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The Journal of Immunology, 2003, 171: 5328-5336.
Copyright © 2003 by The American Association of Immunologists

Identification of Novel Genes Regulated by IL-12, IL-4, or TGF-{beta} during the Early Polarization of CD4+ Lymphocytes 1

Riikka Lund2,*,{dagger}, Tero Aittokallio{ddagger}, Olli Nevalainen{ddagger} and Riitta Lahesmaa*

* Turku Centre for Biotechnology, Turku University and Åbo Akademi, {dagger} Turku Graduate School for Biomedical Sciences, and {ddagger} Turku Centre for Computer Science, Turku University, Turku, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Th1 and Th2 cells arise from a common precursor cell in response to triggering through the TCR and cytokine receptors for IL-12 or IL-4. This leads to activation of complex signaling pathways, which are not known in detail. Disturbances in the balance between type 1 and type 2 responses can lead to certain immune-mediated diseases. Thus, it is important to understand how Th1 and Th2 cells are generated. To clarify the mechanisms as to how IL-12 and IL-4 induce Th1 and Th2 differentiation and how TGF-{beta} can inhibit this process, we have used oligonucleotide arrays to examine the early polarization of Th1 and Th2 cells in the presence and absence of TGF-{beta}. In addition to genes previously implicated in the process, we have identified 20 genes with various known and unknown functions not previously associated with Th1/2 polarization. We have also further determined which genes are targets of IL-12, IL-4, and TGF-{beta} regulation in the cells induced to polarize to Th1 and Th2 directions. Interestingly, a subset of the genes was coregulated by IL-12 or IL-4 and TGF-{beta}. Among these genes are candidates that may modulate the balance between Th1 and Th2 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Th1 and Th2 subtypes have an important role in many immune-mediated diseases, such as asthma, allergy, and certain autoimmune diseases (1, 2, 3). To understand the pathogenesis of these diseases at the molecular level, it is crucial to understand how Th1 and Th2 cells are generated and how the balance between these two types of responses is maintained. Th1 and Th2 cells are known to originate from the naive CD4+ precursor cells (Thp) after antigenic activation through the TCR and costimulatory molecules in a suitable cytokine milieu. The main cytokines orchestrating Th1 and Th2 development are IL-12 and IL-4, respectively. Triggering of the TCR and cytokine signaling leads to activation of complex and to a large extent poorly understood downstream signaling networks finally resulting in maturation of the effector Th1 and Th2 cells (4, 5). IL-12 and IFN-{alpha} induce the Th1-type response by activating the STAT4-mediated signaling pathway (6, 7, 8). Some other cytokines such as IFN-{gamma} and IL-18 can also promote Th1 responses, especially in combination with IL-12. Th2 differentiation is induced by IL-4 through the STAT6 signaling pathway (9, 10). GATA-binding protein 3 (GATA-3), avian musculoaponeurotic fibrosarcoma (v-maf), AS42 oncogene homologue (c-maf), and T box expressed in T cells (T-bet) are also among the most important factors regulating the early polarization of Th2 and Th1 cells, respectively (11, 12, 13, 14, 15, 16).

Another important cytokine involved in Th1 and Th2 differentiation is TGF-{beta}. This immunosuppressive cytokine exhibits pleiotrophic activities in various cellular processes and, importantly, can suppress the differentiation of CD4+ cells into the Th1 and Th2 subtypes (17). However, similar to IL-12 and IL-4, the target genes and details of TGF-{beta} downstream signaling are not clear.

To provide a basis for the studies aiming at understanding the mechanism of action and molecular networks involved in the signaling of these cytokines, we have examined the early phase leading to polarization of Th1 and Th2 cells in the presence and absence of TGF-{beta}. As a result, we have identified 40 genes differentially expressed by the cells induced to polarize to Th1 and Th2 subtypes. Importantly, 20 of these genes have not been previously described to be involved in Th1 and Th2 cell differentiation. In addition, we have further clarified which of the genes involved in the early polarization of Th1 and Th2 cells are targets of IL-12 and IL-4 regulation and which of them are also targets of immunosuppressive TGF-{beta}. These genes with both known and unknown functions provide many candidates for further functional studies and for therapeutic approaches to modulate the balance between Th1 and Th2 responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of Th1 and Th2 in vitro polarization

The mononuclear cells were isolated from the human cord blood of healthy neonates using Ficoll-Paque Isolation (Amersham Pharmacia Biotech, Uppsala, Sweden). The CD4+ cells were further purified using magnetic beads (Dynal, Oslo, Norway). The primary activation was performed using plate-bound anti-CD3 (1000 ng/µl for coating) and 500 ng/µl soluble anti-CD28 (Immunotech, Marseille, France). The cells were cultured in the density of 0.5–2 x 106 cells/ml in Yssel’s medium (Irvine Scientific, Santa Ana, CA) containing 1% AB serum (Red Cross, Helsinki, Finland). The polarization of the cells was performed in either Th1 medium containing 2.5 ng/ml IL-12 (R&D Systems, Minneapolis, MN) or in Th2 medium containing 10 ng/ml IL-4 (R&D Systems) in the presence and absence of 3 ng/ml TGF-{beta} (R&D Systems). Both CD3+CD28-activation and the indicated cytokines were given to the cells at the same time. Part of the cells were cultured in "neutral conditions" without any polarizing cytokines. The cells were harvested at the time points of 0 and 48 h for the gene expression analysis and after 7 days for intracellular cytokine staining.

Real-time quantitative RT-PCR

Real-time quantitative RT-PCR was performed to measure gene expression levels of selected genes (Table I) using TaqMan ABI Prism 7700 (Applied Biosystems, Foster City, CA) as described before (18). Housekeeping gene elongation factor 1 {alpha} (EF1a) was used as a reference transcript. The expression of this housekeeping gene remains stable during the differentiation of Th1 and Th2 cells (19). Primers and probes (Table I) used for the quantification of gene expression (MedProbe, Oslo, Norway) were designed using Primer Express software (Applied Biosystems).


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  		Table I. Oligonucleotides used in quantitative real-time RT-PCR

 
Intracellular cytokine staining and flow cytometric analysis

Intracellular staining was performed for the cells after 7 days of polarization to confirm that the TGF-{beta} had been functional and repressed IFN-{gamma} production. Briefly, 0.5–1 x 106 cells were stimulated for 5 h with 5 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 500 ng/ml ionomycin (Calbiochem, San Diego, CA) in the density of 2 x 106/ml to induce the cytokine production. Part of the cells were maintained as unstimulated control population. After 2 h of stimulation, brefeldin A (10 µg/ml) was added and incubation was continued for an additional 3 h. After stimulation, the cells were washed twice with buffer (0.5% BSA and 0.01% azide in PBS). The cells were stained with anti-CD4-PerCP (BD Biosciences, San Jose, CA) for 15 min, after which the cells were washed with buffer. The cells were fixed with 4% paraformaldehyde for 15 min, washed with buffer, and permeabilized with 0.5% saponin plus 0.5% BSA plus 0.01% azide in PBS (pH 7) for 10 min. The cells were washed again with buffer and intracellular cytokine was performed with anti-IFN-{gamma}-FITC (Caltag Laboratories, Burlingame, CA) for 20 min. The cytokine profiles of the cells were studied with FACScan and CellQuest software (BD Biosciences).

Oligonucleotide array studies

The total RNA of the samples was isolated using the TRIzol method (Invitrogen, Carlsbad, CA) and was further purified with Qiagen’s RNAeasy minikit (Qiagen, Valencia, CA). Four to 5 µg of total RNA was used as starting material for the Affymetrix sample preparation. The sample preparation was performed according to the instructions and recommendations provided by the manufacturer (Affymetrix, Santa Clara, CA). The samples were hybridized to HG-U95Av2 arrays containing probes for ~10,000 genes. The data were analyzed on three consecutive levels. At the detection level, each probe was assigned a call of present, absent, or marginal. The comparison level analysis of the cells cultured defines a gene as up-regulated if the signal log ratio between the reference and the target samples is larger than 1 (2-fold increase) and the target sample is present. Similarly, a gene is defined as down-regulated if the signal log ratio is less than -1 (2-fold decrease) and the reference sample is present. At the third level of data analysis, genes that presented a consistent change in two separate experiments were considered as differentially expressed. The gene transcript levels were determined from data images with the algorithms in the GeneChip Microarray Suite software (Affymetrix MAS5).

Validation of the oligonucleotide array results

For validation of the oligonucleotide array results with real-time RT-PCR, additional Th1 and Th2 primary cultures were generated as previously described (18). Briefly, the priming was performed in the presence of 100 ng/ml PHA (Murex Diagnostics, Chatillon, France) and irradiated CD32-B7 transfected fibroblasts (20). The feeder cells were added in the final density of 1 x 106 cells/ml. Th1 cultures were supplemented with 2.5 ng/ml IL-12 (R&D Systems). Th2 cultures were supplemented with 10 µg/ml anti-IL-12 (R&D Systems) and 10 ng/ml IL-4 (R&D Systems). After 48 h of priming, 40 U/ml IL-2 (R&D Systems) was added into the cultures to enhance the proliferation of the lymphocytes. Part of the cells were cultured without any polarizing cytokines in the presence of IL-2 alone. The cultures were generated from four individuals, and during polarization samples were collected at the time points 0 h, 6 h, 24 h, 48 h, or 7 days.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of marker genes IFN-{gamma}, IL-4, T-bet, and GATA-3

Differential expression of known Th1 and Th2 marker genes IFN-{gamma}, IL-4, T-bet, and GATA-3 was studied in human cells, induced to polarize to Th1 and Th2 direction for 48 h, to ensure that the cells had started to differentiate (Fig. 1). As expected, after 48 h of polarization T-bet and IFN-{gamma} were more expressed in the cells induced to polarize to the Th1 direction and IL-4 and GATA-3 were more expressed in the cells induced to polarize to the Th2 direction, indicating that the cells had responded to cytokines IL-12 and IL-4. The levels of IL-4 were strongly repressed in both Th1 and Th2 conditions by TGF-{beta}. However, TGF-{beta} had no effect on the expression of IFN-{gamma}, T-bet, or GATA-3. To ensure that the TGF-{beta} had been functional also at Th1 conditions, the IFN-{gamma} cytokine expression levels were measured from the human cells polarized to the Th1 direction for 7 days in the presence and absence of TGF-{beta} (Fig. 2). As previously demonstrated with mouse cells, the IFN-{gamma} secretion by Th1 cells (68.4–73.3%) was inhibited in the presence of TGF-{beta} (7.1–8.7%) (17, 21, 22, 23).



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  		FIGURE 1. Expression of Th1 and Th2 marker genes. The CD4+ cells were purified from human cord blood and were activated with plate-bound anti-CD3 (1000 ng/µl for coating) and 500 ng/µl soluble anti-CD28. The cells were further polarized with either 2.5 ng/ml IL-12 for Th1 conditions or 10 ng/ml IL-4 for Th2 conditions in the presence and absence of 3 ng/ml TGF-{beta}. Part of the activated cells were cultured in neutral conditions without any polarizing cytokines (Act, activation). The cells were collected at the time points of 0 and 48 h. RNA was isolated from the samples and cDNA was prepared. Expression of known marker genes IFN-{gamma}, IL-4, T-bet, and GATA-3 was measured from the 48-h samples using real-time RT-PCR to ensure the induction of polarization to the Th1 and Th2 direction. The representative data from one of two repeats is shown.

 


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  		FIGURE 2. Repression of IFN-{gamma} secretion by TGF-{beta}. IFN-{gamma} cytokine expression was measured with flow cytometry from the cells polarized for 7 days in Th1 conditions (anti-CD3 + anti-CD28 plus IL-12) in the presence and absence of 3 ng/ml TGF-{beta}. For intracellular cytokine staining, the cells were stimulated for 5 h with 5 ng/ml PMA and 500 ng/ml ionomycin. Part of the cells were maintained as the unstimulated control population. After 2 h of stimulation, brefeldin A (10 µg/ml) was added and incubation was continued for an additional 3 h. To measure the levels of IFN-{gamma} secretion, the cells were stained with anti-IFN-{gamma}-FITC and were analyzed with FACScan and CellQuest software. The marker (M1) indicates the location of the unstimulated Th1 control cell population.

 
Differences in the gene expression profiles between the cells induced to polarize to Th1 or Th2 direction

To elucidate the genes involved in the early polarization and to explore the inhibitory mechanism of TGF-{beta} on the differentiation of Th1 and Th2 cells, we studied the gene expression profiles of the CD4+ cells induced to Th1 or Th2 directions for 48 h in the absence and presence of TGF-{beta}. The samples were hybridized on HG-U95Av2 arrays representing probes for ~10,000 genes. According to oligonucleotide array results, activation via CD3+CD28 had alone induced expression of ~582 genes and repressed ~533 genes (total 1115 probe sets) compared with the Thp cells (Fig. 3A). In Fig. 3, only the genes showing >=16-fold change in comparison between activated and Thp cells are presented. In addition to genes regulated by activation, numerous targets with various functions were identified as being regulated by the cytokines IL-12, IL-4, and TGF-{beta}.




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  		FIGURE 3. The target genes of activation, IL-12, IL-4, and TGF-{beta}. The CD4+ cells were purified from human cord blood and were activated with plate-bound anti-CD3 (1000 ng/µl for coating) and 500 ng/µl soluble anti-CD28. The cells were further polarized with either 2.5 ng/ml IL-12 for Th1 conditions or 10 ng/ml IL-4 for Th2 conditions in the presence and absence of 3 ng/ml TGF-{beta}. Part of the activated cells were cultured in neutral conditions without any polarizing cytokines. The samples were collected at the time points of 0 and 48 h. The cRNAs were prepared for Affymetrix hybridizations and the data were analyzed with the MAS5 software provided by Affymetrix. To identify the genes changed in response to different treatments, the expression profiles of the samples were compared with each other. A, Target genes of activation (Act vs Thp). B, Genes differentially expressed by Th1- and Th2-induced cells (Act + IL-12 vs Act + IL-4). C, Target genes of IL-12 (Act + IL-12 vs Act). D, Target genes of IL-4 (Act + IL-4 vs Act). E, Target genes of TGF-{beta} in Th1 conditions (Act + IL-12 + TGF-{beta} vs Act + IL-12). F, Target genes of TGF-{beta} in Th2 conditions (Act + IL-4 + TGF-{beta} vs Act + IL-4). The color intensities indicate the differences (signal log ratio) between two treatments. All of the nonreproducible results or changes below 2-fold (signal log ratio <1) were excluded from the results. Higher cutoff (signal log ratio >=4) for the target genes of activation was used to reduce the number of the genes. For each comparison, the regulation of the genes in response to other treatments is shown parallel as raw data. The functional groups of the genes are represented as numbers (1, cell surface molecules; 2, cytokines, chemokines, and other ligands; 3, Enzymes and pathway molecules; 4, structural molecules and intracellular trafficking; 5, transcriptional regulation; 6, unclassified).

 
Altogether 40 genes were detected to be differentially expressed by the cells induced to differentiate to Th1 or Th2 directions (Fig. 3B). As previously described, the genes IFNG, IL-2, IL18RAP, CTLA1/GZMB, G0S2, ANXA3, P2RX5, LIF, and BLR1 were preferentially expressed by the cells induced to polarize to the Th1 direction (24, 25, 26). The genes MAF/C-MAF, GATA-3, EBI2, IL10RA, Cox-2, NTRK1, CXCR4, E4BP4/NFIL3, and IL-13 were confirmed to be preferentially expressed by the cells induced to the Th2 direction (15, 16, 24, 25, 27, 28, 29). Moreover, suppressor of cytokine signaling 1 (TIP3/SOCS-1) was significantly more expressed by the cells polarized to the Th2 direction compared with those induced to the Th1 direction.

In addition to the genes previously implicated in the differentiation to Th1 or Th2 cells, 20 genes new in this context and differentially expressed by the cells induced to polarize to the Th1 and Th2 directions were identified. Of these genes, 3 were more expressed by the IL-12-induced cells (OAS2, AF070579, AMPD3) whereas 17 genes were preferentially expressed by IL-4-induced cells (LRRN3, PLA2G4A, VIM, TUBB, FER1L3, DAPK1, DUSP6, SATB1, LAIR2, AL049963, MAPK6, MGC14797, SPINT2, PXMP1, GARP, GNAI1, MAOA).

Genes regulated in response to IL-12 and IL-4

To further explore the mechanism as to how these genes become differentially expressed by the cells cultured in Th1 and Th2 conditions, we next identified which genes were regulated by IL-12 or IL-4 cytokines. IL-12 induced expression of 14 genes and repressed expression of 9 genes compared with the cells activated via CD3 and CD28 alone (Fig. 3C). IL-4 induced expression of 30 genes and repressed expression of 11 genes when compared with the CD3/CD28-activated cells (Fig. 3D). These genes included both known and unknown IL-12- and IL-4-regulated genes. To our knowledge, in CD4+ T cells, regulation of the genes PACE, MRF-1, FLOT1, MTMR1, GOSR2, AF055029, AL050166, AF070528, and SCYC2 by IL-12 and genes KIAA1013, ID2, KIAA0750, STK17B, HMGCS1, AL049940, AUH, BCL2A1, and HIC by IL-4 has not been previously described.

All of the genes regulated by IL-12 and IL-4 when compared with the CD3/CD28-activated cells were not detected to be differentially expressed by the cells induced to polarize in the Th1 or Th2 direction. Of the 13 genes preferentially expressed by the cells induced to the Th1 direction, five genes (IFNG, IL18RAP, CTLA1/GZMB, G0S2, BLR1) were up-regulated by IL-12 and G0S2 was simultaneously down-regulated by IL-4. Of the 27 genes preferentially expressed by the cells induced to the Th2 direction, 15 genes (MAF, NTRK1, DUSP6, Cox-2, TIP-3, PXMP1, PLA2G4A, E4BP4/NFIL3, GATA-3, EBI2, MAOA, FER1L3, SATB1, IL10RA, SPINT2) were up-regulated by IL-4. IL-12-mediated repression explained preferential expression of GARP by the cells induced to differentiate to the Th2 direction.

Genes regulated in response to TGF-{beta}

TGF-{beta} induced expression of ATP1B1, BASP1, and SCYA5/RANTES and repressed expression of E4BP4/NFIL3 in both cell types cultured in either Th1 or Th2 conditions. Furthermore, the presence of TGF-{beta} in Th1 conditions induced the expression of 16 genes and repressed expression of 6 genes (Fig. 3E). These TGF-{beta} target genes included specific targets of TGF-{beta}, but also genes regulated by IL-12 or IL-4. TGF-{beta} antagonized the effects of IL-12 by up-regulating expression of TNFRSF9 and by repressing expression of GZMB/CTLA-1, a gene induced by both IL-12 and IL-4. Interestingly, in Th1 conditions TGF-{beta} also down-regulated the expression of genes NFIL3/E4BP4 and SATB1, preferentially induced by IL-4, and induced expression of VIM, which was preferentially expressed by the cells cultured in Th2 conditions when compared with those cultured in Th1 conditions.

In addition to common genes regulated both in Th1 and Th2 conditions (ATP1B1, BASP1, SCYA5/RANTES, and E4BP4/NFIL3), in Th2 conditions TGF-{beta} induced expression of 16 genes and repressed 15 genes (Fig. 3F). Again TGF-{beta} regulated a specific set of its own targets, but importantly it antagonized the effects of IL-4 by repressing a set of IL-4-inducible genes (ID2, Cox-2, PLA2G4A, BCL2A1, and E4BP4/NFIL3) and expression of GNAI1, which was preferentially expressed in the cells cultured in Th2 conditions when compared with Th1 conditions.

The closer examination of the genes coregulated by TGF-{beta} and IL-12 or IL-4 (Table II) showed that the genes GZMB and E4BP4/NFIL3 are induced by CD3+CD28-activation alone and the expression is further augmented by both IL-12 and IL-4. However, E4BP4/NFIL3 was clearly more induced by IL-4 than IL-12. Interestingly, these genes were repressed by TGF-{beta} both in Th1 and Th2 conditions. This kind of repression in both Th1 and Th2 conditions was also seen for the IL-4-induced SATB1 and IL-12-repressed VIM.


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  		Table II. Genes coregulated by cytokines TGF-{beta} and IL-12 or IL-4

 
Validation of a set of genes using real-time RT-PCR

Oligonucleotide array results were obtained from two independent experiments. Four of the genes (SATB1, DUSP6, E4BP4/NFIL3, and TIP3) identified to be differentially expressed by the cells cultured in Th1 and Th2 conditions were selected for TaqMan RT-PCR analysis to further validate the results obtained with oligonucleotide arrays. The results were concordant with the oligonucleotide array study and all of the genes were observed to be induced by IL-4 (Fig. 4). E4BP4/NFIL3 was repressed by TGF-{beta} both in Th1 and Th2 conditions. SATB1 was repressed by TGF-{beta} in Th1 conditions, but also slightly in Th2 conditions. We also followed the expression kinetics of this set of interesting genes in Th1 or Th2 conditions during the 1 wk of polarization from Thp cells. Again the results were concordant with the oligonucleotide array data (Fig. 5). Importantly, NFIL3/E4BP4, TIP3/SOCS-1, and DUSP6 were identified to be differentially expressed in the cells cultured in Th1 and Th2 conditions already after 6 h of polarization, and the differences were maintained for at least the first 2 days of polarization. Also SATB1 was differentially expressed after 6 h in three of four individuals studied and in all individuals after 1 and 2 days of polarization. DUSP6 is expressed as two alternative splicing variants, and thus expression of both forms was quantitated. According to the results, only the long form is expressed by the cells studied.



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  		FIGURE 4. Validation of a set of genes using real-time RT-PCR. The CD4+ cells were purified from human cord blood and were activated with plate-bound anti-CD3 (1000 ng/µl for coating) and 500 ng/µl soluble anti-CD28. The cells were further polarized with either 2.5 ng/ml IL-12 for Th1 conditions or 10 ng/ml IL-4 for Th2 conditions in the presence and absence of 3 ng/ml TGF-{beta}. Part of the activated cells were cultured in neutral conditions without any polarizing cytokines. The cells were collected at the time points of 0 and 48 h. RNA was isolated from the samples and cDNA was prepared. Expression of selected genes SATB1, DUSP6, TIP3/SOCS1, and E4BP4/NFIL3 was measured from the 48-h samples using real-time RT-PCR to validate the results obtained with oligonucleotide arrays.

 


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  		FIGURE 5. Expression kinetics of SATB1, DUSP6, TIP3/SOCS1, E4BP4/NFIL3, and GADD45{beta} during 1 wk of polarization. To study the expression kinetics of SATB1, DUSP6, TIP3/SOCS1, E4BP4/NFIL3, and GADD45{beta} during 1 wk of polarization, long-term Th1 and Th2 primary cultures were generated from four individuals. The priming was performed in the presence of 100 ng/ml PHA (Murex Diagnostics) and irradiated CD32-B7-transfected fibroblasts. Th1 cultures were supplemented with 2.5 ng/ml IL-12 (R&D Systems). Th2 cultures again were supplemented with 10 µg/ml anti-IL-12 (R&D Systems) and 10 ng/ml IL-4 (R&D Systems). After 48 h of priming, 40 U/ml IL-2 (R&D Systems) was added into the cultures to enhance the proliferation of the lymphocytes. Part of the cells were cultured without any polarizing cytokines in the presence of IL-2 alone. During polarization, samples were collected at time points 0 h, 6 h, 24 h, 48 h, or 7 days. Real-time quantitative RT-PCR was performed to quantitate the gene expression levels of SATB1, TIP3/SOCS1, DUSP6, E4BP4/NFIL3, and GADD45{beta}. The statistical significance of the difference in the gene expression levels between Th1 and Th2 conditions was determined using the paired t test (***, p <= 0.001; **, p <= 0.01; *, p <= 0.05).

 
During the analysis of the results, Affymetrix updated its data analysis program MAS4 with a new version MAS5, in which the algorithm for analysis had been changed. Reanalysis of the results with MAS5 left out one of the genes, GADD45{beta}, that had been shown to be differentially expressed with the previous version of data analysis program (MAS4). The expression of gene GADD45{beta} was studied with RT-PCR. As a result, the gene was verified to be preferentially expressed by the cells cultured in Th1 conditions compared with Th2 conditions as described before (30, 31).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study demonstrates that triggering of the TCR leads to regulation of >1000 genes with various functions. Similar results have also been described before (32). Compared with that number, the group of target genes regulated by cytokines mediating the differentiation process is specific and limited to only <100 genes, at least at this stage of differentiation.

Comparison of the cells cultured in Th1 and Th2 conditions revealed differential expression of 40 genes. Of these genes, DUSP6, E4BP4/NFIL3, SATB1, and TIP3/SOCS-1 were further analyzed using real-Time RT-PCR and were shown to be preferentially induced by IL-4 in Th2 conditions already after 6 h of polarization. This positions these genes as important candidates as upstream regulators of the early differentiation process. The roles of these genes in Th1 and Th2 differentiation are currently unknown. DUSP6 is a phosphatase, which inhibits activity of extracellular signal-regulated kinase 2 (33). E4BP4/NFIL3 is an inducer or repressor of transcription, which can activate IL-3 expression, and a binding site for this factor is also present in the promoter area of IFN-{gamma}. In pro-B lymphocytes, E4BP4/NFIL3 has been found to participate in preventing apoptosis in response to IL-3 through ras-mediated signaling, which involves activation of both phosphatidylinositol 3-kinase and raf/mitogen-activated protein kinase pathways (34, 35). SATB1 is a DNA-binding protein, which is known to be involved in the development of thymic T cells (36). Its cleavage by caspase-6 and resulting dissociation from chromatin is involved in nuclear degradation occurring during early apoptosis of T cells (37). TIP3/SOCS-1 induced by the cytokine/STAT pathway is an inhibitor of cytokine signaling. Previous reports have provided controversial information concerning the role of TIP3/SOCS-1 in Th1 and Th2 cells; on one hand, the protein has been shown to be preferentially expressed by the Th1 cells in mouse. On the other hand, it has been reported that TIP3/SOCS-1 is induced by IL-6 in humans, which promotes the Th2 and inhibits the Th1 commitment (38, 39). DUSP6, SATB1, E4BP4/NFIL3, and TIP3/SOCS-1 are likely to be among those upstream factors that respond first to the polarizing signals and thus are involved in determining the fate of Thp cells during the early stages of polarization.

Our results demonstrate that a subset of target genes regulated by TGF-{beta} are also target genes for IL-12 and especially for IL-4 (TNFSF9, E4BP4/NFIL3, CTLA1/GZMB, ID2, Cox-2, GNAI1, PLA2G4A, and BCL2A1). The antagonizing influence of TGF-{beta} on the expression of these genes regulated by IL-12 or IL-4 could partly explain the inhibitory effect of TGF-{beta} on differentiation. If TGF-{beta} can modulate the differentiation process through these genes, they must be critical factors for the polarization. Enhanced Th2 response is known to contribute to the phenotype and symptoms of asthma (1, 2). On the other hand, studies with mouse models have demonstrated that TGF-{beta}1 can suppress the airway hyperresponsiveness and airway inflammation associated with asthma (40, 41). Thus, the genes through which TGF-{beta} can repress the Th2 differentiation could be potential targets for therapeutic applications to modulate the Th1 and Th2 balance.

It has been previously shown that TGF-{beta} inhibits the differentiation of Th1 and Th2 subtypes (42, 43). Studies with mice have shown that the inhibition of Th1 and Th2 differentiation by TGF-{beta} occurs through suppressing T-bet and GATA-3 expression, respectively (21, 22, 44). In our study with human cells, these two genes were not among the numerous primary genes regulated by TGF-{beta}, indicating that the mechanism of TGF-{beta} regulation on Th1 and Th2 responses is probably more complex than has been previously thought. The contradiction with the previous studies concerning the regulation of T-bet and GATA-3 by TGF-{beta} is probably due to differences between mouse and humans. However, it is also known that the effects of TGF-{beta} can vary depending on the stage of cell differentiation and cytokine environment, leading to differential observations in distinct experimental conditions (45).

To resolve the exact role of the genes identified in the present study for developing Th1 and Th2 cells, several questions remain to be answered: what is the hierarchical order, upstream regulators, and interaction partners of these factors in the signaling pathways involved in the differentiation process; what are the molecular functions of these factors in Th cells; what is the importance of these genes for the differentiation process, and can they be used to modulate Th1 and Th2 responses? Thus, our study provides candidate genes for further functional analysis. It remains to be determined whether this newly identified panel of genes leads to novel strategies to therapeutically modulate Th1 and Th2 responses.


   Acknowledgments
 
We are grateful to Paula Suominen, Miina Miller, Outi Melin, and Anya NiChomrai for technical assistance and Elizabeth Carpelan for reviewing the language of this manuscript.


   Footnotes
 
1 This study was supported by the Turku University Hospital Research Fund, the Academy of Finland, and Turku Graduate School for Biomedical Sciences. Back

2 Address correspondence and reprint requests to Riikka Lund, Turku Centre for Biotechnology, University of Turku and Åbo Akademi, P.O. Box 123, FIN-20520, Turku, Finland. E-mail address: riikka.lund{at}btk.utu.fi Back

Received for publication January 8, 2003. Accepted for publication September 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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