Dendritic cells (DC) are potent inducers of immunity to foreign Ags, but also contribute to self-tolerance by induction of regulatory T cells or deletion/anergy of self-reactive T cells. In this study, we have studied the capacity of DC to activate naturally occurring CD4+CD25+ regulatory T cells as well as the ability of CD4+CD25+ T cells to suppress the DC-mediated activation of CD4+CD25 T cells. Mature bone marrow-derived dendritic cells, but not splenic DC, were able to induce the proliferation of CD4+CD25+ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4+CD25+ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25+ effector cells. Because bone marrow-derived dendritic cells induce proliferation of both CD4+CD25+ and CD4+CD25 T cells in vitro, it was impossible to assay the suppressive function of the CD4+CD25+ T cells using [3H]TdR uptake or CFSE dilution. We therefore measured IL-2 production in cocultures of CD4+CD25+ and CD4+CD25 T cells using the IL-2 secretion assay. Surprisingly, CD4+CD25+ T cells markedly suppressed IL-2 secretion by the CD4+CD25 T cells without inhibiting their proliferation. Collectively, these results suggest that Ag presentation by DC can induce the expansion of CD4+CD25+ T cells while simultaneously activating their ability to suppress cytokine secretion by effector T cells.

Immune tolerance is maintained by several mechanisms that allow the immune system to discriminate between self and nonself Ags. Although the majority of autoreactive T cells are deleted from the repertoire during the T cell ontogeny in the thymus, some autoreactive cells can escape the deletion process and circulate in the periphery where they need to be controlled. CD4+CD25+ T cells have been shown to play a major role in the maintenance of peripheral tolerance (1, 2, 3, 4). One of the major in vitro correlates of the in vivo suppressive functions of CD4+CD25+ T cells is their ability to suppress the activation/proliferation of naive CD4+ and CD8+ T cells in a poorly characterized cell contact-dependent, cytokine-independent manner (5, 6). Although CD4+CD25+ T cells may also suppress immune T cell activation in vivo by a cell contact-dependent mechanism, several studies have shown that some of the suppressive functions of the CD4+CD25+ T cells in autoimmunity and infectious diseases models may also be mediated by their production of suppressive cytokines such as IL-10 and/or TGF-β (7, 8, 9, 10, 11, 12).

Dendritic cells (DC)2 are also involved in the induction of self-tolerance and immunity. They represent a sparse population present in the lymphoid organs (2–3% of total cells) and tissues and have been recently divided into different subsets according to the expression of certain cell surface markers such as CD4, CD8α, and B220 (13, 14). Although DC are the major stimulators of the activation of naive T cells in vivo, it has been proposed that tissue-residing DC that exhibit an “immature” phenotype may anergize autoreactive T cells in the periphery or lead to the induction of regulatory T cells (15, 16). Although CD4+CD25+ T cells are nonresponsive to TCR stimulation in the presence of a heterogenous population of APC such as irradiated T-depleted spleen (TdS), it has previously been reported that CD4+CD25+ T cells proliferate vigorously to stimulation with anti-CD3 in the presence of mature bone marrow-derived DC (BMDC) and that CD25-mediated suppression of T cell proliferation is abrogated when BMDC are used as APC (17). Similarly, a number of studies have shown that CD4+CD25+ T cells from TCR transgenic mice will proliferate following transfer in vivo when stimulated with their cognate Ag presented by DC. These studies have raised questions regarding the in vivo relevance of the in vitro suppressive capacity of CD4+CD25+ T cells. In this study, we have compared the capacity of different DC populations to differentially activate CD4+CD25+ and CD4+CD25 T cells in vitro. We confirm the results of others that only mature BMDC are capable of stimulating the proliferation of CD4+CD25+ T cells, and that CD4+CD25+ T cells fail to suppress the proliferative responses of CD4+CD25 T cells in cocultures when mature BMDC are used as APC. Although the proliferation of CD4+CD25 T cells was not inhibited as measured in [3H]TdR incorporation assays or by CFSE dilution, their capacity to produce IL-2 was markedly inhibited by the CD4+CD25+ T cells as measured in an IL-2 production/capture assay. The implications of these results for the role of IL-2 in the expansion of CD4+CD25+ T cells and for their suppressive functions in vivo will be discussed.

Female BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD). CD80/86−/− mice on the BALB/c background were provided by R. Hodes (National Cancer Institute, Frederick, MD). TCR transgenic mice on an IL-2−/− background were obtained from Taconic Farms. Thy 1.1 congenic BALB/c mice were obtained from R. A. Seder (Vaccine Research Center, Bethesda, MD) and bred at the National Institutes of Health under specific pathogen-free conditions. All mice used were 6–8 wk old.

After isolation, spleens were fragmented and digested for 30 min at 37°C in complete medium (modified RPMI 1640 supplemented by 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, 4 × 10−7 M 2-ME, 1 mM essential amino acids, and 1 mM sodium pyruvate; all obtained from Biofluids) in the presence of Liberase Blendzyme II (Roche Molecular Biochemicals) and DNase (2 μg/ml) (Roche Applied Science). RBC were removed using an ACK lysis buffer (BioSource International). T and NK cells were depleted after addition of PE-conjugated anti-NK (DX5 clone) and anti-CD3 (2C11 clone) Abs (BD Pharmingen) and anti-PE magnetic beads on an autoMACS (Miltenyi Biotec). CD11c+ cells were then isolated by positive selection on MS magnetic columns (Miltenyi Biotec). The purity was around 95–98%. For their activation in vitro, the CD11c+ cells were cultured overnight (16 h) in complete medium supplemented with LPS (Escherichia coli strain 0111:B4; Sigma-Aldrich) at a concentration of 100 ng/ml or CpG at 0.5–1 μM (InvivoGen).

BMDC were prepared from tibias and femurs after removing the bone marrow cells by flushing with complete medium. After RBC lysis, the preparation was depleted of CD11b+ (M1/70 clone), B220+ (RA3-6B2 clone), I-A/I-E+ (M5/114.15.2 clone), and CD90.2+ (G7 clone) cells by a mixture of mAb (all obtained from BD Pharmingen) and anti-rat IgG-conjugated beads on a Dynal magnet (Dynal Biotech). The remaining cells were counted and seeded at 3 × 106 cells/well in 6-well plates in complete medium supplemented with GM-CSF (BioSource International) or GM-CSF (10 ng/ml) + IL-4 (10 ng/ml) (R&D Systems) for 5 or 7 days. The medium was changed on day 3 and on day 5, and the cells were harvested, washed, and plated again. DC were activated with LPS (100 ng/ml) or CpG (0.5–1 μM) on day 6 for 24 h. When grown in GM-CSF alone, cells were harvested from the supernatant and depleted of granulocytes with PE-conjugated mAb anti-Ly6G&C (RB6-8C5 clone; BD Pharmingen) and anti-PE-coupled magnetic beads (Miltenyi Biotec). The purity was ∼95–98%.

After activation, DC were collected and analyzed for their phenotype. They were stained with the following Abs: anti-CD11c biotinylated (HL3 clone) and streptavidin-allophycocyanin, PE-conjugated anti-CD86 (GL1 clone) or anti-CD80 (16-10A1 clone) or anti-CD40 (3/23 clone) or anti-I-A/I-E (M5/114.15.2 clone). Flow cytometry acquisition of the samples was performed on a FACSCalibur (BD Immunocytometry Systems), and they were then analyzed using CellQuest (BD Biosciences) and FlowJo (Tree Star) softwares.

CD4+CD25+ and CD4+CD25 T cells were purified as described previously (6). For most of the experiments, CD4+CD25+ T cells were sorted following enrichment of T cells on columns (R&D Systems) and staining with FITC-conjugated anti-CD4 (RM4-5 clone) and PE-conjugated anti-CD25 (PC61 clone) after Fc receptor blocking (2.4G2 clone; BD Pharmingen). Thy 1.1+ CD4+CD25 T cells were obtained using a PE-coupled depletion mixture: anti-B220 (RA3-6B2 clone), anti-NK (DX5 clone), anti-CD11b (M1/70.15 clone), anti-I-A/I-E (M5/114.15.2 clone), anti-CD8α (53-6.7 clone), anti-CD25 (PC61 clone) (all obtained from BD Pharmingen), and anti-PE microbeads on an autoMACS (Miltenyi Biotec).

Thy 1.1+ CD4+CD25 or Thy 1.2+ CD4+CD25+ T cells were labeled with 2 μM CFSE for 8 min at room temperature and washed three times in FBS and complete medium before being placed in culture.

After activation, splenic or BMDC were collected and washed twice to remove any cytokines. Viable cells were counted after exclusion of dead cells by trypan blue. For the suppression assays, purified CD4+CD25 T cells (1.5 × 105/ml) were cultured in triplicate with DC at a ratio DC:T cell of 1:20 in 96-well flat-bottom plates. An increasing number of CD4+CD25+ T cells were added to the wells. For the proliferation assays, purified CD4+CD25 or CD4+CD25+ T cells (3 × 105/ml) were seeded with different ratios of DC in triplicates in 96-well flat-bottom plates. The cultures were grown for 72 h in the presence of 1 μg/ml anti-CD3 (2C-11 clone) at 37°C. Cells were pulsed in the last 6 h with 1 μCi of [3H]TdR (Amersham Biosciences) before being collected and assessed for radioactivity. All results are expressed as the mean cpm of triplicate cultures. The SE was always <10% of the mean. CFSE-labeled cells (1.5 × 105 cells/ml) were cultured with DC at different stages of maturation and anti-CD3 (1 μg/ml) in 96-well flat-bottom plates. At 72 h, the cells were harvested from 9 to 18 wells according to the experiment, pooled, counted, and assessed for their CFSE dilution by flow cytometry.

For the IL-2 secretion/capture assay, the cells from triplicate cultures were harvested at different time points, pooled, washed twice, and stained in ice according to the manufacturer’s instructions (Miltenyi Biotec). The cells were then incubated at 37°C to allow IL-2 secretion for 45 min. The secreted IL-2 was captured on the cell surface and detected with an allophycocyanin-coupled Ab. Cells were also stained with biotinylated anti-Thy 1.1 and Pecychrome-conjugated streptavidin (BD Pharmingen) and analyzed on a FACSCalibur. For intracellular cytokine staining, the cells were harvested, pooled, and seeded in 24-well plates for 6 h in the presence of plate-bound anti-CD3 and anti-CD28 Abs (3 μg/ml each). Monensin (2 μM) (Calbiochem) was added for the last 2 h. After 6 h, the cells were collected, washed, and stained with biotinylated anti-Thy-1.1 followed by Pecychrome-conjugated streptavidin after Fc receptor blocking. The cells were then fixed in 4% paraformaldehyde, washed, and permeabilized in PBS, 0.1% saponin, 0.1% BSA before staining with allophycocyanin-coupled anti-IL-2 (JES6-5H4 clone) or anti-IL-4 (11B11 clone) or anti-IFN-γ (XMG1.2 clone) or anti-IL-10 (JES5-16E3 clone).

To evaluate the ability of immature and mature DC to function as APC for CD4+CD25+ T cells, we performed proliferation assays using two sources of DC: ex vivo-purified DC from spleens and DC generated in vitro from bone marrow. After ex vivo isolation, splenic DC exhibited low levels of expression of CD86, CD80, and CD40 molecules, but express levels of MHC class II molecules on the cell surface characteristic of a partially immature phenotype (Fig. 1, top panels, solid gray histograms). When splenic DC were cultured overnight in complete medium with or without LPS or CpG, CD86, CD80, and CD40 molecules were up-regulated (Fig. 1, top panel, thick lines), but the production of IL-12 could only be detected after addition of LPS or CpG (data not shown), suggesting that TLR ligand stimulation was required to generate the complete mature phenotype. BMDC generated in GM-CSF for 5 days were used as a source of immature DC. These cells did not express or expressed only at low levels MHC class II and costimulatory molecules (Fig. 1, middle panels, solid gray histograms). Mature BMDC were generated from immature DC by an additional 48 h of culture in GM-CSF and IL-4. These cells expressed high levels of costimulatory molecules (Fig. 1, bottom panels, solid gray histograms), but further up-regulation of costimulatory molecule expression was seen when these cells were stimulated by TLR ligands for 24 h (Fig. 1, bottom panels, thick lines).

FIGURE 1.

Phenotypes of splenic and BM-derived DC at different stages of maturation. In the top panels, DC were isolated from spleens and analyzed ex vivo (solid gray histograms) or after overnight activation with LPS or CpG (thick line histograms) by flow cytometry for their phenotype. In the middle panels, DC were grown in GM-CSF for 5 days, harvested, and analyzed for their phenotype (solid gray histograms). In the bottom panels, DC were grown in GM-CSF and IL-4 for 7 days and activated (thick line histograms) or not (solid gray histograms) on day 6 for 24 h with LPS or CpG.

FIGURE 1.

Phenotypes of splenic and BM-derived DC at different stages of maturation. In the top panels, DC were isolated from spleens and analyzed ex vivo (solid gray histograms) or after overnight activation with LPS or CpG (thick line histograms) by flow cytometry for their phenotype. In the middle panels, DC were grown in GM-CSF for 5 days, harvested, and analyzed for their phenotype (solid gray histograms). In the bottom panels, DC were grown in GM-CSF and IL-4 for 7 days and activated (thick line histograms) or not (solid gray histograms) on day 6 for 24 h with LPS or CpG.

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We cultured CD4+CD25+ and CD4+CD25 T cells with a graded number of unstimulated or TLR-activated splenic DC (Fig. 2,A) or immature, mature, or TLR ligand-activated BMDC (Fig. 2,B) in the presence of soluble anti-CD3, but in the absence of exogenous IL-2. As expected, CD4+CD25 T cells (Fig. 2,A, right panel), but not CD4+CD25+ T cells (Fig. 2,A, left panel), proliferated vigorously in the presence of splenic DC. Splenic DC activated with LPS or CpG were only slightly more effective stimulators than splenic DC activated with medium alone. CD4+CD25 T cells could also be efficiently activated in the presence of both immature and mature BMDC (Fig. 2,B, right panel). In contrast, CD4+CD25+ T cells did not respond to either medium or TLR ligand-activated splenic DC (Fig. 2,A, left panel), but did manifest significant levels of proliferation when stimulated in the presence of mature, but not immature, BMDC (Fig. 2 B, left panel). Higher proliferative responses were seen in the presence of TLR ligand-activated mature BMDC. It should be noted that proliferation of CD4+CD25+ T cells was only observed at high ratios of DC to responder (1:1 or 1:2), whereas the CD4+CD25 T cells proliferated at much lower ratios of DC to responder (1:5 to 1:20). CD4+CD25 T cells responded in the presence of immature BMDC, whereas CD4+CD25+ T cells could not be activated by immature DC, even when high numbers of DC were used. It is likely that the interaction of immature DC with partially activated CD4+CD25 T cells results in completion of their “maturation,” and that activated CD4+CD25+ T cells might hamper this maturation as suggested by Misra et al. (18), or that immature DC lack a signal required to stimulate CD4+CD25+ T cells.

FIGURE 2.

CD4+CD25+ T cells require mature BM-derived DC to proliferate. A, CD4+CD25+ T cells (3 × 104/well; left panel) or CD4+CD25 T cells (3 × 104/well; right panel) were grown in the presence of anti-CD3 (1 μg/ml), and an increasing number of splenic DC activated overnight in medium or medium supplemented with LPS (100 ng/ml) or CpG (0.5 μM). This figure is representative of four different experiments. B, CD4+CD25+ T cells (3 × 104/well; left panel) or CD4+CD25 T cells (3 × 104/well; right panel) were grown in the presence of anti-CD3 (1 μg/ml) and an increasing number of BMDC at different stages of maturation. This figure is representative of four different experiments. C, A representative experiment showing the CFSE profile of CD4+CD25+ T cells after activation with anti-CD3 and immature, mature, LPS- or CpG-activated BMDC at a DC to T cell ratio of 1:2. In the small tables, a indicates the number of divisions; b, the percentage of cells in each CFSE peak; c, the number of cells per division; d, the geometric mean of fluorescence; e, the number of seeded cells; and f, the number of recovered cells after 72 h of culture. D, CD4+CD25+ T cells were stimulated with anti-CD3 (1 μg/ml) and immature, mature, LPS- or CpG-activated BMDC at a DC to T cell ratio of 1:2 or 1:1. After 72 h of culture, the cells were recovered and counted; the cell fold increase was calculated by dividing the number of cells recovered by the number of cells seeded. The graph is representative of four different experiments.    

FIGURE 2.

CD4+CD25+ T cells require mature BM-derived DC to proliferate. A, CD4+CD25+ T cells (3 × 104/well; left panel) or CD4+CD25 T cells (3 × 104/well; right panel) were grown in the presence of anti-CD3 (1 μg/ml), and an increasing number of splenic DC activated overnight in medium or medium supplemented with LPS (100 ng/ml) or CpG (0.5 μM). This figure is representative of four different experiments. B, CD4+CD25+ T cells (3 × 104/well; left panel) or CD4+CD25 T cells (3 × 104/well; right panel) were grown in the presence of anti-CD3 (1 μg/ml) and an increasing number of BMDC at different stages of maturation. This figure is representative of four different experiments. C, A representative experiment showing the CFSE profile of CD4+CD25+ T cells after activation with anti-CD3 and immature, mature, LPS- or CpG-activated BMDC at a DC to T cell ratio of 1:2. In the small tables, a indicates the number of divisions; b, the percentage of cells in each CFSE peak; c, the number of cells per division; d, the geometric mean of fluorescence; e, the number of seeded cells; and f, the number of recovered cells after 72 h of culture. D, CD4+CD25+ T cells were stimulated with anti-CD3 (1 μg/ml) and immature, mature, LPS- or CpG-activated BMDC at a DC to T cell ratio of 1:2 or 1:1. After 72 h of culture, the cells were recovered and counted; the cell fold increase was calculated by dividing the number of cells recovered by the number of cells seeded. The graph is representative of four different experiments.    

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One major problem with the analysis of the in vitro responsiveness of the CD4+CD25+ T cells is that they are unlikely to be a homogeneous population and even could be contaminated with CD4+CD25 effector T cells. It is possible that the proliferative responses of the CD4+CD25+ T cells, detected by the [3H]TdR incorporation assay in the presence of high numbers of mature BMDC, were mediated by a minor population of contaminating effector cells. We therefore performed a CFSE proliferation assay (Fig. 2,C) and showed that the CD4+CD25+ T cells were proliferating in a homogeneous manner. After 3 days, the cells were recovered, and the results paralleled those obtained in the [3H]TdR study in that at a DC to T cell ratio of 1:1, few cells could be recovered when the CD4+CD25+ T cells were activated by immature DC (fold increase <2), whereas much higher numbers were obtained with mature and TLR ligand-activated BMDC (fold increase ≥2; Fig. 2,D). Moreover, the CFSE profiles and the number of recovered number of cells per division (Fig. 2 C, column c) suggested that a significant number of these cells might have been through four to five rounds of division (column a) after activation with mature or TLR-activated BMDC. However, one limitation of this in vitro system is that cells may die before, during, or after division in a nonspecific manner or after activation. For this reason, it was difficult to give a precise quantitative estimate of the precursor frequencies of the divided and undivided populations. CD4+CD25+ T cells did not divide when cultured alone in the presence of LPS or CpG (data not shown) or with immature BMDC in the continuous presence of LPS or CpG. Taken together, these studies demonstrate that activated mature DC are highly efficient stimulators of a large fraction of highly purified CD4+CD25+ T cells in the absence of exogenous cytokines.

Because CD4+CD25+ T cells are generally regarded as being unable to activate their autocrine production of IL-2, it was important to determine the cellular source of the cytokine (if any) that was responsible for driving their proliferative response in the presence of activated BMDC. It has been reported that LPS or CpG-stimulated DC cell lines or BMDC (19, 20) are capable of producing IL-2. However, we were unable to detect any IL-2 production in our BMDC cultures by ELISA (data not shown). As an alternative approach to rule out the possibility that the BMDC were the source of IL-2, we used BMDC from IL-2−/− mice. BMDC generated from wild-type (wT) or IL-2−/− mice were activated for 24 h with LPS, and their maturation status was assessed by FACS analysis. Both wT and IL-2−/− DC expressed comparable levels of costimulatory molecules (data not shown). BMDC generated from wT or IL-2−/− mice were equivalent in their ability to drive the proliferation of CD4+CD25+ T cells (Fig. 3,A) and CD4+CD25 T cells (Fig. 3,B) when used at a DC to T cell ratio of 1:1. As previously observed, CD4+CD25+ T cells did not proliferate as vigorously as CD4+CD25 T cells, but the maximum response obtained with either population of DC was similar. Most importantly, the number of recovered CD4+CD25+ T cells after activation with wT or IL-2−/− BMDC (at a DC to T cell ratio of 1:1) were quite equivalent (Fig. 3,C; fold increase of 8.2 vs 6.1), and similar results were observed for the number of recovered CD4+CD25 T cells after activation with BMDC (DC to T cell ratio of 1:20) derived from wT or IL-2−/− mice (Fig. 3 D; fold increase 7.8 vs 5.9).

FIGURE 3.

The proliferation of the CD4+CD25+ T cells is not dependent on IL-2 produced by BMDC and only partially dependent on IL-2 produced by effector T cells. CD4+CD25+ T cells (A) or CD4+CD25 T cells (B) were cultured in the presence of LPS-activated BMDC derived from wT or IL-2−/− mice at a ratio DC to T cell of 1:1 and anti-CD3 (1 μg/ml). Anti-IL-2 (20 μg/ml) and anti-CD25 (20 μg/ml) mAb were added to these cultures as indicated. [3H]TdR incorporation was determined at 72 h. CD4+CD25+ T cells (C) or CD4+CD25 T cells (D) were cultured in the presence of LPS-activated BMDC derived from wT or IL-2−/− mice at a ratio DC to T cell of 1:1 and 1:20, respectively, and anti-CD3 (1 μg/ml). At 72 h, the cells were recovered and counted: a, the number of seeded cells; b, the number of recovered cells; and the cell fold increase was calculated by dividing the number of cells recovered by the number of cells seeded. CD4+CD25+ (E) and CD4+CD25 (F) T cells were analyzed for their proliferation in the presence of LPS-activated BMDC generated from wT or CD80/86−/− mice at a DC to T cell ratio of 1:1 for CD4+CD25+ T cells and at a ratio DC to T cell ratio of 1:20 for CD4+CD25 T cells. G, After activation for 72 h in the presence of wT-derived BMDC (DC to T cell ratio of 1:1 for CD4+CD25+ and 1:20 for CD4+CD25 T cells), the cells were harvested, re-stimulated with anti-CD3/anti-CD28 for 6 h, and their cytokine production was assessed by intracellular staining.

FIGURE 3.

The proliferation of the CD4+CD25+ T cells is not dependent on IL-2 produced by BMDC and only partially dependent on IL-2 produced by effector T cells. CD4+CD25+ T cells (A) or CD4+CD25 T cells (B) were cultured in the presence of LPS-activated BMDC derived from wT or IL-2−/− mice at a ratio DC to T cell of 1:1 and anti-CD3 (1 μg/ml). Anti-IL-2 (20 μg/ml) and anti-CD25 (20 μg/ml) mAb were added to these cultures as indicated. [3H]TdR incorporation was determined at 72 h. CD4+CD25+ T cells (C) or CD4+CD25 T cells (D) were cultured in the presence of LPS-activated BMDC derived from wT or IL-2−/− mice at a ratio DC to T cell of 1:1 and 1:20, respectively, and anti-CD3 (1 μg/ml). At 72 h, the cells were recovered and counted: a, the number of seeded cells; b, the number of recovered cells; and the cell fold increase was calculated by dividing the number of cells recovered by the number of cells seeded. CD4+CD25+ (E) and CD4+CD25 (F) T cells were analyzed for their proliferation in the presence of LPS-activated BMDC generated from wT or CD80/86−/− mice at a DC to T cell ratio of 1:1 for CD4+CD25+ T cells and at a ratio DC to T cell ratio of 1:20 for CD4+CD25 T cells. G, After activation for 72 h in the presence of wT-derived BMDC (DC to T cell ratio of 1:1 for CD4+CD25+ and 1:20 for CD4+CD25 T cells), the cells were harvested, re-stimulated with anti-CD3/anti-CD28 for 6 h, and their cytokine production was assessed by intracellular staining.

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To further evaluate the role of IL-2 in these cultures, we added a combination of a neutralizing anti-IL-2 (mAb S4B6) and a blocking anti-CD25 (mAb PC61) to these cocultures. Although the proliferation of naive T cells is regarded as being almost completely dependent on IL-2, the addition of anti-IL-2/CD25 only reduced the proliferation of CD4+CD25 T cells by 60% in these cultures at a DC to T cell ratio of 1:1 (Fig. 3,B). Although some reduction of the much weaker response of the CD4+CD25+ T cells was also seen in the presence of anti-IL-2/CD25, the magnitude of the reduction was reproducibly less than that seen with CD4+CD25 T cells (30–40%; Fig. 3,A). Because naive CD4+CD25 T cells require CD28-mediated costimulatory signals to produce IL-2, we also compared the responses of CD4+CD25+ and CD4+CD25 T cells in the presence of activated BMDC from wT and CD80/CD86−/− mice. The response of the CD4+CD25 T cells was maintained when CD80/CD86−/− BMDC were used at a DC to T cell ratio of 1:1 (Fig. 3,F), but reduced by 68% at a DC to T cell ratio of 1:20 (data not shown). In contrast, the response of the CD4+CD25+ T cells was reduced by 50% when CD80/CD86−/− DC were used at a ratio DC to T cell ratio of 1:1 (Fig. 3 E).

Taken together, these results suggest that the proliferative responses of CD4+CD25+ T cells induced in the presence of activated BMDC are partially mediated by CD28-dependent production of IL-2 in the cocultures. One possibility is that activated BMDC activate the majority of the CD4+CD25+ T cells to produce IL-2, as has been proposed by others (17, 21). Alternatively, only a minority of CD4+CD25+ T cells may produce IL-2, and these cells may, in fact, not be bona fide regulatory T cells, but activated effectors that express CD25. To directly measure on a per cell basis the percentage of CD4+CD25+ T cells producing IL-2 in response to stimulation in the presence of activated BMDC, we first evaluated cytokine production by measuring the capacity of the stimulated cells to produce IL-2 as assayed by intracellular staining following re-stimulation with anti-CD3/CD28. CD4+CD25+ T cells were stimulated with wT BMDC (DC to T cell ratio 1: 1), whereas CD4+CD25 T cells were stimulated with wT BMDC (DC to T cell ratio of 1:20) because previous results showed that the number of recovered cells at these ratios was quite comparable (Fig. 3, C and D). Under these conditions, only 2.9% of the CD4+CD25+ T cells produced IL-2, whereas 57.3% of CD4+CD25 T cells were IL-2 producers (Fig. 3,G). CD4+CD25+ T cells activated under these conditions produced only low levels of IL-4, IFN-γ, or IL-10 (Fig. 3 G). Collectively, these studies strongly suggest that the proliferation of CD4+CD25+ T cells in our cultures induced by activated BMDC is mediated, in part, by paracrine production of IL-2 by a small number of CD4+CD25+ T cells that are presumably effector T cells that are contaminating the regulatory T cell population. A major part of the proliferative response of CD4+CD25+ T cells induced by activated BMDC appears to be mediated by an IL-2-independent pathway.

We first attempted to determine whether freshly isolated CD4+CD25+ T cells would suppress the response of CD4+CD25 T cells in the presence of LPS-activated BMDC and anti-CD3. As previously reported by other groups (17, 21), no inhibition of proliferation was observed even in the presence of high numbers of CD4+CD25+ T cells (Fig. 4 A). This result is not surprising because the studies shown above indicate that stimulation of CD4+CD25+ T cells by activated DC under these conditions induces CD4+CD25+ T cell proliferation and is likely to be responsible for the enhancement of the response when high numbers of CD4+CD25+ T cells were added.

FIGURE 4.

Mature BMDC activate CD4+CD25+ T cells to exert their suppressive activity. A, CD4+CD25 T cells (3 × 104/well) were cultured with an increasing number of CD4+CD25+ T cells for 72 h with anti-CD3 (1 μg/ml) and LPS-activated BMDC. This experiment is representative of three. B, CD4+CD25+ T cells were cultured with wT or IL-2−/− LPS-activated BMDC at DC to T cell ratio of 1:1 for 72 h. The cells were harvested, counted, and their suppressive function was evaluated in a suppression assay using HA-specific TCR transgenic CD4+CD25 T cells (5 × 104/well) as responders in the presence of HA peptide (10 μM) and TdS (ratio 1:1).

FIGURE 4.

Mature BMDC activate CD4+CD25+ T cells to exert their suppressive activity. A, CD4+CD25 T cells (3 × 104/well) were cultured with an increasing number of CD4+CD25+ T cells for 72 h with anti-CD3 (1 μg/ml) and LPS-activated BMDC. This experiment is representative of three. B, CD4+CD25+ T cells were cultured with wT or IL-2−/− LPS-activated BMDC at DC to T cell ratio of 1:1 for 72 h. The cells were harvested, counted, and their suppressive function was evaluated in a suppression assay using HA-specific TCR transgenic CD4+CD25 T cells (5 × 104/well) as responders in the presence of HA peptide (10 μM) and TdS (ratio 1:1).

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One possible interpretation of the study in Fig. 4,A is that the suppressive ability of CD4+CD25+ T cells is abrogated when they undergo a proliferative response in the presence of activated DC. To evaluate this possibility, we cultured CD4+CD25+ T cells from normal BALB/c mice with wT or IL-2−/− BMDC and anti-CD3 for 72 h and then tested their ability to exert nonspecific suppressor effector function. We added the preactivated cells to a second culture containing CD4+CD25 T cells from mice expressing a transgenic TCR specific for an hemagglutinin (HA) peptide. CD4+CD25+ T cells preactivated in the presence of wT or IL-2−/− DC were equally efficient at suppressing the response of the transgenic T cells when stimulated by peptide and TdS cells (Fig. 4 B). Thus, activation of CD4+CD25+ T cells in the presence of DC does not simply abrogate their ability to differentiate into efficient suppressors.

Because activated DC would be the most relevant APC population involved in the activation of CD4+CD25 T cells during an inflammatory response in vivo, one might conclude from the studies presented above that CD4+CD25+ T cells would be very poor suppressors of T cell activation under such conditions. However, it is also clear that the use of T cell proliferation to measure the suppressive function of CD4+CD25+ T cells is inadequate because CD4+CD25+ T cells proliferate in the presence of activated BMDC. To address these concerns, we directly measured the ability of CD4+CD25+ T cells to inhibit IL-2 production by CD4+CD25 T cells using an IL-2 capture assay to measure IL-2-producing cells. CD4+CD25 T cells were isolated from Thy-1.1+ BALB/c congenic mice, labeled with CFSE, and cultured in the presence of anti-CD3 and LPS-activated BMDC at a DC to T cell ratio of 1:20. CFSE-labeled, Thy-1.2+ CD4+CD25+ T cells were added at a suppressor to responder ratio of 1:1. IL-2 secretion from both populations was assayed after 24 and 48 h of culture. The percentage of IL-2-secreting cells in the CD4+CD25+ subset was never higher than 1.18% during the time course of the experiment (Fig. 5,A). This result is consistent with our intracellular staining studies (Fig. 3,G), which were performed at a DC to T cell ratio of 1:1. In contrast, almost 100% of the CD4+CD25 T cell population secreted IL-2 after 48 h of stimulation. In the cocultures, IL-2 production by the CD4+CD25 T cells was markedly inhibited (96 to 0.59% at 48 h). Similar results were obtained when IL-2 production was measured by intracellular staining at 72 h (Fig. 5,B). Although IL-2 secretion was inhibited in the cocultures when using a DC to T cell ratio of 1:20, both subsets appeared to proliferate as detected by dilution of CFSE and the number of recovered cells (Fig. 5 C). Because the suppressive activity of the CD4+CD25+ T cells was not immediate, the IL-2 produced in the first few hours by the CD4+CD25 population might have favored their survival and their proliferation. These results demonstrate that CD4+CD25+ T cells remain potent suppressors of cytokine production when stimulated with activated BMDC.

FIGURE 5.

CD4+CD25+ T cells suppress cytokine production by CD4+CD25 T cells but do not block their proliferative response. A, CD4+CD25+ Thy 1.2+ T cells (3 × 104/well) or CD4+CD25 Thy 1.1+ T cells (3 × 104/well) were cultured alone or together in the presence of LPS-activated BMDC (at a DC to T cell ratio of 1:20) with or without anti-CD3 (1 μg/ml). The cells were harvested after 24 h and 48 h, and IL-2 production was measured in the IL-2 capture assay. B, CD4+CD25+ Thy 1.2+ T cells (3 × 104/well) or CD4+CD25 Thy 1.1+ T cells (3 × 104/well) were cultured alone or together in the presence of LPS-activated BMDC (at a DC to T cell ratio of 1:20) with or without anti-CD3 (1 μg/ml). The cells were harvested after 72 h of culture, restimulated with anti-CD3/anti-CD28 for 6 h, and IL-2 production measured by intracellular staining. C, The CFSE profile was determined after 72 h of culture as described previously: a, the number of seeded cells; and b, the number of recovered cells after 72 h of culture. The percentage of cells per division is indicated in bold on the graph and the geometric mean of fluorescence in italic. The dot plot represents the percentage of CD4+CD25+ Thy 1.2+ and CD4+CD25 Thy 1.1+ T cells in the cocultures at 72 h. This is a representative experiment.

FIGURE 5.

CD4+CD25+ T cells suppress cytokine production by CD4+CD25 T cells but do not block their proliferative response. A, CD4+CD25+ Thy 1.2+ T cells (3 × 104/well) or CD4+CD25 Thy 1.1+ T cells (3 × 104/well) were cultured alone or together in the presence of LPS-activated BMDC (at a DC to T cell ratio of 1:20) with or without anti-CD3 (1 μg/ml). The cells were harvested after 24 h and 48 h, and IL-2 production was measured in the IL-2 capture assay. B, CD4+CD25+ Thy 1.2+ T cells (3 × 104/well) or CD4+CD25 Thy 1.1+ T cells (3 × 104/well) were cultured alone or together in the presence of LPS-activated BMDC (at a DC to T cell ratio of 1:20) with or without anti-CD3 (1 μg/ml). The cells were harvested after 72 h of culture, restimulated with anti-CD3/anti-CD28 for 6 h, and IL-2 production measured by intracellular staining. C, The CFSE profile was determined after 72 h of culture as described previously: a, the number of seeded cells; and b, the number of recovered cells after 72 h of culture. The percentage of cells per division is indicated in bold on the graph and the geometric mean of fluorescence in italic. The dot plot represents the percentage of CD4+CD25+ Thy 1.2+ and CD4+CD25 Thy 1.1+ T cells in the cocultures at 72 h. This is a representative experiment.

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CD4+CD25+ T cells have been shown in numerous studies to be the major population of regulatory/suppressor T cells responsible for control of virtually all immune responses in vitro and in vivo. The most prominent characteristics of this population of regulatory T cells is their nonresponsiveness to TCR stimulation in vitro in the presence of TdS as APC and their capacity to suppress the response of CD4+CD25 T cells in cocultures under the same culture conditions. However, recent studies have demonstrated that when mature BMDC were used as APC, the “anergic” state of the CD4+CD25+ was broken even in the absence of exogenous IL-2, and suppression of the proliferation of CD4+CD25 was abrogated. CD4+CD25+ T cells expressing a transgenic TCR specific for a foreign Ag also proliferate when stimulated with Ag-pulsed DC in vivo. Because mature DC are likely to represent the major population of APC during inflammatory states and are also likely to be activated by TLR ligands, one must therefore question the relevance of the anergic/suppressor phenotype of CD4+CD25+ T cells that has been described in vitro when a heterogeneous population of TdS are used as APC. The goals of the present study were to carefully analyze the capacity of different populations of DC to trigger the proliferation of CD4+CD25+ T cells, determine the cytokines or costimulatory molecules involved in this response, and to examine the relationship between DC-induced proliferation of CD4+CD25+ T cells and their capacity to mediate suppression of the activation of CD4+CD25 T cells.

We initially compared freshly isolated splenic DC, TLR ligand-activated splenic DC, immature BMDC, mature BMDC, and TLR ligand-activated BMDC to stimulate CD4+CD25+ and CD25 T cells. Not surprisingly, all populations of DC tested were able to trigger proliferation of CD4+CD25 T cells in the presence of anti-CD3. Even immature BMDC were capable of activating CD4+CD25 T cells, most likely reflecting in vitro activation of the BMDC by partially activated CD4+CD25 T cells. CD4+CD25+ T cell proliferation could only be triggered by mature BMDC at very high ratios of DC to T responder T cells, and their stimulatory capacity could be enhanced by prior TLR-ligand activation. TLR-activated splenic DC were ineffectual stimulators, even though they expressed levels of the tested costimulatory molecules comparable to those expressed by mature BMDC.

One important question that has been difficult to address is the mechanism responsible for proliferation of the CD4+CD25+ T cells. In general, IL-2 functions as the major, if not exclusive, growth factor for driving proliferation of naive CD4+CD25 T cells. However, one of the fundamental characteristics of CD4+CD25+ T cells is their inability to activate IL-2 gene transcription. It is possible that stimulation in the presence of high numbers of DC does induce the majority of CD4+CD25+ T cells to activate IL-2 mRNA transcription. This conclusion was drawn by Fehervari and Sakaguchi (21) who demonstrated that CD4+CD25+ T cells produced low levels of IL-2 mRNA and that low levels of IL-2 could be detected by an ELISA in culture supernatants. These results differ from our results obtained by intracellular staining or in the IL-2 production/capture assay in which we could detect a maximum of 3% of the CD4+CD25+ T cells as IL-2 producers. We do not believe these IL-2-producing T cells are regulatory/suppressor cells, but actually represent activated effector cells contaminating our highly purified regulatory cells. The IL-2 produced by these effector cells appears to be responsible for 30–40% of the proliferative response of the CD4+CD25+, as determined by the ability of a potent neutralizing anti-IL-2/blocking anti-CD25 mAb to inhibit proliferation. A similar effect of anti-CD25 on the proliferation of CD4+CD25+ T cells was noted by Yamazaki et al. (17). However, the great excess of CD4+CD25+ suppressors present in the cultures should have blocked the small amount of IL-2 produced by these purported contaminating effector cells. One possibility is that the suppressive effects of the CD4+CD25+ T cells are overcome by the very high strength of Ag signal transmitted to these effector cells by the high numbers of DC present in the culture. Similar conclusions regarding the ability of strong antigenic signals to overcome suppression have been drawn in other studies (17).

The capacity of mature BMDC to trigger proliferation of the CD4+CD25+ T cells was also partially dependent on the expression of CD80/86 by the DC. Similar results were observed in the studies of Yamazaki et al. (17). The CD80/86-derived costimulatory signal could have a direct effect either on the CD4+CD25+ or the contaminating CD25+ effector cells. TLR ligand-activated BMDC could also produce cytokines that could trigger the proliferation of the CD4+CD25+ T cells. Pasare and Medzhitov (22) have proposed that TLR-induced activation of DC induced the production of IL-6 and an unidentified factor that induced the proliferation of CD4+CD25 T cells and masked the ability to see CD25-mediated suppression in the same manner as that seen when IL-2 is added to the cultures (23). However, these DC-derived factors did not act on the regulatory T cells because they did not induce proliferation of the CD4+CD25+ T cells and also stimulated proliferation of CD4+CD25 T cells cultured in the absence of CD4+CD25+ T cells. Similarly, Fehervari and Sakaguchi (21) demonstrated that neither IL-6 nor IL-15 was able to break regulatory T cell anergy in the presence of conventional APC, and that IL-6−/− DC were very effective stimulators of CD4+CD25+ T cell proliferation. In contrast, Kubo et al. (24) have claimed that addition of the combination of IL-1 and IL-6 reversed CD4+CD25+ anergy in the presence of mature DC that were not activated by TLR ligands. Although they conclude that IL-1/IL-6 acted directly on the regulatory T cells, they did not exclude the possibility that these cytokines augmented IL-2 production by the few contaminating effector cells that were potentially present in their preparations of regulatory cells. Lastly, these data suggest that TLR-activated BMDC may express unique costimulatory molecules that are capable of inducing proliferation of CD4+CD25+ T cells in vitro by an IL-2-independent pathway. The nature of these costimulatory molecules remains unknown, although members of the TNF family (GITR-L and 4-1BB-L) represent potential candidates. Although GITR-L-transfected cells induce proliferation of CD4+CD25+, significant responses are only seen in the presence of exogenous IL-2. Thus far, we have been unable to inhibit the DC-induced proliferation of CD4+CD25+ T cells with a blocking anti-GITR-L Ab (data not shown).

The phrase “breaking T regulatory cell-mediated suppression” is misleading and must be defined in mechanistic terms. We have recently shown that highly significant suppression of the induction of IL-2 mRNA production is maintained in cocultures of CD4+CD25+ and CD4+CD25 T cells when a high concentration of IL-2 is added to the culture and in the presence of vigorous proliferation of both the CD25+ and CD25 T cells. Thus, the presence of IL-2 and perhaps DC-derived factors masks the suppressive effects of the CD25+ T cells as assayed in either [3H]TdR incorporation or CFSE dilution assays, but does not reverse the potent suppressive effects of the CD4+CD25+ on production of IL-2 by the CD4+CD25 T cells. The gold standard assay in our hands for the in vitro suppressive activity of CD4+CD25+ is therefore inhibition of IL-2 mRNA transcription as measured by real-time PCR. However, this assay does not allow one to quantify IL-2 production on a per cell basis. In this study, we have used the IL-2 capture/secretion assay to quantitatively measure IL-2 production on a per cell basis (25). Indeed, in our hands BMDC are highly effective activators of IL-2 secretion measured after 48 h, and we can routinely demonstrate that >90% of CD4+CD25 T cells produce IL-2 under these conditions. To measure the ability of CD4+CD25+ T cells to inhibit IL-2 secretion, we used a ratio of DC to responder T cells of 1:20. Marked suppression of IL-2 production was seen in the presence of vigorous proliferative responses by both the CD4+CD25 and CD4+CD25+ T cells. Our results differ from those of Kubo et al. (24) who failed to see inhibition of IL-2 production as measured in an ELISA on supernatants in cocultures of CD4+CD25+ and CD4+CD25 T cells in the presence of BMDC. However, these studies were performed at a DC to T cell ratio of 1:3. It remains possible that the strength of the signal transmitted to the responder cells overcame the suppressive effect of the CD25+ T cells. In fact, when we performed the experiments at a DC to T cell ratio of 1:1, the magnitude of suppression of IL-2 production was greatly diminished (data not shown).

At present, we can only speculate on the implications of these in vitro studies on the effects of DC on regulatory T cell function in vivo. One might question whether an in vivo equivalent of TLR ligand-stimulated mature BMDC exists, and, if so, where does it function. Indeed, CD4+CD25+ T cells have been shown to suppress certain immune responses induced by fully competent DC in vivo (26). One might also question the physiological relevance of studies in vitro that involve the use of DC to T cell ratios of 1:1 and suppressor to effector ratios of 1:1. However, dynamic imaging of T cell-DC interactions (27) in vivo certainly suggests that activation of Ag-specific T cells is observed after a one-to-one association with the Ag-bearing DC in the draining lymph node. No in vivo imaging studies are yet available that analyze the cellular interactions of the suppressors with the effectors and the DC, but it is also possible that interaction of a single effector with a single suppressor on the “platform” of a single DC may occur in vivo. Although IL-2 is the major growth factor responsible for T cell proliferation in vitro, it appears to play almost no role in the Ag-specific expansion of CD4+ T cells in vivo. CD4+ TCR transgenic T cells from IL-2−/− mice expand as efficiently as wT CD4+ T cells when challenged with Ag in vivo. If the major effects of CD4+CD25+ regulatory T cells are to suppress IL-2 production by effector cells and IL-2 is not a relevant growth factor in vivo, how do regulatory T cells mediate their suppressive effects? Some studies have observed moderate suppression in early expansion of effector T cells in vivo in the presence of Ag-specific regulatory T cells (28), but inhibition of the expansion of effectors was not seen in another report (29). The most important conclusion to be drawn from this study and our previous studies is that expansion of regulatory T cells by either a DC-derived signal or by exogenous IL-2 does not inhibit their capacity to inhibit IL-2 production by effector T cells. If IL-2 does not play a role in vivo in effector T cell function, the most logical targets for the suppressive effects of CD4+CD25+ T cells are the major polarizing cytokines, IFN-γ and IL-4, that are critical for induction of effector T cells. Studies are now in progress to determine the effects of CD4+CD25+ T cells on the induction of Th1 and Th2 effectors in vitro in the presence of exogenous IL-2 and in vivo in models of Th1- and Th2-mediated organ-specific autoimmunity.

We thank Sarah Tanksley, Thomas Moyer, and Carol Henry for their help in the sorting of CD4+CD25+ and CD4+CD25 T cells, Richard DiPaolo for the breeding of the Thy 1.1 congenic mice, and Denis Bruniquel for the helpful discussions about the IL-2 secretion/capture assay.

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.

2

Abbreviations used in this paper: DC, dendritic cell; TdS, T-depleted spleen; BMDC, bone marrow-derived dendritic cell; wT, wild-type; HA, hemagglutinin.

1
Sakaguchi, S., N. Sakaguchi.
1989
. Organ-specific autoimmune disease induced in mice by elimination of T cell subsets. V. Neonatal administration of cyclosporin A causes autoimmune disease.
J. Immunol.
142
:
471
-480.
2
Sakaguchi, S., T. H. Ermack, M. Toda, L. J. Berg, W. Ho, B. F. Destgroth, P. A. Peterson, N. Sakaguchi, M. M. Davis.
1994
. Induction of autoimmune-disease in mice by germline alteration of the T cell receptor gene-expression.
J. Immunol.
152
:
1471
-1484.
3
Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda.
1995
. Immunological self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.
J. Immunol.
155
:
1151
-1164.
4
Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi.
2000
. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4.
J. Exp. Med.
192
:
303
-309.
5
Piccirillo, C. A., E. M. Shevach.
2001
. Control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells.
J. Immunol.
167
:
1137
-1140.
6
Thornton, A. M., E. M. Shevach.
1998
. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production.
J. Exp. Med.
188
:
287
-296.
7
Cohen, J. L., A. Trenado, D. Vasey, D. Klatzmann, B. L. Salomon.
2002
. CD4+CD25+ immunoregulatory T cells: new therapeutics for graft-versus-host disease.
J. Exp. Med.
196
:
401
-406.
8
Mottet, C., H. H. Uhlig, F. Powrie.
2003
. Cure of colitis by CD4+CD25+ regulatory T cells.
J. Immunol.
170
:
3939
-3943.
9
Salomon, B. L., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe, J. A. Bluestone.
2000
. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes.
Immunity
12
:
431
-440.
10
Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie.
1999
. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation.
J. Exp. Med.
190
:
995
-1004.
11
Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, F. Powrie.
2003
. CD4+CD25+ TR cells suppress innate immune pathology through cytokine-dependent mechanisms.
J. Exp. Med.
197
:
111
-119.
12
Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman.
1996
. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlow CD4+ T cells.
J. Exp. Med.
183
:
2669
-2674.
13
Vremec, D., K. Shortman.
1997
. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes.
J. Immunol.
159
:
565
-573.
14
Vremec, D., J. Pooley, H. Hochrein, L. Wu, K. Shortman.
2000
. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen.
J. Immunol.
164
:
2978
-2986.
15
Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch, R. M. Steinman, M. C. Nussenzweig.
2001
. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo.
J. Exp. Med.
194
:
769
-779.
16
Probst, H. C., J. Lagnel, G. Kollias, M. van den Broek.
2003
. Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance.
Immunity
18
:
713
-720.
17
Yamazaki, S., T. Iyoda, K. Tarbell, K. Olson, K. Velinzon, K. Inaba, R. M. Steinman.
2003
. Direct expansion of functional CD25+CD4+ regulatory T cells by antigen-processing dendritic cells.
J. Exp. Med.
198
:
235
-247.
18
Misra, R. A., J. Bavry, S. Lacroix-Desmazes, M. D. Kazatchkine, S. V. Kaveri.
2004
. Human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells.
J. Immunol.
172
:
4676
-4680.
19
Granucci, F., C. Vizzardelli, N. Pavelka, S. Feau, M. Persico, E. Virzi, M. Rescigno, G. Moro, P. Ricciardi-Castagnoli.
2001
. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis.
Nat. Immunol.
2
:
882
-888.
20
Granucci, F., S. Feau, V. Angeli, F. Trottein, P. Ricciardi-Castagnoli.
2003
. Early IL-2 production by mouse dendritic cells is the result of microbial-induced priming.
J. Immunol.
170
:
5075
-5081.
21
Fehervari, Z., S. Sakaguchi.
2004
. Control of Foxp3+CD25+CD4+ regulatory cell activation and function by dendritic cells.
Int. Immunol.
16
:
1769
-1780.
22
Pasare, C., R. Medzhitov.
2003
. Toll-pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells.
Science
299
:
1033
-1036.
23
Thornton, A. M., E. E. Donovan, C. A. Piccirillo, E. M. Shevach.
2004
. IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function.
J. Immunol.
172
:
6519
-6523.
24
Kubo, T., R. D. Hatton, J. Olivier, X. Liu, C. O. Elson, C. T. Weaver.
2004
. Regulatory T cell suppression and anergy are differentially regulated by proinflammatory cytokines produced by TLR-activated dendritic cells.
J. Immunol.
173
:
7249
-7258.
25
Sojka, D., D. Bruniquel, R. H. Schwartz, N. J. Singh.
2004
. IL-2 secretion by CD4+ T cells in vivo is rapid, transient and influenced by TCR-specific competition.
J. Immunol.
172
:
6136
-6143.
26
Oldenhove, G., M. de Heusch, G. Urbain-Vansanten, J. Urbain, C. Maliszewski, O. Leo, M. Moser.
2003
. CD4+CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo.
J. Exp. Med.
198
:
259
-266.
27
Stoll, S., J. Delon, T. M. Brotz, R. N. Germain.
2002
. Dynamic imaging of T cell-dendritic cell interactions in lymph node.
Science
296
:
1873
-1876.
28
Walker, L. S. K., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas.
2003
. Antigen-dependent proliferation of CD4+CD25+ regulatory T cells in vivo.
J. Exp. Med.
198
:
249
-258.
29
Klein, L., K. Khazaie, H. Von Boehmer.
2003
. In vivo dynamics of antigen-specific regulatory T cells not predicted from behavior in vitro.
Proc. Natl. Acad. Sci. USA
100
:
8886
-8891.