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The Journal of Immunology, 2005, 175: 8051-8059.
Copyright © 2005 by The American Association of Immunologists

Human CD4+ T Cells Express TLR5 and Its Ligand Flagellin Enhances the Suppressive Capacity and Expression of FOXP3 in CD4+CD25+ T Regulatory Cells1

Natasha K. Crellin*, Rosa V. Garcia*, Omeed Hadisfar*, Sarah E. Allan*, Theodore S. Steiner{dagger} and Megan K. Levings2,*

* Department of Surgery and {dagger} Department of Medicine, University of British Columbia and Immunity and Infection Research Centre, Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25– T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Germline encoded pattern recognition receptors (PRRs),3 such as TLRs, provide a critical link between the innate and adaptive immune systems. This link is generally believed to involve APCs, which become capable of stimulating effector T cell responses following ligation of one or more PRRs (1). Interestingly, there is growing evidence to suggest that, in addition to their well characterized indirect effects mediated by APCs, stimulation via PRRs may also directly regulate the function of T cells (2, 3, 4). This interaction could be meaningful in the context of host-pathogen interactions and in intestinal homeostasis.

Immune responses are tightly controlled by a subset of T cells known as CD4+CD25+ T regulatory (Treg) cells, which have a central role in establishing and maintaining peripheral tolerance and immune homeostasis (5, 6, 7). Evidence for the role of CD4+CD25+ Treg cells in preventing autoimmunity and allergy, and promoting allograft acceptance is compelling (6, 7, 8). In addition, these cells have a key role in regulating immune responses to a variety of pathogenic and nonpathogenic infectious agents. For example, CD4+CD25+ Treg cells can prevent the development of colitis induced by the transfer of CD4+ effector T cells (9, 10) or by Helicobacter hepaticus (11), and even reverse established colitis (12). CD4+CD25+ Treg cells are also required for the persistence of several parasites such a Leishmania major (13), Mycobacterium tuberculosis, (14), and Candida albicans (15). Thus the interactions between CD4+CD25+ Treg cells and microorganisms, and the mechanisms that control tolerance vs immunity in this setting are of considerable interest.

Recent reports suggest that direct stimulation of CD4+CD25+ Treg cells via TLRs could modulate immune regulation. For example, mice lacking TLR2 have decreased numbers of peripheral CD4+CD25+ Treg cells, indicating that this molecule may influence expansion and/or maintenance of suppressive T cell populations (16). Furthermore, murine CD4+CD25+ Treg cells express high levels of mRNA for TLR1, 2, 4, 5, 7, and 8, and LPS, which acts through TLR4, has been shown to enhance their suppressive capacity (4). Finally, mice lacking MyD88, an adaptor molecule crucial for much of TLR-mediated signaling, exhibit increased susceptibility to dextran-sulfate-induced colitis (17), consistent with the concept that PRRs may also have anti-inflammatory effects in the intestine.

TLR5 is one of two known TLRs that are activated by proteins (18). Currently, the only characterized ligand for TLR5 is flagellin, a well-known pathogen-associated molecular pattern (PAMP) protein that autopolymerizes to form the flagella of motile bacteria (19). Flagellin has an important role in regulating virulence of intestinal bacteria (20, 21), and is required for the development of Salmonella-induced colitis (20). Interestingly, flagellin is also a dominant Ag in Crohn’s disease (22), suggesting that this protein may direct intestinal immune responses at multiple levels.

Because CD4+ T cell subsets encounter flagellin in the intestinal environment, we investigated whether human CD4+CD25+ Treg and/or CD4+CD25– T cells can directly respond to this bacterial element. Our results demonstrate that stimulation via TLR5 can directly influence the phenotype and function of both effector and suppressive T cells. This capacity of adaptive immune system cells to directly respond to PAMPs may represent an important new mechanism by which pathogens regulate immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell purification

Peripheral blood was obtained from healthy volunteers following informed consent and upon approval of the protocol by the University of British Columbia Clinical Research Ethics Board. PBMCs were isolated by density centrifugation over Ficoll (StemCell Technologies), and CD4+ T cells were subsequently purified by negative selection with magnetic beads (Miltenyi Biotec or StemCell Technologies). As TLR ligands are known to potently activate APCs, which could be an erroneous source of cytokines in some experiments, we rigorously ensured the purity of our isolated T cell populations. Following purification, CD4+ T cells were 95–98% pure and free of detectable CD14+ monocytes or CD19+ B cells (Fig. 1A). CD25+ cells were subsequently purified by positive selection (Miltenyi Biotec), and were passed over 2 MS columns to ensure 90–95% purity (Fig. 1B). The flow-through from the CD25+ selection was retained and used as CD25– cells.



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  		FIGURE 1. Purity of CD4+ T cell subsets. A, CD4+ T cells were purified by negative selection from PBMCs and analyzed for expression of CD4, CD14, and CD19. B, CD4+CD25+ Treg cells were isolated by positive selection and purity was assessed by analysis of CD4 and CD25 expression. Results are representative of >20 individual purifications.

 
In some cases, APCs were prepared in parallel by depletion of CD3+ T cells from PBMCs using magnetic beads (StemCell Technologies). Monocyte-derived dendritic cells (DCs) were generated from the adherent fraction of PBMCs by culture in the presence of IL-4 (10 ng/ml; R&D Systems) and GM-CSF (50 ng/ml; R&D Systems) as previously described (23). Cells were either left immature, or matured with LPS (100 ng/ml) for 48 h. Purity and maturation were verified by monitoring expression of CD1a, CD14, CD83, and HLA-DR (BD Pharmingen). CD14+ monocytes were purified by positive selection using CD14 magnetic beads (StemCell Technologies).

T cell lines and clones

CD4+ T cells were stained for CD4 (BD Pharmingen) and CD25 (Miltenyi Biotec) and FACS sorted into CD25high (top 2–3%) and CD25low fractions on a BD FACSAria. T cell lines were generated by stimulation with anti-CD3/CD28 coupled beads (Dynal Biotech) at a 1:1 ratio of cells to beads in T cell medium (X-VIVO 15, 5% human serum (North American Biologicals), 1x penicillin/streptomycin (Invitrogen Life Technologies), 1x glutamax (Invitrogen Life Technologies)) in the presence of rhIL-2 (100 U/ml; Chiron). Alternatively, sorted cells were cloned by limiting dilution as previously described (24). CD4+CD25+ T cell lines and clones were monitored at the end of every cycle to ensure preservation of their suppressive capacity.

Quantitative RT-PCR

For quantitative RT-PCR, amounts of TLR4, TLR5, and FOXP3 mRNA were determined using custom TaqMan probe and primer sets (Applied Biosystems). Oligonucleotides were as follows: human TLR4 sense 5'-TCC ATG AAG GTT TCC ATA AAA GC-3', antisense 5'-CCA GCG GCT CTG GAT GAA-3'; human TLR5 sense 5'-GCC TTG AAG CCT TCA GTT ATG C-3', antisense 5'-CCA ACC ACC ACC ATG ATG AG-3'; and FOXP3 sense 5'-TCA CCT ACG CCA CGC TCA T-3', antisense 5'-TCA TTG AGT GTC CGC TGC TT-3'. Probe sequences were as follows: human TLR4 5'-AAA GGT GAT TGT TGT GGT GTC CCA GCA-3'; human TLR5 5'-CAG GGC AGG TGC TTA TCT GAC CTT AAC AGT G-3'; and FOXP3 5'-TGG GCC ATC CTG GAG GCT CCA-3'. Amounts of GAPDH mRNA were determined using Assay on Demand real-time PCR kits (Applied Biosystems). All PCR were performed with TaqMan Master Mix (Qiagen) on an ABI 5700 Real-Time PCR machine. All samples were run in triplicate, and relative expression of FOXP3, TLR5, or TLR4 was determined by normalizing to GAPDH to calculate a fold change in value.

Source of flagellin and LPS

Flagellin was purified as previously described (19). In brief, recombinant His-tagged flagellin was expressed in BL21(DE3)pLysS bacteria and purified under native conditions by metal affinity chromatography. Contaminating LPS was removed by purification over a polymyxin B column (Detoxi-Gel; Pierce). The resulting flagellin was tested in a Limulus assay (Cambrex) and found to have <0.06 EU of LPS when tested at a concentration of 25 µg/ml. Biological activity was verified by stimulation of IL-8 release from Caco cells, as previously described (19). To further rule out possible effects of contaminants such as LPS or TLR2 ligands in the flagellin preparation, a noninflammatory variant of the Escherichia coli H18 flagellin generated by random mutagenesis (19) was tested in parallel and shown not to effect proliferation of, or cytokine production by, CD4+CD25– T cells (data not shown). Ultra pure LPS extracted according to the methods of Manthey et al. (25) was purchased from List Biological Laboratories. This LPS was biologically active and resulted in potent maturation of monocyte-derived immature DCs. In some experiments, a less pure form of LPS (Sigma-Aldrich), which is likely contaminated with other bacterially derived molecules, was also tested.

Flow cytometry

Expression of TLR5 on the cell surface was detected by incubation with a 1/200 dilution of an anti-TLR5 Ab (Alexis) followed by addition of a FITC-conjugated rabbit anti-goat secondary Ab (Sigma-Aldrich) on nonfixed nonpermeabilized cells. Total TLR5 expression (intracellular and extracellular) was determined following staining with a 1/50 dilution of an anti-TLR5 mAb directly conjugated to FITC (IMG-663F; IMGENEX) after the cells were fixed with 2% formaldehyde in PBS and permeabilized by saponin. Expression of FOXP3 was detected using PE anti-human FOXP3 staining kit (eBioscience) according to the manufacturer’s instructions. Samples were read on a BD FACSCanto and analyzed with FCS Express v.2 (DeNovo software).

ELISA

To determine amounts of IL-2, capture ELISAs (BD Pharmingen) were performed on supernatants after activation with the indicated concentration of immobilized anti-CD3 alone or with soluble anti-CD28 (1 µg/ml), in the absence or presence of flagellin (10–1000 ng/ml) or LPS (1 µg/ml) for 24 h.

Western blotting

To determine relative amounts of TLR5 expression, whole cell lysates from purified populations of cells were prepared by sonication in lysis buffer containing 1% SDS, 10 mM HEPES, and 2 mM EDTA (pH 7.4). Approximately 150 µg of protein was loaded per lane and subjected to SDS-PAGE electrophoresis. Following transfer to nitrocellulose membranes, expression of TLR5 was determined by immunoblotting with an anti-TLR5 mAb (IMG-664; IMGENEX) used at a 1/500 dilution, followed by goat anti-mouse-HRP (DAKO). The specificity of the Ab was confirmed with 293T cells transfected with a TLR5-encoding expression vector (data not shown). Membranes were stripped and reprobed with anti-p38 Abs (Santa Cruz Biotechnology) to assess loading equivalency.

Activation, proliferation, and suppression of T cells

To activate CD4+CD25+ Treg cells or CD4+CD25– T cells via TCR stimulation, cells were stimulated with anti-CD3/CD28-coupled beads (1 bead/2.5 cells; Dynal Biotech), or the indicated concentration of immobilized anti-CD3 mAbs in the presence or absence of soluble anti-CD28 mAbs (1 µg/ml). To test the proliferative capacity of CD4+CD25+ Treg cells, cells were plated (8000 cells/well in 200 µl) in 96-well round-bottom plates and stimulated with anti-CD3/CD28 beads (1 bead/6 cells).

To test for suppressive capacity in the absence of APCs, CD4+CD25– T cells were plated at 8000 cells/well (in 200 µl) in 96-well round-bottom plates, and stimulated with anti-CD3/CD28-coupled beads (1 bead/6 cells) in T cell medium (optimum bead to cell ratio varies slightly per lot of beads). CD4+CD25+ Treg cells were added in decreasing amounts, starting at a ratio of 1:1. Proliferation was assessed at day 6, after addition of [3H]thymidine (1 µCi per well; Amersham).

To test for suppressive capacity in the presence of APCs, CD4+CD25– T cells (50,000 cells/well in 200 µl) were stimulated with soluble anti-CD3 mAbs (OKT3, 1 µg/ml) in the presence of APCs (CD3-depleted PBMCs, irradiated 5000 rad, 50,000 cells/well). CD4+CD25+ Treg cells were added in decreasing amounts, and suppression was assessed after 4 days by determining the amount of [3H]thymidine incorporation. For all proliferation and suppression experiments, flagellin or LPS was added at the initial time of culture in amounts shown in figures.

Statistical analysis

All analysis for statistically significant differences was performed with the Student’s paired t test. Values of p < 0.05 were considered significant. All cultures were performed in triplicate and error bars represent the SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4+CD25+ Treg cells express high levels of TLR5 mRNA

We investigated whether human CD4+CD25+ Treg or CD4+CD25– T cells expressed TLR5 using quantitative RT-PCR. Although TLR5 mRNA was detected in both populations, the amounts in CD4+CD25+ Treg cells were significantly (p = 0.0048) higher than in CD4+CD25– T cells (Fig. 2A). Because polyclonal populations of human CD4+CD25+ cells contain a mixture of activated effector T cells and Treg cells (24), we confirmed this finding at the clonal level. Similar to freshly isolated cells, CD4+CD25+ Treg cell clones were found to express higher levels of mRNA for TLR5 than their nonsuppressive counterparts (Fig. 2A). We were also interested to know whether human CD4+CD25+ Treg cells expressed high levels of mRNA for TLR4, as previously described in the mouse (4). As shown in Fig. 2B, both ex vivo isolated CD4+CD25+ Treg cells and T cell clones expressed more TLR4 mRNA than their CD4+CD25– counterparts; however in ex vivo cells, this result was variable and not statistically significant.



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  		FIGURE 2. CD4+CD25+ Treg cells express more TLR5 and TLR4 mRNA in comparison to CD4+CD25– T cells. A, RNA was isolated from ex vivo purified CD4+CD25+ Treg and CD4+CD25– T cells or suppressive (Treg) and nonsuppressive (Th) CD4+CD25+ T cell clones. Expression of TLR5 was determined by quantitative RT-PCR. B, Levels of TLR4 mRNA were tested in parallel. C, Levels of TLR5 mRNA in CD4+CD25– T cells and CD4+CD25+ Treg cells were compared with those in ex vivo CD14+ monocytes and DCs in an immature (Imm) or mature (Mat) state. Data are expressed as relative amounts compared with the CD4+CD25– T cells (A and B) or mature DCs (C) and are normalized to amounts of GAPDH. A and B, Each point represents an individual experiment done with different donors or clones, and the horizontal line represents the average. C, Results are representative of three experiments with different donors.

 
We next investigated whether the levels of TLR5 mRNA in CD4+ T cells were comparable to those in innate immune cells known to be highly TLR5 responsive. Relative levels of TLR5 mRNA in both CD4+CD25+ and CD4+CD25– T cells were within the range of those in CD14+ monocytes, and slightly higher than levels in immature and mature DCs (Fig. 2C). Thus, TLR5 mRNA appears to be expressed in human CD4+ T cells at physiologically relevant levels.

TLR5 protein is expressed on the cell surface of CD4+ T cells at physiologically relevant levels

We next investigated the relative levels of TLR5 expression at the protein level. Although some TLRs, particularly those that bind to nucleic acids, are held in intracellular reservoirs (26), it has been reported that TLR5 must be at the cell surface for signaling to occur (27). We therefore determined the level of TLR5 surface expression by flow cytometry. Interestingly, we did not observe any significant difference in TLR5 protein expression between CD4+CD25– effector T cells and CD4+CD25+ Treg cells (Fig. 3A). The average difference between the mean fluorescence intensity of the secondary alone control vs cells stained with TLR5 was 4.4 ± 0.3 for CD4+CD25– T cells compared with 3.9 ± 0.8 for CD4+CD25+ Treg cells (p = NS). The amount of cell surface TLR5 in both populations of T cells, however, was comparable to that on monocytes, immature, and mature DCs (Fig. 3A).



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  		FIGURE 3. CD4+ T cells express physiologically relevant levels of TLR5 protein. A, TLR5 expression was determined by flow cytometry on CD4+CD25+ Treg or CD4+CD25– T cells, CD14+ monocytes, and immature (Imm) DCs or LPS-matured (Mat) DCs. A, Levels of cell surface TLR5. B, Total TLR5 expression as determined by intracellular staining. The thin dotted line represents secondary Ab alone controls, whereas the thick line represents anti-TLR5 staining. C, Whole cell lysates from CD4+CD25+ Treg, CD4+CD25– T cells, and CD14+ monocytes were resolved by SDS-PAGE and immunoblotted with anti-TLR5 mAbs. The membrane was reprobed with anti-p38 Abs to assess equivalency of loading. D, CD4+ T cells were stained, gated on FOXP3+ and FOXP3– populations, and amounts of total TLR5 were compared between the two populations. The black line represents FOXP3+ cells and the gray line represents FOXP3– cells. Results in A and B are representative of four individual experiments and of two experiments in C and D.

 
Faced with the lack of correlation between cell surface TLR5 protein and mRNA levels in CD4+CD25+ Treg cells and CD4+CD25– cells, we asked whether intracellular stores of TLR5 protein might differ between these populations. We therefore performed intracellular staining on the same populations of cells using an Ab raised against the intracellular portion of TLR5. As shown in Fig. 3B, there was no significant difference between the total amount of TLR5 in CD4+CD25+ Treg cells compared with CD4+CD25– T cells. Similar to our findings with extracellular staining only, the amounts of total TLR5 in T cells were comparable to those in monocytes and DCs. The similarity in the level of TLR5 expression between T cell subsets was also confirmed by Western blot analysis, which further confirmed that the levels of expression were comparable to those in innate immune cells (Fig. 3C).

It is now well accepted that the FOXP3 transcription factor is a more specific marker than CD25 for Treg cells (28, 29). We therefore repeated the intracellular staining on total CD4+ T cells gating on the FOXP3 positive and negative populations. CD4+FOXP3+ T cells and CD4+FOXP3– T cells expressed equal levels of TLR5 (Fig. 3D). Similar results were obtained when ex vivo CD4+ T cells were gated on the CTLA-4+ or CTLA-4– populations (data not shown). Thus, despite higher levels of mRNA, there was no difference in TLR5 protein expression between CD4+CD25– T cells and CD4+CD25+ Treg cells.

We also attempted to examine the cell surface expression of TLR4 on CD4+ T cell subsets, but found these levels to be very low and often undetectable (data not shown). This finding is consistent with a previous report, which found that TLR4 was present on the surface of CD4+ T cells only after activation with anti-CD3 mAbs and IFN-{alpha} (2).

Expression of TLR5 on CD4+CD25+ Treg and CD4+CD25– T cells following activation

CD4+CD25+ Treg cells constitutively express high levels of markers such as CD25, HLA-DR, GITR, and CTLA-4, which are also transiently up-regulated on activated effector T cells (24, 30). It was therefore important to determine the levels of TLR5 following TCR-mediated activation, to determine whether TLR5 is another activation marker for CD4+ T cells. CD4+CD25+ Treg cells or CD4+CD25– T cells were activated with anti-CD3/CD28 mAbs, and RNA was isolated after 0, 24, or 48 h. Levels of TLR5 mRNA dropped dramatically in both CD4+CD25+ Treg cells (average decrease 95% ± 6, n = 3; p = 0.0006) and CD4+CD25– T cells (average decrease 89% ± 10, n = 3; p = 0.002) following activation (Fig. 4A).



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  		FIGURE 4. Changes in TLR5 expression following activation. A, CD4+CD25+ Treg or CD4+CD25– T cells were activated with anti-CD3/CD28-coupled beads, and RNA was collected after 0, 24, and 48 h. Amounts of TLR5 mRNA were determined by quantitative RT-PCR and normalized to GAPDH. Data are expressed as relative amounts compared with the lowest sample. B, Total CD4+ cells were activated with anti-CD3/CD28-coupled beads and cell surface or total (intracellular) expression of TLR5 was determined by flow cytometry at the times indicated. Numerical values indicate the change in mean fluorescence intensity ({Delta}MFI) between secondary only controls and TLR5 staining. Results are representative of three individual experiments in A and four individual experiments in B.

 
We also examined whether TCR-mediated activation altered the expression of cell surface and/or total TLR5 protein in these T cell subsets. Amounts of total TLR5 expression transiently increased (between 12 and 24 h) and then rapidly declined by 48–72 h, with the exact kinetics depending on the donor (Fig. 4B), in both T cell subsets. In contrast, cell surface TLR5 expression gradually declined over the time course in both T cell subsets. Together with the decrease in mRNA expression, these data suggest that following T cell activation, the amount of functional TLR5 on CD4+ T cells, and thus their capacity to respond to flagellin, is decreased.

Flagellin fails to reverse hyporesponsiveness of CD4+CD25+ Treg cells, but is a costimulatory molecule for CD4+CD25– T effector cells

One of the defining characteristics of CD4+CD25+ Treg cells is their hyporesponsiveness to polyclonal stimuli (24). We therefore investigated whether costimulation with flagellin, the only known ligand for TLR5, affected the proliferative capacity of CD4+ T cells. CD4+CD25+ Treg or CD4+CD25– T cells were stimulated with anti-CD3/CD28 mAb coupled beads, in the absence or presence of increasing amounts of flagellin (Fig. 5A). Addition of flagellin over a wide range of concentrations failed to break the anergy of CD4+CD25+ Treg cells.



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  		FIGURE 5. Flagellin does not alter CD4+CD25+ Treg cell hyporesponsiveness but it increases CD4+CD25– T cell proliferation and IL-2 production. A, Ex vivo isolated CD4+CD25+ Treg or CD4+CD25– T cells (8000 cells/well) were stimulated with anti-CD3/CD28-coupled beads in the absence or presence of flagellin or LPS (1 µg/ml), and amounts of [3H]thymidine incorporation were measured after 6 days. B, CD4+CD25– T cells (100,000 cells/well) were stimulated with immobilized anti-CD3 mAbs alone or in combination with anti-CD28 mAbs (1 µg/ml), in the absence or presence of flagellin or LPS (1 µg/ml), and amounts of [3H]thymidine incorporation were measured after 3 days. C, Supernatants were collected in parallel after 24 h, and IL-2 was measured by ELISA. Note that the scale is different between the left and right panels. A single representative experiment of at least three performed is shown.

 
In contrast, flagellin significantly enhanced the proliferation of CD4+CD25– T cells in response to stimulation with anti-CD3 mAbs in a dose-dependent manner (Fig. 5B). Maximal costimulation occurred at 100 ng/ml flagellin with anti-CD3 mAbs (1.79 ± 0.22-fold increase, n = 3; p = 0.026, compared with anti-CD3 (1 µg/ml) alone), and proliferation reached levels that were equal to those in the presence of anti-CD28 mAbs (Fig. 5B, compare anti-CD3 alone with control in right panel). Moreover, addition of flagellin further enhanced proliferation in response to anti-CD3 mAbs in the presence of anti-CD28 mAbs (1.52 ± 0.09-fold increase, n = 3; p = 0.04, as compared with anti-CD3/CD28 alone). This strong proproliferative effect of flagellin suggested that, like TLR2 (2, 16), TLR5 may act as a costimulatory molecule.

We also compared the biological effects of flagellin stimulation with those of highly purified LPS. We found that LPS had no effect on the proliferation of CD4+CD25+ Treg cells and tended to inhibit the proliferation of CD4+CD25– T cells (Fig. 5, A and B). When less pure LPS, which was potentially contaminated with other TLR ligands, was used, CD4+CD25– T cells displayed an increased proliferation only in the presence of high concentrations (10 µg/ml). However, even this less-pure preparation of LPS did not affect the proliferation of CD4+CD25+ Treg cells (data not shown).

We next investigated the effects of flagellin on IL-2 production. CD4+CD25+ Treg or CD4+CD25– T cells were stimulated with anti-CD3 mAbs in the presence or absence of anti-CD28 mAbs alone, or with increasing concentrations of flagellin or LPS. After 24 h the amounts of IL-2 in supernatants were determined (Fig. 5C). Consistent with its failure to break anergy, flagellin had no effect on the inability of CD4+CD25+ Treg cells to produce IL-2 (data not shown). However, costimulation with flagellin significantly enhanced production of IL-2 by CD4+CD25– T cells in a dose-dependent manner. Upon stimulation with anti-CD3, addition of flagellin (100 ng/ml) increased IL-2 production an average of 2.2 ± 0.6-fold (n = 4; p = 0.027). In combination with anti-CD28 costimulation, addition of flagellin further increased IL-2 production an average of 2.1 ± 0.2-fold (n = 4; p = 0.02). The drop in IL-2 detectable with 1000 ng/ml flagellin is likely due to cellular consumption. Therefore TLR5, but not TLR4, acted as a costimulatory molecule and enhanced IL-2 production and proliferation in effector T cells.

Flagellin enhances the suppressive capacity of CD4+CD25+ Treg cells

The most important functional characteristic of Treg cells is their ability to suppress effector T cell responses. It has previously been shown that TLR4-stimulated DC produce cytokines, which act on effector T cells and make them resistant to the suppressive effects of CD4+CD25+ Treg cells (31, 32). However, in these studies it was difficult to determine whether TLR ligands may also directly act on the CD4+CD25+ Treg cells themselves. We therefore developed a modified version of the suppression assay first reported by Viglietta et al. (33) to rule out any indirect APC-induced effects and investigate whether flagellin altered the functional capacity of CD4+CD25+ Treg cells. Accordingly, low numbers of CD4+CD25– effector T cells (8000 cells/well) were stimulated with anti-CD3/CD28 mAb-coupled beads, in the absence of APCs, for 6 days. As expected, addition of increasing numbers of CD4+CD25+ Treg cells suppressed proliferation, and significant suppression continued to be observed even at a ratio of 1:81 (Treg cell to T cell effector) (Fig. 6A). At a ratio of 1:81, addition of flagellin increased suppression by an average of 27 ± 2.5% (n = 3; p = 0.0005) (Fig. 6B). Importantly, in the presence of flagellin, significant suppression continued to be observed even at a 1:243 ratio, when it was no longer detectable in controls. As expected, addition of exogenous IL-2 reversed suppression, even in the presence of flagellin (data not shown).



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  		FIGURE 6. Effects of flagellin on the suppressive capacity of CD4+CD25+ Treg cells in the presence or absence of APCs. A and B, CD4+CD25– T cells were stimulated with anti-CD3/CD28-coupled beads in the presence of increasing numbers of CD4+CD25+ Treg cells, and with or without flagellin (10 ng/ml) or LPS (10 ng/ml, similar results obtained with 1 µg/ml). [3H]Thymidine incorporation was measured after 6 days. A, Depicts one representative experiment. B, The average increase in suppression in the presence of flagellin or LPS at a ratio of 1:81 (Treg cell to T cell effector) in three independent experiments. C and D, CD4+CD25– T cells were stimulated with APCs (autologous, irradiated CD3-depleted PBMCs) and soluble anti-CD3 mAbs (1 µg/ml) in the presence of increasing numbers of CD4+CD25+ Treg cells, and with or without flagellin (100 ng/ml) or LPS (1 µg/ml). [3H]Thymidine incorporation was measured after 4 days. C, One representative experiment is shown. D, The average decrease in the percentage of suppression at a 1:2 ratio (Treg cell to T cell effector) from three independent experiments.

 
Addition of LPS to these cultures did not affect the outcome, and in only one of three experiments caused a slight enhancement in the suppression by CD4+CD25+ Treg cells (Fig. 6B). Moreover, this appeared to be attributable to the antiproliferative effects of LPS on the CD4+CD25– T cells as documented in Fig. 5B.

We also investigated whether stimulation of TLR5 in the presence of APCs altered the suppressive capacity of CD4+CD25+ Treg cells. CD4+CD25– T cells were stimulated with irradiated autologous APCs and soluble anti-CD3 mAbs (Fig. 6C), in the absence or presence of CD4+CD25+ Treg cells. As expected, proliferation of effector T cells was suppressed by 70–80% at a 1:2 ratio of Treg cells to T cell effectors. However, addition of flagellin resulted in a consistent decrease in the capacity of CD4+CD25+ Treg cells to suppress proliferation (Fig. 6C). At a 1:2 ratio, flagellin decreased the percentage of suppression by an average of 15 ± 4.7% (n = 3; p = 0.015) (Fig. 6D). In contrast the effects of LPS on suppression were variable and not significant. This latter result is in contrast to the findings of Pasare and Medzhitov (31), but consistent with Peng et al. (34), and possibly due to the fact that the APCs in our experiments were monocytes rather than fully differentiated DCs.

Flagellin enhances expression of FOXP3 in activated CD4+CD25+ Treg cells

Human CD4+CD25+ Treg cells express high levels of FOXP3 (35), and this transcription factor is essential for their normal development in mice (29, 36, 37). In an attempt to determine the molecular basis for the enhanced suppressive capacity of CD4+CD25+ Treg cells upon stimulation of TLR5, we investigated whether flagellin altered the expression of FOXP3. CD25+CD4+ Treg cells were activated with anti-CD3/CD28 mAbs, without or with flagellin or LPS for 48 h (Fig. 7). As expected, CD4+CD25+ Treg cells expressed ~100-fold more FOXP3 than CD4+CD25– T cells (35). Following activation, expression of FOXP3 mRNA in CD25+CD4+ Treg cells was consistently decreased by ~4-fold. In contrast, when CD4+CD25+ Treg cells were activated in the presence of flagellin, levels of FOXP3 mRNA did not decrease, but rather increased by 6 ± 2.6-fold (n = 5, p = 0.006) as compared with the 48-h activated control (Fig. 7B). Flagellin did not alter the minimal activation-induced expression of FOXP3 in CD4+CD25– T cells (Fig. 7A). Addition of LPS had no significant effect on levels of FOXP3 mRNA in either CD4+CD25+ Treg cells or CD4+CD25– T cells. These data indicate that flagellin may enhance the suppressive capacity of CD4+CD25+ Treg cells by influencing expression of this key transcription factor.



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  		FIGURE 7. Flagellin increases FoxP3 expression in CD4+CD25+ Treg cells following TCR-mediated activation. Ex vivo isolated CD4+CD25+ Treg or CD4+CD25– T cells were stimulated with anti-CD3/CD28-coupled beads in the absence of presence of flagellin or LPS (10 µg/ml) for 48 h. RNA was collected and quantitative RT-PCR was performed to determine expression of FOXP3. Data are expressed as relative amounts compared with ex vivo CD4+CD25– T cells and are normalized to amounts of GAPDH. A, A single representative experiment is shown. B, Each symbol represents the fold increase in FOXP3 mRNA in the presence of flagellin (FL, 100 ng/ml) compared with the 48 h-activated control in individual experiments. The horizontal line represents the average.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown that human CD4+CD25+ Treg and CD4+CD25– T cells express TLR5, and that flagellin can directly influence their function. Importantly, levels of TLR5 mRNA and protein on T cells were similar to those observed in monocytes and DCs, indicating a physiologically relevant level of expression. Although flagellin did not overcome the anergy of CD4+CD25+ Treg cells, it acted as a costimulatory molecule on effector T cells. Remarkably, flagellin potently increased the suppressive capacity of CD4+CD25+ Treg cells, even under conditions in which proliferation of the target CD4+CD25– T cells was enhanced. The finding that flagellin can induce a significant increase in FOXP3 expression in CD4+CD25+ Treg cells suggests that, in addition to indirect effects mediated by innate immune cells, TLR ligands may also have the capacity to directly regulate adaptive immune responses.

Surprisingly, although we consistently found that levels of TLR5 mRNA were significantly higher in ex vivo purified CD4+CD25+ Treg cells, T cell lines, and T cell clones than levels in their CD4+CD25– counterparts, there was no corresponding increase in protein expression. The increased expression of TLR4 and TLR5 mRNA we observed in CD4+CD25+ Treg cells is consistent with previous studies that did not go on to investigate expression at the protein level (4). Our data highlight the importance of examining both mRNA and protein expression whenever possible. Very little is known about the correlation between TLR5 mRNA and protein expression, and it is not clear whether this discordance is a common phenomenon. There was also a disparity between mRNA and protein expression following TCR-mediated activation when there was a transient increase in total TLR5 protein expression despite a concurrent dramatic decrease in TLR5 mRNA. It is possible that preexisting TLR5 mRNA may be subjected to posttranscriptional regulation following T cell activation providing a template for this transient increase in protein expression. Alternatively, there may be a similar transient increase in TLR5 mRNA expression at a time point earlier than 24 h. The overall conclusion is that following activation, T cells likely rapidly lose their capacity to respond to flagellin, possibly as part of a negative feedback mechanism.

Costimulation with flagellin, or LPS, did not alter the anergic state of human CD4+CD25+ Treg cells. These data contrast with a previous report that showed that LPS can directly increase the proliferation of murine CD4+CD25+ Treg cells (4). This discrepancy may be the result of inherent differences between mouse and human cells, although both expressed high levels of TLR4 mRNA, or due to the use of different sources of LPS. Other groups have also failed to find a proproliferative effect of LPS on murine CD4+CD25+ Treg cells (2, 3). TLR5 is known to form homodimers, but can also heterodimerize with TLR4 (38). Therefore, it is possible that TLR4 on CD4+ T cells functions primarily through the formation of heterodimers with TLR5.

In contrast, flagellin directly enhanced proliferation and IL-2 production by CD4+CD25– T cells, providing a costimulatory stimulus as strong as that of CD28. Note that LPS had no effect on the proliferation of CD4+CD25– T cells, strongly supporting the conclusion that flagellin directly affects CD4+ T cells, rather than acting indirectly via small numbers of contaminating cells. These findings are consistent with a recent report that flagellin has a stimulatory effect on memory CD4+ T cells (39). Together with evidence that lipopeptide, a TLR2 ligand, results in a similar effect (2, 16), these data indicate that TLRs may represent a new class of costimulatory molecules.

Importantly, in the absence of APCs, flagellin significantly increased the capacity of CD4+CD25+ Treg cells to block effector T cell proliferation, and suppression continued to be observed even at an ~1:250 (Treg cell to T cell effector) ratio. This effect did not appear to be related to alterations in Treg cell-associated surface molecules (data not shown), but did correlate with enhanced expression of FOXP3. Flagellin not only prevented activation-induced down-regulation of FOXP3, but also enhanced its expression compared with resting levels. Because flagellin did not increase the proliferative capacity of CD4+CD25+ Treg cells, it is unlikely this increased FOXP3 was due to a preferential increase in their numbers relative to contaminating effector T cells (24). Moreover, because flagellin actually enhanced IL-2 production in effector T cells, it is also unlikely that TLR5 stimulation drove the de novo differentiation of CD4+CD25+ Treg cells from contaminating non-Treg cells. Thus, we favor the hypothesis that signals downstream of TLR5 can interfere with the apparently negative effects of TCR stimulation to enhance FOXP3 expression.

Considerable evidence is accumulating that suggests that PAMPs regulate CD4+CD25+ Treg cell numbers and function in health and disease. For example, mice deficient in TLR2 have decreased numbers of CD4+CD25+ Treg cells, and TLR2 ligands have a protective effect on their capacity for survival in vitro (16). TLR2-deficient mice are resistant to infection with C. albicans, suggesting that the normal interaction between TLR2 and Treg cells promotes immunosuppression (16). Further, ligation of TLR4 on murine CD4+CD25+ Treg cells enhances their suppressive capacity in some situations (4). In contrast, it has recently been reported that TLR8 ligands act directly on CD4+CD25+ Treg cells and reverse their suppressive capacity (34). Thus it appears likely that different TLR ligands have differential effects on the function of Treg cells.

In support of a role of TLRs in regulating intestinal inflammation, mice deficient in MyD88, a critical signaling molecule downstream of most TLRs, are more susceptible to dextran-sulfate colitis (17). In addition, mutations in the NOD2 PRR are associated with susceptibility to Crohn’s disease. These findings support the hypothesis that the lack of an appropriate PRR signal can lead to loss of regulation (40, 41).

We therefore propose the model as shown in Fig. 8. First, in the context of the early inflammatory response to invading microorganisms, APCs would become activated by PRRs and deliver a stimulatory signal to CD4+ T cells that is further enhanced by direct interactions between CD4+ T cells and PAMPs. Together, this process would result in cytokine release, strong CD4+ T cell responses that are resistant to suppression by CD4+CD25+ Treg cells (31, 32), and a proinflammatory antimicrobial response. Concurrently, the same PAMPs would act directly on the CD4+CD25+ Treg cells to preserve FOXP3 expression and their suppressive capacity. With time, or in the presence of noninvasive commensal bacteria, the cytokine milieu would shift toward a tolerogenic environment in which CD4+ effector T cells are more easily suppressed by CD4+CD25+ Treg cells. Moreover, because CD4+CD25+ Treg cells can directly suppress effector T cells (42), it is possible that recently activated CD4+CD25+ Treg cells could interact with and suppress effector T cells in the absence of TLR-matured APCs.



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  		FIGURE 8. Model of how flagellin may interact with CD4+CD25+ Treg cells during an immune response. We propose a model in which during an immune response, CD4+CD25+ Treg cells would be attracted to sites of inflammation and innate stimuli would preserve and possibly enhance their local numbers and/or function. However, early during this response, production of counter-regulatory cytokines, expression of costimulatory molecules, and strong activation of effector T (Te) cells would make the response "unsuppressible". With time, and reduction in inflammation, the effector cells would become resusceptible to suppression, and the Treg cells would be ready to dampen the response.

 
The capacity of PRRs to directly enhance the numbers and function of CD4+CD25+ Treg cells ensures a mechanism to reduce the potentially harmful effects of uncontrolled inflammation to the host. Further evidence to support this model comes from the finding that CD4+CD25+ Treg cells exert their suppressive effects at the end of an immune response (5) and that these cells are well equipped with specific homing receptors to travel to sites of inflammation (43). If our hypothesis is correct, the capacity of PRRs to directly influence Treg cell responses would represent a new point of integration between the innate and the adaptive immune systems.


   Acknowledgments
 
We gratefully acknowledge the support of Barrett Benny, Michael Barnett, and Raewyn Broady and the Cell Separator Unit at Vancouver General Hospital for providing PBMCs. We thank Jan Dutz, Andrew Hall, and Paul Orban for critical reading of the manuscript, and Claire Baecher-Allan for technical advice on the bead suppression assays.


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


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

1 This work is supported by Grant MOP57834 from the Canadian Institutes for Health Research and British Columbia Transplant Society (to M.K.L.). M.K.L. holds a Canada Research Chair in Transplantation and is a Michael Smith Foundation for Health Research scholar. N.K.C. holds a Michael Smith Foundation for Health Research Senior Graduate Studentship award. Back

2 Address correspondence and reprint requests to Dr. Megan K. Levings, Department of Surgery, 2660 Oak Street, Vancouver, British Columbia, Canada V6H 3Z6. E-mail address: mlevings{at}interchange.ubc.ca Back

3 Abbreviations used in this paper: PRR, pattern recognition receptor; DC, dendritic cell; PAMP, pathogen-associated molecule pattern; Treg, T regulatory. Back

Received for publication April 6, 2005. Accepted for publication October 7, 2005.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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