of May 18, 2011This information is current as

2007;178;3648-3660J Immunol and Riitta LahesmaaTahvanainen, Joonas Scheinin, Tiina Henttinen, Omid RasoolDixon, Zhi Chen, Helena Ahlfors, Soile Tuomela, Johanna Riikka J. Lund, Maritta Löytömäki, Tiina Naumanen, Craig DifferentiationInvolved in Early Th1 and Th2 Cell Genome-Wide Identification of Novel Genes

References

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Genome-Wide Identification of Novel Genes Involved in EarlyTh1 and Th2 Cell Differentiation1

Riikka J. Lund,2* Maritta Loytomaki,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 type1 diabetes and asthma, respectively. Defining the molecular networks and factors that direct Th1 and Th2 cell differentiation willhelp to understand the pathogenic mechanisms causing these diseases. Some of the key factors regulating this differentiation havebeen 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. Basedon our current and previous studies, 288 genes or expressed sequence tags, representing �1–1.5% of the human genome, areregulated in the process during the first 2 days. These transcriptional profiles revealed genes coding for components of certainpathways, such as RAS oncogene family and G protein-coupled receptor signaling, to be differentially regulated during the earlyTh1 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, wedemonstrate 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 processtogether with the previously known factors and are potential targets for developing therapeutics to modulate Th1 and Th2responses. The Journal of Immunology, 2007, 178: 3648–3660.

T helper cell subtypes, Th1 and Th2, originate from com-mon naive precursor cells (Thp) in response to Ag andcytokine stimulation. Although Th cells have a crucial

role in host defense against intracellular and extracellular patho-gens, disturbances in the balance between Th1 and Th2 responsescan promote or lead to pathogenesis of immune-mediated diseases.Enhanced Th2 response is involved in atopic diseases, such asasthma, whereas a dominating Th1 response is implicated in cer-tain autoimmune diseases, like type 1 diabetes or rheumatoid ar-thritis (1). To understand the molecular mechanisms driving thepathogenesis of these diseases, it is important to elucidate the earlydifferentiation process of Th1 and Th2 cells in detail.

A number of the central factors involved in directing the differ-entiation process have been identified. IL-12/STAT4 and IFN-�/

STAT1 signaling are important in driving Th1 polarization,whereas IL-4/STAT6 signaling directs Th2 polarization (2). Tran-scription factors TBX21 (T-bet) and GATA3 are also among thekey factors required for the Th1 and Th2 differentiation, respec-tively (3–6). Although many of the players implicated in the reg-ulation of differentiation have been recognized, the current modelas such is still too simple to explain the process in detail, and otheryet unknown factors are likely to be involved.

Recently, an increasing number of studies have used DNA mi-croarrays to identify new factors involved in the Th1 and Th2polarization in humans and mice (7–14). However, all of thesestudies have focused on studying a limited number of primarilyknown genes. We have previously elucidated the regulation of�9300 genes, most with known functions, during the early differ-entiation of human Th1 and Th2 cells (10, 15). In the presentstudy, we have extended the previous work by exploring the reg-ulation of the rest of the genes in the human genome. Based on thecombination of our current and previous studies, 288 genes orexpressed sequence tags (ESTs),4 representing �1–1.5% of thehuman genome, are differentially regulated during the first 2 daysof Th1 and Th2 cell polarization. Moreover, we demonstrate thata panel of these genes or ESTs are induced by IL-4 through theSTAT6 signaling or play a role in regulation of IFN-� production.

Materials and MethodsIn vitro differentiation of Th1 and Th2 cells

Induction of human Th1 and Th2 cell differentiation was performed aspreviously described (10). Briefly, CD4� T cells isolated (Ficoll IsolationPaque; Amersham Biosciences and Dynal Biotech) from cord blood (Turku

*Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku,Finland; †Graduate School of Biomedical Sciences, University of Turku, Turku, Fin-land; ‡The National Graduate School in Informational and Structural Biology, ÅboAkademi University, Turku, Finland; and §Drug Discovery Graduate School, Uni-versity of Turku, Turku, Finland

Received for publication March 9, 2005. Accepted for publication December21, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Academy of Finland, Sigrid Juselius Foundation,National Technology Agency of Finland, Turku Graduate School of Biomedical Sci-ences, National Graduate School in Informational and Structural Biology, Drug Dis-covery Graduate School, Ida Montin Foundation, Finnish Society of Allergology andImmunology, Pulmonary Association Heli, Jenny and Antti Wihuri Foundation,Vaino and Laina Kivi Foundation, Allergy Research Foundation of South-WesternFinland, and Turku University Hospital Fund.2 R.J.L. and M.L. made an equal contribution to this work.3 Address correspondence and reprint requests to Prof. Riitta Lahesmaa, Turku Centrefor Biotechnology, University of Turku and Åbo Akademi University, P.O. Box 123,FIN-20521, Turku, Finland. E-mail address: riitta.lahesmaa@btk.fi

4 Abbreviations used in this paper: EST, expressed sequence tag; CT, threshold cycle;shRNA, short-hairpin RNA.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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University Central Hospital, Turku, Finland) were activated with plate-bound anti-CD3 (500–1000 ng/ml for coating) and 500 ng/ml soluble anti-CD28 (both from Immunotech). Th1 polarization was induced with 2.5ng/ml IL-12 and Th2 differentiation with 10 ng/ml IL-4 (both from R&DSystems). A subset of the cells was cultured in “neutral conditions” withoutpolarizing cytokines. In the indicated experiments, the cultures weresupplemented with 3 ng/ml TGF-� (R&D Systems). TGF-�-mediatedsuppression of IFN-� production by Th1 cells in these conditions hasbeen previously described (10). The samples were collected after 0, 2,6, or 48 h of polarization and were processed for Affymetrix hybrid-izations or Western blotting.

For validation of the oligonucleotide array results with real-time RT-PCR, additional Th1 and Th2 primary cultures were generated as describedin our previous study (11). Briefly, cord blood CD4� T cells were activatedwith 100 ng/ml PHA (Murex) and irradiated CD32-B7-transfected fibro-blasts (16). Th1 cultures were supplemented with 2.5 ng/ml IL-12, whereasTh2 cultures were supplemented with 10 mg/ml anti-IL-12 and 10 ng/mlIL-4 (all from R&D Systems). After 48 h of priming, 40 U/ml IL-2 (R&DSystems) was added into the cultures. A subset of the cells was culturedwithout any polarizing cytokines in the presence of IL-2 alone. The cul-tures were generated from four individuals. Samples were collected at timepoints 0, 6, 24, and 48 h or 7 days.

Oligonucleotide array studies

Human genome U133A and B arrays recognizing �33,000 transcripts wererehybridized with the previously prepared samples (10, 15). Briefly, afterconfirming the successful polarization of the cells with real-time RT-PCR,the sample preparation and data analysis were performed according to theinstructions and recommendations provided by the manufacturer (Af-fymetrix). Total RNA (4–5 �g) pooled from different individuals was usedas starting material for the Affymetrix sample preparation. Two biologicalrepeats for each microarray experiment were performed. GeneChip Mi-croarray Suite software version 5 (MAS5; Affymetrix), GeneSpring(SiliconGenetics), and Microsoft Access for Windows software were used toevaluate the quality of the data and for routine data analysis and processing.The microarray data was filtered according to the statistical classifications per-formed by the MAS5 software as previously described (10, 15). Genes thatpresented a consistent change (�2-fold) in two separate biological repeatswere considered as differentially expressed. All the genes, which fulfilledthese criteria in at least one of the comparisons and one of the time points,were selected for further analysis where the expression of the genes wasexplored parallel in different conditions without fold change threshold. Thegene annotations were obtained from NetAffx database (17). The normal-ized microarray raw data have been deposited in the Gene Expression

Table I. Primers and probes used in the real-time RT-PCR

Public ID Gene Symbol

1) 5�-6(FAM)-PROBE-(TAMRA)-3�

2) 5�-PRIMER 1–3�

3) 5�-PRIMER 2–3�

AW152437 1)2) 5�-AGCTGAGAAGAATGAAGAGGACATA-3�3) 5�-GTTCACAGCCCCTATGG-3�

AW139719 1)2) 5�-AGACTGGTTTGTTTTCACTTGAGGT-3�3) 5�-GTTTTCCCAGGAGTCTGAGGC-3�

R98767 1)2) 5�-TTTGCCTCAAATCCATTACCAA-3�3) 5�-AACTAGTCAAGTGTGATATAATCAGATTTGC-3�

AA489100 1)2) 5�-GAAAGCAATATGTTTAGCAGCTGTTT-3�3) 5�-ACATTTATGCCTGGATTAAATAACAATAGT-3�

BF056901 1)2) 5�-AAATAAGGCCTAGGTCCGTCTATTG-3�3) 5�-CGGCCTCGACCTTCAAAGA-3�

AI494573 1)2)5�-GCTGGGAAATCTACAAGTCACCTTA-3�3)5�-TTGCTGGCCATTTTATTGTTGAG-3�

AA088177 1)2) 5�-ATGCAAGGTGGAATTTTGGG-3�3) 5�-GTATTGCAGTTGTGGAAGAGCAGA-3�

BE748563 1)2) 5�-CCTTTCACTGCCATGGAATGA-3�3) 5�-AGAAAATGCAAGCTCCCCATAA-3�

AI674404 1)2) 5�-CTGGAACCTTGAGGCCTTCA-3�3) 5�-GCCCCAATCTATGGACAGACA-3�

AL389942 1) 5�-TGTGACAATGATTCTTTAGC-3�2) 5�-CACGGGATCAGAGGGAACTAATA-3�3) 5�-ACCAGAAGCCTCAGCAGTCC-3�

AA237039 1) 5�-TTTATGCAGCGCACTGTCAGACTTCCAA-3�2) 5�-GATGCATCTGCTTTTAACCCTTTT-3�3) 5�-TTACGGAATGCTGTGGTACTCAA-3�

AW629527 FLJ41238 1) 5�-ACCCATTTTGATTGAGACCTACACAGGGC-3�2) 5�-TGGAAGAGGAAAGAAACTGATGGT-3�3) 5�-GCGGTTTCCAATGAATGACA-3�

J04617 EF1� 1) 5�-AGCGCCGGCTATGCCCCTG-3�2) 5�-CTGAACCATCCAGGCCAAAT-3�3) 5�-GCCGTGTGGCAATCCAAT-3�

NM_016651 DACT1 1) 5�-CACGAACTCGCCCTCGCTCACACT-3�2) 5�-AGCAGAGCAATTACACCACCAA-3�3) 5�-AGTCTGGACAAACTGGGACCAA-3�

NM_005815 ZNF443 1) 5�-TCTCTTGGCTCACTTGCTTTCTACGACATG-3�2) 5�-GAATGTAAGGAATGTGGGAAAGC-3�3) 5�-CATAGGATTTCTCTCTCATGTGAATTCT-3�

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Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) of the National Center forBiotechnology 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 de-scribed before (11, 16). The housekeeping gene EF1� was used as a referencetranscript (16). Primers and probes (Table I) used for the quantification ofgene expression (MedProbe or DNA Technology) were designed usingPrimer Express software (Applied Biosystems). The quantitative value ob-tained from TaqMan real-time RT-PCR is a threshold cycle (CT). The folddifferences between different conditions can be calculated from the nor-malized CT values (CT gene X � CT housekeeping gene), �CT values,with the formula: fold difference � 2(I�CT1 � �CT2I). Statistical signifi-cances between the differences in gene expression were evaluated with ttests.

Western blot analysis

CD4� 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-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 celllysates were loaded on the gel. Alternatively, cells were lysed in HEPESbuffer (20 mM HEPES, 0.2% Tween 20, and 1 mM DTT) containing Com-plete Protease Inhibitor (Roche). From the centrifuged lysates, the super-natants were discarded and a second buffer (20 mM HEPES, 420 mMNaCl, 20% glycerol, and 1 mM DTT; Roche Complete Protease Inhibitor)was mixed with the cell pellets. After 1 h of incubation on ice, the sampleswere recentrifuged and supernatants containing nuclear proteins were ob-tained. The protein concentrations were then quantified from the samplesby Bio-Rad protein assay. Equal amounts of protein were subject to SDS-PAGE and transferred to Hybond ECL membrane (Amersham Bio-sciences). Uniform protein transfer was verified with Ponceau S staining(Sigma-Aldrich). The membranes were probed for TBX21 (sc-21749;Santa Cruz Biotechnology) and GATA3 (sc-268) to confirm successfulinduction of Th1/Th2 polarization. Western blotting was then performedfor DACT1 (ab5977-100; Abcam), RAB11FIP1 (RCP 11-A; Alpha Diag-nostic), FOSL2/Fra-2 (sc-604), CISH (sc-1529), ZF9 (sc-7158), FLT1 (sc-9029), and RAB27B (sc-22991) all from Santa Cruz Biotechnology.

Short-hairpin RNA (shRNA) mediated gene knockdown duringthe early polarization of human Th cells

The NDFIP2 shRNA plasmid construct, targeting (5�-GAGGAAGAGTGTCCACCAAGA-3�), was generated by cloning the NDFIP2 shRNA oligo-nucleotide into the BglII and XhoI sites of the previously modified pSuper-H2K-pIRES2 plasmid, which contains a truncated H2K cell surface selectionmarker (18). Similarly, a pSuper-H2K-pIRES2-scramble1-shRNA, (5�-AATTCTCCGAACGTGTCACGT-3�), was designed. In addition, two syntheticsmall interfering RNA oligos (one of which targeting the same sequence as theNDFIP2 shRNA shown above and the other targeting the 5�-CUGGAUAUUUCAAUGGACAUU-3� NDFIP2 sequence; Sigma-Aldrich) were used toknock down NDFIP2. For the STAT6 knockdown studies, the previously pre-pared pSuper-H2K-STAT6-shRNA plasmid, targeting (5�-GAATCAGTCAACGTGTTGTCAG-3�), and the pSuper-H2K-scramble2-shRNA (5�-GCGCGCTTTGTAGGATTCG-3�) were used (18). Furthermore, another pSuper-H2K-STAT6-shRNA plasmid, targeting 5�-CAGTTCCGCCACTTGCCAAT-3�, was used in one replicate culture. Cell transfections, dead cell removal,enrichments (�98%), and differentiations were performed as recently de-scribed (18). The cells were activated with anti-CD3 plus anti-CD28 and wereinduced to polarize to Th1 and Th2 direction as described above. For RT-PCRanalysis the cells were harvested at 24 or 48 h of polarization from three to fourbiological replicates. Cells harvested at 7 days of polarization were washed,restimulated, and cytokines secreted in the supernatant after 24 h of restimu-lation were measured (see the section below). For RT-PCR, the total RNAswere isolated either with an RNA Easy Minikit (Qiagen) or a PicoPureTMRNA Isolation kit (Arcturus). Consequently, cDNAs were prepared with aTranscription First Strand cDNA Synthesis kit (Roche).

Cytokine secretion assay

To measure the IFN-� production by the control cells or cells transfectedwith NDFIP2-shRNA, the cells were incubated with or without 5 ng/mlPMA (Calbiochem) and 500 ng/ml ionomycin (Sigma-Aldrich). The cul-ture medium from each well was collected after 24 h of restimulation withPMA and ionomycin. The culture medium was separated from the cells by

centrifugation and stored at �70°C until used for cytokine determination.Secreted cytokines were measured using Luminex assay and multiplexbead kits from LINCO Research and from Bio-Rad. The assays were con-ducted in duplicate according to the manufacturer’s instructions. Measure-ments and data analysis were performed with the Bio-Plex system in com-bination with the Bio-Plex Manager software (Bio-Rad).

ResultsTo identify novel genes involved in the initiation and early polar-ization of Th1 and Th2 cells at the genome scale, transcriptomeanalysis was conducted using Affymetrix U133A and B oligonu-cleotide arrays. To identify genes regulated by IL-12 or IL-4 inactivated Th cells, the cells induced to polarize to Th1 (CD3 plusCD28 plus IL-12) or Th2 (CD3 plus CD28 plus IL-4) direction for2, 6, or 48 h were compared with each other and to the CD3 plusCD28-activated cells cultured without polarizing cytokines (Th0).Furthermore, genes coregulated by TGF-� and IL-12 or IL-4 dur-ing the early Th1 and Th2 cell differentiation were identified. Inaddition to the previously identified genes, an additional 171 geneswere found to be regulated by IL-12 or IL-4 (Fig. 1A) during thefirst 2 days of Th1 and Th2 differentiation (see Table II for all theIL-12- and IL-4-regulated genes) (10, 15).

The slow response to IL-12

Consistent with our previous observations, IL-12 had minimal orno effect on the polarization process after 2 or 6 h (15). After 2 hof Th1 polarization, there were no genes regulated by IL-12. After6 h, �2-fold induction by IL-12 was seen in the expression of keyregulator of Th1 differentiation TBX21 and GTPase GBP5 (3, 4).After 48 h, the effects of IL-12 were clear and altogether 41 genesbecame regulated by IL-12 (Table II). Among these were only afew genes, such as SOCS3, CEBPB, and IL-7R that have a previ-ously described role in Th1 and Th2 cell responses (13, 19, 20).

FIGURE 1. Summary of the genes regulated by CD3 plus CD28 acti-vation, IL-12, and IL-4. Expression profiles of the cells activated withanti-CD3 plus anti-CD28 alone (Th0) or induced to differentiate to Th1(anti-CD3 plus anti-CD28 plus IL-12) or Th2 (anti-CD3 plus anti-CD28plus IL-4) direction were studied with oligonucleotide arrays after 0, 2, 6,and 48 h of polarization. A, The total number of genes regulated by CD3plus CD28 activation, IL-12, and/or IL-4 during the early Th1 and Th2 celldifferentiation. B, The number of genes up- (1) or down-regulated (2) byIL-4 at 2, 6, and/or 48 h of Th2 cell differentiation.

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

Public ID Gene

Fold Changea Fold Change Fold Change Fold Change

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

2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h

Genes regulated by both IL-12 and IL-41. Enzyme regulator activity (2)

NM_003955 SOCS3 3.1 2.4 2.6 �2 �9.2 �13.9 �5.5NM_021158 TRIB3 (5)b 1.4 1.8 1.8 1.6 3.9

2. Signal transducer activity (2)NM_006137 CD7 2.1 2BE217880 IL7R 2.3 �1.4 �2.5 �25.1

3. Transcription regulator activity (4)NM_013351 TBX21 1.6 �1.5 3.2 10.2 2.8NM_024508 ZBED2 3.7 2.9 3.7 �2.9 10.9 16AB059408 HOP 2.9 3.3AL564683 CEBPB 1.4 1.7 �2.7

4. Structural molecule activity (1)NM_005572 LMNA 2.5 1.5 1.8 27.9 7

5. Catalytic activity (4)NM_021158 TRIB3 (1) 1.4 1.8 1.8 1.6 3.9BG545653 GBP5 1.4 �1.8 2.5 2.7AB000888 PPAP2A �1.5 2.4 �2 �2.1 4.4AI631159 SLC2A3 1.7 1.6 �6.5 �2

6. Other binding molecules (5)NM_018326 GIMAP4 2.1 �1.7 �2.4 1.9 2.7 2.5AI674404 SYTL3 1.8 3.2 2.8 �5.1 �1.9 �4.3 2.4AW574798 KLHL6 1.6 2.3 4.1 2.6AI524095 LY9 �1.7 �2.5 1.9 �2.9 �1.9AI005043 WASPIP �1.7 �2.1

7. Miscellaneous (2)AI042152 TncRNA 2.2 1.9 �1.5 �1.6AW290956 NDFIP2 1.3 1.9 1.5 3 �2.2 �1.5 �2.3 16 48.5 142

8. Unknown (4)AK026646 SURF4 1.6 2.2 2.1 2.9 1.8AI214996 LOC284018 1.6 4.8 �5.1 8.3BF968270 SLC35F3 �2.8 �1.8 �1.6 11.7 13.9BF209337 MGC4677 �1.7 1.5 �1.9 �1.6 2.7 9.2 9.5

Genes regulated by IL-121. Enzyme regulator activity (1)

NM_001629 ALOX5AP �1.2 �1.9 1.92. Signal transducer activity (2)

NM_016081 KIAA0992 8AL121985 SLAMF7 1.8 12.1

3. Catalytic activity (3)Y13786 ADAM19 2.7 4.3AW150720 RDH10 1.5 10.2 3.2AL354872 CTH 2.2 3.6

4. Other binding molecules (4)AF176013 DNAJC12b 4.3 16.6AK021850 FBXO16 /// ZNF395 2 �3.5 �4.4NM_001311 CRIP1 �2.1 �2.8 5.3 4.1NM_002961 S100A4 �2.1 �2.4 �2.5 �9.8

5. Miscellaneous (3)AV713773 MCOLN2 2.3 8.3AA195074 TIFA 1.6 1.7 2.2 �2.3 4J03223 PRG1 �1.9 �2.4 3.2 2.9

6. Unknown (5)AF142573 CRISPLD1 2.9BG110811 LOC340061 2.6 �3.5AA831661 MGC29814 1.7 �5.5 �5.9H99792 �4.6 8BE222344 �2.5 1.9 3 5.3 4.3

Genes regulated by IL-41. Enzyme regulator activity (4)

NM_003807 TNFSF14 (2.2) �1.5 2.5 �6.1 430.5 163.1AI798823 PSCD1 5.9 �4.3 �5.1 �2.5NM_000100 CSTB 1.4 �2.1 2.2 2.6NM_004369 COL6A3 (4) 13.5 1.9 �6.3 12.1

2. Signal transducer activity (17)2.1 Receptor activity (10)

AU159276 CYSLTR1b 2.7 �3AA830854 FLJ31951 (5.1) 2.2 �2 2.1

(Table continues)

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Table II. (Continued)

Public ID Gene

Fold Changea Fold Change Fold Change Fold Change

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

2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h

AF312230 HRH4 3.9 �3.5AA149648 FLT1b (5.2) 2.8 �3.1 8BG170541 MET (5.2) 2.7 �5.1AW193698 TGFBR3 2.5 �2AF053712 TNFSF11 (2.2) 1.7 �1.6 �1.7 4.6 7.2AL121905 PTPRA (5.3) 2.1NM_001838 CCR7 �2 1.5 2.1 �4.3 �1.9AF261135 GPR18 �1.6 �2.2 �2.4

2.2 Receptor binding (5)NM_003807 TNFSF14 (1) �1.5 2.5 �6.1 430.5 163.1NM_000572 IL10 2.5AF053712 TNFSF11 (2.1) 1.7 �1.6 �1.7 4.6 7.2NM_014751 MTSS1 �2.6 �2.1U57059 TNFSF10 �1.9 �2.8 3.4

3. Transcription regulator activity (7)AI924426 ELL2 1.9 1.8 2.6 �1.8 �1.7 3.2NM_005815 ZNF443 4 2.5 �3.2 �2.5AK022280 PHF20L1b 2.5 2.3 �2.5 �2.1BG485129 LOC360030 2.1 �1.5AK023816 EZH2 1.8 �2.1AI670862 FOSL2 2.2 2.9 �1.9 �2.5 �3AW299558 FRBZ1 �4.1 4.1

4. Structural molecule activity (4)AK022771 TUBD1 1.9 �2.1NM_004369 COL6A3 (1) 13.5 1.9 �6.3 12.1AF101051 CLDN1 2.6 �1.5 �1.9 16 12.1 2AF308301 MRPS26 3.5 �3.1

5. Catalytic activity (37)5.1 Ligase (6)

NM_017831 RNF125 2.4 4.1 �2.4 �4.4 �4.6 �6.5 �4.8AW070573 RNF146 3.9 �2.8NM_021178 CCNB1IP1 2.5 �1.9 2.9AA830854 FLJ31951 (2.1) 2.2 �2 2.1AB029316 RNF19 1.4 3.1 �1.6 �2.1 4.8 2.4BG171548 NCE2 1.9 �3 2.5 1.6

5.2 Transferase (13)R98767 CAMK2D 3 1.7 �4.4 �2.1AI221707 STK17B 2.6 �2.7BF739767 DKFZp761P0423 2.5NM_017719 SNRK 2.2 �2.6 �3.5 �4.4AA149648 FLT1b (2.1) 2.8 �3.1 8BG170541 MET (2.1) 2.7 �5.1AI742057 LOC129607 2.5 �2.1AI890347 GALNT4 2 �2.8AA922068 CDK6 1.7 �2 2.5AI823980 RBKS 4AA352113 ST8SIA4 2.3 2.9 3.5AL563795 RP11-311P8.3 2.1 �2 1.5 2.2D50925 PASK �2 1.7 �2.5 �7.7 �8.3

5.3 Hydrolase (6)AW294640 RAB30 3 �3.4 �2.2BF670447 RHOQ 2.1 �2.8BF438386 RAB27B 2.5 �2.1NM_006226 PLCL1 1.5 �4.6AL121905 PTPRA (2.1) 2.1AL136680 GBP3 �2.1 2.1

5.4 Transporter activity (8)AF225986 SCN3A 3.9 3 �6.7 �3.4AF112972 ATP6V0A2 1.6 3 �4 2.5 4.6AL117381 BCL2L1 2.2 5.7 8AA551075 KCTD12 1.6 �3.2AL136583 SLC37A3 2.5 6.3 �2.5 �4.3AL574184 HPGD 2.2 �3.2 �18.4NM_002668 PLP2 �1.4 1.5 1.7 �1.5 3.4 2.1AA854943 ANKH �1.4 5.9

6. Other binding molecules (11)AL118798 CD47b 2.7 1.6 1.8 �2.6 �1.7 �1.9 �1.7 �2.2NM_016837 RBMS1 1.4 2 �1.7 �3

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Table II. (Continued)

Public ID Gene

Fold Changea Fold Change Fold Change Fold Change

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

2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h

AK025100 SNTB1 2.5 �2.2AB003476 AKAP12 4.6 �5.1BC001247 EPLINb 2.9 �4.9BC002827 TPM4 2.1 �1.7AL110164 LIMS1 1.6NM_024711 GIMAP6 �4 4.1 �2.3 �2.1AW273811 HNRPLL �2.9 2.5 8.9 13AA858297 GIMAP7 �2.5 2.9 �2.1NM_023037 13CDNA73 �2 1.9

7. Miscellaneous (17)NM_013324 CISHb 14.4 4 1.4 �13 �2.8 2.9 7.5NM_016651 DACT1 2.9 2.5 1.9 �2.5 �1.9 �2.1BG540494 AKAP2b 2.5 3.1 2.4 �2.3 �2.9 �1.8NM_025151 RAB11FIP1b 2.8 2.1 �3.6 �1.8 3.2BE748563 LOC93081 2.1 2.5 �2.4 �2.2AV725328 PRNP 2.1 1.7 �2.1 �1.5 4.9 2.9AL138717 RRAGD 1.9 2.2 �1.7NM_013322 SNX10 1.9 �2.1 3.1BE676408 HECA 2.1 �3.4 �10.6 �10.9AU150319 VAMP1 �1.5 �2.5 2.4 3 �2.5AI681671 DPH2L1 �2.1 21.9 9.2NM_001924 GADD45A �1.7 1.3 �1.7 �3 �6.1AL035541 TMEPAI �1.6 1.4 10.2AA736452 PGM2L1 �2.3 1.9 8.3 7.7BF589359 PAG �2.1 1.7 �2.3 �1.9 �2.4AF000426 LST1 �4.9NM_022873 G1P3 �2.8

8. Unknown function (46)BF674052 TMEM49 2.9 1.7 3.2 �3.9 �1.8 5.7 7.5 4.6AI969697 3.2 17.8 7.5 �2.5 �5.9 �10.9 �5.9 �30.9 �8.9AB033106 KIAA1280b 1.7 3.4 �1.6 �2.8R14890 3.2 2.7 �3.1 �2.8 33.1 6.3AL109935 KIAA1434 2.9 2.5 �2.6 �2.5 �2.5 �5.1AI797353 FLJ31340 2.3 2.5 �2.6BF056901 MGC16044 3.6 2.3 �3 �2.6AI768674 2.3 2.2AA489100 5.3 2.1 �2 �4.8 �2.6 �3.6AI610684 4.1 1.9 �4.3 �2AC004010 AMIGO2 3.2 1.9 �3.7 �1.9 2.5 3AA088177 KIAA1913 1.7 1.9 �1.9AW152437 3.7 1.6 �4 �1.7 5.9AW139719 3.4 1.6 �3.2 �1.9 19.7AA002140 2.8 2.1 �3.4AL048542 AMICA1 2 3.6 �1.8 �1.9 �3.9 9.8 2AW135176 4.3 �2.8 2.7AI494573 3.7 �3.5BF968097 3.5 �11.7 �2.7AA429262 3 �4.1NM_024048 MGC3020 2.7 �3AI888503 2.4 �2.5 �1.5 �2.2 �2.1AA034012 2.4 �2.8 �2.8 �4.4AW292872 2.1 �2.1 �1.6 1.5AI458439 1.8 �2.4 �2.8 2.8W72060 1.5 �1.6 1.8AW629527 FLJ41238 8.3 10.9 �7 �10.6 29.9AI310524 NIPA1 2.4 �2.6 2.8BE670307 2.2 �2.3AI825833 LOC284801 1.7 1.4BF037662 1.5 �1.7 �2 �7.7 �1.3BF739885 LOC284262 5.9NM_016008 D2LIC 2.5 �2.8AW591809 3.4 �3.6AI342543 FLJ33069 2.8 �2.5 2.5 �2.1AW204712 C10orf128 2.5 �2.1BF969397 LOC387882 2.2 �2.7 1.7AU134977 TncRNA 2 �1.9AI870617 KAI1 �2.4 �1.9 2 1.4 6.5 27.9 3.5AI654547 �1.9 1.6 �1.6 11.3 6.1

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Most of the genes regulated by IL-12 were also regulated by IL-4and/or activation alone (Fig. 1A), although there were differencesin the kinetics and magnitude in the changes induced by thesedifferent treatments. Similarly to the key regulator of Th1 and Th2cell differentiation, TBX21, a subset of genes, including GIMAP4,IL-7R, PPAP2A, MGC4677, and GBP5, was regulated in an op-posite manner by IL-12 and IL-4, although as for TBX21, thechanges were small.

IL-4 rapidly regulates numerous genes during early Th2 celldifferentiation

In contrast to IL-12 signaling, the effects of IL-4 were clearly seenwithin 2 h of Th2 polarization. IL-4 regulated the expression ofaltogether 153 genes during early Th2 differentiation (Fig. 1).Genes AI969697, NDFIP2, CD47, AKAP2, CISH, TMEM49,DACT1, and ELL2 were up-regulated by IL-4 at all time points(Table III). A subset of the 153 genes regulated by IL-4, includingCD47, PRNP, SOCS3, FOSL2, COL6A3, PTPRA, TNFSF10, IL-10, CCR7, CEBPB, IL-7R, GBP3, and TBX21 has been previouslydescribed to have a role in the process (3, 8, 12, 13, 19–23). Im-portantly, most of the IL-4-regulated genes identified were new inthis context.

Genes regulated by TGF-� during the early Th1 or Th2differentiation

To study the effects of TGF-� on the early polarization of Th cells,cells cultured in Th1 or Th2 conditions were compared with thosesimilarly cultured, but in the presence of TGF-�. In addition to thepreviously identified genes, an additional 110 genes were regulated

by TGF-� (data not shown) (10, 15). These included 63 genes thatwere coregulated by TGF-� and the polarizing cytokines IL-12 orIL-4. Altogether 25 genes were coregulated by TGF-� and IL-12(Table IV). Expression of five of these genes was enhanced oraccelerated in the presence of TGF-�, at least to some extent.Importantly, 19 of the IL-12-regulated genes were antagonized byTGF-� in Th1 conditions. Similarly, the effects of IL-4 on theexpression of 20 genes were enhanced or accelerated in the pres-ence of TGF-�, whereas expression of 25 genes was antagonizedby TGF-� (Table V).

A subset of genes (ZBED2, LMNA, NDFIP2, TncRNA, SYTL3,SLC2A3, and TRIB3), coregulated by TGF-� and the polarizingcytokines, was up-regulated by both IL-12 and IL-4 at least to someextent. Of these, the expression of ZBED2, LMNA, and NDFIP2 wasfurther increased in the presence of TGF-� in both Th1 and Th2conditions. In contrast, TncRNA, SYTL3, SLC2A3, and TRIB3, up-regulated by IL-12 and IL-4, were repressed by TGF-� in both Th1and Th2 conditions.

Putative or known functions of the genes involved during earlyTh1 and Th2 differentiation

To elucidate the putative or known functions of the factors in-volved in the early Th1 and Th2 cell differentiation, these geneswere analyzed with the gene ontology annotation tool in NetAffx(17). Concordant with our previous studies, most of the genes reg-ulated during the early differentiation of Th1 and Th2 cells codefor factors involved in the signal transduction from cell surface tonucleus (10, 15). Among the IL-4-regulated genes, the most out-standing functional group consisted of genes coding for proteins

Table II. (Continued)

Public ID Gene

Fold Changea Fold Change Fold Change Fold Change

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

2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h 2 h 6 h 48 h

AL389942 LOC285628 �3.4 �1.7 2.5 66.3 477.7 16.6AI923633 �2.7 1.9 1.6 2.1AI380089 �2.4 2 10.2AW340595 �2.2 1.6 �3.1AI631833 �2.1 1.9 1.3AI701905 �2.1 1.6

a Average fold change values for the genes that were differentially expressed �2-fold in comparisons of Th1 and Th2 cells to each other or to Th0 cells in at least one ofthe time points. Also, for these selected genes, the statistically significant changes �2-fold are shown in parallel conditions. Missing values indicate that the change was notsignificant according to the statistical data analysis by MAS5.

b Gene is found also in the indicated section of the table.

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

Public ID Gene Known or Putative Function

Fold Changea Fold Change

Th2 vs Th0 Th0 vs Thp

2 h 6 h 48 h 2 h 6 h 48 h

AI969697 Unknown 3.3 17.8 7.5 �5.9 �30.9 �8.9AL118798 CD47 Cell-matrix adhesion 2.7 1.6 1.8 �1.7 �2.2NM_016651 DACT1 2.9 2.5 1.9 �2.1BG540494 AKAP2 G protein signaling 2.5 3.1 2.4 �1.8BF674052 TMEM49 Unknown 2.9 1.7 3.3 5.7 7.5 4.6AW290956 NDFIP2 Regulation of I�B/NF-�B cascade 1.9 1.5 3.0 16.0 48.5 142.0AI924426 ELL2 Transcriptional regulator 1.9 1.8 2.6 3.3NM_013324 CISH Intracellular signaling 14.4 4.0 1.4 2.9 7.5

a Average fold change values for the genes that were differentially expressed �2-fold in comparisons of Th1 and Th2 cells to each other or to Th0cells in at least one of the time points. Also, for these selected genes, the statistically significant changes �2-fold are shown in parallel conditions. Missingvalues indicate that the change was not significant according to the statistical data analysis by MAS5.

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with catalytic activity including genes involved in RAS oncogenefamily signaling (RAB27B, RAB30, and RHOQ). Another predom-inant functional group consisted of genes coding for receptors orreceptor binding proteins. These included components of G protein-coupled receptor signaling (CYSLTR1, HRH4, CD47, CCR7, andGPR18). Numerous transcriptional regulators, such as ELL2,ZNF443, PHF20L1, LOC360030, EZH2, ZBED2, FOSL2, andFRBZ1 became differentially regulated during the early Th1 and Th2differentiation. As expected, IL-12 and/or IL-4 regulated several genesinvolved in the immune response, such as IL-10, CCR7, CD7,CEBPB, and IL-7R. Other common functional groups consisted ofgenes involved in apoptosis, proliferation, adhesion, metabolism, mo-tility, cell cycle, protein localization, transport, and regulation of pro-tein activity. The function of the majority of the genes differentiallyregulated during early Th1 and Th2 cell differentiation was unknown.Among these were numerous ESTs that were rapidly up-regulated byIL-4 in the cells induced to polarize to Th2 direction. A set of thesegenes or ESTs was selected for further studies. As IL-12 did not havemuch effect at the initiation of the differentiation, our special interestwas on the genes or ESTs regulated by IL-4.

Validation of the oligonucleotide array results with real-timeRT-PCR

Expression of 14 ESTs or genes with poorly characterized func-tion was further studied with real-time RT-PCR during 7 daysof Th1 and Th2 cell polarization. These included AA088177,AA237039, AA489100, AI494573, AW139719, AW152437,AW629527, AL389942, BF056901, R98767, BE748563,

AI674404, ZNF443, and DACT1. Based on the gene array re-sults, all of these genes and ESTs were rapidly regulated byIL-4 at the initiation of Th2 cell differentiation. RT-PCR con-firmed IL-4-mediated regulation of 12 of 14 genes or ESTs (Fig.2). Regulation of transcripts by IL-4 was primarily limitedwithin the first 2 days of Th2 differentiation. For BF056901 andAI674404, the Affymetrix results could not be confirmed in allthree individuals studied.

FOSL2 is preferentially expressed during the early Th2 cellpolarization at the protein level

Systematic validation of the differences detected at the mRNAlevel was performed at the protein level for those genes that com-mercial Abs were available. Most of the genes identified in thisstudy are poorly characterized and therefore a limited number ofAbs were obtained. The proteins studied included DACT1,RAB11FIP1, FOSL2, CISH, KLF6, FLT1, and RAB27B. As canbe seen from Fig. 3, FOSL2 was indeed differentially expressed atthe protein level, with higher expression in Th2 cells detected at48 h. TBX21 and GATA3 were used as control genes to confirmthe polarization of the cells to Th1 and Th2 direction. For the otherproteins we were not able to detect any differences in the expres-sion levels during early polarization of Th1 or Th2 cells (datanot shown). Proteomics studies in progress will be used to elu-cidate the differences in the expression of the possible variantsof these and other proteins during early stages of Th1 and Th2differentiation.

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

Public ID Gene Symbol Known or Putative Function

Fold Changea Fold Change

Th1�TGFb vs Th1 Th1 vs Act

2 h 6 h 48 h 2 h 6 h 48 h

NM_006137 CD7 Receptor 2.1a �1.6 2.1TGF-� enhances the

effects of IL-12NM_024508 ZBED2b Transcriptional regulator 1.7 3.7AW290956 NDFIP2b Signal transducer 1.7 1.3Y13786 ADAM19 Metal ion binding 3.4 2.7NM_005572 LMNAb Cytoskeletal 2.1 2.5AW574798 KLHL6 Miscellaneous 1.4 1.6

TGF-� antagonizes theeffects of IL-12

AL121985 SLAMF7b Receptor �2.5 1.8AB059408 HOP Transcriptional regulator �7 2.9NM_003955 SOCS3b Protein kinase inhibitor �2 3.1AW150720 RDH10 Oxidoreductase �2.3 1.5NM_021158 TRIB3 Protein kinase �2.5 1.4NM_001629 ALOX5AP Enzyme activator 2.2 �1.2AI631159 SLC2A3 Glucose transporter �2.5 1.7NM_018326 GIMAP4b Miscellaneous �1.9 2.1AK021850 FBXO16 /// ZNF395 Miscellaneous �2.1 2NM_001311 CRIP1b Miscellaneous 3 �2.1BG110811 LOC340061 Unknown �1.5 2.6AV713773 MCOLN2 Unknown �4 2.3AI042152 TncRNA Unknown �2.5 2.2AI674404 SYTL3b Unknown �3.2 1.8AA831661 MGC29814 Unknown �2.1 1.7AI214996 LOC284018b Unknown �1.6 1.6AA195074 TIFAb TRAF-interacting protein �1.6 1.6BF209337 MGC4677b Unknown 1.9 1.7 �1.7BE222344 —b Unknown 1.7 5.1 �2.5

a Average fold change values for the genes that were differentially expressed �2-fold in comparisons of Th1 and Th2 cells to each other or to Th0cells in at least one of the time points. Also, for these selected genes, the statistically significant changes �2-fold are shown in parallel conditions. Missingvalues 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.

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AW629527, AA088177, AA489100, ZNF443, and DACT1 areinduced by IL-4 in a STAT6-dependent manner

To further study whether a subset of poorly characterized genes in-duced by IL-4 is regulated through STAT6 signaling, human periph-eral blood-derived CD4� cells were nucleofected with two differentpSuper-H2K-STAT6-shRNA plasmids or a pSuper-H2K-scramble-shRNA plasmid. To verify the functionality of the pSuper-H2K-STAT6-shRNA, we demonstrated that STAT6 and GATA3 mRNAlevels were down-regulated by these shRNAs (Fig. 4). To determineeffects of this STAT6 knockdown on the expression of a subset of

selected genes or ESTs regulated by IL-4, mRNA expression wasmeasured from these samples with quantitative real-time RT-PCR.The results demonstrated that the induction of AW629527, AA088177,AA489100, ZNF443, and DACT1 genes or ESTs by IL-4 was clearlydependent on STAT6. No effect by STAT6-shRNA on the expressionof AA237039 or AW152437 was observed (data not shown).

NDFIP2 promotes IFN-� production by Th1 cells

NDFIP2 was selected for further functional studies as it was highlyinduced by CD3 plus CD28 activation alone and the expression

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

Public ID Gene Symbol Known or Putative Function

Fold Changea Fold Change

Th2�TGF� vs Th2 Th2 vs Th0

2 h 6 h 48 h 2 h 6 h 48 h

AF261135 GPR18b GPCR signaling 1.5 �1.9 �1.6TGF-� enhances the

effects of IL-4AA149648 FLT1b Receptor, enzyme 1.4 2.8AF053712 TNFSF11b Receptor 1.7 1.7AI924426 ELL2b Transcriptional regulator 2.3 1.9 1.8 2.5NM_024508 ZBED2b Transcriptional regulator 2.2 2.9 3.7AL513759 RBPSUHb Transcriptional regulator 2.4 1.8AW290956 NDFIP2b Signal transducer 1.3 1.9 1.5 3.0NM_001924 GADD45Ab Intracellular signaling �2.2 �1.7U57059 TNFSF10 Intracellular signaling �2.4 �1.9AA029441 CAMK2Db Enzyme 1.3 2.2NM_004369 COL6A3b Enzyme inhibitor, cell adhesion 3.1 13.5BF439618 CD47b Cell-matrix adhesion 1.6 2.3AL118798 CD47 Cell-matrix adhesion 1.5 2.7 1.6 1.8AI524095 LY9b Cell adhesion �1.9 �2.5AF101051 CLDN1b Cell adhesion 1.3 2.5NM_005572 LMNAb Cytoskeletal 3.1 1.5AL110164 LIMS1 Miscellaneous 2.5 1.6W72060 —b Unknown 1.5 1.5AA088177 KIAA1913b Unknown 2.5 1.7 1.9AL048542 AMICAb Unknown 1.6 2.0 3.6BF209337 MGC4677b Unknown 1.5 1.6 1.5AW591809 —b Unknown 2.8 3.4

TGF-� antagonizes theeffects of IL-4

AL564683 CEBPB Transcriptional regulator �2.1 1.7NM_018664 SNFTb Transcriptional regulator �2.0 4.3AL353944 RUNX2b Transcriptional regulator �1.6 2.7AB000888 PPAP2Ab Enzyme �1.9 �1.9 2.4AI823980 RBKS Enzyme �2.5 4.0NM_021158 TRIB3 Protein kinase �2.5 1.8BG171548 NCE2b Enzyme �1.7 1.9AA352113 SIAT8D Enzyme �1.4 2.3NM_002668 PLP2b Transporter 1.5 1.8 1.6 �1.4 1.5AA854943 ANKH Transporter 1.3 1.6 �1.4AI798823 PSCD1b Transporter �3.6 5.9AI631159 SLC2A3 Glucose transporter �1.6 1.6BC002827 TPM4b Cytoskeletal �1.4 2.1AW135176 —b Miscellaneous �2.2 4.3AL035541 TMEPAIb Miscellaneous 1.8 2.7 �1.6AW152437 —b Unknown �1.4 3.7 1.6AI654547 —b Unknown 1.3 1.5 �1.9 1.6AI825833 LOC284801 Unknown �2.7 1.7AI674404 SYTL3b Unknown �2.3 �4.1 3.2 2.8AW629527 FLJ41238 Unknown �1.5 8.3 10.9AU134977 — Unknown �2.3 2.0AI042152 TncRNA Unknown �2.1 1.9AI342543 FLJ33069b Unknown �1.9 2.8AL563795 MGC23937b Unknown �1.6 2.1BF968270 SLC35F3b Unknown 2.1 �1.8

a Average fold change values for the genes that were differentially expressed �2-fold in comparisons of Th1 and Th2 cells to each other or to Th0cells in at least one of the time points. Also, for these selected genes, the statistically significant changes �2-fold are shown in parallel conditions. Missingvalues 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.

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was further enhanced by IL-4 during the early Th2 differentiationat all of the time points studied. This regulation of NDFIP2 wasconfirmed with RT-PCR (Fig. 5A). In contrast to the oligonucle-otide array results, where high induction of NDFIP2 expression byCD3 plus CD28 activation alone was observed (Table III), activa-

tion with PHA plus CD32-B7-transfected feeder cells induced onlya modest short-term increase in NDFIP2 expression as measuredwith RT-PCR (Fig. 5A). Because the role of NDFIP2 in the dif-ferentiation of Th cells was previously unknown, the effect ofNDFIP2 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 inIFN-� production by Th1 cells (Fig. 5B) in two biological repli-cates. In addition, a study with a similar experimental set-up, butusing cord blood CD4� cells and two different NDFIP2-specificsynthetic siRNA oligos was conducted. The latter study resulted incytokine secretion profiles consistent to those obtained using plas-mid-based shRNA knockdown (data not shown). The effect ofNDFIP2 knockdown on IL-4 production could not be estimatedreliably (data not shown), due to the low levels of the IL-4production by these cells, as is commonly observed after thefirst round of human Th2 cell polarization in these conditions(24, 25). Most importantly, our experiment demonstrates thatNDFIP2 has a function in the Th1 cell differentiation process bypromoting secretion of the hallmark cytokine of Th1 cell dif-ferentiation, IFN-�.

FIGURE 3. FOSL2 protein is preferentially expressed by cells polarizedto Th2 direction at early stages of differentiation. Human cord blood CD4�

T cells were either cultured in neutral conditions (Th0) or polarized to Th1or Th2 for 2, 6, and 48 h. Nuclear lysates were subjected to Westernblotting with TBX21 and GATA3 expression to validate successful Th1and Th2 polarization and then probed for FOSL2.

FIGURE 2. Validation of the oligonucle-otide array results with real-time RT-PCR.CD4� T cells were isolated from humancord blood. The cells were activated withPHA and CD32-B7-transfected feeder cells.The cells were either cultured under “neutralconditions” (Th0) or polarized to Th1 or Th2for 7 days. During the polarization expres-sion of selected genes was studied at the in-dicated time points with real-time RT-PCR.�, Statistically significant differences in geneexpression between the Th2 and Th0 cellswere determined with a t test.

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DiscussionWe have conducted a genome-wide screening to identify novelgenes involved in early Th1 and Th2 differentiation (13, 15). In-terestingly, numerous factors with known or unknown functionwere identified to be implicated in the process. A number of thesegenes could be positioned into specific intracellular signaling cas-cades. This study provides an overview of the intracellular signal-ing events in the cells at the initiation of Th1 and Th2 celldifferentiation.

Thp cells are not responsive to IL-12, which can be seen in slowchanges in gene expression profiles in response to IL-12 duringearly Th1 cell differentiation. Our results at whole genome scaleconfirm the previous consensus that indicates activation of IFNsignaling to be the first event during Th1 differentiation. This isdemonstrated by the early up-regulation of key regulators of Th1differentiation IFNG, TBX21, and IFN-regulated genes GBP1 andGBP5 (3, 4, 15, 26, 27). TBX21 is induced by T cell activation andits expression is enhanced by IFN-� during early Th1 polarization(28–31). The role of GBP1 and GBP5 in Th1 polarization is notknown. As these genes are induced by IFNs, it is likely that, sim-ilar to TBX21, they are induced due to enhanced IFN-� signaling.Activation of IFN-� signaling and TBX21 expression during earlydifferentiation is essential for Th1 differentiation, probably becauseit enables IL-12 signaling (4, 28, 32, 33). The genes coding forreceptor components for IL-12 and IL-18 (IL12RB2 and IL18RAP)are up-regulated within 48 h, enabling and enhancing the respon-siveness of the developing Th1 cells to polarizing cytokines (10).Consistently, after 48 h of Th1 polarization several genes wereregulated by IL-12. The roles of most of these genes or ESTs inTh1 differentiation are not known.

Thp cells are responsive to IL-4, which can be seen in the rapidregulation of numerous genes and ESTs with known and unknownfunction. A subset of these genes was demonstrated to be regulatedby IL-4 in a STAT6-dependent manner. Most of the IL-4-regulatedgenes displayed only temporary changes at some of the time points

studied. However, a group of genes, including NDFIP2, CD47,CISH, ELL2, AI969697, AKAP2, DACT1, and TMEM49, were reg-ulated by IL-4 throughout the first 2 days. Previously, we observedsimilar expression patterns for the key mediators of Th2 differen-tiation GATA3 and MAF. Similar regulation for a panel of genes,such as BCL6, NFIL-3, SATB1, SOCS1, DUSP6, IL10RA, andCXCR4, with unknown or less clear functions in Th1 and Th2 celldifferentiation was also demonstrated in our previous studies (10,15). Maintenance of the IL-4-mediated regulation of these genesthroughout the early Th2 cell differentiation suggests them to be

FIGURE 4. AW629527, AA088177, AA489100, ZNF443, and DACT1are induced by IL-4 in a STAT6-dependent manner. Human peripheralblood-derived CD4� cells were nucleofected with a pSuper-H2K-STAT6-shRNA or nonfunctional pSuper-H2K-scramble-shRNA control vectors.The transfected cells (purity of �98%) were induced to polarize to Th2(anti-CD3 plus anti-CD28 plus IL-4) direction. The cells were harvestedafter 24 or 48 h of polarization. Real-time RT-PCR was used to measureexpression of selected IL-4-regulated genes in three to four biological rep-licates. The genes with statistically significant difference between STAT6-shRNA and scramble-shRNA transfected cells are presented in the figure(paired t test: p � 0.05). Fold changes in gene expression between the Th2cells expressing STAT6-shRNA and scramble-shRNA are indicated in thefigure.

FIGURE 5. NDFIP2 promotes IFN-� production by human Th1 cells.CD4� T cells were isolated from human cord blood. The cells activatedwith PHA and CD32-B7 transfected feeder cells were cultured either under“neutral conditions” (Th0) or polarized to Th1 or Th2 for 7 days. Real-timeRT-PCR was used to confirm the differential expression of NDFIP2 duringthe Th cell differentiation (A). Peripheral blood-derived CD4� cells werenucleofected with a pSuper-pIRES2-H2K-NDFIP2-shRNA or pSuper-pIRES2-H2K-scramble-shRNA control vector. The transfected cells (pu-rity of �98%) were induced to polarize to Th1 (anti-CD3 plus anti-CD28plus IL-12) or Th2 (anti-CD3 plus anti-CD28 plus IL-4) direction for 7days. To measure cytokine production, cells were washed and restimulatedwith PMA and ionomycin (stimulation). IFN-� secretion by the nontrans-fected Th1 and Th2 cells as well as cells expressing scramble-shRNA orNDFIP2-shRNA was measured using a Luminex assay. Representativedata from three biological replicates for IFN-� secretion is shown in B.Abbreviation OOR (out of range) in the figure means that the cytokinesecretion has been over the detection limit of the assay.

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important for the process. However, as demonstrated in currentand previously published study (15), not all of these differencesmay be detected at the protein level.

Components involved in well-known intracellular signaling cas-cades, such as RAS and GPCR seem to be preferentially inducedduring the early Th2 cell differentiation. Such genes includeRAB27, RAB30, RHOQ, and the genes identified in our previousstudy, RASGRP1, RASA3, and SOS1, which are induced by IL-4within 2 or 6 h (15). This is consistent with previous reports show-ing that the RAS pathway promotes IL-4R signaling and is essen-tial for Th2 responses in vivo (34–36). Based on our current andprevious studies also, a panel of factors involved in GPCR signal-ing is rapidly regulated in response to Th2-polarizing stimuli.Genes including GNAI1, CD47, CXCR4, PTGER2, CYSLTR1,EDG1, EBI2, FLJ11856, and HRH4 become up-regulated within 2or 6 h and GPRK6 within 48 h. In contrast, genes CD97, PTGER4,ADORA2A, CCR7, GPR18, GPRK5, and P2Y5 are rapidly re-pressed by IL-4 (10, 15). GPCR signaling is implicated in theregulation of Th1 and Th2 responses and plays a role in Th2-mediated diseases such as asthma (37). Induction of GPCR sig-naling may also enhance the activation of RAS pathway, thus pro-moting IL-4 signaling.

TGF-� is an immunosuppressive factor able to inhibit Th1 andTh2 cell differentiation. We hypothesized that the genes differen-tially regulated by the Th1- or Th2-inducing cytokines and TGF-�are important candidates for influencing in the differentiation ofthese subtypes. Therefore, the target genes of TGF-� during theearly differentiation were identified. In agreement with our previ-ously published studies (10, 15), we identified a subset of genesthat was regulated in an opposite manner by TGF-� and IL-12 orIL-4. TGF-� antagonized the effects of IL-12 on genes includingSOCS3, HOP, GIMAP4, SLAMF7, TIFA, CRIP1, and effects ofIL-4 on numerous genes, such as SYTL3, PPAP2A, and AU134977.Previously identified genes that behave in a similar manner andshow inhibition by TGF-� include GZMB, NFIL3, TNFRSF9,VIM, SATB1, BCL2A1, ID2, PTGS2, PLA2G4A, GNAI1, ID3,LAMA3, CCL20, RTP801, and R32184_3 (10, 15). The func-tional role of most of these genes in Th cell differentiation isunknown and deserves further characterization. The genes reg-ulated by IL-12 or IL-4 and antagonized by TGF-� are likely toparticipate in the mechanism by which TGF-� inhibits Th1 andTh2 differentiation.

NDFIP2 was one of the eight genes induced by IL-4 at all thetime points studied during the early Th2 cell differentiation. Dif-ferential expression by the Th1 and Th2 cells polarized for 7 dayswas observed as well. NDFIP2 was highly induced in response toCD3 plus CD28 activation alone as previously described in T cells,but induction in response to PHA plus feeder cell activation wasmodest and temporary (38). NDFIP2 has shown to be able to in-teract with several NEDD4-family proteins. NDFIP2 localizes inthe Golgi network and seems to play a role in the regulation ofprotein trafficking (38, 39). As nothing was previously known onthe role of NDFIP2 in Th differentiation, it was selected for furtheranalysis to determine, whether this factor has a functional role inthe polarization process. The NDFIP2 knockdown during the earlyTh1 cell differentiation demonstrated that NDFIP2 promotes pro-duction of the hallmark cytokine of Th1 cell differentiation, IFN-�.The reason why NDFIP2 is preferentially induced during earlyTh2 differentiation and regulates IFN-� production also by Th1cells is unclear. Also, the exact mechanism of this enhancementremains to be elucidated. One of the possible mechanisms mayinvolve the NF-�B pathway, as NDFIP2 has been suggested toactivate NF-�B signaling (40). Alternatively, NDFIP2 may regu-

late cytokine production through some novel mechanism related toprotein trafficking.

The preferential induction of FOSL2 during early Th2 differen-tiation is also interesting and was confirmed at the protein level.FOSL2 is a member of the AP-1 family of transcription factors andan immediate early gene (41) induced during the differentiation ofCD4� or CD8� T cells from immature double-negative precursors(42). It has been reported that during the Th1/Th2 differentiationprocess, high levels of AP-1 complexes are accumulated, and thatthese complexes are able to induce high levels of AP-1 tran-scriptional activity in Th2, but not in Th1 cells (43). Thesereports together with our observations provide the basis for fur-ther studies to dissect the role of these proteins in Th2 celldifferentiation.

In conclusion, based on our current and previous studies, 288genes, �1–1.5% of the genes in human genome, are differentiallyregulated by cytokines involved in early Th1 and Th2 polarizationduring the first 2 days (10, 15). These novel genes are likely toinclude factors that are critical regulators of Th1 and Th2 differ-entiation process, together with the previously identified factors,such as STAT4, TBX21, STAT6, and GATA3. This study pro-vides a detailed overview of the gene regulation during the earlyTh1 and Th2 cell differentiation and numerous new candidates forfurther studies. Although all of these genes may not be importantfor the differentiation process, these findings imply that the regu-lation of the Th1 and Th2 differentiation process is likely to bemuch more complex than current models suggest.

AcknowledgmentsWe are grateful for Paula Suominen, Outi Melin, Sarita Heinonen, andMarjo Hakkarainen for their valuable technical assistance. Dr. RobertMoulder is acknowledged for the language review of the manuscript. Wealso thank Prof. John Eriksson’s group at Turku Centre for Biotechnologyfor assistance in the use of GraphPad Prism software for graphics.

DisclosuresThe authors have no financial conflict of interest.

References1. Romagnani, S. 1996. Th1 and Th2 in human diseases. Clin. Immunol. Immuno-

pathol. 80: 225–235.2. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune

system: the T helper lymphocyte grows up. Genes Dev. 14: 1693–1711.3. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher.

2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655–669.

4. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, andL. H. Glimcher. 2002. Distinct effects of T-bet in TH1 lineage commitment andIFN-� 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. Transcriptionfactor GATA-3 is differentially expressed in murine Th1 and Th2 cells and con-trols Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272:21597–21603.

6. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is neces-sary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587–596.

7. Hamalainen, H., H. Zhou, W. Chou, H. Hashizume, R. Heller, and R. Lahesmaa.2001. Distinct gene expression profiles of human type 1 and type 2 T helper cells.Genome Biol. 2: RESEARCH0022.1–RESEARCH0022.11.

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-opment of human T helper cells using oligonucleotide arrays. Nat. Genet. 25:96–101.

9. Lu, B., P. Zagouras, J. E. Fischer, J. Lu, B. Li, and R. A. Flavell. 2004. Kineticanalysis of genomewide gene expression reveals molecule circuitries that controlT cell activation and Th1/2 differentiation. Proc. Natl. Acad. Sci. USA 101:3023–3028.

10. Lund, R., T. Aittokallio, O. Nevalainen, and R. Lahesmaa. 2003. Identification ofnovel genes regulated by IL-12, IL-4, or TGF-� during the early polarization ofCD4� lymphocytes. J. Immunol. 171: 5328–5336.

11. Lund, R. J., E. K. Ylikoski, T. Aittokallio, O. Nevalainen, and R. Lahesmaa.2003. Kinetics and STAT4- or STAT6-mediated regulation of genes involved inlymphocyte polarization to Th1 and Th2 cells. Eur. J. Immunol. 33: 1105–1116.

3659The Journal of Immunology

on May 18, 2011

ww

w.jim

munol.org

Dow

nloaded from

12. Chen, Z., R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, and R. Lahesmaa.2003. Identification of novel IL-4/Stat6-regulated genes in T lymphocytes. J. Im-munol. 171: 3627–3623.

13. Chtanova, T., R. A. Kemp, A. P. Sutherland, F. Ronchese, and C. R. Mackay.2001. Gene microarrays reveal extensive differential gene expression in bothCD4� and CD8� type 1 and type 2 T cells. J. Immunol. 167: 3057–3063.

14. Hoey, T., S. Zhang, N. Schmidt, Q. Yu, S. Ramchandani, X. Xu, L. K. Naeger,Y. L. Sun, and M. H. Kaplan. 2003. Distinct requirements for the naturally oc-curring splice forms Stat4� and Stat4� in IL-12 responses. EMBO J. 22:4237–4248.

15. Lund, R., H. Ahlfors, E. Kainonen, A. M. Lahesmaa, C. Dixon, and R. Lahesmaa.2005. Identification of genes involved in the initiation of human Th1 or Th2 cellcommitment. Eur. J. Immunol. 35: 3307–3319.

16. Hamalainen, H. K., J. C. Tubman, S. Vikman, T. Kyrola, E. Ylikoski, J. A.Warrington, and R. Lahesmaa. 2001. Validation of endogenous reference genes forprofiling of lymphocyte differentiation by quantitative real-time RT-PCR. Anal. Bio-chem. 299: 63–70.

17. Liu, G., A. E. Loraine, R. Shigeta, M. Cline, J. Cheng, V. Valmeekam, S. Sun,D. Kulp, and M. A. Siani-Rose. 2003. NetAffx: Affymetrix probe sets and an-notations. Nucleic Acids Res. 31: 82–86.

18. Tahvanainen, J., M. Pykalainen, T. Kallonen, H. Lahteenmaki, O. Rasool, andR. Lahesmaa. 2006. Enrichment of nucleofected primary human CD4� T cells:a novel and efficient method for studying gene function and role in human pri-mary T helper cell differentiation. J. Immunol. Methods 310: 30–39.

19. Seki, Y., H. Inoue, N. Nagata, K. Hayashi, S. Fukuyama, K. Matsumoto,O. Komine, S. Hamano, K. Himeno, K. Inagaki-Ohara, et al. 2003. SOCS-3regulates onset and maintenance of Th2-mediated allergic responses. Nat. Med. 9:1047–1054.

20. Li-Weber, M., O. Laur, I. Davydov, C. Hu, P. Salgame, and P. H. Krammer.1997. What controls tissue-specific expression of the IL-4 gene? Immunobiology198: 170–178.

21. Avice, M. N., M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati. 2000. CD47ligation selectively inhibits the development of human naive T cells into Th1effectors. J. Immunol. 165: 4624–4631.

22. Rincon, M., and R. A. Flavell. 1999. Reprogramming transcription during thedifferentiation of precursor CD4� T cells into effector Th1 and Th2 cells. Mi-crobes Infect. 1: 43–50.

23. Nagai, S., S. Hashimoto, T. Yamashita, N. Toyoda, T. Satoh, T. Suzuki, andK. Matsushima. 2001. Comprehensive gene expression profile of human acti-vated Th1- and Th2-polarized cells. Int. Immunol. 13: 367–376.

24. Sornasse, T., P. V. Larenas, K. A. Davis, J. E. de Vries, and H. Yssel. 1996.Differentiation and stability of T helper 1 and 2 cells derived from naive humanneonatal CD4� T cells, analyzed at the single-cell level. J. Exp. Med. 184:473–483.

25. Cousins, D. J., T. H. Lee, and D. Z. Staynov. 2002. Cytokine coexpression duringhuman Th1/Th2 cell differentiation: direct evidence for coordinated expression ofTh2 cytokines. J. Immunol. 169: 2498–2506.

26. Bradley, L. M., D. K. Dalton, and M. Croft. 1996. A direct role for IFN-� inregulation of Th1 cell development. J. Immunol. 157: 1350–1358.

27. Wenner, C. A., M. L. Guler, S. E. Macatonia, A. O’Garra, and K. M. Murphy.1996. Roles of IFN-� and IFN-� in IL-12-induced T helper cell-1 development.J. Immunol. 156: 1442–1447.

28. Afkarian, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang,T. L. Murphy, and K. M. Murphy. 2002. T-bet is a STAT1-induced regulator ofIL-12R expression in naive CD4� T cells. Nat. Immunol. 3: 549–557.

29. Grogan, J. L., M. Mohrs, B. Harmon, D. A. Lacy, J. W. Sedat, and R. M. Locksley.2001. Early transcription and silencing of cytokine genes underlie polarization of Thelper cell subsets. Immunity 14: 205–215.

30. Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino,D. M. Livingston, A. L. Kung, N. Cereb, T. P. Yao, S. Y. Yang, and S. L. Reiner.2001. Role of T-bet in commitment of TH1 cells before IL-12-dependent selec-tion. Science 292: 1907–1910.

31. Lighvani, A. A., D. M. Frucht, D. Jankovic, H. Yamane, J. Aliberti, B. D. Hissong,B. V. Nguyen, M. Gadina, A. Sher, W. E. Paul, and J. J. O’Shea. 2001. T-bet israpidly induced by interferon-� in lymphoid and myeloid cells. Proc. Natl. Acad. Sci.USA 98: 15137–15142.

32. Tau, G. Z., T. von der Weid, B. Lu, S. Cowan, M. Kvatyuk, A. Pernis,G. Cattoretti, N. S. Braunstein, R. L. Coffman, and P. B. Rothman. 2000. Inter-feron � signaling alters the function of T helper type 1 cells. J. Exp. Med. 192:977–986.

33. Gollob, J. A., H. Kawasaki, and J. Ritz. 1997. Interferon-� and interleukin-4regulate T cell interleukin-12 responsiveness through the differential modulationof high-affinity interleukin-12 receptor expression. Eur. J. Immunol. 27:647–652.

34. Yamashita, M., M. Kimura, M. Kubo, C. Shimizu, T. Tada, R. M. Perlmutter, andT. Nakayama. 1999. T cell antigen receptor-mediated activation of the Ras/mi-togen-activated protein kinase pathway controls interleukin 4 receptor functionand type-2 helper T cell differentiation. Proc. Natl. Acad. Sci. USA 96:1024–1029.

35. Yamashita, M., M. Katsumata, M. Iwashima, M. Kimura, C. Shimizu, T. Kamata,T. Shin, N. Seki, S. Suzuki, M. Taniguchi, and T. Nakayama. 2000. T cell re-ceptor-induced calcineurin activation regulates T helper type 2 cell developmentby modifying the interleukin 4 receptor signaling complex. J. Exp. Med. 191:1869–1879.

36. Shibata, Y., T. Kamata, M. Kimura, M. Yamashita, C. R. Wang, K. Murata,M. Miyazaki, M. Taniguchi, N. Watanabe, and T. Nakayama. 2002. Ras activa-tion in T cells determines the development of antigen-induced airway hyperre-sponsiveness and eosinophilic inflammation. J. Immunol. 169: 2134–2140.

37. Johnson, E. N., and K. M. Druey. 2002. Heterotrimeric G protein signaling: rolein asthma and allergic inflammation. J. Allergy Clin. Immunol. 109: 592–602.

38. Cristillo, A. D., L. Nie, M. J. Macri, and B. E. Bierer. 2003. Cloning and char-acterization of N4WBP5A, an inducible, cyclosporine-sensitive, Nedd4-bindingprotein in human T lymphocytes. J. Biol. Chem. 278: 34587–34597.

39. Shearwin-Whyatt, L. M., D. L. Brown, F. G. Wylie, J. L. Stow, and S. Kumar.2004. N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicu-lar bodies and the Golgi, and has a potential role in protein trafficking. J. Cell Sci.117: 3679–3689.

40. Matsuda, A., Y. Suzuki, G. Honda, S. Muramatsu, O. Matsuzaki, Y. Nagano,T. Doi, K. Shimotohno, T. Harada, E. Nishida, et al. 2003. Large-scale identifi-cation and characterization of human genes that activate NF-�B and MAPKsignaling pathways. Oncogene 22: 3307–3318.

41. Nishina, H., H. Sato, T. Suzuki, M. Sato, and H. Iba. 1990. Isolation and char-acterization of fra-2, an additional member of the fos gene family. Proc. Natl.Acad. Sci. USA 87: 3619–3623.

42. Chen, F., D. Chen, and E. V. Rothenberg. 1999. Specific regulation of fos familytranscription factors in thymocytes at two developmental checkpoints. Int. Im-munol. 11: 677–688.

43. Rincon, M., B. Derijard, C. W. Chow, R. J. Davis, and R. A. Flavell. 1997.Reprogramming the signalling requirement for AP-1 (activator protein-1) acti-vation during differentiation of precursor CD4� T-cells into effector Th1 and Th2cells. Genes Funct. 1: 51–68.

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