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The Journal of Immunology, 2007, 178: 3648-3660.
Copyright © 2007 by The American Association of Immunologists, Inc.

Genome-Wide Identification of Novel Genes Involved in Early Th1 and Th2 Cell Differentiation1

Riikka J. Lund2,*, Maritta Löytömäki2,*,{dagger}, Tiina Naumanen*, Craig Dixon*, Zhi Chen*, Helena Ahlfors*,{ddagger}, Soile Tuomela*,{dagger}, Johanna Tahvanainen*,§, Joonas Scheinin*, Tiina Henttinen*, Omid Rasool* and Riitta Lahesmaa3,*

* Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland; {dagger} Graduate School of Biomedical Sciences, University of Turku, Turku, Finland; {ddagger} The National Graduate School in Informational and Structural Biology, Åbo Akademi University, Turku, Finland; and § Drug Discovery Graduate School, University of Turku, Turku, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 ~1–1.5% of the human genome, are 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-{gamma} production by the polarized human Th1 lymphocytes. Among the novel genes identified, there may be many factors that play a crucial role in the regulation of the differentiation process together with the previously known factors and are potential targets for developing therapeutics to modulate Th1 and Th2 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T helper cell subtypes, Th1 and Th2, originate from common naive precursor cells (Thp) in response to Ag and cytokine stimulation. Although Th cells have a crucial role in host defense against intracellular and extracellular pathogens, disturbances in the balance between Th1 and Th2 responses can promote or lead to pathogenesis of immune-mediated diseases. Enhanced Th2 response is involved in atopic diseases, such as asthma, whereas a dominating Th1 response is implicated in certain autoimmune diseases, like type 1 diabetes or rheumatoid arthritis (1). To understand the molecular mechanisms driving the pathogenesis of these diseases, it is important to elucidate the early differentiation process of Th1 and Th2 cells in detail.

A number of the central factors involved in directing the differentiation process have been identified. IL-12/STAT4 and IFN-{gamma}/STAT1 signaling are important in driving Th1 polarization, whereas IL-4/STAT6 signaling directs Th2 polarization (2). Transcription factors TBX21 (T-bet) and GATA3 are also among the key factors required for the Th1 and Th2 differentiation, respectively (3, 4, 5, 6). Although many of the players implicated in the regulation of differentiation have been recognized, the current model as such is still too simple to explain the process in detail, and other yet unknown factors are likely to be involved.

Recently, an increasing number of studies have used DNA microarrays to identify new factors involved in the Th1 and Th2 polarization in humans and mice (7, 8, 9, 10, 11, 12, 13, 14). However, all of these studies have focused on studying a limited number of primarily known genes. We have previously elucidated the regulation of ~9300 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 regulation of the rest of the genes in the human genome. Based on the combination of our current and previous studies, 288 genes or expressed sequence tags (ESTs),4 representing ~1–1.5% of the human genome, are differentially regulated during the first 2 days of Th1 and Th2 cell polarization. Moreover, we demonstrate that a panel of these genes or ESTs are induced by IL-4 through the STAT6 signaling or play a role in regulation of IFN-{gamma} production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
In vitro differentiation of Th1 and Th2 cells

Induction of human Th1 and Th2 cell differentiation was performed as previously described (10). Briefly, CD4+ T cells isolated (Ficoll Isolation Paque; Amersham Biosciences and Dynal Biotech) from cord blood (Turku 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 2.5 ng/ml IL-12 and Th2 differentiation with 10 ng/ml IL-4 (both from R&D Systems). A subset of the cells was cultured in "neutral conditions" without polarizing cytokines. In the indicated experiments, the cultures were supplemented with 3 ng/ml TGF-beta (R&D Systems). TGF-beta-mediated suppression of IFN-{gamma} production by Th1 cells in these conditions has been previously described (10). The samples were collected after 0, 2, 6, or 48 h of polarization and were processed for Affymetrix hybridizations or Western blotting.

For validation of the oligonucleotide array results with real-time RT-PCR, additional Th1 and Th2 primary cultures were generated as described in our previous study (11). Briefly, cord blood CD4+ T cells were activated with 100 ng/ml PHA (Murex) and irradiated CD32-B7-transfected fibroblasts (16). Th1 cultures were supplemented with 2.5 ng/ml IL-12, whereas Th2 cultures were supplemented with 10 mg/ml anti-IL-12 and 10 ng/ml IL-4 (all from R&D Systems). After 48 h of priming, 40 U/ml IL-2 (R&D Systems) was added into the cultures. A subset of the cells was cultured without any polarizing cytokines in the presence of IL-2 alone. The cultures were generated from four individuals. Samples were collected at time points 0, 6, 24, and 48 h or 7 days.

Oligonucleotide array studies

Human genome U133A and B arrays recognizing ~33,000 transcripts were rehybridized with the previously prepared samples (10, 15). Briefly, after confirming the successful polarization of the cells with real-time RT-PCR, the sample preparation and data analysis were performed according to the instructions and recommendations provided by the manufacturer (Affymetrix). Total RNA (4–5 µg) pooled from different individuals was used as starting material for the Affymetrix sample preparation. Two biological repeats for each microarray experiment were performed. GeneChip Microarray Suite software version 5 (MAS5; Affymetrix), GeneSpring (SiliconGenetics), and Microsoft Access for Windows software were used to evaluate the quality of the data and for routine data analysis and processing. The microarray data was filtered according to the statistical classifications performed by the MAS5 software as previously described (10, 15). Genes that presented a consistent change (≥2-fold) in two separate biological repeats were considered as differentially expressed. All the genes, which fulfilled these 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 was explored parallel in different conditions without fold change threshold. The gene annotations were obtained from NetAffx database (17). The normalized microarray raw data have been deposited in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information and are accessible through GEO Series accession 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{alpha} 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 normalized CT values (CT gene X – CT housekeeping gene), {Delta}CT values, with the formula: fold difference = 2(I{Delta}CT1 – {Delta}CT2I). Statistical significances between the differences in gene expression were evaluated with t tests.


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

 
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 polarization 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 Complete Protease Inhibitor (Roche). From the centrifuged lysates, the supernatants were discarded and a second buffer (20 mM HEPES, 420 mM NaCl, 20% glycerol, and 1 mM DTT; Roche Complete Protease Inhibitor) was mixed with the cell pellets. After 1 h of incubation on ice, the samples were recentrifuged and supernatants containing nuclear proteins were obtained. The protein concentrations were then quantified from the samples by Bio-Rad protein assay. Equal amounts of protein were subject to SDS-PAGE and transferred to Hybond ECL membrane (Amersham Biosciences). 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 successful induction of Th1/Th2 polarization. Western blotting was then performed for DACT1 (ab5977-100; Abcam), RAB11FIP1 (RCP 11-A; Alpha Diagnostic), 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 during the early polarization of human Th cells

The NDFIP2 shRNA plasmid construct, targeting (5'-GAGGAAGAGTGTCCACCAAGA-3'), was generated by cloning the NDFIP2 shRNA oligonucleotide into the BglII and XhoI sites of the previously modified pSuper-H2K-pIRES2 plasmid, which contains a truncated H2K cell surface selection marker (18). Similarly, a pSuper-H2K-pIRES2-scramble1-shRNA, (5'-AATTCTCCGAACGTGTCACGT-3'), was designed. In addition, two synthetic small interfering RNA oligos (one of which targeting the same sequence as the NDFIP2 shRNA shown above and the other targeting the 5'-CUGGAUAUUUCAAUGGACAUU-3' NDFIP2 sequence; Sigma-Aldrich) were used to knock down NDFIP2. For the STAT6 knockdown studies, the previously prepared 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 described (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 analysis the cells were harvested at 24 or 48 h of polarization from three to four biological replicates. Cells harvested at 7 days of polarization were washed, restimulated, and cytokines secreted in the supernatant after 24 h of restimulation were measured (see the section below). For RT-PCR, the total RNAs were isolated either with an RNA Easy Minikit (Qiagen) or a PicoPureTM RNA Isolation kit (Arcturus). Consequently, cDNAs were prepared with a Transcription First Strand cDNA Synthesis kit (Roche).

Cytokine secretion assay

To measure the IFN-{gamma} production by the control cells or cells transfected with NDFIP2-shRNA, the cells were incubated with or without 5 ng/ml PMA (Calbiochem) and 500 ng/ml ionomycin (Sigma-Aldrich). The culture medium from each well was collected after 24 h of restimulation with PMA 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 multiplex bead kits from LINCO Research and from Bio-Rad. The assays were conducted in duplicate according to the manufacturer’s instructions. Measurements and data analysis were performed with the Bio-Plex system in combination with the Bio-Plex Manager software (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To identify novel genes involved in the initiation and early polarization of Th1 and Th2 cells at the genome scale, transcriptome analysis was conducted using Affymetrix U133A and B oligonucleotide arrays. To identify genes regulated by IL-12 or IL-4 in activated Th cells, the cells induced to polarize to Th1 (CD3 plus CD28 plus IL-12) or Th2 (CD3 plus CD28 plus IL-4) direction for 2, 6, or 48 h were compared with each other and to the CD3 plus CD28-activated cells cultured without polarizing cytokines (Th0). Furthermore, genes coregulated by TGF-beta and IL-12 or IL-4 during the early Th1 and Th2 cell differentiation were identified. In addition to the previously identified genes, an additional 171 genes were found to be regulated by IL-12 or IL-4 (Fig. 1A) during the first 2 days of Th1 and Th2 differentiation (see Table II for all the IL-12- and IL-4-regulated genes) (10, 15).


Figure 1
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  		FIGURE 1. Summary of the genes regulated by CD3 plus CD28 activation, IL-12, and IL-4. Expression profiles of the cells activated with anti-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-CD28 plus 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 CD3 plus CD28 activation, IL-12, and/or IL-4 during the early Th1 and Th2 cell differentiation. B, The number of genes up- ({uparrow}) or down-regulated ({downarrow}) by IL-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

 

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  		Table IIA. (Continued)

 

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  		Table IIB. (Continued)

 

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  		Table IIC. (Continued)

 
The slow response to IL-12

Consistent with our previous observations, IL-12 had minimal or no effect on the polarization process after 2 or 6 h (15). After 2 h of Th1 polarization, there were no genes regulated by IL-12. After 6 h, <2-fold induction by IL-12 was seen in the expression of key regulator of Th1 differentiation TBX21 and GTPase GBP5 (3, 4). After 48 h, the effects of IL-12 were clear and altogether 41 genes became regulated by IL-12 (Table II). Among these were only a few genes, such as SOCS3, CEBPB, and IL-7R that have a previously described role in Th1 and Th2 cell responses (13, 19, 20). Most of the genes regulated by IL-12 were also regulated by IL-4 and/or activation alone (Fig. 1A), although there were differences in the kinetics and magnitude in the changes induced by these different treatments. Similarly to the key regulator of Th1 and Th2 cell differentiation, TBX21, a subset of genes, including GIMAP4, IL-7R, PPAP2A, MGC4677, and GBP5, was regulated in an opposite manner by IL-12 and IL-4, although as for TBX21, the changes were small.

IL-4 rapidly regulates numerous genes during early Th2 cell differentiation

In contrast to IL-12 signaling, the effects of IL-4 were clearly seen within 2 h of Th2 polarization. IL-4 regulated the expression of altogether 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, including CD47, PRNP, SOCS3, FOSL2, COL6A3, PTPRA, TNFSF10, IL-10, CCR7, CEBPB, IL-7R, GBP3, and TBX21 has been previously described to have a role in the process (3, 8, 12, 13, 19, 20, 21, 22, 23). Importantly, most of the IL-4-regulated genes identified were new in this context.


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

 
Genes regulated by TGF-beta during the early Th1 or Th2 differentiation

To study the effects of TGF-beta on the early polarization of Th cells, cells cultured in Th1 or Th2 conditions were compared with those similarly cultured, but in the presence of TGF-beta. In addition to the previously identified genes, an additional 110 genes were regulated by TGF-beta (data not shown) (10, 15). These included 63 genes that were coregulated by TGF-beta and the polarizing cytokines IL-12 or IL-4. Altogether 25 genes were coregulated by TGF-beta and IL-12 (Table IV). Expression of five of these genes was enhanced or accelerated in the presence of TGF-beta, at least to some extent. Importantly, 19 of the IL-12-regulated genes were antagonized by TGF-beta in Th1 conditions. Similarly, the effects of IL-4 on the expression of 20 genes were enhanced or accelerated in the presence of TGF-beta, whereas expression of 25 genes was antagonized by TGF-beta (Table V).


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

 

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

 
A subset of genes (ZBED2, LMNA, NDFIP2, TncRNA, SYTL3, SLC2A3, and TRIB3), coregulated by TGF-beta and the polarizing cytokines, was up-regulated by both IL-12 and IL-4 at least to some extent. Of these, the expression of ZBED2, LMNA, and NDFIP2 was further increased in the presence of TGF-beta in both Th1 and Th2 conditions. In contrast, TncRNA, SYTL3, SLC2A3, and TRIB3, up-regulated by IL-12 and IL-4, were repressed by TGF-beta in both Th1 and Th2 conditions.

Putative or known functions of the genes involved during early Th1 and Th2 differentiation

To elucidate the putative or known functions of the factors involved in the early Th1 and Th2 cell differentiation, these genes were analyzed with the gene ontology annotation tool in NetAffx (17). Concordant with our previous studies, most of the genes regulated during the early differentiation of Th1 and Th2 cells code for factors involved in the signal transduction from cell surface to nucleus (10, 15). Among the IL-4-regulated genes, the most outstanding functional group consisted of genes coding for proteins with catalytic activity including genes involved in RAS oncogene family signaling (RAB27B, RAB30, and RHOQ). Another predominant functional group consisted of genes coding for receptors or receptor binding proteins. These included components of G protein-coupled receptor signaling (CYSLTR1, HRH4, CD47, CCR7, and GPR18). Numerous transcriptional regulators, such as ELL2, ZNF443, PHF20L1, LOC360030, EZH2, ZBED2, FOSL2, and FRBZ1 became differentially regulated during the early Th1 and Th2 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 genes involved in apoptosis, proliferation, adhesion, metabolism, motility, cell cycle, protein localization, transport, and regulation of protein activity. The function of the majority of the genes differentially regulated during early Th1 and Th2 cell differentiation was unknown. Among these were numerous ESTs that were rapidly up-regulated by IL-4 in the cells induced to polarize to Th2 direction. A set of these genes or ESTs was selected for further studies. As IL-12 did not have much effect at the initiation of the differentiation, our special interest was on the genes or ESTs regulated by IL-4.

Validation of the oligonucleotide array results with real-time RT-PCR

Expression of 14 ESTs or genes with poorly characterized function was further studied with real-time RT-PCR during 7 days of 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 results, all of these genes and ESTs were rapidly regulated by IL-4 at the initiation of Th2 cell differentiation. RT-PCR confirmed IL-4-mediated regulation of 12 of 14 genes or ESTs (Fig. 2). Regulation of transcripts by IL-4 was primarily limited within the first 2 days of Th2 differentiation. For BF056901 and AI674404, the Affymetrix results could not be confirmed in all three individuals studied.


Figure 2
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  		FIGURE 2. Validation of the oligonucleotide array results with real-time RT-PCR. CD4+ T cells were isolated from human cord blood. The cells were activated with PHA and CD32-B7-transfected feeder cells. 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 expression between the Th2 and Th0 cells were determined with a t test.

 
FOSL2 is preferentially expressed during the early Th2 cell polarization at the protein level

Systematic validation of the differences detected at the mRNA level was performed at the protein level for those genes that commercial Abs were available. Most of the genes identified in this study are poorly characterized and therefore a limited number of Abs were obtained. The proteins studied included DACT1, RAB11FIP1, FOSL2, CISH, KLF6, FLT1, and RAB27B. As can be seen from Fig. 3, FOSL2 was indeed differentially expressed at the protein level, with higher expression in Th2 cells detected at 48 h. TBX21 and GATA3 were used as control genes to confirm the polarization of the cells to Th1 and Th2 direction. For the other proteins we were not able to detect any differences in the expression levels during early polarization of Th1 or Th2 cells (data not shown). Proteomics studies in progress will be used to elucidate the differences in the expression of the possible variants of these and other proteins during early stages of Th1 and Th2 differentiation.


Figure 3
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  		FIGURE 3. FOSL2 protein is preferentially expressed by cells polarized to Th2 direction at early stages of differentiation. Human cord blood CD4+ T cells were either cultured in neutral conditions (Th0) or polarized to Th1 or Th2 for 2, 6, and 48 h. Nuclear lysates were subjected to Western blotting with TBX21 and GATA3 expression to validate successful Th1 and Th2 polarization and then probed for FOSL2.

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

To further study whether a subset of poorly characterized genes induced by IL-4 is regulated through STAT6 signaling, human peripheral blood-derived CD4+ cells were nucleofected with two different pSuper-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 mRNA levels were down-regulated by these shRNAs (Fig. 4). To determine effects of this STAT6 knockdown on the expression of a subset of selected genes or ESTs regulated by IL-4, mRNA expression was measured 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 clearly dependent on STAT6. No effect by STAT6-shRNA on the expression of AA237039 or AW152437 was observed (data not shown).


Figure 4
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  		FIGURE 4. AW629527, AA088177, AA489100, ZNF443, and DACT1 are induced by IL-4 in a STAT6-dependent manner. Human peripheral blood-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 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 ≤ 0.05). Fold changes in gene expression between the Th2 cells expressing STAT6-shRNA and scramble-shRNA are indicated in the figure.

 
NDFIP2 promotes IFN-{gamma} production by Th1 cells

NDFIP2 was selected for further functional studies as it was highly induced by CD3 plus CD28 activation alone and the expression was further enhanced by IL-4 during the early Th2 differentiation at all of the time points studied. This regulation of NDFIP2 was confirmed with RT-PCR (Fig. 5A). In contrast to the oligonucleotide array results, where high induction of NDFIP2 expression by CD3 plus CD28 activation alone was observed (Table III), activation with PHA plus CD32-B7-transfected feeder cells induced only a modest short-term increase in NDFIP2 expression as measured with RT-PCR (Fig. 5A). Because the role of NDFIP2 in the differentiation of Th cells was previously unknown, the effect of 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-{gamma} production by Th1 cells (Fig. 5B) in two biological replicates. In addition, a study with a similar experimental set-up, but using cord blood CD4+ 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 first round of human Th2 cell polarization in these conditions (24, 25). Most importantly, our experiment demonstrates that NDFIP2 has a function in the Th1 cell differentiation process by promoting secretion of the hallmark cytokine of Th1 cell differentiation, IFN-{gamma}.


Figure 5
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  		FIGURE 5. NDFIP2 promotes IFN-{gamma} production by human Th1 cells. CD4+ T cells were isolated from human cord blood. The cells activated with PHA and CD32-B7 transfected feeder cells were cultured either under "neutral conditions" (Th0) or polarized to Th1 or Th2 for 7 days. Real-time RT-PCR was used to confirm the differential expression of NDFIP2 during the Th cell differentiation (A). Peripheral blood-derived CD4+ cells were nucleofected with a pSuper-pIRES2-H2K-NDFIP2-shRNA or pSuper-pIRES2-H2K-scramble-shRNA control vector. The transfected cells (purity of >98%) were induced to polarize to Th1 (anti-CD3 plus anti-CD28 plus IL-12) or Th2 (anti-CD3 plus anti-CD28 plus IL-4) direction for 7 days. To measure cytokine production, cells were washed and restimulated with PMA and ionomycin (stimulation). IFN-{gamma} secretion by the nontransfected Th1 and Th2 cells as well as cells expressing scramble-shRNA or NDFIP2-shRNA was measured using a Luminex assay. Representative data from three biological replicates for IFN-{gamma} secretion is shown in B. Abbreviation OOR (out of range) in the figure means that the cytokine secretion has been over the detection limit of the assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have conducted a genome-wide screening to identify novel genes involved in early Th1 and Th2 differentiation (13, 15). Interestingly, numerous factors with known or unknown function were identified to be implicated in the process. A number of these genes could be positioned into specific intracellular signaling cascades. This study provides an overview of the intracellular signaling events in the cells at the initiation of Th1 and Th2 cell differentiation.

Thp cells are not responsive to IL-12, which can be seen in slow changes in gene expression profiles in response to IL-12 during early Th1 cell differentiation. Our results at whole genome scale confirm the previous consensus that indicates activation of IFN signaling to be the first event during Th1 differentiation. This is demonstrated by the early up-regulation of key regulators of Th1 differentiation IFNG, TBX21, and IFN-regulated genes GBP1 and GBP5 (3, 4, 15, 26, 27). TBX21 is induced by T cell activation and its expression is enhanced by IFN-{gamma} during early Th1 polarization (28, 29, 30, 31). The role of GBP1 and GBP5 in Th1 polarization is not known. As these genes are induced by IFNs, it is likely that, similar to TBX21, they are induced due to enhanced IFN-{gamma} signaling. Activation of IFN-{gamma} signaling and TBX21 expression during early differentiation is essential for Th1 differentiation, probably because 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 responsiveness of the developing Th1 cells to polarizing cytokines (10). Consistently, after 48 h of Th1 polarization several genes were regulated by IL-12. The roles of most of these genes or ESTs in Th1 differentiation are not known.

Thp cells are responsive to IL-4, which can be seen in the rapid regulation of numerous genes and ESTs with known and unknown function. A subset of these genes was demonstrated to be regulated by IL-4 in a STAT6-dependent manner. Most of the IL-4-regulated genes 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 regulated by IL-4 throughout the first 2 days. Previously, we observed similar expression patterns for the key mediators of Th2 differentiation GATA3 and MAF. Similar regulation for a panel of genes, such as BCL6, NFIL-3, SATB1, SOCS1, DUSP6, IL10RA, and CXCR4, with unknown or less clear functions in Th1 and Th2 cell differentiation was also demonstrated in our previous studies (10, 15). Maintenance of the IL-4-mediated regulation of these genes throughout the early Th2 cell differentiation suggests them to be important for the process. However, as demonstrated in current and previously published study (15), not all of these differences may be detected at the protein level.

Components involved in well-known intracellular signaling cascades, such as RAS and GPCR seem to be preferentially induced during the early Th2 cell differentiation. Such genes include RAB27, RAB30, RHOQ, and the genes identified in our previous study, RASGRP1, RASA3, and SOS1, which are induced by IL-4 within 2 or 6 h (15). This is consistent with previous reports showing that the RAS pathway promotes IL-4R signaling and is essential for Th2 responses in vivo (34, 35, 36). Based on our current and previous studies also, a panel of factors involved in GPCR signaling 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 2 or 6 h and GPRK6 within 48 h. In contrast, genes CD97, PTGER4, ADORA2A, CCR7, GPR18, GPRK5, and P2Y5 are rapidly repressed by IL-4 (10, 15). GPCR signaling is implicated in the regulation of Th1 and Th2 responses and plays a role in Th2-mediated diseases such as asthma (37). Induction of GPCR signaling may also enhance the activation of RAS pathway, thus promoting IL-4 signaling.

TGF-beta is an immunosuppressive factor able to inhibit Th1 and Th2 cell differentiation. We hypothesized that the genes differentially regulated by the Th1- or Th2-inducing cytokines and TGF-beta are important candidates for influencing in the differentiation of these subtypes. Therefore, the target genes of TGF-beta during the early differentiation were identified. In agreement with our previously published studies (10, 15), we identified a subset of genes that was regulated in an opposite manner by TGF-beta and IL-12 or IL-4. TGF-beta antagonized the effects of IL-12 on genes including SOCS3, HOP, GIMAP4, SLAMF7, TIFA, CRIP1, and effects of IL-4 on numerous genes, such as SYTL3, PPAP2A, and AU134977. Previously identified genes that behave in a similar manner and show inhibition by TGF-beta include GZMB, NFIL3, TNFRSF9, VIM, SATB1, BCL2A1, ID2, PTGS2, PLA2G4A, GNAI1, ID3, LAMA3, CCL20, RTP801, and R32184_3 (10, 15). The functional role of most of these genes in Th cell differentiation is unknown and deserves further characterization. The genes regulated by IL-12 or IL-4 and antagonized by TGF-beta are likely to participate in the mechanism by which TGF-beta inhibits Th1 and Th2 differentiation.

NDFIP2 was one of the eight genes induced by IL-4 at all the time points studied during the early Th2 cell differentiation. Differential expression by the Th1 and Th2 cells polarized for 7 days was observed as well. NDFIP2 was highly induced in response to CD3 plus CD28 activation alone as previously described in T cells, but induction in response to PHA plus feeder cell activation was modest and temporary (38). NDFIP2 has shown to be able to interact with several NEDD4-family proteins. NDFIP2 localizes in the Golgi network and seems to play a role in the regulation of protein trafficking (38, 39). As nothing was previously known on the role of NDFIP2 in Th differentiation, it was selected for further analysis to determine, whether this factor has a functional role in the polarization process. The NDFIP2 knockdown during the early Th1 cell differentiation demonstrated that NDFIP2 promotes production of the hallmark cytokine of Th1 cell differentiation, IFN-{gamma}. The reason why NDFIP2 is preferentially induced during early Th2 differentiation and regulates IFN-{gamma} production also by Th1 cells is unclear. Also, the exact mechanism of this enhancement remains to be elucidated. One of the possible mechanisms may involve the NF-{kappa}B pathway, as NDFIP2 has been suggested to activate NF-{kappa}B signaling (40). Alternatively, NDFIP2 may regulate cytokine production through some novel mechanism related to protein trafficking.

The preferential induction of FOSL2 during early Th2 differentiation is also interesting and was confirmed at the protein level. FOSL2 is a member of the AP-1 family of transcription factors and an immediate early gene (41) induced during the differentiation of CD4+ or CD8+ T cells from immature double-negative precursors (42). It has been reported that during the Th1/Th2 differentiation process, high levels of AP-1 complexes are accumulated, and that these complexes are able to induce high levels of AP-1 transcriptional activity in Th2, but not in Th1 cells (43). These reports together with our observations provide the basis for further studies to dissect the role of these proteins in Th2 cell differentiation.

In conclusion, based on our current and previous studies, 288 genes, ~1–1.5% of the genes in human genome, are differentially regulated by cytokines involved in early Th1 and Th2 polarization during the first 2 days (10, 15). These novel genes are likely to include factors that are critical regulators of Th1 and Th2 differentiation process, together with the previously identified factors, such as STAT4, TBX21, STAT6, and GATA3. This study provides a detailed overview of the gene regulation during the early Th1 and Th2 cell differentiation and numerous new candidates for further studies. Although all of these genes may not be important for the differentiation process, these findings imply that the regulation of the Th1 and Th2 differentiation process is likely to be much more complex than current models suggest.


   Acknowledgments
 
We are grateful for Paula Suominen, Outi Melin, Sarita Heinonen, and Marjo Hakkarainen for their valuable technical assistance. Dr. Robert Moulder is acknowledged for the language review of the manuscript. We also thank Prof. John Eriksson’s group at Turku Centre for Biotechnology for assistance in the use of GraphPad Prism software for graphics.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Academy of Finland, Sigrid Jusélius Foundation, National Technology Agency of Finland, Turku Graduate School of Biomedical Sciences, National Graduate School in Informational and Structural Biology, Drug Discovery Graduate School, Ida Montin Foundation, Finnish Society of Allergology and Immunology, Pulmonary Association Heli, Jenny and Antti Wihuri Foundation, Väinö and Laina Kivi Foundation, Allergy Research Foundation of South-Western Finland, and Turku University Hospital Fund. Back

2 R.J.L. and M.L. made an equal contribution to this work. Back

3 Address correspondence and reprint requests to Prof. Riitta Lahesmaa, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, P.O. Box 123, FIN-20521, Turku, Finland. E-mail address: riitta.lahesmaa{at}btk.fi Back

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

Received for publication March 9, 2005. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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