The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 3578 -3587
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oberle, N.
Right arrow Articles by Suri-Payer, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oberle, N.
Right arrow Articles by Suri-Payer, E.
Right arrowPubmed/NCBI databases
*Substance via MeSH

Rapid Suppression of Cytokine Transcription in Human CD4+CD25– T Cells by CD4+Foxp3+ Regulatory T Cells: Independence of IL-2 Consumption, TGF-beta, and Various Inhibitors of TCR Signaling1

Nina Oberle2,*, Nadine Eberhardt*, Christine S. Falk{dagger}, Peter H. Krammer* and Elisabeth Suri-Payer*

* Division of Immunogenetics, Tumorimmunology Program, German Cancer Research Center, Heidelberg, Germany; and {dagger} Immunomonitoring Unit, National Center for Tumor Diseases and Institute for Immunology, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+CD25high forkhead box P3+ regulatory T cells (Treg) are critical mediators of peripheral self-tolerance and immune homeostasis. Treg suppress proliferation and cytokine production of conventional T cells (Tcon). The exact mechanism of suppression, however, is still unknown. To gain a better understanding of Treg function, we investigated the kinetics of cytokine suppression in Tcon reisolated from cocultures with preactivated human Treg. Treg inhibited induction of Th1 cytokine mRNA as early as 1 h after stimulation, whereas induction/suppression of Th2 cytokines was delayed to 10–15 h. We show that immediate cytokine mRNA suppression in Tcon was neither dependent on TGF-beta/IL-10 or IL-2 consumption, nor on induction of the transcriptional-repressor forkhead box P3 or other anergy-related genes (e.g., gene related to anergy, transducer of ErbB-2, forkhead homolog-4, repressor of GATA, inducible cAMP early repressor). In contrast, lymphocyte activation gene 3, suppressor of cytokine signaling 1, and suppressor of cytokine signaling 3 mRNA were strongly up-regulated in Tcon in the presence of Treg. However, protein analysis did not confirm a role for these proteins in early suppression. Thus, the identification of a fast inhibitory mechanism in Tcon induced by Treg constitutes an important step for future efforts to unravel the entire elusive suppressive mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system has evolved to defend the organism against pathogens while maintaining tolerance to self proteins. Because tolerance can be broken and the frequency of autoimmune diseases is rising, we need to better understand the mechanisms that maintain and break self-tolerance. Studies in experimental animals have shown that the presence of CD4+CD25high forkhead box P3 (Foxp3+)3 regulatory T cells (Treg) is crucial for preventing organ-specific autoimmune diseases (1). Treg can further inhibit graft rejection, avert graft-vs-host disease as well as overwhelming organ pathology after infection, but can also dampen the immune response against cancer (2).

Although the importance of Treg for homeostasis of the immune system is well documented, the exact mechanism of action of these cells is not known. In vivo, several immunosuppressive cytokines such as TGF-beta and IL-10 were shown to be important for inhibiting colitis or experimental allergic encephalomyelitis, but seem to be unnecessary for prevention of autoimmune gastritis (3, 4, 5). Because various factors might contribute to suppression in different diseases in vivo, it might be easier to clarify the inhibitory mechanism(s) in vitro. Therefore, many groups investigated direct suppression of conventional T cells (Tcon) by Treg and revealed a contact-dependent inhibitory mechanism independent of soluble cytokines (5, 6, 7). Because Treg display membrane-bound TGF-beta on their surface, the role of TGF-beta in suppression is still a subject of debate (1, 8, 9).

The role of IL-2 consumption in the suppressive mechanism of Treg is also under dispute. Treg express all three components of the high-affinity IL-2R, CD25, CD122, and CD132, with CD25 being expressed at a high cell surface density. This is essential for Treg homeostasis in vivo and for their own activation, resulting in efficient suppression (6, 10, 11). Thus, Treg might compete with Tcon for IL-2, consume it, and inhibit Tcon proliferation (11, 12). Proliferation of Tcon can be restored by the addition of exogenous IL-2 (11, 13); however, IL-2 mRNA production is still suppressed in murine cocultures in the presence of APC (13). The question of IL-2 consumption in direct Tcon-Treg cultures has not been evaluated with human cells.

To investigate the suppressive mechanism of Treg, we focused on differences in early signaling patterns of suppressed vs nonsuppressed Tcon. Because it was shown that Treg need to be stimulated via the TCR to develop their suppressive potential (14), we had to dissociate the Treg activation phase from the later suppressive phase to directly study the suppressive mechanism. Therefore, we used preactivated Treg in our assays. This allowed us for the first time to determine the exact kinetics of Treg-mediated cytokine suppression at the mRNA and protein levels in cocultured and reisolated Tcon. Using this approach, we detected immediate suppression of IL-2 and IFN-{gamma} mRNA transcription in suppressed Tcon. Th2 cytokines were also suppressed, although at slower kinetics. To gain insight into the suppressive mechanism, we did the following: 1) revisited the role of suppressive cytokines and IL-2 consumption, and 2) examined the transcription of various inhibitors of TCR signaling that have been linked to T cell anergy or suppression of IL-2 gene transcription in the steady state (15, 16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human samples

Peripheral blood was obtained from healthy adult donors after informed consent. The blood donations were approved by the University of Heidelberg ethics committee according to the Declaration of Helsinki.

Abs, reagents, and FACS staining

mAb against human CD4 and HLA-A2 were obtained from BD Pharmingen; anti-CD25 Ab was from Miltenyi Biotec. The anti-CD3 (OKT3) and anti-CD28 (15E8) mAb were purified from hybridoma supernatants. Annexin V-Alexa Fluor 488 was purchased from Molecular Probes; anti-CD223 (lymphocyte activation gene 3 (LAG-3)) mAb from Santa Cruz Biotechnology; anti-LAG-3 mAb (17B4) from Axxora; human rTGF-beta1 (rhTGF-beta1) from Promo Cell; anti-TGF-beta-1, -2, -3 mAb and anti-human-IL-10R{alpha} mAb from R&D Systems; SB431542 from Sigma-Aldrich; and anti-human IL-2 mAb from BioSource International. FACS was performed with a Canto cytometer, and data were analyzed with Diva software (BD Biosciences).

Lymphocyte separation

Human peripheral blood leukocytes were purified from peripheral blood by Biocoll (Biochrom) gradient centrifugation, followed by plastic adherence to deplete monocytes. Blood from HLA-A2+ donors was used for isolation of Treg and from HLA-A2– donors for isolation of Tcon. We first enriched PBLs for CD25+ cells using anti-CD25 MACS beads (Miltenyi Biotec). CD4+CD25high Treg (1–4% of human CD4 cells) (17) were subsequently sorted from the CD25+ fraction, and CD4+CD25– Tcon were sorted from the CD25– fraction using the FACS-Diva cell sorter (BD Biosciences).

Cell proliferation assay and cytokine detection

Tcon (1 x 104) were incubated in 96-well flat-bottom plates (Nunc) alone or in coculture with Treg (1 x 104, or respective dilution) together with irradiated syngeneic T cell-depleted PBMC (1 x 105) and were stimulated with anti-CD3 mAb (0.2 µg/ml) and anti-CD28 mAb (0.005 µg/ml) in X-Vivo-15 medium (Cambrex) supplemented with 1% glutamine (Invitrogen Life Technologies). After 4 days at 37°C in 5% CO2, 1 µCi of [3H]thymidine/well was added for additional 16 h, and proliferation was measured in cpm using a scintillation counter. Inhibitory capacity (%) of Treg in coculture experiments was defined as follows: (1 – [3H]thymidine uptake (cpm) of coculture of Treg with Tcon divided by cpm of Tcon alone) x 100%. Alternatively, CFSE-labeled Tcon (1 x 105) were incubated in 96-well round-bottom plates (Nunc) alone or in coculture with Treg (1 x 105 or respective dilution) and were stimulated with soluble anti-CD3 (0.2 µg/ml) and anti-CD28 (0.5 µg/ml) mAb. After 4 days at 37°C in 5% CO2, proliferation was detected by measuring the CFSE dilution in Tcon using a FACS Canto. Triplicate wells were used in all experiments. To harvest supernatants daily, six coculture and control wells were set up, and 50 µl supernatants/well were harvested and pooled from three wells on days 1 and 3 and the supernatants of the remaining three wells on days 2 and 4. Cytokine content was analyzed using multiplex technology (Bioplex; Bio-Rad).

Inhibition assay for mRNA detection

Freshly isolated HLA-A2– CD4+CD25– (Tcon) and HLA-A2+ CD4+CD25high (Treg) cells were cultured alone or together in a 1:1 ratio and stimulated with soluble anti-CD3 (0.2 µg/ml) and anti-CD28 (0.5 µg/ml) mAb. When indicated, rhIL-2 (50 U/ml), anti-human IL-2 mAb (10 µg/ml), rhTGF-beta-1 (5 ng/ml), anti-TGF-beta-1, -2, -3 mAb (50 µg/ml), anti-IL-10R{alpha} mAb (10 µg/ml), isotype control Ab, or anti-LAG-3 mAb (17B4) (15 µg/ml) were added to the culture. After the stimulation period, the cell mix was labeled with anti-human HLA-A2 FITC Ab, followed by anti-FITC beads (MACS), and passed over a magnetic column (MACS). The flow through contained HLA-A2– Tcon (>98% pure). HLA-A2+ Treg were retained on the column and eluted thereafter (>75% pure). Cell death was assessed by annexin V positivity and the forward-to-sideward-scatter profile in the FACS.

RNA preparation and quantitative RT-PCR

Total RNA was isolated using the Absolutely RNA Microprep kit (Stratagene), and cDNA was prepared using random oligo(dT) primers (Invitrogen Life Technologies). Gene message was quantified by detection of incorporated SYBR Green using the ABI Prism 5700 sequence detector system (Applied Biosystems). The relative expression level was determined by normalization to GAPDH, and results are presented as fold induction over unstimulated Tcon mRNA levels set to 1. Primer sequences were as follows: GAPDH, 5'-GCA AAT TCC ATC CTC CTT TCC (forward) and 5'-TCG CCC CAC TTG ATT TTG G (reverse); IL-2, 5'-CAA CTG GAG CAT TTA CTG CTG G (forward) and 5'-TCA GTT CTG TGG CCT TCT TGG (reverse); IL-4, 5'-CAC AAG CAG CTG ATC CGA TTC (forward) and 5'-TCT GGT TGG CTT CCT TCA CAG (reverse); IL-10, 5'-GGC GCT GTC ATC GAT TTC TT (forward) and 5'-CAC TCA TGG CTT TGT AGA TGCC (reverse); IL-13, 5'-TGG AAT CCC TGA TCA ACG TGT (forward) and 5'-AAA CTG CCC AGC TGA GAC CTT (reverse); IFN-{gamma}, 5'-TTC AGC TCT GCA TCG TTT TGG (forward) and 5'-TCC GCT ACA TCT GAA TGA CCTG (reverse); FOXP3, 5'-AGC TGG AGT TCC GCA AGA AAC (forward) and 5'-TGT TCG TCC ATC CTC CTT TCC (reverse); inducible cAMP early repressor (ICER), 5'-TGA CGA GGT CCG CTA CGT AAA (forward) and 5'-TTT TGG CCA GTC TGA GTC TGC (reverse); repressor of GATA (ROG), 5'-AAG CAT CAG ATG GAG ACG CA (forward) and 5'-TTG GTC ATG GCC GAG AAGT (reverse); gene related to anergy (GRAIL), 5'-ACA CGA ATT TCA CGG TGC C (forward) and 5'-GAT GGA TCT TGT CTG CGA AGG (reverse); lung Krüppel-like factor (LKLF), 5'-GGA AAA GAC CAC GAT CCT CCT (forward) and 5'-AAG GCA TCA CAA GCC TCG AT (reverse); transducer of ErbB-2 (TOB), 5'-TTC CCA GGA GAC GTG TCA ACA (forward) and 5'-AAC CCC GAT CCT TTG TAT GGC (reverse); forkhead homolog-4 (FOXJ1), 5'-TCG GCC ATC TAC AAG TGG ATC (forward) and 5'-CGA GGC ACT TTG ATG AAG CAC (reverse); LAG-3, 5'-TGC CAC TGT CAC ATT GGC A (forward) and 5'-CCA CAC AAA GCG TTC TTG TCC (reverse); suppressor of cytokine signaling (SOCS)1, 5'-AGC TCC TTC CCC TTC CAG ATT (forward) and 5'-CCA CAT GGT TCC AGG CAA GTA (reverse); and SOCS3, CCT TCA ATT CCT CAG CTT CCC (forward) and 5'-GAG CAA ACA AGT TCC GTT GGA (reverse).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immediate suppression of Tcon cytokine production and transcription

Purification of human Treg requires special care because only CD25high cells are devoid of contaminating CD25+ effector T cells (Teff) (17, 18). Nonadherent PBMC were pre-enriched for CD25-expressing cells using magnetic beads and then FACS sorted for CD25high cells (R1, upper 1–2% of total PBMC). CD4+CD25– Tcon (R2) were sorted from the CD25-depleted PBMC fraction (Fig. 1a). Unless stated otherwise, Treg were preactivated for 16 h with anti-CD3 mAb and IL-2. Fig. 1b depicts a proliferation assay, confirming that our Treg preparations were very pure. Treg did not proliferate by themselves and inhibited Tcon proliferation by 90% at a 1:1 ratio with a proportional reduction of suppression with decreasing Treg-Tcon ratios.


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 1. CD4+CD25high T cells rapidly suppress cytokine secretion and transcription. a, Human peripheral blood leukocytes were pre-enriched with MACS-CD25 beads for CD25+ cells and then FACS sorted into CD4+CD25high cells (R1 = Treg, left panel). CD25-negative cells were FACS sorted into CD4+CD25– cells (R2 = Tcon, right panel). b, Proliferation assay: Tcon were stimulated with syngeneic irradiated T cell-depleted PBMC in the presence or absence of graded numbers of fresh autologous Treg. Cell proliferation was measured by assessing [3H]TdR incorporation. To control for nonspecific inhibition due to crowding in the well, 2x Tcon were used. c, Cytokine secretion: Tcon were stimulated with anti-CD3/anti-CD28 mAb for 4 days with or without Treg in a 1:1 ratio. On each day, supernatants were collected, and the levels of IL-2, IFN-{gamma}, IL-4, and IL-10 from Tcon (•) and cocultures of Tcon and Treg ({circ}) were determined by the BioPlex method. Treg alone secreted no/very low levels of cytokines (IL-2, IFN-{gamma}, IL-4, and IL-10), which are not depicted for clarity. Data shown are representative of two experiments using anti-CD3/anti-CD28 and are similar to two additional experiments using anti-CD3 and APC stimulation. d, Kinetic of cytokine mRNA suppression: Tcon were stimulated with anti-CD3/anti-CD28 mAb for 0, 3, 5, 10, 15, and 23 h in the presence or absence of Treg at a 1:1 ratio. IL-2, IFN-{gamma}, IL-10, or IL-4 transcription was analyzed by real-time PCR in the purified Tcon. Graphs show relative mRNA expression of control Tcon cultured alone (•) or of Tcon cocultured with Treg ({circ}). Due to limited amounts of fresh Treg, the results of two assays (0, 3, and 5 h, and 5, 10, 15, and 23 h) were combined in one graph. Mean values and SD of triplicates normalized to GAPDH are shown. The value for unstimulated Tcon was set to 1, and fold induction was calculated. Data are representative of at least three independent experiments for every time point.

 
To measure inhibition of Tcon at earlier time points than proliferation assays allow, we analyzed cytokine production from days 1 to 4 in Tcon cultures and Tcon/Treg cocultures in the absence (Fig. 1c) and presence of APC (data not shown). Except for IL-10, all cytokines tested were strongly suppressed in cocultures, whereas their concentration increased over time in uninhibited Tcon cultures (IL-2, IFN-{gamma}, IL-4, and IL-10 are shown in Fig. 1c; IL-13, IL-1, TNF-{alpha}, IL-3, IL-6, IL-8, and GM-CSF are not shown). Although at day 1 the amount of secreted cytokines was very low, inhibition was already measurable (67% ± 18 for IL-2, 63% ± 16 for IFN-{gamma}, and 70% ± 10 for IL-4; T cells from some donors did not show IL-4 secretion at this early time point; n = 4).

Because we detected inhibition of protein secretion already on day 1, we analyzed cytokine mRNA levels starting as early as 1 h after activation of Tcon with anti-CD3 and anti-CD28 mAb. At various points in time after activation, HLA-A2+ Treg were separated from the HLA-A2– Tcon. The resulting Tcon populations were 98–99.8% pure, allowing analysis of Tcon mRNA without significant contamination by Treg mRNA. In some donors, we already detected an increase of IL-2 and IFN-{gamma} mRNA 1 h after stimulation in control Tcon, and this increase was prevented in the presence of Treg (data not shown). Overall, IL-2 and IFN-{gamma} mRNA were induced after 3 h of activation in most of the donors, reaching a peak at 5–10 h and declining thereafter (Fig. 1d). In all cases, IL-2/IFN-{gamma} mRNA induction was immediately suppressed by Treg. Because not all time points could be analyzed in one single experiment due to the low numbers of Treg, data of two donors were combined. Although the kinetics of Tcon activation varied slightly between donors, immediate suppression was seen in all cases. The mean IL-2 mRNA suppression was 63% ± 25 at 1 h (n = 5); 76% ± 14.3 at 3 h (n = 6), and 85% ± 12.8 at 5 h (n = 10); for IFN-{gamma} it was 60% ± 17 at 1 h (n = 4), 75% ± 14.1 at 3 h (n = 5), and 97% ± 10.8 at 5 h (n = 7), respectively. Relative mRNA levels for the Th2 cytokines IL-4, IL-10, and IL-13 were lower and transcription was induced at later time points (10–15 h) than in Th1 cytokines. Induction of these three cytokine genes was suppressed in the presence of Treg (Fig. 1d and data not shown). IL-2/IFN-{gamma} mRNA analysis after 5 h of stimulation was chosen for further studies because at this time point significant IL-2/IFN-{gamma} mRNA induction and concomitant suppression by Treg were reliably found in all donors.

We further compared the capacity of freshly sorted vs preactivated Treg to suppress freshly isolated Tcon. After 3 and 5 h of coculture, both types of Treg were able to inhibit IL-2 transcription in cocultured Tcon to approximately the same degree (data not shown). In addition to the cytokine synthesis, cell surface expression of CD25 on Tcon was also suppressed by Treg to approximately half compared with control Tcon at various time points (data not shown). Because CD25 expression is not completely blocked on Tcon (see also Ref. 19), it is conceivable that the initial IL-2-independent up-regulation of CD25 on Tcon is not affected by Treg inhibition, whereas further up-regulation of CD25 via the IL-2 feedback loop is inhibited in the presence of Treg. In some situations, Treg have been described to inhibit Tcon via induction of cell death, especially when preactivated Treg were used (20, 21). However, in our study, neither expression of the apoptosis marker annexin V at 5–48 h, the analysis of the forward/side light scatter ratio, nor assessment by light microscopy indicated increased cell death of Tcon in the presence of Treg (data not shown).

Suppression of cytokine mRNA is independent of IL-2

Many studies have shown that addition of IL-2 to suppression assays restores proliferation (10, 11, 17). Because Treg lose their anergic state and proliferate in the presence of IL-2, restoration of proliferation might potentially be due to division of Treg in the cocultures. Therefore, detection of proliferation in cocultures does not necessarily indicate loss of Treg-suppressive capacity. By analyzing the proliferation of CFSE-labeled Tcon, we found that Tcon indeed proliferate when IL-2 and Treg are present in the culture, although a small inhibitory effect of Treg was still observed (Fig. 2a).


Figure 2
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 2. Cytokine suppression mediated by fresh human Treg is independent of IL-2. To measure proliferation, CFSE-labeled Tcon were stimulated with anti-CD3/anti-CD28 mAb for 4 days in the presence or absence of Treg at a 1:1 ratio with or without exogenous rhIL-2 (50 U/ml) (a). To measure cytokine mRNA Tcon were stimulated with anti-CD3/anti-CD28 mAb for 5 h in the presence or absence of Treg at a 1:1 ratio with or without exogenous rhIL-2 (50 U/ml) or anti-IL-2 mAb (10 µg/ml) (b and c). IL-2 (b) and IFN-{gamma} (c) mRNA were analyzed by real-time PCR in the purified Tcon population. Graphs show relative mRNA expression of control Tcon cultured alone ({blacksquare}), or of Tcon cocultured with Treg ({square}). Mean values and SD of triplicates normalized to GAPDH are shown. Data are representative of three independent experiments.

 
Because exogenous IL-2 might replace the endogenous IL-2 produced by activated Tcon, we directly measured early IL-2 mRNA in the presence or absence of human rIL-2 in the culture supernatant. As expected, addition of IL-2 to Tcon increased IL-2 mRNA production 2-fold most probably due to positive feedback of IL-2 on its own production. Nevertheless, the presence of Treg in the culture still led to an almost complete block of IL-2 mRNA production (Fig. 2b). Inhibition of IL-2 mRNA by Treg was neither affected by addition of IL-2 together with anti-IL-2 mAb (data not shown) nor by addition of anti-IL-2 mAb alone (Fig. 2b). The same result was obtained for IFN-{gamma} mRNA (Fig. 2c). In summary, our data show the following: 1) suppression of Tcon is independent of the presence or absence of IL-2, and 2) once Treg are active, they do not need further IL-2 for their suppressive function.

TGF-beta and IL-10 do not contribute to suppression of Tcon cytokine mRNA by Treg

Due to the longstanding dispute about the role of TGF-beta in Treg-mediated inhibition of CD4+ T cells, we also tested its role in our system. We hypothesized that TGF-beta might play a role in the Treg activation phase that normally takes place within the suppressive cultures, but is excluded in our fast inhibition assays with preactivated Treg. Addition of anti-TGF-beta mAb to the cultures solely containing Tcon and APC led to an increased proliferation (Fig. 3, a and b, left side). This shows that Tcon themselves (or the APC) produce TGF-beta, which inhibits their proliferation (22). Accordingly, proliferation in Tcon/Treg cocultures in the presence of anti-TGF-beta mAb is higher than in those containing only Tcon and Treg (Fig. 3a, left side). However, when we calculated suppression for the cocultures compared with the Tcon single cultures both in the absence and presence of anti-TGF-beta, we observed no significant difference (Fig. 3b, right side). It is possible that an anti-TGF-beta mAb does not have access to the space between interacting Tcon and Treg. Therefore, we also used SB431542, which blocks TGF-beta signaling by inhibiting ALK-5 (23). Titration experiments showed that this inhibitor is only effective in inhibiting TGF-beta signaling at 5 µg/ml. Lower concentrations of SB431542 do not inhibit the TGF-beta signal (23), and higher concentrations are toxic to human T cells, as revealed by cell death and increased annexin V staining, reduction in blast formation, and inhibition in diverse cytokine mRNAs (data not shown). Blockage of TGF-beta signaling with SB431542 resulted in ~35% reduction of the suppressive activity of Treg (data not shown).


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 3. Cytokine suppression mediated by fresh human Treg is only partially dependent on TGF-beta. a, On the left side, a representative proliferation assay with Tcon, Treg, and Tcon/Treg cocultures in the presence of anti-TGF-beta (50 µg/ml) mAb, anti-IL-10R mAb (10 µg/ml), and isotype control Ab (50 µg/ml) is shown. The results for the isotype control were identical with no Ab addition (data not shown). Mean values and SD of triplicate cultures are shown. On the right side, a representative quantitative PCR (SYBR Green) for IL-2 of unstimulated and stimulated control Tcon, and Tcon cocultured with Treg in a 1:1 ratio in the presence of anti-TGF-beta (50 µg/ml) mAb or rhTGF-beta (5 ng/ml) is shown. Data are representative of five independent experiments. b, Summary of the effects of rhTGF-beta (5 ng/ml), anti-TGF-beta Ab (50 µg/ml), and Treg (1:1 ratio) on the proliferation of Tcon. Percentage of proliferation relative to control Tcon (left side) or percentage of abrogation of suppression in the presence of Treg (right side) is shown. Tcon proliferation induced with anti-CD3 and irradiated APC was set to 100% (n = 5). c, Summary of the effects of rhTGF-beta, anti-TGF-beta mAb, and Treg (1:1 ratio) on IL-2 mRNA expression of Tcon. Percentage of IL-2 mRNA relative to control Tcon (left panel) or percentage of abrogation of suppression (right panel) is shown. IL-2 mRNA levels of Tcon induced with anti-CD3 and anti-CD28 were set to 100%. Mean values and SD of five independent experiments are shown.

 
Direct addition of rhTGF-beta inhibited Tcon proliferation alone by ~50%, whereas Treg suppressed >75% of Tcon proliferation independently of further addition of rhTGF-beta (Fig. 3b).

Because TGF-beta might have a different effect on cytokine transcription than on proliferation, we also analyzed its influence on early IL-2 and IFN-{gamma} mRNA induction. Addition of anti-TGF-beta mAb to purified Tcon resulted in increased IL-2 transcription, but the suppression of IL-2 induction by Treg was hardly altered (Fig. 3, a and c). Similarly, blocking TGF-beta signaling with SB431542 did not significantly reduce Treg-mediated suppression of IL-2 mRNA (data not shown). Although exogenous TGF-beta had some direct inhibitory effect on IL-2 transcription, it did not alter the high suppressive capacity of Treg (Fig. 3, a, right side, and c). Similar data were obtained for IFN-{gamma} mRNA analysis (data not shown).

Addition of anti-IL-10R mAb to proliferation or cytokine mRNA inhibition assays did not reveal any difference to the controls (Fig. 3a, left side, and data not shown). Together, these data exclude a role for TGF-beta and IL-10 in in vitro suppression of Tcon cytokine production by Treg, whereas in some donors TGF-beta may contribute moderately to the suppression of Tcon proliferation.

Inhibition of IL-2 mRNA production is not due to induction of FOXP3 or other genes implicated in T cell quiescence or anergy

To assess the possibility that the inhibitory mechanism includes changes in gene transcription and translation, we inhibited transcription via cyclohexamide and observed a reduction of IFN-{gamma} suppression (data not shown). We then analyzed the induction of various factors that have been implicated in suppression of IL-2 transcription. FOXP3 (scurfin) is expressed in Treg and is responsible for suppression of IL-2 in these cells (24). We used FOXP3-specific real-time PCR to investigate whether Treg induced FOXP3 gene expression in suppressed Tcon at early time points of culture. As depicted in Fig. 4a, Treg contain 100- to 300-fold more FOXP3 mRNA than resting Tcon. Upon stimulation, Tcon increased FOXP3 expression 5- to 10-fold compared with unstimulated Tcon. Addition of Treg to Tcon and their subsequent separation led to a small increase (2- to 3-fold) in FOXP3 mRNA in the Tcon preparation (Fig. 4a). Because this increase was remarkably constant at all time points measured, we suppose that a minimal contamination of 1% Treg in the Tcon after separation of the two cell populations is very likely responsible for this effect. Because we did not observe an increase in FOXP3 mRNA in the cocultured cells above the level detected in separately stimulated Tcon, we conclude that Treg do not induce FOXP3 expression in Tcon within the first 24 h of stimulation. Apart from FOXP3, other Forkhead transcription factors can regulate T cell activation. Thus, deficiency in FOXJ1 was shown to lead to enhanced cytokine production and autoimmunity. FOXJ1 inhibits NF-{kappa}B, and may thus prevent IL-2 mRNA transcription (25). Contrary to our expectations, FOXJ1 was increased in activated cells and suppressed in the presence of Treg (Fig. 4b).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. Suppression of cytokine mRNA is not due to induction of FOXP3 or other anergy-related genes in the suppressed Tcon. a, Tcon were stimulated with anti-CD3/anti-CD28 mAb in the presence or absence of Treg in a 1:1 ratio. FOXP3 transcription levels were analyzed by real-time PCR in control Tcon (•) and in the purified Tcon population ([circo) after 0, 3, 5, 10, 15, and 23 h of stimulation; unstimulated Treg are also shown as a control ({blacktriangleup}). Again, data obtained from two different experiments were combined, as explained in Fig. 1, and are representative of three independent experiments. b, FOXJ1, LKLF, TOB, GRAIL, ICER, and ROG transcription levels were measured by real-time PCR in unstimulated Tcon (Ø), 5-h stimulated control Tcon ({blacksquare}), and 5-h stimulated purified cocultured Tcon ({square}). Mean values of triplicates were normalized to GAPDH, and the value of unstimulated Tcon was set to 1. SD of triplicates are indicated, and data are representative of three independent experiments.

 
Next, we considered whether any of the inhibitory molecules or transcriptional repressors that have been described to play a role in inhibition of IL-2 production in anergic or quiescent T cells are induced in suppressed Tcon within 5 h (Fig. 4b). T cell quiescence is an active process regulated by specific genes rather than a default state resulting from a lack of activation. Both TOB and LKLF are expressed in steady-state T cells and are down-regulated upon T cell activation (16). TOB inhibits cyclin D1 expression, IL-2 transcription, and translation, and is also induced in anergic cells (26, 27). In concordance with the literature, both TOB and LKLF mRNAs were reduced in Tcon after activation (Fig. 4b). However, activation of Tcon in the presence of Treg did not induce either transcription factor. GRAIL and other E3 ubiquitin ligases (e.g., cbl-b) are expressed in anergic cells (15), GRAIL overexpression inhibits IL-2 and IL-4 production (28), and an increase in GRAIL mRNA has been reported in Tcon after coculture with Treg (29). Upon stimulation of Tcon, GRAIL mRNA was reduced by half compared with the resting level, and this was not changed in the presence of Treg (Fig. 4b). Furthermore, cbl-b expression was not significantly changed by TCR stimulation or suppression of Tcon (data not shown). Displacement of transcription factors such as CREB and NF-AT on the IL-2 promoter can also inhibit IL-2 production. Using this mechanism, the ICER inhibits various cytokines in human thymocytes or T cells (30). We found that during T cell activation or coactivation with Treg ICER mRNA levels remained constant (Fig. 4b). ROG is another early response gene in T cells that inhibits Th1 and Th2 cytokine expression via NF-{kappa}B inhibition (31). We therefore reasoned that Treg might lead to increased transcription of ROG, and thus limit cytokine production in suppressed T cells. We confirmed that fast up-regulation of ROG occurs after T cell stimulation, but the presence of Treg inhibited ROG transcription (Fig. 4b).

Although most of the genes tested were regulated by T cell activation (up-regulated, FOXP3, FOXJ1, and ROG; down-regulated, LKLF, TOB, and GRAIL), Treg brought about further changes in FOXJ1 and ROG in Tcon. However, they were inhibited rather than induced, and as such, none of the genes tested seem to be involved in the early suppression of Tcon cytokine mRNA by Treg.

Treg led to early induction of LAG-3, SOCS1, and SOCS3 mRNA in cocultured Tcon

LAG-3 (CD223), a CD4-related MHC class II-binding protein, was shown to be expressed on Treg and activated T cells, and engagement of LAG-3 on activated T cells was reported to reduce TCR signaling and cytokine production (32). Furthermore, LAG-3 regulates expansion of T cells in mice, and LAG-3 expressed on Treg may contribute to Tcon suppression (33, 34). Therefore, we reasoned that this molecule might play a role in suppression of Tcon by Treg. We found that Tcon contained low levels of LAG-3 mRNA in the steady-state, up-regulated LAG-3 mRNA upon stimulation and further increased LAG-3 transcription when cocultured with Treg (Fig. 5a). This LAG-3 induction was already detectable after 1 h of coculture and increased over the next 4 h. The expression of LAG-3 mRNA by Treg varied slightly between donors, but was never higher than in cocultured Tcon, and thus, increases of LAG-3 mRNA in Tcon cannot be explained by contamination with Treg. To determine whether this mRNA induction in Tcon cocultured with Treg is reflected in surface expression of LAG-3 protein, through which an inhibitory signal might be transduced, we stained cells with anti-LAG-3 mAb and analyzed them by flow cytometry. Resting unstimulated Tcon were LAG-3–/low, whereas freshly isolated Treg were LAG-3+. Upon stimulation, Treg gradually lost LAG-3 expression (Fig. 5b), probably due to shedding (35). Similarly, Tcon cultured alone or in combination with Treg slightly decreased rather than increased LAG-3 expression, as suggested by the mRNA data (Fig. 5b).


Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 5. Treg lead to early induction of LAG-3, SOCS1, and SOCS3 mRNA in cocultured Tcon. a, LAG-3 mRNA levels were measured in unstimulated (Ø) and 5-h stimulated control Tcon ({blacksquare}), purified cocultured Tcon ({square}), and purified cocultured Treg (Figure 5). Mean values of triplicates were normalized to GAPDH, and the value of unstimulated Tcon was set to 1. b, FACS staining of Tcon and Treg for LAG-3 expression. Tcon were either left untreated or stimulated with anti-CD3/anti-CD28 alone or in a 1:1 coculture with Treg for 4 h or 4 days. In the coculture, Tcon and Treg were also stained with anti-HLA-A2 to distinguish the two T cell subsets. Graphs for LAG-3 expression on the gated Tcon and Treg are shown in the third row. c, SOCS1 mRNA levels in unstimulated (Ø) or for 1- and 5-h stimulated control Tcon ({blacksquare}), purified cocultured HLA-A2-negative Tcon ({square}) and purified 5-h cocultured HLA-A2-positive Treg (Figure 5). d, SOCS3 mRNA levels in unstimulated (Ø) or 1-, 3-, and 8-h stimulated control Tcon ({blacksquare}), purified cocultured HLA-A2-negative Tcon ({square}), and purified cocultured HLA-A2-positive Treg (Figure 5). Mean values of triplicates were normalized to GAPDH, and the value of unstimulated Tcon was set to 1. e, Western blot for SOCS1, SOCS3, and ERK with unstimulated Tcon or with 5-h stimulated control Tcon and HLA-A2-negative cocultured Tcon or HLA-A2-positive cocultured Treg. Data are representative of six independent experiments.

 
We also tested the influence of a blocking anti-LAG-3 mAb (17B4) (34, 36) on the suppression of IL-2 transcription. However, the addition of this mAb to the assay did not abrogate IL-2 suppression in Tcon cocultured with Treg (data not shown). Thus, we deem it unlikely that a negative signal emanating from ligation of cell surface LAG-3 on Tcon has a function in suppression of Tcon.

SOCS proteins have initially been reported to suppress signals downstream of cytokine receptors and to inhibit IL-2 and IFN-{gamma} production (37). In addition, SOCS3 was shown to suppress CD28-mediated IL-2 production (38) and inhibit activation of NF-AT after TCR signaling (39). Because SOCS3 protein can be produced within 30 min of activation (40), we reasoned that it might be involved in suppression of Tcon by Treg. Indeed, we found a specific and strong induction of SOCS1 (Fig. 5c) and SOCS3 (Fig. 5d) mRNA 1 h following Tcon stimulation in the presence of Treg, whereas Tcon stimulation alone did not increase SOCS1 and SOCS3 mRNA. The expression of SOCS1 and SOCS3 mRNA by Treg varied slightly between donors, but was in most cases equal or lower than in cocultured Tcon (Fig. 5, c and d). Again, this rules out that the increase of SOCS mRNA is simply due to contamination by Treg. The transcription of other SOCS family members, SOCS 2, 4, 5, 6, and cytokine-inducible SH-2 domain protein (CIS), was not altered by Treg addition or by cell activation (data not shown). Western blot analysis revealed the presence of small amounts of SOCS1 protein in Tcon and higher levels in the preactivated Treg. However, TCR/CD28 stimulation of Tcon did not increase SOCS1 protein, nor did the coincubation with Treg (Fig. 5e). Similarly, SOCS3 protein was readily detectable in Treg. Depending on the donor, SOCS3 protein was not (n = 8, Fig. 5e) or barely (n = 5, data not shown) detectable in resting Tcon. Again, it was not up-regulated in activated or suppressed Tcon (Fig. 5e). We conclude that SOCS1/3 proteins are not instrumental in the early suppression of IL-2 production.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite intense research into the mechanism by which Treg suppress activation of Tcon, no molecular pathway for this cell contact-dependent inhibition has been found to date. It is conceivable that different mechanisms may come into play that might influence the following: 1) Treg activation and 2) Tcon cytokine production and proliferation. To reduce experimental variables, we used preactivated Treg in a direct Tcon-Treg inhibition assay and searched for immediate signs of Tcon activation and inhibition. Using this system, we show for the first time that Treg suppress transcription and secretion of IL-2 and IFN-{gamma} immediately after stimulation. There is low steady-state mRNA present for these cytokine genes, and an increase in IL-2 and IFN-{gamma} mRNA can be detected as early as 1 h after TCR stimulation. This early induction is already suppressed by Treg in cocultures. Th1 cytokine transcription and its suppression are reliably detectable in all donors ~5 h after activation. Therefore, we used this time point for most of our experiments. Cell death, IL-2 consumption, TGF-beta, and IL-10 can be ruled out as contributing significantly to the early suppression. Transcriptional suppressors that have been implicated in T cell quiescence or anergy are also not responsible for suppression. Although LAG-3 and SOCS1/3 transcription were induced in Tcon during Treg coculture, LAG-3 and SOCS1/3 proteins were not found to be responsible for early suppression. A role for later unresponsiveness and subsequent suppressive activity of Tcon inhibited in this manner is still possible (41).

Little is known about the kinetics of Tcon suppression by Treg. Two points should be considered in this context, as follows: 1) the time after TCR stimulation until Treg have reached their suppressive capacity, and 2) the time during which initial Tcon activation can still be abrogated. The estimates for the latter assume an interval from 12 to 38 h (42, 43). To prevent initial Tcon activation, we used Treg preactivated for 16 h. Consistent with the study by de la Rosa et al. (11), our initial observations showed that bead-purified preactivated human Treg in most donors were more potent suppressors of Tcon proliferation and cytokine secretion than freshly isolated bead-purified Treg (data not shown). However, when analyzing the kinetics of suppression of cytokine mRNA using highly pure flow cytometric sorted human Treg, we did not observe a big difference between freshly isolated and preactivated Treg. This is consistent with recently published data showing similar potency and kinetics of suppression by murine fresh and 6-h preactivated Treg. However, recent reports in the murine system indicated that suppression of IL-2 mRNA and protein occurs between 6 and 10 h. Initial IL-2 production and incomplete suppression were observed in these reports (12, 43). In contrast, we detected very fast and almost complete inhibition for Th1 cytokines, in some donors already at 1 h. Tcon of other donors started to up-regulate Th1 cytokine mRNA at 3–5 h, which was also prevented by Treg. Thus, our data suggest that either human Treg suppress faster than murine Treg, that sorted Treg may be cleaner than bead-purified Treg and therefore suppress faster, or that the sorting procedure itself leads to preactivation of Treg.

Th2 cytokines (IL-4 and IL-13) are mainly secreted by day 2, and transcription is induced at 10–15 h. These events are also suppressed by Treg. Although IL-10 mRNA is suppressed in Tcon, its presence is increased in coculture supernatants, probably due to IL-10 induction in Treg (11, 12, 23).

We agree with the data obtained in the murine system indicating that uptake of IL-2 by Treg may be essential for their activation (10, 11). However, we clearly show the following: 1) Treg after activation do not need IL-2 to suppress, and 2) the presence of IL-2 does not abrogate suppression of cytokine transcription in Tcon. Because wild-type Treg can suppress lymphoproliferative disease in IL-2-deficient mice (44), it is likely that other cytokines can induce Treg activation in the absence of IL-2.

The role of TGF-beta in the suppression of Tcon by Treg is highly controversial. Most in vitro experiments in the murine as well as in the human system have shown that various anti-TGF-beta Abs or Abs blocking its preform latency-associated peptide do not yield any or only minimal effects on inhibition of T cell proliferation in Treg cocultures (7, 42, 45, 46). We confirm and support these data. However, we did not find a significant effect on suppression of IL-2 and IFN-{gamma} mRNA. Furthermore, inhibition of cytokine transcription after 3–5 h is more pronounced upon addition of Treg than when rTGF-beta is added to Tcon cultures. In addition, in Tcon suppressed by Treg, we found no evidence of induction of TGF-beta-dependent genes such as TGF-beta itself and CXCR4 (data not shown). In summary, our data clearly argue against a major role of TGF-beta in the suppressive mechanism of human Treg in vitro and support earlier observations in the murine system that demonstrated suppression of smad3-deficient and dominant-negative TGF-betaRII T cells (7), as well as suppression conferred by TGF-beta1–/– Treg (7, 8). Our conclusions do not eliminate the possibility that under specific circumstances in vivo, e.g., inflammation or cancer, multiple suppressive mechanisms may work in concert (3, 8, 47), and that other means of Treg stimulation such as via TLR ligands might lead to the involvement of IL-10 and TGF-beta in suppression (48).

The resting or anergic state of T cells has been associated with a variety of proteins that can inhibit cytokine transcription. FOXP3/Scurfin is thought to be the main repressor of IL-2 transcription in Treg, and multiple publications have shown that Tcon can up-regulate FOXP3 mRNA and protein after TCR/CD28 costimulation (49, 50). Using almost pure FACS-sorted cell populations, we detected only a minimal increase in FOXP3 mRNA in Tcon cultures. This is consistent with recent reports indicating partial and transient increases in FOXP3 mRNA and protein levels in human Tcon that do not confer suppressive ability (51, 52). A 3-fold increase in FOXP3 mRNA in the suppressed and reisolated Tcon as we have observed is probably due to contamination with 1% Treg. This small increase is below FOXP3 mRNA levels of control Tcon. Therefore, we can rule out the possibility that induction of FOXP3 serves as a mechanism to inhibit cytokine production in Tcon at this early time point of suppression.

Furthermore, Treg coculture does not induce transcription of many factors thought to be responsible for the anergic or quiescent state of T cells. Among all the genes tested (FOXJ1, FOXO3, GRAIL, cbl-b, TOB, LKLF, CREB, CREM, ROG, BTLA, and ETS2; some data not shown), we found none that was specifically induced by Treg. As shown in Fig. 4b, the regulation of LKLF, TOB, ICER, and ROG after TCR stimulation of Tcon alone confirmed published data (15, 16). However, we did not detect the reported up-regulation of GRAIL seen in murine T cells after Treg coculture (29). This could indicate that initial suppression of IL-2 transcription takes place in the absence of GRAIL, whereas induction of GRAIL at later time points cannot be excluded. Furthermore, although Lin et al. (25) reported down-regulation of FOXJ1 mRNA at 24 h after T cell stimulation, we observed up-regulation of FOXJ1 mRNA 5 h after Tcon activation. This difference could be due to the following: 1) the depletion of Treg in our Tcon preparation, 2) the different time points analyzed, 3) different means of stimulation (plate-bound vs soluble anti-CD3), and 4) differences between the murine and the human system.

Interestingly, we observed a fast up-regulation of LAG-3, SOCS1, and SOCS3 mRNA in Tcon during Treg coculture. LAG-3 protein is present on the cell surface of Treg, and LAG-3 cross-linking on activated Tcon was reported to reduce TCR signaling (32, 34). In addition, T cells of LAG-3–/– mice showed increased proliferation in comparison with wild-type T cells (33). Our data of reduced cell surface LAG-3 expression on Treg and Tcon after T cell activation as well as the observation that an anti-LAG-3 mAb did not influence the degree of inhibition by Treg speak against an involvement of cell-bound LAG-3 in suppression. The reduction after stimulation of cell surface LAG-3 could be due to cleavage (35). Therefore, it is possible that cleaved soluble LAG-3 binds to MHC class II or not yet defined receptors on Tcon, and thereby induces a block of cytokine transcription. Cytokine production in T cells is suppressed by a negative feedback mechanism involving SOCS1 and SOCS3, which dampen IL-2 production after cytokine receptor triggering (37). It is possible that these proteins also play an inhibitory role after TCR/CD28 stimulation (38, 39). We detected strong induction of SOCS1 and SOCS3 mRNA in suppressed Tcon shortly after initiation of cocultures with Treg. Western blot analysis, however, did not show a detectable SOCS1/3 induction after 5 h. Therefore, it is unlikely that these SOCS proteins are involved in the mechanism of early suppression. It remains possible that SOCS1 and SOCS3 proteins are increased at later time points, even though IL-2 reportedly induces SOCS3 protein expression in PBMC already within 30 min (40).

In summary, already within the first 5 h after TCR stimulation, cytokine genes, as well as FOXJ1 and ROG (and to a lesser extent ICER and FOXP3) were inhibited by Treg, whereas LAG-3, SOCS1, and SOCS3 were clearly and strongly up-regulated on Tcon in the presence of Treg. Sukiennicki and Fowell (19) published data from gene chip arrays obtained from murine Tcon in comparison with suppressed Tcon at 12 and 36 h. In their study, they see differential gene expression in suppressed Tcon for most genes not before 36 h (including IL-2 and IL-4). Because we analyzed genes at 5 h, it is not possible to directly compare their and our data. In addition, most of the genes we studied were not regulated in their analyses. Furthermore, our data show that up-regulation on the transcriptional level does not necessarily also lead to up-regulation on the protein level, and more importantly, that up-regulated genes might not be relevant functionally with respect to Treg-mediated suppression. Nevertheless, one interesting commonality between our study and the one of Sukiennicki and Fowell (19) is that the genes regulated in suppressed Tcon differ from genes regulated in anergic cells and only partly overlap with genes regulated in cells treated with TGF-beta. The molecular mechanism for this differential gene regulation is not known at present. A future analysis of promoter regions and common transcription factor sites may shed light on the pathways involved.

The rapid kinetics of suppression suggest that Treg may have a direct influence on the TCR and/or CD28 signaling cascade. Our data also indicate that later changes in gene regulation do not affect suppression of Tcon activation itself, but rather the suppressive or inactive state of the suppressed cells that develops after longer coculture (41). The study of early signal transduction pathways in suppressed Tcon is difficult for several reasons, as follows: 1) physiological low TCR stimuli amenable to suppression are too weak to be measured by available test systems, and 2) only low numbers of purified Treg can be obtained from a particular blood donor. Nevertheless, our demonstration of a rapid suppression may be an important step in the endeavor to elucidate the molecular pathway(s) of suppression.


   Acknowledgments
 
D. Vobis, S. Quick, and M. Rutz are gratefully acknowledged for technical assistance, and K. Hexel for cell sorting. We further thank A. Cerwenka, R. Arnold, K. Gülow, H. Weyd, L. Dörner, B. Fritzsching, and E. M. Weiss for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the "Landesstiftung Baden-Württemberg" Germany (P-LS-AL/13) to E.S.-P. and SFB 405 to P.H.K. Back

2 Address correspondence and reprint requests to Dr. Nina Oberle, Division of Immunogenetics (D030), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg. E-mail address: N.Oberle{at}dkfz.de Back

3 Abbreviations used in this paper: FOXP3, forkhead box P3; FOXJ1, forkhead homolog-4; GRAIL, gene related to anergy; ICER, inducible cAMP early repressor; LAG-3, lymphocyte activation gene 3; LKLF, lung Krüppel-like factor; rh, recombinant human; ROG, repressor of GATA; SOCS, suppressor of cytokine signaling; Tcon, conventional T cell; TOB, transducer of ErbB-2; Treg, regulatory T cell. Back

Received for publication February 16, 2007. Accepted for publication July 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
  2. Zou, W.. 2006. Regulatory T cells, tumor immunity and immunotherapy. Nat. Rev. Immunol. 6: 295-307. [Medline]
  3. Izcue, A., J. L. Coombes, F. Powrie. 2006. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol. Rev. 212: 256-271. [Medline]
  4. Shevach, E. M., R. S. McHugh, A. M. Thornton, C. Piccirillo, K. Natarajan, D. H. Margulies. 2001. Control of autoimmunity by regulatory T cells. Adv. Exp. Med. Biol. 490: 21-32. [Medline]
  5. Suri-Payer, E., B. Fritzsching. 2006. Regulatory T cells in experimental autoimmune disease. Springer Semin. Immunopathol. 28: 3-16. [Medline]
  6. Baecher-Allan, C., D. A. Hafler. 2006. Human regulatory T cells and their role in autoimmune disease. Immunol. Rev. 212: 203-216. [Medline]
  7. Piccirillo, C. A., J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura, H. Mizuhara, E. M. Shevach. 2002. CD4+CD25+ regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness. J. Exp. Med. 196: 237-246. [Abstract/Free Full Text]
  8. Nakamura, K., A. Kitani, I. Fuss, A. Pedersen, N. Harada, H. Nawata, W. Strober. 2004. TGF-beta1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J. Immunol. 172: 834-842. [Abstract/Free Full Text]
  9. Ochi, H., M. Abraham, H. Ishikawa, D. Frenkel, K. Yang, A. S. Basso, H. Wu, M. L. Chen, R. Gandhi, A. Miller, et al 2006. Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+. Nat. Med. 12: 627-635. [Medline]
  10. Thornton, A. M., C. A. Piccirillo, E. M. Shevach. 2004. Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34: 366-376. [Medline]
  11. De la Rosa, M., S. Rutz, H. Dorninger, A. Scheffold. 2004. Interleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2480-2488. [Medline]
  12. Barthlott, T., H. Moncrieffe, M. Veldhoen, C. J. Atkins, J. Christensen, A. O’Garra, B. Stockinger. 2005. CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production. Int. Immunol. 17: 279-288. [Abstract/Free Full Text]
  13. Thornton, A. M., E. E. Donovan, C. A. Piccirillo, E. M. Shevach. 2004. Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J. Immunol. 172: 6519-6523. [Abstract/Free Full Text]
  14. Shevach, E. M.. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2: 389-400. [Medline]
  15. Mueller, D. L.. 2004. E3 ubiquitin ligases as T cell anergy factors. Nat. Immunol. 5: 883-890. [Medline]
  16. Yusuf, I., D. A. Fruman. 2003. Regulation of quiescence in lymphocytes. Trends Immunol. 24: 380-386. [Medline]
  17. Baecher-Allan, C., J. A. Brown, G. J. Freeman, D. A. Hafler. 2001. CD4+CD25high regulatory cells in human peripheral blood. J. Immunol. 167: 1245-1253. [Abstract/Free Full Text]
  18. Fritzsching, B., N. Oberle, N. Eberhardt, S. Quick, J. Haas, B. Wildemann, P. H. Krammer, E. Suri-Payer. 2005. In contrast to effector T cells, CD4+CD25+FoxP3+ regulatory T cells are highly susceptible to CD95 ligand- but not to TCR-mediated cell death. J. Immunol. 175: 32-36. [Abstract/Free Full Text]
  19. Sukiennicki, T. L., D. J. Fowell. 2006. Distinct molecular program imposed on CD4+ T cell targets by CD4+CD25+ regulatory T cells. J. Immunol. 177: 6952-6961. [Abstract/Free Full Text]
  20. Gondek, D. C., L. F. Lu, S. A. Quezada, S. Sakaguchi, R. J. Noelle. 2005. Cutting edge: contact-mediated suppression by CD4+CD25+ regulatory cells involves a granzyme B-dependent, perforin-independent mechanism. J. Immunol. 174: 1783-1786. [Abstract/Free Full Text]
  21. Grossman, W. J., J. W. Verbsky, W. Barchet, M. Colonna, J. P. Atkinson, T. J. Ley. 2004. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity 21: 589-601. [Medline]
  22. Li, M. O., Y. Y. Wan, S. Sanjabi, A. K. Robertson, R. A. Flavell. 2006. Transforming growth factor-beta regulation of immune responses. Annu. Rev. Immunol. 24: 99-146. [Medline]
  23. Oida, T., L. Xu, H. L. Weiner, A. Kitani, W. Strober. 2006. TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling. J. Immunol. 177: 2331-2339. [Abstract/Free Full Text]
  24. Rudensky, A. Y., M. Gavin, Y. Zheng. 2006. FOXP3 and NFAT: partners in tolerance. Cell 126: 253-256. [Medline]
  25. Lin, L., M. S. Spoor, A. J. Gerth, S. L. Brody, S. L. Peng. 2004. Modulation of Th1 activation and inflammation by the NF-{kappa}B repressor Foxj1. Science 303: 1017-1020. [Abstract/Free Full Text]
  26. Okochi, K., T. Suzuki, J. Inoue, S. Matsuda, T. Yamamoto. 2005. Interaction of anti-proliferative protein Tob with poly(A)-binding protein and inducible poly(A)-binding protein: implication of Tob in translational control. Genes Cells 10: 151-163. [Abstract/Free Full Text]
  27. Tzachanis, D., G. J. Freeman, N. Hirano, A. A. van Puijenbroek, M. W. Delfs, A. Berezovskaya, L. M. Nadler, V. A. Boussiotis. 2001. Tob is a negative regulator of activation that is expressed in anergic and quiescent T cells. Nat. Immunol. 2: 1174-1182. [Medline]
  28. Anandasabapathy, N., G. S. Ford, D. Bloom, C. Holness, V. Paragas, C. Seroogy, H. Skrenta, M. Hollenhorst, C. G. Fathman, L. Soares. 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18: 535-547. [Medline]
  29. Ermann, J., V. Szanya, G. S. Ford, V. Paragas, C. G. Fathman, K. Lejon. 2001. CD4+CD25+ T cells facilitate the induction of T cell anergy. J. Immunol. 167: 4271-4275. [Abstract/Free Full Text]
  30. Bodor, J., L. Feigenbaum, J. Bodorova, C. Bare, M. S. Reitz, Jr, R. E. Gress. 2001. Suppression of T-cell responsiveness by inducible cAMP early repressor (ICER). J. Leukocyte Biol. 69: 1053-1059. [Abstract/Free Full Text]
  31. Kang, B. Y., S. C. Miaw, I. C. Ho. 2005. ROG negatively regulates T-cell activation but is dispensable for Th-cell differentiation. Mol. Cell. Biol. 25: 554-562. [Abstract/Free Full Text]
  32. Hannier, S., M. Tournier, G. Bismuth, F. Triebel. 1998. CD3/TCR complex-associated lymphocyte activation gene-3 molecules inhibit CD3/TCR signaling. J. Immunol. 161: 4058-4065. [Abstract/Free Full Text]
  33. Workman, C. J., L. S. Cauley, I. J. Kim, M. A. Blackman, D. L. Woodland, D. A. Vignali. 2004. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J. Immunol. 172: 5450-5455. [Abstract/Free Full Text]
  34. Huang, C. T., C. J. Workman, D. Flies, X. Pan, A. L. Marson, G. Zhou, E. L. Hipkiss, S. Ravi, J. Kowalski, H. I. Levitsky, et al 2004. Role of LAG-3 in regulatory T cells. Immunity 21: 503-513. [Medline]
  35. Li, N., Y. Wang, K. Forbes, K. M. Vignali, B. S. Heale, P. Saftig, D. Hartmann, R. A. Black, J. J. Rossi, C. P. Blobel, et al 2007. Metalloproteases regulate T-cell proliferation and effector function via LAG-3. EMBO J. 26: 494-504. [Medline]
  36. Avice, M. N., M. Sarfati, F. Triebel, G. Delespesse, C. E. Demeure. 1999. Lymphocyte activation gene-3, a MHC class II ligand expressed on activated T cells, stimulates TNF-{alpha} and IL-12 production by monocytes and dendritic cells. J. Immunol. 162: 2748-2753. [Abstract/Free Full Text]
  37. Kubo, M., T. Hanada, A. Yoshimura. 2003. Suppressors of cytokine signaling and immunity. Nat. Immunol. 4: 1169-1176. [Medline]
  38. Matsumoto, A., Y. Seki, R. Watanabe, K. Hayashi, J. A. Johnston, Y. Harada, R. Abe, A. Yoshimura, M. Kubo. 2003. A role of suppressor of cytokine signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated interleukin 2 production. J. Exp. Med. 197: 425-436. [Abstract/Free Full Text]
  39. Banerjee, A., A. S. Banks, M. C. Nawijn, X. P. Chen, P. B. Rothman. 2002. Cutting edge: suppressor of cytokine signaling 3 inhibits activation of NFATp. J. Immunol. 168: 4277-4281. [Abstract/Free Full Text]
  40. Cohney, S. J., D. Sanden, N. A. Cacalano, A. Yoshimura, A. Mui, T. S. Migone, J. A. Johnston. 1999. SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation. Mol. Cell. Biol. 19: 4980-4988. [Abstract/Free Full Text]
  41. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, A. H. Enk. 2002. Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J. Exp. Med. 196: 255-260. [Abstract/Free Full Text]
  42. Baecher-Allan, C., V. Viglietta, D. A. Hafler. 2002. Inhibition of human CD4+CD25+high regulatory T cell function. J. Immunol. 169: 6210-6217. [Abstract/Free Full Text]
  43. Sojka, D. K., A. Hughson, T. L. Sukiennicki, D. J. Fowell. 2005. Early kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity. J. Immunol. 175: 7274-7280. [Abstract/Free Full Text]
  44. Malek, T. R., A. Yu, V. Vincek, P. Scibelli, L. Kong. 2002. CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice: implications for the nonredundant function of IL-2. Immunity 17: 167-178. [Medline]
  45. Godfrey, W. R., D. J. Spoden, Y. G. Ge, S. R. Baker, B. Liu, B. L. Levine, C. H. June, B. R. Blazar, S. B. Porter. 2005. Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function. Blood 105: 750-758.
  46. Levings, M. K., R. Sangregorio, C. Sartirana, A. L. Moschin, M. Battaglia, P. C. Orban, M. G. Roncarolo. 2002. Human CD25+CD4+ T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells. J. Exp. Med. 196: 1335-1346. [Abstract/Free Full Text]
  47. Chen, M. L., M. J. Pittet, L. Gorelik, R. A. Flavell, R. Weissleder, H. von Boehmer, K. Khazaie. 2005. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc. Natl. Acad. Sci. USA 102: 419-424. [Abstract/Free Full Text]
  48. Lewkowicz, P., N. Lewkowicz, A. Sasiak, H. Tchorzewski. 2006. Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J. Immunol. 177: 7155-7163. [Abstract/Free Full Text]
  49. Walker, M. R., D. J. Kasprowicz, V. H. Gersuk, A. Benard, L. M. Van, J. H. Buckner, S. F. Ziegler. 2003. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+. J. Clin. Invest. 112: 1437-1443. [Medline]
  50. Mantel, P. Y., N. Ouaked, B. Ruckert, C. Karagiannidis, R. Welz, K. Blaser, C. B. Schmidt-Weber. 2006. Molecular mechanisms underlying FOXP3 induction in human T cells. J. Immunol. 176: 3593-3602. [Abstract/Free Full Text]
  51. Gavin, M. A., T. R. Torgerson, E. Houston, P. DeRoos, W. Y. Ho, A. Stray-Pedersen, E. L. Ocheltree, P. D. Greenberg, H. D. Ochs, A. Y. Rudensky. 2006. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc. Natl. Acad. Sci. USA 103: 6659-6664. [Abstract/Free Full Text]
  52. Yagi, H., T. Nomura, K. Nakamura, S. Yamazaki, T. Kitawaki, S. Hori, M. Maeda, M. Onodera, T. Uchiyama, S. Fujii, S. Sakaguchi. 2004. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int. Immunol. 16: 1643-1656. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
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]

Home page
 J. Immunol.Home page
M. B. Ahmed, N. Belhadj Hmida, N. Moes, S. Buyse, M. Abdeladhim, H. Louzir, and N. Cerf-Bensussan
IL-15 Renders Conventional Lymphocytes Resistant to Suppressive Functions of Regulatory T Cells through Activation of the Phosphatidylinositol 3-Kinase Pathway
J. Immunol., June 1, 2009; 182(11): 6763 - 6770.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. H. Wang, G.-H. Chen, Y. Fan, M. Van Antwerp, and J. R. Baker Jr.
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Inhibits Experimental Autoimmune Thyroiditis by the Expansion of CD4+CD25+ Regulatory T Cells
Endocrinology, April 1, 2009; 150(4): 2000 - 2007.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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]

Home page
 J. Immunol.Home page
H. Inaba, M. Steeves, P. Nguyen, and T. L. Geiger
In Vivo Suppression of Naive CD4 T Cell Responses by IL-2- and Antigen-Stimulated T Lymphocytes in the Absence of APC Competition
J. Immunol., September 1, 2008; 181(5): 3323 - 3335.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oberle, N.
Right arrow Articles by Suri-Payer, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oberle, N.
Right arrow Articles by Suri-Payer, E.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS