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Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(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, CD8
, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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.
Splenic and BMDC purification and in vitro activation
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 x 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 x 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%.
FACs analysis of the DC phenotype
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.
T cell purification
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-CD8 (53-6.7 clone), anti-CD25 (PC61 clone) (all obtained from BD Pharmingen), and anti-PE microbeads on an autoMACS (Miltenyi Biotec).
CFSE labeling
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.
T cell suppression and proliferation assays
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 x 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 x 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 x 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.
IL-2 secretion capture assay and intracellular cytokine staining
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-
(XMG1.2 clone) or anti-IL-10 (JES5-16E3 clone).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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).
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2; Fig. 2D). Moreover, the CFSE profiles and the number of recovered number of cells per division (Fig. 2C, 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. 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
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. 3A) and CD4+CD25– T cells (Fig. 3B) 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. 3C; 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. 3D; fold increase 7.8 vs 5.9).
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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. 3G). CD4+CD25+ T cells activated under these conditions produced only low levels of IL-4, IFN-, or IL-10 (Fig. 3G). 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.
Mature BMDC activate CD4+CD25+ T cells to exert their suppressive activity
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. 4A). 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.
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CD4+CD25+ T cells suppress IL-2 production by CD4+CD25– T cells, but do not block their proliferative response
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. 5A). This result is consistent with our intracellular staining studies (Fig. 3G), 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. 5B). 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. 5C). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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- 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.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 Address correspondence and reprint requests to Dr. Ethan Shevach, Laboratory of Immunology/National Institutes of Health, Building 10, Room 11N315, 10 Center Drive, MSC 1892, Bethesda, MD 20892. E-mail address: eshevach{at}niaid.nih.gov 
2 Abbreviations used in this paper: DC, dendritic cell; TdS, T-depleted spleen; BMDC, bone marrow-derived dendritic cell; wT, wild-type; HA, hemagglutinin.
Received for publication May 12, 2005. Accepted for publication September 21, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155: 1151-1164. [Abstract]
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. This article has been cited by other articles:
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  Q. Chen, T. S. Davidson, E. N. Huter, and E. M. Shevach Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival J. Immunol., October 1, 2009; 183(7): 4458 - 4466. [Abstract] [Full Text] [PDF]
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 S. Piconese, G. Gri, C. Tripodo, S. Musio, A. Gorzanelli, B. Frossi, R. Pedotti, C. E. Pucillo, and M. P. Colombo Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation Blood, September 24, 2009; 114(13): 2639 - 2648. [Abstract] [Full Text] [PDF]
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D. Q. Tran, D. D. Glass, G. Uzel, D. A. Darnell, C. Spalding, S. M. Holland, and E. M. Shevach Analysis of Adhesion Molecules, Target Cells, and Role of IL-2 in Human FOXP3+ Regulatory T Cell Suppressor Function J. Immunol., March 1, 2009; 182(5): 2929 - 2938. [Abstract] [Full Text] [PDF]
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S. Yamazaki, D. Dudziak, G. F. Heidkamp, C. Fiorese, A. J. Bonito, K. Inaba, M. C. Nussenzweig, and R. M. Steinman CD8+CD205+ Splenic Dendritic Cells Are Specialized to Induce Foxp3+ Regulatory T Cells J. Immunol., November 15, 2008; 181(10): 6923 - 6933. [Abstract] [Full Text] [PDF]
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 C. Brinster and E. M. Shevach Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells J. Leukoc. Biol., August 1, 2008; 84(2): 480 - 487. [Abstract] [Full Text] [PDF]
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A. Ouabed, F.-X. Hubert, D. Chabannes, L. Gautreau, M. Heslan, and R. Josien Differential Control of T Regulatory Cell Proliferation and Suppressive Activity by Mature Plasmacytoid versus Conventional Spleen Dendritic Cells J. Immunol., May 1, 2008; 180(9): 5862 - 5870. [Abstract] [Full Text] [PDF]
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J. Hanig and M. B. Lutz Suppression of Mature Dendritic Cell Function by Regulatory T Cells In Vivo Is Abrogated by CD40 Licensing J. Immunol., February 1, 2008; 180(3): 1405 - 1413. [Abstract] [Full Text] [PDF]
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J.-G. Chai, D. Coe, D. Chen, E. Simpson, J. Dyson, and D. Scott In Vitro Expansion Improves In Vivo Regulation by CD4+CD25+ Regulatory T Cells J. Immunol., January 15, 2008; 180(2): 858 - 869. [Abstract] [Full Text] [PDF]
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S. Yamazaki, A. J. Bonito, R. Spisek, M. Dhodapkar, K. Inaba, and R. M. Steinman Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3 precursors Blood, December 15, 2007; 110(13): 4293 - 4302. [Abstract] [Full Text] [PDF]
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R. J. DiPaolo, C. Brinster, T. S. Davidson, J. Andersson, D. Glass, and E. M. Shevach Autoantigen-Specific TGFbeta-Induced Foxp3+ Regulatory T Cells Prevent Autoimmunity by Inhibiting Dendritic Cells from Activating Autoreactive T Cells J. Immunol., October 1, 2007; 179(7): 4685 - 4693. [Abstract] [Full Text] [PDF]
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H. R. Turnquist, G. Raimondi, A. F. Zahorchak, R. T. Fischer, Z. Wang, and A. W. Thomson Rapamycin-Conditioned Dendritic Cells Are Poor Stimulators of Allogeneic CD4+ T Cells, but Enrich for Antigen-Specific Foxp3+ T Regulatory Cells and Promote Organ Transplant Tolerance J. Immunol., June 1, 2007; 178(11): 7018 - 7031. [Abstract] [Full Text] [PDF]
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H. S. Adler, S. Kubsch, E. Graulich, S. Ludwig, J. Knop, and K. Steinbrink Activation of MAP kinase p38 is critical for the cell-cycle-controlled suppressor function of regulatory T cells Blood, May 15, 2007; 109(10): 4351 - 4359. [Abstract] [Full Text] [PDF]
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D. Zhang, W. Yang, N. Degauque, Y. Tian, A. Mikita, and X. X. Zheng New differentiation pathway for double-negative regulatory T cells that regulates the magnitude of immune responses Blood, May 1, 2007; 109(9): 4071 - 4079. [Abstract] [Full Text] [PDF]
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 J. Andersson, I. Stefanova, G. L. Stephens, and E. M. Shevach CD4+CD25+ regulatory T cells are activated in vivo by recognition of self Int. Immunol., April 1, 2007; 19(4): 557 - 566. [Abstract] [Full Text] [PDF]
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S. Wan, C. Xia, and L. Morel IL-6 Produced by Dendritic Cells from Lupus-Prone Mice Inhibits CD4+CD25+ T Cell Regulatory Functions J. Immunol., January 1, 2007; 178(1): 271 - 279. [Abstract] [Full Text] [PDF]
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A. Toda and C. A. Piccirillo Development and function of naturally occurring CD4+CD25+ regulatory T cells J. Leukoc. Biol., September 1, 2006; 80(3): 458 - 470. [Abstract] [Full Text] [PDF]
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S. Rutella, S. Danese, and G. Leone Tolerogenic dendritic cells: cytokine modulation comes of age Blood, September 1, 2006; 108(5): 1435 - 1440. [Abstract] [Full Text] [PDF]
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F. Billiard, E. Litvinova, D. Saadoun, F. Djelti, D. Klatzmann, J. L. Cohen, G. Marodon, and B. L. Salomon Regulatory and Effector T Cell Activation Levels Are Prime Determinants of In Vivo Immune Regulation J. Immunol., August 15, 2006; 177(4): 2167 - 2174. [Abstract] [Full Text] [PDF]
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