Propensity of Adult Lymphoid Progenitors to Progress to DN2/3 Stage Thymocytes with Notch Receptor Ligation This information is current as Jiaxue Huang, Karla P. Garrett, Rosana Pelayo, Juan Carlos of September 29, 2021. Zúñiga-Pflücker, Howard T. Petrie and Paul W. Kincade J Immunol 2005; 175:4858-4865; ; doi: 10.4049/jimmunol.175.8.4858 http://www.jimmunol.org/content/175/8/4858 Downloaded from References This article cites 45 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/175/8/4858.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Propensity of Adult Lymphoid Progenitors to Progress to DN2/ 3 Stage Thymocytes with Notch Receptor Ligation1 Jiaxue Huang,*† Karla P. Garrett,* Rosana Pelayo,* Juan Carlos Zu´n˜iga-Pflu¨cker,‡ Howard T. Petrie,§ and Paul W. Kincade2* Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and .slowly produced TCR؉ and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages Downloaded from were infrequent. Lin؊c-KitlowGFP؉IL-7R␣؉/؊ prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow- derived progenitors with Ig DH-JH rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with VH-DHJH rearrangement products. The Journal of Immunology, 2005, 175: 4858–4865. he Notch family of receptor/transcription factors has at- and CD8 subsets (9–11). Additionally, most of the studies to date http://www.jimmunol.org/ tracted great interest because of its potential importance have used fetal lymphoid progenitors and direct comparisons with T in establishment of the immune system, stem cell self- their counterparts in adult bone marrow have not been reported. renewal, and lineage fate decisions (1). Knockout and overexpres- One of the most unique and important features of hemopoietic sion experiments have generated an impressive body of data indi- stem cells (HSCs)3 is their ability to extensively self-renew while cating that Notch determines whether lymphoid progenitors retaining the ability to give rise to multiple specialized types of generate B or T lineage lymphocytes (2, 3). Moreover, two labo- blood cells. Understanding how this process is regulated may ratories demonstrated that the behavior of normal progenitors someday facilitate expansion of stem cells in culture, opening pos- could be modified by contact with ligand modified stromal cells (4, sibilities for genetic engineering and other therapeutic applica- 5). Indeed, experimental models were described that support all tions. In that context, it is interesting that human stem cells were by guest on September 29, 2021 stages of T lymphopoiesis and even when embryonic stem cell expanded 10-fold in culture by ligation of Notch receptors (12), lines were used to initiate the cultures (6). and stem cell lines were established by introduction of constitu- However, the range of target cells that can respond to Notch tively active Notch 1 (13). Therefore, it is possible that Notch receptor ligation with a lineage choice decision and the extent to receptor ligation might trap cells in a proliferative mode and block which partially committed progenitors can be reprogrammed re- their differentiation. If so, lymphohematopoietic cells might be ex- main unclear. Indeed, we need to rigorously test whether there are panded and maintained for prolonged periods on Delta 1-trans- single branch points where progenitors at a discrete stage are ir- duced stromal cells. reversibly diverted into a particular pathway. Moreover, it is not We have now used RAG-1/GFP-knockin mice, cell sorting, and known if B lineage inhibition and T lineage induction represent coculture with Notch ligand-transduced stromal cells to address completely separate or related events (7, 8). Controversy remains these questions. Unlike their fetal counterparts, multiple subsets of about the role of Notch in determining the relative production of adult bone marrow cells were able to generate long-term, Notch TCR␣␤ vs TCR␥␦ lymphocytes and biasing the formation of CD4 ligand, and IL-7-dependent cell lines paused at the CD4ϪD8Ϫthymocyte (DN)2/3 thymocyte stage. Progression of adult progenitors in the T lineage was blocked in the presence of *Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 5 ng/ml IL-7. These findings provide a basis for further investiga- Oklahoma City, OK 73104; †Department of Microbiology and Immunology, Univer- sity of Oklahoma Health Sciences Center, Oklahoma City, OK 73190; ‡Department tion of adult lymphoid progenitors in short- and long-term cultures. of Immunology, University of Toronto, Sunnybrook and Women’s Research Institute, Toronto, Ontario, Canada; and §The Scripps/Florida Research Institute, Jupiter, FL 33458 Materials and Methods Received for publication May 18, 2005. Accepted for publication July 25, 2005. Mice and embryos The costs of publication of this article were defrayed in part by the payment of page RAG-1/GFP-knockin mice have been described previously (14). Female charges. This article must therefore be hereby marked advertisement in accordance B6.PL-Thy1a/CyJ (Thy-1/CD90.1) congenic, and C57BL/6 mice were with 18 U.S.C. Section 1734 solely to indicate this fact. bought from The Jackson Laboratory. Mating homozygous male RAG-1/ 1 This work was supported by National Institutes of Health Grants AI20069, AI58162, GFP-knockin mice with wild-type C57BL/6 strain female mice generated AI33940, and AI53739 and National Center for Research Resources Grant P20 RR15577. J.C.Z.-P. is supported by an Investigator Award from the Canadian Insti- tutes of Health Research. 3 Abbreviations used in this paper: HSC, hemopoietic stem cell; CLP, common lym- 2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobi- phoid progenitor; DN, CD4ϪD8Ϫ thymocyte; DP, CD4ϩCD8ϩ thymocyte; ELP, ology and Cancer Program, Oklahoma Medical Research Foundation, 825 N.E. 13th early lymphoid progenitor; LSP, L-selectin-positive progenitor; OP9-DL1, murine Street, Oklahoma City, OK 73104. E-mail address: [email protected] Delta-like 1-transduced OP9 cell. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 4859 heterozygous RAG-1/GFP embryos, as well as mice that were studied as selection with the MACS cell separation system (Miltenyi Biotec). These adults. Mating homozygous male RAG-1/GFP-knockin mice with female partially lineage-depleted cells were further stained with rat anti-mouse B6.PL-Thy1a/CyJ mice yielded heterozygous RAG-1/GFP animals bearing lineage markers (biotin-Gr-1, Mac-1, CD2, CD3⑀, CD8, Ter-119, CD45R, the Thy-1/CD90.1 Ag. Timed pregnancies were determined by designating DX-5, and CD19), as well as allophycocyanin-labeled c-Kit and PE-Sca-1. the day of vaginal plug observation as day 0.5 postcoitus (E0.5). For ex- Lineage-positive cells were electronically gated out, and lineage negative periments with adult mice, 5- to 10-wk-old male mice were used. All of the fractions were sorted on the basis of RAG-1/GFP, c-Kit, and Sca-1 ex- studies were reviewed and approved by an institutional review committee. pression with a MoFlo (DakoCytomation). Similar separations were done to isolate the HSC (LinϪSca-1ϩc-KithighCD90.1low), L-selectin-positive Antibodies progenitors (LSPs, LinϪCD90.1ϪSca-1ϩc-KithighCD62Lϩ), early lym- Ϫ ϩ high ϩ ⑀ ␣ phoid progenitors (ELPs, Lin Sca-1 c-Kit RAG-1 ), and common FITC-conjugated CD45R/B220 (RA3-6B2), CD3 (145-2C11), CD8 (53- Ϫ low low ␣ϩ ⑀ lymphoid progenitors (CLPs, Lin Sca-1 c-Kit IL-7R ), as described 6.7), CD43, CD25, and Mac-1 Abs, as well as PE-conjugated CD3 (145- in Fig. 6. These previously described fractions (17–20) were sorted on a 2C11), TCR␥␦, NK1.1(PK136), CD45R/B220 (RA3/6B2), CD19 (ID3), ␣ FACSAria (BD Biosciences) after thoroughly lineage depleting bone mar- and IL-7R (SB/199) Abs were all purchased from BD Pharmingen. The row cells and then staining with biotin-CD90.1 revealed by PE-TR, allo- same source was used for biotinylated-CD106/VCAM-1, biotinylated- phycocyanin-Cy7-CD62L, PE-IL-7R␣, PE-Cy-5-Sca-1, and allophycocya- CD19 (clone 1D3), CD90.1 (OX-7) and allophycocyanin-conjugated ␤ nin-c-Kit.