ADAM10 Overexpression Shifts Lympho- and Myelopoiesis by Dysregulating Site 2/Site 3 Cleavage Products of Notch

This information is current as David R. Gibb, Sheinei J. Saleem, Dae-Joong Kang, Mark of October 4, 2021. A. Subler and Daniel H. Conrad J Immunol 2011; 186:4244-4252; Prepublished online 2 March 2011; doi: 10.4049/jimmunol.1003318 http://www.jimmunol.org/content/186/7/4244 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2011/03/02/jimmunol.100331 Material 8.DC1 http://www.jimmunol.org/ References This article cites 45 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/186/7/4244.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 4, 2021

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ADAM10 Overexpression Shifts Lympho- and Myelopoiesis by Dysregulating Site 2/Site 3 Cleavage Products of Notch

David R. Gibb,*,1 Sheinei J. Saleem,*,1 Dae-Joong Kang,* Mark A. Subler,† and Daniel H. Conrad*

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b+Gr-1+ cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncou- Downloaded from pling the highly regulated of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage2Sca-1+c-Kit+ cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage2Sca-1+c-Kit+ cells, respectively. In contrast, blockade of g-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell http://www.jimmunol.org/ accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination. The Journal of Immunology, 2011, 186: 4244–4252.

isintegrin and (ADAMs) regulate cell substrates contributes to the pathogenesis of multiple disease states, signaling pathways by cleaving the extracellular domains including allergy, cancer, and inflammation (1, 2). Accordingly, D of membrane-bound receptors and ligands. Consequently, there is growing interest in ADAM10 as a pharmacological target these serve as initiators for signaling pathways that require for these conditions. However, determination of the physiologic regulated intramembrane proteolysis (RIP) of receptor:ligand consequences of ADAM10-mediated cleavage events has been

complexes. In vitro-based assays have revealed that ADAM10 is an limited by lethality of ADAM10-null murine embryos (3). by guest on October 4, 2021 important mediator of ectodomain shedding and RIP of numerous Production of ADAM10-deficient embryos and conditional substrates, such as the low-affinity IgE receptor CD23 and the knockout mice has demonstrated a critical role for ADAM10 in Notch ligand Delta-like 1 (DL1). Proteolytic processing of these developmental pathways, including thymocyte and marginal zone B cell development (4, 5). Each report concluded that impaired de- *Department of Microbiology and Immunology, Virginia Commonwealth University, velopment in the absence of ADAM10 was the result of diminished Massey Cancer Center, School of Medicine, Richmond, VA 23298; and †Depart- Notch signaling, which depends on RIP for signal activation. ment of Human and Molecular Genetics, Virginia Commonwealth University, Massey Many studies have demonstrated the importance of Notch sig- Cancer Center, School of Medicine, Richmond, VA 23298 naling in lymphocyte development. The is 1D.R.G. and S.J.S. contributed equally to this work. highly conserved, consisting of four families of receptors (Notch1– Received for publication October 7, 2010. Accepted for publication February 1, 2011. 4) that interact with ligands (Jagged and Delta) expressed by This work was supported by National Institute of Allergy and Infectious Diseases/ neighboring cells (6–8). Following ribosomal synthesis, the Notch National Institutes of Health (Grants R01AI18697 and U19AI077435) (to D.H.C.), receptor undergoes a furin-mediated maturation at site 1 in the the American Heart Association (Grant 0815066E) (to D.R.G.), and National Insti- tutes of Health-National Cancer Institute Center Support Grant P30 CA016059 for Golgi apparatus prior to trafficking to the cell surface. At the the Virginia Commonwealth University Transgenic and Flow Cytometry Facility. surface, Notch is expressed as an integral membrane , Address correspondence and reprint requests to Dr. Daniel H. Conrad, Department of consisting of both extracellular (NEXT) and intracellular domains Microbiology and Immunology, Virginia Commonwealth University, Massey Cancer (NICD). Once engaged with its ligand, the extracellular domain Center, School of Medicine, P.O. Box 980678, Richmond, VA 23298. E-mail address: [email protected] undergoes an ADAM10-mediated cleavage at site 2 (S2). This The online version of this article contains supplemental material. event generates a substrate for g-secretase complex to perform Abbreviations used in this article: A10Tg, ADAM10 transgenic; CLP, common lym- a final cleavage of Notch at site 3 (S3), releasing the transcrip- phoid progenitor; CMP, common myeloid progenitor; DL1, Delta-like 1; ETP, early tionally active NICD (Fig. 6) (9). Several studies have reported the thymocyte progenitor; GMP, granulocyte-macrophage progenitor; GSI, g-secretase inhibitor; HSC, hematopoietic stem cell; LSK, Lineage2Sca-1+c-Kit+; LT-HSC, accumulation of intact receptor and the S2 product as a result of long-term hematopoietic stem cell; MDSC, myeloid-derived suppressor cell; MEP, ADAM10 and g-secretase blockade, respectively (10, 11). Al- megakaryocyte-erythroid progenitor; MPP, multipotent progenitor population; though inhibition of both prevents NICD activation, the NEXT, Notch extracellular domain; NICD, Notch intracellular domain; PLN, periph- eral lymph node; PS, presenilin; RIP, regulated intramembrane proteolysis; S2, site 2; consequences of accumulation of these different cleaved products S3, site 3; ST-HSC, short-term hematopoietic stem cell; T-ALL, T cell acute lym- on hematopoiesis remains to be determined. phocytic leukemia; WT, wild-type. Notch is critical for T cell lineage commitment and maturation as Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 well as marginal zone B cell development (6, 12). Notch1 signaling www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003318 The Journal of Immunology 4245 in common lymphoid progenitors (CLPs) is required for early Virginia Commonwealth University Institutional Care and Use thymocyte development (13), and enforced signaling enhances Committee. T cell development (12). In humans, mutations in the negative Flow cytometry and cell sorting regulatory region of the Notch1 receptor allow ligand-independent cleavage, resulting in excessive Notch1 signaling and the forma- Cell isolation and labeling was conducted as described previously (5). In addition, peritoneal fluid cells were obtained by flushing the peritoneal tion of T cell acute lymphocytic leukemia (T-ALL) (9). Several cavity with PBS (5 ml). BM cells were isolated by flushing excised tibias groups report the use of g-secretase inhibitors (GSIs) as a means and femurs with complete RPMI 1640 medium. Single-cell suspensions of of suppressing the expansion of T-ALL cells and thus as a poten- peripheral lymph node (PLN) cells, thymocytes, and splenocytes were tial method for treating T-ALL and other Notch-dependent lym- created by disrupting inguinal, brachial, axillary lymph nodes, thymus, and spleens, respectively, with glass slides. Cells were labeled following RBC phoproliferative disorders (14, 15). However, GSI treatment does lysis and filtration through 40 mM cell strainers. Abs included anti-mouse not completely abrogate Notch signaling, because it does not af- unlabeled 2.4G2, biotinylated CD135 (A2F10); FITC-conjugated IL-7R fect the initiation of the signaling pathway mediated by ADAMs. (A7R34), CD44 (IM7), B220 (RA3-6B2), and Gr-1 (RB6-8C5); PE- GSI treatment results in the accumulation of the S2 cleavage conjugated B220, CD8 (53-6.7), Gr-1, Ter-119, Thy1.2, (30-H12) CD11b ε product, and its pathologic consequences remain to be elucidated (M1/70), and CD3 (2C11); allophycocyanin-labeled B220, CD4 (RM4-5), CD5 (53-7.3), CD45.2 (104), and c-Kit (2B8); PE/Cy7-conjugated CD11b (8, 16). and sca-1 (D7), PE/Cy5 CD34 (A2F10), APC/Cy7-conjugated CD19 (6D5) In contrast to T cell development, Notch signaling prevents and CD45.1 (A20), and PerCP/Cy5.5-conjugated IgM (RMM-1) and IL-7R commitment of CLPs to the B cell lineage (12). Furthermore, from BioLegend; CD34-FITC (RAM34), Ly6G-FITC (1A8), c-Kit–PE enforced expression of constitutively active NICD or Notch target (2B8), Sca-1-PE/Cy7, and APC-conjugated CD3 (145-2C11),B220 (RA3- 6B2), Ly6C/Ly6G (RB6-8C5), CD11b (M1/70), and TER-119 from BD in BM progenitors abrogates B lineage commitment and

Biosciences; and ADAM10-FITC (FAB946) from R&D Systems. Anti-mouse Downloaded from + + promotes noncell autonomous expansion of CD11b Gr-1 mye- FcgRII/III (in-house) and anti-mouse Ly6D (49-H4) (BD Biosciences) loid cells in transplant recipients (17, 18). were biotinylated with sulfosuccinimidobiotin (EZ Link Sulfo-NHS-Biotin, Although the effect of Notch signaling on lymphopoiesis has Pierce), followed by dialysis to remove free Sulfo-NHS-Biotin. Streptavi- din-PE-Texas Red (Beckman Coulter) was used for secondary labeling of been well established, its role in myelopoiesis remains contro- 2 biotinylated-Ly6D and FcgRII/III-labeled cells. Flow cytometric analysis versial. Several studies have shown that Notch signaling promotes was performed using a Canto or AriaII (BD Biosciences), and data analysis expansion of undifferentiated myeloid cells (19), abrogates B cell was conducted with FCS Express V3 software. Histogram overlays were development, and promotes immature myeloid cell formation generated in SigmaPlot 10.0 as line plots and smoothed using the http://www.jimmunol.org/ (17). In addition, mice deficient in downstream Notch effectors SMOOTH transform. For FACS of LSKs, lineage-positive cells were de- pleted with a lineage cell depletion (MACS; Miltenyi Biotec/StemCell exhibit defective B cell and myeloid development (20). Collec- Technologies). Remaining LSKs (Lin2IL7R2c-kithisca-1hi) were sorted tively, these observations suggest that Notch-mediated alterations with an Aria II. Lineage-positive cells include CD3ε, Gr-1–, CD11b-, in lymphocyte development could serve to modulate myelopoi- B220-, and Ter-119–positive cells. In Fig. 4, B220 was excluded from the + + esis. This supposition is supported by recent data demonstrating lineage. Total spleen MDSCs (CD11b Gr-1 ) were sorted for cytologic that myeloid potential is retained in developing lymphocytes (21, analysis. The purity of sorted cells exceeded 95%. For photomicrographs of sorted spleen MDSCs, cells were cytospun on glass slides and stained with 22). Nonetheless, other studies either have failed to detect mye- the HEMA 3 stain set (Fisher Scientific). Photographs were taken with loid alterations under the same conditions as the aforementioned a BIOQUANT NOVA camera attached to an Olympus BH-2 microscope. by guest on October 4, 2021 studies (23) or have indicated that Notch1 and Notch2 are capable Adoptive transfer and BM chimeras of inhibiting myeloid differentiation (24). Adding to this ambi- guity is the observation that mice with diminished g-secretase Recipient CD45.2+ A10Tg (F240) and CD45.1+ wild-type (WT) mice were 137 activity exhibit splenomegaly as a result of the accumulation of irradiated with 950 rad using a Cs source (Mark I, Model 68-0146; JL Shepherd and Associates). Donor LSK cells were isolated from mouse myeloid cells (25). The presence of multiple conflicting data tibia, femur, and humerus by magnetic cell sorting with a lineage cell suggests that myeloid differentiation may depend on both the depletion kit. (MACS; Miltenyi Biotec). Twenty-four hours after irradia- strength and the temporal stage at which Notch signaling occurs. tion, recipient CD45.2+ A10Tg and CD45.1+ WT mice were injected i.v. 6 + 6 Many disease states are characterized by the excessive pro- with 2.5 3 10 CD45.1 WT and 5 3 10 A10Tg LSK cells, respectively. For generation of mixed BM chimeras, a mixture of LSK cells from duction of ADAM10 cleavage products (2). Inspired by the + 6 + 6 CD45.2 A10Tg (2.5 3 10 ) and CD45.1 WT (1.25 3 10 ) mice were successful exploitation of conditional knockout mice in previous injected i.v. into irradiated CD45.1+ WT mice. Cell populations were an- studies, we generated a strain of ADAM10 transgenic mice alyzed 42 and 63 d after reconstitution. (A10Tg) overexpressing ADAM10 at early stages of lympho- and LSK cultures myelopoiesis. The increased ADAM10 activity in these severely impaired B2 cell development and promoted the strik- BM-derived LSK cells isolated via magnetic cell sorting and FACS were ing expansion of myeloid-derived suppressor cells (MDSCs) via cultured in the presence of IL-7 (1 ng/ml; PeproTech) and Flt3 ligand (5 ng/ 2 + + ml; R&D Systems) as described previously (26). LSK differentiation was a cell-intrinsic mechanism. Lineage Sca-1 c-Kit (LSK) hema- examined via flow cytometric analysis, and differentiated cells were passed topoietic stem cell (HSC) culture assays indicated that selec- onto freshly plated OP9 cells with additional cytokines every 4–5 d. OP9- tive blockade of S2 and S3 cleavage exerts a differential effect GFP and OP9-DL1 cells were provided by J. C. Zuniga-Pflucker (Uni- on Notch signaling and hematopoiesis in vitro. Furthermore, versity of Toronto, Toronto, Ontario, Canada). Compound E (100 nM; m ADAM10 overexpression was found to alter hematopoiesis by Alexis Biochemicals) and GI254023X (5 M; GlaxoSmithKline) were used for blockade of g-secretase and ADAM10 activity, respectively. dysregulating RIP-dependent Notch signaling. Taken together, these observations underscore the importance of ADAM10 in Statistical analysis Notch-mediated signaling for both lympho- and myelopoiesis. The p values were calculated using unpaired two-tailed Student t tests. Error bars represent the SEM between samples.

Materials and Methods Mice Results Generation of A10Tg mice A10Tg mice were generated as described in the supplemental methods. C57BL/6 and congenic CD45.1+ (B6-Ly5.2) mice were purchased from To examine the role of ADAM10 in hematopoiesis, we generated the National Cancer Institute. All mouse protocols were approved by the A10Tg mice that overexpress murine ADAM10 cDNA under con- 4246 ADAM10 OVEREXPRESSION DISRUPTS HEMATOPOIESIS trol of the H-2Kb promoter and the IgH enhancer region (Sup- (31, 32). Accordingly, A10Tg mice have reduced levels of total, plemental Fig. 1A). These transcriptional regulatory units were double-negative, and double-positive thymocytes and a small thy- previously used to generate multiple transgenic mouse lines, in- mus. However, numbers of single-positive thymocytes and peri- cluding TCR, CD23, and bkl transgenics. Thus, this vector allows pheral T cells in the PLN and the spleen were not altered in A10Tg expression in early lymphocyte progenitors (28–30). Inclusion of mice (Fig. 1D). the IgH enhancer results in preferential expression on B lineage cells. Two founder lines, F240 and F258, were generated, and MDSC accumulation in A10Tg mice Southern blot analysis of genomic DNA from F2 progeny dem- The blockade of B2 cell development was expected to result in onstrated that both lines contain similar copy numbers of the reduced levels of total splenocytes. However, the spleens of A10Tg transgene (Supplemental Fig. 1B). Because progeny of both lines mice were noticeably enlarged, weighing an average of 2.5-fold have nearly identical phenotypes, the following data are presented more than LM spleens (Fig. 2A), containing twice as many nu- from line F240, unless otherwise stated. cleated cells (data not shown). The forward and side scatter pat- tern of A10Tg splenocytes indicated the presence of large granular ADAM10 overexpression prevents B2 lymphocyte development myeloid cells (Fig. 2B). Further analysis, as shown in Fig. 2C, and inhibits development of thymocyte progenitors revealed that ∼63% of transgenic splenocytes were CD11b+Gr-1+ Western blot and flow cytometric analysis demonstrated that cells, compared with 5.3% of LM splenocytes. Although the transgene expression resulted in elevated levels of ADAM10 in BM majority of WT CD11b+Gr-1+ cells differentiate into mature + 2 cells, including pro-/pre-B cells (B220 IgM ) (Supplemental Fig. myeloid cells prior to exiting the BM, A10Tg CD11b+Gr-1+ cells 1C–E). Unexpectedly, overexpression markedly reduced the levels expanded in BM, constituting 93.5% of BM cells, and entered the

hi + Downloaded from of pro-/pre-B cells and immature B cells (B220 IgM )inBM spleen and the PBL at dramatically high levels (Fig. 2C). CD11b+ (Supplemental Fig. 1C). This led to a near complete loss of Gr-1+ cells outside the bone marrow (BM) are classified as peripheral B cells in peripheral organs including blood, lymph MDSCs, consisting of monocytic (CD11b+Gr-1intLy6G2)and nodes, and spleen (Fig. 1A; data not shown). Analysis of B cells granulocytic subsets (CD11b+Gr-1hiLy6G+) (33). Light micro- int + from peritoneal fluid revealed that levels of B1a (B220 CD11b graphs of sorted A10Tg CD11b+Gr-1+ splenocytes and flow cy- + int + 2 CD5 ) and B1b cells (B220 CD11b CD5 ) in A10Tg mice were tometry confirmed that A10Tg mice contain both monocytic and not significantly altered compared with littermate (LM) controls, granulocytic MDSCs in the PBL and spleen (Fig. 2D–F). http://www.jimmunol.org/ whereas B2 cells (B220hiCD11b2CD52) were nearly absent (Fig. 1B). Thus, the block in B cell development was specific to BM- Hematopoietic expression of ADAM10 alters development via derived B2 cells. In addition, transgene expression suppressed a cell-intrinsic mechanism development of thymocyte progenitors. A 10-fold reduction To confirm that the observed phenotype was the result of transgene was observed in Lin2CD44+c-Kit+ early thymocyte progenitors expression by hematopoietic cells and not a dysregulation in the (ETPs) in A10Tg BM compared with LM (Fig. 1C), which re- BM stromal environment, mixed BM assays were conducted. As sulted in reduced levels of A10Tg thymic ETPs (data not shown) controls, irradiated CD45.2+ A10Tg hosts were reconstituted with by guest on October 4, 2021

FIGURE 1. ADAM10 overexpression prevents B2 cell development and suppresses development of thymocyte progenitors. A and B, Flow cytometric analysis of T cells (CD3+) and B cells (B220+) in spleen (A) and B cell subsets in peritoneal fluid (B). Lower two panels are gated on B220+ cells in upper histograms. B2 cells, B220hiCD11b2CD52; B1a cells, B220intCD11b+CD5+; and B1b cells, B220intCD11b+CD52. C,13 representative thymi from in- dicated mice. BM cells were analyzed for Lin2CD252CD44+c-Kit+ ETPs. Lineage mixture includes B220, Ter-119, CD11b, Gr-1, CD3, CD4, and CD8. D, Amount of thymocyte subsets and CD3+ T cells in the spleen and PLN; n = 4, mean 6 SEM, DN: CD42CD82, DP: CD4+CD8+, CD4: CD4+CD82, CD8: CD42CD8+. Dot plots and histograms are representative of six (A), four (B), and three (C) independent experiments. Numbers on plots indicated percentage of gated cells within box. *p , 0.05. The Journal of Immunology 4247 Downloaded from http://www.jimmunol.org/

FIGURE 2. ADAM10 overexpression causes the expansion of MDSCs. A,13 representative spleens and average spleen weight of indicated mice, n =4, mean 6 SEM. Flow cytometric analysis of forward scatter (FS) versus side scatter (SS) of splenocytes (B), the percentage of CD11b+Gr-1+ MDSCs present in the BM, PBL, and spleen (SPL) (C), and the percentage of CD11b+Ly6-G+ and CD11b+Ly6-G2 MDSCs in PBL (E) and spleen (F) of indicated mice (E and F). D, 340 and 3100 photomicrographs of sorted CD11b+Gr-1+ splenocytes from A10Tg mice. Flow cytometry plots and photomicrographs are representative of four independent experiments. Numbers on plots indicate the percentage of cells in the upper right (C) as well as upper right and lower right quadrants (E) and (F). by guest on October 4, 2021

LSK cells from WT CD45.1+ congenic mice, and irradiated WT which would cause WT MDSC expansion. They also illustrate that CD45.1+ congenic hosts were reconstituted with CD45.2+ A10Tg ADAM10 overexpression on hematopoietic cells causes MDSC LSK BM cells. Despite being in a WT host, A10Tg BM re- expansion via an intrinsic cell autonomous mechanism. capitulated the observed altered hematopoiesis of A10Tg mice. In addition, WT BM cells demonstrated normal cell differentiation, ADAM10 prevents commitment of CLPs to the B cell lineage even in an A10Tg host (data not shown). This finding indicated Expansion of MDSCs in conjunction with blockade of B2 cell that altered cell differentiation in A10Tg mice was due to alter- development indicated that ADAM10 regulates the commitment of ations in signaling pathway(s) within hematopoietic cells rather BM progenitors to myeloid or lymphoid lineages. According to the than the microenvironment. To further confirm this, mixed BM classical model of hematopoiesis, HSCs, which are LSK cells in chimeras were performed. Irradiated CD45.1+ WT hosts were the BM, develop into common myeloid progenitors (CMPs) or reconstituted with a mixture of LSK BM cells from CD45.2+ CLPs, giving rise to early thymocyte progenitors or pro-B cells A10Tg and CD45.1+ WT donors. This resulted in similar re- (34). Supplemental Fig. 1C illustrates that a small percentage of constitution of host BM by A10Tg and WT donor cells 42 and B220+ cells was present in A10Tg BM. However, further analysis 63 d after cell transfer. However, development from BM into revealed that the few B220+CD19+ BM cells in A10Tg mice also peripheral lymphoid organs was less efficient in A10Tg cells, expressed the myeloid markers CD11b and Gr-1 (data not shown), as ∼66 6 5.1% (SE) and 81 6 6.2% of recipient spleen and indicating that alterations in hematopoiesis occur prior to the pro- PLN cells, respectively, developed from WT BM at day 42. The B cell stage. Thus, to determine the stage at which ADAM10 over- selective development of WT thymocytes was most striking, as expression alters hematopoiesis, levels of LSKs, CLPs, and CMPs 98.1 6 1.0% of thymocytes at day 63 were of WT origin (data were examined. Analysis of BM lineage-positive cells (Ter119, not shown). This result supports the diminished thymocyte de- CD3ε, CD11b, and Gr-1) and CD19+ cells demonstrated the near velopment in A10Tg mice (Fig. 1C,1D). Despite these differ- absence of Lin2CD19+ B cells in A10Tg mice. However, the ences, hematopoietic development of CD45.1+ WT cells was percentage of Lin2CD192 cells was similar to LM levels (Fig. similar to development in LM control mice, and development of 4A). Lin2CD192 LM cells contain B220+ B cell precursors, CD45.2+ A10Tg cells closely mimicked development in A10Tg which were absent from A10Tg BM (Fig. 4B). Analysis of IL-7R+ mice. MDSCs only expanded from A10Tg BM cells, and B lin- cells revealed a modest decrease in CLPs (Lin2CD192B2202IL eage cells predominantly differentiated from WT BM (Fig. 3). 7R+c-Kitintscaint) in A10Tg BM (Fig. 4C). In addition, Inlay et al. These results demonstrate that ADAM10-mediated MDSC ex- (35) recently demonstrated that Ly6D+ CLPs are committed to the pansion is not the indirect result of abrogated B cell development, B cell lineage, whereas Ly6D2 CLPs are uncommitted lymphoid trans-cleavage of BM stromal cell ligands, or cytokine secretion, progenitors. Accordingly, 30% of LM CLPs expressed high levels 4248 ADAM10 OVEREXPRESSION DISRUPTS HEMATOPOIESIS

FIGURE 3. ADAM10 alters hematopoie- sis by a cell-autonomous intrinsic mecha- nism. Flow cytometric analysis of myeloid (A) and lymphocyte differentiation (B)in mixed BM chimeras generated as described in Materials and Methods 42 d after cell transfer. CD45.1+- and CD45.2+-gated cells differentiated from WT and A10Tg LSK BM, respectively. Data are representative of three independent experiments, except PBL data are representative of six independent experiments; numbers on dot plots indicate the percentage of CD45.1- or CD45.2-gated cells within boxes. Downloaded from of Ly6D (Fig. 4D). However, only 0.16% of A10Tg CLPs were (Lin2c-Kithisca2) in A10Tg mice was ∼2-fold greater than the Ly6D+. These findings indicate that hematopoietic alterations in level in LM mice. It was previously shown that myeloid progen- A10Tg mice occur prior to the commitment of CLPs to the B cell itors can also be subdivided into three distinct populations based lineage, and it explains the near absence of B cell precursors in on CD34 and low-affinity IgG FcRs (FcgRII/RIII). These include the BM. CD34+FcgRII/IIIlo CMPs, CD34+FcgRII/IIIhi granulocyte-macro- phage progenitors (GMPs), and CD342FcgRII/IIIlo megakaryocyte-

ADAM10 alters myeloid but not LSK development http://www.jimmunol.org/ erythroid progenitors (MEPs) (36). As shown in Fig. 5B, there is To further determine the state at which A10 overexpression altered a striking difference in the myeloid compartment of A10Tg and cell differentiation, we analyzed numerous subsets of LSKs and LM BM. GMP and MEP populations are nearly absent from A10Tg myeloid precursors. Multiple studies have shown that BM LSKs mice. In addition, ∼80% of the A10Tg myeloid progenitors are in can be subdivided into various distinct populations based on CD34 the CMP stage compared with 31.6% in the LM. This finding may 2 2 and Flt3 expression: CD34 Flt3 long-term HSCs (LT-HSCs), account for the systemic expansion of CD11b+Gr-1+ MDSCs in + 2 + + CD34 Flt3 short-term HSCs (ST-HSCs), and finally CD34 Flt3 A10Tg mice. multipotent progenitor populations (MPPs) (36–38). We therefore analyzed these distinct populations within the LSK compartment ADAM10 overexpression alters hematopoiesis by dysregulating by guest on October 4, 2021 in A10Tg mice. Notch signaling Although the percentage of LSK cells was slightly lower in Although the mechanism by which ADAM10 regulates hemato- A10Tg BM (Fig. 5A), we did not observe any differences in the poiesis remains ambiguous, recent studies have demonstrated its levels of LT-HSCs, ST-HSCs, or MPPs between LM and A10Tg essential role in performing S2 cleavage of Notch receptors (see mice (Fig. 5C). However, the percentage of myeloid progenitors Discussion for model) during embryonic, thymocyte, and marginal

FIGURE 4. Overexpression of ADAM10 alters hematopoiesis prior to CLP commitment to the B cell lineage. Flow cytometric analysis of Lineage- negative (Lin2) versus CD19-labeled BM cells (A), B220 and IL-7R (IL7R) expression by Lin2CD192-gated cells from A (B), c-Kit and sca-1 expression of Lin2CD192B2202IL7R+ cells from B (C). D, Expression of Ly6D by CLPs of LM and A10Tg mice. Numbers on dot plots and histograms indicate the percentage of gated cells. The cell lineage includes Ter-119–, CD11b-, Gr-1–, and CD3ε-positive cells. Plots are representative of three independent experiments. CLPs are defined as Lin2CD192B2202IL7R+c-Kitintsca-1int. The Journal of Immunology 4249 Downloaded from

FIGURE 5. ADAM10 overexpression alters development of myeloid progenitors but not LSK subsets. Flow cytometric analysis of Lin2 BM cells (A). B, CD34 and FcgRII/III expression on CMP gate from A. C, CD34 and Flt3 expression in the LSK-gated cells from A. Numbers on dot plots indicate the percentage of gated BM cells. Cell lineage mixture includes B220, Ter-119–, CD11b–, Gr-1–, and CD3-positive cells. Plots are representative of three independent experiments. LSKs were gated as Lin2sca-1hic-Kit+ and subdivided to LT-HSCs, CD342Flt32; ST-HSCs, CD34+Flt32; and MPPs, CD34+ Flt3+. The c-Kit+ myeloid cells were gated and defined as CMPs, CD34+FcgRII/IIIlo; GMPs, CD34+FcgRII/IIIhi; and MEPs, CD342FcgRII/IIIlo. http://www.jimmunol.org/ zone B cell development (3–5). Thus, to determine the effect of B cell and thymocyte progenitors. Interestingly, high expression of ADAM10 overexpression on Notch signaling in cell differentia- DL1 on OP9-DL1 cells prevented myeloid expansion of A10Tg tion, we cultured purified LSK cells on OP9 stromal cells that LSKs (Fig. 7A). This result suggests that ADAM10 overexpres- either did not express Notch ligands (OP9-GFP) or expressed sion may cause myeloid development by dysregulating Notch sig- a primary Notch ligand, DL1 (OP9-DL1). The addition of IL-7 naling in the absence of sufficient ligand. and Flt3 ligand promote LSK differentiation into T cells on OP9- To further test whether ADAM10 overexpression deregulates DL1 cells and B cells on OP9-GFP cells (26). OP9-GFP cultures Notch signaling, we examined the effects of inhibiting Notch re- showed that although LM LSKs developed into B cells, A10Tg ceptor cleavage in LM and A10Tg LSK cells. Inhibition of by guest on October 4, 2021 LSK cells only generated CD11b+Gr-1+ myeloid cells (Fig. 6A). Notch signaling with the ADAM10 inhibitor (GI254023X) or the OP9-DL1 cultures demonstrated that A10Tg LSK cells are capa- g-secretase inhibitor (compound E) (14) in OP9-DL1 cultures ble of T cell differentiation (Fig. 6B). However, their development prevented LM and A10Tg T cell development (Fig. 6C). In ac- was delayed compared with LM LSK cells. This further supports cordance with reports of ADAM10’s critical role in Notch S2 the adverse effect of ADAM10 overexpression on development of cleavage in other cell types (3–5, 8, 16, 39), this result indicates

FIGURE 6. Inhibition of g-secretase or ADAM10 activity prevents Notch-dependent T cell development. A and B, Flow cyto- metric analysis of differentiated LSKs co- cultured with OP9-GFP (A) or OP9-DL1 (B) stromal cells for 8, 17, and 29 d; represen- tative of four independent experiments. C,T cell development of LSKs cocultured with OP9-DL1 cells for 29 d in the presence of a g-secretase inhibitor, compound E, or an ADAM10 inhibitor, GI254023X; represen- tative of two independent experiments. 4250 ADAM10 OVEREXPRESSION DISRUPTS HEMATOPOIESIS

FIGURE 7. ADAM10 overexpression and g-secretase inhibition disrupt Notch signaling and direct myeloid cell development. Flow cytometric analysis of LSK differentiation af- ter 29 d of LSK coculture with OP9-DL1 (A) or OP9-GFP (B) stromal cells as described in Materials and Methods. Compound E, GI254023X, or control DMSO was added to media to inhibit g-secretase or ADAM10 ac- tivity, respectively. DMSO-treated plots are re- presentative of four independent experiments; plots of inhibitor-treated cultures are represen- tative of two independent experiments. Num- bers on plots indicate the percentage of gated live cells based on forward and side scatter. Downloaded from

that ADAM10 also mediates S2 cleavage in developing hemato- myeloid differentiation of A10Tg cells in OP9-GFP cultures (Fig. poietic precursors. Intriguingly, although both inhibitors blocked 7). In addition, GSI treatment of LM OP9-GFP cultures had no Notch-dependent T cell development, their effects on develop- effect on LSK development. This demonstrates that GSI treatment ment of myeloid and B lineage cells were distinct. ADAM10 in- only directs myeloid development following Notch ligand binding http://www.jimmunol.org/ hibition in OP9-DL1 cultures resulted in B cell development of and ADAM-mediated S2 cleavage, resulting in accumulation of LM and A10Tg LSK cells. In addition, ADAM10 inhibition in the S2 cleavage product. Thus, both inhibition of the re- OP9-GFP cultures not only resulted in enhanced LM B cell de- sponsible for degrading the S2 product (g-secretase) and over- velopment but also it rescued B cell development of A10Tg cells expression of the protease that produces the S2 product (ADAM10) and prevented myeloid differentiation. caused myeloid development. Collectively, these results indicate In contrast to ADAM10 inhibition, GSI treatment of OP9-DL1 that ADAM10 overexpression may also direct myeloid develop- cultures caused myeloid development that was strikingly similar to ment by generating excessive S2 product (see Fig. 8 model). by guest on October 4, 2021

FIGURE 8. A model of differential effects of Notch S2/S3 cleavage in hematopoietic differentiation. In the presence of ligand, the Notch receptor undergoes both S2 and S3 cleavage, resulting in two cleaved products and ultimately promoting T cell development (left panel). In the absence of ligand or in the case of blocked S2 cleavage, the Notch receptor remains intact and B cell development results (middle panel). However, S2 cleavage without S3 because of increased ADAM10 activity results in myelopoiesis (right panel). The Journal of Immunology 4251

Discussion that B2 cells and expanded myeloid cells develop from common ADAM10-mediated S2 cleavage is required for the initiation of the progenitor(s), whereas thymocytes may develop from a unique canonical Notch signaling pathway. Accordingly, we hypothesized precursor. that ADAM10 activity would also regulate differentiation of early The data presented are consistent with the hypothesis that excess hematopoietic progenitors. Our experimental observations dem- ADAM10 causes ligand-independent S2 cleavage of Notch. It is onstrate that ADAM10 overexpression attenuates the development possible that, in the absence of ligand, g-secretase complex is not of thymocytes, abrogates B2 cell development, and promotes ex- recruited to the cell membrane, thus leading to reduced S3 pansion of functional MDSCs via a cell-intrinsic mechanism. Fur- cleavage and accumulation of the S2 product. This would then thermore, the S2 and S3 cleavage products of Notch are shown to result in enhanced myeloid development concurrent with di- exert a differential effect on hematopoietic cell fate determination. minished numbers of T cell progenitors. Given the high ligand Although effects of Notch signaling in B and T cell lineage expression levels in OP9-DL1 cells, we expected proper T cell commitment has been extensively described, the activity of differentiation in A10Tg LSKs as any excess S2 product would be ADAM10 in cell differentiation and early B lineage commit- processed by recruited g-secretase. Indeed, even though T cell ment was uncharacterized previously. We demonstrate that over- development in A10Tg LSK cells was delayed, it was comparable expression of ADAM10 prevented B2 cell commitment from CLPs to LM at day 29. In addition, complete g-secretase inhibition of and that ADAM10 inhibitors in OP9 cultures enhanced B cell LM and A10Tg OP9-DL1 cultures, a condition previously shown differentiation of LM and A10Tg LSKs. Thus, ADAM10 exerts to cause S2 product accumulation (8, 16), mimicked myeloid adverse effects on B cell development. Further analysis of LSK, development of A10Tg LSKs in the absence of ligand. myeloid, and lymphocyte compartments demonstrates the stage at These findings support a model through which Notch cleavage Downloaded from which ADAM10 overexpression alters hematopoiesis. Given that mediates cell fate determination (Fig. 8). In the presence of li- the LSK compartment is not affected, ADAM10 overexpression gand, the Notch receptor undergoes cleavage at both S2 and S3 influences differentiation post-LSK subset differentiation but prior sites, resulting in two products and ultimately promoting T cell to mature lineage commitment (B cells, Ly6D+ CLPs, T cells development. In the absence of Notch ligand or in the case of [ETPs], CMPs, GMPs, and MEPs), which are dramatically af- blocked S2 cleavage, the Notch receptor remains intact and B cell fected in the A10Tg mice. The precise stage at which ADAM10 development results. However, S2 cleavage without S3 cleavage, overexpression affects LSK commitment is likely to be very as observed during ADAM10 overexpression or g-secretase in- http://www.jimmunol.org/ transient and remains to be determined. Although our data show hibition, induces myelopoiesis and delayed T cell development. a generalized overexpression of ADAM10 in BM, this over- This is in agreement with other studies that have shown g-secretase expression is limited to B220+ cells. Thus, this transient phase blockade to result in accumulation of the S2 product (8, 16), which may include a fraction of B220+ cells. Following differentiation of could direct myeloid development. Indeed, diminished presenilin 2 2 2 these B220+ cells into non-B cells, the transgene is no longer (PS)-dependent g-secretase activity in PS1+/ PS2 / mice results overexpressed, likely because the IgH enhancer is not used by in myeloproliferative disease, characterized by accumulation of other non-B cells. Mac-1/CD11b+Gr-l+ cells, causing splenomegaly (25). The differential effects of S2 and S3 blockade on WT Notch

The shift in favor of CMP development (Fig. 5B) could explain by guest on October 4, 2021 the robust accumulation of MDSCs in the periphery. In addition, signaling have significant implications for the treatment of Notch- because CMPs have been shown to retain erythromyeloid poten- related diseases. Many reports have proposed the use of GSIs for tial, it is not surprising that despite the decreased MEP and GMP the treatment of T-ALL and B cell lymphoma (14, 44); however, compartments, erythropoieis remains unaffected. Furthermore, the our findings indicate that GSI treatment could cause MDSC ex- presence of comparable NK cells (data not shown) and T cells in pansion that would ultimately induce immunosuppression and the periphery of A10Tg mice (Fig. 1D) suggests that although enhance tumor growth. Thus, studies of GSI treatment in mice and CLPs are unable to give rise to B cells, their ability to differentiate clinical trials should include careful monitoring of myeloid cell into ETPs and NK cells is retained. development. This study indicates that pharmacologic blockade of It should be noted that upon lineage depletion, comparable BM S2 cleavage with ADAM10 inhibitors may be a more advanta- cell numbers were recovered from both A10Tg and LM mice. geous strategy. Therefore, the percentage of ETPs obtained is reflective of absolute Our data demonstrate that the proteolytic activity of ADAM10 regulates the lineage commitment of B2 cells and the expansion of numbers of thymocyte precursors in the BM. Although peripheral functional MDSCs in a cell-intrinsic manner. Moreover, the data T cell levels are comparable, the observed reduction in ETPs is afford an in vivo model for further examination of MDSC ex- nonetheless very intriguing and consistent with the diminished pansion and MDSC-mediated immune suppression in the absence thymus seeding of A10Tg BM cells. This explains the reduced of confounding tumor-derived factors. Given that these factors also thymus size, relative inability of A10Tg thymocyte precursors to regulate immune responses (45), A10Tg animals provide a tool develop in mixed BM chimeras, and delayed T cell development in with which specific pathways leading to MDSC expansion may be OP-DL1 cultures. elucidated in a controlled fashion. The singular expression of B1 The classical model of hematopoiesis describes the initial di- cells in the absence of B2 cells likewise makes this mouse useful chotomous differentiation of LSKs into CLPs or CMPs. However, for the study of B1-mediated humoral immunity. recent studies demonstrating that ETPs possess myeloid potential Furthermore, our observations support the developing myeloid- have challenged this model (21, 22). Additional studies demon- based model of hematopoiesis, leading us to propose a novel strated that B cell progenitors, including CLPs, also retain mye- mechanism through which the Notch S2 and S3 cleavage products loid potential (40, 41), whereas other progenitors that lack T cell differentially regulate cell fate determination. potential can develop into B cells or macrophages (42). For this reason, a myeloid-based model of LSK development is beginning Acknowledgments to emerge (34, 43). In A10Tg mice, the moderate effects on We thank S. Barbour, H. Bear, M. Manjili, and J. Tew for comments, thymocyte development in combination with the more pronounced L. Graham, J. Elenesky, and J. Gomez-Arroyo for technical assistance, effects on B lineage commitment and myeloid expansion indicate and the Virginia Commonwealth University Transgenic/Knockout Mouse 4252 ADAM10 OVEREXPRESSION DISRUPTS HEMATOPOIESIS

Core Facility for the generation of the mice and the flow cytometry facility 21. Bell, J. J., and A. Bhandoola. 2008. The earliest thymic progenitors for T cells for both flow analysis and sorting. Paul Saftig (Christian-Albrechts-Univer- possess myeloid lineage potential. Nature 452: 764–767. 22. Wada, H., K. Masuda, R. Satoh, K. Kakugawa, T. Ikawa, Y. Katsura, and sita¨t zu Kiel, Kiel, Germany) is acknowledged for providing the ADAM10 H. Kawamoto. 2008. Adult T-cell progenitors retain myeloid potential. Nature cDNA. 452: 768–772. 23. Stier, S., T. Cheng, D. Dombkowski, N. Carlesso, and D. T. Scadden. 2002. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and Disclosures favors lymphoid over myeloid lineage outcome. Blood 99: 2369–2378. The authors have no financial conflicts of interest. 24. Bigas, A., D. I. Martin, and L. A. Milner. 1998. Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines. Mol. Cell. Biol. 18: 2324–2333. 25. Qyang, Y., S. M. Chambers, P. Wang, X. Xia, X. Chen, M. A. Goodell, and References H. Zheng. 2004. Myeloproliferative disease in mice with reduced presenilin 1. Blobel, C. P. 2005. ADAMs: key components in EGFR signalling and de- dosage: effect of g-secretase blockage. Biochemistry 43: 5352–5359. velopment. Nat. Rev. Mol. Cell Biol. 6: 32–43. 26. de Pooter, R., and J. C. Zu´n˜iga-Pflu¨cker. 2007. T-cell potential and development 2. Crawford, H. C., P. J. Dempsey, G. Brown, L. Adam, and M. L. Moss. 2009. in vitro: the OP9-DL1 approach. Curr. Opin. Immunol. 19: 163–168. ADAM10 as a therapeutic target for cancer and inflammation. Curr. Pharm. Des. 27. Weskamp, G., J. W. Ford, J. Sturgill, S. Martin, A. J. Docherty, S. Swendeman, 15: 2288–2299. N. Broadway, D. Hartmann, P. Saftig, S. Umland, et al. 2006. ADAM10 is 3. Hartmann, D., B. de Strooper, L. Serneels, K. Craessaerts, A. Herreman, W. Annaert, a principal “” of the low-affinity immunoglobulin E receptor CD23. L. Umans, T. Lu¨bke, A. Lena Illert, K. von Figura, and P. Saftig. 2002. The Nat. Immunol. 7: 1293–1298. disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for 28. Pircher, H., T. W. Mak, R. Lang, W. Ballhausen, E. Ru¨edi, H. Hengartner, a-secretase activity in fibroblasts. Hum. Mol. Genet. 11: 2615–2624. R. M. Zinkernagel, and K. Bu¨rki. 1989. T cell tolerance to Mlsa encoded anti- 4. Tian, L., X. Wu, C. Chi, M. Han, T. Xu, and Y. Zhuang. 2008. ADAM10 is gens in T cell receptor Vb8.1 chain transgenic mice. EMBO J. 8: 719–727. essential for proteolytic activation of Notch during thymocyte development. Int. 29. Malek, S. N., D. I. Dordai, J. Reim, H. Dintzis, and S. Desiderio. 1998. Ma- Immunol. 20: 1181–1187. lignant transformation of early lymphoid progenitors in mice expressing an 5. Gibb, D. R., M. El Shikh, D. J. Kang, W. J. Rowe, R. El Sayed, J. Cichy,

activated Blk tyrosine kinase. Proc. Natl. Acad. Sci. USA 95: 7351–7356. Downloaded from H. Yagita, J. G. Tew, P. J. Dempsey, H. C. Crawford, and D. H. Conrad. 2010. 30. Payet, M. E., E. C. Woodward, and D. H. Conrad. 1999. Humoral response ADAM10 is essential for Notch2-dependent marginal zone B cell development suppression observed with CD23 transgenics. J. Immunol. 163: 217–223. and CD23 cleavage in vivo. J. Exp. Med. 207: 623–635. 31. Sambandam, A., I. Maillard, V. P. Zediak, L. Xu, R. M. Gerstein, J. C. Aster, 6. Saito, T., S. Chiba, M. Ichikawa, A. Kunisato, T. Asai, K. Shimizu, T. Yamaguchi, W. S. Pear, and A. Bhandoola. 2005. Notch signaling controls the generation and G. Yamamoto, S. Seo, K. Kumano, et al. 2003. Notch2 is preferentially expressed differentiation of early T lineage progenitors. Nat. Immunol. 6: 663–670. in mature B cells and indispensable for marginal zone B lineage development. 32. von Boehmer, H. 2005. Notch in lymphopoiesis and T cell polarization. Nat. Immunity 18: 675–685. Immunol. 6: 641–642. 7. Radtke, F., N. Fasnacht, and H. R. Macdonald. 2010. Notch signaling in the 33. Movahedi, K., M. Guilliams, J. Van den Bossche, R. Van den Bergh, C. Gysemans, immune system. Immunity 32: 14–27. A. Beschin, P. De Baetselier, and J. A. Van Ginderachter. 2008. Identification of http://www.jimmunol.org/ 8. van Tetering, G., P. van Diest, I. Verlaan, E. van der Wall, R. Kopan, and discrete tumor-induced myeloid-derived suppressor cell subpopulations with dis- M. Vooijs. 2009. Metalloprotease ADAM10 is required for Notch1 site 2 tinct T cell-suppressive activity. Blood 111: 4233–4244. cleavage. J. Biol. Chem. 284: 31018–31027. 34. Kawamoto, H., and Y. Katsura. 2009. A new paradigm for hematopoietic cell 9. Kopan, R., and M. X. Ilagan. 2009. The canonical Notch signaling pathway: lineages: revision of the classical concept of the myeloid-lymphoid dichotomy. unfolding the activation mechanism. Cell 137: 216–233. Trends Immunol. 30: 193–200. 10. Mumm, J. S., E. H. Schroeter, M. T. Saxena, A. Griesemer, X. Tian, D. J. Pan, 35. Inlay, M. A., D. Bhattacharya, D. Sahoo, T. Serwold, J. Seita, H. Karsunky, W. J. Ray, and R. Kopan. 2000. A ligand-induced extracellular cleavage reg- S. K. Plevritis, D. L. Dill, and I. L. Weissman. 2009. Ly6d marks the earliest ulates g-secretase–like proteolytic activation of Notch1. Mol. Cell 5: 197–206. stage of B-cell specification and identifies the branchpoint between B-cell and 11. Brou, C., F. Logeat, N. Gupta, C. Bessia, O. LeBail, J. R. Doedens, A. Cumano, T-cell development. Genes Dev. 23: 2376–2381. P. Roux, R. A. Black, and A. Israe¨l. 2000. A novel proteolytic cleavage involved 36. Pronk, C. J., D. J. Rossi, R. Ma˚nsson, J. L. Attema, G. L. Norddahl, C. K. Chan, in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol. Cell M. Sigvardsson, I. L. Weissman, and D. Bryder. 2007. Elucidation of the phe- by guest on October 4, 2021 5: 207–216. notypic, functional, and molecular topography of a myeloerythroid progenitor 12. Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, cell hierarchy. Cell Stem Cell 1: 428–442. T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expression in 37. Wilson, A., and A. Trumpp. 2006. Bone-marrow haematopoietic-stem-cell early lymphopoiesis influences B versus T lineage determination. Immunity 11: niches. Nat. Rev. Immunol. 6: 93–106. 299–308. 38. Luc, S., N. Buza-Vidas, and S. E. Jacobsen. 2007. Biological and molecular 13. Tanigaki, K., and T. Honjo. 2007. Regulation of lymphocyte development by evidence for existence of lymphoid-primed multipotent progenitors. Ann. N. Y. Notch signaling. Nat. Immunol. 8: 451–456. Acad. Sci. 1106: 89–94. 14. Real, P. J., V. Tosello, T. Palomero, M. Castillo, E. Hernando, E. de Stanchina, 39. Jorissen, E., J. Prox, C. Bernreuther, S. Weber, R. Schwanbeck, L. Serneels, M. L. Sulis, K. Barnes, C. Sawai, I. Homminga, et al. 2009. g-Secretase A. Snellinx, K. Craessaerts, A. Thathiah, I. Tesseur, et al. 2010. The disintegrin/ inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leu- ADAM10 is essential for the establishment of the brain cortex. kemia. Nat. Med. 15: 50–58. J. Neurosci. 30: 4833–4844. 15. Sanda, T., X. Li, A. Gutierrez, Y. Ahn, D. S. Neuberg, J. O’Neil, P. R. Strack, 40. Balciunaite, G., R. Ceredig, S. Massa, and A. G. Rolink. 2005. A B220+CD117+ C. G. Winter, S. S. Winter, R. S. Larson, et al. 2010. Interconnecting molecular CD192 hematopoietic progenitor with potent lymphoid and myeloid de- pathways in the pathogenesis and drug sensitivity of T-cell acute lymphoblastic velopmental potential. Eur. J. Immunol. 35: 2019–2030. leukemia. Blood 115: 1735–1745. 41. Rumfelt, L. L., Y. Zhou, B. M. Rowley, S. A. Shinton, and R. R. Hardy. 2006. 16. Bozkulak, E. C., and G. Weinmaster. 2009. Selective use of ADAM10 and Lineage specification and plasticity in CD192 early B cell precursors. J. Exp. ADAM17 in activation of Notch1 signaling. Mol. Cell. Biol. 29: 5679–5695. Med. 203: 675–687. 17. Kawamata, S., C. Du, K. Li, and C. Lavau. 2002. Notch1 perturbation of he- 42. Montecino-Rodriguez, E., H. Leathers, and K. Dorshkind. 2001. Bipotential mopoiesis involves non–cell-autonomous modifications. J. Immunol. 168: 1738– B-macrophage progenitors are present in adult bone marrow. Nat. Immunol. 2: 1745. 83–88. 18. Kawamata, S., C. Du, K. Li, and C. Lavau. 2002. Overexpression of the Notch 43. Chi, A. W., J. J. Bell, D. A. Zlotoff, and A. Bhandoola. 2009. Untangling the target genes Hes in vivo induces lymphoid and myeloid alterations. Oncogene T branch of the hematopoiesis tree. Curr. Opin. Immunol. 21: 121–126. 21: 3855–3863. 44. Lan, K., M. Murakami, B. Bajaj, R. Kaul, Z. He, R. Gan, M. Feldman, and 19. Schroeder, T., and U. Just. 2000. Notch signalling via RBP-J promotes myeloid E. S. Robertson. 2009. Inhibition of KSHV-infected primary effusion lympho- differentiation. EMBO J. 19: 2558–2568. mas in NOD/SCID mice by g-secretase inhibitor. Cancer Biol. Ther. 8: 2136– 20. Schroeder, T., H. Kohlhof, N. Rieber, and U. Just. 2003. Notch signaling induces 2143. multilineage myeloid differentiation and up-regulates PU.1 expression. J. 45. Ostrand-Rosenberg, S., and P. Sinha. 2009. Myeloid-derived suppressor cells: Immunol. 170: 5538–5548. linking inflammation and cancer. J. Immunol. 182: 4499–4506.