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The Journal of Immunology, 2007, 179, 1893 -1900
Copyright © 2007 by The American Association of Immunologists, Inc.
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Forkhead Box P3 Regulates TLR10 Expression in Human T Regulatory Cells1

Michael P. Bell, Phyllis A. Svingen, Meher K. Rahman, Yuning Xiong and William A. Faubion, Jr.2

Mayo Clinic, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4+CD25–FOXP3– T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The existence of a T cell subset with a regulatory phenotype in humans is now well established. Although the field of T regulatory (Treg)3 cells is rapidly expanding and frequently contradictory, three points of consensus relevant to this report have been reached. First, naturally occurring, thymus-derived CD4+CD25+ forkhead box P3+ (FOXP3+) Treg cells exist in the peripheral circulation in humans, and the biology of these human Treg cells has far-reaching implications to the field of autoimmunity and cancer (1). Second, Treg cells have been clearly shown to express functionally relevant TLRs, germline-encoded receptor proteins specific for pathogen-associated molecular patterns. These receptors have functional relevance because ligation of human Treg cells through TLR5 and TLR8 has been shown to affect proliferation and suppressive function (2, 3). Third, the transcription factor FOXP3 appears to be critically important for Treg cell development and function. Mutation of FOXP3 in humans results in the immune proliferative syndrome of immunodysregulation polyendocrinopathy enteritis and X-linked, and transduction of non-Treg cells with FOXP3 imparts suppressive function in both in vitro suppression assays and murine models of autoimmunity (4, 5). Thus, human Treg cells are critical to immune homeostasis, express functionally relevant TLRs, and are dependent upon the transcription factor FOXP3 for development and function.

In our studies of TLR expression in human Treg cells, we demonstrate that the transcription factor FOXP3 is required for expression of TLR10. FOXP3 directly binds DNA-binding elements proximal to the transcription start site (TSS) of TLR10 and exhibits positive promotional control. Furthermore, FOXP3 and NF-AT act as cotranscriptional activators of TLR10 expression. These data advance our understanding of the biology of TLR expression in human Treg cells and provide additional insights on Treg cell-specific genes regulated by the cooperative complex of FOXP3 and NF-AT.


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

All studies were reviewed and approved by the Institutional Review Board of the Mayo Clinic.

Cell isolation and flow cytometry

Human Treg cells were isolated from healthy control blood donors by Ficoll separation and magnetic bead sorting (CD4+CD25+ Treg cell isolation kit, 130-091-301; Miltenyi Biotec). Only cells of the highest CD25 expression (CD252+) were selected through incubation with a limiting quantity of anti-CD25 Ab beads, as described (2 µl of anti-CD25 beads/107 cells) (6). Nonregulatory CD25– cells were selected by collecting flow-through from saturating amounts of anti-CD25 Ab (20 µl of anti-CD25 beads/107 cells). Cells of intermediate CD25 expression (CD25+) used for immunoblotting for TLR10 were collected using 10 µl of anti-CD25 beads/107 cells. CD252+ cells are ≥60% FOXP3 positive (range 60–75%), and CD25– cells are ≤3% positive for FOXP3 upon intracellular staining and flow cytometry. The suppressive phenotype of CD252+ cells was confirmed through in vitro suppression assays. On average, the suppression of responder cell proliferation at a 1:1 ratio with Treg cells is >75%. For primary T cell transfection studies and chromatin immunoprecipitation (ChIP) assays, both CD4+CD252+ T cells and CD4+CD25– T cells were expanded over 10 days using anti-CD3/anti-CD28-conjugated beads (DynaBeads CD3/CD28 T Cell Expander, 111.31; Dynal Biotech) and IL-2 (7).

Flow cytometry was performed using the FACScan (BD Biosciences) and four-color staining. Commercially available Abs for TLR10 (Imgenex), FOXP3 (BioLegend), CD4 (eBioscience), and CD25 (eBioscience) were used per manufacturer’s specifications.

Western blotting

Protein was extracted from whole cell lysates derived from freshly isolated Treg cells and non-Treg cells. The cells were lysed in mild lysis buffer, as previously published (8). Protein (20 µg) was run on 10% gel over 6.5 h at 10mAmps. Upon transfer overnight to nitrocellulose, the membrane was incubated with anti-TLR10 polyclonal Ab (Imgenex) in 5% milk for 1 h. TLR10 protein was detected with 1/1000 dilution of anti-rabbit-HRP conjugate and ECL luminescence detection kit (SuperSignal West Femto Chemiluminescent Kit; Pierce).

EMSA

Nuclear extracts were made according to the method described by Dignam et al. (9) from Jurkat cells transfected with PCI-human FOXP3 full length. Briefly, 15 million cells were transfected by electroporation with 30 µg of plasmid DNA. Cells were harvested 20 h later, and nuclear extracts were isolated and quantitated. EMSAs were performed on 3 µg of extracts, 2 µg of poly(dI-dC), and 30,000 cpm of labeled oligonucleotide corresponding to the TLR-10 promoter. The coding strand sequence is as follows: 5'-ATTTGTTTGTTTGTTTATTTATTTATTTATTTATTT-3'.

Samples were allowed to react 20 min at room temperature before loading on a 6% nondenaturing polyacrylamide gel. Gels were dried and exposed on film for autoradiography. To demonstrate specificity of the complex, polyclonal rabbit anti-FOXP3 (Abcam ab10563) or rabbit IgG was preincubated on ice for 20 min before addition of labeled probe.

RT-PCR and ChIP assay

To define the TSS, we first selected the seven available transcripts for TLR10 from the three most commonly used databases for sequence information (Refseq, Ensembl, MGC). All transcripts started within 500 bp of one another; thus, an estimate of the TSS was made ~829 bp upstream of exon 1. RNA for PCR was isolated using Qiagen Rneasy kit 74104 per manufacturer’s protocols. Quantitative PCR was performed using Applied Biosystem’s 7900HT real-time PCR instrument. Detection was Power SYBR Green from Applied Biosystems. ChIP was performed using Treg cells expanded in vitro over 10 days, as described above. Approximately 2.5 million cells in 13 ml of medium were cross-linked with 270 µl of 37% formaldehyde. Cells were washed twice in ice-cold PBS with inhibitors. Samples were lysed in 500 µl of SDS lysing buffer with inhibitors for 10 min on ice. Samples were then subjected to sonication at 45 W at 10-s bursts for a total of eight times. DNA gel fractionation confirmed DNA shearing to ~200 kb. The samples were incubated with isotype control 0.5 µg of rat Ab (BD Pharmingen 553927) or 0.5 µg of anti-FOXP3 (eBioscience clone PCH101 14-4776-82) for 18 h. Ab was then captured with protein A-Sepharose beads over 3 h and subsequently eluted with two 250-µl aliquots of elution buffer, as per manufacturer’s protocol (Upstate Biotechnology 17-295). Concomitant eluted DNA was amplified with either primers directed at the putative FOXP3 binding site or control primers developed to bind 2.5 kb upstream. PCR product from each reaction was resolved by gel fractionation as was input DNA to ensure equal template. The four oligonucleotide primers used in ChIP assay for FOXP3 and NF-AT are as follows: forkhead (FKH) forward primer, 5'-CTCTCTGGCACAAGTTACGCT-3'; FKH reverse primer, 5'-AAACCACCATGGCATGTGTAT-3'; control forward, 5'-ACTGCCAGGGTCCTATCAAGC-3'; and control reverse, 5'-TCAGGTCAAGACTCGGGTGAA-3'.

Transfection and luciferase assays

Primary human CD4+ T cells were transfected using previously described methodology (10). Briefly, isolated cells were electroporated with 332 V at 1-pulse, 10-pulse length/ms and transfected with a PCI vector (Promega) containing the coding sequence for human FOXP3. Jurkat T cell transfections were performed using electroporation on a BTX Model 820 square wave electroporator at 310 V with 1 pulse at 10 ms and a PCI vector containing either FOXP3 or mutant FOXP3 contructs.

Luciferase assays were performed through concomitant transfection of the pT8 luciferase reporter construct (American Type Culture Collection) with putative FOXP3-binding elements inserted. The luciferase reading was made after overnight incubation of transfected cells on a Berthold Lumat LB 9507 luminometer and controlled for cell death with internal standard renilla, as per protocols previously described (Promega Dual Luciferase Reporter Assay system technical manual).

Gene silencing

To produce FOXP3-specific targeting short hairpinned RNA (shRNA) molecules, complementary oligonucleotides to human FOXP3 were synthesized. Each oligonucleotide pair contains a 5' BglII and 3' HindIII overhang, an RNA polymerase III start and termination sequence, and 19 nt (N19) of FOXP3-specific sequence separated by a 9-nt loop. The invariant nucleotide sequences of both the upper and lower oligonucleotide strands are 5'-GATCCCC(N19)ttcaagaga(61N)TTTTTGGAAA-3' and 3'-GGG (N19)aagttctct(61N)AAAAACCTTTTCGA-5'. The specific targeting sequence (N19) for FOXP3 was subsequently subjected to BLAST search algorithm against the human expressed sequence tag database to confirm targeting specificity. The three sequences for FOXP3 targeted in this study are as follows: FOXP3 A, 5'-GGCACTGACCAAGGCTTCA-3'; FOXP3 B, 5'-CTCCCCCATGATATCCTTT-3'; and FOXP3 C, 5'-GTGAGAAGGTCTTCGAAGA-3'. The shRNA sequences were cloned into the mammalian expression vector PCMS3-HIP-EGFP (sequence selection and expression vector provided by D. Billadeau, Mayo Clinic, Rochester, MN).

Upon transfection with FOXP3, Jurkat T cells were cotransfected using electroporation with FOXP3-targeting shRNA plasmids A, B, or C. Pooled FOXP3-specific small interfering RNA (siRNA) obtained from Dharmacon was also transfected as a positive control.

Relative FOXP3 suppression was determined by immunoblot 24 h after transfection. The most effective FOXP3-targeting sequence (shRNA C) was used in subsequent experiments of FOXP3 gene silencing.

Statistical analysis and data reproducibility

All experiments were performed a minimum of three times. In experiments with results that can be quantified (PCR and luciferase), experiments were performed in triplicate with the mean and SD reported. ChIP assays were performed six times with four different blood donors during protocol optomization, and the result shown is representative of the last three experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TLR10 is constitutively expressed in human Treg cells

Primary human Treg cells were isolated from the peripheral blood of healthy blood donors, as described in Materials and Methods. Quantitative PCR performed on extracted RNA from Treg cells demonstrated 200-fold expression of TLR10 mRNA in human Treg cells compared with CD4+CD25– non-Treg cells. The mRNA expression of TLR10 is similar to that of TLR8, and greater than that of TLR5, the only two TLRs to date shown to be functionally relevant in human Treg cells (Fig. 1A) (2, 3). These data were confirmed by flow cytometry, demonstrating constitutive expression of TLR5, TLR6, TLR8, and TLR10 in freshly isolated human Treg cells (Fig. 1B). Other than the established expression of TLR5 (2), CD4+CD25– non-Treg cells did not constitutively express TLR protein by flow cytometry (Fig. 1B). TLR10 protein could also be detected in Treg cells, but not CD4+CD25– non-Treg cells by Western blotting (Fig. 1C). In subsequent experiments of transcriptional regulation, we chose to focus on TLR10, because this receptor showed consistently the greatest degree of constitutive mRNA expression and protein expression by flow cytometric analysis. The transcription factor FOXP3 has been shown to induce CTLA-4 and CD25 (11), two other cell surface receptors constitutively expressed by Treg cells; thus, we next examined the 5' genomic DNA upstream of TLR10 for potential FOXP3-binding elements.


Figure 1
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  		FIGURE 1. TLR10 is constitutively expressed in human CD4+CD25+ Treg cells. The constitutive expression of both TLR10 mRNA (A) and protein (B and C) is demonstrated in freshly isolated, resting human Treg cells. A, Quantitative PCR demonstrates the fold expression of TLR mRNA isolated from Treg cells compared with non-Treg cells. The mean cycle threshold values for beta-actin, TLR8, and TLR10 representing three independent experiments are demonstrated in the inset. B, Identification of TLR protein expression by flow cytometry in both Treg and non-Treg cells is shown. The shaded, closed histogram represents isotype control, whereas the heavy line represents TLR-specific mAb. C, The presence of TLR10 protein in human Treg cells is further confirmed by Western blotting. CD252+ refers to the CD4+ T cells of highest CD25 expression, and CD25+ refers to cells of intermediate expression obtained through limiting dilutions of anti-CD25 Ab and magnetic bead separation (Materials and Methods). Two positive controls include a lymph node lysate (LN Control) commercially available with the anti-TLR10 Ab and the Raji B cell line. Data represent experiments repeated at least three times.

 
FOXP3 associates with binding elements in proximity to the TSS of TLR10

The upstream 5-kb genomic DNA sequence 5' of the first exon of TLR10 was screened for the established forkhead consensus-binding element as follows: T (G/A) TT (T/G) (G/A) (T/C) (11). Eight forkhead consensus-binding elements (demonstrated in box; Fig. 2B) were identified in overlapping tandem over a 38-nt region within 2.2 kb of the suspected TSS.


Figure 2
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  		FIGURE 2. FOXP3 associates with binding elements in proximity to the TSS of TLR10. A, In this EMSA, clear bands are demonstrated in lanes containing nuclear extract from FOXP3-transfected Jurkat T cells (FOXP3, FOXP3 plus rabbit IgG), but not from lane vector, which contains nuclear extract from cells transfected with vector alone. Specificity for FOXP3 is demonstrated by the disruption of the complex through preincubation with a polyclonal Ab specific for the DNA binding domain of FOXP3 (+anti-FOXP3). The addition of competing unlabeled probe (+cold probe) likewise disrupts complex identification. This EMSA is representative of three experiments. B, This 38-nt sequence is ~2.2 kb upstream of the TLR10 TSS and incorporates eight perfect FOXP3-binding elements (boxed sequence). ChIP assay with FOXP3-binding element-specific primers (FKH primers) or control primers (designed 2.5 kb upstream, CTRL primers) demonstrates clear binding of FOXP3 to DNA in proximity to the TLR10 TSS.

 
A 36-bp radiolabeled probe was synthesized incorporating the suspected FOXP3-binding elements of the TLR10 promoter region (see Materials and Methods). Nuclear extract from Jurkat T cells transiently transfected with FOXP3 or empty vector was prepared and used in EMSA. Fig. 2A demonstrates probe-transcription factor complex formation in lanes containing nuclear extract from FOXP3-transfected (FOXP3; Fig. 2A), but not vector-alone-transfected (vector, Fig. 2A) Jurkat cells. All lanes with the exception of the first (vector) represent nuclear extract derived from Jurkat cells transfected with FOXP3. Upon preincubation of the nuclear extract with a FOXP3-specific polyclonal Ab (specific for the C terminus, DNA binding region), complex with the DNA probe is blocked (+anti-FOXP3; Fig. 2). Control rabbit IgG (+rabbit IgG; Fig. 2A) does not affect complex formation, and competition with 2 µM unlabeled probe depletes the band as well (+cold probe). These findings suggest that probe:protein complex formation is a result of direct binding of FOXP3 to DNA and not the result of a FOXP3-induced transcription factor:probe interaction. To further support this hypothesis, we performed ChIP experiments assaying the proximal elements of this potential TLR10 regulatory region. In primary human Treg cells, we performed ChIP assays using FOXP3-specific Ab and primers designed to amplify the region of DNA incorporating the suspected FOXP3-binding elements proximal to TLR10.

Primers were designed to anneal in proximity to this suspected forkhead-binding element (FKH primer; Fig. 2B) or irrelevant DNA ~2.5 kb further upstream (control primers, CTRL; Fig. 2B). Fig. 2B demonstrates that upon immunoprecipitation with FOXP3-specific Ab (right two lanes), but not an isotype control rat IgG (left two lanes), the primer pair specific for the genomic region rich with potential FOXP3-binding elements (boxed nucleotide sequence) produces a strong band. The control primer pair designed to irrelevant DNA upstream did not produce a PCR product (labeled CTRL primers). Reactions consisting of input DNA (preprecipitation) as template produced equivalent product with both primer pairs (data not shown). We conclude that FOXP3 binds to this potential regulatory region upstream of TLR10.

FOXP3 binding to the suspected promoter region of TLR10 results in transcriptional activation

To test whether the DNA binding established in the EMSA and ChIP assays results in transcriptional regulation, we cloned two DNA fragments into separate luciferase reporter constructs. The first, a 640-bp DNA fragment, incorporated the suspected regulatory region depicted in Fig. 2B (referred to as luciferase-full length). We then deleted 38 nt containing the overlapping FKH consensus-binding elements (boxed sequence in Fig. 2B) from this 640-bp DNA fragment to create the luciferase-deletion construct. Transient transfections were performed into Jurkat T cells with wild-type FOXP3 or a mutated form, truncated to remove the DNA binding domain (FOXP3-DBDmt) (12). Our luciferase reporter assays demonstrate that the FOXP3-binding element functions as a cis-regulatory region as transfection of wild-type FOXP3, but not FOXP3-DBDmt, into Jurkat T cells results in luciferase expression (Fig. 3A). Deletion of the suspected FOXP3-binding element in the luciferase reporter vector (luciferase-deletion; Fig. 3B) abrogates transcriptional activity of FOXP3, strengthening the evidence supporting the regulatory role of FOXP3 over TLR10 gene expression.


Figure 3
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  		FIGURE 3. Binding of FOXP3 to the suspected TLR10 promoter region results in transcriptional activity. A, Luciferase activity is recorded from Jurkat T cells cotransfected with 0.1 µg of FOXP3-expressing plasmid and a luciferase reporter vector containing the suspected FOXP3-binding elements (exact sequence shown in Fig. 2B). FOXP3-DBDmt refers to cells transfected with 0.1 µg of the FOXP3 DNA-binding mutant. B, Mutation of the luciferase reporter vector by deleting the 38 nt incorporating the FOXP3-binding elements (Luciferase-deletion) eliminates the transcriptional activity seen upon transfection with FOXP3. These experiments were performed in triplicate and repeated three times.

 
To further support specificity of FOXP3 to this apparent transcriptional activity, we developed shRNA constructs to selectively silence FOXP3. Three shRNA constructs were designed with a validated selection algorithm (13) and tested for the ability to silence FOXP3 by Western blot analysis (Fig. 4A). Pooled siRNA (Dharmacon) was also tested for the ability to silence FOXP3 (Fig. 4A). Jurkat T cells were cotransfected with wild-type FOXP3 expression vector (FOXP3; Fig. 4A) and one of three shRNA constructs predicted to silence FOXP3 (shRNA A, B, or C; Fig. 4A). The most effective construct (shRNA C) was then used in the following experiment. Cotransfection of shRNA plasmid C inhibits FOXP3-dependent transcriptional activity of the TLR10 promoter luciferase construct (Fig. 4B). We conclude that FOXP3 binds to regulatory elements proximal to human TLR10, and in this in vitro system promotes expression. We next studied the potential of FOXP3 to regulate transcription of TLR10 in primary human T cells.


Figure 4
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  		FIGURE 4. FOXP3 gene silencing abrogates transcriptional activity. A, Jurkat T cells were cotransfected with the FOXP3 expression vector (labeled FOXP3) or empty vector (labeled vector) and one of three shRNA constructs designed to silence FOXP3. Silencing of protein expression is demonstrated most strongly for shRNA construct C. Commercially available siRNA (siRNA, 100 pmol) is demonstrated as a positive control. B, Cotransfection of FOXP3 and the shRNA C construct abrogates the TLR10 promoter luciferase reporter activity. PCMS3 vector represents the empty shRNA vector.

 
The transcription factor FOXP3 is sufficient for expression of TLR10 in primary human T cells

Primary human FOXP3– CD4+CD25– T cells were isolated from the peripheral blood of healthy blood donors and expanded in vitro for 10 days using anti-CD3/CD28-conjugated beads in the presence of IL-2 (14). Aliquots of expanded cells were transfected through electroporation with either FOXP3 or empty vector. Twenty-four hours after transfection, mRNA extracted from transfected cells was analyzed by quantitative PCR for expression of FOXP3 and TLR10 mRNA. CD4+ T cells ectopically expressing FOXP3 demonstrated a greater than 100-fold change in TLR10 mRNA when compared with T cells transfected with vector alone (Fig. 5A). TLR8, which showed high mRNA expression in Fig. 1, was not greatly expressed in non-Treg FOXP3-transfected cells. Exploration of the genomic region 5' to the TSS of TLR8 found only two widely spaced putative FOXP3 binding sites compared with the eight overlapping sites, as demonstrated for TLR10, an observation that may explain the apparent differential regulation. To confirm the mRNA expression data with protein expression of TLR10, we performed transient transfection of primary, freshly isolated CD4+CD25– T cells with FOXP3 + GFP (GFP, cloned into PCI mammalian expression vector; Promega) or empty vector + GFP. Twenty-four hours after transfection, GFP-positive cells were assessed for TLR10 protein expression by flow cytometry. Fig. 5B demonstrates the mean fluorescent intensity for TLR10 to be significantly greater among cells transfected with FOXP3-GFP vs those transfected with GFP alone (mean fluorescence intensity 19.45 ± 0.71 vs 12.08 ± 0.28; p < 0.05). Each data point represents one of six experiments from six different healthy individuals. Thus, the transcription factor FOXP3 appears to be sufficient for TLR10 gene transcription. As recent data demonstrate the ability of FOXP3 and NF-AT to form cooperative complexes promoting the transcription of genes critical to Treg function (i.e., CTLA-4 and CD25) (11), we next examined potential cotranscriptional control of TLR10 by FOXP3 and NF-AT.


Figure 5
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  		FIGURE 5. Introduction of the transcription factor FOXP3 is sufficient for the expression of TLR10 in human non-Treg cells. A, CD4+CD25– primary non-Treg cells were isolated, expanded, and transfected with the FOXP3 expression vector. Twenty-four hours after transfection, TLR10 mRNA is evident by quantitative PCR. Data shown are the mean and SE of three independent experiments using three different healthy control subjects. One experiment demonstrated several thousand-fold expression of TLR10 mRNA, thus producing the large error bar. B, Freshly isolated FOXP3– CD4+CD25– non-Treg cells were transfected with either FOXP3 plus GFP or GFP alone. Twenty-four hours after transfection, TLR10 protein expression is evident by flow cytometry. The data shown are the mean fluorescent intensities for each of six experiments of six different healthy control subjects. The mean of these experiments is represented by a line.

 
NF-AT coassociates with FOXP3 and regulates expression of human TLR10

Recent data demonstrate that NF-AT and FOXP3 form cooperative complexes with composite sites on the IL-2 promoter, and in the absence of NF-AT, FOXP3 binds quite poorly to target DNA elements (11). Indeed, NF-AT consensus-binding elements exist, flanking the suspected FOXP3-binding elements on TLR10 (GGAA, 6 nt upstream and 28 nt downstream). To examine the role of NF-AT in FOXP3-dependent TLR10 gene transcription, we performed three experiments. First, the effect of TCR stimulation on TLR10 protein expression was studied. TCR stimulation of freshly isolated Treg cells, but not CD4+CD25– non-Treg cells, results in up-regulation of TLR10 protein by flow cytometry (Fig. 6A), and this augmentation of protein expression is inhibited by preincubation of Treg cells with the calcineurin inhibitor, cyclosporine A (Fig. 6A). Preincubation with cyclosporine A does not affect resting expression of TLR10 in Treg cells (Fig. 6A). As the transcription factor NF-AT represents the immediate downstream effector molecule of calcium-dependent TCR signals, we next examined the direct interaction between NF-AT and FOXP3 on the TLR10 promoter sequence. NF-AT binding to chromatin in proximity to the FOXP3-binding elements was established through a second ChIP assay. Fig. 6B shows the results of this second ChIP assay using precisely the same primers as the study of Fig. 2B, but probing with an NF-AT-specific Ab. Thus, this result demonstrates binding of NF-AT in the same region of chromatin as FOXP3.


Figure 6
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  		FIGURE 6. FOXP3 and NF-AT form a cooperative complex resulting in TLR10 transcriptional activity. A, Stimulation of freshly isolated Treg cells, but not non-Treg cells through the TCR (plate-bound anti-CD3 Ab, 0.5 µg of clone UCHT1, overnight; BD Pharmingen) results in enhanced TLR10 protein expression as determined by flow cytometry. Non-Treg cells do not express significant TLR10 protein under any conditions (left panel). Cyclosporine A (lightly shaded histogram) does not affect resting levels of TLR10 expression (dark histogram) in Treg cells (center panel). Shown on the right panel, the augmented expression (dark, open histogram) is inhibited (lightly shaded histogram) upon preincubation for 30 min with cyclosporine A (1 µg/ml). As in all panels, the isotype control Ab is represented in the open, hatched histogram, and the resting TLR10 expression is demonstrated by the darker shaded histogram. B, Immunoprecipitation for the transcription factor NF-AT and DNA amplification using the same FKH and CTRL primers of Fig. 2B was performed. The presence of PCR product demonstrated in the lane precipitated with anti-NF-AT, but not isotype control Ab suggests NF-AT DNA binding to this region of DNA. Control primers designed to anneal to DNA 2.2 kb upstream of our region of interest demonstrated no significant PCR product (CTRL). This ChIP assay was repeated three times with three separate human samples. C, Cotransfection experiments into Jurkat T cells were performed using the FOXP3 expression vector or the WWRR mutant FOXP3 expression vector. In the left panel, the luciferase reporter construct is the ARRE2 IL-2 promoter (NF-AT:AP-1-binding element), and in the right panel, the luciferase reporter construct is the TLR10 promoter sequence used in the previous experiments of FOXP3 transcriptional regulation. The WWRR mutation disrupts FOXP3 repressor function of the NF-AT:AP-1-dependent IL-2 reporter (left panel) and the FOXP3-dependent transcriptional activation of the TLR10 reporter (right panel), evidence for the requirement of FOXP3:NF-AT synergistic complex formation to promote the expression of TLR10. These experiments were performed in triplicate and repeated three times.

 
Finally, to establish the physiologic relevance of a FOXP3:NF-AT cooperative complex in the transcriptional regulation of TLR10, we created point mutations in FOXP3 predicted to interfere with FOXP3 and NF-AT association. Point mutations predicted by crystal structure to interfere with FOXP3:NF-AT complex formation have recently been published (11). We chose to mutate human FOXP3 through four point mutations duplicating the murine WWRR FOXP3 mutant (11). As recently published, disruption of salt bridge formation with the fg loop of NF-AT1 through E->R mutations (E399R E401R) and steric disruption of FOXP3 and NF-AT shape complementarity through tryptophan insertion (T359W N361W), result in a mutant FOXP3 protein (WWRR mutant) capable of binding DNA, but incapable of NF-AT coassociation (11). Using an IL-2 ARRE2 promoter reporter assay, we first tested the capacity of our point mutations to disrupt the cooperative FOXP3:NF-AT complex. It is well established that FOXP3 inhibits NF-AT:AP-1-dependent IL-2 gene transcription, and this inhibitory function appears to be dependent upon FOXP3:NF-AT complex formation (11). This inhibitory effect is demonstrated in Fig. 6C (upper panel) as transfected wild-type FOXP3 inhibits IL-2 reporter activity in stimulated Jurkat T cells. Transfection of the WWRR mutant results in complete loss of IL-2 repressor function, confirmation of the disruption of FOXP3:NF-AT complex formation in this mutation of human FOXP3. Fig. 6C, lower panel, demonstrates that transfection of the WWRR mutant form of FOXP3 does not result in TLR10 reporter activity, indicating the requirement for FOXP:NF-AT complex formation in the transcriptional regulation of TLR10.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report three key findings. First, TLR10 is expressed on human Treg cells. Second, the transcription factor FOXP3 is sufficient for TLR10 expression in human T cells, and third, this transcriptional regulation appears to be dependent upon FOXP3:NF-AT cooperative complex formation.

The biologic relevance of TLR expression on Treg cells is considered first. TLR10, an orphan receptor without a murine homologue, is most closely related to TLR1 and TLR6, has been previously identified on plasmacytoid dendritic cells and B cells, and appears to signal in a MyD88-dependent fashion (8). Defining the functional relevance of TLR10 expression in human Treg cells is problematic given the current lack of an established TLR10 ligand. However, the robust expression of TLR10 unique to Treg cells and the significant up-regulation upon TCR stimulation argue the importance of this pathogen recognition receptor to human Treg cell biology. Indeed, polymorphisms in TLR10 have been recently linked to asthma, highlighting the importance of this TLR in human immune-mediated disease (15).

Other TLRs are clearly relevant to human Treg cell biology. We and others have shown TLR5, TLR6, TLR8, and TLR10 to be abundantly expressed on human Treg cells (2, 3). TLR2 appears to be expressed to varying degrees, yet functionally relevant (16). TLR5, one of two TLRs activated by proteins, is the receptor for flagellin, a common bacterial Ag present on most motile enteric bacteria. A strong adaptive immune response to a specific family of flagellins has been identified in both murine models of inflammatory bowel disease and patients with Crohn’s disease (17, 18, 19), suggesting a selective impairment in Crohn’s disease of Treg cell responsiveness to specific bacterial Ags. In support of this model, flagellin stimulation of Treg cells through TLR5 results in enhanced suppressor function in vitro (2). TLR8 (along with TLR3 and TLR7) are intracellular receptors that recognize ssRNA and dsRNA in which modifications such as methylation have occurred (20). In a study of Ag-specific Treg cells isolated from tumor-infiltrating lymphocyte lines, TLR8 ligation regulated Treg cell-suppressive function in vitro. Although these data support a mechanism linking TLR8 to Treg cell function, the physiologic relevance to normal immune responses in vivo remains to be determined (3). TLR6 and TLR10 are most closely related to TLR1 and appear to signal in heterodimeric units. TLR1/2 heterodimers and TLR2/6 heterodimers recognize tri- and diacetylated lipopeptides, respectively (21). There are yet no data regarding the functional relevance of TLR6 or TLR10 on human Treg cells.

The requirement of FOXP3 for TLR10 expression is considered next. FOXP3-dependent genes have been the subject of two recent publications (22, 23). Using ChIP for FOXP3 in murine primary Treg cells or a FOXP3-transduced murine T cell hybridoma followed by DNA microarrays, FOXP3 was estimated to associate with ~1200 genes (22, 23). Although no murine TLRs were identified as FOXP3 regulatable in these microarrays, these two studies are relevant to our data for three reasons. First, precisely the same forkhead DNA-binding motif (T G/A TTTGT) evident in the TLR10 regulatory region of our study was found to significantly discriminate between FOXP3-bound chromatin and unbound chromatin (p < 10–41), adding to the evidence that this region is a functional binding element for human TLR10. Second, consistent with our data of FOXP3-induced TLR10 expression, FOXP3 transcriptional programs demonstrated significant transcriptional activation, an observation contrary to the common view that FOXP3 acts as a transcriptional repressor. Third, due to the stringent criteria used to designate differential expression, numerous known targets of FOXP3 were not identified in these arrays, including CTLA-4, Bcl-10, Cd53, Rbpsuh, and Rgs, supplemental data (23). Thus, these data are in agreement with our evidence that FOXP3, binding to a highly conserved element, commonly activates gene transcription.

Little is established regarding the transcriptional regulation of TLR genes in human cells. Indeed adding to the complexity is the disparate literature due to different cell types studied and interspecies variation (mouse vs human). A general theme is that TLRs that activate the type I IFN response (TLR3, TLR4, TLR7, TLR8, and TLR9) are induced in a feedback loop by IFN-{alpha}/beta through IFN regulatory factor family transcription factors (24, 25, 26). Similarly, murine TLR2, which signals through the transcription factor NF-{kappa}B, can be induced through NF-{kappa}B. In this manner, TLR2 is rapidly expressed upon proinflammatory stimuli (27). In humans, TLR2 appears to be alternatively regulated by the Sp1 family of transcription factors (27). There are no other published data germane to the transcriptional regulation of TLR5, TLR6, or TLR10.

The cooperative complex formation of FOXP3 and NF-AT is considered last. The crystal structure of the tricomplex of FOXP3, NF-AT, and DNA has been recently solved. Using this model, the investigators predicted key amino acid substitutions that would disrupt this cooperative complex. The murine FOXP3 mutant WWRR completely disrupted FOXP3/NF-AT coassociation, yet maintained FOXP3 DNA-binding ability (11). Because murine FOXP3 and human FOXP3 are highly homologous, particularly so for the domains critical to FOXP3 function (DNA binding domain, leucine zipper region, nuclear targeting sequence, and NF-AT association sites), we constructed the identical WWRR mutation in human FOXP3. FOXP3 has been previously thought to be a negative regulator of NF-AT-dependent gene transcription (28), and indeed those genes dependent upon consensus NF-AT- and AP-1-binding elements such as IL-2 are negatively regulated by FOXP3 (11). However, genes critical to Treg function such as CTLA-4 and CD25 have been shown to be positively regulated through consensus NF-AT/FOXP3-binding elements (11). Our study is the first to demonstrate a TLR gene with consensus NF-AT/FOXP3-binding elements that exert positive promotional control.

In conclusion, human Treg cells express substantial TLR10, and this expression is enhanced through TCR activation. The Treg cell-defining transcription factor FOXP3 in cooperation with NF-AT promotes TLR10 gene expression. Whether FOXP3 regulation of TLR family members in human Treg cells is a general effect is currently under study. The import of TLRs in general and TLR10 to asthma in particular deem significant the further study of TLR regulation and significance to Treg function in human immune-mediated disease.


   Acknowledgments
 
We acknowledge Dr. Cox Terhorst for his mentorship and Drs. David J. McKean and Paul J. Leibson for their mentorship and critical review of these data.


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


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

1 This work was made possible through the Dana Foundation Human Immunology Award and National Institutes of Health Grant DK64194-04. Back

2 Address correspondence and reprint requests to Dr. William A. Faubion, Jr., Mayo Clinic, Department of Internal Medicine, Division of Gastroenterology and Hepatology, 200 1st Street SW, Rochester, MN 55905. E-mail address: Faubion.William{at}mayo.edu Back

3 Abbreviations used in this paper: Treg, T regulatory; ChIP, chromatin immunoprecipitation; FKH, forkhead; FOXP3, forkhead box P3; shRNA, short hairpinned RNA; siRNA, small interfering RNA; TSS, transcription start site. Back

Received for publication March 5, 2007. Accepted for publication May 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Tang, Q., J. A. Bluestone. 2006. Regulatory T-cell physiology and application to treat autoimmunity. Immunol. Rev. 212: 217-237. [Medline]
  2. Crellin, N. K., R. V. Garcia, O. Hadisfar, S. E. Allan, T. S. Steiner, M. K. Levings. 2005. Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells. J. Immunol. 175: 8051-8059. [Abstract/Free Full Text]
  3. Peng, G., Z. Guo, Y. Kiniwa, K. S. Voo, W. Peng, T. Fu, D. Y. Wang, Y. Li, H. Y. Wang, R. F. Wang. 2005. Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science 309: 1380-1384. [Abstract/Free Full Text]
  4. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4: 330-336. [Medline]
  5. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4: 337-342. [Medline]
  6. Beyer, M., M. Kochanek, K. Darabi, A. Popov, M. Jensen, E. Endl, P. A. Knolle, R. K. Thomas, M. von Bergwelt-Baildon, S. Debey, et al 2005. Reduced frequencies and suppressive function of CD4+CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine. Blood 106: 2018-2025. [Abstract/Free Full Text]
  7. Hoffmann, P., R. Eder, L. A. Kunz-Schughart, R. Andreesen, M. Edinger. 2004. Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells. Blood 104: 895-903. [Abstract/Free Full Text]
  8. Hasan, U., C. Chaffois, C. Gaillard, V. Saulnier, E. Merck, S. Tancredi, C. Guiet, F. Briere, J. Vlach, S. Lebecque, et al 2005. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J. Immunol. 174: 2942-2950. [Abstract/Free Full Text]
  9. Dignam, J. D., R. M. Lebovitz, R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11: 1475-1489. [Abstract/Free Full Text]
  10. Bell, M. P., C. J. Huntoon, D. Graham, D. J. McKean. 2001. The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis. Nat. Med. 7: 1155-1158. [Medline]
  11. Wu, Y., M. Borde, V. Heissmeyer, M. Feuerer, A. D. Lapan, J. C. Stroud, D. L. Bates, L. Guo, A. Han, S. F. Ziegler, et al 2006. FOXP3 controls regulatory T cell function through cooperation with NFAT. Cell 126: 375-387. [Medline]
  12. Lopes, J. E., T. R. Torgerson, L. A. Schubert, S. D. Anover, E. L. Ocheltree, H. D. Ochs, S. F. Ziegler. 2006. Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor. J. Immunol. 177: 3133-3142. [Abstract/Free Full Text]
  13. Upshaw, J. L., L. N. Arneson, R. A. Schoon, C. J. Dick, D. D. Billadeau, P. J. Leibson. 2006. NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1 intermediate and phosphatidylinositol-3-kinase in human natural killer cells. Nat. Immunol. 7: 524-532. [Medline]
  14. Godfrey, W. R., Y. G. Ge, D. J. Spoden, B. L. Levine, C. H. June, B. R. Blazar, S. B. Porter. 2004. In vitro-expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood 104: 453-461. [Abstract/Free Full Text]
  15. Lazarus, R., B. A. Raby, C. Lange, E. K. Silverman, D. J. Kwiatkowski, D. Vercelli, W. J. Klimecki, F. D. Martinez, S. T. Weiss. 2004. TOLL-like receptor 10 genetic variation is associated with asthma in two independent samples. Am. J. Respir. Crit. Care Med. 170: 594-600. [Abstract/Free Full Text]
  16. Zanin-Zhorov, A., L. Cahalon, G. Tal, R. Margalit, O. Lider, I. R. Cohen. 2006. Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling. J. Clin. Invest. 116: 2022-2032. [Medline]
  17. Mow, W. S., E. A. Vasiliauskas, Y. C. Lin, P. R. Fleshner, K. A. Papadakis, K. D. Taylor, C. J. Landers, M. T. Abreu-Martin, J. I. Rotter, H. Yang, S. R. Targan. 2004. Association of antibody responses to microbial antigens and complications of small bowel Crohn’s disease. Gastroenterology 126: 414-424.
  18. Targan, S. R., C. J. Landers, H. Yang, M. J. Lodes, Y. Cong, K. A. Papadakis, E. Vasiliauskas, C. O. Elson, R. M. Hershberg. 2005. Antibodies to CBir1 flagellin define a unique response that is associated independently with complicated Crohn’s disease. Gastroenterology 128: 2020-2028.
  19. Sitaraman, S. V., J. M. Klapproth, D. A. Moore, III, C. Landers, S. Targan, I. R. Williams, A. T. Gewirtz. 2005. Elevated flagellin-specific immunoglobulins in Crohn’s disease. Am. J. Physiol. 288: G403-G406.
  20. Kariko, K., M. Buckstein, H. Ni, D. Weissman. 2005. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23: 165-175. [Medline]
  21. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  22. Zheng, Y., S. Z. Josefowicz, A. Kas, T. T. Chu, M. A. Gavin, A. Y. Rudensky. 2007. Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature 445: 936-940. [Medline]
  23. Marson, A., K. Kretschmer, G. M. Frampton, E. S. Jacobsen, J. K. Polansky, K. D. Macisaac, S. S. Levine, E. Fraenkel, H. von Boehmer, R. A. Young. 2007. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature 445: 931-935. [Medline]
  24. Guo, Z., S. Garg, K. M. Hill, L. Jayashankar, M. R. Mooney, M. Hoelscher, J. M. Katz, J. M. Boss, S. Sambhara. 2005. A distal regulatory region is required for constitutive and IFN-beta-induced expression of murine TLR9 gene. J. Immunol. 175: 7407-7418. [Abstract/Free Full Text]
  25. Siren, J., J. Pirhonen, I. Julkunen, S. Matikainen. 2005. IFN-{alpha} regulates TLR-dependent gene expression of IFN-{alpha}, IFN-beta, IL-28, and IL-29. J. Immunol. 174: 1932-1937. [Abstract/Free Full Text]
  26. Honda, K., H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, T. Taniguchi. 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434: 772-777. [Medline]
  27. Haehnel, V., L. Schwarzfischer, M. J. Fenton, M. Rehli. 2002. Transcriptional regulation of the human Toll-like receptor 2 gene in monocytes and macrophages. J. Immunol. 168: 5629-5637. [Abstract/Free Full Text]
  28. Rudensky, A. Y., M. Gavin, Y. Zheng. 2006. FOXP3 and NFAT: partners in tolerance. Cell 126: 253-256. [Medline]




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