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TLRs are a class of conserved pattern recognition receptors that are used by cells of the innate immune system. Recent studies have demonstrated the expression of TLRs on both human and mouse T cells raising the possibility that TLRs play a direct role in adaptive immunity. TLR2 is activated primarily by bacterial wall components including peptidoglycan and lipoproteins. Several studies have shown that mouse regulatory T (Treg) express TLR2 and claimed that engagement of TLR2 by synthetic ligands reversed their suppressive function. In contrary, enhancement of Treg function was observed following engagement of TLR2 on human Treg. We have re-examined the expression and function of TLR2 on mouse Treg purified from Foxp3-GFP knock in mice. TLR2 ligation by TLR2 agonist, the synthetic bacterial lipoprotein (BLP) Pam3CSK4, enhanced the proliferative responses of both conventional T cells and Treg in response to TLR stimulation in the absence of APC. Treatment of Foxp3+ Treg with Pam3CSK4 did not alter their suppressive function in vitro or in vivo and did not reduce their level of Foxp3 expression. An additional effect of TLR2 stimulation of Treg was induction of Bcl-xL resulting in enhanced survival in vitro. Treatment of mice with the TLR2 agonist enhanced the antigen-driven proliferation of Treg in vivo, but did not abolish their ability to suppress the development of EAE. Development of methods to selectively stimulate TLR2 on Treg may lead to a novel approaches for the treatment of autoimmune diseases.
Microbes are recognized by cells of innate immune system in the host via pattern recognition receptors (PRRs), with the TLR-family the best characterized. Currently, 13 TLRs have been identified including 10 human TLRs (TLR1-10) and 12 murine TLRs (TLR1-9 and 11–13), which recognize different pathogen-associated molecular patterns (PAMPs3) (1). TLRs were originally thought to be expressed in cells involved in innate immunity such as macrophages, dendritic cells (DCs), epithelial cells, endothelial cells, as well as organ parenchymal cells (2, 3). However, more recent studies have shown that certain TLRs are also expressed in CD4+ and CD8+ T lymphocytes. Flow cytometric analysis indicates human TLR2, 3, 4, 5 and 9 proteins are expressed intracellularly in non-stimulated T cells (4). TLR1, 2, 3, 6 and 7 mRNA are readily detected in murine CD4+ T cells by RT-PCR (5, 6). Several studies have suggested that TLRs can directly modulate T cell functions as novel co-stimulatory receptors. For example, TLR2 is shown to regulate both murine and human CD4+ T cell functions (5, 7, 8). Poly(I:C) and CPG DNA enhance the survival, but not the proliferation of activated murine CD4+ T cells by up-regulating the expression of the anti-apoptotic protein Bcl-xL and augmenting the activation of NF-κB (9).
Foxp3+ Treg are critical for the maintenance of peripheral T cell tolerance and their depletion leads to organ-specific autoimmune diseases. Treg suppress CD4+ and CD8+ T lymphocytes via contact-dependent mechanisms and the secretion of suppressor cytokines (10–13). Although mouse Treg express TLR1, 2, 4, 5, 6, 7 and 8 mRNA (14), the function of TLRs on Treg is controversial. Engagement of either TLR4 (6) or TLR5 (15) has been shown to enhance Treg suppressive activity, while TLR2 ligation on mouse (5, 16) or TLR8 ligation on human Treg (17) are claimed to reverse Treg suppressive function. One of the major problems in the analysis of TLR function on Treg is the difficulty in obtaining highly purified populations of Foxp3+ Treg. As TLRs are also expressed on monocytes, B cells and DCs, an analysis of the effects of TLR engagement on Treg function must carefully exclude the presence of contaminating cells including Foxp3−CD25+CD4+ and Foxp3−CD25−CD4+ T cells. We have re-examined the effects of TLR2 agonist, Pam3CSK4, on Foxp3+ Treg. Unlike previous studies, we isolated highly purified populations of Treg by FACS sorting GFP+ cells from mice expressing a GFP–Foxp3 fusion protein (18). We demonstrate that freshly-isolated Treg express only low levels of TLR2 mRNA that can be up-regulated by TCR stimulation and further increased upon TLR2 ligation. Contrary to previous studies (5, 16), we demonstrate that Treg pre-treated and activated with Pam3SCK4 in vitro maintain Foxp3 expression and have normal suppressive activity when co-cultured with TLR2−/− responder T cells. Using a model of experimental autoimmune encephalomyelitis (EAE) in TLR2−/− mice, we further conclude that administration of Pam3CSK4 in vivo does not alter the ability of wild type (WT) Treg to modulate the induction of disease. The major effect of TLR2 ligation on Treg is to reduce their threshold for activation and significantly enhance their survival via up-regulating anti-apoptotic molecule Bcl-xL.
Foxp3gfp mice were a gift of Dr. A. Rudensky (University of Washington, Seattle), bred at the National Institute of Allergy and Infectious Diseases (NIAID) contract facility at Taconic Farms, Inc. (Germantown, N.Y.) and maintained on a mixed C57BL/6 × 129 background. TLR2−/− mice on the C57BL/6 background were purchased from Jackson laboratories (Bar Harbor, ME) and bred in our facility. OVA-specific TCR transgenic OT-II mice (C57BL/6 background) and wild-type C57BL/6 were obtained from Taconic Farms. Female heterozygous B6.Cg-Foxp3sf/J (Scurfy) mice were purchased from Jackson Laboratories and bred to C57BL/6 WT male mice to generate hemizygous male B6 [Cg-Foxp3sf/Y (Scurfy)] offspring. All animals used for this study were female, 6 to 8 wk of age. They were housed and handled according to NIH institutional guidelines under an approved animal protocol.
APCs were purified from mouse spleen by magnetic sorting the CD90− fraction and then irradiated (3000 rads) prior to use. T cells were obtained from pooled mouse spleen and lymph nodes and the CD4+ fraction was first purified by autoMACs Cell Separator using anti-mouse CD4 beads (Miltenyi Biotec, Auburn, CA). The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+CD25−, CD4+GFP− or CD4+GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA). The postsort purity for each cell type was higher than 98% and the Foxp3 purity for Treg was higher than 95%.
Single cell suspensions were stained using the following conjugated antibodies according to the manufacturer's protocol: PE or APC anti-mouse CD4 (L3T4), PE anti-mouse CD25 (PC61.5), APC-Alexa Fluor® 750 anti-mouse CD45.2 (104), Biotin anti-mouse CD45.1 (A20), PE-Texas Red-Streptavidin, APC or Pacific Blue anti-mouse Foxp3 (FJK-16s), PE anti-Vα2+ (B20.1), (all from eBioscience, San Diego, CA) and Biotin anti-Vβ5+ (MR 9-4) (BD Pharmingen, San Diego, CA). For staining of Foxp3 or PE anti-mouse BCL-2 (BD Pharmingen, San Diego, CA), the cells were fixed and permeabilized using a Fixation/Permeabilization kit (eBioscience, San Diego, CA). Acquisition was done on a LSR II (BD Biosciences) equipped with a FACS-DiVa digital upgrade. Analysis was done using FlowJo software (Treestar, Inc., Ashland, OR).
T cells were stimulated with plate-bound anti-mouse ε CD3 MAb (clone 145-11, PharMingen) at the concentrations shown in figure legends. Unless indicated otherwise, TLR ligands were used at the following concentrations: Poly I:C (1µg/ml), CPG (0.5µg/ml), CL097 (0.5µg/ml), LTA-SA (0.5µg/ml), Pam2CSK4 (50ng/ml) and Pam3CSK4 (1µg/ml) (all from invivogen, San Diego, CA). RPMI 1640 media (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 5 0µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), 1% sodium pyruvate, 1% nonessential amino acids, 1% HEPES, 100U/ml penicillin and 100µg/ml streptomycin, and 2mM L-glutamine (all from Invitrogen) was used as culture medium. Where indicated, cultures were supplemented with 100U/ml rmIL-2 (Hoffman-LaRoche, Nutley.NJ). The cells were incubated at 37°C and under 5% CO2 for the indicated time.
The proliferation of GFP+ Treg was assayed by culturing GFP+ Treg (5×104/well) in 96-well flat-bottomed plates (Corning Life Sciences) in 0.2 ml of medium in the presence of plate-bound anti-CD3 at the concentration indicated in the figure legends. Treg suppression assays were performed by culturing responder T cells (5×104) in the presence of irradiated T-depleted spleen cells (5×104), soluble anti-CD3 (0.25µg/ml) and varying numbers of Treg. After 72h, the cultures were pulsed for 6 h with 3H-TdR (1µCi/well), harvested and the incorporated radioactivity was measured by standard techniques.
A CFSE stock (10mM in DMSO; Invitrogen) stored at −20 °C, was thawed and diluted in phosphate-buffered saline (PBS) to the desired working concentrations. Purified T cells were re-suspended in PBS (0.1% BSA) at 2×106 cells/ml and incubated with CFSE (final concentration: 2µM) for 8 min at 37 °C. Cells were washed and re-suspended in culture medium for 15 min to stabilize the CFSE staining. Following 72h of culture, CFSE dilution of responder T cells was determined by flow cytometry.
Cells were washed in PBS and re-suspended at a concentration of 1×106 cells/ml in Annexin V Binding Buffer (BD PharMingen, San Diego, CA) and stained with Annexin V-PE (BD PharMingen, San Diego, CA) and 7-amino actinomycin D (7-AAD) (Sigma-Aldrich, St. Louis, MO), according to the manufacturer's protocol. Alternatively, apoptosis was also quantified by a reduction in forward scatter (FSC) and an increase in side scatter (SSC) as determined by flow cytometry.
Total RNA was extracted from cells (1×106 per sample) using RNeasy Plus Kit (Qiagen, Valencia, CA). cDNAs were generated from 1 µg of total RNA using SuperScript II RNase H-reverse transcriptase (Invitrogen). The resulting cDNA was subjected to a 14 cycle PCR amplification using the TaqMan Universal 2× master mix and performed in triplicate on the ABI/PRISM 7900 Sequence Detector System (Applied Biosystems, Foster City, CA). The ready-made primer and probe sets for TLR2 were ordered from Applied Biosystems. (Catalog #: Tlr2: Mm00442346_m1). The amount of TLR2 mRNA expression was normalized to the 18S rRNA and calculated according to the comparative cycle threshold (Ct) method as described by Applied Biosystems.
GFP+ Treg (1 × 106) were left un-stimulated or stimulated for 24 or 48 h with plate-bound anti-CD3 (0.5µg/ml) and IL-2 (100U/ml) in the presence or absence of Pam3CSK4 (1µg/ml). The recovered cells were then lysed with cell lysis buffer (30 mM Tris-Cl pH 7.5, 150 mM NaCl and 1% CHAPS). Total protein (50µg) was resolved on 10% SDS-PAGE gels and transferred to Immuno Blot Polyvinylidene Fluoride membranes (GE Osmonics Labstore, Minnetonka, MN). Blotting was performed with BCL-xL Ab (Cell Signaling Technology, Beverly, MA) and anti-mouse β-Actin (Sigma-Aldrich, St. Louis, MO). Bands were visualized with secondary HRP-conjugated Ab (Bio-Rad, Hercules, CA) and the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
Treg from OT-II transgenic mice were isolated by FACS purification of CD4+CD25+ T cells. The post-sort Foxp3 purity was higher than 90%. The Treg were then CFSE labeled (2µM) and injected into TLR2−/− mice via tail vein (1×106 per mouse). 24h later, mice were immunized with OVA323–339 peptide (50µg, synthesized by the peptide synthesis facility, NIAID) in IFA or injected together intraperitoneally (i.p.) with 100µg Pam3CSK4. Five days later, the draining lymph nodes from mice were taken and stained with APC anti-CD4+, PE anti-Vα2+ and Biotin anti-Vβ5+ followed by PE-Texas Red-Streptavidin. The cells were then fixed and permeabilized for staining with Pacific Blue-anti-Foxp3 and analyzed by flow cytometry.
Male RAG−/− mice (five per group) were reconstituted with 5×106 total lymph node and spleen cells from 7-day-old Scurfy mice alone or co-transferred with either 1×106 Treg. Histopathological evaluation was performed as previously described (19).
Mice (five per group) were immunized subcutaneously with myelin oligodendrocyte glycoprotein (MOG35–55) peptide in CFA. Pertussis toxin (200ng) was injected i.p. on days 0 and 2 to induce EAE. CD4+GFP+ Treg (1×106) were transferred into TLR2−/− mice 24h before immunization. Mice were treated with Pam3CK4 (20µg, i.p.) on days −1, 1, 3 and 5. Mice were then monitored daily for disease until day 30. Clinical assessment of EAE was performed according to the following criteria: (0), no disease; (1), tail paralysis; (2), hind-leg weakness; (3), full hind-leg paralysis; (4), complete hind-limb paralysis plus front-limb paraparesis; (5), death. The data shown are mean clinical scores of each group.
A previous study (16) suggested that CD4+CD25+ Treg expressed TLR2 protein as determined by flow cytometry. To more accurately characterize TLR2 expression in Treg, we sorted GFP+ T cells from Foxp3-GFP knock in mice. We first attempted to examine TLR2 protein expression by flow cytometry, but could not observe a positive signal above control staining using commercially available mAbs (data not shown). Levels of TLR mRNA are indicative of protein expression, as TLR proteins are tightly regulated by ubiquitin-mediated degradation (20). We therefore determined the level of TLR2 mRNA by RT-PCR. TLR2 mRNA levels were compared to the levels expressed in epithelial cells from TLR2−/− mice as a negative control, to levels in bone marrow-derived macrophages from WT mice as a positive control. TLR2 mRNA was detectable in non-activated CD4+GFP− T cells, but not in GFP+ T cells (Fig. 1A). As TCR activation has been reported to induce TLR2 expression in different T cell subsets (5, 8, 14), we activated Treg with plate-bound anti-CD3 in the presence of IL-2; TLR2 mRNA expression in Treg was detectable after 16h and was further elevated 10-fold by adding Pam3CSK4 to the culture. However, the levels of TLR2 in the stimulated Treg were still 20-fold less than those detected in macrophages. Of note, TCR stimulation resulted in slightly enhanced TLR2 mRNA expression in GFP−Foxp3− T cells, but the levels were only slightly increased by addition of Pam3CSK4 (Fig. 1B). Thus, up-regulation of TLR2 expression in Treg is dependent on TCR stimulation and can be further modulated by TLR2 ligation.
As other TLR ligands have been reported to have direct effects on Treg (6, 7, 15–17), we next compared several different TLR agonists for their capacity to modulate Treg proliferation. When Treg were stimulated with soluble anti-CD3 in the presence of irradiated T-depleted splenocytes or with plate-bound anti-CD3 in the absence of APC, none of TLR agonists, including Poly I:C (TLR3), CpG (TLR9), CL097 (TLR7/8) and Pam3CSK4 (TLR2/TLR1) were able to stimulate the proliferation of the Treg (Fig. 2A,B). Since the proliferation of Foxp3+Treg can be induced by TCR stimulation in the presence of exogenous IL-2, we also tested the effects of the TLR agonists on Treg in response to stimulation with a suboptimal concentration of anti-CD3 in the presence of IL-2 and APC (Fig. 2C). Slight enhancement (~ 2-fold) of Treg proliferation was observed when CpG and CL097 were added, while Pam3CSK4 increased Treg proliferation approximately 4-fold. In order to determine whether the TLR agonists were acting on the APC or on the Treg, we stimulated the Treg in the absence of APC with plate-bound anti-CD3 and IL-2 (Fig. 2D). Under these conditions, a significant enhancement of T cell proliferation (~ 8-fold) was only seen in the presence of Pam3CSK4. Taken together, it appears that the enhancement of proliferation induced by CpG and CL097 is mediated indirectly via acting on the APC, while the synthetic TLR2 agonistic Pam3CSK4 is able to enhance Treg proliferation by directly acting on Treg.
The co-stimulatory effects of Pam3CSK4 were most prominent when the Treg were stimulated with a low concentration of anti-CD3 in the presence of IL-2 and no enhancement of proliferation was observed at the higher concentration (Fig. 2E). Pam3CSK4 also appeared to directly enhance the proliferation of conventional GFP− T cells stimulated with a low concentration of anti-CD3 in the absence of exogenous IL-2 (Fig. 2F). Thus, TLR2 ligation on both Treg and conventional T cells results in a co-stimulatory signal and appears to lower the threshold for TCR activation.
Pam3CSK4 is a synthetic tripalmitoylated lipopeptide that mimics the acylated amino terminus of bacterial lipoprotein. Recognition of this synthetic TLR ligand is mediated by the TLR2/TLR1 heterodimer complex through their cytoplasmic domains (21). TLR2 homodimer or TLR2/TLR6 heterodimer can recognize other microbial components (2, 3). We therefore compared the ability different TLR2 complex ligands to costimulate the proliferation of Treg induced by plate-bound anti-CD3 and IL-2 (Fig. 3). LTA-SA (TLR2/TLR2 homodimer) and the synthetic ligand, Pam2CSK4 (TLR6/TLR2 heterodimer) only slightly enhanced GFP+ Treg proliferation (~2-fold). In contrast, Pam3CK4 induced a dose-dependent, 4-fold increase in Treg proliferation suggesting that signal transduction via the TLR2/TLR1 heterodimer results in the most effective co-stimulus for Treg proliferation.
As TLR2 engagement on Treg enhances their proliferation in vitro, it is important to clarify whether TLR2 agonists modulate Treg function. Several reports have claimed that engagement of TLR2 on Treg transiently reverses their suppressive function secondary to reduced Foxp3 expression (5, 16). We initially examined the influence of the TLR2 agonist on Foxp3 expression by flow cytometric analysis. GFP+ Treg were expanded for 3 days in vitro by stimulation with anti-CD3 and IL-2 in the presence or absence of Pam3CSK4. No decrease in the levels of GFP (Fig. 4A, left panels) or Foxp3 expression (Fig. 4A, right panels) was observed when Pam3CSK4 was present during the 3 day culture.
To determine whether Pam3CSK4 would alter the suppressive functions of Treg in vitro, we mixed responder T cells and APCs from TLR2−/− mice with varying numbers of Treg from WT GFP+Foxp3+ mice. The continuous presence of Pam3CSK4 during the 3-day suppression assay had no effect on capacity of WT Treg to suppress the proliferation of T responder cells from TLR2−/− mice induced by soluble anti-CD3 (Fig. 4B). We also pre-activated the Treg for 3 days with plate-bound anti-CD3 and IL-2 in the presence of Pam3CSK4 and added these cells to the suppressor assay in the absence of additional Pam3CSK4. No difference in suppressive capacity was observed between Treg that had been pre-activated in the presence or absence of Pam3CSK4 (Fig. 4C). Furthermore, the addition of Pam3CSK4 to the suppressor assay in the presence of the pre-treated Treg had no effect on Treg suppressor function.
As an initial approach to determining the mechanism of action of Pam3CSK4 on Treg function, we stimulated Treg with a low concentration of plate-bound anti-CD3, IL-2 and Pam3CSK4. After 7 days of culture, an increased percentage of viable cells was noted on FSC/SSC analysis of recovered cells in cultures containing Pam3CSK4 (Fig. 5A, top panel). Similarly, the percentage of 7-AAD and Annexin-V double negative cells increased from 11% to 25% when the cells were cultured in the presence of Pam3CSK4 (Fig. 5A, bottom panel). All of the gated live cells maintained GFP-Foxp3 expression and no difference in the level of Foxp3 expression was seen when the cells were cultured with Pam3CSK4 (Fig. 5A, middle panel).
We next examined whether Pam3CSK4 treatment modulates the levels of anti-apoptotic proteins in Treg. The intracellular expression of Bcl-2 was first examined by flow cytometry, but no differences in the levels of Bcl-2 were seen in cells cultured with or without Pam3CSK4 during the indicated time periods (Fig. 5B). However, marked enhancement of Bcl-xL protein expression was observed both after 24 and 48h of stimulation when the Treg were cultured in the presence of Pam3CSK4 (Fig. 5C). Therefore, TLR2 ligation potentiates Treg survival and is associated with the up-regulation of anti-apoptotic protein Bcl-xL.
To address the influence of TLR2 engagement on Treg proliferation in vivo, we transferred CFSE-labeled Treg purified from OT-II TCR transgenic mice to non-transgenic TLR2−/− mice and then immunized the recipients with their cognate peptide in IFA. The mice were simultaneously injected i.p. with Pam3CSK4. As previously reported (22), TCR stimulation alone can induce vigorous proliferation of Treg in vivo and treatment of the mice with Pam3CSK4 appeared to further enhance Treg proliferation (Fig. 6A) as measured by increased dilution of CFSE and in the absolute recovery of Foxp3+ T cells (Fig. 6B). Thus, engagement of TLR2 also co-stimulates Treg proliferation in vivo in the absence of exogenous cytokines.
To test whether TLR2 engagement alters the capacity of Treg to prevent the development of autoimmune disease, we used a previously characterized model (19) where T cells from Scurfy mice are transferred into RAG−/− mice. Approximately 4 wk after transfer of total lymph node and spleen cells from 7-day-old Scurfy to RAG−/− mice, all the recipients develop lymphosplenomegaly, skin inflammation, and marked lymphocyte infiltration into the skin and liver. Co-transfer of natural Treg or TGF-β-induced Treg (19) prevents most of the manifestations of disease. Treg were pre-cultured with anti-CD3 and IL-2 for 5 days in the presence or absence of pam3CSK4 and co-transferred with Scurfy T cells into RAG−/− recipients. Treg that had been pre-treated with Pam3CSK4 were as efficient as non-treated Treg in suppressing both skin and live disease 4 wk after transfer (Fig. 7).
One problem with the interpretation of the Scurfy transfer study is that Treg were pre-treated with the TLR2 ligand and may have recovered their suppressor function after transfer. To determine whether direct TLR2 signaling in vivo can modulate Treg function, we used a model of Treg-mediated suppression of EAE originally described by Kohm et al (23), where supplementation of normal mice with polyclonal Treg prior to disease induction modulates the severity of disease. TLR2−/− mice were reconstituted with GFP+ Treg one day before EAE induction and then treated with four injections of Pam3CSK4. TLR2−/− mice that were not reconstituted with Treg all developed severe EAE. In contrast, mice reconstituted with Treg had a marked reduction in disease severity (Fig. 8). Treatment of the reconstituted mice with Pam3CSK4 had no effect on the ability of the transferred Treg to modulate the induction of disease. Thus, brief treatment of mice with a TLR2 agonist does not reverse the suppressive function of Treg.
Most studies on TLR signaling have been focused on their function in cells of the innate immune system. Studies from a number of groups (4, 6, 14, 24) have demonstrated that TLRs can be expressed by conventional CD4+ and CD8+ T cells, as well as by Foxp3+ Treg. Stimulation of TLRs on conventional T cells in all studies results in enhancement of T cell activation and prolongation of TCR survival in vitro and in vivo. In most, but not all studies (25), T cells require activation as a prerequisite to TLR responsiveness. This requirement for prior TCR activation would prevent non-specific activation of T cells by TLR ligands. A confusing picture has emerged from the analysis of the function of TLRs on both human and mouse Treg cells. Some studies (5, 7, 16) have shown that engagement of TLR2 on mouse Treg or TLR5 on human Treg results in enhancement of their suppressive functions, while other studies have claimed that engagement of TLR2 on mouse Treg (5, 16) of TLR8 in human Treg (17) reverses Treg suppressive function. Two major problems exist in the interpretation of these studies. First, a consensus view (26) has yet to emerge as to the mechanisms used by Treg to suppress their targets in vivo or in vitro. It is therefore difficult to determine how TLRs modulate suppression either positively or negatively. Secondly, as the TLRs are widely expressed on both conventional T cells and innate immune cells, contamination of the Treg preparation with non-Treg may have been responsible for the differences between the studies.
To avoid the problems encountered when Treg are isolated based on CD25 expression, we have re-examined the effects of TLR expression on murine Treg using highly purified Treg isolated from mice expressing eGFP under the control of the Foxp3 promoter. We initially tested TLR2, TLR3, TLR7/8, and TLR9 agonists to determine whether they could enhance the proliferation of Treg in culture when stimulated with anti-CD3. No changes were seen in the presence of anti-CD3 alone, but TLR2, TLR7/8, and TLR9 agonists all augmented the proliferative responses of Treg in the presence of soluble anti-CD3 and APC, while significant enhancement of proliferation to plate-bound anti-CD3 and IL-2 was only seen with the TLR2 agonist, Pam3CSK4. We concluded from these studies that the augmentation of Treg response by TLR7/8 and TLR9 agonists was likely to be indirect and mediated via the APC, while TLR2 stimulation had a direct effect on the Treg. We therefore focused our studies on the potential role of TLR2 in modulation of Treg function. Augmentation of proliferation was associated with enhanced Treg survival in culture and enhanced expression of the anti-apoptotic protein, Bcl-xL. A similar increase in Bcl-xL, but not Bcl-2 levels, was seen when conventional CD4+ T cells were treated with poly(I:C) or CpG DNA (9).
In contrast to previous studies, we were unable to detect TLR2 expression on the cell surface using the commercially available anti-TLR2 antibodies. Low levels of TLR2 mRNA expression were detected in un-activated Foxp3− T cells, while TLR2 mRNA could not be detected in freshly isolated Foxp3+ T cells. Following TCR activation, a modest increase in TLR2 mRNA expression was seen both in Foxp3+ and Foxp3− T cells, but the level of TLR2 mRNA was augmented 10-fold in the Foxp3+ T cells in the presence of the TLR2 agonist suggesting a positive feedback on TLR2 expression by stimulation in the presence of the TLR2 agonist. A similar positive effect of a TLR agonist on TLR expression was observed (27) when human γδ T cells were stimulated with poly(I:C). It has also been reported that TLR2 transcription is increased during infection with P. carinii where the host utilizes TLR2 to recognize major surface glycoprotein of the infecting organism (28). Although TLR2 can exist as a homodimer or heterodimerize with TLR1 and TLR6, the TLR2/TLR1 complex appears to be the most critical for costimulation of Treg proliferation, as significant enhancement of proliferation was only seen with the TLR2/TLR1 ligand, Pam3CSK4 and not with the TLR2/TLR6 ligand, Pam2CSK4, or with the natural ligand for TLR2/TLR2 homodimers, LTA-SA. It will therefore be interesting to determine whether activation of TLR2/TLR1 heterodimeric complex on Treg can modulate immune responses to different microbial pathogens.
Both of the previous studies (5, 16) on the function of mouse Treg also observed augmentation of Treg proliferation in the presence of TCR activation and Pam3CSK4. They concluded from this observation that the enhanced proliferative responses of the Treg should result in abrogation of their suppressive ability. However, abrogation of Treg suppressive function is not seen under conditions where Treg are actively proliferating. Addition of a high concentration of IL-2 to co-cultures of Treg and responders masks the suppressive effects of Treg on responder cell proliferation, but does not reverse the capacity of the Treg to inhibit the production of IL-2 or IFNγ by the responder T cells (29, 30). Proliferation of Treg can also be induced in the presence of activated bone marrow derived DCs and inhibition of responder T cell proliferation is also not seen under those conditions. However, suppression of IL-2 production by the responder cells is not reversed (31).
As TLR2 is expressed by both responder T cells and APC, analysis of the effects of TLR2 engagement on Treg suppressive ability is only possible in assays using wild type Treg and responder T cells/APCs from TLR2−/− mice. In contrast to previous studies, addition of Pam3CSK4 to these co-cultures had no effect on Treg-mediated suppression of proliferation over a broad range of Treg to responder ratios. Previous studies also claimed that overnight exposure of Treg to a TLR2 agonist also abrogated their suppressive capacity, while in our studies continuous exposure of the Treg to the agonist for 3 days had no effect on their ability to suppress when they were added immediately to co-cultures in the absence of a rest period or even when the agonist was also added to the co-cultures. There are several potential reasons for the differences between our results and those reported previously. First, as noted above, Treg in our studies were purified based on Foxp3 expression and were therefore much less likely to be contaminated with CD4+CD25+Foxp3− T effector cells. In our hands, the TLR2 agonist markedly enhanced the proliferation of conventional T cells. Secondly, in both the studies of Sutmiller et al (16) and Liu et al (5), very low levels of proliferation were observed in cultures of responder cells alone, high ratios (1:1 or 1:2) of Treg to responder cells were tested, and the magnitude of the reversal of suppression (30–40%) was not impressive. Liu et al (5) also claimed that the transient increase in Foxp3 mRNA induced by TCR activation of Treg was blocked by exposure to the TLR2 agonist, but did not analyze Foxp3 protein expression. In our hands, culture of the Treg for several days in the continuous presence of Pam3CSK4 has absolutely no effect on the expression of Foxp3 protein expression.
Conclusions based solely on in vitro studies with Treg must be interpreted with caution, as Treg function in vivo may be mediated by different mechanisms. Treatment of mice with Pam3CSK4 resulted in a moderate increase in the proliferation of antigen-specific Treg in response to immunization with antigen. Similar results were seen in the studies of Sutmiller et al (16) although they did not observe significant proliferation in the presence of antigen alone. It should be noted that Treg suppression of the expansion of antigen-specific T cells in vivo may be accompanied by expansion of the Treg (32). Thus, proliferation of Treg in vivo as in vitro does not indicate abrogation of Treg suppressive capacity. We used two different models to determine whether TLR2 engagement inhibited Treg function in vivo. The first model was similar to the one used by Liu et al (5) and involved pretreatment of the Treg with Pam3CSK4 prior to transfer in vivo. One of the most sensitive assays for Treg function in vivo is the ability of Treg to suppress the transfer of a global autoimmune syndrome by Scurfy T cells to RAG−/− mice (19). Treg expanded and treated with Pam3CSK4 were as suppressive as Treg expanded in the absence of the TLR2 ligand. In contrast, Liu et al (5) claimed that a brief exposure to Pam3CSK4 delayed the ability of Treg to treat IBD and completely inhibited the ability of the animals to clear L. major. We also used the system originally described by Kohm et al (23) in which supplementation of normal mice with Treg from WT mice modulates the induction of EAE induced by immunization with a MOG peptide. When TLR2−/− recipient mice were supplemented with WT Treg and treated with multiple injections of Pam3CSK4 during the induction of EAE, the decrease in disease severity was identical to that seen in mice not treated with the TLR2 ligand. The treatment regimen used was very similar to the one used by Sutmiller et al (16) in their studies of mice infected with C. albicans. However, they observed an enhancement of the magnitude of colonization of the mouse with C. albicans when WT Treg were transferred to TLR2−/− mice, and abrogation of this enhancement when the mice were treated with Pam3CSK4. Again, it is very difficult to determine the reasons for the differences between these results. Our studies used two well-characterized models of Treg mediated modulation of autoimmune disease, while the other studies used models of immune responses to pathogens. In any case, it appears that TLR2 stimulation does not reproducibly modulate Treg function in vivo.
One of the most interesting questions raised by these studies is the significance of expression of TLRs on Treg. Taken together, our studies and those of Crellin et al (15) and Zhorov et al (7) indicate that stimulation of Treg via TLR2 or TLR5 either enhances their suppressor function or their expansion and survival. Superficially, enhancement of Treg function should be deleterious to the immune response to a TLR ligand expressing pathogen. In many respects, one would have predicted that TLR ligands should actually abrogate Treg function in response to pathogens as suggested by some studies. Alternatively as proposed by Crellin et al (15), early in the immune response, the TLR signal delivered to APCs results in cytokine production that together with the direct action of the TLR ligands on responder T cells renders them resistant to suppression. The simultaneous stimulation of Treg with the TLR ligands would both result in enhancement of Treg viability and proliferation. When the acute response to the pathogen begins to subside, TLR-mediated enhanced Treg function can then play a critical role in the prevention of immune pathology or in maintaining low levels of pathogens that are needed for maintenance of immunologic memory (33). Further detailed analysis of the different signaling pathways modulated by TLR ligands in Treg compared to T effector cells may offer insights to the development of agents that can be used to selectively expand Treg in culture for use in cellular biotherapy or to enhance Treg function in vivo as a component of the treatment of autoimmune disease.
We thank the NIAID Flow Cytometry Section, particularly Bishop Hague and Elina Stregevsky for cell sorting.
1These studies were supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
3Abbreviations used in this paper: BLP, bacterial lipoprotein; DCs, dendritic cells; PRRs, pattern recognition receptors; Treg, regulatory T; PAMPs, pathogen-associated molecular patterns; LTA-SA, lipoteichoic acid from Staphylococcus aureus; WT, wild type; GITR, glucocorticoid-induced TNF receptor family-related protein; FSC, forward scatter; SSC, side scatter; 7-AAD, 7-amino actinomycin D; SC, scurfy mice; EAE, experimental autoimmune encephalitis; MOG, myelin oligodendrocyte glycoprotein.
The authors have no financial conflict of interest.