Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021 is online at: average * The Journal of Immunology , 16 of which you can access for free at: 2014; 192:2667-2676; Prepublished online 14 from submission to initial decision 4 weeks from acceptance to publication February 2014; doi: 10.4049/jimmunol.1302605 http://www.jimmunol.org/content/192/6/2667 Differential Requirement for Nfil3 during NK Cell Development Cyril Seillet, Nicholas D. Huntington, Pradnya Gangatirkar, Elin Axelsson, Martina Minnich, Hugh J. M. Brady, Meinrad Busslinger, Mark J. Smyth, Gabrielle T. Belz and Sebastian Carotta J Immunol cites 37 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2014/02/14/jimmunol.130260 5.DCSupplemental This article http://www.jimmunol.org/content/192/6/2667.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of October 5, 2021. The Journal of Immunology Differential Requirement for Nfil3 during NK Cell Development Cyril Seillet,*,† Nicholas D. Huntington,*,† Pradnya Gangatirkar,* Elin Axelsson,‡ Martina Minnich,‡ Hugh J. M. Brady,x Meinrad Busslinger,‡ Mark J. Smyth,{ Gabrielle T. Belz,*,† and Sebastian Carotta*,† NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow–derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of + 2 Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL Eomes NK cells develop Downloaded from independently of Nfil3. Loss of Nfil3 during the development of bone marrow–derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil32/2 progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3. The Journal of Immunology, 2014, 192: 2667–2676. he innate immune system provides a front-line defense cells in vitro (2). Using a reporter mouse model in which GFP is http://www.jimmunol.org/ against invading pathogens and cells undergoing malig- expressed under the control of the transcription factor Id2 (3, 4), T nant transformation. NK cells are an essential component we purified an Id2-GFP+ NKP subset that efficiently gave rise to of this rapid-response armory, largely because of their ability to NK cells and had lost T cell potential (3). Subsequently, Fathman detect and kill target cells without prior sensitization. The majority et al. (5) demonstrated that a pure NKP population could be iso- of NK cells in the adult mouse are thought to develop in the bone lated without the use of the ID2-GFP reporter mouse model by marrow (BM) from common lymphoid progenitors (CLP) that give selecting Lin2CD27+CD244+CD122+IL7Ra+Flt32 cells from the rise to committed NK cell precursors (NKP) (1). Until recently, the BM. Developing from NKP, immature NK (iNK) cells acquire NKP was thought to be the earliest known committed NK cell NK1.1 expression and subsequently give rise to CD122+NK1.1+ by guest on October 5, 2021 within the BM. NKP are identified through the expression of IL- NKp46+CD49b+ mature NK (mNK) cells that gain effector function 2b-chain (CD122) and the lack of NK cell surface markers NK1.1 and circulate throughout the body. These conventional NK cells and CD49b (2). Despite this, it is clear that this population is (cNK) express high levels of CD49b (DX5) and secrete high levels heterogeneous, because ,30% of cells can differentiate into NK of IFN-g and IL-13 (1, 6). In addition to BM, the liver and thymus are sites of NK cell development (1). Thymic NK cells are derived from early thymic precursors and differ from cNK cells in that they *Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; †Department of Medical Biology, University of Melbourne, Parkville retain high levels of IL-7Ra, depend on the transcription factor 3010, Australia; ‡Research Institute of Molecular Pathology, Vienna Biocenter, x Gata-3, and are competent cytokine producers but have poor cyto- 1030 Vienna, Austria; Department of Life Sciences, Imperial College, London + { toxicity (7). In addition to liver-resident CD49b cNK cells, a pop- SW7 2AZ, United Kingdom; and Queensland Institute of Medical Research, 2 Brisbane, Queensland 4006, Australia ulation of CD49b NK cells that constitutively express TRAIL + Received for publication September 27, 2013. Accepted for publication December exists (8, 9). TRAIL NK cells constitute the main NK cell pop- 16, 2013. ulation in fetal and neonatal mice and decrease with age, but a This work was supported by a project grant from the National Health and Medical stable population is retained during adulthood in liver. Function- Research Council of Australia (APP1027472), the Victorian State Government Op- ally, TRAIL+ NK cells are poor secretors of IFN-g and IL-13, and erational Infrastructure Support, and the Australian Government National Health and Medical Research Council of Australia Independent Research Institutes Infrastruc- they were reported to possess memory-like properties (10, 11). ture Support Scheme. Research by the Busslinger group was supported by Boehringer The transcriptional network that regulates NK cell commitment Ingelheim and the Austrian Genome Research in Austria initiative. S.C. was sup- and controls the different NK cell fates is not well understood (12). ported by a National Health and Medical Research Council of Australia Career Development Fellowship, N.D.H. was supported by a C.J. Martin Fellowship, and Recent evidence suggests that different transcription factor net- G.T.B. was supported by an Australian Research Council Future Fellowship. works drive the formation of these distinct subsets. The tran- Address correspondence and reprint requests to Dr. Sebastian Carotta and Dr. Gabrielle scription factors T-bet and Eomes were described to coordinately T. Belz, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, control the generation of BM-derived CD49b+ NK cells, whereas VIC 3052, Australia. E-mail addresses: [email protected] (S.C.) and belz@wehi. + edu.au (G.T.B.) Eomes expression is dispensable for TRAIL NK cell develop- The online version of this article contains supplemental material. ment (13). The downstream molecular events and transcription Abbreviations used in this article: ALP, all lymphoid progenitor; BLP, B cell–biased factors that induce the expression of Eomes have not been iden- progenitor; BM, bone marrow; CLP, common lymphoid progenitor; cNK, conven- tified. In addition to its role in thymic NK cell development, Gata- tional NK; iNK, immature NK; mNK, mature NK; NKP, NK cell precursor; WT, 3 is implicated in the homing of NK cell progenitors to the liver wild-type. (7, 14). Id2 and Nfil3 are accepted to be important regulators of Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 cNK cell development; however, how they orchestrate this, as well www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302605 2668 TRANSCRIPTIONAL REGULATION OF NK DEVELOPMENT as their role in the development of alternative NK cell subsets, is following TaqMan assays were used: IL-21R (Mm00600319_m1), Lyl1 not clear. Deletion of either Id2 or Nfil3 results in a severe re- (Mm00493219_m1), Sell (Mm00441291_m1), CXCR4 (Mm01292123_m1), duction in cNK cells in the BM, spleen, liver, and lung (15–19). It and CXCR6 (Mm02620517_s1). Primer sequences are available upon request. was proposed that Nfil3 regulates the expression of Id2, because Nfil3-deficient NK cell progenitors were reported to exhibit re- Solexa deep sequencing duced Id2 expression compared with controls, and ectopic ex- 2/2 The transcriptome of the indicated samples was determined as described pression of Id2 partially rescued Nfil3 NK cell development (21). (15). In both Id2- and Nfil3-deficient mice, the generation of CD122+NK1.12CD49b2 NKP was unaffected, yet iNK cells were Statistical analysis strongly reduced. However, given that only ∼30% of NKP possess GraphPad Prism software (GraphPad Software) was used for statistical NK cell potential, it is not clear precisely when Id2 and Nfil3 act analysis. The two-tailed Mann–Whitney U test or unpaired Student t test , to control NK cell commitment. was used for comparisons. A p value 0.05 was regarded as significant. All results are expressed as mean 6 SEM. In this article, we show that cNK cells depend on Nfil3 and that it is critical for the development of thymic NK cells. Strikingly, we found that the generation of TRAIL+ liver NK cells in adult mice Results and neonates occurs independently of Nfil3. Furthermore, we Nfil3 expression during early NK cell development show that Nfil3 promotes cNK cell development by regulating the Nfil3 was shown to be expressed at low levels in Lin2 hemato- expression of the transcription factor Eomes.