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The Journal of Immunology, 2007, 178: 1635-1644.
Copyright © 2007 by The American Association of Immunologists, Inc.

Suppression of Early IL-4 Production Underlies the Failure of CD4 T Cells Activated by TLR-Stimulated Dendritic Cells to Differentiate into Th2 Cells1

Jie Sun and Edward J. Pearce2

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-{gamma}. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)3 play a central role in directing CD4 T cell (Th cell) response polarization (1, 2). Following activation through TLRs by bacterial or protozoal pathogens, DCs up-regulate MHC II and costimulatory molecules and become capable of activating naive CD4 T cells. In addition, activation of DCs via TLRs leads to the production of IL-12 (3), which promotes Th1 response development (4) and delivers a negative signal for Th2 cell differentiation by suppressing the expression of GATA-3 (5), a transcription factor that is essential for Th2 cell development (4, 6, 7). As a result, it has been believed that the Th1/Th2 choice is imposed by DCs primarily through the production of IL-12 (1). However, the fact that IL-12–/– mice infected with various pathogens fail to mount default Th2 responses (8, 9, 10) suggests that there are additional mechanisms involved in this process.

Among the factors that promote Th2 cell development, the most important appears to be IL-4, which signals through the IL-4R-STAT6 pathway to enhance expression of the Th2 lineage-specific transcription factor GATA-3 (5). As a result, mice deficient in IL-4R or STAT6 fail to mount efficient Th2 responses (4). IL-4 produced by CD4 T cells is essential and sufficient for Th2 response development (11, 12). Recent reports have shown that naive Th cells can rapidly produce IL-4 in response to TCR/CD28 stimulation, and it is believed that this early IL-4, working in an autocrine fashion through a STAT6-dependent pathway, is critical for the up-regulation of GATA-3 expression and commitment to the Th2 lineage (13). New findings indicate that IL-2 signaling through STAT5 plays an essential role in early IL-4 production (14).

Recently, we showed that DCs stimulated through TLRs deliver a potent MyD88 dependent and IL-12/IL-18/IL-23/IL-27-independent negative signal for Th2 cell development (15). In this study we show that the suppression of Th2 cell development by TLR-activated DCs is associated with a failure of stimulated Th cells to respond to IL-2 and initiate the production of early IL-4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

C57BL/6 (B6) or BALB/c IL-12p40–/– mice and T-bet–/– and IL-4–/– mice were purchased from The Jackson Laboratory. OT-II, IL-4–/– OT-II, T-bet–/– OT-II, DO11.10, and 4get/DO11.10 transgenic mice were bred at the University of Pennsylvania (Philadelphia, PA). Heat-killed Propionibacterium acnes (Pa) was purchased from the Van Kampen Group. LPS, Escherichia coli serotype 0111:B4, and polyinosinic-polycytidylic acid (poly(I:C)) were obtained from Sigma-Aldrich. CpG ODN 1826 was obtained from Coley Pharmaceuticals. Peptidoglycan (PGN) was provided by Dr. Y. Choi (University of Pennsylvania School of Medicine, Philadelphia, PA). All cytokines were recombinant and from R&D Systems. GATA-3 and T-bet-specific Abs were from Santa Cruz Biotechnology. Other Ab were from BD Pharmingen unless otherwise stated. Sterile endotoxin-free OVA protein was prepared as described (15). The OVA323–339 peptide was synthesized by Invitrogen Life Technologies.

DC preparation and DC/CD4 T cell coculture

DCs were grown from bone marrow as previously described (16). IL-12–/– DCs were harvested and pulsed with OVA (1 mg/ml) with or without Pa (10 µg/ml), LPS (200 ng/ml), PGN (10 µg/ml), CpG (1 µg/ml), poly(I:C) (5 µg/ml), or rIFN-{alpha} (5000–10000 U/ml) in DC medium with 5 ng/ml GM-CSF for ~=18 h, washed, and mixed with CD4 T cells that had been negatively depleted with a MACS CD4 kit (Miltenyi Biotech) to which, in some experiments, was added an Ab to CD25 (clone 7D4). We used a ratio of 2 x 104 DCs to 2 x 105 CD4 T cells per well of a round-bottom, 96-well plate (15). In some experiments (Figs. 1–5), 30 U/ml human IL-2 was added to the culture. We found that without blocking mouse IL-2, human IL-2 addition does not affect IL-4 production and Th cell differentiation (Fig. 6). Th1 conditions were created by the addition of 50 ng/ml rIL-12 and 30 µg/ml anti-IL-4 (clone 11B11), while Th2 conditions included 10 ng/ml IL-4 and 50 µg/ml anti-IFN-{gamma} Ab (XMG6). Where necessary, IL-2, IL-4, and IL-12 were neutralized by the inclusion of 20 µg/ml S4B6, 11B11, or C17.8, respectively, and IL-10 function was blocked by adding anti-IL-10R (clone 1B1.3a) Ab at 10 µg/ml. In some experiments DCs were mixed with CD4 T cells and the OVA323–339 peptide was added at various concentrations. At different time points cells were stimulated with PMA (100 ng/ml) and ionomycin (1 µg/ml) in the presence of brefeldin A (10 µg/ml), fixed, and permeabilized and stained for intracellular cytokines using fluorochrome-labeled Ab and flow cytometry (FACS), as described (15). When 4get/DO11.10 T cells were used, GFP expression was detected directly by FACS. IL-2 was measured by capture ELISA.


Figure 1
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  		FIGURE 1. TLR-activated DCs inhibit early IL-4 production. A, OT-II Th cells were stimulated with OVA or Pa/OVA pulsed IL-12–/– DCs in the absence (upper panel) or presence (lower panel) of anti-IL-4 Ab for 5 days. The anti-IFN-{gamma} Ab was present throughout these cultures. Th cells were stimulated with PMA/ionomycin and stained for IFN-{gamma} and IL-4. B, Upper panel, OT-II Th cells were stimulated with OVA pulsed wild-type (WT) or IL-4–/– DCs in the presence of neutralizing anti-IL-12 Ab; lower panel, OT-II Th cells or IL-4–/– OT-II Th cells were stimulated with OVA pulsed IL-12–/– DCs. After 5 days, Th cells were stimulated with PMA/ionomycin and stained for IL-4 or IL-5. KO, knockout. C, OT-II Th cells were stimulated with OVA- or Pa/OVA-pulsed IL-12–/– DCs for 2 days and then restimulated with PMA/ionomycin and stained for IL-4. D, 4get/DO.11.10 Th cells were stimulated with OVA- or Pa/OVA-pulsed IL-12–/– DCs for 2 days in the absence (upper panel) or presence (lower panel) of anti-IL-4 Ab. GFP expression in KJ1-26 (clonotype-specific Ab)-positive cells was analyzed. E, OT-II Th cells were stimulated with OVA and TLR ligand-pulsed IL-12–/– DCs for 2 days as shown. CD69+ cells were then sorted and IL-4 and IFN-{gamma} mRNA levels were measured by quantitative RT-PCR. Percentages of cytokine or GFP+ cells within gates/quadrants are shown. Data are from one experiment and typical of three.

 

Figure 2
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  		FIGURE 2. DCs activated by TLR ligands, but not IFN-{alpha}, block early IL-4 production. A, IL-12–/– DCs were pulsed with OVA plus TLR ligands or cytokine as indicated for 18 h. CD86 expression on DCs was measured by flow cytometry. B, DCs were used to stimulate 4get/DO.11.10 Th cells. After 2 days in culture, CD69 expression on KJ1-26 positive cells was measured as an indication of cell activation. C and D, After 2 (C) or 5 (D) days in culture, GFP expression in CD69+ (C) or CD44high (D) activated Th cells was analyzed by flow cytometry. The numbers show the percentages of Th cells that are CD69+ (B) or the percentages of activated CD4 cells that are GFP+. E, IL-12–/– DCs were pulsed with OVA plus TLR ligands and mixed with OT-II Th cells in the absence or presence of an anti-IL-10R Ab. After 4 days in culture Th cells were restimulated with PMA/ionomycin to stain for the intracellular IL-4. Numbers are the percentages of IL-4+ cells within the activated CD4 population. Pam3, Pam3Cys. F, IL-12–/– DCs were pulsed with OVA plus LPS or TNF-{alpha} and mixed with 4get/DO.11.10 Th cells. After 5 days in culture, GFP expression in activated Th cells was analyzed by flow cytometry. The numbers show the percentages of GFP+ cells in activated Th cells. Data are from one experiment and typical of three.

 

Figure 3
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  		FIGURE 3. The inhibition of early IL-4 production and Th2 cell development by TLR-activated DCs occurs in an Ag-dose independent manner. A, 4get/DO.11.10 Th cells were stimulated with IL-12–/– DCs or IL-12–/– Pa/DCs in the presence of different concentrations of soluble OVA323–339 peptide. After 2 (A) or 4 (B) days in culture, GFP expression in Th cells was measured by flow cytometry. C, Top row, IL-12–/– DCs were pulsed with Pa, CpG, or rIFN-{alpha} for 18 h and MHC II expression was measured by flow cytometry. Middle and bottom rows, 4get/DO.11.10 Th cells were stimulated with IL-12–/– DCs pulsed with TLR ligands or cytokine as shown in the presence of 10 ng/ml soluble OVA323–339 peptide. After 2 days (middle row) or 4 days (bottom) in culture, GFP expression in Th cells was measured by flow cytometry. The numbers show the percentages of Th cells that are the percentages of activated CD4 cells that are GFP+. Data are from one experiment and typical of three.

 

Figure 4
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  		FIGURE 4. Suppression of early IL-4 production and Th2 cell development by TLR-activated DCs is T-bet independent. A and B, OT-II Th cells were stimulated with OVA- or Pa/OVA- pulsed IL-12–/– DCs or with OVA-pulsed IL-12–/– DCs with superimposed Th1 conditions (Th1). After 2 days in culture, T-bet mRNA (A) or protein levels (B) in CD69+ cells were measured by real-time RT-PCR or intracellular staining, respectively. The numbers in B show the mean florescence intensity of T-bet staining. C and D, OT-II (upper panels) and T-bet–/– OT-II (lower panels) Th cells were stimulated with OVA- or Pa/OVA-pulsed IL-12–/– DCs. After 2 (C) or 4 (D) days in culture, Th cells were stimulated with PMA/ionomycin and stained for IFN-{gamma} and IL-4. Percentages of Th cells positive for IL-4 and IFN-{gamma} are shown. KO, Knockout; WT, wild type. E and F, OT-II Th cells were stimulated with OVA- or Pa/OVA-pulsed IL-12–/– DCs in the presence of anti-IFN-{gamma} Ab alone (top panel) or with anti-IL-4 Ab or IL-4 as indicated. After 2 days (2d) (E) or 4 days (4d) (F) in culture, GATA-3 expression levels were measured by intracellular cytokine staining; numbers show mean florescence intensity. Data are from one experiment and typical of at least three.

 

Figure 5
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  		FIGURE 5. The inhibition of early IL-4 production by T cells by TLR-activated DCs occurs before cell division. A, OT-II Th cells were labeled with CFSE and stimulated with OVA and Pa- or CpG-pulsed IL-12–/– DCs as shown. Thirty-six hours later, cell proliferation was analyzed using flow cytometry to measure CFSE. B, CD69+ cells were sorted and IFN-{gamma}, IL-4, GATA-3, and c-Maf mRNA levels measured by quantitative RT-PCR. RNA samples from CD4 T cells grown under Th1 or Th2 conditions for 5 days were used for positive controls. Data are from one experiment and representative of two.

 

Figure 6
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  		FIGURE 6. CD4 T cells activated by TLR-stimulated DCs produce IL-2 but are less capable of phosphorylating STAT5 in response to this cytokine. A, OT-II CD4 cells were stimulated with OVA, Pa/OVA, or rIFN-{alpha}/OVA pulsed IL12–/– DCs for 24 h in the presence or absence of an anti-IL-2 Ab. After this, supernatants were removed for IL-2 measurements using capture ELISA (A) and cells were fixed, permeablized, and stained for CD25 and pSTAT5. B, Staining for CD25 and pSTAT5. Numbers inside the boxes are the percentages of total T cells that are CD25high/pSTAT5high (upper box) or CD25low/pSTAT5low (lower box); numbers outside the boxes are the percentages of CD25+ T cells that are CD25high/pSTAT5high. C, Twenty-four hours after initiating cultures, cells were washed and returned to culture in medium lacking IL-2 and containing anti-murine IL-2 to rest for 4 h before adding human IL-2 (100 U/ml). Twenty-five minutes later cells were recovered and stained for pSTAT5. Numbers are mean fluorescence intensity of pSTAT5 staining in cells to which human IL-2 was added (dark line). Dotted line represents pSTAT5 staining in cells that remained in IL-2-free medium. Light line shows the isotype control staining of cells to which human IL-2 was added. Data shown are from an individual experiment and are typical of results from three or more experiments.

 
T-bet, GATA-3, and phosphorylated STAT5 (pSTAT5) staining

Staining for T-bet, GATA-3, and STAT5 was based on a previously described protocol (17) with modifications. Briefly, CD4 T cells were harvested, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS with 0.5% BSA. For T-bet and GATA-3 staining, mouse IgG1 (BD Pharmingen) was used as blocking Ab and isotype control followed by primary unlabeled anti-T-bet or GATA-3 Ab (Santa Cruz Biotechnology). After incubation and washing, Cy-5 donkey F(ab')2 anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used to detect bound Ab. For pSTAT5 staining, fixed and permeabilized cells were blocked with rat IgG (Sigma-Aldrich) and stained with an Alexa Fluor 647-conjugated pSTAT5 Ab (clone 47; BD Pharmingen) or Alexa Fluor 647-conjugated isotype control Ab.

Real-time RT-PCR

RNA was extracted using RNeasy (Qiagen) from purified CD69+ cells and treated with DNase I (Invitrogen Life Technologies). Random hexamers and SuperScript II reverse transcriptase (Invitrogen Life Technologies) were used to synthesize first strand cDNAs from equivalent amounts of RNA from each sample. Real-time RT-PCR was performed in the SmartCycler system (Cepheid) with SYBR Green RT-PCR master mix (Qiagen). Data were generated with the comparative threshold cycle ({Delta}CT) method by normalizing to hypoxanthine phosphoribosyltransferase. The sequences of primers used in the studies are available on request.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR-activated DCs suppress early IL-4 production in responding Th cells

We have shown previously that when stimulated with OVA-pulsed IL-12–/– DCs (OVA/DCs), a large percentage of OT-II CD4 T cells differentiate into Th2 cells (15) (unless otherwise stated, experiments in this report use DCs made from the bone marrow of IL-12–/– mice). However, Th2 cell development is inhibited when OVA-pulsed DCs are costimulated with Pa (Pa/OVA/DCs) or other TLR-agonists, even when IFN-{gamma} is neutralized (15). IL-4 has been shown to be a critical factor for Th2 cell development by CD4 cells activated with anti-CD3/anti-CD28 (4). Likewise, in our system IL-4 is needed for Th2 cell development because neutralizing anti-IL-4 Ab blocks the development of Th2 cells in cocultures of OVA-pulsed DCs and OT-II CD4 T cells (Fig. 1A). In this system the IL-4 necessary for Th2 cell differentiation is produced by the CD4 cells themselves and not by DCs, because IL-4–/– OT-II CD4 T cells were unable to develop into Th2 cells when cocultured with IL-4 sufficient DCs, but WT OT-II CD4 T cells were able to become Th2 cells when activated by OVA pulsed IL-4–/– DCs (Fig. 1B). Taken together, these results indicate that IL-4 derived from CD4 T cells, but not DCs, is needed for Th2 cell development.

CD4 T cells up-regulate both IL-4 and IFN-{gamma} production at early times postactivation with anti-CD3/anti-CD28, and IL-4 produced in this process is sufficient to drive Th2 cell development (11, 12, 13, 18). We hypothesized that Pa-activated DCs might inhibit Th2 cell development by suppressing early IL-4 production by CD4 T cells. To test this possibility, OT-II CD4 T cells were stimulated with OVA/DCs or Pa/OVA/DCs and intracellular staining of IL-4 was performed on day 2 instead of day 5. We found that after 2 days in culture there were small percentages (~5% of CD44high cells) of CD4 cells producing IL-4 and that this early IL-4 production was not apparent in CD4 T cells stimulated by Pa/OVA/DCs (Fig. 1C). We obtained the same finding using RAG–/–/OT-II Th cells, indicating that this early IL-4 is made by newly activated naive Th cells and not by previously activated memory cells (data not shown).

To avoid possible artifacts associated with restimulating CD4 T cells with PMA/ionomycin, we repeated these experiments using CD4 T cells from 4get/DO.11.10 mice (19); in these cells, IL-4 expression is reported by the production of GFP. As was the case for the OT-II CD4 T cells, 4get/DO.11 CD4 T cells produced IL-4/GFP within 2 days of activation by OVA/DCs, but GFP+ cells were not evident when Pa/OVA/DCs were used as APCs. When neutralizing anti-IL-4 Ab was added to cultures, the percentage of GFP+ cells decreased, suggesting that IL-4 had been made even in this early stage of T cell differentiation and had worked in an autocrine fashion to promote further IL-4/GFP expression (Fig. 1D). The data also suggest that early IL-4 production by CD4 T cells is in part IL-4 independent, because the percentage of GFP+ CD4 cells evident in culture where IL-4-induced signaling is blocked is still significantly higher than in cultures where CD4 T cells were activated by Pa/OVA/DCs. This correlates well with reported findings (13). To further verify these observations, we next performed quantitative real-time RT-PCR of IL-4 and IFN-{gamma} mRNAs in sorted CD69+ CD4 T cells. We found that while Pa/OVA/DCs could promote up-regulation of the IFN-{gamma} message ~2-fold, they inhibited IL-4 transcription 10-fold compared with OVA/DCs. (Fig. 1E). Together, these results suggest that in the absence of IL-12, TLR-activated DCs suppress the early production of IL-4 by activated CD4 T cells.

TLR agonists, not cytokines, activate DCs to suppress early IL-4 production and Th2 cell development

TLR ligands induce DC maturation, evident as the increased surface expression of MHC II and costimulatory molecules. In contrast, Th2 Ags usually do not induce DC maturation (2). Consequently, it seemed likely that maturation per se could lead to the expression of a signal that inhibits Th2 cell development. To address this possibility, we compared the ability of DCs exposed to a variety of TLR ligands (Pa, LPS, CpG, poly(I:C), and PGN) or to a cytokine stimulus of maturation (IFN-{alpha}) to suppress IL-4 production. All of these maturation signals induced increased surface expression of CD86 (Fig. 2A) and of MHC II and CD80 (data not shown). In cocultures with 4get/DO.11 CD4 cells for 2 days, all matured DCs were more effective at activating CD4 cells (as indicated by increased numbers of CD69+ cells) than were DCs pulsed with OVA alone (Fig. 2B). Nevertheless, whereas all TLR-activated OVA-pulsed DCs suppressed both early IL-4 expression (Fig. 2C) and late Th2 cell development (Fig. 2D), this was not the case for IFN-{alpha}/OVA/DCs. Rather, IFN-{alpha}/OVA/DCs tended to promote the development of Th2 cells (Fig. 2D). In addition, DCs exposed to TNF-{alpha}/OVA or IFN-{gamma}/OVA also failed to suppress Th2 cell development (Fig. 2F and data not shown).

Pa contains a TLR2 ligand and PGN is a defined TLR2 ligand, and both activated DCs to suppress Th2 cell development. This is intriguing because there are reports that S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH,trihydrochloride (Pam3Cys), another TLR2 ligand, favors Th2 cell differentiation perhaps by enhancing production of IL-10 by DCs (20). To examine this issue, we stimulated DCs with Pam3Cys and cocultured them with OT-II Th cells in the presence or absence of a blocking anti-IL10R Ab. We found that Pam3Cys was as capable as PGN and Pa of inhibiting Th2 cell differentiation whether IL-10 function was blocked or not (Fig. 2E). Together, our data suggest that signals induced generally by TLR activation in DCs suppress Th2 cell development. These signals appear not to be generated following the activation of DCs by cytokines.

Inhibition of Th2 cell development by TLR-activated DCs is Ag-dose independent

The Ag dose has been shown to be an important factor in Th1/Th2 differentiation driven by DCs (21). We reasoned that the suppression of Th2 cell development by TLR-activated DCs might be due to the high concentration of Ag presented by virtue of enhanced Ag-processing and increased MHC II expression. To examine this issue, we stimulated 4get/DO.11.10 CD4 T cells with DCs or Pa/DCs in the presence of OVA323–339 peptide over a dilution series from 1 µg/ml to 1 ng/ml. In the absence of TLR stimulation we observed Th2 commitment at 1 and 10 ng/ml peptide, but noted that at higher peptide concentrations (1 µg/ml and 100 ng/ml) there was a relative failure of early IL-4 production and Th2 cell development (Fig. 3, A and B), a result that is in agreement with an earlier report (21). We found that Pa/DCs could suppress early IL-4 production and later Th2 development at every Ag dose tested (data not shown and Fig. 3, A and B). In a second series of experiments, we stimulated 4get/DO.11.10 CD4 T cells with DCs, Pa/DCs, CpG/DCs, or IFN-{alpha}/DCs in the presence of the 10 ng/ml concentration of the OVA323–339 peptide that is optimal for Th2 cell development (Fig. 3, A and B). In this setting, rIFN-{alpha}-activated DCs up-regulated MHC II expression to the same extent as TLR-activated DCs but, in contrast to the TLR-activated DCs, failed to suppress either early IL-4 production or later Th2 development (Fig. 3C). In summary, these results suggest that although the Ag dose plays a role in the decision process that results in Th2 cell development, the ability of TLR-activated DCs to block commitment to the Th2 lineage is Ag dose independent.

Neither up-regulation of T-bet expression nor early suppression of GATA-3 expression accounts for the failure to commit to the Th2 lineage

T-bet–/– mice spontaneously exhibit multiple symptoms characteristic of asthma, indicating that T-bet is a potent negative regulator of Th2 cell development (22). Expression of T-bet is induced by signaling through TCR, IFN-{gamma}/STAT-1, and possibly Notch (4, 23). We reasoned that TLR-activated DCs could be suppressing Th2 cell development by inducing T-bet. To examine this possibility, we measured T-bet expression in OT-II CD4 T cells after 2 days of culture with OVA/DCs or Pa/OVA/DCs in the absence of IL-12, at which time suppression of IL-4 production is evident (Fig. 1), but found little evidence for increased levels of T-bet mRNA (Fig. 4A) or protein (Fig. 4B). In contrast, T-bet levels were high in CD4 cells stimulated by OVA/DCs or Pa/OVA/DCs in Th1 conditions (Fig. 4, A and B). These results suggest that the suppression of early IL-4 production by TLR-activated DCs occurs independently of induced T-bet expression. To address this directly, we stimulated T-bet–/– OT-II CD4 T cells with OVA/DCs or Pa/OVA/DCs and examined IL-4 production. As anticipated, after 2 days more IL-4-producing CD4 cells emerged in cocultures of T-bet–/– Th cells and OVA/DCs than was the case when T-bet sufficient CD4 T cells were used (Fig. 4C). To a greater extent this was also true at day 4 (Fig. 4D). Nevertheless, at day 2 T-bet–/– CD4 T cells activated by TLR-stimulated DCs failed to make IL-4 (Fig. 4C). By day 4 a population of Th2 cells had emerged in cocultures of Pa/OVA/DCs and T- bet–/– CD4 T cells, but it was only one-third the size of that which developed when OVA/DCs were used as APCs (Fig. 4D).

GATA-3 is up-regulated in response to IL-4/STAT-6 and TCR-induced signals (4, 24). It seemed feasible that a block in GATA-3 expression could underlie the failure of CD4 T cells to commit to the Th2 lineage. However, we found that after 2 days this transcription factor could be detected at equivalently low levels in responding (CD44high) CD4 T cells in cultures with OVA/DCs or Pa/OVA/DCs, (Fig. 4E); these levels were ~4-fold higher than in CD44low cells from the same cultures (not shown). After 4 days in culture, CD4 T cells stimulated with OVA/DCs exhibited increased expression of GATA-3, whereas CD4 T cells stimulated with Pa/OVA/DCs did not (Fig. 4E). Neutralizing IL-4 in the cultures resulted, by day 2, in reduced GATA-3 levels in CD44high CD4 T cells (Fig. 4E), although these levels did not drop to those observed in CD44low CD4 T cells (data not shown), suggesting that TCR signaling is the main pathway in the induction of GATA-3 expression at this early time. Neutralization of IL-4 strongly inhibited GATA-3 expression in CD4 cells that had been cultured with OVA/DCs for 4 days, reducing it to levels equivalent to those in CD4 T cells stimulated with Pa/OVA/DCs (Fig. 4F). The addition of IL-4 promoted GATA-3 expression at days 2 and 4, but to a lesser extent in CD4 cells stimulated by Pa/OVA/DCs than by OVA/DCs (Figs. 4, E and F). These data suggest that the difference in GATA-3 expression at day 4 between CD4 cells stimulated with OVA/DCs or Pa/OVA/DCs is the downstream effect of differences in early IL-4 production, and they point to suppression of early IL-4 as the key event initiated by TLR-activated DCs that results in the abortion of Th2 cell development.

The suppression of early IL-4 production by TLR-activated DCs is independent of cell proliferation

To examine whether the suppression of IL-4 production and Th2 cell development reflects a negative instructional signal from TLR-activated DCs or is due to the selective inhibition of the outgrowth of IL-4+ CD4 cells, we used real-time RT-PCR to measure IL-4 and IFN-{gamma} mRNA levels in CD4 cells that had been activated only 36 h earlier and which had yet to divide (Fig. 5A). At this early time point CD4 T cells stimulated with OVA/DCs contained appreciable levels of IL-4 mRNA. Nevertheless, this very early burst of IL-4 production was substantially inhibited when OVA-pulsed DCs were also stimulated by Pa or CpG (Fig. 5B). In these cultures where IL-12 was absent, the IFN-{gamma} mRNA levels in CD4 T cells stimulated with OVA/DCs, Pa/OVA/DCs, or CpG/OVA/DCs were approximately equivalent (Fig. 5B). Given our failure to detect differences in GATA-3 levels in CD4 cells that had been stimulated 2 days earlier by OVA/DCs vs Pa/OVA/DCs, we examined whether the more sensitive RT-PCR technique might reveal a difference in the early expression of this transcription factor at 36 h. We also examined c-Maf transcript levels because, along with GATA-3, this transcription factor plays a role in Th2 cell commitment (25), but we found no significant differences in the expression of either transcription factor in CD4 T cells stimulated by OVA/DCs vs Pa/OVA/DCs (Fig. 5B).

T cells stimulated with TLR-activated DCs are less responsive to IL-2

It has become clear recently that the ability of newly activated naive CD4 T cells to respond to signals from IL-2 is essential for Th2 differentiation (14, 26, 27). It is possible then that TLR-activated DCs inhibit early IL-4 production by inhibiting IL-2 production or responsiveness to this cytokine. To examine this issue, we first measured IL-2 levels in the supernatants of OT-II CD4 T cells stimulated with OVA/DCs, Pa/OVA/DCs, or rIFN-{alpha}/OVA/DCs. We found that CD4 T cells stimulated by TLR-activated DCs or by cytokine-activated DCs had made more IL-2 after 24 h than had CD4 T cells stimulated by DCs pulsed with OVA alone (Fig. 6A). Next, we used direct staining for pSTAT5 to examine whether CD4 T cells stimulated with Pa/DCs are responsive to IL-2. We found that the level of pSTAT5 expression is tightly correlated with the level of CD25 (IL-2R{alpha}) expression on CD4 T cells, with CD25high cells having the highest levels of pSTAT5 (Fig. 6B). We reproducibly observed small populations of CD25highpSTAT5+ CD4 T cells in cocultures of OT-II CD4 T cells with OVA/DCs or rIFN-{alpha}/OVA/DCs and noted the absence of this population when OT-II CD4 T cells were stimulated with Pa/OVA/DCs (Fig. 6B). In these experiments, the existence of the CD25highpSTAT5high cell populations is dependent on IL-2 signaling, because inclusion of neutralizing anti-IL-2 Ab blocked the development of these cells (Fig. 6B). Taken together, these results suggest that CD4 T cells stimulated by TLR-activated DCs are less responsive to IL-2 during the initial 24 h following activation than are CD4 T cells that have been stimulated by OVA/DCs or rIFN-{alpha}/OVA/DCs. To formally address this question, we initiated DC-CD4 T cell cocultures and, after 24 h, rested cells in medium containing anti-murine IL-2 before pulsing them with human IL-2 and measuring pSTAT5 phosphorylation. We found that STAT5 was phosphorylated in response to human IL-2 in CD4 T cells that had been activated by OVA/DCs or IFN-{alpha}/OVA/DCs and, to a much lesser extent, in CD4 T cells activated by Pa/OVA/DCs (Fig. 6C). Interestingly, CD4 T cells activated by TLR-stimulated DCs proliferate at least as effectively as those activated by DCs pulsed with Ag alone (15), supporting the view that IL-2 signaling through STAT5 is not important for the entry of CD4 T cells into the cell cycle.

To directly link CD25/pSTAT5 levels to early IL-4 production, we stimulated OT-II Th cells with OVA/DCs or Pa/OVA/DCs for 24 h and sorted the CD25high cells (which we knew to be pSTAT5high; Fig. 6B) and CD25low cells by FACS; we used real time RT-PCR to measure IL-4 transcripts in these cells. We found that the expression of early IL-4 in the CD25highpSTAT5high cells was almost 10-fold more than in CD25low cells (Fig. 7A). These results indicate that the CD25highpSTAT5high cells are those making abundant IL-4 mRNA, which is consistent with the findings that STAT5 is an important transcription factor for IL-4 production.


Figure 7
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  		FIGURE 7. TLR-activated DCs suppress early IL-4 production by T cells by inhibiting responsiveness to IL-2. A, OT-II Th cells were left unstimulated or stimulated with OVA- or Pa/OVA-pulsed IL-12–/– DCs. After 24 h in culture, CD25 expression in live CD4 cells was measured by flow cytometry. Then CD25high or CD25low T cells were FACS sorted and the level of IL-4 mRNA was measured by real-time RT-PCR. Numbers are the percentages of CD25high or CD25low cells in the CD4 population. Rel. exp. level, relative expression level. B, CD4 T cells were stimulated with DCs as shown for 2 days under conditions in which murine IL-2 was active, neutralized, or neutralized but to which human IL-2 (300 U/ml) was added as indicated. Cells were stimulated with PMA and ionomycin and IL-4 production by CD4 cells was assessed by intracellular cytokine staining and flow cytometry. Numbers represent percentages of CD4 cells making IL-4. C and D, Th cell stimulation and intracellular staining were performed as in A. CD25 expression and IL-4/IFN-{gamma} production were measured by flow cytometry. Numbers are the percentages of T cells that are CD25+ (C) or the percentages of CD25+ cells that are CD25high (D, left panel) or the percentages of CD25high or CD25low cells that are IL-4+ or IFN-{gamma}+ (D, right panel, as indicated). Data shown are from one experiment and are typical of results from two or more experiments.

 
To further confirm that IL-2 signaling is essential for early IL-4 production, we examined IL-4 production in CD4 T cells stimulated under conditions in which mouse IL-2 was either active or neutralized. We found that the numbers of T cells producing IL-4 at day 2 following activation by OVA/DCs or IFN-{alpha}/OVA/DCs were reduced when mouse IL-2 was neutralized in the cultures and that this effect was reversed by the inclusion of human IL-2 (Fig. 7B). However, the inclusion of human IL-2 in the cultures did not induce early IL-4 production by T cells stimulated with Pa/OVA-pulsed DCs (Fig. 7B), indicating that Pa-activated DCs inhibit the downstream consequences of IL-2 signaling that are linked to IL-4 production in CD4 T cells. We next examined CD25 expression and early IL-4 production by CD4 T cell 2 days following stimulation with OVA/DCs, IFN-{alpha}/OVA/DCs, or Pa/OVA/DCs. We found that both IFN-{alpha}- and Pa- activated DCs stimulated more T cells to express CD25 (Fig. 7C) than did OVA-pulsed DCs. However, fewer CD25high cells were apparent when CD4 T cells were activated by Pa/OVA/DCs (Fig. 7D). IL-2 neutralization only slightly decreased the overall percentages of CD25+ cells regardless of the stimulus used for CD4 T cell activation in all three groups, supporting the idea that IL-2 signaling is not essential for T cell activation (27). However, IL-2 neutralization significantly diminished the percentages of CD4 T cells that became CD25high following stimulation with OVA/DCs or IFN-{alpha}/OVA/DCs. Inclusion of human IL-2 reversed the inhibition of CD25high cell development that occurred when murine IL-2 was neutralized. These results suggested that CD25high cells result from IL-2 signaling and that TLR-activated DCs suppress the development of CD25high cells by suppressing IL-2 signaling. Importantly, only CD25high cells, but not CD25low cells, expressed IL-4 in response to OVA/DCs or IFN-{alpha}/OVA/DCs stimulation (data not shown and Fig. 7D). In contrast, IFN-{gamma} expression was equivalent in CD25high and CD25low populations (Fig. 7D).

TLR-activated DCs fail to inhibit IL-4 production by Th2 polarized cells

Our data show that TLR-activated DCs profoundly suppress early IL4 production by newly activated naive CD4 T cells. To examine the possibility that TLR-activated DCs are additionally capable of inhibiting IL-4 production by polarized Th2 cells, we stimulated 4get/DO.11 T cells with OVA/DCs under Th2 conditions for 6 days and then examined IL-4 production in response to restimulation with OVA/DCs, Pa/OVA/DCs or IFN-{alpha}/OVA/DCs. After culturing in Th2 condition for 6 days, >75% percent of 4get/DO.11 CD4 T cells had expressed GFP and become Th2 polarized. We found that Pa/OVA/DCs were at least as capable as OVA/DCs or IFN-{alpha}/OVA/DCs in restimulating these Th2 cells to make IL-4 (Fig. 8).


Figure 8
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  		FIGURE 8. TLR-activated DCs fail to inhibit IL-4 production by fully committed Th2 cells. 4get/D0.11.10 T cells were stimulated with OVA pulsed IL-12–/– DCs under Th2 condition with rIL-4 and anti-IFN-{gamma} for 6 days. Cells were then harvested, washed and restimulated with OVA, Pa/OVA, or IFN-{alpha}/OVA pulsed IL-12–/– DCs or IL-12–/– DCs without Ag (No Ag) as indicated. After 3 days, cells were harvested and analyzed for IL-4 expression by either directly measuring GFP expression by flow cytometry (A) or by measuring intracellular IL-4 following restimulation with PMA/ionomycin (B). Data shown are from an individual experiment and are typical of results from three experiments. MFI, Mean fluorescence intensity.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In a previous report we established that, independently of promoting Th1 cell development, TLR-activated DCs deliver potent negative signals for Th2 cell development (15). In this report we provide evidence that this negative signal functions by inhibiting early IL-4 production by responding naive CD4 T cells. IL-4 has been shown to play an essential role in driving Th2 cell differentiation both in vitro and in vivo (28). IL-4 promotes Th2 cell development at three different levels: 1) it promotes expression of GATA-3, the transcription factor that orchestrates Th2 commitment (5); 2) it enhances Th2 cell proliferation and survival (29); and 3) it inhibits IFN-{gamma} expression and thereby counteracts the inhibitory effects of IFN-{gamma} on Th2 cell development (30). Consistent with this, in our DC/CD4 T cell cocultures IL-4 is clearly needed for optimal GATA-3 expression and Th2 cell development (Figs. 1 and 4). Moreover, the IL-4 that plays this role is derived from newly activated naive CD4 T cells themselves, consistent with previous findings that IL-4 derived from T cells is sufficient for Th2 cell development (11, 12). By inhibiting the early production of IL-4 by responding CD4 T cells, TLR-activated DCs undermine the Th2 development pathway and effectively prevent the outgrowth of this type of effector cell.

It has been shown recently that naive CD4 T cells produce both IFN-{gamma} and IL-4 very soon after activation (13). This occurs independently of signaling through STAT6/STAT4 (13) and, thus, at this early stage of T cell activation IL-4 and IFN-{gamma} production are not a reflection of the delivery of signals by IL-4 and/or IL-12, but rather would appear to be an inherent component of the CD4 T cell activation program. However, our results indicate that this process is susceptible to regulation by cells of the innate system because it is early IL-4 production, occurring independently of IL-4 signaling (Fig. 1D), that is effectively suppressed in CD4 T cells that are responding to Ag being presented by TLR-activated DCs. The nature of the signals that allow early IL-4 production are not clear, but it is likely that the strength of the activation signal is important in this context, because at high peptide concentrations in the absence of TLR-stimulation we were unable to measure early IL-4/GFP production by 4get/DO11 CD4 T cells, while at lower doses early IL-4 production and Th2 cell commitment were pronounced (Fig. 3, A and B).

TLR activation of DCs increases Ag processing and presentation and up-regulates costimulatory molecule expression (31). Together, these would be expected to result in the delivery of a stronger activation signal to CD4 T cells. Consistent with this, we observed that significantly greater percentages of CD4 T cells became activated when cocultured with TLR-activated, OVA-pulsed DCs rather than with DCs pulsed with OVA alone (Fig. 2). Although it is feasible that this increased strength of activation signal provided by TLR-activated DCs underlies the failure of CD4 T cells to make early IL-4, we believe that additional factors must be at play because, first, TLR-activated DCs can suppress early IL-4 production by CD4 T cells at every Ag dose tested (Fig. 3) and, second, despite the fact that IFN-{alpha}-activated DCs up-regulate MHC II and costimulatory molecule expression and are more effective at activating CD4 T cells than are DCs pulsed with Ag alone, they do not inhibit early IL-4 production or Th2 commitment (Figs. 2 and 3C). These results suggest that unique signals/molecules emanating from TLR-activated DCs, but not from DCs activated by immune system intrinsic signals (such as IFN-{alpha}, IFN-{gamma}, TNF-{alpha}, thymic stromal lymphopoietin (32), etc.) inhibit early IL-4 production by responding CD4 T cells and thus inhibit Th2 cell development. Our data indicating that TLR 2 ligands profoundly inhibit Th2 cell development are contradictory to those findings that have suggested the role of TLR2 ligands in the induction of Th2 responses (20, 33, 34, 35, 36, 37, 38). Nevertheless, there is a large body of published data that is consistent with our findings showing that TLR2 ligands, including Pam3Cys, either promote Th1 responses or inhibit Th2 responses (39, 40, 41, 42, 43, 44, 45, 46, 47). The differences between these two groups of reports are hard to reconcile but may be due to the different experimental settings used. Further studies are needed to clarify the exact physiological roles of TLR2 ligands in Th cell responses during infections. In this setting, however, we should keep in mind that the natural TLR2-stimulating Gram-positive bacteria usually induce Th1 responses but not Th2 responses (31).

T-bet is the master regulator of Th1 development (4). In addition to promoting the expression of lineage-defining cytokine genes, this transcription factor inhibits the expression of Th2 genes (48). For example, under steady state conditions, T-bet deficient mice spontaneously develop asthma, a Th2-cell mediated disease (22). Moreover, T-bet overexpression is able to suppress IL-4 and IL-5 expression in developing and mature Th2 cells through a mechanism that is independent of IFN-{gamma} signaling (48). T-bet expression has also been reported to inhibit early IL-4 production (49). In our hands when stimulated with OVA/DCs, T-bet-deficient OT-II CD4 T cells expressed more IL-4 and more cells committed to Th2 development than was the case with WT CD4 T cells (Fig. 4 and data not shown). Nevertheless, TLR-activated DCs inhibited both early IL-4 production and, to a great extent, Th2 cell development in CD4 T cells that lacked T-bet (Fig. 4). This result emphasizes that the strong negative signal for Th2 cell development delivered by TLR-activated DCs can function independently of the major regulator of Th1 commitment.

GATA-3 in Th2 cells is the transcription factor counterpart of T-bet in Th1 cells (4). GATA-3 has been shown to be necessary for both early IL-4 production and later Th2 cell lineage maintenance (7, 14). GATA-3 expression is induced by both TCR signaling and then further through the IL-4/STAT6 signaling pathway, with the latter being the major pathway through which Th2 commitment is enforced (6). We were able to measure GATA-3 transcripts and protein in equivalent levels in CD4 T cells recently activated by OVA or OVA/Pa-pulsed DCs, suggesting that the inhibition of early IL-4 production by TLR-activated DCs is not a reflection of diminished early GATA-3 expression. Consistent with the importance of IL-4 signaling for Th2 commitment, GATA-3 levels were significantly lower in CD4 T cells that had been stimulated with Pa/OVA/DCs vs OVA/DCs 4 days previously.

IL-2, signaling through STAT5, has been strongly implicated in IL-4 expression and Th2 cell development, and this appears to be due to the importance of STAT5 for early IL-4 production (27). Our finding that an IL-2 blockade in DC/CD4 T cell cocultures impairs early IL-4 production (Fig. 7) is consistent with this. Given the importance of IL-2 signaling in early IL-4 production, we reasoned that the suppression of IL-4 production by TLR-activated DCs could be due to the selective inhibition of this pathway. In support of this view, we found very few pSTAT5+ CD4 T cells in cocultures with TLR-stimulated DCs, whereas distinct pSTAT5+ CD4 T cell populations were evident in cocultures with OVA/DCs or with IFN-{alpha}/OVA/DCs (Fig. 6). The lack of pSTAT5+ cells following activation by Pa/OVA/DCs reflects a failure of IL-2 signaling rather than a failure to produce IL-2 (Fig. 6 and data not shown). We are currently investigating the underlying mechanism for the failure of signaling downstream of the IL-2R in CD4 T cells that have been stimulated by TLR-activated DCs. Others have reported that high doses of peptide Ag inhibit STAT5 phosphorylation and early IL-4 production through the induction of ERK phosphorylation (14), and we are exploring the possibility that alterations in ERK signaling underlie our observations. An alternative explanation is that TLR-activated DCs suppress IL-2 signaling by suppressing expression of the IL-2R{alpha}-chain, CD25, on responding CD4 T cells. Although we did not see reduced overall levels of expression of CD25 on CD4 T cells that would fail to become Th2 cells, we did, as expected, note that pSTAT5 levels in CD4 T cells were tightly linked to the expression levels of CD25, and in our experiments pSTAT5high cells were those exhibiting a CD25high phenotype; CD25highpSTAT5high CD4 T cells were absent in cocultures with TLR-activated DCs. However, IL-2 blockade inhibited the development of CD25high CD4 T cells in cocultures with OVA/DCs or IFN-{alpha}/OVA/DCs, suggesting that the difference in CD25 expression in CD4 T cells stimulated with TLR-activated OVA-pulsed DCs vs with DCs pulsed with Ag alone is the downstream effect of the difference in IL-2 signaling, rather than an effect on IL-2R expression directly (50). These findings indicate that IL-2 signaling is responsible for the further up-regulation of CD25 expression in CD4 T cells after TCR engagement and that TLR-activated DCs selectively inhibit this up-regulation by inhibiting IL-2 signaling. Given the importance of STAT5 in IL-4 transcription and the fact that CD25high cells are those cells with higher pSTAT5 levels, it is thus not surprisingly that the early IL-4 producers are exclusively CD25high cells. This result is in part consistent with previous findings that the level of CD25 expression is significantly higher in developing Th2 cells than in developing Th1 cells during in vitro T cell differentiation (51). Interestingly, CD4 T cells activated by TLR-stimulated DCs proliferate at least as effectively as those activated by DCs pulsed with Ag alone (15), supporting the view that IL-2 signaling through STAT5 phosphorylation is not crucial for CD4 T cell proliferation (27) (data not shown).

Our findings support the following view of Th2 cell differentiation. Naive CD4 T cells activated by DCs that have processed appropriate Ags but have not received a TLR-initiated maturation signal transcribe IFN-{gamma}, IL-4, and GATA-3. In the absence of IL-12, early IL-4 is able to signal via STAT6 to induce up-regulation of GATA-3 and c-maf expression and suppression of IFN-{gamma} expression. The lineage-specific transcription factors GATA-3 and c-maf stabilize the Th2 cytokine locus, transcribe Th2 cytokines, and repress Th1 cytokine expression, thereby consolidating commitment to the Th2 lineage. Consistent with the view that lineage commitment involves stable epigenetic modifications to signature cytokine gene loci (52), we have found that TLR-activated DCs cannot prevent IL-4 production by committed Th2 cells (Fig. 8). In contrast, DCs that have processed Ag in the context of TLR-initiated signals favor Th1 response initiation by not only providing IL-12, which promotes IFN-{gamma} expression to facilitate T-bet expression (53), represses GATA-3 and Th2 cytokine expression (5), and promotes the growth and survival of IFN-{gamma} producing cells (54), but also, as described here, by inhibiting Th2 cell development. This block of Th2 development is not a consequence of commitment of the Th cells to Th1 differentiation, because it can occur in the absence of IL-12 or T-bet. Rather, it reflects an inability of the responding cells to respond to autocrine/paracrine IL-2 and up-regulate early IL-4 production, a process that was only recently recognized as being important for Th2 differentiation (14). The rapid production of IL-4 before cell division and the subsequent responsiveness to IL-4 are essential for CD4 T cells to increase the GATA-3 expression initiated by TCR/CD28 ligation and to commit to the Th2 lineage.

Our data show that the factors that regulate early IL-4 production are subject to tight regulation by DCs and provide an explanation for why, under specific conditions of infection in the absence of IL-12 or T-bet, there is no default Th2 response (8, 9, 10, 55). Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth. We believe that understanding how TLR-activated DCs are able to accomplish this will help the development of new therapies for asthma, allergy, and Th2-mediated autoimmune pathologies.


   Acknowledgments
 
We thank Markus Mohrs for 4get mice and advice, Euihye Jung for technical assistance, and Yonwong Choi, Christopher Hunter, Phillip Scott, Andrew Wells, and members of the laboratory for constructive discussion. E.J.P. is the recipient of a Burroughs Wellcome Fund Scholar in Molecular Parasitology Award.


   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 a National Institutes of Health Grant AI53825 to (E.J.P.). Back

2 Address correspondence and reprint requests to Dr. Edward J. Pearce, University of Pennsylvania, 216 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: ejpearce{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; Pa, Propionibacterium acnes; Pam3Cys, S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH,trihydrochloride; PGN, peptidoglycan; poly(I:C), polyinosinic/polycytidylic acid; pSTAT5, phosphorylated STAT5. Back

Received for publication June 27, 2006. Accepted for publication November 14, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Moser, M., K. M. Murphy. 2000. Dendritic cell regulation of TH1-TH2 development. Nat. Immunol. 1: 199-205. [Medline]
  2. Kapsenberg, M. L.. 2003. Dendritic-cell control of pathogen-driven T-cell polarization. Nat. Rev. Immunol. 3: 984-993. [Medline]
  3. Trinchieri, G.. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat. Rev. Immunol. 3: 133-146. [Medline]
  4. Murphy, K. M., S. L. Reiner. 2002. The lineage decisions of helper T cells. Nat. Rev. Immunol. 2: 933-944. [Medline]
  5. Ouyang, W., S. H. Ranganath, K. Weindel, D. Bhattacharya, T. L. Murphy, W. C. Sha, K. M. Murphy. 1998. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 9: 745-755. [Medline]
  6. Mowen, K. A., L. H. Glimcher. 2004. Signaling pathways in Th2 development. Immunol. Rev. 202: 203-222. [Medline]
  7. Zhu, J., B. Min, J. Hu-Li, C. J. Watson, A. Grinberg, Q. Wang, N. Killeen, J. F. Urban, Jr, L. Guo, W. E. Paul. 2004. Conditional deletion of Gata3 shows its essential function in Th1-Th2 responses. Nat. Immunol. 5: 1157-1165. [Medline]
  8. Schijns, V. E., B. L. Haagmans, C. M. Wierda, B. Kruithof, I. A. Heijnen, G. Alber, M. C. Horzinek. 1998. Mice lacking IL-12 develop polarized Th1 cells during viral infection. J. Immunol. 160: 3958-3964. [Abstract/Free Full Text]
  9. Brombacher, F., A. Dorfmuller, J. Magram, W. J. Dai, G. Kohler, A. Wunderlin, K. Palmer-Lehmann, M. K. Gately, G. Alber. 1999. IL-12 is dispensable for innate and adaptive immunity against low doses of Listeria monocytogenes. Int. Immunol. 11: 325-332. [Abstract/Free Full Text]
  10. Jankovic, D., M. C. Kullberg, S. Hieny, P. Caspar, C. M. Collazo, A. Sher. 2002. In the absence of IL-12, CD4+ T cell responses to intracellular pathogens fail to default to a Th2 pattern and are host protective in an IL-10(–/–) setting. Immunity 16: 429-439. [Medline]
  11. Schmitz, J., A. Thiel, R. Kuhn, K. Rajewsky, W. Muller, M. Assenmacher, A. Radbruch. 1994. Induction of interleukin 4 (IL-4) expression in T helper (Th) cells is not dependent on IL-4 from non-Th cells. J. Exp. Med. 179: 1349-1353. [Abstract/Free Full Text]
  12. Noben-Trauth, N., J. Hu-Li, W. E. Paul. 2000. Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation. J. Immunol. 165: 3620-3625. [Abstract/Free Full Text]
  13. Grogan, J. L., M. Mohrs, B. Harmon, D. A. Lacy, J. W. Sedat, R. M. Locksley. 2001. Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets. Immunity 14: 205-215. [Medline]
  14. Yamane, H., J. Zhu, W. E. Paul. 2005. Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment. J. Exp. Med. 202: 793-804. [Abstract/Free Full Text]
  15. Sun, J., M. Walsh, A. V. Villarino, L. Cervi, C. A. Hunter, Y. Choi, E. J. Pearce. 2005. TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development. J. Immunol. 174: 742-751. [Abstract/Free Full Text]
  16. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 77-92. [Medline]
  17. Bocek, P., Jr, G. Foucras, W. E. Paul. 2004. Interferon {gamma} enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production. J. Exp. Med. 199: 1619-1630. [Abstract/Free Full Text]
  18. Kamogawa, Y., L. A. Minasi, S. R. Carding, K. Bottomly, R. A. Flavell. 1993. The relationship of IL-4- and IFN {gamma}-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 75: 985-995. [Medline]
  19. Mohrs, M., K. Shinkai, K. Mohrs, R. M. Locksley. 2001. Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15: 303-311. [Medline]
  20. Dillon, S., A. Agrawal, T. Van Dyke, G. Landreth, L. McCauley, A. Koh, C. Maliszewski, S. Akira, B. Pulendran. 2004. A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells. J. Immunol. 172: 4733-4743. [Abstract/Free Full Text]
  21. Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, A. O’Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197: 101-109. [Abstract/Free Full Text]
  22. Finotto, S., M. F. Neurath, J. N. Glickman, S. Qin, H. A. Lehr, F. H. Green, K. Ackerman, K. Haley, P. R. Galle, S. J. Szabo, J. M. Drazen, G. T. De Sanctis, L. H. Glimcher. 2002. Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science 295: 336-338. [Abstract/Free Full Text]
  23. Minter, L. M., D. M. Turley, P. Das, H. M. Shin, I. Joshi, R. G. Lawlor, O. H. Cho, T. Palaga, S. Gottipati, J. C. Telfer, L. Kostura, A. H. Fauq, K. Simpson, K. A. Such, L. Miele, T. E. Golde, S. D. Miller, B. A. Osborne. 2005. Inhibitors of {gamma}-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat. Immunol. 6: 680-688. [Medline]
  24. Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, A. Antonellis, G. A. Koretzky, K. Gardner, P. L. Schwartzberg. 2004. SAP regulates Th2 differentiation and PKC-{theta}-mediated activation of NF-{kappa}B1. Immunity 21: 693-706. [Medline]
  25. Rengarajan, J., S. J. Szabo, L. H. Glimcher. 2000. Transcriptional regulation of Th1/Th2 polarization. Immunol. Today 21: 479-483. [Medline]
  26. Zhu, J., J. Cote-Sierra, L. Guo, W. E. Paul. 2003. Stat5 activation plays a critical role in Th2 differentiation. Immunity 19: 739-748. [Medline]
  27. Cote-Sierra, J., G. Foucras, L. Guo, L. Chiodetti, H. A. Young, J. Hu-Li, J. Zhu, W. E. Paul. 2004. Interleukin 2 plays a central role in Th2 differentiation. Proc. Natl. Acad. Sci. USA 101: 3880-3885. [Abstract/Free Full Text]
  28. Seder, R. A., W. E. Paul. 1994. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu. Rev. Immunol. 12: 635-673. [Medline]
  29. Zhu, J., L. Guo, B. Min, C. J. Watson, J. Hu-Li, H. A. Young, P. N. Tsichlis, W. E. Paul. 2002. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 16: 733-744. [Medline]
  30. Tanaka, T., J. Hu-Li, R. A. Seder, B. Fazekas de St Groth, W. E. Paul. 1993. Interleukin 4 suppresses interleukin 2 and interferon {gamma} production by naive T cells stimulated by accessory cell-dependent receptor engagement. Proc. Natl. Acad. Sci. USA 90: 5914-5918. [Abstract/Free Full Text]
  31. Iwasaki, A., R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987-995. [Medline]
  32. Zhou, B., M. R. Comeau, T. De Smedt, H. D. Liggitt, M. E. Dahl, D. B. Lewis, D. Gyarmati, T. Aye, D. J. Campbell, S. F. Ziegler. 2005. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat. Immunol. 6: 1047-1053. [Medline]
  33. Pulendran, B., P. Kumar, C. W. Cutler, M. Mohamadzadeh, T. Van Dyke, J. Banchereau. 2001. Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J. Immunol. 167: 5067-5076. [Abstract/Free Full Text]
  34. van der Kleij, D., E. Latz, J. F. Brouwers, Y. C. Kruize, M. Schmitz, E. A. Kurt-Jones, T. Espevik, E. C. de Jong, M. L. Kapsenberg, D. T. Golenbock, et al 2002. A novel host-parasite lipid cross-talk: schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization. J. Biol. Chem. 277: 48122-48129. [Abstract/Free Full Text]
  35. Agrawal, S., A. Agrawal, B. Doughty, A. Gerwitz, J. Blenis, T. Van Dyke, B. Pulendran. 2003. Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J. Immunol. 171: 4984-4989. [Abstract/Free Full Text]
  36. van der Kleij, D., A. H. van den Biggelaar, Y. C. Kruize, K. Retra, Y. Fillie, M. Schmitz, P. G. Kremsner, A. G. Tielens, M. Yazdanbakhsh. 2004. Responses to Toll-like receptor ligands in children living in areas where schistosome infections are endemic. J. Infect. Dis. 189: 1044-1051. [Medline]
  37. Chisholm, D., L. Libet, T. Hayashi, A. A. Horner. 2004. Airway peptidoglycan and immunostimulatory DNA exposures have divergent effects on the development of airway allergen hypersensitivities. J. Allergy Clin. Immunol. 113: 448-454. [Medline]
  38. Redecke, V., H. Hacker, S. K. Datta, A. Fermin, P. M. Pitha, D. H. Broide, E. Raz. 2004. Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J. Immunol. 172: 2739-2743. [Abstract/Free Full Text]
  39. Akdis, C. A., F. Kussebi, B. Pulendran, M. Akdis, R. P. Lauener, C. B. Schmidt-Weber, S. Klunker, G. Isitmangil, N. Hansjee, T. A. Wynn, et al 2003. Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides. Eur. J. Immunol. 33: 2717-2726. [Medline]
  40. Sieling, P. A., W. Chung, B. T. Duong, P. J. Godowski, R. L. Modlin. 2003. Toll-like receptor 2 ligands as adjuvants for human Th1 responses. J. Immunol. 170: 194-200. [Abstract/Free Full Text]
  41. Weigt, H., P. F. Muhlradt, M. Larbig, N. Krug, A. Braun. 2004. The Toll-like receptor-2/6 agonist macrophage-activating lipopeptide-2 cooperates with IFN-{gamma} to reverse the Th2 skew in an in vitro allergy model. J. Immunol. 172: 6080-6086. [Abstract/Free Full Text]
  42. Zhu, X., T. V. Ramos, H. Gras-Masse, B. E. Kaplan, L. BenMohamed. 2004. Lipopeptide epitopes extended by an Nepsilon-palmitoyl-lysine moiety increase uptake and maturation of dendritic cells through a Toll-like receptor-2 pathway and trigger a Th1-dependent protective immunity. Eur. J. Immunol. 34: 3102-3114. [Medline]
  43. Watanabe, T., A. Kitani, P. J. Murray, W. Strober. 2004. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat. Immunol. 5: 800-808. [Medline]
  44. Revets, H., G. Pynaert, J. Grooten, P. De Baetselier. 2005. Lipoprotein I, a TLR2/4 ligand modulates Th2-driven allergic immune responses. J. Immunol. 174: 1097-1103. [Abstract/Free Full Text]
  45. Patel, M., D. Xu, P. Kewin, B. Choo-Kang, C. McSharry, N. C. Thomson, F. Y. Liew. 2005. TLR2 agonist ameliorates established allergic airway inflammation by promoting Th1 response and not via regulatory T cells. J. Immunol. 174: 7558-7563. [Abstract/Free Full Text]
  46. Visser, L., H. Jan de Heer, L. A. Boven, D. van Riel, M. van Meurs, M. J. Melief, U. Zahringer, J. van Strijp, B. N. Lambrecht, E. E. Nieuwenhuis, J. D. Laman. 2005. Proinflammatory bacterial peptidoglycan as a cofactor for the development of central nervous system autoimmune disease. J. Immunol. 174: 808-816. [Abstract/Free Full Text]
  47. Bafica, A., C. A. Scanga, C. G. Feng, C. Leifer, A. Cheever, A. Sher. 2005. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 202: 1715-1724. [Abstract/Free Full Text]
  48. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669. [Medline]
  49. Miller, A. T., H. M. Wilcox, Z. Lai, L. J. Berg. 2004. Signaling through Itk promotes T helper 2 differentiation via negative regulation of T-bet. Immunity 21: 67-80. [Medline]
  50. Nakajima, H., X. W. Liu, A. Wynshaw-Boris, L. A. Rosenthal, K. Imada, D. S. Finbloom, L. Hennighausen, W. J. Leonard. 1997. An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor {alpha} chain induction. Immunity 7: 691-701. [Medline]
  51. Hwang, E. S., I. A. White, I. C. Ho. 2002. An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines. Proc. Natl. Acad. Sci. USA 99: 13026-13030. [Abstract/Free Full Text]
  52. Ansel, K. M., D. U. Lee, A. Rao. 2003. An epigenetic view of helper T cell differentiation. Nat. Immunol. 4: 616-623. [Medline]
  53. Afkarian, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang, T. L. Murphy, K. M. Murphy. 2002. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat. Immunol. 3: 549-557. [Medline]
  54. 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, S. L. Reiner. 2001. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science 292: 1907-1910. [Abstract/Free Full Text]
  55. Way, S. S., C. B. Wilson. 2004. Cutting edge: immunity and IFN-{gamma} production during Listeria monocytogenes infection in the absence of T-bet. J. Immunol. 173: 5918-5922. [Abstract/Free Full Text]



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