Differentiation Involved in Early Th1 and Th2 Cell Genome-Wide

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 September 28, 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 Downloaded from http://www.jimmunol.org/content/178/6/3648

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/

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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 on our current and previous studies, 288 genes or expressed sequence tags, representing ϳ1–1.5% 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. http://www.jimmunol.org/ Among the novel genes identified, there may be many factors that play a crucial role in the regulation of the differentiation process 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- by guest on September 28, 2021 can promote or lead to pathogenesis of immune-mediated diseases. ulation of differentiation have been recognized, the current model 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 differentiation 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 Vaïnoänd 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) Downloaded from 2)5Ј-GCTGGGAAATCTACAAGTCACCTTA-3Ј 3)5Ј-TTGCTGGCCATTTTATTGTTGAG-3Ј AA088177 1) 2) 5Ј-ATGCAAGGTGGAATTTTGGG-3Ј 3) 5Ј-GTATTGCAGTTGTGGAAGAGCAGA-3Ј BE748563 1) 2) 5Ј-CCTTTCACTGCCATGGAATGA-3Ј

3) 5Ј-AGAAAATGCAAGCTCCCCATAA-3Ј http://www.jimmunol.org/ AI674404 1) 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 2) 5Ј-TGGAAGAGGAAAGAAACTGATGGT-3Ј by guest on September 28, 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) were activated with plate- Oligonucleotide array studies bound anti-CD3 (500–1000 ng/ml for coating) and 500 ng/ml soluble anti- ϳ CD28 (both from Immunotech). Th1 polarization was induced with 2.5 Human genome U133A and B arrays recognizing 33,000 transcripts were ng/ml IL-12 and Th2 differentiation with 10 ng/ml IL-4 (both from R&D rehybridized with the previously prepared samples (10, 15). Briefly, after Systems). A subset of the cells was cultured in “neutral conditions” without confirming the successful polarization of the cells with real-time RT-PCR, polarizing cytokines. In the indicated experiments, the cultures were the sample preparation and data analysis were performed according to the supplemented with 3 ng/ml TGF-␤ (R&D Systems). TGF-␤-mediated instructions and recommendations provided by the manufacturer (Af- suppression of IFN-␥ production by Th1 cells in these conditions has fymetrix). Total RNA (4–5 ␮g) pooled from different individuals was used been previously described (10). The samples were collected after 0, 2, as starting material for the Affymetrix sample preparation. Two biological 6, or 48 h of polarization and were processed for Affymetrix hybrid- repeats for each microarray experiment were performed. GeneChip Mi- izations or Western blotting. croarray Suite software version 5 (MAS5; Affymetrix), GeneSpring For validation of the oligonucleotide array results with real-time RT- (SiliconGenetics), and Microsoft Access for Windows software were used to PCR, additional Th1 and Th2 primary cultures were generated as described evaluate the quality of the data and for routine data analysis and processing. in our previous study (11). Briefly, cord blood CD4ϩ T cells were activated The microarray data was filtered according to the statistical classifications per- with 100 ng/ml PHA (Murex) and irradiated CD32-B7-transfected fibro- formed by the MAS5 software as previously described (10, 15). Genes that blasts (16). Th1 cultures were supplemented with 2.5 ng/ml IL-12, whereas presented a consistent change (Ն2-fold) in two separate biological repeats Th2 cultures were supplemented with 10 mg/ml anti-IL-12 and 10 ng/ml were considered as differentially expressed. All the genes, which fulfilled IL-4 (all from R&D Systems). After 48 h of priming, 40 U/ml IL-2 (R&D these criteria in at least one of the comparisons and one of the time points, Systems) was added into the cultures. A subset of the cells was cultured were selected for further analysis where the expression of the genes was without any polarizing cytokines in the presence of IL-2 alone. The cul- explored parallel in different conditions without fold change threshold. The tures were generated from four individuals. Samples were collected at time gene annotations were obtained from NetAffx database (17). The normal- points 0, 6, 24, and 48 h or 7 days. ized microarray raw data have been deposited in the Gene Expression 3650 EARLY Th1 AND Th2 CELL DIFFERENTIATION

Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information and are accessible through GEO Series acces- sion number GSE2770. Real-time quantitative RT-PCR To validate the oligonucleotide array results for the selected genes, either probe-based or SYBR Green-based real-time quantitative RT- PCR (TaqMan ABI Prism 7700; Applied Biosystems) was performed as described before (11, 16). The housekeeping gene EF1␣ was used as a reference transcript (16). Primers and probes (Table I) used for the quantiﬁcation of gene expression (MedProbe or DNA Technology) were designed using Primer Express software (Applied Biosystems). The quantitative value obtained from TaqMan real-time RT-PCR is a threshold cycle (CT). The fold differences between different conditions can be calculated from the nor- malized CT values (CT gene X Ϫ CT housekeeping gene), ⌬CT values, with the formula: fold difference ϭ 2(I⌬CT1 Ϫ⌬CT2I). Statistical signiﬁ- cances between the differences in gene expression were evaluated with t tests. Western blot analysis CD4ϩ T cells were isolated as described above and polarized to the Th0,

Th1, or Th2 direction. The cells harvested after 2, 6, and 48 h of polar- Downloaded from ization were lysed in SDS buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM DTT). Equal protein amounts of whole cell lysates were loaded on the gel. Alternatively, cells were lysed in HEPES buffer (20 mM HEPES, 0.2% Tween 20, and 1 mM DTT) containing Com- plete Protease Inhibitor (Roche). From the centrifuged lysates, the super- FIGURE 1. Summary of the genes regulated by CD3 plus CD28 acti- natants were discarded and a second buffer (20 mM HEPES, 420 mM vation, IL-12, and IL-4. Expression profiles of the cells activated with NaCl, 20% glycerol, and 1 mM DTT; Roche Complete Protease Inhibitor) anti-CD3 plus anti-CD28 alone (Th0) or induced to differentiate to Th1 http://www.jimmunol.org/ was mixed with the cell pellets. After1hofincubation on ice, the samples (anti-CD3 plus anti-CD28 plus IL-12) or Th2 (anti-CD3 plus anti-CD28 were recentrifuged and supernatants containing nuclear proteins were ob- plus IL-4) direction were studied with oligonucleotide arrays after 0, 2, 6, tained. The protein concentrations were then quantified from the samples and 48 h of polarization. A, The total number of genes regulated by CD3 by Bio-Rad protein assay. Equal amounts of protein were subject to SDS- plus CD28 activation, IL-12, and/or IL-4 during the early Th1 and Th2 cell PAGE and transferred to Hybond ECL membrane (Amersham Bio- differentiation. B, The number of genes up- (1) or down-regulated (2)by sciences). Uniform protein transfer was verified with Ponceau S staining (Sigma-Aldrich). The membranes were probed for TBX21 (sc-21749; IL-4 at 2, 6, and/or 48 h of Th2 cell differentiation. Santa Cruz Biotechnology) and GATA3 (sc-268) to confirm successful induction of Th1/Th2 polarization. Western blotting was then performed for DACT1 (ab5977-100; Abcam), RAB11FIP1 (RCP 11-A; Alpha Diag- centrifugation and stored at Ϫ70°C until used for cytokine determination. nostic), FOSL2/Fra-2 (sc-604), CISH (sc-1529), ZF9 (sc-7158), FLT1 (sc- Secreted cytokines were measured using Luminex assay and multiplex by guest on September 28, 2021 9029), and RAB27B (sc-22991) all from Santa Cruz Biotechnology. bead kits from LINCO Research and from Bio-Rad. The assays were conducted in duplicate according to the manufacturer’s instructions. Measure- Short-hairpin RNA (shRNA) mediated gene knockdown during ments and data analysis were performed with the Bio-Plex system in com- the early polarization of human Th cells bination with the Bio-Plex Manager software (Bio-Rad). The NDFIP2 shRNA plasmid construct, targeting (5Ј-GAGGAAGAGTGT CCACCAAGA-3Ј), was generated by cloning the NDFIP2 shRNA oligo- Results nucleotide into the BglII and XhoI sites of the previously modified pSuper- To identify novel genes involved in the initiation and early polar- H2K-pIRES2 plasmid, which contains a truncated H2K cell surface selection ization of Th1 and Th2 cells at the genome scale, transcriptome Ј marker (18). Similarly, a pSuper-H2K-pIRES2-scramble1-shRNA, (5 -AATT analysis was conducted using Affymetrix U133A and B oligonu- CTCCGAACGTGTCACGT-3Ј), was designed. In addition, two synthetic small interfering RNA oligos (one of which targeting the same sequence as the cleotide arrays. To identify genes regulated by IL-12 or IL-4 in NDFIP2 shRNA shown above and the other targeting the 5Ј-CUGGAUAU activated Th cells, the cells induced to polarize to Th1 (CD3 plus UUCAAUGGACAUU-3Ј NDFIP2 sequence; Sigma-Aldrich) were used to CD28 plus IL-12) or Th2 (CD3 plus CD28 plus IL-4) direction for knock down NDFIP2. For the STAT6 knockdown studies, the previously pre- 2, 6, or 48 h were compared with each other and to the CD3 plus pared pSuper-H2K-STAT6-shRNA plasmid, targeting (5Ј-GAATCAGTCA ACGTGTTGTCAG-3Ј), and the pSuper-H2K-scramble2-shRNA (5Ј-GCGC CD28-activated cells cultured without polarizing cytokines (Th0). GCTTTGTAGGATTCG-3Ј) were used (18). Furthermore, another pSuper- Furthermore, genes coregulated by TGF-␤ and IL-12 or IL-4 dur- H2K-STAT6-shRNA plasmid, targeting 5Ј-CAGTTCCGCCACTTGCCAAT- ing the early Th1 and Th2 cell differentiation were identified. In 3Ј, was used in one replicate culture. Cell transfections, dead cell removal, addition to the previously identified genes, an additional 171 genes Ͼ enrichments ( 98%), and differentiations were performed as recently de- were found to be regulated by IL-12 or IL-4 (Fig. 1A) during the scribed (18). The cells were activated with anti-CD3 plus anti-CD28 and were induced to polarize to Th1 and Th2 direction as described above. For RT-PCR first 2 days of Th1 and Th2 differentiation (see Table II for all the analysis the cells were harvested at 24 or 48 h of polarization from three to four IL-12- and IL-4-regulated genes) (10, 15). biological replicates. Cells harvested at 7 days of polarization were washed, restimulated, and cytokines secreted in the supernatant after 24 h of restimu- The slow response to IL-12 lation were measured (see the section below). For RT-PCR, the total RNAs were isolated either with an RNA Easy Minikit (Qiagen) or a PicoPureTM Consistent with our previous observations, IL-12 had minimal or RNA Isolation kit (Arcturus). Consequently, cDNAs were prepared with a no effect on the polarization process after 2 or 6 h (15). After 2 h Transcription First Strand cDNA Synthesis kit (Roche). of Th1 polarization, there were no genes regulated by IL-12. After Ͻ Cytokine secretion assay 6h, 2-fold induction by IL-12 was seen in the expression of key regulator of Th1 differentiation TBX21 and GTPase GBP5 (3, 4). To measure the IFN-␥ production by the control cells or cells transfected After 48 h, the effects of IL-12 were clear and altogether 41 genes with NDFIP2-shRNA, the cells were incubated with or without 5 ng/ml PMA (Calbiochem) and 500 ng/ml ionomycin (Sigma-Aldrich). The cul- became regulated by IL-12 (Table II). Among these were only a ture medium from each well was collected after 24 h of restimulation with few genes, such as SOCS3, CEBPB, and IL-7R that have a previ- PMA and ionomycin. The culture medium was separated from the cells by ously described role in Th1 and Th2 cell responses (13, 19, 20). The Journal of Immunology 3651

Table II. Genes regulated by IL-12 and/or IL-4 during the early Th1 and Th2 cell differentiation

Fold Changea Fold Change Fold Change Fold Change

Th1 vs Th0 Th2 vs Th0 Th1 vs Th2 Th0 vs Thp

Public ID Gene 2h 6h 48h 2h 6h 48h 2h 6h 48h 2h 6h 48h

Genes regulated by both IL-12 and IL-4 1. Enzyme regulator activity (2) NM_003955 SOCS3 3.1 2.4 2.6 Ϫ2 Ϫ9.2 Ϫ13.9 Ϫ5.5 NM_021158 TRIB3 (5)b 1.4 1.8 1.8 1.6 3.9 2. Signal transducer activity (2) NM_006137 CD7 2.1 2 BE217880 IL7R 2.3 Ϫ1.4 Ϫ2.5 Ϫ25.1 3. Transcription regulator activity (4) NM_013351 TBX21 1.6 Ϫ1.5 3.2 10.2 2.8 NM_024508 ZBED2 3.7 2.9 3.7 Ϫ2.9 10.9 16 AB059408 HOP 2.9 3.3 AL564683 CEBPB 1.4 1.7 Ϫ2.7 4. Structural molecule activity (1) NM_005572 LMNA 2.5 1.5 1.8 27.9 7

5. Catalytic activity (4) Downloaded from NM_021158 TRIB3 (1) 1.4 1.8 1.8 1.6 3.9 BG545653 GBP5 1.4 Ϫ1.8 2.5 2.7 AB000888 PPAP2A Ϫ1.5 2.4 Ϫ2 Ϫ2.1 4.4 AI631159 SLC2A3 1.7 1.6 Ϫ6.5 Ϫ2 6. Other binding molecules (5) NM_018326 GIMAP4 2.1 Ϫ1.7 Ϫ2.4 1.9 2.7 2.5 AI674404 SYTL3 1.8 3.2 2.8 Ϫ5.1 Ϫ1.9 Ϫ4.3 2.4 AW574798 KLHL6 1.6 2.3 4.1 2.6 http://www.jimmunol.org/ AI524095 LY9 Ϫ1.7 Ϫ2.5 1.9 Ϫ2.9 Ϫ1.9 AI005043 WASPIP Ϫ1.7 Ϫ2.1 7. Miscellaneous (2) AI042152 TncRNA 2.2 1.9 Ϫ1.5 Ϫ1.6 AW290956 NDFIP2 1.3 1.9 1.5 3 Ϫ2.2 Ϫ1.5 Ϫ2.3 16 48.5 142 8. Unknown (4) AK026646 SURF4 1.6 2.2 2.1 2.9 1.8 AI214996 LOC284018 1.6 4.8 Ϫ5.1 8.3 BF968270 SLC35F3 Ϫ2.8 Ϫ1.8 Ϫ1.6 11.7 13.9

BF209337 MGC4677 Ϫ1.7 1.5 Ϫ1.9 Ϫ1.6 2.7 9.2 9.5 by guest on September 28, 2021 Genes regulated by IL-12 1. Enzyme regulator activity (1) NM_001629 ALOX5AP Ϫ1.2 Ϫ1.9 1.9 2. Signal transducer activity (2) NM_016081 KIAA0992 8 AL121985 SLAMF7 1.8 12.1 3. Catalytic activity (3) Y13786 ADAM19 2.7 4.3 AW150720 RDH10 1.5 10.2 3.2 AL354872 CTH 2.2 3.6 4. Other binding molecules (4) AF176013 DNAJC12b 4.3 16.6 AK021850 FBXO16 /// ZNF395 2 Ϫ3.5 Ϫ4.4 NM_001311 CRIP1 Ϫ2.1 Ϫ2.8 5.3 4.1 NM_002961 S100A4 Ϫ2.1 Ϫ2.4 Ϫ2.5 Ϫ9.8 5. Miscellaneous (3) AV713773 MCOLN2 2.3 8.3 AA195074 TIFA 1.6 1.7 2.2 Ϫ2.3 4 J03223 PRG1 Ϫ1.9 Ϫ2.4 3.2 2.9 6. Unknown (5) AF142573 CRISPLD1 2.9 BG110811 LOC340061 2.6 Ϫ3.5 AA831661 MGC29814 1.7 Ϫ5.5 Ϫ5.9 H99792 Ϫ4.6 8 BE222344 Ϫ2.5 1.9 3 5.3 4.3 Genes regulated by IL-4 1. Enzyme regulator activity (4) NM_003807 TNFSF14 (2.2) Ϫ1.5 2.5 Ϫ6.1 430.5 163.1 AI798823 PSCD1 5.9 Ϫ4.3 Ϫ5.1 Ϫ2.5 NM_000100 CSTB 1.4 Ϫ2.1 2.2 2.6 NM_004369 COL6A3 (4) 13.5 1.9 Ϫ6.3 12.1 2. Signal transducer activity (17) 2.1 Receptor activity (10) AU159276 CYSLTR1b 2.7 Ϫ3 AA830854 FLJ31951 (5.1) 2.2 Ϫ2 2.1 (Table continues) 3652 EARLY Th1 AND Th2 CELL DIFFERENTIATION

Table II. (Continued)

Fold Changea Fold Change Fold Change Fold Change

Th1 vs Th0 Th2 vs Th0 Th1 vs Th2 Th0 vs Thp

Public ID Gene 2h 6h 48h 2h 6h 48h 2h 6h 48h 2h 6h 48h

AF312230 HRH4 3.9 Ϫ3.5 AA149648 FLT1b (5.2) 2.8 Ϫ3.1 8 BG170541 MET (5.2) 2.7 Ϫ5.1 AW193698 TGFBR3 2.5 Ϫ2 AF053712 TNFSF11 (2.2) 1.7 Ϫ1.6 Ϫ1.7 4.6 7.2 AL121905 PTPRA (5.3) 2.1 NM_001838 CCR7 Ϫ2 1.5 2.1 Ϫ4.3 Ϫ1.9 AF261135 GPR18 Ϫ1.6 Ϫ2.2 Ϫ2.4 2.2 Receptor binding (5) NM_003807 TNFSF14 (1) Ϫ1.5 2.5 Ϫ6.1 430.5 163.1 NM_000572 IL10 2.5 AF053712 TNFSF11 (2.1) 1.7 Ϫ1.6 Ϫ1.7 4.6 7.2 NM_014751 MTSS1 Ϫ2.6 Ϫ2.1 U57059 TNFSF10 Ϫ1.9 Ϫ2.8 3.4

3. Transcription regulator activity (7) Downloaded from AI924426 ELL2 1.9 1.8 2.6 Ϫ1.8 Ϫ1.7 3.2 NM_005815 ZNF443 4 2.5 Ϫ3.2 Ϫ2.5 AK022280 PHF20L1b 2.5 2.3 Ϫ2.5 Ϫ2.1 BG485129 LOC360030 2.1 Ϫ1.5 AK023816 EZH2 1.8 Ϫ2.1 AI670862 FOSL2 2.2 2.9 Ϫ1.9 Ϫ2.5 Ϫ3 AW299558 FRBZ1 Ϫ4.1 4.1 4. Structural molecule activity (4) http://www.jimmunol.org/ AK022771 TUBD1 1.9 Ϫ2.1 NM_004369 COL6A3 (1) 13.5 1.9 Ϫ6.3 12.1 AF101051 CLDN1 2.6 Ϫ1.5 Ϫ1.9 16 12.1 2 AF308301 MRPS26 3.5 Ϫ3.1 5. Catalytic activity (37) 5.1 Ligase (6) NM_017831 RNF125 2.4 4.1 Ϫ2.4 Ϫ4.4 Ϫ4.6 Ϫ6.5 Ϫ4.8 AW070573 RNF146 3.9 Ϫ2.8 NM_021178 CCNB1IP1 2.5 Ϫ1.9 2.9

AA830854 FLJ31951 (2.1) 2.2 Ϫ2 2.1 by guest on September 28, 2021 AB029316 RNF19 1.4 3.1 Ϫ1.6 Ϫ2.1 4.8 2.4 BG171548 NCE2 1.9 Ϫ3 2.5 1.6 5.2 Transferase (13) R98767 CAMK2D 3 1.7 Ϫ4.4 Ϫ2.1 AI221707 STK17B 2.6 Ϫ2.7 BF739767 DKFZp761P0423 2.5 NM_017719 SNRK 2.2 Ϫ2.6 Ϫ3.5 Ϫ4.4 AA149648 FLT1b (2.1) 2.8 Ϫ3.1 8 BG170541 MET (2.1) 2.7 Ϫ5.1 AI742057 LOC129607 2.5 Ϫ2.1 AI890347 GALNT4 2 Ϫ2.8 AA922068 CDK6 1.7 Ϫ2 2.5 AI823980 RBKS 4 AA352113 ST8SIA4 2.3 2.9 3.5 AL563795 RP11-311P8.3 2.1 Ϫ2 1.5 2.2 D50925 PASK Ϫ2 1.7 Ϫ2.5 Ϫ7.7 Ϫ8.3 5.3 Hydrolase (6) AW294640 RAB30 3 Ϫ3.4 Ϫ2.2 BF670447 RHOQ 2.1 Ϫ2.8 BF438386 RAB27B 2.5 Ϫ2.1 NM_006226 PLCL1 1.5 Ϫ4.6 AL121905 PTPRA (2.1) 2.1 AL136680 GBP3 Ϫ2.1 2.1 5.4 Transporter activity (8) AF225986 SCN3A 3.9 3 Ϫ6.7 Ϫ3.4 AF112972 ATP6V0A2 1.6 3 Ϫ4 2.5 4.6 AL117381 BCL2L1 2.2 5.7 8 AA551075 KCTD12 1.6 Ϫ3.2 AL136583 SLC37A3 2.5 6.3 Ϫ2.5 Ϫ4.3 AL574184 HPGD 2.2 Ϫ3.2 Ϫ18.4 NM_002668 PLP2 Ϫ1.4 1.5 1.7 Ϫ1.5 3.4 2.1 AA854943 ANKH Ϫ1.4 5.9 6. Other binding molecules (11) AL118798 CD47b 2.7 1.6 1.8 Ϫ2.6 Ϫ1.7 Ϫ1.9 Ϫ1.7 Ϫ2.2 NM_016837 RBMS1 1.4 2 Ϫ1.7 Ϫ3 (Table continues) The Journal of Immunology 3653

Table II. (Continued)

Fold Changea Fold Change Fold Change Fold Change

Th1 vs Th0 Th2 vs Th0 Th1 vs Th2 Th0 vs Thp

Public ID Gene 2h 6h 48h 2h 6h 48h 2h 6h 48h 2h 6h 48h

AK025100 SNTB1 2.5 Ϫ2.2 AB003476 AKAP12 4.6 Ϫ5.1 BC001247 EPLINb 2.9 Ϫ4.9 BC002827 TPM4 2.1 Ϫ1.7 AL110164 LIMS1 1.6 NM_024711 GIMAP6 Ϫ4 4.1 Ϫ2.3 Ϫ2.1 AW273811 HNRPLL Ϫ2.9 2.5 8.9 13 AA858297 GIMAP7 Ϫ2.5 2.9 Ϫ2.1 NM_023037 13CDNA73 Ϫ2 1.9 7. Miscellaneous (17) NM_013324 CISHb 14.4 4 1.4 Ϫ13 Ϫ2.8 2.9 7.5 NM_016651 DACT1 2.9 2.5 1.9 Ϫ2.5 Ϫ1.9 Ϫ2.1 BG540494 AKAP2b 2.5 3.1 2.4 Ϫ2.3 Ϫ2.9 Ϫ1.8 NM_025151 RAB11FIP1b 2.8 2.1 Ϫ3.6 Ϫ1.8 3.2

BE748563 LOC93081 2.1 2.5 Ϫ2.4 Ϫ2.2 Downloaded from AV725328 PRNP 2.1 1.7 Ϫ2.1 Ϫ1.5 4.9 2.9 AL138717 RRAGD 1.9 2.2 Ϫ1.7 NM_013322 SNX10 1.9 Ϫ2.1 3.1 BE676408 HECA 2.1 Ϫ3.4 Ϫ10.6 Ϫ10.9 AU150319 VAMP1 Ϫ1.5 Ϫ2.5 2.4 3 Ϫ2.5 AI681671 DPH2L1 Ϫ2.1 21.9 9.2 NM_001924 GADD45A Ϫ1.7 1.3 Ϫ1.7 Ϫ3 Ϫ6.1 AL035541 TMEPAI Ϫ1.6 1.4 10.2 http://www.jimmunol.org/ AA736452 PGM2L1 Ϫ2.3 1.9 8.3 7.7 BF589359 PAG Ϫ2.1 1.7 Ϫ2.3 Ϫ1.9 Ϫ2.4 AF000426 LST1 Ϫ4.9 NM_022873 G1P3 Ϫ2.8 8. Unknown function (46) BF674052 TMEM49 2.9 1.7 3.2 Ϫ3.9 Ϫ1.8 5.7 7.5 4.6 AI969697 3.2 17.8 7.5 Ϫ2.5 Ϫ5.9 Ϫ10.9 Ϫ5.9 Ϫ30.9 Ϫ8.9 AB033106 KIAA1280b 1.7 3.4 Ϫ1.6 Ϫ2.8 R14890 3.2 2.7 Ϫ3.1 Ϫ2.8 33.1 6.3

AL109935 KIAA1434 2.9 2.5 Ϫ2.6 Ϫ2.5 Ϫ2.5 Ϫ5.1 by guest on September 28, 2021 AI797353 FLJ31340 2.3 2.5 Ϫ2.6 BF056901 MGC16044 3.6 2.3 Ϫ3 Ϫ2.6 AI768674 2.3 2.2 AA489100 5.3 2.1 Ϫ2 Ϫ4.8 Ϫ2.6 Ϫ3.6 AI610684 4.1 1.9 Ϫ4.3 Ϫ2 AC004010 AMIGO2 3.2 1.9 Ϫ3.7 Ϫ1.9 2.5 3 AA088177 KIAA1913 1.7 1.9 Ϫ1.9 AW152437 3.7 1.6 Ϫ4 Ϫ1.7 5.9 AW139719 3.4 1.6 Ϫ3.2 Ϫ1.9 19.7 AA002140 2.8 2.1 Ϫ3.4 AL048542 AMICA1 2 3.6 Ϫ1.8 Ϫ1.9 Ϫ3.9 9.8 2 AW135176 4.3 Ϫ2.8 2.7 AI494573 3.7 Ϫ3.5 BF968097 3.5 Ϫ11.7 Ϫ2.7 AA429262 3 Ϫ4.1 NM_024048 MGC3020 2.7 Ϫ3 AI888503 2.4 Ϫ2.5 Ϫ1.5 Ϫ2.2 Ϫ2.1 AA034012 2.4 Ϫ2.8 Ϫ2.8 Ϫ4.4 AW292872 2.1 Ϫ2.1 Ϫ1.6 1.5 AI458439 1.8 Ϫ2.4 Ϫ2.8 2.8 W72060 1.5 Ϫ1.6 1.8 AW629527 FLJ41238 8.3 10.9 Ϫ7 Ϫ10.6 29.9 AI310524 NIPA1 2.4 Ϫ2.6 2.8 BE670307 2.2 Ϫ2.3 AI825833 LOC284801 1.7 1.4 BF037662 1.5 Ϫ1.7 Ϫ2 Ϫ7.7 Ϫ1.3 BF739885 LOC284262 5.9 NM_016008 D2LIC 2.5 Ϫ2.8 AW591809 3.4 Ϫ3.6 AI342543 FLJ33069 2.8 Ϫ2.5 2.5 Ϫ2.1 AW204712 C10orf128 2.5 Ϫ2.1 BF969397 LOC387882 2.2 Ϫ2.7 1.7 AU134977 TncRNA 2 Ϫ1.9 AI870617 KAI1 Ϫ2.4 Ϫ1.9 2 1.4 6.5 27.9 3.5 AI654547 Ϫ1.9 1.6 Ϫ1.6 11.3 6.1 (Table continues) 3654 EARLY Th1 AND Th2 CELL DIFFERENTIATION

Table II. (Continued)

Fold Changea Fold Change Fold Change Fold Change

Th1 vs Th0 Th2 vs Th0 Th1 vs Th2 Th0 vs Thp

Public ID Gene 2h 6h 48h 2h 6h 48h 2h 6h 48h 2h 6h 48h

AL389942 LOC285628 Ϫ3.4 Ϫ1.7 2.5 66.3 477.7 16.6 AI923633 Ϫ2.7 1.9 1.6 2.1 AI380089 Ϫ2.4 2 10.2 AW340595 Ϫ2.2 1.6 Ϫ3.1 AI631833 Ϫ2.1 1.9 1.3 AI701905 Ϫ2.1 1.6

Most of the genes regulated by IL-12 were also regulated by IL-4 by TGF-␤ (data not shown) (10, 15). These included 63 genes that and/or activation alone (Fig. 1A), although there were differences were coregulated by TGF-␤ and the polarizing cytokines IL-12 or Downloaded from in the kinetics and magnitude in the changes induced by these IL-4. Altogether 25 genes were coregulated by TGF-␤ and IL-12 different treatments. Similarly to the key regulator of Th1 and Th2 (Table IV). Expression of ﬁve of these genes was enhanced or cell differentiation, TBX21, a subset of genes, including GIMAP4, accelerated in the presence of TGF-␤, at least to some extent. IL-7R, PPAP2A, MGC4677, and GBP5, was regulated in an op- Importantly, 19 of the IL-12-regulated genes were antagonized by posite manner by IL-12 and IL-4, although as for TBX21, the TGF-␤ in Th1 conditions. Similarly, the effects of IL-4 on the

changes were small. expression of 20 genes were enhanced or accelerated in the pres- http://www.jimmunol.org/ ence of TGF-␤, whereas expression of 25 genes was antagonized IL-4 rapidly regulates numerous genes during early Th2 cell by TGF-␤ (Table V). differentiation A subset of genes (ZBED2, LMNA, NDFIP2, TncRNA, SYTL3, In contrast to IL-12 signaling, the effects of IL-4 were clearly seen SLC2A3, and TRIB3), coregulated by TGF-␤ and the polarizing within2hofTh2polarization. IL-4 regulated the expression of cytokines, was up-regulated by both IL-12 and IL-4 at least to some altogether 153 genes during early Th2 differentiation (Fig. 1). extent. Of these, the expression of ZBED2, LMNA, and NDFIP2 was Genes AI969697, NDFIP2, CD47, AKAP2, CISH, TMEM49, further increased in the presence of TGF-␤ in both Th1 and Th2 DACT1, and ELL2 were up-regulated by IL-4 at all time points conditions. In contrast, TncRNA, SYTL3, SLC2A3, and TRIB3, up- (Table III). A subset of the 153 genes regulated by IL-4, including regulated by IL-12 and IL-4, were repressed by TGF-␤ in both Th1 by guest on September 28, 2021 CD47, PRNP, SOCS3, FOSL2, COL6A3, PTPRA, TNFSF10, IL- and Th2 conditions. 10, CCR7, CEBPB, IL-7R, GBP3, and TBX21 has been previously described to have a role in the process (3, 8, 12, 13, 19–23). Im- Putative or known functions of the genes involved during early portantly, most of the IL-4-regulated genes identiﬁed were new in Th1 and Th2 differentiation this context. To elucidate the putative or known functions of the factors involved in the early Th1 and Th2 cell differentiation, these genes ␤ Genes regulated by TGF- during the early Th1 or Th2 were analyzed with the gene ontology annotation tool in NetAffx differentiation (17). Concordant with our previous studies, most of the genes reg- To study the effects of TGF-␤ on the early polarization of Th cells, ulated during the early differentiation of Th1 and Th2 cells code cells cultured in Th1 or Th2 conditions were compared with those for factors involved in the signal transduction from cell surface to similarly cultured, but in the presence of TGF-␤. In addition to the nucleus (10, 15). Among the IL-4-regulated genes, the most out- previously identiﬁed genes, an additional 110 genes were regulated standing functional group consisted of genes coding for proteins

Table III. Genes regulated by IL-4 throughout the early Th2 cell differentiation (regulation by activation alone is shown parallel)

Fold Changea Fold Change

Th2 vs Th0 Th0 vs Thp

Public ID Gene Known or Putative Function 2h 6h 48h 2h 6h 48h

AI969697 Unknown 3.3 17.8 7.5 Ϫ5.9 Ϫ30.9 Ϫ8.9 AL118798 CD47 Cell-matrix adhesion 2.7 1.6 1.8 Ϫ1.7 Ϫ2.2 NM_016651 DACT1 2.9 2.5 1.9 Ϫ2.1 BG540494 AKAP2 G protein signaling 2.5 3.1 2.4 Ϫ1.8 BF674052 TMEM49 Unknown 2.9 1.7 3.3 5.7 7.5 4.6 AW290956 NDFIP2 Regulation of I␬B/NF-␬B cascade 1.9 1.5 3.0 16.0 48.5 142.0 AI924426 ELL2 Transcriptional regulator 1.9 1.8 2.6 3.3 NM_013324 CISH Intracellular signaling 14.4 4.0 1.4 2.9 7.5

Table IV. Genes coregulated by IL-12 and TGF-␤

Fold Changea Fold Change

Th1ϩTGFb vs Th1 Th1 vs Act

Public ID Gene Symbol Known or Putative Function 2h 6h 48h 2h 6h 48h

NM_006137 CD7 Receptor 2.1a Ϫ1.6 2.1 TGF-␤ enhances the effects of IL-12 NM_024508 ZBED2b Transcriptional regulator 1.7 3.7 AW290956 NDFIP2b Signal transducer 1.7 1.3 Y13786 ADAM19 Metal ion binding 3.4 2.7 NM_005572 LMNAb Cytoskeletal 2.1 2.5 AW574798 KLHL6 Miscellaneous 1.4 1.6 TGF-␤ antagonizes the effects of IL-12 AL121985 SLAMF7b Receptor Ϫ2.5 1.8 AB059408 HOP Transcriptional regulator Ϫ7 2.9 NM_003955 SOCS3b Protein kinase inhibitor Ϫ2 3.1 AW150720 RDH10 Oxidoreductase Ϫ2.3 1.5

NM_021158 TRIB3 Protein kinase Ϫ2.5 1.4 Downloaded from NM_001629 ALOX5AP Enzyme activator 2.2 Ϫ1.2 AI631159 SLC2A3 Glucose transporter Ϫ2.5 1.7 NM_018326 GIMAP4b Miscellaneous Ϫ1.9 2.1 AK021850 FBXO16 /// ZNF395 Miscellaneous Ϫ2.1 2 NM_001311 CRIP1b Miscellaneous 3 Ϫ2.1 BG110811 LOC340061 Unknown Ϫ1.5 2.6 AV713773 MCOLN2 Unknown Ϫ4 2.3 AI042152 TncRNA Unknown Ϫ2.5 2.2 http://www.jimmunol.org/ AI674404 SYTL3b Unknown Ϫ3.2 1.8 AA831661 MGC29814 Unknown Ϫ2.1 1.7 AI214996 LOC284018b Unknown Ϫ1.6 1.6 AA195074 TIFAb TRAF-interacting protein Ϫ1.6 1.6 BF209337 MGC4677b Unknown 1.9 1.7 Ϫ1.7 BE222344 —b Unknown 1.7 5.1 Ϫ2.5

a Average fold change values for the genes that were differentially expressed Ն2-fold in comparisons of Th1 and Th2 cells to each other or to Th0 cells in at least one of the time points. Also, for these selected genes, the statistically significant changes Ͻ2-fold are shown in parallel conditions. Missing values indicate that the change was not significant according to the statistical data analysis by MAS5. b Differential expression by the cells induced to polarize to Th1 or Th2 direction. by guest on September 28, 2021 with catalytic activity including genes involved in RAS oncogene AI674404, ZNF443, and DACT1. Based on the gene array re- family signaling (RAB27B, RAB30, and RHOQ). Another predom- sults, all of these genes and ESTs were rapidly regulated by inant functional group consisted of genes coding for receptors or IL-4 at the initiation of Th2 cell differentiation. RT-PCR con- receptor binding proteins. These included components of G protein- firmed IL-4-mediated regulation of 12 of 14 genes or ESTs (Fig. coupled receptor signaling (CYSLTR1, HRH4, CD47, CCR7, and 2). Regulation of transcripts by IL-4 was primarily limited GPR18). Numerous transcriptional regulators, such as ELL2, within the first 2 days of Th2 differentiation. For BF056901 and ZNF443, PHF20L1, LOC360030, EZH2, ZBED2, FOSL2, and AI674404, the Affymetrix results could not be confirmed in all FRBZ1 became differentially regulated during the early Th1 and Th2 three individuals studied. differentiation. As expected, IL-12 and/or IL-4 regulated several genes involved in the immune response, such as IL-10, CCR7, CD7, CEBPB, and IL-7R. Other common functional groups consisted of FOSL2 is preferentially expressed during the early Th2 cell genes involved in apoptosis, proliferation, adhesion, metabolism, mo- polarization at the protein level tility, cell cycle, protein localization, transport, and regulation of pro- Systematic validation of the differences detected at the mRNA tein activity. The function of the majority of the genes differentially level was performed at the protein level for those genes that com- regulated during early Th1 and Th2 cell differentiation was unknown. mercial Abs were available. Most of the genes identified in this Among these were numerous ESTs that were rapidly up-regulated by study are poorly characterized and therefore a limited number of IL-4 in the cells induced to polarize to Th2 direction. A set of these Abs were obtained. The proteins studied included DACT1, genes or ESTs was selected for further studies. As IL-12 did not have RAB11FIP1, FOSL2, CISH, KLF6, FLT1, and RAB27B. As can much effect at the initiation of the differentiation, our special interest be seen from Fig. 3, FOSL2 was indeed differentially expressed at was on the genes or ESTs regulated by IL-4. the protein level, with higher expression in Th2 cells detected at 48 h. TBX21 and GATA3 were used as control genes to confirm Validation of the oligonucleotide array results with real-time the polarization of the cells to Th1 and Th2 direction. For the other RT-PCR proteins we were not able to detect any differences in the expres- Expression of 14 ESTs or genes with poorly characterized func- sion levels during early polarization of Th1 or Th2 cells (data tion was further studied with real-time RT-PCR during 7 days not shown). Proteomics studies in progress will be used to elu- of Th1 and Th2 cell polarization. These included AA088177, cidate the differences in the expression of the possible variants AA237039, AA489100, AI494573, AW139719, AW152437, of these and other proteins during early stages of Th1 and Th2 AW629527, AL389942, BF056901, R98767, BE748563, differentiation. 3656 EARLY Th1 AND Th2 CELL DIFFERENTIATION

Table V. Genes coregulated by TGF-␤ and IL-4

Fold Changea Fold Change

Th2ϩTGF␤ vs Th2 Th2 vs Th0

Public ID Gene Symbol Known or Putative Function 2h 6h 48h 2h 6h 48h

AF261135 GPR18b GPCR signaling 1.5 Ϫ1.9 Ϫ1.6 TGF-␤ enhances the effects of IL-4 AA149648 FLT1b Receptor, enzyme 1.4 2.8 AF053712 TNFSF11b Receptor 1.7 1.7 AI924426 ELL2b Transcriptional regulator 2.3 1.9 1.8 2.5 NM_024508 ZBED2b Transcriptional regulator 2.2 2.9 3.7 AL513759 RBPSUHb Transcriptional regulator 2.4 1.8 AW290956 NDFIP2b Signal transducer 1.3 1.9 1.5 3.0 NM_001924 GADD45Ab Intracellular signaling Ϫ2.2 Ϫ1.7 U57059 TNFSF10 Intracellular signaling Ϫ2.4 Ϫ1.9 AA029441 CAMK2Db Enzyme 1.3 2.2 NM_004369 COL6A3b Enzyme inhibitor, cell adhesion 3.1 13.5 BF439618 CD47b Cell-matrix adhesion 1.6 2.3

AL118798 CD47 Cell-matrix adhesion 1.5 2.7 1.6 1.8 Downloaded from AI524095 LY9b Cell adhesion Ϫ1.9 Ϫ2.5 AF101051 CLDN1b Cell adhesion 1.3 2.5 NM_005572 LMNAb Cytoskeletal 3.1 1.5 AL110164 LIMS1 Miscellaneous 2.5 1.6 W72060 —b Unknown 1.5 1.5 AA088177 KIAA1913b Unknown 2.5 1.7 1.9 AL048542 AMICAb Unknown 1.6 2.0 3.6 BF209337 MGC4677b Unknown 1.5 1.6 1.5 http://www.jimmunol.org/ AW591809 —b Unknown 2.8 3.4 TGF-␤ antagonizes the effects of IL-4 AL564683 CEBPB Transcriptional regulator Ϫ2.1 1.7 NM_018664 SNFTb Transcriptional regulator Ϫ2.0 4.3 AL353944 RUNX2b Transcriptional regulator Ϫ1.6 2.7 AB000888 PPAP2Ab Enzyme Ϫ1.9 Ϫ1.9 2.4 AI823980 RBKS Enzyme Ϫ2.5 4.0 NM_021158 TRIB3 Protein kinase Ϫ2.5 1.8 b BG171548 NCE2 Enzyme Ϫ1.7 1.9 by guest on September 28, 2021 AA352113 SIAT8D Enzyme Ϫ1.4 2.3 NM_002668 PLP2b Transporter 1.5 1.8 1.6 Ϫ1.4 1.5 AA854943 ANKH Transporter 1.3 1.6 Ϫ1.4 AI798823 PSCD1b Transporter Ϫ3.6 5.9 AI631159 SLC2A3 Glucose transporter Ϫ1.6 1.6 BC002827 TPM4b Cytoskeletal Ϫ1.4 2.1 AW135176 —b Miscellaneous Ϫ2.2 4.3 AL035541 TMEPAIb Miscellaneous 1.8 2.7 Ϫ1.6 AW152437 —b Unknown Ϫ1.4 3.7 1.6 AI654547 —b Unknown 1.3 1.5 Ϫ1.9 1.6 AI825833 LOC284801 Unknown Ϫ2.7 1.7 AI674404 SYTL3b Unknown Ϫ2.3 Ϫ4.1 3.2 2.8 AW629527 FLJ41238 Unknown Ϫ1.5 8.3 10.9 AU134977 — Unknown Ϫ2.3 2.0 AI042152 TncRNA Unknown Ϫ2.1 1.9 AI342543 FLJ33069b Unknown Ϫ1.9 2.8 AL563795 MGC23937b Unknown Ϫ1.6 2.1 BF968270 SLC35F3b Unknown 2.1 Ϫ1.8

AW629527, AA088177, AA489100, ZNF443, and DACT1 are selected genes or ESTs regulated by IL-4, mRNA expression was induced by IL-4 in a STAT6-dependent manner measured from these samples with quantitative real-time RT-PCR. To further study whether a subset of poorly characterized genes in- The results demonstrated that the induction of AW629527, AA088177, duced by IL-4 is regulated through STAT6 signaling, human periph- AA489100, ZNF443, and DACT1 genes or ESTs by IL-4 was clearly eral blood-derived CD4ϩ cells were nucleofected with two different dependent on STAT6. No effect by STAT6-shRNA on the expression pSuper-H2K-STAT6-shRNA plasmids or a pSuper-H2K-scramble- of AA237039 or AW152437 was observed (data not shown). shRNA plasmid. To verify the functionality of the pSuper-H2K- ␥ STAT6-shRNA, we demonstrated that STAT6 and GATA3 mRNA NDFIP2 promotes IFN- production by Th1 cells levels were down-regulated by these shRNAs (Fig. 4). To determine NDFIP2 was selected for further functional studies as it was highly effects of this STAT6 knockdown on the expression of a subset of induced by CD3 plus CD28 activation alone and the expression The Journal of Immunology 3657

FIGURE 2. Validation of the oligonucleotide array results with real-time RT-PCR. CD4ϩ T cells were isolated from human cord blood. The cells were activated with PHA and CD32-B7-transfected feeder cells. Downloaded from The cells were either cultured under “neutral conditions” (Th0) or polarized to Th1 or Th2 for 7 days. During the polarization expression of selected genes was studied at the indicated time points with real-time RT-PCR. Statistically signiﬁcant differences in gene ,ء http://www.jimmunol.org/ expression between the Th2 and Th0 cells were determined with a t test. by guest on September 28, 2021

was further enhanced by IL-4 during the early Th2 differentiation tion with PHA plus CD32-B7-transfected feeder cells induced only at all of the time points studied. This regulation of NDFIP2 was a modest short-term increase in NDFIP2 expression as measured confirmed with RT-PCR (Fig. 5A). In contrast to the oligonucle- with RT-PCR (Fig. 5A). Because the role of NDFIP2 in the dif- otide array results, where high induction of NDFIP2 expression by ferentiation of Th cells was previously unknown, the effect of CD3 plus CD28 activation alone was observed (Table III), activa- NDFIP2 knockdown on the cytokine production by the polarized (7 days) Th1 and Th2 cells was studied by Luminex assay. shRNA-mediated down-regulation of NDFIP2 led to a decrease in IFN-␥ production by Th1 cells (Fig. 5B) in two biological replicates. In addition, a study with a similar experimental set-up, but using cord blood CD4ϩ cells and two different NDFIP2-specific synthetic siRNA oligos was conducted. The latter study resulted in cytokine secretion profiles consistent to those obtained using plasmid-based shRNA knockdown (data not shown). The effect of NDFIP2 knockdown on IL-4 production could not be estimated reliably (data not shown), due to the low levels of the IL-4 production by these cells, as is commonly observed after the FIGURE 3. FOSL2 protein is preferentially expressed by cells polarized to Th2 direction at early stages of differentiation. Human cord blood CD4ϩ first round of human Th2 cell polarization in these conditions T cells were either cultured in neutral conditions (Th0) or polarized to Th1 (24, 25). Most importantly, our experiment demonstrates that or Th2 for 2, 6, and 48 h. Nuclear lysates were subjected to Western NDFIP2 has a function in the Th1 cell differentiation process by blotting with TBX21 and GATA3 expression to validate successful Th1 promoting secretion of the hallmark cytokine of Th1 cell dif- and Th2 polarization and then probed for FOSL2. ferentiation, IFN-␥. 3658 EARLY Th1 AND Th2 CELL DIFFERENTIATION

FIGURE 4. AW629527, AA088177, AA489100, ZNF443, and DACT1 are induced by IL-4 in a STAT6-dependent manner. Human peripheral blood-derived CD4ϩ cells were nucleofected with a pSuper-H2K-STAT6- shRNA or nonfunctional pSuper-H2K-scramble-shRNA control vectors. The transfected cells (purity of Ͼ98%) were induced to polarize to Th2 Downloaded from (anti-CD3 plus anti-CD28 plus IL-4) direction. The cells were harvested after 24 or 48 h of polarization. Real-time RT-PCR was used to measure expression of selected IL-4-regulated genes in three to four biological replicates. The genes with statistically significant difference between STAT6- shRNA and scramble-shRNA transfected cells are presented in the figure (paired t test: p Յ 0.05). Fold changes in gene expression between the Th2 http://www.jimmunol.org/ cells expressing STAT6-shRNA and scramble-shRNA are indicated in the figure.

Discussion We have conducted a genome-wide screening to identify novel genes involved in early Th1 and Th2 differentiation (13, 15). In- terestingly, numerous factors with known or unknown function were identified to be implicated in the process. A number of these by guest on September 28, 2021 genes could be positioned into specific intracellular signaling cas- cades. This study provides an overview of the intracellular signaling events in the cells at the initiation of Th1 and Th2 cell ␥ differentiation. FIGURE 5. NDFIP2 promotes IFN- production by human Th1 cells. CD4ϩ T cells were isolated from human cord blood. The cells activated Thp cells are not responsive to IL-12, which can be seen in slow with PHA and CD32-B7 transfected feeder cells were cultured either under changes in gene expression profiles in response to IL-12 during “neutral conditions” (Th0) or polarized to Th1 or Th2 for 7 days. Real-time early Th1 cell differentiation. Our results at whole genome scale RT-PCR was used to confirm the differential expression of NDFIP2 during confirm the previous consensus that indicates activation of IFN the Th cell differentiation (A). Peripheral blood-derived CD4ϩ cells were signaling to be the first event during Th1 differentiation. This is nucleofected with a pSuper-pIRES2-H2K-NDFIP2-shRNA or pSuper- demonstrated by the early up-regulation of key regulators of Th1 pIRES2-H2K-scramble-shRNA control vector. The transfected cells (pu- differentiation IFNG, TBX21, and IFN-regulated genes GBP1 and rity of Ͼ98%) were induced to polarize to Th1 (anti-CD3 plus anti-CD28 GBP5 (3, 4, 15, 26, 27). TBX21 is induced by T cell activation and plus IL-12) or Th2 (anti-CD3 plus anti-CD28 plus IL-4) direction for 7 its expression is enhanced by IFN-␥ during early Th1 polarization days. To measure cytokine production, cells were washed and restimulated ␥ (28–31). The role of GBP1 and GBP5 in Th1 polarization is not with PMA and ionomycin (stimulation). IFN- secretion by the nontrans- fected Th1 and Th2 cells as well as cells expressing scramble-shRNA or known. As these genes are induced by IFNs, it is likely that, sim- ␥ NDFIP2-shRNA was measured using a Luminex assay. Representative ilar to TBX21, they are induced due to enhanced IFN- signaling. data from three biological replicates for IFN-␥ secretion is shown in B. ␥ Activation of IFN- signaling and TBX21 expression during early Abbreviation OOR (out of range) in the figure means that the cytokine differentiation is essential for Th1 differentiation, probably because secretion has been over the detection limit of the assay. it enables IL-12 signaling (4, 28, 32, 33). The genes coding for receptor components for IL-12 and IL-18 (IL12RB2 and IL18RAP) are up-regulated within 48 h, enabling and enhancing the respon- studied. However, a group of genes, including NDFIP2, CD47, siveness of the developing Th1 cells to polarizing cytokines (10). CISH, ELL2, AI969697, AKAP2, DACT1, and TMEM49, were reg- Consistently, after 48 h of Th1 polarization several genes were ulated by IL-4 throughout the first 2 days. Previously, we observed regulated by IL-12. The roles of most of these genes or ESTs in similar expression patterns for the key mediators of Th2 differen- Th1 differentiation are not known. tiation GATA3 and MAF. Similar regulation for a panel of genes, Thp cells are responsive to IL-4, which can be seen in the rapid such as BCL6, NFIL-3, SATB1, SOCS1, DUSP6, IL10RA, and regulation of numerous genes and ESTs with known and unknown CXCR4, with unknown or less clear functions in Th1 and Th2 cell function. A subset of these genes was demonstrated to be regulated differentiation was also demonstrated in our previous studies (10, by IL-4 in a STAT6-dependent manner. Most of the IL-4-regulated 15). Maintenance of the IL-4-mediated regulation of these genes genes displayed only temporary changes at some of the time points throughout the early Th2 cell differentiation suggests them to be The Journal of Immunology 3659 important for the process. However, as demonstrated in current late cytokine production through some novel mechanism related to and previously published study (15), not all of these differences protein trafficking. may be detected at the protein level. The preferential induction of FOSL2 during early Th2 differen- Components involved in well-known intracellular signaling cas- tiation is also interesting and was confirmed at the protein level. cades, such as RAS and GPCR seem to be preferentially induced FOSL2 is a member of the AP-1 family of transcription factors and during the early Th2 cell differentiation. Such genes include an immediate early gene (41) induced during the differentiation of ϩ ϩ RAB27, RAB30, RHOQ, and the genes identified in our previous CD4 or CD8 T cells from immature double-negative precursors study, RASGRP1, RASA3, and SOS1, which are induced by IL-4 (42). It has been reported that during the Th1/Th2 differentiation within 2 or 6 h (15). This is consistent with previous reports show- process, high levels of AP-1 complexes are accumulated, and that ing that the RAS pathway promotes IL-4R signaling and is essen- these complexes are able to induce high levels of AP-1 tran- tial for Th2 responses in vivo (34–36). Based on our current and scriptional activity in Th2, but not in Th1 cells (43). These previous studies also, a panel of factors involved in GPCR signal- reports together with our observations provide the basis for fur- ing is rapidly regulated in response to Th2-polarizing stimuli. ther studies to dissect the role of these proteins in Th2 cell Genes including GNAI1, CD47, CXCR4, PTGER2, CYSLTR1, differentiation. EDG1, EBI2, FLJ11856, and HRH4 become up-regulated within 2 In conclusion, based on our current and previous studies, 288 ϳ or 6 h and GPRK6 within 48 h. In contrast, genes CD97, PTGER4, genes, 1–1.5% of the genes in human genome, are differentially ADORA2A, CCR7, GPR18, GPRK5, and P2Y5 are rapidly re- regulated by cytokines involved in early Th1 and Th2 polarization pressed by IL-4 (10, 15). GPCR signaling is implicated in the during the first 2 days (10, 15). These novel genes are likely to regulation of Th1 and Th2 responses and plays a role in Th2- include factors that are critical regulators of Th1 and Th2 differ- Downloaded from mediated diseases such as asthma (37). Induction of GPCR sig- entiation process, together with the previously identified factors, naling may also enhance the activation of RAS pathway, thus pro- such as STAT4, TBX21, STAT6, and GATA3. This study promoting IL-4 signaling. vides a detailed overview of the gene regulation during the early TGF-␤ is an immunosuppressive factor able to inhibit Th1 and Th1 and Th2 cell differentiation and numerous new candidates for Th2 cell differentiation. We hypothesized that the genes differen- further studies. Although all of these genes may not be important tially regulated by the Th1- or Th2-inducing cytokines and TGF-␤ for the differentiation process, these findings imply that the regu- http://www.jimmunol.org/ lation of the Th1 and Th2 differentiation process is likely to be are important candidates for influencing in the differentiation of much more complex than current models suggest. these subtypes. Therefore, the target genes of TGF-␤ during the early differentiation were identified. In agreement with our previously published studies (10, 15), we identified a subset of genes Acknowledgments that was regulated in an opposite manner by TGF-␤ and IL-12 or We are grateful for Paula Suominen, Outi Melin, Sarita Heinonen, and IL-4. TGF-␤ antagonized the effects of IL-12 on genes including Marjo Hakkarainen for their valuable technical assistance. Dr. Robert Moulder is acknowledged for the language review of the manuscript. We SOCS3 HOP GIMAP4 SLAMF7 TIFA CRIP1 , , , , , , and effects of also thank Prof. John Eriksson’s group at Turku Centre for Biotechnology

IL-4 on numerous genes, such as SYTL3, PPAP2A, and AU134977. for assistance in the use of GraphPad Prism software for graphics. by guest on September 28, 2021 Previously identified genes that behave in a similar manner and show inhibition by TGF-␤ include GZMB, NFIL3, TNFRSF9, Disclosures VIM, SATB1, BCL2A1, ID2, PTGS2, PLA2G4A, GNAI1, ID3, The authors have no financial conflict of interest. LAMA3, CCL20, RTP801, and R32184_3 (10, 15). The functional role of most of these genes in Th cell differentiation is References unknown and deserves further characterization. The genes reg- 1. Romagnani, S. 1996. Th1 and Th2 in human diseases. Clin. Immunol. Immuno- ulated by IL-12 or IL-4 and antagonized by TGF-␤ are likely to pathol. 80: 225–235. participate in the mechanism by which TGF-␤ inhibits Th1 and 2. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune system: the T helper lymphocyte grows up. Genes Dev. 14: 1693–1711. Th2 differentiation. 3. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. NDFIP2 was one of the eight genes induced by IL-4 at all the 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655–669. time points studied during the early Th2 cell differentiation. Dif- 4. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, and ferential expression by the Th1 and Th2 cells polarized for 7 days L. H. Glimcher. 2002. Distinct effects of T-bet in TH1 lineage commitment and was observed as well. NDFIP2 was highly induced in response to IFN-␥ production in CD4 and CD8 T cells. Science 295: 338–342. 5. Zhang, D. H., L. Cohn, P. Ray, K. Bottomly, and A. Ray. 1997. Transcription CD3 plus CD28 activation alone as previously described in T cells, factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and con- but induction in response to PHA plus feeder cell activation was trols Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272: 21597–21603. modest and temporary (38). NDFIP2 has shown to be able to in- 6. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is neces- teract with several NEDD4-family proteins. NDFIP2 localizes in sary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89: the Golgi network and seems to play a role in the regulation of 587–596. 7. Hamalainen, H., H. Zhou, W. Chou, H. Hashizume, R. Heller, and R. Lahesmaa. protein trafficking (38, 39). As nothing was previously known on 2001. Distinct gene expression profiles of human type 1 and type 2 T helper cells. the role of NDFIP2 in Th differentiation, it was selected for further Genome Biol. 2: RESEARCH0022.1–RESEARCH0022.11. analysis to determine, whether this factor has a functional role in 8. Rogge, L., E. Bianchi, M. Biffi, E. Bono, S. Y. Chang, H. Alexander, C. Santini, G. Ferrari, L. Sinigaglia, M. Seiler, et al. 2000. Transcript imaging of the devel- the polarization process. The NDFIP2 knockdown during the early opment of human T helper cells using oligonucleotide arrays. Nat. Genet. 25: Th1 cell differentiation demonstrated that NDFIP2 promotes pro- 96–101. ␥ 9. Lu, B., P. Zagouras, J. E. Fischer, J. Lu, B. Li, and R. A. Flavell. 2004. Kinetic duction of the hallmark cytokine of Th1 cell differentiation, IFN- . analysis of genomewide gene expression reveals molecule circuitries that control The reason why NDFIP2 is preferentially induced during early T cell activation and Th1/2 differentiation. Proc. Natl. Acad. Sci. USA 101: Th2 differentiation and regulates IFN-␥ production also by Th1 3023–3028. 10. Lund, R., T. Aittokallio, O. Nevalainen, and R. Lahesmaa. 2003. Identification of cells is unclear. Also, the exact mechanism of this enhancement novel genes regulated by IL-12, IL-4, or TGF-␤ during the early polarization of remains to be elucidated. One of the possible mechanisms may CD4ϩ lymphocytes. J. Immunol. 171: 5328–5336. ␬ 11. Lund, R. J., E. K. Ylikoski, T. Aittokallio, O. Nevalainen, and R. Lahesmaa. involve the NF- B pathway, as NDFIP2 has been suggested to 2003. Kinetics and STAT4- or STAT6-mediated regulation of genes involved in activate NF-␬B signaling (40). Alternatively, NDFIP2 may regu- lymphocyte polarization to Th1 and Th2 cells. Eur. J. Immunol. 33: 1105–1116. 3660 EARLY Th1 AND Th2 CELL DIFFERENTIATION

12. Chen, Z., R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, and R. Lahesmaa. 29. Grogan, J. L., M. Mohrs, B. Harmon, D. A. Lacy, J. W. Sedat, and R. M. Locksley. 2003. Identification of novel IL-4/Stat6-regulated genes in T lymphocytes. J. Im- 2001. Early transcription and silencing of cytokine genes underlie polarization of T munol. 171: 3627–3623. helper cell subsets. Immunity 14: 205–215. 13. Chtanova, T., R. A. Kemp, A. P. Sutherland, F. Ronchese, and C. R. Mackay. 30. Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino, 2001. Gene microarrays reveal extensive differential gene expression in both D. M. Livingston, A. L. Kung, N. Cereb, T. P. Yao, S. Y. Yang, and S. L. Reiner. CD4ϩ and CD8ϩ type 1 and type 2 T cells. J. Immunol. 167: 3057–3063. 2001. Role of T-bet in commitment of TH1 cells before IL-12-dependent selec- 14. Hoey, T., S. Zhang, N. Schmidt, Q. Yu, S. Ramchandani, X. Xu, L. K. Naeger, tion. Science 292: 1907–1910. Y. L. Sun, and M. H. Kaplan. 2003. Distinct requirements for the naturally oc- 31. Lighvani, A. A., D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B. D. Hissong, curring splice forms Stat4␣ and Stat4␤ in IL-12 responses. EMBO J. 22: B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, and J. J. O’Shea. 2001. T-bet is 4237–4248. rapidly induced by interferon-␥ in lymphoid and myeloid cells. Proc. Natl. Acad. Sci. 15. Lund, R., H. Ahlfors, E. Kainonen, A. M. Lahesmaa, C. Dixon, and R. Lahesmaa. USA 98: 15137–15142. 2005. Identification of genes involved in the initiation of human Th1 or Th2 cell 32. Tau, G. Z., T. von der Weid, B. Lu, S. Cowan, M. Kvatyuk, A. Pernis, commitment. Eur. J. Immunol. 35: 3307–3319. G. Cattoretti, N. S. Braunstein, R. L. Coffman, and P. B. Rothman. 2000. Inter- ␥ 16. Hamalainen, H. K., J. C. Tubman, S. Vikman, T. Kyrola, E. Ylikoski, J. A. feron signaling alters the function of T helper type 1 cells. J. Exp. Med. 192: Warrington, and R. Lahesmaa. 2001. Validation of endogenous reference genes for 977–986. ␥ profiling of lymphocyte differentiation by quantitative real-time RT-PCR. Anal. Bio- 33. Gollob, J. A., H. Kawasaki, and J. Ritz. 1997. Interferon- and interleukin-4 chem. 299: 63–70. regulate T cell interleukin-12 responsiveness through the differential modulation 17. Liu, G., A. E. Loraine, R. Shigeta, M. Cline, J. Cheng, V. Valmeekam, S. Sun, of high-affinity interleukin-12 receptor expression. Eur. J. Immunol. 27: D. Kulp, and M. A. Siani-Rose. 2003. NetAffx: Affymetrix probe sets and an- 647–652. notations. Nucleic Acids Res. 31: 82–86. 34. Yamashita, M., M. Kimura, M. Kubo, C. Shimizu, T. Tada, R. M. Perlmutter, and 18. Tahvanainen, J., M. Pyka¨laïnen, T. Kallonen, H. La¨hteenma¨ki, O. Rasool, and T. Nakayama. 1999. T cell antigen receptor-mediated activation of the Ras/mi- R. Lahesmaa. 2006. Enrichment of nucleofected primary human CD4ϩ T cells: togen-activated protein kinase pathway controls interleukin 4 receptor function a novel and efficient method for studying gene function and role in human pri- and type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA 96: mary T helper cell differentiation. J. Immunol. Methods 310: 30–39. 1024–1029. 35. Yamashita, M., M. Katsumata, M. Iwashima, M. Kimura, C. Shimizu, T. Kamata,

19. Seki, Y., H. Inoue, N. Nagata, K. Hayashi, S. Fukuyama, K. Matsumoto, Downloaded from O. Komine, S. Hamano, K. Himeno, K. Inagaki-Ohara, et al. 2003. SOCS-3 T. Shin, N. Seki, S. Suzuki, M. Taniguchi, and T. Nakayama. 2000. T cell re- regulates onset and maintenance of Th2-mediated allergic responses. Nat. Med. 9: ceptor-induced calcineurin activation regulates T helper type 2 cell development J. Exp. Med. 1047–1054. by modifying the interleukin 4 receptor signaling complex. 191: 1869–1879. 20. Li-Weber, M., O. Laur, I. Davydov, C. Hu, P. Salgame, and P. H. Krammer. 36. Shibata, Y., T. Kamata, M. Kimura, M. Yamashita, C. R. Wang, K. Murata, 1997. What controls tissue-speciﬁc expression of the IL-4 gene? Immunobiology M. Miyazaki, M. Taniguchi, N. Watanabe, and T. Nakayama. 2002. Ras activa- 198: 170–178. tion in T cells determines the development of antigen-induced airway hyperre- 21. Avice, M. N., M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati. 2000. CD47 sponsiveness and eosinophilic inﬂammation. J. Immunol. 169: 2134–2140. ligation selectively inhibits the development of human naive T cells into Th1

37. Johnson, E. N., and K. M. Druey. 2002. Heterotrimeric G protein signaling: role http://www.jimmunol.org/ effectors. J. Immunol. 165: 4624–4631. in asthma and allergic inflammation. J. Allergy Clin. Immunol. 109: 592–602. 22. Rincon, M., and R. A. Flavell. 1999. Reprogramming transcription during the ϩ 38. Cristillo, A. D., L. Nie, M. J. Macri, and B. E. Bierer. 2003. Cloning and char- differentiation of precursor CD4 T cells into effector Th1 and Th2 cells. Mi- acterization of N4WBP5A, an inducible, cyclosporine-sensitive, Nedd4-binding crobes Infect. 1: 43–50. protein in human T lymphocytes. J. Biol. Chem. 278: 34587–34597. 23. Nagai, S., S. Hashimoto, T. Yamashita, N. Toyoda, T. Satoh, T. Suzuki, and 39. Shearwin-Whyatt, L. M., D. L. Brown, F. G. Wylie, J. L. Stow, and S. Kumar. K. Matsushima. 2001. Comprehensive gene expression profile of human acti- 2004. N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicu- vated Th1- and Th2-polarized cells. Int. Immunol. 13: 367–376. lar bodies and the Golgi, and has a potential role in protein trafficking. J. Cell Sci. 24. Sornasse, T., P. V. Larenas, K. A. Davis, J. E. de Vries, and H. Yssel. 1996. 117: 3679–3689. Differentiation and stability of T helper 1 and 2 cells derived from naive human ϩ 40. Matsuda, A., Y. Suzuki, G. Honda, S. Muramatsu, O. Matsuzaki, Y. Nagano, neonatal CD4 T cells, analyzed at the single-cell level. J. Exp. Med. 184: T. Doi, K. Shimotohno, T. Harada, E. Nishida, et al. 2003. Large-scale identifi- 473–483. cation and characterization of human genes that activate NF-␬B and MAPK

25. Cousins, D. J., T. H. Lee, and D. Z. Staynov. 2002. Cytokine coexpression during signaling pathways. Oncogene 22: 3307–3318. by guest on September 28, 2021 human Th1/Th2 cell differentiation: direct evidence for coordinated expression of 41. Nishina, H., H. Sato, T. Suzuki, M. Sato, and H. Iba. 1990. Isolation and char- Th2 cytokines. J. Immunol. 169: 2498–2506. acterization of fra-2, an additional member of the fos gene family. Proc. Natl. 26. Bradley, L. M., D. K. Dalton, and M. Croft. 1996. A direct role for IFN-␥ in Acad. Sci. USA 87: 3619–3623. regulation of Th1 cell development. J. Immunol. 157: 1350–1358. 42. Chen, F., D. Chen, and E. V. Rothenberg. 1999. Speciﬁc regulation of fos family 27. Wenner, C. A., M. L. Guler, S. E. Macatonia, A. O’Garra, and K. M. Murphy. transcription factors in thymocytes at two developmental checkpoints. Int. Im- 1996. Roles of IFN-␥ and IFN-␣ in IL-12-induced T helper cell-1 development. munol. 11: 677–688. J. Immunol. 156: 1442–1447. 43. Rincon, M., B. Derijard, C. W. Chow, R. J. Davis, and R. A. Flavell. 1997. 28. Afkarian, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang, Reprogramming the signalling requirement for AP-1 (activator protein-1) acti- T. L. Murphy, and K. M. Murphy. 2002. T-bet is a STAT1-induced regulator of vation during differentiation of precursor CD4ϩ T-cells into effector Th1 and Th2 IL-12R expression in naive CD4ϩ T cells. Nat. Immunol. 3: 549–557. cells. Genes Funct. 1: 51–68.