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Proc Natl Acad Sci U S A. Oct 23, 2007; 104(43): 17034–17039.
Published online Oct 17, 2007. doi:  10.1073/pnas.0708426104
PMCID: PMC2040448
Immunology

Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes

Abstract

Recently, a new lineage of CD4+ T cells has been described in the mouse that specifically secretes IL-17 [T helper (Th) 17]. This discovery has led to a revision of the hypothesis that many autoimmune diseases are predominantly a Th1 phenomenon and may instead be critically dependent on the presence of Th17 cells. Murine Th17 cells differentiate from naïve T cell precursors in the presence of TGF-β and IL-6 or IL-21. However, given their putative importance in human autoimmunity, very little is known about the pathways that control the expression of IL-17 in humans. Here we show that the factors that determine the expression of IL-17 in human CD4+ T cells are completely different from mice. IL-6 and IL-21 were unable to induce IL-17 expression in either naïve or effector T cells, and TGF-β actually inhibited IL-17 expression. The expression of IL-17 was maximally induced from precommitted precursors present in human peripheral blood by cell–cell contact with Toll-like receptor-activated monocytes in the context of T cell receptor ligation. Furthermore, unlike IFN-γ, IL-17 expression was not suppressed by the presence of FOXP3+ regulatory CD4+ T cells. Taken together, these data indicate that human and mouse Th17 cells have important biological differences that may be of critical importance in the development of therapeutic interventions in diseases characterized by aberrant T cell polarization.

Keywords: autoimmunity, IL-17, T lymphocytes

It has been shown by several groups that murine naïve T cells (T helper precursors, Thp) can be induced to secrete IFN-γ [T helper (Th) 1], IL-4 (Th2), or IL-17 (Th17) in a mutually exclusive manner. The differentiation of both mouse and human Th1 and Th2 cells has been elucidated previously with IL-4 signaling through STAT-6, the critical cytokine in inducing Th2 cell differentiation (1), and IL-12 signaling via STAT-4, the central cytokine for commitment toward the Th1 lineage (2). Recently it was demonstrated that TGF-β and dendritic cell-derived IL-6 induce murine Thp to undergo commitment to the Th17 lineage in vitro (35). Exposure of Thp to TGF-β in the absence of IL-6 induces the generation of a regulatory T cell (Treg) population that is characterized by the surface expression of CD4 and CD25 and the expression of the transcription factor Foxp3 (6, 7). However, so far it remains to be established whether a similar Th17 differentiation process occurs in humans.

Lineage-defining transcription factors have been identified in Th1 and Th2 cells in both mouse and human. T-bet acts as an important regulator of Th1 lineage commitment, and deficiency of this transcription factor in mice causes resistance to many autoimmune diseases and susceptibility to infectious diseases (810). It was recently suggested that IL-17 production is enhanced by T-bet absence and IL-4 blockade, when cells are stimulated in the presence of accessory cells (11). However, it is unresolved whether this is a direct effect of T-bet or a consequence of the reduced IFN-γ production observed in T-bet−/− T cells. The generation and survival of Th2 cells are controlled by GATA-3, although loss-of-function experiments in vivo have been limited because of the fact that deletion is embryonic-lethal and that it has a nonredundant role in T cell development (1214). Foxp3 has been shown to be critical in the generation and function of Tregs and is specifically expressed in this cell type in mice (15). Loss-of-function mutations in Foxp3 cause immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome in humans (16). However, in humans Foxp3 expression is not entirely restricted to Tregs, because it is also expressed in activated CD4+ T cells (17). Recently the transcription factor RORγT has been implicated in the generation of both murine (18) and human (19, 20) IL-17+ cells.

Given that IL-17 is a cytokine produced in a number of human autoimmune and inflammatory diseases (reviewed in refs. 21 and 22) and that important differences exist between human and murine effector T cell differentiation, we sought to determine the pathways that lead to the generation of IL-17 production in human CD4+ T cells. Our results demonstrate that in humans naïve T cell stimulation in the presence of IL-6 and TGF-β does not lead to Th17 differentiation. Instead, large numbers are readily induced by stimulating CD4+CD45RO+ T cells through the T cell receptor in the presence of Toll-like receptor (TLR)-activated monocytes. Importantly, CD4+CD25+FoxP3+ Tregs, which are known inhibitors of IFN-γ-producing Th1 cells, do not suppress IL-17 production and in fact induce an increase in the percentage of IL-17+ CD4+ T cells.

Results

Optimal Th17 Induction in Human CD4+ T Cells Requires Cell Contact with Activated Monocytes and Is Inhibited by the Addition of TGF-β.

To investigate whether Th17 cells could be induced in humans, CD4+ T cells were stimulated under various conditions in the absence or presence of IL-6 and TGF-β at different time points. In the presence of monocytes (irradiated, live, or LPS-activated) distinct populations of IL-17+ and IFN-γ+ cells were induced, as were, interestingly, some double positive cells (Fig. 1A). The highest percentage of IL-17+ cells was found when T cells were stimulated in the presence of LPS-activated monocytes. Addition of IL-6 and TGF-β to the cultures consistently led to a decrease of IL-17+ as well as IFN-γ+ cells. Although IL-17+ T cells could be produced in a monocyte-free system using anti-CD3/CD28 mAb stimulation (Fig. 1A and data not shown), the percentage was substantially higher in the presence of monocytes. No positive staining for IL-17 in monocytes was observed (data not shown). We also stimulated CD4+ T cells with allogeneic mature dendritic cells in the absence or presence of IL-6 and TGF-β. No induction of IL-17+ T cells [supporting information (SI) Fig. 5] or IL-17 production in supernatants (data not shown) were observed in these conditions, suggesting that the type or maturation stage of antigen-presenting cells (APC) may be important for Th17 induction in humans.

Fig. 1.
Th17 induction in human CD4+ T cells is promoted in the presence of monocytes but inhibited by the addition of IL-6 and TGF-β. (A) Bulk CD4+ T cells (1 × 106 per well) were cultured with anti-CD3/CD28 microbeads or with a 1:1 ratio of ...

To determine whether the IL-17-producing cells were derived from the naïve or memory T cell population, CD4+ T cells were separated into CD45RA+ and CD45RO+ T cells. When the naïve CD4+ T cell population was stimulated in the presence of anti-CD3 mAb with either LPS-activated monocytes or IL-6 and TGF-β, very few IL-17+ T cells (<1%) were found (Fig. 1B). Long-term repetitive stimulation (up to 3 weeks) of naïve T cells with either LPS-activated monocytes and anti-CD3 or immobilized anti-CD3/CD28 mAbs did not lead to significantly enhanced IL-17 expression, nor did experiments using human cord blood CD4+ T cells yield significant Th17 induction (data not shown). In contrast, coculture of memory CD4+ T cells with LPS-activated monocytes led to a distinct IL-17+ T cell population of ≈9%, which was dramatically reduced to <1% by the addition of IL-6 and TGF-β (Fig. 1B) (P = 0.03). This was coupled to a decrease in IL-17 production in the supernatants (data not shown). Additional experiments demonstrated that these effects were due to TGF-β rather than IL-6 (SI Fig. 6). Addition of exogenous IL-21 (up to 100 ng/ml) in either the monocyte/LPS or APC-free system did not lead to enhancement of IL-17+ cells, as was recently shown in mice (2325), further highlighting differences between the mouse and human system (data not shown).

To investigate whether the IL-17+ cells were induced from a noncommitted CD4+ memory T cell population or reflected simply an expansion of a preexisting population, we isolated CD4+CD45RO+ T cells from peripheral blood and measured the baseline IFN-γ and IL-17 expression levels directly ex vivo (Fig. 1C). On average 11% of the cells were IFN-γ+ (n = 5), 5.4% were IL-4+ (n = 2; data not shown), but only 0.4% were IL-17+ (n = 5). This makes expansion of preexisting IL-17+ T cells an unlikely scenario given that the average percentage of IL-17+ T cells at day 3 was 8–10% and the final cell count was comparable in all of the culture conditions (data not shown); this would imply four to five rounds of divisions in 3 days, which is not supported by general cell proliferation kinetics on memory T cells. We therefore favor the explanation that the IL-17+ T cells are newly induced from uncommitted memory T cells or, as suggested by the presence of the IFN-γ+/IL-17+ population (Fig. 1), from (semi) committed Th1 cells.

To investigate how critical the addition of monocytes and/or LPS was for the induction of IL-17+ T cells, memory or naive T cells were activated without or with monocytes in the absence or presence of LPS. Similar to the data in Fig. 1, only very low levels of IL-17+ T cells were generated from naïve T cells (Fig. 2A). When memory T cells were stimulated with anti-CD3 mAb in the absence of monocytes, <0.5% IL-17+ T cells were generated, and this was not substantially enhanced by the addition of LPS. In contrast, the addition of monocytes clearly increased the percentage (3 ± 1%), and this was further enhanced by the addition of LPS (7 ± 1%) (Fig. 2A). LPS was not the only TLR ligand to induce this effect, because monocytes activated by other TLR ligands also had enhancing effects on the percentage of IL-17+ cells and/or IL-17 production (Fig. 2C).

Fig. 2.
Optimal Th17 induction in human CD4+ T cells requires cell contact with activated monocytes. Naïve or memory T cells were cultured for 3 days in the presence of anti-CD3 under various conditions and then stained as described in Fig. 1. (A) T cells ...

We next investigated whether the induction of the IL-17+ pool depended on cell contact with monocytes by performing transwell assays in which monocytes were cultured in the upper well and memory T cells in the lower well. The number of IL-17+ cells was significantly decreased when cell contact between monocytes and memory T cells was prevented (from 9 ± 2% to 2 ± 0.3%) (Fig. 2B). The requirement for cell contact suggested that the increase in IL-17+ cells in the presence of activated monocytes could not be solely attributed to monocyte/macrophage-derived cytokines such as IL-23 and/or IL-1β, as suggested recently (20, 2628). To further investigate this we cultured memory CD4+ T cells with monocytes, LPS, and anti-CD3 mAb in the absence or presence of additional human recombinant (hr) IL-23 (10 ng/ml) for 3 days. No increase in either the percentage of IL-17+ cells or IL-17 production was observed during this short-term culture period (SI Fig. 7). However, in longer-term cultures with IL-23 (>5 days) an increase in the number of IL-17+ cells was observed (data not shown), and so we do not exclude the possibility IL-23 may promote survival or expansion of IL-17+ T cells during longer-term cultures. We also tested whether the addition of supernatants taken from activated monocyte cultures could induce IL-17-producing cells, but we found that this was not effective in inducing IL-17+ T cells in the memory cells (data not shown). Together these data indicate that the induction of Th17 cells is not solely cytokine-dependent but rather requires cell contact with activated monocytes.

Th1 vs. Th2 Skewing Conditions Have Opposite Effects on the Induction of Human Th17 Cells.

To further investigate which conditions may affect the conversion of memory CD4+ T cells to IL-17+ cells in humans, we cultured CD4+CD45RO+ and CD4+CD45RA+ T cells with monocytes, anti-CD3, and LPS under Th1 (hrIL-12 and anti-IL-4 mAb) and Th2 (hrIL-4 and anti-IFN-γ mAb) skewing conditions. After 3 days, the presence of IL-17- and IFN-γ-producing cells was measured by intracellular cytokine staining and ELISA. Interestingly, the percentage of IL-17+ cells in CD45RO+ T cells was decreased under Th1 conditions but increased under Th2 conditions (Fig. 3A). Although only very small percentages of IL-17+ T cells were generated from naïve T cells, in agreement with the data in Figs. 1 and and2,2, we did observe a similar pattern with Th1 conditions consistently decreasing (from 0.06 ± 0.01 to 0.03 ± 0.01%) and Th2 conditions increasing the percentage of IL-17+ T cells (from 0.06 ± 0.01 to 0.16 ± 0.9; n = 3). Fig. 3 B and C shows the average of n = 7 and n = 6 experiments, respectively, using CD45RO+ cells and confirms that Th1 skewing conditions significantly inhibited IL-17+ T cells with a concomitant reduction in IL-17 protein as measured in the cell culture supernatant, whereas Th2 skewing had the opposite effect. This was confirmed at the mRNA level by real-time PCR (Fig. 3D). The percentage of IFN-γ/IL-17 double positive cells (3.4 ± 0.7%) was also down-regulated by Th1 conditions (2.2 ± 0.7%) but maintained under Th2 conditions (3.4 ± 0.6%) (n = 7; data not shown). As expected, Th1 skewing significantly increased IFN-γ at the intracellular as well as the secreted protein level, whereas Th2 skewing significantly down-regulated this cytokine. To demonstrate that the effect on IL-17 induction was not caused by the actions of Th1/Th2 conditions on monocytes, an APC-free environment was used with plate-bound anti-CD3 and anti-CD28 mAb. Although lower levels of Th17 cells were found with this system, the same pattern in the effects of Th1/Th2 skewing on IL-17 was observed (SI Fig. 8A). In addition, using this system we confirmed that Th1 vs. Th2 skewing induced T-bet vs. GATA-3 mRNA expression, respectively (SI Fig. 8B). Together these data indicate that Th1 skewing blocks Th17 induction whereas Th2 skewing conditions promote the generation of IL-17+ T cells.

Fig. 3.
Th1 vs. Th2 skewing conditions have opposite effects on the induction of human Th17 cells. Memory CD4+ T cells (0.5 × 106 cells per well) were cultured with anti-CD3, monocytes, and LPS in the absence or presence of either Th1 (hrIL-12 plus anti-IL-4 ...

To elucidate whether the increase in IL-17+ T cells under Th2 conditions was due to IFN-γ blockade or the addition of IL-4, CD45RO+CD4+ T cells were cultured with monocytes, LPS, and anti-CD3, or in the APC-free system, in the presence of anti-IFN-γ or IL-4, or both. Relative to the Th2 conditions where both IL-4 and anti-IFN-γ were added, the addition of IL-4 alone consistently reduced the number of IL-17+ cells, whereas the addition of anti-IFN-γ increased IL-17 production and secretion (Fig. 3E and data not shown). The addition of other Th2 cytokines (IL-5 or IL-13) did not increase the induction of Th-17 cells in either system (data not shown). Together these data demonstrate that IFN-γ blockade rather than the promotion of Th2 cells via IL-4, IL-5, or IL-13 addition is responsible for the induction of Th17 cells.

CD4+CD25+Foxp3+ Tregs Promote the Induction of IL-17+ T Cells by Suppressing Th1 Responses.

The results so far indicated that Th17 induction in humans can occur when memory CD4+ T cells come into contact with activated monocytes and can be enhanced when IFN-γ signaling is blocked. One cell type known to efficiently suppress IFN-γ is the CD4+CD25+Foxp3+ Treg population (29, 30). We therefore wanted to investigate the effects of Tregs on Th17 induction in humans. CD4+CD25+Foxp3+ Tregs were isolated by MACS (SI Fig. 9) and labeled with CFSE or the far red dye DDAO to exclude them from our FACS analysis when measuring intracellular IL-17 production in the effector memory T cells. Fig. 4 shows that the addition of CD4+CD25+Foxp3+ Tregs suppressed the production of IFN-γ but did not down-regulate the production of IL-17. Instead, the addition of Tregs resulted in a small but significant increase in the number of IL-17+ T cells in the responder cells (on average a 19% increase; n = 8) (Fig. 4 C and D and SI Fig. 10).

Fig. 4.
CD4+CD25+Foxp3+ Tregs promote the induction of IL-17+ T cells by suppressing Th1 responses. Memory CD4+ T cells (0.5 × 106 cells per well) were cultured with anti-CD3, monocytes, and LPS in the absence or presence of CFSE- or DDAO-labeled CD4 ...

Discussion

Although the role of Th17 cells in human disease is not yet firmly established, experimental data from animal models of inflammation and autoimmunity indicate that these cells are critical players in immune-mediated pathology. Understanding the generation of these cells is therefore vitally important. Our report indicates that the generation of Th17 cells from naïve CD4+ T cells in humans is not readily achieved by using protocols recently described for mouse Th17 cells (35, 2325, 31). We tested several different stimulation conditions, including multiple rounds of stimulation, but the percentage of IL-17+ cells derived from human naïve CD4+ T cells never reached substantial levels (<2%) whether or not IL-6/TGF-β or IL-21 was added, as shown in mice. Instead we observed profound induction of IL-17+ cells (up to 28%) when memory T cells were stimulated with anti-CD3 mAb in the presence of monocytes that were triggered via TLR ligands. Because <0.5% of CD4+ T cells were found to be IL-17+ directly ex vivo it seems unlikely that this IL-17+ population resulted from expansion of preexisting Th17 cells. Monocytes alone are enough to drive this population, but activation of the monocyte with TLR ligands increases the yield dramatically, as was also shown in a recent paper (20). Our data show that LPS acts directly on the monocyte, which in turn facilitates, via cell contact, the induction of a Th17 population. The requirement for cell contact suggests that the increase in IL-17+ cells in the presence of activated monocytes is not solely due to monocyte/macrophage-derived cytokines such as IL-23, IL-6, and/or IL-1β, as suggested recently (20, 28). Importantly, addition of TGF-β dramatically reduced the percentage of IL-17+ cells in the memory T cell pool, which confirms recent reports that in humans this cytokine, rather than being essential, may in fact inhibit Th17 induction (20, 28).

We examined how Th1 or Th2 skewing conditions influence the presence of Th17 cells in humans and found that Th1 skewing decreases Th17 induction, suggesting that in humans Th1 and Th17 cells are inversely correlated, which is supported by their different chemokine receptor profiles (19, 32). We also found that IL-17+ cells are increased in a Th2 environment, which is predominantly because of blocking IFN-γ rather than the induction of Th2 cells via IL-4 (or other Th2 cytokines). Because our data show that the induction of human Th17 cells is much more efficient using memory than naïve CD4+ T cells, it is possible that many of these cells arise from precommitted T cells. Indeed, T cell polarity is flexible in human CD4+ T cells (33), raising the possibility that blockade of IFN-γ signaling in the presence of LPS-activated monocytes is causing a switch in lineage commitment from previously committed effector cells. This hypothesis is supported by the presence of a substantial percentage of IFN-γ/IL-17 double positive cells in humans (Fig. 1) (20, 34), which suggests that Th1 and Th17 cells share a common pathway.

IL-17 has been suggested to play a detrimental role in inflammatory disease, particularly experimental and rheumatoid arthritis (reviewed in refs. 21 and 22). IL-17 is found in the synovium and synovial fluid of rheumatoid arthritis patients where it is thought to act as a major contributor to tissue inflammation and bone destruction via its stimulatory effects on proinflammatory mediators, angiogenesis, neutrophil maturation and recruitment, and osteoclastogenesis. Interestingly, synovial tissue and fluid of inflamed joints contain a high number of activated monocytes/macrophages as well as memory T cells (35, 36). These conditions are very similar to our in vitro culture system, where we found the highest induction of IL-17+ cells, i.e., when human memory T cells were stimulated in the presence of activated monocytes. In addition to activated monocytes and memory T cells, a high percentage of functional CD4+CD25+Foxp3+ Tregs is found in the synovial tissue and/or fluid of rheumatoid arthritis patients (3739). Because these cells are known to be potent suppressors of T cell proliferation, IL-2, TNF-α, and IFN-γ production (40, 41), these findings have led to the puzzling question as to why inflammation persists despite the increased presence of functional Tregs. Our results may offer an explanation for this conundrum because we show that Tregs are unable to regulate Th17 cells: in the presence of Tregs, IL-17 production is maintained and the percentage of IL-17+ T cells is enhanced, whereas the production of IFN-γ is suppressed. This is not because of the kinetics of IL-17 vs. IFN-γ production because IL-17+ cells appear after IFN-γ+ cells (data not shown), making it unlikely that the observed effect was due to a temporal disparity. Importantly, this finding suggests that CD4+CD25+Foxp3+ Tregs in an ongoing inflammatory context may contribute to tissue inflammation rather than resolve it, and this may in part explain the paradoxical finding that human autoimmunity persists despite the presence of CD4+CD25+Foxp3+ Tregs at the site of inflammation (3739, 42, 43). Naturally this may have important implications for therapies aimed at transferring or inducing Tregs in chronic inflammatory disease. Indeed, a recent study in mice showed that cotransfer of CD25+ Tregs at the disease induction stage resulted in complete disease prevention due to suppression of pathogenic T cell priming and expansion, but this was not successful when Tregs were transferred at a later stage. Strikingly, similar to our experiments, no suppression of IL-17 production was observed, but rather an increase in IL-17+ T cells was seen (44). These data illustrate that the proinflammatory effects of IL-17 can persist even in the presence of functional Tregs and that Treg transfer in established autoimmune disease in humans may be of limited success. It remains to be determined whether Th17 cells in humans are intrinsically resistant to suppression by CD4+CD25+ Tregs and/or whether Tregs actively promote Th17 generation.

In conclusion, we demonstrate that optimal Th17 induction in humans requires T cell receptor ligation in the context of TLR-activated monocytes. Th17 induction is further increased by blockade of IFN-γ signaling, which may occur either through a Th2 environment or via the suppressive effects of CD4+CD25+Foxp3+ Tregs on Th1 cells. Together these findings highlight the delicate balance between inflammation and regulation and indicate that dampening Th1 cells or promoting Treg activity in established disease may exacerbate, rather than reduce, inflammation due to concomitant effects on IL-17-producing cells. Understanding the pathways that regulate the Th17 subset in humans, particularly where this differs from those operative in mice, is clearly critical for the rational translation of these findings into safe and effective human therapeutics.

Materials and Methods

Peripheral Blood Cell Isolation.

Ethical approval for the use of peripheral blood from healthy donors was obtained from the King's College Research Ethics Committee. After informed consent peripheral blood mononuclear cells were isolated by using density gradient centrifugation (lymphocyte separation media; PAA, Pasching, Austria). Purification of cell subsets was performed by magnetic cell sorting (Miltenyi Biotec, Bergisch-Gladbach, Germany) and confirmed by flow cytometry. Monocytes (>90% pure) were isolated by positive selection using anti-CD14 microbeads. CD4+CD25hi Tregs were isolated from CD4+ T cells (99% pure) using the CD4+CD25+ Regulatory T Cell Isolation Kit and magnetic separation over two consecutive MS selection columns, resulting in >95% purity of CD4+CD25+ T cells with >90% Foxp3+ expression (SI Fig. 9). Memory CD4+CD25 T cells contained in the effluent were positively selected via CD45RO+ microbeads (>85% pure). The remaining naïve T cell population was further depleted of any remaining CD45RO+ cells by a second depletion round (>99% CD45RA+ T cells).

Cell Culture.

Bulk CD4+ T cells (1 × 106 per milliliter) were cultured in 24-well plates in the absence or presence of monocytes in RPMI medium 1640 supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% heat-inactivated FCS (Cambrex, Nottingham, U.K.). Cultures were stimulated with 100 ng/ml anti-CD3 (OKT3; Ortho Biotech, Bridgewater, NJ) and, where indicated, 100 ng/ml LPS, 5 μg/ml zymosan (Sigma, St. Louis, MO), 4 μg/ml LTA, or 0.3 μg/ml flagellin (InvivoGen, San Diego, CA). When naïve or memory CD4+ T cells were used, 0.5 × 106 monocytes were cultured at a 1:1 ratio with CD4+CD45RO+ or CD4+CD45RA+ T cells for 3 days (or 6 days for Fig. 1A) in 24-well plates. For transwell assays CD4+ T cell subsets and monocytes were separated by an insert containing a 0.4-μm semipermeable membrane (Corning Costar, Cambridge, MA), and anti-CD3 and/or LPS were added to both compartments. In the APC-free stimulation cells were cultured at 2 × 106 per milliliter in 24-well plates, stimulated for 48 h with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) mAbs (Invitrogen, Paisley, U.K.), and then cultured for a further 5 days in RPMI medium 1640 supplemented with hrIL-2 (200 units/ml). For skewing of CD4+ T cell subsets the following cytokines or mAbs were added: for Th1, hrIL-12 (10 ng/ml) and anti-IL-4 mAb (10 μg/ml); for Th2, hrIL-4 (10 ng/ml) and anti-IFN-γ mAb (10 μg/ml); and for Th17, hrIL-6 (20 ng/ml), hrIL-23 (10 ng/ml), hrIL-21 (100 ng/ml), and hrTGFβ1 (1–3 ng/ml). All reagents were supplied by R & D Systems (Abingdon, U.K.).

Coculture Experiments.

CD4+CD25CD45RO+ responder T cells (0.5 × 106 per well in 24-well plates) were cocultured at a 1:1 ratio with monocytes in the presence or absence of 0.5 × 106 CD4+CD25+Foxp3+ Tregs. The Tregs were labeled with 2 μM DDAO or 0.5 μM CFSE (Invitrogen) to distinguish the responder T cells from the Tregs during intracellular cytokine staining. As a control, parallel experiments were carried out in the presence of CFSE- or DDAO-labeled CD4+CD25Foxp3 effector T cells.

Flow Cytometry.

The following antibodies were used: anti-CD3-FITC, anti-CD4-phycoerythrin (PE)-Cy5, anti-CD8-PE-Cy5, anti-CD14-PE-Cy5, and anti-CD19-PE (all Beckman Coulter, Fullerton, CA), anti-CD25-PE (Miltenyi Biotec), anti-CD45RO FITC, and CD45RA allophycocyanin (a kind gift from Arne Akbar, University College London, London, U.K.). Cells were stained for 30 min at 4°C, fixed in 2% paraformaldehyde, and analyzed on a FACSCalibur using CellQuest software (both from Becton Dickinson, San Jose, CA) or FlowJo software (TreeStar). Foxp3 expression was assessed by using anti-Foxp3 allophycocyanin (eBiosciences, San Diego, CA).

Intracellular Cytokine Staining.

Cells were restimulated at day 3, day 5, or day 6 for 4–6 h with PMA (50 ng/ml; Sigma–Aldrich) and ionomycin (750 ng/ml; Sigma–Aldrich) in the presence of Golgistop (Becton Dickinson) during the last 3 h. Cells were stained for cell surface markers, fixed in 2% paraformaldehyde, and permeabilized with 0.5% saponin, then labeled with either anti-IgG1 FITC and anti-IgG1 PE (Beckman Coulter) or anti-IFN-γ FITC and anti-IL-17 PE (eBiosciences). For ex vivo analysis freshly isolated CD4+CD45RO+ T cells were stimulated with PMA/ionomycin for 5 h and immediately subjected to intracellular cytokine staining.

ELISA.

Supernatants were collected after stimulation and stored at −80°C until use. The presence of IL-17 and IFN-γ (Invitrogen) was measured by ELISA according to the manufacturer's instructions. The detection limit was 15 pg/ml.

Real-Time PCR.

RNA was extracted by using TRIzol reagent (Invitrogen), and cDNA was transcribed by using an iScript kit (Bio-Rad, Hemel Hempstead, U.K.), both according to the manufacturers' instructions. A total of 2 μl of cDNA was then used in real-time PCR in a reaction mix consisting of TaqMan Universal PCR Master Mix and specific primers for human IL-17, T-bet, and GATA-3 (Applied Biosystems, Warrington, U.K.) labeled with a reporter (FAM) and quencher dye. Reactions were normalized to β-actin (VIC-labeled) in a multiplex reaction performed in a PTC-200 Chromo 4 DNA Engine (MJ Research/Bio-Rad).

Statistical Analysis.

Significant differences were calculated with Prism 4.02 software (GraphPad, San Diego, CA) using a nonparametric matched-pairs test.

Supplementary Material

Supporting Figures:

Acknowledgments

This work was supported by a Ph.D. studentship funded by the Medical Research Council (to H.G.E.) and a studentship funded by the Biotechnology and Biological Sciences Research Council/GlaxoSmithKline (to T.S.). G.M.L. was supported by a Medical Research Council Clinician Scientist Grant (G108/380) and funding from the Department of Health via the National Institute for Health Research Comprehensive Biomedical Research Centre Award to Guy's and St. Thomas' National Health Service Foundation Trust in partnership with King's College London.

Abbreviations

TLR
Toll-like receptor
Treg
regulatory T cell
APC
antigen-presenting cell
hr
human recombinant
Th
T helper
PE
phycoerythrin.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708426104/DC1.

References

1. Nelms K, Keegan AD, Zamorano J, Ryan JJ, Paul WE. Annu Rev Immunol. 1999;17:701–738. [PubMed]
2. Gately MK, Renzetti LM, Magram J, Stern AS, Adorini L, Gubler U, Presky DH. Annu Rev Immunol. 1998;16:495–521. [PubMed]
3. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. Immunity. 2006;24:179–189. [PubMed]
4. Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, Hatton RD, Wahl SM, Schoeb TR, Weaver CT. Nature. 2006;441:231–234. [PubMed]
5. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK. Nature. 2006;441:235–238. [PubMed]
6. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. J Exp Med. 2003;198:1875–1886. [PMC free article] [PubMed]
7. Zheng SG, Wang J, Wang P, Gray JD, Horwitz DA. J Immunol. 2007;178:2018–2027. [PubMed]
8. Peng S, Szabo S, Glimcher L. Proc Natl Acad Sci USA. 2002;99:5545–5550. [PMC free article] [PubMed]
9. Neurath MF, Weigmann B, Finotto S, Glickman J, Nieuwenhuis E, Iijima H, Mizoguchi A, Mizoguchi E, Mudter J, Galle PR, et al. J Exp Med. 2002;195:1129–1143. [PMC free article] [PubMed]
10. Glimcher LH, Townsend MJ, Sullivan BM, Lord GM. Nat Rev Immunol. 2004;4:900–911. [PubMed]
11. Rangachari M, Mauermann N, Marty RR, Dirnhofer S, Kurrer MO, Komnenovic V, Penninger JM, Eriksson U. J Exp Med. 2006;203:2009–2019. [PMC free article] [PubMed]
12. Zheng W, Flavell R. Cell. 1997;89:587–596. [PubMed]
13. Pai S-Y, Truitt ML, Ting C-N, Leiden JM, Glimcher LH, Ho IC. Immunity. 2003;19:863–875. [PubMed]
14. Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, Killeen N, Urban JF, Guo L, Paul WE. Nat Immunol. 2004;5:1157–1165. [PubMed]
15. Kim JM, Rudensky A. Immunol Rev. 2006;212:86–98. [PubMed]
16. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD. Nat Genet. 2001;27:20–21. [PubMed]
17. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK. Int Immunol. 2007;19:345–354. [PubMed]
18. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR. Cell. 2006;126:1121–1133. [PubMed]
19. Acosta-Rodriguez EV, Rivino L, Geginat J, Jarrossay D, Gattorno M, Lanzavecchia A, Sallusto F, Napolitani G. Nat Immunol. 2007;8:639–646. [PubMed]
20. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Nat Immunol. 2007;8:942–949. [PubMed]
21. Miossec P. Arthritis Rheum. 2003;48:594–601. [PubMed]
22. Afzali B, Lombardi G, Lechler RI, Lord GM. Clin Exp Immunol. 2007;148:32–46. [PMC free article] [PubMed]
23. Korn T, Bettelli E, Gao W, Awasthi A, Jager A, Strom TB, Oukka M, Kuchroo VK. Nature. 2007;448:484–487. [PMC free article] [PubMed]
24. Nurieva R, Yang XO, Martinez G, Zhang Y, Panopoulos AD, Ma L, Schluns K, Tian Q, Watowich SS, Jetten AM, Dong C. Nature. 2007;448:480–483. [PubMed]
25. Zhou L, Ivanov II, Spolski R, Min R, Shenderov K, Egawa T, Levy DE, Leonard WJ, Littman DR. Nat Immunol. 2007;8:967–974. [PubMed]
26. Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ. J Exp Med. 2005;201:233–240. [PMC free article] [PubMed]
27. Hoeve MA, Savage NDL, de Boer T, Langenberg DML, de Waal Malefyt R, Ottenhoff THM, Verreck FAW. Eur J Immunol. 2006;36:661–670. [PubMed]
28. Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, et al. Nat Immunol. 2007;8:950–957. [PubMed]
29. Stephens LA, Mottet C, Mason D, Powrie F. Eur J Immunol. 2001;31:1247–1254. [PubMed]
30. DiPaolo RJ, Glass DD, Bijwaard KE, Shevach EM. J Immunol. 2005;175:7135–7142. [PubMed]
31. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Immunity. 2006;24:677–688. [PubMed]
32. Sato W, Aranami T, Yamamura T. J Immunol. 2007;178:7525–7529. [PubMed]
33. Messi M, Giacchetto I, Nagata K, Lanzavecchia A, Natoli G, Sallusto F. Nat Immunol. 2003;4:78–86. [PubMed]
34. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E, Fili L, Ferri S, Frosali F, et al. J Exp Med. 2007;204:1849–1861. [PMC free article] [PubMed]
35. Kinne RW, Brauer R, Stuhlmuller B, Palombo-Kinne E, Burmester G-R. Arthritis Res. 2000;2:189–202. [PMC free article] [PubMed]
36. Kohem C, Brezinschek R, Wisbey H, Tortorella C, Lipsky P, Oppenheimer-Marks N. Arthritis Rheum. 1996;39:844–854. [PubMed]
37. Cao D, Malmstrom V, Baecher-Allan C, Hafler D, Klareskog L, Trollmo C. Eur J Immunol. 2003;33:215–223. [PubMed]
38. van Amelsfort JMR, Jacobs KMG, Bijlsma JWJ, Lafeber FPJG, Taams LS. Arthritis Rheum. 2004;50:2775–2785. [PubMed]
39. Ruprecht CR, Gattorno M, Ferlito F, Gregorio A, Martini A, Lanzavecchia A, Sallusto F. J Exp Med. 2005;201:1793–1803. [PMC free article] [PubMed]
40. Sakaguchi S. Cell. 2000;101:455–458. [PubMed]
41. Shevach EM. Nat Rev Immunol. 2002;2:389–400. [PubMed]
42. Cao D, van Vollenhoven R, Klareskog L, Trollmo C, Malmstrom V. Arthritis Res Ther. 2004;6:R335–R346. [PMC free article] [PubMed]
43. Feger U, Luther C, Poeschel S, Melms A, Tolosa E, Wiendl H. Clin Exp Immunol. 2007;147:412–418. [PMC free article] [PubMed]
44. Lohr J, Knoechel B, Wang JJ, Villarino AV, Abbas AK. J Exp Med. 2006;203:2785–2791. [PMC free article] [PubMed]

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