Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells

Zoltán Fehérvári1 and Shimon Sakaguchi1,2,3

1 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in 53, Sakyo-ku, Kyoto 606-8507, Japan
2 Laboratory of Immunopathology, Research Center for Allergy and Immunology, The Institute for Physical and Chemical Research (RIKEN), Yokohama 230-0045, Japan
3 Core Research for Evolutional Science and Technology (CREST) Program, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Correspondence to: Z. Fehervari; E-mail: zed72{at}frontier.kyoto-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Naturally occurring CD4+CD25+ regulatory T (TR) cells play crucial roles in normal immunohomeostasis. CD4+CD25+ TR cells exhibit a number of interesting in vitro properties including a ‘default state’ of profound anergy refractory to conventional T cell stimuli. We investigated the in vitro activation requirements of CD4+CD25+ TR cells using bone marrow-derived DC, which as professional antigen presenting cells (APC) can support the activation of normal naïve T cells. Comparison of different APC types revealed that LPS-matured DC were by far the most effective at breaking CD4+CD25+ TR cell anergy and triggering proliferation, and importantly their IL-2 production. Examination of Foxp3, a key control gene for CD4+CD25+ TR cells, showed this to be stably expressed even during active proliferation. Although CD4+CD25+ TR cell proliferation was equivalent to that of CD25 cells their IL-2 production was considerably less. Use of IL-2–/– mice demonstrated that the DC stimulatory ability was not dependent on IL-2 production; nor did IL-15 appear crucial but was, at least in part, related to costimulation. DC also blocked normal CD4+CD25+ TR cell-mediated suppression partially via IL-6 secretion. DC therefore possess novel mechanisms to control the suppressive ability, expansion and/or differentiation of CD4+CD25+ TR cells in vivo.

Keywords: anergy, CD25+CD4+ TR, DC, LPS, regulation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A rapidly growing body of data highlights the critical role played by naturally occurring CD4+CD25+ regulatory cells (TR) in the control of normal immune homeostasis (1,2). Although primarily responsible for the maintenance of self-tolerance and the consequent prevention of destructive autoimmunity, recent evidence has seen their natural functional repertoire extending also to the control of tumour and pathogen immunity (36). The identification of specific markers such as the IL-2 receptor {alpha}-chain, CD25, has facilitated the experimental manipulation and characterization of TR cells (7). Most recently the forkhead-winged helix family transcription factor, Foxp3, has been shown to be specifically expressed in murine CD25+CD4+ TR cells and as such appears to be a ‘master gene’ controlling the development and suppressive function of these cells (810). Thus CD4+CD25+ TR cells constitute a functionally and developmentally unique subpopulation of T cells.

Foxp3-expressing CD25+CD4+ TR cells demonstrate a number of interesting properties, among them a profound state of in vitro and in vivo anergy, refractory to conventional T cell stimulation (5,11,12). Despite this unresponsiveness they are able to effectively suppress the activation and proliferation of CD4+ and CD8+ T cells through contact-dependent mechanisms; moreover the anergy is tightly coupled to their suppressive ability (5,12,13). TCR stimulation together with anti-CD28 costimulation, exogenous IL-2, or exposure to a lymphopenic environment is still able to break normal CD25+CD4+ TR cell anergy (5,11,14). Furthermore, the expression of TLR4 (Toll-like receptor) by CD4+CD25+ TR cells apparently renders them responsive to bacterial lipopolysaccharide (LPS), inducing both direct proliferation and synergistic enhancement of suppressive functions (15). Little is known however about the antigen presenting cells (APC) and the interactions that can influence the development and/or peripheral expansion of CD25+CD4+ TR cells and the manner in which this may occur, although evidence certainly suggests that their role may be important (1619).

We therefore set out to examine the role played by various APC in the potential activation of CD25+CD4+ TR cells. Using TCR-transgenic cells responsive to ova peptide, we observe that only dendritic cells (DC) can readily break the normal CD25+CD4+ TR cell-associated anergy, trigger their proliferation and production of IL-2, and dampen their suppressive functions while maintaining stable expression of Foxp3. CD25+CD4+ TR cell proliferation appears equivalent to that of CD25 cells but their production of IL-2 is far lower at both the protein and mRNA level. LPS-matured DC are the most effective at stimulation of CD25+CD4+ TR cells and their ability is, at least in part, dependent on the co-stimulatory molecules they express. DC are therefore important cells in the normal control of CD25+CD4+ TR cell function and expansion.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
IL-2– knock-out, BALB/c, and DO11.10 TCR transgenic mice specific for ovalbumin peptide (ova) were bred in the Institute of Frontier Medical Sciences animal facility. IL-6 knockout mice were backcrossed to BALB/c more than eight generations and bred as above (20). All mice were maintained under specific pathogen-free conditions according to institutional guidelines for animal welfare.

MAbs, reagents and media
The following mAbs were all purchased from PharMingen (San Diego, CA): anti-CD3{varepsilon}–FITC and azide-free anti-CD3{varepsilon} (145-2C11), anti-CD4–FITC (RM4-5), CD11c–PE (HL3), CD19–PE (1D3), anti-CD25–PE (7D4), CD40–FITC (3/23), CD80–FITC (16-10A1), CD86 (GL1), anti-IAd–FITC (AMS32.1) and anti-DX5–FITC. Anti-CD8 (3.155) and anti-CD24 (J11D) were used as neat tissue culture supernatants from the respective hybridomae. The anti-rat IgG used for panning was purchased from Cedarlane (Ontario, Canada). T cells were cultured in RPMI-1640, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µM 2-ME (all purchased from Sigma, St Louis, MO). Dendritic cells were grown in IMDM (Gibco/BRL, Gaithersburg, MD) supplemented as above but with the addition of rmGM-CSF and rmIL-4 (Peprotech, London, UK). LPS from Salmonella enteritidis was purchased from Sigma. Recombinant mIL-6 and mIL-15 were from Peprotech (London, UK) and IL-2 was a kind gift from Shionogi Co., Hyogo, Japan.

Preparation of CD4+ cells, DC and B cells
Six to fifteen-week old DO11.10 or BALB/c mice were used as sources of CD25+CD4+ TR cells. Initially, lymphocyte suspensions were prepared from pooled spleen and lymph nodes (axillar, branchial, cervical, inguinal and mesenteric) by forcing through a 100 µM nylon mesh followed by red blood cell lysis with an ammonium chloride-based buffer. Cells were then stained with anti-CD8 (clone 3.155) and anti-CD24 (clone J11D). Cells were next washed and plated onto 10 cm sterile non-tissue culture-treated petri-dishes pre-coated with 50 µg/ml anti-rat IgG monoclonal antibody allowing the removal of stained cells by means of panning. The now largely CD4+ (~80%) population was stained with anti-CD25–PE and anti-CD4–FITC. CD25+ cells were then sorted either magnetically by positive selection using anti-PE MACS beads and LS columns (Miltenyi Biotech, Sunnyvale, CA) or by means of an AltraTM Flow sorter (Beckman-Coulter, Miami, FL) yielding typical purities >=96% or >=99%, respectively. After CD25+ selection, conventional CD25 T cells were positively selected by anti-CD4 beads or purified by flow sorting. All incubations with mAbs and other reagents were carried out on ice for 30 min. Bone marrow derived DC were prepared according to established protocols (21). Briefly, bone marrow progenitor cells were flushed from femurs and tibiae of BALB/c, or IL-2–/– mice and dispersed into a single cell suspension. DC precursors were then grown in IMDM containing 10% FCS with the addition of rmGM-CSF and rmIL-4 (10 ng/ml final concentration for both cytokines). On day 3, supernatants and any non-adherent cells were aspirated and replaced by fresh cytokine supplemented medium. Immature DC were harvested on day 6 or 7 of culture to then be used in assays as required. Mature DC were generated by the overnight addition of 1 µg/ml of LPS (S. enteritidis; Sigma) on either day 5 or 6 of culture. DC prepared in this way were typically 60–70% CD11c+ but, where higher purities were required, cells were sorted on CD11c by MACS beads or by flow sorting to yield ~90% purity. BALB/c B cells were purified by staining whole splenocytes with anti-CD3{varepsilon}–FITC and anti-DX5–FITC. B cells were then negatively selected using anti-FITC MACS beads and an LD depletion column, yielding >90% CD19+ cells. Activated B cells were generated by overnight pre-incubation with 1 µg/ml of LPS in 24-well plates. Immature B cells were left unstimulated. All purified cells were routinely analysed by flow cytometry using a FACSCaliburTM (BD Biosciences, San Jose, CA).

Cell culture
For antigenic stimulation using TCR transgenic DO11.10 T cells and APC, 2.5 x 104 CD25+ or CD25 responders were mixed with 2.5 x 104 DC or 5 x 104 B cells in round bottom 96-well tissue culture plates (Falcon, NJ). The stimulatory ovalbumin (ova) peptide (amino acids 323–339) for the DO11.10 strain was generally added in a titrated dose to the cultures (10–0.1 µM). In some experiments DC were pre-pulsed with ova for 2 h in unsupplemented RPMI at 37°C prior to setting up in culture. All APC were washed thoroughly three times and irradiated with 18 Gy. For stimulation with anti-CD3, round bottom 96-well plates were pre-coated for >1 hour at 37°C with 5 µg/ml of antibody and 2.5 x 104 CD25 or CD25+ responder T cells were added to the wells. All T cell stimulation cultures involving the use of mDC were set-up in the presence of 20 µg/ml polymixin B to neutralize any potential LPS contaminants.

Proliferation and cytokine measurement
Cell proliferation was determined at 72 h. Wells were pulsed with 37 kBq of [3H]thymidine (DuPont-NEN, MA) for the last 6 h of culture before harvesting. For IL-2 measurement, culture supernatants were harvested after ~36 h and concentration assessed by an IL-2 sandwich ELISA (BioSource International, Camarillo, CA). TGF-ß and IL-10 were analysed from cell supernatants after ~72 h by sandwich ELISA (Promega, Madison, WI; and Biosource, respectively). The limits of IL-2, IL-10 and TGF-ß detection were approximately 3, 31 and 16 pg/ml, respectively.

Semi-quantitative and real-time PCR
Approximately 2.5 x 105–3 x 106 target cells, MACS or flow-sorted T cells and DC were stored in Isogen reagent (Nippon Gene, Tokyo, Japan). Samples were supplemented with 10 µg glycogen and total RNA was extracted using standard techniques. RNA was reverse transcribed using Superscript II reverse transcriptase and oligo(dT)12–18 primer (Life Technologies, MD) in a final volume of 20 µl. For semi-quantitative PCR cDNA levels were normalized by titration of samples and analysed for HPRT. PCR reaction mixture (40 µl) contained 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 µM forward and reverse primers, 1 U Taq, in PCR buffer (Promega). For HPRT amplification PCR consisted of 2.5 min denaturation at 94°C followed by 28 cycles as follows: 94°C for 30 s, 60°C for 30 s, 72°C for 30 s. Foxp3 PCR was carried out as above except the annealing step was 57°C and the cycle number was 30–32. IL-2 PCR also used similar conditions except the annealing step was 58°C, the extension step was for 45 s and 27 cycles were used. Primer sequences were as follows; HPRT: 5'-GTTGGATACAGGCCACACTTTGTTG-3', and 5'-GAAGGGTAGGCTGGCCTATAGGCT-3', Foxp3: 5'-CAGCTGCCTACAGTGCCCCTAG-3', and 5'-CATTTGCCAGCAGTGGGTAG-3', IL-2: 5'-AAGATGAACTTGGACCTCTGCGG-3', and 5'-CCTTATGTGTTGTAAGCAGGAGG-3'. Reactions were carried out on a BioRad iCycler (Bio-Rad). Real-time RT–PCR were carried out on an ABI/Prism 7700 sequence detection system (PE Applied Biosystems, CA). Reactions consisted of primers, an internal fluorescent TaqMan probe specific for Foxp3, HPRT or IL-2 and the QuantiTect Probe PCR kit (Qiagen, Japan). PCRs contained 0.4 µM primer, 0.2 µM TaqMan Probe, and had a 10 min denaturation step at 95°C followed by 45 cycles of 15 s at 95°C and 60 s at 60°C. The primer and probe sequences were as follows; Foxp3 primers: 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCACTTG-3'; Foxp3 probe: 5'-FAM-ATCCTACCCACTGCTGGCAAATGGAGTC-3'; HPRT primers: 5'-TGAAGAGCTACTGTAATGATCAGTCAAC-3', and 5'-AGCAAGCTTGCAACCTTAACCA-3', HPRT probe: 5'-VIC-TGCTTTCCCTGGTTAAGCAGTACAGCCC-3'. IL-2 primers: 5'-CCTGAGCAGGATGGAGAATTACA-3', and 5'-TCCAGAACATGCCGCAGAG-3', IL-2 probe: ACTCCCCAGGATGCTCACCTTCAAATTT-3'. Normalized mRNA values were calculated by dividing by the associated HPRT values and setting the value of Foxp3 from purified CD25+CD4+ samples to 100. All samples were run in triplicate.

Statistical analysis
Differences in cytokine production and proliferation were analysed using Student's t-test. Data were considered significant at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DC are able to stimulate CD25+CD4+ TR cell proliferation and perturb their normal suppressive capacity
We first characterized the antigen presenting abilities of bone marrow-derived ‘immature’ DC and ‘mature’ DC (hereafter referred to as iDC and mDC, respectively) in our experimental system. Freshly isolated CD25CD4+ TR cells from TCR transgenic DO11.10 mice were used as responder cells and stimulated by specific ova peptide. mDC were generated by the addition of 1 µg/ml LPS (from S. enteritidis) on day 5 of culture and incubated for ~24 h, whereas iDC were left unmanipulated. mDC always showed elevated levels of the activation/co-stimulatory molecules CD40, CD80, CD86 and MHC class II, typically with a 2-fold increase over their iDC counterparts (see also Fig. 5A). Furthermore, the respective maturation status of each DC population was also supported by their known abilities to phagocytose latex beads, with the iDC being consistently more efficient than mDC (data not shown). A titrated number of iDC or mDC was added to 25 000 DO11.10 CD25CD4+ T cells, corresponding to a T cell:DC ratio of 2.5:1, 1:1 or 0.5:1, and stimulatory ova peptide was added at a fixed concentration of 1 µM. All such comparisons between mDC and iDC were conducted in the presence of polymixin B to neutralize any potential LPS contamination from the mDC cultures.



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Fig. 5. Comparison of purified B cells and DC APC in the stimulation of CD25+CD4+ TR cells. B cells and DC were used unstimulated (naïve B cell and iDC respectively) or matured overnight with 1 µg/ml LPS (activated B cells or mDC). CD25+CD4+ TR cells were mixed with either 25 000 DC or 50 000 B cells. (a) Activation and co-stimulation marker expression by naïve B cells, activated B cells, iDC and mDC (shaded histograms) compared to isotype stained controls (unshaded histograms). Gated on either CD11c+ DC or CD19+ B cells. Values denote % positive and MFI (in parenthesis). (b) Proliferation of CD25+CD4+ TR cells stimulated by either naïve B cell, activated B cell, iDC or mDC. APC pre-pulsed with 0.1 µM or 1 µM ova peptide. Values expressed as means ± SD. *Significant differences (P < 0.05). Representative of two separate experiments.

 
mDC were significantly more efficient at stimulating T cell proliferation than iDC over a range of T cell:APC ratios, even when increased numbers of iDC were used relative to mDC (Fig. 1A). Given the relatively high costimulatory marker expression of mDC and the known ability of strong T cell stimuli such as anti-CD3 plus anti-CD28 to break normal CD25+CD4+ TR cell anergy (5), we assessed the role of mDC as APC for CD25+CD4+ TR cells. mDC readily broke the normal in vitro CD25+CD4+ TR cell anergy and were significantly more effective at triggering proliferation of CD25+CD4+ TR cells than iDC (Fig. 1B). Importantly, when mDC were used as APC, CD25+CD4+ TR cells proliferated to at least the same extent as their CD4+CD25 counterparts (Fig. 1C) as shown by [3H]thymidine incorporation. Similar results were seen when DC were sorted from iDC and mDC to give highly purified CD11c+ populations (data not shown). The suppressive ability of CD25+CD4+ TR cells was also assessed in the presence of either iDC or mDC (Fig. 1D). When using irradiated splenic APC (i.e. composed primarily of B cells) in standard suppression assays, the CD25+CD4+ TR cells themselves do not proliferate, in effect allowing the examination of CD25CD4+ cells in isolation. However, the potent CD25+CD4+ TR cell stimulatory capacity of mDC, and to a lesser extent iDC, complicated their use in such assays, since a summation of both CD25 and CD25+ cell proliferation was measured (data not shown). Therefore, CD25+CD4+ TR cells were treated with mitomycin C to render them mitotically inert, before using them in suppression assays. Figure 1(D) shows that such mitomycin C-treated CD25+CD4+ TR exhibited intact suppression in the presence of splenic APC (which were mainly B cells) but perturbed suppression in the presence of either iDC or mDC. Finally, the breakage of anergy and the extensive proliferation did not affect mRNA expression of the CD25+CD4+ TR cell-specific transcription factor Foxp3 for a variety of stimulation times (up to 1 week), suggesting that its expression was stable after/during activation by mDC (Fig. 1E). Conversely, the stimulation of conventional CD25CD4+ cells with iDC/mDC did not appear to induce the de novo expression of Foxp3 (data not shown).



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Fig. 1. In vitro stimulation of CD25CD4+ and CD25+CD4+ TR cells by bone marrow derived DC. mDC and iDC pulsed with ova peptide at varying concentrations were used as APC to stimulate 25 000 flow-sorted CD25+ or CD25CD4+ TCR transgenic DO11.10 T cells. (a) Proliferation of DO11.10 CD25CD4+ cells and a titrated number of DC pulsed with 1 µM ova. (b) Proliferation of DO11.10 CD25+CD4+ TR cells stimulated with peptide pulsed iDC or mDC. (c) Comparison of CD25 and CD25+ CD4+ T cell proliferation stimulated with peptide pulsed mDC. (d) CD25+CD4+ TR cell suppression assay. Proliferation of 25 000 CD25CD4+ cells with titrated numbers of mitotically arrested CD25+CD4+ TR cells in the presence of either iDC, mDC or splenocyte APC and 1 µM ova. (e) Real-time RT–PCR of Foxp3 expression of mDC-stimulated (1 µM ova) CD25+CD4+ TR cells at various time points. *Significant differences (P < 0.05). Values expressed as means ± SD. Representative of at least four separate experiments.

 
CD25+CD4+ TR cells stimulated by mDC and to a lesser extent iDC produce low levels of IL-2
Figure 1 demonstrates that DC could effectively support CD25+CD4+ TR cell proliferation, as measured by [3H]thymidine incorporation, to at least the same extent as their normal CD25CD4+ T cell counterparts. We therefore next examined whether the CD25+CD4+ TR cells were capable of producing IL-2. Closely mirroring the proliferation data, Fig. 2(A) demonstrates that DO11.10 CD25+CD4+ TR cells are capable of producing IL-2 in response to DC stimulation, and mDC are significantly more effective at triggering its production. Although IL-2 protein production by CD25+CD4+ TR cells was readily detectable, the levels were actually far lower (almost 10-fold) than those produced by normal CD25CD4+ cells (Fig. 2B). It was unclear, however, whether this significantly lower level of IL-2 reflected an actual reduction in protein synthesis per se or was due to a ‘sinking effect’ attributable to the high IL-2R expression by CD25+CD4+ TR cells. We therefore examined endogenous levels of expression by measuring IL-2 mRNA. DO11.10 CD25+ and CD25 CD4+ T cells were stimulated by mDC and 1 µM ova for ~36 h or left unstimulated. Semi-quantitative PCR showed strong upregulation of IL-2 mRNA after stimulation of CD25CD4+ but not CD25+CD4+ TR cells (Fig. 2C), although a faint level of IL-2 transcript could be detected in CD25+CD4+ TR cells at higher cycle numbers (data not shown). mDC prior to setting up the assay, or after being incubated with the T cells, similarly failed to show detectable levels of IL-2 by PCR (Fig. 2C and data not shown). Quantitative real-time RT–PCR yielded similar results and demonstrated that mDC-stimulated CD25+CD4+ TR cells transcribed ~6-fold less IL-2 than their CD25CD4+ counterparts; a value broadly in accordance with the protein data (Fig. 2D). The CD25high population (representing only ~1–2% of CD4+ cells) was sorted in these IL-2 studies, which should exclude most, if not all, conventional activated T cells, although a tiny contamination could not be ruled out completely (22). To assess this possibility, we used DO11.10 CD25+ CD4 single positive thymocytes which we speculated should not contain activated cells, in contrast to those potentially generated peripherally. Such sorted thymocytes were also seen to produce IL-2 on stimulation by mDC plus ova (Fig. 2E). Regulatory cytokines such as IL-10 and TGF-ß, which have been suggested by some to be produced by CD25+CD4+ TR cells in some other models (23,24), could not be detected in supernatants from mDC-stimulated CD25+CD4+ TR cultures after ~3 days (data not shown).



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Fig. 2. IL-2 production by CD25 and CD25+CD4+ T cells. (a) IL-2 protein production by DO11.10 CD25+CD4+ TR cells stimulated with peptide pulsed iDC or mDC. (b) Comparison of IL-2 production by DO11.10 CD25 and CD25+ CD4+ T cells stimulated by peptide pulsed mDC. (c) Semi-quantitative PCR measurement of IL-2 mRNA from mDC, unstimulated and 1 µM ova stimulated CD25 or CD25+ CD4+ T cells. (d) As in (c) but using real-time RT–PCR. (e) Measurement of IL-2 production by DO11.10 CD25+CD4+ (single positive) thymocytes following ova stimulation (1 µM). IL-2 measured at ~36 h. *Significant differences (P < 0.05). Values expressed as means ± SD.

 
Production of IL-2, IL-6 and IL-15 by DC is not required for CD25+CD4+ TR cell proliferation but IL-6 can modulate suppressive functions
Several recent reports have demonstrated the production of IL-2 from mouse DC in response to various microbial stimuli (2527). Since IL-2 and TCR stimulation is known to break CD25+CD4+ TR cell in vitro anergy we reasoned that its potential production by mDC may be responsible, at least in part, for the dramatic stimulation observed with CD25+CD4+ TR cells (5). Similarly, DC-produced IL-2 may also account for a proportion of the IL-2 seen in the mDC-stimulated CD25+CD4+ TR cultures above. Initially, we directly examined LPS-stimulated (1 µg/ml) DC cultures for production of IL-2 at either the protein or mRNA level. We were however unable to detect any IL-2 at a variety of time points (2, 4, 8, ~24, ~48 or ~72 h post-stimulation) by either ELISA (sensitivity 3 pg/ml) or RT–PCR (data not shown). To address this issue further, we decided to use IL-2–/– mice as a source of iDC and mDC to determine their effectiveness as APC for CD25+CD4+ TR cells. mDC derived from IL-2–/– mice were just as effective as those from wild-type littermates at stimulation of CD25+CD4+ TR cell proliferation and IL-2 production (Fig. 3A and B). Importantly, using the IL-2–/– DC enabled us to definitively confirm that the IL-2 detected from CD25+CD4+ TR cell cultures originated from the T cells themselves rather than the APC.



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Fig. 3. Stimulation requirements of CD25+CD4+ TR cells. CD25+CD4+ TR cells were stimulated by mDC generated from either IL-2 deficient BALB/c mice (IL-2–/–) or wild-type littermates (wt). (a) Proliferation and (b) IL-2 production by CD25+CD4+ TR cells. (c) Effect of exogenous IL-15 on the proliferation of CD25+CD4+ TR cells in the presence of various APC (iDC, mDC or whole spleen APC). DC or whole splenic APC were pre-pulsed with 0.1 µM ova peptide. Cultures were set up with the indicated addition of exogenous cytokines; IL-15 (12.5–50 ng/ml), IL-2 (100 U). Role of DC-derived IL-6 in direct stimulation of purified TR cells (d) and regulation of CD25 by TR cells (e). Purified CD25+CD4+ TR cells were set up with ova pulsed (1 µM) mDC from wild-type (Wt), IL-6 knock-out (IL-6–/–) DC or whole spleen (APC). Exogenous IL-6 (50 ng/ml) or IL-2 (100 U/ml) were added as indicated. Values expressed as means ± SD. Representative of three separate experiments.

 
We also assessed the potential role of the IL-2 related cytokine, IL-15, which shares similar biological properties (28). Although IL-15 transcripts could readily be detected in bone-marrow derived DC (data not shown), the addition of the exogenous cytokine could not break CD25+CD4+ TR cell anergy at all when stimulated by splenic APC plus peptide, nor could it boost iDC-driven stimulation to the levels of that seen with mDC (Fig. 3C). Similar results were observed when IL-15 was added to purified DO11.10 CD25+CD4+ TR cells alone or together with anti-CD3 and APC stimulation (data not shown), in contrast with an effective breakage of anergy by exogenous IL-2 (Fig. 3C).

A recent report suggested the importance played by DC secretion of IL-6 in the modulation of CD25+CD4+ TR cell suppression (19). We investigated the role played by DC-derived IL-6 both in the stimulation of CD25+CD4+ TR cells and in its ability to block suppression, by utilizing both IL-6–/– DC and exogenous cytokine (Fig. 3D and E). Unlike IL-2, IL-6 played no obvious role in the stimulation and breakage of CD25+CD4+ TR cell in vitro anergy, with IL-6–/– DC still acting as capable stimulators. Similarly, the addition of exogenous cytokine in the presence of conventional splenic APC was unable to stimulate CD25+CD4+ TR cell proliferation (Fig. 3D). On the other hand, and in accordance with a previous report, we observed a partial requirement for IL-6 in the blockade of CD25+CD4+ TR cell suppression, with a reasonable recovery of suppression in the presence of IL-6–/– DC.

Salmonella enteritidis LPS has no direct effects on CD25+CD4+ TR cells
Use of LPS to maturate DC made us concerned about its influence as a potential contaminant in the CD25+CD4+ TR cultures. This was especially relevant since a recent report demonstrated that LPS was able to directly stimulate CD25+CD4+ TR cells through their expression of Toll-like receptors (15). We therefore ensured that DC were thoroughly washed and subsequent cultures set-up in the presence of polymixin B, which could completely block LPS-mediated B cell proliferation and dendritic cell upregulation of costimulation markers (data not shown). To further obviate any confounding influence of LPS, we determined its direct effects on CD25+CD4+ TR cells. CD25CD4+ and CD25+CD4+ cells from normal BALB/c mice were stimulated with either plate-bound anti-CD3 or splenocyte APC plus anti-CD3, with or without the presence of exogenously added LPS (10 µg/ml). Figure 4(A and B) confirms that CD25+CD4+ TR cells were anergic. Furthermore, LPS was unable to directly trigger CD25+CD4+ TR cell proliferation, nor did it show any synergistic effect with the plate-bound anti-CD3 or APC plus soluble anti-CD3.



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Fig. 4. Effects of LPS on CD25+CD4+ TR cells. CD25+ or CD25 CD4+ T cells were stimulated by either plate-bound anti-CD3 (5 µg/ml) (a) or whole splenocyte APC plus soluble anti-CD3 (1 µg/ml) (b) with or without the presence of 10 µg/ml LPS (S. enteritidis). ‘No stim.’ Denotes stimulated with PBS alone. Values expressed as means ± SD. Representative of at least three separate experiments.

 
iDC and mDC are far more effective APC for triggering CD25+CD4+ TR cell proliferation than activated B cells
By comparing iDC and mDC with highly purified B cells we attempted to determine whether DC, and in particular mDC, were unique in their effectiveness to stimulate the proliferation of CD25+CD4+ TR cells. The B cells were either activated by overnight stimulation with 1 µg/ml LPS (‘activated’), or left overnight in plain medium and used as ‘naïve’ cells. Figure 5(A) shows flow cytometric comparison of the different APC populations. B cells showed relatively high constitutive expression of CD40, and very high levels of MHC class II, both of which could be increased on activation. CD86 was significantly increased from low basal levels on B cell activation but CD80 remained largely unchanged after overnight stimulation. In contrast, iDC showed low expression of CD40, which was increased on mDC, and moderate levels of MHC class II, which increased only slightly on activation. CD86, and in particular CD80, showed higher expression than that seen on B cells and both markers could be significantly upregulated on stimulation. When these different cell populations were then used as APC, both ova pulsed iDC and mDC were significantly superior to naïve B cells and activated B cells at inducing the proliferation of CD25+CD4+ TR cells (Fig. 5B). Importantly, even iDC were significantly more effective than activated B cells, despite the latter's generally high level of costimulation marker and Class II expression.

DC costimulatory molecules are important for CD25+CD4+ TR cell stimulation in vitro
To investigate the parameters of CD25+CD4+ TR cell activation, we decided to sort iDC and mDC according to their expression of costimulatory molecules. This would also enable us to ask whether there were any inherent differences in stimulatory ability between iDC and mDC in addition to costimulatory marker expression. We opted to use CD86 as a candidate costimulatory marker since its crucial role in naïve T cell activation is well documented (29), and BM-DC show a discrete CD86high population which was amenable to cell sorting. iDC also express a small CD86high population, typically between 10 and 20% even when grown in the presence of polymixin B (data not shown). iDC and mDC were then sorted into CD11c+CD86low and CD11c+CD86high populations (Fig. 6A). Each population thus sorted was >92% double-positive according to the desired sort criteria and of similar MFI between iDC and mDC. Sorted DC were then plated-out, incubated overnight, and used to stimulate highly purified DO11.10 CD25+CD4+ TR cells the following day with the addition of ova peptide. CD86high cells derived from both iDC and mDC were significantly better at stimulating CD25+CD4+ TR cells than CD86low DC, although the latter were still able to appreciably support proliferation (Fig. 6B). Interestingly, no differences could be observed between iDC CD86high or CD86low cells and their mDC counterparts.



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Fig. 6. Role of the co-stimulatory molecule CD86 in the stimulation of CD25+CD4+ TR cells. CD11c+CD86low or CD11C+CD86high cells were flow-sorted from iDC and mDC as indicated in (a). Sorted DC populations were used to stimulate DO11.10 CD25+CD4+ TR cells with 1 µM ova peptide (‘Stim.’) or left unstimulated (‘No stim.’) (b). Values expressed as means ± SD. *Significant differences (P < 0.05). Representative of two separate experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD25+CD4+ TR cells have been classically characterized as having a ‘default state’ of suppression and anergy in vitro and in vivo, yet are still able to expand in vivo under specific conditions such as lymphopenia (5,11). It now seems clear that CD25+CD4+ TR cells are also critical for immunohomeostasis, as evidenced by the uncontrolled lymphoproliferation and autoimmunity seen in the absence of functional Foxp3 (810). On the other hand, a continuous suppression by CD25+CD4+ TR would be similarly detrimental since protective immune responses would be hampered from proceeding. Clearly, a control of TR cell behaviour, fine-tuned according to prevailing in vivo conditions, is critical to the survival of an organism. In trying to understand such issues, we have looked particularly at the role of DC and attempted to shed some light on the nature of the CD25+CD4+ TR cell–DC interaction, and suggest that such a process is important to the normal control of CD25+CD4+ TR cell suppressive function and expansion. The role of DC in CD25+CD4+ TR cell stimulation presented here is broadly in accord with that recently shown by Yamazaki et al. (16).

Using bone marrow derived DC we observed in vitro that these cells are highly capable of breaking CD25+CD4+ TR cell anergy and effectively perturb their normal suppressive functions (Fig. 1). Not only can CD25+CD4+ TR cells proliferate in response to DC, they also produce detectable levels of IL-2 (Fig. 2), a cytokine never observed in CD25+CD4+ TR cells using standard forms of TCR stimulation (5,13). [3H]thymidine incorporation suggested that mDC-stimulated CD25+CD4+ TR proliferate to similar extents as their conventional CD25 T cell counterparts. This proliferation may not be sustained however, as suggested by CFSE labelling (16). We therefore speculate that CD25+CD4+ TR may undergo an unsustained burst of proliferation following mDC stimulation.

Several lines of evidence collectively suggest that IL-2 expression is attributable to the CD25+CD4+ TR cells themselves. (i) The cells were highly purified using cell-sorting (usually >98–99% CD25+CD4+). (ii) No IL-2 could be detected in/from DC after LPS stimulation using RT–PCR or ELISA (data not shown). (iii) CD25+CD4+CD8 thymocytes also produce detectable levels of IL-2, and such cells should obviate the possibility of contamination by peripherally activated T cells. (iv) IL-2 can be detected from CD25+CD4+ TR cells stimulated by IL-2–/– DC (Fig. 3). Although proliferative responses of CD25+CD4+ TR cells are equivalent to their CD25CD4+ counterparts, interestingly their levels of IL-2 production are significantly lower (typically as much as 10-fold). This difference appears to originate at the stage of production itself, as opposed to simply an IL-2 ‘sinking’ effect, since IL-2 mRNA levels were also diminished in CD25+CD4+ TR cells (~6-fold by quantitative PCR). CD25+CD4+ TR cells thus appear to be ‘programmed’ to produce lower amounts of IL-2. The reason for this reduced IL-2 synthesis/secretion is uncertain, but it may be to limit the paracrine stimulation of effector T cells and yet provide enough growth factor for supplying the autocrine expansion of the CD25+CD4+ TR cells when required. The robust proliferation of CD25+CD4+ TR cells stimulated by mDC but in the presence of significantly reduced IL-2, may reflect both their elevated levels of adhesion molecules (including the CD25 molecule itself) (30), as well as a heightened sensitivity of the IL-2R to its ligand. Although the latter possibility is difficult to reconcile with the known selective expression of SOCS (suppressors of cytokine signalling) 1 and 2 by CD25+CD4+ TR cells (11,31), nothing is yet known of SOCS3 in this context. SOCS3 has recently been shown to be the most critical of such molecules in the control of IL-2-driven T cell activation/proliferation, and its role in modulating CD25+CD4+ TR cell stimulation sensitivity would therefore be of great interest (32). Finally, the breakage of CD25+CD4+ TR cell anergy and their resultant active proliferation, had no effect on Foxp3 expression (Fig. 1E). Importantly, this suggests that the regulatory cell is a stably differentiated state, and is consistent with the reversion of these cells back to an anergic and suppressive phenotype once potent stimulation such as exogenous IL-2 or DC is withdrawn (5,12,16).

Some recent data have demonstrated a direct stimulatory action of LPS on highly purified CD25+CD4+ TR cells (15). We therefore took great care throughout to avoid any experimental artefacts attributable to LPS contamination. Not only were mDC washed thoroughly, but all culturing was performed in the presence of polymixin B to neutralize trace or cell membrane-adsorbed LPS. As a final confirmation we added LPS directly to T cell cultures to observe its effects. Contrary to the previously published data, we could find no readily apparent stimulatory or synergistic properties with CD25+CD4+ TR cells (Fig. 4). This result proved convenient for our experiments since it excluded LPS as a confounding factor. Although the reasons for our discrepancy with Caramalho et al. (15) are unclear, the differences in the sources and purity of the LPS may well have a bearing on this issue (both species and source differed).

We cannot at this stage claim with certainty that DC are the only APC type capable of breaking TR cell anergy, but they appeared to be far superior to purified immature (naïve) and LPS-matured B cells (activated), even though the latter expressed high levels of CD40, CD86 and MHC class II (Fig. 5A). Moreover, activated B cells had superior expression of several markers when compared to iDC and yet were still notably inferior as APC (Fig. 5B). Another group has additionally shown peritoneal exudate cells (composed primarily of macrophages) to be incapable of effective stimulation of CD25+CD4+ TR cells (16), which together with the data here suggests that DC may well be natural controllers of CD25+CD4+ TR cells. Although mDC were the most effective APC for CD25+CD4+ TR cells, always triggering significantly more proliferation and IL-2 production than iDC, a similar result was obtained with highly purified CD11c+ cells, suggesting that the differences between iDC and mDC cannot be explained simply by differences in the number of differentiated DC between the two groups. The difference may in part be related to co-stimulatory molecule expression rather than another parameter specifically associated with mDC, since CD86high sorted iDC appeared indistinguishable as APC from the mDC CD86high (Fig. 6). Conversely, sorting mDC CD86low cells showed them to be relatively poor APC for CD25+CD4+ TR cells, again equivalent to iDC. Interestingly, the CD86high iDC are always present in small numbers even when cultures are grown in the presence of polymixin B, and appear to represent a distinct spontaneously matured population probably identical to their mDC counterparts, phenotypically and functionally. Whether CD86 itself or something segregating with it, such as CD80, is important remains to be determined since CD86high DC also tend to be positive for a variety of other costimulatory markers. With regard to expression markers examined, the only major difference between B cells and DC was that of CD80, which was always low on the former at the time points examined (Fig. 5A). The seemingly redundant functions of CD80 and CD86 is puzzling, but may potentially be resolved by their relative abilities to stimulate CD25+CD4+ TR cells as suggested here and by others (29,33). There may of course be hitherto unrecognized, or as yet poorly characterized, molecules such as GITRL (34) which are uniquely or more highly expressed only by professional APC. Such molecules may be specifically responsible for the control of CD25+CD4+ TR cells, and we are currently trying to investigate this possibility (Nishioka et al., manuscript in preparation).

Addition of exogenous IL-2 has been shown to break CD25+CD4+ TR cell anergy, but the use of IL-2–/– DC here demonstrated its production by the APC was not necessary for the robust stimulation of CD25+CD4+ TR cells (Fig. 3). This is partially consistent with Yamazaki et al. who observed only a minor reduction in stimulation with IL-2–/– DC under very particular conditions, and similarly, transwell experiments have previously demonstrated a requirement for cell-surface interactions for DC stimulation of CD25+CD4+ TR cells (16). Collectively, our data and those published previously suggest a more significant role for cell contact in the activation, at least, of CD25+CD4+ TR cells. The IL-2-related cytokine IL-15, while detectable at the mRNA level in DC, did not appear to play a significant role in CD25+CD4+ TR cell stimulation since high levels of exogenous IL-15 were unable to break CD25+CD4+ TR cell anergy under a variety of stimulation conditions, nor did it show any synergistic effects with iDC (Fig. 3, and data not shown). Certainly, IL-15 was far inferior to the dramatic effects seen here with IL-2 and reported elsewhere (5,13,35) (Fig. 3). Similar to a recent report by Pasare and Medzithov (19), we also observed a consistent blockade of CD25+CD4+ TR cell suppressive functions in the presence of mDC and to a lesser extent iDC (Fig. 1D and 3E) which was partially dictated by the presence of DC-derived IL-6. IL-6 itself though, was not responsible for the breakage of CD25+CD4+ TR cell in vitro anergy. Other factors, perhaps uniquely associated with DC, are also critical for modulating suppression since splenic APCs plus exogenous IL-6, at a dose sufficient to restore T cell responses in the presence of IL-6–/– DC, were unable to perturb suppression to any extent (Fig. 3E).

We have shown that DC, and in particular mDC, are able to break CD25+CD4+ TR cell anergy and trigger significant proliferation and detectable IL-2 production, and that the presence of DC prevents normal CD25+CD4+ TR regulatory functions. These results, coupled with related data, suggest that DC may be important regulators of CD25+CD4+ TR cells, predominantly through cell–cell interaction, inhibiting or facilitating their function as required for productive immunity (16,19).


    Acknowledgements
 
Z.F. would like to thank Noriko Sakaguchi for invaluable assistance and Shohei Hori, Takashi Nomura, Masahiro Ono, Takeshi Takahashi and Sayuri Yamazaki for helpful discussion. This work was supported by a Japan Society for the Promotion of Sciences (JSPS) scholarship and by grants-in-aid from the Ministry of Education, Sports and Culture and the Japanese Ministry of Human Welfare.


    Abbreviations
 
DC   dendritic cell
iDC   immature dendritic cell
mDC   mature dendritic cell
ova   ovalbumin
TLR   Toll-like receptor
TR   regulatory T cell

    Notes
 
Transmitting editor: A. Cooke

Received 14 November 2003, accepted 24 September 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Shevach, E. M. 2000. Regulatory T cells in autoimmmunity. Annu. Rev. Immunol. 18:423.[CrossRef][ISI][Medline]
  2. Takahashi, T. and Sakaguchi, S. 2003. The role of regulatory T cells in controlling immunologic self-tolerance. Int. Rev. Cytol. 225:1.[ISI][Medline]
  3. Shimizu, J., Yamazaki, S. and Sakaguchi, S. 1999. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J. Immunol. 163:5211.[Abstract/Free Full Text]
  4. Onizuka, S., Tawara, I., Shimizu, J., Sakaguchi, S., Fujita, T. and Nakayama, E. 1999. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res. 59:3128.[Abstract/Free Full Text]
  5. Takahashi, T., Kuniyasu, Y., Toda, M., Sakaguchi, N., Itoh, M., Iwata, M., Shimizu, J. and Sakaguchi, S. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract]
  6. Belkaid, Y., Piccirillo, C. A., Mendez, S., Shevach, E. M. and Sacks, D. L. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502.[CrossRef][ISI][Medline]
  7. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  8. Hori, S., Nomura, T. and Sakaguchi, S. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.[Abstract/Free Full Text]
  9. Khattri, R., Cox, T., Yasayko, S. A. and Ramsdell, F. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4:337.[CrossRef][ISI][Medline]
  10. Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[CrossRef][ISI][Medline]
  11. Gavin, M. A., Clarke, S. R., Negrou, E., Gallegos, A. and Rudensky, A. 2002. Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat. Immunol. 3:33.[CrossRef][ISI][Medline]
  12. Thornton, A. M. and Shevach, E. M. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
  13. Thornton, A. M. and Shevach, E. M. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  14. Walker, L. S., Chodos, A., Eggena, M., Dooms, H. and Abbas, A. K. 2003. Antigen-dependent proliferation of CD4+ CD25+ regulatory T cells in vivo. J. Exp. Med. 198:249.[Abstract/Free Full Text]
  15. Caramalho, I., Lopes-Carvalho, T., Ostler, D., Zelenay, S., Haury, M. and Demengeot, J. 2003. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J. Exp. Med. 197:403.[Abstract/Free Full Text]
  16. Yamazaki, S., Iyoda, T., Tarbell, K., Olson, K., Velinzon, K., Inaba, K. and Steinman, R. M. 2003. Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells. J. Exp. Med. 198:235.[Abstract/Free Full Text]
  17. Fallarino, F., Grohmann, U., Hwang, K. W. et al. 2003. Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4:1206.[CrossRef][ISI][Medline]
  18. Cederbom, L., Hall, H. and Ivars, F. 2000. CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30:1538.[CrossRef][ISI][Medline]
  19. Pasare, C. and Medzhitov, R. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033.[Abstract/Free Full Text]
  20. Hata, H., Sakaguchi, N., Yoshitomi, H. et al. 2004. Distinct contribution of IL-6, TNF-alpha, IL-1 and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice. J. Clin. Invest. 114:582.[Abstract/Free Full Text]
  21. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. and Steinman, R. M. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract]
  22. Kuniyasu, Y., Takahashi, T., Itoh, M., Shimizu, J., Toda, G. and Sakaguchi, S. 2000. Naturally anergic and suppressive CD25(+)CD4(+) T cells as a functionally and phenotypically distinct immunoregulatory T cell subpopulation. Int. Immunol. 12:1145.[Abstract/Free Full Text]
  23. Powrie, F., Carlino, J., Leach, M. W., Mauze, S. and Coffman, R. L. 1996. A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells. J. Exp. Med. 183:2669.[Abstract]
  24. Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L. and Powrie, F. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190:995.[Abstract/Free Full Text]
  25. Granucci, F., Vizzardelli, C., Pavelka, N., Feau, S., Persico, M., Virzi, E., Rescigno, M., Moro, G. and Ricciardi-Castagnoli, P. 2001. Inducible IL-2 production by dendritic cells revealed by global gene expression analysis. Nat. Immunol. 2:882.[CrossRef][ISI][Medline]
  26. Granucci, F., Feau, S., Angeli, V., Trottein, F. and Ricciardi-Castagnoli, P. 2003. Early IL-2 production by mouse dendritic cells is the result of microbial-induced priming. J. Immunol. 170:5075.[Abstract/Free Full Text]
  27. Granucci, F., Zanoni, I., Feau, S. and Ricciardi-Castagnoli, P. 2003. Dendritic cell regulation of immune responses: a new role for interleukin 2 at the intersection of innate and adaptive immunity. EMBO J. 22:2546.[Abstract/Free Full Text]
  28. Fehniger, T. A. and Caligiuri, M. A. 2001. Interleukin 15: biology and relevance to human disease. Blood 97:14.[Free Full Text]
  29. Sansom, D. M., Manzotti, C. N. and Zheng, Y. 2003. What's the difference between CD80 and CD86? Trends Immunol. 24:314.[ISI][Medline]
  30. Itoh, M., Takahashi, T., Sakaguchi, N., Kuniyasu, Y., Shimizu, J., Otsuka, F. and Sakaguchi, S. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
  31. McHugh, R. S., Whitters, M. J., Piccirillo, C. A., Young, D. A., Shevach, E. M., Collins, M. and Byrne, M. C. 2002. CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[ISI][Medline]
  32. Yu, C. R., Mahdi, R. M., Ebong, S., Vistica, B. P., Gery, I. and Egwuagu, C. E. 2003. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J. Biol. Chem. 278:29752.[Abstract/Free Full Text]
  33. Zheng, Y., Manzotti, C. N., Liu, M., Burke, F., Mead, K. I. and Sansom, D. M. 2004. CD86 and CD80 differentially modulate the suppressive function of human regulatory T cells. J. Immunol. 172:2778.[Abstract/Free Full Text]
  34. Tone, M., Tone, Y., Adams, E., Yates, S. F., Frewin, M. R., Cobbold, S. P. and Waldmann, H. 2003. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc. Natl Acad. Sci. USA 100:15059.[Abstract/Free Full Text]
  35. Thornton, A. M., Piccirillo, C. A. and Shevach, E. M. 2004. Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34:366.[CrossRef][ISI][Medline]