The Transcription Factor E74-Like Factor 4 Suppresses Differentiation of Proliferating CD4+ T Cells to the Th17 Lineage This information is current as Ping-Hsien Lee, Monica Puppi, Kimberly S. Schluns, of September 27, 2021. Li-Yuan Yu-Lee, Chen Dong and H. Daniel Lacorazza J Immunol published online 20 November 2013 http://www.jimmunol.org/content/early/2013/11/19/jimmun ol.1301372 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 27, 2021 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 20, 2013, doi:10.4049/jimmunol.1301372 The Journal of Immunology The Transcription Factor E74-Like Factor 4 Suppresses Differentiation of Proliferating CD4+ T Cells to the Th17 Lineage Ping-Hsien Lee,*,† Monica Puppi,*,† Kimberly S. Schluns,‡ Li-Yuan Yu-Lee,x Chen Dong,‡ and H. Daniel Lacorazza*,†,{ The differentiation of CD4+ T cells into different Th lineages is driven by cytokine milieu in the priming site and the underlying transcriptional circuitry. Even though many positive regulators have been identified, it is not clear how this process is inhibited at transcriptional level. In this study, we report that the E-twenty six (ETS) transcription factor E74-like factor 4 (ELF4) suppresses the differentiation of Th17 cells both in vitro and in vivo. Culture of naive Elf42/2 CD4+ T cells in the presence of IL-6 and TGF-b (or IL-6, IL-23, and IL-1b) resulted in increased numbers of IL-17A–positive cells compared with wild-type controls. In contrast, Downloaded from the differentiation to Th1, Th2, or regulatory T cells was largely unaffected by loss of ELF4. The increased expression of genes involved in Th17 differentiation observed in Elf42/2 CD4+ T cells suggested that ELF4 controls their programming into the Th17 lineage rather than only IL-17A gene expression. Despite normal proliferation of naive CD4+ T cells, loss of ELF4 lowered the requirement of IL-6 and TGF-b signaling for IL-17A induction in each cell division. ELF4 did not inhibit Th17 differentiation by promoting IL-2 production as proposed for another ETS transcription factor, ETS1. Elf42/2 mice showed increased numbers of Th17 cells in the lamina propria at steady state, in lymph nodes after immunization, and, most importantly, in the CNS following http://www.jimmunol.org/ experimental autoimmune encephalomyelitis induction, contributing to the increased disease severity. Collectively, our findings suggest that ELF4 restrains Th17 differentiation in dividing CD4+ T cells by regulating commitment to the Th17 differentiation program. The Journal of Immunology, 2014, 192: 000–000. he main function of CD4+ T cells is to provide help to APC activate naive CD4+ T cells via TCR recognition, co- cells of the innate and adaptive immune system. There are stimulation, and secretion of cytokines that induce differentiation different Th cell lineages with unique features (i.e., Th1, into effector cells. These cytokines imprint unique molecular T + by guest on September 27, 2021 Th2, and Th17 cells) that modulate immune responses against signatures on CD4 T cells by creating a transcriptional program infections and tumor growth by promoting the effector functions that controls lineage commitment and effector functions. In the and memory formation of CD8+ T cells, inducing Ab class switching case of Th17 cells, potent inducers of tissue inflammation, IL-6 of B cells, and enhancing the activity of phagocytes (1, 2). Lineage (or IL-21), and TGF-b initiate the differentiation program in ac- differentiation of CD4+ T cells is driven by TCR activation and tivated CD4+ T cells by inducing expression of the master regu- a specific cytokine milieu. Alterations in the balanced generation of lator RAR-related orphan receptor g t(RORgt), which subsequently different Th cells often lead to immunodeficiencies or autoimmune turns on IL-17A and other Th17-associated genes (3–6). Differen- disorders (2). Although many studies have focused on identifying tiating Th17 cells are not fully functional until receiving IL-23 specific regulators of CD4+ T cell differentiation, the cell-intrinsic signal, which stabilizes and expands in vivo Th17 responses in mechanisms downstream of the TCR and cytokine receptors are a STAT3-dependent manner (7). Consequently, transcription factors largely unknown. are important cell-intrinsic mediators that translate environmental cues into effector functions of Th17 cells. However, negative reg- ulators that prevent excessive Th17 responses are ill defined. *Department of Pathology & Immunology, Baylor College of Medicine, Houston, E74-like factor 4 (ELF4) is a member of the E-twenty six (ETS) TX 77030; †Department of Pathology, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030; ‡Department of Immunology, M.D. Anderson Cancer family transcription factors with a highly conserved ETS domain x Center, Houston, TX 77030; Immunology, Allergy and Rheumatology Section, De- that mediates protein–DNA and protein–protein interactions (8). partment of Medicine, Baylor College of Medicine, Houston, TX 77030; and {De- partment of Pediatrics, Baylor College of Medicine, Houston, TX 77030 Because ETS proteins generally work in concert with other cor- egulatory proteins by forming supramolecular complexes, both Received for publication May 22, 2013. Accepted for publication October 19, 2013. protein–DNA and protein–protein interactions contribute to their This work was supported by National Institutes of Health Grants R01AI077536 and R01AI077536-02S1. transcriptional activity and specificity (8, 9). Some ETS family Address correspondence and reprint requests to Dr. H. Daniel Lacorazza, Department proteins have been linked to carcinogenesis because of their roles of Pathology & Immunology, Baylor College of Medicine, One Baylor Plaza, Hous- in cellular proliferation, differentiation, and apoptosis (8–11). ton, TX 77030. E-mail address: [email protected] Given that certain ETS transcription factors such as ETS1 and Abbreviations used in this article: EAE, experimental autoimmune encephalomyeli- PU.1 are involved in Th cell differentiation (12–16), we decided to tis; ELF4, E74-like factor 4; ETS, E-twenty six; IRF4, IFN regulatory factor 4; LTi, investigate the role of ELF4 in this process. ELF4 is widely lymphoid tissue inducer; MOG35–55, myelin oligodendrocyte gp35–55; mTOR, mam- malian target of rapamycin; NICD, Notch1 intracellular domain; RORgt, RAR- expressed in several tissues, including bone marrow, thymus, and related orphan receptor g t; RUNX, Runt-related transcription factor; Treg, regula- the spleen (17). ELF4 regulates cell cycle progression in hema- tory T; WT, wild-type. topoietic stem cells and endothelial cells and has both tumor Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 suppressor and oncogenic activity (18–21). In the immune system, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301372 2 ELF4 INHIBITS DIFFERENTIATION OF Th17 CELLS ELF4 plays important roles in both innate and adaptive immune proliferation was determined by CFSE dilution using flow cytometry. For + 2 cells, as embryonic deletion of ELF4 resulted in impaired lytic differentiation assays, CD4 CD25 T cells were stimulated with 10 mg/ml activity of NK cells as well as aberrant proliferation and traf- plate-bound anti-CD3 and 2 mg/ml soluble anti-CD28 Ab in the presence of + indicated cytokines and neutralizing Ab. Th0: 10 ng/ml IL-2 (PeproTech, ficking of CD8 T cells (22, 23). Given that ELF4 is generally Rocky Hill, NJ), 5 mg/ml anti–IFN-g (XMG1.2; BioLegend), and 5 mg/ml considered a transcriptional activator, its aforementioned effects anti–IL-4 (11B11; BioLegend); Th1: 10 ng/ml IL-2, 40 ng/ml IL-12 on NK cells and CD8+ T cells were caused at least in part by direct (PeproTech), and 5 mg/ml anti–IL-4; Th2, 10 ng/ml IL-2, 50 ng/ml IL-4 regulation of the Prf1 and Klf4 genes, respectively (22, 23). We (PeproTech), and 5 mg/ml anti–IFN-g; regulatory T (Treg), 10 ng/ml IL-2, 5 ng/ml TGF-b (PeproTech), 5 mg/ml anti–IFN-g,and5mg/ml anti–IL-4; previously showed that TCR activation leads to rapid downregu- Th17, 30 ng/ml IL-6 (PeproTech), 0.1 ng/ml TGF-b,5mg/ml anti–IFN-g, + lation of ELF4 transcripts in naive CD4 T cells (24), suggesting and 5 mg/ml anti–IL-4, unless otherwise indicated. Cells were analyzed a regulatory role of ELF4 in TCR-mediated biological processes for intracellular levels of IFN-g, IL-4, IL-17A, or Foxp3 after 3 d. such as T cell differentiation. Inhibition of Notch1 signaling during Th17 differentiation was ach- In this work, we report that loss of ELF4 specifically enhanced ieved using the g-secretase inhibitor compound E (Enzo Life Sciences, Farmingdale, NY). Th17 differentiation both in vitro and in vivo. ELF4 did not sig- nificantly affect the proliferation or survival of CD4+ T cells but RNA isolation and quantitative RT-PCR instead regulated commitment to the Th17 differentiation program + Total RNA was extracted from CD4 T cells using RNeasy Plus Mini kit downstream of STAT1, STAT3, STAT5, and SMAD2/3 proteins, (Qiagen, Valencia, CA), and cDNA was synthesized from 100–500 ng RNA most likely by suppressing Notch1 signaling.