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Human CD4⁺CD25^high Regulatory T Cells Modulate Myeloid but Not Plasmacytoid Dendritic Cells Activation

Roch Houot^1,*, Ivan Perrot^1,, Eric Garcia, Isabelle Durand and Serge Lebecque^2,,

^* Department of Hematology and Department of Pneumology, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; Schering-Plough Research Institute, Laboratory for Immunological Research, Dardilly, France; and Institut National de la Santé et de la Recherche Médicale Unité 503 IFR128, Université Claude Bernard Lyon 1, Lyon, France

	Abstract

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Human CD4⁺CD25⁺ regulatory T cells (Treg) play an essential role in the prevention of autoimmune diseases. However, the mechanisms of immune suppression and the spectrum of cells they target in vivo remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they have been shown to inhibit the Ag-presenting functions of macrophage- and monocyte-derived dendritic cells (DC). We have now studied the maturation of human blood-derived myeloid DC and plasmacytoid DC activated with TLR ligands in the presence of Treg. Preactivated Treg suppressed strongly TLR-triggered myeloid DC maturation, as judged by the blocking of costimulatory molecule up-regulation and the inhibition of proinflammatory cytokines secretion that resulted in poor Ag presentation capacity. Although IL-10 played a prominent role in inhibiting cytokines secretion, suppression of phenotypic maturation required cell-cell contact and was independent of TGF- and CTLA-4. In contrast, the acquisition of maturation markers and production of cytokines by plasmacytoid DC triggered with TLR ligands were insensitive to regulatory T cells. Therefore, human Treg may enlist myeloid, but not plasmacytoid DC for the initiation and the amplification of tolerance in vivo by restraining their maturation after TLR stimulation.

	Introduction

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Mouse CD4⁺CD25⁺ regulatory T cells (Treg)³ are critical for maintaining peripheral self-tolerance (1), and their human counterparts have been described recently (2, 3). However, the mechanisms by which CD4⁺CD25⁺ Treg mediate their suppressive effects in vivo are poorly understood and, although conventional T cells (Tconv) represent a major target of Treg, non-T cells might also be censored (4). In particular, while Treg have a direct suppressive action in vitro on Tconv (5, 6), this does not eliminate the possibility of an action on APCs as well. In previous reports, Treg were found not to alter (5, 7) or to suppress (8, 9, 10, 11) the expression of costimulatory molecules by APCs. Furthermore, the two last studies have shown that Treg-conditioned human monocytes (11) or monocyte-derived dendritic cells (DC) (10) present Ags poorly to T cells. Recently, mouse CD4⁺CD25⁺ T cells were shown to drive suppressive functions of mouse splenic DCs both in vitro and in vivo (12). In human, myeloid DC (mDC) and plasmacytoid DC (pDC) represent the two main subpopulations that differ in many phenotypic and functional aspects (13, 14). We have now studied whether human Treg could directly modulate the maturation of human blood mDC and pDC stimulated by TLR ligands.

	Materials and Methods

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TLR ligands

A total of 25 ng/ml LPS from Escherichia coli (Sigma-Aldrich), 10 µg/ml CpG 2216 (MWG Biotec), and 10 µM R848 (Schering-Plough) was used to stimulate mDC, pDC, or both, respectively.

Blocking Abs

A total of 20 µg/ml rabbit anti-human pan-specific TGF- ((R&D Systems), 10 µg/ml mouse anti-human CTLA-4 (clone AS32; Sigma-Aldrich), and 10 µg/ml mouse anti-human IL-10R (clone 3C5.2b; Schering-Plough) was used as blocking Abs. Mouse control isotype (10 µg/ml) and 20 µg/ml normal rabbit IgG (R&D Systems) were used as control Abs.

Cell sorting and cultures

CD4⁺CD25^high and CD4⁺CD25^– T cells were purified from normal human peripheral blood using a FACSVantage cell sorter (BD Biosciences). Briefly, PBMC were isolated after centrifugation on a Ficoll gradient, and CD4⁺ T cells were enriched by immunomagnetic depletion using Dynabeads (Dynal Biotech), using anti-CD8 (OKT8), anti-CD14 (MOP9.25), anti-CD19 (4G7) ascites and anti-CD16 (ION16; Beckman Coulter), anti-CD35 (CR1; Beckman Coulter), anti-CD56 (NKH1; Beckman Coulter), and anti-glycophorine A (JC159; DakoCytomation) purified mAbs. Enriched T cells were stained with Cy5-coupled anti-CD4 (Beckman Coulter) and PE-coupled anti-CD25 (M-A251; BD Biosciences) Abs. CD4⁺CD25^high and CD4⁺CD25^– populations were sorted using a FACSVantage (BD Biosciences) to a final purity >95%. Both populations were activated separately for 20 h at 5 x 10⁵ cells/ml in round-bottom 96-well plates coated with 1 µg/ml anti-CD3 (UCHT1; BD Pharmingen) in the presence of 2 µg/ml soluble anti-CD28 (15E8; R&D Systems).

DC were enriched from normal volunteer PBMC following the same magnetic beads depleting protocol supplemented with anti-CD3 (OKT3) ascites. Enriched DC were stained with Cy5-coupled anti-CD4 mAb and PE-coupled anti-CD11c (Leu-M5; BD Biosciences) mAb for mDC and BDCA4 (Miltenyi Biotec) mAb for pDC. mDC and pDC were sorted by FACS to a purity of >98 and >96%, respectively. DC were cultured separately during 20 h in round-bottom 96-well plates at 2.5 x 10⁵ cells/ml, alone or with various concentrations of CD4⁺CD25^high or CD4⁺CD25^– T cells, and immediately activated with TLR ligands.

For some experiments, culture of DC and T cells was performed in Transwells. A total of 5 x 10⁵ sorted DC was incubated in 24-well plates in 300 µl of complete RPMI 1640 medium with 10 ng/ml LPS. Anti-CD3 + anti-CD28-preactivated Treg or Tconv were then placed in 0.2-µm pore-size Transwell chambers of DC-containing well.

Flow cytometry analysis of DC phenotypic changes

After 24-h activation, mDC and pDC were collected and stained with allophycocyanin-coupled anti-CD11c (BD Biosciences), or anti-CD123 (Miltenyi Biotec) and FITC-coupled Abs for activation markers: CD80, CD86 and HLA-DR (BD Biosciences), CD83 (Beckman Coulter), CCR7 (R&D Biosystems), and CD40 (Caltag Laboratories). Cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences).

Cytokines quantification

Supernatants of 72-h T cell and 20-h activated DC cultures were collected, and IFN-, IL-2, IL-5, and IL-10 or IL-12p40, IL-6, TNF-, and IL-10, respectively, were quantified by ELISA using the OptEIA kits (BD Pharmingen). IFN- was quantified with the Cell Com IFN- kit (Beckman Coulter).

T cell proliferation and MLR assay

A total of 2.5 x 10⁴ CD4⁺CD25^– sorted T cells/well was cultured for 72 h with or without various concentrations of CD4⁺CD25^high sorted T cells in round-bottom 96-well plates coated with anti-CD3 and with soluble anti-CD28 mAbs. For MLR, mDC activated with LPS alone or in the presence of Treg or Tconv were harvested and purified from T cells using MACS CD33-coupled microbeads (Miltenyi Biotec). The purity of DC was quantified by flow cytometry using FITC-coupled CD11c (BD Biosciences) and anti-CD3 (DakoCytomation) mAbs. mDC were cocultured at various concentrations with 2 x 10⁴ allogenic CD4⁺CD45RA⁺ T cells for 2 or 3 days. Cultures were pulsed for additional 16 h with 1 µCi/well [³H]thymidine (Amersham Biosciences), and proliferation was measured with a TopCount apparatus (PerkinElmer).

Statistical analysis

Paired t test was used to analyze Treg inhibition with or without blocking Abs data.

	Results

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Activated human Treg inhibit TLR-stimulated phenotypic maturation of mDC, but not pDC

Human Treg are defined by a combination of phenotypic and functional features: they express CD4 and high level of CD25 (15), proliferate poorly in the absence of exogenous IL-2, and exert a contact-dependent suppression on Tconv in vitro. As expected (15), sorted human blood CD4⁺CD25^high T cells activated with anti-CD3 and anti-CD28 mAbs did not proliferate and suppressed the proliferation and cytokine secretion of cocultured CD4⁺CD25^– Tconv in a dose-dependent manner (Fig. 1 and data not shown). These sorted and preactivated Treg and Tconv were added separately to freshly isolated allogenic blood mDC or pDC together with LPS, CpG oligodesoxynucleotide (ODN) 2216, or R-848. Those ligands were used to trigger the maturation of mDC, pDC, or both through TLR4, TLR9, and TLR7/8, respectively (14, 16). After 20 h of culture, the up-regulation of B7 costimulatory molecules on mDC by either TLR4 or TLR8 ligands was strongly inhibited by preactivated Treg, with maximal inhibition observed at a ratio of 3 Treg for 1 mDC (data not shown). Therefore, this ratio was used in the following experiments, unless specified differently. The percentage of mDC acquiring CD80 was reduced 5-fold (Fig. 2A), while the CD80 mean fluorescence intensity (MFI) was decreased by 65 and 50% after LPS and R848 stimulation, respectively, and the CD86 MFI was decreased by 35% after LPS stimulation (Fig. 2B). On the contrary, both the percentage of positive mDC and the level of expression of CD80 were significantly increased by the presence of Tconv after LPS, but not R848 stimulation (Fig. 2, A and B). Interestingly, mDC activated by TLR ligands in the presence of Tconv expressed lower level of CCR7 (55% decrease of MFI), when compared with mDC activated alone or in presence of Treg (Fig. 2B). In contrast, preactivated Treg had no effect on the up-regulation of CD80 and CD86 expression by pDC triggered with TLR9 or TLR7 ligands (Fig. 2, C and D), while Tconv increased the expression of the costimulatory molecules. Furthermore, Treg did not reduce the level of CCR7 on TLR-stimulated pDC, and Tconv even increased the expression after CpG stimulation (Fig. 2D). No significant difference in the expression of CD83, HLA-DR, or CD40 by mDC or pDC activated in presence of Treg or Tconv was observed (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Blood-purified CD4+CD25high T cells strongly inhibit the proliferation of blood-purified conventional CD4+CD25– T cells. Proliferation of Tconv activated with anti-CD3 and anti-CD28 mAbs alone or in coculture with Treg at the indicated ratios. The proliferation measured by [3H]thymidine incorporation is expressed as percentage of proliferation of Tconv activated alone. Data are representative of seven independent experiments.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Treg modulate the expression of costimulatory molecules and CCR7 induced by TLR on mDC, but not pDC. mDC or pDC were cultured alone or with preactivated Treg or Tconv, and simultaneously exposed to LPS or CpG 2216, respectively. DC were analyzed after 24 h of culture (A). The percentage of CD80-positive mDC after LPS activation is decreased by Treg. B, Treg inhibit the up-regulation of B7 costimulatory molecules on mDC, but do not down-regulate CCR7 expression. C, Treg do not decrease the percentage of CD80-positive pDC after activation with CpG ODN 2216. D, Treg do not affect the up-regulation of B7 molecules and CCR7 on pDC. A and C, Representative of three independent experiments. B and D, Each graph represents more than three independent experiments. *, Indicates significant differences (p < 0.05).

To further characterize the effect of Treg on maturing DC, cytokines were measured in the supernatants of the cocultures described above. In line with the inhibition of B7 molecule up-regulation, Treg dramatically decreased the secretion of IL-12p40, TNF-, and IL-6 by mDC activated with LPS and to a lesser, but still significant extent with R848 (Fig. 3A). The average inhibition was 95% for IL-12p40, 93% for TNF-, and 50% for IL-6 after LPS activation, and 38, 35, and 38%, respectively, after R848 stimulation. In contrast, the production of these cytokines by mDC was greatly increased by the presence of activated Tconv (Fig. 3A). Remarkably, when added to TLR-stimulated mDC, Treg did not suppress IL-10 production (Fig. 3B). Like for the phenotype, Treg did not block the TLR7- or TLR9-stimulated secretion of IFN-, TNF-, and IL-6 by pDC (Fig. 3C). Of note, only Tconv increased the low level of IL-10 produced by TLR-stimulated pDC (Fig. 3D).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Activated Treg inhibit the secretion of proinflammatory cytokines by TLR-activated mDC, but not pDC. Culture conditions were as in Fig. 2 for 20 h, and cytokines in supernatants were quantified by ELISA. A, Treg strongly suppress, whereas Tconv enhance LPS-induced secretion of IL-12p40, TNF-, and IL-6 by mDC. B, IL-10 production by mDC is not suppressed by Treg. C, Treg do not inhibit IFN-, TNF-, and IL-6 secretion by CpG-stimulated pDC. D, IL-10 production by pDC is very low and not affected by Treg. Each graph represents more than three independent experiments. *, Indicates significant differences (p < 0.05).

Given the impairment of maturation of mDC, but not pDC, we concentrated our next analysis on the activity of Treg on mDC exclusively. Several publications have shown that Treg suppress Tconv only if they were preactivated (17, 18). Indeed, resting Treg had no effect on the up-regulation of CD80 and CD86 and on the down-regulation of CCR7 by LPS-stimulated mDC (Fig. 4A). In contrast, even nonactivated Treg blocked IL-12p40 and TNF- (but not IL-6) secretion (Fig. 4B), suggesting different mechanisms for suppressing those two aspects of mDC maturation.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Treg inhibit phenotypic maturation and cytokine secretion of mDC through different mechanisms. mDC were cultured as in Fig. 2 with either nonactivated or preactivated Treg or Tconv: A, their phenotype was analyzed by FACS; B, cytokines were measured in supernatant. C, mDC were cultured as in Fig. 2 with blocking or control mAb. Each graph represents three independent experiments. *, Indicates statistically significant differences (p < 0.05).

To investigate how Treg suppress mDC, we first separated TLR-stimulated mDC from preactivated Treg in Transwells. Without cell-cell contact, neither phenotypic modulation nor inhibition of cytokines production was observed (data not shown). TGF- and CTLA-4 present at the surface of Treg (2, 19) have both been implicated in their suppressive activity. In addition, IL-10 is a well-recognized immunosuppressive cytokine that can drive DC into tolerogenic APCs (20). None of the blocking mAbs directed against these three molecules could significantly reverse the modulation by Treg of CD80, CD86, and CCR7 mDC expression (data not shown). In contrast, anti-IL-10R mAb, but neither anti-TGF- nor anti-CTLA-4 blocking Abs restored strongly IL-12p40 and IL-6 production and partially TNF- secretion by mDC activated with LPS in presence of Treg (Fig. 4C). Those data suggest that Treg prevent the costimulatory molecules up-regulation on mDC through contact-dependent mechanisms, while the modulation of cytokines secretion appears to be also mediated by IL-10.

Treg-conditioned mDC have a reduced ability to trigger naive T cell proliferation

In view of the capacity of Treg to inhibit costimulatory molecules expression and proinflammatory cytokines secretion by TLR-activated mDC, we examined how such Treg-conditioned mDC would activate naive T cells in a MLR. After 20-h coculture with either Treg or Tconv, CD11c⁺ mDC were efficiently purified with magnetic beads (Fig. 5A). Either Treg- or Tconv-conditioned mDC were then cultured with CD4⁺CD45RA⁺ naive T cells for 3 days, before DC survival started to decline significantly. At the ratio of 1 DC/10 naive T cells, Treg-conditioned mDC were approximately three times less efficient than unconditioned mDC or Tconv-conditioned mDC to induce the proliferation of naive T cells (Fig. 5B). Of note, viability of T cells and mDC measured by trypan blue exclusion did not differ significantly between MLR driven either by control or Treg-conditioned mDC (data not shown). The role of CD3⁺ Treg contaminating the Treg-conditioned mDC was evaluated by sorting and adding them to the secondary MLR. As shown on Fig. 5C, inhibition of the secondary MLR was detected only when over three recovered Treg were added per stimulating mDC. Therefore, the 6% Treg contaminating the Treg-conditioned mDC (Fig. 5A) cannot account for the inhibition observed in the secondary MLR. Altogether, these data demonstrate that mDC activated in the presence of Treg have a decreased capacity to trigger the proliferation of allogenic naive T cells.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. Treg-conditioned mDC have decreased Ag presentation ability. A, mDC cultured with LPS for 24 h alone or in the presence of preactivated Treg or Tconv were analyzed by FACS for CD11c and CD3 expression before and after purification with CD33-coupled magnetic beads. B, mDC purified after culture alone, with Treg or with Tconv, were seeded at indicated ratio with allogenic CD4+CD45RA+ T cells for 3 days. [3H]Thymidine incorporation was measured 16 h after the pulse. C, Freshly purified mDC were cultured with allogenic CD4+CD45RA+ T cells only or with the indicated ratios of Treg purified, as in A. Data are representative of two independent experiments with similar results.

	Discussion

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Regarding the mDC, the present results are in agreement and extend previous reports in mice (8, 9, 12) as well as in humans, showing the lack of maturation of monocytes/macrophages (11) and monocyte-derived DC (10) cocultured with activated Treg. Indeed, we have now demonstrated that, in addition to the T cell-dependent activation, the TLR-triggered maturation of mDC that is likely to occur first in peripheral tissues can also be blocked by activated Treg. Furthermore, we show that such suppression also apply to the most relevant blood-derived mDC. Suppression of mDC by Treg encompassed the phenotypic maturation, the proinflammatory cytokines secretion, and the resulting Ag presentation ability.

First, in regard to phenotypic maturation, inhibition of CD80 and CD86, but not CD40 and HLA DR up-regulation after TLR stimulation is similar with what has been published with monocyte-derived DC/Treg cocultures. The decreased expression of CCR7 observed after DC/Tconv interaction suggests that, in vivo, such interaction may terminate mDC migration. Therefore, the lack of CCR7 down-regulation on TLR-stimulated mDC after coculture with Treg might indicate that those DC could retain their ability to move to secondary lymphoid organs, in vivo, but this hypothesis will require further demonstration. Second, Treg suppressed the secretion of proinflammatory cytokines TNF-, IL-6, and IL-12p40 induced by TLR4 and TLR8 ligands. Likewise, after coculture with Treg, monocytes/macrophages secreted reduced amounts of TNF- and IL-6 when exposed to LPS. The higher susceptibility of mDC to Treg suppression after LPS vs R848 activation does not merely correlate with the intensity of the activating signals and may therefore reflect differences in signal transduction pathway between TLR4 and TLR8. In contrast with proinflammatory cytokines, but similar to what had been reported with monocytes/macrophages cocultured with Treg, Treg-conditioned mDC secreted unaltered amounts of IL-10 upon TLR activation. Although both mDC and Treg could produce IL-10, almost no IL-10 was detected in the supernatant of preactivated Treg and Treg-Tconv cocultures (data not shown) and of Treg-pDC coculture (Fig. 3D). This indirectly suggests that, in our coculture conditions, Treg did not produce significant amounts of IL-10 and that mDC was the main source of this cytokine. This conclusion is further supported by previous report showing that the number of IL-10-secreting monocyte-derived DC is increased 2-fold when cocultured with Treg vs Tconv (10).

Third, the Ag-presenting function of TLR-stimulated mDC was significantly reduced by Treg, as shown for monocyte-derived DC and monocytes/macrophages cocultured with Treg (10, 11). The fate of T cells exposed to Treg-conditioned mDC remains to be analyzed, as they could become anergic or suppressive. Indeed, Tconv activation requires two signals provided by the antigenic peptide associated with MHC molecules and the costimulatory molecules expressed by APC, respectively (21). But TCR engagement without or with low level of costimulatory signal leads to anergic T cells (21). Furthermore, the limited secretion of cytokines except IL-10 combined with poor ability to drive naive T cell proliferation are reminiscent of the semimature DC with tolerogenic functions in vivo (22) that drive the development of suppressive IL-10-producing CD4⁺ T cells, in vitro (17) as well as in vivo (23). Moreover, additional work will be required to establish the importance of the inhibition of IL-6 secretion, a cytokine necessary to render Tconv resistant to Treg suppression (24), and to determine whether, like mouse DC (12), human mDC are driven to immunosuppressive tryptophan catabolism by Treg.

Although the precise mechanisms remain to be elucidated, cell-cell contact appears required for both the modulation of costimulatory molecule up-regulation and the suppression of cytokine secretion, the latter being critically dependent on IL-10 as well. In agreement with our data, the importance of cellular contact and the minimal contribution of IL-10 and TGF- had been described previously for the inhibition by Treg of the Ag-presenting function of human monocyte-derived DC (10). The requirement of both Treg preactivation and cell contact suggests more stringent conditions for the inhibition of phenotypic maturation of mDC than for the suppression of cytokines secretion.

The inability of activated Treg to suppress the phenotypic maturation and to inhibit the secretion of IFN-, TNF-, and IL-6 by pDC activated with either TLR7 or TLR9 ligand contrasts with the sensitivity of mDC. The insensitivity of TLR-activated pDC to Treg suppression might have several nonexclusive explanations. The limited up-regulation of B7 molecule expression by pDC treated with R848 or CpG ODNs does not suggest that the TLRs trigger a stronger activation of pDC that would be more difficult for Treg to override. However, pDC may not express the surface molecule(s) apparently required for mDC to be sensitive to Treg suppression, and they may have a limited sensitivity to IL-10. Whichever the reasons for their resistance to Treg suppression, the physiological consequence is unclear, as pDC have been shown to suppress both CD4⁺ (25) and CD8⁺ (6) T cell activation.

In conclusion, our results provide the first evidence of a direct inhibition of human blood-derived mDC, but not pDC maturation by CD4⁺CD25⁺ Treg. The phenotypic and functional features of Treg-conditioned mDC suggest that, in the context of cancer, tumor-infiltrating Treg (26) could not only hamper the maturation of tumor-infiltrating DC (27), but also confer them tolerogenic functions at the tumor site (28), and, thanks to the persistence of CCR7, in the draining lymph nodes. Further studies are now required to validate this model.

	Acknowledgments

 
We acknowledge Catherine Massacrier for expert technical assistance, and Christophe Caux, Giorgio Trinchieri, and Alain Vicari for reviewing the manuscript.

	Disclosures

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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 R.H. and I.P. contributed equally to this work.

² Address correspondence and reprint requests to Dr. Serge J. Lebecque, Centre Hospitalier Lyon Sud, 69310 Pierre-Benite, France. E-mail address: serge.lebecque{at}chu-lyon.fr

³ Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; mDC, myeloid DC; MFI, mean fluorescence intensity; ODN, oligodesoxynucleotide; pDC, plasmacytoid DC; Tconv, conventional T cell.

Received for publication September 19, 2005. Accepted for publication January 26, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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