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Surface Expression of Notch1 on : Correlation with the Double-Negative to Double-Positive Transition This information is current as of October 9, 2021. Eugene Y. Huang, Alena M. Gallegos, Sabrina M. Richards, Sophie M. Lehar and Michael J. Bevan J Immunol 2003; 171:2296-2304; ; doi: 10.4049/jimmunol.171.5.2296

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Surface Expression of Notch1 on Thymocytes: Correlation with the Double-Negative to Double-Positive Transition1

Eugene Y. Huang, Alena M. Gallegos, Sabrina M. Richards, Sophie M. Lehar, and Michael J. Bevan2

Notch1 plays a critical role in regulating T lineage commitment during the differentiation of lymphoid precursors. The physio- logical relevance of Notch1 signaling during subsequent stages of differentiation has been more controversial. This is due in part to conflicting data from studies examining the overexpression or targeted deletion of Notch1 and to difficulties in distin- guishing between the activities of multiple Notch family members and their ligands, which are expressed in the . We employed a polyclonal antiserum against the extracellular domain of Notch1 to study surface expression during thymopoiesis. We found high levels of Notch1 on the cell surface only on double negative (DN) stage 2 through the immature single-positive stage of development, before the double-positive (DP) stage. The , as read out by Deltex1 expression Downloaded from levels, is highly active in DN thymocytes. When an active Notch1 transgene, Notch1IC, is exogenously introduced into thymocytes of recombinase-activating gene 2-deficient mice, it promotes proliferation and development to the DP stage following anti-CD3 treatment without apparently affecting the intensity of pre-TCR signaling. In addition, a line expressing the Notch ligand, Delta-like-1, promotes the in vitro expansion of wild-type DN3 thymocytes in vitro. Consistent with other recent reports, these data suggest a role for Notch1 during the DN to DP stage of thymocyte maturation and suggest a cellular mechanism by which Notch1IC oncogenes could contribute to thymoma development and maintenance. The Journal of Immunology, 2003, 171: http://www.jimmunol.org/ 2296Ð2304.

s T cells develop in the thymus, TCR signals provide (6). Studies with transgenic mouse models and retrovirally trans- critical checkpoints as cells transit through the various duced stem cells have shown that expression of the active intra- A stages of maturation. For example, a pre-TCR signal is cellular portion of Notch1, Notch1IC, can modify the survival, necessary for the most immature thymocyte subset, termed double proliferation, and maturation of thymocytes and potentially mod- negative (DN),3 to develop into double-positive (DP) thymocytes, ulate TCR signaling (6–11). In addition, pharmacological inhibi- expressing both CD4 and CD8 (1). The assembly and surface ex- tion of Notch signaling in thymic organ culture systems impairs pression of CD3, pre-T␣, and a functionally rearranged TCR␤- thymocyte development (12, 13). Specific gene deletion of Notch1 by guest on October 9, 2021 chain mediate this checkpoint, termed ␤ selection. After successful at the earliest lymphoid precursors obviates further development pre-TCR signaling, DN thymocytes undergo many rounds of di- into the T lineage (14, 15). vision and multiple phenotypic changes. In addition to genes that The Notch1 protein belongs to a family of receptors that upon encode pre-TCR components, a number of other genes regulate interaction with ligand releases the intracellular fragment, maturation. These genes either affect pre-TCR signaling indirectly Notch1IC, via a proteolytic cleavage event. Notch1IC translocates or are required for the numerous cellular changes seen during the into the nucleus, binds C promoter binding factor 1, and activates DN to DP transition (2–5). (16). Target genes induced by Notch1 in T cell lines The Notch1 signaling pathway has been proposed to play a role include Hes family members, Deltex1, and pre-T␣ (6, 17, 18). during various stages of T cell development. Notch response genes Multiple homologues of the Notch1 and a number of dif- such as Deltex1 are regulated during thymocyte development. Del- ferent Notch ligands are expressed throughout the normal thymus. tex1 is highly expressed in DN, down-regulated in DP cells, and The four Notch family members share homology in their intracel- up-regulated in mature CD8 and CD4 single-positive cells (SP) lular domains, and all have been shown to bind to C promoter binding factor 1. Therefore, intracellular signaling of the family members may converge on similar target genes. For example, Department of Immunology, Howard Hughes Medical Institute, University of Wash- ington, Seattle, WA 98195 overexpression of the active intracellular forms of Notch1, Notch2, Received for publication February 14, 2003. Accepted for publication July 1, 2003. and Notch3 is able to up-regulate Hes family members (19–21). The mammalian Notch1 receptor was originally identified in a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance subset of human T cell acute lymphoblastic (T-ALL), with 18 U.S.C. Section 1734 solely to indicate this fact. where a chromosomal translocation resulted in a Notch1 trunca- 1 This work was supported by National Institutes of Health Grants AI29802 and tion, ostensibly Notch1IC, brought under the control of a T cell- T32GM07270 and the Howard Hughes Medical Institute. specific promoter (22). In mice this has been recapitulated by the 2 Address correspondence and reprint requests to Dr. Michael J. Bevan, Howard expression of intracellular fragments of both human and mouse Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195-7370. E-mail address: [email protected] Notch1 and mouse Notch3 (23–25). Tumor development with 3 Abbreviations used in this paper: DN, double negative; DP, double positive; EGF, these constructs, even when expressed in all hemopoietic cells, epidermal growth factor; ERK, extracellular signal-regulated kinase; HPRT, hypo- occurs exclusively in immature thymocytes (23, 24). In addition, in xanthine phosphoribosyltransferase; ISP, immature single positive; RAG, recombi- nase-activating gene; SP, single positive; T-ALL, T cell acute lymphoblastic vivo mouse mammary tumor insertional mutagenesis studies . identified intracellular Notch1 constructs as putative collaborators

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 2297 with c-Myc transformation in the majority of the isolated tumors To induce DN to DP development in RAG2-deficient mice, 100 ␮gof (26). However, the cellular mechanisms that are targeted by 2C11, anti-CD3⑀, mAb was delivered in one i.p. injection. Mouse thymi Notch1IC signaling during thymomagenesis have not been well were harvested and analyzed at various time points after injection, as in- dicated. 2C11 mAb was immunoaffinity purified from ascites and dialyzed characterized. against sterile PBS. Notch1 has been clearly implicated in promoting T lineage choice from common lymphoid progenitors, although its role in later development is less clear (15, 27). Inducible gene ablation of Transcript expression analysis Notch1 via -Cre-mediated deletion suggests its role at least Sorted subsets. Thymocytes were isolated from 4- to 6-wk-old B6 through the DN stages (14). However, ablation of Notch1 at the mice and sorted on a FACSVantage (BD Biosciences, San Diego, late DN stages via CD4/Cre-mediated gene deletion allows normal CA) into phenotypic thymocyte subsets; unsorted thymocytes, DN thymocyte development (28). On the other hand, overexpression of (CD3Ϫ, CD8Ϫ, CD4Ϫ, B220Ϫ), DP (CD8ϩ, CD4ϩ), CD4 (CD4ϩ, Notch1IC, pharmacological inhibition of Notch signaling, and the CD3ϩ), CD8 (CD8ϩ, CD3ϩ), and DN subsets as previously de- pattern of Notch target gene expression suggest a role at the later scribed (32); unsorted DN (CD3Ϫ, CD8Ϫ, CD4Ϫ, B220Ϫ), stages of DP to SP development (6, 10, 12, 13). In light of the DN1(DN and CD44ϩ, CD25Ϫ), DN2 (DN and CD44ϩ, CD25ϩ), conflicting data on the physiological role of Notch1 during differ- DN3 (DN and CD44-, CD25ϩ), and DN4 (DN and CD44Ϫ, ent stages of thymocyte development, we sought to determine at CD25Ϫ). RNA and cDNA were prepared using RNA STAT 60 what stages of development is biologically accessible Notch1 re- (Tel-Test, Friendswood, TX) and SuperScript II (Invitrogen, Carls- ceptor expressed on thymocytes and to correlate it with Notch bad, CA) according to the manufacturer’s instructions. target gene expression. Notch ligand family members Delta and

The cDNA samples were analyzed in triplicate by real-time Downloaded from Jagged, have been shown to be expressed throughout the thymus, PCR with sequence-specific probes on an ABI 7700 sequence de- and all are able to interact with Notch1 (29, 30). This suggests that tector (PE Applied Biosystems, Foster City, CA). Primers and the surface expression of Notch proteins may play an important probes were designed to span exon/intron borders to prevent am- role in regulating whether a cell is capable of receiving a Notch plification of genomic sequence: hypoxanthine phosphoribosyl- signal. We show that specific staining of surface Notch1 is appar- transferase (HPRT): forward, 5Ј-TGG AAA GAA TGT CTT GAT ent on immature DN thymocytes and is down-regulated as the cells TGT TGA A; reverse, 5Ј-AGC TTG CAA CCT TAA CCA TTT http://www.jimmunol.org/ mature to DP and later stages. Using a recombinase-activating TG; probe, 5Ј-FAM-CAA ACT TTG CTT TCC CTG GTT AAG gene (RAG)-deficient model and anti-CD3 to provide the pre-TCR CAG TAC AGC-TAMRA; Notch1: forward, 5Ј-GAG ACC AAG signal, we show that enforced expression of Notch1IC augments AAG TTC CGG TTT G; reverse, 5Ј-CTT CAC TGT TGC CTG the DN to DP transition. These data suggest a role for Notch1 TCT CAA GG; probe, 5Ј-FAM-CAA TGT TCG AGG ACC AGA signaling in the proliferative expansion and differentiation of DN TGG CTT CAC A-TAMRA; Deltex1: forward, 5Ј-TGA GGA ␤ cells following -selection. TGT GGT TCG GAG GT; reverse, 5Ј-CCC TCA TAG CCA GAT GCT GTG; probe, 5Ј-FAM-CGC CTG ATG AGG ACT GTA Materials and Methods CCA TTT GCA T-TAMRA; and pre-Ta: forward, 5Ј-CTG CTT Cell lines CTG GGC GTC AGG T; reverse, 5Ј-TGC CTT CCA TCT ACC by guest on October 9, 2021 Human 293 HEK cells were transfected with full-length murine Notch1 AGC AGT; probe, 5Ј-FAM-CCT TTC CGT CTC TGG CTC CAC (FLN1/293) inserted into the pcDNA3 vector (Stratagene, La Jolla, CA) or CCA-TAMRA. In a 50-␮l reaction cDNA was incubated with 20 with vector alone. Clones were selected in DMEM with 10% BSA and 1 ␮M forward and reverse primers (Invitrogen), 10 ␮M5Ј,6-FAM- mg/ml G418, and the expression of murine Notch1 was confirmed by 3Ј-TAMRA fluorogenic probe (Biosearch Technologies, Novato, Western analysis with Abs to both intracellular and extracellular portions ϫ of mNotch1. CA), and TaqMan 2 universal PCR Mastermix (PE Applied Bio- systems). No template controls and no reverse transcriptase con- Ab and cell staining trols were included for each primer/probe set and cDNA set, re- The anti-Notch1ec reagent is an immunoaffinity-purified rabbit IgG raised spectively. Transcript levels were normalized to HPRT levels against the rat Notch1 epidermal growth factor (EGF) 10-EGF22, residues before determining the relative fold difference in the gene of in- 381–853 (31) (Upstate Biotechnology, Lake Placid, NY), which cross- terest. RT-PCR analysis of the Notch1IC transgene was performed reacts with mouse Notch1. Cells were stained using a tertiary protocol with a biotinylated goat anti-rabbit Ab, followed by a streptavidin-conjugated as described previously (6). fluorochrome. Purified normal rabbit IgG (Upstate Biotechnology) was also included as a staining control. Fluorochrome-conjugated Abs for CD4, CD8, CD24 (heat shock Ag), CD44, CD25, CD69, B220, CD3 (BD Histology PharMingen (San Diego, CA) and eBioscience (San Diego, CA)) were used to phenotypically characterize and sort cell populations by FACS Analyses of 6- to 8-wk-old B6 mouse thymuses used an indirect enzyme analysis. immunohistochemical procedure (33). Briefly, frozen sections of tissue GST corresponding to the murine extracellular region were serially incubated with optimal dilutions of anti-N1ec preincubated recognized by anti-N1ec, residues 381–853 (mN1ec-GST), and GST alone with mN1ec-GST or GST as a control. Notch1 was detected by sequential were made as blocking agents and controls for surface staining. Recom- exposure to digoxigenin-conjugated goat anti-rabbit IgG and peroxidase- conjugated Fab of goat anti-digoxigenin Abs. Peroxidase activity was re- binant proteins were produced in BL-21 Gold (DE3) bacteria (Stratagene, Ј La Jolla, CA) and purified using the pGEX2T protocol (American Bio- vealed with 3,3 -diaminobenzidine in the presence of hydrogen peroxide. sciences, Piscataway, NJ). Anti-N1ec Ab was preincubated with 5 ␮g/ml of mN1ec-GST or GST for 15 min at room temperature before the FACS staining protocol. Cell cycle analysis Mice Cells were stained with the DNA-binding dye 7-amino actinomycin D to determine DNA content (BD Biosciences). Briefly, cells were stained for C57BL/6 (B6) mice were obtained from The Jackson Laboratory (Bar Har- cell surface Ags, permeabilized, fixed, and stained with 0.25 ␮g of 7-amino bor, ME). Notch1IC (N1ic) mice that express the intracellular portion of actinomycin D/1 ϫ 106 cells before FACS analysis. Cells were gated on murine Notch1 under the Lck-proximal thymic promoter (6) were crossed the appropriate thymic subsets as defined by CD8 and CD4 cells surface Ͼ10 generations onto the B6 background and mated to RAG2-deficient expression and were analyzed for the percentage of cells with Ͼ2 N DNA ϫ mice on a B6 background (The Jackson Laboratory) to obtain Notch1IC content. B6 DP thymocytes, which are mostly in G0, were used to define RAG2Ϫ/Ϫ and RAG2Ϫ/Ϫ littermate control mice. 2 N gates. 2298 NOTCH1 ACTIVITY IN IMMATURE THYMOCYTES

Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation Thymocytes were pooled from 3- to 5-wk-old Notch1IC ϫ RAG2Ϫ/Ϫ or RAG2Ϫ/Ϫ mice. Cells were stimulated with 5 ng/ml PMA at 37°Cat1E7 cells/ml in HBSS with 1% BSA for the indicated times. Cells were lysed ␮ in TBS, 1% Triton 0.2 mM Na3VO4, 50 mM NaF, 10 g/ml leupeptin, 10 ␮g/ml aprotinin, and 1 mM PMSF. Supernatant from 2.5 ϫ 106 thymocyte equivalents was resolved by SDS-PAGE on a 10% gel and transferred to Optitran nitrocellulose membrane (Schleicher & Schuell, Keene, NH) Im- munoblot analysis with anti-phospho-ERK Ab (p44/42 MAPK; Cell Sig- naling, Beverly, MA) was performed according to the manufacturer’s pro- tocol. The blot was subsequently stripped in 100 mM 2-ME, 2% SDS, and 62.5 mM Tris (pH 6.8) at 50°C for 30 min; reblocked; and immunoblotted for total ERK (ERK2; Santa Cruz , Santa Cruz, CA) as a loading control. Proteins were detected by HRP-conjugated secondary Abs (Santa Cruz Antibodies) and ECL according to the manufacturer’s protocol (Am- ersham Pharmacia Biotech, Piscataway, NJ). OP9 monolayer cultures DP and DN3 thymocytes were isolated from 4- to 6-wk old mice. CD8ϩCD4ϩCD69low (DP) thymocytes were isolated by FACS sorting. For isolation of DN3 thymocytes, the total DN population was enriched by Downloaded from immunomagnetic separation using biotinylated Abs to CD4 and CD8 (Dy- nal, Oslo, Norway) according to the manufacturer’s instructions, and the DN3 population was sorted on a FACS vantage. Total DN (CD4ϪCD8Ϫ) FIGURE 1. Characterization of the anti-N1ec IgG. A, Schematic of the thymocytes were negatively gated for lineage markers (CD4, CD8, CD3, Notch1 receptor. EGF, EGF repeats; Nlin, Nlin domain; RAM/ANK, RAM B220, Mac1, Gr1, and Ter114), and the DN3 fraction was identified as domain and ankyrin repeats; O, OPA sequence; P, PEST domain. The Ϫ ϩ ϫ 5 CD44 CD25 . Sorted thymocyte subsets (1 10 ) were plated on mono- underlined region indicates the extracellular EGF repeats used as an im- layers of OP9 or OP9 cells expressing the Notch ligand, Delta-like 1 (OP9- munogen and also corresponds to the murine GST fusion protein (mN1ec- Dl1) (34), in 24-well plates in DMEM (Life Technologies, Gaithersburg, http://www.jimmunol.org/ GST) that was made as a blocking reagent. B, 293 cells transfected with MD) supplemented with 15% FCS, penicillin/streptomycin, L-glutamine, and 5 ng/ml of rIL-7 and Flt3L (PeproTech, Rocky Hill, NJ). The growth full-length murine Notch1 (FLN1/293) were stained with anti-N1ec (black and viability of the thymocytes were assessed by Trypan Blue staining. line) or with normal rabbit IgG (gray line). 293 cells transfected with vector alone were stained with anti-N1ec ( ). C, FLN1/293 cells stained with Results anti-N1ec (black line), GST (gray line), and mN1ec-GST fusion protein Anti-N1ec Ab specifically stains surface mouse Notch1 (dashed line). Cells were stained without primary anti-N1ec Ab ( )asa negative control. To date there has been little characterization of Notch1 surface expression in mammalian systems, including the thymus. We first

sought to identify and characterize an Ab that is able to specifically by guest on October 9, 2021 stain surface Notch1. We used a rabbit immunoaffinity-purified and subcapsular zone of the thymus (Fig. 2). This staining IgG against the extracellular portion of rat Notch1, corresponding is blocked in the presence of mN1ec-GST fusion protein. The ex- to EGF10-EGF22 anti-N1ec, for and pression appears to be intercellular and suggests cell surface ex- FACS staining (Fig. 1A). Specificity for surface mouse Notch1 pression of Notch1. In the medulla, where mature SP thymocytes was determined by three assays. Firstly, 293 cells stably express- are thought to reside before export, Notch1 staining is less appar- ing full-length murine Notch1, FLN1/293, were generated. The ent (Fig. 2). This pattern of staining is consistent with expression transfected cells stained positively using anti-N1ec compared with by early thymocytes. T cell progenitors are thought to enter the control cell lines transfected with vector alone (cDNA3/293) and thymus at the cortical-medullary junction and migrate to the sub- Ab controls (Fig. 1B). Secondly, anti-N1ec was preadsorbed on capsular zone during DN development. Specifically at the subcap- FLN1/293 or control cDNA3/293 monolayers, and specific stain- sular zone late DN populations, DN3 and DN4 thymocytes, are ing of Notch1 on the FLN1/293 and thymocytes was compared. present (35). Preadsorption on FLN1/293, but not on control cDNA3/293 re- To identify the population of thymocytes expressing surface moved all staining activity (data not shown). Finally, a GST fusion Notch1, we used anti-N1ec in FACS analysis of cells isolated from protein containing the murine residues corresponding to the im- normal C57BL/6 mice. We detected Notch1 surface expression in

munogen (i.e., EGF10–22), mN1ec-GST, was able to block staining the majority of DNs and on a subset of DP and CD8 SP cells (Fig. on FLN1/293 cells and thymocytes when preincubated with anti- 3B). Staining of CD8 SP cells was surprising given the lack of N1ec relative to GST controls (Figs. 1C, 2, and 3). staining in the thymus medulla (Fig. 2). This apparent discrepancy was resolved when we analyzed Notch 1 surface expression on Early thymocytes express Notch1 at the cell surface immature CD8 SPs (ISPs), which express high levels of CD24 and The expression of endogenous Notch1 during thymocyte develop- are an intermediate step between DN and DP stage, vs staining on ment has not been well characterized. The majority of published mature, CD24low, CD3highCD8 SP thymocytes. The data in Fig. reports have examined mRNA, and these results have been incon- 3C show that only the immature, pre-DP CD8ϩ T cells stain at the sistent. The expression of Notch1 at the cell surface requires sev- surface for Notch 1. eral post-transcriptional modifications, including proteolytic pro- We further examined Notch1 surface expression on the DN sub- cessing, assembly in the , and transport to populations by costaining for CD44 and CD25. We found surface the cell surface before ligand-induced signaling. To address which expression of Notch1 up-regulated at the DN2 stage, when cells cells may be capable of receiving a Notch1 signal, we assayed for become fully committed to the T lineage, and remained throughout the expression of Notch1 in the thymus by immunohistochemistry. the DN and ISP compartments until down-regulated at the DP Using anti-N1ec to stain C57BL/6 thymus sections, we found stage (Fig. 3, B and C, and Fig. 4B) (36). The early expression in that specific Notch1ec staining is localized primarily to the outer DN thymocytes was further confirmed by real-time cDNA analysis The Journal of Immunology 2299

FIGURE 2. Immunohistochemical localization of Notch1 in B6 thymus Downloaded from sections. Notch1 was detected using anti-N1ec preincubated with GST (left panels) or mN1ec-GST as a control (right panels). M, medulla; C, cortex; SCZ, subcapsular zone. Magnification: A, ϫ100; B, ϫ200.

of DN, DP, and mature CD4 and CD8 SP subsets (Fig. 3D). We http://www.jimmunol.org/ found that the Notch1 transcript is highly expressed in the DN subset relative to the levels in DP and SP subsets. While surface Notch1 expression is necessary for conventional activation of the Notch1 signaling pathway, it does not indicate which cells have actually received a Notch1 signal. To determine at what stage of T cell maturation the Notch1 pathway is normally FIGURE 3. Surface expression of Notch1 on thymocyte subsets. B6 activated, we examined the expression of Deltex1 mRNA in DN thymocytes were stained with anti-N1ec preincubated with GST (black thymic subsets. Previous data from thymoma cell lines and trans-

line) or mN1ec-GST ( ). A, Thymic subsets were defined by CD4 and by guest on October 9, 2021 genic mice showed Deltex1 is highly induced by active Notch1IC CD8 expression with the indicated gates and analyzed for Notch1 surface (7). We found that Deltex1 expression is increased at the DN3 staining. B, Anti-N1ec staining of the four major thymocyte subsets. C, stage before being down-regulated at the DN4 stage (Fig. 4C) con- Anti-N1ec staining of immature SP CD8ϩ HSAhi cells (ISP) and mature sistent with a recent PCR analysis of Deltex1 expression (37). The CD8ϩ HSAlow thymocytes. D, cDNA isolated from thymocyte subsets decrease in Deltex1 expression relative to the high surface Notch1 were analyzed by real-time TaqMan PCR for Notch1 transcripts. Fold dif- expression in DN4 subsets suggests that Deltex1 expression may ferences were calculated after normalizing cDNA levels of HPRT tran- not fully reflect activation of the Notch pathway. It is clear that not scripts between samples. all Notch-responsive genes, such as Hes1 and Meltrin ␤, are reg- ulated to the same extent (6). This may be due to additional control by other transcriptional regulators and other Notch family mem- of other genes that affect survival and cell cycle, TCR signaling, or bers. These data suggest a role for Notch1 and activation of the transcriptional activation, such as p53, Lck, ␤-catenin, and FADD, Notch signaling pathway during immature thymocyte develop- permit development to the DP stage independent of pre-TCR sig- ment, specifically during the late DN transition. This is consistent naling. (39–42) To determine whether Notch1 activation alone with a previous report that intracellular Notch1 expression is high- would cause DN to DP development in the absence of a pre-TCR est in DN thymocytes (38). signal, we analyzed thymi from mice expressing an active form of Notch1, Notch1IC, under the thymus-specific Lck-proximal pro- Notch1 activation during the DN-to-DP transition increases moter on a RAG2-deficient background, Notch1IC ϫ RAG2Ϫ/Ϫ. thymic cellularity and cell cycling Activated Notch1IC is unable to overcome the RAG developmen- Given the expression pattern of Notch1 at the cell surface and the tal block, as thymus cellularity is similar to RAG2Ϫ/Ϫ controls, evidence for activation of the pathway, as indicated by target gene and the cells remain at the DN3 stage (Fig. 5A). To check that the expression, we investigated the function of Notch1 during early Notch1IC transgene is expressed at this early stage, transgene- thymocyte development. During the DN to DP transition there are specific primers were used in semiquantitative RT-PCR assay to several distinguishing hallmarks of thymocyte development. Upon show the presence of the NotchIC transcript. As expected from the functional rearrangement of the TCR␤, a pre-TCR signal is deliv- Lck-proximal promoter, Notch1IC is expressed in the RAG2-de- ered, allowing transition from the DN3 to the DP stage and a ficient background, although at lower levels than in the RAG-suf- coincident proliferative expansion. We asked what role the expres- ficient thymocytes, which are predominately DP cells (Fig. 5B). sion of an active Notch1 transgene would play during this devel- This result is consistent with the failure of Notch1IC retrovirally opmental stage. introduced into RAG-deficient hemopoietic stem cells to develop The lack of RAG recombinase machinery prevents TCR rear- into DP cells (43). Additionally, the Notch1IC ϫ RAG2Ϫ/Ϫ mice rangement and thus precludes ␤-selection. However, dysregulation or retrovirally transduced hemopoietic stem cells do not generate 2300 NOTCH1 ACTIVITY IN IMMATURE THYMOCYTES

FIGURE 5. Notch1IC transgene does not rescue the RAG2 knockout (RAG2KO) developmental block at the DN3 stage. A, Thymocytes from Downloaded from RAG2KO (upper panels) and Notch1IC ϫ RAG2KO mice were stained for CD4, CD8, CD44, and CD25 expression. CD4/CD8 profiles of total thymocytes (left panels) and CD44/CD25 profiles of gated DN cells (right panels) are shown. Numbers indicate the total cellularity. B, RT-PCR anal- ysis of the Notch1IC transgene. cDNA prepared from total thymocytes from N1IC mice, RAG2KO, and Notch1IC ϫ RAG2KO, were serially

diluted 3-fold and analyzed with transgene-specific primers. Analysis of http://www.jimmunol.org/ HPRT transcripts was used as a control.

anti-CD3 Ab over time or complete filling of the thymic compart- ment (data not shown). FIGURE 4. Surface expression of Notch1 and activation of the Notch1 pathway in DN subsets. B6 thymocytes were stained with anti-N1ec. A,DN Notch1 activation has been implicated in both preventing apo- thymocyte (CD4Ϫ, CD8Ϫ, B220Ϫ, CD3Ϫ) subsets were defined by CD44 ptosis and promoting proliferation (7–9). To assess which cellular mechanism may explain the increased cell numbers found in the

and CD25 expression with the indicated gates. B, DN subsets stained with by guest on October 9, 2021 Ϫ Ϫ anti-N1ec preincubated with GST (black line) or mN1ec-GST ( ). C, anti-CD3-treated, Notch1IC ϫ RAG2 / mice, we examined an- cDNA isolated from DN subsets was analyzed by real-time TaqMan PCR nexin V staining as a measure of and DNA content to for Deltex1, a target gene of Notch activation. Fold differences were cal- determine the percentage of cycling cells. Annexin V staining of culated after normalizing cDNA levels of HPRT transcripts between thymocytes after anti-CD3 treatment revealed little difference be- samples. tween anti-CD3-treated Notch1IC ϫ RAG2Ϫ/Ϫ mice and controls (data not shown). However, the percentage of cycling cells with Ͼ2 N DNA content, was increased in the DN, ISP, and DP pop- Ϫ Ϫ thymomas normally associated with enforced Notch1IC expres- ulations from the Notch1IC ϫ RAG2 / mice (Fig. 6D). These sion, suggesting a downstream pathway relative to the pre-TCR data suggest that constitutive Notch1 activation promotes the in- signal for Notch1IC signaling in thymomagenesis. crease in cell number by enhancing cell proliferation. Pre-TCR signaling can be induced in RAG-deficient mice by injection of anti-CD3 Ab. This mimics ␤-selection and results in Pre-TCR signals are not affected by Notch1 activation proliferation and developmental progression from the DN to the To determine whether the increase in cellularity and the fraction of DP stage (44). To determine whether constitutive activation of the cycling cells in anti-CD3-treated Notch1IC ϫ RAG2Ϫ/Ϫ mice is Notch1 pathway can influence early thymocyte development after due to an effect of Notch1 activation on pre-TCR signaling, we ␤-selection, we injected anti-CD3 into Notch1IC ϫ RAG2Ϫ/Ϫ analyzed three components of the pre-TCR signaling cascade. Be- mice and RAG2Ϫ/Ϫ littermate controls. Two days after anti-CD3 cause the pre-T␣ gene is a target of Notch1 activation, we analyzed treatment, Notch1IC ϫ RAG2Ϫ/Ϫ mice show an increased rate of the expression of pre-T␣ and surface CD3 levels to determine progression from DN to DP and increased thymic cellularity com- whether the difference in response could be attributed to differ- pared with their RAGϪ/Ϫ littermates similarly injected with anti- ences in the levels of the TCR components. We saw no increase in CD3 (Fig. 6, A and B). The greatest difference is seen in the ISP basal levels of surface CD3 by FACS analysis in Notch1IC ϫ CD8ϩ compartment, with a 3-fold increase compared with RAG2Ϫ/Ϫ mice compared with littermate controls (data not RAG2Ϫ/Ϫ treated with anti-CD3 (Fig. 6C). The time course after shown). Similarly, using real-time PCR to measure mRNA tran- anti-CD3 treatment shows an increase in cellularity as early as 2 scripts, we found no significant increase in pre-T␣ expression in days until 7 days after injection. In addition, the kinetics of devel- Notch1IC ϫ RAG2Ϫ/Ϫ thymocytes (Fig. 7A). We also performed opment, as measured by the percentage of cells that have left the a kinetic analysis of ERK phosphorylation in response to PMA DN compartment, appear to be increased in the Notch1IC ϫ stimulation to determine whether proximal phosphorylation events RAG2Ϫ/Ϫ relative to controls from day 2 to day 4 (Fig. 6A and of the TCR pathway were modulated by an active Notch1 signal. data not shown). Time points after 14 days showed similar cellu- This kinetic analysis allowed us to determine whether changes in larity at ϳ300 ϫ 106 thymocytes, presumably due the depletion of the absolute levels or in the duration of signals were affected by The Journal of Immunology 2301 Downloaded from http://www.jimmunol.org/

FIGURE 7. TCR signaling pathways are not modulated by the concur- rent activation of Notch1. A, cDNA isolated from untreated RAG knockout (RAG2KO), Notch1IC ϫ RAG2KO, and B6 thymocytes was analyzed by real-time TaqMan PCR for pre-T␣ transcripts. Fold differences were cal- culated after normalizing cDNA levels of HPRT transcripts between sam- ples. B, Thymocytes from RAG2KO or Notch1IC ϫ RAG2KO mice were incubated with 5 ng/ml PMA for the indicated times and analyzed by Western blot for phospho-ERK protein. The lower panel is the same blot as above, which was stripped and probed for total ERK protein as a loading by guest on October 9, 2021 control. C, CD69 expression after in vivo anti-CD3 treatment. RAG2KO (black line) and Notch1IC ϫ RAG2KO (gray line) were treated with 100 ␮g of anti-CD3e or PBS i.p. ( ; Notch1IC ϫ RAG2KO). After 24 h thymocytes were stained for CD69 expression. The percentage of CD69- positive cells, as defined by the bracket, is indicated. Levels of CD69 in thymocytes from PBS controls with RAG2KO were similar to Notch1IC ϫ RAG2KO injected with PBS (not shown).

RAG2Ϫ/Ϫ mice, and we observed no significant difference in mean FIGURE 6. Cellular analysis of thymocytes from Notch1IC ϫ RAG fluorescence of CD69 staining or the percentage of CD69-positive knockout (RAG2KO) and RAG2KO mice after injection of anti-CD3. cells, suggesting that there was no difference in the perceived level Mice were treated with 100 ␮g of anti-CD3e or PBS injected i.p. After 2 of TCR signal due to the Notch1IC transgene (Fig. 7C). days thymocytes were isolated from the mice and analyzed for CD4 and These results indicate that both proximal and distal points of the CD8 expression and DNA content. A, CD4 and CD8 expression of thy- pre-TCR signaling cascade are unaffected by concurrent activation mocytes. Numbers indicate the percentage of total thymocytes that fall with in the indicated quadrant. B, Total thymocyte numbers from RAG2KO and of the Notch1 pathway, suggesting that Notch1IC affects prolifer- Notch1IC ϫ RAG2KO mice treated with anti-CD3 or PBS. C, Numbers of ation by augmenting a parallel pathway to the pre-TCR cascade or DN, ISP, and DP thymocyte subsets. D, Percentage of cells expressing Ͼ2 via other downstream effectors of the pre-TCR cascade outside the N DNA. ERK signaling branch.

Delta-like-1-dependent proliferation of DN in vitro enforced activation of Notch1. We found that ERK phosphoryla- To examine the role of Notch signaling in a more physiological tion is induced and sustained after stimulation with the same ki- setting, i.e., without an active NotchIC transgene, we turned to the netics in Notch1IC ϫ RAG2Ϫ/Ϫ and control RAG2Ϫ/Ϫ thymo- recent description of T in vitro driven by a stromal cytes (Fig. 7B). cell line expressing the Notch ligand, Dl1 (34). The OP9 stromal Up-regulation of CD69 at the cell surface is an early marker of cell line can support development from fetal or bone TCR stimulation and reflects the intensity of TCR signaling re- marrow precursors, and Schmidt and Zuniga-Pflucker (34) showed ceived by the T cell (45, 46). Mice injected with anti-CD3 were that following expression of full-length Dl1 in OP9, T, but not B, analyzed for CD69 up-regulation on thymocytes after 24 h. CD69 cell development ensued. To determine whether Notch-Notch li- levels were up-regulated in both controls and Notch1IC ϫ gand signaling would enhance the expansion of DN cells from 2302 NOTCH1 ACTIVITY IN IMMATURE THYMOCYTES

cultures (12, 13). In these studies the inhibitors block ␥-secretase/ presenilin-dependent cleavage, a step shared by all Notch family members for ligand-dependent signaling of the Notch receptors. Finally, multiple ligands that interact with all the Notch receptors are expressed throughout the thymus (data not shown), allowing the potential for ligand-dependent Notch signaling at a variety of developmental stages. These findings suggest that the effect of Notch1IC on SP development may be interpreted as activating other Notch pathways or target genes that may be physiologically FIGURE 8. DN3 thymocytes proliferate in response to Notch signals. important during SP development. Sorted DN3 (F and E;CDϪCD8ϪCD44ϪCD25ϩ)orDP(f and Ⅺ; In a recent study Wolfer et al. (14) reported that deletion of ϩ ϩ CD4 CD8 CD60low) thymocytes were plated on monolayers of OP9 (E Notch1 at the DN2 stage via the expression of Lck-Cre resulted in and Ⅺ) or OP9-Dl1 (F and Ⅺ). Culture medium was supplemented with an impairment in the rate of VDJ recombination at the TCR␤ lo- IL-7 and Flt3L, and the total number of cells recovered per well was mon- cus. They also noted an accumulation of aberrant DN4 thymocytes itored over the 6-day culture period. that lacked a functionally rearranged TCR␤-chain. To explain the latter observation they hypothesized that signaling through Notch1 is required to eliminate DN thymocytes that have failed TCR␤ normal mice, we FACS-purified DN3 and DP cells from normal selection (14). In line with this, our results show that Notch1 is B6 thymus, plated them on monolayers of control OP9 and OP9- highly expressed at the cell surface from the DN2 to ISP stage, and Dl1, and followed their progress by cell counts. The results pre- the levels of Deltex1 mRNA are highest at the DN3 stage. Breed- Downloaded from sented in Fig. 8 complement our previous conclusions on the role ing the Notch1IC transgenic mice to RAG2Ϫ/Ϫ mice allowed us to of Notch signaling at the DN to DP transition. Thus, DN3 cells ask what effect constitutive Notch signaling would have on early plated on OP9-Dl1 expanded dramatically, while following plating thymocytes. We found no evidence that enforced Notch signaling on control OP9 monolayers, they only maintained the starting increased the turnover rate or apoptosis of RAG2Ϫ/Ϫ thymocytes number. Also in line with our Notch1 expression analysis, that do not express a functional ␤-chain. Staining RAG2Ϫ/Ϫ con- CD69low DP thymocytes showed no difference in response when trols vs Notch1IC ϫ RAG2Ϫ/Ϫ thymocytes for DNA content or for http://www.jimmunol.org/ plated on control or Dl1-expressing OP9 monolayers. apoptotic cells showed no difference between the two (data not shown). Discussion Enforced Notch1 signaling also did not alter the phenotype of Overexpression of Notch1IC in cells of the T lineage by the use of the RAG2Ϫ/Ϫ thymus; cellularity was unchanged, and most cells transgenes or retroviral transduction has lead to a variety of alter- remained arrested at the DN3 stage (Fig. 5). This establishes a ations in thymic phenotypes, mostly on the development of DP to hierarchy of Notch1 signaling relative to pre-TCR signaling. For mature SP cells (6, 10, 11). However, deletion of Notch1 at the example, constitutive activation of Lck and ␤-catenin in RAG- early DP stage via the CD4-Cre transgene reveals no aberrations in deficient mice results in DN to DP transition, suggesting that de- by guest on October 9, 2021 the differentiation of SP cells (28). In contrast, deletion of Notch1 velopment in these mice is pre-TCR-independent and that Lck and at the DN2/DN3 stage via an Lck-Cre transgene shows impairment ␤-catenin are proposed downstream targets after initiation of the in further development, decreased DP development, and an overall pre-TCR signal (41). Conversely, the DN3 block seen in decrease in cellularity in thymic subsets beyond the DN stage (14). Notch1IC ϫ RAG2Ϫ/Ϫ suggests that Notch1 signaling at this stage Because immunoblot or mRNA analyses have shown Notch1, is upstream or separate from the pre-TCR signaling cascade. Pre- Notch2, and Notch3 plus all four ligands of the Notch receptors to viously, two groups using Notch1IC and Notch3IC have shown be expressed in the thymus, it has been difficult to establish the that rapid leukemia development requires pre-TCR signaling. physiological relevance of the Notch1 protein specifically. Diffi- From these experiments it is unclear whether NotchIC requires a culties in interpretation arise due to genetic complementation and specific stage development, i.e., after ␤-selection, to induce trans- redundancy or due to the activation of multiple Notch pathways by formation or whether NotchIC plays a role in affecting pre-TCR overexpression of the highly conserved intracellular region of signaling, which leads to transformation. The developmental time- Notch1IC. Here we present expression analysis of endogenous line in our experiments evaluates developmental and cellular Notch1 at the cell surface that reads out the level of biologically changes from a few hours to a few days after ␤-selection, i.e., accessible Notch1. We also correlate Notch1 surface expression proximal to the pre-TCR signal (25, 43). with the expression levels of transcriptional targets. These results Mimicking pre-TCR signaling by injection of anti-CD3 Ab al- suggest that Notch1 plays a role during DN development and is lowed us to examine whether the Notch1IC transgene would have less involved in the maturation of SP thymocytes. an effect on the kinetics of the DN to DP transition that follows Our staining results are in general agreement with previous ␤-selection. Notch1IC increases the rate of development of DN3 work by Hasserjian et al. (38). In that work, intracellular staining cells to the ISP and then the DP compartment. Thus, in addition to of BALB/c mouse thymocytes using an Ab against the cytoplasmic the role for Notch1 signaling in driving TCR␤ rearrangement (14), portion of human Notch1 suggested that all thymus subsets express we propose that Notch signaling may play a positive role following Notch1, compared with the staining with an anti-GST-negative pre-TCR signaling. One explanation for the enhanced development control. They reported highest levels in DN subsets and lower of Notch1IC ϫ RAG2Ϫ/Ϫ thymocytes following anti-CD3 injec- levels in DP and SP subsets. tion could have been that the DN3 cells in these mice express a We have previously shown that Notch target genes are up-reg- higher level of CD3 and would therefore receive a stronger signal ulated in both CD4 and CD8 SP compared with the levels in their from the anti-CD3 Ab. Relevant to this is the fact that pre-T␣ has DP precursors (6, 7). Thus, it remains possible that other Notch been shown to be a target of Notch signaling in thymomas and family members are important for DP to SP development. This thymocytes (7). Even in the absence of a TCR␤-chain, it is con- possibility is supported by data showing that low levels of prese- ceivable that increased levels of pre-T␣ could up-regulate the sur- nilin inhibitors can block SP development in fetal thymic organ face expression of CD3. Our expression analysis, however, The Journal of Immunology 2303 showed no heightened expression of pre-T␣ nor any increase in 10. Robey, E., D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans, G. Weinmaster, detectable levels of surface CD3 (data not shown) in the Notch1IC and P. Salmon. 1996. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87:483. transgenic thymocytes. Furthermore, by studying the degree and 11. Izon, D. J., J. A. Punt, L. Xu, F. G. Karnell, D. Allman, P. S. Myung, N. J. Boerth, J. C. Pui, G. A. Koretzky, and W. S. Pear. 2001. Notch1 regulates maturation of kinetics of ERK phosphorylation and CD69 up-regulation in stim- ϩ ϩ Ϫ/Ϫ ϫ Ϫ/Ϫ CD4 and CD8 thymocytes by modulating TCR signal strength. 14: ulated thymocytes from RAG2 and Notch1IC RAG2 253. mice, we found no evidence that enforced Notch signaling mod- 12. Doerfler, P., M. S. Shearman, and R. M. Perlmutter. 2001. Presenilin-dependent ulated the pre-TCR signaling cascade. Finally, the comparison of ␥-secretase activity modulates thymocyte development. Proc. Natl. Acad. Sci. USA 98:9312. the in vitro growth of DN3 thymocytes from control B6 mice on 13. Hadland, B. K., N. R. Manley, D. Su, G. D. Longmore, C. L. Moore, M. S. Wolfe, OP9 vs OP9-Dl1 stromal cells strongly supports a role for Notch E. H. Schroeter, and R. Kopan. 2001. ␥-Secretase inhibitors repress thymocyte signals in the DN to DP expansion (Fig. 8). development. Proc. Natl. Acad. Sci. USA 98:7487. 14. Wolfer, A., A. Wilson, M. Nemir, H. R. MacDonald, and F. Radtke. 2002. In- The effect of Notch1IC on proliferation may shed light on the activation of Notch1 impairs VDJ␤ rearrangement and allows pre-TCR-indepen- cellular mechanisms that Notch1IC targets as an oncogene in the dent survival of early ␣␤ lineage thymocytes. Immunity 16:869. maintenance of thymoma growth and tumorigenesis (47). Re- 15. Wilson, A., H. R. MacDonald, and F. Radtke. 2001. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194:1003. cently, Aster’s group (48) has shown that pharmacological inhibi- 16. Allman, D., J. A. Punt, D. J. Izon, J. C. Aster, and W. S. Pear. 2002. An invitation tion of the Notch pathway in Notch1IC-induced T-ALL lines re- to T and more: Notch signaling in lymphopoiesis. Cell 109(Suppl.):S1. 17. Jarriault, S., C. Brou, F. Logeat, E. H. Schroeter, R. Kopan, and A. Israel. 1995. sults in cell cycle arrest and induction of apoptosis. This suggests Signalling downstream of activated mammalian Notch. Nature 377:355. that constitutive Notch1 activation is required for the growth po- 18. Reizis, B., and P. Leder. 2002. Direct induction of T -specific gene tential of Notch1IC-induced thymomas in addition to any effects expression by the mammalian Notch signaling pathway. Genes Dev. 16:295. 19. Hsieh, J. J., D. E. Nofziger, G. Weinmaster, and S. D. Hayward. 1997. Epstein- Notch1IC may have on differentiation of the tumor.

Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentia- Downloaded from Physiological Notch signaling as well as oncogenic Notch1IC tion. J. Virol. 71:1938. signaling may be important in up-regulating genes that promote 20. Beatus, P., J. Lundkvist, C. Oberg, and U. Lendahl. 1999. The Notch 3 intracel- lular domain represses Notch 1-mediated activation through Hairy/Enhancer of proliferation, such as the bHLH gene, Hes1. Hes1 is up-regulated split (HES) promoters. Development 126:3925. in DN subsets, and deletion of Hes1 results in an autonomous 21. Mizutani, T., Y. Taniguchi, T. Aoki, N. Hashimoto, and T. Honjo. 2001. Con- defect in the ability of immature thymocytes to proliferate (49, 50). servation of the biochemical mechanisms of signal transduction among mamma- lian Notch family members. Proc. Natl. Acad. Sci. USA 98:9026. In vivo Hes1 deficiency blocks development at the DN2 and DN3 22. Ellisen, L. W., J. Bird, D. C. West, A. L. Soreng, T. C. Reynolds, S. D. Smith, stages, which correlates with Notch1 pathway activity (Fig. 4C). and J. Sklar. 1991. TAN-1, the human homolog of the Drosophila Notch gene, is http://www.jimmunol.org/ broken by chromosomal translocations in T lymphoblastic . Cell 66: Although the molecular mechanism for Hes1 involvement in pro- 649. liferation is not well characterized, there is evidence that Hes1 23. Pear, W. S., J. C. Aster, M. L. Scott, R. P. Hasserjian, B. Soffer, J. Sklar, and inhibits E protein activity by binding E47 and preventing tran- D. Baltimore. 1996. Exclusive development of T cell neoplasms in mice trans- planted with expressing activated Notch alleles. J. Exp. Med. 183: scriptional repression. In line with this, the loss of E47 activity 2283. results in early DN proliferation and tumorigenesis (51). We hy- 24. Aster, J., W. Pear, R. Hasserjian, H. Erba, F. Davi, B. Luo, M. Scott, pothesize that enforced expression of Notch1IC results in an in- D. Baltimore, and J. Sklar. 1994. 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