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Regulation of Differentiation in during Emergency Myelopoiesis

This information is current as Hao Liu, Jie Zhou, Pingyan Cheng, Indu Ramachandran, of September 29, 2021. Yulia Nefedova and Dmitry I. Gabrilovich J Immunol published online 5 July 2013 http://www.jimmunol.org/content/early/2013/07/04/jimmun ol.1300714 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 5, 2013, doi:10.4049/jimmunol.1300714 The Journal of Immunology

Regulation of Dendritic Cell Differentiation in Bone Marrow during Emergency Myelopoiesis

Hao Liu,1 Jie Zhou,1,2 Pingyan Cheng, Indu Ramachandran,3 Yulia Nefedova,3 and Dmitry I. Gabrilovich3

Although accumulation of dendritic cell (DC) precursors occurs in bone marrow, the terminal differentiation of these cells takes place outside bone marrow. The signaling, regulating this process, remains poorly understood. We demonstrated that this process could be differentially regulated by Notch ligands: Jagged-1 (Jag1) and Delta-like ligand 1 (Dll1). In contrast to Dll1, Jag1, in vitro and during induced myelopoiesis in vivo, prevented DC differentiation by promoting the accumulation of their precursors. Although both ligands activated Notch in hematopoietic progenitor cells, they had an opposite effect on Wnt signaling. Dll1 activated Wnt pathways, whereas Jag1 inhibited it via downregulation of the expression of the Wnt receptors Frizzled (Fzd). Jag1 suppressed fzd

expression by retaining histone deacetylase 1 in the complex with the transcription factor CSL/CBF-1 on the fzd promoter. Our Downloaded from results suggest that DC differentiation, during induced myelopoiesis, can be regulated by the nature of the Notch ligand expressed on adjacent stroma cells. The Journal of Immunology, 2013, 191: 000–000.

endritic cells (DCs) are professional APCs critically played by different Notch ligands in defining the biological effects important for the induction of immune responses (1, 2). of Notch. At present, two major Notch ligand families, Delta (Dll1, D The regulation of DC differentiation is a complex, spa- Dll3, and Dll4) and Jagged (Jag1 and Jag2), have been described http://www.jimmunol.org/ tially controlled process. Although it is initiated in bone marrow in mammals (6). Although these ligands activate Notch signaling, (BM), most DCs became terminally differentiated cells in the in recent years, evidence has emerged of the contrasting effects of peripheral lymphoid organs or tissues. There are only a few dif- Delta and Jagged on different cells (7–13). However, the mecha- ferentiated, functionally competent DCs in BM (3). This plays an nisms these effects remain largely unclear. important biological role by limiting the possible inflammatory re- It is known that the Notch pathway is involved in the differ- action in BM. The molecular mechanisms regulating this process entiation and function of DCs. However, its role remains contro- remained largely unclear. versial. Most of the studies with “gain-of-function” experiments DC differentiation in BM is controlled by a complex network of have demonstrated an upregulation of DC differentiation, whereas soluble factors and cell surface–bound molecules. Among the many studies with “loss of function,” using knockout (KO) mice, by guest on September 29, 2021 latter, the Notch family of transcriptional regulators plays a major showed either a lack of the effect or an inhibition of DC differ- role. Notch signaling is initiated by the binding of the Notch re- entiation (reviewed in Ref. 14). Dll1 was shown to potently pro- ceptor to specific ligands that result in the proteolytic cleavage of mote DC differentiation (11, 15, 16), whereas Jag1 prevented the intracellular domain (ICN), followed by the ICN translocation the terminal DC differentiationinvitrobyinducinganaccu- to the nucleus, where it interacts with the transcriptional repressor mulation of immature myeloid cells (IMCs) and DC precursors CSL/CBF-1 (4, 5). One of the most puzzling questions is the role (11). The mechanism of such an opposite effect remained un- clear. The goal of this study was to understand the mechanisms that prevent DC differentiation in BM and possible role of Jag1 Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, in this process. Tampa, FL 33612 1H.L. and J.Z. contributed equally to this work. Materials and Methods 2Current address: Institute of Human Virology, Zhongshan School of Medicine, Sun Mice Yat-Sen University, Guangzhou, China. 3 All mouse experiments were approved by the University of South Florida Current address: Wistar Institute, Philadelphia, PA. Institutional Animal Care and Use Committee. Female C57BL/6 mice (aged Received for publication March 15, 2013. Accepted for publication June 6, 2013. 6–8 wk) were obtained from the National Cancer Institute. Mx1-Cre mice b flox+ This work was supported by National Institutes of Health Grants CA141438 (to D.I.G.). (mouse strain number 003556), -catenin mice (mouse strain number + and CA130923 (to Y.N.) 004152 on C57BL/6 background), and CD45.1 congenic mice (B6.SJL- PtrcaPep3b/BoyJ) were purchased from The Jackson Laboratory. Jagged- Address correspondence and reprint requests to Dr. Dmitry I. Gabrilovich at the 1flox+ mice were provided by Dr. J. Lewis (London Research Institute, current address: Wistar Institute, Room 118C, 3601 Spruce Street, Philadelphia, PA b 19104-4265. E-mail address: [email protected] Cancer Research UK, London, U.K.) (10). The conditional -cat or Jag1 KO mice were generated by crossing homozygous floxed b-cat or Jag1 mice with The online version of this article contains supplemental material. Mx1-Cre mice, and the offspring carrying a floxed b-cat or Jag1 allele and Abbreviations used in this article: BM, bone marrow; BMS, stroma from bone mar- Mx1-Cre were backcrossed to the homozygous floxed b-cat or Jag1 mice. row; ChIP, chromatin immunoprecipitation; DC, dendritic cell; Dll, Delta-like ligand; Homozygous floxed b-cat or Jag1 mice carrying Mx1-Cre transgene were Fzd, Frizzled; HDAC, histone deacetylase; HGT, hydrodynamic gene transfer; HPC, selected and were referred to as b-catfl/flCre+/2 or Jag1fl/flCre+/2 mice. hematopoietic progenitor cell; ICN, intracellular domain; IMC, immature myeloid Jag1fl/flCre/2 or b-catfl/flCre2/2 mice were used as controls. To induce the cell; Jag, Jagged; KO, knockout; LEF, lymphoid enhancer factor; LN, lymph node; b m MF, ; PB, peripheral ; poly(I:C), polyinosinic:polycytidylic acid; -cat or Jag1 deletion, 250 g polyinosinic:polycytidylic acid [poly(I:C)] siRNA, small interfering RNA; SPL, spleen; SPS, spleen from stroma; TCF, T cell were injected i.p. every other day for three or five times. Mice were used factor; TSA, trichostatin A; WT, wild-type. within 3 wk after their last poly(I:C) injection. For adoptive transfer experiments, Jag1fl/fl mice were further backcrossed for eight generations Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 with C57BL/6 mice.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300714 2 DCs, Notch, AND EMERGENCY MYELOPOIESIS

Reagents cation of endogenous cyclophilin or hprt was used as an internal control. Primers sequence was reported previously (18). The analysis of gene ex- Abs against mouse I-A/E, CD11b, CD86 (B7-2), CD45, CD45.1, CD11c, pression by immunoblotting was performed as described previously (11). Gr1, B220, and isotype control Abs were obtained from BD Pharmingen For immunoprecipitation, 500 mg precleared cell lysate was incubated with (San Diego, CA). Siglec H, DEC-205, and DCIR-2 (33D1) Abs were from specific Ab for overnight and the Ag–Ab complex was precipitated by eBioscience (San Diego, CA). F4/80 Ab was purchased from Serotec using protein A/G–agarose beads (Santa Cruz Biotechnology, Dallas, TX) (Raleigh, NC). Recombinant murine GM-CSF and IL-4 were obtained and then subjected to electrophoresis in 8% NaDodSO4-polyacrylamide from Research Diagnostics (Flanders, NJ), and IL-3, FLT-3, M-CSF, gels. The specific bands were visualized by an ECL detection kit (Amer- a recombinant human cytokines GM-CSF, TNF- , and mouse Wnt3a were sham Life Sciences, Arlington Heights, IL). obtained from R&D Systems (Minneapolis, MN). Jagged-IgG was from Alexis Biochemical. SB216763, human IgG, and anti-human IgG were Chromatin immunoprecipitation assay from Sigma-Aldrich (St. Louis, MO). b-Catenin, Jagged-1, and histone deacetylases (HDACs) 1, 2, 5, and 7 Abs were from Cell Signaling Tech- Chromatin immunoprecipitation (ChIP) assay was performed with the b reagents and protocol from Millipore (Billerica, MA). In brief, we fixed 2 3 nology (Beverly, MA). Abs against Frizzled (Fzd) 6, 7, and 10, -actin, 6 Delta-1, CBF1, and siRNA for Jagged-1, HDAC1, Delta, and CBF-1 were 10 32D cells by adding formaldehyde into culture media to a final con- obtained from Santa Cruz Biotechnology (Santa Cruz, CA). CBF1 reporter centration of 1%. The cells were then lysed and sonicated to shear the and short hairpin RNA for mouse b-catenin were from SABiosciences chromatin DNA. Cell lysate was precleared with protein G–agarose before (Frederick, MD), dual-luciferase assay kit was from Invitrogen (Carlsbad, immunoprecipitation overnight at 4˚C with 2 mg specific Ab or normal CA), and primers for Real-time RT-PCR were synthesized by Integrated mouse IgG. The precipitated Ab–chromatin complex was collected by DNA Technologies (Coralville, IA). The monomeric form of the Notch li- incubation with protein G–agarose for 1 h at 4˚C and subsequently washed gand Delta-1 was made by fusing the extracellular domain of Delta-1 to a and eluted in elution buffer. DNA, in the precipitated samples, was re- series of myc epitopes Delta-1ext-myc28 and was a gift from Dr. I. Bernstein covered by reverse cross-linking at 65˚C for 4 h. Ten percent of lysate, b before Ab precipitation, was used as the input. Real-time PCR was carried (Fred Hutchinson Cancer Research Center, Seattle, Washington). -cat-GFP Downloaded from plasmid was provided by Dr. I. Mellman (Genentech, South San Francisco, CA). out with Fzd10-specific primers. Amplification of cyclophilin from the in- TheHDACactivityassaykitwasfromCaymanChemical(AnnArbor,MI). put was used as the loading control. Cell lines, cell culture, and flow cytometric analysis Primers used in ChIP of Fzd10 BM and spleen (SPL) stroma cells were generated as described previously The following primers were used (boldface suggests the CBF-1 binding (11). The NIH3T3 cell lines transfected with Delta-1, Jagged-1, or control site): CHIP primer set 1, 59-ACCTATGCAGTTGGGGGAGT-39 and 59- plasmids were described previously (11). Mouse hematopoietic progenitor ACCGCAGTCCTTAGATGTCC-39; CHIP primer 2, 59-GGTTGAAGCT- http://www.jimmunol.org/ cells (HPCs) from mouse BM were enriched with a lineage cell depletion CTGTGTCCTTATC-39 and 59-GGCTGGTGGTTTCTCCAA-39;CHIP kit from Miltenyi Biotec (Auburn, CA). Enriched HPCs, from naive BM, primer 3, 59-CCCTAAACTCCGGCTTCC-39 and 59-GGGATTTGCGTTT- were placed onto 3T3 cells or primary stromal cells the following day and TATGACC-39; CHIP primer 4, 59-GCAGGTGATGATAGGCTCGT-39 and cultured in RPMI 1640 medium supplemented with 10% FBS and 20 ng/ml 59-AACACAGGAAGAAGCGAAGG-39; Gapdh, 59-TACTAGCGGTTT- GM-CSF for various times. HPC progenies were transferred to new wells TACGGGCG-39 and 59-TCGAACAGGAGGAGCAGAGAGCGA-39;and and coated with corresponding cell lines every 2–3 d. CD45+ cells were putative CBF-1 binding sites: site 1, 59-GCTCTATTCCCAGGCCAG-39; gated so that hematopoietic cells could be distinguished from contaminated site 2, 59-GGTAGTTTCCCAGACACT-39; site 3, 59-GGCTGGTTCCC- stroma. For the analysis of gene and protein expression, hematopoietic cells ACGCAGA-39; site 4, 59-TCCGGCTTCCCAAGTCCC-39; and site 5, 59- were isolated with biotin-conjugated CD45 Ab and magnetic beads (Miltenyi TGGAGATTCCCATGTGCA-39. Biotec) (17). 32D cells (murine hematopoietic cell line) were cultured in

RPMI 1640 medium with 10% FBS and 20% conditioned medium con- Allogeneic MLR by guest on September 29, 2021 taining IL-3 (WEHI-3B cell line). The incubation of 32D cells with different The C57BL/6 BM HPCs were cultured on immobilized Jag-1, on plate, in 3T3 cell lines was similar to HPCs. Cells was analyzed by flow cytometry on the presence of SB216763 plus GM-CSF or GM-CSF only for 5 d. The HPC a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) or progenies were cocultured with T cells (105/well) from allogeneic BALB/c LSR II (BD Biosciences), and data were analyzed with the CellQuest pro- mice for 4 d, in triplicate, in U-bottom 96-well plates at different ratios. gram (BD Biosciences) or FlowJo program (Tree Star, Ashland, OR). [3H]Thymidine (1 mCi) was added to each well 18 h prior to cell harvesting. The immobilization of Notch ligands on plastic was performed, as described 3 m T cell proliferation was measured by [ H]thymidine incorporation in a liq- previously (18). In brief, 20 g/ml Ab against human IgG was placed into uid scintillation counter (Packard Instrument, Meriden, CT). each well of a 24-well plate and incubated for 30 min at 37˚C. Wells were blocked with complete culture medium for 30 min and then incubated for 3 h Statistical methods at 37˚C with 7.5 mg/ml Dll1, Jag1, or IgG in complete culture medium. The data were analyzed with a two-tailed Student t test using GraphPad Plasmid construction and transfection Software. A p value , 0.05 was considered to be statistically significant. The 59-regulatory sequence (21233 to +235 bp) of the mouse fzd10 gene (NM_175284) was cloned into pGL3 enhancer vector (Promega, Madison, Results 9 WI), using the following pair of oligonucleotides: forward primer, 5 - Jag1 negatively regulates DC differentiation in BM CGACGCGTGATTAGCCCAAAACCAACCA-39; and reverse primer, 59- CCGCTCGAGGTCCTTGCACATGGGAATCT-39. To evaluate the role of Jag1 in DC differentiation, we used mice Transient transfections were performed on 32D cells using the Gene- with conditional KO of this protein. Jag1fl/fl mice (10) were crossed PORTER2 transfection agent (Genlantis, San Diego, CA) and following the with Mx1-Cre mice, and the Jag1 deletion was induced by re- manufacturer’s guidelines. For the reporter activity assay, Renilla plasmid fl/fl 2/2 was cotransfected with luciferase plasmids and relative light units of lu- peated injections of poly(I:C). As a control, we used Jag1 Cre minescence were measured 48 h after transfection with a dual-luciferase littermates treated with poly(I:C). To simplify abbreviation, in reporter assay system (Promega). HPCs were transfected with mouse mac- this paper, Jag1fl/flCre2/+ with inducible deletion of Jag1 are F rophage (M ) buffer and Y01 program at Amaxa nucleofector (Amaxa, referred as Jag1 KO and control mice as wild-type (WT). We Gaithersburg, MD). generated stroma from BM (BMS) and SPL (SPS) with inducible Hydrodynamic gene transfer deletion of Jag1 (Supplemental Fig. 1) and evaluated their effect Hydrodynamic gene transfer (HGT) was performed as described previously on DC differentiation from enriched HPCs in the presence of (19). Briefly, GM-CSF plasmid (20) was purified with an Endofree plasmid GM-CSF. SPS supported DC differentiation substantially better m mega kit (Qiagen). Plasmid (80 g), resuspended in 1.6 ml saline, was than BMS (control in Fig. 1A versus Fig. 1B). The absence of injected i.v. within 5 s. Jag1 in SPS did not affect DC differentiation (Fig. 1A). In con- Quantitative real-time PCR, immunoprecipitation, and trast, lack of Jag1 on BMS significantly (p , 0.05) promoted DC immunoblotting differentiation (Fig. 1B). Absence of Jag1 on BMS had no effect PCR was performed by using SYBR Green RT-PCR reagent kit (Applied on differentiation of DCs generated in the presence of FLT3L Biosystems, Foster City, CA) and target gene–specific primers. Amplifi- (Fig. 1C). The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Jag1 negatively regulates DC differentiation on BMS and CFA-treated mice. (A and B) CD45.1+ HPCs were cultured in the presence of 20 ng/ml GM-CSF for 5 d on the monolayer of CD45.2+ SPS (A) or BMS (B) generated from Jag1 KO or WT mice. Cells were labeled with mixture of indicated Abs and examined by flow cytometry on day 5. Only CD45.1+ cells were evaluated. Data are presented as mean 6 SEM from three independent experiments. *p , 0.05; **p , 0.01. (C) CD45.1+ HPCs were cultured in the presence of 100 ng/ml FLT3L for 10 d on the BM stromal from Jag1 KO or control mice. CD45.1+cells were evaluated by FACS. Data are presented as mean 6 SEM from three independent experiments. (D and E) The kinetic of Gr- 1+CD11b+ IMCs (D) and CD11c+ (E) in PB of WT and Jag1 KO mice at different time points after CFA injection. Mean 6 SEM from five mice per group are shown. (F) The total cell numbers of CD11c+I-A/E+ DCs in 1 ml PB of WT and Jag1 KO mice on day 8 after CFA injection mean 6 SEM from five mice per group are shown. *p , 0.05. (G) Jag1 KO and control mice were injected with CFA. The proportions and absolute number of indicated populations of cells in BM of mice on day 8 after CFA injection are shown. Individual data from mice (seven mice per group) are shown. (H) Allogeneic MLR. BM cells from Jag1 KO or WT mice were stimulated with 100 ng/ml LPS for 16 h and cultured at different ratio with allogeneic (BALB/c) T cells for 5 d. Cell proliferation was measured in triplicate by [3H]thymidine uptake in the last 16 h. Data are presented as mean 6 SEM (n = 3 mice/group). (I) The pro- portions of indicated cell populations in LNs of mice on day 8 after CFA injection. Numbers in each figure indicated p value. Individual data from mice (seven mice per group) are shown.

Next, we evaluated the DC differentiation in Jag1-deficient mice in vitro experiments showed that Jag12/2 BMS had an effect on in vivo. In steady state, no differences in the total number or the DC differentiation only in the presence of GM-CSF, which is proportions of different DC populations in BM, SPL, or lymph associated with emergency hemtopoiesis, we evaluated the role of nodes (LNs) were found (Supplemental Fig. 2A). Because our Jag1 in a model of inflammation-induced myelopoiesis caused by 4 DCs, Notch, AND EMERGENCY MYELOPOIESIS injection of CFA. In both, WT and Jag1 KO mice, CFA induced The total number of cells in WT and Jag1 KO mice was the same rapid and equal increase of IMCs in peripheral blood (PB) (Fig. (Supplemental Fig. 2B), and as a result, the total number of DCs 1D). In contrast, Jag1 KO mice had significantly higher numbers showed the same changes as the proportion of the cells. of CD11c+ cells than WT mice (Fig. 1E). CD11c+ cells (21) are Similar effect of Jag1 was observed in the different model of composed of DCs (CD11c+MHC II+) (3), DC progenitors (mainly induced myelopoiesis caused by HGT of GM-CSF plasmid in vivo. CD11c+MHCII2) (22), and few activated leukocytes (reviewed in An equal increase in IMCs in the PB of both WT and Jag1KO mice Ref. 23). Analysis of the population of CD11c+MHC II+ DCs in was seen (Fig. 2A). In Jag1 KO mice, GM-CSF effect on DC PB confirmed significant increase in the presence of these cells presence in PB (Fig. 2B), BM (Fig. 2C), and LNs (Fig. 2D) was in Jag1 KO mice (Fig. 1F). These results might reflect differences similar to the effect of CFA. There were no difference in the ab- in the mobilization of DCs. To test this possibility, we evaluated solute cell number between WT and Jag1 KO BM. DCs in BM 8 d after CFA injection (at the peak of DC presence in To clarify the role of Jag1 in DC differentiation, we used an PB). The Jag1 KO mice had a significantly higher proportion and adoptive transfer of BM cells from congenic CD45.1+ WT mice to absolute number of plasmacytoid DCs and CD11c+MHC II+ DCs, lethally irradiated Jag1 KO or WT CD45.2+ mice. Jag1 deletion but not MFs, than their control littermates (Fig. 1G). An increased was induced by the poly(I:C) injections. Three weeks later, mice presence of DCs in BM of Jag1 KO mice was confirmed in al- were irradiated, and congenic BM cells were transferred. Six logeneic MLR, a hallmark of DC activity (Fig. 1H). Jag1 KO mice weeks after the transfer, recipient mice were injected with CFA also had a significantly higher presence of DCs in LNs (Fig. 1I). and evaluated 8 d later. Jag1 KO and WT mice were equally Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Jag1 negatively regulates DC differentiation in BM after GM-CSF stimulation. (A and B) The kinetic of IMCs (A) and CD11c+ I-A/E+ DCs (B) in PB of Jag-1 KO and WT mice after treatment with GM-CSF HGT. Each group included three to four mice. *p , 0.05. (C) The proportion and absolute number of indicated populations of cells in BM of mice obtained on day 4 after HGT. Mean 6 SEM from five mice per group are shown. *p , 0.05. (D) Absolute number of indicated cell populations in LNs of the same mice as in (C). (E) Lethally irradiated CD45.2+ Jag1 KO or WT mice were reconstituted with 106 CD45.1+ donors’ HPCs from WT mice. After 6 wk, mice were treated with CFA as described in the legend to Fig.1G, and the presence in BM of indicated donor’s cells was evaluated 8 d after CFA injection. Data are presented as mean 6 SEM (n = 5 mice/group). *p , 0.05. The Journal of Immunology 5 reconstituted with donor’s BM (Supplemental Fig. 3). However, 15, 16, 27), Dll1 promoted DC differentiation from HPCs (Fig. Jag1 KO mice had a significantly higher presence of donor’s DCs 5A). Addition of Jag1 to Dll1 completely abrogated this effect in BM (Fig. 2E, Supplemental Fig. 3) than WT mice. Thus, taken (Fig. 5A). We evaluated the effect of activation of Wnt pathway together these results indicated that Jag1 in BM inhibited DC downstream of Fzd receptors on the DC differentiation. Wnt path- differentiation. way was activated by overexpression of a constitutively active b-cat mutant (28) (Fig. 5B) or by inhibition of the GSK-3b kinase, Dll1 and Jag1 have opposite effect on wingless (Wnt) signaling which is critically important for activation of Wnt signaling, using in HPCs specific inhibitor SB216763 (29) (Fig. 5C). In both cases, acti- What could be the mechanism of Jag1 negatively regulating the DC vation of Wnt pathway abrogated the inhibitory effect of Jag1 on differentiation? Activation of the canonical Wnt pathway in HPC DC differentiation (Fig. 5B, 5C) and restored the proportion of positively regulates DC differentiation; and the Dll1 effect on DC DCs to the control level. It also restored the ability of cells dif- differentiation could be mediated via activation of Wnt signaling ferentiated in the presence of Jag1 to stimulate allogeneic T cells, (17). Canonical Wnt pathway is activated by Wnt proteins binding the function attributed to DCs (Fig. 5D). to Fzd receptors and coreceptors, which leads to stabilization and Next, we investigated the consequences of downregulation of translocation of b-catenin to the nuclei to form coactivators with Wnt signaling for DC differentiation, using mice with a conditional T cell factor (TCF)/lymphoid enhancer factor (LEF) to regulate KO of the b-cat gene. b-catfl/fl mice were crossed with Mx1-Cre the target genes (24–26). We asked whether Wnt signaling is in- mice, and b-cat deletion was achieved by repeated injections of volved in Jag1-mediated effects on DC differentiation. Enriched poly(I:C) (Fig. 5E). These mice were further referred as b-cat KO.

BM HPCs were incubated for 18 h on a monolayer of NIH3T3 BM cells from b-cat KO mice and Cre- WT littermates were Downloaded from fibroblasts overexpressing Dll1, Jag1, or the corresponding control adoptively transferred into lethally irradiated congeneic CD45.1 vectors (11). Jag1 cells did not significantly increase expression of mice. Four weeks after the transfer, the mice were treated with any Fzd receptors but inhibited the expression of number of them GM-CSF HGT or with a control vector, as described in Fig. 2C, (Fig. 3A). This was in contrast to the effect of Dll1, which dra- and analyzed on day 4. No differences in DC differentiation be- matically upregulated members of Fzd family (17). The down- tween recipients of WT and b-cat KO BM cells were seen in mice

regulation of several Fzd receptors was confirmed by Western treated with the control vector. GM-CSF HGT caused significant http://www.jimmunol.org/ blotting (Fig. 3B). The inhibition of Wnt signaling was deter- increase in the presence of WT donors’ DCs in BM, which was mined by downregulation of b-catenin (Fig. 3C), Wnt target genes consistent with the recent paper (30). In contrast, no increase was (Fig. 3D), and TCF/LEF reporter activity (Fig. 3E). To avoid observed in recipients of b-cat KO BM (Fig. 5F). MF differen- a possible confounding effect of fibroblasts, we used an experi- tiation was not impaired in the b-cat KO BM (Fig. 5F). mental system where Notch signaling was activated by Notch To evaluate DC differentiation from their precursors, BM Gr-1+ ligands directly immobilized on plastic in the presence of re- CD11b+ IMCs were isolated from WT and b-cat KO mice combinant Wnt3a. Dll1 and Jag1 activated the Notch pathway as (CD45.2+) and transferred to sublethally irradiated CD45.1+ was measured by the activity of CBF-1 reporter and expression of recipients. Donors’ cells were evaluated 4 d later. The proportion hes5 (Fig. 3F, 3G), suggesting that both ligands are able to activate and absolute number of splenic cDCs (MHCII+CD11chi) but not by guest on September 29, 2021 the conventional Notch signaling. Jag1 inhibited the expression of MFs generated from b-cat KO IMCs was significantly lower than fzd10 and Wnt target genes (Fig. 3G), decreased the amount of that from WT IMCs (Fig. 5G). The differentiation of CD8a- cDC Fzd6 and Fzd10 protein (Fig. 3H), and downregulated the activity subset was also significantly impaired, which was consistent with of TCF/LEF reporter (Fig. 3I). In contrast, Dll1 induced upregu- previous observations that the development of splenic CD8a2 lation of Wnt signaling (Fig. 3G, 3I). cDCs was dramatically blocked in CBF-1 KO mice (31). The We asked whether Jag1 can neutralize activating effect of Dll1 culture of enriched HPCs from b-cat KO mice with GM-CSF and on Wnt signaling. Dll1-IgG and Jag1-IgG were mixed at a 1:1 ratio IL-4 produced smaller proportion of CD11c+CD11b+F4/802 DCs and immobilized on plastic. As a control, Dll1-IgG were mixed at than that of WT HPC (Fig. 5H). Treatment of mice with selective a 1:1 ratio with control IgG. Jag1 did not inhibit Dll1-inducible Wnt inhibitor ICG-001 (32) reduced the proportion of DCs in BM upregulation of hes5 expression, indicating that mix of these of Jag1 KO mice treated with CFA but not in control mice two ligands did not affect Notch signaling (Fig. 4A). In contrast, (Supplemental Fig. 4). Thus, these data support the hypothesis that the presence of Jag1 dramatically reduced the expression of fzd6 Jag1 regulates DC differentiation in emergency hematopoiesis via and fzd10, as well as wisp1 and wisp2 induced by Dll1 (Fig. 4A). inhibition of Wnt signaling. Jag1 also decreased the activity of the TCF/LEF reporter induced by Dll1 (Fig. 4B). Thus, Jag1 was able to neutralize upregulation Molecular mechanism of opposing effects of Dll1 and Jag1 on of Wnt pathway caused by Dll1. Wnt signaling Incubation of HPC on BMS generated from Jag1 KO mice HDAC is a major component of the constitutive CSL/CBF-1 re- resulted in increased expression of Wnt–targeted genes (Fig. 4C). pressor complex. We explored the possible role of HDAC in the HPCs isolated from BM of CFA-treated Jag1 KO mice (described regulation of fzd expression by Jag1. HDAC inhibitor trichostatin in Fig. 1) had a significantly higher expression of Wnt target genes A (TSA) completely abrogated the Jag1-inducible inhibition of and fzd10 as well as b-cat and Fzd10 proteins than the HPCs from fzd6 and fzd10 expression (Fig. 6A) and restored the expression of control mice (Fig. 4D). These results were confirmed in experi- the Wnt target genes (Fig. 6B). We made a luciferase reporter ments in vitro using BMS with Jag1 downregulated with specific construct by cloning the fzd10 promoter region into a pGL3 en- siRNA (Fig. 4E). Thus, Jag1 in contrast to Dll1 substantially re- hancer vector. 32D myeloid progenitor cells were transfected with duced Wnt signaling in HPC via downregulation of Fzd receptors. this vector and placed on fibroblasts, overexpressing different Notch ligands. Dll1 significantly upregulated the reporter (Fig. Jag1 negatively regulate DC differentiation via downregulation 6C), whereas Jag1 inhibited it (Fig. 6D). The TSA slightly up- of Wnt signaling regulated the fzd10 reporter in the cells cultured on Dll1 but Next, we investigated the mechanism of Jag1 mediated regulation completely restored its activity in 32D cells cultured on Jag1 (Fig. of DC differentiation. Consistent with previous observations (11, 6C, 6D). 6 DCs, Notch, AND EMERGENCY MYELOPOIESIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Notch ligands differentially regulate Wnt signaling in HPCs. (A) Enriched HPCs were incubated on a monolayer of NIH3T3 fibroblasts overexpressing Jag1, or control MSCV-vectors in medium containing 20 ng/ml GM-CSF. CD45+ hematopoietic cells were isolated using magnetic beads after 18 h culture and expression of fzd gene family were evaluated using quantitative RT-PCR. Relative gene expression was calculated by normalizing to internal control (cyclophilin). Mean 6 SEM of three experiments performed in triplicates are shown. (B and C) The amounts of selected Fzd proteins (B) and b-catenin protein (C) evaluated by Western blot after 3 d culture. Typical examples of three performed experiments are shown. (D) mRNA level of Wnt target gene were by measured by quantitative RT-PCR after 18 h culture. Mean 6 SEM of three experiments performed in triplicates are shown. Please note differences in the scale. (E) HPCs were transfected with TCF/LEF reporter plasmid and control Renilla plasmid and then cultured on fibroblast cells for 48 h. Reporter activity was measured using dual-luciferase reporter assay. Three experiments were performed in duplicates. (F–I) The effect of immobilized Notch ligands on Notch signaling in HPCs. (F) Enriched HPCs were transfected with luciferase CBF-1 reporter plasmid and control Renilla plasmid. Cells were cultured for 24 h on plates coated with 7.5 mg/ml control IgG, Dll1, or Jag1. Luciferase activity was measured in triplicates in dual-luciferase assay. Results of two experiments are shown. (G) Enriched HPCs were cultured on a 24-well plate coated with 7.5 mg/ml D-IgG, Jag-IgG, or control IgG protein in the presence of 100 ng/ml recombinant Wnt3A. Expression of fzd and Wnt-targeted genes evaluated using quantitative RT-PCR after 24 h culture. Fold changes in relative gene expression over the control IgG levels was calculated. The negative values indicate downregulation of genes expression. Mean 6 SEM of three experiments performed in triplicates are shown. (H) The amount of selected Fzd proteins was evaluated in Western blotting using indicated Abs. (I) HPCs were transfected with TCF/LEF reporter plasmid and control Renilla plasmid before cultured on ligands coated plate. Reporter activity was measured 48 h later. Mean 6 SEM of three experiments performed in duplicates are shown. *p , 0.05.

To clarify these findings, ChIP assays were performed in 32D between HDAC1 and CBF-1. HPCs were cultured on immobilized cells using Ab specific for acetylated histone H3 and primers Dll1 or Jag1 in the presence of GM-CSF for 4 h. Cell lysates were specific for fzd10 promoter. Dll1 upregulated the association of precipitated with CBF-1 Ab and then probed with HDAC1 Ab. The acetylated H3 with the promoter, but it was significantly inhibited amount of HDAC1 coprecipitated with the CBF-1 in HPC incu- by Jag1 (Fig. 6E). The opposite effect was observed when chro- bated on Dll1 was substantially lower than in HPC cultured on Jag1 matin was precipitated with HDAC1 Ab (Fig. 6F). Thus, in con- (Fig. 6G). To test whether HDAC1 association with the fzd10 trast to Dll1, Jag1 did not cause the displacement of HDAC1 from promoter depends on the presence of CBF-1, HPCs were trans- the fzd10 promoter, which could result on silencing of the gene fected with CBF-1 siRNA (Fig. 6H, inset). The downregulation of expression. CBF-1 completely abrogated the Jag1-inducible HDAC1 associ- Because both Jag1 and Dll1 induced CBF-1 activation, we asked ation with the fzd10 promoter (Fig. 6H), indicating that HDAC1 whether these ligands differently regulated the physical interaction interacts with the fzd10 promoter in complex with CSL. HDAC1 The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Jag1 neutralized the effect of Dll1 on Wnt signaling. (A) HPCs were cultured for 24 h on 24-well plates coated with Dll1 or 1:1 mixture of Dll1 with control IgG or Jag1 in the presence of 100 ng/ml Wnt3a. Expression of fzd and Wnt-targeted genes was measured by quantitative RT-PCR. Mean 6 p , B SEM of three experiments performed in triplicates are shown. * 0.05, statistically significant differences between IgG+Dll1 and Jag1+Dll1 groups. ( ) by guest on September 29, 2021 Enriched HPCs were transfected with luciferase TCF/LEF reporter plasmid and control renilla plasmid. Cells then were cultured for 24 h on plates coated with control IgG, Dll1, or 1:1 mix of Dll1 with IgG or Dll1 with Jag1 protein. Luciferase activity was measured in HPCs in triplicates in dual-luciferase assay. Mean 6 SEM of three experiments are shown. *p , 0.05. (C) CD45.1+ HPCs were cultured for 24 h on the monolayer of Jag1+/+ or Jag12/2 BMS cells in the presence of 20 ng/ml GM-CSF. Total mRNA was extracted from isolated CD45.1+ cells and expression of Wnt target genes was evaluated in quantitative PCR. Each experiment was performed in triplicates. Cumulative results from three experiments are shown as mean 6 SEM. *p , 0.05 between groups. (D) mRNA and whole-cell lysate (inset) were isolated from BM HPCs of Jag1KO or control mice treated as described in legend to Fig. 1G. Expression of fzd and Wnt-targeted genes was evaluated by quantitative RT-PCR in triplicates. Each group included three mice. Fold increase of gene expression in Jag1KO over control mice is shown. All differences were significant (p , 0.05). Indicated proteins were detected by Western blotting (inset). (E) BMS prepared from CD45.1+ congenic mice were transfected with control or Jag1 siRNA for 24 h. Enriched HPCs from CD45.2+ mice were cultured on BMS for 48 h. Ex- pression of fzd and Wnt-targeted genes in CD45.2+ HPCs was measured by quantitative RT-PCR. Inset, Protein level in BMS by Western blotting dem- onstrating specificity of Jagged-1 knockdown. was knocked down in 32D cells by using siRNA (Fig. 6I, inset). effect on the differentiation and enhanced generation of myeloid This did not affect the Dll1-inducible upregulation of the ex- progenitors during stress hematopoiesis (34). The fact that the pression of fzd10 and Wnt target genes. However, the Jag1- effect of Jag1 deletion on DCs was confined largely to BM could inducible downregulation of these genes was abrogated (Fig. 6I). be explained by the fact that Jag1 expression is predominant in BMS but not in SPS (11). Jag1 was also previously shown to be the Discussion main Notch ligand that is expressed in the HSC niche in BM (35). It This study suggests the novel mechanism of regulation of DC appears that Jag1 may play an important role in the inhibition of DC differentiation in BM, which depends on the expression of the differentiation from the precursors inside BM. When DC precursors Notch ligand, Jag1. Under steady-state conditions Jag1 was dis- leave BM, they undergo a terminal differentiation in tissues where pensable for DC differentiation. However, it was important in the the expression of Jag1 is substantially reduced. conditions of forced myelopoiesis. Among several models of The main focus of this study was on the mechanism of the emergency myelopoiesis, we used two: inflammation-related opposite effect of Jag1 and Dll1 on DC differentiation. Both are caused by the injection of CFA; and induced by the administra- ligands for the same receptors and both are able to activate Notch tion of GM-CSF. Although both treatments caused an equal in- signaling. One of the possible explanations is the different strength crease of IMCs in WT and Jag1 KO mice, the presence of DCs in of the Notch signaling, mediated by Jag1 and Dlll. It was shown blood and BM was significantly higher in Jag1 KO than in WT that Jag1 had the weakest ability to activate Notch1, whereas, Dll4 mice. These data were consistent with previous observations that was the strongest Notch1 activator, followed by Dll1 and Jag2 (12). Notch signaling had no effect on long-term, repopulating HSC self- The different effect of Jag1 and Dlll was also associated with the renewal when assessed during homeostasis (33) but had a strong Lunatic-Fringe–mediated glycosylation of Notch1. Glycosylation 8 DCs, Notch, AND EMERGENCY MYELOPOIESIS Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Jag1 negatively regulate DC differentiation via downregulation of Wnt signaling. (A) Enriched HPCs were cultured on 24-well plates coated with a 1:1 mixture of Dll1 and Jag1 or IgG proteins in the presence of Wnt3a (100 ng/ml) for 5 d in medium with 20 ng/ml GM-CSF. Mean 6 SEM of three experiments are shown. *p , 0.05, statistically significant differences between IgG+Dll1 and Jag1+Dll1 groups. (B) Enriched HPCs were transfected with b-catenin-GFP or GFP vector and then cultured on monolayer of fibroblasts for 5 d. The percentage of the indicated population of cells was calculated within gated CD45+GFP+ cells. Mean 6 SEM of three experiments are shown. *p , 0.05 between Jag1 and Jag1+b-cat groups. (C) Enriched HPCs were cultured on plates coated with IgG or Jag1-IgG for 4 d. SB216763 (10 nM) was added on day 0. The percentage of the indicated population of cells was evaluated on day 5. Cumulative results of two performed experiments. *p , 0.05, statistically significant differences between Jag1 and Jag1+SB216763 groups. (D) Allogeneic MLR. Cells from experiments described in (C) were cultured at different ratios with T cells isolated from allogeneic BALB/c mice for 4 d. Cell proliferation was measured in triplicate by [3H]thymidine uptake. Values are the mean 6 SEM. (E) b-Catenin expression in from b-catfl/flMx1-Cre+/2 KO or control b-catfl/fl Mx1-Cre2 (control) mice. (F) Lethally irradiated CD45.1+ recipients were reconstituted with 1.5 3 106 cells BM from CD45.2+ b-cat KO or WT mice. Four weeks after the adoptive transfer, mice were treated with GM-CSF HGT or control vector and evaluated 4 d later. Data show the number of indicated populations of donors cells presented as mean 6 SEM (n = 5 mice/group). *p , 0.05, statistically significant differences between WT and b-cat KO mice; (G)83 106 CD45.2+Gr-1+ cells were isolated from BM of b-cat KO or WT mice and transferred i.v. into sublethally irradiated CD45.1+ mice. The presence of indicated population of cells among donors cells were evaluated in spleens 4 d after the transfer. Mean 6 SEM from four mice per group are shown. *p , 0.05, statistically significant differences from control. (H) Differentiation of DCs in the presence of GM-CSF and IL-4 in serum free medium for 6 d from 1.5 3 105 enriched BM HPCs from b-cat KO or WT mice. Representative FACS profiles from four individual experiments and cumulative results of the total number of F4/802CD11c+CD11b+ DCs are shown. **p , 0.01 between groups. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Jag1 regulates the expression of fzd10 via HDAC. (A and B) HPCs were incubated on monolayer of NIH3T3 fibroblasts expressing either Jag- 1 or control vectors for 18 h, followed by treatment with 10 nM TSA for 12 h. Expression of fzd (A) or Wnt target (B) genes was measured in HPCs by quantitative RT-PCR. Mean 6 SEM of three experiments performed in triplicates are shown. *p , 0.05. (C and D) 32D cells were transfected with fzd10- Luc reporter plasmid, followed by incubation with fibroblasts expressing Dll1, Jag1, or their control vectors for 18 h and then treated with 10 nM TSA for another 12 h. Renilla plasmid was used as internal control. Luciferase activity was measure using dual-reporter assay system. Mean 6 SEM of three experiments are shown. *p , 0.05. (E and F) ChIP assay in 32D cells cultured on different fibroblasts with Abs against acetylated H3 histone (E)orHDAC1 (F). Association of Fzd10 promoter with acetylated histone 3 or HDAC1 was evaluated by quantitative PCR and presented as the fold increase over input DNA. Two experiments in triplicates were performed. (G) HPCs were cultured on plates with immobilized Dll1, Jag1, or control IgG for 4 h. Cell lysates were pulled down with CBF-1 Ab followed by immunoblotting with HDAC1 Ab. The level of CBF-1 and b-actin was measured in cell lysates. Intensity of HDAC-1 band normalized to CBF-1 is shown in the bottom panel. Three experiments with similar results were performed. (H) Enriched HPCs were transfected with control or CBF-1 siRNA and then were placed on MSCV or Jag1 fibroblasts for 36 h. ChIP assay was performed with HDAC1 Ab. Association of Fzd10 promoter with HDAC1 was evaluated by quantitative PCR and presented as the fold increase over input DNA. Two experiments in triplicate with the same results were performed. Different primers sets were used. Their location in promoter region of Fzd10 is shown in supplementary data. Inset, Protein level by western blot demonstrating downregulation of CBF-1 in HPCs. (I) 32D cells were transfected with control siRNA or HDAC1 siRNA. Twenty-four–hour posttransfection cells were incubated with different fibroblasts for another 18 h. Expression of fzd and Wnt-targeted genes was determined by quantitative RT-PCR. Mean 6 SEM of three experiments performed in triplicates are shown. Inset, Protein level demonstrating specificity of downregulation of HDAC1. *p , 0.05, statistically significant differences from control siRNA. of Notch1 potentiated Notch signaling through Dlll ligands and shown that Notch2 enhanced the rate of formation of HSCs while Jag2, in contrast to Jag1 (12, 36). Upon glycosylation of Notch, delaying myeloid differentiation in BM, during nonhomeostatic Dll4-Notch signaling was enhanced, whereas Jag1 had a weak conditions, including after chemotherapy or during marrow re- signaling capacity (7). Individual Notch receptors have different generation after stem cell transplantation. However, both Jag1 transcriptional activity (37). Jagged family members, in the and Dll1 activated Notch2 in the HSC-enriched population that presence of Fringe, can activate Notch2 (38‑40). It was recently inhibited myeloid differentiation and enhanced the generation of 10 DCs, Notch, AND EMERGENCY MYELOPOIESIS myeloid progenitors during stress hematopoiesis (34). In our 2. Domı´nguez, P. M., and C. Ardavı´n. 2010. Differentiation and function of mouse -derived dendritic cells in steady state and inflammation. Immunol. study, lack of Jag1, during emergency hematopoiesis, did not af- Rev. 234: 90–104. fect the total number of myeloid cells. No decrease in the presence 3. Sapoznikov, A., Y. Pewzner-Jung, V. Kalchenko, R. Krauthgamer, I. Shachar, of IMCs or MFs was seen. This suggested that deletion of Jag1 and S. Jung. 2008. Perivascular clusters of dendritic cells provide critical sur- vival signals to B cells in bone marrow niches. Nat. Immunol. 9: 388–395. did not result in a modified expansion of progenitor cells but rather 4. Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. 1999. Notch signaling: cell influenced the specific differentiation of DCs in the compartment fate control and signal integration in development. Science 284: 770–776. with prevalent Jag1 expression (BM). Jag1 was able to actively 5. Allman, D., J. A. Punt, D. J. Izon, J. C. Aster, and W. S. Pear. 2002. An invitation to T and more: notch signaling in . Cell 109(Suppl.): S1–S11. neutralize the effect of Dll1 on DC differentiation. These data 6. Osborne, B., and L. Miele. 1999. Notch and the immune system. Immunity 11: argue that the inhibitory effect of Jag1 on DC differentiation was 653–663. unlikely, because of its inability to reach the required Notch signal 7. Benedito, R., C. Roca, I. So¨rensen, S. Adams, A. Gossler, M. Fruttiger, and R. H. Adams. 2009. The notch ligands Dll4 and Jagged1 have opposing effects strength threshold to induce DC differentiation. on angiogenesis. Cell 137: 1124–1135. Our data suggest a novel mechanism of a negative regulation of 8. Bugeon, L., L. M. Gardner, A. Rose, M. Gentle, and M. J. Dallman. 2008. Cutting edge: Notch signaling induces a distinct cytokine profile in dendritic DC differentiation by Jag1 that involves Wnt signaling. Previous cells that supports T cell-mediated regulation and IL-2‑dependent IL-17 pro- studies have implicated the Wnt pathway in DC differentiation and duction. J. Immunol. 181: 8189–8193. activation (17) (28, 41). Dll1 promoted DC differentiation via up- 9. Kwon, S. M., M. Eguchi, M. Wada, Y. Iwami, K. Hozumi, H. Iwaguro, H. Masuda, A. Kawamoto, and T. Asahara. 2008. Specific Jagged-1 signal from regulation of the expression of the Fzd family of Wnt receptors and bone marrow microenvironment is required for endothelial progenitor cell de- activation of Wnt signaling (17). This effect was different from the velopment for neovascularization. Circulation 118: 157–165. recently reported posttranslational inhibition of b-catenin in stem 10. Brooker, R., K. Hozumi, and J. Lewis. 2006. Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development 133: 1277– and colon cancer cells by membrane-bound Notch (42). To our 1286. Downloaded from surprise, Jag1 directly inhibited Wnt signaling in HPCs via the 11. Cheng, P., Y. Nefedova, C. A. Corzo, and D. I. Gabrilovich. 2007. Regulation of transcriptional downregulation of the expression of several Wnt dendritic-cell differentiation by bone marrow stroma via different Notch ligands. Blood 109: 507–515. receptors Fzd. Jag1 was able to neutralize the Dll1-inducible up- 12. Van de Walle, I., G. De Smet, M. Ga¨rtner, M. De Smedt, E. Waegemans, regulation of Fzd, despite the fact that Dll1 has a much stronger B. Vandekerckhove, G. Leclercq, J. Plum, J. C. Aster, I. D. Bernstein, et al. 2011. D effect on Notch signaling. Jagged2 acts as a -like Notch ligand during early hematopoietic cell fate decisions. Blood 117: 4449–4459.

These data suggest that Wnt signaling could affect the differen- 13. Ramasamy, S. K., and N. Lenka. 2010. Notch exhibits ligand bias and maneuvers http://www.jimmunol.org/ tiation of DCs. However, recipients of b-cat KO BM cells, in steady stage-specific steering of neural differentiation in embryonic stem cells. Mol. Cell. Biol. 30: 1946–1957. state, had no defects in DC differentiation. This was consistent with 14. Cheng, P., and D. Gabrilovich. 2008. Notch signaling in differentiation and a previous paper (43). However, when recipients of b-cat KO BM function of dendritic cells. Immunol. Res. 41: 1–14. were treated with GM-CSF by HGT, they failed to upregulate DC 15. Hoshino, N., N. Katayama, T. Shibasaki, K. Ohishi, J. Nishioka, M. Masuya, b Y. Miyahara, M. Hayashida, D. Shimomura, T. Kato, et al. 2005. A novel role for production in BM, which suggested that -cat may play an im- Notch ligand D-1 as a regulator of human development from portant role in DC differentiation during emergency hematopoiesis. blood . J. Leukoc. Biol. 78: 921–929. In the absence of Notch activation, CBF-1 exists in nuclei as 16. Olivier, A., E. Lauret, P. Gonin, and A. Galy. 2006. The Notch ligand D-1 is a hematopoietic development cofactor for plasmacytoid dendritic cells. Blood a repressor complex, in association with the transcriptional core- 107: 2694–2701. pressors SMRT, NCoR, CIR, SHARP, KyoT2, and Skip. Some of 17. Zhou, J., P. Cheng, J. I. Youn, M. J. Cotter, and D. I. Gabrilovich. 2009. Notch by guest on September 29, 2021 these corepressors (SMRT, CIR, and SHARP) are shown to recruit and wingless signaling cooperate in regulation of dendritic cell differentiation. Immunity 30: 845–859. HDACs to Notch target genes (44, 45). After activation, cleaved 18. Ohishi, K., B. Varnum-Finney, D. Flowers, C. Anasetti, D. Myerson, and ICN displaces these corepressors and HDACs, from CBF-1, to form I. D. Bernstein. 2000. Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, D-1. a transcription-activating complex (46). In addition, ICN, binding to Blood 95: 2847–2854. CBF-1, recruits mastermind (MAML) to the complex, which can 19. Liu, F., Y. Song, and D. Liu. 1999. Hydrodynamics-based transfection in animals recruit HDACs to the activation complex (47, 48). It appears that by systemic administration of plasmid DNA. Gene Ther. 6: 1258–1266. 20. Takebe, Y., M. Seiki, J. Fujisawa, P. Hoy, K. Yokota, K. Arai, M. Yoshida, and the interaction of Notch with Dll1 in HPCs follows this scheme. As N. Arai. 1988. SR alpha promoter: an efficient and versatile mammalian cDNA a result, expression of fzd and Wnt signaling is dramatically in- expression system composed of the simian virus 40 early promoter and the R-U5 creased. However, Jag1 fails to displace HDAC1 from corepressor segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8: 466–472. complexes on fzd promoters that shut-down fzd expression and 21. Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, and inhibited Wnt signaling and terminal DC differentiation. This effect R. M. Steinman. 1990. The distinct leukocyte integrins of mouse spleen dendritic was not global, because Jag1 activated transcription of hes1 and cells as identified with new hamster monoclonal antibodies. J. Exp. Med. 171: 1753–1771. hes5 target genes. The detailed mechanism of this process needs to 22. del Hoyo, G. M., P. Martı´n, H. H. Vargas, S. Ruiz, C. F. Arias, and C. Ardavı´n. be further elucidated. However, it is possible that the number of 2002. Characterization of a common precursor population for dendritic cells. Nature 415: 1043–1047. CBF-1 binding sited in different genes can be important. Our data 23. Hashimoto, D., J. Miller, and M. Merad. 2011. Dendritic cell and macrophage may help to explain the diversity and context-dependent nature of heterogeneity in vivo. Immunity 35: 323–335. Notch-mediated effects and provide a novel model of regulation of 24. Reya, T., and H. Clevers. 2005. Wnt signalling in stem cells and cancer. Nature 434: 843–850. the DC differentiation in various tissue compartments. 25. Katoh, M., and M. Katoh. 2007. WNT signaling pathway and stem cell signaling network. Clin. Cancer Res. 13: 4042–4045. 26. Mikels, A. J., and R. Nusse. 2006. Wnts as ligands: processing, secretion and Acknowledgments reception. Oncogene 25: 7461–7468. 27. Ohishi, K., B. Varnum-Finney, R. E. Serda, C. Anasetti, and I. D. Bernstein. 2001. We thank Drs. I. Mellman and I. Bernstein for providing the reagents and The Notch ligand, D-1, inhibits the differentiation of monocytes into Dr. Lewis for providing the Jag1fl/fl mice. but permits their differentiation into dendritic cells. Blood 98: 1402–1407. 28. Jiang, A., O. Bloom, S. Ono, W. Cui, J. Unternaehrer, S. Jiang, J. A. Whitney, J. Connolly, J. Banchereau, and I. Mellman. 2007. Disruption of E-cadherin‑mediated Disclosures adhesion induces a functionally distinct pathway of dendritic cell maturation. Im- The authors have no financial conflicts of interest. munity 27: 610–624. 29. Owen, R., and P. R. Gordon-Weeks. 2003. Inhibition of glycogen synthase kinase 3b in sensory neurons in culture alters filopodia dynamics and microtubule distribution in growth cones. Mol. Cell. Neurosci. 23: 626–637. References 30. Li, H. S., C. Y. Yang, K. C. Nallaparaju, H. Zhang, Y. J. Liu, A. W. Goldrath, and 1. Shortman, K., and W. R. Heath. 2010. The CD8+ dendritic cell subset. Immunol. S. S. Watowich. 2012. The signal transducers STAT5 and STAT3 control expres- Rev. 234: 18–31. sion of Id2 and E2-2 during dendritic cell development. Blood 120: 4363–4373. The Journal of Immunology 11

31. Caton, M. L., M. R. Smith-Raska, and B. Reizis. 2007. Notch-RBP-J signaling 39. Yang, L. T., J. T. Nichols, C. Yao, J. O. Manilay, E. A. Robey, and controls the homeostasis of CD8‑ dendritic cells in the spleen. J. Exp. Med. 204: G. Weinmaster. 2005. Fringe glycosyltransferases differentially modulate 1653–1664. Notch1 proteolysis induced by D1 and Jagged1. Mol. Biol. Cell 16: 927–942. 32. Emami, K. H., C. Nguyen, H. Ma, D. H. Kim, K. W. Jeong, M. Eguchi, 40. Hicks, C., S. H. Johnston, G. diSibio, A. Collazo, T. F. Vogt, and G. Weinmaster. R. T. Moon, J. L. Teo, H. Y. Kim, S. H. Moon, et al. 2004. A small molecule 2000. Fringe differentially modulates Jagged1 and D1 signalling through Notch1 inhibitor of b-catenin/CREB-binding protein transcription [corrected]. Proc. and Notch2. Nat. Cell Biol. 2: 515–520. Natl. Acad. Sci. USA 101: 12682–12687. 41. Staal, F. J., T. C. Luis, and M. M. Tiemessen. 2008. WNT signalling in the 33. Maillard, I., U. Koch, A. Dumortier, O. Shestova, L. Xu, H. Sai, S. E. Pross, immune system: WNT is spreading its wings. Nat. Rev. Immunol. 8: 581–593. J. C. Aster, A. Bhandoola, F. Radtke, and W. S. Pear. 2008. Canonical notch 42. Kwon, C., P. Cheng, I. N. King, P. Andersen, L. Shenje, V. Nigam, and signaling is dispensable for the maintenance of adult hematopoietic stem cells. D. Srivastava. 2011. Notch post-translationally regulates b-catenin protein in Cell Stem Cell 2: 356–366. stem and progenitor cells. Nat. Cell Biol. 13: 1244–1251. 34. Varnum-Finney, B., L. M. Halasz, M. Sun, T. Gridley, F. Radtke, and 43. Cobas, M., A. Wilson, B. Ernst, S. J. Mancini, H. R. MacDonald, R. Kemler, and I. D. Bernstein. 2011. Notch2 governs the rate of generation of mouse long- and F. Radtke. 2004. b-Catenin is dispensable for hematopoiesis and lymphopoiesis. short-term repopulating stem cells. J. Clin. Invest. 121: 1207–1216. J. Exp. Med. 199: 221–229. 35. Calvi, L. M., G. B. Adams, K. W. Weibrecht, J. M. Weber, D. P. Olson, 44. Oswald, F., M. Winkler, Y. Cao, K. Astrahantseff, S. Bourteele, W. Kno¨chel, and M. C. Knight, R. P. Martin, E. Schipani, P. Divieti, F. R. Bringhurst, et al. 2003. T. Borggrefe. 2005. RBP-Jk/SHARP recruits CtIP/CtBP corepressors to silence Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425: 841–846. Notch target genes. Mol. Cell. Biol. 25: 10379–10390. 36. Kato, T. M., A. Kawaguchi, Y. Kosodo, H. Niwa, and F. Matsuzaki. 2010. Lu- 45. Hsieh, J. J., S. Zhou, L. Chen, D. B. Young, and S. D. Hayward. 1999. CIR, natic fringe potentiates Notch signaling in the developing brain. Mol. Cell. a corepressor linking the DNA binding factor CBF1 to the histone deacetylase Neurosci. 45: 12–25. complex. Proc. Natl. Acad. Sci. USA 96: 23–28. 37. Ong, C. T., H. T. Cheng, L. W. Chang, T. Ohtsuka, R. Kageyama, G. D. Stormo, 46. Miele, L. 2006. Notch signaling. Clin. Cancer Res. 12: 1074–1079. and R. Kopan. 2006. Target selectivity of vertebrate notch proteins: collaboration 47. Nam, Y., A. P. Weng, J. C. Aster, and S. C. Blacklow. 2003. Structural between discrete domains and CSL-binding site architecture determines acti- requirements for assembly of the CSL.intracellular Notch1.Mastermind-like 1 vation probability. J. Biol. Chem. 281: 5106–5119. transcriptional activation complex. J. Biol. Chem. 278: 21232–21239. 38. Visan, I., J. B. Tan, J. S. Yuan, J. A. Harper, U. Koch, and C. J. Guidos. 2006. 48. Fryer, C. J., E. Lamar, I. Turbachova, C. Kintner, and K. A. Jones. 2002. Regulation of T lymphopoiesis by Notch1 and Lunatic fringe-mediated com- Mastermind mediates chromatin-specific transcription and turnover of the Notch Downloaded from petition for intrathymic niches. Nat. Immunol. 7: 634–643. enhancer complex. Genes Dev. 16: 1397–1411. http://www.jimmunol.org/ by guest on September 29, 2021