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Blimp-1 Attenuates Th1 Differentiation by Repression of Ifng, Tbx21, and Bcl6 Gene Expression

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Blimp-1 Attenuates Th1 Differentiation by Repression of Ifng, Tbx21, and Bcl6 Gene Expression Blimp-1 Attenuates Th1 Differentiation by Repression of ifng, tbx21, and bcl6 Gene Expression This information is current as Luisa Cimmino, Gislaine A. Martins, Jerry Liao, Erna of September 24, 2021. Magnusdottir, Gabriele Grunig, Rocio K. Perez and Kathryn L. Calame J Immunol 2008; 181:2338-2347; ; doi: 10.4049/jimmunol.181.4.2338 http://www.jimmunol.org/content/181/4/2338 Downloaded from References This article cites 68 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/181/4/2338.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 24, 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Blimp-1 Attenuates Th1 Differentiation by Repression of ifng, tbx21, and bcl6 Gene Expression Luisa Cimmino,* Gislaine A. Martins,† Jerry Liao,† Erna Magnusdottir,‡ Gabriele Grunig,† Rocio K. Perez,‡ and Kathryn L. Calame1*§ T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-␥ production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-␥, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Downloaded from Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes. The Journal of Immunology, 2008, 181: 2338–2347. ifferentiation of the CD4 Th cell lineages is dictated IL-13 genes (7) and can autoactivate its own expression both by the strength of antigenic stimulation, cytokine en- independently and as a result of IL-4-mediated STAT-6 signal- D vironment, and the complement of transcription fac- ing (8). How T-bet and GATA-3 compete to drive Th1 or Th2 http://www.jimmunol.org/ tors activated by these processes. T-bet and GATA-3 transcrip- differentiation has been intensively studied and has laid the tion factors are master regulators of Th1 and Th2 lineage foundation for understanding how other transcription factors differentiation, respectively; Foxp3 is critical for regulatory T influence the way CD4 lineage decisions are made. cell development and function, and, more recently, ROR␥t (ret- The transcriptional repressor B lymphocyte-induced maturation ␥ 2 inoid-related orphan receptor t) has been shown to act as a protein-1 (Blimp-1) has recently been identified as a regulator of master regulator of Th17 development (1–3). Additionally, T cell homeostasis and function (9, 10). Encoded by the prdm1 complicated regulatory pathways and feedback loops cooperate gene, Blimp-1 is well established as a master regulator of plasma with the master regulators to establish and maintain CD4 dif- cell differentiation and maintenance (11, 12). Blimp-1 also plays a ferentiation states. role in multiple developmental checkpoints in nonlymphoid lin- by guest on September 24, 2021 Th polarization into Th1 and Th2 lineages can be divided into eages including germ cell formation during embryogenesis (13– hierarchical steps of initiation, reinforcement, and maintenance 15), myeloid differentiation (16), keratinocyte maturation (17), and (1). Initiation of lineage differentiation begins upon T cell recog- sebocyte differentiation (18). nition of Ag presented by APCs and response to the initial cyto- In the T cell lineage Blimp-1 is strongly induced upon TCR kine environment. Reinforcement of commitment is driven by the activation (10), with IL-2 playing an important role in the induc- up-regulation of cytokine production, positive feedback loops, and tion (19). In two independent studies, T cell-specific deletion of repression of genes necessary for the alternative fate. Maintenance Blimp-1 in mice caused spontaneous colitis (10) and a multiorgan of the differentiated state is conferred by heritable epigenetic inflammatory disease (9). These autoimmune pathologies were at- changes. Transcription factors activated by TCR-signaling such tributed in part to altered homeostasis within the CD4 T cell lin- ␬ as AP-1, NF- B, and NFAT can differentially induce both eage, including hyperproliferation in response to TCR stimulation, ␥ IFN- and IL-4 expression directly or via the induction of T-bet increased IL-2 and IFN-␥ production, and decreased IL-10 pro- and GATA-3, respectively (1, 4). In the case of Th1 differen- duction, possibly contributing to diminished regulatory T cell ␥ tiation, T-bet can be induced both by IFN- or IL-12 through function in vivo (9, 10). the transcription factors STAT-1 and STAT-4, respectively, to Blimp-1 is a transcriptional repressor. Interestingly, most of the ␥ directly reinforce expression of the IFN- gene while simulta- known direct targets of Blimp-1 are also transcription regulators, neously repressing GATA-3 function (2, 5, 6). GATA-3 acti- explaining in part its ability to control extensive developmental vates expression at the Th2 cytokine locus of IL-4, IL-5, and programs (11). In B cells Blimp-1 directly represses myc, spiB, CIITA, id3, and pax5. In the epidermis it represses myc, fos, nfat5, *The Institute of Human Nutrition, †Department of Microbiology, ‡Department of and dusp16 (17). It has also been shown to repress p53 in some Biological Sciences, and §Department of Biochemistry and Molecular Biophysicis, tumor cell lines (20). College of Physicians and Surgeons, Columbia University, New York, NY 10032 Received for publication March 18, 2008. Accepted for publication June 9, 2008. 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 2 Abbreviations used in this paper: Blimp-1, B lymphocyte-induced maturation pro- with 18 U.S.C. Section 1734 solely to indicate this fact. tein-1; Bcl-6, B cell lymphoma-6; ChIP, chromatin immunoprecipitation; CNS, con- 1 Address correspondence and reprint requests to Dr. Kathryn Calame, Department of served noncoding sequence; IP, immunoprecipitation; MFI, mean fluorescence inten- Biochemistry and Molecular Biophysicis, College of Physicians and Surgeons, Co- sity; NP-KLH, 4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin. lumbia University, 701 168th Street, Hammer Health Science Center 1202, Manhat- tan, NY 10032. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 2339 Direct targets for Blimp-1 in T cells have not yet been identified. In vitro CD4 Th cell differentiation However, it is known that B cell lymphoma-6 (Bcl-6) mRNA is For in vitro polarization, naive CD4 T cells were seeded at 1 ϫ 106 cells/ml elevated in Blimp-1-deficient CD4 T cell effectors (10). Mutual in 24-well plates coated with 5 ␮g/ml anti-CD3, 2.5 ␮g/ml anti-CD28 (BD repression by Bcl-6 and Blimp-1 in B cells is important for main- Pharmingen), and 25 U/ml of recombinant human IL-2 in RPMI 1640 with taining mutually exclusive programs of germinal center B cells and the addition of murine IL-12 (10 ng/ml) and anti-IL4 (10 ␮g/ml) or murine ␥ ␮ plasma cells (21). In T cells, Bcl-6 repression is involved in Th1 IL-4 (1000 U/ml) and anti-IFN- (10 g/ml). differentiation by repressing Th2 cytokine expression. It decreases GATA-3 protein levels (22), represses IL-5 transcription (23), and Western blot analysis competes for binding at STAT-6 recognition sequences in IL-4- CD4 T cells were cultured for 2 wk under polarizing conditions de- responsive genes (24, 25). scribed above, lysed in RIPA buffer (50 mM HEPES (pH 7.6), 1 mM EDTA, 0.7% sodium deoxycholate, 1% Nonidet P-40, and 0.5 M The goal of the present study was to learn how Bcl-6 and ␮ LiCl2), and 50 g of protein from the whole-cell extract was separated Blimp-1 interact during Th1/Th2 development and to determine by 8% SDS-PAGE and Western blotted using mouse monoclonal anti- the molecular mechanism(s) by which Blimp-1 alters expres- Blimp-1 (26), anti-GATA-3, and anti-␤-tubulin Abs (Santa Cruz sion of cytokines important for Th1 vs Th2 differentiation and Biotechnology). function. We show that Blimp-1 is most highly expressed in Th2 cells and that mice lacking Blimp-1 in CD4 T cells exhibit Quantitative RT-PCR impaired humoral Th2 responses, establishing a role for Total RNA was isolated according to the manufacturer’s instructions by Blimp-1 in Th2 differentiation. We also show that ifng, tbx21, using TRIzol reagent (Invitrogen) from in vitro-activated CD4 T cells and bcl6 are direct targets of Blimp-1-dependent repression in or sorted CD4 T cell subsets, and reverse transcription was performed Downloaded from CD4 T cells, providing a molecular basis for the idea that using SuperScript III (Invitrogen) following the manufacturer’s instruc- Blimp-1 helps to oppose Th1 differentiation during Th2 lineage tions.
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