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International Immunology Advance Access originally published online on February 7, 2007
International Immunology 2007 19(3):227-237; doi:10.1093/intimm/dxl139
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Dendritic cells partially abrogate the regulatory activity of CD4+CD25+ T cells present in the human peripheral blood

Justin S. Ahn*, Deepa K. Krishnadas* and Babita Agrawal

Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

Correspondence to: B. Agrawal; E-mail: bagrawal{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The factors that influence the functionality of human CD4+CD25+ regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4+CD25+ T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4+CD25+(High) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4+CD25– or CD4+CD25+(Med–low) T cells. Intracellular Foxp3 was equivalently expressed on CD4+CD25+(All), CD4+CD25+(High), CD4+CD25+(Med–low) and CD4+CD25– T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4+CD25+ T cells were isolated and then cultured in vitro with CD4+CD25– responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the Th1 cytokine IFN-{gamma}, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-ß, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4+CD25+ regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-{gamma} and IL-2 production or inhibition of IL-10 and/or TGF-ß production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.

Keywords: CD4+25+ T cells, dendritic cells, human T cells, regulatory T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Immune homeostasis requires a delicate balance between activity and tolerance. Imbalance can lead to dangerous over- or under-responsiveness to both self- and foreign antigens. For the past 30 years, there has been a search for a distinct population of cells responsible for regulating the immune system. The search was apparently completed when Sakaguchi et al. (1) found that adoptive transfer of CD4+ T cells constitutively expressing the IL-2R{alpha} chain (CD25) could effectively prevent autoimmune diseases in mice. These CD4+CD25+ T cells (Treg cells) have since been postulated to play a regulatory role in maintaining peripheral tolerance to self-antigens as well as potential roles in mediating immune responses to cancer, infectious diseases and transplanted organs (2–4).

Since their original discovery, additional populations of Treg cells have been identified. The most prevalent are natural Treg cells, which develop in the thymus and exist in anergic state, thus failing to secrete IL-2 or respond to anti-CD3-mediated TCR stimulation (5). They suppress the activation and expansion of CD4+CD25– T cells in vitro through contact-dependent mechanisms and also in vivo through IL-10, transforming growth factor (TGF)-ß production and/or contact-dependent mechanisms (2, 5). In addition to CD25, the most characteristic markers of human and murine Treg cells includes: CTL-associated antigen 4 (CD152) and GITR (glucocorticoid-induced tumor necrosis factor receptor family-related protein) (6, 7). However, these markers are also up-regulated upon T cell activation and thus, make it difficult to identify and isolate legitimate Treg cells (8). Murine Treg cells are also characterized by expression of Foxp3, a gene which encodes Scurfy, a forkhead/winged helix transcriptional repressor. Although its function is not certain, Foxp3 mutants and knockouts in mice result in the development of organ-specific autoimmunity (9). Retroviral transfection of Foxp3 into Foxp3– T cells induces Treg cell-like phenotype and suppressive activity, making Foxp3 the most unique marker for Treg cells to date (10). While mutations in the human homolog, Foxp3, have also been associated with the autoimmune disease IPEX, Foxp3 expression in human CD4+CD25+ T regulatory cells is debatable (9–13). The majority of regulatory T cell data has been developed in inbred murine systems and as such murine Treg cells have been readily distinguished. Conversely, humans are more difficult models to work with because of the diversity, complexity and exposure to numerous microbial pathogens. Thus, human Treg cells are less defined than in mice and a number of discrepancies have been identified between the two species (14–21).

It has been demonstrated that regulatory T cells perform their functions by contact-dependent mechanisms and cytokine production (2, 5). However, the mechanism of function of regulatory T cells and their regulation is poorly understood. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) capable of inducing tolerance or immunity against specific self- and non-self-antigens. However, the role of human DCs in modulating regulatory T cell functions is not clear. It has been suggested that in mouse models, DCs possess ability to control the suppressive ability, expansion and/or differentiation of CD4+CD25+ Treg cells (22). However, DC-mediated control of natural Treg cells present in human peripheral blood has not been demonstrated yet. As suggested before, Treg cell function is much like a double-edged sword which can lead to an outcome that is context dependent (23). Under these circumstances, a greater understanding of modulation and regulation of regulatory T cells is critical, in order to understand their biological role in maintaining immune homeostasis, regulating immune response to pathogens, autoimmune diseases and also to utilize their potential for therapeutic purposes.

We sought to examine the relationship between human Treg cells and DCs and examine whether DCs can modulate regulatory T cell functions. We identified and characterized a distinct CD4+CD25+ T cell population obtained from peripheral blood of human donors according to current literature. We observed that a higher proportion of CD4+CD25+ T cells expressed surface GITR and CTL-associated antigen 4 than CD4+CD25– T cells. Expression of intracellular Foxp3 was not distinguishable between CD4+CD25+ or CD4+CD25– T cells. We then isolated these T cells and examined their effect on proliferation of autologous CD4+CD25– responder T cells under anti-CD3 TCR stimulation and co-stimulation provided by immature dendritic cells (iDCs), LPS-matured DCs (LPS-mDCs), polyI:C-matured DCs (polyI:C-mDCs), PBMCs or PBMCs plus anti-CD28 antibodies. In addition, we examined the cytokines ({gamma}-IFN, IL-2, IL-10 and TGF-ß) secretion in these cultures. The results obtained in our studies suggest that human DCs are capable of reversing the suppression mediated by CD4+CD25+ T cells, independent of cytokine secretion. Our data suggest that the function of regulatory T cells can be fine-tuned by DCs such that the expansion of effector T cells may be modulated without any effect on cytokine production, a process which may affect paracrine and/or autocrine function of the effector cells. These results further emphasize the complex and interrelated factors that modulate Treg function, thus providing a delicate balance in cellular immune responses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Isolation of lymphocytes from human blood
Peripheral blood samples were obtained from donors 30–60 years of age of both sexes after informed consent. Use of human blood samples was approved by institutional Health Research Ethics Board at the University of Alberta, Canada. Blood obtained from human donors was separated using differential centrifugation and Lymphocyte Separation Medium (Cellgro, Herndon, VA, USA). The intermediate buffy layer containing PBMCs was extracted, washed twice in warm PBS and then re-suspended in DC media: RPMI 1640 supplemented with 1.0% penicillin–streptomycin, 1.0% sodium pyruvate (Invitrogen, Carlsbad, CA, USA) and 1% human AB serum (Sigma, St. Louis, MO, USA). Cells were seeded into six-well plates at 3 x 107 cells per well and then incubated for 2 h in a humidified chamber at 37°C and 5% CO2 (all incubations were done under these conditions and will hereafter be referred to as 37°C). After incubation, non-adherent cells (NACs) were collected from the wells for cell sorting (see below). NACs isolated after removing adherent cells were used as to purify various CD4+CD25+ and CD4+CD25– T cells. NACs comprised of >80% CD3+ T cells. Adherent cells bound to plate bottoms were differentiated into iDCs using 10 ng ml–1 IL-4 and 50 ng ml–1 granulocyte macrophage colony-stimulating factor (Peprotech, Ottawa, ON, Canada) and 6–7 days of incubation at 37°C. PolyI:C (12.5 µg ml–1) or LPS (100 ng ml–1) (Sigma) was added on the 5–6th day for 18 h to induce maturation and obtain mature dendritic cells (mDCs). We have previously reported the differentiation and maturation of DCs obtained from peripheral blood monocytes (24–26).

Staining for flow cytometry
Cell suspensions containing 4 x 105 NACs were stained with 5 µl of the following mAbs: anti-CD4 (Quantum Red) from Sigma, anti-GITR (FITC) and anti-CD45RA (FITC) from eBioscience (San Diego, CA, USA), anti-CTL-associated antigen 4 (FITC) from R&D Systems (Hornby, ON, Canada) and anti-CD25 (PE) and isotype control mouse IgG (QR, PE or FITC) from Becton Dickinson (Mississauga, ON, Canada). For intracellular staining, cells were first pre-incubated in 100 µl of cold permeabilization buffer (PBS + 2% FBS + 0.3% Saponin + 5% normal human serum) for 5 min on ice (4°C) then, allophycocyanin-conjugated anti-Foxp3 antibody (eBioscience) or allophycocyanin-mouse IgG1 isotype control was added. After incubation on ice for 30 min, cells were washed twice with cold FACS wash extracellular (PBS + 1% sodium azide + 2% FBS) or FACS wash intracellular (PBS + 2% FBS + 0.1% saponin) and then fixed with FACS fix (PBS + 1% sodium azide + 1% PFA). Samples were analyzed using FACScan, FACS-CANTO and Cell Quest software (Becton Dickinson).

CD4+CD25+ and CD4+CD25– T cell isolation
NACs from fresh blood or cells thawed after being cryopreserved were sorted using either FACS or MACS (magnetic cell sorting) (Miltenyi Biotec, Auburn, CA, USA). In different experiments, we used these two methods and found similar results (data not shown).

For sorting using FACSAria (Becton Dickinson), NACs were stained prior to isolation to calculate the volume of antibodies needed, depending on the proportion of each population. Approximately 1.5 x 108 NACs were re-suspended in 500 µl of cold FACS buffer (PBS + 2% FBS + 1% penicillin/streptomycin) and pre-incubated on ice for 30 min. Cells were stained with antibodies and incubated for an additional 30 min on ice. NACs (1 x 106) were stained for the single and unstained controls. After incubation, samples were washed at least two times in FACS buffer (PBS + 2% FBS + 1% penicillin/streptomycin + 0.5 mM EDTA), re-suspended to at least 1 x 107 cells ml–1 and then mixed to create a single-cell suspension. Cells were added in 1 ml aliquots into FACS tubes. Using FACSAria, CD4+CD25+, CD4+CD25(High) and CD4+CD25– T cells were sorted at >97% purity.

MACS columns were used to purify human CD4+CD25+ regulatory T cells according to manufacturer's instructions. In brief, ~2 x 108 NACs were re-suspended in 1.8 ml MACS buffer (PBS + 0.5% BSA + 2 mM EDTA, pH 7.2) and incubated with 200 µl of biotin-antibody cocktail for 10 min on ice. Then, 400 µl of anti-biotin microbeads were added and further incubated for 15 min on ice. The cells were washed and re-suspended in 1 ml of MACS buffer and applied onto the LD column. The cells which passed through the column contained the unlabeled pre-enriched CD4+ T cell fraction. These cells were then incubated on ice with CD25 microbeads for 15 min. The cells were washed and re-suspended in 500 µl of MACS buffer and applied onto the MS column. The cells which passed through the column contained CD4+CD25– and the cells that bound to the column were flushed out with 1 ml buffer and contained CD4+CD25+. These isolated populations were found to be at >97% purity by flow cytometry.

In vitro T cell proliferation
Autologous PBMCs, iDCs and mDCs were irradiated with 18 Gy before use. Viability was assessed using trypan blue staining and hemocytometer counts. All cells were washed prior and re-suspended in AIM-V medium (Invitrogen). Autologous PBMCs (2 x 105), iDCs or mDCs (1 x 104) were added to 96-well plates along with responder CD4+CD25– T cells (5 x 103). Anti-CD28 mAbs (eBioscience) were also used in concentrations of 1 µg ml–1. OKT3 (anti-CD3 mAbs) was generated from a hybridoma culture (OKT3, American Type Tissue Collection) and 1–10 µg ml–1 was added to the cell cultures (concentration of OKT3 was determined using a standard curve, 10 µg ml–1 was found to be optimal). Various concentrations (1–10 µg ml–1) of anti-CD3 antibody were used in several experiments, and no difference was found in the cultures with or without CD4+CD25+ T cells and therefore, all of the data shown in the manuscript is with 10 µg ml–1 anti-CD3. CD4+CD25+ T cells were added to responder T cells in the ratios 1:1, 1:2, 1:5, 1:10 and/or 1:50 (CD4+CD25+ T cells to CD4+CD25– responder T cells, number of the responder cells was kept constant at 5000 per well). As controls, CD4+CD25– T cells were added in the same ratios instead of CD4+CD25+ T cells. As a proliferation control, CD4+CD25– T cells were also stimulated with 1 µg ml–1 PHA (Sigma). Additional controls included individual cultures and cultures lacking either T cells, Treg cells, iDCs, mDCs, anti-CD28 antibodies and/or OKT3. After 4 days of incubation at 37°C, 75 µl of supernatant was carefully collected from each well for cytokine analysis. Wells were then pulsed with 0.5 µCi ml–1 [3H] TdR (Amersham, Piscataway, NJ, USA) for 18 h, harvested and analyzed using a microbeta liquid scintillation counter (Wallac).

Cytokine secretion
Detection of secreted cytokines was carried out using IL-10, IFN-{gamma}, IL-2 and TGF-ß sandwich ELISA kits purchased from Biosource International (Camarillo, CA, USA). The assay was performed as outlined in the instructions. A dilution of 1:5 to 1:10 was used for the samples and the standards ranged from 15.6 to 2000 pg ml–1.

Statistical analysis
Student's t-test was performed for proliferation and cytokine assays using SPSS v.13 software.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Phenotype of CD4+CD25+ and CD4+CD25– T cell populations present in human peripheral blood
In order to identify the regulatory T cells in the peripheral blood of healthy human donors, CD4+CD25+ T cells were examined. Staining of non-adherent lymphocytes from peripheral blood showed distinct CD4+CD25+ and CD4+CD25– populations (Fig. 1A). The mean fluorescence intensity of CD4 was considerably higher than CD25, but both levels were distinctly expressed above background (Fig. 1B). The proportion of CD4+CD25+ T cells was highly variable between donors (n = 12) ranging from 0.2 to 8.4% of NACs with an average of ~3.12 ± 2.71% (Fig. 1C).


Figure 1
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  		Fig. 1. Staining of human peripheral blood non-adherent cells for CD4+CD25+ T cells. (A) Dot plot shows a distinct CD4+CD25+ population constituting 4.13% of non-adherent cells. (B) Histograms representing the mean fluorescence intensity of CD25 and CD4 expression in (A). The shaded histograms represent the stained samples while the unshaded histograms represent the unstained controls. The data is representative of 12 independent experiments from individual donors. (C) Presence of CD4+CD25+ T cell population in the peripheral blood of 12 individual donors.

 
First, the expression of intracellular Foxp3 protein in CD4+CD25+ T cells was examined. Three-color flow cytometry was performed to determine the Foxp3 expression in CD4+CD25+ or CD4+CD25– T cells. Small, resting lymphocytes were gated based on the forward and side scatter profiles to analyze only the non-activated lymphocytes and exclude large activated T lymphocytes (Fig. 2A). They were grouped into four subsets (CD4+CD25+ (All), CD4+CD25+ (High), CD4+CD25+ (Med–low) and CD4+CD25–) based on the expression levels of CD25 (Fig. 2B). And in each of these subsets, the expression level of intracellular Foxp3 was analyzed (Fig. 2C). We found that the histograms for intracellular Foxp3 expression between the different CD4+CD25+/– T cell subsets exactly overlapped in terms of the percentage of positive cells and mean fluorescence intensity (Fig. 2C). Mouse isotype controls showed that the antibodies were binding specifically to Foxp3 and that the mean fluorescence intensity was above background. The peak of each histogram correlates to the size of the T cell subset i.e. the peak with CD4+CD25– T cells is the highest, indicating that they are a larger population than CD4+CD25+ T cells whereas CD4+CD25+(High) T cells represent the lowest peak, simply because they are the smallest population. However, the intensity of Foxp3 expression in all subsets is equivalent because all histograms end at a common point on the x-axis (Fig. 2C). This clearly indicates that Foxp3 is not exclusively or selectively expressed on ‘CD4+CD25+’ or ‘Treg’ cells.


Figure 2
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  		Fig. 2. Phenotype of CD4+CD25+ and CD4+CD25–T cell populations. (A) Lymphocytes gated on forward and side scatter properties to exclude dead and/or activated cells. (B) Further gating of CD4+ T cells based on CD25 expression. (C) Histogram shows the percentage of intracellular Foxp3 expression in CD4+CD25+ T cell subsets (CD4+CD25+(All)-red, CD4+CD25+(High)-green, CD4+CD25+(Med–low)-brown) and in CD4+CD25–(blue) T cells. Isotype control is indicated in black dotted lines. The data is representative of three independent experiments.

 
We further characterized Foxp3 expression in CD4+CD25+ T cells expressing various markers associated with regulatory T cells such as CD45RA (naive population), extracellular CTL-associated antigen 4 and GITR. For these experiments, four-color flow cytometry was performed. Table 1 indicates the percentage of cells expressing extracellular CD45RA, CTL-associated antigen 4 and GITR among the single-positive CD4+CD25– and double-positive CD4+CD25+ T cell subsets and the percentage of intracellular Foxp3 in these triple-positive populations (CD4+CD25+CD45RA+, CD4+CD25+CTL-associated antigen 4+, CD4+CD25+GITR+). Interestingly, the percentage of both GITR and CTL-associated antigen 4-positive cells were higher in CD4+CD25+ (High) T cells compared with CD4+CD25+ (All), CD4+CD25+ (Med–low) and CD4+CD25–, whereas the percentage of CD45RA-positive cells were lower in CD4+CD25+ (High) T cells compared with CD4+CD25+ (All), CD4+CD25+ (Med–low) and CD4+CD25–. However, in comparison to the CD4+CD25– T cells, CD4+CD25+(All) T cells have a higher proportion of CTL-associated antigen 4+, an equivalent proportion of GITR+ and a lower proportion of CD45RA+. Upon examining intracellular Foxp3, each of these triple-positive CD4+CD25+CD45RA+, CD4+CD25+CTL-associated antigen 4+ and CD4+CD25+GITR+ populations of T cells expressed equivalent levels of intracellular Foxp3 compared with their respective CD4+CD25+/– T cell populations (Table 1). Interestingly, the proportion of Foxp3+ cells in the CD4+CD25+(High)GITR+ T cell subset was less than half of the CD4+CD25+(Med–low)GITR+ T cell subset. There was no apparent difference in the proportion of Foxp3+ cells between CD4+CD25+(Med–low) T cells and CD4+CD25+(High) T cells in the other triple-positive populations.


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  		Table 1. Comparison of expression profiles of extracellular GITR, CTL-associated antigen 4, CD45RA and intracellular Foxp3 between CD+CD25– T cells and various CD4+CD25+ T cell subsets

 
The presence of iDCs and mDCs can reverse CD4+CD25+ T cell-mediated suppression
In order to examine the effect of professional APCs (DCs) on the ability of CD4+CD25+ T cells (Treg) to suppress the in vitro proliferation of CD4+CD25– T cells (Tresp), varying ratios of CD4+CD25+ to CD4+CD25– T cells were stimulated with anti-CD3 and co-stimulation provided by irradiated PBMCs, PBMCs plus anti-CD28, irradiated iDCs, LPS-mDCs or polyI:C-mDCs (Fig. 3A). The results obtained show that T cell proliferation was suppressed only when PBMCs were used as source of co-stimulation (P = <0.05, at each of the ratios of Treg:Tresp except at 1:50). Upon adding soluble co-stimulatory anti-CD28 mAb, the Treg-mediated suppression of T cell proliferation was not affected (P = <0.05, at each ratios of Treg:Tresp except at 1:10). However, in the presence of iDCs or mDCs, the effect of Treg on the responder T cell (Tresp) proliferation was reversed except at very high supra-physiological Treg:Tresp (CD4+CD25+:CD4+CD25–) ratio (1:1) (with mDCs, P = >0.1, at each ratios of Treg:Tresp except at 1:1 where P = <0.05, whereas with iDCs P = >0.08 at each ratio of Treg:Tresp). Interestingly, in the cultures with LPS-mDCs, the cultures with CD4+CD25+ T cells proliferated more than the cultures without (Fig. 3), whereas in the presence of iDCs as well as polyI:C-mDCs, there was no increase in proliferation. In order to determine whether DCs lead to increased proliferation of Treg themselves, equal numbers of the CD4+CD25+ T cells or CD4+CD25– T cells alone were cultured with iDCs, PBMCs or PBMCs + anti-CD28 and stimulated with anti-CD3 (Fig. 3B). In each of these cultures, the CD4+CD25+ T cells proliferated significantly less than the CD4+CD25– T cells, suggesting that the iDCs were actually not able to break the partial anergy of CD4+CD25+ T cells, but likely modified the proliferation of responder T cells in the presence of regulatory T cells. In addition, proliferation of CD4+CD25– T cells noticeably increases as the strength of co-stimulation increases (PBMCs->PBMCs + anti-CD28->iDCs). The CD4+CD25+ T cells also showed ~2-fold increase in proliferation in the presence of PBMCs + anti-CD28 compared with PBMCs alone, probably due to increased IL-2 levels and autocrine regulation of proliferation. All irradiated PBMCs and DCs were incubated with anti-CD3 and anti-CD28 to verify that they could not proliferate and that the [3H] TdR was only incorporated by the purified T cells. Control groups that had cultured cells without TCR stimulation or without co-stimulation did not proliferate [counts per minute (c.p.m) <1000] as well as cells that were cultured individually—only CD4+CD25+ T cells, only CD4+CD25– T cells, only PBMCs, only iDCs or only mDCs also did not show any proliferation (cpm <200) (data not shown).


Figure 3
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  		Fig. 3. Dendritic cells reverse CD4+CD25+ T cells-mediated suppression of CD4+CD25–T cell proliferation. (A) T cells were stimulated using 10 µg ml–1anti-CD3 antibodies and co-stimulation provided by either PBMCs (without or with 1 µg ml–1anti-CD28 antibodies) or immature DCs or mature DCs (stimulated with 100 ng ml–1LPS or 12.5 µg ml–1PolyI:C). In the experiment, varying numbers of CD4+CD25+ T cells (open bars) or CD4+CD25–T cells (filled bars) were added to a fixed number of responder CD4+CD25–(5000 per well) T cells in the specified ratios. The data represents the average of triplicate wells. Bars represent standard deviation. (B) CD4+CD25+ T cells proliferate less than CD4+CD25–T cells irrespective of the co-stimulation. CD4+CD25+ (open bars) or CD4+CD25–(filled bars) T cells (2500 per well) were cultured in the presence of OKT3 + PBMCs, OKT3 + PBMCs + anti-CD28 or OKT3 + iDCs for 4 days followed by [3H]TdR incorporation assay. All data shown is representative of two to four repeated experiments.

 
The presence of iDCs and mDCs does not reverse the suppression of cytokine production mediated by the CD4+CD25+ T cells
Cytokines IFN-{gamma} and IL-2 are secreted by active, proliferating Th1 cells. The secretion of these two cytokines in the proliferation co-cultures, consisting of varying ratios of CD4+CD25+ regulatory T cells and CD4+CD25– responder T cells and fixed numbers of co-stimulatory cells ± anti-CD28, was determined by ELISA (Fig. 4A and B). It was observed that the iDCs or mDCs matured either by LPS or polyI:C did not lead to reversal of suppression in IFN-{gamma} production induced by CD4+CD25+ T cells (P = <0.05, at Treg:Tresp of 1:2 and 1:1, and P = >0.05 at Treg:Tresp of 1:50, 1:10 and 1:5, for all of the groups). Interestingly, however, in the cultures with PBMCs and anti-CD28 co-stimulation, IL-2 production was not reduced in cultures with CD4+CD25+ T cells compared with cultures without CD4+CD25+ T cells (Fig. 4B). In fact, the IL-2 production was higher in the anti-CD28 + PBMC-stimulated cultures with CD4+CD25+ T cells in comparison to the cultures without. IL-2 was not produced to a detectable level in culture with co-stimulation provided by PBMCs alone. However, in the cultures with iDC or mDCs, IL-2 was produced at a lower level in the cultures with Treg compared with the ones without Treg (P = <0.05, at Treg:Tresp of 1:2 and 1:1, and P = >0.05 at Treg:Tresp of 1:50, 1:10 and 1:5, for all of the groups).


Figure 4
Figure 4
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  		Fig. 4. CD4+CD25+ T cells modulate cytokine production depending on the co-stimulation source. Cytokine secretion by T cells stimulated with 10 µg ml–1anti-CD3 antibodies and co-stimulation provided by PBMCs (without or with 1 µg ml–1anti-CD28 antibodies), immature DCs or mature DCs (stimulated with LPS or Poly I:C). All concentrations are shown in picogram per milliliter. IFN-{gamma} (A), IL-2 (B) and IL-10 (C) secretion were examined in co-cultures at varying ratios of CD4+CD25+ T cells (open bars) or CD4+CD25–T cells (filled bars) (as controls) to responder CD4+CD25–T cells (5000 per well). (D) TGF-ß secretion was examined in co-cultures at 1:1 ratio. All data shown are representative of three independent experiments.

 
The immunoregulatory cytokine IL-10 has been postulated as a suppression mechanism utilized by Treg cells in vivo, but its role in in vitro suppression remains uncertain. Overall, we found that cultures produced less IL-10 than IFN-{gamma}. In cultures with PBMCs and PBMCs with anti-CD28, IL-10 was not detected (<15 pg ml–1, undiluted culture supernatants) in the presence or absence of Treg (data not shown). Interestingly, upon stimulation with iDCs IL-10 production was not discriminated between the cultures with or without regulatory T cells. However, in the cultures with LPS and polyI:C-stimulated DCs, there was higher IL-10 production in the groups with CD4+CD25+ T cells except in the 1:1 ratios but these differences were not significant (Fig. 4C) (P = >0.1 at each of the Treg:Tresp ratios with all of the co-stimulations used). These results suggest that the DCs did not reverse the suppressive effect of CD4+CD25+ T cells by inhibiting IL-10 production. Upon culturing of iDC, LPS-mDCs or polyI:C-mDCs alone (1 x 104 per well per 200 µl), IL-10 was not detected (<15 pg ml–1), suggesting that the IL-10 detected in the experiments described above was not produced by the DCs.

Secretion of TGF-ß, another potent immunoregulatory cytokine reported to be an effector mechanism of suppression mediated by regulatory T cell, was also determined (Fig. 4D). At ratios of 1:1 (Treg:Tresp) TGF-ß levels were produced at higher levels in the cultures with CD4+CD25+ T cells, compared with the cultures without CD4+CD25+ T cells, whereas at lower Treg:Tresp ratios, detectable levels of TGF-ß were not observed. TGF-ß production was also not inhibited by the co-stimulation with iDCs or mDCs. In all of the groups, upon stimulation with iDCs, LPS-DCs, PBMCs or PBMCs with anti-CD28, considerably higher levels of TGF-ß was produced in the cultures with CD4+CD25+ T cells, compared with the cultures without. However, in the culture stimulated with polyI:C-mDCs, the overall TGF-ß levels were reduced compared with the cultures stimulated with iDCs or LPS-mDCs. Upon culturing of iDC, LPS-mDCs or polyI:C-mDCs alone (1 x 104 per well per 200 µl), TGF-ß was not detected within the assay range (<15 pg ml–1).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The adaptive immune system allows one to react and defend against a pathogen while also remaining quiescent or tolerant against self-non-pathogenic antigens. However, in certain circumstances this quiescence against self-antigens can be broken resulting in autoimmune diseases. The central tolerance to self-antigens is brought about by clonal deletion and clonal anergy/suppression. It has been suggested that an extrinsic form of dominant peripheral tolerance takes the form of natural Treg cells circulating in the peripheral blood to actively maintain homeostasis (27, 28). Various accessory or co-stimulatory molecules, cells and cytokines are key in controlling and modulating the function of natural CD4+CD25+ regulatory T cells. However, it is not clear how these factors and signals are integrated or disintegrated in order to actively regulate Treg functions. There must be plasticity in the function of regulatory T cells in order to avoid a sub-optimal immune response to an invading pathogen.

In human studies, there has been strong evidence supporting the existence of regulatory T cells but the factors that regulate their function are poorly understood. We found that CD4+CD25+ T cells constituted ~3.12 ± 2.71% of all NACs, corresponding to ~4–10% of CD4+ T cells. This agrees with reports from other groups which found that 1–10% of CD4+ T cells are CD25+ and thus, it is most likely that we have isolated the same population (14–21).

Valmori et al. (18) showed that CD4+CD25+CD45RA+ is a subset of naive Treg in human peripheral blood. We found that CD45RA+CD4+CD25+ T cells constitute only a smaller (~45.1%) population of CD4+CD25+ T cells and that a higher percentage of CD4+CD25– T cells expressed CD45RA (65.3%). In addition to CD45RA, we also examined the surface GITR and CTL-associated antigen 4 as well as intracellular Foxp3 expression. While GITR is up-regulated upon human CD4+ T cell activation, it has been reported to be preferentially expressed on CD4+CD25+ T cells (29). Likewise, in mice, CD4+CD25+ T cells also constitutively express GITR and up-regulate it upon activation (7, 30–32). However, GITR expression on CD4+CD25– T cells is believed to interact with GITR ligand on APCs which somehow renders them resistant to CD4+CD25+ T cell-mediated suppression (30). Similarly, removal of GITR+ T cells or addition of blocking anti-GITR monoclonal antibodies resulted in organ-specific autoimmunity in mice (7, 31). Uraushihara et al. (32) have reported that murine CD4+GITR+ T cells, regardless of CD25 expression are capable of suppressing T cell proliferation in vitro and preventing wasting disease and colitis in vivo. Thus, GITR appears to have a role in regulating immunosuppression. When we tested human T cells for GITR expression, we found that a greater proportion of CD4+CD25+(High) T cells were GITR+ but overall, the proportions were equal between CD4+CD25+(All) T cells and CD4+CD25– T cells (Table 1). Similar to GITR, we found that a greater proportion of CD4+CD25+(High) T cells express extracellular CTL-associated antigen 4 (Table 1). However, unlike GITR, the proportion of CTL-associated antigen 4 expressed by CD4+CD25+(All) T cells is nearly double that of CD4+CD25– T cells. CTL-associated antigen 4 is a co-inhibitory molecule that is reported to be constitutively expressed on human and murine CD4+CD25+ T cells and/or up-regulated upon activation (6, 14, 18–21). However, its role has been somewhat inconsistent. Kataoka et al. (33) recently found that murine CD4+CD25+ T cells still possessed regulatory activity with a mutant-truncated CTL-associated antigen 4. Addition of anti-CTL-associated antigen 4 blocking antibodies has been controversial with some reporting no effects on suppression (5, 20) while others report that inhibiting CTL-associated antigen 4 interaction reverses or partially reverses CD4+CD25+ T cell activity (21, 34).

Foxp3 is a member of forkhead or the winged helix family of transcription factors. Foxp3 has been identified as a master regulatory gene for Treg lineage commitment or developmental differentiation of Treg cells (9, 10). This association has been quite stringent in mouse Treg cells. In our studies, we first wanted to identify an association of Foxp3 expression within CD4+CD25+ regulatory T cell populations, in order to more stringently purify human natural Treg cells, and examine their modulation by DCs. Therefore, intracellular Foxp3 expression was examined in various CD4 T cell populations by multicolor staining and intracellular staining for Foxp3 (Fig. 2, Table 1). First in three-color staining, it was observed that Foxp3 was equivalently expressed in all of the CD4 populations examined (CD4+CD25+(All), CD4+CD25+(High), CD4+CD25+(Med–low) and in CD4+CD25–). Then four-color flow cytometry was performed to examine whether any specific association of Foxp3 expression existed among putative CD4+CD25+ regulatory T cell populations expressing CD45RA, CTL-associated antigen 4 or GITR (Table 1). Although the percentage of Foxp3+ T cells fluctuated depending on the associated marker (i.e. CD45RA, CTL-associated antigen 4 or GITR), the percentage within their respective CD4+CD25+ T cell and CD4+CD25– T cell subsets fell within one standard deviation of the other. Thus, in multiple experiments with independent donors, there did not appear to be any correlation of expression of Foxp3 to the CD4+CD25+ regulatory T cell populations. These studies, although contradictory to the idea that Foxp3 is a distinguishable marker for Treg, are in line with previous observations made in human Treg cells where mRNA for Foxp3 was not found to be confined to the CD4+CD25+ regulatory T cell populations (13). Therefore, we performed purification of T regulatory cells based on CD4+CD25+ expression by FACS or MACS. Both of these methods provided highly pure populations of CD4+CD25+ and CD4+CD25– T cells and were used invariably in our experiments.

DCs are professional APCs. Non-activated iDCs reside in various tissues of the body and are capable of recognizing and capturing antigens bearing pathogen-associated molecular patterns. Upon activation, the DCs up-regulate co-stimulatory molecules, process and present antigens in context of MHC molecules, and produce cytokines to provide efficient priming and stimulation of antigen-specific naive T cells (35). In addition to their sentinel duties, DCs also play a major role in peripheral tolerance and homeostasis. In the peripheral blood of humans, there are two distinct populations of DCs or DC precursors which can be distinguished based on phenotypical and morphological characteristics. Myeloid DCs (MDCs) were thought to be from myeloid origin and plasmacytoid DCs (PDCs) from lymphoid origin. However, more recent evidence suggests that MDCs and PDCs may be interrelated and may not represent separate lineages because both PDC and MDCs can be generated from lymphoid as well as myeloid progenitors expressing Flt3, and PDCs can differentiate into MDCs following viral infection (36). The PDCs uniquely express TLR7 and TLR9 which allows them to recognize viral single-stranded RNA and CpG DNA, respectively. The PDCs produce IFN-{alpha} and IFN-ß upon activation which can induce anti-viral immune responses. High production of type-1 interferons by PDCs promotes their survival and increases MHC expression on neighboring APCs, thus enhancing anti-viral immunity. The MDCs selectively express TLR4 but lack TLR7 and TLR9 and thus, are able to respond to LPS stimulation but not to viral danger molecules (37, 38). The local cytokine environment contributes significantly to the establishment of DC phenotype. Although the DCs obtained from differentiation of monocytes are heterogeneous, we used two different stimuli i.e. LPS or polyI:C to obtain maturation and activation of DCs through TLR4 and TLR3, respectively, and test the ability of these differentially mDCs in modulating the suppressive action of CD4+CD25+ T cells.

Freshly isolated CD4+CD25+ T cells were combined with CD4+CD25– responder T cells in increasing ratios along with anti-CD3 monoclonal antibodies for TCR stimulation and iDCs, LPS-mDCs, polyI:C-mDCs, PBMCs or PBMCs plus anti-CD28 antibody to provide co-stimulation (Fig. 3A). We observed that in the cultures with PBMCs, presence of Treg cells led to suppression of proliferation of responder T cells. Suppression of responder T cell proliferation by regulatory T cells was reversed by the presence of iDCs or mDCs. However, providing co-stimulation by anti-CD28 in the presence of PBMCs did not reverse the suppressive effect. The reversal of the suppression was not due to higher proliferation of Treg cells in the presence of DCs, as in cultures where CD4+CD25+ T cells or CD4+CD25– T cells were individually stimulated by DCs or PBMCs, CD4+CD25+ T cells showed less proliferation than CD4+CD25– T cells (Fig. 3B).

Cytokine analyses showed that cultures with CD4+CD25+ T cells produced significantly less IFN-{gamma} than cultures without CD4+CD25+ T cells. The inhibition of IFN-{gamma} production due to the presence of CD4+CD25+ T cells was not reversed upon providing co-stimulation with iDCs, LPS-mDCs, polyI:C-mDCs or PBMCs + anti-CD28. The production of IFN-{gamma} did not correspond to the effect of these stimulations on proliferation, suggesting that DCs that modulate the suppressive effect of Treg on proliferation have no effect on IFN-{gamma} production. It was previously reported that Treg cells induce DCs to secrete IFN-{gamma}, which stimulates the DCs to produce indoleamine 2,3-dioxygenase, an enzyme that effectively degrades tryptophan and reduces T cell function and/or viability (39). However, we did not find that high IFN-{gamma} levels correlate to reduced T cell proliferation.

Determination of IL-2 production presented a similar interesting scenario. The levels of IL-2 being produced in the cultures with Treg were significantly reduced compared with the cultures without, and stimulation by DCs did not lead to a reversal of suppression in IL-2 production. Interestingly, IL-2 suppression was reversed in the presence of PBMC and anti-CD28, apparently due to increased stability of the IL-2 mRNA. These results suggested that the reversal of suppression by DCs was also not connected to reversal in IL-2 production, and DCs' mediated modulation of Treg is independent of IL-2 production. This is an important observation. In previous studies, IL-2 was suggested to be a target for Treg cells where IL-2 production by responder T cells was inhibited in order to provide suppression (28). This contrasted with the previous observations as well as our results which showed that providing co-stimulation through CD28 induced bolus IL-2 production, but no reversal in suppression mediated by Treg. The independence of IL-2 production to the function of regulatory T cells has also been suggested recently (40). Therefore, DC derived signals do not reverse the capacity of the regulatory T cells to inhibit IL-2 production by responder T cells.

IL-10 has been suggested to be an important regulatory cytokine produced by regulatory T cells (14). It has been shown that stimulation of naive CD4+ T cells by iDCs results in IL-10 producing regulatory T cells (14). In our studies, IL-10 was not detected in the cultures where PBMCs and PBMC + anti-CD28 were used as co-stimulation in the cultures with or without CD4+CD25+ T cells. Interestingly, in the cultures with iDCs, significantly higher levels of IL-10 were produced, but were independent of the presence or absence of regulatory CD4+CD25+ T cells. However, in the presence of LPS or polyI:C-mDCs, IL-10 was produced in higher amounts in the presence of CD4+CD25+ T cells compared with the cultures without CD4+CD25+ T cells (except at supra-physiological 1:1 ratios). These results suggest that LPS and polyI:C-mDCs, in contrast to iDCs, induce more IL-10 production by co-cultures with Treg cells compared with co-cultures without Treg cells. Nonetheless, IL-10 production is independent of the suppressive effect of regulatory T cells since IL-10 was not detected in proliferation cultures where suppression was seen (PBMCs ± anti-CD3).

With respect to TGF-ß, secretion was higher in the presence of CD4+CD25+ T cells compared with cultures with only CD4+CD25– T cells. In addition, TGF-ß levels were not suppressed by the presence of DCs, which led to modulation of suppressive action by regulatory T cells.

In conclusion, we have provided the first direct evidence in humans that iDCs and mDCs are capable of modulating the suppressive effect of regulatory T cells on responder T cell proliferation. Our results demonstrate that this modulation of regulatory T cell activity by DCs is not due to a decrease in IL-2 and/or IFN-{gamma},or an increase in TGF-ß and/or IL-10 production. It is possible that the role of DCs in modulating Treg mediated suppression is related to the interaction of various co-stimulatory molecules expressed on the DCs with the ligands expressed on Treg, such as CD80/CD28, CD86/CTL-associated antigen 4, CD27/CD70 and/or CD40/CD40L. In our study, responder T cells exhibited suppressed proliferation when incubated with CD4+CD25+ T cells and PBMCs. Although the addition of anti-CD28 antibodies did not reverse suppression, the level of proliferation was greater than incubation with PBMCs alone (compare y-axis in Fig. 3A). Addition of DCs possibly provided a stronger co-stimulation source allowing the complete reversal of suppression and proliferation equal to or greater than cultures without CD4+CD25+ T cells. In addition, we found that a higher proportion of CD4+CD25+(High) T cells expressed CTL-associated antigen 4 compared with other CD4+CD25+/– T cell subsets. It may be possible that with a weak co-stimulation source, CTL-associated antigen 4 expressed on CD4+CD25+(High) T cells may effectively suppress responder T cell proliferation through negative signaling. However, strong co-stimulation expressed on DCs may outcompete CTL-associated antigen 4 on CD4+CD25+(High) T cells allowing the responder T cells to proliferate. We are currently examining the role of co-stimulation and contact dependence in DC-mediated modulation of regulatory T cell activity. It is also possible that this effect is mediated by soluble effectors other than those studied in this report.

Irrespective of the exact mechanism of the modulation of Treg function by DCs, our results support the notion that Treg activity can be modulated at several checkpoints independently in order to finely tune the outcome of adaptive immune responses. Immune regulation is a complex network of interactions between immune cells throughout the body, and the identification of distinct pathways to modulate regulatory T cells would help develop novel treatments for fighting infectious diseases, cancer, autoimmune diseases and increasing the success of organ transplantations.


   Acknowledgements
 
The authors sincerely thank Jie Li for much appreciated help in the lab and Dorothy Rutkowski for cell sorting. Chris Tse and Jeff Konowalchuk in the Agrawal lab are thanked for helpful discussions. This work was supported by an operating grant from the Canadian Institutes of Health Research (Canada, EOP 79327) and Canadian Foundation for Innovation New Opportunities Funds to B.A. J.S.A is a recipient of summer studentship awards from the Alberta Heritage Foundation for Medical Research and Northern Alberta Clinical Trials and Research Centre. B.A. is a recipient of the Alberta Heritage Foundation for Medical Research (Canada) Medical Scholar Award and Establishment grant.


   Abbreviations
 
APC, antigen-presenting cell
DC, dendritic cell
GITR, glucocorticoid-induced tumor necrosis factor receptor family-related protein
LPS-mDC, LPS-matured dendritic cell
IDC, immature dendritic cell
MACS, magnetic cell sorting
mDCs, mature dendritic cells
MDCs, myeloid dendritic cells
NAC, non-adherent cell
PDCs, plasmacytoid dendritic cells
polyI:C-mDCs, polyI:C-matured dendritic cell
TGF, transforming growth factor

   Notes
 
* These authors contributed equally to this work. Back

Transmitting editor: A. Falus

Received 25 September 2006, accepted 30 November 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, 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]
  2. Fehervari Z and Sakaguchi S. (2004) Development and function of CD25+CD4+ regulatory T cells. Curr. Opin. Immunol. 16:203.[CrossRef][Web of Science][Medline]
  3. Fontenot JD and Rudensky AY. (2005) A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat. Immunol. 6:331.[CrossRef][Web of Science][Medline]
  4. Belkaid Y and Rouse BT. (2005) Natural regulatory T cells in infectious disease. Nat. Immunol. 6:353.[CrossRef][Web of Science][Medline]
  5. Thornton AM and Shevach EM. (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]
  6. Takahashi T, Tagami T, Yamazaki S, et al. (2000) Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
  7. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S. (2002) Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3:135.[CrossRef][Web of Science][Medline]
  8. Sakaguchi S. (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6:345.[CrossRef][Web of Science][Medline]
  9. Fontenot JD, Gavin MA, Rudensky AY. (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[CrossRef][Web of Science][Medline]
  10. Hori S, Nomura T, Sakaguchi S. (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.[Abstract/Free Full Text]
  11. Bennett CL, Christie J, Ramsdell F, et al. (2001) The immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27:20.[CrossRef][Web of Science][Medline]
  12. Walker MR, Kasprowicz DJ, Gersuk VH, et al. (2003) Induction of Foxp3 and acquisition of T regulatory activity by stimulated human CD4+CD25– T cells. J. Clin. Invest. 112:1437.[CrossRef][Web of Science][Medline]
  13. Morgan ME, Van Bilsen JHM, Bakker AM, et al. (2005) Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans. Hum. Immunol. 66:13.[Web of Science][Medline]
  14. Jonuleit H, Schmitt E, Schuler G, Knop J, Enk AH. (2000) Induction of interleukin 10-producing nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
  15. Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH. (2001) Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:285.
  16. Taams LS, Smith J, Rustin MH, Salmon M, Poulter LW, Akbar AN. (2001) Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis-prone population. Eur. J. Immunol. 31:1122.[CrossRef][Web of Science][Medline]
  17. Taams LS, Vukmanovic-Stejic M, Smith J, et al. (2002) Antigen-specific T cell suppression by CD4+CD25+ regulatory T cells. Eur. J. Immunol. 32:1621.[CrossRef][Web of Science][Medline]
  18. Valmori D, Merlo A, Souleimanian NE, Hesdorffer CS, Ayyoub MA. (2005) Peripheral circulating compartment of natural naive CD4 Tregs. J. Clin. Invest. 115:1953.[CrossRef][Web of Science][Medline]
  19. Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. (2001) Ex vivo isolation and characterization of CD4+CD25+ T cells with regulatory properties from human blood. J. Exp. Med. 193:1303.[Abstract/Free Full Text]
  20. Levings MK, Sangregorio R, Roncarolo MG. (2001) Human CD25+CD4+ T regulatory cells suppress naïve and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295.[Abstract/Free Full Text]
  21. AnnunziatoCosmi FL, Lotta F, Lazzeri E, et al. (2002) Phenotype, localization, and mechanism of suppression of CD4+CD25+ human thymocytes. J. Exp. Med. 196:379.[Abstract/Free Full Text]
  22. Fehevari Z and Sakaguchi S. (2004) Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells. Int. Immunol. 16:1769.[Abstract/Free Full Text]
  23. Editorial. (2005) Essence of harmony. Nat. Immunol. 6:325.[CrossRef][Web of Science][Medline]
  24. Ahn JS and Agrawal B. (2005) IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells. Int. Immunol. 17:1337.[Abstract/Free Full Text]
  25. Li W, Li J, Tyrrell DL, Agrawal B. (2006) Expression of hepatitis C virus-derived core or NS3 antigens in human dendritic cells leads to induction of pro-inflammatory cytokines and normal T-cell stimulation capabilities. J. Gen. Virol. 87:61.[Abstract/Free Full Text]
  26. Li W, Krishnadas DK, Li J, Tyrrell DL, Agrawal B. (2006) Induction of primary human T cell responses against hepatitis C virus-derived antigens NS3 or core by autologous dendritic cells expressing hepatitis C virus antigens: potential for vaccine and immunotherapy. J. Immunol. 176:6065.[Abstract/Free Full Text]
  27. Schwartz RH. (2005) Natural regulatory T cells and self-tolerance. Nat. Immunol. 6:327.[CrossRef][Web of Science][Medline]
  28. Sakaguchi S. (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6:345.[CrossRef][Web of Science][Medline]
  29. Li Z, Mahesh SP, Kim BJ, Buggage RR, Nussenblatt RB. (2003) Expression of glucocorticoid induced TNF receptor family related protein (GITR) on peripheral T cells from normal human donors and patients with non-infectious uveitis. J. Autoimmun. 21:83.[CrossRef][Web of Science][Medline]
  30. Stephens GL, McHugh RS, Whitters MJ, et al. (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J. Immunol. 173:5008.[Abstract/Free Full Text]
  31. McHugh RS, Whitters MJ, Piccirillo CA, et al. (2002) CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[CrossRef][Web of Science][Medline]
  32. Uraushihara K, Kanai T, Ko K, et al. (2003) Regulation of murine inflammatory bowel disease by CD25+ and CD25– CD4+ glucocorticoid induced TNF receptor family-related gene+ regulatory T cells. J. Immunol. 171:708.[Abstract/Free Full Text]
  33. Kataoka H, Takahashi S, Takase K, et al. (2005) CD25+CD4+ regulatory T cells exert in vitro suppressive activity independent of CTL-associated antigen 4. Int. Immunol. 17:421.[Abstract/Free Full Text]
  34. Read S, Malmstrom V, Powrie F. (2000) Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  35. Adams S, O'Neill DW, Bhardwaj N. (2005) Recent Advances in dendritic cell biology. J. Clin. Immunol. 25:87.[CrossRef][Web of Science][Medline]
  36. Zuniga EI, McGavern DB, Pruneda-Paz JL, Teng C, Oldstone MB. (2004) Bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection. Nat. Immunol. 5:1227.[CrossRef][Web of Science][Medline]
  37. Coccia EM, Severa M, Giacomini E, et al. (2004) Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocyte-derived dendritic cells. Eur. J. Immunol. 34:796.[CrossRef][Web of Science][Medline]
  38. Ito T, Wang YH, Liu YJ. (2005) Plasmacytoid dendritic cell precursors/type I interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9. Springer Semin. Immunopathol. 26:221.[CrossRef][Web of Science][Medline]
  39. Fallarono F, Grohmann U, Hwang KW, et al. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4:1206.[CrossRef][Web of Science][Medline]
  40. Brinster C and Shevach EM. (2005) Bone marrow-derived dendritic cells reverse the anergic state of CD4+CD25+ T cells without reversing their suppressive function. J. Immunol. 11:7332.

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