Growth Factor Independence-1B Expression Leads to Defects in T Cell Activation, IL-7 Receptor α Expression, and T Cell Lineage Commitment This information is current as of September 26, 2021. Loretta L. Doan, Mary Kate Kitay, Qing Yu, Alfred Singer, Sabine Herblot, Trang Hoang, Susan E. Bear, Herbert C. Morse III, Philip N. Tsichlis and H. Leighton Grimes J Immunol 2003; 170:2356-2366; ; doi: 10.4049/jimmunol.170.5.2356 Downloaded from http://www.jimmunol.org/content/170/5/2356 References This article cites 41 articles, 16 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/170/5/2356.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 September 26, 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Growth Factor Independence-1B Expression Leads to Defects in T Cell Activation, IL-7 Receptor ␣ Expression, and T Cell Lineage Commitment1 Loretta L. Doan,2*† Mary Kate Kitay,2* Qing Yu,‡ Alfred Singer,‡ Sabine Herblot,¶ Trang Hoang,¶ Susan E. Bear,ʈ Herbert C. Morse III,§ Philip N. Tsichlis,# and H. Leighton Grimes3*† T cell differentiation in the thymus is dependent upon signaling through the TCR and is characterized by the resulting changes in expression patterns of CD4 and CD8 surface coreceptor molecules. Although recent studies have characterized the effects of proximal TCR signaling on T cell differentiation, the downstream integration of these signals remains largely unknown. The growth factor independence-1 (GFI1) and GFI1B transcriptional repressors may regulate cytokine signaling pathways to affect Downloaded from lymphocyte growth and survival. In this study, we show that Gfi1 expression is induced upon induction of the T cell program. Gfi1B expression is low and dynamic during T cell development, but is terminated in mature thymocytes. Transgenic expression of GFI1 and GFI1B in T cells allowed us to determine the functional consequences of constitutive expression. GFI1 potentiates response to TCR stimulation and IL-2, whereas GFI1B-transgenic T cells are defective in T cell activation. Moreover, GFI1B- transgenic thymocytes display reduced expression of the late-activation marker IL-7R␣, and a decrease in CD4؊8؉ single-positive http://www.jimmunol.org/ T cells that can be mitigated by transgenic expression of BCL2 or GFI1. These data show that GFI1 and GFI1B are functionally unique, and implicate a role for GFI1 in the integration of activation and survival signals. The Journal of Immunology, 2003, 170: 2356–2366. cell differentiation in the thymus is dependent upon sig- positive (CD4 SP) or CD4Ϫ8ϩ (CD8 SP) T cells is triggered by the naling through the TCR and is characterized by the re- engagement of TCRs on the immature TCRhighCD4ϩCD8ϩ thy- T sulting changes in expression patterns of CD4 and CD8 mocytes by self-peptide/MHC complex on thymic epithelial cells surface coreceptor molecules. Early thymocyte precursors are (1, 2). Negative selection eliminates immature DP thymocytes Ϫ Ϫ 4 CD4 CD8 (double negative; DN) and are signaled to differen- through clonal deletion of those T cells that have high affinity for by guest on September 26, 2021 ϩ ϩ tiate first into CD4 CD8 (double positive; DP) thymocytes. Fur- self-peptide and thus are potentially autoreactive (3). Positive se- ϩ Ϫ ther differentiation of DP thymocytes into mature CD4 8 single- lection occurs when low-affinity TCR-ligand interactions trigger a signal for survival and results in termination of one or the other CD4 or CD8 coreceptor molecule. The choice of which coreceptor *Institute for Cellular Therapeutics and Department of Surgery, University of Lou- to extinguish is referred to as lineage commitment. isville School of Medicine, Louisville, KY 40202; †Department of Biochemistry and Molecular Biology, ‡Experimental Immunology Branch, National Cancer Institute, The growth factor independence-1 (GFI1) and GFI1B proteins and §Laboratory of Immunopathology, National Institute of Allergy and Infectious are closely related nuclear oncoproteins that may regulate cytokine Diseases, National Institutes of Health, Bethesda, MD 20892; ¶Clinical Research Institute of Montreal, Quebec, Canada; ʈKimmel Cancer Center, Thomas Jefferson pathways. Gfi1 was originally identified as the gene up-regulated University, Philadelphia, PA 19107; and # Molecular Oncology Research Institute, by insertion of Moloney murine leukemia virus in a thymic lym- Tufts-New England Medical Center, Boston, MA 02111 phoma that was selected for its ability to grow in the absence of the Received for publication April 30, 2002. Accepted for publication December T cell cytokine IL-2 (4). Forced expression of GFI1 in the IL-2- 17, 2002. dependent parental cell line potentiates the outgrowth of IL-2-in- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance dependent cell lines, without inducing IL-2 (4, 5). Gfi1B was iden- with 18 U.S.C. Section 1734 solely to indicate this fact. tified by low stringency hybridization screening with a cDNA 1 This work was supported by Hope Street Kids, the Jennifer Sacco Memorial Fund, probe encoding the zinc-finger region of Gfi1 (6). GFI1 and GFI1B the University of Louisville School of Medicine Grant-in-Aid, a University of Lou- isville Research Initiation Grant, and in part by the Commonwealth of Kentucky are 97% homologous in the carboxyl-terminal 165 aa that code for Research Challenge Trust Fund and the Jewish Hospital Foundation. L.L.D. is sup- six Cys-His zinc fingers. An amino-terminal 20-aa snail and Gfi1 ported by a National Science Foundation Graduate Research Fellowship. This work was supported in part by PHS CA56110 (to P.N.T.). (SNAG) domain, responsible for nuclear localization and tran- scriptional repressor function, is also highly conserved (5). In con- 2 L.L.D. and M.K.K. contributed equally to this work. trast, the 236 intervening amino acids between the GFI1 SNAG 3 Address correspondence and reprint requests to Dr. H. Leighton Grimes, Institute for Cellular Therapeutics, University of Louisville, Baxter Biomedical Research and zinc-finger domains bear no homology to the corresponding Building, Suite 404-F, 570 South Preston Street, Louisville, KY 40202-1760. E-mail 145 aa of GFI1B. Both proteins bind to virtually identical DNA address: [email protected] consensus sequences and function as transcriptional repressors in a 4 Abbreviations used in this paper: DN, CD4ϪCD8Ϫ (double negative); DP, CD4ϩCD8ϩ (double positive); CD4 SP, CD4ϩ8Ϫ single positive; CD8 SP, CD4Ϫ8ϩ SNAG-dependent manner (5, 6). GFI1 is mildly antiapoptotic and single positive; GFI1, growth factor independence-1; MFI, mean fluorescence inten- inhibits growth arrest of IL-2-dependent T cell lines under condi- sity; ISP, intermediate single positive; PϩI, phorbal-12-myristate-13-acetate and ionomycin; SNAG, snail and Gfi1 repressor domain; GH, human growth hormone; tions of limiting IL-2 (5, 7), while GFI1B inhibits both IL-6-in- RAG, recombination-activating gene; IRF, IFN regulatory factor; WT, wild type. duced differentiation and growth arrest of M1 myelomonocytic Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 2357 cells (6). Mice deleted for GFI1 have altered inflammatory re- perfusion with 10 ml of medium 199. Both thymocyte and splenocyte cell sponses and differentiation in the myeloid lineage (8), while mouse debris was depleted by passage through Nytex nylon-mesh screens. embryos deleted for GFI1B die in utero due to a lack of definitive Splenocytes were treated with ammonium chloride-potassium bicarbonate solution (150 mM NH4Cl and 10 mM KHCO3) to lyse RBCs. For exper- erythropoiesis (9). iments requiring isolation of T cells, splenic cell preps were depleted of Gfi1 and Gfi1B are differentially expressed in lymphoid com- other cell types by the use of T cell enrichment columns (R&D Systems, partments. Northern analysis reveals that Gfi1 is expressed in the Minneapolis, MN). All cells were counted with a Coulter Counter model bone marrow and thymus, with low-level expression in the spleen, Z2 (Coulter, Miami, FL), and viability was assayed by trypan blue exclusion. whereas Gfi1B is expressed in the bone marrow and spleen, with low-level expression in the thymus (6). Both Gfi1 and Gfi1B show Flow cytometric analysis regulated expression during T cell development, but Gfi1B expres- sion is terminated in mature thymocytes. Gfi1 message is not ex- Cell surface staining was performed by incubating 1 ϫ 106 cells with pressed in G splenic T cells, but is induced upon T cell activation mAbs at varying concentrations in FACS medium (HBSS with 0.1% BSA, 0 0.1% sodium azide, and 0.036% sodium bicarbonate) for 20 min on ice. (4, 10). Transgenic expression of GFI1 and GFI1B in T cells al- Stained cells were washed twice with FACS medium and fixed in 1% lowed us to determine the functional basis for differential expres- formaldehyde (Polysciences, Warrington, PA). For intranuclear staining, sion of these factors. Transgenic expression of GFI1 potentiates T cells were fixed in 2% formaldehyde in PBS and permeabilized and stained cell activation.