Notch1 Signaling and Regulatory Function Naoki Asano, Tomohiro Watanabe, Atsushi Kitani, Ivan J. Fuss and Warren Strober This information is current as of September 29, 2021. J Immunol 2008; 180:2796-2804; ; doi: 10.4049/jimmunol.180.5.2796 http://www.jimmunol.org/content/180/5/2796 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Notch1 Signaling and Function

Naoki Asano,* Tomohiro Watanabe,*† Atsushi Kitani,* Ivan J. Fuss,* and Warren Strober1*

Previous studies have shown that the Notch1 and TGF-␤ signaling pathways are mutually re-enforcing. Given recent evidence that regulatory T cell (Treg) effector function is mediated by TGF-␤ signaling, we investigated whether Notch1 signaling also partic- ipated in Treg effector function. Initial studies showed that Notch1 ligands, particularly Jagged1, are present on Tregs and that, indeed, blockade of Notch1 signaling with an anti-Jagged1 or a blocking anti-Notch1 Ab inhibits Treg suppressor function in vitro. We then showed that a signaling component generated by Notch1 activation (Notch1 intracellular domain) of dendritic cells physically interacts with a signaling component generated by TGF-␤ signaling (pSmad3). Furthermore, this interaction has functional downstream effects because over-expression of Notch1 intracellular domain facilitates pSmad3 translocation to the nucleus and enhances pSmad3 transcriptional activity of a Smad-sensitive promoter linked to a luciferase reporter. Finally, we showed that blockade of TGF-␤ signaling and Notch signaling did not have additive inhibitory effects on Treg suppressor function. Downloaded from These results are consistent with the conclusion that Notch1 signaling facilitates TGF-␤-mediated effector function of Tregs. The Journal of Immunology, 2008, 180: 2796–2804.

he CD4ϩCD25ϩ regulatory T cells (Tregs)2 play an im- The is another pathway that may be portant role in regulating the by suppress- involved in Treg suppressor function. This possibility arises from T ing self-reactive T cells that have escaped negative selec- several reports that overexpression of Notch can induce http://www.jimmunol.org/ tion in the as well as hyperactive T cells that are induced regulatory cells (13–15); in addition, it has recently been shown during excessive immune responses in peripheral lymphoid tissues that Tregs with membrane-bound TGF-␤ stimulate the Notch path- (1–3). Over the past several years, strong evidence has accumu- way and that such stimulation is necessary for the induction of lated supporting the idea that the suppressive function of Tregs is tolerance to respiratory Ags in the airway (16). Notch is a phyloge- mediated by TGF-␤, an evolutionarily conserved that is netically old molecule that was first identified as a signaling compo- known to have a suppressive function as one of its numerous prop- nent essential for the differentiation of the embryonic nervous system erties (4). Perhaps the most important of these with respect to Treg of (17, 18). Later studies showed that Notch was also function is the fact that Tregs secrete TGF-␤ and express the latter

involved in mammalian differentiation processes including those gov- by guest on September 29, 2021 on the cell surface, as well as the fact that TGF-␤ inhibits APC erning the development of the immune system (19). The latter in- function, the main focus of Treg suppression (5–9). Thus, when cluded evidence for a role of Notch signaling in the intrathymic dif- TGF-␤ binds to its , the receptor phosphorylates the sig- ferentiation of T cells (20–22) as well as Th cell development in the naling molecule Smad3 that then forms a heterodimeric or hetero- periphery (23–27). Notch is, like TGF-␤, an evolutionarily conserved trimeric complex with Smad4; this complex then translocates to molecule but, unlike TGF-␤, is a type I transmembrane receptor rather the nucleus where it plays a negative role in the transcription of than a receptor ligand. Upon binding with its ligands (Jagged1 and 2 ␤ various (10). It should be noted, however, that TGF- can and Delta-like1, 3, and 4), it releases its intracellular domain into the also initiate positive signaling pathways including one pathway cytoplasm which then translocates to the nucleus (19). Recently, it has that is important to Treg function. This involves the ability of been shown that the released signaling models arising from Notch1 ␤ ϩ TGF- to induce naive CD4 cells to differentiate into Tregs and, activation enhance Smad signaling function in fibroblast and other thus, to facilitate the expansion of peripheral Treg populations (8, mesenchymal cells (28), suggesting that Notch1 signaling could in- 11, 12). The molecular mechanisms underlying this positive func- teract with the TGF-␤ signaling occurring in Treg function. tion, however, are poorly understood. In this study, we show that Tregs preferentially express cell mem- brane Notch ligands and that blocking Notch signaling initiated by such ligands inhibits Treg suppressor function. We then showed that *Mucosal Section, Laboratory of Host Defenses, National Institute of Al- phosphorylated Smad3 arising from TGF-␤ signaling binds to Notch1 lergy and Infectious , National Institutes of Health, Bethesda, MD 20892; and intracellular domain (NICD) arising from Notch signaling and that the †Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan complex formed facilitates the translocation of phosphorylated Smad3 Received for publication June 14, 2007. Accepted for publication December 13, 2007. to the nucleus. These findings suggest that Treg suppressor function The costs of publication of this article were defrayed in part by the payment of page does involve Notch signaling, but the effect of the latter is channeled charges. This article must therefore be hereby marked advertisement in accordance through the suppressor signal mediated by TGF-␤. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Warren Strober, National Insti- tute of and Infectious Diseases, National Institutes of Health, Building 10- Clinical Research Center, Mail Stop Code 1890, 10 Center Drive, Bethesda, MD Materials and Methods 20892. E-mail address: [email protected] Mice 2 Abbreviations used in this paper: Treg, regulatory T cell; DC, ; NICD, Notch1 intracellular domain; MLEC-PAI-1, luciferase reporter construct mink lung A group of 5–7-wk-old BALB/c female mice were purchased from The epithelial cells stably expressing plasminogen activation inhibtor-1 luciferase reporter Jackson Laboratory and maintained in the animal facility of the National construct; GSI, ␥-secretase inhibitor. Institutes of Health. www.jimmunol.org The Journal of Immunology 2797

FIGURE 1. Notch ligands are ex- pressed on CD4ϩCD25ϩ T cells. Na- ive CD4ϩCD25ϩ T cells and CD4ϩ CD25Ϫ T cells were isolated from of BALB/c mice. The cells were stained for Notch ligands, Notch1 and Foxp3, and their expres- sion was assessed by flow cytometry. Downloaded from http://www.jimmunol.org/

Cells with calcium chloride, magnesium chloride, and magnesium sulfate) and then cut into small pieces with a carbon steel surgical blade (Miltex). The HT29 cells, THP-1 cells, and were purchased from American pieces were then incubated in Collagenase D solution for 30 min at Type Cell Culture (ATCC) and maintained according to the ATCC proto- 37°C and passed through a 70 ␮m cell strainer (BD Falcon). The cells were col. MLEC-PAI cells (mink lung epithelial cells stably expressing plas- then subjected to magnetic selection with CD11c microbeads (Miltenyi minogen activation inhibtor-1 (PAI-1) luciferase reporter construct origi- Biotec). Isolated cells were then stained with appropriate Ab and analyzed nally developed by Abe et al. (29)) was obtained from Dr. L. van de Water

by flow cytometry. by guest on September 29, 2021 (Massachusetts General Hospital, Boston, MA) and maintained as previ- ously described (30). Human -derived DCs Abs and reagents were elutriated from the peripheral of healthy donors and were cultured in 6-well plates (1 ϫ 106/ml) in 5 ml of complete medium Anti-mouse CD3e Ab was purchased from BD Pharmingen. Notch-1 Ab-1 (RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% FCS) (Clone A6) was purchased from Neomarkers; this Ab has been shown in supplemented with rGM-CSF (20 ng/ml) and rIL-4 (20 ng/ml). After 3 days of previous studies to be a blocking Ab that inhibits Notch1 signaling (16, culture, half of the medium in each well was exchanged. After 6 days of 31). Abs for Notch ligands, multispecies TGF-␤1, 2, and 3 mAb (Clone culture, at the time of their use, Ͼ90% of the cells expressed characteristic 1D11), mouse IgG1, mouse IgG2b control, and recombinant DC-specific markers (CD1a and HLA-DR), as determined by flow cytometry. TGF-␤1 were purchased from R&D Systems. pSmad3 Ab and anti-cleaved Notch1 Ab was purchased from Technology. Affinity purified Generation of Jagged1 expressing Jurkat cell line agarose immobilized V5 Ab was purchased from Bethyl Laboratories, and A/G plus-agarose immunoprecipitation reagent was purchased Jurkat cells were transfected with Jagged1 expressing pcDNA3.1/Hygro plas- from Santa Cruz Technology. Hygromycin B solution for cell selection mid (gift from Dr. Doreen Kacer, Maine Medical Center Research Institute; was from Mediatech. Recombinant human GM-CSF and IL-4 were pur- Ref. 32), and positive clones were selected by limiting dilution using 1000 ␮ chased from PeproTech. Collagenase D was from Roche. g/ml hygromycin. The obtained Jagged1 expressing Jurkat cells will be re- ferred to as Jag-Jurkat cells. Suppressor assays Immunoprecipitation and Western blotting ϩ ϩ ϩ Ϫ CD4 CD25 T cells and CD4 CD25 T cells were isolated from the ϫ 6 spleen of the BALB/c mice using a mouse CD4ϩ T cell enrichment column A total of 1 10 THP1 cells were cultured in 6-well plates filled with 2.5 ϩ ϩ ml complete RPMI 1640 medium (10% FCS, 2 mM L-glutamine, 100 U/ml (R&D Systems) and a mouse CD4 CD25 Treg isolation kit purchased ␮ from Miltenyi Biotec. A total of 5 ϫ 104 CD4ϩCD25Ϫ T cells were cul- penicillin, and 100 g/ml streptomycin). The cells were transfected with tured with 3000 rad irradiated splenocytes (as APCs), various ratios of either cleaved Notch1 expression vector pN1ICD-V5 or empty pcDNA4.0- CD4ϩCD25ϩ T cells, and 1 ␮g/ml anti-CD3e in 96-well round-bottom V5/his version A vector (gifts from Dr. Doreen Kacer; Ref. 32). A total of ␮ 3 48 h after transfection, the cells were cultured with or without 10 ng/ml plates for 72 h. A total of 1 Ci/well [ H]thymidine was added to the ␤ wells during the last8hofculture. The cells were then harvested and rTGF- for 30 min, washed twice with PBS, and lysed with RIPA buffer assessed for thymidine incorporation in a liquid scintillation counter. In (150 mM NaCl, 50 mM Tris, and 1% NP40) containing a protease inhibitor the studies in which suppression was blocked with Abs to TGF-␤, mixture (Roche). The cell lysates were then collected and mixed with aga- Notch1, Jagged1, or Delta-like4, the Abs were added to the culture at a rose-immobilized anti-V5 on a rotary shaker at 4°C overnight, after which final concentration of 50 ␮g/ml. Blocking studies were controlled with the agarose beads were washed three times with RIPA buffer. The eluted the use of isotype-control (mouse IgG1 and mouse IgG2b) at a final protein was then subjected to SDS-PAGE followed by transfer to nitrocellu- concentration of 50 ␮g/ml. lose membrane (Amersham Biosciences). In experiments using human mono- cyte-derived DCs, 6 ϫ 106 human monocyte-derived DCs were cultured in Mouse splenic dendritic cells (DCs) 6-well plates filled with 3 ml of complete medium with or without 10 ng/ml rTGF-␤ and with or without either 2 ϫ 104 Jurkat or Jag-Jurkat cells. A total Spleens obtained from 5–7-wk-old BALB/c female mice were injected of 4 h after transfection, the cells were washed twice with PBS and lysed with with Collagenase D solution (containing 2 mg/ml Collagenase D in HBSS RIPA buffer containing a protease inhibitor mixture (Roche) and phosphatase 2798 NOTCH1 SIGNALING AND REGULATORY T CELL FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Notch1 is expressed on DCs. a, Mouse splenic CD11c cells were purified by Ab-coated beads, following which the cells obtained were stained with Abs recognizing Notch ligands and Notch1 and analyzed by flow cytometry. b, Human monocyte-derived DCs were stained with Abs recognizing Notch ligands and Notch1 and the surface expression of these molecules was detected by flow cytometry.

inhibitor mixture (Roche). The cell lysates were then collected and mixed with Nuclear extraction and Western blotting anti-cleaved Notch1 on a rotary shaker at 4°C overnight. Protein A/G plus- ϫ 5 agarose immunoprecipitation reagent (Santa Cruz Technology) was then A total of 2.5 10 HT29 cells were cultured in 6-well plates filled with added to the mixture and the samples were mixed on a rotary shaker at 4°C 2.5 ml complete DMEM/F-12 medium (10% FCS, 2 mM L-glutamine, 100 ␮ for another 2 h, after which the samples were centrifuged and supernatants U/ml penicillin, and 100 g/ml streptomycin). The cells were transfected were collected. The agarose beads were then washed three times with RIPA with either pN1ICD-V5 or empty pcDNA4.0-V5/his version A vector us- buffer and the eluted protein was subjected to SDS-PAGE followed by transfer ing TransIT-LT1 transfection reagent (Mirus Bio Corporation) according to nitrocellulose membrane (Amersham Biosciences). After blocking with 5% to the manufacturer’s protocol. A total of 36 h after transfection, the me- skim milk, the membrane was incubated with appropriate primary Abs at 4°C dium was changed to serum-free DMEM/F-12 containing 1% BSA, and overnight and then washed and incubated with appropriate secondary Abs for 48 h after transfection, the cells were cultured with or without 1 ng/ml ␤ 1 h at room temperature. Finally, after incubation, the membrane was washed rTGF- for 30 min, after which nuclear extraction was performed using a and the were visualized by chemiluminescence according to the man- transfactor extraction kit (Clontech Laboratories). The nuclear extracts ufacturer’s protocol (Pierce). In the experiments with human DCs, the col- were subjected to SDS-PAGE, transfer to the nitrocellulose membrane, and lected supernatant was also subjected to SDS-PAGE and assessed for pSmad3 chemiluminescence as indicated above. and actin. Luciferase assay Statistical analysis Data are expressed as mean Ϯ SD. Statistical significance was valued by A total of 2.5 ϫ 105 MLEC-PAI cells were transfected with either empty Student’s t test. pcDNA4.0-V5/his vector or Notch1 expression vector pN1ICD-V5 (gifts from Dr. Doreen Kacer; Ref. 32) together with pSV-␤-galactosidase con- trol vector (Promega) in a 96-well flat-bottom plate. A total of 48 h after Results transfection, the cells were cultured with various concentrations of recom- ϩ ϩ ϩ Ϫ binant TGF-␤ for 8 h, after which cell lysates were prepared using a Glo- CD4 CD25 T cells, but not CD4 CD25 T cells, express cell Lysis (Promega) or ␤-gal kit (Applied Biosystems). Luciferase activity in surface Notch ligand Jagged1 the lysates was measured by Wallac Victor3 luminometer (PerkinElmer) and relative luciferase activity was calculated by dividing luciferase units In initial studies, we assessed the expression of Notch ligands on ϩ ϩ ϩ Ϫ with ␤-gal units. freshly isolated splenic CD4 CD25 T cells and CD4 CD25 T The Journal of Immunology 2799 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Blocking Notch1 inhibits suppression by CD4ϩCD25ϩ T cells. Isolated CD4ϩCD25Ϫ T cells, CD4ϩCD25ϩ T cells were mixed at various ratios with irradiated splenocytes (3000 rad) and 1 ␮g/ml anti-CD3 Ab. Anti-Notch1 neutralizing Ab (a), anti-Jagged1 Ab (b), anti-Jagged1 and anti- p Ͻ 0.01 compared with ,ءء ;p Ͻ 0.05 ,ء .Delta-like4 Ab (c), or isotype control IgG (d) were added where indicated. Results are shown as means Ϯ SD the proliferation of the CD4ϩCD25Ϫ cells without added Abs. Data shown is representative of three independent experiments.

cells by two-color flow cytometry. As shown in Fig. 1, ϳ90% of Blocking Notch1 signaling partially inhibits the regulatory isolated CD4ϩCD25ϩ cells expressed Foxp3, indicating that this function of CD4ϩCD25ϩ T cells cell population was indeed composed largely of Tregs (and can be Because the Notch ligands, especially Jagged1, are preferentially referred to as such). As also shown in Fig. 1, Jagged1 and, to a expressed on Tregs and Notch1 is expressed on DCs, it seemed lesser extent, Delta-like4 was expressed on Tregs whereas such ϩ Ϫ possible that Notch signaling was involved in the regulatory ac- expression was minimal on CD4 CD25 T cells. Jagged2 or Delta- tivity exerted by these cells. To test this possibility, we performed like1 was not expressed on either cell type. Both Tregs and CD4ϩ Ϫ an in vitro suppression assay in which we determined the ability of CD25 T cells expressed Notch1. a blocking anti-Notch Ab to inhibit suppressor activity at various CD25ϩ:CD25Ϫ cell ratios. In this assay, irradiated splenocytes served as APCs. As shown in Fig. 3a, blocking anti-Notch1 (50 DCs express Notch1 on their surface ␮g/ml) did in fact inhibit suppression, but only in cultures that In parallel studies, we assessed the expression of Notch1 or Notch1 contained a 4:1 and greater ratio of CD25Ϫ T cells to CD25ϩ T ligands on splenic CD11c DCs by flow cytometry. As shown in cells. Similar, albeit weaker, inhibitory effects were noted in sup- Fig. 2a, unstimulated murine splenic DCs express Notch1, but not pression assays in which blocking anti-Jagged1 Ab was added to Notch ligands, on their surface. As shown in Fig. 2b, similar re- the cultures (Fig. 3b). In an additional study, we also determined sults were obtained with human monocyte-derived DCs. whether a greater inhibition of suppression could be obtained with 2800 NOTCH1 SIGNALING AND REGULATORY T CELL FUNCTION

FIGURE 5. Activated Notch1 (NICD) enhances translocation of pSmad3 to the nucleus. HT29 cells were transfected with either empty vector or NICD-expressing vector. The cells were then cultured with TGF-␤ for 30 min and then processed to obtain nuclear extracts. The latter were then subjected to Western blotting with anti-pSmad3.

CD25Ϫ cells in the absence of Tregs (data not shown), possibly because GSI affects many proteins that are cleaved by ␥-secre-

tase (33). Downloaded from

An intracellular Notch1 component released upon Notch1 activation binds to phosphorylated Smad3 Previous studies have shown that Treg suppressor activity is me- diated by membrane-bound TGF-␤ (30). We therefore hypothe-

sized that the above participation of Notch signaling in Treg ef- http://www.jimmunol.org/ fector function may be due to its ability to enhance TGF-␤ signaling in APCs. To investigate this possibility, we initially de- termined whether a major TGF-␤ signaling intermediate (Smad3) physically interacts with a Notch signaling intermediate, NICD, in TGF-␤-stimulated cells. To this end, we transfected the human cell line, THP1 cells, with either empty vector or with V5-tagged NICD and stimulated the transfected cells with TGF-␤. We then obtained whole cell lysates of the stimulated cells at 30 min after stimulation, the time of peak expression of phosphory- by guest on September 29, 2021 lated Smad3. We performed immunoprecipitation using anti-V5- coated agarose beads and assessed the expression of pSmad3. As shown in Fig. 4a, immunoprecipitation of the lysates with anti- V5-coated agarose beads followed by SDS-PAGE and immuno- blotting with anti-pSmad3 Ab yield a strong pSmad3 band in TGF- ␤-stimulated NICD-transfected cells as well as a weak pSmad3 band in nonstimulated NICD-transfected cells, the latter probably FIGURE 4. Activated Notch1 binds to pSmad3. a, THP1 cells were due to the TGF-␤ present in the cell culture medium. Similar re- transfected with either empty vector or NICD-expressing vector and sults were obtained in studies of NICD-transfected HT29 cells then stimulated with TGF-␤. Cell lysates were collected and subjected (data not shown). to immunoprecipitation with anti-V5 followed by immunoblotting anti- In further studies, we determined whether the Smad/NICD in- pSmad3. b, Jurkat cells or Jurkat cells stably transfected with a teraction also occurs in untransfected primary APCs. To this end, Jagged1-expression plasmid (Jag-Jurkat) were stained with anti- Jagged1 and then analyzed for surface Jagged1 expression by flow cy- we developed a Jurkat cell line stably transfected with Jagged1 tometry. c, Human monocyte-derived DCs were cultured alone or (Jag-Jurkat) (see Materials and Methods) and, as shown in Fig. 4b, cocultured with Jurkat or Jag-Jurkat cells with or without TGF-␤. After expressing Jagged1 on its surface to about the same extent as 4 h of culture, cells were lysed and the whole cell lysates obtained were Tregs; untransfected Jurkat cells, however, exhibited only weak subjected to immunoprecipitation with anti-NICD Ab followed by im- Jagged1 expression. Next, using the Jag-Jurket cells to simulate munoblotting anti-pSmad3. Tregs, we cocultured the Jag-Jurkat cells (or Jurkat cells) with human monocyte-derived DCs in the presence of TGF-␤ and after 4 h lysed the cells to obtain whole cell lysates for immunoprecipi- a mixture of anti-Jagged1 and anti-Delta-like4 present in equal tation studies of Notch-Smad interactions. As shown in Fig. 4c, concentration. As shown in Fig. 3c, the presence of both Abs was immunoprecipitation of the lysates with anti-NICD followed by only marginally better than anti-Jagged1 Ab alone in the reversal immunoblotting with anti-pSmad3 yielded a strong pSmad3 band of suppression. In control studies shown in Fig. 3d, addition of in lysates of cells cocultured with Jag-Jurkat and stimulated with isotype control IgG did not inhibit suppression in the suppressor TGF-␤ as well as a weak pSmad3 band in cells cocultured with assay. Jurkat, the latter presumably due to the small amount of Jagged1 We also attempted to evaluate the ability of an inhibitor of expressed on the surface of Jurkat cells. These findings offer strong Notch signaling, ␥-secretase inhibitor (GSI), to influence Treg evidence that NICD and pSmad3 physically associate in TGF-␤- function in a suppressor assay. This proved to be infeasible stimulated cells subjected to Notch1 signaling, such as DCs inter- because GSI itself had a profound anti-proliferative effect on acting with Tregs. The Journal of Immunology 2801

Activated Notch1 enhances TGF-␤-induced PAI-1 luciferase activity In a second approach to the question of whether NICD/pSmad3 binding has a functional consequence, we determined the influ- ence of NICD on TGF-␤ signaling in MLECs stably transfected with a Smad-sensitive PAI-1 promoter linked to a luciferase reporter cassette. In particular, we transfected the MLECs with either empty vector or NICD vector, together with a control pSV-␤-galactosidase vector, and then stimulated the transfected cells with TGF-␤. As shown in Fig. 6, transfection of the NICD vector enhanced TGF-␤-induced PAI-1 luciferase activity over a range of TGF-␤ concentrations and especially at 500 pg/ml, FIGURE 6. Activated Notch1 (NICD) enhances TGF-␤-induced Smad- the highest concentration tested. These data suggest that Notch mediated transcription. MLEC-PAI cells were transfected with either signaling and generation of NICD leads to augmentation of empty vector or NICD expressin-vector. The cells were then cultured with TGF-␤ transactivation. TGF-␤ for 30 min and then processed for measurement of relative lucif- erase activity. Each data point shown is the mean of values from 12 sam- ples Ϯ SD. Data shown is representative of three independent experiments. Notch1 suppresses the proliferation of CD4ϩCD25Ϫ cells p Ͻ 0.001 compared with the relative luciferase activity through TGF-␤ Downloaded from ,ءء ;p Ͻ 0.01 ,ء of the cells transfected with empty vector. Taken together, the above findings relating to TGF-␤/Notch1 sig- naling are consistent with the idea that Notch1 signaling affects Treg effector function via its effect on TGF-␤ signaling rather than via a TGF-␤-independent mechanism. To test this hypothesis, we Activated Notch1 enhances pSmad3 translocation into the reasoned that if the TGF-␤ and Notch1 signaling effects on Treg nucleus http://www.jimmunol.org/ effector function were independent then blockade of suppres- We next determined whether the above physical interaction be- sion by blocking Abs in an in vitro suppressor assay would be tween NICD and pSmad3 had functional consequences. Because additive, whereas if the effects were dependent then blockade of activated Notch1 (NICD) translocates into the nucleus, one such suppression would not be additive. As shown in Fig. 7, under consequence could be the facilitation of pSmad3 nuclear translo- conditions in which blockade achieved with anti-TGF-␤ is sub- cation. To test this possibility, we transfected an increasing amount optimal (to be able to monitor the possibility of an additive of NICD vector into HT29 cells and stimulated the cells with effect of anti-Notch1) an additive effect was not seen at any TGF-␤. We then isolated nuclear extracts from the cells and per- effector:suppressor cell ratio. Thus, it is likely that the two path- formed Western blots with anti-pSmad3. As shown in Fig. 5, trans- ways are dependent. In addition, because blockade of either by guest on September 29, 2021 fection of NICD led to a dose-dependent increase in intranuclear pathway has an equally inhibitory effect on suppression, it is pSmad3. Thus, another mechanism of enhancement of TGF-␤ sig- likely that Notch signaling is a necessary accompaniment of naling by Notch activation is facilitation of intranuclear transport. TGF-␤ signaling in Treg effector function.

FIGURE 7. Blocking anti-Notch1 and anti-TGF-␤ Abs do not have additive inhibitory effect on Treg activity. Isolated CD4ϩCD25Ϫ T cells, CD4ϩ CD25ϩ T cells were mixed in various ratios with irradiated splenocytes and 1 ␮g/ml anti-CD3 Ab as described in Fig. 1. A total of 50 ␮g/ml anti-Notch1 neutralizing Ab and/or 50 ␮g/ml anti-TGF-␤ Ab was added where indicated. Results shown are representative of three independent studies. Results are .p Ͻ 0.01 compared with the proliferation of the CD4ϩCD25Ϫ cells without any Abs ,ءء ;p Ͻ 0.05 ,ء .shown as means Ϯ SD 2802 NOTCH1 SIGNALING AND REGULATORY T CELL FUNCTION

Discussion TGF-␤-induced responses, indicating that Notch activation aug- In previous studies, we and other investigators established that ments Smad-mediated transcription at Smad-sensitive promoters. either cell surface TGF-␤ (in the form of TGF-␤ linked to latency These studies make it apparent that Notch1 activation can enhance associated protein, i.e., latent TGF-␤ or latency associated protein) TGF-␤ signaling both at the level of Smad translocation into the or secreted TGF-␤ is a key component of the effector mechanism nucleus and at the level of Smad transactivation at promoter sites. underlying the suppressor activity of naturally occurring Tregs. In a final set of studies, we showed that blockade of TGF-␤ sig- These studies show, for instance, that Tregs have TGF-␤ on their naling and Notch signaling in a suppression assay do not have cell surface and that by blocking such TGF-␤ the in vitro sup- additive effects. Taken together, this set of findings is consistent pressive ability of Tregs is greatly diminished (30, 34). In ad- with the conclusion that while TGF-␤ signaling is essential for dition, they show that cells lacking the ability to respond to Treg effector function, Notch signaling plays an important and TGF-␤ signaling cannot be suppressed by Tregs, either in vitro perhaps essential role in facilitating TGF-␤ signaling in such ef- or in vivo (30, 35). Finally, they show that T cell populations fector function. Furthermore, it is highly likely that this conclusion depleted of cells bearing membrane-bound TGF-␤ or T cells applies to TGF-␤ signaling mediated by membrane-bound TGF-␤ from mice with T cell-specific inability to produce TGF-␤ can- on Tregs in as much as we have previously shown that such TGF-␤ not mediate Treg function in vivo (36). These and other findings induces robust and rapid Smad activation (5, 34). settled the question of the involvement of TGF-␤ in Treg ef- Although the conclusion above is the most likely, the data at fector function and, at the same time, opened the door to other hand could still support the notion that TGF-␤ signaling is en- questions relating to how TGF-␤ signaling actually leads to hancing Notch-mediated effector function, rather than the other Downloaded from suppression of immune function. way around. This arises from that fact that while in these studies With regard to these latter questions, recently it was shown that we showed that Notch signaling enhances TGF-␤ function, in cells bearing surface TGF-␤ (but not cells that lacked this prop- other studies cited above the enhancement was mutual (28). This erty) could activate the Notch1 signaling pathway in potential tar- latter possibility would be supported by evidence that Notch sig- get cells (16). More importantly, the activity of regulatory cells naling has an inherently negative effect on T cell and/or APC func- that prevent the development of experimental could be tion; however, this does not seem to be the case. Thus, while a blocked in the whole animal by the administration of a blocking number of studies of the effect of Notch signaling on T cell pro- http://www.jimmunol.org/ anti-Notch1 Ab (16). These results thus suggested that Notch sig- liferation have disclosed that such signaling has a negative effect naling was also involved in Treg effector function, albeit in the on cell proliferation (38–40), a number of other studies have in- context of TGF-␤ because only cells bearing TGF-␤ could activate dicated a positive effect (41, 42). In addition, it has been shown the Notch pathway. It should be noted, however, that these studies that Notch signaling may selectively influence T cell differentia- of the possible role of Notch signaling in regulatory T cell function tion depending, in some studies, on the type of Notch ligand in- were grounded in earlier studies seeking to establish an interrela- ducing Notch activation (24–27); this type of Notch signaling tionship between TGF-␤ signaling and Notch signaling at the bio- function could be considered positive or at least neutral because T

chemical level. The first hint of such an interrelationship came cell differentiation occurs within a positive proliferative milieu. by guest on September 29, 2021 from studies that showed that TGF-␤ signaling leads to rapid up- Finally, with respect to the effect of Notch signaling on APCs, regulation of Hes-1, a major target of Notch signaling in keratin- there is clear evidence that stimulation of APCs (DCs) via Jagged1 ocytes (37). Later, more definite evidence of “cross-talk” between enhances APC function from the point of view of maturation the two pathways has come from studies showing TGF-␤ signaling markers, cytokine production, and differentiating effects on T cells in neural stem cells in vivo and in myoblasts in vitro led to (43). Overall, therefore, whether one considers the T cell or the Notch-dependent Hes-1 expression (28). In addition, intracel- APC the main target of Treg suppression, there is no consistent lular signaling molecules intrinsic to the TGF-␤ (Smad3) and evidence that Notch signaling has the required negative effects that Notch1 (NICD) signaling pathways synergize in the activation one would expect of a mediator of suppression. This, taken to- of Notch- and TGF-␤-specific synthetic promoters. Finally, it gether with the fact TGF-␤ does have an inherently negative effect was shown that Smad3 and NICD physically interact and that on cell function and the fact mentioned above that cells lacking the such interaction led to recruitment of Smad3 to Notch target ability to be signaled by TGF-␤ cannot be suppressed, puts the genes via binding to CSL, a key DNA-binding protein of the TGF-␤ pathway firmly in the primary position with respect to Treg Notch pathway. effector function. These previous findings led us to determine whether similar Yet another consideration is whether TGF-␤ can act in the ab- TGF-␤/Notch interactions governed the TGF-␤ signaling under- sence of Notch1 signaling to mediate Treg suppression. In the lying Treg function. We found that, indeed, the machinery for study shown in Fig. 7, the capacities of anti-TGF-␤ and anti- activation of the Notch pathway in Treg function was in place in Notch1 to inhibit suppression were equivalent; however, in several that Tregs expressed surface Jagged1 (and lesser amounts of Delta- other studies, including several in which the inhibitory capacities like4) and potential target cells expressed Notch; moreover, we of these Abs were compared by CFSE dilution measurement, the found that this machinery “worked” in relation to Treg regulatory anti-TGF-␤ was able to inhibit at a higher CD25Ϫ:CD25ϩ ratio function in that blockade of Notch1 (and to a lesser extent, of (data not shown); it is therefore likely that the TGF-␤ signaling Jagged1) impaired Treg suppressor function in an in vitro suppres- pathway in Treg effector function is the more primary pathway, but sion assay. In further studies to understand the basis of these find- additional studies will be necessary to firmly establish this ings, we determined that Smad3 generated by TGF-␤ signaling of conclusion. human monocyte-derived DCs (or THP1 cells (a human macro- Finally, the data reported here showing that Notch ligands are phage cell line)) associates with NICD, the Notch fragment de- found on Tregs and Notch is found on APCs implies that the Notch rived from activated cell surface Notch1. In addition, we showed signaling pathway in relation to suppression occurs mainly as a that transfection of NICD leads to enhanced nuclear translocation result of regulatory T cells acting on and inhibiting the function of of pSmad3 and that NICD transfection of reporter cells responding APCs. There is now ample evidence for this mode of regulatory T to TGF-␤ signaling with a luciferase response led to enhanced cell activity (44–47) including one report in which two-photon The Journal of Immunology 2803 laser-scanning microscopy was used to show that Tregs form sta- T cells following overexpression of a Notch ligand by human B . ble associations with DCs, but not with helper T cells (44). In J. Virol. 77: 10872–10880. 15. Kared, H., H. Adle-Biassette, E. Fois, A. Masson, J. F. Bach, L. Chatenoud, addition, it has been shown that Tregs cocultured with DCs en- E. Schneider, and F. Zavala. 2006. Jagged2-expressing hematopoietic progenitors hance IL-10 and suppress IL-6 production by the DCs, whereas promote regulatory T cell expansion in the periphery through notch signaling. Immunity 25: 823–834. naive T cells have the opposite effect at least under noninflamma- 16. Ostroukhova, M., Z. Qi, T. B. Oriss, B. Dixon-McCarthy, P. Ray, and A. Ray. tory conditions (45). This said, it remains possible that Treg 2006. Treg-mediated involves activation of the Notch-HES1 function involving Notch signaling also involves direct effects axis by membrane-bound TGF-␤. J. Clin. Invest. 116: 996–1004. 17. Xu, T., I. Rebay, R. J. Fleming, T. N. Scottgale, and S. Artavanis-Tsakonas. 1990. on T cells as suggested by previous studies showing Hes-1 ac- The Notch locus and the genetic circuitry involved in early Drosophila neuro- ϩ tivation in CD4 T cells under in vivo tolerogenic conditions genesis. Genes Dev. 4: 464–475. (16). It is also important to consider the possibility that the 18. Artavanis-Tsakonas, S., M. A. Muskavitch, and B. Yedvobnick. 1983. Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster. Notch signaling pathway may be involved in negative signals Proc. Natl. Acad. Sci. USA 80: 1977–1981. delivered by DCs to T cells rather than vice versa as considered 19. Osborne, B. A., and L. M. Minter. 2007. Notch signalling during peripheral T-cell so far. This is possible because while Notch ligands were not activation and differentiation. Nat. Rev. Immunol. 7: 64–75. 20. Pear, W. S., and F. Radtke. 2003. Notch signaling in . Semin. detected on resting DCs in the studies reported here, there is Immunol. 15: 69–79. ample evidence that activated DCs express such ligands (48– 21. Robey, E. 1999. Regulation of T cell fate by Notch. Annu. Rev. Immunol. 17: 283–295. 50). In addition, in a recent study it was shown that interactions 22. Valdez, P., and E. Robey. 1999. Notch and the CD4 versus CD8 lineage decision. between DCs and T cells are bi-directional under conditions of Cold Spring Harbor Symp. Quant. Biol. 64: 27–31. cell activation (48). It is not clear, however, whether the Notch 23. Maekawa, Y., S. Tsukumo, S. Chiba, H. Hirai, Y. Hayashi, H. Okada, K. Kishihara, and K. Yasutomo. 2003. Delta1-Notch3 interactions bias the func- Downloaded from signal in the DC to T cell direction is suppressive; while in one tional differentiation of activated CD4ϩ T cells. Immunity 19: 549–559. study it was shown that APCs that overexpress Notch ligand 24. Amsen, D., J. M. Blander, G. R. Lee, K. Tanigaki, T. Honjo, and R. A. Flavell. have suppressive qualities, in another study it was shown under 2004. Instruction of distinct CD4 fates by different notch ligands on -presenting cells. Cell 117: 515–526. more physiologic conditions that inhibition of Notch signaling 25. Minter, L. M., D. M. Turley, P. Das, H. M. Shin, I. Joshi, R. G. Lawlor, led to decreased cytokine production (IFN-␥) by interacting T O. H. Cho, T. Palaga, S. Gottipati, J. C. Telfer, et al. 2005. Inhibitors of ␥-secre- cells (48, 51). Thus, while we recognize that mature DCs can be tase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat. 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