Genome-Wide Identification of Novel Genes Involved in Early Th1 and Th2 Cell Differentiation This information is current as Riikka J. Lund, Maritta Löytömäki, Tiina Naumanen, Craig of October 2, 2021. Dixon, Zhi Chen, Helena Ahlfors, Soile Tuomela, Johanna Tahvanainen, Joonas Scheinin, Tiina Henttinen, Omid Rasool and Riitta Lahesmaa J Immunol 2007; 178:3648-3660; ; doi: 10.4049/jimmunol.178.6.3648 http://www.jimmunol.org/content/178/6/3648 Downloaded from References This article cites 43 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/178/6/3648.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 October 2, 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Genome-Wide Identification of Novel Genes Involved in Early Th1 and Th2 Cell Differentiation1 Riikka J. Lund,2* Maritta Lo¨yto¨ma¨ki,2*† Tiina Naumanen,* Craig Dixon,* Zhi Chen,* Helena Ahlfors,*‡ Soile Tuomela,*† Johanna Tahvanainen,*§ Joonas Scheinin,* Tiina Henttinen,* Omid Rasool,* and Riitta Lahesmaa3* Th cell subtypes, Th1 and Th2, are involved in the pathogenesis or progression of many immune-mediated diseases, such as type 1 diabetes and asthma, respectively. Defining the molecular networks and factors that direct Th1 and Th2 cell differentiation will help to understand the pathogenic mechanisms causing these diseases. Some of the key factors regulating this differentiation have been identified, however, they alone do not explain the process in detail. To identify novel factors directing the early differentiation, we have studied the transcriptomes of human Th1 and Th2 cells after 2, 6, and 48 h of polarization at the genome scale. Based ϳ on our current and previous studies, 288 genes or expressed sequence tags, representing 1–1.5% of the human genome, are Downloaded from regulated in the process during the first 2 days. These transcriptional profiles revealed genes coding for components of certain pathways, such as RAS oncogene family and G protein-coupled receptor signaling, to be differentially regulated during the early Th1 and Th2 cell differentiation. Importantly, numerous novel genes with unknown functions were identified. By using short- hairpin RNA knockdown, we show that a subset of these genes is regulated by IL-4 through STAT6 signaling. Furthermore, we demonstrate that one of the IL-4 regulated genes, NDFIP2, promotes IFN-␥ production by the polarized human Th1 lymphocytes. Among the novel genes identified, there may be many factors that play a crucial role in the regulation of the differentiation process http://www.jimmunol.org/ together with the previously known factors and are potential targets for developing therapeutics to modulate Th1 and Th2 responses. The Journal of Immunology, 2007, 178: 3648–3660. helper cell subtypes, Th1 and Th2, originate from com- STAT1 signaling are important in driving Th1 polarization, mon naive precursor cells (Thp) in response to Ag and whereas IL-4/STAT6 signaling directs Th2 polarization (2). Tran- T cytokine stimulation. Although Th cells have a crucial scription factors TBX21 (T-bet) and GATA3 are also among the role in host defense against intracellular and extracellular patho- key factors required for the Th1 and Th2 differentiation, respec- gens, disturbances in the balance between Th1 and Th2 responses tively (3–6). Although many of the players implicated in the reg- can promote or lead to pathogenesis of immune-mediated diseases. ulation of differentiation have been recognized, the current model by guest on October 2, 2021 Enhanced Th2 response is involved in atopic diseases, such as as such is still too simple to explain the process in detail, and other asthma, whereas a dominating Th1 response is implicated in cer- yet unknown factors are likely to be involved. tain autoimmune diseases, like type 1 diabetes or rheumatoid ar- Recently, an increasing number of studies have used DNA mi- thritis (1). To understand the molecular mechanisms driving the croarrays to identify new factors involved in the Th1 and Th2 pathogenesis of these diseases, it is important to elucidate the early polarization in humans and mice (7–14). However, all of these differentiation process of Th1 and Th2 cells in detail. studies have focused on studying a limited number of primarily A number of the central factors involved in directing the differ- known genes. We have previously elucidated the regulation of entiation process have been identified. IL-12/STAT4 and IFN-␥/ ϳ9300 genes, most with known functions, during the early differ- entiation of human Th1 and Th2 cells (10, 15). In the present study, we have extended the previous work by exploring the reg- *Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, ulation of the rest of the genes in the human genome. Based on the Finland; †Graduate School of Biomedical Sciences, University of Turku, Turku, Fin- ‡ combination of our current and previous studies, 288 genes or land; The National Graduate School in Informational and Structural Biology, Åbo 4 ϳ Akademi University, Turku, Finland; and §Drug Discovery Graduate School, Uni- expressed sequence tags (ESTs), representing 1–1.5% of the versity of Turku, Turku, Finland human genome, are differentially regulated during the first 2 days Received for publication March 9, 2005. Accepted for publication December of Th1 and Th2 cell polarization. Moreover, we demonstrate that 21, 2006. a panel of these genes or ESTs are induced by IL-4 through the The costs of publication of this article were defrayed in part by the payment of page STAT6 signaling or play a role in regulation of IFN-␥ production. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by the Academy of Finland, Sigrid Juse´lius Foundation, National Technology Agency of Finland, Turku Graduate School of Biomedical Sci- In vitro differentiation of Th1 and Th2 cells ences, National Graduate School in Informational and Structural Biology, Drug Dis- Induction of human Th1 and Th2 cell differentiation was performed as covery Graduate School, Ida Montin Foundation, Finnish Society of Allergology and ϩ Immunology, Pulmonary Association Heli, Jenny and Antti Wihuri Foundation, previously described (10). Briefly, CD4 T cells isolated (Ficoll Isolation Va¨ino¨and Laina Kivi Foundation, Allergy Research Foundation of South-Western Paque; Amersham Biosciences and Dynal Biotech) from cord blood (Turku Finland, and Turku University Hospital Fund. 2 R.J.L. and M.L. made an equal contribution to this work. 4 Abbreviations used in this paper: EST, expressed sequence tag; CT, threshold cycle; 3 Address correspondence and reprint requests to Prof. Riitta Lahesmaa, Turku Centre shRNA, short-hairpin RNA. for Biotechnology, University of Turku and Åbo Akademi University, P.O. Box 123, FIN-20521, Turku, Finland. E-mail address: riitta.lahesmaa@btk.fi Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 3649 Table I. Primers and probes used in the real-time RT-PCR 1) 5Ј-6(FAM)-PROBE-(TAMRA)-3Ј 2) 5Ј-PRIMER 1–3Ј Public ID Gene Symbol 3) 5Ј-PRIMER 2–3Ј AW152437 1) 2) 5Ј-AGCTGAGAAGAATGAAGAGGACATA-3Ј 3) 5Ј-GTTCACAGCCCCTATGG-3Ј AW139719 1) 2) 5Ј-AGACTGGTTTGTTTTCACTTGAGGT-3Ј 3) 5Ј-GTTTTCCCAGGAGTCTGAGGC-3Ј R98767 1) 2) 5Ј-TTTGCCTCAAATCCATTACCAA-3Ј 3) 5Ј-AACTAGTCAAGTGTGATATAATCAGATTTGC-3Ј AA489100 1) 2) 5Ј-GAAAGCAATATGTTTAGCAGCTGTTT-3Ј 3) 5Ј-ACATTTATGCCTGGATTAAATAACAATAGT-3Ј BF056901 1) 2) 5Ј-AAATAAGGCCTAGGTCCGTCTATTG-3Ј 3) 5Ј-CGGCCTCGACCTTCAAAGA-3Ј AI494573 1) Ј Ј 2)5 -GCTGGGAAATCTACAAGTCACCTTA-3 Downloaded from 3)5Ј-TTGCTGGCCATTTTATTGTTGAG-3Ј AA088177 1) 2) 5Ј-ATGCAAGGTGGAATTTTGGG-3Ј 3) 5Ј-GTATTGCAGTTGTGGAAGAGCAGA-3Ј BE748563 1) 2) 5Ј-CCTTTCACTGCCATGGAATGA-3Ј 3) 5Ј-AGAAAATGCAAGCTCCCCATAA-3Ј AI674404 1) http://www.jimmunol.org/ 2) 5Ј-CTGGAACCTTGAGGCCTTCA-3Ј 3) 5Ј-GCCCCAATCTATGGACAGACA-3Ј AL389942 1) 5Ј-TGTGACAATGATTCTTTAGC-3Ј 2) 5Ј-CACGGGATCAGAGGGAACTAATA-3Ј 3) 5Ј-ACCAGAAGCCTCAGCAGTCC-3Ј AA237039 1) 5Ј-TTTATGCAGCGCACTGTCAGACTTCCAA-3Ј 2) 5Ј-GATGCATCTGCTTTTAACCCTTTT-3Ј 3) 5Ј-TTACGGAATGCTGTGGTACTCAA-3Ј AW629527 FLJ41238 1) 5Ј-ACCCATTTTGATTGAGACCTACACAGGGC-3Ј 5Ј-TGGAAGAGGAAAGAAACTGATGGT-3Ј 2) by guest on October 2, 2021 3) 5Ј-GCGGTTTCCAATGAATGACA-3Ј J04617 EF1␣ 1) 5Ј-AGCGCCGGCTATGCCCCTG-3Ј 2) 5Ј-CTGAACCATCCAGGCCAAAT-3Ј 3) 5Ј-GCCGTGTGGCAATCCAAT-3Ј NM_016651 DACT1 1) 5Ј-CACGAACTCGCCCTCGCTCACACT-3Ј 2) 5Ј-AGCAGAGCAATTACACCACCAA-3Ј 3) 5Ј-AGTCTGGACAAACTGGGACCAA-3Ј NM_005815 ZNF443 1) 5Ј-TCTCTTGGCTCACTTGCTTTCTACGACATG-3Ј 2) 5Ј-GAATGTAAGGAATGTGGGAAAGC-3Ј 3) 5Ј-CATAGGATTTCTCTCTCATGTGAATTCT-3Ј University Central Hospital, Turku, Finland)