Does Not Alter T Cell Fate Specification but Notch Signaling In

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Transgenic Expression of Numb Inhibits Notch Signaling in Immature Thymocytes But Does Not Alter T Cell Fate Specification

This information is current as Michelle B. French, Ute Koch, Rachel E. Shaye, Melanie A. of September 29, 2021. McGill, Sascha E. Dho, Cynthia J. Guidos and C. Jane McGlade J Immunol 2002; 168:3173-3180; ; doi: 10.4049/jimmunol.168.7.3173

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Transgenic Expression of Numb Inhibits Notch Signaling in Immature Thymocytes But Does Not Alter T Cell Fate Speciﬁcation1

Michelle B. French,2* Ute Koch,2† Rachel E. Shaye,* Melanie A. McGill,* Sascha E. Dho,* Cynthia J. Guidos,† and C. Jane McGlade3*

The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identiﬁed a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during Downloaded from T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage ,in thymocyte development (CD4؊CD8؊CD3؊ cells that were CD44؉CD25؉ or CD44؊CD25؉; double-negative 2/3). Moreover bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our

results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling http://www.jimmunol.org/ at this stage does not alter ␥␦/␣␤ or CD4/CD8 T cell fate speciﬁcation. The Journal of Immunology, 2002, 168: 3173Ð3180.

unctionally distinct mature T cells develop in the thymus development (7, 8), suggesting that activation of Notch-1 signaling from multipotent progenitor cells that migrate from the in multipotential hemopoietic progenitors controls T vs B cell lin- F bone marrow. The majority of mature T cells bear the ␣␤ eage determination. Washburn and colleagues (9) have implicated TCR along with either CD4 or CD8 coreceptors, whereas a small Notch-1 signals in regulating the choice between the ␣␤ vs ␥␦ T population of mature T cells bears the ␥␦ TCR. ␣␤ T cells develop cell lineages during T cell development in the thymus. Activated Ϫ Ϫ in the thymus from CD4 CD8 (double-negative, DN)4 cells forms of Notch-1 also promote the survival and maturation of DP ϩ ϩ through an immature CD4 CD8 (double-positive, DP) stage into thymocytes and influence the DP-SP transition (10Ð13). Collec- by guest on September 29, 2021 ϩ ϩ mature CD4 or CD8 (single-positive, SP) cells. Thymocytes tively, these data indicate that Notch-1 may influence the survival commit to the ␣␤ vs ␥␦ lineages very early in T cell development and lineage commitment of T cell progenitors at several discrete Ϫ Ϫ at the CD44 CD25 DN (DN4) stage (1). Signals emanating from stages. the pre-TCR and ␣␤ TCR play well-defined roles in ␣␤ T cell Notch activation in Drosophila is regulated by the temporal and development at the DN-DP and DP-SP transitions, respectively. spatial distribution of Notch ligands, Delta and Serrate, as well as Members of the Notch family play critical roles in the determi- by the expression of signaling modulators such as Numb (2). Ex- nation of cell fate and in the maintenance of progenitors during periments, primarily in Drosophila, indicate that Numb acts up- development of a number of different cell types in a variety of stream of Notch action to inhibit Notch signaling (14Ð17). For different organisms (2, 3). Notch signals are essential for the de- example, during the development of the sensory organ in Dro- velopment of T lineage cells (4Ð6), whereas they prevent B cell sophila, loss-of-function mutations in dNumb mimic gain-of-function mutations in Notch, suggesting that Numb inhibits Notch activity (14). Numb may prevent the translocation of the Notch *Arthur and Sonia Labatt Brain Tumor Research Center and †Program in Develop- intracellular domain to the nucleus following ligand binding (15, mental Biology, Hospital for Sick Children, Toronto, Ontario, Canada 17). However, the direct effect of Numb on the expression of Received for publication May 11, 2001. Accepted for publication January 22, 2002. Notch target genes in vivo has not been reported. The costs of publication of this article were defrayed in part by the payment of page The mechanisms regulating Notch activation in T cell progen- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. itors are not well defined. The Notch ligands, Jagged-1 and 1 This work was supported by grants from the National Cancer Institute of Canada Jagged-2, are expressed in the murine (18, 19) and rat (20) thymus, (NCIC) with funds from the Canadian Cancer Society (to C.J.M.) and the Medical and development of ␥␦ T cells is slightly impaired, but not blocked Research Council of Canada (to C.J.G.). U.K. is supported by a NCIC postdoctoral in mice with a mutation in Jagged-2 (21). Mammalian homologues fellowship award, C.J.G. is a Medical Research Council of Canada Scientist, and C.J.M. is a Research Scientist of the NCIC, supported with funds from the Canadian of dNumb have been identified and are widely expressed in em- Cancer Society. bryos and in adult tissue, including the thymus (22Ð25). The func- 2 M.B.F. and U.K. contributed equally to this work. tion of mammalian Numb (mNumb) remains unclear, but mNumb 3 Address correspondence and reprint requests to Dr. C. Jane McGlade, Hospital for is thought to influence neurogenesis in vertebrates (17, 22, 26). Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. E-mail Mice with a null mutation in the mNumb gene display multiple address: [email protected] developmental defects involving the nervous system as well as 4 Abbreviations used in this paper: DN, double negative; dNumb, Drosophila numb; DP, double positive; mNumb, mammalian Numb; PI, propidium iodide; PTB, phos- other cell lineages resulting in embryonic cell death before day photyrosine binding; SP, single positive; Tg, transgenic. E11.5 (26, 27).

Structurally, mNumb resembles an adapter protein. It has an MO) for 30 min. Following the incubation, sections were washed three N-terminal phosphotyrosine-binding (PTB) domain (22, 23, 28), a times for 15 min each with 1ϫ PBS. To permeabilize the tissue, slides were ϫ proline-rich region containing several putative Src-homology 3 incubated with a solution of 1 PBS containing 0.2% Triton X-100 at room temperature for 30 min. The slides were washed three times for 10 binding sites (23), and an Eps 15 homology domain-binding motif min each with 1ϫ PBS. To prevent nonspecific binding of the secondary at the carboxyl terminus (29). Consistent with the notion that Ab used in the detection of Numb and AP-2, slides were incubated with a Numb acts as adapter protein, a number of mNumb-binding part- solution of 1ϫ PBS containing 5% goat serum (The Jackson Laboratory, ners have been identified (29Ð31), including Notch-1 (22). Bar Harbor, ME) at 37¡C for 30 min. Slides were then washed as described above. To detect the Numb protein, slides were treated with a 1/10 dilution There are four different isoforms of mNumb (p65, p66, p71, and of a 1 mg/ml stock solution of rabbit anti-Numb polyclonal anti-Numb-C p72), which arise from alternative splicing of mNumb RNA, and are Ab (23) for 24 h at 4¡C. The sections were washed three times for 10 min characterized by the presence or absence of an 11-aa insert in the PTB each and treated with a 1/300 dilution of a stock 2 mg/ml goat anti-rabbit domain and the presence or absence of a 48-aa insert in the proline- Ab conjugated to AlexaFlour 488 (Molecular Probes, Eugene, OR) for 2 h rich region. All four isoforms of mNumb are expressed in the thymus, at room temperature. Slides were washed and incubated with a 1/100 dilution of a stock 1 mg/ml mouse anti-AP-2 mAb (Affinity BioReagents, although the p65 and p66 isoforms are predominant (25, 32). This Golden, CO) for 24 h at 4¡C. Slides were washed and treated with a 1/300 observation, together with the reports that dNumb inhibits Notch ac- dilution of a stock 2 mg/ml goat anti-mouse Ab conjugated to CY3 tivity and that Notch-1 influences T cell development, prompted us to (Molecular Probes) for2hatroom temperature. Slides were cover- determine whether transgenic (Tg) overexpression of the p66 isoform slipped (Baxter Diagnostics, McGraw Park, IL) with fluorescent mount- ing media (DAKO Diagnostics, Carpinteria, CA) and analyzed using a of mNumb would antagonize Notch signaling in thymocytes and alter confocal microscope. T cell development. RT-PCR analysis of endogenous Numb, Tg Numb, and Notch-1 Downloaded from Materials and Methods target genes in TgϪ and Tgϩ thymus Generation of Tg mice RNA from thymi from Tgϩ and TgϪ littermates that were 3- to 4-wk-old The complete coding sequence of rat Numb cDNA (nt 196-2007) (23) was was prepared using the TRIzol system (Life Technologies, Grand Island, cloned into the BamHI site of p1017, which contains the Lck proximal NY). Superscript II reverse transcriptase (Invitrogen, San Diego, CA) was promoter and portions of the human growth hormone gene, including a used to prepare cDNA, which was then normalized for equivalent template

polyadenylation sequence (33). In the resulting plasmid, the cDNA for amounts by serial dilution and amplification using primers specificto␤- http://www.jimmunol.org/ Numb was flanked by the mouse Lck proximal promoter at the 5Ј end and actin(5Ј-GTCGTACCACAGGCATTGTGATGG-3Јand5Ј-GGTGGTACA portions of the human growth hormone gene, including introns and a poly- TGGGTCCGTAACG-3Ј). RT-PCR analysis was conducted as described adenylation sequence at the 3Ј end. The rat Numb cDNA that was used previously (11). Briefly, normalized cDNA amplified for 35 cycles (95¡C, encodes the p66 mNumb isoform with the 11-aa insert in the PTB domain, 1 min; 60¡C, 30 s; 72¡C, 1 min) using primers specific for all isoforms of but does not contain the 48-aa insert in the proline-rich region of the endogenous mNumb (5Ј-CTACGGCAAAGCTTCAGGAGA-3Ј and 5Ј- molecule (23). The amino acid sequence of the protein encoded is TCAGCAACTTTTCACTAATCC-3Ј) and Tg mNumb (5Ј-AGAAGTGT identical to the murine p66 isoform of Numb (25). A SpeI fragment CAAAGAGTGTGCG-3Ј and 5Ј-CAG GCTTTTTGACAACGCTAT-3Ј). from the plasmid consisting of the entire transgene was purified and To analyze specific Notch-1 target genes, normalized cDNA was serially ϫ ϩ injected into (C57BL/6 SJL)F2 embyros. Tg founder mice were diluted 3-fold and amplified for 35Ð60 cycles (95¡C, 1 min; 55¡C, 1 min; identified by PCR amplification of tail DNA using primers that spanned 72¡C, 1 min) using Ifi-D3 (5Ј-ACTTCCTCTGTGTTAGAGGCTGC-3Ј the junction between the Lck proximal promoter and mNumb (5Ј primer, and 5Ј-AAAGCTGTCATTTAGAGGTG-3Ј), Hes-1 (5Ј-GCCAGTGT by guest on September 29, 2021 ATG TCT CCC AGG TAG TCC CC and 3Ј primer, GTG CAT TCC TCT CAACACGACACCGG-3Ј and 5Ј-TCACCTCGTTCATGCACTCG-3Ј), TGA CTC ATC). Tgϩ founders were bred to C57BL/6. The majority of the and preT␣ (5Ј-TGGCTGCAACTGGGTCATGCTTC-3Ј and 5Ј-GGCT analysis was performed on mice from two lines of founder mice that had CAGAGGGGTGGGTAAGATC-3Ј) primers. PCR products were sepa- been backcrossed three to four times to C57BL/6. rated by electrophoresis through a 0.8% agarose gel. Immunoprecipitation, in vitro binding, and Western blotting Abs and flow cytometry Protein extracts of thymus, spleen, and lymph nodes from Tgϩ and non-Tg Ϫ Single-cell suspensions were prepared from thymus, spleen, lymph node, and (Tg ) mice were made by homogenizing the tissues in lysis buffer (50 mM bone marrow; stained (2 ϫ 106 cells/sample) with various Ab; and analyzed by HEPES, 150 mM NaCl, 1.5 mM MgCl , 1 mM EGTA, 10 mM sodium 2 flow cytometry. The following mAb, which were generated in the laboratory pyrophosphate, 100 mM NaF, 10% glycerol, 1% Triton X-100, and pro- unless otherwise indicated, were used: anti-CD4 PE; anti-CD4 FITC (GK1.5); tease inhibitors (Roche Molecular Biochemicals, Laval, Quebec, Canada)). anti-CD4 biotin (YTS191.1); anti-CD8 APC; anti-CD8 FITC (53-6.7); anti- Lysates were centrifuged at 14,000 rpm at 4¡C to pellet insoluble matter. CD8 biotin (YTS169.4); anti-B220 FITC (6B2); anti-B220 biotin; anti-B220 For each immunoprecipitation, 0.5 mg of protein was made up to a volume APC (Cedarlane, Hornby, Ontario, Canada); anti-CD3⑀ biotin (YCD3); anti- of 0.5 ml with lysis buffer and incubated for 2Ð4 h with Ab and protein TCR␤ FITC; anti-TCR␤ biotin (H57-597); anti-TCR ␥␦ biotin (GL3; BD A-Sepharose beads. Immune complexes bound to protein A-Sepharose PharMingen, San Diego, CA); anti-CD25 FITC (7D4; BD PharMingen); anti- were washed four times with lysis buffer and eluted by boiling in SDS CD44 PE (IM781); and anti-IgM FITC (R6-60.2; BD PharMingen). Biotin- sample buffer. Proteins were separated by SDS-PAGE, transferred onto ylated Ab were detected either with streptavidin-PE or streptavidin-Cy5PE. polyvinylidene difluoride membrane (Immobilon-P), and immunoblotted Where required, Ab directed against CD45.1 and/or CD45.2 (FITC or PE with primary Ab for 1Ð2 h at room temperature. Bound Ab were visualized conjugated; BD PharMingen) were included. All FACS analysis was per- using HRP-conjugated protein A, followed by ECL. mNumb was immu- formed on a FACSCalibur flow cytometer with CellQuest software (BD noprecipitated using affinity-purified anti-Numb-A Ab and blotted with anti- Biosciences, Mountain View, CA). Dead cells were excluded by gating for Numb-C Ab. Anti-Numb-A is specific for mNumb and does not recognize ϩ Ϫ forward scatter and propidium iodide (PI) exclusion. For each sample, the related protein, Numb-like (25). DN cells from Tg and Tg mice were 50,000Ð125,000 cells were analyzed. Cell cycle analysis was performed by lysed in lysis buffer, and 50 ␮g of whole cell lysate was separated by PI staining of ethanol-fixed, RNase-treated cells, followed by flow cyto- SDS-PAGE and analyzed by Western blot with Numb-C Ab. Blots were metric analysis of DNA content, as previously described (34). reprobed with ␤-tubulin Ab (Amersham, Arlington Heights, IL) to confirm equal protein loading. Purification of DN2/3 thymocytes and peripheral T cells Immunohistochemistry DN2/3 thymocytes from Tgϩ and TgϪ mice were isolated and purified as Thymic tissue from Tgϩ mice was rapidly removed, flash frozen to Ϫ80¡C, follows. Thymocytes expressing CD4, CD8, and/or CD3 surface receptors and sectioned using a cryostat modular cryosection apparatus. The 5-␮m were removed using purified Abs directed against CD4, CD8, and CD3, thymic sections were mounted on positively charged slides (Fisher Scien- and sheep anti-rat IgG magnetic beads (Dynal, Great Neck, NY). tific, Mississauga, Canada) and stored at Ϫ20¡C. A dual immunofluores- CD44ϩCD25ϩ (DN2) and CD44ϪCD25ϩ (DN3) cells were then isolated cent staining procedure was used to demonstrate Numb membrane colo- (99% pure) by cell sorting. Peripheral lymph node cells were enriched for calization with the AP-2 protein. Thymic sections were fixed via incubation T cells (peripheral T cells) by magnetic bead depletion of cells that express at room temperature with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, B220, Mac-1, and Gr-1 cell surface receptors. The Journal of Immunology 3175

Generation of mixed bone marrow chimeras Drosophila peripheral nervous system (23). p66 mNumb is the Mixed bone marrow chimeras were generated using an adaptation of the predominant isoform expressed in the mouse thymus (25), and engraftment model (35). T cell-depleted bone marrow was prepared from therefore we chose to examine the effects of p66 mNumb overex- Ly-5.2ϩ mNumb Tgϩ or TgϪ littermates and Ly-5.1ϩ C57BL/6 mice pression on T cell development. We generated Tg mice with the a (B6.SJL-ptprc /BoAiTac; Taconic Farms, Germantown, NY). T cells were p66 mNumb expressed under the control of the Lck proximal pro- depleted from bone marrow by magnetic depletion of Thy-1.2-expressing cells. A total of 5 ϫ 106 cells was injected i.v. into RAG-2Ϫ/Ϫ mice moter. This promoter becomes active coincident with T cell com- Ϫ ϩ (B10.D2/nSnJTac-Rag2tm1 N10; Taconic Farms) that had been gamma- mitment in CD44 CD25 DN (DN3) thymocytes, and remains irradiated with a lethal dose (1000 cGy, Gammacell 137C source; Atomic active during the DP thymocyte stage (36). Accordingly, we as- Energy, Ottawa, Canada) 4Ð18 h before injection. Chimeras were analyzed sessed the affect of mNumb overexpression on ␣␤ vs ␥␦ TCRϩ T 4Ð5 wk following reconstitution. cell and CD4ϩ vs CD8ϩ T cell development. Four lines of Tg mice Cell culture were generated. Most of the experiments were done using the two Thymocytes (3 ϫ 106 cells/ml) from Tgϩ or TgϪ mice were cultured in founder lines, A and B, which had the highest levels of thymic RPMI 1640 supplemented with 12.5% FBS, 25 mM HEPES (pH 7), 50 ␮M mNumb expression. Similar results were obtained with each line; 2-ME, and 2 mM L-glutamine for 24 or 40 h. Cell cycle analysis and the therefore, the results have been combined. degree of apoptosis were determined by FACS analysis of the DNA content, as described above. Immunoprecipitation and Western blot experiments demon- strated that mNumb protein was dramatically overexpressed in Results thymocytes (Fig. 1A). This was evident at the early DN stage of

Previously, we have shown that misexpression of the p66 isoform thymic development, since Numb was detected by Western blot in Downloaded from of mNumb is sufﬁcient to produce cell fate transformations in the Tgϩ DN, but not in TgϪ DN cells (Fig. 1B). mNumb was also http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. mNumb is overexpressed in Tgϩ mice. A, Overexpression of mNumb protein in the thymus, spleen, and lymph nodes of Tgϩ mice. mNumb was immunoprecipitated from equal amounts of lysate (0.5 mg of protein per immunoprecipitation) from thymus, spleen, and lymph nodes of two lines of Tgϩ mice (TgϩA and TgϩB) and their TgϪ littermates using anti-Numb-A Ab and Western blotted with anti-Numb-C Ab (23). Arrowhead, mNumb. B, Overexpression of mNumb protein in Tgϩ DN thymocytes. DN thymocytes purified by magnetic bead depletion (97% purity) from Tgϩ and TgϪ littermates were lysed, and equivalent amounts of total protein were separated by SDS-PAGE and Western blotted with anti-Numb-C Ab. Blots were reprobed with anti-tubulin Ab. CÐE, Transgenically expressed mNumb is membrane associated and colocalizes with AP-2. Thymic sections from Tgϩ (A) animals were fixed 4% paraformaldehyde and permeabilized in a solution of 1ϫ PBS containing 0.2% Triton X-100. Slides were preblocked in PBS containing 5% goat serum and coimmunostained with mouse anti-AP-2 mAb, followed by goat anti-mouse Ab conjugated to CY3 (red) and affinity-purified rabbit anti-Numb-C polyclonal Ab (23), followed by goat anti-rabbit Ab conjugated to AlexaFlour 488 (green). Images showing AP-2 staining (C), Numb staining (D), and the merged images (E) were obtained using confocal microscopy. F, Expression of endogenous and Tg mNumb during T cell development. RNA extracted from depleted and FACS-sorted populations of cells from CD44ϩCD25ϩ and CD44ϪCD25ϩ (DN2/3) and DP thymocytes, and peripheral T cells (PT) from 3- to 4-wk-old Tgϩ and TgϪ mice were used to prepare cDNA. cDNA pools were normalized for equivalent template amounts by amplification using primers specificto␤-actin. PCR amplifications were performed using primers specific for endogenous mNumb and Tg mNumb. 3176 NUMB OVEREXPRESSION DOES NOT ALTER T CELL FATE SPECIFICATION elevated in the spleen and lymph node cells of Tgϩ mice in comparison with their TgϪ littermates (Fig. 1A). Using anti-Numb an- tisera recognizing different regions of the Numb protein, we con- firmed that the transgenically expressed mNumb protein is full length (data not shown). Confocal microscopy revealed that Tg mNumb was localized to thymic cell membranes and colocalized with AP-2, a marker of clathrin-coated pits and early endosomes (Fig. 1, CÐE). This is in agreement with previous studies showing that mNumb proteins interact with components of the early endocytic machinery and localize to endocytic vesicles (37) (S. E. Dho, C. A. Smith, and C. J. McGlade, manuscript in preparation). To further examine the timing of Tg mNumb and endogenous mNumb expression during thymocyte development, we performed RT- PCR on RNA extracted from DN2/3 and DP subpopulations of thymocytes and from mature peripheral T cells isolated from 4-wk-old mice. Two primer pairs, one specific for endogenous mNumb and the other specific for Tg mNumb, were used in the reaction. Both endogenous mNumb and Tg mNumb mRNA was detected in all of the subpopulations of cells (Fig. 1F). Therefore, Downloaded from Tg mNumb protein was expressed during thymocyte development before ␤- and ␣-chain rearrangement and before CD4/CD8 lineage commitment, and it localized correctly within the cells. To determine whether Tg expression of Numb inhibited Notch-1 signaling in the thymus, we examined the expression of Hes-1, preT␣, and Ifi-D3, which have previously been shown to be up- http://www.jimmunol.org/ regulated in the presence of constitutively active Notch-1 (11). Total RNA from Tgϩ or TgϪ littermates was isolated and used in semiquantitative PCR. The expression of all three of these genes was reduced in the Tg thymi as compared with controls (Fig. 2A). ϩ Ϫ Similar results were obtained from three separate littermate pairs FIGURE 3. Phenotypic profile of thymocytes from mNumb Tg and Tg mice. A, Phenotypic profile of total thymocytes. Two-parameter 7% probabil- of animals at 3 wk of age (data not shown). Expression of Hes-1 ϩ ity contour plots depict surface expression of CD4 vs CD8 on thymocytes from and preT␣ was also reduced in thymocytes from 5-wk-old Tg ϩ Ϫ Ϫ representative 4-wk-old Tg and Tg mice. The percentages of DN, DP, CD4 mice compared with Tg littermates (data not shown). To further SP, and CD8 SP in each sample are indicated. The histograms depict expres- by guest on September 29, 2021 delineate the developmental stages at which Notch-1 signaling was sion of TCR␤ on total thymocytes. B, Phenotypic profile of immature DN decreased by Tg Numb, we examined the expression of Ifi-D3 by thymocytes. Two-parameter 7% probability contour plots depict the expression of CD44 vs CD25 (top panels) and CD44 vs B220 (bottom panels)inDN semiquantitative RT-PCR of RNA prepared from sorted popula- ϩ Ϫ tions of DN2/3 and DP thymocytes and peripheral T cells. Ifi-D3 thymocytes of Tg and Tg mice. DN thymocytes were identified by lack of ϩ staining with Ab specific for CD4, CD8, CD3, and TCR␤. The numbers within expression was significantly decreased in Tg cells in comparison the quadrants represent the percentage of live cells gated by PI exclusion. with TgϪ cells in all subpopulations (Fig. 2B). Cells isolated from the thymus, spleen, and lymph nodes of Tgϩ and TgϪ littermates were analyzed by flow cytometry to examine mocytes stained for CD4, CD8, and TCR␤ (A), and immature DN the effect of mNumb expression on the development of T cells and thymocytes stained for CD44, CD25, and B220 (B), and indicates other lineages. Fig. 3 shows representative analysis of total thy- the similar percentages of cells positive for these surface markers

FIGURE 2. Expression pattern of Hes-1, preT␣, and Ifi-D3 is altered by Tg mNumb expression. A, RNA extracted from the thymi of 3-wk-old Tgϩ and TgϪ littermates was used to prepare cDNA. cDNA pools were normalized for equivalent template amounts by serial dilution and amplification using primers specificto␤-actin. PCR amplification of Notch-1 target genes was performed using primers specific for Ifi-D3, Hes-1, and preT␣. PCR products were separated by electrophoresis through a 0.8% agarose gel. Lanes 1Ð4, Serial dilution of TgϪ thymic cDNA; lanes 5Ð8, serial dilution of Tgϩ thymic cDNA. Representative results obtained from three independent pairs of Tgϩ and TgϪ and littermates are shown. B, RNA was prepared from DN2/3 and DP thymocytes and peripheral T cells (PT) from 3- to 4-wk-old Tgϩ and TgϪ mice and used to make cDNA. The cDNA was amplified with Ifi-D3 and ␤-actin-specific primers, and PCR products were separated as above. The Journal of Immunology 3177

FIGURE 5. Cell cycle analysis of total, SP/DP, and DN thymocytes from mNumb Tgϩ and TgϪ mice. DNA content of thymocytes from Tgϩ and TgϪ mice was determined by PI staining and ﬂow cytometric analysis. Downloaded from The percentages of cells in G0 and G1 or in S and G2-M phases of the cell cycle in total, CD4/CD8 SP and DP, or DN thymocytes are shown. DN cells were analyzed by gating on FITC-negative thymocytes stained with anti-CD4 and anti-CD8 mAbs. The results are representative of three independent experiments.

FIGURE 4. Phenotypic profile of splenocytes and lymphocytes from mNumb Tgϩ and TgϪ mice. A, The histograms depict expression of B220 http://www.jimmunol.org/ on splenocytes (top panels). Two-parameter 7% probability contour plots formed cell cycle analysis on thymocytes (total, SP/DP, and show surface expression of CD4 vs CD8 on splenocytes (bottom panels). DN) from Tgϩ and TgϪ mice (Fig. 5). The proportion of cells B, Staining profile of lymphocytes using two-parameter 7% probability in G0 and G1 or in S and G2-M phases of the cell cycle was contour plots. The numbers within the quadrants represent the percentage similar when each subpopulation of thymocytes from Tgϩ and of live cells. TgϪ was compared. We considered that Tg overexpression of mNumb might have more subtle effects on T cell development that might be revealed in Tgϩ vs TgϪ mice. There was no increased generation of intra- ϩ by competitive repopulation experiments. Precedent for this hy- thymic B cells in Tg mice, an observation that has been made in by guest on September 29, 2021 pothesis comes from experiments designed to examine the effect of mice with conditional inactivation of Notch-1 in bone marrow pre- reduced Notch-1 levels on T cell development. In these studies, cursors (4), and in mice overexpressing Lunatic Fringe, a negative ϩ Ϫ mice with one mutant Notch-1 allele (Notch-1 / ) were normal. regulator of Notch-1 signaling, in cells before T/B lineage com- ϩ ϩ ϩ Ϫ However, when bone marrow from Notch-1 / and Notch-1 / mitment (6). Fig. 4 shows representative FACS analysis of spleno- Ϫ Ϫ mice was mixed and transferred to RAG-2 / recipients, differ- cytes stained for B220, CD4, and CD8 (Fig. 4A) and lymphocytes ences in the relative contributions made by these cells to different stained for CD4 and CD8 (Fig. 4B). Again, we observed similar T cell lineages were observed. Specifically, there was an increase proportions of cells positive for these cell markers in the two in the proportion of ␥␦ T cells derived from progenitors with re- groups of mice. Other cell lineages examined were: ␥␦ TCRϩ ϩ ϩ ϩ duced levels of Notch-1 (9). Using a similar strategy, bone marrow thymocytes; B220 , IgM , and ␥␦ TCR splenocytes; and ␣␤ ϩ ϩ Ϫ ϩ cells from Ly-5.2 mNumb Tg or Tg littermates and Ly-5.1 TCRϩ and ␥␦ TCRϩ lymphocytes. The proportions of cells in ϩ wild-type mice were mixed in a 1:1 ratio, injected into irradiated these different lineages were similar when the results from Tg Ϫ Ϫ RAG-2 / recipients, and analyzed at day 35 or 43. Based on and TgϪ mice were compared (data not shown). In addition, the previous studies, we expected that there would be an increase in total number of cells in the thymus, spleen, and lymph nodes of ϩ ϩ Ϫ the contribution of mNumb Tg cells to the ␥␦ T cell lineage Tg and Tg littermates was similar (Table I). From these anal- ϩ and/or a decrease in the contribution of mNumb Tg cells to the yses, we conclude that Tg overexpression of the p66 isoform of ϩ CD8 T cell lineage in the presence of mNumb inhibition of mNumb, and the resulting down-regulation of Notch-1 target Notch-1 activity. genes has no obvious effect on ␥␦/␣␤ or CD4/CD8 lineage commitment. To assess the effect of Tg mNumb on the rate of entry and exit from the DP stage of thymocyte development, we per- Table II. Percentage of Tgϩ or TgϪ cells in the thymus, spleen, or lymph nodes of recipient mice reconstituted with mixtures of Tgϩ or TgϪ and Ly5.1ϩ wild-type cellsa Table I. Total number of cells in the thymus, spleen, and lymph nodes ϩ Ϫ of Tgϩ and TgϪ micea %Tg or%Tg Cells Tgϩ and Ly5.1ϩ TgϪ and Ly5.1ϩ Tissue Tgϩ TgϪ Tissue wild type wild type

Thymus 116 ϫ 106 Ϯ 20 ϫ 106 139 ϫ 106 Ϯ 45 ϫ 106 Thymus 35 Ϯ 12.8 (n ϭ 30) 42 Ϯ 2.2 (n ϭ 8) Spleen 65 ϫ 106 Ϯ 16 ϫ 106 68 ϫ 106 Ϯ 21 ϫ 106 Spleen 47.2 Ϯ 8.8 (n ϭ 30) 58 Ϯ 1.6 (n ϭ 8) Lymph nodes 55 ϫ 106 Ϯ 12 ϫ 106 47 ϫ 106 Ϯ 10 ϫ 106 Lymph nodes 47.9 Ϯ 7.7 (n ϭ 24) 58 Ϯ 2.5 (n ϭ 8)

a Values are the mean Ϯ SD. Six mice per group were analyzed. a Values are the mean Ϯ SD. n, number of tissues analyzed in each group. 3178 NUMB OVEREXPRESSION DOES NOT ALTER T CELL FATE SPECIFICATION

Table III. Percentage of cell death of thymocytes from Tgϩ and TgϪ mice in culture for 24 and 40 ha

% Cell Death Time in Culture (h) Tgϩ TgϪ

24 48 Ϯ 10 44.4 Ϯ 4.1 40 62 Ϯ 7.3 60.2 Ϯ 1.9

a Mean Ϯ SD, n ϭ 5 per group. Mice were 6 wk of age.

marrow cells (Fig. 6). However, these differences were also observed in the control recipients that were injected with mixtures of TgϪ and wild-type Ly-5.1ϩ cells. Therefore, it is unlikely that these differences reﬂect an effect of Tg mNumb overexpression. Data in Fig. 6 and Table II were pooled from groups of mice analyzed at day 35 or day 43 after reconstitution. There was no difference in the results from the two time points, indicating no

obvious kinetic difference in ␣␤, ␥␦, DP, and SP production from Downloaded from Tgϩ vs TgϪ bone marrow cells. In previous work by others, Tg expression of a constitutively active form of Notch-1 also promoted survival of thymocytes in culture (10). Therefore, we determined whether thymocytes with Tg overexpression of mNumb would undergo apoptosis more

readily in culture. We observed similar amounts of cell death in http://www.jimmunol.org/ cultures of Tgϩ and TgϪ thymocytes (Table III), suggesting the Tg mNumb does not increase apoptosis in vitro.

Discussion In Drosophila, Numb is a cell fate determinant thought to function by antagonizing Notch signaling by, presumably, reducing Notch nuclear activity and Notch-dependent gene expression (16, 38Ð 42). In vertebrates, evidence points to a similar role for Numb in antagonizing the effects of aberrantly activated Notch. For exam- by guest on September 29, 2021 ple, Numb promotes neurite outgrowth that has been suppressed by overexpression of activated Notch in primary neuron cultures, and can interfere with the ability of an active form of Notch to inhibit neuronal differentiation in the avian CNS (14Ð17). However, in these experimental systems, the direct effects of Numb on the expression Notch target genes and/or on Notch nuclear activity have not been measured. Therefore, the observed effects cannot be at- tributed directly to down-regulation of Notch signaling, nor do they address the role of Numb on endogenous Notch activity. FIGURE 6. Transgenic overexpression of mNumb does not alter lym- We analyzed two different lines of Tg mice to determine the phocyte development. Mixtures of T cell-depleted bone marrow cells from ϩ ϩ Ϫ effect of mNumb overexpression on endogenous Notch-1 activity Ly-5.2 mNumb Tg mice (left panel)orTg littermates (right panel) and and on T cell development. High levels of functional p66 mNumb Ly-5.1ϩ wild-type mice were injected into irradiated RAG-2Ϫ/Ϫ mice. The protein were expressed in thymocytes of Tg mice, including DN thymus (A), spleen (B), and lymph nodes (C) of the recipient mice were analyzed by FACS 4Ð5 wk after reconstitution. The number of cells in a cells. Overexpression of mNumb resulted in reduced expression of ϩ ␣ specific subset is represented as a percentage of the number of Ly-5.2 or three genes, Hes-1, preT , and Ifi-D3, previously shown to be Ly-5.1ϩ cells in each tissue. Each dot represents the value from one re- up-regulated by activated Notch (11), providing evidence that cipient mouse, and the bar represents the average. Numb antagonizes endogenous Notch-1 signaling. This reduction was evident as early as the DN2/3 stage of development coincident with commitment to the T cell lineage. Despite this evidence for a The percentages of Tgϩ or TgϪ cells contributing to the thymus, direct effect of Numb on Notch-1 signaling, we were unable to detect spleen, and lymph nodes were similar in recipient mice that re- any changes in ␥␦/␣␤ or CD4/CD8 lineage commitment or thymo- ceived mixtures of Tgϩ and wild-type Ly-5.1ϩ bone marrow or cyte cell cycle or survival. Moreover, the lymphopoietic activity of those that received control mixtures of TgϪ and wild-type Ly-5.1ϩ bone marrow from mNumb Tgϩ mice was equivalent to that of TgϪ bone marrow (Table II). In addition, the proportions of Tgϩ and bone marrrow in competitive repopulation experiments. wild-type Ly-5.1ϩ cells contributing to DN, DP, CD4ϩ, and CD8ϩ In support of our results, it was recently reported that Notch-1 SP and ␥␦ TCRϩ subsets in these tissues were similar. There was signaling is not required after T cell commitment for thymocyte a small increase in the proportion of Tgϩ B cells (B220ϩ) and a survival, differentiation, or fate specification in mice with a con- small decrease in the proportion of Tgϩ T cells (CD4ϩ SP or ditional, loss-of-function mutation of Notch-1 (43). There is the CD8ϩ SP) in the spleen and lymph nodes of the recipient mice possibility that other Notch genes may have acted redundantly in reconstituted with mixtures of Tgϩ and wild-type Ly-5.1ϩ bone place of Notch-1 in that study, since downstream consequences of The Journal of Immunology 3179 the conditional Notch-1 deletion were not directly measured (43). References ϩ However, results from our analyses of Numb Tg mice, in which 1. Zuniga-Pflucker, J. C., and M. J. Lenardo. 1996. Regulation of thymocyte de- Notch signaling is down-regulated through Numb overexpression, velopment from immature progenitors. Curr. Opin. Immunol. 8:215. 2. Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. 1999. Notch signaling: cell support the conclusion that Notch-1 signaling is not required in fate control and signal integration in development. Science 284:770. thymocytes for ␥␦/␣␤ or CD4/CD8 fate specification. 3. Osborne, B., and L. Miele. 1999. Notch and the immune system. Immunity 11: In contrast, previous studies on the role of Notch-1 signaling in 653. 4. Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. MacDonald, T cell development, in which a truncated, constitutively active and M. Aguet. 1999. Deficient T cell fate specification in mice with an induced form of Notch-1 was overexpressed in thymocytes, led to the con- inactivation of Notch1. Immunity 10:547. clusion that Notch-1 signaling plays a critical role in ␥␦/␣␤ and 5. Tomita, K., M. Hattori, E. Nakamura, S. Nakanishi, N. Minato, and R. Kageyama. 1999. The bHLH gene Hes1 is essential for expansion of early T CD4/CD8 lineage decisions during T cell development (12, 13). cell precursors. Genes Dev. 13:1203. This form of Notch is known to be oncogenic in several cell types, 6. Koch, U., T. A. Lacombe, D. Holland, J. L. Bowman, B. L. Cohen, S. E. Egan, and C. J. Guidos. 2001. Subversion of the T/B lineage decision in the thymus by including T cells, and may have produced a nonphysiologic effect lunatic fringe-mediated inhibition of Notch-1. Immunity 15:225. (44Ð46). It is also possible that activated Notch-1 may have al- 7. Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, tered the activity or expression of proteins that are normally in- T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity fluenced by other Notch genes. The role of these other Notch fam- 11:299. ily members was not addressed in experiments in which Notch-1 8. Wilson, A., H. R. MacDonald, and F. Radtke. 2001. Notch1-deficient common was constitutively deleted (43), and the effect of mNumb on the lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194:1003. 9. Washburn, T., E. Schweighoffer, T. Gridley, D. Chang, B. J. Fowlkes, D. Cado, activity of other Notch genes is not known. and E. Robey. 1997. Notch activity influences the ␣␤ versus ␥␦ T cell lineage Reduction of Notch-1 levels in bone marrow cells results in an decision. Cell 88:833. Downloaded from ␥␦ ϩ 10. Deftos, M. L., Y. W. He, E. W. Ojala, and M. J. Bevan. 1998. Correlating notch enhancement in the development of TCR T cells, which can signaling with thymocyte maturation. Immunity 9:777. be observed in competitive reconstitution experiments with mix- 11. Deftos, M. L., E. Huang, E. W. Ojala, K. A. Forbush, and M. J. Bevan. 2000. tures of Notch-1 heterozygous mutant and normal bone marrow Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes. Immunity 13:73. (9). We used a similar strategy to study the effect of Tg overex- 12. Robey, E., D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans, G. Weinmaster, pression of mNumb on the ␣␤ TCR vs ␥␦ TCR lineage commit- and P. Salmon. 1996. An activated form of Notch influences the choice between ment. Transgenic mNumb was readily expressed in DN2/3 thymo- CD4 and CD8 T cell lineages. Cell 87:483. http://www.jimmunol.org/ 13. Izon, D. J., J. A. Punt, L. Xu, F. G. Karnell, D. Allman, P. S. Myung, N. J. Boerth, cytes, and the expression of the Notch target gene, Ifi-D3, was J. C. Pui, G. A. Koretzky, and W. S. Pear. 2001. Notch1 regulates maturation of significantly reduced in this subpopulation. Thus, an effect of CD4ϩ and CD8ϩ thymocytes by modulating TCR signal strength. Immunity 14: ␥␦␣␤ 253. mNumb on lineage commitment, if present, should have 14. Guo, M., L. Y. Jan, and Y. N. Jan. 1996. Control of daughter cell fates during been observed at this developmental stage; however, we found that asymmetric division: interaction of Numb and Notch. Neuron 17:27. mNumb Tgϩ and TgϪ cells contributed equally to the different cell 15. Frise, E., J. A. Knoblich, S. Younger-Shepherd, L. Y. Jan, and Y. N. Jan. 1996. The Drosophila Numb protein inhibits signaling of the Notch receptor during lineages in repopulation experiments at the different time points cell-cell interaction in sensory organ lineage. Proc. Natl. Acad. Sci. USA 93: analyzed. Therefore, Tg overexpression of mNumb and down-reg- 11925. ulation of Notch signaling at this stage of development do not 16. Spana, E. 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sibling neuron cell fates. Neuron 17:21. by guest on September 29, 2021 mimic the effect of reduced Notch-1 levels in competitive repopu- 17. Wakamatsu, Y., T. M. Maynard, S. U. Jones, and J. A. Weston. 1999. NUMB lation experiments. It is possible that the inhibition of Notch by localizes in the basal cortex of mitotic avian neuroepithelial cells and modulates neuronal differentiation by binding to NOTCH-1. Neuron 23:71. Numb overexpression in our experiments was insufficient or per- 18. Luo, B., J. C. Aster, R. P. Hasserjian, F. Kuo, and J. Sklar. 1997. Isolation and haps the reduction in Notch-1 may be required earlier, since in the functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for experiments with heterozygous Notch-1 mutants (9), Notch-1 ex- the Notch1 receptor. Mol. Cell. Biol. 17:6057. 19. Felli, M. P., M. Maroder, T. A. Mitsiadis, A. F. Campese, D. Bellavia, A. Vacca, pression would be reduced at all stages of development. R. S. Mann, L. Frati, U. Lendahl, A. Gulino, and I. Screpanti. 1999. Expression In mammals, there are four isoforms of mNumb, and several pattern of Notch1, 2, and 3 and Jagged1 and 2 in lymphoid and stromal thymus lines of evidence imply that the Numb isoforms may have distinct components: distinct ligand-receptor interactions in intrathymic T cell development. Int. Immunol. 11:1017. cellular functions (25, 32). It is currently unknown whether the 20. Shawber, C., J. Boulter, C. E. Lindsell, and G. Weinmaster. 1996. Jagged2:a Numb isoforms differ in their capacity to antagonize Notch-1, or serrate-like gene expressed during rat embryogenesis. Dev. Biol. 180:370. 21. Jiang, R., Y. Lan, H. D. Chapman, C. Shawber, C. R. Norton, D. V. Serreze, whether they interact with other Notch family members. In this G. Weinmaster, and T. Gridley. 1998. Defects in limb, craniofacial, and thymic study, we have shown that the p66 isoform of Numb is able to development in Jagged2 mutant mice. Genes Dev. 12:1046. suppress expression of Notch-1 target genes in vivo. We cannot 22. Zhong, W. M., J. N. Feder, M. M. Jiang, L. Y. Jan, and Y. N. Jan. 1996. Asym- metric localization of a mammalian Numb homolog during mouse cortical neu- exclude the possibility that a different isoform of Numb may have rogenesis. Neuron 17:43. a different effect on Notch-1 signaling or on other Notch genes 23. Verdi, J. M., R. Schmandt, A. Bashirullah, S. Jacob, R. Salvino, C. G. Craig, expressed in the thymus, which would alter T cell development. A. E. Program, H. D. Lipshitz, and C. J. McGlade. 1996. Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate. However, we are aware of unpublished results in which the p65 Curr. Biol. 6:1134. isoform of mNumb was transgenically expressed using the same 24. Zhong, W. M., M. M. Jiang, G. Weinmaster, L. Y. Jan, and Y. N. Jan. 1997. Differential expression of mammalian Numb, Numblike and Notch1 suggests promoter, and like our results, no alterations in T cell development distinct roles during mouse cortical neurogenesis. Development 124:1887. were observed (Y.-R. Zou and D. R. Littman, personal 25. Dho, S. E., M. B. French, S. A. Woods, and C. J. McGlade. 1999. Characteriza- communication). tion of four mammalian numb protein isoforms: identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain. J. Biol. Conditional targeting of Notch-1 much earlier in development Chem. 274:33097. has revealed an essential role in specification of T vs B lineage 26. Zhong, W., M. M. Jiang, M. D. Schonemann, J. J. Meneses, R. A. Pedersen, precursors (4, 7, 8). Therefore, during the course of lymphocyte L. Y. Jan, and Y. N. Jan. 2000. Mouse numb is an essential gene involved in cortical neurogenesis. Proc. Natl. Acad. Sci. USA 97:6844. development, there is an apparent transition between Notch-1 de- 27. Zilian, O., C. Saner, L. Hagedorn, H.-Y. Lee, E. Sauberli, U. Suter, L. Sommer, pendence and independence. As such, a role for Numb in lympho- and M. Auget. 2001. Multiple roles of mouse Numb in tuning developmental cell cyte development will likely be revealed by modulating its levels fates. Curr. Biol. 11:494. 28. Bork, P., and B. Margolis. 1995. A phosphotyrosine interaction domain. Cell at earlier stages of hemopoiesis. Our results, in keeping with re- 80:693. cently published work (43), support the conclusion that in imma- 29. Salcini, A. E., S. Confalonieri, M. Doria, E. Santolini, E. Tassi, O. Minenkova, ␥␦ ␣␤ G. Cesareni, P. G. Pelicci, and P. P. Di Fiore. 1997. Binding specificity and in ture thymocytes, Notch-1 signaling is not essential for / or vivo targets of the EH domain, a novel protein-protein interaction module. Genes CD4/CD8 T cell lineage commitment or survival. Dev. 11:2239. 3180 NUMB OVEREXPRESSION DOES NOT ALTER T CELL FATE SPECIFICATION

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