Serrate1-induced Notch signalling regulates the decision between immunity and tolerance made by peripheral CD4+ T cells

Gerard F. Hoyne, Isabelle Le Roux1, Marta Corsin-Jimenez, Karen Tan, Jenny Dunne2, Lynn M. G. Forsyth, Margaret J. Dallman3, Michael J. Owen2, David Ish-Horowicz1 and Jonathan R. Lamb

Respiratory Medicine Unit, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK
1 Developmental Genetics and
2 Lymphocyte Molecular Biology Laboratories, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK
3 Department of Biology, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK

Correspondence to: G. F. Hoyne


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Signals derived from antigen-presenting cells (APC) influence the functional differentiation of CD4+ T cells. We report here that Serrate1 (Jagged1), a ligand for the Notch1 receptor, may contribute to the differentiation of peripheral CD4+ T cells into either helper or regulatory cells. Our findings demonstrate that antigen presented by murine APC overexpressing human Serrate1 induces naive peripheral CD4+ T cells to become regulatory cells. These cells can inhibit primary and secondary immune responses, and transfer antigen-specific tolerance to recipient mice. Our results show that Notch signalling may help explain `linked' suppression in peripheral tolerance, whereby tolerance induced to one epitope encompasses all epitopes on that antigen during the course of an immune response.

Keywords: antigen-presenting cells, Notch signalling, peripheral tolerance, regulatory T cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T lymphocytes regulate the functional activity of many aspects of both innate and adaptive immunity. The ability of these cells to promote productive immunity through cytokine secretion and the provision of cognate interactions with B cells is well documented. It is now emerging that selected CD4+ T cells as part of the normal peripheral repertoire can exert inhibitory effects and prevent autoimmunity (14), maintain tolerance to transplantation antigens (e.g. 5), and regulate mucosal tolerance to non-pathogenic dietary and inhaled antigens (e.g. 6). These inhibitory CD4+ T cells, termed regulatory cells, therefore play an important role in peripheral tolerance. A characteristic feature of peripheral tolerance is that it can spread to encompass all epitopes within an antigen which is referred to as `linked suppression'. Alternatively, it has been noted by several groups that tolerance can spread to influence T cell responses to unrelated third-party antigens and this latter phenomenon is referred to as `bystander suppression' (711). Although the induction of regulatory T cells depends upon antigen presentation by antigen-presenting cells (APC), the nature of the signals, which enable naive CD4+ T cells to differentiate to become regulatory cells in preference to helper cells, is unknown.

During development cell–cell interactions are critical in enabling equivalent precursor cells to adopt alternate cell fates. One signalling pathway which has the capacity to regulate such cell fate decisions in various tissues, and in a diverse range of organisms, is controlled by the Notch receptor (1213). Notch is an evolutionarily conserved transmembrane protein that was first described as the product of a neurogenic gene in Drosophila. Vertebrate Notch homologues have now been identified in various tissues and it is anticipated that they will play a role not only in embryogenesis, but also in the regulation of cell growth and differentiation of adult tissues (14). Notch can bind to two separate ligands, Delta and Serrate (15), of which there are vertebrate homologues, and the receptor and its ligands are co-expressed within the same tissues (1620). In the immune system, cell fate decisions occur within the thymus, where T cell precursors choose between the TCR{alpha}ß and TCR{gamma}{delta} lineages, and then again when TCR{alpha}ß cells differentiate into CD4+ and CD8+ single-positive cells (2122). The studies of Robey and colleagues suggest that Notch signalling is required at both of these stages of T cell development (23, 24). The analysis of mice with induced deletion of Notch1 reveals that the pathway functions at an early stage in T cell selection and in its absence precursor cells are directed towards a B cell fate (25). It has been reported that Hes-1, a transcriptional target of the Notch1 pathway, is required for functional silencing of the CD4 gene (26) and Notch1 signalling enhances the survival of CD4+CD8+ thymocytes enabling them to differentiate in CD4+ or CD8+ cells (27). These results confirm a role for Notch1 signalling in the selection of single-positive cells. However, at present, little is known of the physiological function of the Notch ligands in mediating the cell fate decisions of thymocytes.

Peripheral CD4+ T cells also make cell fate decisions resulting in the development of either helper cells which promote antigen-specific immune responses or regulatory cells which suppress such responses and confer antigen-specific tolerance. The decision between the helper or regulatory cell fate depends, in part, upon the signals they receive from APC at the time of antigen recognition. The Notch1 gene is expressed in peripheral lymphoid tissues including T cells and, therefore, similar to other situations in embryonic development it is possible that signalling through this receptor may influence CD4+ T cell differentiation in the periphery (2830).

Here we report that transcripts for murine Notch1 and the ligand Serrate1 (also known as Jagged1) are present in T cells and APC in the spleen of naive mice. However, we do not know the physiological circumstances which might modulate Serrate1 expression or whether it is present on only selected subsets of APC. Therefore, we have constitutively expressed Serrate1 on APC, which are required for the activation of naive T cells (reviewed in 31), using retroviral-mediated gene transfer and investigated the functional consequences of this on Serrate1 expression by APC in the regulation of peripheral immune responses. We demonstrate that antigen presented by APC overexpressing human Serrate1 results in the differentiation of antigen-specific CD4+ T cells into regulatory cells which can transfer tolerance to naive mice. Thus, the functional phenotype of the CD4+ T cells obtained after antigen priming by Serrate1+ APC is comparable to that observed in tolerance mediated by peptide delivered via the respiratory mucosa (10). Collectively, our data suggest that Serrate1/Notch signalling between naive peripheral CD4+ T cells and APC may enable these T cells to choose between alternate cell fates, and biases the development of T cells towards the regulatory pathway.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female inbred C57BL/6J (H-2b) mice were purchased from Harlan Olac (Bicester, UK) and the Medical Microbiology Transgenic Unit at the University of Edinburgh. All experiments were performed in accordance to the animal ethics regulations of the Home Office in the United Kingdom.

Antigens
Affinity-purified Der p 1 and synthetic peptides (p 1, 110–131 and p 1, 81–102) were prepared as previously described (32). Chicken egg ovalbumin (OVA) grade V crystalline was purchased from Sigma (St Louis, MO).

Antibodies
The purified rat anti-mouse CD4 or CD8 mAb used for complement-mediated lysis of T cells were provided by Professor H. Waldmann (University of Oxford). They were purified on a Protein G column (Pharmacia, Amersham Pharmacia, St Albans, UK) from culture supernatants following the manufacturer's instructions. Anti-CD4, anti-CD8, anti-CD19 and anti-CD11c antibodies coupled to magnetic beads were purchased from Miltenyi Biotech (Bisley, UK), and used for cell purification following the manufacturer's instructions.

Immunizations
All mice were immunized s.c. at the base of tail with 50 µg affinity-purified Der p 1 or OVA in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (Difco, Detroit MI) or 50 µg OVA in CFA.

Retroviruses
For construction of Serrate1 retrovirus, a BamHI–EcoRI fragment containing the full-length human Serrate1 gene was cloned into the pBABEneo. Replication deficient pseudotype retroviruses were made by transfecting PA327 cells with the human Serrate1 neo vector. Virus culture was initiated by transfection of cells with the vesicular stomatitis virus envelope under a cytomegalovirus promoter. Virus containing supernatants were added to spleen dendritic cells (DC).

Preparation and infection of DC-enriched APC populations
Expansion of splenic DC in vitro was performed following a similar method to that described by Ridge et al. (33). Single-cell suspensions were prepared from the spleens of naive mice. Adherent cells were isolated by culturing spleen cells at 37°C for 2–3 h and after the non-adherent cells were removed by extensive washing they were cultured in the presence of 50 ng/ml murine granulocyte macrophage colony stimulating factor (PharMingen, San Diego, CA). After 24 h in culture the splenic DC-enriched populations (APC) were infected with Serrate1 or control virus for a further 2 days and then pulsed with 100 µg/ml p1, 110–131 over the last 2 h of culture. Non-adherent APC were collected, washed twice and injected i.v. to naive mice (105 cells/mouse). Titration in vivo revealed that 105 peptide-pulsed Serrate1+ APC/mouse was the most effective dose for inhibiting T cell responses (>90% inhibition) and was, therefore, used in the experiments reprinted here. FACS analysis revealed that ~80% of the cells in the final cell preparation were CD11c and MHC class II+. The efficiency of retroviral infection ranged from 70 to 80% in these experiments and was assessed by in situ hybridization using a digoxigenin-labelled antisense RNA probe specific for the human Serrate1 cDNA (17).

Conditions for cell culture
Lymph node (LN) or spleen cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 2% FCS (Gibco), 50 mM 2-mercaptoethanol (Sigma), 2 mM L-glutamine (Sigma) and 20 µg/ml penicillin/streptomycin. LN cells were pressed through a stainless steel wire mesh, washed and cultured in triplicate at 4x105 cells/well in 200 µl flat-bottom 96-well tissue culture plates (Nunc, Roskilde, Denmark). Protein or peptides were added at different concentrations and the cells were incubated at 37°C for 24 or 48 h. Supernatants were collected and stored at –20 °C until required for the analysis of cytokines IL-2 and IFN-{gamma}, and were measured as previously described (10).

Separation of cell populations
DC, T cells and B cells were purified from the spleen of naive mice by positive selection using specific MACS microbeads for CD4+, CD8+, CD19+ or CD11c+ supplied by the manufacturer (Miltenyi Biotech). Briefly, the spleens were removed and single-cell suspensions prepared. For the separation of DC, spleens were injected with 500 µl of collagenase D at 1 mg/ml and incubated at 37°C for 1 h. Cells were resuspended in PBS supplemented with 10% BSA at 1x108 cells/100 µl and incubated with the appropriate MACS antibody at 4°C for 15 min. The cells were then passed over a MACS column and the cells retained on the column after washing were collected. Separated cell populations were analysed for purity by FACS or were processed for mRNA extraction. The level of purity for CD4+, CD8+ T cells and B cell populations ranged was >95% while DC were 80% CD11c+ and MHC class II+.

Adoptive transfer of CD4+ T cells
LN were collected from DC-injected mice and cell suspensions prepared. CD4+-enriched T cells were prepared by negative selection using antibody-mediated complement lysis of either CD4+ or CD8+ T cells. CD4+ or CD8+ T cell populations were transferred to naive mice i.p. at 2x107, 5x106 or 1x106 cells/mouse. Mice were immunized at the same time with either 50 µg Der p 1/CFA or 50 µg OVA/CFA. One week later LN cells were collected and assayed in vitro for responses to the relevant antigen.

RT-PCR
RT-PCR was performed using the Access RT-PCR kit (Promega, Southampton, UK); 50 ng of total RNA was used per reaction, and primers specific for murine Notch1, Serrate1 or c-actin were used at 50 pmol and amplified for 35 cycles. The annealing temperature for all sets of primers was 58°C. Samples were analysed by gel electrophoresis on a 2% agarose gel. A 1 kb DNA marker (Gibco/BRL, Paisley, UK) was run with each PCR reaction. The sequence of Serrate1 forward primer is: 5'-GGG GGT CAC TGT CAG AAT GA-3'. The sequence of Serrate1 reverse primer is: 5'-AGA TAT ACC GCA CCC CTT CAG-3'. Serrate1 primers amplify a product of 289 bp. The sequence of Notch1 forward primer is: 5'-TGT TAA TGA GTG CAT CTC CAA-3'. The sequence of Notch1 reverse primer is: 5'-CAT TCG TAG CCA TCA ATC TTG TCC-3'. Notch1 primers amplify a sequence of 638 bp. The sequence of actin forward primer is: 5'-TCA CCA ACT GGG ACG ACA TG-3'. The sequence of actin reverse primer is: 5'-GTC TCA AAC ATG ATC TGG GTC-3'. c-actin primers amplify a sequence of 151 bp.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Notch1 and Serrate1 are expressed on T cells and APC
The expression of murine Notch1 and Serrate1 was analysed by RT-PCR using RNA isolated from purified CD4+ and CD8+ T cells, B cells and DC prepared from the spleens of naive mice. PCR products of 638 and 289 bp specific for Notch1 and Serrate1 respectively could be readily observed in DC, CD4+ and CD8+ T cells. B cells expressed Serrate1 but transcripts for Notch1 were apparent but seemingly at a lower level compared with the other cell populations. DC-enriched populations were obtained from the spleen (hereafter referred to as APC); we have used these cells to examine the functional relevance of Serrate1 expression on the regulation of peripheral immune responses.

Serrate1+ DC-enriched populations prevent peripheral T cell immunity in vivo
Mice of the H-2b haplotype are high responders to the house dust mite protein Der p 1 and recognize four distinct CD4+ T cell epitopes on the protein (32). However, the outcome of this antigen presentation depends upon the route and dosage of peptide administration. Systemic administration of the immunodominant peptide, p 1, 110–131 in adjuvant induces CD4+ T cells that support productive immunity, but the same peptide when delivered mucosally results in peripheral tolerance through induction of regulatory T cells (34). We have observed that signals delivered by APC are necessary for the selection of regulatory cells and subsequent induction of tolerance (10,35). Peripheral T cell function is dependent on signals from APC and having established that Notch1 is expressed on T cells (Fig. 1Go) we were prompted to examine whether or not overexpression of Serrate1 affects CD4+ T cell responses to the HDM peptide p 1, 110–131 peptide. Thus, cultured splenic APC (33) were transfected with human Serrate1 by retroviral mediated gene transfer and then used to immunize recipient mice.



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Fig. 1. Expression of Notch1 and Serrate1 in the spleen of naive mice. B cells (lane 1), CD4+ (lane 2), CD8+ (lane 3) and DC (lane 4) were purified from the spleens of naive mice using antibody-coated magnetic microbeads. The total RNA was extracted and quantified by optical density; 50 ng were used in each reaction and control amplification using c-actin primers confirmed approximate equivalence of all samples. Amplified products were analysed by gel electrophoresis on a 2% agarose gel. Product sizes are 151 bp c-actin; 638 bp, Notch1; 289 bp, Serrate1.

 
Naive mice were injected with either Serrate1+ or control APC (105/mouse) pulsed with p1, 110–131 and left for 2 weeks before immunization with Der p 1. LN cells from the mice receiving control APC displayed strong proliferative responses in vitro when re-stimulated with the intact Der p 1 protein. In contrast, injection of Serrate1+ APC causes a striking reduction of T cell proliferative responses in LN, leading to >90% inhibition when cells were stimulated in vitro with higher doses of protein (10 µg/ml) (Fig. 2AGo). Inhibition was antigen dependent and occurred only in mice which received Serrate1+ APC pulsed with peptide. Transfer of Serrate1+ APC alone that had not been pulsed with peptide had no inhibitory effect on the ability of mice to respond to Der p 1 in vivo. Although the mice were treated with only a single peptide on the Serrate1-expressing APC, we have consistently observed that responses to all epitopes on the antigen are inhibited. Mice exposed to Serrate1-expressing APC also fail to mount a proliferative response to the subdominant epitope of Der p 1, p1, 81–102 (Fig. 2A and BGo). This finding is consistent with the phenomenon of `linked' suppression which we and others have demonstrated during the induction of mucosal tolerance mediated by intranasal administration of peptide (7,10,34,3637). Analysis of cytokine secretion in these assays revealed that both IL-2 and IFN-{gamma} secretion were down-regulated in CD4+ T cells injected with Serrate1+ APC (Fig 2C and DGo). The studies here indicate that antigen presentation by Serrate1+ APC can inhibit the induction of T cell immunity to a foreign antigen in vivo.



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Fig. 2. Serrate1+ APC inhibit antigen-specific T cell immunity. (A) APC were infected with Serrate1 (•) or control virus ({square}) virus, pulsed with p1, 110–131 peptide and injected into naive C57BL/6J mice and 2 weeks later mice were immunized with 50 µg Der p 1/CFA. LN cells were cultured in vitro with Der p 1 and proliferation was measured and the results presented as mean c.p.m. ± SD of four mice per group. (B) LN cells from mice primed as described above [Serrate1+ APC (shaded bars) or a control APC (closed bars)] were cultured in vitro with HDM peptides p 1, 110–131 or p 1, 81–102 at 10 µg/ml and proliferation measured. The supernatants from these assays were collected at 24 h and assessed for IL-2 production (C), while 48 h supernatants were assessed for the presence of IFN-{gamma} (D).

 
It was important to determine if antigen presentation by Serrate1+ APC leads to a global suppression of T cell immunity or whether it is specific for the antigen used in the initial immunization regimen. Mice were injected with either control or Serrate1+ APC pulsed with the HDM peptide and subsequently immunized with either Der p 1 or the unrelated antigen, OVA. Only in mice receiving HDM peptide-pulsed Serrate1+ APC prior and rechallenged with Der p 1 was inhibition of T cell proliferation observed (Fig. 3AGo). The response of these mice to the irrelevant antigen OVA was unaffected (Fig. 3BGo). Taken together these results suggest that immunization with Serrate1+ APC bearing specific peptide does not affect the response to unrelated third party antigens.



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Fig. 3. Immunization with Serrate1+ APC pulsed with specific peptide induces antigen specific but not global suppression of immunity. (A) APC were infected with Serrate1 (•) or control ({square}) virus pulsed with p1, 110–131 and 2 weeks later the mice were immunized with 50 µg Der p 1/CFA. LN cells were cultured in vitro with Der p 1 and proliferation was measured and the results presented as mean c.p.m. ± SD of four mice per group. (B) Mice were injected with Serrate1+ APC pulsed with p1, 110–131 as described above but then immunized with OVA/CFA. LN cells were re-stimulated with OVA in vitro and proliferation measured as above.

 
Established immunity is inhibited by Serrate1+ APC
Experiments were designed to determine whether or not Serrate1+ APC are able to inhibit established antigen-specific immune responses. Mice previously immunized with Der p 1 were injected with either control or Serrate1+ APC pulsed with p 1, 110–131 and then rechallenged with intact protein. T cells from control mice could mount strong Der p 1-specific proliferative responses when stimulated in vitro, whereas T cells from mice injected with Serrate1+ APC displayed a 75% reduction in their response to the same antigen (Fig. 4Go).



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Fig. 4. Serrate1+ APC inhibit established immune responses. Naive mice were immunized with 50 µg Der p1/CFA and 3 weeks later they were injected with p1, 110–131-pulsed DC infected with either Serrate1 (•) or control ({square}) virus. Two weeks later mice were reimmunized with 50 µg Der p 1/incomplete Freund's adjuvant and the proliferative response of LN cells to re-stimulation with Der p 1 measured as described in the legend to Fig. 2(A)Go.

 
Inhibition of T cell immunity induced by Serrate1+ APC is long lived
A feature of antigen-induced peripheral tolerance is that it is long lived and therefore experiments were performed to determine the longevity of the specific unresponsiveness we observe in mice immunized with peptide pulsed Serrate1+ APC. Mice were immunized with Der p 1 at 2, 6 or 12 weeks after having been injected with control or Serrate1+ APC pulsed with the HDM peptide. At each of the time points tested T cells from mice receiving control APC could still mount strong proliferative responses to Der p 1 (Fig. 5AGo–C). In contrast, Der p 1-specific proliferation LN T cells was markedly decreased, even at 12 weeks, in those mice that had received the Serrate1+ APC (Fig. 5Go). The above results demonstrate that antigen presentation by Serrate1+ APC can inhibit antigen-specific T cell immunity in vivo, however, they do not address the underlying cellular mechanism(s).



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Fig. 5. Inhibition of CD4+ T cells responses induced by Serrate1+ APC is long lived. Peptide-pulsed APC infected with either Serrate1+ (•) or control ({square}) virus were injected into naive C57BL/6J mice and (A) 2, (B) 6 or (C) 12 weeks later mice were immunized with Der p 1/CFA and proliferation determined as described in the legend to Fig. 2(A) Go.

 
Tolerance induced by Serrate1+ APC can be transferred by CD4+ T cells
It is possible that direct cell–cell interactions between Notch1 expressed on naive T cells (23,28,38) and Serrate1 on APC at the time of initial antigen recognition, even in the presence of co-stimulation, prevents clonal expansion of these T cells through activation of the Notch signalling pathway. Alternatively, Serrate1–Notch signalling may alter the outcome of antigen presentation such that naive T cells differentiate into CD4+ regulatory rather than Th cells. If the latter situation were to arise, it should be possible to transfer tolerance to naive mice using CD4+ T cells from donor mice which had received peptide-pulsed Serrate1+ APC. In order to differentiate between these two possible mechanisms, mice were injected with either control or Serrate1+ APC pulsed with p 1, 110–131 and CD4+ or CD8+ T cells were transferred from the mice to naive recipients which were then immunized with Der p 1. CD4+ or CD8+ T cells from donor mice that had received control APC had no inhibitory effect on Der p 1 specific T cell responses in the recipient mice. In contrast, we observed a 70% decrease in the proliferative responses of LN cells from recipient mice who had received CD4+ T cells from Serrate1+ APC injected donors (Fig. 6A and BGo). The most effective inhibition of T cell proliferative responses was observed following the transfer of 2x107 CD4+ T cells/mouse (Fig. 6BGo). Transferring CD8+ T cells had no inhibitory effect on Der p 1-induced proliferative responses (Fig. 6AGo).



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Fig. 6. Antigen-specific tolerance induced by Serrate1+ APC can be transferred to naive mice by CD4+ T cells. (A) Peptide-pulsed APC infected with either Serrate1 or control virus were injected into naive C57BL/6J mice and 2 weeks later CD4+ or CD8+ T cells were isolated from spleens and adoptively transferred to naive mice at 2x107/mouse. On the same day mice were immunized with 50 µg Der p 1/CFA and 1 week later LN cells were cultured in vitro with Der p 1. Results are presented for proliferation (mean c.p.m. ± SD of four mice per group) measured at 72 h in response to re-stimulation with 10 µg/ml Der p 1. Transfer of T cells from mice injected with Serrate1+ (open and shaded bars) or control (solid and grey bars) DC is shown. (B) Peptide-pulsed APC infected with Serrate1 (grey bars) or control (solid bars) virus were injected into naive C57BL/6J mice and 2 weeks later CD4+ or CD8+ T cells were isolated from spleens and transferred to naive mice. In addition, 2x107 control CD4+ T cells or 2x107, 5x106 or 1x106 CD4+ T cells from Serrate1+ APC injected mice were transferred to naive mice and immunized with 50 µg Der p 1/CFA on the same day. Results are presented for proliferation of LN cells (mean c.p.m. ± SD of four mice per group) measured at 72 h in response to re-stimulation with 10 µg/ml Der p 1. (C) Peptide-pulsed APC infected with Serrate1 (open bars) or control (solid bars) virus were injected into naive C57BL/6J mice and 2 weeks later CD4+ T cells were isolated from spleens and transferred to naive mice. 2x107 control CD4+ T cells or CD4+ T cells from Serrate1+ APC injected mice were transferred to naive mice which were immunized with 50 µg OVA/CFA on the same day. Results are presented for proliferation of LN cells (mean c.p.m. ± SD of four mice per group) measured at 72 h in response to re-stimulation with 800 µg/ml OVA.

 
Having established that Serrate1+ APC could induce CD4+ T cells were capable of transferring tolerance, their antigen specificity was investigated in vivo. CD4+ T cells from mice treated with either control or Serrate1+ APC pulsed with p 1, 110–131 were injected into recipients which were immunized at the same time with either Der p 1 or OVA (Fig. 6CGo). CD4+ T cells from mice receiving p 1, 110–131-specific Serrate1+ APC could inhibit the Der p 1-specific response in recipient mice by as much as 80%. However, these same T cells had no inhibitory effect on the OVA response, confirming that CD4+ T cells induced by Serrate1+ APC mediate antigen-specific tolerance in vivo.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is well documented that APC are required for the differentiation of naive CD4+ T cells. Cell contact between APC and T cells mediated by adhesion molecules, ligation of TCR by peptide–MHC class II complexes together with the delivery of co-stimulatory signals leads to T cell activation and clonal expansion (reviewed in 39). Despite extensive knowledge of these early events in the induction of CD4+ Th cells, information on the signals provided by APC necessary for the differentiation of regulatory T cells is limited. In this study we have analysed the functional role of Serrate1, a ligand for the receptor Notch, in the regulation of peripheral T cell immunity. We find that the following recognition of antigen presented on the surface of APC overexpressing Serrate1, peripheral naive CD4+ T cells differentiate into regulatory cells that are capable of transferring antigen-specific tolerance to naive recipients.

For Notch signalling to regulate the choice between helper and regulatory CD4+ T cells in the periphery, the receptor and its ligand(s) should be expressed in lymphoid tissues. We and others (40) have observed expression of Notch1 and Serrate1 T cells and APC populations. Furthermore, we now have evidence that administration of high-dose peptide leads to increased expression of Serrate1 on draining lymph DC compared to untreated mice. The mechanisms for this increased expression remains to be resolved. However, the primary aim of this study was to evaluate the possible effects of Notch signalling on peripheral T cell responses. Therefore, we used retroviral gene-mediated transfer to express high levels of Serrate1 on APC, pulsed them within an immunogenic peptide and used these cells to immunize recipient mice.

The injection of Serrate1+ APC pulsed with a specific peptide of HDM was sufficient to render mice profoundly unresponsive to an immunogenic challenge with the intact Der p 1 protein. We have observed a corresponding decrease in both proliferation and cytokine production (e.g. IL-2 and IFN-{gamma}) by T cells from mice receiving Serrate1+ APC when the cells were stimulated in vitro with specific antigen. This inhibition was characterized by a loss of T cell responses to all epitopes on the protein antigen, which deserves comment since the mice received only the immunodominant T cell epitope of Der p 1 presented on the surface of Serrate1+ APC. This finding is consistent with other models of peripheral tolerance in which `linked' suppression has also been observed (7,9,10,41).

Several mechanisms could explain our findings and the phenomenon of `linked' suppression. It is possible that p 1, 110–131-specific regulatory cells transmit an inhibitory signal(s) to the naive Der p 1-reactive T cells clustered on the same or adjacent APC that are expressing both the major (p 1, 110–131) and minor (e.g. p 1, 81–102) epitopes. Alternatively, the regulatory T cells may signal APC such that they develop a tolerogenic phenotype and inhibit the growth of T cells reactive with the minor epitopes. `Linked' suppression in either case could arise from direct contact between the appropriate cell populations or through the production of inhibitory cytokines (e.g. transforming growth factor-ß or Th2-type cytokines) which have been demonstrated in some models of peripheral T cell tolerance (3,89). We have failed to detect the secretion of inhibitory cytokines in peptide-induced peripheral tolerance (10), which is mediated by regulatory CD4+ T cells. Therefore, we favour direct cell contact as a mechanism of `linked' suppression; our results suggest that it may involve the Notch signalling pathway. Regulatory T cells have been described as a natural component of the peripheral immune system and although they can inhibit T cell growth by direct cell contact, the mechanisms underlying this as yet have not been characterized (4,42).

Collectively, the findings reported here, i.e. that Serrate1+ APC can induce tolerance which is associated with `linked' suppression, the ability to transfer tolerance with CD4+ T cells and that these cells are maintained in the peripheral circulation for long periods, all provide support for the concept that the presentation of antigens on the surface of Serrate1+ APC promotes the differentiation of CD4+ T cells as regulatory cells. At present we have no knowledge of the signals required to maintain these regulatory CD4+ T cells in the periphery. It is possible that a proportion of the cells which have become regulatory cells as a result of initial antigen priming by Serrate1+ APC enter the memory pool. The signals that lead to the reactivation of memory regulatory CD4+ T cells are likely to differ from those that are required in their induction and may be independent of Serrate1-mediated signalling.

We cannot examine directly requirements for Serrate1 or Notch1 in regulating cell fate decisions between helper and regulatory CD4+ T cells because homozygous mice mutant for these genes die before the development of the peripheral immune system (43,44). In addition, mice expressing a constitutively active Notch1 gene have an altered T cell repertoire biased towards CD8+ T cells due to preturbation of normal thymic T cell maturation (2324). In the case of mice with deleted Notch1 there is an absence of mature T cells (25). The findings presented here suggest that when Serrate1+ APC present antigen to naive T cells they influence the cell fate of the CD4+ T cells allowing them to differentiate into regulatory cells at the expense of Th cells. In Caenorhabditis elegans vulva precursor cells can give rise to two distinct lineages known as the ventral uterine cell, a primary fate, or the anchor cell, a secondary fate. The choice between these lineages is regulated by a cell contact-dependent process involving signalling through the Notch receptor Lin-12 and its ligand Lag-2 (45). Therefore, by analogy to this model of Notch signalling, the differentiation of Th cells would represent a primary cell fate choice while formation of regulatory T cells is a secondary fate.

In the case of CD4+ T cells, which provide cognate interactions with B cells to induce primary antibody responses, it will be of interest to examine what role Serrate1–Notch signalling may have on the cross-talk between these cell populations. The ability of CD4+ T cells to signal APC via the Notch pathway may act as a physiological mechanism of negative feedback in the control of productive immunity. Furthermore, it will be intriguing to examine in more detail the functional consequences of Serrate1 expression by CD8+ T cells. From our findings with CD4+ T cells, we can only speculate that maybe Serrate1–Notch signalling in naive CD8+ T cells may also be important in controlling their effector activity in vivo. Studies emerging from the role of Notch signalling in the regulation of haematopoiesis (4650) suggest that Notch signalling can influence the differentiation of haematopoietic precursors in the presence of different cytokines (46). Therefore, it is likely that Serrate1–Notch signalling does not act alone in the induction of regulatory T cells in vivo but also changes the profile of cytokine mediated signalling. This is supported by the finding that IL-10 can induce regulatory CD4+ T cells that are capable of down-regulating Th1 responses in vivo (5153). Furthermore, DC exposed to IL-10 adopt a phenotype that is able to inhibit antigen-dependent T cell proliferation (54). Whether Notch signalling contributes to IL-10 secretion or IL-10 itself modulates Serrate1 expression on either APC or T cells at present is not known; however, if so, this may allow amplification of the tolerogenic signal (54).

In summary, we find that Serrate1 expression on APC can induce naive CD4+ T cells to differentiate into a regulatory phenotype and once committed to this fate these cells can both prevent and down-regulate ongoing immunity (Fig. 4Go). We do not suggest that Serrate–Notch interaction is the only one which leads to the generation of long-lived regulatory T cells (Fig. 5Go), and, indeed, other workers have described alternative mechanisms for the induction and maintenance of peripheral T cell tolerance (e.g. 4,39,51,53,55). However, due to the nature of the signalling molecules involved, it is possible that this is a fundamental mechanism of tolerance induction which may be amplified by, or interact with, other pathways in the generation of immune regulation.


    Acknowledgments
 
We thank Chris Haslett, Ita Askonas, Charles Janeway and Mairi Stewart for discussion and critical review of the manuscript, and Nigel Savage and Gill Hall for preparation of Der p 1. This work was supported by the Medical Research Council, Wellcome Trust, British Lung Foundation, Imperial Cancer Research Fund and a Sir Henry Wellcome Commemorative Award for Innovative Research to G. F. H. D. I. H. is supported by a grant from the Howard Hughes Medical Institute through the International Programme.


    Abbreviations
 
APC antigen-presenting cell
CFA complete Freund's adjuvant
DC dendritic cell
LN lymph node
OVA ovalbumin

    Notes
 
Transmitting editor: A. McMichael

Received 6 September 1999, accepted 14 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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