect on proliferation is seen in the absence of Notch when strong TCR signals are induced, it would be of value to examine other aspects of the system. Notch signalling can rescue DO11.10 hybridoma cells from TCR induced apoptosis by inhibiting activity of the orphan nuclear steroid receptor Nur77 (Jehn et al., 1999). Expression of Nur77 is a feature of activation-induced cell death (van den Brink et al., 1999), a key mechanism in the termination of T cell responses (Green et al., 2003). TCR mediated stimulation can result in proliferation or apoptosis depending on additional signals present at the time of T cell activation (van den Brink et al., 1999). Enhanced Notch signalling may promote survival of activated T cells, maintaining immune responses.

7. Notch and T effector function The effector function of CD4+ T cells reflects their ability to activate and direct activities of other immune system cells. This is predominantly mediated through cytokine secretion. Since Notch is capable of manipulating TCR signal transduction, specifically NF␬B activity, it seems reasonable that Notch signalling may influence polarisation of Th1/Th2/T regulatory cell phenotypes. The first publication detailing how Notch signalling could influence T cell function utilised Serrate1 transfected dendritic cells as APCs. Mice were immunised with either Serrate1 (Jagged1) transfected or control dendritic cells pulsed with the house dust mite antigen Der p1 (Hoyne et al., 2000). Antigen presentation in the context of Serrate resulted in tolerance, which was transferred by CD4+ T cells, suggesting generation of antigen-specific regulatory T cells. Unfortunately, these cells have not been characterised in terms of cytokine secretion or expression of Notch components, so it is difficult to say whether Notch signalling directly induced T regulatory cells or whether it altered APC function and hence activation of the T cells. Our own data suggest that Notch signalling may be important for tolerance since anti-CD3 and anti-28 antibody activated CD4+ T cells treated with ␥-secretase inhibitors failed to secrete IL-10 (Benson et al., submitted manuscript). IL-10 has several immunoregulatory functions including inhibiting the function of APCs, eosinophils and macrophages, and acting directly on T cells to inhibit their activation. IL-10 is also associated with the generation and function of Tr1 regulatory CD4+ T cells (Groux, 2001). Consistent with Notch signalling inducing tolerance, use of allogeneic EBV-lymphoblastoid B cells over-expressing Jagged1 reduced allo-induced proliferation of CD4+ T cells and was associated with reduced levels of IFN␥, IL-2 and IL-5 (most likely due to cell numbers) (Yvon et al., 2003). CD4+ T cells activated in this way produced increased levels of TGF␤. IL-10 secretion appeared unchanged even in the absence of proliferation, perhaps suggesting that more was produced per cell. This cytokine phenotype most likely relates to generation of an antigen-specific Th3 regulatory population, capable of transferring hyporesponsiveness to fresh cultures yet not affecting responses to third party antigens (Yvon et al., 2003). These regulatory cells also expressed higher levels of hes1 transcripts than cells stimulated with normal allogeneic cells, indicating that they had received increased Notch signal transduction. Mice transgenic for a lck promoter-driven constitutively active intracellular domain of Notch3 develop T lymphomas (Bellavia et al., 2000). Transgenic thymocytes failed to down-regulate CD25, pre-T␣ and had elevated levels of NF␬B activity (Bellavia et al., 2000). These mice then developed aggressive T cell lymphoblastic lymphomas. However, the transgene provided protection against induction of experimental autoimmune diabetes (Anastasi et al., 2003). NICD3 transgenic spleens contained significantly

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higher levels of CD25+ CTLA-4+ CD4+ T cells, a phenotype shared by naturally occurring T regulatory cells. The NICD3 CD25+ CD4+ T cells were also found to express higher amounts of IL-10, at both transcript and protein levels (Anastasi et al., 2003). Additionally, adoptive transfer of NICD3 transgenic CD4+ T cells could prevent recipient mice developing diabetes. Whether these cells do represent the naturally occurring thymus derived T regulatory cells is uncertain. Stimulation of transgenic spleens also induced elevated levels of IL-4, so perhaps protection against diabetes in this system is due to lack of Th1 polarisation (Anastasi et al., 2003). The data however do strengthen the link between Notch and CD25 expression. It might be speculated that differential Notch signals are induced when cells undergo altered negative selection, a process thought to generate naturally occurring CD25+ CD4+ T regulatory cells. Whether this signal is directly responsible for generating T regulatory cells is not clear; however, differential expression of Notch pathway associated genes has been observed in both human and mouse CD25+ CD4+ T regulatory cells (Ng et al., 2001; Anastasi et al., 2003; Benson, unpublished observation). The use of a recombinant Delta1–Fc fusion protein has been reported to inhibit secretion of pro-inflammatory cytokines by activated CD4+ T cells. Delivery of Notch signalling in this context enhances secretion of IL-10, with the resulting polarised population having a cytokine profile reminiscent of Tr1 cells, but it is unclear whether these cells have regulatory capacity (Mckenzie et al., 2003). In a different system, high doses of Delta1–Fc inhibited proliferation of CD4+ T cells. This aspect of Delta induced Notch signalling has not been fully explored but seems not to be IL-10 or TGF␤ mediated (Maekawa et al., 2003). Interestingly, cells activated in the presence of this Delta1–Fc protein developed Th1 like cytokine profiles with high IFN␥ secretion (Maekawa et al., 2003). Delta1–Fc could even induce CD4+ T cell secretion of IFN␥ in unstimulated cultures and under Th2 promoting conditions (Maekawa et al., 2003). This phenotype was mimicked by over-expression of the Notch3 intracellular domain (Maekawa et al., 2003) in direct contrast to a previous report detailing that Notch3 signalling promoted regulatory T cell generation (Anastasi et al., 2003). Palaga et al., (2003) showed that the ␥-secretase inhibitor IL-CHO reduced secretion of IFN␥ by murine CD8+ T cells. This was related to a lower level of NF␬B activity in the absence of Notch signalling. Our own data also suggest that IFN␥ secretion by anti-CD3 antibody activated CD4+ T cells is reduced when Notch signalling is attenuated; however, levels are restored on addition of CD28 co-stimulation, perhaps reflecting the ability of this pathway to increase NF␬B activity (Benson et al., submitted manuscript). However, our data show that this is also true for TNF␣, IL-4 and IL-5 secretion (Benson et al., submitted manuscript), suggesting that Notch signalling promotes secretion of a number of cytokines.

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Certain trends are becoming evident pertaining to the role of Notch as a co-stimulatory molecule. The instructions delivered by this signalling pathway likely reflect involvement of specific receptors with specific ligands. Indeed, based on the available literature, one might say that induction of Notch signalling by Jagged promotes T regulatory cell generation, whereas Delta promotes Th1 cell development. Understanding the role played by regulators of Notch signalling will prove vital in developing a clear picture of Notch signalling in T cells. One such family of Notch regulators, the activity of the Fringe family of glycosyltransferases (Haines and Irvine, 2003), may determine the difference between immune response and tolerance by influencing the ability of Notch receptors to bind specific ligands. Other regulators of Notch signalling, such as Numb, Dishevelled and Deltex, may also function to direct T cell activation. What is clear is that Notch signalling is important to the function of mature CD4+ T cells and as such presents many new possibilities for control of the immune response.

8. Shh signalling Hedgehog (Hh) proteins are a highly conserved family of intercellular signalling molecules (Hammerschmidt et al., 1997; Ingham, 1998). Originally identified as a Drosophila segment polarity gene required for embryonic patterning (Nusslein-Volhard and Wieschaus, 1980), several vertebrate homologues have now been discovered—Indian (Ihh), Desert (Dhh) and Sonic Hedgehog (Shh), the most extensively characterised. Shh is synthesised as a 45 kDa precursor protein which undergoes autoproteolysis to yield a ∼20 kDa N-terminal domain (Shh-N) and a ∼25 kDa C-terminal domain (Shh-C) (Fig. 3a). The signalling activity of Shh resides only in the N-terminal domain, whilst the C-terminal domain is responsible for the autoprocessing (Lee et al., 1994; Bumcrot et al., 1995). Shh-C is thought to act as a cholesterol transferase during the processing, allowing a cholesterol modification of Shh-N at its C terminus. Palmitoylation also takes place at the N-terminus of Shh-N to give Shh-Np; in this way, Shh-Np can remain membrane-associated and function as a short range signalling molecule interacting with neighbouring cells (Pepinsky et al., 1998). There is also evidence for a freely diffusible form of Shh-Np (termed s-Shh-Np) that mediates long range signalling. The release of the cholesterol modified and therefore hydrophobic Shh-Np from the cell membrane is thought to involve a multimerisation step in order to bury its lipid moieties in the hydrophobic surface of an adjacent Shh protein. A transmembrane protein Dispatched (Disp) has been shown to be required for the release of the cholesterol modified Shh-Np (Porter et al., 1995; Burke et al., 1999; Williams et al., 1999). Shh interacts with a receptor complex comprised of two transmembrane proteins patched (ptc) and smoothened (smo) (Fig. 3b). Ptc is the Shh ligand-binding sub-unit and

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contribute to lymphoid cell development and differentiation (Outram et al., 2000; Bhardwaj et al., 2001). Recently, it has also been demonstrated that Shh-mediated signalling is a physiological component of both murine and human peripheral T cell responses (Lowrey et al., 2002; Stewart et al., 2002). Finally, the Shh signalling pathway has been found to be involved with several immunopathological disorders (Stewart et al., 2003; Wang et al., 2003). It is the association of Shh with these different aspects of immune function that is reviewed here.

10. Shh in the development of T lymphocytes

Fig. 3. Shh biosynthesis (A) and signalling pathway (B). See text for details.

Smo is the signal transduction component. In the absence of the Shh ligand, ptc interacts with and inhibits smo. However, once Shh binds ptc, this inhibition is released allowing smo to transduces the Shh signal, which is then mediated by the Gli family of zinc finger transcription factors (Murone et al., 1999; Theil et al., 1999).

9. Shh signalling in immunity Shh signalling in vertebrates has been shown to regulate a wide range of developmental processes in a number of tissues including CNS, gut, lung, limbs, pituitary gland, pancreas and skin (Hammerschmidt et al., 1997; Fan and Khavari, 1999; Thomas et al., 2000; Treier et al., 2001; van den Brink et al., 2001). Although there is considerably less information available than for the Notch signalling pathway, the Shh signalling pathway has also been shown to

Outram et al. (2000) investigated the role for Shh in T cell development in the thymus. Components of the Shh signalling pathway, including Shh, ptc, smo and Gli1-3, were expressed in the murine adult and foetal thymus. Both RT-PCR on sorted thymocyte population immunocytochemistry confirmed that Shh was only detected in thymic epithelial cells and not present on any of the thymocyte populations in the adult mouse. During development in the thymus, T cells pass through a series of stages defined by CD4 and CD8 expression (Fig. 1). The most immature CD4− CD8− double negative (DN) thymocytes progress to a double positive (DP) stage and then mature to either a CD4+ or CD8+ single positive (SP) T cell (Haks et al., 1999). ptc RNA was detected in DN, DP and CD8 SP cells. However, smo RNA and protein were only present in the DN thymocyte population. This DN thymocyte population can be sub-divided into several developmental stages defined by CD44 and CD25 expression. The earliest cells are CD44+ CD25− , then CD44+ CD25+ followed by CD44− CD25+ , they then lose both markers becoming CD44− CD25− DN before maturing to DP then CD4+ or CD8+ SP (Haks et al., 1999). Smo was found to be negative in the earliest DN population, cells which are not committed to the T cell lineage, with the highest expression seen in the subsequent CD44+ CD25+ DN population, cells which are committed to the T cell lineage and undergoing rearrangement of the TCR␤ chain gene. The authors conclude that these are the cells which are responding to the Shh signal provided by the thymic epithelium (Outram et al., 2000). Outram et al. (2000) found that treatment of thymus explants with a neutralising anti-Shh antibody 5E1 (which recognises an epitope that overlaps the ptc binding site on Shh and thus blocks signalling by preventing Shh binding to its receptor (Ericson et al., 1996)), increased differentiation from DN to DP thymocytes. Conversely, treatment with the recombinant Shh protein inhibited this differentiation, arresting thymocytes as CD25+ DN cells after initiation of TCR␤ gene rearrangement. The authors suggest that Shh may function to maintain the DN thymocytes in a non-proliferative state as they rearrange their TCR␤ genes. The progression of a DN to a DP thymocyte requires the expression of a functional pre-TCR complex (von Boehmer

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et al., 1999)—this can be overcome by stimulation with anti-CD3 antibodies in vitro (Levelt et al., 1995). Rag1−/− thymocytes arrest at the CD25+ DN stage as they cannot rearrange their TCR␤ genes and so cannot express the pre-TCR complex (Mombaerts et al., 1992). Outram et al. (2000) used this model to demonstrate that ligation of CD3 (i.e. pre-TCR signalling) on Rag1−/− thymocytes causes down-regulation of smo, thus preventing Shh signalling, and the DN thymocyte is released to proliferate and differentiate into the DP stage. Therefore, their findings suggest that Shh signalling plays an important role in T cell development, regulating the progression from a DN thymocyte to a DP thymocyte. Another aspect of the development of the immune system in which Shh has been shown to play a role is haematopoiesis. Bhardwaj et al. (2001) detected genes encoding Shh, ptc, smo and Gli1-3 on primitive stem cells isolated from human blood, and on cells comprising the haematopoietic microenvironment such as bone marrow stromal cells. Shh and its receptors ptc and smo were also detected on committed sub-sets of myeloid and lymphoid (both B and T) cell populations. The differentiation of progenitors into different lineages is controlled by several haematopoietic cytokines such as c-kit ligand, IL-3, -6, -7 and GM-CSF (Dieterlen-Lievre et al., 1998; Domen and Weissman, 1999). Bhardwaj et al. (2001) found a functional role for Shh signalling in the proliferation and differentiation of human primitive stem cells. The primitive stem cells were cultured with an optimised combination of haematopoietic cytokines previously shown to promote proliferation of these stem cells into differentiated cells. It was shown that blocking Shh function via addition of neutralising anti-Shh antibody inhibited this cytokine-induced stem cell proliferation whilst maintaining them in an undifferentiated state. The effect seen with addition of anti-Shh antibody suggests that human primitive stem cells normally produce endogenous Shh via an autocrine or paracrine mechanism. Conversely, it was shown that addition of exogenous Shh enhanced the cytokine-induced proliferation of primitive stem cells. However, this was not at the expense of differentiation, as it was shown that Shh had a proliferative effect on progenitors and in vivo repopulating pluripotent stem cells. Therefore, the authors conclude that Shh signalling regulates the proliferation and differentiation of human stem cells as responsiveness to haematopoietic cytokines is dependent on the presence of soluble or membrane bound Shh protein functioning in an autocrine or paracrine fashion. 11. Shh signalling in CD4+ T lymphocytes The Shh signalling pathway has also been found to play a role in the peripheral immune system both in murine and human T cell effector function (Lowrey et al., 2002; Stewart et al., 2002). Expression of Shh and ptc has been found

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in both resting and activated murine and human peripheral CD4+ T cells (Lowrey et al., 2002; Stewart et al., 2002), human macrophages and CD8+ T cells (Stewart et al., 2002) and secondary lymphoid tissue (Lowrey et al., 2002). Lowrey et al. (2002) showed that addition of exogenous Shh peptide significantly increased the proliferation of anti-CD3/CD28 activated peripheral CD4+ T cells. Cell cycle analysis demonstrated that this was not an anti-apoptotic effect as the percentage of live T cells present in the culture was very similar with and without Shh added. Rather, it was shown that Shh exerted this effect by promotion of CD4+ T cells into the proliferative S/G2 phase of the cell cycle. Shh showed no effect on resting CD4+ T cells. This effect has been reported by several other groups. Shh has been found to induce an increased number of both keratinocytes (Fan et al., 1997) and neuronal precursors (Kenney and Rowitch, 2000) into the S/G2 phase of the cell cycle. They also found that Shh had no effect on resting cells and could only sustain cell cycle progression. It has previously been shown that Shh causes an increased expression of cyclin D1, D2, E, and cdk-2 and cdk-4—all important regulators of cell cycle progression (Fan et al., 1997; Kenney and Rowitch, 2000; Duman-Scheel, 2002). It would be interesting to investigate this in peripheral T cells. Conversely, it was shown that addition of the neutralising anti-Shh antibody 5E1 to activated CD4+ T cell cultures resulted in a dose-dependent inhibition of proliferation (Lowrey et al., 2002). The effect of this blocking antibody suggests that Shh is endogenously produced by activated CD4+ T cells and that this Shh plays a physiological role in the clonal expansion of the T cells. Cell cycle analysis demonstrated that again the percentage of live cells present in the T cell cultures was very similar with and without the addition of the anti-Shh antibody. This blocking antibody exerts its effect by blocking the entry of CD4+ T cells into S/G2 phase of the cell cycle with the majority of cells arresting at G1 phase. We also demonstrated that Shh promoted the up-regulation of bcl2 expression in activated T cells, previously shown by Fan et al. (1997). As this is a necessary step in the generation of memory cells rather than apoptosis of effete effector cells following antigen activation (Mueller et al., 1996; Garcia et al., 1999), it may be that Shh plays a role in the cell fate decision necessary for memory cell differentiation. Therefore, it would appear that endogenously produced Shh may play a role in maintaining normal CD4+ T cell proliferation and survival, and exogenously added Shh enhances this response. In a separate study from our laboratory, this enhanced proliferative effect of Shh was also seen on human peripheral CD4+ T cells (Stewart et al., 2002). Again, no effect was seen on resting CD4+ T cells. Addition of exogenous Shh to activated CD4+ T cells significantly enhanced surface expression of T cell activation antigens CD25 and CD69 and the production of IL-2, IL-10 and IFN-␥. Conversely, addition of the blocking anti-Shh antibody 5E1 to activated CD4+ T cell cultures reduced CD25 and CD69 expression

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and inhibited IL-2 and IFN-␥ production, although IL-10 secretion was unaffected. Thus, in both murine and human CD4+ T cells, Shh-mediated signalling is a physiological component of peripheral T cell responses able to modulate CD4+ T cell effector functions, such as clonal expansion and cytokine production. The fact that Shh had no effect on resting CD4+ T cells suggests that it acts as a co-factor to potentiate T cell activation following ligation of TCR and co-stimulatory receptors. Indeed, we have shown using peripheral CD4+ T cells activated with anti-CD3 and anti-CD28 antibodies and comparing with those activated with anti-CD3 antibody and exogenously added Shh, that Shh can compensate for CD28 in vitro (Lowrey et al., manuscript in preparation). CD28 is a regulator of T cell proliferation, and this function is mediated mainly by the ability of CD28 to co-stimulate production of IL-2 and cell cycle progression (Lenschow et al., 1996; Noel et al., 1996; Kane and Weiss, 2003). The fact that we have demonstrated an up-regulation in both cell cycle progression (Lowrey et al., 2002), CD25 expression and IL-2 production (Stewart et al., 2002; Lowrey et al., manuscript in preparation) with addition of Shh to activated CD4+ T cells suggests that Shh could exert an effect on some component of the CD28 signalling pathway. Indeed, Shh has been shown to increase PI3-kinase activity and phosphorylate AKT (two components in the CD28 signalling pathway) in murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs) (Kanda et al., 2003).

12. Shh and chronic lung inflammation As well as regulation of T cell survival and function, we have also found Shh to be associated with chronic lung inflammation. This is particularly exciting as Shh is necessary for normal lung development (Bellusci et al., 1997; Pepicelli et al., 1998) and this may illustrate an interaction between Shh signalling in two distinct tissue types. In interstitial lung disease (ILD), the complex alveolar structure of the lung is continually damaged and remodelled. This involves interactions between various cell types including epithelial and endothelial cells, fibroblasts, and both resident and recruited cells of the immune system, in which the tissue attempts to replace damaged with new epithelium derived from alveolar type II cells. The continuous remodelling leads to interstitial fibrosis and an accompanying infiltrate composed predominantly of T and B cells (Lympany and du Bois, 1997). In most cases of ILD, the damaging agent is unknown (Turner-Warwick, 1991), but an immunological origin has been presumed (Crystal et al., 1984; Tuder, 1996). It is known that both TGF-␤ and Shh play a role in lung morphogenesis and epithelialisation (Bellusci et al., 1997; Pepicelli et al., 1998; McKarns and Kaminski, 2000). It is also well known that TGF-␤1 is involved in fibrosis (Khalil

et al., 1996; Gauldie et al., 1999). Shh has also been reported to induce epithelial hyperplasia (Bellusci et al., 1997; Fan and Khavari, 1999). However, little is known regarding Shh and damaged epithelia. Stewart et al. (2002) investigated whether the Shh signalling pathway could be involved, like TGF-␤1, in human fibrotic lung disease. They used biopsies from patients with cryptogenic fibrosing alveolitis (CFA) (the most common form of ILD) and bronchiectasis. They also studied two experimental models of lung disease—a murine model of ILD (i.e. a fibrotic lung disease) induced by intra-tracheal installation of fluoroscein isothiocyanate (FITC) and a murine model of allergic airway inflammation (i.e. non-fibrotic) induced by the major house dust mite allergen Der p1. Immunocytochemistry demonstrated that ptc was expressed by alveolar macrophages and infiltrating lymphocytes in all four models examined, and constitutive expression was found at mRNA and protein levels on circulating peripheral blood CD4+ and CD8+ T cells. Furthermore, murine lung epithelial cells express ptc and this pattern does not alter in chronic lung inflammation. In contrast, Shh expression was confined to damaged epithelium at sites of tissue remodelling and fibrosis, but not at sites of normal lung tissue in the models of fibrotic disease. Shh expression was minimal in non-fibrotic inflammation (i.e. the Der p1-induced lung inflammation model). These results suggest that the Shh signalling pathway may play a role in the remodelling of damaged lung epithelium. The fact that the Shh receptor ptc was present on both infiltrating and normal circulating blood lymphocytes suggests that Shh may allow immune cells to react to signals derived from damaged epithelial cells at the site of inflammation. This may further promote chronic inflammation if Shh induces bcl2 expression in T cells present in the lung, thus promoting local survival of activated T cells.

13. Shh and tumour associated inflammation Recently, a study by Wang et al. (2003) has also implicated the immune system in the pathogenesis of medulloblastoma, a human tumour of the CNS. The Shh pathway is essential for normal cerebellum development by stimulating proliferation of granule neuron progenitor cells (Dahmane and Altaba, 1999). Dysregulation of the Shh pathway, however, via ptc mutations, or over-expression of Shh predisposes to medulloblastoma (Weiner et al., 2002). Wang et al. (2003) were studying the function of STAT2 using transgenic mice (termed GIFN/STAT2−/− ) with CNS-specific production of IFN-␣. A surprising finding was that these mice died prematurely of medulloblastoma. Wang et al. (2003) found IFN-␣ stimulated up-regulation of IFN-␥ in the brains of these mice and the immune pathology in the tumour was found to consist of an up-regulation of CD4+ and CD8+ T cells expressing IFN-␥ mRNA. As the Shh pathway is important in medulloblastoma, the authors investigated whether

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dysregulated Shh signalling could be the under-lying factor in the development of disease in these mice. Using in situ hybridisation (ISH), expression of both Shh and Gli1 genes was significantly increased in the brains of these mice and the transcripts co-localised in the granule neurons and medulloblastoma cells. Furthermore, Shh gene expression in neurons from these mice was induced by IFN-␥. A role for viruses has been implicated in the aetiology of human medulloblastoma (Del Valle et al., 2002; Fine, 2002). Wang et al. (2003) surmise that an anti-viral immune response may lead to IFN-␥ production in the brain. Progression to medulloblastoma may require loss of STAT2 that may function as a tumour repressor (Huang et al., 2002). Some viruses are known to inhibit STAT2 and this would lead to dysregulated IFN-␥ production in the brain, in turn leading to dysregulated Shh signalling and therefore medulloblastoma.

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