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Commitment in Adult Mouse Thymus Fine-Scale Staging of T Cell Lineage

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Commitment in Adult Mouse Thymus Fine-Scale Staging of T Cell Lineage Fine-Scale Staging of T Cell Lineage Commitment in Adult Mouse Thymus Mary A. Yui, Ni Feng and Ellen V. Rothenberg This information is current as J Immunol 2010; 185:284-293; Prepublished online 11 June of October 1, 2021. 2010; doi: 10.4049/jimmunol.1000679 http://www.jimmunol.org/content/185/1/284 Downloaded from References This article cites 54 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/185/1/284.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 1, 2021 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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Fine-Scale Staging of T Cell Lineage Commitment in Adult Mouse Thymus Mary A. Yui, Ni Feng, and Ellen V. Rothenberg T cell development is marked by the loss of alternative lineage choices accompanying specification and commitment to the T cell lineage. Commitment occurs between the CD4 and CD8 double-negative (DN) 2 and DN3 stages in mouse early T cells. To determine the gene regulatory changes that accompany commitment, we sought to distinguish and characterize the earliest committed wild- type DN adult thymocytes. A transitional cell population, defined by the first downregulation of surface c-Kit expression, was found to have lost the ability to differentiate into dendritic cells and NK cells when cultured without Notch-Delta signals. In the presence of Notch signaling, this subset generates T lineage descendants in an ordered precursor–product relationship between DN2, with the highest levels of surface c-Kit, and c-Kit–low DN3 cells. These earliest committed cells show only a few differences in regulatory gene expression, compared with uncommitted DN2 cells. They have not yet established the full expression of Notch-related and T cell differentiation genes characteristic of DN3 cells before b selection. Instead, the downregulation of select stem cell and Downloaded from non-T lineage genes appears to be key to the extinction of alternative lineage choices. The Journal of Immunology, 2010, 185: 284–293. arly T cell development is a prolonged process in the clear delineation of the earliest events in b and gd selection and thymus marked by loss of alternative lineage choices the manipulation of these cells (9–11). E accompanying T cell specification and commitment to the Multiple studies have shown that DN3 cells are committed to the http://www.jimmunol.org/ gd or ab T lineages. Although much progress has been made in T lineage but the DN2 population is not, with possible alternative understanding the gene expression patterns and regulatory net- fates still accessible, including myeloid cells, dendritic cells (DCs), works involved in the early stages of T cell development (1–5), the NK cells, and mast cells (12–16). Fetal thymic DN2 cells from specific regulatory events that result in T lineage commitment pLck-GFP transgenic (Tg) mice were found to be heterogenous in have not yet been elucidated. developmental potential: GFP+ DN2 cells could not differentiate To identify the critical gene expression changes leading to T cell into DCs and made NK cells very poorly in comparison with commitment, the event must be accurately timed. Commitment is GFP2 DN2 cells (17), implying that regulatory events establishing scored by loss of alternative developmental potential, and is thus T lineage commitment may occur close to the DN2 stage, at least by guest on October 1, 2021 dependent on refined purification of staged precursor cells. Mouse in fetal cells, well before the cell cycle arrest characteristic of the T cell precursors are CD4, CD8 double negative (DN) cells lacking DN3 stage b selection checkpoint (9). a TCR, which were initially subdivided into DN1, DN2, DN3, and Between the DN2 and DN3 stages is a period of major de- DN4 stages based upon CD25 and CD44 expression (6). Sub- velopmental change at molecular and cellular levels, triggered by sequently, additional markers were found to more precisely define unknown intrinsic and/or extrinsic events. This is a time of these populations, especially for the DN1 stage, which includes markedly decreased proliferation, increased RAG-mediated TCR both T cell precursors and irrelevant cells when defined solely on gene rearrangement, upregulation of Notch target gene expression, the basis of CD44 and CD25 expression (7). High levels of c-Kit shifted growth and survival requirements crucial for b selection (CD117), the receptor for stem cell factor, were found to mark the checkpoint enforcement, and upregulated expression of many crit- pluripotent early thymic precursor (ETP) subset of DN1 cells as ical T cell genes [reviewed in Ref. (18)]. DN2 cells also express well as DN2 progenitor cells (8). Furthermore, cell size and CD27 many “legacy” stem cell genes and genes associated with alterna- expression were found to subdivide adult DN3 cells into pre- and tive lineages, many of which decline precipitously between the post-b selection cells (DN3a and DN3b, respectively), allowing DN2 and DN3 stages (1–3, 5, 16, 19). Although the expression of many transcription factors, T cell identity genes, and non-T cell genes changes dramatically from the ETP to the DN3 stage, it is Division of Biology, California Institute of Technology, Pasadena, CA 91125 not clear which of these gene changes are causally linked to T lineage commitment. For this answer, a more precise ordering Received for publication February 26, 2010. Accepted for publication April 25, 2010. of the gene expression changes in relationship to the commitment This work was supported by National Institutes of Health Grant R01 AI064590 (to M.A.Y.), the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foun- event is required. dation, and the Albert Billings Ruddock Professorship (to E.V.R.). In this study, we found that the first phenotypic shift in c-Kit Address correspondence and reprint requests to Dr. Mary A. Yui or Dr. Ellen surface expression can be used in wild-type adult mouse thymo- V. Rothenberg, Division of Biology, MC 156-29, California Institute of Technology, cytes as a marker to reveal the earliest committed cells that precede 1200 E. California Boulevard, Pasadena, CA 91125. E-mail addresses: yui@caltech. edu or [email protected] the phenotypic and functional changes defining the DN3a stage. Abbreviations used in this paper: bHLH, basic helix-loop-helix; DC, dendritic cell; Two inbred mouse strains were used to confirm the generality of DN, double negative; DP, double positive; ETP, early thymic precursor; FSC, forward these findings: C57BL/6 (B6) mice, the most commonly studied light scatter; MFI, mean fluorescence intensity; MHC-II, MHC class II; nd, not strain for immunological studies, and NOD mice, an autoimmu- determined; Tg, transgenic. nity-prone strain with a defect in early T cell checkpoint control Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (20). Gene expression analysis reveals that commitment is most www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000679 The Journal of Immunology 285 closely associated, in both strains, with downregulation of a spe- the exception of Lyl1 (FOR 59-CAGGACCCTTCAGCATCTTC-39; REV cific set of progenitor cell or non-T lineage regulatory genes, that 59-ACGGCTGTTGGTGAACACTC-39). Results were calculated using the it precedes major increases in Notch-dependent gene expression, DCt method, normalizing all samples to Actb expression. and that only a select group of T lineage transcription factors is upregulated before commitment. Results Surface c-Kit levels distinguish a developmentally intermediate population between DN2 (DN2a) and DN3 cells Materials and Methods Mice DN2 cells are CD25-positive cells that also express hematopoietic progenitor-associated CD44 and c-Kit, both of which decline as the C57BL/6J, NOD.Shi/J, NOD.B6-(D6Mit254-D6Mit289)/CarJ (NOD. cells progress to the DN3 stage (22) (Fig. 1A,1B). High c-Kit NK1.1), and Em-Bcl-2-25 Tg (Bcl2Tg) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). These strains were bred and main- expression is critical to define the DN2 stage (23, 24), but we had + tained in the Caltech Laboratory Animal Facility. Female mice were used previously noted that CD25 cells with a small reduction in c-Kit at 4–6 wk of age. All animal protocols were reviewed and approved by the levels were distinguishable in their expression of several develop- Animal Care and Use Committee of the California Institute of Technology. mentally important genes (3). We sought to determine if this sub- Ab staining, cell sorting, and flow cytometric analysis set could provide a useful developmental-intermediate population to stage the loss of non-T cell potential and for analysis of the To isolate DN populations, mice were sacrificed, their thymuses were re- accompanying gene expression changes. moved, and single-cell suspensions were made. Mature cells were depleted by staining with biotinylated Abs to CD8a (53-6.7), TCRgd (GL3), TCRb Fig. 1 shows the phenotypic dissection and gating of T cell (Η572597), Gr1 (R86.8C5), Ter119 (Ter119), CD122 (5H4), NK1.1 precursor stages from B6 and NOD mouse thymuses.
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