The CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the control of peripheral tolerance by directly inhibiting conventional T cell proliferative and effector functions. However, the mechanisms by which Treg regulate the homeostasis of lymph nodes remain unclear. In this study, we show in a mouse model that Treg control two major checkpoints dictated by the interaction between self-reactive CD4+ T cells and resident dendritic cell (DC) in secondary lymphoid organs. First, Treg inhibit the production of CCR5 ligands, limiting the CCR5-dependent recruitment of DC in the lymph nodes. Second, Treg prevent the DC exposure of IL-15Rα, markedly interfering in the DC-mediated NK cell proliferation in vivo. Therefore, the DC/T cell autoreactivity leading to NK cell triggering could potentially be controlled by the coinhibition of both IL-15Rα and CCR5 in autoimmune disorders in which NK cells play a deleterious role.

The CD4+CD25+ regulatory T cells (Treg)3 are a unique population of T cells that maintain immune tolerance (1, 2, 3). They contribute to exert a dominant tolerance during infections (4, 5), tumor progression (6, 7), and allogeneic transplantations (8). Treg keep in check several components of the immune system to maintain homeostasis. The forkhead box transcription factor Foxp3, the most specific marker of Treg cells, is involved in their development (9, 10, 11). Scurfy mice, a mouse model lacking Foxp3, develop a fatal lymphoproliferative disease essentially due to the absence of Treg (12, 13). In Foxp3 knock-in mice, Treg elimination in neonates induces an overt autoimmune syndrome similar to what is observed in scurfy mice (3, 14). Ablation of Treg in adult mice also results in the development of a fatal autoimmune disease (3), supporting the notion that Treg are required to maintain peripheral tolerance and homeostasis of the immune system. Indeed, Treg can suppress the effector functions of cells involved in adaptive immunity, such as CD4+ T cells, CD8+ T cells, B cells, NK cells, and dendritic cells (DC) (15, 16, 17, 18, 19). Treg can act directly on CD4+ or CD8+ T cells (20, 21, 22) or indirectly by inhibiting DC maturation and Ag presentation (23). In a recent model system, Kim et al. (3) described the deleterious effects of Treg depletion on the lymph node (LN) composition, highlighting a 7- to 9-fold increase in resident NK and DC. We have demonstrated in a previous work that Treg inhibit NK cells in vitro and in vivo. In scurfy mice, devoid of Treg, or in (WT) mice treated with PC61 or cyclophosphamide (CTX), high proliferation of NK cells occurs (18). But the mechanisms involve in the control of DC and NK cell homeostasis in LN in mice lacking Treg remain to be understood.

In this study, we demonstrate that the absence of Treg results in the activation of self-reactive CD4+ T cells in LN responsible for: 1) DC recruitment in a CCR5-dependent manner and 2) DC maturation leading to IL-15Rα-dependent NK cells proliferation. This is the first observation demonstrating the regulatory role of CD4+CD25+Foxp3+ T cells in DC/NK cross-talk at the steady state in LN.

Female C57BL/6 (H-2b) mice were obtained from Charles River Laboratories and the Centre d’Elevage Janvier (Le Genest-st-Isle, France) and maintained in the Institut Gustave Roussy animal facilities according to the Animal Experimental Ethics Committee Guidelines. CCR5-deficient mice on the C57BL/6 (H-2b) background were bred in the Pitié-Salpétrière animal facility (Nouvelle Animalerie Commune of Pitié-Salpêtrière, Paris, France). IL-15Rα−/− mice were provided by Prof. A. Ma (24). CD11c-DTR/GFP-transgenic mice on the C57BL/6 (H-2b) background were bred in the Institut Gustave Roussy animal facility (25).

CD4+CD25 T cells (Tconv) and CD4+CD25+ T cells (Treg) were obtained from splenocytes by magnetic separation using a CD4+CD25+ isolation kit (Miltenyi Biotec) according to manufacturer’s procedure. For MLR, 1 × 105 Tconv from C57BL/6 mice were cultured with 2 × 104 irradiated BALB/c splenocytes during 4 days in the presence or absence of Treg (purified from PBS- or CTX-treated mice) at different Tconv:Treg ratios (1:1, 2:1, 4:1, and 8:1). One microcurie of [3H]thymidine was added per well in the last 18 h. [3H]Thymidine incorporation was measured by liquid scintillation counting after harvesting the cells on glass fiber filters using an automatic cell harvester (Tomtec).

Mouse bone marrow derived-DC (BM-DC) were cultured as previously described (26). Briefly, bone marrow progenitor cells were grown in IMDM culture medium (Sigma-Aldrich) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM l-glutamine, 10% decomplemented FCS (Invitrogen Life Technologies), 50 μM 2-ME (Sigma-Aldrich), and 30% J558-mouse GM-CSF culture supernatants for 10–12 days. The phenotype of BM-DC was analyzed by flow cytometry using anti-mouse CD11c, I-Ab, CD80, CD86, and CD40 mAb (BD Pharmingen).

For in vitro functional assays, DC were cocultured at day 0 with Tconv at a final ratio of 50:1 (Tconv:DC) in the presence or absence of Treg. Tconv:Treg ratios were as follows: 1:1, 2:1, 4:1, and 8:1. At day 7, supernatants were harvested and RANTES/CCL5 and MIP-1α/CCL3 were quantified using an ELISA kit (R&D Systems). Where indicated in the figure legend, neutralizing anti-TGFβ1,2,3 was added in the coculture (R&D Systems). For IL-15Rα staining, cells were harvested and stained by FITC-anti-IAb, PE-anti-CD11c, and biotinylated anti- IL-15Rα (R&D Systems) and analyzed by flow cytometry.

Mice received 100 mg/kg CTX or 100 μg of anti-CD25 (PC61) or 150 μg of anti-CD4 (GK1.5) or PBS i.p. at day 0. At day 7, mice were sacrificed and popliteal, inguinal, axillary LN were removed. LN were mechanically minced. Mononuclear LN cells were enumerated using trypan blue exclusion before staining with PE-anti-CD11c, allophycocyanin-anti-CD11c, FITC-anti-I-Ab, PerCp-anti-CD3, allophycocyanin-anti-NK1.1, FITC-anti-CD4, FITC-anti-CD8, allophycocyanin-anti-CD25, PE-anti-CD80, PE-anti-CD40 Abs (BD Pharmingen), and biotinylated-anti-IL-15-Rα (R&D Systems). Biotinylated Ab (anti-IL-15-Rα) was visualized by streptavidin-allophycocyanin. For intracellular staining, cells were stained with anti-IFN-γ (BD Pharmingen) according to the manufacturer’s instruction after short stimulation with PMA and ionomycin (1 μg/ml).

For BrdU incorporation assays, 1.5 mg of BrdU solution per mice was injected i.p. at day 6. The day after, LN DC and NK cells were analyzed in flow cytometry, respectively, using CD11c/I-Ab and NK1.1/CD3 staining. BrdU incorporation was revealed using allophycocyanin anti-BrdU Ab according to the BrdU Flow Kit (BD Pharmingen) and analyzed by flow cytometry (FACScan; BD Biosciences).

In CD11c-DTR/GFP mice, 100 mg/kg CTX or PBS was injected i.p. at day 0. At day 4, 5 μg/kg diphtheria toxin (DT; Sigma-Aldrich) was injected i.p. At day 7, mice were sacrificed and LN were analyzed. CD11c+ cell depletion was checked by staining splenocytes with anti-CD11c Ab.

Results are expressed as means ± SEM. Groups were compared by using ANOVA followed by multiple comparisons of means with Fisher’s least-significance procedure. We evaluated statistical significance with Prism software (GraphPad Software). Values of p < 0.05 were considered significant.

To investigate the role of Treg in keeping in check LN homeostasis, we conducted two different approaches: one aimed at depleting Treg using the PC61 Ab (27) and the other one aimed at inhibiting Treg-suppressive functions using one dose of CTX (28, 29). In the former case, systemic injection of 100 μg of anti-CD25 Ab/mouse promoted the complete ablation of splenic (Fig. 1,A) and LN (data not shown) CD4+CD25+ cells. In the latter case, systemic administration of 100 mg/kg of CTX/mouse resulted in the maintenance of the CD4+CD25+Foxp3+ Treg pool in all lymphoid organs (Fig. 1,A and data not shown) but those Treg completely lost their suppressive functions on TCR-stimulated Tconv (Fig. 1,B). Seven days after injection, we observed in both cases a 2-fold increase in the percentages of LN-resident DC defined as CD11chigh I-Ab+ cells (2.1± 0.3% before CTX vs 3.2± 0.3% at day 7 after CTX treatment; Fig. 1,C). Similar results were obtained with depletion using the PC61 mAb (Fig. 1,C). The absolute number of DC was also increased by 2-fold after Treg depletion (82,680 ± 8,304 DC vs 196,098 ± 26,586 DC in the absence of Treg, p < 0.0001). The augmentation of DC in LN could be assigned to an enhanced proliferation and/or recruitment of the DC. To label dividing cells, we injected BrdU 6 days after CTX or PC61 pretreatment. The BrdU incorporation in LN DC was not significantly boosted by treatment of mice with CTX or PC61 mAb, indicating that the increase in DC number in the absence of Treg was due to recruitment rather than a proliferation (Fig. 1,D). The phenotype of resident DC was slightly modified with enhanced cell surface expression of MHC class II molecules (Fig. 1,E) but no up-regulation of CD80, CD86, or CD40 costimulatory signals. Because DC are known to express the CCR5 chemokine receptor at the immature stage (30), we hypothesized that in the absence of Treg, DC could be recruited in LN in a CCR5-dependent manner. Indeed, DC failed to accumulate in CCR5 loss-of-function animals (CCR5−/− mice) deprived of Treg (Fig. 2,A). Based on a previous report suggesting that DC activation and recruitment induced by Treg depletion could rely upon self-reactive T cells (3), we depleted conventional CD4+ T cells along with Treg using the GK1.5-depleting mAb (31) before assessing LN DC proportion and number. Indeed, in hosts depleted of both Treg and Tconv, DC recruitment into LN was compromised (Fig. 2,B). These results suggest that in the absence of Treg, the uncontrolled autoreactive T cells elicit the release of CCR5 ligands and the subsequent trafficking and accumulation of DC into LN. To test this hypothesis, the autoreactive cross-talk between immature DC and polyclonal autologous CD4+ Tconv was reconstituted in vitro at a 1:50 DC:T cell ratio in the presence of increasing amounts of autologous Treg. Although neither DC nor Tconv alone could secrete MIP-1α/CCL3 or RANTES/CCL5, the coculture of both DC and Tconv allowed the significant release of both MIP-1α/CCL3 and RANTES/CCL5 in vitro (Fig. 2, C and D). Most strikingly, Treg completely abolished the production of CCR5 ligands by the autoreactive DC:Tconv cell cross-talk at a 1:1 up to 1:4 Treg:Tconv ratio (Fig. 2, C and D).

FIGURE 1.

Functional or physical ablation of Treg induces DC recruitment in LN. A, The PC61 Ab depletes CD4+CD25+ Treg. Splenocytes from C57BL/6 mice treated with PBS, CTX, or PC61 7 days before were stained by anti-CD4, anti-CD25, and anti-Foxp3 mAb. Flow cytometry analyses on gated CD3+CD4+ cells are shown for a representative experiment of three yielding identical results. B, CTX suppresses the inhibitory effects of Treg on Tconv. C57BL/6 CD4+CD25 Tconv were cultured with allogeneic BALB/c splenocytes at a ratio of 1:5. Treg purified from PBS- or CTX-treated mice were added in the coculture at different ratios as indicated. Proliferation indices were monitored by the incorporation of tritiated thymidine at day 5 after an overnight pulsing of tritiated thymidine. The percentages of inhibition of CD4+CD25 proliferation by Treg are depicted for a representative experiment of three yielding similar results. C, Percentage of CD11c+/I-Ab+ DC in LN in the absence of functional Treg. Mice were sacrificed 7 days after administration of either CTX or PC61 mAb, and LN were harvested. Mononuclear LN cell numbers were determined by trypan blue exclusion assays before staining with anti-CD11c and anti-I-Ab mAb and flow cytometry analysis. Data from three independent experiments were pooled and depicted. D, DC fail to proliferate in the absence of functional Treg. Same experimental settings as in C, but mice received BrdU solution at day 6. LN cells were subjected to a CD11c/I-Ab staining and BrdU incorporation was revealed as described in Materials and Methods. The percentage of DC incorporating BrdU is shown for all six animals of two independent experiments (right panel). ∗, p < 0.05. E, Enhanced MHC class II expression in LN resident DC. Flow cytometry analysis of MHC class II (I-Ab) cell surface expression on CD11c-positive cells before and after CTX administration. Similar data obtained using PC61 (data not shown).

FIGURE 1.

Functional or physical ablation of Treg induces DC recruitment in LN. A, The PC61 Ab depletes CD4+CD25+ Treg. Splenocytes from C57BL/6 mice treated with PBS, CTX, or PC61 7 days before were stained by anti-CD4, anti-CD25, and anti-Foxp3 mAb. Flow cytometry analyses on gated CD3+CD4+ cells are shown for a representative experiment of three yielding identical results. B, CTX suppresses the inhibitory effects of Treg on Tconv. C57BL/6 CD4+CD25 Tconv were cultured with allogeneic BALB/c splenocytes at a ratio of 1:5. Treg purified from PBS- or CTX-treated mice were added in the coculture at different ratios as indicated. Proliferation indices were monitored by the incorporation of tritiated thymidine at day 5 after an overnight pulsing of tritiated thymidine. The percentages of inhibition of CD4+CD25 proliferation by Treg are depicted for a representative experiment of three yielding similar results. C, Percentage of CD11c+/I-Ab+ DC in LN in the absence of functional Treg. Mice were sacrificed 7 days after administration of either CTX or PC61 mAb, and LN were harvested. Mononuclear LN cell numbers were determined by trypan blue exclusion assays before staining with anti-CD11c and anti-I-Ab mAb and flow cytometry analysis. Data from three independent experiments were pooled and depicted. D, DC fail to proliferate in the absence of functional Treg. Same experimental settings as in C, but mice received BrdU solution at day 6. LN cells were subjected to a CD11c/I-Ab staining and BrdU incorporation was revealed as described in Materials and Methods. The percentage of DC incorporating BrdU is shown for all six animals of two independent experiments (right panel). ∗, p < 0.05. E, Enhanced MHC class II expression in LN resident DC. Flow cytometry analysis of MHC class II (I-Ab) cell surface expression on CD11c-positive cells before and after CTX administration. Similar data obtained using PC61 (data not shown).

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FIGURE 2.

CCR5-dependent recruitment of DC in the LN in the absence of Treg. A, DC recruitment is dependent on CCR5 ligands. Same experimental setting as in Fig. 1 C but using CCR5−/− and WT C57BL/6 mice, but DC absolute number is shown. B, DC recruitment in the absence of Treg is dependent on CD4+ self-reactive T cells. DC percentage (left panel) and absolute number (right panel) are depicted in Treg-depleted (PC61) or both Tconv and Treg-depleted (GK1.5) mice. A and B, Experiments included three to four mice per group and were repeated twice with identical results. Pooled data are shown. C and D, CD4+CD25+ Treg inhibit MIP-1α and RANTES secretion induced by the DC/CD4+ self-reactive T cell cross-talk. Treg and Tconv were purified from splenocytes as described in Materials and Methods. Tconv were cocultured with BM-DC at a ratio of 50:1. Treg were added at various and indicated Tconv:Treg ratios. After 7 days of coculture, accumulated levels of CCL3 and CCL5 were measured in the supernatant using ELISA (DuoSet; R&D Systems). Means ± SE of triplicate wells are presented for a representative experiment of three. ∗, p < 0.05.

FIGURE 2.

CCR5-dependent recruitment of DC in the LN in the absence of Treg. A, DC recruitment is dependent on CCR5 ligands. Same experimental setting as in Fig. 1 C but using CCR5−/− and WT C57BL/6 mice, but DC absolute number is shown. B, DC recruitment in the absence of Treg is dependent on CD4+ self-reactive T cells. DC percentage (left panel) and absolute number (right panel) are depicted in Treg-depleted (PC61) or both Tconv and Treg-depleted (GK1.5) mice. A and B, Experiments included three to four mice per group and were repeated twice with identical results. Pooled data are shown. C and D, CD4+CD25+ Treg inhibit MIP-1α and RANTES secretion induced by the DC/CD4+ self-reactive T cell cross-talk. Treg and Tconv were purified from splenocytes as described in Materials and Methods. Tconv were cocultured with BM-DC at a ratio of 50:1. Treg were added at various and indicated Tconv:Treg ratios. After 7 days of coculture, accumulated levels of CCL3 and CCL5 were measured in the supernatant using ELISA (DuoSet; R&D Systems). Means ± SE of triplicate wells are presented for a representative experiment of three. ∗, p < 0.05.

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In conclusion, in the absence of Treg, DC are recruited into LN in a CCR5-dependent manner. Moreover, Treg are able to inhibit MIP-1α/CCL3 and RANTES/CCL5 secretion induced during CD4+ self-reactive T cell/DC cross-talk in vitro, suggesting that Treg could control DC recruitment in LN at the steady state by inhibiting MIP-1α/CCL3 and RANTES/CCL5 secretion by resident LN cells.

We have shown that, during stimulation by LPS or IL-12, NK cell effector functions can be directly blunted by Treg in a TGF-β-dependent manner (18). In scurfy mice devoid of Treg or in WT mice treated with PC61 or CTX, the homeostatic proliferation of NK cells is markedly enhanced (18). However, the mechanisms involved in NK cell proliferation in mice lacking Treg remain unclear. As shown in Fig. 3, in the absence of Treg, the NK cell percentage was enhanced while the percentage of CD3+ T lymphocytes did not increase. We also observed a 2-fold increase of the NK cell number after Treg depletion (54,152 ± 6,292 NK cells vs 97,897 ± 13,287 NK cells in the absence of Treg; p = 0.0094). This increasing number of NK cells was due to a proliferation as demonstrated by BrdU incorporation (Fig. 3 C). Thus, blunting Treg function selectively induced the proliferation of NK cells in secondary lymphoid organs.

FIGURE 3.

Treg control the IL-15Rα -dependent NK cell proliferation. A, Percentage of NK cells after CTX or PC61 treatment. Identical settings as in Fig. 1,C, but LN cells were subjected to a NK1.1/CD3 double staining. Data of three independent experiments were pooled and are depicted. on this graph. B, Percentage of CD3+ T cells after CTX or PC61 treatment. Same experimental settings as in A and B, but the percentage of CD3+/NK1.1 cells is shown. C, NK cells proliferate in the absence of functional Treg. Same experimental setting as in Fig. 1 D, but cells were subjected to a NK1.1/CD3 staining. Histograms represent means ± SEs of the percentages of BrdU-incorporating cells. D, NK cell proliferation in the absence of functional Treg depends on IL-15Rα. Same experimental settings as in Fig. C but using WT and IL-15Rα−/− mice. Histograms represent means ± SEs of the percentages of BrdU-incorporating NK cells. E, NK cell proliferation in the absence of Treg is dependent on CD11chigh myeloid DC. CD11c-DTR/GFP mice and WT counterparts received CTX at day 0. At day 4, mice were treated with DT as described in Materials and Methods. Mice received BrdU at day 6 and were sacrificed at day 7. LN cells were stained as described in C and D. Data are representative of two independent experiments. ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.

FIGURE 3.

Treg control the IL-15Rα -dependent NK cell proliferation. A, Percentage of NK cells after CTX or PC61 treatment. Identical settings as in Fig. 1,C, but LN cells were subjected to a NK1.1/CD3 double staining. Data of three independent experiments were pooled and are depicted. on this graph. B, Percentage of CD3+ T cells after CTX or PC61 treatment. Same experimental settings as in A and B, but the percentage of CD3+/NK1.1 cells is shown. C, NK cells proliferate in the absence of functional Treg. Same experimental setting as in Fig. 1 D, but cells were subjected to a NK1.1/CD3 staining. Histograms represent means ± SEs of the percentages of BrdU-incorporating cells. D, NK cell proliferation in the absence of functional Treg depends on IL-15Rα. Same experimental settings as in Fig. C but using WT and IL-15Rα−/− mice. Histograms represent means ± SEs of the percentages of BrdU-incorporating NK cells. E, NK cell proliferation in the absence of Treg is dependent on CD11chigh myeloid DC. CD11c-DTR/GFP mice and WT counterparts received CTX at day 0. At day 4, mice were treated with DT as described in Materials and Methods. Mice received BrdU at day 6 and were sacrificed at day 7. LN cells were stained as described in C and D. Data are representative of two independent experiments. ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.

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Since IL-15Rα has been implicated in NK cell survival, proliferation (24, 32, 33, 34, 35), as well as in the DC-dependent NK cell priming (36, 37), we hypothesized that IL-15 transpresentation by DC could be implicated in NK cell proliferation in the absence of Treg. Since a substantial frequency of NK cells remains detectable in IL-15Rα−/− mice (0.125 ± 0.012%), we could study their proliferation capacity in the absence of Treg. In IL-15Rα−/−-treated mice, the proportion of NK cells remained stable (0.13 ± 0.015%) and NK cells failed to proliferate in IL-15Rα-deficient hosts treated with CTX (Fig. 3,D). It is interesting to note that NK cells could proliferate in IL-15Rα−/− mice upon administration of IL-2 (data not shown). To address the role of DC in the potential transpresentation of IL-15Rα/IL-15 to NK cells, we took advantage of CD11c-DTR-transgenic mice expressing the DT receptor under the control of the CD11c promoter, in which treatment with DT exerts a conditional and transient depletion of conventional myeloid DC (25). In these hosts indeed, in the absence of CD11chigh myeloid DC, NK cell proliferation failed to occur in CTX-treated mice (Fig. 3 E).

Thus, the presence of myeloid DC is required for the IL-15Rα-dependent proliferation of NK cells induced in the absence of Treg.

Immature DC are not supposed to express IL-15Rα which has been shown to be tightly regulated by type 1 IFN and maturation stimuli (37, 38, 39). Indeed, at the steady state, LN-resident DC did not harbor IL-15Rα molecules on the cell surface (Fig. 4,A). We addressed whether ablation of Treg could turn on IL-15Rα expression on LN DC. Indeed, an up-regulation of IL-15Rα expression was observed in the absence of Treg, whereas expression of costimulatory molecules such as CD40 or CD80 did not change (Fig. 4, A and B). This phenomenon was abolished if both Treg and Tconv were depleted (following administration of the GK1.5 mAb) (Fig. 4,B). Furthermore, a 4-fold increase in the numbers of DC exhibiting cell surface expression of IL-15Rα could be monitored 7 days after injection of the PC61 mAb (44,070 ± 10,720 without Treg compared with 11,590 ± 3,772 with Treg; p = 0.0079) (Fig. 4,B). In vitro cocultures revealed that IL-15Rα surface exposure on DC resulted from a productive interaction between immature DC with autologous polyclonal Tconv (Fig. 4,C) that could be inhibited by Treg in a TGF-β-dependent fashion (Fig. 4 D).

FIGURE 4.

IL-15Rα expression on DC is controlled by Treg. A, IL-15Rα expression on LN DC is enhanced after Treg depletion unlike CD40 or CD80 expression. Flow cytometry analysis of IL-15Rα, CD80, and CD40 cell surface expression on CD11c+/I-Ab+ cells before (upper panel) and after PC61 (lower panel) administration. Shaded histograms represent isotype control; open histograms represent IL-15Rα, CD80, or CD40 staining. B, IL-15Rα expression on DC is enhanced in the absence of Treg and is dependent on CD4+ self-reactive T cells. Same experimental settings as in Fig. 1,C, but Treg (PC61) or Treg and Tconv (GK1.5) were depleted. Cells were subjected to a CD11c/I-Ab/IL-15Rα triple staining. C and D, In vitro, Treg keep in check IL-15Rα expression on DC in a TGF-β-dependent manner. Same experimental settings as in Fig. 2, C and D, but after 7 days of coculture, cells were removed and subjected to a CD11c/I-Ab/IL-15Rα staining. D, Anti-TGF-β Ab is added to the coculture. A representative FACS analysis (C) and a graph (D) show the results (means ± SE) obtained from two independent experiments. ∗, p < 0.05 and ∗∗, p < 0.01.

FIGURE 4.

IL-15Rα expression on DC is controlled by Treg. A, IL-15Rα expression on LN DC is enhanced after Treg depletion unlike CD40 or CD80 expression. Flow cytometry analysis of IL-15Rα, CD80, and CD40 cell surface expression on CD11c+/I-Ab+ cells before (upper panel) and after PC61 (lower panel) administration. Shaded histograms represent isotype control; open histograms represent IL-15Rα, CD80, or CD40 staining. B, IL-15Rα expression on DC is enhanced in the absence of Treg and is dependent on CD4+ self-reactive T cells. Same experimental settings as in Fig. 1,C, but Treg (PC61) or Treg and Tconv (GK1.5) were depleted. Cells were subjected to a CD11c/I-Ab/IL-15Rα triple staining. C and D, In vitro, Treg keep in check IL-15Rα expression on DC in a TGF-β-dependent manner. Same experimental settings as in Fig. 2, C and D, but after 7 days of coculture, cells were removed and subjected to a CD11c/I-Ab/IL-15Rα staining. D, Anti-TGF-β Ab is added to the coculture. A representative FACS analysis (C) and a graph (D) show the results (means ± SE) obtained from two independent experiments. ∗, p < 0.05 and ∗∗, p < 0.01.

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Therefore, in vivo expression of IL-15Rα on DC involves CD4+ T cells and is controlled by Treg. Moreover, in vitro IL-15Rα is induced on DC during a cross-talk with CD4+ T cells. These results suggest that at the steady-state Treg prevent a DC-CD4+ T cell interaction leading to the induction of IL-15Rα on DC.

In this study, we provide the first evidence that Treg control an aberrant DC/NK cell cross-talk by keeping in check CD4+ self-reactive T cells in vivo. We addressed the mechanisms by which Treg maintain the homeostasis in mouse secondary lymphoid organs. Following a functional or physical loss of Treg, DC became capable of interacting with CD4+ self-reactive T cells that resulted in a CCR5-dependent recruitment of DC into the LN (Fig. 2,A) and in an aberrant IL-15Rα-dependent NK cell proliferation and activation (Fig. 3,D and putative schema, Fig. 5). These two events might be linked and/or concomitant in that in vitro studies demonstrated that the direct CD4+T/DC self-reactive contact led to the secretion of CCR5 ligands (CCL3, CCL5; Fig. 2, C and D) and to IL-15Rα expression on the DC plasma membrane (Fig. 4,C), both events being markedly controlled by Treg (Figs. 2, C and D, and Fig. 4 C).

FIGURE 5.

Putative scenario and ménage à quatre between Treg/Tconv/DC/NK partners. At the steady state (upper panel), LN homeostasis is controlled by Treg which prevent the DC/CD4+ self-reactive T cell cross-talk. In the absence of Treg (lower panel), the DC-CD4+ T cell interaction occurs (1) and promotes CCR5 ligand secretion (2) responsible for DC recruitment (3). CD4+ T cells induce DC maturation characterized by I-Ab and IL-15Rα expression (4). IL-15Rα expression on DC triggers NK cell proliferation (5).

FIGURE 5.

Putative scenario and ménage à quatre between Treg/Tconv/DC/NK partners. At the steady state (upper panel), LN homeostasis is controlled by Treg which prevent the DC/CD4+ self-reactive T cell cross-talk. In the absence of Treg (lower panel), the DC-CD4+ T cell interaction occurs (1) and promotes CCR5 ligand secretion (2) responsible for DC recruitment (3). CD4+ T cells induce DC maturation characterized by I-Ab and IL-15Rα expression (4). IL-15Rα expression on DC triggers NK cell proliferation (5).

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These conclusions were drawn using two different experimental approaches leading to either Treg depletion (in the case of administration of anti-CD25 mAb) or Treg functional inhibition (using appropriate dosing of CTX). Using an elegant transgenic mouse model where the DT receptor was expressed under the control of the Foxp3 promoter, Kim et al. (3) were the first to show that after DT injection and the subsequent ablation of Foxp3-expressing cells, a dramatic increase (>5-fold) in conventional DC and NK cells occurred. Our data corroborate these findings and bring up the critical role played by IL-15Rα and CCR5 ligands during autoimmune reactions.

Interestingly, in the absence of Treg, the percentages (Fig. 3,B) and absolute numbers of CD3+ T cells (data not shown) did not augment and CD3+ T cells did not enter cell cycle (Fig. 3,C). The most important quantitative variations involved both DC and NK cells through enhanced homing or proliferation, respectively. Inasmuch as both DC and NK cells elaborate productive and bilateral interactions, the homeostasis of LN became markedly imbalanced. The enforced DC-NK cell interaction achieved following Flt3-L administration could break the tumor-induced tolerance inducing NK cell-dependent antitumor effects (40). The DC/NK cell cross-talk resulted in NK cell recruitment/proliferation and activation in LN (36, 41, 42), where it played a key role in promoting Th1 differentiation (42). In this study, we show that ablation of Treg also promotes NK cell triggering in LN. Because no proliferation of NK cells was observed in CD11chigh+ depleted mice (Fig. 3), we can rule out a possible involvement of IL-15Rα+ cells other than DC, such as epithelial cells. Moreover, NK cells do not incorporate BrdU in nude mice that lack Treg (our data not shown), confirming the critical role of T cells in this phenomenon.

Transpresentation of IL-15 by IL-15Rα exposed on DC is a key regulatory component of NK cell functions (36). IL-15 is essential for NK cell development and survival in vivo (33, 34, 43). IL-15 produced by DC is also an essential factor for the survival and proliferation of NK cells (38). Indeed, IL-15/IL-15Rα plays a key role in the DC-mediated NK cell priming in vitro (36, 37) and in vivo (36). TLR ligands or agonistic anti-CD40 mAb could both induce the expression of IL-15Rα on DC in vivo (36). However, cytokines such as IFN-α or IFN-γ have been identified as inducers of IL-15/IL-15Rα (36, 39, 44, 45, 46, 47). The precise mechanism by which Treg ablation and the subsequent CD4+ T-DC interaction promoted the up-regulation of IL-15Rα on DC in our system is unclear. In the absence of TLR stimulation, the activation of CD4+ Tconv could provide CD40 ligand and/or IFN-γ which could theoretically contribute to IL-15Rα expression on LN resident DC.

Once NK cells are activated, NK cells become capable of either killing or activating DC in vitro (48, 49, 50) and in vivo (51, 52, 53). The pathophysiological consequences of overt and selective DC killing or DC maturation in the LN remain unclear. The absence of Treg appears to favor DC maturation (rather than NK cell-mediated killing of the resident DC) as shown by the enhanced surface expression of MHC class II and IL-15Rα on the DC surface. The NK cell-induced DC activation may act as a positive feedback loop on NK cell triggering.

The CD4+ T cell-dependent recruitment of DC in the absence of Treg (Fig. 2) was induced by the unleashed release of CCR5 ligands in LN. In vitro, we showed that CCL3/MIP-1α and CCL5/RANTES were indeed secreted during the autoreactive interaction between DC and CD4+ Tconv. This result does not rule out the possibility that such CCR5 ligands can be secreted directly by NK cells, for instance activated by IL-2 released from CD4+ Tconv. Then, NK cells could contribute to a paracrine feedback loop of chemokine secretion, leading not only to DC recruitment but also to NK cell homing to LN. CCL3 and, to a lesser extent, CCL5 are powerful chemokines involved in transendothelial migration of immature murine BM-DC (54). In this study, we show that immature BM-DC characterized by low expression of CD40 and CD86 molecules (data not shown) failed to produce detectable amounts of CCL3 and CCL5 (Fig. 2, C and D). However, CCR5 ligands are secreted during the DC/self-reactive CD4+ T cell cross-talk in the absence of Treg. Conversely, Treg could significantly reduce this production in a dose-dependent manner (Fig. 2, C and D). It is conceivable that TGF-β is a critical component accounting for the inhibitory effects of Treg on CCR5 ligands production for the following reasons. First, TGF-β is an essential mediator used by Treg to suppress T (55, 56) and NK cell effector functions (18, 19). Second, this cytokine could inhibit the transcription of genes involved in chemotaxis such as CCL3/MIP-1α and CCL5/RANTES in microgial cells (57). Third, the inhibition of TGF-β restored the expression of IL-15Rα on DC cocultured with Tconv in the presence of Treg. Indeed, we showed that Treg could keep in check the IL-15Rα expression on DC through a TGF-β-dependent mechanism (Fig. 4 D).

In the present study, it remains unclear whether Treg acted on the level of DC or CD4+ Tconv to prevent the productive CD4+ T-DC interactions. Three mechanisms have been described to date. First, Treg can directly inhibit CD4+ T cells via cell-cell contact or regulatory cytokine secretions (58). Second, Treg can modulate the maturation stage of DC and down-regulate their Ag presentation capacity compared with stimulation by Tconv (23, 59). Third, Treg formed long-lasting conjugates with DC, preventing the priming of autoreactive T cells (60). Now, we show that Treg can prevent IL-15Rα exposure on DC, thereby limiting NK cell and potentially CD8+ T cell functions.

Treg can control NK cell effector functions when NK cells are activated through IL-12 but not through IL-2Rγ chain-dependent cytokines (18). Additional levels of control of NK cells need then to be unraveled. Our study highlights the capacity of Treg to suppress transpresentation of IL-15 by DC required for NK cell triggering and underscores the indirect inhibitory role of Treg on NK cells.

NK cells have been involved in the development of some autoimmune disease. In two different type I diabetes models, in NOD and in BDC2.5-TCR-transgenic NOD.NK1.1 mice, depletion of NK cells, respectively, by anti-asialo GM1 or anti-NK1.1 Ab markedly reduced the severity of the disease (61, 62). Furthermore, NK cells have been involved in the transition between insulitis and overt diabetes (62). In experimental autoimmune myasthenia gravis, depletion of NK1.1+ cells reduced the magnitude of the disease and decreased T cell activity (63). Because NK cells have been implicated in some autoimmune disorders and because the vast majority of the NK cell pool resides in secondary lymphoid organs, it appears critical to establish whether the two checkpoints controlled by Treg (i.e., CCR5 ligands and IL-15Rα expression) leading to NK cell triggering could synergistically contribute to the pathogenicity of autoimmune diseases. Blocking CCR5 with Met-RANTES did not significantly induce retardation of gastric atrophy in the Biermer autoimmune gastritis (64). However, it remains unclear whether NK cells play any role in the pathogenicity of autoimmune gastritis (65). In a model of streptozotocin-induced diabetes, blockade of IL-15 by soluble IL-15Rα attenuated the development of the disease (66). Moreover, administration of anti-CCR5- neutralizing Abs in NOD mice prevented the destruction of pancreatic islets (67). Therefore, the concomitant blockade of both IL-15Rα and CCR5 could represent a suitable strategy to improve NK cell-dependent autoimmune disorders (according to schema Fig. 5) such as diabetes. However, the role of NK cells in autoimmune disorders remains controversial, since in different mouse models such as colitis or experimental autoimmune encephalomyelitis, NK cell depletion increased disease incidence (68, 69). Furthermore, a novel regulatory role for NK cells in controlling activated CD4+ T cells through an interaction involving Qa-1/CD94-NKG2A has recently been highlighted (70). Harnessing this ability to eliminate autoreactive CD4+ T cells could also be conceivable in autoimmune disorders.

The authors have no financial conflict of interest.

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.

3

Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; CTX, cyclophosphamide; LN, lymph node; WT, wild type; Tconv, conventional T cell; BM-DC, bone marrow-derived DC; DT, diphtheria toxin; DTX, DT receptor.

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